key: cord- -mkv jc u authors: chen, yirong; badaruddin, hishamuddin; lee, vernon j.; cutter, jeffery; cook, alex r. title: the effect of school closure on hand, foot, and mouth disease transmission in singapore: a modeling approach date: - - journal: am j trop med hyg doi: . /ajtmh. - sha: doc_id: cord_uid: mkv jc u singapore implements a school closure policy for institutional hand, foot, and mouth disease (hfmd) outbreaks, but there is a lack of empirical evidence on the effect of closure on hfmd transmission. we conducted a retrospective analysis of , cases of hfmd over the period – at the national level and of , cases in , institutional outbreaks over the period – in singapore. the effects of school closure due to ) institutional outbreaks, ) public holidays, and ) school vacations were assessed using a bayesian time series modeling approach. school closure was associated with a reduction in hfmd transmission rate. during public holidays, average numbers of secondary cases having onset the week after dropped by % ( % credible interval – %), and during school vacations, the number of secondary cases dropped by % ( % credible interval – %). schools being temporarily closed in response to an institutional outbreak reduced the average number of new cases by , ( % credible interval , – , ). despite the positive effect in reducing transmission, the effect of school closure is relatively small and may not justify the routine use of this measure. hand, foot, and mouth disease (hfmd) is a common pediatric disease that is endemic in east and southeast asia , and increasingly found in north america , and europe. when caused by coxsackieviruses, hfmd usually leads to relatively mild infections with symptoms that are self-limiting. however, hfmd caused by human enterovirus may lead to complications involving the nervous system and result in reduced cognitive function, delayed neurodevelopment, and motor impairment, or death [ ] [ ] [ ] ; in china, an estimated - children die of hfmd annually. because of this potential severity, the ministry of health in singapore imposes strict control policies, especially in preschools. in preschools, daily routine health checks for all children and isolation of suspected cases are implemented for early detection and control of outbreaks. should transmission continue within an outbreak, school closure is enforced. this policy was implemented following an outbreak in in which several children died from enterovirus complications , ; the details have evolved over time, but until recently, if a school has more than cases or an attack rate more than % with a transmission period more than days, the school will be required to close for a period of days. in the most updated guideline, the policy has been relaxed somewhat, and ministry of health will consider the predominant circulating strain when assessing closure in addition to the trigger. this change provides an avenue to assess the impact of this policy. school closure as a form of social distancing intervention to mitigate transmission during an infectious disease outbreak is often found in countries' pandemic preparedness plans. the influenza literature shows that school closure may effectively reduce the spread, [ ] [ ] [ ] and school vacations have a significant impact in limiting transmission. , analyses also suggest that school closure combined with the use of antiviral agents is cost-effective and is a justifiable strategy for mitigating influenza pandemics. , there is, however, a lack of empirical evidence on the effect of school closure on hfmd transmission: one exception is a review of hfmd in hong kong that revealed fewer hfmd consultations than expected during the severe acute respiratory syndrome and the influenza pandemic, which was attributed to various control measures including school closure. , this article aimed to assess the effect of school closure on hfmd transmission. in singapore, hfmd is endemic with year-round transmission and is legally notifiable by physicians and childcare teachers, as well as actively screened for in preschool-aged children. these policies provide data that enable us to obtain three sources of information on the effect of school closure: ) the reduction in the numbers of cases after a public holiday, when childcare centers and schools close; ) the reduction during school vacations; and ) the impact within childcare centers of school closure in response to an ongoing outbreak. singapore's school closure policy for hfmd, which has been implemented for over a decade, in tandem with a comprehensive hfmd surveillance system, therefore, provides a unique opportunity to assess the impact of this important method of outbreak control. source of data. ministry of health, singapore, actively monitors and publishes the incidence of hfmd, which was made a notifiable disease in the year . two sets of data on hfmd were extracted from the ministry's records for this study. the first dataset contains aggregate reported hfmd cases from to , with the number of daily cases with onset of symptoms, stratified by age. the second dataset contains information on all hfmd outbreaks in childcare centers and kindergartens in singapore, during the period - . this provides the cumulative number of cases per day in each preschool with an outbreak, together with the school type (childcare centers or kindergarten), enrollment size, whether the schools were closed because of the outbreak, and, if so, dates of closure and reopening. data were retained at a daily resolution for two analyses but aggregated to weekly for the analysis of vacations. data were collected under singapore's infectious disease act, and because aggregate non-identifiable data were used, institutional review board approval was not deemed necessary for this study. statistical analysis. separate statistical analyses were performed to investigate the ) public holiday effect, ) school vacation effect, and ) school closure effect on hfmd transmission, as described in the following paragraphs. public holiday effect. there are typically public holidays in singapore each year, as detailed in supplemental table ; these are a mix of secular and religious holidays, some of which rotate around the year following the lunar calendar. if a public holiday falls on sunday, the following monday will be a public holiday. we derived the dates of all public holidays from to from the official listing of the ministry of manpower. the effect of public holiday on hfmd transmission was measured by quantifying the reduction attributable to the public holiday. because time points not immediately preceding or following a public holiday contribute little information to the effect of the public holiday, rather than considering a time series model, we developed a bayesian model of the time points surrounding public holidays. the number of cases in the week before the holiday i is modeled as x i ∼ poissonðα i × μÞ, and the number the week after as y i ∼ poissonðα i × ½ μ + θμÞ, or y i ∼ poissonðα i × ½ μ + θμÞ for two-day-long holidays. here, μ represents the average number of cases on a typical day; estimates of θ and a % credible interval provide a measure of the public holiday effect (i.e., À θ is the reduction in the number of infections on a public holiday compared with that on a normal day); and α i ∼ Γða,aÞ is an individual week effect which has an expected value of and allows for autocorrelation in the time series. the window length of week was selected to ensure balance in the number of weekdays in each window and to correspond roughly to the assumed incubation period of hfmd of around - days. this model assumes that the difference between the number of cases prior or after the public holidays is purely because of the difference in the number of infections happening on the public holiday, which is unobservable but indirectly represented by the change in the number of symptomatic cases in the following weeks. to assess whether there was any longer term impact, we repeated the analysis comparing incidence in the second week (days [ ] [ ] [ ] [ ] [ ] [ ] [ ] following the holiday with the week before it, as well as in the third week (days [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the main analysis does not treat public holidays that were less than days apart specially. in sensitivity analysis, we removed data corresponding to both holidays from the fitting procedure if they were less than days apart. sensitivity analysis was also conducted by applying different forms of the non-informative prior distributions for the parameters to test the robustness of the inference. school vacation effect. in singapore, kindergartens, primary schools, and secondary schools have school vacations in march for week, in june for weeks, in september for week, and in november and december for - weeks. to measure the effect of school vacations on hfmd transmission, we built time series models, fit bayesianly, for the weekly number of children with hfmd aged years and younger. this has some similarities with an autoregressive time series, but the bayesian approach afforded greater flexibility in the model specification. we assumed a negative binomial distribution for the number of hfmd cases, y t , observed in week t, with mean and shape parameter p, where h t = if week t is a school holiday and otherwise, and a, b, d, p, are model parameters that needed to be estimated: b accounts for autocorrelation, whereas d determines the effect of school vacation. a negative binomial distribution is used to allow more flexibility to capture the observed variability in the data than that obtained using the poisson model originally considered. a constant b that does not vary with time was used because singapore has very little seasonality that would add forcing to the timing of epidemics of hfmd, and because it would hamper identifiability of the vacation effect. the adequacy of using a constant b was assessed and confirmed by examining the distribution of the residual of -week-ahead model forecasts. the model was fit using bayesian methods (markov chain monte carlo) with non-informative prior distributions, as described in the supplemental file. for each draw from the posterior distribution, we iteratively derived the median number of cases for the following week based on the number of cases in the current week using the formula in the aforementioned model. the number of cases in the first week of the simulations was taken to be the observed average weekly number of cases across the time horizon. this was done for weeks ( years) and only the latter weeks were kept as the number of weekly cases for a typical year. for each set of posterior values, , such simulations were performed. based on these , simulations of a typical year scenario using each of the , sets of posterior values, % prediction and credible intervals were derived for each of the time points. median values for all , simulated numbers for each set of posterior values ( , median values in total) were calculated. school closure during outbreaks effect. for each preschool that had an outbreak during the time horizon - , a period of days from the first day of case onset was considered; this was enough to span the duration of most outbreaks. the number of incident cases on day j in outbreak i, x i,j , was modeled by where c i,j is the cumulative number of cases within week before day j in outbreak i, s i,j is the number of children not yet symptomatically infected during the current outbreak by day j of outbreak i, and w i,j is the indicator for school closure on day j in outbreak i (w i,j = if the school was closed on day j in outbreak i, and otherwise). we smoothed the effect λ k of the current outbreak size k using the formula the posterior samples were used to project the number of cases that were avoided by forcing schools to close when they hit the trigger. for each draw from the posterior, simulations were performed to each of the schools with closures in our study period. in each simulation, the number of new incident cases on each day after the closure day was calculated using the model formulation and w i,j was set to be . the total number of cases was then compared with the observed number of cases when schools close and the total number of additional cases due to not closing schools for each simulation/set of posterior values was recorded. median values for all simulated numbers for each set of posterior values ( , median values in total) were calculated. we use bayesian methods to fit the models as the bayesian paradigm was considered more flexible than its frequentist analog. throughout, non-informative prior distributions were selected (detailed in supplemental file ) and models were fit, using markov chain monte carlo algorithms with burn-in periods of , and , iterations, in the r statistical environment. the posterior samples were then used to obtain posterior distributions of derived quantities. throughout, equal-tailed % credible intervals are used. model convergence was evaluated by using the gelman diagnostic tests and model validity assessed by comparing posterior predictions or simulations with the actual observed data. a total of , hfmd cases were notified to the ministry of health, singapore, from to . table shows the demographics of all notified cases. children aged less than years accounted for about % of all hfmd cases, and about % of all children with hfmd were aged years and younger; % of the cases were male, whereas % were ethnic chinese, % malay, % indian, and % others: the indian ethnic group is substantially underrepresented among cases compared with the general population ( % of singapore residents aged less than years have indian ethnicity). table shows the summary statistics for the bayesian models for the public holiday effect: the average of hfmd cases a day was reduced by % ( % confidence interval [ci]: [ %, %]) in the week following a public holiday, but only by % ( % ci: [ %, %)]) in the second week, whereas there was no reduction in the third week ( %, % ci: [− %, %]). alternative forms of non-informative prior distribution did not affect the results (not shown). during school vacations, weekly number of cases were modeled to be reduced to % ( figure shows the simulations for a typical year based on the modeled effects of school vacations on hfmd transmission, using typical timing of school holidays. both overall analysis and separate models for different age groups showed similar results and are consistent with the patterns of the data for the -year period. the temporally structured distribution of residuals in the -week-ahead model forecasts is shown in supplemental figure . no trends were observed, suggesting that the model with a constant b is adequate. from to , there were totally , school-level outbreaks involving a total of , hfmd cases in childcare centers and kindergartens, of which led to closure (table ). in all, , outbreaks including closures were included in our model: schools without accurate enrolment sizes were removed and four schools with closure falling beyond days after the first day of outbreak were categorized as no closure (detailed in supplemental file ) . the bayesian poisson model shows that the expected number of new cases decreases to % ( % ci: - %) if a school was closed on that day compared with a normal school day, after adjusting for size and duration of the outbreak. one-dayahead predictions based on the previous day's observed number of cases, shown in figure for arbitrarily selected outbreaks (more outbreaks are presented in supplemental file ), both with and without short-term school closure, demonstrate that the fitted model adequately captures the observed outbreak patterns. figure show the effect of school closure by showing the cumulative number of cases if there were no school closure for four arbitrarily selected outbreaks with closure (more outbreaks are presented in supplemental file ). the percentage of cases avoided from school closure for all closures is shown in figure . the majority of school closure events (> %) were associated with a less than % difference between observed and modeled cases, regardless of outbreak size at closure. closures that prevented more than % of total school size were generally in bigger schools (of size more than ). overall, the modeled number of infections prevented through the school closure policy from to was , ( % ci: [ , , , ] ), that is, an average % reduction compared with the total number of cases during that period. convergence of all bayesian models used was achieved according to the gelman diagnostic test. model validity assessed by comparing posterior predictions or simulations school closure is a common control measure in pandemic preparedness plans and in response to actual outbreaks, which may possibly be a high impact method of controlling an outbreak of a severe or potentially severe infectious disease. its effectiveness has been assessed in modeling studies, , , which posit that because children have closer social networks and high contact rates in schools, closing schools may substantially reduce transmission, if they do not compensate by having greater contact outside of school during a closure. the burden of school closure on families which may need to make alternative childcare arrangements means that it is imperative to have real-world evidence supporting its effectiveness. relatively little such evidence is available, though some studies in japan, france and the united states have assessed school closures in response to influenza outbreaks. , in light of this, the evidence on the effect of closure from singapore's long-standing routine school closures to control hfmd may prove valuable. this study demonstrated a consistent reduction in average numbers of hfmd cases when schools were closed, regardless of the reason for the closure. there was an estimated decrease of % in the number of cases in the week after a public holiday than the week before it, corresponding to a reduction in the number of infections that occurs on the holiday itself of %, although the effect of the holiday declines in the weeks following as infections return to baseline levels. a similar reduction in the number of infections during school vacations was also estimated (also about %). this may be an underestimate of the actual effect size because many preschool-aged children still attend preschools during school holidays. this hypothesis is supported by the slightly accentuated effect in children who have started formal schooling, with a reduction in risk around % for children aged years and older. although the reduction was statistically significant for all age groups, the difference in effect between age groups was not statistically significant. the data on school closure in response to outbreaks allowed a quasi-experimental analysis because recently the school closure policy has been reinterpreted to allow more discretion by the ministry of health whether to close the affected school or not. this led to a decrease in the number of school closures in the later periods of the study (table ) . although not randomized, this change allowed some overlap in the exposure (closure) and response (outbreak growth). the analysis showed that as with holidays and public holidays, school closure during an outbreak had the intended effect of orange line shows the cumulative number of cases if the school was not closed during the closure period. light pink and dark pink shades are % and % confidence intervals, respectively. this figure appears in color at www.ajtmh.org. mitigating transmission, but the effect was relatively small, and we estimated that only ∼ , cases were averted over the years analyzed. the high asymptomatic rate , and seroprevalence of the main causative viruses may mean that a substantial fraction of children are no longer susceptible by the time of closures in response to large outbreaks, which is currently triggered when more than cases or % of children are symptomatic. the fraction of cases prevented by outbreak-induced closure was modeled to be small in general, but larger for the larger preschools, which we attribute to the structure of the thresholds for closure: this is effected when either the fraction or the number of children in the school is notified, meaning that closure of larger schools occurs when the threshold for the number of cases is hit but the fraction is still low. given the disruptions to parents/families from unplanned closures, , outbreak-driven closures may, therefore, cause more problems than closure because of holidays. in light of the limited effect, it is not clear that this policy should be continued to be used routinely for hfmd outbreaks, but the effect of closures because of holidays suggests that a school closure policy may still be valuable for pandemic preparedness plans and needs to be carried out when there are serious outbreaks or when the outbreak is due to novel pathogens of unknown severity. although this was a retrospective study, with hfmd being a legally notifiable disease in singapore, incident data were collected in a standard way through preschools, clinics, and hospitals, and as such, the data used for this study are largely reliable indicators of symptomatic cases. however, some other limitations exist. several assumptions were made in the modeling process: the public holiday effect analysis assumed that the observed difference in the number of cases before and after the public holiday was solely attributable to the public holiday. in temperate countries, this assumption may not be tenable because of seasonal changes that drive transmission. singapore, however, lies close to the equator and has almost no seasonality-having year-round high temperature and humidity-with the effect that some virus transmission is effectively aseasonal. for the same reason, the assumption that there are no other temporal effects in the school vacation analysis may be more robust than it would in a temperate setting. these three analyses provide evidence that school closure-whether in response to an outbreak, or because of a public holiday or longer school vacation-reduces the transmission of hfmd. although we found that school closure in response to outbreaks does have a positive effect in reducing transmission, the effect is small, and given the high incidence and the fact that most of the cases of hfmd are mild and selflimiting, the evidence from this study suggests that policymakers in singapore should evaluate whether routine use of school closure outside of public health emergencies justifies the impact on families. epidemiology and control of hand, foot and mouth disease in singapore the epidemiology of hand, foot and mouth disease in asia: a systematic review and analysis notes from the field: severe hand, foot, and mouth 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with universal enterovirus and ev -specific primers mitigation of infectious disease at school: targeted class closure vs school closure effect of short-term school closures on the h n pandemic in japan: a comparative case study analysis of the effectiveness of interventions used during the a/h n influenza pandemic estimating the impact of school closure on social mixing behaviour and the transmission of close contact infections in eight european countries effect of winter school breaks on influenza-like illness school closure and mitigation of pandemic (h n ) , hong kong economic analysis of pandemic influenza mitigation strategies for five pandemic severity categories the cost effectiveness of pandemic influenza interventions: a pandemic severity based analysis changing epidemiology of hand, foot, and mouth disease in hong kong transmission of hand, foot and mouth disease and its potential driving factors in hong kong randomness of dengue outbreaks on the equator r: a language and environment for statistical computing general methods for monitoring convergence of iterative simulations projecting social contact matrices in countries using contact surveys and demographic data closure of schools during an influenza pandemic clinical features and risk factors of pulmonary oedema after enterovirus- -related hand, foot, and mouth disease seroepidemiology of enterovirus infection prior to the season in children in shanghai severity and burden of hand, foot and mouth disease in asia: a modeling study dynamic modelling of costs and health consequences of school closure during an influenza pandemic acknowledgments: we thank the data extraction team from communicable disease division, ministry of health, singapore, for their help in extracting data for our analysis. authors' addresses: yirong chen and alex r. cook, saw swee hock school of public health, national university of singapore and national university health system, singapore, e-mails: chenyirong @gmail. com and alex.richard.cook@gmail.com. hishamuddin badaruddin, vernon j. lee, and jeffery cutter, ministry of health, singapore, e-mails: drphisham@gmail.com, vernonljm@hotmail.com, and jeffery_cutter@ moh.gov.sg.this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. key: cord- -zgdvo n authors: biglan, anthony; elfner, karen; garbacz, s. andrew; komro, kelli; prinz, ronald j.; weist, mark d.; wilson, dawn k.; zarling, amie title: a strategic plan for strengthening america’s families: a brief from the coalition of behavioral science organizations date: - - journal: clin child fam psychol rev doi: . /s - - - sha: doc_id: cord_uid: zgdvo n despite significant progress in research on the treatment and prevention of psychological, behavioral, and health problems, the translation of this knowledge into population-wide benefit remains limited. this paper reviews the state of america’s children and families, highlighting the influence of stressful contextual and social conditions on child and family well-being and the concentration of disadvantage in numerous neighborhoods and communities throughout the nation. it then briefly reviews the progress that has been made in pinpointing policies that can reduce stressful contextual conditions such as poverty, discrimination, and the marketing of unhealthful foods and substances. it also describes numerous family and school interventions that have proven benefit in preventing psychological and behavioral problems as diverse as tobacco, alcohol, and other drug use; depression; antisocial behavior; academic failure; obesity prevention; and early childbearing. we argue that progress in translating existing knowledge into widespread benefit will require a nationwide effort to intervene comprehensively in neighborhoods and communities of concentrated disadvantage. we present a strategic plan for how such an effort could be organized. the first step in this organizing would be the creation of a broad and diverse coalition of organizations concerned with advancing public health and well-being. such a coalition could increase public support both for the policies needed to focus on these disadvantaged areas and the research needed to incrementally improve our ability to help these areas. this paper presents a strategic plan for reducing the prevalence of concentrated disadvantage in the usa. concentrated disadvantage consists of a tangle (sampson ) of inter-related environmental and behavioral problems such as poverty, discrimination, unemployment, substance use, single parenting, and academic failure that result in intergenerational poverty and the development of multiple psychological, behavioral, and health problems often resulting in premature death (national academy of sciences b). over the past fifty years, the prevalence of such disadvantage has increased dramatically in the usa (kristof and wudunn ; putnam ) . reversing this trend and thereby reducing the prevalence of these problems will require an unprecedented organization of our national resources over an indefinite period of time. we hope that this paper will contribute to such a nationwide effort. the paper begins with a brief summary of what we know about the contextual conditions that undermine family wellbeing and the resulting patterns of family life that contribute to life-long intergenerational psychological, behavioral, and health problems. it then summarizes the extensive evidence regarding programs and policies that have proven benefit in preventing or ameliorating one or more aspects of disadvantage, as well as the research that is needed to translate existing knowledge into widespread success in reducing concentrated disadvantage and its sequelae. we then present a strategy for building a nationwide effort to strengthen family well-being with special attention to neighborhoods and communities of concentrated disadvantage. the well-being of america's families can be gauged by the quality of the contextual conditions that affect families and by the quality of family life itself. even a brief summary of the evidence indicates that many families live in stressful social contexts and have high levels of family dysfunction. among the harmful contextual conditions that have been verified by previous large-scale studies are family poverty, homelessness, contacts with the criminal justice system, discrimination, and the marketing of health-compromising behaviors in youth (e.g., unhealthful food and substances). according to the u.s. census bureau (fontenot et al. ) , , , american families were living in poverty in . according to the organization for economic co-operation and development (oecd family database ), as of , the usa had the third-highest rate of poverty among developed countries ( . %). the federally defined poverty level for a family of four is $ , (families usa ). as of , % of children lived in homes with income below the poverty level, and . % were in homes below % of the poverty level (child trends databank ). a major reason for these rates of poverty is the high rate of single parenting. more than % of poor children live in single-mother families, and . % live in single-father families. a meta-analysis by stringhini et al. ( ) of studies involving . million people showed that socioeconomic status was associated with higher rates of mortality even when the researchers controlled for well-established risk factors for disease. children raised in poverty had significantly greater risk of most psychological, behavioral, and health problems (jarjoura et al. ; matthews and gallo ; pampel et al. ) and shorter life expectancy (galobardes et al. (galobardes et al. , miller et al. ) . distinct from the effects of poverty, economic inequality is a risk factor for multiple psychological, behavioral, and health problems. pickett and wilkinson, as well as other investigators (pickett and pearl ; pickett and wilkinson ; kaplan ) , have shown that premature death, obesity, infant mortality, depression, social anxiety, property crimes, and homicide are greater in economically unequal countries. many of these disparities in health are found among all but the very highest-earning segments of a population. the deleterious effects of inequality appear to be due to the fact that people living in economically unequal communities are more likely to have stressful interactions with people who are above or below them in the economic hierarchy (pickett and wilkinson ; wilkinson and pickett ) . according to a national system for estimating homelessness, there were about , homeless families with children on any given night in (national alliance to end homelessness ). homeless children have more developmental delays, poorer academic development, and more physical illnesses and conditions, such as stunted growth, anemia, asthma, lead poisoning, and infectious diseases (hart-shegos ) . the criminal justice system can have a devastating impact on families. the usa has the highest rate of incarceration in the world (wagner and walsh ) , and there is a much higher rate of incarceration of black people, a result of disparities at every point in the criminal justice process, from arrest through sentencing (alexander ) . a survey of american adults indicated that % of black people indicated that a member of their family had been incarcerated; the rate was % for whites and % for hispanics (enns et al. ) . families with a parent in prison are likely to experience poverty, homelessness, and residential instability (peterson et al. ) . parental imprisonment often contributes to children's behavior problems, depression, poor academic performance, and antisocial behavior (parke and clarke-stewart ; peterson et al. ) . children may also experience trauma through involvement with the criminal justice system at other points (e.g., if they witness the arrest of a parent or before or during visits with a parent in prison or jail; eddy and poehlmann ). discrimination is widespread in the usa (national public radio, robert wood johnson foundation, & harvard t.h. chan school of public health ) . in addition to welldocumented effects on the health of individuals (pascoe and smart richman ) , discrimination affects family wellbeing due to discriminatory hiring and pay and residential segregation. however, the most damaging aspect of discrimination may simply be the frequent exposure to experiences that are threatening or aversive and that contribute to inflammatory disease (american psychological association ). the marketing of tobacco, alcohol, and unhealthful food and beverages harms families. although the makers of these products often argue that their marketing is not to blame for underage use of tobacco and alcohol or childhood obesity, empirical research shows that marketers target children, their messages reach children, and these messages influence youths' purchases and consumption (biglan a; nestle ; pechmann et al. ) . moreover, the ready availability of illicit drugs in neighborhoods of concentrated disadvantage contributes to drug use disorders among both parents and children (ford et al. ; storr et al. ). each of the just-described conditions contributes to the development of psychological, behavioral, and health problems that undermine children's life chances and contribute to premature death. however, the risk of such outcomes is increased when multiple disadvantaging conditions are present. evidence for this comes from studies showing that the greater number of adverse experiences that children have, the more likely that their behavioral and physical health will be affected (anda et al. (anda et al. , felitti et al. ) . this underscores the necessity of targeting neighborhoods and communities of concentrated disadvantage if the nation is going to significantly reduce the number of children who are destined for multiple problems, impeded potential and premature death. a recent monograph by acevedo-garcia et al. documents the extent to which american communities have neighborhoods with extensive disadvantage (acevedo-garcia et al. ). the just-described contextual conditions contribute to the undermining of effective parenting. stressors such as poverty, economic inequality, and discrimination undermine parents' time, ability, and motivation to provide warm, responsive support of their children's development. poverty, discrimination, unemployment, and homelessness also make depression more likely. maternal depression is a risk factor for socio-emotional and cognitive delays in children (bernard-bonnin, society, health, & committee ) . there is also evidence that neighborhoods that are low in socioeconomic status have higher rates of some types of substance use (karriker-jaffe ). and, according to the substance abuse and mental health services administration (samhsa), about . % of children ( . million) live in homes where at least one parent has a substance use disorder (lipari and van horn ) . parental substance use is associated with a higher likelihood of child abuse (institute of medicine & national research council ) and later substance use by the child (anda et al. ) . finally, as the proportion of marriageable men declines due to unemployment and poverty, family stability is undermined, and the rate of single parenting increases. sawhill ( ) reported that, across ethnic groups, the rate of single parenting has increased in recent years among those without a college education. schools in high poverty neighborhoods and rural communities, generally have fewer resources than schools in more affluent areas, even though it is in high poverty areas that more resources are needed (duncombe ) . high poverty schools need more special education, more incentives for skilled teachers, and more effective supports for social and emotional learning. the absence of these resources is one of the reasons that students perform less well in high poverty communities (garcía and weiss ) . evidence across a wide variety of sub-areas of behavioral science converge in a consilience about how stressful social conditions result in a "fast life" strategy where threatening environments make such a strategy more likely to result in successful reproduction (wilson ) , but frequently culminate in premature death. figure depicts the impact of a stressful social context on parents and schools and the subsequent impact of all of these conditions on child and adolescent development. the conditions increase the likelihood that parents will have multiple problems and that, as a result, families will have frequent coercive interactions (dishion and snyder ) . it is well established that coercive interactions in families contribute to children's failure to develop self-regulation and to the development of aggressive and uncooperative behavior that puts them on a trajectory to fail in school and be rejected by peers dishion and snyder ; patterson et al. ). this rejection, in turn, leads to their joining with similarly rejected peers to form deviant peer groups. these groups become a training ground for the constellation of psychological and behavioral problems that include delinquency, substance use, risky sexual behavior, depression, and school dropout (debaryshe et al. ; patterson et al. patterson et al. , . this analysis is supported by research from a diversity of sub-disciplines (biglan et al. in press) . these include a wealth of evidence showing that multiple, interconnected problem behaviors result from a common set of adverse environmental conditions (biglan et al. ) . it is also supported by evolutionary analyses of the expression of fast life strategies that appear to have survival value in a dangerous environment (ellis and bjorklund ; wilson ) . it is shown by the evidence of epigenetic changes that make these strategies more likely (bergen et al. ). the analysis is also consistent with social psychological research contrasting the development of materialistic vs. prosocial values and the impact of threat on materialism and the association of materialism with diminished wellbeing (kasser ) . finally, there is clinical psychological research showing the benefits to human well-being of psychological flexibility, which is a pragmatic way of living that involves living intentionally according to one's values, a life strategy that benefits the individual and those around that individual (hayes ) . in addition to family influences, the quality of schools affects this developmental trajectory. as noted above, schools in high poverty neighborhoods and communities have higher rates of punitive practices and fewer resources for preventing the development of problem behavior (american psychological association, n.d.). there is one other point to be made about this developmental trajectory because it is so important for redirecting the expenditure of resources in our healthcare system. the u.s. per capita cost of healthcare is at least twice that of most other developed nations even though we lag behind these nations in health and longevity (biglan ) . a major yet frequently overlooked reason for these disparities is that ill health and premature death are made more likely by stressful social interactions in childhood (miller et al. ) . redirecting some of our healthcare expenditures to the prevention of stressful social relations in childhood is likely to make a bigger contribution to american's health than any advance in the treatment of chronic disease (biglan a) . when coupled with evidence from prevention research, these findings suggest a unifying framework about the kind of environments that are needed to nurture successful development (biglan a; biglan et al. ) . figure depicts the way in which nurturing conditions contribute to the development of prosocial behavior. by prosocial behavior, we refer to constellation of behaviors that contribute to not only the individual well-being, but the well-being of those around the person. these include caring, compassion, and an orientation toward helping others (kasser ) . they also include the development of a wide range of academic and social skills, as well as skill in art, music, and science. the evidence indicates that the development of prosocial behavior is promoted by environments that (a) minimize socially toxic coercive interactions, (b) richly reinforce all types of prosocial behavior, (c) limit both opportunities for problem behavior (such as unsupervised involvement with highrisk peers) and influences toward problem behavior such as the marketing of tobacco and alcohol, and (d) promote psychological flexibility, which is a values focused, mindful, and pragmatic way of living. thus, in the next section, we summarize the research suggesting that this problematic fast life trajectory can be prevented through the promotion of these nurturing conditions. the figure also indicates that we need to be concerned not only with the proximal family and school influences on development but on the neighborhood and community conditions that affect families and schools and often directly affect the child. as just suggested, focusing only on the proximal family and school influences on development is insufficient. we need to be concerned with multiple systems that can improve family well-being. in this section, we review evidence on interventions designed to affect the contextual conditions that we described above. in addressing family poverty, we rely on a recent nam report, a roadmap for reducing child poverty (nasem a), which thoroughly reviewed the evidence on the impact of poverty on children and the policies that have been shown to reduce it. the report identified ten policies or programs shown to reduce family poverty. contextual factors that have been instrumental in understanding family wellbeing involve many systems. we focus on policies below that could have a fundamental impact on these contextual factors. four policies both increase income and promote employment. the report deemed promotion of employment to be as valuable as increasing income because employment raises the probability that a family will continue to prosper over time. this view is consistent with our priorities in using a multi-level and systemic approach to improving family well-being. this policy supplements low paid workers' income by providing a tax credit on income they have earned. for example, people receive a refund of money previously withheld from their taxes. the credit is refundable in the sense that a worker may receive more money for their work than they would have had to pay in taxes. research on the eitc shows it reduces poverty, increases workforce participation (nichols and rothstein ), and increases the long-term health of family members. markowitz et al. ( ) found an association of higher tax credits with higher birth weights and longer gestation periods. unfortunately, states do not offer a state eitc and an additional six states have a nonrefundable eitc (tax policy center, nd.). a nonrefundable eitc can only offset state income taxes, so the benefit is limited for low-income families with little taxable income. also, many families who are eligible for federal and state eitcs do not apply for them, thus indicating a need to increase education and access to these opportunities. these subsidies impact family income, and at the same time, they increase workforce participation because they enable family members to work who might otherwise be providing childcare. the nam report estimated that the average annual cost for childcare for a young child (age through ) is $ , . the current federal minimum wage is $ . an hour, although states and the district of columbia have higher minimum wages. the nam report concluded that raising the wage could reduce the availability of jobs in some places if employers reduce their workforce in response, but also cited evidence that a higher minimum wage helps lift families out of poverty. an analysis by the robert wood johnson foundation ( ) concluded that minimum wage increases are associated with improved health. specifically, adult women who became pregnant and experienced an increase in the minimum wage had a reduced likelihood of smoking and fewer low birth weight babies. increases in state-level minimum wages are associated with decreased infant mortality, suicide rates, and heart disease death rates (kaufman et al. ; komro et al. ; van dyke, komro, shah et al. ) . at least two workforce development programs have been shown to increase employment and earnings for poor unemployed or underemployed workers. workadvance (schaberg ) was reviewed in the nam report. this program works with potential employers to identify the skills they need and then trains workers on those skills. four workforce development organizations ran randomized trials evaluating the program, and though not all replicated effects, the program was shown to increase employment, earnings, and job satisfaction. in addition, the arnold foundation, which funds and monitors results of randomized controlled trials evaluating government-funded interventions, reported that a randomized trial of quest, a job training program for low-income individuals, showed that it increased recipients' annual income % more than the control group over a nine-year follow-up period (straight talk on evidence ). like workadvance, quest works with employers to identify skill sets that companies need and provides training in those skills. based on this evidence, it appears that workforce-training programs focused on training for job skills that are in demand should be one component of improving family income. additional randomized controlled trials of such programs should take place and should include ongoing monitoring of their impact. the national academy of medicine report also examined the impact of a set of policies that would directly increase family income. one is housing vouchers. the federal government provides vouchers to families with very low incomes to subsidize rent, thus reducing expenditures and often enabling moves to better neighborhoods. a randomized trial of the impact of housing vouchers showed that they contributed to lower levels of obesity and depression (ludwig et al. ) and increased later earnings of those who moved to more affluent neighborhoods before the age of (chetty et al. ). the nam report (nasem b) indicated that increasing housing vouchers could result in moving % of children who currently live in poverty out of poverty. the national academy committee on poverty reduction also concluded that an increase in the number of families receiving assistance would contribute to reducing the proportion of children living in poverty by . %. the committee also estimated that this supplement to the income of poor families could reduce the number of children living in poverty by . %. note that while the nam report focused primarily on how these policies would increase family income and labor force participation, most have also improved health and wellbeing. for example, the report notes that the eitc improves children's educational and health outcomes. there is evidence that when people work together, each contributing to the group's success, prejudice diminishes. the most extensive research on this strategy involves cooperative learning procedures in schools, wherein small groups of students learn to work toward a common goal. this strategy has been shown to reduce prejudice and bullying (van ryzin and roseth a). paluck and green ( ) note that very little of this research has been done in real-world settings. given the extent of discrimination in the usa, its impact on public health, and the size of the workforce, it is imperative that foundations and the nih fund research to identify more effective ways to reduce prejudice in schools, work settings, and neighborhoods and communities. jurisdictions across the usa are beginning to recognize and address the harms that criminal justice involvement can cause in families. for example, some efforts are underway to reduce sentence length, eliminate incarceration, and use alternative sentences (e.g., enhanced community supervision) for certain nonviolent crimes. yet the country can do much more. the usa has not only a far higher rate of incarceration than other developed countries, it also has a much higher recidivism rate (fazel and wolf ) . comprehensive reform of the criminal justice system will require that its leaders make their primary goal a reduction in the incidence of crime. the pursuit of this goal will require a bigger investment in prevention, which, in turn, will require the system to become more involved in and supportive of the other sectors of society, including schools, family services, and healthcare. it will also require the adoption of evidence-based practices that can reduce recidivism. among the reforms that are likely to remedy this problem are the following. punitive practices in schools, such as zero-tolerance policies, often escalate suspensions and expulsions. this results in children's separation from school, extra-curricular activities, and peers and increases the likelihood of further offending as well as dropping out of school (american psychological association-zero tolerance task force ; insley ; martinez ; mitchell ) . moreover, school and justice policies that bring together at-risk youth amplify the development of antisocial behavior (dishion et al. ). these practices need to be replaced with evidence-based therapeutic interventions (lipsey ; lipsey et al. ) . involvement of parents in the criminal justice system can be traumatizing to their children at the same time that it reduces family income and the availability of a parent (peterson et al. ) . promising evidence points to enhanced family relationships through interventions such as parenting skills training, well-managed visitations, and improved communications (dallaire et al. ) . however, the evidence is not sufficient to justify widespread implementation. further research on how to reduce the harm to families affected by incarceration should be a high priority. in addition, immigration policy is currently doing substantial harm to children and families. the current highly punitive policies need to be examined in terms of the harm that they do. policy reform must start with the goal of enhancing rather than undermining family well-being-a reform which is unlikely under the current administration. as noted above, the marketing of tobacco, alcohol, and unhealthful food harms children's health and undermines parents' efforts to protect it. some progress is being made in regulating cigarette marketing because the evidence is so clear that such marketing contributes to underage smoking (biglan b ) and, ultimately, to the deaths of about a third of smokers (centers for disease control and prevention ) . similarly, the marketing of alcohol has been shown to increase underage drinking (sargent and babor ) . food marketing needs similar research. to the extent that research helps to determine whether marketing is a causal influence on alcohol or unhealthful food consumption, governments should regulate such marketing in the same way it regulates tobacco marketing. it is also clear that taxing tobacco and alcohol significantly reduces the proportion of young people who smoke or drink (komro et al. ) . and the evidence is beginning to emerge that taxation on sugar-sweetened beverages, can reduce the consumption of these products (redondo et al. ) . the harm that consumption of tobacco, alcohol, and unhealthful food does to children is substantial. this problem could be reduced if federal and state governments implement inflation-adjustable taxes on alcohol and unhealthful food and that those taxes are commensurate with taxes on tobacco products. in this section, we present a brief overview of the family and school interventions that have been shown to prevent problem development in youth. the evidence documents numerous family and school interventions that make these environments more nurturing. while it is true that addressing the above-described contextual conditions is likely to improve family well-being significantly, it is unlikely that reducing problems like poverty and discrimination will ensure that every family and school adopt the most effective ways of nurturing children's development . interventions are needed at both levels. research over the past thirty years has identified a broad array of family interventions that are available across the developmental spectrum. these interventions can reduce coercive interactions, promote parental skills in reinforcing prosocial behavior, set limits on risky behavior, and promote resilience in the face of challenges that stressful environments can pose. the strategies focus on two main areas: ( ) family promotion of children's social, emotional, and behavioral well-being and ( ) family promotion of children's healthy lifestyle behaviors to lower risk of obesity and other chronic disease conditions. this section briefly summarizes progress in each area and discusses what strategies can help achieve broad positive parenting and family support in society. over five decades, clinical and prevention professionals have developed a broad array of parenting and family support programs to foster social, emotional, and behavioral well-being of children. hundreds of studies document the benefits of such programs for families and children from infancy through adolescence. parenting and family support programming, if broadly implemented, has the potential to impact child mental health, child abuse, academic failure, bullying, antisocial or violent behavior, substance misuse, obesity, sexually transmitted disease in adolescence, teen parenthood. these programs increase positive and nurturing parent-child interactions, kindness, cooperation in peer interactions, and safe family environments many of these programs have common elements (prinz ) , including the following: • adopting a positive frame: no matter the circumstances, effective programs convey an optimistic and positive approach to each family. • action focus: parents actually do things instead of simply talking with a staff member. • problem-solving orientation: emphasis on solving current problems, not on casting blame. • specific, concrete, and practical parenting strategies: programs offer menus of strategies to address parenting challenges. • collaborative goal setting: parents and staff members collaborate to set programmatic goals for each family. • consultative rather than prescriptive attitude: programs favor providing useful consultation to parents without giving them orders. such programs reflect a well-honed set of parenting practices that are adaptable to children's ages and that offer parents many ways to foster nurturance, such as. • giving rules and instructions to children before situations begin. • paying frequent attention to positive behaviors. • providing supportive prompts, coaching, and correction. • setting clear limits and boundaries. • ignoring harmless but annoying behaviors so children don't get an "audience" for these. • recognizing and encouraging behaviors that are the opposite of any identified problem behaviors. • rearranging situations so that positive behaviors result in enjoyable activities. • expressing affection and caring frequently. • developing effective ways of solving daily and recurring problems. • envisioning the future and setting specific and attainable goals. families can positively influence youth health behaviors by providing a warm and supportive setting for learning, growth, and development (kitzman-ulrich et al. ; wilson et al. ) . it is increasingly apparent that positive parent-child communication is fundamental in promoting healthy behaviors in children. specifically, a supportive family climate has a positive association with adolescent health behaviors and healthy weight status (berge et al. ; halliday et al. ; st. george et al. ; wilson and sweeney ) . research shows that higher levels of family functioning (defined as communication, joint problem solving, closeness, and appropriate behavioral control) are related to healthier body mass index, nutrition, and physical activity among adolescents. haines and colleagues (haines et al. ) found similar results. they discovered relationships between higher family functioning, lower obesity risk, and less sedentary behavior. other evidence indicates that improving family climate through positive interventions reduces deleterious effects of unsupportive parenting on youth weight-related outcomes, with benefits that can be detected nearly years later (brody et al. ; wilson et al. ) . family programs that improve children's self-regulation also promote healthy lifestyles from early childhood through adolescence. for example, self-monitoring, which involves prompting families to keep a record of a specified behavior (e.g., in an electronic or written diary), is one of the most effective strategies for eliciting changes in diet and physical activity (michie et al. ). another common strategy is prompting specific goal setting. this includes details such as the frequency, intensity, or duration of behavior, as well as specifying when, where, and how the child plans to achieve the goal (gollwitzer ) . action planning, including encouraging families to identify potential barriers to performing a behavior and planning supportive ways to overcome them, is another often-used technique for promoting healthy lifestyle changes (ayotte et al. ; bandura ) . these behavior-change strategies have been increasing in family contexts to promote health-related behaviors among lowincome and ethnic minority populations. another approach for promoting healthy behaviors among youth focuses on supporting their autonomy, rather than telling them what they should do. motivational interviewing (mi) is designed to increase motivation for behavior change by listening to the child about their concerns and joining them around their personal desires to change their health or weight (miller and rose ) . rather than providing advice or urging specific actions, clinicians and parents use this strategy to elicit the motivation to change from individuals themselves using strategies through reflective listening and shared decision-making. the counselor or parent can then provide the child (or adolescent) with specific behavioral skills, including identifying goals, creating an action plan, anticipating potential barriers, and monitoring children's health behaviors (resnicow and mcmaster ) . ample evidence shows mi is an effective strategy when coupled with behavioral skills training for reducing weight problems and obesity (for a review, see armstrong et al. ) and for helping parents facilitate weight loss in their overweight children (resnicow et al. ; spear et al. ). in the mi framework, shared decision-making and autonomy support are critical skills for parents as youth transition from childhood to adolescence (ryan et al. ) . shared decision-making involves parents and children making decisions together in a way that facilitates sharing in ownership over plans. this encourages motivation for and enjoyment of healthy behaviors (ryan et al. ) . studies have demonstrated positive associations between parental autonomy support and adolescent intrinsic motivation on improving healthy lifestyle-related behaviors and adhering to weight-loss treatment (mâsse et al. ) . parents can also use this strategy to model problem solving and troubleshooting of barriers. research has shown that these behavioral and motivational approaches are effective for improving health behaviors from early childhood through adolescence (kahan et al. ; st. george et al. ; wilson and sweeney ; wilson et al. ) . however, in the case of these health behavior interventions, a gap between research and practice exists that limits the implementation of evidence-based interventions outside of research programs. this gap exists because we have not yet developed effective strategies for getting nonresearch settings to adopt these strategies. given the deleterious effects of parental substance use on children, interventions to address parental substance use are needed. the evidence for the value for children of treating parental substance use is currently limited. calhoun et al. ( ) reviewed four randomized trials of the impact of treating parental substance use on children. each of these studies provided some evidence that parental treatment could benefit children, either by reducing the likelihood of the children's substance use or by improving children's psychological or behavioral functioning. research on evidence-based methods for reducing family violence indicates promising effects of behavioral interventions. although much more research is required for strong recommendations about how to prevent child maltreatment, studies have shown positive effects of parenting programs that reduce risk factors such as parental depression and stress, parents' inappropriate attitudes toward child-rearing, abusive parenting behaviors, insufficient parenting skills, and minimal knowledge about child development (chen and chan ) . recent research on domestic violence interventions also indicates promising new efforts using cognitive-behavioral interventions. an intervention based on acceptance and commitment therapy is now being used in the state of iowa for all individuals convicted of domestic violence. the intervention joins an abusing partner around their own prosocial goals and helps them learn to step back from anger-inducing thoughts, and feelings in a self-compassionate way. in this context, they become better able to control anger and develop more nurturing ways of interacting with their family members. research on the program shows preliminary evidence that it is effective in reducing repeat domestic assault, in both community corrections and incarcerated populations (berta and zarling ; zarling et al. ) . evidence-based family interventions are essential but not sufficient for a successful impact on the well-being of children and their families. broad population reach is critical as well. reaching one family at a time (e.g., home visitation programs) has utility but does not preclude the use of collective and cost-efficient strategies to provide parenting and family support for large numbers of families. critical to population reach are multiple access points for programming, an array of delivery modalities including media, the involvement of personnel from several service sectors, de-stigmatized programming capitalizing on a range of intervention intensities, and recognition of how to take into account major social determinants of health (kaplan ; prinz ). substantial progress has also been made on school interventions that promote prosocial behavior and prevent the development of multiple problems. these programs have in common a focus on helping schools to replace punitive discipline practices with practices that promote prosocial behavior. here we briefly describe four programs that have proven benefit. pbis is a schoolwide system for promoting prosocial behavior. a team of staff members establishes and monitors pbis implementation. they establish a small number of clear rules for appropriate behavior. common rules are be responsible, be respectful, and be safe. students are then taught examples of behavior consistent with these rules in all of the venues of the school (classrooms, hallways, playground, buses). a system for increasing praise and rewards for appropriate behavior is put in place throughout the school. the pbis system monitors disruptive or inappropriate behavior throughout the school and uses the data to pinpoint settings or individuals with high rates of inappropriate behavior. this enables the pbis team to put in place more intensive interventions for particular students or settings in the school. bradshaw et al. ( ) reported a group randomized trial of the impact of positive behavior intervention and support and elementary schools. they found significant reductions in behavior and concentration problems and improved social-emotional functioning, and prosocial behavior. children in pbis schools were % less likely to be referred to the office for inappropriate behavior. pbs is now in more than , schools throughout the nation. this is a program that teaches students to engage in positive actions throughout the school. washburn et al. (washburn et al. ) reported on three randomized trials evaluating positive action in elementary schools. in all three studies, students who were randomized to the positive action intervention showed smaller declines in positive actions than those in controls. the program appears to have arrested the typical decline in positive actions that is seen as students enter early adolescence. the randomized trial conducted in chicago elementary schools showed that the students receiving positive action had significantly more positive affect and life satisfaction, and significantly lower levels of depression and anxiety (lewis a (lewis , b . cooperative learning was originally developed as a method of increasing student learning, especially among students who were performing poorly. this strategy involves having small teams of students work together in ways that require each student to make a contribution to the group's product. numerous studies have shown the benefit of cooperative learning in increasing academic success (roseth et al. ) . it has also shown a clear benefit in reducing intergroup prejudice (pettigrew and tropp ) . recently, a randomized trial of cooperative learning in middle schools has shown that the program significantly reduced bullying and victimization and that students in the cooperative learning condition reported less stress, fewer emotional problems, and less use of alcohol and tobacco (van ryzin and roseth b). like cooperative learning, the good behavior game involves small teams of students working together cooperatively. teams that meet the criteria for appropriate behavior receive rewards such as extra time for recess. extensive research over a period of years has shown that the good behavior game significantly reduces disruptive behavior (embry ) . a randomized trial of the benefit of the good behavior game among students in early elementary school in baltimore showed that students who played the game were significantly less likely to smoke or be arrested by middle school and that by young adulthood they were significantly more likely to graduate high school and attend college and significantly less likely to have problems with substance abuse, antisocial behavior, or suicidality (kellam et al. ). the paxis institute developed a version of the good behavior game that combines it with a set of simple behavior influence techniques called kernels. that version of the game has generally replicated the benefits of the program that were shown in previous studies (johannsson, biglan, & embry, under revision) . at this writing, more than , teachers have been trained in the implementation of this version of the game. there is also evidence of the benefit of increasing collaboration between families and schools. jeynes ( ) conducted a meta-analysis of the impact of a variety of parental involvement programs on academic success and found they increased the performance of students of all ages. similarly, sheridan, smith, moorman kim, beretvas, and park ( ) conducted a meta-analysis of studies on the impact of various types of family-school collaboration on students' social-emotional functioning and found significantly better social-behavioral competence and mental health among students of all ages; effects were strongest for african american children. the components of collaboration that made a difference included two-way communication between parents and teachers and trust between parents and teachers. also important was school provision of enrichment activities for the home, parents volunteering in the classroom, and specific help to parents on how to model and reinforce desirable behavior. despite the benefits of family-school collaboration, the collaborative practices sheridan et al. ( ) identified remain largely aspirational in typical school environments (garbacz et al. ) . perpetuating the gap between research and practice is a lack of trust families and educators have for each other. mistrust has intensified over time through certain practices, such as the disproportionate use of discipline (townsend ) . to promote family well-being, evidence-based family-school systems and practices (sheridan et al. ) should be aligned and integrated within existing school programs and initiatives (garbacz ) . such alignment and integration will provide the necessary context, through two-way communication, culturally sensitive practices, and family-centered services for parents and educators to build the skills and relationships necessary to strengthen families and support children. to advance this line of work, research is needed that examines the impact of strategies for promoting collaboration on (a) the level of parent-teacher collaboration; (b) the impact on teachers' attitudes and beliefs about parents and collaboration with them, and teacher burnout/morale; and (c) factors that explain the impact of a strategy on an outcome. in addition, an examination of the implementation process should consider alignment and integration in school and community systems; parent, youth, and teacher characteristics and experiences; the school infrastructure; and the external environment (feldstein and glasgow ). this brief review of family and school interventions documents the availability of numerous programs that can contribute to the well-being of families and the prosocial development of children. at the same time, additional research is needed if we are going to translate this body of knowledge into a widespread benefit. continued investment in strengthening these interventions is likely to improve their effectiveness. although there is evidence of the benefit of family and school interventions for poor and minority populations, further research on reaching and affecting this population is needed (van ryzin et al. ) . moreover, research on combining interventions at multiple levels and on how to get these interventions widely and effectively implemented will enhance our ability to make them available to neighborhoods and communities of concentrated disadvantage. the evidence presented thus far establishes three things. first, a large proportion of children and families are living in circumstances that undermine their well-being. indeed, over the past fifty years, conditions for families have deteriorated in the u.s. (kristof and wudunn ; putnam ) , and we have fallen behind other economically developed nations (unicef ) . second, those at greatest risk for future psychological, behavioral, and health problems are living in neighborhoods or communities of concentrated disadvantage. our impact on child and family well-being will be limited if we do not focus policies to alleviate structural barriers to well-being and how to further provide resources and assist these neighborhoods and communities. third, there is ample evidence that changes in public policy and the wider implementation of family, school, and community interventions can prevent or ameliorate many of the factors that undermine development. in principle, we have the knowledge needed to significantly improve wellbeing in american communities. we are, however, a long way from effectively addressing family well-being. despite the availability of policies and programs that could increase the number of families that are thriving, there appears to be no systematic plan for steadily improving well-being. the national academy's report fostering healthy mental, emotional, and behavioral development in children and youth (nasem b) called for a decade-long effort focused on promoting healthy development. the report further called attention to the fact that seven other academy reports had advocated for greater attention to the problems of poverty and concentrated disadvantage because these conditions constitute the greatest obstacle to successful development. in keeping with this focus, the remainder of this paper presents a strategic plan for how we might create an unprecedented effort to reduce concentrated disadvantage and thereby increase the proportion of families that support the successful development of children and adolescents. there are various sectors and policymaking bodies that are trying to address the challenges to family well-being. however, there is not a comprehensive and sufficiently cogent plan to fully impact family well-being in the u.s. for this reason, this strategic plan is addressed primarily to the key policymaking bodies, leaders in business, ngos, and foundations, child and family advocates, other key opinion leaders, and consumer groups. the proposed goals for this effort would be as follows. . increase the proportion of american families that meet an empirically defensible public health standard of wellbeing. . identify, monitor, and reduce the number of neighborhoods and communities of concentrated disadvantage. . promote the adoption and implementation of policies and programs that advantage families. . increase the amount of research relevant to reducing family disadvantage. this would include research on a. establishing a national standard for family wellbeing in the form of an index of family well-being. b. experimentally evaluating comprehensive strategies for reducing disadvantage in neighborhoods of concentrated disadvantage. there are two facets of our plan for furthering these goals. the first concerns organizing an unprecedented coalition of all sectors of society to make increasing the well-being of american families a national priority along the lines of what the nasem report on the promotion of mental, emotional, and behavioral health called for. the second is the creation of research agenda that would advance family well-being, especially in neighborhoods and communities of concentrated disadvantage. every sector of society has a role to play in reversing the deterioration of family well-being that has occurred over the past years (putnam ) . in each sector we need to identify practices that undermine family well-being and those that enhance well-being. the foundational value for this effort is a commitment to ensuring the well-being of every person. it is only when a growing number of people do so that we will have the support for the norms and public policies that select beneficial practices and diminish harmful ones (biglan a) . we envision a process in which each sector is prompted to examine whether its practices are contributing to the reduction of concentrated disadvantage. in particular, it would look for ways in which the sector undermines well-being and the degree to which it makes use of evidence-based policies and programs that enhance well-being. higher education is critical to a nationwide effort to reduce concentrated disadvantage. over the past forty years, a consilience has emerged in the human sciences regarding the conditions that people need to thrive and those that contribute to the development of psychological and behavioral problems and premature death (biglan (biglan , a biglan and embry ; biglan et al. ; biglan et al. ; catalano et al. ; dishion and snyder ; hawkins et al. ; institute of medicine ; kasse kasse , miller et al. ; nasem a nasem , b national research council & institute of medicine ; wilson et al. ) . the report of the national academy of medicine summarized the evidence this way: "the scientific foundation has been created for the nation to begin to create a society in which young people arrive at adulthood with the skills, interests, assets, and health habits needed to live healthy, happy, and productive lives in caring relationships with others." the research and training functions of our universities need to further develop strategies for translating what we know into effective interventions for disadvantaged families. according to the society for prevention research, there are only seven ph.d. programs in prevention science in the usa; there are seven masters level programs (society for prevention research ). increasing their numbers is a first step in advancing effective prevention. many other areas of the human sciences have a role to play in strengthening supports for family well-being. every university should be encouraged to examine how well it is preparing the scientists and practitioners that are needed to address the needs of families. higher education can also affect the trajectory of children living in disadvantaged communities. they can work with those communities to increase the number of children who attend college. they can reduce the costs of higher education for disadvantaged children. our healthcare system needs to shift from a primary emphasis on treating diseases once they develop to preventing them in the first place. here is just one example. there is an epidemic of childhood obesity in the usa and with it, a growing tendency for children to develop what used to be called adult-onset diabetes (u.s. department of health and human services ). a common practice among pediatricians is to screen children for obesity and counsel them and their parents (perrin et al. ). however, there is no evidence that intervention with an already obese child affects their trajectory (homer ). on the other hand, there is extensive evidence about the impact of food industry marketing on obesity and of the benefit of increased taxation on unhealthful food (biglan b) . restricting marketing and increasing the price of these foods are the practices that are needed. the same is true for the prevention of smoking and alcohol use (biglan c; pechmann et al. ) . strengthening the ability of the public health segment of the healthcare system to practice population health promotion will have a significant impact in reducing the incidence of a wide variety of disease that we currently wait to treat. the role of the public health sector in addressing the coronavirus epidemic may increase support for public health. in any case, this is the sector of the healthcare system that can lead the reforms that are needed. recent developments in business include a movement to evaluate business practices in terms of their contribution not just to investors, but to employees, customers, suppliers, and the society as whole. the business roundtable recently embraced these values and the b corp and conscious capitalism (business roundtable, n.d.; b corporation, n.d.; mackey and sisodia ) movements have been promoting them for some time. concrete steps that businesses can take include: (a) identifying and eliminating practices that harm, such as the marketing of harmful products (biglan b; c; d; e; f; g) ; (b) assessing the well-being of employees, customers, and the communities that they operate in; and (c) adopting practices that steadily increase family income and supports for child-rearing such as high quality day care and paid parental leave. a company that truly embraces the goal of ensuring that its employees are thriving, would strive to ensure that their wages are above the poverty level. the allies in reform of business norms and practices are the leaders of the conscious capitalism and b corp movements. as discussed above, the criminal justice system can reduce harm to families by reducing the use of imprisonment and investing more heavily in rehabilitation and the prevention of juvenile crime. there are numerous organizations working to reform this system (kerman, n.d.) . they are natural allies in any effort to reduce family disadvantage. we reviewed the array of family interventions that are available to help families reduce conflict and promote prosocial skills. every community has such services. they are increasingly guided by empirical evidence about effective practice. we need to encourage the transition from reactive intervention to instances of child abuse and neglect to a system of family check-ups and provision of support that prevents problems from developing. we also described some of the school-based programs that are available to schools. here too the challenge is to increase the proportion of schools that have the resources to make use of these programs. both the reform and the effectiveness of each of these sectors will be enhanced if organizations working in any given sector form a coalition with other sectors (biglan a) there are three reasons. first, the explicit embrace of the goal of enhancing population well-being by any give sector will build support for this goal in other sectors. for example, when people in criminal justice speak of reducing the incidence of juvenile crime and increasing the prevalence of prosocial youth it encourages those in other sectors to think in terms of the well-being of the entire population they serve rather than only those who seek or are remanded to intervention. second, progress in any sector makes the job easier for any other sector. for example, to the extent that criminal justice and family services improve their support of families, schools will have fewer students who lack self-regulation, engage in disruptive behavior, and fail academically. conversely, to the extent that schools are nurturing prosocial behavior, they will prevent crime. the third reason a coalition is needed is so that, speaking with one voice, the organizations in each of these sectors can look beyond a narrow focus on the problems each addresses and work to address contextual conditions that contribute to most psychological and behavioral problems as well as poor health. our systems for dealing with human problems have naturally evolved out of the need to address an emergent problem-a crime, an injury, and illness. as such they are reactive. however, the evidence presented above documents the impact of poverty, discrimination, homelessness, and concentrated disadvantage have on the entire range of problems. each sector has a stake in reducing these problems. speaking with one voice about the need to address these problems would contribute to greater support for the policies needed to address them. coalition building will need to proceed at the local, state, and national levels. increasingly community interventions are making use of the collective impact model to bring the sectors of the community together around a shared agenda (kania and karmer ) . at the same time, success at the local level will be enhanced to the extent that sector leaders at the state and national level are working together to achieve state and federal support for such efforts. in describing each sector, we mentioned groups within that sector that are working on reform. at the same time, there are numerous foundations and advocacy organizations that are working on the issues we address in this paper. two behavioral science organizations that are specifically working to advance effective action are the coalition of behavioral science organizations (cbso) which supported the writing of this paper and the national prevention science coalition, which is a member of the cbso and which has more than sixty national organizations affiliated with it. a formal coalition among all of these organizations is an important next step for advancing the goals we have proposed. the process of creating such a coalition would begin by inviting an initial group of organizations to formally create the coalition. the major impetus for changes in public policy and practice are the reports of the national academy of science, engineering, and medicine, surgeon generals' reports, monographs of the national institutes of health, and reports of the centers for disease control and prevention. with the publication of the recent nasem reports on poverty reduction and the promotion of mental, emotional, and behavioral health, the coalition of behavioral science organizations and the national prevention science coalition are working to publicize and build on the recommendations of these reports. behavioral scientists are beginning to do more to educate the public about how we can evolve a society that enhances everyone's well-being. npsc is regularly doing congressional briefings and publishing oped pieces in major national outlets. however, it is no longer possible to rely solely on mainstream media. since the advent of social media, it has become possible for numerous actors to reach a large audience with views, misinformation, and hateful rhetoric that would never have been widely disseminated previously (marantz ) . this fact has only recently come to the attention of people working mainstream media (haidt and rose-stockwell ) . it is imperative that those of us who are trying to influence movement in the direction of supporting everyone's well-being, make more and better use of social media to promote understanding of what is needed to improve human well-being. we believe that the two most important priorities for research are the development of a national system for monitoring both family well-being and neighborhood and community disadvantage and the development and testing of comprehensive interventions to reduce disadvantage and enhance family well-being. if the nation is going to be guided to steadily increase family well-being and reduce community disadvantage, then it needs valid and widely accepted indices of each type of well-being. ultimately, these indices should be reported annually by the federal government in the same way the indices of community economic well-being are monitored and reported. with respect to child and family well-being, the urban institute recently reported well-being and basic needs survey that is an example of the type of survey system that is needed. at the same time, systems are being created to track conditions in neighborhoods and communities. acevedo-garcia et al. ( ) have a system that reports on the conditions in communities that are likely to enhance or hinder family well-being. ultimately every community should be able to track well-being in their neighborhoods. the covid- pandemic has exposed virtually the entire nation to the practice of tracking well-being down to the level of the community. because of the danger of a high rate of covid- infections, people watch with great interest whether case rates were increasing or decreasing. states track what was working in other states and adopt practices that seem to be working. these same procedures are relevant to achieving changes in other aspects of well-being. granted the outcomes that would be focused on with a system that was monitoring family and community well-being will not attract as much attention as one that is tracking deaths due to a virus. however, that is in part because the public has not been educated about the fact that family and community well-being are critical for the prevention of premature death and virtually all of the psychological and behavioral problems that costs our nation so much. community-level monitoring of child and family wellbeing and the contextual conditions that affect families will provide the criteria by which a community can assess its progress and can maintain or modify its practices in light of the data. this process can shape the practices of each sector. for example, reductions in juvenile crime should result from adoption of evidence-based school, family, and juvenile justice practices. although it will not be possible to precisely attribute reductions in juvenile crime to one of these systems, each system can monitor how many youth and families it is reaching with evidence-based practices and can assess the impact of their services. evidence of the value of both economic development efforts and programs targeting families and schools suggests it is time to test comprehensive interventions that simultaneously address all risk factors for problems for which we have some effective solutions. interventions that only address family and school environments will leave many families in poverty. interventions that target only economic well-being will not ameliorate patterns of conflict in families, nor school cultures that undermine development and well-being . a variety of intervention strategies and evaluation methods have been used, but most have no connection to other work on this problem. there is research on workforce development, economic development, and family and school interventions that could contribute to reducing intergenerational poverty. there are community interventions focused on preventing adolescent problem behavior (hawkins et al. ) , efforts to strengthen supports for child and adolescent development, and collective impact approaches to bringing all sectors of communities together to address challenges. however, a truly comprehensive framework for addressing the tangle of problems will likely require all of these strategies. the principles of community organizing (hawkins et al. ; minkler ) and collective impact (cabaj and weaver ) are foundational for a comprehensive intervention. each of the sectors of the community needs to engage in a participatory process in which the community identifies the actions they want to take to improve wellbeing. as we indicated above, the efforts of each sector to enhance well-being will benefit from success of other sectors. to the extent that each sector supports other sectors efforts, synergistic benefits are likely. a second line of research that is essential for any comprehensive intervention consists of efforts to enhance economic well-being in the neighborhood and communities. the brookings institution (shambaugh and nunn ) reviewed research on place-based strategies to improve economic well-being in communities or neighborhoods of concentrated disadvantage. these included job training, tax benefits for investment in neighborhoods, subsidized employment, involving research universities in economic development, and extending the reach of existing health and nutrition programs. because of the limited number of experimental evaluations of these strategies, evidence for the benefit of programs such as enterprise and opportunity zones is, at best, weak. a third component of such interventions would be the implementation of family and school interventions that have proven benefit in enhancing family and school supports for successful development. table indicates activities that promote prosociality at every stage of development, from preconception through young adulthood. to our knowledge, no community has systematically implemented a comprehensive lifespan intervention and carefully assessed its impact. the closest we have found is the harlem children's zone, whose results are encouraging (whitehurst and croft ) . there are also likely to be synergistic effects of efforts at different phases of development. perhaps a child did not develop cognitive problems because her parents did everything they could to ensure her well-being in utero. as a result, a kindergarten class may have one less disruptive and uncooperative child. one more child will be able to read at grade level by third grade. if that child encounters adversity later in childhood (e.g., if a parent dies), she will benefit from good programs such as the one sandler and colleagues (sandler et al. ) developed to aid in coping with a death in the family. most evaluations of family and school programs with demonstrated benefits have been in contexts without other programs in place. and although the benefits of many interventions are significant, the size of their impact is generally small (jones et al. ) , and there is no evidence that their effectiveness has been increasing over time . one reason may be that these interventions are implemented in isolation both from many of the contextual conditions that affect behavior and from ameliorative interventions at other stages of development. it is likely that ensuring supports for families and schools at every stage of development can produce a larger impact on population well-being than any of these interventions in isolation. in addition to the many programs and policies with the potential to enhance well-being in communities, it may be beneficial to promote compassion and kindness. people who have faced numerous adverse experiences are likely to be hyper-vigilant and to read others as threatening; this is an adaptive orientation in such an environment . a community-wide effort to promote patient, caring, and kind interactions may contribute to reducing aversive interactions that not only maintain suspicion and distrust but lead to high levels of stress-related physiological processes that contribute to premature death (miller et al. ) . for example, imagine a family moving into a community that has increasingly organized around nurturing values, and that has explicit goals and procedures for promoting prosocial behavior. imagine this family has a long history of stressful contact with schools, the criminal justice system, healthcare providers, and perhaps neighbors. such a history is common in communities of concentrated disadvantage. the family's first experience in the community is likely to be enrolling their children in school, which may make them feel nervous and defensive. but suppose the school has a program to invite new families to discover how it supports children's prosocial behavior. suppose that staff convey that they like and admire the children. the family relaxes a bit, which is likely to promote positive interactions at home. now imagine that in every encounter in the community over the next few weeks, they meet people who show an interest in them and are welcoming and helpful. each positive encounter can contribute to family members becoming more comfortable while also conveying prosocial norms and reinforcing behavior. the complexities involved in evaluating comprehensive community interventions have hampered progress in helping neighborhoods of concentrated disadvantage. here we discuss what could strengthen this work. randomized trials are expensive and difficult to conduct in whole communities, especially if the intervention to be tested is multi-faceted. and, given the complexity of the multiple problems that need to be addressed, interventions need to be multi-faceted. randomized trials are useful for evaluating impact across cases, and they have demonstrated the value of numerous family and school interventions. however, they are not the best way to tease out functional relationships between environments and behavior. the family and school interventions that have been validated by randomized trials grew out of single-case experimental designs that revealed functional relationships between parents' and children's behavior (kazdin ) . similarly, before we can test complex, multi-faceted interventions through randomized trials in multiple communities, we need to show they are working reliably in individual communities. moreover, randomized trials require the assumption that all communities are alike in their needs and with regard to what will work. this makes it difficult to tailor an intervention to the needs of the community and still maintain consistency of the intervention across communities. greater use of interrupted time series designs would enhance research on communities (biglan et al. a, b) . these designs involve obtaining repeated measures of an individual behavior or group action and assessing the impact of a well-defined intervention or independent variable on that time series. such multiple baseline designs could help to evaluate the impact of comprehensive interventions on the wide range of aspects of child and family well-being, including economic well-being, quality of family interactions, and psychological and behavioral functioning of children and parents. by monitoring well-being in three communities or neighborhoods and implementing a comprehensive intervention in one of the communities, while continuing monitoring, but not intervening in the other two communities, we can assess whether the intervention affects measures of well-being. if an effect is detected in the first community over, for example, two years, it can be implemented in a second community. if its effects are replicated in the second community, it can be implemented in a third. such a design provides a valid experimental test of the impact of the intervention (biglan et al. a, b) . these methods are also useful for discovering the impact of individual interventions. for example, a community might implement the pax good behavior game (jiang et al. ) in a series of three elementary schools and track its impact on disruptive behavior and students' behavioral functioning. the community might initially evaluate introduction of an evidence-based family intervention in a multiple baseline across four families. it could similarly stage and evaluate a $ , per child allowance in terms of its impact on family relations. these assessments of individual programs would immediately show evidence of their efficacy, which could promote wider adoption. if they did not show benefits, the community could abandon the interventions or modify them until they worked. the beauty of these designs is that providing immediate experimental assessment of the impact of an intervention can enable its improvement. rather than requiring that each community get the same thing, these designs enable continuous quality improvement, so that successive communities receive an intervention modified by the improvements made in earlier implementations. although we believe that interrupted time series designs are the most efficient and effective strategy for developing effective interventions in neighborhoods of concentrated disadvantage, we also believe that it is impossible to say precisely what experimental strategies will be most productive. it would be a mistake to dictate that all interventions or all experimental procedures should have the same features. one experimental method that should also be tried is the most design (guastaferro and collins ). these factorial designs are valuable for efficiently identifying which components of an intervention and which combination of components are most effective. for example, an important question in neighborhood interventions is the relative and combined impact of economic development interventions and interventions focused on changing behavior in families and schools. a factorial design would efficiently address this question. it would involve randomly assigning neighborhoods to one of four conditions: (a) a combination of economic development and school and family interventions; (b) economic development interventions alone; (c) school and family strategies alone; or (d) no intervention. this design would allow assessment of the impact of each component, as well as the impact of the comprehensive intervention. we hope that this paper has made the case for a new initiative to address the problem of concentrated disadvantage in america. the extent of the problem is clear. its impact on the health and well-being of people living in these places is well-established. its harm to the rest of the nation is measured in the costs of poor health, drug use disorders, overdose deaths, and lost productivity. the policies and programs that can ameliorate these conditions are clear. this conclusion is being written on the day in which the u.s. became the country with the greatest number of covid- cases. the nation and the world are getting a lesson in how to think about public health. the entire world has been mobilized to do everything it can to prevent people from becoming infected. in the process millions of people have learned to think about the entire population. the incidence and prevalence of problems in entire populations has become as important as the individual case. this gives us an opportunity to bring attention to many other problems in society that would be best dealt with through public health strategies. every significant psychological, behavioral, and health problem can be thought of in terms of its incidence and prevalence. moreover, these problems are intertwined. this means that where we have high levels of one problem we are likely to high levels of others. and, specifically, these problems are concentrated in neighborhoods and communities that are high in poverty, substance use disorders, and social conflict. thus, efforts to greatly reduce the number of families that are affected by disadvantage will have limited success if we do not focus on reducing the prevalence of highly disadvantaged places. making a difference in these places will require policies and programs that reduce harmful contextual conditions such as poverty, homelessness, and discrimination, as well as programs that directly assist families and schools in adopting the most nurturing practices. the evidence that we can affect these problems is substantial. however, the challenge is to develop and evaluate comprehensive efforts to address multiple risk factors at every stage of development. putting in place such efforts will require a coalition of all sectors of society. if we can learn to speak with one voice about the harm that is being done by allowing children to be raised in concentrated disadvantage and the fact that we have policies and programs that could greatly reduce disadvantage, we may be able to achieve unprecedented improvements in the well-being of children and families in the usa. conflict of interest the authors declare that they have no conflict of interest. ethical approval this article does not contain any data from any studies with human participants that were performed by any of the authors. the geography of child opportunity: why 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approach to education the spirit level: why greater equality makes societies stronger behavioral medicine and integrated care: efficient delivery of the most effective treatments promoting social nurturance and positive social environments to reduce obesity in high-risk youth this view of life evolving the future: toward a science of intentional change. behavioral and brain science evaluation of acceptance and commitment therapy for domestic violence offenders. psychology of violence key: cord- -bgl xzbi authors: smith, louise e; woodland, lisa; amlôt, richard; rubin, antonia; rubin, g james title: a cross-sectional survey of parental perceptions of covid- related hygiene measures within schools and adherence to social distancing in journeys to and from school date: - - journal: bmj paediatr open doi: . /bmjpo- - sha: doc_id: cord_uid: bgl xzbi during the early stages of the covid- pandemic, schools in england were only kept open to children of ‘key workers’ and, from june, to children in reception, year and year . our cross-sectional survey of parents found low rates of attendance (< %) in both groups. many parents whose children attended school reported low adherence to hygiene measures (eg, not maintaining distance from others during school drop-off) and doubted that their school was adhering to covid- guidelines. this must be improved if parents are to feel confident about a more comprehensive return to school, as planned for september. the covid- pandemic has led to schools closing across the world, with substantial psychological and educational costs. reopening schools when safe to do so is a priority. in england, schools were kept open to children of 'key workers'. children in reception (aged - years), year ( - years) and year ( - years) were encouraged to return to school from june . the return was voluntary for both groups, and the government required schools to put measures in place to reduce the likelihood of disease transmission. these included: frequent hand cleaning; children mixing in groups of or fewer; maintaining physical distancing where possible; minimising parent contact at the school gates; and limiting use of public transport. additional steps were also recommended by some schools, including advising parents to wash children's clothes daily and asking children to have their temperature taken daily. protective measures are important both to reduce infection rates and to give parents confidence that it is safe for their children to return. if parents report that infection control measures are not in place, then this should be taken as a warning that hygiene practices need to be improved, that communication with parents needs to be improved or both. bmg research recruited participants on our behalf using active sampling from its existing panel to conduct an on-line poll to investigate the parental perception of the measures being implemented in schools and the number of physical contacts parents had while taking children to and from school. four hundred thirty-seven participants were excluded (dropouts, non-eligibility and quality control). participants were paid on average £ . . data were collected from to june, week after schools opened to children in reception, year and year . the sample fell within five percentage points of the known distribution of school children in england by child gender, school year and school they attended (fee paying or state-funded). the research was approved by the king's college london psychiatry, nursing and midwifery research ethics subcommittee (lrs- / - ). a school trustee assisted in designing the questionnaire and coauthored this report. a total of parents completed the survey in full ( responses were coded as missing data because of sample grouping inconsistencies): did not have a child eligible for school; had children in reception, year or year ('eligible year groups'); parents reported that they or their spouse was a key worker and that they did not have a child in an eligible year group. of children in eligible year groups, only ( %) had attended school for at least day in the past week and ( %) had not (n= 'do not know'). of children of key worker parents, only ( %) had attended school and ( %) had not (n= 'do not know'). the experiences and perceptions of parents whose child had attended school are presented in table . parents reported suboptimal levels of almost every hygiene practice that we asked about, including limited handwashing facilities on the way into schools or classrooms, class sizes that breached the recommended limit of and not maintaining physical distance from other people during the school run. given the urgent need for information, our data are based on a non-probability sample generated according to market research best practice and therefore care is required in interpretation because of likely selection bias. nonetheless, the results are noteworthy. if school attendance is to increase, then parents must have confidence that good hygiene practices are in place. urgent action is required to provide this by schools assuring parents of the hygiene measures that have been implemented and reiterating the social distancing guidance. contributors les designed the survey with input from all authors. lw and les analysed the results. all authors contributed to survey design and approved the final draft. table experience and perceptions of parents (n= / ) in england whose child had attended school in the past week (data collection: to june ) mental health effects of school closures during covid- -implementingprotective-measures-in-education-and-childcare-settings/ coronavirus-covid- -implementing-protective-measures-ineducation-and-childcare-settings key: cord- - ysogr authors: twining, peter; butler, deirdre; fisser, petra; leahy, margaret; shelton, chris; forget-dubois, nadine; lacasse, michel title: developing a quality curriculum in a technological era date: - - journal: educ technol res dev doi: . /s - - - sha: doc_id: cord_uid: ysogr there is considerable rhetoric internationally around the need for national curricula to reflect the changes that are taking place in the world outside school. this raises questions about what a quality curriculum in a technological era should look like, and equally challenging issues about how to achieve the necessary changes in schooling in order for such a curriculum to be realised. this paper summarises the views of experts from seven countries. it introduces a sociocultural framework that highlights the complexity of achieving alignment between policies and practice spanning the national to local school to classroom levels. three key issues that underpin alignment are then explored, each of which link with the issue of trust: stakeholders engagement; teacher professionalism; summative assessment. by exploring and exemplifying these three issues the paper indicates potential ways of addressing them and provides ‘tools to think with’ to enhance future curriculum development initiatives. we live in a rapidly changing world that is struggling "to respond to economic, environmental and social transformations -including technological advances, climate change and migration" (oecd , p. ). these transformations, and in particular the impact of digital technologies, have increased demand for higher order skills, competencies and knowledge. they have also led to uncertainty about the future, for example: the potential impact of artificial intelligence on some workplaces; how to keep up with an accelerating pace of change; or how to manage global competition for skills . within this global context, the school sector is expected not only to meet the demand for new, higher-order skills and competencies but also to prepare students to thrive in a future where the competencies and knowledge they will need to succeed are constantly changing. this is not about learning about digital technology per se, it is about how our digital era impacts on all aspects of schooling. indeed, focussing on one aspect of the system, such as digital technology, without considering the system as a whole is unlikely to bring about sustained change (butler et al. ) . in an effort to meet these challenges, many countries are re-designing their national curricula so as to better prepare young people now and in the future (wolfenden et al. ). this begs the question 'what does a quality curriculum look like in a digital era?' that is the question that is the focus of this paper and that technical working group (twg ) at edusummit set out to answer. in so doing they needed to consider what educational visions, policies and practices might be most appropriate (butler et al. ) . this involved discussion of: (a) what knowledge, competencies, attitudes and values today's school students need to thrive and shape their world (e.g. see erstad and voogt ) . (b) how school systems can develop such knowledge, competencies, attitudes and values effectively (e.g. oecd ). however key to these discussions was developing an understanding of what is understood when we use the term 'curriculum' and how 'quality' is defined. the starting point for any consideration of 'quality curriculum' for schools must include a discussion of what is understood by the concept 'curriculum'. the word comes from latin, where 'curriculum' (related to the verb currere, i.e. running) refers to a 'course' or 'track' to be followed. in the context of schooling, where learning is the central activity, the most obvious interpretation of the word curriculum is then to view it as a course, trajectory, or 'plan for learning ' (cf. taba ) . however, this is a rather narrow view. van den akker ( ) differentiates between different levels of curriculum: • supra level: international debates or agreements on aims and quality of education • macro level: curriculum at the system/society/nation/state level (e.g. national syllabi or core objectives) • meso level: curriculum at the school/institution level (e.g. school-specific curriculum) • micro level: curriculum at the classroom level (e.g. textbooks, instructional materials) • nano level: curriculum at the individual/personal level thus the process of curriculum development can be seen as narrow (developing a specific curricular product) or broad (a long term, ongoing process of curriculum improvement, often including many related aspects of educational change, such as teacher education, school development, and examinations). building on un sustainable develoment goal (united national general assembly ) and butler et al. ( ) , a quality curriculum would be a 'plan for learning' which supports young people in acquiring the knowledge, competences and dispositions needed to be successful in the digital era. an important aspect of the digital era is growing global interconnectedness, and thus it makes sense to examine curriculum developments at the international (supra) level. in a review of international trends and practices in curriculum development walsh ( ) identified that one of the key characteristics of international practices (at the supra level) was the preparation of curriculum frameworks rather than prescribed curricula, allowing flexibility in interpretation and enactment at a national (macro) and school (meso) level. such frameworks provide an insight into the key issues underlying curriculum development debates internationally. two such curriculum frameworks which were explicitly developed in the light of understandings of the digital era are outlined below. the oecd learning framework (oecd ), which is summarised in fig. , explicitly addresses the needs of individuals and of society. it is underpinned by a vision of "helping every learner develop as a whole person, fulfil his or her potential and help shape a shared future built on the well-being of individuals, communities and the planet" (p. ). this framework merges different views of knowledge (including 'knowledge about'/ content) with understanding different ways of making sense of the world and knowing how people act in different disciplines. this is combined with skills ('knowledge of') in the form of the application of knowledge to enable students to act in appropriate/valued ways in particular contexts. attitudes and values are seen as mediating these different forms of knowledge. the combination of these three elements ('knowledge about', 'knowledge of', and values and attitudes) result in what the oecd refer to as competencies. this contrasts with traditional curricula in many school systems, which have tended to focus on 'knowledge about' (biesta ) . importantly, the oecd framework recognises that the development of competencies requires changes in pedagogy, with a particular focus on increasing student agency. the eu's key competences for lifelong learning includes eight key competences (see fig. ), which are identified as being "essential to citizens for personal fulfilment, a healthy and sustainable lifestyle, employability, active citizenship and social inclusion" (european commission , p. ). as with the oecd framework there is a focus on both individual and societal needs, and a recognition that learning needs to go beyond content to include skills and attributes. these supra level frameworks provide a useful starting point for discussions of 'quality curriculum' at the national (macro) level. both the eu key competences framework and the oecd learning framework are underpinned by views about the purposes of education that are encapsulated in their aims. in both cases these encompass individual performance and collective well being, which may be seen as being in tension with each other. indeed, both frameworks see the purposes of education in terms of capacities, competencies or capabilities, so articulation is not "in terms of what the students should learn but in terms of what they should become" (biesta and priestley , p. ). this provides a stimulus for national curricula to think more broadly about what knowledge, competencies and dispositions school students need to thrive and shape their world (e.g. see erstad and voogt ) and thus should be included in a quality curriculum; i.e. a 'plan for learning' which supports young people in acquiring the knowledge, competences and dispositions needed to be successful in the digital era. having outlined the context, this paper will now examine the importance of purpose before exploring issues that arise when creating a quality curriculum in the digital era. the aim being to inform and hence enhance future curriculum development. it has long been recognised that being clear about the purpose of schooling is critical (e.g. goodlad ). the importance of defining the purpose of schooling however should not be confined to international frameworks but should be central to the development of curriculum at a national level. for example, as part of the ongoing redesign of the irish primary school curriculum, walsh ( ) reviewed international trends and practices in curriculum development and stressed the need for clarity of purpose, aims and principles: central to the revised overview must be an articulation of the purpose, aims and principles of the curriculum in ireland. the inclusion of these elements will distinguish the curriculum from a syllabus, where the primary focus is on subject content. being clear about this central purpose is imperative to ensure that all other elements of the curriculum, including the content, become subservient to this fundamental driver. (walsh , p. ) there is wide ranging support for the view that curriculum development essentially involves making decisions around what are considered to be the most important values and purposes for inclusion from the culture of a society (lawton ) . kelly ( ) advocated that curriculum developers should place their energies on articulating the ideological position and broad principles rather than defining specific content. similarly, biesta and priestley ( ) noted that having explicit aims and purpose in the curriculum provides a rationale for the inclusion or exclusion of particular elements in a transparent manner. furthermore, there is evidence from a cross-national study of nine jurisdictions that countries considered to have high performing education systems had clear and well thought through aims, principles and goals of education (creese et al. ) . however, it is clear that there are differences of views about the purposes of schooling across and within educational jurisdications (butler et al. ) . thus, the starting point for any discussion of curriculum needs to be an agreement about the purposes of schooling. while this idea is not new (e.g. biesta and priestly ; kelly ; lawton ; tedesco et al. ) and may seem self-evident, in some instances it has not been the case. for example, the english national curriculum's aims were added a number of years after the curriculum content and, as alexander ( , p. ) points out, were little more than "a high-sounding statement you attach to the curriculum after you've determined its content and whose function is therefore cosmetic". in contrast, having a clear rationale, which informs your purpose, provides the major orientation point, to which other components of the curriculum should be linked. this applies at each level of the system. a way to present this visually is to arrange them as a spider's web (van den akker ) (fig. ) . this curricular spider web not only illustrates the many interconnections between the components, but also its vulnerability. it illustrates that although the emphasis of curriculum design on specific components may vary over time, eventually some kind of alignment has to occur to maintain coherence, as each element is tied to the rationale at the centre. this coherence and alignment is often not evident within national curricula as outlined in the next section. as a starting point for their discussions members of twg individually created blog posts that set out their personal views about what an ideal curriculum for the digital age would look like. they each then compared their 'ideal curriculum' with the reality of the curricula in their own country, which included: australia, canada (quebec), england, ireland, sri lanka, slovakia, and the netherlands. the data were analysed inductively, using emergent theme analysis (wong and blandford ) . this analysis foregrounded two issues: • differences in the purposes and thus curriculum elements that group members were focussing on when thinking about their blog posts; • a clear disconnect between the visions of a quality curriculum within the blog posts and the realities of the curricula within the twg members' countries. each of these is explored below. analysis of group members' blog posts highlighted differences in the underpinning purposes that group members were focussing on when thinking about their blog posts. for example, whilst some posts looked at the overarching aims of schooling, others focussed more narrowly on aspects of the curriculum explicitly linked to digital technology. in the subsequent group discussions it was agreed that the focus should be on that broader view. building on the work of edusummit (butler et al. ) , members of twg adopted the yin-yang vision framework (fig. ). this sets out what the ultimate purpose of schooling should be and key elements that are necessary for the vision to be realised. this framework is underpinned by a sociocultural theoretical perspective (twining et al. ) which drew upon sociocultural theory as set out by lave ( ) , within which the concepts of identity, agency and participation are key. the items on the left of the figure relate to individual fulfilment. the items on the right focus on universal wellbeing. the items in the middle are the capabilities that are necessary to achieve the vision as a whole, providing a bridge between the individual (identity) and the collective (participation). all of these dispositions and capabilities are developed through engagement with content. this content should reflect key concepts -powerful ideas that have relevance in multiple contexts and enable one to make sense of the world. for example, the notion of 'systems', which has relevance in biology (environmental systems, nervous system), wellbeing (health systems), business and it (computer systems), and many other areas. within this conception of curriculum the development of knowledge, skills and dispositions is seen as fig. the yin-yang vision framework (twining a) happening through engagement with key concepts which span discipline boundaries. this contrasts with traditional school curricula which tend to compartmentalise 'content' into discrete subjects that are often taught in isolation. underpinning this framework is a recognition that in order to achieve a vision of individual fulfilment and universal wellbeing you need to be able to apply your understandings in appropriate ways. this means that you need knowledge, capabilities and dispositions. we need to move away from a binary debate about content vs skills. using the yin-yang vision framework, we believe could act as a bridge between supra (international) and macro (national) level curriculum debates, highlighting the importance of purpose, and providing a tool to think with at the macro and meso (school) levels. the disconnect between twg members' visions and the curricula realities in their school systems seemed to reflect tensions around the purposes of schooling in our rapidly changing world. this is mirrored in the gap between the dominant rhetoric of ' st century skills' and the reality of predominatly knowledge based curricula aligned with high stakes testing (e.g. naplan) and international assessments (e.g. pisa) that cannot assess many of these critical skills and dispositions (etag ). butler et al. ( ) explored the issue of alignment, noting that this required alignment of purpose, policy (including curriculum, assessment, accountability and teacher professional learning), and practice at three levels (macro/national, meso/school, and micro/ teacher). twining ( ) extended this analysis using a sociocultural framework (see fig. ). this framework has three levels, • the constitutive order -the broader context, including cultural norms, values and beliefs, as well as more explicit policies, rules and regulations. this includes any nationally specified curriculum. • the arena -the enduring elements of the school context which are taken up from the constitutive order. for example, how policies and expectations at the national level are interpreted and enshrined in the school expectations, policies and facilities. this includes any school specified curriculum (e.g. school schemes of work). the arena provides opportunities for action. • the setting -the local context (e.g. the classroom) in which practice is implemented. at this level of analysis, the actors (e.g. teacher and students) perceive what is possible within the context of the school arena in the light of their identities. as illustrated by the shaded box within the setting in fig. actors 'take up' some of those perceived possibilities in their iterative interactions with the other people in their setting. achieving alignment between and within each of these levels (constitutive order, school arena, and setting) is complex. this reflects a tension in finding a reasonable balance between curriculum freedom and regulation when developing a quality curriculm. having agreed on the overarching yin yang vision framework twg focussed on three key issues that need to be addressed in order to achieve alignment within and between levels. these key issues are stakeholder ownership, teacher professionalism, and assessment, each of which is explored below. curriculum development and alignment is a process with many stakeholders. even though the stakeholders might have different values and interests in relation to the curriculum, it is important to involve them in a timely and authentic manner, to ensure commitment and ownership. in relation to broad curriculum development, van den akker ( ) states that this is usually a long and cyclical process with many stakeholders and participants; in which motives and needs for changing the curriculum are formulated; ideas are specified in programmes and materials; and efforts are made to realise the intended changes in practice. members of twg agreed that in order to ensure alignment between the specified and enacted curricula it was critical that all stakeholders should be involved in co-constructing the curriculum; including agreeing the purposes that the curriculum is intended to meet. table illustrates the key stakeholders at each level of alignment. at every level there are large numbers of stakeholders. the nature of those stakeholders changes between levels, with their foci becoming more local and immediate as you move from the constitutive order to arena to setting. teachers are a particularly important group because ultimately they enact a curriculum at the setting level. involving them in the curriculum development process can help address the challenge of clarity and hold: • clarity, in the sense of having a shared understanding about what needs to be learnt, and • hold in the sense of ensuring alignment between the specified curriculum and what is enacted in the setting. the consequence of misalignment between curriculum purposes and curriculum practices can be seen in donaldson's ( ) report on the national curriculum in wales. donaldson's suggestions for improving the curriculum were based on an analysis of the problems of the previous curriculum in wales. donaldson notes that a highly prescriptive curriculum combined with powerful legislative and accountability mechanisms led to "much of the curriculum as experienced by children and young people has become detached from its avowed aims and too focused on the short-term" (p. ). donaldson's approach to addressing these problems and to align curriculum, assessment and accountability was to "involve as many people in wales as possible" ( , p. ). however, how to organise involving as many stakeholders as possible is a thorny issue. so it is useful to learn and build on what other countries have tried as outlined in the example below from the netherlands. the netherlands is an example which illustrates a range of typical approaches to tackling the problems and tensions of trying to address the shifting priorities of society particularly in relation to the use of digital technologies and how to accommodate their use in the school curriculum. however, what is unique is how the focus came round to having a national discussion on the future of schooling. since the s/ s, 'information science' and 'informatics' was part of the national curriculum in the netherlands, this was: (a) particularly the case in secondary schooling and; (b) primarily focused on understanding and being able to work with computers and programming. these subjects eventually proved very difficult to implement and they disappeared in from the curriculum (voogt and ten brummelhuis ) . the discussion about ict in schooling gradually changed from learning about ict to using ict for learning, and more and more attention was paid to the integration of ict in schooling as a "tool" for teachers. however a report from the royal netherlands academy of arts and sciences (knaw ) stated that the increasing digitisation of information and communication in society required new skills but that these skills (digital literacy) were not getting sufficient attention in schooling. this report ignited the discussion again with regard to the role and use of digital technologies in schools. this focus, coupled with a range of other issues which were gaining attention (such as twenty-first century skills, equity, and the perceived overload of the current curriculum), led to a broad national discussion about the future of schooling in the netherlands. in november , the state secretary for education, culture and science of the netherlands officially launched an online countrywide consultation about the future of primary and secondary schooling. everyone in the netherlands had the opportunity to take part. the board consultation process outlined in fig. was the start of a process of curriculum design. this consultative process was an effort to garner the views of as wide a range of stakeholders as possible to begin the process of reaching an agreement on a shared common purpose for the curriculum (rationale in terms of the spider web). for example, via social media over , people contributed their ideas on what the young students of today should learn if they are to be productive members of society in the year . based on the consultation an independent commission, platform onderwijs , wrote an advisory report about future-oriented education in the netherlands which they presented in january to the state secretary, this was meant as an overall rationale for the ongoing curriculum review. next to the importance of specific content domains such as language, science, numeracy and social studies they concluded that citizenship and digital literacy should also be part of the formal curriculum (platform onderwijs ). conscious of building a shared understanding of a common purpose to underpin the curriculum, it took a year to discuss the report with pupils, teachers, parents, school leaders, administrators, scientists, representatives of social and cultural organizations, business and political parties (platform onderwijs ). based on these discussions parliament agreed that a new curriculum for the primary and secondary school sector was needed and in the process to develop this was started. based on the consultative process this new curriculum would consist of nine subjects: dutch, arithmetic/mathematics, english/ modern foreign languages, exercise & sport, art & culture, human & nature, human & society, citizenship, and digital literacy. the next stage of the curriculum design process is particularly difficult in a co-constructive process. there is always a challenge to get a balance between involving everyone and making sure you have a workable situation. what the dutch example illustrates is what a government and the coordination group accomplished by letting the teacher design teams do the work and giving all others the opportunity to react. as a starting point, teachers and school leaders were recruited to work on the new curriculum. for each subject a teacher design team (tdt) was formed to work on a subject. the tdt worked together in six three-day sessions. after each three-day session, the documents they had produced were open for online consultation. everyone with an interest in the various subjects was able to react to the documents. next to the general consultation rounds, all documents were discussed with the specific "subject associations". these associations are groups of experts from a specific subject. the reactions/feedback was not taken for granted. everything was read and either included in the documents or -when decided that it should not be included -arguments were given for why it was omitted. in addition, a special working group on "coherence" with experts from the national expertise centre for curriculum development analysed all documents from all working groups to see if the big ideas and the key stages or learning trajectories were a) in line with the rationale and b) in line with each other. taking digital literacy as an example, a teacher design team consisting of teachers, two school leaders, and two members of the national institute for curriculum development built a vision for the subject, designed big ideas, and corresponding learning trajectories for the big ideas (ontwikkelteam digitale geletterdheid ). the tdt defined digital literacy as the skills that relate to using ict effectively, efficiently and responsibly. they involve a combination of ict (basic) skills, computational thinking, media literacy and information literacy (thijs et al. ) . big ideas (also known as essential understandings) are broad statements that frame what students will learn (government of alberta ). eight big ideas were described by the tdt: ( ) data and information, ( ) safety and privacy, ( ) using and controlling, ( ) communication and cooperation, ( ) digital citizenship, ( ) digital economy, ( ) applying and designing, and ( ) sustainability. in order to help teachers from primary and secondary schooling to teach the concepts of digital literacy key stages or learning trajectories needed to be designed. learning trajectories were defined as "a reasoned structured set of intermediate objectives and content leading to a certain core objective" (strijker , p. ). learning trajectories not only describe what students should learn, providing clarity about the core objective, but also afford the opportunity to personalise learning by adjusting learning goals and related learning activities to the needs of the learners. the tdt concluded that digital literacy should be both a separate subject and embedded as a cross-curricular theme across all disciplines. taking all the feedback at each stage into consideration, the tdt for digital literacy published their final version of the vision, big ideas and learning trajectories in june . the products of all tdts were combined into a final report that was sent to parliament in october . after discussions in parliament the decision was made to start with the next stage of curriculum development for primary and lower secondary schools: making the learning trajectories and goals part of the legal and mandatory framework for primary and lower secondary schooling in the netherlands. working with teacher design teams again, teachers, school leaders and teacher trainers will work together, supported by experts from the national expertise centre for curriculum development to complete this next stage of curriculum development. it is expected that the new curriculum will be put into practice in . there are a wide range of groups and individuals who can make a strongly justified claim to be a key stakeholder in schooling and thus have a right and a responsibility to contribute to the curriculum design process. while the approach taken in the netherlands ensures that teacher design teams are at the core of the curriculum reform, we need to pay particular attention to the wider body of teachers who, although given the opportunity to contribute to consultations, may have been less directly involved in the overall curriculum design. these teachers form a particularly significant stakeholder group because of their direct control over how curriculum intentions and expectations are actualised in classroom teaching and learning activities (at the setting level). the second key challenge relates to the role and professional status of the school teacher in the enactment of any new curriculum at the setting level. accepting that teachers are central to the adoption of any curriculum reform (kärkkäinen ; schoenfeld ), does not just require teacher 'buy in' but recognition of what it takes to develop ownership by the teaching profession of any new curriculum or curriculum innovation. in considering the schooling system as a whole (constitutive order, school arena, setting), the challenge is therefore how to capture and build on the expertise of teachers so that they become invested and genuinely participative in curriculum reform, thus ensuring that intended curriculum aims are achieved in practice. the extent to which teachers adhere to the specified curriculum and have control over classroom activities will vary between countries, local school systems, and within individual schools. this relates to the professional status of the teacher in their context and directly impacts on the effectiveness of any attempt at curriculum reform. the talis (oecd ) teacher survey suggests that although teachers in most countries have a large degree of autonomy over their practice in the classroom, they still have significant challenges ahead of them. only % of teachers in the countries surveyed say that policy makers in their country/region value their view, and only % believe that they can influence education policy. this reflects a lack of trust of teachers, which emerged as active distrust in the later part of the twentieth century followed by a focus on achievement data and top-down measures that emphasised performance targets and public scrutiny (hargreaves and shirley ) . the implications according to schleicher ( , p. ) are that teachers "rarely own their professional standards to the extent other professionals do, and rarely work with the level of autonomy and in the collaborative culture that people in other knowledge-based professions take for granted". decades of research show that curriculum reform does not automatically result in sustainable changes in the everyday practices of schools (fullan and miles ) and that how reform is implemented plays a key role in determining whether an intended curriculum achieves its desired outcomes (mclaughlin ; tuinamuana ) . it is also well documented that teachers have a central role in the successful adoption of any curriculum reform (kärkkäinen ; schoenfeld ) and that new curricula are often not implemented as planned. this lack of implementation as planned is due in large part to conflicts between the principles of a new curriculum and teachers' existing beliefs and practices about teaching (orafi and borg ) . a new curriculum may demand significant shift in thinking and practice which according to prendergast and treacy ( ) leads to concerns among teachers on issues such as the reasoning behind the reform, the implications for classroom practices and students, as well as their ability to implement the changes. thus, educators may implement the reform with fidelity by following the curriculum as prescribed, adapt the curriculum to the local context while adhering to its core principles, comply with the curriculum by only implementing surface-level changes, co-opt the curriculum to fit with existing practices, or not implement the curriculum at all (berman and mcloughlin ; tichnor-wagner et al. ; both cited in taguma and barrera ). there are two possible conceptions of the role of the teacher in transacting a curriculum: to view teachers as technicians whose job is to deliver what others have decided, or to treat teachers as professionals who are best placed to ensure that schooling meets the aims that society has agreed on. the first view, a top-down, centralised approach where policymakers or curriculum designers seek compliance from implementers on the ground (e.g. teachers) to carry out their directives without any alterations to the intended curriculum design, can lead to teacher de-professionalisation as the judgement of teachers is seen to no longer be valuable (ball ) . it can be enacted through attempts to 'teacher-proof' the curriculum via overly-prescriptive lesson plans or scripts based on a "theoretical assumption that … the behaviour of teachers needs to be controlled and made consistent and predictable across different schools and student populations" (giroux , p. ) . however, such attempts can have negative consequences, including reducing teachers to 'task managers' rather than 'concept/skill builders' who are concerned with the pupils' deep learning of knowledge and skills (twistleton ) . it leads to the erosion of teacher autonomy, along with their personal and professional identities (day ) . ultimately teachers' knowledge and professional practices are diminished (giroux ; fitzgerald and knipe ; mclaren and farahmandpur ) . as pollard ( ) suggests, pedagogy is 'impoverished' (p. ) if teachers are unable to engage in curriculum development. the de-professionalisation of teaching has happened in many countries (rubin ; rubtcova et al. ) . according to priestley et al. ( , p. ) this control over schooling "could be seen as the result of such systems having been subject for at least two decades to the combined influence of prescriptive national curricula and the use of outcomes steering, both backed by rigorous inspection regimes and the quantitative use of attainment data". in the uk for example, in his summary of findings from the cambridge primary review, alexander ( , p. ) states that "in many primary (uk) schools a professional culture of excitement, inventiveness and healthy scepticism has been supplanted by one of dependency, compliance and even fear; and the approach may in some cases have depressed both standards of learning and the quality of teaching". in portugal, teodoro and estrela ( , p. ) report that teacher autonomy was "progressively removed with the emergence of administrative control of the curriculum". while in the usa, au ( , p. ) claims that 'teachers' power' [is] being increasingly usurped through policy and curriculum structure. this erosion of the teaching profession and deprofessionalising of the teacher now presents as a serious challenge and both reflects and undermines trust in teachers. recent trends in curriculum development have placed greater emphasis on the formulation of curricula in terms of competences and capacities and on the teacher as a central agent in curriculum development (priestley and biesta ; sinnema and aitken ) . the focus on twenty-first century skills and competences has been identified in various comparative analyses of curriculum (e.g. creese et al. ) . trends towards school-based curriculum within a central framework (kärkkäinen ) and an outcomes-based educational approach (biesta et al. ) have also been idenified. many countries have accordingly prepared curriculum frameworks rather than prescribed curricula. such frameworks allow flexibility in interpretation and enactment at teacher level. for example, priestley and sinnema ( ) found that current curriculum policies in new zealand and scotland are characterised by flexibility at both national and school levels. similarly, in quebec, the ministry of education's digital action plan measures for how digital technology might be used are presented as suggestions, although the use of digital technologies in teaching and learning is required. ireland also has a digital learning framework (department of education and skills a, b) which is comprised of standards and statements of practice for embedding digital technologies in teaching. however, it is up to schools and teachers to decide which statements to focus on. these changes represent a shift in emphasis from teachers as 'curriculum implementers' to 'curriculum developers and co-constructors'. however, this shift not only places the teacher at the centre of curriculum development but accentuates the importance of teacher capacity and autonomy (biesta et al. ) . the challenge is how to gain the 'buy-in' of teachers so that they become invested in and develop ownership of the reform. while it is suggested that embracing a culture of trust and autonomy, and promoting teachers' agency over the curriculum reform, increases teachers' engagement in the reform work (mccharen et al. ; priestley ) ; it is accepted that teachers are often not fully engaged in the consultation process when decisions are made in relation to curriculum (e.g. elliott ; williamson ) . this is significant when one considers that teachers' expertise and attitudes, including their understanding of the reform, contributes to the way the reform is implemented (tikkanen et al. ). in addition, tensions continue to exist in this form of curriculum development between the autonomy proffered to teachers as curriculum developers and the control and accountability mechanisms of the wider system (biesta and priestley ). this raises questions about how to re-professionalise teaching and hence increase trust of teachers. fundamentally this is about ensuring that teachers are treated and act like profesionals. the key characteristics of professionals include: • being knowlegable about the underpinning principles and theories in your field; • having expertise in curriculum and assessment enactment; • engaging in regular professional learning and re-acreditation; • being the creators and curators of knowledge in your field; • working the hours necessary to perform their role rather than a fixed number of hours; • having a high degree of autonomy, within the bounds of agreed professional standards; • having a independent professional body that oversees the professional standards. the first four of these are closely linked to pre-service and continuing professional learning. based on evidence from the literature twining and henry ( ) identitied key characteristics of effective professional learning (see fig. )-it should be: • strategic-involving the senior management team, linking with the school development plan, and being integrated with the school's self-review and/or performance management processes; • impact focussed -in terms of enhancing curriculum, pedagogy, and/or learning outcomes; • context relevant -related to the needs of the staff and students and using resources/ equipment available in the school; • informed by both internal expertise (e.g. school staff) and external expertise (e.g. consultants); • collaborative -supporting sharing and professional dialogue; • experimental and reflective -trying out new ways of working and reflecting collectively on practice; • sustained rather than one-off; • evaluated in relation to planned impact. practitioner research can encapsulate all of those elements, and positions teachers as the creators of their professional knowledge base (twining and henry ) . the last three characteristics of professionals relate to the regulation and autonomy of the field. the key challenge here is how to uncouple education policy (at the constitutive order level) from short-term political control. finland, which is widely recognised as being one of the more successful education systems in the world, has a model in which schools have considerable autonomy (world bank , p. ). they note two key factors that underpin the success of the finnish education system: "the two most important factors explaining the success of the finnish education system are: (i) education has been a national priority for decades, and (ii) the system operates on trust." trust (or lack of it) and summative assessment are closely linked. as stated in the introduction, the word curriculum is related to the latin verb currere, which means course or track to be followed. in schooling contexts summative assessment is sometimes seen as the finish line of a race. indeed, summative assessment generally drives practice in schools irrespective of what the curriculum specifies (e.g. conseil supérieur de l'éducation ; etag ) and thus, where something is not being assessed it is likely not to be a priority (robinson and aronica ) . the pressure to improve student performance on mandated tests diminishes teachers' authority and control over what and fig. characteristics of effective teacher professional learning (twining b) how they teach (au ; wills and sandholtz ) . fundamentally, there are two main issues with current forms of summative assessment. the first issue is that summative assessments are often used not only to establish what students have learnt, but also as indicators of the quality of the educational provision (e.g. klenowski ) . thus, teachers focus on summative assessment because that is what they are held accountable against (etag ) . berliner ( ) highlights that this can have a range of negative educational impacts. such impacts include: off-rolling students; holding students back by a year; focussing on students who are on grade boundaries; cheating; narrowing the curriculum; and teaching to the test. this problem could be overcome by separating out the evaluation of the quality of school provision from the assessment of what young people have learnt. the second issue is that traditional forms of summative assessment are unable to capture evidence of many of the competences and dispostions that are valued as being essential today if schools are going to succeed in enhancing individual fulfilment and universal wellbeing. the fact that summative assessments, especially national assessments, are determinant for pupils' access to higher education (e.g. a levels in england; hscs in australia; the leaving certificate in ireland) results in too narrow a focus on academic understanding at the expense of other critical competences and dispositions. high stakes testing replaces trust in teachers' professional judgment. summative assessment is of course important for selection and certification purposes. the challenge is how to assess the competences and dispositions that are seen as being essential, as well as the knowledge that current high stakes assessments focus on. we need to develop new forms of summative assessment that complement existing ones, and align with the purposes of schooling. whilst this would increase the validity of school assessment in the sense of measuring the things that are important, achieving this raises many challenges as well as opportunities (webb and ifenthaler ) , including potentially reducing construct validity and reliability. thus, for example, including teacher judgements of practical performance, such as in a science experiment, would increase the validity of a science assessment. however, it would also increase the risk of inter-marker inconsistency, which would need to be mitigated by some form of moderation process. this highlights three key issues that are explored further below: • criteria for evaluating summative assessment • approaches to complementing traditional forms of assessment including emerging possibilities • teachers professional learning to enable them to design assessment tasks twining ( a) set out criteria for evaluating summative assessment of young people's learning. these include being: • relevant -this is an aspect of validity: summative assessment needs to measure things that are important (i.e. the goals identified in the curriculum) • credible -this combines aspects of validity (measuring what it claims to measure) and reliability (being both accurate and consistent) • practical -it must be feasible to implement, and ideally integral to the learning process • scalable -it must be able to be used with large numbers of learners • formative -assessment should inform future learning as well as providing a summary of current knowledge, competences and dispositions • positive -it should be constructive, and focus on success rather than failure • ethical, including: -it should enable everyone to succeed by being at an appropriate level and/or providing alternative pathways to success. norm based assessment is unethical because it requires that some people fail in order for others to succeed -it should be transparent -both in the sense of not being covert and being clear why particular judgements have been made -it should be unbiased -it should not favour particular individuals • able to be reported concisely -this is important because recruiters and admissions officers typically don't have time to read through copious documentation about student performance a number of approaches to complementing traditional forms of assessment have been developed, including: portfolios, eexams and digital badges. portfolios, can support reflection and self-assessment. at the junior cycle ( year olds) in ireland there has been a shift towards the use of portfolios in place of traditional examinations. whilst they can provide rich evidence of achievement, that can be mapped to standards, they are time consuming to mark or evaluate. digital badges, and other forms of micro credentials, can provide certification for competencies and dispostions which may have been developed either within or outside formal schooling. they suffer from a lack of standardisation, which potentially undermines their credibility. eexams, which use the power of digital technology, potentially enable students to demonstrate competences and dispositions (etag ) , for example through the use of simulations. however, the pragmatics of moving to eexams are considerable. for example, in a school context where all students studying a particular subject need to take their exam at the same time and in 'exam conditions', as is common for high stakes tests, most schools do not have the necessary infrastructure (e.g. hundreds of devices and robust internet). approaches to addressing this challenge have been developed, based on a bring your own (byo) device model (e.g. fluck and hillier ), but have not gained traction in schools. there are also a number of emerging possibilities, including: • point of learning (pol) which is a new approach to collecting compelling evidence of competences and dispositions in the form of 'claims' based on observations of practice by multiple people over prolonged periods (twining, rix and sheehy ) . • social learning analytics which utilises ai, and in particular data mining techniques to assess learning in online environments (e.g. see twining c). • the use of online 'living cvs' which include endorsements and recommendations from members of the learner's community both inside and outside school (e.g. twining b). this reflects how adults use linkedin to develop their professional profiles and networks. one way to start to develop new approaches to assessment would be to ensure that teachers are better educated to design assessment tasks. whilst in some countries teacher education provision does focus on assessment, this tends to be formative rather than summative assessment. in other jurisdictions, such as quebec, there are very few courses dedicated to assessment in teachers' professional learning programmes, and what there are tend to be disconnected from courses about teaching subjects (conseil supérieur de l'éducation ). unless solutions are found to 'the assessment problem', the gap between the rhetoric of national curricula and the reality of practice in schools will remain. there is a growing consensus that current school curricula are not meeting the needs of individuals or society. if we are going to strive for a vision of individual fulfilment and universal wellbeing then the curriculum needs to prepare young people to influence the world outside school and to tackle the wicked challenges that humanity faces. in short, the curriculum needs to follow purpose. this means that we need to change the predominantly content focus of traditional curricula so that they encompass knowledge, competences and dispositions. that of course has knock on implications for many other aspects of educational provision, as highlighted in the spider web of inter-relationships (fig. ) . we also need to recognise that a curriculum that genuinely focusses on learners having the ability to act outside school will require not just a change in curriculum but also in pedagogy. however a change in pedagogy won't be enough if the teacher is not recognised and respected as a professional who has an active role in determining the curriculum and how learning is to be assessed. furthermore, we need to recognise that it doesn't matter what the curriculum says if it is not aligned with summative assessment and does not have the buy in of key stakeholders. in exploring the development of a quality curriculum in the digital era, this paper has clarified that a quality curriculum is one that supports young people in acquiring the knowledge, competences and dispositions needed to be successful in the digital era. the yin-yang vision framework was introduced, as a bridge between supra (international) and macro (national) level curriculum debates, that highlights the importance of purpose, and provides a tool to think with at the macro and meso (school) levels. however, in tandem with highlighting the importance of purpose as a critical starting point for developing a quality curriculum, understanding the issue of alignment was identified as essential. using a socio-cultural framework (fig. ) this paper illustrates the complexity of alignment of purpose, policy (including curriculum, assessment, accountability and teacher professional learning) and practice between and within each level (constitutive order, school arena, and setting). three key challenges in achieving alignment were discussed, namely: stakeholder engagement, enhancing the professionalism of teachers, and developing better forms of summative assessment. understanding and addressing these issues is essential in order to achieve alignment between purpose, policy and practice across levels of the school system. the recent covid- crisis has opened up debates and started to crystalise these issues. for example, clarifying one key purpose of schooling being to provide child care so that parents (and in particular 'essential workers') can go to work. similarly, the replacement of high stakes tests in many countries (e.g. england, scotland, ireland, australia, slovakia) with alternative approaches to summatively assessing school leavers raises questions about the role of teacher assessments and opens the door for a wider debate about teacher professionalism and how best to judge students' learning. running through all three of the key issues explored in this paper is a tension between flexibility and control of the curriculum, and fundamentally the extent to which teachers are trusted. perhaps the time is right, to move from what hargreaves and shirley ( ) describe as the third way-trust through control and accountability-to what they call the fourth way-trust based on a shared purpose, partnership, and teacher professionalism. children, their world, their education: final report and recommendations of the cambridge primary review the best that has been thought and said teaching under the new taylorism: high-stakes testing and the standardization of the st century curriculum performativity, privatisation, professionals and the state rational responses to high stakes testing: the case of curriculum narrowing and the harm that follows against learning: reclaiming a language for education in an capacities and the curriculum the role of beliefs in teacher agency. teachers and teaching: theory and practice education systems in the digital age: the need for alignment Évaluer pour que ça compte vraiment, rapport sur l'état et les besoins de l'éducation comparing international curriculum systems: the international instructional systems study school reform and transitions in teacher professionalism and identity en/schoo ls-colle ges/infor matio n/ infor matio n-commu nicat ions-techn ology -ict-in-schoo ls/digit al-learn ing-frame work-prima ry en/schoo ls-colle ges/infor matio n/infor matio n-commu nicat ions-techn ology 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educational change bringing about curriculum innovations the curriculum: theory and practice questioning the validity of multiple uses of naplan data education, culture and the national curriculum cognition in practice: mind, mathematics and culture in everyday life school innovation: the mutual impacts of organizational learning and creativity the pedagogy of oppression: a brief look at 'no child left behinds the rand change agent study revisited: macro perspectives and micro realities the future of education and skills: education education at a glance : oecd indicators teachers and school leaders as valued professional-sparis: talis concept-eindproduct van het ontwikkelteam digitale geletterdheid intentions and realities in implementing communicative curriculum reform ons onderwijs : eindadvies [our education -final recommendations platform onderwijs professionalism and pedagogy: a contemporary opportunity. a commentary by tlrp and gtce curriculum reform in irish secondary schools? a focus on algebra schools, teachers, and curriculum change: a balancing act teacher agency in curriculum making: agents of change and spaces for manoeuvre reinventing the curriculum: new trends in curriculum policy and practice downgraded curriculum? an analysis of knowledge in new curricula in scotland and new zealand creative schools: the grassroots revolution that's transforming education digitale geletterdheid in het voortgezet onderwijs the disheartened teacher: living in the age of standardisation, high-stakes assessments, and no child left behind (nclb) deprofessionalisation as a performance management dysfunction: the case of inclusive education teachers in russia what makes for powerful classrooms, and how can we support teachers in creating them? a story of research and practice teachers' use of research to improve practice: why should we, how could we leerlijnen en vocabulaires in de praktijk (learning trajectories and vocabularies in practice) curriculum development: theory and practice oecd future of education and skills : curriculum analysis the curriculum debate: why it is important today (ibe working papers on curriculum issues global agendas and regional and national reconfigurations e eeuwse vaardigheden in het curriculum van het funderend onderwijs primary determinants of a large-scale curriculum reform: national board administrators' perspectives teacher professional standards, accountability, and ideology: alternative discourses educational alignment (and sociocultural theory). halfbaked.education blog what should be learnt? halfbaked characterisitcs of effective cpd ai and assessment -mining learning outcomes characteristics of effective summative assessment. halfbaked.education blog. https ://halfb aked.educa tion/chara cteri stics -of-effec tive-summa tive-asses sment from standardised testing to living cvs enhancing 'ict teaching' in english schools: vital lessons np -new purposes, new practices, new pedagogy: meta-analysis report. society for educational studies _devel oping _point _of_learn ing_an_innov ative _appro ach_to_enhan cing_profe ssion al_learn ing resolution / : transforming our world: the agenda for sustainable development curriculum perspectives: an introduction information literacy in the netherlands: rise, fall and revival towards an overview of a redeveloped primary school curriculum: learning from the past, learning from others. dublin: national council for curriculum and assessment section introduction: using information technology for assessement: issues and opportunities constrained professionalism: dilemas of teaching in the face of testbased accountability education workforce initiative: initial research systems approach for better education results -finland -school autonomy and accountability publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.peter twining is professor of education (innovation in schooling and educational technology) at the university of newcastle (australia). he has been professor of education (futures) at the open university (uk), co-editor in chief for computers & education, and the lead on a number of major projects looking at the use of digital technology both inside and outside formal education.deirdre butler is a professor in dcu's institute of education, whose passion in life is exploring what being digital in learning can mean and what skills or competencies are needed to live in today's complex globally connected world, challenging us to examine how we learn and questioning our assumptions about "traditional" models of schooling. she has designed and managed a range of projects / school-based initiatives which focus on sustainable, scalable models of teacher professional learning and creative uses of digital technologies.petra fisser is currently working as educational advisor at the college of health care of roc van twente, a regional training centre for vocational education and training and adult education. projects she is involved in are related to educational innovation of secondary vocational education and training (vet), adult education, and tailor-made courses for sme's. before working at roc van twente, petra worked at the national institute for curriculum development and the university of twente in the netherlands, where she was engaged in projects, knowledge development and professional development in the field of curriculum development and evaluation, internationalization, digital literacy and tpack.margaret leahy is head of the school of stem education, innovation and global studies at the institute of education, dcu university. she has extensive experience in the theory and practice of digital learning, the design and development of professional learning programmes for teachers and has been involved in a range of innovations/research projects, all of which focus on ways digital technologies can be used to create more powerful learning environments.chris shelton is head of education within the institute of education, health and social sciences at the university of chichester where he is responsible for education and teacher education programmes. chris has a range of research interests relating to technology, computing and digital literacy in schools and universities. his recent work has included publications about teacher's thinking about technology in higher education; the pedagogy of teaching computing; and the curriculum.nadine forget-dubois has a ph.d. in anthropology from the université de montréal and is a research agent at the conseil supérieur de l'éducation du québec, attached to the production of the report on the states and needs of education.michel lacasse is a doctoral candidate in education administration and policy at université laval. his thesis subject concerns the technological competence of school directors and, between theory and practice, he participates in various research projects in the field of education, which include school governance, alternative pedagogies, educational technologies and the training of teachers and administrators of education. key: cord- -pnjhi cu authors: foreman, stephen; kilsdonk, joseph; boggs, kelly; mouradian, wendy e.; boulter, suzanne; casamassimo, paul; powell, valerie j. h.; piraino, beth; shoemaker, wells; kovarik, jessica; waxman, evan(jake); cheriyan, biju; hood, henry; farman, allan g.; holder, matthew; torres-urquidy, miguel humberto; walji, muhammad f.; acharya, amit; mahnke, andrea; chyou, po-huang; din, franklin m.; schrodi, steven j. title: broader considerations of medical and dental data integration date: - - journal: integration of medical and dental care and patient data doi: . / - - - - _ sha: doc_id: cord_uid: pnjhi cu dental health insurance coverage in the united states is either nonexistent (medicare and the uninsured), spotty (medicaid) and limited (most employer-based private benefit plans). perhaps as a result, dental health in the united states is not good. what public policy makers may not appreciate is that this may well be impacting medical care costs in a way that improved dental benefits would produce a substantial return to investment in expanded dental insurance coverage. have been rising at double digit rates. most employers have been dropping health care coverage rather than expanding it ( kaiser family foundation ) . medicare trust funds are bankrupt (social security and medicare boards of trustees ). adding coverage would exacerbate an already alarming problem. medicaid funding is a major source of state government defi cits. many states are slashing medicaid coverage during this time of crisis (wolf ). improving medicaid dental coverage during times of budget crisis would meet substantial political resistance. strikingly, strong and increasing evidence suggests relationships between oral health and a range of chronic illnesses. for example, recent fi ndings show relationships between periodontal infl ammatory conditions and diabetes, myocardial infarction, coronary artery disease, stroke, preeclampsia and rheumatoid arthritis. this suggests that improved oral health may well have the potential to reduce the incidence of chronic diseases as well as their complications. if chronic disease incidence is reduced it may be possible to avoid medical care costs related to treating them. it would be important to know more about the extent to which improved oral health could reduce health care costs and improve lives. there are few, if any, studies of the costs of providing medicare dental benefi ts, the costs of improving the medicaid dental benefi t or the cost of providing dental insurance to the uninsured. there are a few studies that indicate that periodontitis increases medical care costs, perhaps by as much as % (ide et al. ; albert et al. ) . ideally there should be a controlled study to assess the benefi t of providing dental coverage through a government payer system. for a preliminary inquiry we can consider work already done and using some cost and benefi t estimates, determine whether it is possible that benefi ts of extending dental coverage may outweigh costs. the failure of medicare to cover dental care has engendered some (albeit not much) public debate. in , congress enacted the medicare prescription drug, improvement, and modernization act (medicare part d). by medicare provided $ . billion in benefi t payments for outpatient prescription drugs and medicaid paid . billion for outpatient prescription drugs (center for medicare and medicaid services ) . benefi ciaries provided billions more in the form of monthly part d premiums. the expense of the medicare prescription drug program and the controversy surrounding its enactment may well have eroded public support for increased medicare coverage. so while there has been no shortage of effort paid to improving medicare, the one common theme in all of the recent initiatives is that dental care has been conspicuously a new study by hedlund, jeffcoat, genco and tanna funded by cigna of patients with type ii diabetes and periodontal disease found that medical costs of patients who received maintenance therapy were $ . per year lower than patients who did not. cigna, research from cigna supports potential association between treated gum disease and reduced medical costs for people with diabetes, http://newsroom.cigna.com/newsreleases/research-from-cigna-supports-potential-association-between-treated-gum-disease-and-reduced-medical-costs-for-people-with-diabetes. omitted. as a result, million medicare recipients in (us census bureau ) continue to have no dental insurance coverage through medicare. medicaid dental coverage is an optional benefi t that states may or may not elect to provide. in medicaid, both the state and the federal government provide funds to cover healthcare services to eligible patients. the bulk of the money comes from the federal government. because the medicaid dollars are limited and coverage for systemic diseases has precedence, medicaid coverage of dental care has been spotty. even where it has been provided, payments to dental providers have been so low as to make it diffi cult or impossible for medicaid benefi ciaries to obtain adequate dental care (broadwater ) . the recession increased the number of medicaid eligible individuals nationwide. further, the federal budget defi cits of the past few years have reduced the federal contribution to state medicaid programs. the combination of increases in the number of benefi ciaries and diminished revenues has caused a number of states to eliminate or curtail medicaid dental coverage (ehow ; mullins et al. ) . the result, million medicaid benefi ciaries in the us (us census bureau ) in either had no dental insurance coverage or inadequate coverage. approximately million people in the united states do not have health insurance (kaiser family foundation ) . presumably, they have no dental insurance either. further, not every employer provides dental insurance. a cdc survey found that . % of adults do not have dental insurance coverage (centers for disease control ) . a montana survey found that % of employers who offer health insurance do not offer dental insurance coverage (montana business journal ) . in there were approximately million people enrolled in health insurance plans (us census bureau ) . if half (a rough combination of the cdc and montana percentages) of them do not have dental insurance it is likely that an additional million (nonelderly, non-poor) people in the us do not have dental insurance coverage. finally, the term "dental insurance" is actually a misnomer. dental policies cover routine treatments, offer discounts for more complex treatment and impose a low yearly on total payments. in fact, it has been called "part insurance, part prepayment and part large volume discount" (manski ) . effectively, many (if not most) people who have dental insurance fi nd it coverage to be quite restrictive. for example, many impose a small yearly cap ($ , is common) or large coinsurance amounts ( % for orthodontia, for example) (rubenstein ) . even with discounts it is easy for many people to exceed the annual limit. given the lack of dental insurance coverage it is not surprising that the status of oral health in the us is not particularly good. in approximately . % of adults between the ages of and had untreated caries, % had decayed, missing and fi lled tooth surfaces and more than one-half of adults had gingival bleeding (dental, oral and craniofacial data resource center of the national institute of dental and craniofacial research ) . three fourths of adults in the us have gingivitis and % have periodontitis (mealey and rose ) . if these levels of untreated disease were applied to most systemic diseases, there would be public outcry. over the past decade evidence has been building that there is a relationship between dental disease, particularly periodontal disease, and chronic illnesses. mealey and rose note that there is strong evidence that "diabetes is a risk factor for gingivitis and periodontitis and that the level of glycemic control appears to be an important determinant in this relationship" (mealey and rose ) . moreover, diabetics have a six times greater risk for worsening of glycemic control over time compared to those without periodontitis and, periodontitis is associated with an increased risk for diabetic complications. for example, in one study more than % of diabetics with periodontitis experienced one or more major cardiovascular, cerebrovascular or peripheral vascular events compared to % of the diabetic subjects without periodontitis (thorstensson et al. ) . also, a longitudinal study of type diabetics found that the death rate from ischemic heart disease was . times higher in subjects with severe periodontitis and the death rate from diabetic nephropathy was . times higher (saremi et al. ) . clinical trials have demonstrated that treatment of periodontal disease improved glycemic control in diabetics (miller et al. ) . moreover, investigations have found an association between periodontal disease and the development of glucose intolerance in non-diabetics (saito et al. ) . while it is diffi cult to establish causality and it is possible that other factors infl uence periodontal disease and medical complications, these studies suggest that treatment of periodontitis substantially improves health and greatly reduces medical complications related to diabetes. similarly, periodontitis is associated with cardiovascular disease and its complications including ischemia, atherosclerosis, myocardial infarction and stroke. a study by slade and colleagues found both a relationship between periodontitis and elevated serum c-reactive protein levels (systemic marker of infl ammation and documented risk factor for cardiovascular disease) as well as a relationship among body mass index, periodontitis and crp concentrations (slade et al. ) . hung and colleagues evaluated the association between baseline number of teeth and incident tooth loss and peripheral arterial disease. they determined that incident tooth loss was signifi cantly associated with pad, particularly among men with periodontal disease potentially implying an oral infection-infl ammation pathway (hund et al. ) . the same group of researchers used the population enrolled in the health professionals' follow-up study ( , men free of cardiovascular disease and diabetes at baseline) to assess the relationship between tooth loss and periodontal disease and ischemic stroke. controlling for a wide range of factors including smoking, obesity, and dietary factors, the researchers found a "modest" association between baseline periodontal disease history and ischemic stroke . as early as destefano and colleagues found that among subjects, those with periodontitis had a % increased risk of coronary heart disease relative to those without. the association was particularly high among young men. the authors questioned whether the association was causal or not, suggesting that it might be a more general indicator of personal hygiene and possibly health care practices (destefano et al. ) . in wu and colleagues used data from the first national health and nutrition examination survey and its epidemiologic follow-up study to examine the association between periodontal disease and cerebrovascular accidents. the study found that periodontitis was a signifi cant risk factor for total cva, in particular, for non-hemorrhagic stroke (wu et al. ) . in addition to diabetes and coronary artery disease, associations have been found between periodontal disease and rheumatoid arthritis and respiratory disease. this is not surprising given the role of periodontal disease in the production of infl ammation related proteins. dissick and colleagues conducted a pilot study of the associate ion between periodontitis and rheumatoid arthritis using multivariate regression and chi square tests. they found that periodontitis was more prevalent in patients with rheumatoid arthritis than in the control group and that patients who were seropositive for rheumatoid factor were more likely to have moderate to severe periodontitis than patients who were rf negative and also that patients who were positive for anti-cyclic citrullinated peptide antibodies were more likely to have moderate to severe periodontitis (redman et al. ) . paju and scannapeico investigated the association among oral biofi lms, periodontitis and pulmonary infections. they noted that periodontitis seems to infl uence the incidence of pulmonary infections, particularly nosocomial pneumonia in high-risk subjects and that improved oral hygiene has been shown to reduce the occurrence of nosocomial pneumonia. they found that oral colonization by potential respiratory pathogens, for possibly fostered by periodontitis and possibly by bacteria specifi c to the oral cavity contribute to pulmonary infections (paju and scannapeico ) . the implications for these fi ndings are profound. professionally, they suggest that managing patients with chronic illness and periodontal disease will require teamwork and a deeper knowledge base for dentists and for physicians (mealey and rose ) . dentists will need to be alert for early signs of chronic illness among their patients and physicians will need to be alert for signs of dental disease. both will need to consider wider treatment options than their specialty indicates. dentistry and medicine have operated as professional silos in the past. the relationship between dental disease and chronic medical conditions suggests that continued separation is detrimental to patient centered care. beyond treatment implications, there are extremely important health policy concerns. if treatment of periodontitis and other dental problems leads to reduced incidence of chronic illness, fewer complications from chronic diseases and reduced morbidity among chronically ill patients, increased access to dental services could signifi cantly reduce health care costs. the diseases associated with periodontitis are among the most common illnesses, the fastest growing and the most expensive diseases that we treat. a recent robert wood johnson report notes that approximately million americans have one or more chronic conditions, that the number of people with chronic conditions is expected to increase by % per year for the foreseeable future and that the most common chronic conditions include hypertension, disorders of lipid metabolism, upper respiratory disease, joint disorders, heart disease, diabetes, cardiovascular disorders, asthma and chronic respiratory infections (anderson ) (see fig . . ). one in four americans has multiple chronic conditions. ninety-one percent of adults aged and older have at least one chronic condition and % have two or more of them (anderson ) . people with chronic conditions account for % of all healthcare spending. seventy eight percent of private health insurance spending is attributable to the % of privately insured persons with chronic conditions. seventy three percent of healthcare spending for the uninsured is for care received by the one third of uninsured people who have chronic conditions. seventy nine percent of medicaid spending goes to care for the % of non-institutionalized benefi ciaries who have chronic conditions (anderson ) (see fig. . ). further, health care spending increases with the number of chronic conditions (anderson ) (see fig. . ). more than three fi fths of healthcare spending (two thirds of medicare spending) goes to care for people with multiple chronic conditions. those with multiple chronic conditions are more likely to be hospitalized, fi ll more prescriptions, and have more physician visits (anderson ) . in the american diabetes association estimated direct medical expenditures for diabetes at $ . billion: $ . billion for diabetes care, $ . billion for chronic complications and $ . billion for excess prevalence of general medical conditions. approximately % of direct medical expenditures were incurred by people over . indirect expenditures included lost workdays, restricted productivity mortality and permanent disability -a total of $ . billion. all told, diabetes was found to be responsible for $ billion of $ billion in total expenditures. per capita medical expenditures totaled $ , annually for people with diabetes and $ for people without diabetes (hogan et al. ) . more recently, dall and colleagues estimated that the us national economic burden of prediabetes and diabetes had reached $ billion in , $ million in higher medical costs and $ billion in reduced productivity. annual cost per case was estimated at $ , for undiagnosed diabetes and , for type diabetes (dall et al. ) . the costs of caring for people with diabetes have risen both because the numbers of diabetics has been increasing and because the per capita costs of care have increased. the number of diabetics increased from . million on to . million in (ashkenazy and abrahamson ) . a recent report by the unitedhealth group center for health reform & modernization provides a dire estimation -that more than % of adult americans could have diabetes ( %) or prediabetes ( %) by at a cost of $ . trillion over the decade. this compares with current estimates of % of the population with diabetes and % with prediabetes, or %. these estimates conclude that diabetes and prediabetes will account for % of total healthcare spending in at an annual cost of $ billion, up from an estimated $ billion in (unitedhealth center for health reform and modernization ) . average annual spending over the next decade by payer type is $ billion for private health insurance, $ billion for medicare, $ billion for medicaid and $ . billion for the uninsured. what about cardiovascular disease and rheumatoid arthritis? among the top ten health conditions requiring treatment for medicare benefi ciaries in approximately % of benefi ciaries suffered from hypertension, % from heart conditions, % had hyperlipidemia % had copd, % had osteoarthritis and % had diabetes (thorpe et al. ) . the american heart association estimates the cost of cardiovascular disease and stroke to be $ billion in direct expenditures and $ . billion for productivity losses due to morbidity and $ . billion in lost productivity due to mortality (present value of lost wages at %) (lloyd- ) . the centers for disease control estimates that during - million americans had selfreported doctor diagnosed arthritis, million of them with activity limitations (cheng et al. ) . cisternas and colleagues estimated that total expenditures by us adults with arthritis increased from $ billion in to $ billion in . most of the increase was attributable to people who had co-occurring chronic conditions (cisternas et al. ) . the cisternas study appears to aggregate all medical care expenditures by people with arthritis (which would include expenditures to treat diabetes and cardiovascular disease). an earlier cdc study focused on the direct and indirect costs in attributable to arthritis that estimated $ . billion in direct costs (medical expenditures) and $ billion in indirect costs (lost earnings) (yelin et al. ) . in short, current cost estimates for direct health care expenditures (excluding productivity losses) related to diabetes are approximately $ billion, for cardiovascular treatment, $ billion, and for rheumatoid arthritis, approximately $ billion (estimating that the $ . billion in costs have grown approximately % per year), a total of $ billion of the $ . trillion that will be spent in the us in . moreover, given current growth in the prevalence of diabetes, the unitedhealth estimate of $ million in spending for diabetes alone is not unreasonable. if health care costs attributable to diabetes, cardiovascular disease and rheumatoid arthritis only increase by % over the next decade (even given added demand produced by the aging baby boomer population), annual costs of these chronic diseases will exceed $ . trillion in . if we use the unitedhealth estimates for the proportions of diabetes costs paid by private insurance ( %), medicare ( %), medicaid ( %) and the uninsured ( %) and estimate total costs based on the studies projecting a % increase in years and a % increase in years we can obtain an estimate of future costs for treating diabetes, cardiovascular disease and arthritis. table . set forth below, summarizes these cost estimates. by medicare costs for these chronic illnesses would be approximately $ billion. the estimated costs to medicaid will be approximately $ billion. the costs for the uninsured will be approximately $ billion. any intervention that has the potential to substantially reduce these costs will produce meaningful results. unfortunately, even though there had been a substantial numbers of studies that show relationships between dental disease and chronic illness that are have been very few studies that actually test whether improved dental treatment reduces the incidence of chronic illness and complications due to chronic illness. the potential for large health care cost savings through an active and aggressive program of dental care is so large that such studies are clearly indicated. suppose, for example, that % of all medical care costs required to treat diabetes, cardiovascular disease and arthritis could be avoided through an active aggressive program of dental care. what this would mean is that in private health insurers could see a $ billion reduction in healthcare costs, medicare would see a $ . billion reduction and medicaid pay $ . billion reduction. recent health reform has provided for the issuance of health insurance to the uninsured by state exchanges. aggressive dental care that saved % of costs attributable to diabetes, cardiovascular disease and arthritis could save the exchanges $ billion per year. and, if greater proportions of costs can be saved or if the estimates of costs are low, potential benefi ts will be even larger. once again, it would be important to know whether aggressive dental care could produce such savings and how much. ide and colleagues found that people who were treated for periodontitis incurred % higher health care costs than those who were free of periodontal disease (ide et al. ) . similarly, albert, et al., found medical costs associated with diabetes, cardiovascular disease and cerebrovascular disease were signifi cantly higher for enrollees who were treated for periodontitis than for other dental conditions (albert et al. ) . additional studies of this nature would be important to support a measured approach to expanding dental coverage. so what do we mean by an aggressive dental treatment plan? suppose we were to provide dental insurance to all medicare benefi ciaries at the level of current private dental insurance coverage and strongly encourage benefi ciaries to receive dental treatment. suppose we were to provide for medicaid payment for all benefi ciaries at the level of current private dental insurance coverage. suppose health care insurers provided dental coverage in order to reduce their costs and that such coverage was consistent with current private dental insurance coverage. suppose health insurance companies, understanding the benefi ts from dental care, were to require their private employer customers to cover the costs of dental care. how much would all of this cost? how would it compare to the benefi ts that may be available? in order to estimate the potential costs of providing enhanced coverage for dental care we start use the cms estimates of national health care spending for dental services and statistical abstract of the us estimates for medicare enrollment, medicaid enrollment, private health insurance enrollment and uninsured persons. based on the estimate that half of private employers with health insurance provided dental insurance coverage we estimate that of the private health insurance enrollment one half would have dental insurance coverage and one half would not. table . sets forth the national health care expenditures for dental services in millions and enrollment in private dental plans, medicare, medicaid, the uninsured without health insurance and dental insurance, the uninsured with health insurance and dual eligibles. from this we derive a cost per enrollee for private dental insurance, medicare dental benefi ts and medicaid dental benefi ts. in order to estimate the annual cost of providing full dental coverage to medicare benefi ciaries we subtracted dual eligibles (who receive some dental insurance) from total medicare enrollees to determine the number of persons who would need coverage. in our example there were million medicare benefi ciaries including million dual eligibles. accordingly, the estimates would cover the million medicare benefi ciaries that are not dual eligible at a cost equal to the per capita cost of private dental insurance ($ . ) less amounts that medicare is already paying for dental services ($ . per person). the result provides an estimate of the cost of covering all medicare benefi ciaries for dental services at a level equivalent to private health insurance. using the example the cost of providing full dental insurance coverage to medicare benefi ciaries would have been $ . billion. in addition, we used the cms national health expenditure fi gures to determine administrative costs for private health insurance, medicare and medicaid as a percentage of program expenditures for medical care. we found that the administrative costs of the medicare program were . % on average for - . in order to fully estimate the cost of medicare dental coverage we added . % to the cost health insurers will be in the same position as medicare and medicaid regarding dental coverage. if quality dental coverage saves health care costs attributable to diabetes, cardiovascular disease and rheumatoid arthritis then the exchanges will have an incentive to provide quality dental coverage to reduce costs. accordingly, we estimated the cost of providing dental coverage equivalent to private dental insurance coverage through the exchanges. again we assume that the costs of such coverage will be equivalent to the number of uninsured persons multiplied by the annual per capita cost of coverage. for the example, this would refl ect coverage for million people at $ . per person, a total of $ . billion. with administrative costs, the cost of providing dental insurance coverage to the uninsured at a level equivalent to private dental coverage would be $ . billion. finally, given the evidence that improved dental care has the potential to reduce health care costs private health insurers may wish to expand health insurance to cover dental care. here, we estimate the cost of providing dental insurance to the % of the workforce whose employers currently do not provide dental insurance benefi ts. once again, we multiply the number of covered lives by the estimated annual per capita cost. for the example we estimate million adults will receive dental coverage at $ per person: $ billion for dental services and $ . billion for administrative costs or a total of $ . billion. of course, as noted a number of times above, these estimates are based on providing full "universal" dental insurance coverage at levels equivalent to current benefi t levels for private dental insurance. it may be that an appropriate package of dental services that deals specifi cally with periodontitis can be provided for less than the full cost of private dental insurance. once again, further research should provide better information. the health reform law does not attempt to provide coverage to all million people without health insurance. estimates are that only million people will be covered by the bill. even though this is the case we prepare our estimates using all million uninsured americans. indeed, the failure of % of employers to cover dental services may well constitute a classic externality in the market for health insurance. internalizing this externality may well provide better effi ciency. it is also possible that dental care for persons with greater incidence of chronic illness as is the case with medicare benefi ciaries may require even higher levels of spending per benefi ciary. again, it would be good to know scientifi cally if this is the case. as noted in sect. above, costs for diabetes, cardiovascular disease and arthritis will be $ billion for private health insurance, $ billion for medicare, $ billion for medicaid and $ billion for the uninsured. costs of providing "full" dental coverage will be $ . billion for medicare, $ . billion for medicaid, $ . billion for the uninsured and $ . billion for private health insurance. given this, if . % or more of the medicare costs can be "saved" through improved dental care, medicaid dental insurance will pay for itself and will provide a positive return on investment. see table . . similarly, private health insurers could justify providing dental insurance coverage to employees who do not have it so long as they spend . % or more of their chronic care costs for diabetes, cardiovascular disease and arthritis. on the other hand, it would appear that medicaid expansion would require cost savings of approximately % and that health care insurance coverage of the uninsured would require savings of approximately % in order to justify coverage. while it is possible, it may not be likely that full dental coverage would be justifi ed for these programs. of course, these estimates do not consider indirect costs in the form of lost wages or premature death. these costs are externalities to the health insurance programs. to the extent that they represent a social benefi t that a national dental insurance program might internalize, it would be appropriate to consider their impact in the cost-benefi t analysis. in any event, better understanding of the potential for deriving savings in health insurance costs related to chronic diseases like diabetes, cardiovascular disease and arthritis would be crucial to any determination whether to expand insurance coverage for dental care. heretofore the case for expanding medicare coverage to include dental care has taken the form of "benefi t" to patients rather than benefi t to health insurance programs and society and has been cast in emotional and political terms. for example, oral health america grades "america's commitment to providing oral health access to the elderly" (oral health america ) . in truth, there is no american commitment to providing oral health access to any age group, much less the elderly. rubenstein notes that "at least one commentator has suggested that the dental profession should join with senior citizen groups when the time is right to ask congress to expand medicare to cover oral health" (rubenstein ) . rubenstein emphasizes that "calls for action" are "mere words" unless they are accompanied by political actions that health policy professionals and the dental profession must help promote (rubenstein ) . another commentator has suggested that "as soon as the debate over medicare prescription drug coverage and, the debate to provide dental care coverage for the elderly may soon begin" (manski ) . rubenstein, again suggests that "the dental community must convince americans, and particularly aging boomers, that oral health is integral to all health, and for that reason, retiree dental benefi ts are an important issue". in truth, a decade of defi cit spending and public distaste for out of control program costs in the medicare and medicaid programs as well as the unpopularity of the process that was used to provide medicare prescription drug coverage (with perceived abuses by the health insurance and drug lobbies) and national health reform makes it unlikely that the public would be willing to approve expansions in insurance coverage for dental care "for its own sake" or "as the right thing" or to "benefi t seniors." what this political climate has produced is an arena in which a good idea that could provide appropriate return on investment for society might well be rejected out of hand based on political history of health insurance coverage. as a result, it is incumbent on policymakers, medical and dental research scientists and health economists to investigate and confi rm the potential savings that expansion of dental insurance coverage has the potential to produce and to develop hard evidence regarding potential costs of the expansion prior to, not as a part of, political efforts aimed at dental coverage expansion. a responsible, well informed effort to expand dental coverage may well go far to restore public confi dence in the health policy process. joseph kilsdonk and kelly boggs the adage of "putting your money where your mouth is" is often referenced when being challenged about public statements or claims. in this instance, we use it literally. in health care costs in us were $ . trillion. there have been numerous reports on health disparities, the burden of chronic diseases, increasing healthcare costs and the need for change. long-term economic benefi ts associated with the cost of care are dependent upon integrating oral health with medicine. this is particularly true as it relates to the management of those conditions which impact the economics of healthcare the most. as examples, % of medicare costs and % of medicaid costs are in managing chronic health conditions (partnership for solutions national program offi ce ) . more than % of the u.s. population has one or more chronic condition (cartwright-smith ) and in , % of medicare spending was on patients with fi ve or more chronic diseases (swartz ) . effective management of health care resources and information are critical to the economic well-being of our healthcare system. we can no longer afford to manage care in isolation. integration of care between medicine and dentistry holds much promise in terms of reducing the cost of care and an integrated medical-dental electronic healthcare record (iehr) is the vehicle that will lead to downstream cost savings. in the united states the center for medicare & medicaid services (cms) has conducted demonstration projects around chronic disease management. section of the benefi ts improvement and protection act of mandated cms to conduct a disease management demonstration project. april , , as an effort to reduce the cost of care and improve quality associated with chronic diseases, cms partnered with ten premier health systems to effectively manage chronic diseases in a medicare physician group practice demonstration (pgp). it was the fi rst pay-for-performance initiative for physicians under the medicare program (center for medicare and medicaid services ) . it involved giving additional payments to providers based on practice effi ciency and improved management of chronically ill patients. participants included ten multispecialty group practices nationwide, with a total of more than , physicians, who care for more than , medicare benefi ciaries (frieden ) . the chronic diseases that were targeted were based on occurrence in the population and included diabetes, heart failure, coronary artery disease, and hypertension (frieden under the pgp, physician groups continued to be paid under regular medicare fee schedules and had the opportunity to share in savings from enhancements in patient care management. physician groups could earn performance payments which were divided between cost effi ciency for generating savings and performance on quality measures phased in during the demonstration as follows: year , measures, year , measures and years and having quality measures. for each of the years only the university of michigan faculty group practice and marshfi eld clinic, earned performance payments for improving the quality and cost effi ciency of care. a large part of the success of this project was attributed to being able to extract, evaluate, and monitor key clinical data associated with the specifi c disease and to manage that data through an electronic health record (table . ). during the third year of the demonstration project marshfi eld clinic, using a robust electronic health record succeeded in saving cms $ million dollars; that's one clinic system in year. as a result of such demonstration projects and as of this writing, cms is looking to establish accountable care organization's as the medical front runners to new care delivery methods for quality and cost control. accountable care organization (aco) is a term used to describe partnerships between healthcare providers to establish accountability and improved outcomes for the patients. in a cms workshop on october , , don berwick, the administrator of cms, stated "an aco will put the patient and family at the center of all its activities…" an emerging model of an aco is the patient-centered medical home (pcmh). pcmh is at the center of many demonstration projects. acos were derived from studies piloted by cms. since funds provided by cms, do not cover routine dental care as part of the patient management or quality and cost objectives cms aco studies are limited if they become models for the pcmh, due to the exclusion of dental. more recently, organizations representing the major primary care specialtiesthe american academy of family practice, the american academy of pediatrics, the american osteopathic association, and the american college of physicianshave worked together to develop and endorse the concept of the "patient-centered medical home," a practice model that would more effectively support the core functions of primary care and the management of chronic disease (fisher ) . in geisinger health system, kaiser permanente, mayo clinic, intermountain healthcare and group health cooperative announced they will be creating a project called the care connectivity consortium. this project is intended to exchange patient information. although progressive in their approach their project does not include dental. these benefi ts however, are yet to be adapted in the arena of oral health. as of this writing, dentistry remains largely separate from medical reimbursement mechanisms such as shared billing, integrated consults, diagnosis, shared problem lists, and government coverage. for example, cms does not cover routine dental care. dentistry is also working to establish its own "dental home" with patients. however to reap the economic benefi ts of integrated care, a primary care "medical-dental" home is what needs to be created. according to an institute of oral health report ( ) it is widely accepted across the dental profession that oral health has a direct impact on systemic health, and increasingly, medical and dental care providers are building to bridge relationships that create treatment solutions. the case for medical and dental professionals' comanaging patients has been suggested for almost the past century, in william gies reported that "the frequency of periodic examination gives dentists exceptional opportunity to note early signs of many types of illnesses outside the domain of dentistry" (gies ) . as described by dr. richard nagelberg, dds "the convergence of dental and medical care is underway. our patients will be the benefi ciaries of this trend. for too long, we have provided dental care in a bubble, practicing -to a large degree -apart from other health-care providers. even when we consulted with our medical colleagues, it was to fi nd out if premedication was necessary, get clearance for treatment of a medically compromised patient, or fi nd out the hba c level of a diabetic individual, rather than providing true patient co-management. we have made diagnoses and provided treatments without the benefi t of tests, reports, metrics, and other information that predict the likelihood of disease development and progression, as well as favorable treatment outcomes. we have practiced in this manner not due to negligence, but because of the limitations of tools that were available to us" (nagelberg ) . integrated medical/dental records need to be a tool in a providers' toolbox. in the case of marshfi eld clinic, dental was not included in their past cms demonstration project as dental is not a cms covered benefi t, and thus not part of the demonstration. however, as a leader in healthcare, the marshfi eld clinic recognizes the importance of data integration for both increased quality and cost savings. "marshfi eld clinic believes the best health care comes from an integrated dental/medical approach," said michael murphy, director, business development for cattails software. integration enhances communication between providers and can ultimately lead to better management of complex diseases with oral-systemic connection, avoidance of medical errors, and improved public health. while the cms pgp and other demonstration projects along with independent studies have shown to improve quality and reduce costs through integration, greater results may be afforded if studies are not done in isolation from dental data. in fact, if healthcare does not fi nd a way to manage the systemic nature of the pathogens known to the oral cavity the economic impact and cost savings around chronic disease management will hit a ceiling. the economic opportunity of having clinical data for integrated decision making is readily identifi ed by the insurance industry. the effective management of clinical data around chronic and systemic oral and medical disease as part of an iehr is the greatest healthcare cost savings opportunity associated with such a tool. the insurance industry sustains itself through risk management [obtaining best outcomes] using actuarial analysis [data] and controlling costs [reduction of costs] in order to ensure coverage [profi tability]. as such they have pursued the economic and outcome benefi ts of integrated medical -dental clinical decision making. as an example, in there was a study conducted by the university of michigan, commissioned by the blue cross blue shield of michigan foundation ( ) , the study included , blue cross blue shield of michigan members diagnosed with diabetes who had access to dental care, and had continuous coverage for at least year. with regular periodontal care, it was observed diabetes related medical costs were reduced by %. when compounding chronic health complications were also examined, the study showed a % reduction in cost related to the treatment of cardiovascular disease in patients with diabetes and heart disease. a % reduction in cost related to treatment of kidney disease for patients with diabetes and kidney disease. and a % reduction in costs related to treating congestive heart failure for patients with diabetes and congestive heart failure. according to a joint statement by lead researchers, and blue cross blue shield of michigan executives, "our results are consistent with an emerging body of evidence that periodontal disease…it addresses quality of care and health care costs for all michigan residents." also, at the institute for oral health conference in november joseph errante, d.d.s., vice president, blue cross blue shield of ma reported that blue cross blue shield of massachusetts claims data showed medical costs for diabetics who accessed dental care for prevention and periodontal services averaged $ /month, while medical costs for diabetics who didn't get dental care were about $ /month (errante ) . similarly insured individuals with cardiovascular diseases who accessed dental care had lower medical costs, $ /month lower than people who did not seek dental treatment (errante ) . the cost is $ less per visit for those diabetics who accessed prevention and periodontal services. those savings could be translated into access to care or additional benefi ts for more individuals. in the case of neonatal health there is similar research. over % of all births in the u.s. are delivered preterm, with many infants at risk of birth defects ( martin et al. ) . according to a january statement issued by cigna, announcing their cigna oral health maternity program, "the program was launched in response to mounting research indicating an increased probability of preterm birth for those with gum disease. these research-based, value-added programs are designed to help improve outcomes and reduce expense" (cigna ) . the program was initially designed to offer extended dental benefi ts free of charge to members who were expecting mothers, citing "research supporting the negative and costly impact periodontal disease has on both mother and baby." according to research cited by cigna, expecting mothers with chronic periodontal disease during the second trimester are seven times more likely to deliver preterm (before th week), and the costs associated with treating premature newborns is an average of times more during their fi rst year, and premature newborns have dramatically more healthcare challenges throughout their life. cigna also cited the correlation between periodontal disease and low birth weight, pre-eclampsia, gestational diabetes as additional rationale to support extended dental benefi ts to expecting mothers. six months later cigna initiated well aware for better health, an extended benefi ts free of charge program for diabetic and cardiovascular disease patients aimed at "turning evidence into action by enhancing dental benefi ts for participants in disease management" programs. it is interesting to note, not only does cigna offer extended dental benefi t to targeted groups, they also reimburse members for any out-of-pocket expenses associated to their dental care (co-pays, etc.) in , columbia university researchers conducted a -year retrospective study of , aetna ppo members with continuous medical and dental insurance, exhibiting one of three chronic conditions (diabetes mellitus, coronary artery disease, and cerebrovascular disease) (aetna ) . researchers found members who received periodontal treatments incurred higher initial per member per month medical costs, but ultimately achieved signifi cantly lower health screening (episode risk group/erg) risk scores than peers receiving little or no dental care. convinced by the data and understanding lower risk scores ultimately leads to healthier people and cost savings, aetna initiated the dental/medical integration (dmi) program in . aetna's dmi program offers enhanced benefi ts in the form of free-of-charge extended benefi t dental care to aetna's . million indemnity, ppo and managed choice medical plan members, specifi cally targeting members deemed at-risk, including those who are pregnant, diabetic, and/or have cardiovascular disease and have not been to a dentist in year as a result of various outreach methods during the pilot, % of at-risk members who had not been to a dentist in the previous months, sought dental care (aetna ) . "the fi ndings from this latest study we conducted continue to show that members with certain conditions who are engaged in seeking preventive care, such as regular dental visits, can improve their overall health and quality of life," said alan hirschberg, head of aetna dental (aetna ) . delta dental of wisconsin understands the connection between oral and systemic health and has created a program that is designed to offer members with certain chronic health conditions the opportunity to gain additional benefi ts. more than , groups now offer delta dental of wisconsin's evidence-based integrated care plan (ebicp) option (delta dental of wisconsin ) . ebicp provides expanded benefi ts for persons with diseases and medical conditions that have oral health implications. these benefi ts include increased frequency of cleanings and/or applications of topical fl uoride. they address the unique oral health challenges faced by persons with these conditions, and can also play an important role in the management of an individual's medical condition. ebicp offers additional cleanings and topical fl uoride application for persons who are undergoing cancer treatment involving radiation and/or chemotherapy, persons with prior surgical or nonsurgical treatment of periodontal disease and persons with suppressed immune systems. the ebic offers additional cleanings for persons with diabetes and those with risk factors for ie, persons with kidney failure or who are on dialysis and for women who are pregnant. the iehr provides the insurance industry in partnership with the healthcare industry an integrated tool to facilitate these health and subsequently economic outcomes across medicine and dentistry. in addition to the anticipated savings through better outcomes using integrated clinical data, an example of a positive economic outcome associated with an integrated record as related to increased effi ciency and patient safety is found in the united states veterans administration (va) hospitals and clinics. the va is one of the few institutions that have implemented the shared electronic medical-dental record successfully. the va has the ability to be the "one stop shop" for their patients. an april press release published on the department of veterans affairs website highlighted the success of va's health information technology in terms of cost reductions and "improvements in quality, safety, and patient satisfaction" (department of veterans affairs ) . the press release spotlighted a recent study conducted by the public health journal, health affairs, which focused on va's health it investment from to . the study confi rmed that while va has spent $ billion on their technology initiative, a conservative estimate of cost savings was more than $ billion. after subtracting the expense of the it investment, there was a net savings of $ billion for the va during the years covered by the study (mcbride ) . furthermore, the study estimated that "more than percent of the savings were due to eliminating duplicated tests and reducing medical errors. the rest of the savings came from lower operating expenses and reduced workload." independent studies show that the va system does better on many measures, especially preventive services and chronic care, than the private sector and medicare. va offi cials say "its [integrated] technology has helped cut down hospitalizations and helped patients live longer" (zhang ) . recently, the journal of obstetrics and gynecology reported on a tragic loss of life due to the systemic nature of oral health. a study found oral bacteria called fusobacterium nucleatum was the likely culprit in infecting a -year-old woman's fetus through her bloodstream (carroll ) . the doctors determined that the same strain of oral bacteria found in the woman's mouth was in the deceased baby's stomach and lungs. integrated records would provide critical data to the obstetrician including oral health issues and when the patient had her last dental exam. how does one measure the economic impact of a life not lived and another derailed by such tragedy? in a randomized controlled study, lopez et al. ( ) determined that periodontal therapy provided during pregnancy to women with periodontitis or gingivitis reduced the incidence of preterm and of low birth weight. the institute of medicine and national academies estimate that preterm births cost society at least $ billion annually . data integration of the iehr enables the effective management between the dentist and obstetrician to ensure proper periodontal therapy has been provided during pregnancy. such management based on the lopez et al. study, will have direct impact in reducing the prevalence per preterm births leading to reduced health care costs. there have also been studies indicating a correlation between poorer oral hygiene or defi cient denture hygiene and pneumonia or respiratory tract infection among elderly people in nursing homes or hospitals (rosenblum ; ghezzi and ship ; scannapieco ) . one such study of elderly persons in two nursing homes in japan (adachi et al. ) concluded that "the number of bacteria silently aspirating into the lower respiratory tract was lower in the group who received professional oral care, which resulted in less fatal aspiration pneumonia in that group." over the month period of the study, of the patients receiving professional oral care, % died of pneumonia versus . % of the patients that died of the same cause who maintained their own oral hygiene. lack of access is certainly a key factor to consider. however, lack of available data respective to the interrelationship between oral health and systemic health also contributed to the apathy in these cases. as identifi ed above, complications are correlated to cost. as conditions compound, costs go up. marshfi eld clinic, as part of their iehr is creating a shared problem list that identifi es both oral and medical conditions and history to recent visits and medication lists for monitoring at point of care [be it a medical or dental visit], such cross access to clinical data and care management milestones serves as a tool to prevent conditions from compounding and escalating costs such as those described above. several other areas of economic impact will be seen as iehr's become broadly deployed. some of these are listed as follows: medication management. a great deal of provider and allied support time is • spent obtaining medication information between dentistry and medicine [and vice versa] including current medications, contraindications, tolerances, etc. marshfi eld clinic cattails software has created a dashboard that readily identifi es this for both the medical and dental providers. not is time saved but chances for complications or escalation of conditions is reduced [both of which impact cost]. for example an integrated record allows medical providers treating respiratory infections to include or exclude oral fl ora as the possible source of the infection which would lead to more knowledgeable prescribing decision on the antibiotic used. coordination of care has a direct impact on cost for the system and the patient. • for example, in . % of the us population aged years and older that was diagnosed diabetes had been to the dentist in the past year (healthy people ( )). the us government's program healthy people includes an initiative to increase the proportion of people with diagnosed diabetes who have at least an annual dental examination. the american diabetes association recommends that diabetic patients be seen semi-annually and more if bleeding gums or other oral issues are present. the american diabetes association also recommends the consultation between the dentist and doctor to decide about possible adjustments to diabetes medicines, or to decide if an antibiotic is needed before surgery to prevent infection. the target from the healthy people is a % improvement at . %. integrated medical/dental records could allow for the coordination of efforts between providers to include communication of treatment plan and services leading to quicker resolution, increased patient compliance, and less patient time away from work or home and potentially less travel. similarly, integrated records also create a platform to integrate clinical appointing • between medicine and dentistry. as such, combative patients or severely disabled patients needing anesthesia in order for care to be delivered can be treated with one hospital sedation vs. multiple sedations. family health center of marshfi eld, inc. (fhc) dental clinics shares an iehr with marshfi eld clinic and uses it integrated scheduling feature to complete dental care, lab work, ent care, woman's health, preventive studies, all in one visit. follow up care management can be more focused and coordinated. for example, • without the knowledge or dental conditions, medical providers could spend months attempting to control diabetes with periodontal disease. however, with access to an iehr, the practitioner or allied care manager can determine patient's oral health status immediately to determine possible infl uence of periodontal disease. similarly an iehr with a shared patient data dashboard brings to light history • and physical examination data without having to have patients be the historian to their physician on their last dental visit or for the dentist to have to rely on the patient's recall of medications or medical diagnosis. for example, if an integrated record saved providers min per hour of patient care, that would be min per day. imagine giving a physician or dentist min more a day. in a capitated system, this allows for more patients to be seen in a day for roughly the same amount of expenditure. in a production based clinic this allows more patients to be seen and more charges per day. in either case, the investment into informatics is covered. in an underserved area, more patients get care quicker, which creates the opportunity for quicker resolution, which can lead to a healthier society, which in turn may lead them back to a productive livelihood sooner. an iehr results in one system for acquisition, orientation, training and support. • pc based owners who also own a mac and mac owners who also have to operate a pc can relate. need we say more? imagine if your pc function just like a mac [or your mac function just a pc]. no cross learning of software quirks. not having to purchase two separate units to begin with. reduced costs, increased space. not having to jump from one computer to the other computer to get data from one data from another to create a report. not having to call two separate computer companies for service or updates. third party coordination. having an iehr creates a platform for interfacing • with third party payers. a common system and language for timely reimbursement. in part, the result of an iehr is driving the diagnostic coding for dentistry. such an integrated interface provides a tool to bridge with healthcare payors that historically kept payment as segregated as the oral and medical health professions. the iehr overcomes that limitation. timely payment, consolidation of payment, expansion of covered patient and provider benefi ts based on clinical integration, and a viable system for interfacing are all potential economic benefi ts of iehr clinical data. the iehr creates new horizons for research that will lead to cost saving discov-• eries. as example, knowing the benefi ts of research, marshfi eld clinic research foundation (mcrf) has created an oral and systemic health research project (oshrp). the creation of oshrp, led by dr. murray brilliant, will allow mcrf to capitalize on its existing and growing strengths in the areas of complex disease interactions and personalized health care (phc) to advance oral health and the health of the rest of the body. the oshrp has three specifi c goals: understand the connections between oral and systemic health (diabetes, heart disease, pre-term births) understand the causes of oral diseases and determine the effect of genetics, diet, water source (well/city + fl uoridation) and microbiome. understand how improving oral health aids systemic health (comparative effectiveness) and bring personalized health care (phc) to the dental arena. the oshrp research resource will be unique in the nation. as mcrf has done • with other projects, it will share this resource with qualifi ed investigators at other academic institutions both within and outside of wisconsin. oshrp will advance scientifi c knowledge, improve healthcare and prevention, reduce the cost of oral healthcare, and create new economic opportunities. such knowledge will have a direct economic impact on the cost of care and care management. the iehr creates an ability to have an integrated patient portal to comprehen-• sively maintain their health. portals are becoming more and more popular in the healthcare industry as a means to helping maintain compliance with care management recommendations and preventative procedures. portals provide patents a tool to stay up to date on their care and recommendations. portals can take iehr clinical data, adapt it through programming, and provide creative visual reinforcement for patients as they monitor their health status. the more patients engage in owning their health status, the more preventative services are followed through with. the more medicine and dentistry can leverage the prevention potential [which insurance companies have come to realize] the more likely costly conditions can be avoided. the link between oral health and systemic health is well documented. the separation of dental and medical is not a sustainable model in modern healthcare delivery. a new model of integrated care is necessary. aristotle said, "the whole is greater than the sum of its parts." increased access to combined medical and dental histories and diagnosis at the providers' fi ngertips makes vital information available. shared diagnosis between physicians and dentists could aid in formulating interventions and to accelerate decision making abilities by allowing for prioritizing of medical/ dental procedures. clinical management and treatment of the patient would be expedited with immediate access to both records. quality could be improved through a complete picture of the patient through the dashboard. all of which have a direct or indirect economic benefi t. the iehr will be the tool that facilitates such delivery and the studies and scenarios described in these pages point to signifi cant economic benefi ts to patients, payors, and providers. if increased access, multi-provider monitoring, shared problems lists with enhanced decision making abilities from iehr could reduce healthcare costs. the greatest cost reduction will be with using the iehr to manage chronic disease. a combined dental-medical electronic record with a shared data informatics platform is most likely to yield the best long-term economic solution while maintaining or enhancing positive patient outcomes. this section reveals viewpoints from a variety of medical and dental providers. one section focuses on optimal use of ophthalmic imaging, which should show how that the challenges of clinical data integration go beyond those encountered in the effort to bring oral health and systemic health together. wendy e. mouradian , suzanne boulter , paul casamassimo , and valerie j. harvey powell oral health is an important but often neglected part of overall health. historically separate systems of education, financing and practice in medicine and dentistry fuel this neglect, contributing to poorer health outcomes for vulnerable populations such as children, while increasing costs and chances for medical error for all patients. advances in understanding the impact of oral health on children's overall health, changing disease patterns and demographic trends strengthen the mandate for greater integration of oral and overall healthcare, as reviewed in two recent institute of medicine reports (iom a, b ) . the pediatric population could realize substantial benefit from oral disease prevention strategies under a coordinated system of care enhanced by integrated electronic health records (ehr). this approach would benefit all children but especially young children and those from low socioeconomic, minority and other disadvantaged groups who are at higher risk for oral disease and difficulties accessing dental care. this section focuses on the pediatric population and the need for close collaboration of pediatric medical and dental providers. first we consider how a child's developmental position and their parents' level of understanding might affect oral health outcomes. next we address the importance of children's oral health and the urgency of seizing missed opportunities to prevent disease. we then briefl y outlines some steps to preventing early childhood oral disease utilizing some of the many health providers that interact with families. finally we examine one pediatric hospital's approach to choosing an integrated ehr technology. children have unique characteristics which distinguish their needs from those of adults. children's developmental immaturities may increase their risks for poor oral health outcomes ( fig. all children, but especially young children, are limited in their ability to care for their own health and must depend upon adults. a child's parent/caregiver may also lack basic oral health knowledge and an awareness of their child's oral health needs, and/or suffer from poor oral health themselves. low oral health literacy is prevalent among patients and health professionals alike in america; individuals of low socioeconomic status or from ethnically diverse backgrounds may be at particular risk for low oral health literacy (iom a ) . without appropriate education, a parent…. may not correctly interpret a child's symptoms or signs of oral disease • may not know that caries is an infectious disease that can be spread to a child by • sharing spoons, for example, may not know the potential value of chewing gum with xylitol, • may not fully grasp the importance of good oral health hygiene habits, • may not grasp the consequences of a child consuming quantities of sugared • foods or beverages, may have diffi culty controlling the child's consumption of sugared foods or bev-• erages in or out of the home, may not realize the consequences of chronic use of sugared medications, • may not know the potential for systemic spread of disease from a toothache, or • for liver damage due to overuse of acetaminophen or other analgesics, may not grasp the long-term consequences of early childhood caries, • may live in a community without fl uoride in the tap water and not know about • alternative sources of fl uoride, may overlook oral health due to the stress of living in poverty, • may be fearful of dentists or oral health care due to their own experiences, • may have diffi culty locating a dental provider accepting public insurance, or • have other problems navigating the health care system. parents in turn depend on access to medical and dental providers with current understanding of the most effective ways to prevent caries and promote the child's oral and overall health. an important element in helping families is the provision of culturally-sensitive care to a diverse population. children are the most diverse segment of the population with % from minority backgrounds compared with % of the overall population (us census bureau ) . the separation of medical and dental systems and the lack of shared information can create additional barriers for families, especially for those with low health literacy or facing linguistic or cultural barriers. all pediatric health professionals have increased ethical and legal responsibilities to promote children's health, including advocacy for them at the system level (mouradian ) . although many factors can infl uence children's oral health outcomes, caries is largely a preventable disease. despite this, national trends and other data on broader considerations of medical and dental data integration children's oral health attest to this persistent national problem ) . some important facts include the following…. caries is the most prevalent chronic disease of childhood, • caries is a preventable disease unlike many chronic diseases of childhood, • yet according to (nicdr ) % of children - have had dental caries in • their primary teeth; % of children - have untreated dental caries. further, " % of children - have had dental caries in their permanent teeth; % of children - have untreated decay." overall "[c]hildren - have an average of . decayed primary teeth and . decayed primary surfaces," the latest epidemiologic evidence shows increasing rates of caries for young-• est children, reverse from the healthy people goal of decreasing caries. according to (nicdr ), overall "dental caries in the baby teeth of children - declined from the early s until the mid s. from the mid s until the most recent ( ) ( ) ( ) ( ) ( ) ( ) ) national health and nutrition examination survey, this trend has reversed: a small but signifi cant increase in primary decay was found. this trend reversal was more severe in younger children." disparities in children's oral health and access to care persist by age, income • level, race and ethnicity, and parental education level (edelstein and chinn ) . of concern, the latest increase was actually in a traditionally low-risk group of young children (dye and thornton-evans ) . the human and economic costs of early childhood caries are substantial • (casamassimo et al. ) . according to catalanotto ( ) , health consequences include… extreme pain, spread of infection/facial cellulitis, even death (otto - ) diffi culty chewing, poor weight gain falling off the growth curve (acs et al. - ) risk of dental decay in adult teeth (broadbent et al. - ; li and wang ) crooked bite (malocclusion) -children with special health care needs (cshcn) may be at higher risk for oral • disease and diffi culties accessing care. analyzing data from the national survey of children with special health care needs, (lewis ) found that "cshcn are more likely to be insured and to receive preventive dental care at equal or higher rates than children without special health care needs. nevertheless, cshcn, particularly lower income and severely affected, are more likely to report unmet dental care need compared with unaffected children." children who were both low-income and severely affected had . times the likelihood of unmet dental care needs, dental care is the highest unmet health care need of children; . million children • had unmet dental care needs because families could not afford care compared with . million with unmet medical needs for the same reasons (cdc ) , according to the national survey of children's health, children are . times as • likely to lack dental as medical insurance (lewis et al. ) , there is evidence that children who get referred to a dentist early may have lower • costs of care and disease. savage et al. ( ) reported that children "who had their fi rst preventive visit by age were more likely to have subsequent preventive visits but were not more likely to have subsequent restorative or emergency visits" and concluded that preschool "children who used early preventive dental care incurred fewer dentally related costs," ramos- gomez and shepherd ( • ) , in their "cost-effectiveness model for prevention of early childhood caries," conclude that preventive ecc interventions could reduce ecc by - % for a particularly vulnerable population of children, and that part of the costs of interventions will be offset by savings in treatment costs. as these facts convey, and the deaths of more than one child from consequences of untreated caries make painfully clear, there is an urgent need for more attention to the oral health needs of children. a more coordinated system for oral health care including integrated ehr would be an important advance. a glance at table . , an ideal model, reveals that intervention should begin before birth and that a range of medical and oral health professionals can contribute to the child's oral health. early intervention is necessary because of the transmissibility of cariogenic bacteria from mother/caregiver to infant, and importance of oral health practice in preventing disease. the following professionals may be involved: • pediatric medical provider family physician pediatrician pediatric nurse nurse practitioner in pediatric/family practice physician assistant in pediatric /family practice - other appropriate allied health professionals • the availability of some of these professionals can be affected socioeconomic status, health insurance, place of residence, or by a child's special health care need. one obvious limitation on developing a "relay" as in table . , with a "hand-off" from family care to obstetric care to pediatric care is the education of the medical providers. as part of pre-conception and perinatal healthcare, providers should address oral health, but may lack the knowledge to do so. additionally, as noted by ressler-maerlaender et al. ( ) , "some women may believe that they or their table . timeline of some oral health interventions to prevent early childhood caries (ecc) -birth to years age (marrs et al. ; lannon et al. ; han et al. ; ezer et al. ; aap ; mouradian et al. ) child's age intervening professional(s) planning conception, prenatal and perinatal family physician the physician and/or obstetric provider educates mother-to-be about good maternal oral hygiene and infant oral health issues, including transmissibility of caries. mother's dentist assesses and treats caries, gingivitis or other oral health problems and educates the mother-to-be obstetrician/nurse midwife obstetric nurse general dentist obstetric nurse obstetric nurse advises new mother to chew xylitol gum, limit salivary contact between mother and infant, and help child avoid sugar intake (exposure) while asleep and from common sugar sources (medicines, sugared water, bottle feeding on demand at night with fl uid other than water -following tooth eruption, certain foods), and to schedule dental exam at year age months pediatric medical provider first dental examination recommended by aapd when the fi rst tooth comes in, usually between to months pediatric/general dentist educate mother about optimal fl uoride levels . assess the woman's oral health status, oral health practices, and access to a dental home; . discuss with the woman how oral health affects general health; . offer referrals to oral health professionals for treatment; . educate the woman about oral health during pregnancy, including expected physiological changes in the mouth and interventions to prevent and relieve discomfort; and . educate the woman about diet and oral hygiene for infants and children and encourage breastfeeding a combination of anticipatory guidance, with continuity from prenatal and perinatal care to pediatric care, can help move infant oral health from "missed opportunities" to "seized opportunities." others who may be of assistance to families in closing these gaps are professionals at the women, infants and children's (wic) supplemental nutrition program, early head start/head start and neurodevelopmental/birth to three programs. together medical, dental and community professionals can help create a system of care to improve maternal and child oral health. for the envisioned model in table . to be realized, the mother requires access to a general dentist with accurate information on her oral health during pregnancy and on her infant's oral health, including the need for an early dental visit. the mother and child then need access to a pediatric medical provider who will provide oral health screening/counseling, and who will guide the family to establishing the child's dental home by age . success in dental referral requires access to a pediatric or general dentist willing and able to provide infant oral health. (dela cruz et al. ) , in a discussion of the referral process mentioned that among the factors in assessing the likelihood of a dental referral were the medical providers' "level of oral health knowledge, and their opinions about the importance of oral health and preventive dental care." since young children are much more likely to access medical than dental care, the medical provider plays an important role in promoting children's oral health. (catalanotto ) recommends, as part of a pediatric well child checkup: an oral screening examination, • a risk assessment, including assessment of the mother's/caregiver's oral health, • application of fl uoride varnish • anticipatory guidance (parental education) including dietary and oral hygiene • information, attempted referral to a dental home. • the aap recommends that child healthcare providers be trained to perform an oral health risk assessment and triage all infants and children beginning by months of age to identify known risk factors for early childhood caries (ecc). the oral health component of pediatric care is integrated into the aap's "recommendations for preventive pediatric health care (periodicity schedule)" (aap ) . to what extent are medical and dental and providers aware of recommendations for a fi rst dental visit for a child by age one, as recommended by the aap, the american academy of pediatric dentistry (aapd), and the american dental association? (wolfe et al. ) reported that % of licensed general dentists in iowa were familiar with the aapd age dental visit recommendation and that most obtained the information through continuing education; % believed that the fi rst dental visit should occur between and months of age. however, according to (caspary et al. ) , when pediatric medical residents were asked the age for the fi rst dental visit, the average response was . years, while % reported received no oral health training during residency. in a national survey of pediatricians ) reported that less than % of had received oral health education in medical school, residency, or continuing education. finally (ferullo et al. ) surveyed allopathic and osteopathic schools of medicine and found that . % reported offering less than h of oral health curriculum, while . % offered no curriculum at all. other workforce considerations relevant to preventing early childhood caries include the training of dentists in pediatric oral health (seale et al. ) , the number and diversity of the dental workforce, the number of pediatric dentists, and the use of alternative providers such as dental therapists, expanded function dental assistants and dental hygienists (mertz and mouradian ; nash ) . examples of integrated care models do exist, such as that presented by (heuer ) involving school-linked and school-based clinics with an "innovative health infrastructure." according to heuer, "neighborhood outreach action for health (noah)" is staffed by two nurse practitioners and a part-time physician to provide "primary medical services to more than , uninsured patients each year" in scottsdale, arizona. heuer counts caries among the "top ten" diagnoses every year. mabry and mosca ( ) described community public health training of dental hygiene students for children with neurodevelopmental/intellectual disabilities. they mentioned that the dental hygiene students had worked together with school nurses and "felt they had impacted the school nurses' knowledge of oral disease and care." the decision to acquire an integrated ehr as pediatric clinicians (both medical and dental) work more closely together, they require appropriate ehr systems that integrate a patient's medical and dental records. following is a set of local "best practices" from nationwide children's hospital in columbus, ohio, which may help other children's hospitals in planning acquisition of an integrated pediatric ehr system. integrated (medical-dental) ehr technologies are becoming more widely available outside the federal government sector (see integrated models e and e in fig. . ). nationwide children's 'drivers' for the acquisition process were, in : . minimize registration and dual databases . patient registration takes time and requiring both a stand-alone dental and a medical patient registration inhibits cost-effective fl ow of services. integration allows for the use of single demographics information for all clinics in the comprehensive care system serving the patient. clinicians always have an updated health history on patients, if they have been a patient of record. if not, and for a dental clinic that sees walk-ins, a brief "critical" dental health history can be completed on paper by a parent and scanned into the emr. in designing an integrated medical-dental record for patients of record, the system can sort essential health history elements into a brief focused dental history without the detail needed by other medical specialty clinics. kioskdriven electronic health histories for those children who are new to clinic similar to those used in airline travel could be considered if feasible in busy clinics. . for charting, no more key/mouse strokes than with paper . some commercial dental record products try to accomplish too much. moving from paper to electronics should be driven in part by effi ciencies. the tooth chart, which is an essential part of any dental record, must be such that examination fi ndings can be transferred quickly and accurately to either paper or electronic capture. a helpful exercise is visualization of the functionality of the charting process, including both the different types of entries (caries, existing restorations and pathology) and how these are entered in the paper world. if charting will be able to be used for research the system should be able to translate pictures to numerical values, often a complex programming function. dental practitioners and faculty may want to use drawings of teeth or graphics of surfaces because that is their current comfort level. a true digital charting is possible with no images of teeth, but some habits are hard to change. . maximizing drop downs with drop down building possible . duplication of paper chart entries using drop downs which can be upgraded as more clinical entities are found is a staple of an emr. the paper process usually relies on a clinician's wealth of medical-dental terms since inclusion of every possible, or even the most common fi ndings, is prohibitive on a paper chart. the emr drop down requires front-end loading of the most common clinical fi ndings with opportunity for free-hand additions. being able to add terms to any drop down is a needed capability. . don't design a system for uncommon contingencies, but for your bulk of work . a pediatric dental record should be primarily designed around dental caries, with secondary emphases on oral-facial development (orthodontics) and a lesser capability to record traumatic injuries and periodontal fi ndings. these second and third level characteristics can be hot-buttoned and should not drive the design of the basic system which is caries charting for % of our patients. sadly in most dental schools, the chart is slave to every teaching form, few of which ever exit with the dds into practice! these forms may have little relationship to patient care and only create "signature black holes" that need to be addressed, usually after treatment is completed. . progress notes should be designed for the routine entries with free-hand modification possible . student learners tend to write too much and a carefully crafted progress note format with standard entries in required fi elds helps patient fl ow and record completion. in federally funded clinics and residencies, attending reconciliation of student/resident service delivery is a compliance requirement. a well-designed emr system can "stack" required co-signing tasks on a computer screen, offer standard entries as well as free-hand options, and create a process far faster than paper records for an attending's validation (same as reconciliation?). . tie examination results to treatment planning and treatment planning into billing . a good system allows easy transfer of clinical fi ndings needing treatment into some problem "basket" and ideally in a tabulated format. an alternative is a split screen that allows a clinician to visualize clinical fi ndings, radiographic fi ndings while compiling a treatment plan. again, in clinical settings where compliance to medicaid/medicare regulations is required, the design of the record should give attention to auditing principles and security. a good emr system allows portals of entry for billing and compliance personnel. . plan for users of different skill levels and different periods of exposure . the teaching hospital or dental school environment often involves learners and attendings with varying skill levels and computer experience who may be there for brief periods of time. this reality adds signifi cant security and userfriendliness issues. some medical record systems are far too complex for shortterm or casual users. a well-integrated medical-dental emr allows navigation of the depths of the medical side should a user want to explore, but should focus on the dental portion. some suggestions in design: initial opening or logging into the dental portion for dental users, rather than • opening into the medical portion, clearly indicated options for exploration of medical portions, • orientation of major dental component (examination, radiographs, treatment • plan) in a logical dental treatment fl ow to replicate the way dentistry works rather than trying to reshape dentistry's normal fl ow to the record, minimization of seldom-used functions on the main dental screen, such as • specialty medical clinics, old laboratory tests and hyperfunctionalities like letter writing, clear identifi cation of existing non-caries dental portions like orthodontics or • trauma, so a novice user need not randomly search to see if a patient has any of these records. unfortunately, many pediatric hospitals do not yet have an ehr system that supports convenient communication among a pediatric patient's medical and dental providers. evidence of this state of affairs was provided unintentionally by (fiks et al. ) . some pediatric hospitals may have an awkward mix of systems serving physicians, dentists, and orthodontists and their shared patients. this section demonstrates how closely medical and dental professionals must collaborate to deliver appropriate oral health care for infants and children. such collaboration is especially important given the developmental vulnerabilities of children and the urgency of the oral health needs of many children, especially those from underserved populations. collaboration is made more diffi cult by the long-standing separation of medical and dental systems and poor oral health literacy of parents and medical professionals alike. teamwork in the delivery of pediatric care requires appropriate electronic patient record technology to facilitate sharing of patient information, to avoid patient record discrepancies between systems, and to create effi ciencies by maintaining only a single repository for patient demographics. only comparatively recently have appropriate integrated systems become available to support a range of clinical sites from pediatric special needs clinics to the largest children's hospitals. nationwide children's has given practical examples of effi cient decision-making in identifying an integrated system to acquire. much more work will be needed to develop the means to move towards integrating offi ce and community-based care for children through the sharing of electronic health records. oral health is an oft neglected area in the care of patients who have chronic kidney disease. furthermore, the provision of care by dentists and physicians to the same patient is fragmented as communication between the two health care providers is scant. emerging data suggesting the periodontal disease is closely linked to chronic kidney disease highlights the importance of proper oral health and the importance of communication between dentists and physicians in the care of the patient. investigators used data from nhanes iii, including information on , adults who had an oral examination by a dentist who categorized each patient as having no periodontal disease, periodontal disease or edentulous to examine the relationship between numerous risk factors for moderate to severe chronic kidney disease, as determined by calculation of estimated gfr through use of the mdrd formula (fisher et al. ) . no chronic kidney disease was defi ned as an estimated gfr of ml per min per . m . three percent of the patients had ckd, . % were hypertension and . % had diabetes ( . % with glycated hemoglobin of % or higher). four models were constructed to examine the potential relationship between periodontal disease and ckd. in model one adults with either periodontal disease or edentulous had an adjusted odds ratio of . (with % confi dence intervals of . - . ) of having ckd, independent of the other risk factors for ckd including of age above years, ethnicity, hypertension, smoking status, female gender and c-reactive protein elevation. the fourth model contained potential risk factors including the periodontal disease score and for every -unit increase in the score, the risk of having ckd increased by % controlling for the other risk factors. the authors hypothesized from their results that the relationship between periodontal disease and ckd was bidirectional in that ckd may increase the risk of periodontal disease which in turn increases the risk of ckd. grubbs et al. ( ) also used nhanes data to look more closely at the relationship between periodontal disease and ckd, using dental examinations obtained from to (n = , adults, - years) (grubbs v, et al. ) . in this analysis edentulous subjects were excluded and those with albuminuria were included in the defi nition of ckd. in the entire population ckd was present in . %, but in those with moderate to severe periodontal disease this increased to . %. other associations with moderate to severe periodontal disease were being older, male, nonwhite, less educated and poor. there was a strong relationship between periodontal disease and ckd ( . unadjusted odds ratio). when adjusted for age, gender, tobacco use, hypertension, diabetes, ethnicity, poverty and educational attainment, the odds ratio for the association of periodontal disease and ckd was still signifi cant ( . ). in some groups (mexican american, poor, and poorly educated) dental care was not received on an annual basis in the majority of this segment of the population. periodontal disease has been associated with an increased risk of death in hemodialysis patients (kshirsagar et al. ). this relationship has been poorly studied in peritoneal dialysis patients. this requires further study but it appears possible that periodontal disease might hasten loss of residual kidney function and perhaps contribute to atherosclerosis in dialysis patients and therefore, contribute to the high mortality in this population. patients who desire a kidney transplant are required to undergo a thorough evaluation beforehand including an oral examination by a dentist. some patients on dialysis have inadequate insurance which does not cover dental care, leading to a situation in which a kidney transplant is denied because the patient cannot afford the dental examination. communications between dentists and physicians in the care of the patient is scant. if oral surgery is required in a dialysis patient, the surgeon generally requires a brief summary from the nephrologist with recommendations. these might include suggestions for prophylactic antibiotics, avoidance of vasoconstrictor agents to an excess locally (which can elevate blood pressure) and the increased risk of bleeding of a dialysis patient. for more routine dental examinations no information is requested which could potentially lead to drug interactions or a dangerous situation. most nephrologists and health care providers in the dialysis unit do not inquire of the patient concerning dental health and examination of the mouth is quite uncommon. although the dialysis patient is seen monthly at a minimum, there is little conversation or documentation of oral health. connecting the electronic health records of in-patient care, the out-patient dialysis unit and the dentists' offi ce could potentially have a large impact in improving the care of those with end stage kidney disease. integrating medical and dental records in ehr's may or may not be the "golden ring." first, we need to integrate the clinical thinking…something we both realize is important, but not likely to be solved by an inert computer. i also think that integrated records will be very cumbersome, given the fact that the language used by the separate disciplines is so different, and the kind of detail required to support good decisions and good work is so different. it could be done…but for many professionals on either "side," they would never open the other module. to me, a more sensible solution may be to have a condensed "nugget" of information that could cross populate. "moderate periodontal disease" may be what the medical doctor needs to know, plus know what a treatment plan may include. she won't need to know the number of the teeth with the deepest pockets and erosions but will need to support the patient's determination to follow through. on the other hand, if the patient has shown remarkable initiative in gum care and has successfully migrated to a lower severity index, that would be important for congratulation and reinforcement…and also to encourage similar diligence in managing, let's say, the hypertension that is not optimally controlled. in the other direction, the dentist should know that a patient has been erratic in clinical follow up, does not self-test blood glucose, uses hypoglycemic drugs only intermittently, and has failed several appointments for eye exams. this would lead to a rather different set of approaches from a highly motivated grandmother who is enrolled in a community cultural center's senior exercise club, and is learning to become a lay community teacher for diabetes. right now, i don't think even this superfi cial degree of information is exchanged. we need to support each other's efforts, but we probably do not need to share minute details. the benefi ts of an electronic health record are well described. ehrs allow for legible standardized documentation and easier sharing of patient data between providers at multiple locations. they are less prone to loss and require much less space to store. they have the potential to result in a reduction in the cost of health care. a distinct disadvantage of the ehr, in its current confi guration, is the problem of information overload. simply put, there is often too much information presented in a way that is diffi cult to review and digest. the ehr equivalent of thumbing through a chart quickly is not yet available. as a result we frequently see practitioners look only at the last note or two as they review a patient's history. we require a way to communicate information directly relevant to patient diagnosis, treatment and prognosis among subspecialists and primary care providers. we require a way to identify subclinical cerebrovascular disease in a patient, independent of blood pressure and other traditional risk factors. we require a way to recognize which patients with cerebrovascular disease are two to four times more likely than average to develop a stroke in the next years. we have a way -retinal imaging. the eye is the one place in the body we can directly observe arteries, veins and a cranial nerve in a noninvasive manner. routine imaging of the retina and optic nerve could allow primary care providers to assess retinal, and by proxy systemic, end organ damage from atherosclerosis in an effi cient manner. the key to optimal use of the medical record and effi cient yet effective communication among providers may lie with the familiar adage; a picture is worth a , words. traditionally, when ophthalmologists communicate with primary care providers they send brief letters regarding the fi ndings seen during a yearly dilated examination and the presence, absence or progression of diabetic retinopathy. these letters end by exhorting the virtues of improved blood sugar, blood pressure and lipid control, a sentiment that the primary care provider likely shares. this system of communication does not provide particularly useful information for the primary care provider, except to serve as a notice that the standard of care screening guidelines have been met. the box has been checked. if primary care providers, cardiologists, nephrologists had access to routine ophthalmic imaging, they would be able to directly visualize the effect that suboptimal blood sugar control is having on their diabetic patients. as importantly, they would be equipped with information directly predictive of congestive heart failure, stroke, and cardiovascular mortality for their patient with hypertension, hyperlipidemia and for those who smoke. large clinical studies have shown that assessment of retinal vascular changes such as retinal hemorrhages, microaneurysms and cotton wool spots provides important information for vasculopathy risk stratifi cation. as an example, wong et al. showed that the presence of retinopathy indicates susceptibility to and onset of preclinical systemic vascular disease, independent of and qualitatively different from measuring blood pressure or lipids (wong and mcintosh ) . in the atherosclerosis risk in communities (aric) study, individuals with hypertensive retinopathy signs such as cotton wool spots, retinal hemorrhages and microaneurysms were two to four times more likely to develop a stroke within years, even when controlling for the effects of blood pressure, hyperlipidemia, cigarette smoking and other risk factors (wong et al. ) . in a recent study by werther et al., patients with retinal vein occlusions were found to have a two-fold increased risk of stroke compared to controls (werther et al. ) . in addition, the aric study group reported that individuals with retinopathy were twice as likely to develop congestive heart failure as individuals without retinopathy, even after controlling for pre-existing risk factors (wong et al. a ) . interestingly, even among individuals without pre-existing coronary artery disease, diabetes or hypertension, the presence of hypertensive retinopathy was associated with a three-fold increased risk of congestive heart failure events (wong et al. a ) . in the beaver dam eye study, cardiovascular mortality was almost twice as high among individuals with retinal microaneurysms and retinal hemorrhages as those without these signs ( wong et al. a, b ) . the aric and beaver dam eye studies have also shown that, independent of other risk factors, generalized retinal arteriolar narrowing predicts the incidence of type ii diabetes among individuals initially free of the disease (wong et al. a (wong et al. , b . a primary care provider with access to patients' retinal photographs may therefore have the evidence needed to suggest which patient with either established systemic vascular disease or preclinical systemic vascular disease requires a more aggressive treatment and risk factor modifi cation. they could do this without wading through the electronic equivalent of piles of records. one photograph could refl ect both acute changes in blood pressure (retinal hemorrhages, microaneurysms and cotton wool spots) and chronic changes resulting from cumulative damage from hypertension (av nicking and generalized arteriolar narrowing) (sharrett et al. ; wong et al. a ; leung et al. ) . in brown et al. out of patients, excluding those with known diabetes, that presented with a single cotton wool spot or a predominance of cotton wool spots on examination of the retina were found to have underlying systemic disease (brown et al. ) . systemic work-up revealed diagnoses including previously undiagnosed diabetes, hypertension, cardiac valvular disease, severe carotid artery obstruction, leukemia, metastatic carcinoma, systemic lupus erythematosus, aids and giant cell arteritis (brown et al. ) . these fi ndings illustrate the importance of retinal fi ndings on a systemic level. the utilization and integration of ophthalmic imaging may serve to achieve more effective communication among subspecialists and primary care providers and ultimately to provide improved diagnosis and treatment for delivery of optimal quality of patient care. moreover, the improved integration and maximal use of resources may serve to reduce overall health care cost and perhaps decrease provider frustration with the electronic health record (fig. . ). there are cotton wool spots, exudates, intraretinal dot-blot hemorrhages and microaneurysms. av nicking is also present especially along the superior arcade just as the vessel leaves the optic nerve ( fig. . ) . av nicking, tortuosity of vessels, intraretinal hemorrhages and dry exudates are seen ( fig. . ) . there is edema of the optic nerve head, with cotton wool spots and fl ame shaped hemorrhage along the disc margin. there are several cotton wool spots along the vascular arcades and scattered dot hemorrhages throughout the posterior pole and periphery ( fig. . ) . notice the cholesterol plaque in the vessel just as it exits the optic nerve head and the pallor in the superior macula corresponding to retinal ischemia and edema ( fig. . ). the cholesterol embolus has resulted in lack of blood fl ow to the superior arcade ( fig. . ) . there is pooling of subretinal blood just superior to the optic disc with a central fi brin clot and associated vitreous hemorrhage (fig. . ) . optic disc edema, fl ame hemorrhages and venous congestion are seen in a patient with severe hypertension. biju cheriyan in clinical practice, an otolaryngologist often needs a dental consult not only because of the topographically adjacent nature of the structures but also because most structures are supplied by the same neurovascular bundle and therefore there is overlapping of symptoms. the converse scenario can also apply. apart from this, there are many systemic medical conditions (for example: bleeding diatheses, diabetes) a hypertensive optic neuropathy dentist encounters throughout his or her practice which can determine the outcome of a successful treatment. sometimes, providers may observe a cluster of diagnostic criteria which may have to a single source. in the sections below, i will explore a few of these scenarios and conditions, and indicate where and how an integrated electronic health record (ehr) could optimize delivery of health care by dentists and otolaryngologists. cleft palate/cleft lip : cleft lip and cleft palate (cl/cp) are congenital conditions that require multidisciplinary management by dentists, oral and maxillofacial surgeons, orthodontists, otolaryngologists, speech pathologists and plastic surgeons a number of studies report that a multidisciplinary approach is essential for better treatment outcomes (wangsrimongkol and jansawang ) and for post operative rehabilitation (furr et al. ). these multidisciplinary approaches may lead to new ways to manage and treat cl/cp patients (salyer et al. ). hutchinson's teeth : notching of the upper two incisors is typically seen in individuals infl icted with congenital syphilis. macroglossia refers to enlarged tongue in relation to oral cavity. macroglossia is an important sign. it can indicate important systemic diseases like systemic amyloidosis, congenital hypothyroidism, acromegaly, or down syndrome. a common complaint that dentists and otolaryngologists encounter in their practice is the common headache. because of the special nature of the neurovascular bundle of the head and neck this symptom can be presented to both dentists and otolaryngologists (ram et al. ). any sinus pathology can present as a headache to an otolaryngology practice. since the maxillary sinus fl oor is in close proximity to the maxillary premolars and molars, it is imperative to obtain a dental evaluation in persistent cases of headache. there are a number of causes for headache from the dental and otolaryngology perspective. a mal-aligned denture patient with chronic headache, whom i saw in my practice was shuttled between departments and an array of investigations only to fi nd at the end that an ill-fi tting denture caused the intractable headache. in these cases, an integration of fi ndings is extremely important in providing quality treatment to the patient and also saves money and time for the whole health care system. hence it is important to have an integrated patient record for this particular symptom alone. trigeminal neuralgia is facial pain of neurogenic origin experienced along the distribution of the trigeminal nerve(fi fth cranial nerve). it can present as a dental pain and can also be triggered by brushing teeth among other trigger factors. as a result, patients with dental pain without obvious causes are required to have a physicians' consultation to rule out this obscure condition. sometimes it is diagnosed by omission (aggarwal et al. ; rodriguez-lozano et al. ; spencer et al. ). any tumor of the nasal sinuses (specifi cally maxillary and ethmoids) can erode the lower bony wall and present in the oral cavity (usually the maxillary arch) as dental pain, loose tooth, etc. therefore, these are areas of interest to both dentists and otolaryngologists. such tumors most commonly present fi rst to a dentist or could also be an accidental fi nding. cancers of the naso/oro/laryngo pharynx can also present as toothache to a dentist as these structures have a common nerve supply from cranial nerves , and . therefore, an integration of the patient record may even help in early diagnosis of the tumor. the same principle applies to all oral tumors, tumors of the nasopharynx, the oropharynx etc. this is especially true of malignant lesions of the oral cavity as these may help in early detection and treatment of cancer. in these cases, an early biopsy and histopathology can save the life of the patient. therefore, it is imperative to say that a collaborative patient record can save patients' lives. ulcers of the oral cavity from aphthous ulcers to carcinomas can present both to a dentist and an otolaryngologist. oral ulcers can be of dental origin. contact ulcers from sharp edges of a mal-aligned tooth can result in intractable ulcers, where a simple smoothing of sharp edges may eradicate the ulcer and terminate it as a chronic condition and can even prevent the ulcer turning into a malignancy. if you have an integrated electronic health record (ehr) these problems are immediately addressed and managed. otherwise, the condition will consume valuable time of both the patient and the physician concerned. in addition to this, there are a few conditions which require special attention: aphthous stomatitis (canker sore), which may indicate oral manifestation of defi ciencies of iron, vitamin b , folate deficiency and oral candidiasis, which can be a sign of diabetes mellitus or of an immunocompromised patient (e.g. aids). temperomandibular joint (tmj) disorders can present in a variety of symptoms to both dentists and otolaryngologists. they can present as a headache, earache, toothache, or as facial pain. there can be a number of causes for this including osteoarthritis of the tmj, recurrent dislocation, bruxism, or even an ill fi tting denture. there have been cases where patients have been subjected to removal of teeth for chronic toothache only to discover at the end that the symptom was a referred pain from tmj! therefore, an integrated ehr can prevent misdiagnoses and resulting impairment or disability to patients. trismus (lock jaw) can indicate important diagnoses such as tetanus and rabies.it is due to a spasm of muscles of mastication, which is an important oral manifestation of widespread muscle spasm. apart from these conditions, other causes of trismus are peritonsillar abscesses, and scleroderma. other problems dentists and otolaryngologists encounter in clinical practice are concurrent systemic diseases (patients with multiple problems): patients with bleeding diatheses, diabetes mellitus and a hidden primary malignancy. a non-healing ulcer in the oral cavity may hide a primary malignancy behind it. in these cases, you have to look for it specifi cally. similarly, one has to be aware of oral manifestations of internal pathology. some of them are crohn's disease, ulcerative colitis and gastro-intestinal tract malignancies. often dentists see patients after a tooth extraction with intractable bleeding to fi nd that they have a bleeding diathesis. so, this may be the fi rst presentation of these patients' bleeding disorder. when this patient undergoes any elective procedure in future, it will be a great help to surgeons to be aware of this information to prevent any inadvertent complications. therefore an integrated ehr can prevent unwanted complications where a patient's life may be in jeopardy. the source of otalgia or earache can be from a number of sites other than ear itself. technically ear lobe and ear canal are supplied by four different cranial nerve branches ( th, th, th, th). therefore, an area with a common nerve supply can present as earache. common dental problems which present as referred otalgia are ( ) dental caries ( ) oro-dental diseases or abscesses ( ) an impacted molar tooth (which is a common cause) ( ) malocclusion ( ) benign and malignant lesions of oral cavity and tongue (kim et al. ) . therefore, it is essential these two departments collaborate with each other in diagnosing and treating these diseases, and one way of facilitating it is through an integrated ehr system. there is a lot of overlap between dentists and otolaryngologists in the diagnosis and treatment of patients with halitosis (delanghe et al. ; bollen et al. ) . poor oral hygiene is the most common cause for this common complaint. oral causes include tooth caries, oral ulcers, periodontal diseases, unhealthy mucosa of the oral cavity. it is interesting to note that a simple oral ulcer can form an abcess eroding the fl oor of mouth and becoming a life-threatening oral cellulitis (ludwig angina). once the cellulitis has developed, it becomes a medical emergency. therefore, it is essential to prevent it before it can progress into a life-threatening condition, which of course is possible. causes pertaining to otolaryngologists include: chronic sinusitis or mucociliary disorder, chronic laryngitis or pharyngitis, pharyngeal pouches-related pathology, tumors or ulcers of naso/oro/laryngopharynx, diseases or conditions that impair normal fl ow of saliva such as salivary gland diseases or stones preventing fl ow of saliva, medications which cause dryness of mouth: antihistamines, antidepressants; local manifestation of systemic disorders: auto immune disorders, sjögren syndrome, dehydration from any cause, diabetes mellitus and gastro esophageal refl ux disorder (gerd). gerd is caused by improper neuro-autonomy of the lower esophageal sphincter (les). the les does not close tightly after food intake which causes gastric content to enter the esophagus. over time this can erode mucosa and cause various diseases even becoming cancerous (friedenberg et al. ). this disorder is attributed to life style. fast food consumption habits (oily fried foods) and eating habits (swallowing food without properly chewing) are partly responsible for this disorder (lukic et al. ; al-humayed et al. ) . here again an early diagnosis can manage the disease process before it is fully developed. at present there are no integrated ehr systems serving these specialties (dentistry and otolaryngology). an integrated ehr would facilitate effi cient communication between a dentist and an otolaryngologist who are providing care to the same patient and addressing a problem with a shared focus between the two disciplines. such integrated communication, may only require consulting the available medical or dental record of the patient, based on the particular circumstance. even enabling this simple communication would avoid duplication of effort, clarify the context of certain symptoms and reduce stress endured by the patient. it also has the potential to reduce healthcare delivery costs, and in some cases, even contribute to saving the patient's life. henry hood, allan g. farman, and matthew holder in this chapter, the authors attempt to put forth a justifi cation for precisely this kind of collaborative approach through a summary and discussion of a series of actual clinical cases. the protocols discussed in the management of each of these clinical cases illustrate the value in providing whole-person, interdisciplinary health care to this complex patient population. there is arguably no single patient population for whom the provision of collaborative, interdisciplinary health care is more challenging than for patients with neurodevelopmental disorders and intellectual disabilities (nd/id). in planning and delivering the generally-accepted standard of health care to this unique population, myriad biomedical, psychosocial and sociopolitical realities converge to create a landscape that is, at best, daunting for patients with these disorders, and for the clinicians who are charged with their care. anecdotal and scientifi c evidence suggest that this landscape has produced a paucity of physicians and dentists who are willing and able to provide care to patients with nd/id, and that american medical and dental schools are providing little training focused on their care (holder et al. ; wolff et al. ) . in february of , th surgeon general david satcher issued a report, which documented that americans with nd/id experience great diffi culty accessing quality health care (thompson ) . in that same report, former health and human services secretary tommy thompson said, "americans with mental retardation and their families face enormous obstacles in seeking the kind of basic health care that many of us take for granted." (thompson ) the disparities identifi ed by dr. satcher and secretary thompson require that physicians and dentists approach this population in a spirit of collaboration, compassion, and teamwork in order to produce positive health outcomes for them. perhaps, an even greater imperative driving the need for collaboration between medicine and dentistry in this arena is the fact that many patients with intellectual disabilities have developed this cognitive impairment as the result of an underlying neurodevelopmental disorder that is often undiagnosed. and it is this neurodevelopmental illness and the constellation of potentially devastating complications associated with that illness that create a biomedical fragility and a vulnerability that neither begins nor ends at the oral cavity, and that leaves these patients at risk in almost every aspect of their daily lives. when, for example, patients with nd/id are dependent upon publicly-funded programs for their health care, and when these systems fail to provide the health services that biomedically complex cases require because they fail to account for and accommodate the link between medical and dental pathologies, the risk of a negative outcome is greatly enhanced. such was the case for an intellectually disabled woman in michigan who, in october of , was unable to access dental services through the state's public medical assistance program, and who fatally succumbed to a systemic bacteremia resulting from an untreated periodontal disease (mich. dent. assoc. ). the american academy of developmental medicine and dentistry (aadmd) defi nes a neurodevelopmental disorder as a disorder involving injury to the brain that occurs at some point between the time of conception and neurological maturationapproximately age or (zelenski et al. ). examples of frequently-encountered neurodevelopmental disorders would include fragile x syndrome, a genetically acquired neurodevelopmental disorder caused by a mutation at the distal end of the long arm of the x chromosome (see fig. . ), trisomy , another genetic disorder, which features extra genetic material at the chromosome site (see fig. . ), and cerebral palsy, a prenatal or perinatal, acquired neurodevelopmental disorder (see fig. . ). patients with neurodevelopmental disorders tend to present clinically with one or more of fi ve frequently-encountered, objective symptom complexes or primary complications. these fi ve, classic primary complications include intellectual disability (aka: mental retardation), neuromotor impairment, seizure disorders, behavioral disturbances, and sensory impairment (aadmd). additionally, multiple secondary health consequences can derive from the fi ve primary complications; and any one of these secondary health consequences, or a combination of them, can produce profound morbidity. an example of a common secondary health consequence seen in patients with nd/id, which is derived from intellectual disability and / or neuromotor impairment, is the patient who is unable to care for his or her own mouth, and who develops ubiquitous caries and advanced periodontal disease as a result (see: fig. . ). another example would be the patient who suffers from the secondary health consequence of gastroesophageal refl ux disease (gerd) as a result of the neuromotor impairment associated with multiple neurodevelopmental disorders; and whose tooth enamel and dentinal tissues become chemically eroded as a result of the chronic intraoral acidity produced by gerd (see: fig. . ) . the diagnosis and management of these secondary health consequences provide dentists and physicians with a unique opportunity to work together to improve the quality of health and quality of life for their patients by implementing a team approach, which crosses the traditional interdisciplinary lines of communication, and which expands each clinician's ability to make meaningful treatment options available. indeed, it is often the case that quality primary care provided in one discipline will provide potentially valuable information to an attending clinician from another discipline. such is the case with the patients featured in figs. . and . . the patient whose intraoral photograph is featured in fig. . is a year-old male patient who presented to a special needs dental clinic accompanied by his mother. the mother indicated that her son was exhibiting hand-mouthing behaviors that she believed suggested he was experiencing mouth pain. a comprehensive radiographic and intraoral exam revealed, among other maladies, notched incisors, multiple diastemas, grossly decayed mulberry molars, and advanced periodontal disease. the patient also exhibited moderate to severe intellectual disability. these fi ndings were all consistent with a diagnosis of congenital syphilis. however, in developing the medical history with the mother, it was learned that no previous diagnosis of syphilis had been discussed with the mother, nor was it included in the health history. in cases like this, a comprehensive dental treatment plan should always include consultation with the primary care physician for purposes of moving forward with confi rmation of the clinical diagnosis by serologic testing, and consultation with a cardiologist to assist in the management of potential cardiovascular sequelae. as the dental treatment plan is being developed, consideration should also be given to human immunodefi ciency virus (hiv) testing for this patient, as coinfection is a common fi nding . this issue could easily be attended to by a primary care physician, an internist or an infectious disease specialist. in the absence of any of these team members, the dentist should feel entirely comfortable ordering hiv testing. the primary care physician and the developmental dentist should continue to advise each other and their respective consultant specialists of any signifi cant developments or new information, which could in any way impact either the medical or the dental treatment plan. as treatment progresses, both the physician and the dentist should expect improvement in the patient's periodontal status, which will likely be refl ected in a decrease in the frequency of immune-related illnesses, and in the maladaptive behaviors produced by chronic oral pain. it is quite often the case in this patient population that, with a reduction in maladaptive behaviors, comes a reduction of the use of psychotropic medications prescribed in a frequently futile attempt to manage behaviors that were born of an undiagnosed medical or dental illness. gerd is defi ned as the refl ux of gastric contents into the esophagus. gerd is primarily associated with incompetence of the lower esophageal sphincter; however there are numerous co-contributors, which may predispose a patient to gerd or exacerbate an existing refl ux problem. these co-contributors include a diet high in fat, neuromotor impairment associated with functional abnormalities such as dysphagia, neuromotor impairment associated with impaired ambulation and prolonged periods of recumbence, and the use of multiple medications including anxiolytics, calcium channel blockers, and anticholinergics. gerd is thought to affect approximately - % of the general us population. it has been established in the literature that the incidence of gerd in patients with intellectual disabilities is signifi cantly higher than in the neurotypical population, and that the relative number of unreported cases of gerd is much higher in patients with a neurodevelopmental diagnosis, as well. patients who have gastric refl ux as a function of a neurodevelopmentally-derived neuromotor impairment and a coexisting intellectual disability are impaired in their ability to voice the complaint that would, in the neurotypical patient, commonly lead to an encounter with either a family physician or a gastroenterologist and, ultimately, to a diagnosis. this inability to voice a complaint can be problematic in that, left untreated, gerd can produce maladaptive and sometimes aggressive behaviors in this population. and, of even greater concern, is the fact that undiagnosed esophageal refl ux can lead to more complex conditions that can produce signifi cant morbidity or even mortality -maladies such as barrett's esophagus or adenocarcinoma of the esophagus. chronic gerd can also produce an acidic intraoral environment, which can lead to the chemical erosion of the enamel and dentinal tissues of the teeth. ali et al. have established a link between erosion of the enamel and dentinal tissues of the teeth and gerd. there is additional anecdotal evidence suggesting a link between tooth enamel erosion and gerd, and related maladies. a special needs dental clinic in the eastern united states serving , patients with nd/id, has reported that, of nine patients referred to gastroenterology who presented for dental exam with a fi nding of either tooth enamel erosion or ubiquitous caries, two cases were diagnosed with gerd, two with barrett's esophagus, three with gastritis, and one with duodenitis. in all cases, medical treatment was required. in light of all that is known about the incidence of gerd and of the gerdrelated risks unique to this patient population; and in light of the link between tooth enamel erosion and gerd, it is incumbent upon any dentist encountering tooth enamel erosion in a patient with an intellectual disability to immediately refer that patient to gastroenterology for a work up, which should include esophagogastroduodenoscopy (egd) and ph monitoring. a dentist encountering gerd in a patient with an intellectual disability must be aware that he or she may be the fi rst and only link between that patient and the diagnosis of a potentially life-threatening illness. phenytoin-induced gingival enlargement can appear as either an infl ammatory lesion or a more dense, fi brotic hyperplastic lesion. the infl ammatory lesion is one in which the gingival tissues are swollen and bleeding, and in which pain is often a component. this type of gingival enlargement is the more acute lesion, frequently seen in patients who are currently taking phenytoin. in advanced cases of infl ammatory gingival enlargement, the tissues can appear botryoid, with a characteristic grape-cluster appearance. in advanced cases of phenytoin-induced gingival enlargement, the lesion can sometimes shroud entire sections of the dentition. phenytoin has long been a common medication used to treat seizure disorders in patients with neurodevelopmental disorders and intellectual disabilities. however, the gingival enlargement it produces, and the obstacle this lesion can pose to effective oral hygiene -especially in a population in which oral hygiene is typically compromised -can, over time, lead to periodontal disease, edentulism, and in advanced cases, systemic bacteremias. gingivectomy performed to reduce phenytoin-induced gingival enlargement will typically fail unless the patient is weaned off the offending medication, and another anti-seizure medication is titrated to effect. multiple alternative anti-seizure medications are currently available, which do not have the side effect profi le of phenytoin, and most patients who are weaned off phenytoin will demonstrate a virtual % resolution of the infl ammatory lesion within a matter of or months. the image in fig. . is of a year-old, microcephalic african-american male with intellectual disability, neuromotor impairment, and a seizure disorder. figure . illustrates the appearance of this patient's gingival tissues while he was currently on phenytoin. figure . features the same patient months after being weaned off phenytoin and placed on topiramate. these images illustrate the dramatic result that can be achieved when a dentist and a physician work in collaboration in the best interests of the patient. it is worth noting that this particular collaboration required only one intervention to achieve this result: the patient was weaned off phenytoin and was placed on a safer alternate anti-seizure medication. any dentist caring for a patient with an intellectual disability who presents with phenytoin induced gingival enlargement should immediately contact either the primary care physician or neurologist managing the patient's seizure disorder, and strongly urge that the patient be weaned off phenytoin and placed on a safer alternative anti-seizure medication. edentulism and bacteremia need not be a side-effect of a seizure management protocol. the patient seen in fig. . is a year old male patient with idiopathic intellectual disability who presented to an outpatient dental clinic for comprehensive dental evaluation and treatment. he was accompanied by his father. his father was referred to the clinic by the staff at his son's day program workshop. the day program staff had observed hand-mouthing behaviors, and they had voiced concern that the patient may be in pain. in the waiting room, the patient exhibited behaviors consistent with neurodevelopmental dysfunction. he was non-communicative, and his gaze aversion and tactile defensiveness were suggestive of autism. he was resistant and somewhat combative when directed to the dental chair, and effective behavior management in both the waiting room and operatory required the combined efforts of his father and two staff fig. . the adult patient suspected of having fragile x syndrome members. the patient's health history was positive for attention defi cit hyperactivity disorder (adhd), and there was no history of seizure or neuromotor impairment. the father indicated that, at age ten, the patient was admitted to an inpatient psychiatric unit for evaluation of his uncontrollable behavior. the following day, the parents were told that managing the patient's behavior was beyond the ability of the psychiatric unit staff, and the parents were asked to take the child home. the father also indicated that the psychiatric unit staff described the child's behavior as overwhelming. the patient was last seen by a dentist years prior to presentation; examination and treatment at that time were carried out in the operating room under general anesthesia. effective oral examination of this patient required utilization of papoose board and molt mouth prop. multiple options for behavior management, including utilization of general anesthesia in the operating room, were discussed with the father, and informed consent to utilize medical immobilization techniques for purposes of this examination was obtained and documented prior to taking the patient into the operatory. in the operatory a dental examination was performed, and a baseline panel of digital radiographs was obtained. the head and facial features of this patient were suggestive of fragile x syndrome (see: fig. . ) . the body of the mandible was somewhat elongated; the nose was prominent; the head had somewhat of a triangular shape, and the patient readily averted his gaze. upon further inquiry, the father reported that the patient also exhibited macroorchidism, although he indicated that no physician or dentist had ever suggested a work up for fragile x. fragile x syndrome is a disorder with which many clinicians are unfamiliar. yet it is the second leading genetic cause of intellectual disability in the united states, and it is the leading known cause of autism in the u.s. in addition to the phenotypic fi ndings noted in this case, there are other frequently-encountered physical characteristics consistent with fragile x that may move a clinician toward this diagnosis. they include pectus excavatum or funnel chest (see fig. . ) and joint laxity (see fig. . ) . gaze aversion, as previously mentioned, is a typical fi nding in autism and in fragile x syndrome. indeed, in conjunction with non-verbal behaviors, gaze aversion is often the fi nding that initially alerts the clinician to the possibility of a neurodevelopmental diagnosis featuring autism as a complication. figure . features a photograph of fi ve children at a school for children with special needs. four of the children have been diagnosed with autism, and a fi fth child is a neurotypical child who was visiting his brother on the day the photograph was taken. the reader is left to decide which child is the neurotypical child. any physician or dentist who encounters a patient with an obvious intellectual disability, who does not have an established underlying neurodevelopmental diagnosis, and who presents with additional fi ndings, which may include gaze aversion, shyness, a prominent chin, pectus excavatum, a large nose or large ears, should suspect a possible fragile x diagnosis. the primary care clinician -physician or dentist -should discuss with the guardian or family member the importance of establishing a neurodevelopmental diagnosis. the family member or guardian should be informed that genetic counseling should be made available to all members of the extended family, since fragile x syndrome is a genetic disorder that can be passed from parents to offspring. once this discussion has taken place, a referral to a geneticist for a complete genetic work up is indicated. both the dentist and physician should feel entirely comfortable making this referral. in remote areas where the services of a geneticist may not be available, the attending physician or dentist may order a high resolution chromosomal analysis and a fragile x dna test, and have those results sent to a remote location for interpretation by a geneticist. consultation with a psychiatrist or a clinical psychologist may also be advisable, as patients with fragile x can sometimes experience enhanced social integration as a benefi t of behavioral therapy. the healthcare access problem for americans with neurodevelopmental disorders and intellectual disabilities is, at its core, a healthcare education problem -an education problem resulting from a long-standing defi ciency in professional training focused on the care of this patient population. and it is clear that the medical and dental professions share equally in responsibility for these defi ciencies. eighty-one percent of america's medical students will graduate without ever having rendered clinical care to a single patient with a neurodevelopmental disorder or intellectual disability; and the graduates of % of america's medical residency programs will graduate from those residencies having had no formal training whatsoever -didactic or clinical -in the care of this patient population. additionally, % of graduating dentists have never treated a single patient with a disability. it is no wonder that patients like those whose cases were discussed in earlier sections of this chapter have such diffi culty accessing quality health care. as robert uchin, dean of nova southeastern university college of dental medicine observed in a speech in to his faculty, "not only do we not have enough doctors to care for these patients; we don't have enough teachers to teach them how to care for them." as a result of these defi ciencies in professional education, few clinicians with any expertise in developmental medicine or developmental dentistry are to be found in communities across america. the experts in developmental medicine and dentistry, for the most part, tend to be physicians and dentists who work at the few remaining intermediate care facilities, and at special needs outpatient clinics, psychiatric hospitals, and nursing homes. these physicians and dentists possess the knowledge and expertise in these disciplines because they are the physicians and dentists with the clinical experience. unfortunately for the patients with neurodevelopmental disorders who are clamoring for quality care, there are too few of these clinicians. national experts in developmental medicine and dentistry, however, have begun to collaborate in the creation of patient care protocols; and they have produced multidisciplinary curricula in both dvd and online format. the aadmd has made available hours of online curriculum in developmental medicine, developmental dentistry, and developmental psychiatry (see: list of urls). the curriculum program is entitled, the continuum of quality care , and it teaches collaborative patient care in three disciplines through an interdisciplinary format. the aadmd, through a grant from the wal mart foundation and the north carolina developmental disabilities council, and in collaboration with the north carolina mountain area health education center and the family medicine education consortium, has also established the national curriculum initiative in developmental medicine. this initiative, which is scheduled for completion in , will develop curriculum standards for physicians in the primary care of adults with nd/id. the curriculum stresses the importance of a collaborative approach, which includes medicine, dentistry, podiatry, optometry, and multiple ancillary health professions. if the disparities in access to healthcare for americans with nd/id are to be resolved, physicians and dentists must be willing to cross professional boundaries and work together to plan and deliver whole-person healthcare to their patients with nd/id. interdisciplinary protocols in the diagnosis of neurodevelopmental disorders and in the management of the secondary health consequences associated with these disorders must be established. additionally, clinicians with expertise in these arenas must be willing to work and teach in our nation's medical and dental schools. the clinicians with expertise must be willing to develop predoctoral and postdoctoral curricula, and the deans of america's professional schools must be willing to include these curricula as part of their larger programs in primary and specialized care. the clinicians with expertise in developmental medicine and dentistry must also be willing to conduct patient-focused, interdisciplinary, clinical research in an effort to solve the myriad problems that create obstacles to the delivery of the standard of care for patients with nd/id. they must be willing to obtain institutional review board approval for this research, and they must be willing to make this research available to their colleagues through publication in peer-reviewed journals and text books, and in professional lecture forums. the patient featured in figs. . and . is a man named james. he is a year old patient with idiopathic intellectual disability who presented to a dental clinic for evaluation of a painful facial swelling. a comprehensive intraoral exam revealed a cellulitis resulting from multiple grossly decayed teeth, and a generalized advanced periodontitis. no fewer than fi ve clinicians became involved in this patient's care. they included a general dentist, two oral surgeons, a family practice physician, and a geneticist. over the course of several months, as the treatment plan was completed, and as the chronic dental and periodontal infections were eliminated, james experienced signifi cant improvement in his overall state of health. a comparison of these two photographs reveals not only signifi cant improvement in his aesthetic appearance, but also in his skin turgor and color. these improvements in the patient's health translated to improvements in his daily life. he found gainful employment, and his caregivers now report that he smiles constantly -at work and at home. these photographs were entered into evidence in before a congressional subcommittee investigating the death of a young african-american boy who died as a result of an untreated dental abscess. the photographs were intended to make the point that patients with intellectual disabilities need not die as a result of medical illnesses derived from untreated dental disease. this patient's case illustrates that, when physicians and dentists are willing to work together toward a common goal of whole-person health for their patients, profoundly positive outcomes can be achieved. in a larger context, if our nation's medical and dental professions are willing to commit to a shared agenda, one which promotes the idea of collaborative, interdisciplinary care as a foundational concept, signifi cant improvements in quality of health and quality of life can be realized, not just for americans with neurodevelopmental disorders, but for every patient seeking quality care. in light of the events of , bioterrorism has become subject of increased attention from all members of society. government agencies, professional associations, academia, etc. have expressed their determination to wage war on such threats by all means available. dentists can also participate in this effort by providing assistance at interested groups and the general public (flores et al. ) . in this chapter we will examine the elements and components that may play a role in the establishment of an electronic network for the dental profession for supporting the fi ght against bioterrorism. in this section we review the threats, the public health system, current electronic surveillance systems, regulations and ethical issues, the computerization of dentistry, and how dentistry can serve in improving biosurveillance efforts. the aftermath of september and the anthrax incidents in october ( lane and fauci ) , made the us government reorganize its priorities and reform its current structure (white house offi ce of the press secretary ) . in response to these incidents, president bush proposed the "health security initiative" (white house letter ) in february nd of . this effort labeled the "bioshield initiative," (white house letter ) has the purpose to stimulate research and development of medical countermeasures against bioterrorism attacks. however, despite all these efforts, terrorist attacks are likely to happen in the future and even the best work from intelligence and security agencies will be unable to prevent such events (betts and richard ; council on foreign relations ; baker and koplan ) . to cope with this threat, a report published by an independent task force sponsored by the council on foreign relations "america-still unprepared, still in danger" (council on foreign relations ) , suggested a series of steps to assist the government in preparing to better protect the country. one of these suggestions is the bolstering of the "public health systems". baker et al. defi ne the u.s. public health system as a system that consists of a broad range of organizations and partnerships needed to carry out the essential public health services, such as hospitals, voluntary health organizations, other non-governmental organizations and the business community (baker and koplan ) which can collaborate with local, state and federal public health entities. after the unfortunate incidents in the public health system was revisited and the realization that "the nation's public health infrastructure is not fully prepared to meet this growing challenge" (frist ) became clear. to address this need, congress and president bush enacted the public law (p.l.) - titled "public health security and bioterrorism preparedness and response act of " (frist ; th congress ) . the main purpose of this law was to improve the public health capacity by means of increasing funding and fostering other measures. frist ( ) , described the law as a "good start" and that "to be prepared for bioterrorism, it is imperative that we develop a cohesive and comprehensive system of ongoing surveillance and case investigations for early detection". in this way, several early detection systems have been implemented with different levels of success among different geographic regions in the us. one of the most important initiatives over the years has been the establishment of the national electronic disease surveillance system (nedss) (baker and koplan ; nedss ) . the national electronic disease surveillance working group establishes that the "nedss is a broad initiative focused on the use of data and information systems standards to advance the development of effi cient, integrated, and interoperable surveillance systems at the state and local levels. the long-term objectives for nedss are the ongoing automatic capture and analyses of data needed for public health surveillance". the purpose of this system is to take into consideration and integrate the information of current public health systems implemented at different health department levels: county, state and fi nally at the centers for disease control and prevention (cdc). another initiative spearheaded by the cdc is biosense (looks ) . the purpose of this program is to develop advance detection capabilities of health related events including disease outbreaks. in addition, its emphasis is to improve situational awareness by integrating advanced analytics to process data generated by different health providers and other entities in the us. now that we have examined the general aspects, we will continue our background review focusing on the aspects that pertain to the specifi cs of the dental profession. this section will provide some perspective of the structure of the dental profession in comparison with its medical counterpart. "there are approximately , active dentists in the united states" (mertz and o'neil ) . in the dentistto-population ratio was of - , . and it is expected that by the year the ratio will be . , which translates into one dentist for every , people. " in contrast, the physician-to-population ratio has been increasing for the past years and now stands at per , , about one physician for every people." eighty percent of the dentists are in general practice. during march and of , the american dental association and the us public health service sponsored the conference "dentistry's role in responding to bioterrorism and other catastrophic events" (palmer ; national institute of dental and craniofacial research ) . this meeting reviewed several aspects of bioterrorism and the dental profession: the nature of biological pathogens and its oral manifestations, what needed to be communicated, how dentists should participate, etc. dr. michael c. alfano described the diffi culties that biological pathogens create for clinicians because "they are so insidious." while discussing the anthrax mailings after september th he pointed out that: "… early symptoms appeared so they resembled the aches, fever, and malaise of fl u so those affected delayed seeking treatment, a delay that has proven fatal in some cases". lieutenant colonel ross h. pastel of the us army medical research institute of infectious disease (usamriid) listed the "category a" pathogens as defi ned by the centers for disease control and prevention, and those are: smallpox, anthrax, plague, botulinum toxin, tularemia and viral hemorrhagic fever. he also described an outbreak of smallpox in yugoslavia in and the measure that had to be taken to control it. dr. michael glick described the oral manifestations of smallpox showing "signs hours before skin rash. these oral signs include tongue swelling, multiple mucosa vesicles, ulceration, and mucosal hemorrhaging. oral signs are also evident in inhalation and gastro-intestinal anthrax. in oropharyngeal anthrax the mucosa appears edematous and congested; there may be neck swelling, fever, and sore throat" . dr. ed thompson, deputy director of the centers for disease control and prevention mentioned that "none of the new counter-bioterrorism measures can be effective unless local health practitioners are vigilant in observing and reporting a possible disease outbreak. such surveillance-knowing what to look for and whom to report to-is critical and applies not only to suspected bioterrorist agents, but to a list of reportable diseases which has grown to include such entities as west nile virus and sever acute respiratory syndrome (sars)." dr. sigurs o. krolls presented the response at the local level and he "stressed the importance of communication and the need for redundant systems", "to keep all the parties informed". he also posed the question "can dentists recognize signs and systems of contagious diseases?", and emphasized that education can be essential. dr. louis depaola made several connotations that can be key in the scope of this paper by saying "dentists can contribute to bioterrorism surveillance by being alert to clues that might indicate a bioterrorism attack. such surveillance would note if there is an infl ux of people seeking medical attention with non-traumatic conditions and fl ulike or possibly neurological or paralytic symptoms… or even specifi c signs of a bioterrorist agent. patterns of school of work absence, appointment cancellations or failures to appear, could also be indicators." dr. depaola made clear that in cases of limited release of bioterrorist agents, dentists "have little to offer" but "a widespread attack can certainly tap into dental professional skills in recognition, isolation and management". in addition, dr. guay ( ) lists all the possible roles in which dentists can participate including "education, risk communication, diagnosis, surveillance and notifi cation, treatment, distribution of medications, decontamination, sample collection and forensic dentistry." dental informatics must pay attention to these and other recommendations, in order to develop integrated systems that take these recommendations into consideration. it is also important to understand that informatics has to work with technologies already in place like the computer-based oral health record and current standards. the fi nal recommendation from the meeting stated that to play an important role in biodefense, a serious amount of coordination and preparation will be required, not only from dentists but from other groups, most likely requiring medical and dental data integration. the cohr as described by rhodes ( ) "can provide a structure for documentation that goes beyond the concept of a blank form on a page, it includes a glossary of dental terminology for the entire content of the form as well as knowledge bases and expert systems that can enhance the practitioner's diagnostic and treatment planning decisions". he also acknowledges that one of the advantages of this type of documentation is that it "is much more transportable". he also recognizes the need for standardized methods for collecting information from dentists. schleyer and eisner ( ) defi ned several scenarios where the cohr is used in a "shared" environment where several healthcare providers interact and information is seamless communicated, improving the decisions made by clinicians. delrose and steinberg ( ) discuss how the "digital patient record" enhances clinical practice by providing "better quality information" to the clinician. although all of these benefi ts sound promising and encouraging some still express concern of the lack of standards among different information systems, which translates in communication breakdowns (schleyer ) . on the other hand, heid and colleagues ( ) mention a list all the steps that are currently being taken by different organizations such as the ada in order to produce a standardized cohr. other examples of standardization can be found in a paper presented by narcisi ( ) where ada's participation as a voting member in the american national standards institute has allowed edi or the cohr to be discussed and improved at a national level. additional infl uences in the standardization of the cohr are the security regulations mandated by hipaa, the health insurance portability and accountability act of . dentists are required to "adopt practices necessary for compliance" (sfi kas ; chasteen et al. ) . these and other regulations (szekely et al. ) will encourage the homogeny among different system vendors. computer ownership, on the other hand, has increased steadily during the last years. according to schleyer et al. ( ) in only % of dental professionals used computers in their practices compared to % in the year . additionally similar trends in internet connectivity where described. the issues mentioned above describe the issues that have to be considered in order to create surveillance system against bioterrorism for the dental profession. this review has tried to be inclusive by covering different aspects starting with the current state of affairs and environment, treats, technology, law, etc. next we present a blueprint for developing a biosurveillance system. the purpose of developing an electronic health surveillance system is to gather information from patients directly ( wagner et al. ) by detecting signs and/or symptoms, or indirectly by obtaining other types of information such as over the counter medication sales, patients' no-shows, usage of internet search engines keywords, etc. in this particular case, the proximity of contact between the dentist and the patient is equivalent to a medical inspection in terms of immediacy and/or closeness. such signs and symptoms can be easily detected if the dentist is properly prompted to search for them. this is just one example of ways how a system could provide assistance in the detection of a bioterrorism incident. but, before describing our proposed system, it would be important to address the fact that current syndromic surveillance systems have certain advantages in terms of its particular technological implementation . the rods laboratory obtains data directly from chief complains in the emergency departments from hospitals. the advantage of this surveillance system is that the implementation has to be made with only a limited number of parties (hospitals, clinics, health systems, etc.). on the other hand, our system would have to deal with thousands of different implementations (one in each dental offi ce). this and other challenges have to be considered when designing the proposed system: the proposed system should work at multiple levels: the system would have to provide a mechanism to alert the dentist if there is • suspicion that a bioterrorist attack may be happening. the mechanism would increase the dentist's awareness in case of fi nding suspicious signs or symptoms in a patient. this can be triggered by the patient's characteristics such as geographic location of residence, etc. automated collection of information from the patient's oral health record. the • system would report to a central database signs or symptoms of interest. the aggregation of this data could generate information that would eventually identify the presence of patterns that may lead to the early detection of such events. collection of additional information, which combined with other sources, can be • useful in terms of detecting or tracing some incident. patients' "no-shows" is the primary example, that, if combined with others such as work or school absenteeism can provide a relevant pattern for public health offi cials. dr. x, who practices in a community min away from capitol city, installed a new clinical management system months ago. among the features that were included in this new clinical management system (cms), a bioterrorism detection module was added. she felt curious because of recent news she read in the newspaper about possible attacks against the us and decided to install such feature. he read about how the module would work in combination with the cms she just bought. the educational information provided with the software instructed dr. x, that in case that a patient victim of a bioterrorism attack happens to be seen in her practice, the software would collect information and would send it to public health offi cials. when installing the software, dr. x was asked if she agreed to share such information with authorities. she was provided the option to receive notifi cation in case some information was sent but she decided not to enforce it. during the last week a patient walked into dr. x's dental offi ce. the patient presented some signs that indicated the presence of a disease; still its origin was not clear. an epidemiologic study later would show that the patient was present at the football stadium when an infectious agent was released (fig. . ) . although, at that time his medical history showed no indication of a systemic disease, the presence of multiple oral vesicles prompted the dentist to make an annotation into the cohr. the system, by using a natural language processing engine, detected such sign and sent this information to a central database. the patient was discharged and instructed to take some support medication to treat the oral ulcers. the next day, the central database pinpointed the presence of an out of the ordinary increase in the number of cases with the same signs and symptoms around that region. when the presence of this peak was detected, the central server sent a request to the dentist computer for additional information. one of the requested elements was if there was any use of medication for treating oral ulcers. fortunately this information was available. the central database crossed this with the information of other surveillance systems together with the information from other patients that happen to have similar clinical signs and/or symptoms. dr. x received an email from a public health offi cial asking her to communicate to the local health department to discuss information about one her patients. the case depicted above simulates the release of smallpox during a football game. in the case of smallpox oral symptoms include tongue swelling, multiple oral mucosal vesicles, ulceration, and mucosal hemorrhaging (national institute of dental and craniofacial research ) . dentists could be alerted by an electronic system to search for such signs or they can be detected automatically. in case of a high incidence within a group of patients, in a confi ned area, public health offi cials get to be notifi ed. in our hypothetical case there are issues that need to be addressed in order to make such detection system feasible: as described by schleyer et al. ( ) , % of dentists in the us use a computer in their practices. this fi gure would generate an estimate of , computers in dental practices. this prevalence of computers represents an opportunity for public health data collection. the creation of a software application for surveillance purposes must rely on existing technology. currently there are approximately major clinical management software packages in the market (dentistry today ) . out of these , clearly permit direct database manipulation. this characteristic can easily allow the creation of a "querying" application that would look for specifi c information within the data stored by those packages. additionally, a natural language processing engine could be embedded into the application in order to detect variations in data input on the computer oral health record. nevertheless, it is necessary to obtain a detailed list of the oral manifestations of diseases that are likely to be found on patients. successful implementations of similar systems have been shown to work successfully (chapman et al. ; ivanov et al. ) and using the same approach for our system seems technically feasible. this collected information later would be send to a central server in order to be analyzed and interpreted. the components of our system would be as follows (fig. . ) : thin client: a software application distributed for data collection. it would be • conformed of a "querying" mechanism, combined with a natural language processing engine and a communication module. this software client should be as thin as possible to reduce the work load on the dentist's equipment and should be embedded as a plug-in for current clinical management systems. vendors should be contacted to ask for their collaboration in the development of such application to ensure maximum compatibility and integrity of data collection. central servers: server software in charge of integrating all the data collected • from dental offi ces. it has to be capable of handling simultaneous requests from multiple users. this server would integrate all the data and would perform an analysis with the intention of detecting anomalies. it would be recommended that redundant servers should be located in different data centers with mirroring capabilities to guarantee their survivability in case of technical diffi culties. communication network: the transmission of information should be done using • the internet. this, of course, would essentially depend on the practitioner's current connectivity. if that is not available, backup connection to the central servers should be established. dentistry uses several standards for transmission of health related information. clinical management systems use standard-based technology to transmit information (narcisi ; chasteen et al. ; szekely et al. ; dentrix dental systems ) . dentists are aware of these standards and use them in a day-to-day basis to transmit information to insurers. additionally, in order to interact with other surveillance systems such as the nedss, our application should rely on the same standards. the software both client and server should be thoroughly verifi ed to be secure in terms of being safe against hacker attacks. on the server side, redundancy should be provided so downtime is reduced from design. the system should be developed so mirrored servers are always up and running. data integrity mechanism should also be considered. privacy and confi dentiality are important issues that need to be incorporated as part of a robust biosurveillance system and distinct regulations such as hipaa require protecting patient information (frist ; chasteen et al. ; bayer and colgrove ; etzioni ; ivanov et al. ) . in our hypothetical case we describe the use of several sources of information for detecting a bioterrorist attack. we described how syndromic information is transmitted to a central database which initially should be de-identifi ed. later, after the suspicion a bioterrorist attack more information is requested (medications) and more inferences are made. this, although technically possible, would require changing our processes and also the will to share clinical information. this leads to the discussion mentioned in the background section about "individual rights" vs. "common good". although hipaa addresses public health , some other implications may arise and the health professionals including dentists, physicians, public health offi cials and patients should discuss and address such issues. as discussed earlier, legislators face a diffi cult task in terms of determining what is best on behalf of the individuals they were asked to represent. legislation may have to be passed in order to guarantee the functioning of such a system. individual freedom and privacy are important values which may pose a confl ict when collecting individuals' information even for their own good. in any case, careful consideration has to be given to which information is required to detect a bioterrorist attack and also, by keeping in mind that it is always important to reduce, as much as possible, the collection and transmission of patients' information over the internet or any other network. a detection algorithm has to be created or adapted in order to determine the presence of a bioterrorist attack. some algorithms have proven their effectiveness (wong et al. a, b ) and it is likely that from these, a new analysis should be done in order to select or create one that addresses the particular needs of our system. a study was conducted to assess the feasibility of using oral manifestations in order to detect disease outbreaks (torres-urquidy et al. ) . it was found that for diseases such as botulism and smallpox it would be feasible to gather data that contains oral manifestations that would allow creating a detection signal using natural language processing followed by the use of statistical methods such as moving average to serve as part of a detection algorithm. the system should also be thoroughly evaluated, before and after implementation. to perform the evaluation before the system implementation computer simulation can be used to assess the effectiveness and likelihood of detection. simulation and modeling techniques (reshetin and regens ) have been used to estimate the effects of a bioterrorist attack. the same techniques can be used to evaluate our system. in case of the study by torres-urquidy ( ) , the investigators utilized synthetic outbreaks to test the performance of different signals. from their evaluation process, they learned, for instance, how many cases would be necessary to occur for the system to reach certain detection thresholds. several dental organizations have shown publicly their support of measures against bioterrorism. the american dental association and the national institute of dental and craniofacial research are two organizations who could play an important role in the development, deployment and ongoing support for our system. local dental societies also would also play an important role in the deployment of the proposed system. similarly, local, state and federal public health agencies should engage in activities that could make these mechanisms for health surveillance feasible. if dentists want to play an active role in the fi ght against bioterrorism, they should commit to collaborate with public health entities as well as to seek a way to integrate their information with the rest of electronic biosurveillance systems. professional organizations such as the american dental association can also participate by endorsing such efforts and by collaborating in the educational process of the dental professionals and their patients. as mentioned by dr. depaola (national institute of dental and craniofacial research ) dentists "have little to offer" in the current biosurveillance state of affairs. however, the integration of different technologies can change this perception. goldenberg et al. ( ) described over-the-counter medication sales as a technique for discovering disease outbreaks and stated that their approach may be "more timely" than traditional medical or public health approaches. medical cases that result from bioterrorism attacks do not produce symptoms until they have fully developed, so it is likely that different patterns can be detected before the patients start reaching the emergency department. as stated earlier (torres-urquidy et al. ) , it may be possible to have dentists participating of biosurveillance efforts, if we solve the proper organizational and technical challenges. dr. john r. lumpkin ( ) states that "hippocrates noted the health of the community was dependent on characteristics of a community and the habits of the people who lived there." dr. krolls (nidcr ) in his fi nal remarks during his presentation at the dentistry's role conference against bioterrorism, said, "dentists may pick up telltale information about what is happening in the community. after all, dentists spend more time with their patients than any other health specialty". kass-hout t, zhang x. biosurveillance: methods and case studies. muhammad f. walji maintaining patient records are essential for both clinical care and research. clinical research often occurs in the context of also providing patient care, yet the systems that are used for each are different and often cannot exchange data. the lack of data exchange between systems pose signifi cant barriers to effi ciently treating patient and conducting clinical research in dentistry. the purpose of this section is to review the benefi ts and challenges of integrating electronic health record (ehr) used for patient care and electronic data capture (edc) which is used for clinical research such as clinical trials. an increasing number of dentists routinely use ehrs (schleyer et al. ) . most dental schools in north america also use ehrs. benefi ts of using ehrs include increased legibility, portability, and improved patient safety (buntin et al. ) . recent federal incentives, although not directly benefi cial to dentists, will also likely spur the adoption of ehr (blumenthal and tavenner ) . clinical researchers, especially those conducting clinical trials, are also discovering benefi ts of using electronic data capture compared to paper. a clinical trial is a process in which new treatments, medications and other innovations are tested to evaluate safety and effi cacy. a standard part of health care, clinical trials are often lengthy and costly due to myriads of regulatory oversight. recent estimates set the cost of drug development in excess of $ million (grabowski et al. ) . accurately documenting data with suffi cient detail is critical for providing patient care and conducting clinical research. while the medical record is the foundation for patient care, the case report form is the foundation in a clinical trial. not all clinical research is clinical trials. clinical trials whose data will be submitted to the fda as a new therapy or device have additional requirements relating to the collection and transmission of the data. similarly for patient care data, ehrs need to meet the privacy and security requirements of hipaa. case report forms (crf) are a medium in which research study sites collect subject data in pre-defi ned formats for communication with clinical trial sponsors (rondel and webb ) many clinical trials data are collected on paper (rondel and webb ) . data measurement, collection, and recording are considered the "most crucial stage" in the data management process (hosking et al. ) . traditionally, study coordinators often record information in a case report form and subsequently mail or fax the crf to the centralized coordinating center. there, data entry staff, sometimes with the aid of optical character recognition systems, input crf data into a computer. errors made during this second data entry process are diffi cult to detect and correct (hosking et al. ) . lengthy guidelines in literature discuss methods for developing paper case report forms to reduce data entry mistakes (hosking ) . a well-designed crf may allow a user to effi ciently collect and record pertinent data. however, forms are often revised and redesigned during a clinical trial due to changes in protocol, unforeseen outcomes, or oversight (singer and meinert ) . there has been a recent drive to use electronic case report forms (ecrf). direct data entry at a study site shortens time to analysis and provides opportunities to audit data at time of entry. this could reduce data errors that might otherwise be caught weeks after submission. for quality control purposes, some studies require double data entry using computers and paper (day et al. ) , though alternative solutions have been explored including the use of data sampling (king and lashley ) and probability statistics to select only those forms likely to contain errors (kleinman ) . ecrfs may also facilitate data collection from existing electronic information systems such as lab systems. however, ecrfs are almost always reside in a separate system that is not linked to a patients record. although many clinical research studies are still being conducted using paper, an increasing number of studies are using ecrfs and electronic data capture (edc). for example, a review of canadian clinical trials found that % use edc (el emam et al. ). studies that are sponsored by a pharmaceutical company and are multicenter appear to use edc at a higher rate than those sponsored by government or a university. the cost of a commercial edc is substantial. recently a freely available edc has become popular amongst universities called redcap. a tool originally developed at vanderbilt university, it is now being used at over a institutions worldwide (harris et al. ). however, such tools are generally not integrated with the institutions ehr. although moving from paper to electronic will afford benefi ts there is a great need to allow data exchange between the patient care and clinical research components of information systems. although ehr and edc are similar, several challenges remain unresolved that prevent integration. one of the major barriers is likely to be different workfl ows for patient care purposes and to collect data for research. research is needed in defi ning an optimal workfl ow that can streamline the tasks associated with patient care and research, while at the same time providing a unifi ed information system that support these activities. also, the data that are collected for care and research are likely to differ. a researcher may require far more granularity of an oral health measurement than a clinician seeking to provide care. in cases when conducting a double blind placebo controlled clinical trial, the investigator may not even know the type of treatment that has been delivered to the patient. due to complexities of each domain, and large differences in goals, to date mutually exclusive workfl ows have arisen. a clinician investigator who sees a patient for both care and research, will likely need to enter data on this same patient twice; once in the ehr and once in the edc system. despite the availability of electronic systems, a major barrier is the integration and compatibility of disparate health information systems to converse with one another. the languages are important because they can help data sharing. clinical trials are not usually conducted in isolation, but are part of conventional medical care. therefore sharing data by clinical trials, patient care and laboratory systems becomes especially important with the adoption of ehrs in dentistry. in biomedical informatics, standardized terminologies are recognized as a critically important area to help better represent and share data for use in electronic systems (cimino ) . the systematized nomenclature of medicine clinical terms (snomed-ct), developed by the college of american pathologists, is the most comprehensive medical terminology (strang et al. ; chute et al. ) and is used in a number of health informatics applications. the us department of health and human services ( ) has also licensed snomed-ct, allowing access throughout the us at no charge. therefore snomed-ct is even more likely to be the vocabulary used in electronic formats of patient records in the future. the medical dictionary for regulatory activities (meddra) is terminology used by the fda and drug development industry to classify, retrieve, present, and communicate medical information throughout the medical product regulatory cycle (brown et al. ) . in particular it is used to record and report adverse drug event data. therefore standard languages are essential in sharing clinical trials data between sites, and also with regulatory agencies. no one single terminology is suited for all tasks. snomed-ct is likely to be more comprehensive to code clinical encounters, while meddra is more suited to help adverse event reporting. however, it is important that terminologies are widely adopted and used for similar purposes. even when standard terminologies are agreed upon, such information needs to be interchanged in standard formats. health level (hl ) is an important organization whose standards are widely adopted in healthcare to exchange information between computer systems. the clinical data interchange standards consortium (cdisc) is also an important group that helps to defi ne different data standards specifi cally for clinical trials research, such as clinical trials or regulatory submissions. one particular challenge in oral health has been the lack of a standardized terminology to describe diagnoses. although icd contains oral health concepts, they are often not granular enough to be useful for some patient care or research purposes. recently a dental diagnostic terminology has been developed by a group of dental schools, and has already been adopted by several institutions and used within dental ehrs (kalenderian et al. ) . the american dental association (ada) has also been developing snodent, but is not yet publically available for clinical use (goldberg et al. ). another link between ehr data and clinical research is the potential to fi nd human subjects. recruiting suffi cient numbers of patients that meet eligibility requirements within an allotted time frame for clinical trials is challenging. as ehrs contain detailed information about patients, they can be used to fi nd patients that meet specifi c inclusion and exclusion criteria. informatics for integrating biology and the bedside (i b ), an open source data warehousing platform, has been found to be a useful tool for cohort selection especially if the source data from an ehr is represented in a structured format (deshmukh et al. ). further, with health information increasingly available to patients through the internet, it is possible interested patients will be more effective in fi nding clinical trials than investigators looking for patients. many clinical trial registers are now available online. the national institutes of health (nih) have made available their database of nih funded research (mccray ) . there is currently no single repository for patients to fi nd all trials studying a health condition. a recent study assessed the comprehensiveness of online trial databases concerning prostate and colon cancer and found that online trial registries are incomplete, especially for industry-sponsored trials (manheimer and anderson ) . a more collaborative effort between government and industry-sponsored research groups to compile and standardize information may be a mutually benefi cial effort. it is not clear how many patients now enroll in clinical trials through online discovery. ehr data originally collected for patient purposes can be potentially used for research. aggregating data from multiple sources can provide a large dataset that could otherwise not be available. electronic health records (ehr) contain a wealth of information and are a promising source to conduct research. data extracted from ehrs differ from other sources such as population surveys or data obtained from payers, as they provide a more detailed and longitudinal view of patients, symptoms, diseases, treatments, outcomes, and differences among providers. therefore ehr data in dentistry can potentially provide valuable insight into oral health diseases, and treatments performed on a large cohort of subjects. ehrs also play an important role in enhancing evidence-based decision-making in dentistry (ebd) and improving clinical effectiveness through decision support (atkinson et al. ; walji et al. ; valenza and walji ; taylor et al. ; spence et al. ; chambers et al. ; langabeer nd et al. ; walji mf et al. ). the consortium of oral health related informatics (cohri) provides an example of how dental ehrs are used for research purposes (schleyer et al. ; stark et al. ) . cohri was formed in by a group of dental schools who used the same ehr platform and who are interested in sharing clinical and education data. through funding from the national library of medicine, four dental schools are participating in a pilot project to develop an inter-university oral health research database by extracting and integrating data from ehrs. one promising area where data repositories derived from ehr data can be used for new discoveries is in the area of comparative effectiveness research. comparative effectiveness research is defi ned as "a rigorous evaluation of the impact of different options that are available for treating a given medical condition for a particular set of patients." (congressional budget offi ce ) further, such research includes focusing on the clinical benefi ts and risks of each option (clinical effectiveness), and an analysis on the costs and benefi ts (cost effectiveness analysis). comparative effectiveness research (cer) is also likely to reduce costs of dental care and increase access to the majority of the population who currently receive no dental care. unfortunately many recent systematic reviews focusing on cer questions in dentistry have been inconclusive due to the lack of existing evidence in the scientifi c literature. secondary analysis of the data that reside in dental electronic health records (ehr) is a particularly appealing approach to facilitate cer and generate new knowledge. ehr data has the potential to provide a comprehensive picture of patients' histories, treatments, and outcomes, and if integrated with similar data from other dental clinics can include a large and diverse set of patients. however, numerous challenges must be solved before ehrs can be used for cer. first, data suitable for cer must actually be collected from ehr systems. second, this data, which often resides in proprietary systems, must be accessible and retrievable. and lastly, this data should be structured in a format that can be integrated with data from other sources or institutions. practice-based research networks (pbrn) are groups of primary care clinicians and practices working together to answer community-based health care questions and translate research fi ndings into practice. pbrns engage clinicians in quality improvement activities and an evidence-based culture in primary care practice to improve the health of all americans. in , the national institute of dental craniofacial research funded three such research networks. the dental pbrn's to date have been conducting both prospective and retrospective research. for example, barasch et al. conducted a case controlled study to investigate risk factors associated with osteonecrosis of the jaws . many prospective studies conducted as part of pbrns still require separate data collection systems for the research data. ehr data contained in practices as part of pbrns are beginning to be used for secondary purposes. for example fellows et al. conducted a retrospective analysis of data contained in electronic health records to estimate incidence rates of osteonecrosis of the jaws ( fellows et al. ) . pbrns provide great promise of how ehr and clinical research data can be used effectively to promote both patient care and new discoveries. another area that intersects both the patient care and research realm are patient registries. patient registries are ways to track groups of patients who have had specifi c diseases or have had certain treatments. while ehr data would contain information on all types of patients, their diseases, and treatments, registries would allow focus on specifi c diseases or treatments of interest. registries would not be as costly in terms of resource requirements like a traditional clinical trial, but would require specifi c eligibility criteria, informed consents, and collection addition to that collected as part of routing care. dentistry has lagged far behind in forming data registries, primarily because dentistry is practiced in small offi ces and not in large hospitals making the process of integrating data very diffi culty. however, dental schools which themselves house large clinical operations are ideally positioned to create disease specifi c registries that can potentially use data collected for patient care and extend for research purposes. there is great potential for providing new insight in oral health by the integration of patient records and clinical research from both a workfl ow and information systems perspective. the technology challenges of developing systems that can exchange data, and use standardized terminologies appear solvable. however, the socio-technical issues such as determining how to incorporate optimal workfl ows for conducing both patient care and research with minimal additional overhead appear to be the greatest challenge before widespread adoption. similarly, there appears to be great potential in using ehr data originally collected for patient care for the secondary use of research and discovery. this will require collaboration between patients, providers and researchers from all healthcare disciples, and institutions with friendly policies for sharing data to improve both patient care and drive new discoveries. amit acharya , andrea mahnke , po-huang chyou , and franklin m. din more recently there has been a strong push from the united states federal government for the adoption of the electronic health record (ehr) within the healthcare industry. as a result, $ . billion is made available to incentivize the physicians, dentists and hospitals for the adoption of the ehr through the health information technology for economic and clinical health (hitech) act. as the nation head towards adoption of the ehrs, there has also been a growing interest with the majority of the u.s. dental schools to implement ehrs within the educational setting. fifty of the fi fty-six u.s. dental schools, as well as dental schools in canada and europe, are either using or in the process of adopting some aspects of a common dental ehr framework (white et al. ) . a group of dental schools known as consortium for oral health-related informatics (cohri) was formed in which used this common dental ehr framework -axium (stark et al. ) . currently there are about dental schools within cohri. the ehr will not only support clinical care, but will also result in training the next generation dental students and to conduct innovative research that was not possible earlier. however, not much is known about how many of these dental schools' electronic dental records are integrated with their respective university's electronic medical record. a common medical-dental ehr model at healthcare universities would enable a holistic approach to providing patient care and provide the much needed electronic infrastructure to study interrelationship between the various oral-systemic diseases. recently a group of researchers from marshfi eld clinic in wisconsin, us conducted a survey to investigate the current states of health information technology and informatics within the dental school in the us. list of us dental schools were identifi ed through the american dental education association (adea) web site. dental schools were contacted to determine who the most appropriate person to take the survey would be. once the list of contact was developed from each dental school, an email was sent to us dental schools with a link to a survey created in surveymonkey. the survey was administered on tuesday march , . reminder survey emails were sent to all recipients on march and march . the survey was closed on march . the anonymous survey was at most questions, depending on how questions were answered. the survey focused on topics such as presence of dental informaticians within the dental schools, use of fi nancial and clinical information systems, interest in federal stimulus support for ehr adoption provided through american recovery and reinvestment act and meaningful use of ehr, relationships with health care entities and bidirectional nature of the dental and medical ehrs. the study was approved as exempt from the marshfi eld clinic institutional review board under section cfr . (b) and waived requirement for an authorization. thirty out of the fi fty fi ve dental schools responded to the survey (response rate of %). however, fi ve of the thirty dental schools representative did not complete the survey and hence their response was not included in the analysis. regarding the question about the presence of a dedicated department or a center for information technology (it) or informatics within the dental school in us, % (n = ) of the responding dental schools had a dedicated it/informatics department or center (p-value of . ). the it or the informatics department size (in terms of the number of personnel) at the dental schools is illustrated in fig. . . thirty fi ve percent (n = ) of the us dental schools that housed an it / informatics departments had personnel with not only it training but also dental informatics training. while % (n = ) of the dental schools were considering integration of dental informatics personnel within their department or center. twenty fi ve percent (n = ) of the dental schools did not have any plans of integrating personnel with dental informatics personnel within their department or center (see fig. . ). partial responses to additional questions in the section of the survey is provided under table . . the majority of the responding dental schools were currently using financial electronic systems (fes) (p-value of < . ) and electronic dental records (edr) (p-value of . ). the use of fes outnumbered the use of edrs in the dental schools (see fig . ) . about % of the dental schools that were currently utilizing the edrs used it in all the clinical modules (p-value of . ), while % of the dental schools used the edrs in some of the clinical modules. when asked about the commercial edr system that the dental schools were using, axium (exan group, canada) was by far the most implemented edr system. two dental schools had salud (two-ten health limited, ireland) implemented and two dental schools had gsd academic (general systems design group, iowa, us) implemented. combinations of two ehr systems (home grown and dentrix) were implemented at two dental schools. one school had a dentrix only implementation, while another had developed its own edr system (home grown) (see fig. . ). there were dental schools which had implemented an edr fi ve or more years ago, dental schools - years ago, dental schools - years ago and dental schools less than a year ago (see fig. . ) (p value of . ). when the dental schools were asked the question as to whether they were expecting to apply for the medicaid meaningful use incentive program, majority ( %) of the dental schools did not know and only % of the dental schools were expecting to apply within the next years (fig. . ) (p-value of . ). challenges or barriers identifi ed by some of the dental schools in complying with the meaningful use objectives were (a). lack of certifi ed edr and information regarding it, (b). issues with getting auxium certifi ed and (c). qualifi cations of the edr as many of the meaningful use objectives do not apply to dentistry and lack of specifi c information about it. only % of the responded dental schools were part of a health system. fifty two percent (n = ) of the responded dental schools had a formal relationship with other health care delivery entities in terms of sharing facilities, patient transfer, training programs. some of the types of relationship mentioned by the dental schools that had a formal relationship with other health care delivery entities included: (a). a gpr program and an emergency dental unit in the hospital, (b). affi liated hospital, (c). affi liation agreements, (d). oral and maxillofacial surgery (omfs), anesthesia and pedodontics all have some portion of education in medical health center, (e). omfs residents are also residents of medical health center, (f). residents providing care under contract with area hospitals, (g). sharing patients wand facilities with the health center, (h). students rotating in the community health centers and (i) collaborative grand programs. eighty fi ve percent of the dental schools that had a formal relationship with the health care delivery entities had routine interaction with them because of their existing relationship (p-value of . ). their usual method for exchanging information was through informal medium such as phones, emails and faxes and formal medium such as memorandums, letters and contracts. when the dental schools were asked about the communication between the health systems' emr and the school's edr, majority of the dental schools did not have any communication ( %) or did not know is such a communication existed ( %) (p-value of < . ) (see fig. . ). out of the % (n = ) of the responded dental schools who's edr did not communicate with the health system's emr, % (n = ) of the dental schools stated that they did not need to exchange patient information electronically as a reason for the non-communication, while % (n = ) dental schools states that they would like to exchange patient information electronically but there were barriers that prevent them from doing so. some of the barriers identifi ed by these dental schools were (a). the hospitals and the dental school are not part of the same medial system and hipaa concerns prevent sharing data, (b). the dental school currently neither did have an edr nor the infrastructure to support one and (c). hospital is not interested and has high and perhaps unrealistic security standards. the remaining % (n = ) of the dental schools expected to exchange patient information electronically in the near future (next years). some of the information categories that were shared between the edr and emr in the small number of dental schools are illustrated in fig . . finally when asked about any research projects under way in their dental school to investigate discrepancies between medical and dental records for the same patient, only ( %) dental school was currently undertaking such project. in all common diseases, including those that affect the oral cavity, both the environment and genetics are pathogenic conspirators. unfortunately, we currently know little about the specifi c mechanisms underlying any common disease; and oral diseases are among the least understood. elucidating the etiology of chronic oral diseases will involve a synthesis of results from careful experiments of environmental exposures such as diet and tobacco use, the oral microbiome, co-morbidities, largescale, well-designed genetic studies, and the various interaction effects. with regard to genetics, the past few decades have witnessed transformative developments in our ability to interrogate the entire genome for genes that contribute to disease. while dramatic advances in experimental designs, statistical approaches, and clinical insights have greatly aided this scientifi c campaign, the central driver of this progress has been the development of high-throughput, inexpensive genetic technologies. following initial molecular studies using variant forms of enzymes, or allozymes, a major breakthrough was the use of highly informative dna-based markers throughout the genome (botstein et al. ) . this idea of directly assaying existing dna variation to conduct linkage and association studies in genetics began a revolution in disease gene mapping. recent interest from commercial entities has produced a feverish pace of technological innovation, markedly reducing cost and expanding the depth of inquiry. previously unfathomable, the testing of over one million single nucleotide polymorphisms (snps) in thousands of patients and controls is now commonplace (wellcome trust case control consortium ; schaefer et al. ) ; and very recently, next generation sequencing technologies have progressed to the point where sequencing of the entire protein-coding portion of the genome (exome) or even the entire genome is a costeffective method to examine disease traits across the entire spectrum of genetic variants in small numbers of affected individuals (ng et al. ) . there is little doubt that soon whole genome sequencing will be applied to nuclear family-based designs, studies among distantly-related affected individuals in extended pedigrees, and case/control studies involving thousands of individuals. this unprecedented scope of inquiry made possible by large-scale genetics, has begun to yield fascinating resulting into predisposition to oral cancers, caries, and periodontal disease that will molecularly redefi ne these pathologies, explicate unique biological connections with related diseases, give impetus to the development of directed therapeutics, and indeed personalize medicine. still, much more genetic focus on oral disease phenotypes is required if we are to realize this medical impact in a timely fashion. as genetic technologies have allowed the progression of interrogating single protein variants to single dna markers to entire genes to markers across the genome, and now to the entire genome sequence, the promises of these large-scale genetic studies have understandably undergone monumental expansion. it may be reasonable to expect the results from whole genome sequencing to decidedly revolutionize medicine within the next two decades. however, this new scientifi c capacity comes at a cost. as genetics, and biology in general, transitions to a data-rich science, practitioners have found themselves woefully unprepared to store and analyze the volume of data generated. once analyzed, interpretation and integration of these abundant and multifaceted results into medical practice will also be an appreciable challenge. insuffi cient assimilation of genetic fi ndings into merged dental and medical records will severely limit the ability of clinicians to appropriately treat patients. inadequately addressing these informatics issues will severely derail efforts in the basic sciences efforts as well as the translational and clinical sciences. this chapter explores the current state of genomics studies, what we have learned from genetic investigations into oral diseases, and where we may be headed. genetic studies have much to offer investigations of disease etiology. why do some acquire diseases and others do not? for those affected, why do some progress more rapidly than others? what causes some patients to respond to therapies, while others suffer from adverse reactions? these are all fundamental questions in both biology and medicine, whether the focus is on the gastrointestinal tract, the hippocampus, the lymphatic system, metabolic disorders, or oral diseases. speaking generally across disease areas, a portion of the answers to these questions often lies in described environmental effects. in numerous chronic diseases, infectious agents are likely contributors to the disease process -periodontitis, for example, is initiated by gram negative anaerobes in susceptible individuals (holt and ebersole ) . surely, unique and latent environmental exposures provide a random component to common disease susceptibility and progression. through twin studies, studies of risk in close relatives, and quantitative traits experiments, it is well-understood that heritable factors, including but not limited to dna variation, are typically responsible for - % of the phenotype variability for common diseases. this section will attempt to cover, at least at a cursory level, the major salient developments affecting genetic insights into chronic and aggressive periodontitis, with some comment on genetic factors infl uencing susceptibility to caries and oral cancers. while it would be extremely naïve to view genetic studies as an immediate panacea for our ills, the discovery of disease-causing genes does illuminate hitherto unknown biological pathways and molecular mechanisms, draws unforeseen connections with other traits, may improve prognostic models applicable for individuals, and suggests specifi c therapeutics. industrialization has brought forth increased lifespan and wellness through vaccination, modern sanitation practices, public health policies, and advances in medical science translated into practice. however, the accompanying physical inactivity coupled with a high calorie diet are probable contributors to an extremely common, chronically infl amed metabolic syndrome (hotamisligil ) that is thought to give rise to a multitude of intimately related disease traits: insulin resistance, compromised insulin signaling, hyperglycemia, obesity, dyslipidemia, hypertension, impaired kidney function, elevated liver enzymes and steatohepatitis, poor wound healing, neurodegeneration, vascular disease, pregnancy complications, accelerated immunosenescence, and periodontal disease (ford et al. ; ferrannini et al. ; eaton et al. ; holvoet et al. ; speliotes et al. ; eckel et al. ; d'aiuto et al. ) . these diseases often co-occur within the same patient and could be considered variable expression complications arising from a state of aberrant caloric fl ux that induces metabolic dysfunction and chronic, systemic infl ammation. these features constitute a disruption in a fundamental homeostatic mechanism with intensifying pathogenic consequences. the rapidly increasing incidence and decreasing age of onset for this pathophysiological state have generated a major source of mortality and morbidity in modern cultures (ford et al. ; ferrannini et al. ; weiss et al. ) . it is becoming increasing clear that many chronic diseases have an infectious component. there is relatively convincing evidence that many systemic, t-cell mediated autoimmune disorders may be initiated by infections. for example, from archaeological data, it is believed that an infectious agent -currently unknown -is necessary for rheumatoid arthritis (firestein ) , and both guillain-barre syndrome and rheumatic fever have well-described pathogeneses triggered by specifi c infections in susceptible individuals (bach ) . in many instances, oncogenesis and tumor progression can be traced to pro-infl ammatory responses at the site of chronic infection (coussens and werb ) , although it is not known whether these effects are mediated through the actions of the immune system, the infectious agents, or a combination thereof. several cancers fall into this category including gastric adenocarcinoma (uemura et al. ) , cervical cancer (walboomers et al. ) , hepatocellular carcinoma (saito et al. ) , and kaposi's sarcoma (dictor ) , all having unequivocal infectious agent etiologies. recent fi ndings of antiinfl ammatory pharmaceuticals, particularly those that inhibit cox- and cox- , reduce the incidence of certain classes of cancers are consistent with this view (dannenberg and subbaramaiah , rothwell rothwell et al. ) . in addition, there is moderate evidence that several bacteria -the most studied is chlamydia pneumoniae -play a role in atherosclerosis and myocardial infarction (saikku et al. ; watson and alp ) , however the studies are not conclusive and antibiotic treatment does not appear to be effective (andraws et al. ) . chronic periodontal disease is fi rmly footed at the intersection of infection, chronic infl ammation, and metabolic dysfunction. chronic periodontitis is characterized by infl ammation of the periodontal membrane, slowly causing gingival recession and eventual bone loss. the proximate cause of periodontitis is the virulent oral microbiome. the involvement of gram negative anaerobes has been fi rmly established for the disease. aside from the known oral pathogenic species p. gingivalis , t. denticola , and t. forsythensis , the so-called "red complex" (holt and ebersole ) , new bacterial species associated with chronic periodontitis have also been described (kumar et al. ) . the advent of an extensive database covering the oral microbiome will surely propel such investigations . numerous studies have shown that periodontal disease covaries with many diseases, presumably due to overlapping molecular etiologies. compelling meta-analyses demonstrate a highly signifi cant synchronicity of obesity and periodontal disease (chaffee and weston ) . in addition, the correlation between periodontal diseases/ alveolar bone loss and frank metabolic syndrome is repetitively observed (nesbitt et al. ; andriankaja et al. ) . extensive work has also shown a strong role for both infl ammation-related genes and circulating infl ammatory markers in periodontal disease (nikolopoulos et al. ; bretz et al. a, b ) . treatment studies further support the link between periodontal disease and immuno-metabolic syndrome. these experiments have demonstrated a signifi cant improvement in intermediate molecular markers of infl ammation when chronic periodontitis in the presence of metabolic syndrome (acharya et al. ) or type diabetes (iwamoto et al. ) was treated. conversely, treatment of periodontal disease with reduction of bacterial load leads to greater glycemic control among diabetic patients (simpson et al. ; stewart et al. ) . given the high prevalence of periodontitis and the co-morbidity of metabolic syndrome with periodontal disease, these treatment experiments appear to suggest that the virulent oral microbiome could play an important role in the pathogenesis of systemic infl ammatory metabolic syndrome, and is exacerbated by the syndrome. certainly, further studies are needed to defi nitively answer this question. as chronic periodontal disease seems to be a critical feature of sustained, systemic dysfunction of both metabolic and infl ammatory networks, uncovering the genetic variants carried by susceptible individuals would not only provide much needed insight into the molecular pathogenesis of chronic periodontal disease, but would also markedly aid our understanding of the infl ammatory metabolic syndrome and how it drives related co-morbidities. such genetic studies may also shed light on the specifi c mechanisms that appear to improve cardiovascular, infl ammatory, and diabetic outcomes when periodontal disease is treated, potentially leading to therapies and medical/dental intervention with greater effectiveness. such studies may also provide clues to which subsets of individuals respond more effectively than others and why they do so. periodontal disease can also present in a rapid manner with aggressive bone loss and early-onset. this is termed aggressive periodontitis (lang et al. ) . in contrast to chronic periodontitis, there is often a greater degree of familial aggregation with aggressive periodontitis, and it is hypothesized that most aggressive cases may affl ict individuals with one or more defective immune genes (zhang et al. ; amer et al. ; machulla et al. ; carvalho et al. ; toomes et al. ; hart et al. ; hewitt et al. ) . mutations in the lysosomal protease, cathepsin c, have been shown to be responsible for some forms of aggressive periodontitis, along with complications associated with other infl ammatory diseases (laine and busch-petersen ) . the specifi c hla variants thought to play a role in aggressive periodontitis, are also involved in infectious disease susceptibility and autoimmunity; and, interestingly, two of the non-mhc-linked regions, fam c and a locus on chromosome p , have been implicated in myocardial infarction (connelly et al. ) and may have action as a tumor suppressor in tongue squamous cell carcinoma (kuroiwa et al. ) . as with chronic periodontal disease, an infectious microbiome is heavily involved. however, in general, microbiome differences could not explain the presence of chronic versus aggressive forms of the disease, although in some aggressive periodontitis patients, a highly leukotoxic a. actinomycetemcomitans strain may contribute to the disease process (mombelli et al. ) . we currently do not fully know the differences between the genetic susceptibility factors for the chronic and aggressive forms of the disease. the most prevalent chronic disease in both children and adults is dental caries (national institute of dental and craniofacial research) . caries formation is a complex disease with several interacting components form the environment and host genetics. similar to gingivitis and periodontitis, caries have an infection-initiating etiology with acidifi cation leading to localized demineralization. epidemiological studies have long shown that diet is a strong predictor of caries formation; and the reduction in ph is exacerbated by high consumption of carbohydrates. the principal pathobacterial species are streptococcus mutans and lactobacillus ( van houte ) . there are also several reports of positive correlations of caries with infl ammatory diseases, although the association is not always repeatable. it is also not clear what proportion of the putative association with infl ammatory disease is due to innate upregulation of immune networks in contrast to the immuno-modulating pharmaceuticals prescribed to those with infl ammatory disease (steinbacher and glick ) . much of the effect is reported to result from lack of saliva volume (steinbacher and glick ) . interestingly, the presence of epilepsy may be associated with higher caries rates (anjomshoaa et al. ) . fluoride is an effective antimicrobial agent that interferes with bacterial growth and metabolism (wiegand et al. ) . hence, topical fl uoride administration as well as ingestion of fl uoridated water inhibits cariogenesis and caries progression (ripa ) . amelogenesis is a key process involved in modifying the rate of caries formation. both common variation and rare mutations in enamel formation genes such as amelogenin and enamelin are involved in caries rates (patir et al. ; kim et al. ; crawford et al. ) , the molecular actions of which are beginning to be revealed (lakshminarayanan et al. ) . over , new cases of cancers affecting the oral cavity and pharynx were expected in the united states for , with deaths numbering , (jemal et al. ) . the majority of these malignancies involved solid tumors originating from cancerous changes in squamous cells of the mouth. again, oral cancers have a complex etiology existing of entangled genetic, epigenetic, infectious, and dietary causes, further modifi ed by tobacco, alcohol and other environmental exposures. as with most cancers, it is reasonable to expect that both germline and somatic genetic changes will be involved in carcinogenesis, tumor growth, and metastasis. promoter hypermethylation of genes central to cellular growth, differentiation, dna fi delity, apoptosis, and metabolic stability is an important facet of these cancers (poage et al. ) . indeed, methylation-mediated silencing of genes involved in tumor suppression (e.g. the cyclin-dependent kinase inhibitor a), detoxifi cation (e.g. mgmt ), and apoptosis (e.g. the death-associated protein kinase- ) are commonly found in oral squamous cell carcinoma samples (ha and califano ) . to quantify the proportion of the variance in a phenotypic trait that is due to variance in genetic factors, population geneticists defi ned the concept of heritability (visscher et al. ; falconer and mackay ) . researchers subsequently developed several methods for estimating heritabilities using the measure of a trait (e.g. occurrence of disease/not-disease) in combinations of relatives (e.g. parentoffspring, or monozygotic-dizygotic twins). in general, the higher the measured heritability of a variable phenotype, the larger the contribution of genetic factors is in comparison to environmental effects. it is fallacious to assume that the heritable variation is composed entirely of alleles residing in the dna sequence, for heritability studies simply examine the covariance between relatives without comment on specifi c molecular mechanisms. hence, any heritable variation such as methylation patterns, vertically-transmitted infectious agents, as well as dna variation can contribute to the heritability measure. heritability results are important because they not only give a rough estimate of the collective effects of heritable factors, but also can provide a measure to quantify how much of the total genetic effect is accounted for by specifi c loci examined. for periodontal disease, four twin-based studies of heritability have been performed (michalowicz et al. ; corey et al. ; michalowicz et al. ; mucci et al. ) . although varying in sample size and methodological details, all four arrived at consistent results, with - % of the variance in periodontal disease being attributed to genetic variability for chronic periodontitis. given the segregation patterns described in the literature, it is reasonable to assume that aggressive periodontal disease exhibits a higher heritability. therefore, given the prevalence of periodontal disease, heritable factors within the population at large are likely appreciable. using twin pairs, bretz and colleagues reported substantial heritability values for multiple traits related to caries ranging from % to % (bretz et al. a, b ) . lastly, mutagen sensitivity studies of head and neck cancer patients suggest a signifi cant effect of genetic factors for the carcinogenesis of oral cancers (cloos et al. ) . hence, there is every reason to believe that a sizable pool of genetic and/or epigenetic factors await discovery for oral diseases. once the development of pcr (saiki et al. ) was applied to the idea of using naturally-occurring dna variation (botstein et al. ) , large-scale dna-based studies of disease underwent a substantial acceleration (schlotterer ) . genotyping of short, tandem repeated sequences (weber and may ) -microsatellites -spurred on a wave of genome-wide linkage studies, which evaluate the co-segregation of disease state with microsatellite markers, for both rare mendelian disorders as well as more common diseases with complex inheritance patterns. while the rarer traits with more coherent transmission patterns generally relinquished their genetic secrets to linkage analysis, more common diseases did not. in the mid-to late s, several theoretical studies had shown that the power to detect disease-causing alleles is higher with association-based designs such as a case/control experiment or association in the presence of a linkage signal as in the transmission/disequilibrium test if the frequency of those alleles is high and the effects are moderate (kaplan et al. ; risch and merikangas ; jones ; long and langley ) . however, to conduct genomewide association studies presented an ominous obstacle for the genetic technologies at the time. the number of markers required to effectively cover the genome was prohibitively large as the chromosomal blocks in population-based samples used in association designs were expected to be small. even within large extended families, the limited number of recombination events generates substantial chromosomal blocks passed through the pedigree, but researchers had both theoretical and empirical evidence that the blocks in population-based samples were on the order of k base pairs for most large human populations. as the reader can imagine, the mean length of blocks that are shared by descent is inversely related to the product of recombination rate, the number of affected individuals and the number of meioses separating the affected individuals. in practice, even very large extended families segregate regions shared by affected members on the order of several million base pairs in length. however, once geneticists seriously considered large-scale studies using a case/control design where individuals are separated by say , meioses, it became clear that to adequately cover the much smaller shared regions across the entire genome, hundreds of thousands of markers would be required (kruglyak ) . utilizing the human genome sequence (venter et al. ; lander et al. ) , a number of studies at celera diagnostics provided an intermediate solution, where approximately , putative functional snps primarily located in genes were assayed through allelespecifi c pcr in a number of common diseases using a staged case/control design. these studies were successful in identifying several gene-centric polymorphisms associated with common diseases (begovich et al. ; cargill et al. ) (fig. . ) . concurrently, several groups had performed extensive sequencing and genotyping across the genome to produce a genome-wide map of haplotype structure (hinds et al. ) , useful in linkage disequilibrium mapping. within years, technology for snp hybridization arrays had advanced so as to enable genome-wide association studies capable of capturing most of the common genetic variation in the genome either through direct genotyping or indirect interrogation using linkage disequilibrium -the term linkage disequilibrium is a measure of the correlation of alleles at closely-linked sites (see fig. . ). these investigations were met with numerous successes (klein et al. ; kathiresan et al. ; graham et al. ; gudmundsson et al. ) . inexpensive genotyping platforms and urging from theoreticians ensured that these genome-wide association studies were, in general, highly powered to detect all but very mild effects from high frequency alleles. these efforts, led by large academic consortia such as the wellcome trust, the international multiple sclerosis genetics consortium, and the broad institute and commercial entities such as decode genetics and perlegen have greatly expanded our understanding of the basic biology of common diseases: we now know, for example, that (i) autophagy-related genes are involved in crohn's disease (rioux et al. ) , (ii) there are a number of genes such as the protein tyrosine phosphatase, ptpn and the interleukin- receptor, il r , that exhibit ample pleiotropic effects among autoimmune conditions (lopez-escamez ; safrany and melegh ) , (iii) in the case of age-related macular degeneration, predictive models using the genetic results enable fairly accurate prognosis of individuals who are at high risk of disease (seddon et al. transcription factor tcf l plays a role in type diabetes (grant et al. ) , and (v) aberrant il- signaling likely contributes to multiple sclerosis susceptibility (gregory et al. ) . the plot shows the tremendous progress in genotyping technology where, a decade ago, very little of the genome was accessible for disease studies using association designs through the current wave of viable sequencing-based whole exome studies ( ) ( ) and whole genome studies ( ) ( ) . in fig. . , the average distance between adjacent genetic markers is plotted as a function of year of introduction to the disease mapping community. impressively, the total number of genetic markers has increased a million-fold over the past decade. although successful in uncovering numerous pathogenic pathways for common diseases, results from the current wave of genome-wide association studies, with a few exceptions, explain little of existing disease heritability. the reasons for this are cryptic and the subject of heavy debate (manolio et al. ) . multiple rare sequence variants generating high levels of allelic heterogeneity, functional de novo mutations, structural mutations such as copy number variants and large deletions, and epigenetic effects constitute four of several possible disease models that could account for the heritability discrepancy. the answer will almost certainly consist of a conglomeration of these and other effects. bringing forth the new genome-wide technologies that illuminate these previously non-or under-interrogated properties of the genome to bear on this enigma is a reasonable next step for all complex traits including oral diseases. the most commonly used measure of ld in a sample of chromosomes is linkage disequilibrium (ld) is a measure of the correlation between alleles at two sites in a sample of chromosomes. for two biallelic sites, if the a allele is always paired with the b allele, and the a allele is always on the same haplotype as the b allele, then the two sites are said to be in perfect ld. successive recombination diminishes ld. interrogating one site for disease association allows investigators to indirectly interrogate other sites in sufficiently high ld with the interrogated site. a key feature explicitly studied in molecular population genetics and implicitly used in disease gene mapping studies is the site frequency spectrum; that is, the distribution of allele frequencies at single sites in the genome that vary in the human population studied. from both diffusion models (kimura ) and coalescent theory (hudson ) in theoretical population genetics, we know that the vast majority of realistic models generate many more rare variants compared to common polymorphisms. this is particularly true for expanding populations. are these rare variants the source of much of the missing heritability? recently, with the application of high-throughput sequencing technology to human studies over the past decade, empirical studies have clearly verifi ed these predictions -the large majority of variants have low frequencies (the international hapmap consortium ) . the distribution of deletions appears to be skewed toward more rare frequencies, presumably due to the deleterious effects of such variants. individual mutations appearing de novo typically are extremely rare events per locus, but collectively are numerous. other types of genetic variability, such as copy number repeats, span both ends of the frequency spectrum with the preponderance of the markers being rare. thus, there is a sizable pool of low-frequency variants in human populations that have yet to be thoroughly investigated. over the past few years it has become increasingly clear that structural variants exist in the human genome at a far higher rate than previously thought. structural variants can exist in a multitude of forms including deletions, copy number variants, and inversions among others. due to the nature of these genetic changes, many are considered to be highly disruptive of molecular function if they lie in functional motifs. indeed, there are several mendelian diseases are caused by fully-penetrant structural variants impacting a chromosomal region (lupski ) . numerous structural variants have recently been reported to be associated with common diseases, particularly in the neurological fi eld (sebat et al. ; stefansson et al. ; elia et al. ) , infectious disease susceptibility (gonzalez et al. ) , and drug metabolism (zackrisson et al. ) . although they have improved dramatically over the past few years, algorithms using snp-based data from hybridization arrays to infer copy number variants have had high error rates, perhaps explaining the rather low rates of replication of structural variation association results for common diseases. nevertheless, given the high frequency of structural variants, their pathogenic potential, and that we are on the precipice of a sequencing revolution in genome-wide studies, examination of these variants should be a high priority for new sequencing-based studies in oral disease susceptibility, progression, and related pharmacogenetic applications. as different technologies examine different portions of the site frequency spectrum (i.e. genome-wide snp scans interrogate variation that is common in the hapmap populations, whereas sequencing-based studies typically interrogate the entire frequency spectrum), where one believes genetic causation is harbored should infl uence the selection of genotyping technology. if common genetic variation contains the vast majority of heritable effects on disease phenotypes, then an investigator would be wise to employ a snp-based experimental design. if, however, there is reason to believe that a signifi cant portion of the genetic load of the disease studied exists in the highly populated portion of the distribution -the rare variants -then a sequencing-based study may be better suited to unravel causative alleles. the studies of heritability discussed previously show that there is heritable variation underlying a substantial portion of the variance observed in oral diseases. as discussed above, sequencing technologies may address many aspects of dna variation including copy number loci, rare haplotypes, inversions, and insertions/deletions, but it is also worthwhile to repeat that the molecular mechanisms for disease heritability are not necessarily limited to variation at the dna level. for a disease state, the covariance between relatives could be driven by co-inherited chromosomal regions or other phenomena. chief alternative heritable mechanisms include dna methylation (hammoud et al. ) , modifi cations to the histones (bestor ) , complex rna zygotic transfer (rassoulzadegan et al. ) , and vertical transmission of infectious agents. additionally, transgenerational effects offer an intriguing class of epigenetic mechanisms (nadeau ) . in a thorough review on epigenetics and periodontitis, gomez et al. make a strong argument for consideration of both cpg dinucleotide methylation and deacetylation actions on cytokine expression as a credible avenue for further investigation in periodontal disease etiology (gomez et al. ) . genome-wide epigenetic studies have been successfully conducted for oral cancers (poage et al. ) . the scale of this study on head and neck squamous cell carcinomas allowed these researchers to show a global pattern of tumor copy number changes signifi cantly correlated with methylation profi les that was not detectable at the individual gene promoter level. with advanced chromatin immunoprecipitation and new methods to study dna methylation, efforts to apply highthroughput epigenetic methods to oral diseases should be accelerated. numerous studies have been conducted in oral disease traits using a candidate gene approach. there are two large reviews of the existing candidate gene results (nikolopoulos et al. ; ) . laine and colleagues have recently put together a comprehensive review article covering gene polymorphisms. there are some suggestive fi ndings for cyclooxygenase- gene, cox- , the cytokineencoding genes, il and il b , the vitamin d receptor, vdr , a polymorphism immediately upstream of cd , and the matrix metalloproteinase- gene, mmp . however, these initial results will require further confi rmation, for the association patterns are inconsistent across independent studies, the statistical signifi cance is moderate, and the posterior probability of disease is decidedly bland. the striking pattern that emerges from the laine et al. summary data is the lack of coherent replication of genetic association for the vast majority of polymorphisms examined. the situation is reminiscent of genetic association studies prior to large-scale snp studies where poor repeatability of results plagued the fi eld. in a pivotal study from , hirschhorn and colleagues (hirschhorn et al. ) examined the state of genetic association studies, fi nding that "of the putative associations that had been studied three or more times, only six have been consistently replicated." the dearth of robust results was largely remedied when large-scale genetic studies were applied to very substantial numbers of well-characterized patients and geneticallymatched controls and stringent statistical criteria enforced. one can only suspect that a similar state of affairs is operating in genetic studies of chronic periodontitis. perhaps efforts to ( ) reduce the heterogeneity of the disease state through detailed clinical and laboratory assessments, ( ) drastically increase sample sizes, and ( ) expand the scope of inquiry to larger numbers of genes/regions, and examine a more comprehensive set of variants/epigenetic effects will improve the current situation. the second large study is a meta-analysis of studies, where nikolopoulos and colleagues analyzed six cytokine polymorphisms linked to il a , il b , il , and tnf-alpha (nikolopoulos et al. ) . two of these, an upstream snp in il a and a snp in il b , exhibited signifi cant association with chronic periodontal disease risk. although the results were not particularly strong, as is typical with complex diseases, the results do suggest the importance of infl ammation-response variability in chronic periodontitis predisposition. perhaps the strongest, most replicable genetic association fi nding with coronary heart disease and myocardial infarction is centered on the short arm of chromosome ( p . ) (mcpherson et al. ; helgadottir et al. ) . two studies of periodontal disease showed that the same alleles at the p . locus confer risk for aggressive periodontitis (schaefer et al. ; ernst et al. ) . the discovery of such a pleiotropic locus may explain a portion of the aggregation of periodontal disease with other co-morbid conditions. further studies investigating overlapping genetic susceptibility factors between periodontitis and cardiovascular disease, diabetes mellitus, metabolic syndrome, rheumatoid arthritis, and other related diseases may be a fruitful strategy for honing in on shared genes affecting these immuno-metabolic disorders. using patients from families from the philippines, the fi rst genome-wide linkage study for caries was completed in (vieira et al. ) . the study identifi ed fi ve loci which exhibit suggestive statistical evidence (lod scores exceeding . ): q . , q . , xq . , q . , and q . . the latter of which overlapped with a quantitative trait locus discovered from mapping work in the mouse. further work is necessary to refi ne these signals and localize the variants that may be driving these linkage signals. aggressive periodontal disease and rarer dental diseases have also been subjected to linkage analysis. results from linkage studies for dentinogenesis imperfecta type i, for example, have gone on to produce the novel gene fi ndings of the dentin sialophosphoprotein-encoding gene on q . being responsible (song et al. ; crosby et al. ) . a linkage study in african american families examining localized aggressive periodontitis found a strong linkage signal in a region covering approximately megabases on chromosome (li et al. ) . several interesting genes are in this region. in a study earlier this year further mapping from carvalho et al. in brazilian families identifi ed haplotypes in this region on q in fam c which were associated with aggressive periodontitis (carvalho et al. ) . the function of the fam c protein is not fully understood. fam c is localized in the mitochondria and it appears to play a role in vascular plaque dynamics and risk of myocardial infarction (laass et al. ) . it should also be noted here that other types of mapping analyses such as homozygosity mapping to identify have yielded gene discoveries. for example, the lysosomal protease cathepsin c gene for the recessively-inherited papillon-lefevre syndrome which is characterized by aggressive and progressive periodontitis was effectively mapped using homozygosity mapping (fischer et al. ; connelly et al. ) . cathepsin c is highly expressed in leukocytes and macrophages and is a key coordinating molecule in natural killer cells (rao et al. ; meade et al. ) . although sparse, these linkage results are undoubtedly encouraging. employing very large extended families subjected to genome-wide genotyping or sequencing will surely shed much needed light on chromosomal regions and genes relevant to oral disease research (fig. . ). for periodontitis, a single study has employed a genome-wide association design in an effort to uncover aggressive periodontal variants (schaefer et al. ) . this study by schaefer and colleagues discovered and replicated an intronic snp, rs , in the glycosyltransferase glt d which is signifi cantly correlated with aggressive periodontal disease in both german and dutch samples. often, seemingly signifi cant results from large studies are due to the effect of reporting the top result from a great many statistical tests -this is called the multiple testing problem. in this situation, the strength of the fi nding, along with the replication across three case/control studies, argues for true association with aggressive periodontal susceptibility. the snp may modulate the binding affi nity of gata- . the association with glt d is currently one of strongest genetic associations for aggressive periodontal disease, testifying to the power of genome-wide studies to generate novel, relevant molecular pathophysiology for complex diseases. it seems unlikely that glt d would be extremely high on a candidate gene list, and it was only through a genome-wide scan that it appeared. like many excellent studies, the fi nding by schaefer et al. raises more questions than it answers and will undoubtedly provide fertile ground for ensuing molecular work. after a somewhat sluggish start, due to a lack of critical mass of investigators aiming to collect large numbers of patient samples and bring high throughput genetic technologies to caries susceptibility, gingivitis, and periodontal disease traits, the future of genetic studies in oral health is bright. scientifi c progress in revealing the molecular pathogenesis of oral diseases is dependent on genome-wide genetic studies; and i have argued that progress in related immuno-metabolic diseases is also dependent on these large-scale genetic studies in periodontal disease. to study sporadic disease, substantial patient collection efforts are required for the application of these technologies. this may involve a combination of new recruitment and consortiumrelationships with existing collections. the beginning of such a collection for sporadic aggressive periodontitis in europe has shown extremely intriguing initial results, but more patients are needed to examine rare variants of moderate effect. both the german/dutch collection of aggressive periodontitis and the brazilian collection have begun to revolutionize the study of periodontal disease susceptibility with the discovery of glt d snps and fam c -linked haplotypes. there is little doubt that subsequent molecular work on these two genes will uncover novel mechanisms for the predisposition to aggressive periodontal disease. focus should also be placed on the collection of extended families segregating these diseases. applying sequencing technologies to large pedigrees can be an effective method of identifying rare variants and structural variants in a highly-refi ned phenotype. furthermore, applying these methods to the entire genome would make for a comprehensive genetic study. several trends in large-scale genomics science hold promise to signifi cantly advance our understanding of oral disease pathogenesis: the sociology of biological sciences has changed over the past years so as to • become more collaborative. essential for association-based designs, consortiumbased genetic research has blossomed over that time period, increasing sample sizes and therefore the power to detect disease-causing variants. there currently is consortium-based research in periodontal disease and oral cancer. further expanding these efforts will enhance subsequent studies, particularly those investigating rare alleles and/or rare epigenetic effects. through over a century of laboratory work, the collective knowledge of bio-• chemical pathways, signal transduction, cell physiology, regulatory mechanisms, and structural biochemistry is weighty. incorporation of this information into etiological models may substantially advance oral disease work as well as the fi eld of complex disease genetics in general. sophisticated analysis techniques are needed to perform this task. recent advances merging results from network science with probability theory within the context of computer science have produced the fi eld of machine learning. this rigorous framework can be used to identify those factors responsible for disease status and can also be used to develop robust predictive models using known biological networks and genetic data. the output from such models, typically the probability of disease, an estimate of disease progression rate, or a probability of adverse reaction, can be used by physicians and dentists to personalize medical care. until relatively recently, population genetics did not contribute a great deal to • human genetics research. that has changed in the past decade where effort spent on association studies surpassed that spent on family-based studies. those investigating disease gene mapping began to collaborate with population geneticists and population geneticists took up a wide-spread interest in fi nding disease alleles. incorporation of population genetics theory into such studies markedly improved association studies on several levels: confounding by population stratifi cation was effectively treated using population genetics, linkage disequilibrium patterns. use of population genetics theory in large-scale oral disease mapping studies may accelerate discoveries. sequencing technology has rapidly progressed over the past decade. currently, • sequencing studies across the exome can be accomplished at reasonable cost and yield data for all known genes in the genome. within the next few years, sequencing costs will depreciate to a point where whole-genome sequencing studies will be commonplace, using both family-based and population designs. application of these technologies to oral disease studies is imperative for comprehensive studies of etiology. high-throughput dna methylation and chromatin immunoprecipitation studies • will enable large-scale epigenetic studies in oral diseases (meade et al. ; ehrich et al. ; bibikova et al. ; ren et al. ; pokholok et al. ) . these have already started to play an important role in delineating mechanisms responsible for oral cancers (poage et al. ) . additional application of these techniques to studies of gingivitis, caries, and periodontal diseases may generate novel fi ndings. molecular biologists and pharmacologists have increasingly become able to • develop and evaluate highly targeted pharmaceuticals based on genetic discoveries. the use of such genetic information may improve the chances of developing effi cacious therapies. geneticists and disease researchers are beginning to realize that oral diseases • both impact and are intrinsically tied to susceptibility and progression of other common diseases. a synthesis of genetic fi ndings from immuno-metaboliclinked disorders would seem to greatly increase the knowledge of these diseases and better pinpoint their respective etiologies. as the new high-throughput genomics and epigenomics technologies become • implemented in oral disease research, the storage, management, analysis, and interpretation of the ensuing colossal amounts of data will be critical to enable clinicians to use these results in daily practice. advances in dental and medical informatics will facilitate these steps. we are in exciting times where advances in genetic technologies will uncover the genetic causes of diseases, including those that affect the oral cavity. with more focus in the area of oral disease genomics and the harnessing of new high-throughput sequencing and epigenetic technologies, novel insights into the pathways driving these diseases are imminent. these discoveries will, in turn, motivate directed therapies, aid in illuminating the molecular etiology of related disorders such as diabetes, and increase the level of personalized medicine. joseph kilsdonk the title of this section reinforces a institute of medicine (iom) report titled "dental education: at the crossroads." to quote yogi berra, a baseball sage: "when you come to a fork in the road, take it." the implication being that dental education must take action and move beyond its crossroads. these crossroads are described in the fi rst third of the section. it includes a summary and recommendations of the iom report and three transitional reports that followed: the surgeon general's report identifying oral health as a silent epidemic, the josiah macy foundation report, and a "pipeline" study funded by both the robert wood johnson and the california foundations. having been at the crossroads for a decade or so, the middle portion of the section highlights educational models that may lead to a more promising future. the later third of this section describes an alternative path of action for dental education which emphasizes the central roles of clinic-based education and dental informatics in dental education curriculum. it is unknown how traditional dental educators may view this model; however, it is effectively a logical conclusion and responsive to the reports. in the institute of medicine (iom) published "dental education at the crossroads" (field ) . the title was apropos as the authors' analysis concluded: ( ) economics surrounding dental education were unsustainable ; ( ) student service learning opportunities and access to care for patients were limited; and ( ) new dental schools were not replacing those forced to close due to the economic climate. the iom report additionally proposed key recommendations to reform dental education and service delivery. fifteen years later, we remain at "the crossroads" as these issues remain largely unresolved. furthermore, these recommendations have retained their validity. their implementation would directly impact structures and services for contemporary models of dental education in the future. the following iom recommendations (field ) are intrinsic to the proposed dental education reform: recommendation : to increase access to care and improve the oral health status of underserved populations… recommendation : to improve the availability of dental care in underserved areas and to limit the negative effects of high student debt… recommendation : to prepare future practitioners for more medically based modes of oral health care and more medically complicated patients, dental educators should work with their colleagues in medical schools and academic health centers to: move toward integrated basic science education for dental and medical • students; require and provide for dental students at least one rotation, clerkship or • equivalent experience in relevant areas of medicine and offer opportunities for additional elective experience in hospitals, nursing homes, ambulatory care clinics and other settings; continue and expand experiments with combined md-dds programs and • similar programs for interested students and residents; increase the experience of dental faculty in clinical medicine so that they, • and not just physicians, can impart medical knowledge to dental students and serve as role models for them. recommendation : to prepare students and faculty for an environment that will demand increasing effi ciency, accountability, and evidence of effectiveness, the committee recommends that dental students and faculty participate in effi ciently managed clinics and faculty practices in which the following occurs: patient-centered, comprehensive care is the norm; • patients' preferences and their social, economic, and emotional circumstances • are sensitively considered; teamwork and cost-effective use of well-trained allied dental personnel are • stressed; evaluations of practice patterns and of the outcomes of care guide actions to • improve both the quality and the effi ciency of such care; general dentists serve as role models in the appropriate treatment and referral • of patients needing advanced therapies; larger numbers of patients, including those with more diverse characteristics • and clinical problems, are served. recommendation : because no single fi nancing strategy exists, the committee recommends that dental schools individually and, when appropriate collectively evaluate and implement a mix of actions to reduce costs and increase revenues. potential strategies, each of which needs to be guided by solid fi nancial information and projections as well as educational and other considerations, include the following: increasing the productivity, quality, effi ciency, and profi tability of faculty • practice plans, student clinics, and other patient care activities; pursuing fi nancial support at the federal, state, and local levels for patient-• centered predoctoral and postdoctoral dental education, including adequate reimbursement of services for medicaid and indigent populations and contractual or other arrangements for states without dental schools to support the education of some of their students in states with dental schools; rethinking basic models of dental education and experimenting with less • costly alternatives; raising tuition for in or out-of-state students if current tuition and fees are low • compared to similar schools; developing high-quality, competitive research and continuing education • programs; consolidating or merging courses, departments, programs, and even entire • schools. in summary, the iom report identifi ed that: ( ) an outdated curriculum continues to be retained which refl ects past dental practice rather than current and emerging practice and knowledge; ( ) clinical education does not suffi ciently incorporate the goal of comprehensive care, with instruction focusing too heavily on procedures; ( ) medical care and dentistry are not integrated; and ( ) the curriculum is crowded with redundant material, often taught in disciplinary silos. the iom's report was followed by the surgeon general's report on oral health in and a subsequent supplement by the surgeon general in called "the national call to action" (u.s. department of health and human services ) . five signifi cant fi ndings and recommendations from the surgeon general's report(s) that have implications pertaining to the envisioned structure and services of new models for dental education include: changing the perception of oral health so that it will no longer be considered • separate from general health; improving oral health care delivery by reducing disparities associated with popu-• lations whose access to dental treatment is compromised by poverty, limited education or language skills, geographic isolation, age, gender, disability, or an existing medical condition; encouraging oral health research, expanding preventive and early detection pro-• grams, and facilitating the transfer of knowledge about them to the general population; increasing oral health workforce diversity, capacity, and fl exibility to overcome • the underrepresentation of specifi c racial and ethnic groups in the dental profession. in this regard, the national call to action urged the development of dental school recruitment programs to correct these disparities and to encourage parttime dental service in community clinics in areas of oral health shortage; increasing collaboration between the private sector and the public sector to cre-• ate the kind of cross-disciplinary, culturally sensitive, community-based, and community-wide efforts to expand initiatives for oral health promotion and dental disease prevention. spurred by the iom report and the surgeon general's report, the josiah macy foundation ( ) conducted a study entitled "new models of dental education." the study was prompted by concerns about declines in dental school budgets and the diffi culties experienced by schools in meeting their educational, research, and service missions. the macy study concluded that: financial problems of dental schools are real and certain to increase. • current responses of schools to these economic challenges are not adequate. • most promising solutions require new models of clinical dental education. • macy study lead researcher dr howard bailit, and his team recently concluded in reference to points one and two above, that: "if current trends (to aforementioned) continue for the next years, there is little doubt that the term crisis will describe the situation faced by dental schools. further, assuming that it will take at least ten or even more years to address and resolve these fi nancial problems, now is the time for dental educators, practitioners, and other interested parties from the private and public sectors to come to a consensus on how to deal with the coming crisis. clearly, these fi nancial problems will not be solved by minor adjustments to the curriculum, modest improvements in the clinical productivity of students or faculty, or even signifi cant increases in contributions from alumni. the solutions 'must involve basic structural changes in the way dental education is fi nanced and organized' (bailit et al. ) ." this statement is supported by the fact that in the past years more dental schools have closed than opened. specifi cally eight schools have closed, whereas to date a couple has opened and a handful is pending. curriculum relevance was also a focus of the study. findings concluded that "changing the curriculums in dental schools to allow students to spend more time in community venues would be highly benefi cial to both society and student. society benefi ted from having underserved patients cared for while students were assessed as being fi ve to ten times more productive, more profi cient, more confi dent, more technically skilled and more competent in treating and interacting with minority patients" (brodeur ) . macy study (formicola et al. ) outcomes represented signifi cant and foundational guideposts for assessing and planning any future models for dental education. their report led to the robert wood johnson foundation pipeline study ( ) , a major research study funded by the robert wood johnson foundation and the california endowment (tce). the goal of the dental pipeline program was to reduce disparities in access to dental care. the pipeline study provided over $ million for the start up or expansion of schools and student clinical programs that incorporated services to underserved extramural clinical settings (primarily community health centers). the following recommendations from the surgeon general's report structured • the goals of the pipeline's initiative: increase the number of under-represented minority and low-income students enrolled in the dental schools participating in the pipeline program so that there would be a voice of minority and low-income students at all the funded schools. provide dental students with courses and clinical experience that would prepare • them for treating disadvantaged patients in community sites. have senior dental students spend an average of days in community clinics • and practices treating underserved patients. increasing the community experience of dental students was expected to have an immediate impact on increasing care to underserved patients (brodeur ) . this third point is pivotal to future success of dental curricula and dental education economics. recently published in a supplemental volume to the journal of dental education , february, , the pipeline study reported the following outcomes: minority recruitment of low-income students increased by %; • the rate of recruitment for under-represented populations was almost twice that • of non-pipeline schools; the length of time dental students spent in extramural rotations increased from a • mean of days to a mean of days over a period of years. procedural profi ciency increased compared to that of their non-extramural peers. of the pipeline-funded programs, only four schools achieved the goal of • days of extramural rotations; through extra funding from tce, the four schools extended extramural rotations to an average of days; based on this publication, it appears that only a handful of pipeline schools defi -• nitely plan to sustain their extended extramural rotations. financial concerns were highlighted as the major problem in sustaining future recruitment and placement of students beyond the timeframe of the study; a survey of program seniors indicated a mean of % [range of - % by • school] were planning to devote ³ % of their practice to serving minority patients. only % [range of - % by school] were planning to practice at community clinics. in the context of these outcomes, discussion indicated that the unwillingness of students to practice in underserved settings was based on several factors: students that participated were already enrolled in traditional programs and were • not necessarily seeking a pipeline experience or a future in community service. concern over future reimbursement as a provider in a community setting; • limited time spent in underserved settings; • limited loan forgiveness scholarship opportunities. • the fact that the large majority of pipelines were unsustainable was attributed to lack of productivity in the school clinics while the students were on rotation at community based clinics. schools generate meager, yet necessary revenue streams on intramural student clinical activity to support the costly clinical and faculty infrastructure. currently, similar economic constraints involved with outsourcing students to serving rural and underserved populations impacts the ability of tradition dental schools to participate in sustained outreach programs. most recently, the pew center on the states national academy for health policy ( ) released "help wanted: a policy makers guide to new dental providers". this report provided an excellent summary outlining workforce needs, access issues, and strategies for dental-related services to help states and institutions develop creative ways to solve oral health access and care issues. the guide proposes the following relevant components and trends for consideration in development of future sustainable school models: dental colleges are willing to bear a large and disproportionate share of the burden • in terms of access to care, particularly during a time of incredibly scarce resources. expanded, extensive, and/or creative extramural rotations have been developed • in recent years under the conceptual umbrella of service-learning. these often involve clinics providing direct or indirect payment to dental schools or clinics managed in some way by dental schools. dental education has certain obligations. first, education must adhere to accreditation standards with the goal of producing competent practitioners. second, education must remain responsive and impact the societal need for care. lastly, the delivery of dental education must be economically sustainable. the macy, rwjf, and iom reports note that improved oral health, sustainable dental education economic models, and competent workforce pipelines converge around community health centers (chc). university of michigan researchers fitzgerald and piskorowski ( ) reaffi rm this conclusion in an evaluation of an ongoing -year program, stating that: (the chc model) is self-sustaining and can be used to increase service to the underserved and increase the value of students' clinical educational experiences without requiring grant or school funding, thus improving the value of dental education without increased cost. self-sustaining contracts with seven federally qualifi ed health centers (fqhcs) have resulted in win-win-win-win outcomes: win for the underserved communities, which experienced increased access to care; win for the fqhcs, which experienced increased and more consistent productivity; win for the students, who increased their clinical skills and broadened their experience base; and win for the school in the form of predictable and continuing full coverage of all program costs (fitzgerald and piskorowski ) (fig. . ). however, unlike medicine that outsources their students to clinical sites, dental education programs retain the majority of the student time within their own "clinical laboratories" as documented by the aforementioned studies, this limits students' exposure to extramural experiences. costs to operate such intramural clinical programs are ever increasing and many schools' clinical operations run defi cits. if that component can be outsourced to community-based resources such as a chc, then the burden of cost is shifted away from the school. an example would be a.t. still university's arizona school of dentistry and oral health (asdoh) which matriculated its fi rst class in . at the prompting of the state's community based clinics, asdoh designed a program that placed students into community-based settings for up to months, an unprecedented length of time for an extramural rotation. they also saw this as an opportunity to use an adjunct centric faculty that signifi cantly reduced traditional education overhead. through this innovation, the school was able to develop a program that was sustained by "fair market" value tuition and trained students where community needs were greatest for up to months (which was then unprecedented). conversely, if the chc can rely on student service-learning to care for patients, the cost of care is reduced. other schools are also advancing with innovative education and care delivery. adea's charting progress (valachovic ) fig. . the synergy between access to care, student competency, and fi nancially sustainable dental education converge around chc/fqhcs little rock, arkansas; and the university of southern nevada in south jordan, utah. western university is planning placement of % of their fourth year class in community health centers, while east carolina is seeking to set up rural clinical campuses as well as clinical partnerships with the state's fqhc. at the time of this publication, several existing schools are expanding or looking to expand including the university of north carolina, marquette university, midwestern university in downer's grove, il. such expansions will contribute to solving the existent access supply and demand issues. however, it was observed even with all the start ups and expansions, graduation numbers will not approach the output of schools in the late s and early s. these creative models establish the foundation for a sustainable clinic structure by generating self-sustaining revenue through student service-learning, which, unlike medical student services, are billable. simultaneously these new models provide access to care for the needy while student exposure to clinical experiences that are often not available in academic patient pools. these models also shift some of the cost of providing clinical education from the dental college to community-based clinics. however, this innovation is not without criticism. schools are dependent on the success of their clinics and clinic partnerships. one author cautions: "however, these creative models also may present potential political strategic risk or confl ict: private practitioners may organize and protest higher than normal reimbursement schemes. potentially, such protests could even jeopardize the very existence of such models (dunning et al. ) ." notably, community health centers have historically received strong bipartisan support. for example, during the bush administration, fqhc funding was doubled and most recently expanded through health reform legislation by the obama administration. according to the institute for oral health, "the group practice of the future is the dentist working with the physician" (ryan ) . the ada reported "multidisciplinary education must become the norm and represent the meaning and purposes of primary care as it applies to dentistry. educational sequences should include rotation strategies across discipline specialties in medicine and dentistry, clerkships and hospital rotations, and experience in faculty and residency clinics." (barnett and brown ) the models alluded to, were school-based attempts at improving educational outcomes. perhaps the proverbial fork in the road regarding the future of dental education leaves two paths for consideration. is it better to travel down a road that leads a school to develop and operate a clinic? or is the road less traveled, where a clinic becomes a school, the better of the two options? the answer, perhaps, is that a combination of both will accomplish the desired outcome. for example, didactic knowledge is measured by examination whereas competency as a practitioner is measured by clinical demonstration. at a minimum, the result must achieve learner competency, quality, and sustainability. however, the road less traveled has not been taken yet. william gies, in his revered report written years ago on the state of american dental education, wrote "dental faculties should show the need…. for integrated instruction in the general principles of clinical dentistry and in its correlations with clinical medicine" (gies ) . basic sciences aside, could a clinicalbased educational training center have an advantage over a school-based clinical center? soon-to-be-implemented new commission on dental accreditation (coda) standards will require schools to demonstrate competency in patient-centered care (valachovic ) . might an enterprise profi cient at running a successful clinical business model have an advantage running a professional, patient-centered clinical training program as compared to a pedagogical business model attempting to run a clinical training model? these questions should challenge us to reexamine why our thinking about educational models should be limited to schools being the starting point for the development of a profession that demands clinical competency, patientcenteredness, and integration as outcomes. the clinic based model may serve as an equivalent starting point and, have some distinct advantages for achieving responsiveness to recommendations and directions cited in this section. beginning in november through august , the family health center (fhc) of marshfi eld, inc, marshfi eld, wisconsin, launched of a broad network of developing dental clinics, targeting dental professional shortage areas with the provision of dental services to the underserved communities whose dental needs were not being adequately met by the existing infrastructure. fhc-marshfi eld is a federally qualifi ed health center (fqhc). as an fqhc, fhc receives cost-based reimbursement for its dental services to medicaid populations. along with the cost-based reimbursement, fqhcs are obligated to provide care to anyone regardless of their ability to pay. presently, fhc is the nation's largest federally qualifi ed dental health center. to date, this network of dental clinics has served over , unique patients, % of whom were under % of poverty. notably, service was provided to a signifi cant number of cognitively and developmentally disabled patients in special stations developed for serving patients with special needs. these patients frequently travel the furthest to get to our dental centers for care. beginning in , fhc stepped up the pace of dental clinic expansion, constructing two new dental centers in , two in , and two more are slated to open in . when fully operational, this will establish capacity to serve , patients annually. each site has proactively included dedicated clinical and classroom training space for dental residents or students, thus laying the framework for clinic-based training of new dental professionals. the plan is to continue to stand up new dental centers until they have the capacity to serve , patients annually or approximately % of the , underserved patients in the rural service area. in addition to the capacity for training residents and students, a dental post-baccalaureate program is being considered in partnership with regional year under graduate campuses. the post-baccalaureate program is aimed at preparing students from rural and underserved areas who desire to practice in rural and underserved areas for acceptance and success in dental schools. presently fhc in partnership with marshfi eld clinic is moving forward with plans to develop dental residencies at these sites and a dental post baccalaureate training program to better prepare pre-doctoral students from rural and/or underserved backgrounds to be successful in dental school as a means to create a dental academic infrastructure responsive to rural environments which have been classically underserved. marshfi eld clinic has a long-standing history in medical student education and multiple medical residency programs. creating access for the underserved population was the major motivational force driving the establishment of the dental clinic network back in . the fi ndings of the iom, macy, and rwjf reports became the foundational framework for developing the vision of a dental education model that would realize the major recommendations found in the reports. by establishing clinical campuses in regional underserved dental health professional shortage areas, access to care where care is needed most was provided. sustainment of a work force for provision of care across the dental clinic network is accomplished by schools contracting with fqhc's for service learning, thus circumventing challenges associated with releasing dental students at traditional dental schools to distant extramural training sites as discussed previously. this model is however not without its own set of challenges including calibration of faculty, supervision and evaluation of students in training, and achieving accreditation acceptance. however, through video connectivity and iehr technology curriculum, learning plans, competency assessment, progression, performance, faculty development, and learner evaluations can be centrally calibrated. additionally, this dental service-learning model based in a community health center setting offers students unique state-of-the-art exposures to alternative access models, cutting-edge informatics (including access to a combined dental-medical record) and a quality-based outcomes-driven practice. given the novelty of such an extended extramural dental clinical training model, there is limited data on the success of rural placement leading to retention to practice in a rural setting. the pipeline study piloted a model for getting students into underserved communities. however, that experiment was limited to -day rotations. outcome driven programs may provide a predictive surrogate for purposes of comparative analysis. for example, the rural medical education "rmed" program of the university of illinois medical school at rockford, has sustained a longstanding program in illinois. over years in duration with over student participants of whom % have been retained as primary care medicine practitioners in rural illinois. rabinowitz et al. ( a ) further reinforced that medical school rural programs have been highly successful in increasing the supply of rural physicians, with an average of - % of graduates choosing to practice in rural areas. they also noted rural retention rates of - % among the programs (rabinowitz et al. a) . recently, the university of wisconsin school of medicine and public health (uwsmph) launched the wisconsin academy for rural medicine (warm program). the warm program places medical students in rural academic medical centers during their third and fourth years in medical school. marshfi eld clinic is one of those sites. warm students affi liating with marshfi eld clinic's system would ultimately share learning experiences with dental students, clinical rotations, team-based rounding, lectures, and exposure to a combined medical-dental patient record. in an analogous manner, the marshfi eld clinic dental education model will incorporate a curriculum that embeds students in rural clinical practice for up to years. a secondary but not insignifi cant outcome of placing residents and students in clinical campuses focused on developing competency and providing care where needs are often greatest is the cost savings to taxpayers associated with the public care of patients. these savings are accomplished through the "service-learning" of the student. for example, in the model described where clinical training is embedded within the fhc clinics, the stipend resident or unpaid student learner provides the patient care as part of their service learning training while requiring oversight from one paid faculty per four to six learners. as a result, an academic based clinical partnership creates a model that reduces the cost for care provided to underserved patients. an additional benefi t to the community based clinic might be realized through tuition assistance by the academic program to help support patient procedures that develop learner competencies. in educational quality and infl uence, dental schools should equal medical schools, for their responsibilities are similar and their tasks analogous (william gies ) . the commission on dental accreditation (coda) notes that one of the learning objectives of an advanced education general dentistry (aegd) residency is to have the graduate function as a "primary care provider". to function competently in this role, the graduate needs to have a strong academic linkage to primary care medicine. at a dental deans forum, years after the gies report, dr polverini made the statement "dentistry has never been linked to the medical network but unless dentistry becomes part of the solution to the challenge of providing comprehensive patient care, it will be looked on as part of the problem, and ultimately, all dental schools will be called into question." (polverini ) the use of dental informatics and an integrated record are elements essential to this competency. on april , , fhc and marshfi eld clinic successfully transitioned all of their dental centers to a new practice management and electronic health record system that fully integrates medical and dental; one of the fi rst such systems in the nation. along with the benefi ts derived in fig. . , chc placement also exposes students to an integrated medical-dental care setting where learners can develop skills in system-based practice to include the interdependence of health professionals, systems, and the coordination of care. on the administrative side, dental and medical appointments can be coordinated to enhance convenience for patients and improve compliance with preventive dental visits. in , marshfi eld clinic's research foundation biomedical informatics research center hired their fi rst dental informatician, dr. amit acharya, bds, ms, phd. with dedicated biomedical informatics and research resource centers, the marshfi eld clinic has laid the groundwork for true medical/dental integration with appropriate electronic health record decision support and is positioned to develop a dental education curriculum capable of implementing the iom recommendations. downstream benefi ts of using such a curriculum are the ability of future practitioners to use informatics to improve quality of care and reduce the burden of disease. according to an institute of oral health report ( ) it is widely accepted across the dental profession that oral health has a direct impact on systemic health, and increasingly, medical and dental care providers are building to bridge relationships to create treatment solutions. as early as , william gies recognized that "the frequency of periodic examination gives dentists exceptional opportunity to note early signs of many types of illnesses outside the domain of dentistry" (gies ) . the following examples show how integration of dental and medical care can impact patient outcomes, underlining the importance of this concept in dental curriculum design. a study of , blue cross blue shield of michigan (bcbs) members with diabetes, who had access to dental care lead researchers, and bcbs executives to conclude that treatment of periodontal disease signifi cantly impacts outcomes related to diabetes care and related costs (blue cross blue shield of michigan ) . another example is found in the context of preterm delivery and miscarriage. according to research cited by cigna ( ) , expecting mothers with chronic periodontal disease during the second trimester are seven times more likely to deliver preterm (before th week), and have dramatically more healthcare challenges throughout their life. cigna also cites the correlation between periodontal disease and low birth weights, pre-eclampsia, gestational diabetes. equally important is the opportunity to develop and implement the team-based curriculum that trains future dentists and physicians in the management of chronic disease as an accountable care organization (aco) in a patient-centered environment. as an example, joseph errante, d.d.s., vice president, blue cross blue shield of ma, reported that medical costs for diabetics who accessed dental care for prevention and periodontal services were signifi cantly lower than those who didn't get dental care (errante ) . these data suggest that team based case management of prevalent chronic health conditions have considerable cost savings opportunities for government payers, third party payers, employers and employees (errante ) . these economic benefi ts to integration as it relates to the iehr are discussed elsewhere in this book, but begin with the ability of providers to function in a team based environment and as such, underscore the importance of training in such an environment. dentists trained in a fqhc iehr integrated educational model will be well positioned to function successfully within an aco model. an aco is a system where providers are accountable for the outcomes and expenditures of the insured population of patients they serve. the providers within the system are charged with collectively improving care around cost and quality targets set by the payor. within this system, care must be delivered in a patient-centered environment. the patient-centered environment according to the national committee for quality assurance (ncqa), is a health care setting that cultivates partnerships between individual patients and their personal physicians and, when appropriate, the patient's family. care is facilitated by registries, information technology, health information exchange and other means to assure that patients receive defi ned, timely and appropriate care while remaining cognizant of cultural, linguistic and literacy needs of the patient being served. the model includes the opportunity to deliver patient care that is patient-centric, incorporates the patient in the care planning, considers the patient's beliefs and views, and incorporates the patient's families as needed. the model allows providers to deliver care that is inclusive of needs, attentive, and accessible. the model equips payers to purchase high quality and coordinated care among teams of providers across healthcare settings. while this describes the medical home, most dental practices also follow this process. many dental practices function in this regard with insured populations and refl ect elements of the model that medicine is creating. william gies would be proud. training in the delivery of accountable and patient-centered medical-dental care must be done purposefully. commenting on the inadequate training relative to the integration of medical and dental education, baum ( ) stated that "we need to design new curricula with meaningful core competencies for the next generation of dentists rather than apply patches to our existing ones." while this statement was made in reference to the basic sciences, the same holds true for patient-centered system-based practice competencies. utilizing state-of-the-art electronic medical records as a tool and the fhc infrastructure as the service venue, meaningful patient-centered system-based practice core competencies achievement becomes possible in a manner highly responsive to societal needs. by defi nition, fqhcs must provide primary medical care, dental care, and behavioral health. fqhc have also historically been utilized as healthcare workforce training centers and the affordable care act of reinforced their role as healthcare training centers. specifi cally, this legislation serves to promote fqhcs as the entity through which the primary care workforce (including dental) will be developed and expanded. in combination, fqhcs and primary care centers are positioned to be the front runners in a medical/-dental home training model which will be essential to preparing future practitioners for practice in an aco. critical to this success is the ability to train these practitioners on an integrated medical-dental record and informatics platform. use of this platform imprints most strongly during the learner's formative years of training; instructing and guiding disease management, decision making, patient care coordination, prevention, and both outcome-based and comprehensive care. training in this hybrid academically orientated clinically integrated setting moves dental education off its crossroads and creates the highway to its future. concerns with the new models extend to their ability to integrate medical and dental disciplines at the clinical and informatics level. while the iom report identifi ed the need to integrate medical and dental curriculum, success at the curricular and technological level within schools, has been limited. three major factors have contributed to the limited progress: access priorities. creating access to care has outranked the need to integrate • care. in part, this refl ects societal need for care and public demand to reduce the burden of the "silent epidemic." schools play an important role as a safety net to care for the uninsured and underinsured through intramural clinical service learning. even though "dental colleges seem to be willing to bearing a large and disproportionate share of the burden in terms of access to care" (dunning et al. ) , schools were challenged as part of iom, surgeon general, and macy reports, to expand that role. while these reports have prompted creative educational solutions to increase access, the reports understate the tremendous opportunity, quality and cost benefi ts that could result from an integration of medicine and dentistry. it is diffi cult to change the culture and structure of existing schools. this is not • unique to dentistry. however, the iom report specifi cally recommended that schools "eliminate marginally useful and redundant courses and design an integrated basic and clinical science curriculum". the challenges with this are many. examples include: some schools may not have other disciplines to draw from to create an inte-grated curriculum; a number of schools use a faculty senate to determine curriculum. this can result in curriculum that preserves the current faculty structure; changing curriculum is associated with expense and can be fi nancially pro-hibitive to some schools physical changes may be needed and represent an expense and/or may, in some instances, may not be practicable based on structure of existing facilities. public school programs may direct the fi nal curriculum, as boards or regent's one or two steps removed from the curriculum often have fi nal authority conversely, private schools may specify business or mission objectives that determine fi nal design. perhaps most germane to this text is the lack of a common technology plat-• form between disciplines in a learning environment. an integrated curriculum requires an integrated platform to accomplish delivery and evaluation. this is particularly essential to clinical management of the patient by professionals in training as part of a healthcare delivery team. some progress in establishing shared basic sciences curricula has been documented in the literature. to date, no single integrated electronic health (medical-dental) record has been meaningfully adapted for educational purposes, including incorporation of assessment of the learner relative to integrated competencies, integrated case-based and problem-based curriculum, and integrated evaluation and assessment. another concern with new educational programs emerging in response to these reports and relative to creating a transformational integrated curriculum is that some of the programs are focusing primarily on creating clinicians with no value or emphasis on integrating training with research and/or scholarly activity. integrated training models counter such concerns. research will be fundamental to measuring the relative benefi ts and outcomes associated with treatment of patients in a shared curriculum setting and will be the catalyst for the development of integrated medical-dental informatics incorporating educational capabilities. additionally, accreditation will also need to evaluate its response to such models. presently it is unclear how accrediting bodies will view an integrated crossdisciplinary curriculum. further, due to its integrated nature, such a curriculum would lie outside of the expertise of a single traditional accrediting body focused on one particular discipline. it has yet to be determined how accrediting bodies will review and appraise such cross-disciplinary competencies. lastly, it is important to recognize that a successful education model with innovative informatics is only successful if its focus is patient care. graduating learners with competency only in the use of informatics will be limited unless adapted to training and delivery programs that result in patient centric care. research and reports over the past years support the need to reform dental education. first steps have been taken and lead the way for continued innovation around clinic-based education and integrated curriculum. the models identifi ed point to a strong partnership and interrelationship with chcs for creative, cost saving, effective and sustainable delivery methods. moreover, chc's must be more involved in a training curriculum integrated with informatics. chcs, in turn, benefi t from residents and students through service-learning to help meet a societal and workforce need, while the learners benefi t from increased competency. in order to train an evidence-based, patient-centered, medical-dental workforce, it is imperative that medical and dental data and record accessibility be incorporated into these training and care delivery initiatives. in order to keep moving away from the crossroads, such integration must become the pathway on which curriculum is developed and implemented. public law - 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doc_id: cord_uid: yfuuirnw disasters are increasing around the world. children are greatly impacted by both natural disasters (forces of nature) and man-made (intentional, accidental) disasters. their unique anatomical, physiological, behavioral, developmental, and psychological vulnerabilities must be considered when planning and preparing for disasters. the nurse or health care provider (hcp) must be able to rapidly identify acutely ill children during a disaster. whether it is during a natural or man-made event, the nurse or hcp must intervene effectively to improve survival and outcomes. it is extremely vital to understand the medical management of these children during disasters, especially the use of appropriate medical countermeasures such as medications, antidotes, supplies, and equipment. skeleton as a result of incomplete calcification and active bone growth centers. protected organs, such as the lungs and heart, may be injured due to overlying fractures. cervical spine injuries can also be pronounced, as in patients with head trauma. in fact, spinal cord injury may be present without any radiographic abnormalities of the spine. finally, vital signs will vary based on the pediatric patient's age. this may be a pitfall during rapid evaluation by any nurse or hcp not accustomed to the care of children. younger pediatric patients have higher metabolic rates and, therefore, higher respiratory rates and heart rates. this can be a distinct disadvantage versus older pediatric patients when encountering similar diseases. an example is inhaled toxins (e.g., nerve agents and lung-damaging agents). infants and children will suffer greater toxicity since they inhale at a faster rate due to higher metabolic demands and thus, distribute the toxin more rapidly to various end-organs. understanding respiratory differences is essential to the management of the acutely ill pediatric patient. the most common etiology for cardiorespiratory arrest in children is respiratory pathology, typically of the upper airway. most of the airway resistance in children occurs in the upper airway. nasal obstruction can lead to severe respiratory distress due to infants being obligate nose breathers. their relatively large tongue and small mouth can lead to airway obstruction quickly, especially when the neuromuscular tone is abnormal such as during sedation or encephalopathy. in infants, physiologic (i.e., copious secretions) and pathologic (i.e., edema, vomitus, blood, and foreign body) factors will exaggerate this obstruction. securing the airway in such events can be quite challenging. typically, the glottis is located more anterior and cephalad. appropriate visualization during laryngoscopy can be further hampered by the prominent occiput that causes neck flexion and, therefore, reduces the alignment of visual axes. the omega or horseshoeshaped epiglottis in young infants and children is quite susceptible to inflammation and swelling. as in epiglottitis, the glottis becomes strangulated in a circumferential manner leading to dangerous supraglottic obstruction. children also have a natural tendency to laryngospasm and bronchospasm. finally, due to weaker cartilage in infants, dynamic airway collapse can occur especially in states of increased resistance and high expiratory flow. along with altered pulmonary compensation and compliance, a child may rapidly progress to respiratory failure and possibly arrest. cardiovascular differences are critical in the pediatric patient. typical physiological responses tend to allow compensation with seemingly normal homeostasis. with tachycardia and elevated systemic vascular resistance, younger pediatric patients can maintain normal blood pressure despite decreased cardiac output and poor perfusion (compensated shock). since children have less blood and volume reserve, they progress to this state quickly. in pediatric patients with multiorgan injury or severe gastrointestinal losses, these compensatory mechanisms are pushed to their limits. the unaccustomed hcp may be lulled into complacency since the blood pressure is normal. all the while, the pediatric patient's organs are being poorly perfused. once these compensatory mechanisms are exhausted, the patient rapidly progresses to hypotension and, therefore, hypotensive shock. if not reversed expeditiously, this may lead to irreversible shock, ischemia, multiorgan dysfunction, and death. pediatric patients with altered mental status pose significant problems. the differential diagnosis will be very broad in the comatose patient based on development alone. for example, younger pediatric patients can present with nonconvulsive status epilepticus (ncse) instead of generalized convulsive status epilepticus (gcse). in fact, ncse is more common among younger pediatric patients than gcse, especially in those from to months of age. furthermore, many of them are previously well without preexisting diseases such as epilepsy. other disease states may include poisoning, inborn errors of metabolism, meningitis, and other etiologies of encephalopathy. using the modified pediatric glasgow coma scale (gcs) is the cornerstone when evaluating the young pediatric patient when they are preverbal. pupillary response, external ocular movements, and gross motor response may be challenging to evaluate in a developmentally young or delayed pediatric patient. pediatric traumatic brain injury is extremely devastating. whether considered accidental (motor vehicle crash) or nonaccidental (abusive head trauma), evaluation of the neurological status of the acutely injured pediatric patient can be problematic, especially the gcs. some prefer to use the avpu system (alert, responds to verbal, responds to pain, and unresponsive). due to the disproportionately larger head and weaker neck muscles, there is more risk of acceleration-deceleration injuries (fall from a significant height, vehicular ejection, and abusive head trauma). furthermore, the softer skull, dural structural differences, and vessel supply will place the pediatric patient at risk for brain injury and intracranial hemorrhage. finally, due to pediatric brain composition, the risk of diffuse axonal injury and cerebral edema is much higher. although spinal cord injury is rare in young pediatric patients, morbidity and mortality are significant. in pediatric patients less than years of age, the most commonly seen injuries are in the atlas, axis, and upper cervical vertebrae. in young pediatric patients, spinal injuries tend to be anatomically higher (cervical) versus adolescents (thoracolumbar). furthermore, congenital abnormalities, such as atlantoaxial abnormalities (trisomy ), may exaggerate the process. the clinical presentation of spinal cord injury varies in young pediatric patients due to ongoing development. laxity of ligaments, wedge-shaped vertebrae, and incomplete ossification centers contribute to specific patterns of injuries. finally, spinal cord injury without radiographic abnormality (sciwora) may result. because of the disproportionately larger head, weaker neck muscles, and elasticity of the spine, significant distraction and flexion injury of the spinal cord may occur without apparent ligament or bony disruption (hilmas et al. ; jacobson and severin ; severin ). motor skills develop from birth. gross and fine motor milestones are achieved in a predictable manner and must be assessed during each hcp encounter. cognitive development will follow a similar pattern of maturation. the development of these skills can often predict injuries and their extent. for example, consider a house fire. a young infant, preschooler, and adolescent are sleeping upstairs in house when a fire breaks out in the middle of the night. the smoke detectors begin to alarm. each child is awoken by the ensuing noise and chaos. based on the development, the adolescent will most likely make it out of the house alive. he will comprehend the threat, run down the stairs, and exit the house without delay. smoke inhalation may be minimal. if it is a middle adolescent, an attempt may be made to jump out of the window leading to multiple blunt trauma with or without traumatic brain injury. the preschooler most likely will be too scared and not understand how to escape. tragically, he may hide under a bed or in a closet. when the firefighters arrive and search the house, the preschooler may remain silent because of fear, especially of strangers in the house. he will most likely succumb to thermal injuries along with the effects of carbon monoxide and die. as far as the infant, he cannot walk, climb, crawl, or run. furthermore, he cannot scream for help or know how to escape. as the smoke engulfs the room, he will most likely suffer severe smoke inhalation injury including extensive carbon monoxide toxicity along with thermal injuries and die. this example also points out another important difference in pediatric patients: their dependence on caregivers. when considering neonates, for example, their entire existence depends on the caregiver, including feeding, changing of diapers, nurturing, and environmental safety. these dynamics are essential to the pediatric patient's health and survival, especially during a disaster. another aspect of development is the attainment of language skills. this, too, develops over time in a predictable fashion. one of the biggest challenges in pediatrics is the lack of the patient's ability to verbally convey complaints. as described above, verbal milestones vary among the different age ranges of the pediatric patient. hcps are often faced with a caregiver's subjective assessment of the problem. although it can be revealing and informative, this may not be available in an acute crisis situation. it will take the astute hcp to determine, for example, if an inconsolably crying infant is in pain from a corneal abrasion or something more life-threatening such as meningitis. this can also be a challenging task in a teenager, especially during middle adolescence. an hcp will have to determine, for example, if the seemingly lethargic middle adolescent is intoxicated with illicit drugs or has diabetic ketoacidosis. finally, the hcp will have to address developmental variances among their pediatric patients and any comorbid features. young pediatric patients can regress developmentally during any illness or injury. this is especially seen in patients with chronic medical conditions (cancer) or during prolonged hospitalization with rehabilitation (multisystem trauma). furthermore, those pediatric patients with developmental and intellectual disabilities, for example, will be difficult to evaluate based on the effects of their underlying pathology. these pediatric patients typically have unique variances in their physical exams (jacobson and severin ; severin ) . please refer to chap. for more detailed information on pediatric development. pediatric patients will often reflect the emotional state of their caregiver. they take verbal and physical cues from their caregiver. at times, this may also occur in the presence of a nurse or hcp. the psychological impact of illness will vary greatly with the child's development and experience. children tend to have a greater vulnerability to post-traumatic stress disorder especially with disaster events. furthermore, they are highly prone to becoming psychiatric casualties despite the absence of physical injury to themselves. and as any pediatric hcp can tell you, the younger pediatric patients tend to also have greater levels of anxiety, especially while preparing for invasive procedures such as phlebotomy and intravenous line placement (hilmas et al. ; jacobson and severin ; severin ) . please refer to chap. for more detailed content on mental health. the world health organization and the pan american health organization define a disaster as "an event that occurs in most cases suddenly and unexpectedly, causing severe disturbances to people or objects affected by it, resulting in the loss of life and harm to the health of the population, the destruction or loss of community property, and/or severe damage to the environment. such a situation leads to disruption in the normal pattern of life, resulting in misfortune, helplessness, and suffering, with adverse effects on the socioeconomic structure of a region or a country and/or modifications of the environment to such an extent that there is a need for assistance and immediate outside intervention" (lynch and berman ). types of disasters usually fall into two broad categories: natural and man-made. natural disasters are generally associated with weather and geological events, including extremes of temperature, floods, hurricanes, earthquakes, tsunamis, volcanic eruptions, landslides, and drought. naturally occurring epidemics, such as the h n , ebola, and novel coronavirus outbreaks, are often included in this category. man-made disasters are usually associated with a criminal attack such as an active shooter incident, or a terrorist attack using weapons such as explosive, biological, or chemical agents. however, man-made disasters can also refer to human-based technological incidents, such as a building or bridge collapse, or events related to the manufacture, transportation, storage, and use of hazardous materials, such as the chernobyl radiation leak and the bhopal toxic gas leak. even though disasters can be primarily placed into any of these two categories, they can often impact each other and compound the magnitude of any disaster incident (united states department of homeland security, office of inspector general ). a prime example is the march tohoku earthquake leading to a tsunami (natural) that triggered the fukushima daiichi nuclear disaster (man-made). disasters can also be characterized by the location of such an event. internal disasters are those incidents that occur within the health care facility or system. employees, physical plant, workflow and operations of the clinic, hospital, or system can be disrupted. external disasters are those incidents that occur outside of the health care facility or system. this impacts the community surrounding the facility, proximally or distally, but does not directly threaten the facility or its employees. as with natural and man-made disasters, internal and external disasters can impact each other. for example, an overflow of patients during a high census period may lead to the shutdown of the hospital to any new patients (internal disaster). this will place the hospital on bypass and possibly stress other hospitals in the community beyond their means (external disaster). a terrorist event, such as the release of sarin in a subway system during a busy morning commute, can lead to massive disruption in the community (external disaster). all the victims of the attack will seek medical care at nearby hospitals, possibly overwhelming the health care staff and depleting critical resources (internal disaster). characterization of disasters by geography (local, state, national, and international) can also be used. again, no matter the site of the incident, a disaster in one area could easily create a disaster in another geographical region. for example, a factory and its community could be ravaged by a hurricane (local disaster). if this is the only factory in the world to produce a certain medication, this could lead to critical shortages to hospitals all around the world (international disaster). the term "disaster preparedness" has been used over the years as a way to describe efforts to manage any disaster event. however, preparedness is only one aspect of the process. the use of the term disaster planning is more appropriate. it considers all aspects needed for an effective effort and is dependent on additional phases, not just preparedness. national preparedness efforts, including planning, are now informed by the presidential policy directive (ppd) that was signed by the president in march and describes the nation's approach to preparedness (united states department of education, office of elementary and secondary education, office of safe and healthy students ; united states department of homeland security b). a recommended method for disaster preparedness efforts is the utilization of an "all-hazards" model of emergency management (adini et al. ; waugh ) . the four overlapping phases of the model include mitigation, preparedness, response, and recovery. the mitigation phase involves "activities designed to prevent or reduce losses from a disaster" (waugh ) . examples include land use planning in flood plains, structural integrity measures in earthquake zones, and deployment of security cameras. the preparedness phase includes the "planning of how to respond in an emergency or a disaster, and developing capabilities for more effective response" (waugh ) . examples include training programs for emergency responders, drills and exercises, early warning systems, contingency planning, and development of equipment and supply caches. up to this point, all planning efforts are proactive and not reactive. often times, a hazard analysis is conducted to delineate areas of strengths and identify potential risks. it helps in "the identification of hazards, assessment of the probability of a disaster, and the probable intensity and location; assessment of its potential impact on a community; the property, persons, and geographic areas that may be at risk; and the determination of agency priorities based on the probability level of a disaster and the potential losses" (waugh ) . after a disaster or emergency incident occurs, the response phase, or "immediate reaction to a disaster", (waugh ) begins. examples include mass evacuations, sandbagging buildings and other structures, providing emergency medical services, firefighting, and restoration of public order. in some situations, the response period may be a short (e.g., house fire), intermediate (e.g., bomb detonation), or extended (e.g., pandemic influenza) duration. after a period of time, the recovery phase follows. these are "activities that continue beyond the emergency period to restore lifelines" (waugh ) . examples include the provision of temporary shelter, restoration of utilities such as power, critical stress debriefing for responders, and victims, job assistance and small business loans, and debris clearance. recovery always seems to be the most unpredictable; it may take days to months to years. as demonstrated with recent hurricanes harvey, irma, and maria in , the most affected regions are still in the phase of recovery and may be along a prolonged track as hurricane katrina in . as mentioned, the early phases of planning (mitigation and preparedness) truly hinge upon the environment or community surrounding the health care site (e.g., clinic, hospital, or long-term care facility). identification of potential hazards and risks is a key step in disaster planning. using a hazard vulnerability assessment (hva) or a threat and hazard identification and risk assessment (thira) can provide a basis for mitigation and prevention tasks. an hva/thira emphasizes which types of natural or man-made disasters are likely to occur in a community (e.g., tornado, flood, chemical release, or terrorist event). they further highlight the impact those disasters may have on the community and any capabilities that are in place that may lessen the effects of the disaster (illinois emergency medical services for children ). a basic principle of the hva methodology is to determine the risk of such an event or attack occurring at a given hospital or hospital system. simply, the risk is a product of the probability of an event and the severity of such an event if it occurs (risk = probability × severity). however, there are many complexities in quantifying terrorism risk (waugh ; woo ) . it is important to note that in some circumstances, exposure may need to be included in the equation (risk = probability × severity × exposure), but usually for operational risk management applications (mitchell and decker ) . at any rate, issues to consider for the probability of an event occurring include, but are not limited to, geographic location and topography, proximity to hazards, degree of accessibility, known risks, historical data, and statistics of various manufacturer/vendor products. severity, on the other hand, is dependent on the gap between the magnitude of an event and mitigation for the given event (severity = magnitude -mitigation). magnitude varies upon the impact of the event to humans, property, and/or business. mitigation varies upon the development of internal and external readiness before a disaster strikes. as one can surmise, if the magnitude of the event outstrips the mitigation, the event is considered a threatening hazard. once the hva is completed, the health care site should immediately prioritize planning efforts for the top - hazards and develop plans accordingly. all other identified hazards must also be addressed to ensure a broad and robust disaster plan. it is important to realize that local and regional entities also perform comprehensive hvas. a concerted analysis among a hospital and key community stakeholders is optimal for a coordinated plan. an hva/thira contains both quantitative and qualitative components. specific tools have been developed through private and public organizations (e.g, fema) that can help in the analysis (united states department of homeland security, federal emergency management agency ). using these tools as a guide, the entity can determine what types of hazards have a high, medium, or low probability of occurring within specific geographic boundaries. typically, these tools do not have components specific to children or other at-risk populations. however, the tools can be adapted either directly by adding children to specific hazards or ensuring considerations specific to children are incorporated into the hva/thira calculations. the hva/thira should be reviewed and updated minimally on an annual basis to identify changing or external circumstances. this includes conducting a pediatric-specific disaster risk assessment to identify where children congregate and their risks (e.g., schools, popular field trip designations, summer camps, houses of worship, and juvenile justice facilities) (illinois emergency medical services for children ). of note, hva techniques have been utilized for pediatric-specific disaster plans. having a separate pediatric hva (phva) is crucial to a well-rounded and robust health care disaster plan. first, it demonstrates the extent of the pediatric population in the community. it is estimated that % of the population fits within the age range of pediatric patients. in some situations, it may be more. during the performance of a phva, it was demonstrated that % of the community was less than years of age (jacobson and severin ) . second, a phva increases the situational awareness of those tasked to plan for disasters that involve children and adolescents. often times, children and adolescents are excluded from local and regional disaster plans. the unique vulnerabilities of pediatric patients will demand appropriate drills, exercises, equipment, medications, and expertise. thirdly, identifying pediatric risks in a community will help prioritize efforts of planning, especially in those hospitals not accustomed to caring for pediatric patients. finally, a phva helps to develop a framework for global pediatric disaster planning. this can extend beyond a local community and actually advance city, state, regional, and national disaster planning efforts. there has been a development of web-based tools to simplify and enhance the phva process (jacobson and severin ) . after an hva/thira has been completed, the results should be used to help direct and plan drills/exercises based on high impact and high probability threats. it is advised to conduct an hva/thira on an annual basis to assess specific threats unique to your organization's physical structure as well as the surrounding geographic environment. it will also provide insight into whether there is an improvement in previous planning efforts. completion of a population assessment that provides a demographic overview of the community with a breakdown of the childhood population is strongly recommended in conjunction with the hva/ thira. collaborating with other community partners, such as local health departments and emergency management agencies, can assist an organization with the conduction of a comprehensive hva/thira (illinois emergency medical services for children ). please see chap. for further information on hospital planning. pediatric supplies, equipment, and medications will be scarce during a disaster. it will become more of an issue if the health care facility is not accustomed to caring for acutely ill pediatric patients. this will be further exacerbated by a massive surge of acutely ill pediatric patients, a widespread or prolonged disaster, and supply line disruptions. to protect the health security of children and families during a public health emergency, the assistant secretary for preparedness and response (aspr) manages and maintains the strategic national stockpile (sns), a cache of medical countermeasures for rapid deployment and use in response to a public health emergency or disaster (fagbuyi et al. ) . various pediatric-specific supplies and countermeasures are included in the sns. maintaining a supply of medications and medical supplies for specific health threats allows the stockpile to respond with the right product when a specific disease or agent is known. if a community experiences a large-scale public health incident in which the disease or agent is unknown, the first line of support from the stockpile is to send a broad-range of pharmaceuticals and medical supplies. place and martin ) . the emergency equipment and supply lists can easily be adapted for any pediatric disaster emergency (place and martin ) or incident requiring pediatric mass critical care (desmond et al. ) . ageappropriate nutrition, hygiene, bedding, and toys/distraction devices should also be available (illinois emergency medical services for children ) (tables . and . ). endotracheal tubes • uncuffed: . and . mm • cuffed or uncuffed: . , . , . , . , and . mm • cuffed: . , . , . , . , and . mm feeding tubes ( f and f) laryngoscope blades curved: and ; straight: , , , and laryngoscope handle magill forceps (pediatric and adult) nasopharyngeal airways (infant, child, and adult) oropharyngeal airways (sizes - ) stylets for endotracheal tubes (pediatric and adult) suction catheters (infant, child, and adult) tracheostomy tubes (sizes . , . , . , . , . , . , and . mm) yankauer suction tip bag-mask device (manual resuscitator), self-inflating (infant size: ml; adult size: ml) clear oxygen masks (standard and nonrebreathing) for an infant, child, and adult masks to fit bag-mask device adaptor (neonatal, infant, child, and adult sizes) nasal cannulas (infant, child, and adult) nasogastric tubes (sump tubes): infant ( f), child ( f), and adult ( f- f) laryngeal mask airway a vascular access arm boards (infant, child, and adult sizes) catheter over-the-needle device ( - gauge) intraosseous needles or device (pediatric and adult sizes) intravenous catheter-administration sets with calibrated chambers and extension tubing and/or infusion devices with ability to regulate rate and volume of infusate umbilical vein catheters ( . f and . f) b central venous catheters ( . f- . f) intravenous solutions to include normal saline, dextrose % in normal saline, and dextrose % in water fracturemanagement devices extremity splints, including femur splints (pediatric and adult sizes) spine-stabilization method/devices appropriate for children of all ages c (continued) laryngeal mask airways could be shared with anesthesia but must be immediately accessible to the ed b feeding tubes (size f) may be used as umbilical venous catheters but are not ideal. a method for securing the umbilical catheter, such as an umbilical tie, should also be available c a spinal stabilization device is one that can stabilize the neck of an infant, child, or adolescent in a neutral position when a pediatric disaster victim presents acutely ill to the hospital, various emergency interventions will be needed to stabilize the patient. evaluation of the pediatric patient should include a primary survey (abcde), secondary survey (focused sample history and focused physical examination), and diagnostic assessments (laboratory, radiological, and other advanced tests). this will guide further therapeutic interventions. particular attention should be given to the identification of respiratory and/or circulatory derangements of the child, including airway obstruction, respiratory failure, shock, and cardiopulmonary failure. interventions will be based on physiologic derangements of the pediatric patient and determined by the scope of practice and protocols, such as standard resuscitation algorithms for neonatal (american academy of pediatrics and american heart association et al. ) and pediatric (american heart association ) victims. the hcp must be knowledgeable of various emergency medications (table . ) used for children, the appropriate dosages and their mechanism of action, any potential side effects, and drug/drug interactions. other medications, such as antibiotics, antidotes, or countermeasures, may be needed as well. pharmacologic therapy should be initiated immediately based on clinical suspicion and not delayed due to pending laboratory tests (e.g., antibiotics for presumed infection/sepsis or antidotes for suspected nerve agents). dosages should be based on the patient's weight or a length-based weight system. (montello et al. ) or hard copy countermeasure manuals may be more practical, especially during a disaster incident when computer service or internet access may be unreliable. in , the centre for research on the epidemiology of disasters (cred) launched the emergency events database (em-dat). em-dat was created with the initial support of the world health organization (who) and the belgian government. the main objective of the database is to serve the purposes of humanitarian action at national and international levels. the initiative aims to rationalize decision-making for disaster preparedness as well as provide an objective base for vulnerability assessment and priority setting. em-dat contains essential core data on the occurrence and effects of over , mass disasters in the world from to the present day. the database is compiled from various sources, including united nation agencies, nongovernmental organizations (ngos), insurance companies, research institutes, and press agencies (cred ). as described in the cred report entitled natural disasters : lower mortality, higher cost, a disaster is entered into the database if at least one of the following criteria is fulfilled: or more people reported killed; or more people reported affected; declaration of a state of emergency; and/or call for international assistance (cred ). in economic losses, poverty and disasters - : cred/unisdr report, the cred defines a disaster as "a situation or event which overwhelms local capacity, necessitating a request at national or international level for external assistance; an unforeseen and often sudden event that causes great damage, destruction and human suffering" (cred ). the cred em-dat classifies disasters according to the type of hazard that triggers them. the two main disaster groups are natural and technological disasters. there are six natural disaster subgroups. geophysical disasters originate from the solid earth and include earthquake (ground movement and tsunami), dry mass movement (rock fall and landslides), and volcanic activity (ash fall, lahar, pyroclastic flow, and lava flow). lahar is a hot or cold mixture of earthen material flowing on the slope of a volcano either during or between volcanic eruptions. meteorological disasters are caused by short-lived, micro-to meso-scale extreme weather and atmospheric conditions that last from minutes to days and include extreme temperatures (cold wave, heat wave, and severe winter conditions such as snow/ice or frost/ freeze), fog, and storms. storms can be extra-tropical, tropical, or convective. convective storms include derecho, hail, lightning/thunderstorm, rain, tornado, sand/dust storm, winter storm/blizzard, storm/surge, and wind. derecho is a widespread and usually fast-moving windstorm associated with convection/convective storm and includes downburst and straight-line winds. hydrological disasters are caused by the occurrence, movement, and distribution of surface/subsurface freshwater and saltwater and include floods, landslides (an avalanche of snow, debris, mudflow, and rockfall), and wave action (rogue wave and seiche). flood types can be coastal, riverine, flash, or ice jam. climatological disasters are caused by longlived, meso-to macro-scale atmospheric processes ranging from intraseasonal to multidecadal climate variability and include drought, glacial lake outburst, and wildfire (forest fire, land fire: brush, bush, or pasture). biological disasters are caused by the exposure to living organisms and their toxic substances or vectorborne diseases that they may carry and include epidemics (viral, bacterial, parasitic, fungal, and prion), insect infestation (grasshopper and locust), and animal accidents. extraterrestrial disasters are caused by asteroids, meteoroids, and comets as they pass near-earth, enter earth's atmosphere, and/or strike the earth, and by changes in the interplanetary conditions that affect the earth's magnetosphere, ionosphere, and thermosphere. types include impact (airbursts) and space weather (energetic particles, geomagnetic storm, and shockwave) events (cred ). there are three technological disaster subgroups. industrial accidents include chemical spills, collapse, explosion, fire, gas leak, poisoning, radiation, and oil spills. a chemical spill is an accidental release occurring during the production, transportation, or handling of hazardous chemical substances. transport accidents include disasters in the air (airplanes, helicopters, airships, and balloons), on the road (moving vehicles on roads or tracks), on the rail system (train), and on the water (sailing boats, ferries, cruise ships, and other boats). miscellaneous accidents vary from collapse to explosions to fires. collapse is an accident involving the collapse of a building or structure and can either involve industrial structures or domestic/nonindustrial structures (cred ). technological disasters are considered man-made, but as suggested by their subgroup, they are accidental and not intentional. the united nations office for disaster risk reduction (unisdr) and cred report, economic losses, poverty, and disasters - , reviews global natural disasters during that time period, their economic impact, and the relationship with poverty. between and , climate-related and geophysical disasters killed . million people and left a further . billion injured, homeless, displaced, or in need of emergency assistance. although the majority of fatalities were due to geophysical events, mostly earthquakes and tsunamis, % of all disasters was caused by floods, storms, droughts, heatwaves, and other extreme weather events. the financial impact was staggering. in - , disaster-hit countries reported direct economic losses valued at us$ billion, of which climate-related disasters caused us$ billion or % of the total. this was up from % (us$ billion) of losses (us$ billion) reported between and . overall, reported losses from extreme weather events rose by % between these two -year periods. in absolute monetary terms, over the last -years, the usa recorded the biggest losses (us$ billion), reflecting high asset values as well as frequent events. china, by comparison, suffered a significantly higher number of disasters than the usa ( vs. ) but lower total losses (us$ billion) (cred ) (figs. . , . , . , . , . , . , . , . and . in , climate-related and geophysical incidents in the world were estimated with , deaths and over million people impacted. indonesia recorded approximately half of the deaths with india accounting for half of those impacted by disasters. notable features of were intense seismic activity in indonesia, a series of disasters in japan, floods in india, and an eventful year for both volcanic activity and wildfires. however, an ongoing trend of lower death tolls from previous years continued into (centre for research on the epidemiology of disasters (cred) and united nations office for disaster risk reduction (unisdr) ) (tables . , . , . , . , . , . and . there are no specific deviations when medically managing children after a natural disaster. according to sirbaugh and dirocco ( ) "small-scale mass casualty incidents occur daily in the united states. few present unusual challenges to the local medical systems other than in the number of patients that must be treated at one time. except in earthquakes, explosions, building collapses, and some types of terrorist attacks, the same holds true for large-scale disasters. sudden violent disaster mechanisms can produce major trauma cases, including patients needing field amputations or management of crush syndrome. for the most part, medicine after a disaster is much the same as it was before the disaster, with more minor injuries, more people with exacerbations of their chronic illnesses, and number of patients seeking what is ordinarily considered primary care. this is true for children and adults." it should be noted, however, that children have a predisposition to illness and injury after natural disasters. the hcp must be able to identify any health problems and treat the child effectively and efficiently while utilizing standard resuscitation protocols as indicated. traumatic injuries may be seen after any natural disaster. the injuries can range from minor scrapes and bruises to major blunt trauma or traumatic brain injury. children are at increased risk for injury since adults are distracted by recovery efforts and may not be able to supervise them closely. the environment may not be safe due to environmental hazards, such as collapsed buildings, sinkholes, and high water levels. dangerous equipment used during relief efforts may be present, such as heavy earth moving equipment, chainsaws, and power generators. hazardous chemicals, such as gasoline and other volatile hydrocarbons, may be readily accessible or taint the environment. without suitable shelter, children are also exposed to weather, animals, and insects (sirbaugh and dirocco ) . infectious diseases may also pose a problem to children after a natural disaster. infectious patterns will persist during a disaster based on the season and time of year. there may be outbreaks or epidemics of highly contagious infections (e.g., influenza, respiratory syncytial virus, streptococcus pyogenes) due to mass sheltering of children and families. poor nutrition or decreased availability of food may lower their resistance against infections. various water-borne or food-borne diseases may cause illnesses in children. poor hygiene and mass shelter environments may exacerbate these illnesses. immunized children should be protected against common preventable diseases after a natural disaster but still could be a problem in mass groups that are not completely or appropriately immunized. after the haiti earthquake, there were increased cases of diarrhea, cholera, measles, and tetanus in children months after the earthquake despite some level of vaccination (sirbaugh and dirocco ) . children are at risk for various environmental emergencies. austere environments will impact children greatly. heat exposure coupled with minimal access to drinkable water may lead to severe dehydration. exposure to the cold may lead to frostbite or hypothermia. children are at risk for carbon monoxide toxicity due to generator use or natural gas poisoning due to disrupted gas lines. there is always a risk for thermal injury due to the use of candles and other flame sources. exposure to animals (snakes) and insects (spiders) may increase the risk of envenomation. submersion injury and drowning incidents may escalate. this will be due to lack of supervision of children around storm drains, newly formed bodies of water, or rushing waters of storm diversion systems (sirbaugh and dirocco ) . mental health issues are often seen in children after natural disasters. even though a child may not be injured, they may become "psychiatric casualties" due to the horrific sights they have seen during or after the disaster. children and adolescents with behavioral or psychiatric problems may experience worsening symptoms and signs due to stress, trauma, disruption of routines, or availability of medications. this is often exacerbated if the parent, guardian, caregiver, or hcp is also having difficulty coping with the stress of the disaster. in general, the most common mental health problem in children is a post-traumatic stress disorder. however, separation anxiety, obsessive-compulsive symptoms, and severe stranger anxiety can also be seen in children after a traumatic event (sirbaugh and dirocco ) . see chap. for more detailed information. terrorism impacts children and families all around the world (tables . and . ). after the events of / , much attention has been given to the possibility of another mass casualty act of terrorism, especially with weapons of mass destruction, that include chemical, biological, nuclear, radiological, and explosive devices (cbnre), or other forms of violence such as active shooter incidents and mass shootings (jacobson and severin ) . since then, other incidents, both foreign and domestic, have involved children and complicates the concept of and the response to terrorism. johnston ( ) said it best in his review of terrorist and criminal attacks targeting children: "one of the more accepted defining characteristics of terrorism is that it targets noncombatants including men, women, and children. however, terrorist attacks specifically targeting children over other noncombatants are uncommon. this is for the same reason that most terrorists have historically avoided mass casualty terrorism: the shock value is so great that such attacks erode support for the terrorists' political objectives. the / attacks represent an increasing trend in mass casualty terrorism. at the same time, policymakers are examining this evolving threat, they must increasingly consider the threat of terrorist attacks targeting children." based on historical events, it is clear infants, toddlers, children, and adolescents have been victims of terrorism. this global trend of terrorists targeting children seems to be escalating (johnston ) . therefore, it is imperative to understand terrorism and ways it impacts the children and families served by the health care community. combs ( ) defines terrorism as "an act of violence perpetrated on innocent civilian noncombatants in order to evoke fear in an audience". however, she goes on to argue that to become an operational definition, there must also be the addition of a "political purpose" of the violent act. therefore, "terrorism, then, is an act composed of at least four crucial elements: ) it is an act of violence, ) it has a political motive or goal, ) it is perpetrated against civilian noncombatants, and ) it is staged to be played before an audience whose reaction of fear and terror is the desired result." (combs ) . there are different typologies of terrorism. at least five types of terror violence have been suggested by feliks gross: "mass terror is terror by a state, where the regime coerces the opposition in the population, whether organized or unorganized, sometimes in an institutionalized manner. dynastic assassination is an attack on a head of state or a ruling elite. random terror involves the placing of explosives where people gather (such as post offices, railroads, and cafes) to destroy whoever happens to be there. focused random terror restricts the placing of explosives, for example to where significant agents of oppression are likely to gather. finally, tactical terror is directed solely against the ruling government as a part of a 'broad revolutionary strategic plan'" (combs ). an additional typology offered is "lone wolf terror which involves someone who commits violent acts in support of some group, movement, or ideology, but who does stand alone, outside of any command structure and without material assistance from any group" (combs ) . martin ( ) reviews eight different terrorism typologies in the ever shifting, multifaceted world of modern terrorism. the new terrorism "is characterized by the threat of mass casualty attacks from dissident terrorist organizations, new and creative configurations, transnational religious solidarity, and redefined moral justifications for political violence" (martin ) . state terrorism is "committed by governments against perceived enemies and can be directed externally against adversaries in the international domain or internally against domestic enemies" (martin ) . dissident terrorism is "committed by nonstate movements and groups against governments, ethno-national groups, religious groups, and other perceived enemies" (martin ) . religious terrorism is "motivated by an absolute belief that an otherworldly power has sanctioned and commanded the application of terrorist violence for the greater glory of the faith…[it] is usually conducted in defense of what believers consider to be the one true faith" (martin ) . ideological terrorism is "motivated by political systems of belief (ideologies), which champion the self-perceived inherent rights of a particular group or interest in opposition to another group or interest. the system of belief incorporates theoretical and philosophical justifications for violently asserting the rights of the championed group or interest" (martin ) . international terrorism "spills over onto the world's stage. targets are selected because of their value as symbols of international interests, either within the home country or across state boundaries" (martin ) . criminal dissident terrorism "is solely profit-driven, and can be some combination of profit and politics. for instance, traditional organized criminals accrue profits to fund their criminal activity and for personal interests, while criminalpolitical enterprises acquire profits to sustain their movement" (martin ) . gender-selective terrorism "is directed against an enemy population's men or women because of their gender. systematic violence is directed against men because of the perceived threat posed by males as potential soldiers or sources of opposition. systematic violence is directed against women to destroy an enemy group's cultural identity or terrorize the group into submission" (martin ) . the all-hazards national planning scenarios are an integral component of dhs's capabilities-based approach to implementing homeland security presidential directive : national preparedness (hspd- ). the national planning scenarios are planning tools and are representative of the range of potential terrorist and natural disasters and the related impacts that face the nation. the federal interagency community has developed all-hazards planning scenarios for use in national, federal, state, and local homeland security preparedness activities. the objective was to develop a minimum number of credible scenarios to establish the range of response requirements to facilitate disaster planning (dhs ) (table . ). twelve of the scenarios represent terrorist attacks while three represent natural disasters or naturally occurring epidemics. this ratio reflects the fact that the nation has recurring experience with natural disasters but faces newfound dangers, including the increasing potential for use of weapons of mass destruction by terrorists. the scenarios form the basis for coordinated federal planning, training, exercises, and grant investments needed to prepare for all hazards. dhs employed the scenarios as the basis for a rigorous task analysis of prevention, protection, response, and recovery missions and identification of key tasks that supported the development of essential all-hazards capabilities (united states department of homeland security, federal emergency management agency ) (table . ). each of the scenarios follows the same outline to include a detailed scenario description, planning considerations, and implications. for each of the terrorismrelated scenarios, fema national preparedness directorate (npd) partnered with dhs office of intelligence and analysis (i&a) and other intelligence community and law enforcement experts to develop and validate prevention prequels. the prequels provide an understanding of terrorists' motivation, capability, intent, tactics, techniques and procedures, and technical weapons data. the prequels also provide a credible adversary based on known threats to test the homeland security community's ability to understand and respond to indications and warnings of possible terrorist attacks (united states department of homeland security, federal emergency management agency ). a chemical agent of terrorism is defined as any chemical substance intended for use in military operations to kill, seriously injure, or incapacitate humans (or animals) through its toxicological effects. chemicals excluded from this list are riot-control agents, chemical herbicides, and smoke/flame materials. chemical agents are classified as toxic agents (producing injury or death) or incapacitating agents (producing temporary effects). toxic agents are further described as nerve agents (anticholinesterases), blood agents (cyanogens), blister agents (vesicants), and lung-damaging agents (choking agents). incapacitating agents include stimulants, depressants, psychedelics, and deliriants (banks ; departments of the army, the navy, and the air force, and commandant, marine corps ). nerve agents are organophosphate anticholinesterase compounds. they are used in various insecticide, industrial, and military applications. military-grade agents include tabun (ga), sarin (gb), soman (gd), cyclosarin (gf), venom x (vx), and the novichok series. these are all major military threats. the only known battlefield use of nerve agents was the iraq-iran war. however, other nerve agent incidents, such as the tokyo subway attack (sarin), the chemical attacks in syria (chlorine, sarin, mustard), and the attempted assassination of sergei skripal in salisbury, uk (novichok), support that civilian threats also exist. nerve agents are volatile chemicals and can be released in liquid or vapor form. however, the liquid form can become vapor depending upon its level of volatility (e.g, g-agents are more volatile than vx). the level of toxicity depends on the agent, concentration of the agent, physical form, route and length of exposure, and environmental factors (temperature and wind) (tables . and . ). nerve agents exert their effects by the inhibition of esterase enzymes. acetylcholinesterase inhibition prevents the hydrolysis of acetylcholine. the clinical result is a cholinergic crisis and subsequent overstimulation of muscarinic and nicotinic receptors throughout the body including the central nervous system. clinical muscarinic responses include sludge (salivation, lacrimation, urination, defecation, gastrointestinal distress, and emesis) and dumbels (diarrhea, urinary incontinence, miosis/muscle fasciculation, bronchorrhea/bronchospasm/bradycardia, emesis, lacrimation, and salivation). nicotinic responses vary by site. preganglionic sympathetic nerve stimulation produces mydriasis, tachycardia, hypertension, and pallor. however, stimulation at the neuromuscular junction leads to muscular fasciculation and cramping, weakness, paralysis, and diaphragmatic weakness. central nervous system presentations range from anxiety and restlessness to seizures, coma, and death (banks ; rotenberg and newmark ; rotenberg b ). pediatric manifestations (table . ) may vary from the classic clinical responses due to their unique vulnerabilities (hilmas et al. ): • children may manifest symptoms earliest and possibly more severe presentations. • could be hospitalized for similarly related illnesses and diseases. • smaller mass. • lower baseline cholinesterase activity. • tendency to bronchospasm. • pediatric airway and respiratory differences. • altered pulmonary compensation. • lower reserves of cardiovascular system and fluids. • isolated central nervous system signs (stupor, coma). • less miosis. • vulnerability to seizures and neurotransmitter imbalances (excitability). • immature metabolic systems. differential diagnoses include upper or lower airway obstruction, bronchiolitis, status asthmaticus, cardiogenic shock, acute gastroenteritis, seizures, and poisonings (carbon monoxide, organophosphates, and cyanide). diagnostic tests include acetylcholinesterase levels, red blood cell cholinesterase levels, and an arterial blood gas. treatment (tables . and . ) includes decontamination (reactive skin decontamination lotion ® [potassium , -butanedione monoximate], soap and water, and . % hypochlorite solution), supportive care, and administration of nerve agent antidotes (atropine, pralidoxime chloride, and diazepam). atropine is a competitive antagonist of acetylcholine muscarinic receptors and reverses peripheral muscarinic symptoms. it does not restore function at the neuromuscular junction nicotinic receptors. it does, however, treat early phases of convulsions. pralidoxime chloride separates the nerve agent from acetylcholinesterase and restores enzymatic function. it also binds free nerve agent. the major goal is to prevent "aging" of the enzyme (e.g., gd). diazepam provides treatment of nerve agent-induced seizures and prevents secondary neurologic injury. typically, associated seizures are refractory to other antiepileptic drugs. the antiseizure effect of diazepam is enhanced by atropine (banks ; cieslak and henretig ; messele et al. ) . potential medical countermeasures include trimedoxime (tmb ), hi- (an h-series oxime), obidoxime, "bioscavengers" (butyrylcholinesterase, carboxylesterase, organophosphorus acid anhydride hydrolase, and human serum paraoxonase), novel anticonvulsant drugs, n-methyl-d-aspartate (nmda) receptor antagonists (ketamine, dexanabinol), and common immunosuppressants such as cyclosporine a (jokanovic ; merrill et al. ; national institutes of health ; united states department of health and human services ). all patients should be observed closely for electroencephalographic changes and neuropsychiatric pathologies. polyneuropathy, reported after organophosphate insecticide poisoning, has not been reported in humans exposed to nerve agents and has been produced in animals only at unsurvivable doses. the intermediate syndrome has not been reported in humans after nerve agent exposure, nor has it been produced in animals. muscular necrosis has occurred in animals after high-dose nerve agent exposure but reversed within weeks; it has not been reported in humans (banks ). on march , , sergei skripal, a former russian double agent, and his daughter, yulia skripal, were found unresponsive on a park bench in salisbury, uk. they were brought to a nearby hospital and treated for signs consistent with a cholinergic crisis due to a nerve agent exposure. analysis of the skripals found the presence of a secret nerve agent called novichok. further testing found high concentrations of the agent on the front-door handle of his home. one of the investigating police officers, detective sergeant nick bailey, unknowingly touched the door-handle and also became ill. all three survived due to rapid recognition of the nerve agent exposure by hospital personnel. four months later, two other people, dawn sturgess and charlie rowley, became ill with identical symptoms in the town of amesbury, miles from salisbury. they were later confirmed to have high concentrations of novichok on their hands from a perfume bottle found in a recycling bin. both were immediately treated, but dawn sturgess later died. charlie rowley survived. it was believed the discarded perfume bottle contained novichok and was discarded by the assailants after the attempt on sergei skripal. on september , , the uk government revealed that their investigation uncovered two suspects from closed circuit television (cctv) footage near the skripal's home. the suspects entered the uk on russian passports using the names alexander petrov and ruslan boshirov, stayed in a london hotel for days, visited salisbury briefly, and then returned to moscow. minute traces of novichok were also found in the london hotel where they had stayed. the uk prime minister, teresa may, said that the suspects are thought to be officers from russia's military intelligence service the glavnoye razvedyvatel'noye upravleniye (gru), and that this showed that the poisoning was "not a rogue operation" and was "almost certainly" approved at a senior level of the russian state. the two suspects later appeared on russian tv denying the accusations and saying they were just "tourists" who had traveled all the way from moscow to salisbury just to see the "famous cathedral". however, cctv of the cathedral area found no evidence of the two men visiting the cathedral, although they were captured on cctv near the skripal's home. in a development in september , one of the men was revealed as actually being a russian intelligence officer named colonel anatoliy chepiga and was a decorated veteran of russian campaigns in chechnya and ukraine. and later in october, the second man was named as dr. alexander mishkin, a naval medical doctor allegedly recruited by the gru (chai et al. ; may ) . novichok (Новичоќ: russian for "newcomer") is a highly potent nerve agent developed from the russian classified nerve agent program known as foliant. almost everything known about these agents is due to a russian defector, vil mirzayanov ( ) who was an analytical chemist at the russian state research institute of organic chemistry and technology (gosniiokht). he has described the details of the novichok program in his book "state secrets: an insider's chronicle of the russian chemical weapons program". the first three nerve agents of the novichok series developed in the program were substance- , a- , and a- (table . ). they were synthesized as unitary agents, like vx, tabun, soman, and sarin. unitary means that the chemical structure was produced at its maximum potency. however, the novichok agents were developed as binary agents: maximum potency when two inert substances are combined together prior to deployment to create the active nerve agent (cieslak and henretig ) . very little is known about the chemistry of these weaponized organophosphate agents. however, they appear to be more potent than current nerve agents. for example, the ld of novichok agents is reported . μg/kg similar to -(dimethylamino)ethyl n,n-dimethylphosphoramidofluoridate (vg), a novel fourth generation nerve agent. furthermore, novichok- is × more effective than vx and novichock- is × more effective than soman (cieslak and henretig ; hoenig ) . clinically, they behave like other organophosphates by binding to acetylcholinesterase preventing the breakdown of acetylcholine thereby leading to a cholinergic crisis. there appears to be a similar "aging" process as seen with other nerve agents. in addition, the novichok agents binding to peripheral sensory nerves distinguishes this class of organophosphates. prolonged or high-dose exposure results in debilitating peripheral neuropathy. exposure to these agents is fatal unless aggressively managed (cieslak and henretig ) . decontamination is essential to prevent ongoing exposure to the patient and medical personnel. clothing should be removed and quickly placed in a sealed bag (prevents ongoing exposure to the emission of vapors) followed by thorough washing with soap and water. application of dry bleach powder should be avoided as it may hydrolyze nerve agents into toxic metabolites that can produce ongoing cholinergic effects. supportive care is essential. antidote therapy should be given as usual for nerve agents, including atropine, diazepam, and pralidoxime chloride (united states department of health and human services, office of the assistant secretary for preparedness and response, national library of medicine ; united states department of health and human services, chemical hazards emergency medical management (chemm) ). of note, the toxicity of the novichok agents may not rely on anticholinesterase inhibition. some have suggested that reactive oximes like potassium , -butanedione monoximate are preferred oximes for antidotal therapy (cieslak and henretig ) . cyanide is a naturally occurring chemical. it can be found in plants and seeds. it is also used in many industrial applications and is a common product of combustion of synthetic materials. typical cyanogens include hydrogen cyanide (ac) and cyanogen chloride (ck). low levels of cyanide are detoxified by a natural reaction in the human body using the rhodanese system. there is reversible metabolism with vitamin b a to vitamin b (cyanocobalamin). an irreversible reaction occurs with sulfanes to produce thiocyanates and sulfates. the former is excreted via the urinary tract. when cyanide overwhelms this natural process, cyanide binds to ( ) a vx = venom x (cieslak and henretig ) cytochrome oxidase within the mitochondria and disrupts cellular respiration. cyanide has an affinity for fe+ in the cytochrome a complex and oxidative phosphorylation is interrupted. cells can no longer use oxygen to produce atp and lactic acidosis ensues from resultant anaerobic metabolism. when inhaled, cyanide produces rapid onset of clinical signs. findings include transient tachypnea and kussmaul breathing (from hypoxia of carotid and aortic bodies), hypertension and tachycardia (from hypoxia of aortic body), and neurologic findings such as seizures, muscle rigidity (trismus), opisthotonus, and decerebrate posturing. other findings include cherry red flush, acute respiratory failure/ arrest, bradycardia, dissociative shock, and cardiac arrest. venous blood samples exhibit a bright red color. arterial blood gas may demonstrate a metabolic acidosis with an increased anion gap due to lactic acid (banks ; cieslak and henretig ; rotenberg a) . pediatric manifestations (table . ) may vary from the classic clinical responses due to their unique vulnerabilities (hilmas et al. ): • thinner integument leading to shorter time from exposure to symptom development. • higher vapor density (ck) and concentration accumulation in living zone of children, • higher minute ventilation and metabolism. • abdominal pain, nausea, restlessness, and giddiness are common early findings. • cyanosis mostly noted other than classic cherry red flushing of the skin. • resilient with recovery even when just using supportive measures alone. differential diagnoses include meningitis, encephalitis, gastroenteritis, ischemic stroke, methemoglobinemia, and poisonings (nerve agents, organophosphates, methanol, hydrogen sulfide, and carbon monoxide). diagnostic tests include arterial blood gas, lactic acid, and thiocyanate levels. treatment (tables . and . ) includes decontamination, supportive care, and administration of cyanide antidote kit (nitrites and thiosulfate). the nitrites facilitate the production of methemoglobinemia (fe+ ) which attracts cyanide molecules forming cyanmethemoglobin. amyl nitrite pearls are crushed into gauze and placed over the mouth/nose or in a mask used for bag/mask ventilation. sodium nitrite is given parenterally and dosed according to the patient's estimated hemoglobin so as to prevent severe methemoglobinemia. since the formation of cyanmethemoglobin is a reversible reaction, and sodium thiosulfate is given to extract the cyanide. dosing is also dependent upon estimated hemoglobin. along with the naturally occurring rhodanese enzymatic system, the irreversible reaction forms thiocyanate. thiocyanate is water soluble and is excreted harmlessly via the kidneys (banks ; cieslak and henretig ). potential medical countermeasures (national institutes of health ; united states army medical research institute of infectious diseases (usamriid) ) include hydroxocobalamin, cobinamide (a cobalamin precursor), dicobalt edetate, cyanohydrin-forming compounds (alpha-ketoglutarate and pyruvate), s-substituted crystallized rhodanese, sulfur-containing drugs (n-acetylcysteine), and methemoglobin inducers ( -dimethylaminophenol and others). blistering agents, or vesicants, promote the production of blisters. typical examples include sulfur mustard (hd), nitrogen mustard (hn), and lewisite (l). these agents, especially sulfur mustard, are considered capable chemical weapons since illness may not occur until hours or days later. vesicants are alkylating agents that affect rapidly reproducing and poorly differentiated cells in the body. however, they can also produce cellular oxidative stress, deplete glutathione stores, and promote immature cognitive function unable to flee emergency immature coping mechanisms inability to discern threat, follow directions, and protect self high risk for developing ptsd bbb blood-brain barrier, bsa body surface area, cns central nervous system, ptsd post-traumatic stress disorder (hilmas et al. ) intense inflammatory responses. clinical findings are initially cutaneous (erythema, pruritus, yellow blisters, ulcers, and sloughing), respiratory (hoarseness, cough, voice changes, pneumonia, respiratory failure, acute lung injury, and acute respiratory distress syndrome), and ophthalmologic (pain, irritation, blepharospasm, photophobia, conjunctivitis, corneal ulceration, and globe perforation) in nature. after exposure through these primary portals of entry, other sites are affected, including the gastrointestinal tract (nausea, vomiting, and mucosal injury), the hematopoietic system (bone marrow suppression), the cardiovascular system (l), reproductive system (hd, hn) , and the central nervous system (lethargy, headache, malaise, and depression) (banks ; yu et al. ) . pediatric manifestations (table . ) may vary from the classic clinical responses due to their unique vulnerabilities (hilmas et al. ): • thinner integument leading to shorter time from exposure to symptom development. • higher vapor density and concentration accumulation in the living zone. • higher minute ventilation and metabolism. • greater pulmonary injury. • ocular findings more frequent (less self-protection and more hand/eye contact). • gastrointestinal manifestations more prominent. • unable to escape and decontaminate. • unable to verbalize complaints (i.e., pain). treatment (tables . and . ) includes decontamination and supportive care. currently, there are no antidotes for mustard toxicity (cieslak and henretig ) . agents under investigation include antioxidants (vitamin e), anti-inflammatory drugs (corticosteroids), mustard scavengers (glutathione, n-acetylcysteine), and nitric oxide synthase inhibitors (l-nitroarginine methyl ester). other therapeutics under investigation include the use of british anti-lewisite (bal), reactive skin protectants, and ocular therapies (national institutes of health ; usamriid ). lung-damaging agents are toxic inhalants and potentially can affect the entire respiratory tract. typical examples include chlorine (cl ), phosgene (carbonyl chloride), oxides of nitrogen, organofluoride polymers, hydrogen fluoride, and zinc oxide. since many of these chemicals are readily available and have multiple industrial applications, they are considered terrorist weapons of opportunity. toxicity is dependent upon agent particle size, solubility, and method of release. large particles produce injury in the nasopharynx (sneezing, pain, and erythema). midsize particles affect the central airways (painful swelling, cough, stridor, wheezing, and rhonchi). small particles cause injury at the level of the alveoli (dyspnea, chest tightness, and rales). highly soluble agents, such as chlorine, dissolve with mucosal moisture and immediately produce strong upper airway reactions. less soluble agents, such as phosgene, travel to the lower airway before dissolving and subsequently causing toxicity. it is important, however, to realize that very few lungdamaging agents affect only the upper or lower airway (e.g., cl ). if the agent is aerosolized, solid or liquid droplets suspend in the air and distribute by size. if it is a gas or vapor release, there is uniform distribution throughout the lungs and toxicity will be based on solubility and reactivity of the agent (banks ; burklow et al. ; cieslak and henretig ) . pediatric manifestations (table . ) may vary from the classic clinical responses due to their unique vulnerabilities (hilmas et al. ): • pediatric airway and respiratory tract issues (obligate nose breathers, relatively small mouth/large tongue, copious secretions, anterior/cephalad vocal cords, omega or horseshoe-shaped epiglottis, tendency of laryngospasm and bronchospasm, and anatomically small, "floppy" airways). • high vapor density and concentration accumulation in the living zone. • unable to verbalize or localize physical complaints. • rapid dehydration and shock secondary to pulmonary edema. • increased minute ventilation and metabolism. differential diagnoses include smoke inhalation injury, cardiogenic shock, heart failure, traumatic injury, asthma, bronchiolitis, and poisoning (cyanide). treatment (tables . and . ) includes decontamination and supportive care. currently, there are no antidotes for lung-damaging agent toxicity (cieslak and henretig ) . potential countermeasures include novel positive-pressure devices, drugs to prevent lung inflammation, and treatments for chemically induced pulmonary edema (beta agonists, dopamine, insulin, allopurinol, and ibuprofen). in addition, drugs are being investigated that act at complex molecular pathways of the lung the centers for disease control and prevention (cdc) has delineated bioterrorism agents and diseases into three categories based on priority. category a agents include organisms with the highest risk because the ease of dissemination or transmission from person-to-person, result in high mortality rates, have the potential for major public health impact, promote public panic and social disruption, and require special action of public health preparedness. these agents/diseases include smallpox (variola major), anthrax (bacillus anthracis), plague (yersinia pestis), viral hemorrhagic fevers (filoviruses [ebola, marburg] and arenaviruses [lassa, macupo]), botulinum toxin (from clostridium botulinum), and tularemia (francisella tularensis). category b agents, the second highest priority, include those that are moderately easy to disseminate, result in moderate morbidity and low mortality rates, and require specific enhancements of diagnostic capacity and enhanced disease surveillance. these agents/diseases include ricin toxin (ricinus communis), brucellosis (brucella species), epsilon toxin of clostridium perfringens, food safety threats (salmonella species, escherichia coli o :h , shigella), glanders (burkholderia mallei), meliodosis (burkholderia pseudomallei), psitticosis (chlamydia psittaci), typhus fever (rickettsia prowazekii), q fever (coxiella burnetii), staphylococcal enterotoxin b, trichothecenes mycotoxin, viral encephalitis (alphaviruses, such as eastern equine encephalitis, venezuelan equine encephalitis, and western equine encephalitis), and water safety threats (vibrio cholera, cryptosporidium parvum). category c agents have the next priority and include emerging pathogens that could be engineered for mass dissemination because of availability, ease of production and dissemination, and have the potential for high morbidity and mortality rates and major health impact. recognition of a biologic attack is essential. there are various epidemiologic clues to consider when determining whether the outbreak is natural or man-made (markenson et al. ; cieslak ; usamriid ) : • the appearance of a large outbreak of cases of a similar disease or syndrome, or especially in a discrete population. • many cases of unexplained diseases or deaths. • more severe disease than is usually expected for a specific pathogen or failure to respond to standard therapy. • unusual routes of exposure for a pathogen, such as the inhalational route for disease that normally occur through other exposures. • a disease case or cases that are unusual for a given geographic area or transmission season. • disease normally transmitted by a vector that is not present in the local area. • multiple simultaneous or serial epidemics of different diseases in the same population. • a single case of disease by an uncommon agent (smallpox, some viral hemorrhagic fevers, inhalational anthrax, pneumonic plague). • a disease that is unusual for an age group. • unusual strains or variants of organisms or antimicrobial resistance patterns different from those known to be circulating. • a similar or identical genetic type among agents isolated from distinct sources at different times and/or locations. • higher attack rates among those exposed in certain areas, such as inside a building if released indoors, or lower rates in those inside a sealed building if released outside. • outbreaks of the same disease occurring in noncontiguous areas. • zoonotic disease outbreaks. • intelligence of a potential attack, claims by a terrorist or aggressor of a release, and discovery of munitions, tampering, or other potential vehicle of spread (spray device, contaminated letter). one should know the cellular, physiological, and clinical manifestations of each biologic agent. furthermore, knowledge of distinct presentation patterns of children will be helpful to diagnosis. in any event, the ten steps in the management of biologic attack victims, pediatric, or otherwise, should be applied (cieslak and henretig ; cieslak ; usamriid smallpox is caused by the orthopoxvirus variola and was declared globally eradicated in . the disease is highly communicable from person-to-person and remains a threat due to its potential for weaponization. the only stockpiles are at the cdc and at the russian state centre for research on virology and biotechnology. however, clandestine stockpiles in other parts of the world are unknown. since the cessation of smallpox vaccination, the general population has little or no immunity. the three clinical forms of smallpox include ordinary, flat, and hemorrhagic. another form, modified type, occurred in those previously vaccinated who were no longer protected. the asymptomatic incubation period is from to days (average days) after exposure. a prodrome follows that lasts for - days and is marked by fever, malaise, and myalgia. lesions start on the buccal and pharyngeal mucosa. the rash then spreads in a centrifugal fashion, and the lesions are synchronous. initially, there are macules followed by papules, pustules, and scabs in - weeks. other clinical features include extensive fluid loss and hypovolemic shock, nausea, vomiting, diarrhea, bacterial superinfections, viral bronchitis and pneumonitis, corneal ulceration with or without keratitis, and encephalitis. death, if it occurs, is typically during the second week of clinical disease. variola minor caused a mortality of % in unvaccinated individuals. however, the variola major type caused death in % and % in those vaccinated and unvaccinated, respectively. flat (mostly children) and hemorrhagic (pregnant women and immunocompromised) types caused severe mortality in those populations infected. the differential diagnoses for smallpox include chickenpox (varicella), herpes, erythema multiforme with bullae, or allergic contact dermatitis. varicella typically has a longer incubation period ( - days) and minimal or no prodrome. furthermore, the rash distributes in a centripetal fashion and the progression is asynchronous (images . and . ). diagnosis of smallpox is mostly clinical (centers for disease control and prevention a). if considered, contact public health immediately. laboratory confirmation (cdc or who) can be done by dna sequencing, polymerase chain reaction (pcr), restriction fragment-length polymorphism (rflp), real-time pcr, and microarrays. these are more sensitive and specific than the conventional virological and immunological approaches (goff et al. ) . generally, treatment is largely supportive (table . ). fluid losses and hypovolemic shock must be addressed. also, due to electrolyte and protein loss, replacement therapy will be required. bacterial superinfections must be aggressively treated with appropriate antibiotics. biologic countermeasures and antivirals against smallpox are under investigation, including cidofovir, brincidovir (cmx- ), and tecovirimat (st- ). these agents have shown efficacy in orthopoxvirus animal models and have been used to treat disseminated vaccinia infection under emergency use. cidofovir has activity against poxviruses in animal studies (in vitro and in vivo) and some humans (eczema vaccinatum and molluscum contagiosum). brincidovir is an oral formulation of cidofovir with less nephrotoxicity and has recently been announced as an addition to the strategic national stockpile (sns) for patients with smallpox. tecovirimat is a potent and specific inhibitor of orthopoxvirus replication. a recent study found that treatment with tecovirimat resulted in % survival of cynomolgus macaques challenged with intravenous variola virus. the disease was milder in tecovirimat-treated survivors and viral shedding was reduced compared to placebo-treated survivors. prophylaxis comes in the form of the smallpox vaccine (vaccinia virus), acam ® , which replaced wyeth dryvax™ in . safety profile of the two vaccines appears to be similar. side effects of vaccination range from low-grade fever and axillary lymphadenopathy to inadvertent inoculation of the virus to other body sites to generalized vaccinia and cardiac events (myopericarditis). rare, but typically fatal complications include progressive vaccinia, eczema vaccinatum, postvaccination encephalomyelitis, and fetal vaccinia. modified vaccinia ankara (mva) smallpox vaccine (bavarian nordic's imvamune ® ) is a live, highly attenuated, viral vaccine that is under development as a future nonreplicating smallpox vaccine (greenberg et al. ; kennedy and greenberg ). passive immunoprophylaxis exists in the form of vaccinia immune globulin (vig) and is used for primarily treating complications from smallpox vaccine. limited information suggests that vig may be of use in postexposure prophylaxis of smallpox if given the first week after exposure and with vaccination. monoclonal antibodies may represent another form of immunoprophylaxis. postexposure administration of human monoclonal antibodies has protected rabbits from a lethal dose of an orthopoxvirus. as mentioned, smallpox is highly communicable person-to-person (table . ). contact precautions with full personal protective equipment (ppe) are required. airborne isolation with the use of an n- mask is needed for baseline protection. an n- mask or powered airpurifying respirator (papr) is recommended for protection during high risk procedures (beigel and sandrock ; goff et al. ; rotz et al. ; pittman et al. ; usamriid ). anthrax is caused by the aerobic, spore-forming, nonmotile, encapsulated gram-positive rod bacillus anthracis. it is a naturally occurring disease in herbivores. humans contract the illness by handling contaminated portions of infected animals, especially hides and wool. infection is introduced by scratches or abrasions on the skin. there is concern for potential aerosol dispersal leading to intentional infection through inhalation: it is fairly easy to obtain, capable of large quantity production, stable in aerosol form, and highly lethal. anthrax spores enter the body via skin, ingestion, or inhalation. the spores germinate inside macrophages and become vegetative bacteria. the vegetative form is released, replicates in the lymphatic system, and produces intense bacteremia. the production of virulence factors leads to overwhelming sepsis. the main virulence factors are encoded on two plasmids. one produces an antiphagocytic polypeptide capsule. the other contains genes for the synthesis of three proteins it secretes: protective antigen, edema factor, and lethal factor. the combination of protective antigen with lethal factor or edema factor forms binary cytotoxins, lethal toxin, and edema toxin. the anthrax capsule, lethal toxin, and edema toxin act in concert to drive the disease. three clinical syndromes occur with anthrax: cutaneous, gastrointestinal, and inhalational. cutaneous anthrax is the most common naturally occurring form. after an individual is exposed to infected material or the agent itself, there is a - day (average days) incubation period. a painless or pruritic papule forms at the site of exposure. the papule enlarges and forms a central vesicle, which is followed by erosion into a coal-black but painless eschar. edema surrounds the area and regional lymphadenopathy may occur. gastrointestinal anthrax is rare. typically, it develops after ingestion of viable vegetative organisms found in undercooked meats of infected animals. the two forms of gastrointestinal anthrax, oropharyngeal and intestinal, have incubation periods of - days. the oropharyngeal form is marked by fever and severe pharyngitis followed by ulcers and pseudomembrane formation. other findings include dysphagia, regional lymphadenopathy, unilateral neck swelling, airway compromise, and sepsis. the intestinal form begins with fever, nausea, vomiting, and abdominal pain. bowel edema develops which leads to mesenteric lymphadenitis with necrosis, shock, and death. endemic inhalational anthrax (woolsorters' disease) is also extremely rare and is due to inhaling spores. therefore, any case of inhalational anthrax should be assumed to be due to intentional exposure until proven otherwise. the incubation period is - days but can be up to days. there is a prodrome of - days consisting of fever, malaise, and cough. within h, the disease rapidly progresses to respiratory failure, hemorrhagic mediastinitis (wide mediastinum), septic shock, multiorgan failure, and death. patients with inhalational anthrax may also have hemorrhagic meningitis. mortality is greater than % in - h despite aggressive treatment of inhalational anthrax. the differential diagnoses of ulceroglandular lesions include antiphospholipid antibody syndrome, brown recluse spider bite, coumadin/heparin necrosis, cutaneous leishmaniasis, cutaneous tuberculosis, ecthyma gangrenosum, glanders, leprosy, mucormycosis, orf, plague, rat bite fever, rickettsial pox, staphylococcal/ streptococcal ecthyma, tropical ulcer, tularemia, and typhus. the differential diagnoses of ulceroglandular syndromes include cat scratch fever, chancroid, glanders, herpes, lymphogranuloma venereum, melioidosis, plague, staphylococcal and streptococcal adenitis, tuberculosis, and tularemia. the differential diagnoses for inhalational anthrax include influenza and influenza-like illnesses from other causes. the differential diagnoses of mediastinal widening include normal variant, aneurysm, histoplasmosis, sarcoidosis, tuberculosis, and lymphoma. the diagnosis of anthrax is by culture and gram stain of the blood, sputum, pleural fluid, cerebrospinal fluid, or skin. specimens must be handled carefully, especially by lab personnel and those performing autopsies. elisa and pcr are available at some reference laboratories. the chest radiograph of inhalational anthrax shows the classic widening of the mediastinum. additional findings include hemorrhagic pleural effusions, air bronchograms, and/or consolidation (purcell et al. ). supportive treatment is indicated, including mechanical ventilation, pleural effusion drainage, fluid and electrolyte support, and vasopressor administration. for inhalational anthrax, antibiotic treatment is unlikely to be effective unless started before respiratory symptoms develop. treatment (table . ) includes ciprofloxacin (or levofloxacin or doxycycline), clindamycin, and penicillin g. raxibacumab, a monoclonal antibody, was approved by the fda in for the treatment of inhalational anthrax in combination with recommended antibiotic regimens and prophylaxis for inhalational anthrax when other therapies are unavailable or inappropriate. it works by inhibiting anthrax antigen binding to cells and, therefore, prevents toxins from entering cells (kummerfeldt ) . the adult dose is mg/kg given iv over h and min. the dose for children is weight based; ≤ kg: mg/kg; > - kg: mg/kg; > kg: mg/kg. premedication with diphenhydramine iv or po is recommended h before the infusion. it can also be used as postexposure prophylaxis in high risk spore exposure cases (cieslak and henretig ; migone et al. ; the medical letter ). obiltoxaximab (anthim) is a recently approved monoclonal antibody treatment for inhalational anthrax in combination with recommended antibiotic regimens and prophylaxis for inhalational anthrax when other therapies are unavailable or inappropriate. adults and children > kg should receive a single obiltoxaximab dose of mg/kg. the recommended dose is mg/kg for children > - kg and mg/kg for those weighing ≤ kg. premedication with diphenhydramine is recommended to reduce risk of hypersensitivity reactions (the medical letter ). in patients with inhalational anthrax, intravenous anthrax immune globulin (anthrasil) should be considered in addition to appropriate antibiotic therapy (mytle et al. ; the medical letter ; usamriid ). postexposure prophylaxis includes ciprofloxacin (or levofloxacin or doxycycline) for days plus administration of vaccine; since spores can persist in human in addition to appropriate antibiotic regimen, monoclonal antibody therapy (see text for dosing) and intravenous anthrax immune globulin should be administered for inhalational anthrax c levofloxacin or ofloxacin may be an acceptable alternative to ciprofloxacin d rifampin or clarithromycin may be acceptable alternatives to clindamycin as a drug that targets bacterial protein synthesis. if ciprofloxacin or another quinolone is employed, doxycycline may be used as a second agent because it also targets protein synthesis e ampicillin, imipenem, meropenem, or chloramphenicol may be acceptable alternatives to penicillin as drugs with good cns penetration f assuming the organism is sensitive, children may be switched to oral amoxicillin ( - mg/kg/d divided q h) to complete a -day course. the first days of therapy of postexposure prophylaxis, however, should include ciprofloxacin or levofloxacin and/or doxycycline regardless of age. vaccination should also be provided; if not, antibiotic course will need to be longer g according to most experts, ciprofloxacin is the preferred agent for oral prophylaxis h ten days of therapy may be adequate for endemic cutaneous disease. a full -day course is recommended in the setting of terrorism, however, because of the possibility of concomitant inhalational exposure tissues for a long time, antibiotics must be given for a longer period if vaccine is not also given. the anthrax vaccine adsorbed (ava biothrax™) is derived from sterile culture fluid supernatant taken from an attenuated strain of bacillus anthracis and does not contain any live or dead organisms. the vaccine is given . ml intramuscularly at and weeks then at , , and months followed by yearly boosters (pittman et al. ; usamriid ) . consult with cdc for current pediatric recommendations. anthrax is not contagious in the vegetative form during clinical illness (table . ). contact with infected animals increases likelihood of spread. therefore, contact should be limited and the use of appropriate ppe in endemic areas is indicated (beigel and sandrock ; purcell et al. ; usamriid ) . plague is caused by yersinia pestis, a nonmotile, nonsporulating gram-negative bacterium. it is a zoonotic disease of rodents. it is typically found worldwide and is endemic in western and southwestern states. humans develop the disease after contact with infected rodents, or being bitten by their fleas. after a rodent population dies off, the fleas search for other sources of blood, namely humans. this is when large outbreaks of human plague occur. pneumonic plague is a very rare disease and when it is present in a patient, it may be highly suspicious for intentional dispersal of this deadly agent. three clinical syndromes occur with plague: bubonic plague ( %), septicemic plague ( %), and primary pneumonic plague ( - %). bubonic plague occurs after an infected flea bites a human. after an incubation period of - days, there is onset of high fever, severe malaise, headache, myalgias, and nausea with vomiting. almost % have abdominal pain. around the same time, a characteristic bubo forms which is tender, erythematous, and edematous without fluctuation. buboes typically form in the femoral or inguinal lymph nodes, but other areas can be involved as well (axillary, intraabdominal). the spleen and liver can be tender and palpable. the disease disseminates without therapy. severe complications can ensue, including pneumonia, meningitis, sepsis, and multiorgan failure. pneumonia is particularly concerning since these patients are extremely contagious. mortality of untreated bubonic plague is %, but % with efficient and effective treatment. septicemic plague is characterized by acute fever followed by sepsis without bubo formation. the clinical syndrome is very similar to other forms of gram-negative sepsis: chills, malaise, tachycardia, tachypnea, hypotension, nausea, vomiting, and diarrhea. in addition to sepsis, disseminated intravascular coagulation can ensue leading to thrombosis, necrosis, gangrene, and the formation of black appendages. multiorgan failure can quickly follow. untreated septicemic plague is almost % fatal versus - % in those treated. pneumonic plague is very rare and should be considered due to an intentional aerosol release until proven otherwise. the incubation period is relatively short at - days. sudden fever, cough, and respiratory failure quickly follow. this form produces a fulminant pneumonia with watery sputum that usually progresses to bloody. within a short period of time, septic shock and disseminated intravascular coagulation develop. ards and death may occur. mortality rate of pneumonic plague is very high but may respond to early treatment. plague meningitis is a rare complication of plague. it can occur in % of patients with septicemia and pneumonic forms and is more common in children. usually occurring a few weeks into the illness, it affects those receiving subtherapeutic doses of antibiotics or bacteriostatic antibiotics that do not cross the blood-brain barrier (tetracyclines). fever, meningismus, and other meningeal signs occur. plague meningitis is virtually indistinguishable from meningococcemia. the differential diagnoses of bubonic plague include tularemia, cat scratch fever, lymphogranuloma venereum, chancroid, scrub typhus, and other staphylococcal and streptococcal infections. the differential diagnoses of septicemic plague should include meningococcemia, other forms of gram-negative sepsis, and rickettsial diseases. the differential diagnosis of pneumonic plague is very broad. however, sudden appearance of previously healthy individuals with rapidly progressive gram-negative pneumonia with hemoptysis should strongly suggest pneumonic plague due to intentional release. diagnosis can be made clinically as previously described. demonstration of yersinia pestis in blood or sputum is paramount. methylene blue or wright's stain of exudates may reveal the classic safety-pin appearance of yersinia pestis. culture on sheep blood or macconkey agar demonstrates beaten-copper colonies ( h) followed by fried-egg colonies ( h). detection of yersinia pestis f -antigen by specific immunoassay is available, but the result is available retrospectively. chest radiograph of patients will demonstrate patchy infiltrates (centers for disease control and prevention a; worsham et al. ) . treatment includes mechanical ventilation strategies for ards, hemodynamic support (fluid and vasopressor administration), and antimicrobial agents (table . ). gentamicin or streptomycin is the preferred antimicrobial treatment. alternatives include doxycycline or ciprofloxacin or levofloxacin or chloramphenicol. in cases of meningitis, chloramphenicol is recommended due to its ability to effectively cross the blood-brain barrier. streptomycin is in limited supply and is available for compassionate use. it should be avoided in pregnant women. postexposure prophylaxis includes doxycycline or ciprofloxacin. no licensed plague vaccine is currently in production. a previous licensed vaccine was used in the past. it only offered protection against bubonic plague but not aerosolized yersinia pestis. the plague bacterium secretes several virulence factors (fraction (f ) and v (virulence) proteins) that as subunit proteins are immunogenic and possess protective properties. recently, an f -v antigen (fusion protein) vaccine developed by usamriid provided % protection in monkeys against high-dose aerosol challenge. there is no passive immunoprophylaxis (i.e., immune globulin) available for pre-or postexposure of plague (usamriid ). use of standard precautions for patients with bubonic and septicemic plague is indicated. suspected pneumonic plague will require strict isolation with respiratory droplet precautions for at least h after initiation of effective antimicrobial therapy, or until sputum cultures are negative in confirmed cases. an n- respirator should be used for baseline protection (table . ). it is also recommended to use an n- respirator or papr for high risk procedures (beigel and sandrock ; ; centers for disease control and prevention ; centers for disease control and prevention b; pittman et al. ; usamriid ) . in a mass casualty setting, parenteral therapy might not be possible. in such cases, oral therapy (with analogous agents) may need to be used b ofloxacin (and possibly other quinolones) may be acceptable alternatives to ciprofloxacin or levofloxacin; however, they are not approved for use in children c concentration should be maintained between and μg/ml. some experts have recommended that chloramphenicol be used to treat patients with plague meningitis, because chloramphenicol penetrates the blood-brain barrier. use in children younger than may be associated with adverse reactions but might be warranted for serious infections d ribavirin is recommended for arenavirus or bunyavirus infections and may be indicated for a viral hemorrhagic fever of an unknown etiology although not fda approved for these indications. for intravenous therapy use a loading dose: kg iv once (max dose, g), then mg/kg iv q h for days (max dose, g), and then mg/kg iv q h for days (max dose, mg). in a mass casualty setting, it may be necessary to use oral therapy. for oral therapy, use a loading dose of mg/kg po once, then mg/kg/day po in divided doses for days viral hemorrhagic fever has a variety of causative agents. however, the syndromes they produce are characterized by fever and bleeding diathesis. the etiologies include rna viruses from four distinct families: arenaviridae, bunyaviridae, filoviridae, and flaviviridae. the filoviridae (includes ebola and marburg) and arenaviridae (includes lassa fever and new world viruses) are category a agents. based on multiple identified characteristics, there is strong concern for the weaponization potential of the viral hemorrhagic fevers. specifically, there has been demonstration of high contagiousness in aerosolized primate models. there are five identified ebola species, but only four are known to cause disease in humans. the natural reservoir host of ebola virus remains unknown. however, on the basis of evidence and the nature of similar viruses, researchers believe that the virus is animal-borne and that bats are the most likely reservoir. four of the five virus strains occur in an animal host native to africa. marburg virus has a single species. geographic distribution of ebola and marburg is africa (fitzgerald et al. ). both diseases are very similar clinically. incubation period is typically - days with a range of - days. symptoms may include fever, chills, headache, myalgia, nausea, and vomiting. there is rapid progression to prostration, stupor, and hypotension. the onset of a maculopapular rash on the arms and trunk is classic. disseminated intravascular coagulation and thrombocytopenia develops with conjunctival injection, petechiae, hemorrhage, and soft tissue bleeding. there is a possible central nervous system and hepatic involvement. bleeding, uncompensated shock, and multiorgan failure are seen. high viral load early in course is associated with poor prognosis. death usually occurs during the second week of illness. mortality rate of marburg is - % and for ebola - %. in a retrospective cohort study of children during the / ebola outbreak in liberia and sierra leone (all less than years with a median age of years with one-third less than years of age), the most common features upon presentation were fever, weakness, anorexia, and diarrhea. about % were initially afebrile. bleeding was rare upon initial presentation. the overall case fatality rate was %. factors associated with death included children less than years of age, bleeding at any time during hospitalization, and high viral load (smit et al. ) . in another retrospective cohort study of children at two ebola centers in sierra leone in (all less than years of age), presenting symptoms included weakness, fever, anorexia, diarrhea, and cough. about % were afebrile on presentation. the case fatality rate was higher in children less than years ( %) versus - years of age ( %) and times more likely to die if child had a higher viral load. signs associated with death included fever, emesis, and diarrhea. interestingly, hiccups, bleeding, and confusion were only observed in children who died (shah et al. ) . lassa virus and new world viruses (junin, machupo, sabia, and guanarito) are transmitted from person-to-person. the vector in nature is the rodent. the incubation period is from to days. the geographical distribution is west africa and south america, respectively. the south american hemorrhagic fevers are quite similar but differ from lassa fever. the onset of the south american viruses is insidious and results in high fever and constitutional symptoms. petechiae or vesicular enanthem with conjunctival injection is common. these fevers are associated with neurologic disease (hyporeflexia, gait abnormalities, and cerebellar dysfunction). seizures portend a poor prognosis. mortality ranges from % to over %. on the contrary, lassa viruses are mild. less than % of infections result in severe disease. signs include chest pain, sore throat, and proteinuria. hemorrhagic disease is uncommon. other features include neurologic disease such as encephalitis, meningitis, cerebellar disease, and cranial nerve viii deafness (common feature). mortality can be as high as %. differential diagnoses include malaria, meningococcemia, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and typhoid fever. diagnosis is through detection of the viral antigen testing by elisa or viral isolation by culture at the cdc. no specific therapy is present and generally involves supportive care, especially mechanical ventilation strategies for ards, hemodynamic support, and renal replacement therapy. for the arenaviridae and bunyaviridae groups, ribavirin may be indicated ( (pittman et al. ) . there is no current vaccine for ebola that is licensed by the fda. an experimental vaccine called rvsv-zebov was found to be highly protective against ebola virus in a trial conducted by the world health organization (who) and other international partners in guinea in . fda licensure for the vaccine is expected in . until then, , doses have been committed for an emergency use stockpile under the appropriate regulatory mechanism in the event and an outbreak occurs before fda approval is received (centers for disease control and prevention b; henao-restrepo et al. ) . another ebola vaccine candidate, the recombinant adenovirus type- ebola vaccine, was evaluated in a phase trial in sierra leone in . an immune response was stimulated by this vaccine within days of vaccination and strict contact precautions (hand hygiene, double gloves, gowns, shoe and leg coverings, and face shield or goggles) and droplet precautions (private room or cohorting, surgical mask within ft) are mandatory for viral hemorrhagic fevers. airborne precautions (negative-pressure isolation room with - air exchanges per h) should also be instituted to the maximum extent possible and especially for procedures that induce aerosols (e.g., bronchoscopy). at a minimum, a fit-tested, hepa filter-equipped respirator (e.g., an n- mask) should be used, but a battery-powered papr or a positive pressure-supplied air respirator should be considered for personnel sharing an enclosed space with, or coming within ft of, the patient. multiple patients should be cohorted in a separate ward or building with a dedicated airhandling system when feasible (table . ). environmental decontamination is accomplished with hypochlorite or phenolic disinfectants (beigel and sandrock ; radoshitzky et al. ; usamriid ; won and carbone ) . francisella tularensis, a small aerobic, nonmotile gram-negative coccobacillus, causes tularemia (rabbit fever). clinical disease is caused by two isolates, biovars jellison type a and b. this organism can be stabilized for weaponization and delivered in a wet or dry form. the incubation period is usually - days (range - days). initial symptoms are nonspecific and mimic the flu-like symptoms or other upper respiratory tract infections. there is acute onset of fever with chills, myalgias, cough, fatigue, and sore throat. the two clinical forms of tularemia are typhoidal and ulceroglandular diseases. typhoidal tularemia ( - %) occurs after inhalational exposure and sometimes intradermal or gastrointestinal exposures. there is abrupt onset of fever, headache, malaise, myalgias, and prostration. it presents without lymphadenopathy. nausea, vomiting, and abdominal pain are sometimes present. untreated, there is a % mortality rate in naturally acquired cases (vs. - % in those treated). it is higher if pneumonia is present. this form would be most likely seen during an aerosol release of the agent. ulceroglandular tularemia ( - %) occurs through skin or mucus membrane inoculation. there is abrupt onset of fever, chills, headache, cough, and myalgias along with a painful papule at the site of exposure. the papule becomes a painful ulcer with tender regional lymph nodes. skin ulcers have heaped up edges. in - %, there is focal lymphadenopathy without an apparent ulcer. lymph nodes may become fluctuant and drain when receiving antibiotics. without treatment, they may persist for months or even years. in some cases ( - %), the primary entry port is the eye leading to oculoglandular tularemia. patients have unilateral, painful, and purulent conjunctivitis with local lymphadenopathy. chemosis, periorbital edema, and small nodular granulomatous lesions or ulceration may be found. oropharyngeal tularemia with pharyngitis may occur in % of patients. findings include exudative pharyngitis/tonsillitis, ulceration, and painful cervical lymphadenopathy. the differential diagnosis is antibiotic unresponsive pharyngitis, infectious mononucleosis, and viral pharyngitis. pulmonary involvement ( - %) is seen in naturally occurring disease. it ranges from mild to fulminant. various processes include pneumonia, bronchiolitis, cavitary lesions, bronchopleural fistulas, and chronic granulomatous diseases. left untreated, % will die. differential diagnoses include those for typhoidal (typhoid fever, rickettsia, and malaria) or pneumonic (plague, mycoplasma, influenza, q-fever, and staphylococcal enterotoxin b) tularemia. diagnosis should be considered when there is a cluster of nonspecific, febrile, systemically ill patients who rapidly progress to fulminant pneumonitis. tularemia can be diagnosed by recovering the organism from sputum (pcr or dfa) or serology at a state health laboratory. chest radiograph is nonspecific with possible hilar adenopathy. treatment is streptomycin or gentamicin (table . ). alternatives include doxycycline, ciprofloxacin, or chloramphenicol. a live-attenuated vaccine (ndbr ) exists and typically used for laboratory personnel working with francisella tularensis. there is no passive immunoprophylaxis. ciprofloxacin or doxycycline can be given as pre-and postexposure prophylaxis (beigel and sandrock ; hepburn et al. ; pittman et al. ; usamriid ) . botulinum neurotoxins (bont) are produced from the spore-forming, gram-positive, obligate anaerobe clostridium botulinum. it is the most potent toxin known to man. a lethal dose is ng per kilogram. it is , times more toxic than sarin (gb). there are seven serotypes of botulinum toxin (a through g). a new serotype (h) has been tentatively identified in a case of infant botulism but has not been fully investigated. most common are serotypes a, b, and e. the toxin acts on the presynaptic nerve terminal of the neuromuscular junction and cholinergic autonomic synapses. this disrupts neurotransmission and leads to clinical findings. there are three forms of botulism: foodborne, wound, and intestinal (infant or adult intestinal). botulinum toxin can also be released as an act of bioterrorism via ingestion or aerosol forms. incubation can be from h after exposure to several days later. clinical findings of botulism include cranial nerve palsies such as ptosis, diplopia, and dysphagia. this is followed by symmetric descending flaccid paralysis. however, the victim remains afebrile, alert, and oriented. death is typically due to respiratory failure. prolonged respiratory support is often required ( - months). differential diagnoses include guillain-barre syndrome, myasthenia gravis, tick paralysis, stroke, other intoxications (nerve gas, organophosphates), inflammatory myopathy, congenital and hereditary myopathies, and hypothyroidism. diagnosis is mostly clinical. laboratory confirmation can be obtained by bioassay of patient's serum. other assays include immunoassays for bacterial antigen, pcr for bacterial dna, and reverse transcriptase-pcr for mrna to detect active synthesis of toxin. cerebrospinal fluid demonstrates normal protein (unlike guillain-barre syndrome). emg reveals augmentation of muscle action potential with repetitive nerve stimulation at - hz. treatment (table . ) is mainly supportive including intubation and ventilator support. tracheostomy may be required due to prolonged respiratory weakness and failure. antibiotics do not play a role in treatment. botulism antitoxin heptavalent [a, b, c, d, e, f, g]-equine (bat) was approved by the fda in . bat was developed at usamriid as one of two equine-derived heptavalent bont antitoxins. bat is approved to treat individuals with symptoms of botulism following a known or suspected exposure. it has the potential to cause hypersensitivity reactions in those sensitive to equine proteins. the safety of bat in pregnant and lactating women is unknown. evidence regarding safety and efficacy in the pediatric population is limited. in , the fda approved botulinum immune globulin intravenous (babybig), a human botulism immune globulin derived from pooled plasma of adults immunized with pentavalent botulinum toxoid. it is indicated for the treatment of infants with botulism from toxin serotypes a and b. immediately after clinical diagnosis of botulism, adults (including pregnant women) and children should receive a single intravenous infusion of antitoxin (bat or, for infants with botulism from serotypes a or b, babybig) to prevent further disease progression. the administration of antitoxin should not be delayed for laboratory testing to confirm the diagnosis. the pentavalent toxoid vaccine (previously for protection against a, b, c, d, and e; but not f or g) is no longer available as of . no replacement vaccine is currently available. standard isolation precautions (table . ) should be followed (beigel and sandrock ; dembek et al. ; pittman et al. ; timmons and carbone ; usamriid ). ricin is a potent cytotoxin derived from the castor bean plant ricinus communis. it is related in structure and function to shiga toxins and shiga-like toxin of shigella dysenteriae and escherichia coli, respectively. it consists of two glycoprotein subunits, a and b, connected by a disulfide bond. the b-chain allows the toxin to bind to cell receptors and gain entrance into the cell. once ricin enters the cell, the disulfide chemical linkage is broken. the free a chain then acts as an enzyme and inactivates ribosomes thereby disrupting normal cell function. cells are incapable of survival and soon die. ricin has a high terrorist potential due to it characteristics: readily available, ease of extraction, and notoriety (maman and yehezkelli ) . three modes of exposure exist: oral, inhalation, and injection. four to eight hours after inhalation exposure, the victim develops fever, chest tightness, cough, dyspnea, nausea, and arthralgias. airway necrosis and pulmonary capillary leak ensues within - h. this is followed quickly by severe respiratory distress, ards, and death due to hypoxemia within - h. injection may cause minimal pulmonary vascular leak. pain at the site and local lymphadenopathy may occur. however, it may be followed by nausea, vomiting, and gastrointestinal hemorrhage. ingestion leads to necrosis of the gastrointestinal mucosa, hemorrhage, and organ necrosis (spleen, liver, and kidney). diagnosis is suspected when multiple cases of acute lung injury occur in a geographic cluster. serum and respiratory secretions can be checked for antigen using elisa. pulmonary intoxication is managed by mechanical ventilation. gastrointestinal toxicity is managed by gastric lavage and use of cathartics. activated charcoal has little value due to the size of ricin molecules. supportive care is indicated for injection exposure. in general, treatment is largely supportive, especially for pulmonary edema that can result from the capillary leak. there is no vaccine available or prophylactic antitoxin for human use. however, there are two ricin vaccines in the development that focus on the ricin toxin a (rta) chain subunit. a mutant recombinant rta chain vaccine, rivax, has been shown to be safe and immunogenic in humans. the other vaccine is another recombinant rta chain vaccine, rvec . it has shown effectiveness in animal models by producing protective immunity against aerosol challenge with ricin in animal models. standard precautions are advised for health care workers (pittman et al. ; roxas-duncan et al. ; traub ; usamriid ). recent events which include the nuclear reactor meltdown at fukushima and international tension between nuclear powers, spark concern over potential devastation from nuclear catastrophes. there are numerous examples of radiation disasters in history. sixty-six thousand people were killed in hiroshima and thirty-nine thousand people were killed in nagasaki from nuclear bombs detonated over these cities in (avalon project-documents in law, history and diplomacy n.d.). many other people suffered from long-term consequences of radiation poisoning. in , , square kilometers of land in russia, ukraine, and belarus were contaminated with radiation from a meltdown at a nuclear power plant in chernobyl, ukraine. one hundred and thirty-five thousand people were permanently evacuated from their homes (likhtarev et al. ) . long-term health consequences included many children who developed thyroid cancer several years later. many of these children died. a tsunami pummeled the east coast of japan in march of . the power outage that ensued at the fukushima power plant led to a failure of the cooling system of the fuel rods, leading to a meltdown of four of the reactors at the plant. a massive quantity of radiation was released into the atmosphere, forcing people to evacuate their homes indefinitely. creative thinking and heroic actions by the tokyo fire department prevented entire populations of cities from being poisoned with radiation. terrorism experts are concerned that terrorist organizations will produce and detonate a radiological dispersion device (rdd), sometimes referred to as a dirty bomb. this is a conventional explosive, loaded with radioactive material which would be dispersed upon detonation. this would likely involve only one radioisotope. fewer people would be exposed and a smaller area would be contaminated than what would transpire with the detonation of a nuclear weapon. spreading fear and panic would be the primary purpose of such a device (mettler jr and voelz ) . radiation is the emission and propagation of energy through space or through a medium in the form of waves. radiation can be ionizing or nonionizing depending on the amount of energy released. most radiation that people encounter is low energy and, therefore, nonionizing with no biological effects. ionizing radiation emits enough energy to strip electrons from an atom, which provokes cellular changes and thereby, results in biological effects. radiation emitted from nuclear decay is always ionizing (radiation emergency assistance center/training site (react/s-cdc) ). atomic nuclei are held together by a very powerful binding energy despite positively charged protons repelling each other. this energy is released from unstable nuclei in the form of electromagnetic waves or particles. when ionizing radiation reaches biological tissue, chemical bonds are disrupted, free radicals are produced, and dna is broken. electromagnetic waves are of two types, x-rays and gamma rays. x-rays are relatively low energy and less penetrating. gamma rays have a shorter wavelength and contain relatively higher energy, making them more penetrating of biological tissue. ionizing radiation in the form of particles consists of alpha particles, beta particles, and neutrons. alpha particles are the largest of the forms of particulate radiation. they are composed of two neutrons and two protons. they do not easily penetrate solid surfaces, including clothes and skin. however, they can cause severe damage to an organism if internalized. in , in the united kingdom, alexander litvienko, an ex kgb agent was poisoned with a radioactive element called polonium (mcphee and leikin ). a small amount of polonium was sprinkled into his food. polonium releases alpha particles when it decays. it was relatively safe for the assassin to carry this element with him because of the relatively poor ability of alpha particles to penetrate clothing and skin. once it is ingested, however, alpha particles have profound biological effects. mr. litvienko became very ill, and ultimately died. beta particles are high energy electrons discharged from the nucleus and are highly penetrating. neutrons emitted from a nucleus are also highly penetrating. in general, neutrons are only released by the detonation of a nuclear weapon. ionizing radiation of any form cannot be detected by our senses. it is not smelled, felt by touch, tasted, or seen. it is possible to be exposed to a lethal dose of radiation without realizing it. in goiania, brazil, in , children found a canister of radioactive cesium ( cs) that had been looted from a medical center and left in the street. the children liked the appearance of the substance but were not able to sense any abnormalities or danger with it. they began to rub it on their bodies because they liked the way it made them glow in the dark. the children all became ill. ultimately, people were exposed to this radioisotope. it took days before physicians recognized that the people had radiation poisoning. four people died of acute radiation syndrome. four factors determine the severity of exposure to ionizing radiation: time, distance, dose, and shielding. time is the time of exposure to the radiation source. distance is the distance from the radiation source. based on the inverse square law, exposure is reduced exponentially with increasing distance from the radiation source. dose is measured by the amount of energy released by the source and is numerically described by how many disintegrations per second occur, in curies (ci) or becquerels (bq). shielding is the efficacy of the barrier to the radioactive source. lead is well-known to be a very effective shield to x-rays. in a radiation exposure, injury to skin from trauma or burns may cause a greater degree of contamination because of loss of the shielding of the skin. there are four important principles for the nurse or hcp to understand with regard to exposure to ionizing radiation: external exposure, external contamination, internal contamination, and incorporation. external contamination occurs when radioactive material is carried on a person after exposure. this person can then contaminate others. removing contaminated clothing eliminates % of the toxin. others are then less vulnerable to exposure. internal contamination is when a radioactive substance enters the body through inhalation, ingestion, or translocation through open skin. incorporation is internalization of the toxin into body organs. incorporation is dependent on the chemical and not the radiological properties of the radioactive toxin. radioactive iodine, i, is taken up by the thyroid gland because iodine enters the gland as part of normal physiology (advanced hazmat life support (ahls) ). ionizing radiation can damage chromosomes directly and indirectly, causing ravaging biological effects. indirect damage comes from the production of h + and oh − . free radical formation upsets biochemical processes and causes inflammation. these effects can take anywhere from seconds to hours to be expressed. clinical changes can take from hours to years to be realized (zajtchuk et al. ). immediately after a major radiation exposure, the clinical matters of most concern are those related to trauma from blast and thermal injuries. these injuries may be life-threatening and must be addressed first. after thermal and traumatic injuries are addressed, attention should be paid to the severity of radiation exposure. severe exposure can cause acute radiation syndrome. "the acute radiation syndrome is a broad term used to describe a range of signs and symptoms that reflect severe damage to specific organ systems and that can lead to death within hours or up to several months after exposure" (national council on radiation protection (ncrp) and measurements ; national council on radiation protection (ncrp) and measurements ). the mechanism of cell death from toxic radiation exposure is related to the inhibition of mitosis. organs with the most rapidly dividing cells are the most susceptible. the gastrointestinal and the hematopoietic are the organ systems most notably affected. the organs of pediatric patient have a higher mitotic index, in general, to those of adults and are more vulnerable to injury from radiation poisoning. the time of onset and the severity of acute radiation syndrome are controlled by the total radiation dose, the dose rate, percent of total body exposed, and associated thermal and traumatic injuries. there is a % death rate (ld ) within days for people exposed to a dose of radiation of . - . gy. the ld is lower for the pediatric population. the acute radiation syndrome is composed of four phases: prodromal, latent, manifest illness, and death or recovery. inflammatory mediator release during the prodromal phase causes damage to cell membranes. this phase occurs during the first h after exposure to radiation. nausea and vomiting and fever can occur during this time. if these symptoms occur during the first h after exposure, there is a poor prognosis. the onset of the latent phase is usually in the first days post exposure but can ensue anytime during the first days thereafter. all cell lines of the hematopoietic system are affected. lymphocytes and platelets, the most rapidly dividing cells of the bone marrow, are most severely affected. the illness phase manifests after days since radiation exposure. infection, impaired wound healing, anemia, and bleeding occur during this time of illness. the hematopoietic, gastrointestinal, central nervous, and integumentary are the organ systems affected. there is a marked reduction of cells from all cell lines of the bone marrow. there is a direct correlation with the drop in absolute lymphocyte count with the dose of radiation received. the absolute lymphocyte count is commonly used to estimate the dose of radiation received. the gastrointestinal (gi) epithelial lining, one of the most rapidly dividing cell lines of the body is the second most vulnerable to radiation poisoning. the radiation dose required to affect the gi system is gy. vomiting, diarrhea, and a capillary leak syndrome for gi tract are common manifestations. hypovolemia and electrolyte instability ensue. translocation of bacteria into the bloodstream, combined with the diminished immunity caused by the decimation of the hematopoietic system, place victims at high risk for septic shock. another organ system affected by the acute radiation syndrome is the central nervous system. this requires a large dose of at least gy. manifestations include cerebral edema, disorientation, hyperthermia, seizures, and coma. acute radiation syndrome that involves the central nervous system is always fatal. the integumentary system is frequently affected by the acute radiation syndrome, especially if the skin is in direct contact with a radioisotope. epilation, erythema, dry desquamation, wet desquamation, and necrosis occur respectively with increasing severity associated with increasing doses of radiation. radiation burns can be distinguished from thermal or chemical burns by their delayed onset. it can take days to weeks for radiation burns to affect victims. thermal and chemical burns cause signs and symptoms more acutely. hospitals that anticipate victims of radiation should prepare areas of triage with decontamination supplies and techniques ready to be deployed. an emergency department (ed) should be divided into "clean" and "dirty" areas. the dirty area is created for the purpose of decontamination to prevent the spread of radioisotopes. all health care personnel should wear ppe including surgical scrubs and gowns, face shields, shoe covers, caps, and two pairs of gloves. the inner pair of gloves is taped to the sleeves of the gown. each health care worker should be monitored for the exposure of the radiation and its dose with a dosimeter worn underneath the gown. the radiation safety officer of the hospital should take a leadership role in health care worker protection and decontamination procedures. consultation from the radiation emergency, assistance center (react/ts) is imperative. react/ts is a subsidiary of the u.s. department of energy. its contact information is as follows: phone number during business hours is - - . the phone number is - - after business hours. the react/ts website is http://orise.orau. gov/reac/ts/. as victims arrive, triage protocols of mass casualty scenarios should be implemented. it should be noted that radiation exposure is not "immediately" lifethreatening. initial clinical management should focus on the abcde (airway, breathing, circulation, disability, and exposure) of basic trauma protocol. the "d" in the above acronym can also be a symbol for decontamination. after airway, breathing, and circulation are addressed, initial phase of decontamination entails careful removal of potentially contaminated clothing. caution should be exercised to remove the clothing gently, while rolling garments outward to prevent the release of dust of radioactive material that could contaminate people in the treatment area. further decontamination procedures take place after initial stabilization. skin decontamination procedures are identical to those of toxic chemical exposure with the following exceptions: • ppe are slightly different as described above. • gentle skin rubbing is done to prevent provocation of an inflammatory response and further absorption of the radioactive toxin. • only soap and water are used. rubbing alcohol and bleach should be avoided. it is advisable to shampoo the hair first, because it is usually the site of the highest level of contamination of the body, and runoff onto the body can then be cleansed during skin decontamination (radiation event medical management (remm) of the u.s. dept. of health and human services n.d.). it should be noted that health care workers are not at risk for contamination if they wear proper ppe during the resuscitation and decontamination process. the lack of knowledge of this point may lead to reluctance to treat patients and increase morbidity and mortality for victims. "no hcp has ever received a significant dose of radiation from handling, treating, and managing patients with radiation injuries and/or contamination."(react/s-cdc ). when initial resuscitation and decontamination have been completed, attention should be paid to ongoing support of ventilation, oxygenation, the management of fluid and electrolytes, and treatment of traumatic and burn injuries. infection control procedures are important due to the impending immunocompromised state of the victims. it is important to ascertain the details of the catastrophic event. data on the nature and size of the exposure and the types of radioactive agents involved are vital for ongoing management and decontamination. after the details of the nature of the exposure are uncovered, diagnostic tests should be done, including serial cbc and cytogenetic analysis of lymphocytes, otherwise known as cytogenic dosimetry (react/s-cdc ). measurements of change in lymphocyte counts and cytogenetic dosimetry are sensitive markers for the dose of radiation received by a victim. measurements of internal decontamination are done by the sampling and analysis of nasal and throat swabs, stool, and h urine. wound samples and irrigation fluid should also be sampled. after initial stabilization, external decontamination, and diagnostic testing, internal decontamination is performed. external decontamination involves removal of clothes and cleaning the skin and hair. internal decontamination removes radioisotopes that are internalized via inhalation, ingestion, and entry into open wounds. because ionizing radiation is being released inside the body, internal decontamination must be performed promptly after initial resuscitation. since radioisotopes behave identically to their nonradioactive counterparts, antidotes are chosen based on the chemical, and not the radiological properties of the element. basic strategies of internal decontamination include chelation, competitive inhibition, enhanced gastrointestinal elimination, and enhanced renal elimination. specific agents are used for chelation of different radioisotopes. dtpa (diethyenetriaminepentaacetic acid) is administered for the elimination of heavy metals such as americium, californium, curium, and plutonium. dtpa comes in two forms, calcium dtpa (ca-dtpa) and zinc-dtpa (zn-dtpa). ca-dtpa is ten times more effective than zn-dtpa. for adults and adolescents, administration is as follows: • g of ca-dtpa iv initially in the first h, followed by g zn-dtpa iv daily for maintenance. • for children less than years of age administer: • fourteen mg/kg ca-dtpa iv initially, followed by fourteen mg/kg of zn-dtpa iv daily thereafter (national council on radiation protection (ncrp) and measurements ). • the initial dose of dtpa may be administered via inhalation to adolescents and adults if the contamination occurred via inhalation. this method of administration is not approved for pediatric use. chelation with dimercaprol (bal) is used to eliminate polonium. bal is a highly toxic drug and should be administered with caution. the dose is . mg per kg im four times a day for days, then twice a day on the third day and once a day for - days, thereafter (national council on radiation protection (ncrp) and measurements ). alkalinization of the urine is renal protective during administration. a less toxic alternative to bal, dimercaptosuccinic acid (dmsa), otherwise known as chemet ® is also available. the dose of dmsa is ten mg per kg po every h for days. the same dose is given every h for days, thereafter (national council on radiation protection (ncrp) and measurements ). another mechanism for internal decontamination is competitive inhibition. the radioisotope, i, is released during a meltdown of a reactor at a nuclear power plant. potassium iodide (ki) is widely recognized as a competitive inhibitor to its radioactive counterpart, i, from being incorporated into the thyroid gland. ki blocks % of i uptake into the thyroid gland if ki is given within the first hour of exposure. it will block % of incorporation if given within h of exposure. its protective effect lasts for h. with administration of this drug, thyroid function should be monitored closely. dosing guidelines (table . ) are included in the table below (u.s. food and drug administration n.d.). gastrointestinal elimination is another mechanism of internal decontamination (table . ). an ion exchanger, prussian blue, (ferric ferrocyanide), binds elements that circulate through the enterohepatic cycle. since it is not absorbed through the gastrointestinal tract, prussian blue carries the toxins into the stool. it is highly effective in the elimination of cs or thallium and was used during the cs incident in goiania, brazil. the dosing of prussian blue is as follows: • infants: . - . mg per kg po three times a day (not fda approved). • children - years of age: g po three times a day. • children ≥ years of age: g po three times a day. • prussian blue is administered for at least days, and can be adjusted based on the degree of poisoning (national council on radiation protection (ncrp) and measurements ). urinary elimination is another useful method of internal decontamination. tritium can be eliminated with excess fluid administration. uranium is eliminated by alkalinizing the urine to a ph of - . sodium bicarbonate is given at a dose of meq/kg iv every - h and is titrated to effect. if renal injury occurs, dialysis may be required. the basic approach to treating acute radiation syndrome is supportive therapy. gi losses from gastrointestinal difficulties are treated with iv fluids and electrolyte replacement. -ht antagonists can be used to suppress vomiting and benzodiazepines for anxiety. a patient suffering from acute radiation syndrome may be severely immunocompromised and requires a room with positive pressure isolation. colony stimulating agents for granulocytes and erythrocytes can be used for bone anemia and leukopenia. bone marrow transplant may be required for severe cases. a patient with skin contamination with radiation should be decontaminated with soap and water. a geiger counter can be helpful to identify areas of contamination. scrubbing is performed in a concentric matter, beginning at the outer layers of contamination and moving into the center since the area of greatest contamination is in the center. in this way, the area of contamination remains contained. attention should be paid to good nutrition and pain control. burn and plastic surgery service should also be consulted. more details on decontamination can be found in chap. . the psychological impact of a radiation catastrophe on the pediatric victims is likely to be devastating (american academy of pediatrics (aap) ). sleep disturbances, social withdrawal, altered play, chronic fear and anxiety, and developmental regression can occur. a correlation between the parent's psychological response and that of the child would occur as with other types of disaster. mental health professionals should be consulted in the event of this type of situation. please refer to chap. for more information. a lot of concern has been expressed over the possibility of terrorist attacks involving explosive devices in recent years (depalma et al. ) . explosive devices are relatively simple to manufacture and easy to detonate. they can injure and kill many people and spread fear over large populations. victims of bomb blasts sustain more body regions injured, have more body injury severity scores, and require more surgeries than victims of nonexplosive trauma incidents. victims of explosives also have a higher mortality (kluger et al. ) . these observations are also true of pediatric victims (daniel-aharonson et al. ) . many factors influence the number of people injured and the severity of the injuries in an explosion. the magnitude of the explosion and its proximity to people and the number of people in the area affect the severity and number of injuries. other factors include the collapse of building or structure from the blast, promptness of the rescue operation, and the caliber and proximity of medical resources in the vicinity. victims who experience explosions in closed spaces are especially vulnerable to more severe injuries. twenty-nine case reports of injuries from terrorist bombings were reviewed (arnold et al. a) . the investigators compared the injury severity of victims of explosions who sustained injuries from structural collapse, closed space explosions without structural collapse, and open space explosions. the mortality rate for these victims was %, %, and %, respectively. hospitalization rates were %, %, and %, respectively. ed visits were %, %, and %, respectively. victims of closed space explosions without structural collapse experienced greater hospitalizations rates than those involved in a structural collapse, because many of the victims involved in the structural collapse experienced immediate death. an explosion is defined as a rapid chemical conversion of a liquid or solid into a gas with energy release. substances that are chemically predisposed to explosion, called explosives, are characterized as low or high order, depending on the speed and magnitude of energy release. low-order explosives release energy at a relatively slow pace and explosions from these substances tend not to produce large air pressure changes or a "blast." the energy release is caused by combustion, producing heat. the involved material "goes up in flames." gunpowder, liquid fuel, and molotov cocktails are examples of low-order explosives (centers for disease control and prevention ). explosions from high-order materials cause a blast with a pressure wave in addition to causing the release of heat and light. the blast pressure wave causes compression of the surrounding medium which is physically transformed in all directions from the exact point of explosion. when an explosion occurs on land, air is the surrounding medium compressed. in bodies of water, the surrounding medium is water. the degree of medium compression and the distance that the energy wave travels is determined by the magnitude of the explosion. the power of the blast is measured in pounds per square inch (psi). the pressure blast wave has distinctive characteristics. the amplitude of the wave reaches its highest point immediately after the blast. the blast wave then rapidly decays as it travels through space. as the blast wave propagates, and compresses the surrounding medium, it leaves a vacuum because of displaced molecules in the surrounding medium and a negative phase of the wave ensues. in a land explosion, air molecules are displaced by the initial positive pressure, after which a negative pressure occurs in the vacated space. a wave that propagates through a confined space rebounds off of the wall and reverberates. it may interact with victims in the confined space many times, causing more severe injuries (stuhmiller et al. ) (fig. . ). four kinds of injury occur in high energy explosions. primary blast injuries occur directly from the pressure wave of the blast. secondary injuries occur from being struck by flying objects from the blast. these injuries can be blunt or penetrating. tertiary injuries occur when victims are displaced from a location and strike other objects or surfaces. all other injuries related to the blast are called quarternary. they include burns, inhalational injuries, toxic exposures, and traumatic injuries from structural collapse. primary injuries from blast waves affect bodily tissues with a tissue gas interface. when a pressure wave enters the body, tissue of gas filled organs compress slower than the air inside the tissue, causing stress in the tissue, possibly damaging it. this baseline positive phase originally described by friedlander, a blast wave consists of a short, high-amplitude overpressure peak followed by a longer depression phase. injury potential depends on the wave's amplitude as well as the slopes of its increase and decrease in pressure. x-axis refers to time and y-axis refers to pressure. (jacobson and severin ) also known as the "spalling effect." as the negative pressure phase of the blast wave propagates through, it causes more stress on the tissue and further damage. in addition to damaging tissues with an air tissue interface, pressure blasts can cause injury to the brain and can lead to limb detachments. despite the fact that primary blast injuries can be ravaging, they are less common than other types of injury from blasts. the tympanic membranes, lungs, and gastrointestinal tract are the most common organs sustaining injury from pressure waves. the tympanic membrane is the most vulnerable of these three organ systems (depalma et al. ; garth ) . five psi, which is considered a weak blast, will rupture % of tympanic membranes. to put this in perspective, c , a commonly used explosive generates a pressure of four million psi. otoscopy can reveal ruptured tympanic membranes. neuropraxia, deafness, tinnitus, and vertigo are symptoms that can be experienced. severe blast injuries of the ear can result in damage to the organ of corti, resulting in permanent hearing loss. the second most common organ injured from a blast wave is the lung. fifteen psi are required to cause injury to this organ. lung injuries are more likely to occur from a blast within a closed space, or when victims sustain burns (burns commonly cause acute lung injury from release of inflammatory mediators). direct alveolar damage, blood vessel with bleeding, and inflammation are the three different manifestations of lung injury from blasts. alveolar damage can cause pneumothorax and pulmonary interstitial emphysema. when air dissects along the bronchovascular sheath, pneumomediastinum, pneumopericardium, and subcutaneous emphysema can occur. air that enters the pulmonary venous system can result in a systemic arterial air embolism, and possibly, a stroke. inflammation of the lungs from direct pressure damage to the tissue, cause acute lung injury and possibly, disseminated intravascular coagulation. clinical signs of lung injury include tachypnea, chest pain, hypoxia, rales, and dyspnea. if there is vascular disruption, hemoptysis can occur. air leaks from alveolar injury can result in diminished breath sounds, subcutaneous crepitance, increased resonance, and tracheal deviation. hemodynamic compromise will occur with tracheal deviation. alveolar damage, leading to air in the pulmonary venous system, can lead to a systemic arterial air embolism. air in the coronary arteries can lead to coronary ischemia with st and/or t waves changes on ecg. air embolism to cerebral arteries leads to cerebral vascular accidents (strokes) with focal neurological deficits. other manifestations of systemic air embolism include mottling of the skin, demarcated tongue blanching, and/or air in the retinal vessels (the most common sign of arterial air embolus). rapid death after initial survival is most often caused by arterial air embolus. initiation of positive pressure ventilation may trigger this event (ho and ling ) . a lung injury from a blast can also precipitate a vagal reflex resulting in bradycardia and hypotension. it is postulated that this occurs from the stimulation of c fibers in the lungs (guy et al. ). the gastrointestinal system is the third most common organ system affected by primary blast injury. physical stress and/or mesenteric infarct leads to weakening of the bowel wall with possible rupture. hemorrhage can also occur (paran et al. ; sharpnack et al. ) . the most common site of injury is the colon. injury to the bowel can be delayed and occur up to several days after the inciting incident. solid organs are spared because of their homogeneity and lack of air tissue interface. brain injury is becoming increasingly recognized as a result of primary blast. shearing injuries of the brain occur as a result of wave reverberation in the skull. hippocampal injury causing cognitive impairment has been shown in animal studies (cernak ; cernak et al. ; singer et al. ) . observations in humans have revealed electroencephalographic abnormalities and attention deficit disorder (born ) . human autopsies have revealed punctate hemorrhages and disintegration of nissl substance in victims who sustained blast injury without direct head trauma (guy et al. ) . research involving yucatan minipigs revealed that the brain sustains neuronal loss in the hippocampus after being subjected to primary blast injury. brain injury also occurred from the inflammation that ensued post blast (goodrich et al. ) . novel therapeutic approaches may be on the horizon for treatment of traumatic brain injury, including that caused by primary blast. intranasal insulin administered to rats subjected to traumatic brain injury resulted in enhanced neuronal glucose uptake and utilization, and subsequently improved motor function and memory. decreased neuroinflammation and preservation of the hippocampus were also noted (brabazon et al. ) . in a different investigation, a neuroprotective nucleotide, guanosine, was administered to rats subjected to traumatic brain injury. the treatment group of rats had better locomotor and cognitive outcomes than did the placebo group. programmed cell death and inflammation were also attenuated in the treatment group (gerbatin et al. ) . the leading cause of death from blast is from flying objects striking victims (secondary blast injury). eyes are particularly vulnerable. injuries resulting from displacement of the victims who strike objects are known as tertiary injuries. lighter weight children are particularly susceptible to this type of injury. burns, toxic exposures, and crush injuries constitute quaternary injuries. crush injuries commonly occur in explosions with structural collapse. the "crush syndrome" can occur when a trapped limb sustains prolonged compromise to the circulation, leading to rhabdomyolysis. tissue destruction and inflammatory response then occur. lifethreatening electrolyte abnormalities including hyperkalemia, renal failure, hyperuricemia, metabolic acidosis, acute respiratory distress syndrome, disseminated intravascular coagulation, and shock can result from crush syndrome (gonzalez ) . the crush syndrome is commonly seen in natural disasters that result in a lot of structural collapse. structural collapse and fires can cause the release of toxic materials such as carbon monoxide and cyanide. knowledge of the details of a blast can greatly enhance the ability of nurses and hcps to care for victims of a blast in a hospital setting. knowledge of whether a blast occurred in a closed or open space, whether structural collapse occurred, or if a victim was rescued from a collapsed area are details that can alert nurses and hcps as to what kind of injuries that they may anticipate. if toxic substances are released with a blast, nurses and hcps can prepare for decontamination techniques and antidote therapies. it would be advantageous for a hospital to be aware of the number of victims that are arriving for care. a mass casualty incident will stress the resources of the institution. hospital personnel should take stock of the resources that are available. the number of available ventilators and o-blood are examples of finite resources that should be considered. advanced trauma life support (atls) principles should be applied to all blast injury victims. abcd of initial resuscitation is applied. the "d" stands for disability as well as decontamination. decontamination techniques should be deployed if there is uncertainty about toxic exposure as described elsewhere in this chapter. on completion of abcd of initial resuscitation a secondary survey is performed, as described by atls protocol. attention should be paid to potential injuries that occur with blast injuries. ruptured tympanic membranes should alert the nurse or hcp of problems from primary blast injury. impaled objects should remain in place and removed in the operating room by surgical staff so that bleeding may be controlled. a thoracoscopy tube should be placed with an open three point seal over a wound on the side of the chest with an open pneumothorax. a hemothorax is also treated with a thoracoscopy tube. an autotransfusion setup can be applied to recirculate the blood from the pleural cavity of a hemothorax (wightman and gladish ) that would help preserve donor blood for other victims. for severe respiratory distress and/or impending respiratory failure, endotracheal intubation should be performed and positive pressure ventilation should be instituted. because lung tissue could be weakened from primary blast injury, caution should be exercised because of a high risk of pneumothorax, hemorrhage, or arterial air embolus. gentle application of positive pressure ventilation should be applied to avoid these complications. if only one lung is injured unilateral lung ventilation can be considered for larger children and adults. this technique is not suitable for babies and small children. supplemental oxygen with an fio of % should be administered to patients suspected of having an arterial air embolus. hyperbaric oxygen therapy could even be considered to help accelerate the removal of air from the arteries. placement of the patient in the left lateral recumbent position may reduce the likelihood of the air lodging in the coronary arteries. victims of blast injuries should be treated identically to those of other types of trauma after initial resuscitation is completed. if primary blast injury occurred, frequent chest and abdominal x-rays should be performed in consideration of the possibility of lung or gastrointestinal injuries. limbs with open fractures should be immobilized and covered with sterile dressings. systemic, broad spectrum antibiotics should be administered to patients with open limb injuries. eyes that sustained chemical injury should be irrigated with water for an hour. all injured eyes should be covered. most ruptured tympanic membranes will heal spontaneously. victims with tympanic membrane injury should be advised to avoid swimming for some time. topical antibiotics are prescribed if dirt or debris is seen in the ear canal. oral prednisone is prescribed for hearing loss. victims with crush injuries should be treated with large volumes of iv fluids to treat inflammatory shock and possibly rhabdomyolysis. electrolytes should be monitored carefully as these patients are at risk for hyperkalemia, hyperphosphatemia, hyperuricemia, hypocalcemia, and acidosis. smoke inhalation, burns, and toxic exposures should be treated according to guidelines of burn, trauma, and toxicology protocols. mass casualty incidents (i.e. mass shootings, active shooter events, bombings, and other multifatality crimes) often attract extensive media coverage as well as the attention of policy makers. many agencies and organizations record and publish data on these incidents. the measurement and reporting does vary based on the absence of a common definition. however, it is clearly evident that mass casualty incidents (mcis) continue to increase in both number and scope (federal bureau of investigation ; office for victims of crime, office of justice programs, u.s. department of justice ). in the u.s., mass shootings are the most common and most closely tracked. the congressional research service (crs) defines mass shootings as events where more than four people are killed with a firearm "within one event, and in one or more locations in close proximity." congress uses the term mass killings and describes these events as "three or more killings in a single incident." the federal bureau of investigation (fbi) uses the term active shooter, which it defines as "an individual actively engaged in killing or attempting to kill people in a populated area." it is important to realize that nongovernmental ( ranking third of all locations for and , seven of the incidents occurred in educational environments resulting in five killed and wounded. two incidents occurred in elementary schools, resulting in two killed (including a firstgrade student) and eight wounded (one teacher shot, three students shot, and four wounded from shrapnel). one incident occurred in a junior/senior high school, resulting in none killed and four wounded (two from shrapnel, all students). four incidents occurred at high schools (one outside a school during prom), resulting in three killed (all students) and seven wounded (all students). fortunately, no incident occurred at institutions of higher learning during or (advanced law enforcement rapid response training (alerrt) center, texas state university and federal bureau of investigation, u.s. department of justice ). notably, two of the incidents occurred in houses of worship, resulting in killed and wounded. one of these incidents occurred at the first baptist church in sutherland springs, texas, and had the third highest number of casualties ( killed and wounded) in . the dead included women, men, children ( girls and boy), and an unborn child (goldman et al. ) . a summary report has also been developed for all active shooter incidents from to , including incidents per year (fig. . ), casualties per year (fig. . ) , and location ( fig. . ) categories (federal bureau of investigation ; federal bureau of investigation ). overall, there was an increase in number of active shooter incidents and casualties per year. location categories with number of incidents and statistics of their contribution were provided: areas of educational environments account for a large portion of locations for active shooter incidents, ranking only second to commercial areas. of the incidents ( . %) occurring at schools, one took place at a nursery (pre-k) school and one incident occurred during a school board meeting that was being hosted on school property but no students were involved (neither perpetrator or victim). the remainder ( incidents) were perpetrated by or against students, faculty, and/or staff at k- schools (federal bureau of investigation ). finally, active shooter incidents ( %) did occur at institutions of higher learning. as a reminder, no incident occurred at institutions of higher learning during or . table . provides a detailed summary of educational environment incidents from to . since the beginning of , other tragic active shooter attacks have occurred in the u.s. and greatly impacted children and adolescents. two of these such events have occurred in educational environments (united states secret service national threat assessment center ). on february , , a gunman opened fire at marjory stoneman douglas high school. fourteen students and three staff members were killed while fourteen others were injured (follman et al. ) . twelve victims died inside the building, three died just outside the building on school premises, and two died in the hospital. the shooter was a former student of the school. another active shooter event occurred on may , at santa fe high school in santa fe, texas. the shooter killed ten individuals including eight students and two teachers while injuring others. the shooter was an enrolled student at the school (follman et al. ) . based on the statistics of active shooter incidents, casualties, and locations, it is vital to prepare schools and plan for such events. national preparedness efforts, including planning, are now informed by the presidential policy directive (ppd) that was signed by the president in march and describes the nation's approach to preparedness. this directive represents an evolution in our collective understanding of national preparedness based on the lessons learned from terrorist attacks, hurricanes, school incidents, and other experiences. ppd- defines preparedness around five mission areas and can be applied to school active shooter incidents. on march , , at : p.m., jeffery james weise, , armed with a shotgun and two handguns, began shooting at red lake high school in red lake, minnesota. before the incident at the school, the shooter fatally shot his grandfather, who was a police officer, and another individual at their home. he then took his grandfather's police equipment, including guns and body armor, to the school. a total of nine people were killed, including an unarmed security guard, a teacher, and five students; six students were wounded. the shooter committed suicide during an exchange of gunfire with police campbell county comprehensive high school (education) on november , , at : p.m., kenneth s. bartley, , armed with a handgun, began shooting in campbell county comprehensive high school in jacksboro, tennessee. before the shooting, he had been called to the office when administrators received a report that he had a gun. when confronted, he shot and killed an assistant principal and wounded the principal and another assistant principal. the shooter was restrained by students and administrators until police arrived and took him into custody pine middle school (education) on march , , at : a.m., james scott newman, , armed with a handgun, began shooting outside the cafeteria at pine middle school in reno, nevada. no one was killed; two were wounded. the shooter was restrained by a teacher until police arrived and took him into custody essex elementary school and two residences (education) on august , , at : p.m., christopher williams, , armed with a handgun, shot at various locations in essex, vermont. he began by fatally shooting his ex-girlfriend's mother at her home and then drove to essex elementary school, where his ex-girlfriend was a teacher. he did not find her, but as he searched, he killed one teacher and wounded another. he then fled to a friend's home, where he wounded one person. a total of two people were killed; two were wounded. the shooter also shot himself twice but survived and was apprehended when police arrived at the scene orange high school and residence (education) on august , , at : p.m., alvaro castillo, , armed with two pipe bombs, two rifles, a shotgun, and a smoke grenade, began shooting a rifle from his vehicle at his former high school, orange high school in hillsborough, north carolina. he had fatally shot his father in his home that morning. one person was killed; two were wounded. the shooter was apprehended by police weston high school (education) on september , , at : a.m., eric jordan hainstock, , armed with a handgun and a rifle, began shooting in weston high school in cazenovia, wisconsin. one person was killed; no one was wounded. the shooter was restrained by school employees until police arrived and took him into custody west nickel mines school (education) on october , , at : a.m., charles carl roberts, iv, , armed with a rifle, a shotgun, and a handgun, began shooting at the west nickel mines school in bart township, pennsylvania. after the shooter entered the building, he ordered all males and adults out of the room. after a -min standoff, he began firing. the shooter committed suicide as the police began to breach the school through a window. five people were killed; five were wounded on april , , at : a.m., su nam ko, aka one l. goh, , armed with a handgun, began shooting inside oikos university in oakland, california. he then killed a woman to steal her car. seven people were killed; three were wounded. the shooter was arrested by police later that day on august , , at : a.m., robert wayne gladden jr., , armed with a shotgun, shot a classmate in the cafeteria of perry hall high school in baltimore, maryland. the shooter had an altercation with another student before the shooting began. he left the cafeteria and returned with a gun. no one was killed; one person was wounded. the shooter was restrained by a guidance counselor before being taken into custody by the school's resource officer sandy hook elementary school and residence (education) on december , , at : a.m., adam lanza, , armed with two handguns and a rifle, shot through the secured front door to enter sandy hook elementary school in newtown, connecticut. he killed students and six adults, and wounded two adults inside the school. prior to the shooting, the shooter killed his mother at their home. in total, people were killed; two were wounded. the shooter committed suicide after police arrived taft union high school (education) on january , , at : a.m., bryan oliver, , armed with a shotgun, allegedly began shooting in a science class at taft union high school in taft, california. no one was killed; two people were wounded. an administrator persuaded the shooter to put the gun down before police arrived and took him into custody new river community college, satellite campus (education) on april , , at : p.m., neil allen macinnis, , armed with a shotgun, began shooting in the new river community college satellite campus in the new river valley mall in christiansburg, virginia. no one was killed; two were wounded. the shooter was apprehended by police after being detained by an off-duty mall security officer as he attempted to flee santa monica college and residence (education) on june , , at : a.m., john zawahri, , armed with a handgun, fatally shot his father and brother in their home in santa monica, california. he then carjacked a vehicle and forced the driver to take him to the santa monica college campus. he allowed the driver to leave her vehicle unharmed but continued shooting until he was killed in an exchange of gunfire with police. five people were killed; four were wounded sparks middle school (education) on october , , at : a.m., jose reyes, , armed with a handgun, began shooting outside sparks middle school in sparks, nevada. a teacher was killed when he confronted the shooter; two people were wounded. the shooter committed suicide before police arrived arapahoe high school (education) on december , , at : p.m., karl halverson pierson, , armed with a shotgun, machete, and three molotov cocktails, began shooting in the hallways of arapahoe high school in centennial, colorado. as he moved through the school and into the library, he fired one additional round and lit a molotov cocktail, throwing it into a bookcase and causing minor damage. one person was killed; no one was wounded. the shooter committed suicide as a school resource officer approached him berrendo middle school (education) on january , , at : a.m., mason andrew campbell, , armed with a shotgun, began shooting in berrendo middle school in roswell, new mexico. a teacher at the school confronted and ordered him to place his gun on the ground. the shooter complied. no one was killed; were wounded: students and an unarmed security guard. the shooter was taken into custody (continued) on june , , at : p.m., aaron rey ybarra, , armed with a shotgun, allegedly began shooting in otto miller hall at seattle pacific university in seattle, washington. he was confronted and pepper sprayed by a student as he was reloading. one person was killed; were wounded. students restrained the shooter until law enforcement arrived reynolds high school (education) on june , , at : a.m., jared michael padgett, , armed with a handgun and a rifle, began shooting inside the boy's locker room at reynolds high school in portland, oregon. one student was killed; teacher was wounded. the shooter committed suicide in a bathroom stall after law enforcement arrived marysville-pilchuck high school (education) on october , , at : a.m., jaylen ray fryberg, , armed with a handgun, began shooting in the cafeteria of marysville-pilchuck high school in marysville, washington. four students were killed, including the shooter's cousin; students were wounded, including one who injured himself while fleeing the scene. the shooter, when confronted by a teacher, committed suicide before law enforcement arrived florida state university (education) on november , , at : a.m., myron may, , armed with a handgun, began shooting in strozier library at florida state university in tallahassee, florida. he was an alumnus of the university. no one was killed; were wounded. the shooter was killed during an exchange of gunfire with campus law enforcement. umpqua community college (education) on october , , at : a.m., christopher sean harper-mercer, , armed with several handguns and a rifle, began shooting classmates in a classroom on the campus of umpqua community college in roseburg, oregon. nine people were killed; were wounded. the shooter committed suicide after being wounded during an exchange of gunfire with law enforcement. madison junior/ senior high school (education) on february , , at : a.m., james austin hancock, , armed with a handgun, allegedly began shooting in the cafeteria of madison junior/senior high school in middletown, ohio. he shot two students before fleeing the building. no one was killed; four students were wounded (two from shrapnel). the shooter was apprehended near the school by law enforcement officers antigo high school (education) on april , , at : p.m., jakob edward wagner, , armed with a rifle, began shooting outside a prom being held at his former school, antigo high school in antigo, wisconsin. two law enforcement officers, who were on the premises, heard the shots and responded immediately. no one was killed; two students were wounded. the shooter was wounded in an exchange of gunfire with law enforcement officers and later died at the hospital townville elementary school (education) on september , , at : p.m., jesse dewitt osborne, , armed with a handgun, allegedly began shooting at the townville elementary school playground in townville, south carolina. prior to the shooting, the shooter, a former student, killed his father at their home. two people were killed, including one student; three were wounded, one teacher and two students. a volunteer firefighter, who possessed a valid firearms permit, restrained the shooter until law enforcement officers arrived and apprehended him on january , , at : a.m., ely ray serna, , armed with a shotgun, allegedly began shooting inside west liberty salem high school, in west liberty, ohio, where he was a student. after assembling the weapon in a bathroom, the shooter shot a student who entered, then shot at a teacher who heard the commotion. the shooter shot classroom door windows before returning to the bathroom and surrendering to school administrators. no one was killed; two students were wounded. school staff members subdued the shooter until law enforcement arrived and took the shooter into custody freeman high school (education) on september , , at : a.m., caleb sharpe, , armed with a rifle and a pistol, allegedly began shooting at freeman high school in rockford, washington, where he was a student. one student was killed; three students were wounded. a school employee confronted the shooter, ordered him to the ground, and held him there until law enforcement arrived and took him into custody rancho tehama elementary school and multiple locations in tehama county, california (education) on november , , at : a.m., kevin janson neal, , armed with a rifle and two handguns, began shooting at his neighbors, the first in a series of shootings occurring in rancho tehama reserve, tehama county, california. after killing three neighbors, he stole a car and began firing randomly at vehicles and pedestrians as he drove around the community. after deliberately bumping into another car, the shooter fired into the car and wounded the driver and three passengers. the shooter then drove into the gate of a nearby elementary school. he was prevented from entering the school due to a lockdown, so he fired at the windows and doors of the building, wounding five children. upon fleeing the school, the shooter continued to shoot at people as he drove around rancho tehama reserve. law enforcement pursued the shooter; they rammed his vehicle, forced him off the road, and exchanged gunfire. the shooter's wife's body was later discovered at the shooter's home; the shooter apparently had shot and killed her the previous day. in total, five people were killed; were wounded, eight from gunshot injuries (including one student) and six from shrapnel injuries (including four students). the shooter committed suicide after being shot and wounded by law enforcement during the pursuit aztec high school (education) on december , , at approximately : a.m., william edward atchison, , armed with a handgun, began shooting inside aztec high school in aztec, new mexico. the shooter was a former student. two students were killed; no one was wounded. the shooter committed suicide at the scene, before police arrived a in a study of active shooter incidents in the united states between and , the fbi identified locations where the public was most at risk during an incident. these location categories include commercial areas (divided into business open to pedestrian traffic, businesses closed to pedestrian traffic, and malls), education environments (divided into schools [prekindergarten through th grade] and institutions of higher learning), open spaces, government properties (divided into military and other government properties), residences, houses of worship, and health care facilities. in , the fbi added a new location category, other location, to capture incidents that occurred in venues not included in the previously identified locations (federal bureau of investigation ). this table only includes educational environments. an entire list of all incidents from to at all locations can be found at https://www.fbi.gov/file-repository/activeshooter-incidents- .pdf/view (federal bureau of investigation prevention means the capabilities necessary to avoid, deter, or stop an imminent crime or threatened/actual mass casualty incident. prevention is the action schools take to prevent a threatened or actual incident from occurring. protection means the capabilities to secure schools against acts of violence and man-made or natural disasters. protection focuses on ongoing actions that protect students, teachers, staff, visitors, districts, networks, and property from a threat or hazard. mitigation means the capabilities necessary to eliminate or reduce the loss of life and property damage by lessening the impact of an event or emergency at the school. it also means reducing the likelihood that threats and hazards will happen. response means the school's or school district's capabilities necessary to stabilize an emergency once it has already happened or is certain to happen in an unpreventable way, establish a safe and secure environment, save lives and property, and facilitate the transition to recovery. recovery means the capabilities necessary to assist schools affected by an event or emergency in restoring the learning environment. it also means teaming with community partners to restore educational programming, the physical environment, business operations, and social, emotional, and behavioral health. the majority of prevention, protection, and mitigation activities generally occur before an incident, although these three mission areas do have ongoing activities that can occur throughout an active shooter incident. response activities occur during an incident, and recovery activities can begin during an incident and occur after an incident (united states department of education, office of elementary and secondary education, office of safe and healthy students ; united states department of homeland security b; united states department of homeland security ). in the k- school security guide, the u.s. department of homeland security (dhs) focuses on prevention and protection since the activities and measures associated with them occur prior to an incident ( ). effective preventative and protective actions decrease the probability that schools (or other facilities) will encounter incidents of gun violence or should an incident occur, it reduces the impact of that incident. the guide emphasizes that the level of security at a facility will be based on hazards relevant to the facility, people, or groups associated with it. it also warns that as new or different threats become apparent, the perception of the relative security changes and insecurity should drive change to reflect the level of confidence of the people of groups associated with the facility. the dhs utilizes a hometown security approach that emphasizes the process of connect, plan, train, and report (cptr) with the objective to realize effective, collaborative outcomes (united states department of homeland security b). the initial phase is connect and occurs by a school or district reaching out and developing relationships in the community, including local law enforcement. having these relationships before an incident or event can help speed up the response when something happens. each school must begin with identification or development of a security team, group, or organization. this phase also emphasizes outreach, collaboration, and building of a coalition. there should be coalition members from within a school and may include district/school administrators, teachers, aides, facility operations personnel, human resources, administrative, counseling, and student groups. external groups directly related to the school might include boards of education, parent organizations, mental health groups/agencies, and teacher and bus driver unions. external groups indirectly related to the school include all responder organizations such as police and fire departments, sheriff's office, emergency medical services, emergency management, and the local dhs protective security advisor (psa). other tangential groups such as volunteer organizations, utility providers, and facilities in close geographic proximity should also be considered. core and advisory members of the coalition are established. a coalition champion is also identified and is the person who owns the majority of the responsibility for achieving a school's security goals. the champion organizes the coalition as it grows and matures (united states department of homeland security b). the next phase is plan. this will bring the coalition together. the guide for developing high quality school emergency operations plans (united states department of education, office of elementary and secondary education, office of safe and healthy students ) is an excellent resource for the coalition. a school security survey for gun violence can be completed and the coalition or user can quickly and effectively determine a facility's security proficiency (united states department of homeland security. ). specific portions of or topics within a school plan should be assigned to individuals, committees, or working groups most qualified to address them. the planning process must be sustainable. the amount of time spent in the planning phase should be commensurate with the amount of effort expended on the other phases (united states department of homeland security b). the next phase of the process is to train on the plan developed by the coalition. determining who is responsible for what and how it should be done is the basic function of planning. in fact, telling various members of the team what is expected of them and when to do that activity is the function of training. it is vital to utilize the curricula development expertise possessed by the k- community. school administrators should take advantage of this skill set and find creative ways to address difficult topics, such as gun violence. it should be carried out in an effective and nontraumatic way. presenting the training in pieces or steps allows for a more comprehensive learning experience. it is important to validate training through exercises and drills, all of which should include the students. the training event should be followed by the completion and implementation of an after-action improvement plan with adjustment of the cptr as indicated (united states department of homeland security b). the final phase in the process is report. the reporting phase is arguably the most important of all the phases. reporting principles underlie the other three phases and have profound prevention and protection impacts by driving forward information. dhs models the reporting phase using the "if you see something, say something ® " campaign (u.s. dhs, ) and the nationwide suspicious activity reporting (sar) initiative (nationwide suspicious activity reporting initiative (nsi) ). "if you see something, say something ® " focuses on empowering anyone who sees suspicious activity to do something about it by contacting local law enforcement, or if an emergency to call - - (united states department of homeland security a). this is a compelling capability when well organized and managed. a good plan for reporting, especially for a k- school, involves training staff and students on what is considered suspicious. there are many methods in which schools can employ to facilitate this, such as dedicated telephone numbers, websites for anonymous reporting, email or text messaging, and mobile phone applications. conducting simple drills for reporters and receivers keeps skills sharp and reinforces the importance of the effort with the goal to save lives. if the plan includes sharing all suspicious activity calls with the local fusion center then the probability of higher fidelity reporting increases (united states department of homeland security b). when making changes to a school's plans, procedures, and protective measures, it is imperative the needs of individuals with special health care needs be addressed throughout the process. planning, training, and execution should always consider accessible alert systems for those who are deaf or hard of hearing; students, faculty, and staff who have visual impairments or are blind; individuals with limited mobility; alternative notification measures; people with temporary disabilities; visitors; people with limited english proficiency; sign cards with text-and picture-based emergency messages/symbols; and involving people with disabilities in all planning (united states department of homeland security, interagency security committee ). it is important to understand that no "profile" exists for an active shooter (united states department of education, office of elementary and secondary education, office of safe and healthy students ). however, research indicates there may be signs or indicators. o'toole ( ) presents an in depth, systematic procedure for school shooter threat assessment and intervention. the model was designed to be used by educators, mental health professionals, and law enforcement agencies. its fundamental building blocks are the threat assessment standards, which provide a framework for evaluating a spoken, written, and symbolic threat, and the fourpronged assessment approach which provides a logical, methodical process to examine the threatener and assess the risk that the threat will be carried out. schools should learn the signs of a potentially volatile situation that may develop into an active shooter situation and proactively seek ways to prevent an incident with internal resources, or additional external assistance (united states department of education, office of elementary and secondary education, office of safe and healthy students ). potential warning signs of a school shooter may include increasingly erratic, unsafe, or aggressive behaviors; hostile feelings of injustice or perceived wrongdoing; drug and alcohol abuse; marginalization or distancing from friends and colleagues; changes in performance at work or school; sudden and dramatic changes in home life or in personality; pending civil or criminal litigation; and observable grievances with threats and plans of retribution (united states department of homeland security b). at a minimum, schools should establish and enforce policies that prohibit, limit, or determine unacceptable behaviors and consequences of weapons possession/use, drug possession/use, alcohol/tobacco possession/use, bullying/harassment, hazing, cyber-bullying/harassment/stalking, sexual assault/misconduct/harassment, bias crimes, social media abuse, and any criminal acts (united states department of homeland security b). in addition to policies and positive school climates, school districts and administrators should establish dedicated teams to evaluate threats, such as a threat assessment team (tat). the team should include mental health professionals (e.g., forensic psychologist, clinical psychologist, and school psychologist) to contribute to the threat assessment process (united states department of homeland security b). it is the responsibility of the tat to investigate and analyze communications and behaviors to make a determination on whether or not an individual poses a threat to him/herself or others (united states department of education, office of elementary and secondary education, office of safe and healthy students ). as well as tats, some schools have even opted to establish social media monitoring teams which look for keywords that may indicate bullying or other concerning statements. if a school opts to create such a team, it should work very closely with the tat to ensure that applicable privacy, civil rights and civil liberties, other federal, state and local laws, and information sharing protocols are followed. please refer to chap. for further information. after an active shooter incident, field triage (e.g., jumpstart) must commence and the patient must be evaluated by an experienced emergency medicine or trauma surgeon, preferably by a pediatric specialist in those disciplines. if an active shooter incident is coupled with detonation of an explosive device, the child must be screened and decontaminated for radiation exposure ("dirty bomb"). triage tags are extremely helpful when multiple victims present in a short period of time. medical response to an active shooter event will focus on control of external hemorrhage along with circulatory stabilization. operative emergencies will be common and receive the highest priority. severe extremity injuries may be controlled with tourniquet application or other forms of hemorrhage control. re-evaluation is paramount to prevent ischemia to distal regions. however, thoracic or abdominal (truncal) injuries will need immediate surgical exploration and intervention. penetrating trauma will cause more vascular injuries than blunt trauma, and vascular surgical trays may be in short supply at a hospital. major procedure or surgical trays may become short in supply based on the increased operative demand. resuscitative blood transfusion therapy may utilize a massive blood transfusion protocol. since whole blood may be short in supply, some will simply use the : : rule (administer one unit of packed cells: one unit of fresh frozen plasma: one unit of platelets). a unit for children may be substituted as an aliquot based on size of the patient (e.g., administer ml/kg of packed cells: ml/kg of fresh frozen plasma: ml/kg of platelets). calcium must also be replaced when there is a large volume transfusion. due to extensive blood product utilization, there may be a heavy impact on institutional or regional blood supplies. plans should be in place to address these problems, including the implementation of allocation of scarce resources. mental health support and staff debriefs are essential and should be included after an active shooter event (hick et al. ). in conclusion, all forms of disasters, whether man-made or natural, impact infants, children, and adolescents throughout the world. effective and efficient interventions remain the cornerstone of sustaining a child's well-being while reducing untoward complications due to all forms of disasters. having a deep understanding of pediatric physiology and pathophysiology is crucial to all levels of disaster diagnostics and therapeutics. all nurses and hcps have an obligation to understand these principles and deliver excellent, compassionate care to the pediatric disaster victim. advanced law enforcement rapid response training evidence-based support for the all-hazards approach to emergency preparedness ahls advanced hazmat life support provider manual active shooter incidents in the united states in and radiation disasters and children-committee on environmental health apls: the pediatric emergency medicine resource textbook of neonatal resuscitation american college of emergency physicians, pediatric committee, and emergency nurses association pediatric committee american college of emergency physicians, national association of ems physicians, pediatric equipment guidelines committee emergency medical services for children (emsc) partnership for children stakeholder group handbook of emergency cardiovascular care for healthcare providers pediatric advanced life support provider manual mass casualty terrorist bombings: a comparison of outcomes by bombing type committee on pediatric emergency medicine and task force on terrorism. the pediatrician and disaster preparedness mass casualty terrorist bombings: a comparison of outcomes by bombing type assistant secretary for preparedness and response, united states department of health and human services avalon project-the atomic bombings of hiroshima and nagasaki fort sam houston, tx: borden institute, us army research institute of chemical defense (usamricd), 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we approach these questions with limited data and past epidemics provide little guidance for covid- . but, we know this: schools will reopen. their closure is too burdensome on parents, communities, and the economy. simply put, we cannot fully re-open society without re-opening schools. this requires that children will be part of the first wave to re-emerge from shelter-in-place policies. with fast-approaching preparations required for a new school year, a collaborative team of clinicians, scientists, and educators developed this commentary to begin to highlight issues that must be considered to ensure a safe and strategically planned re-opening of schools. the american academy of pediatrics also has recently posted considerations important to the re-opening of schools. in the u.s., in an urgent attempt to curb spread of sars-cov- and save lives, the nationwide closure of k- schools occured rapidly. planning for schools re-opening must be more deliberate, delineating precisely how, when, under what conditions, and base the reopening on available data. school re-opening can mitigate risks to children, families, and school personnel only if it is sensitive to community needs. models such as the cdc's whole school, whole community, whole child and the school health index can provide a helpful framework. systematic review of the early chinese experience reveals that patients younger than years accounted for only . % of , confirmed cases. although severe covid- is uncommon in children , a picture of critically ill children in the u.s. is emerging, with up to % having no underlying medical condition and the medical conditions of others seemingly varying by communities served. [ ] [ ] [ ] furthermore, without more community-based data, we don't know whether most children actually evade infection, or if infected, largely are asymptomatic. this uncertainty is dangerous. in children, who frequently require hands-on care, asymptomatic infection may pose a greater risk to susceptible individuals than might exposure to an asymptomatic adult. in addition, current data indicate that children and adults have different infection outcomes. possible mechanisms include changes in innate and adaptive immune responses with maturation, pediatric vaccinations or common infections that stimulate protective innate responses in children or conversely, previous infections with common uri coronaviruses that stimulate deleterious acquired responses in adults, and differences in virus binding and infectivity of host epithelial cells. though sars-cov- community surveillance testing has yet to be standardized, large scale viral nucleic acid and serological testing in children is needed to guide safe school reopening. this testing approach will require activation of non-traditional testing sites, such as homes, schools and "child-friendly" self-collection methods. testing capability is only the first step. the second is understanding the test results in the appropriate context. as large-scale testing is implemented, care must be taken to ensure that these test results are interpreted and communicated appropriately so as to inform, empower, and protect families, school personnel, and communities. the safe re-opening of schools also demands sensitivity to community inequities. all schools present unique challenges for mitigation strategies commonly implemented in the rest of society, eg, physical distancing, face coverings, and good hygiene practice. in addition, many school systems face additional challenges related to food security, safe transportation, healthcare safety nets, and emergency preparedness policies, which also often are within their purview. these interventions will be more difficult for resource-constrained title schools, which often are situated in low-income and minority neighborhoods. how do we recover from the psychological, medical, and economic damage resulting from school closures? schools are a bedrock of the american social network, providing not only education, but also nutritional support, healthcare and social services. the response to pandemicrelated challenges will require augmenting existing school-based programs. the pandemic has impacted students' access to and relationships with healthcare providers, with many children needing more behavioral and medical care services. as schools re-open, school-based health centers should be expanded. these centers improve student health and educational outcomes and reduce healthcare disparities among vulnerable student populations, while providing demonstrable cost savings. in addition, federally assisted school-based meal programs annually benefit nearly million children. student eligibility for these programs, meal distribution practices, and food safety standards must be reviewed and adjusted to meet the unprecedented circumstances. consideration must be given to the availability of these programs to student caregivers and parents, more of whom are unemployed with each passing week. finally, schools and communities must support teachers and staff returning to school. concerns and stress among schoolstaff may exacerbate already high rates of teacher turnover that disproportionately affect students attending under-resourced schools. greater school instability could undermine safe school re-opening. children with chronic conditions will be especially vulnerable during this return to school. although covid- symptoms are mild in most otherwise healthy children, we cannot assume that the estimated - % of children with chronic diseases will be similarly resilient. children with underlying neurologic conditions and medical complexity have been among those severely affected in the u.s. [ ] [ ] [ ] early data from italy suggest that most patients with cystic fibrosis in community settings generally may be avoiding covid- infection through physical distancing. whether this effect can be achieved in schools remains unknown. some data suggest that children with cancer have not had severe morbidity with covid- , however this experience is not universal, and the full effect of immunocompromising therapies on covid- risk and severity has yet to be determined. in addition, concern about covid- transmission could present a disproportionate barrier to school re-entry for children with cystic fibrosis or asthma who frequently have chronic, often productive cough. acute chest syndrome in children with sickle cell disease may be difficult to distinguish from covid- related lung disease . under current conditions of increased sensitivity to possible covid- symptomatology, children with these chronic health disorders risk being socially shunned and unnecessarily removed from school. more research is needed to understand the risks that all children, including those with chronic conditions, may encounter in school settings in the covid- era. even as we curtail physical contact, we must not reflexively abandon after-school activities and physical education. this would exacerbate the more slow-moving pandemic of childhood physical inactivity and obesity, while also denying the physical and mental health enriching benefits of extracurricular activities. early-life physical activity is beneficial for child health and health across the lifespan, and physical fitness in children is associated with improved school learning. conversely, the lack of participation of adolescents in pe classes is associated with social isolation and loneliness. of note, obesity is a serious complicating comorbidity of the covid- pandemic in adults . data preceding the pandemic indicate that obese children admitted to pediatric intensive care units required more invasive therapeutic interventions than non-obese children. preparations for school reopening should include novel approaches to pe, structured recesses, and access to safe after-school activities. finally, we must recognize that re-opening schools raises a number of ethical issues, including safety, privacy, autonomy, vulnerability, proportionality, and health disparities, that impact children and their families, as well as teachers, staff, and administrators. parents and caregivers, as well as viral surveillance and contact tracing teams likely will require more frequent monitoring of the school environment. these activities will influence costs, the educational dynamic in the classroom, risks related to privacy, and the autonomy of older children and adolescents. if wearable devices or smartphones are deployed to collect health information, who will be required to seek and provide consent and at what age will children be asked to assent? what will happen if a child or caregiver refuses to comply with surveillance programs? how do long-standing issues with immigration status and health disparities influence the effective implementation of school reopening plans? in the u.s., states made rapid decisions to shutter k- schools in response to the pandemic. public safety dictated this haste, but safe school re-opening must be more deliberate and carefully planned. re-opening inherently carries risk and solutions that will be disruptive may need to be considered (e.g. staggered start times). it is clear that healthy school re-opening strategies will require creativity and considerable monetary investment to obtain unique pediatric data on symptoms and the dynamics of virus shedding, the ability to test for the presence of serum antibody and to understand its meaning,as well as the capability to perform contact tracing in real time related to inevitable exposures. new educational strategies must be developed, and the generally inadequate allocation of school nurses will need to be reconsidered. school closure and management practices during coronavirus outbreaks including covid- : a rapid systematic review whole child model: a new approach for improving educational attainment and healthy development for students shi | school health index | healthy schools | cdc clinical characteristics and diagnostic challenges of pediatric covid- : a systematic review and meta-analysis severe covid- in children and young adults in the washington, dc metropolitan region clinical characteristics and outcomes of hospitalized and critically ill children and adolescents with coronavirus disease (covid- ) at a tertiary care medical center characteristics and outcomes of children with coronavirus disease (covid- ) infection admitted to us and canadian pediatric intensive care units. jama pediatr school-based health centers in an era of health care reform: building on history impact of covid- on people with cystic fibrosis flash survey on severe acute respiratory syndrome coronavirus- infections in paediatric patients on anticancer treatment covid infection in patients with sickle cell disease covid and obesity. clin obes outcomes and resource use among overweight and obese children with sepsis in the pediatric intensive care unit the community at large will need to view schools as "healthy places" for children and society. this could be accomplished by building public health-focused collaboratives capable of continuous learning and rapid cycles of implementation, as covid- information evolves at breakneck speed. otherwise, we risk further compounding the incalculable damage already incurred by covid- among children across our country and the world. key: cord- - iayu jg authors: nan title: for a calm and secure back-to-school time date: - - journal: bull acad natl med doi: . /j.banm. . . sha: doc_id: cord_uid: iayu jg nan role in instruction and socialization. after the containment and the summer vacations, the need for a return to class, face-to-face teaching and a collective life is essential for all pupils. the efficiency of home-school telework, which is very uneven among the population, justifies an evaluation of learning achievements and pedagogical reinforcement. everything must be done to ensure that the persistence of the covid- epidemic does not disrupt the start of the september school year so eagerly awaited by the majority of children, parents and teachers. it is important to address the legitimate fears that parents still have about the risk to their children. according to recent epidemiological data, pediatric forms of covid- worldwide account for only to % of all cases. children infected with cov- -sars are most often asymptomatic or mildly reached [ ] . in france, only % of hospitalizations for covid- involve children. moreover, studies confirm that children are weak transmitters, especially before the age of years. pupil-to-pupil transmission of sars-cov- is rarely observed in schools, as is intra-school transmission from pupils to adults [ ] . the rare cases of infection observed in students come from the home environment and not from the school. these data need to be recalled in order to approach the new school year with confidence. furthermore, it is imperative to implement from day one the hygiene measures detailed in the ministry of national education's health protocol for students and adults working in schools [ ] . the learning of ''barrier gestures'' by children allows them to be involved as promoters and defenders of prevention. it is also necessary to inform and involve parents, whose participation in the fight against the epidemic in schools is essential. the obligation to wear a mask extends to all students in the ''collège'' and ''lycée'', inside and outside the school, to all school staff and to parents. hand washing with soap, which is preferable to hydro-alcoholic solutions, should be carried out in primary school in the presence of an adult. a distance of one meter between students in classrooms and dining halls should be observed when appropriate. likewise, the mixing of students must be reduced by organizing displacements and avoiding groupings. in addition to these measures, the premises and equipment are regularly ventilated and disinfected several times a day. it should be clear what to do in front of a child with signs suggestive of covid- . the child should stay at home if he or she is feverish (temperature ≥ • c) or if a person is carrying the virus in the family home. in the classroom, it is the responsibility of the school physician to manage screening around a case and appropriate exclusion procedures while avoiding excessive class closures. routine screening for asymptomatic cases is of no interest in children. rt-pcr tests should be performed from primary school onwards in children who are symptomatic or exposed to a positive case. in kindergarten children, symptomatic cases should be distinguished from seasonal infections. the exclusion period for confirmed cases is days but could be reduced. in the current context of health crisis, the national academy of medicine recommends: • not to delay the start of the school year for the education and psycho-social well-being of children; • to reinforce the role of school medicine for information, health education and screening activities; • to set up an investigation protocol in each establishment with rapid access to screening tests, immediately applicable in the event of the occurrence of a case, in conjunction with the regional health agency; • to promote flu vaccination campaigns in schools starting in october. mesures sanitaires pour la réouverture des écoles, collèges, lycées et crèches; [communiqué de l'académie european centre for disease prevention and control. covid- in children and the role of school settings in covid- transmission ministère de l'Éducation nationale, de la jeunesse et des sports. covid- protocole sanitaire des écoles et établissements sco-q laires --année scolaire ministère de l'Éducation nationale, de la jeunesse et des sports the authors declare that they have no competing interest. uncited reference q [ ] . key: cord- -luqvw y authors: levinson, julia; kohl, kid; baltag, valentina; ross, david title: investigating the effectiveness of school health services delivered by a health provider: a systematic review of systematic reviews date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: luqvw y schools are the only institution regularly reaching the majority of school-age children and adolescents across the globe. although at least countries have school health services, there is no rigorous, evidence-based guidance on which school health services are effective and should be implemented in schools. to investigate the effectiveness of school health services for improving the health of school-age children and adolescents, a systematic review of systematic reviews (overview) was conducted. five databases were searched through june . systematic reviews of intervention studies that evaluated school-based or school-linked health services delivered by a health provider were included. review quality was assessed using a modified ballard and montgomery four-item checklist. references were screened and systematic reviews containing primary studies were assessed narratively. interventions with evidence for effectiveness addressed autism, depression, anxiety, obesity, dental caries, visual acuity, asthma, and sleep. no review evaluated the effectiveness of a multi-component school health services intervention addressing multiple health areas. strongest evidence supports implementation of anxiety prevention programs, indicated asthma education, and vision screening with provision of free spectacles. additional systematic reviews are needed that analyze the effectiveness of comprehensive school health services, and specific services for under-researched health areas relevant for this population. and should be implemented in schools. to investigate the effectiveness of school health services for improving the health of school-age children and adolescents, a systematic review of systematic reviews (overview) was conducted. five databases were searched through june . systematic reviews of intervention studies that evaluated school-based or school-linked health services delivered by a health provider were included. review quality was assessed using a modified ballard and montgomery four-item checklist. references were screened and systematic reviews containing primary studies were assessed narratively. interventions with evidence for effectiveness addressed autism, depression, anxiety, obesity, dental caries, visual acuity, asthma, and sleep. no review evaluated the effectiveness of a multi-component school health services intervention addressing multiple health areas. strongest evidence supports implementation of anxiety prevention programs, indicated asthma education, and vision screening with provision of free spectacles. additional systematic reviews are needed that analyze the effectiveness of comprehensive school health services, and specific services for under-researched health areas relevant for this population. the world health organization (who) launched the global school health initiative in with the goal to improve child, adolescent and community health through health promotion and programming in schools [ ] . this initiative is dedicated to promoting development of school health programs and increasing the number of health-promoting schools, characterized by who as "a school constantly strengthening its capacity as a healthy setting for living, learning and working" [ ] . in , who, the united nations educational, scientific and cultural organization (unesco), the united nations children's fund (unicef) and the world bank developed a partnership for focusing resources on effective school health -a fresh start approach [ ] . the fresh framework promotes four pillars: health-related school policies, provision of safe water and sanitation, skills-based health education and school-based health and nutrition services [ ] . while various guidance documents have been published by united nations (un) organizations addressing a range of services from oral health to malaria [ ] [ ] [ ] [ ] [ ] , there is no internationally accepted guideline regarding school health services. this systematic review of systematic reviews, henceforth referred to as an overview, will inform the upcoming development of a who guideline that addresses one pillar of the fresh framework: school health services delivered by a health provider. schools offer a unique platform for health care delivery. in , the global means for the primary and secondary net school enrollment rates were % and %, respectively, thus the potential reach of school health services is wide [ ] . additionally, a recent review found that school-based or school-linked health services already exist in at least countries [ ] . the global accelerated action for the health of adolescents (aa-ha!) implementation guidance calls for the prioritization of school health programs as an important step towards universal health coverage and urges that "every school should be a health promoting school" [ ] . the primary objective of this overview was to explore the effectiveness of school- based or school-linked health services delivered by a health provider for improving the health of school-age children and adolescents. through a comprehensive literature search, the overview aimed to identify health areas and specific school health service interventions that have at least some evidence of effectiveness. it was also designed to suggest further research in areas where recent systematic reviews (srs) exist, but with insufficient evidence. finally, the overview aimed to identify the health areas and specific school health services interventions for which no srs were found, whether because the primary literature does not exist or where there are primary studies but no sr has been conducted. this overview was conducted using the preferred reporting items for systematic reviews and meta-analyses (prisma) [ ] . a protocol was developed a priori that outlined the overview objectives, aims, operational definitions, search strategy, inclusion/exclusion criteria, and quality appraisal methods. this document was followed throughout the review process and is available in s appendix. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] enrolled in schools; (b) interventions were school-based or school-linked health services, involved a health provider (see definitions in s appendix), and were of any duration or length of follow-up; (c) intervention effectiveness was compared to either no intervention, an alternative intervention, the same intervention in a different setting (i.e. not in schools), an active control, or a waitlist control; (d) interventions aimed to improve some aspect of health; and (e) study designs were either randomized controlled trials (rcts), quasi-experimental studies (qes), or other non-randomized intervention studies. there were no date restrictions on publication of included srs. in addition to these criteria for included studies, the srs themselves had to fulfill the following criteria: (a) included the words "systematic review" in the title or abstract; (b) outlined inclusion criteria within the methods section; (c) published in peer-reviewed journals and indexed before june , ; (d) published in the english language. in addition to srs that did not meet these inclusion criteria, srs were excluded if the review was superseded by a newer version. study selection citations identified from the systematic search were uploaded to covidence systematic review software [ ] and duplicates were automatically deleted. two reviewers (kk and jl) screened all titles and abstracts using the inclusion/exclusion criteria and excluded all articles that were definitely ineligible. articles that received conflicting votes (ineligible vs. potentially or probably eligible) were discussed and consensus was reached. the same two reviewers screened the full text of all the potentially or probably eligible articles using a ranked list of the inclusion criteria (s appendix). reasons for exclusion were selected from the ranked list. if consensus was not possible during title/abstract or full text screening, a third reviewer (dr), who had the casting vote, would have been asked to independently screen the article. this was never required as consensus was always reached. data collection one reviewer (jl) extracted summary data from each selected article using a customized standard form with independent data extraction performed for % of included srs by one of the other reviewers (dr or kk). there was % agreement between reviewers for all items within the standard form, with discrepancies only in level of detail . data items included the research design of the sr and primary studies, sample description and setting, intervention characteristics, outcomes, meta-analysis results, quality appraisal, and conclusions. due to the heterogeneity of the srs included in this overview, it was not possible to perform a meta-analysis. outcome measures were collected from included studies. risk of bias within primary studies was recorded in s appendix. risk of bias across srs was determined using ballard and montgomery's four-item checklist for overviews of srs [ ] . these items include: ( ) overlap (see below), ( ) rating of confidence from the amstar checklist [ ] , ( ) date of publication, and ( ) match between the scope of the included srs and the overview itself. an important consideration in overviews is the degree of overlap, or the use of the same primary study in multiple included srs. high overlap can contribute to biased results [ ] . this overview used the corrected covered area (cca), a comprehensive and validated measure, to determine overlap [ ] . the cca is calculated using three variables: the number of "index" publications (r), the number of total publications (n), and the number of srs within the overview (c). an "index" publication is the first appearance of a primary study within an overview. the formula for the cca is: ccas can be interpreted as indicating slight, moderate, high or very high overlap with scores of - , - , - , or > , respectively [ ] . the amstar checklist [ ] was used to appraise quality of included srs. one reviewer (jl) assessed all srs and a second (kk) duplicated appraisal of %, with % agreement and only minor disagreements that did not impact grades of confidence. following the recommendation of the amstar developers [ ] , individual ratings were not combined into an overall score. instead, the authors determined which of the items on the checklist were critical for this overview and which of the items were non-critical. building on a method suggested by shea and colleagues [ ] , grades of confidence in the results of each sr were generated based on critical flaws and non-critical weaknesses. the grading system is available in s appendix. confidence in results ranged from high (three or fewer non-critical weaknesses) to critically low (more than three critical flaws with or without non-critical weaknesses) [ ] . used meta-analysis to combine results [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , whereas the remaining nine srs narratively synthesized results [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . eleven srs included studies located in countries with high- income or upper-middle income economies only [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , ] . six srs included at least one study from countries with lower-middle income or lower income economies [ , , , , , ] . the final three srs either did not state the locations of included studies [ , ] or provided regions rather than specific country locations [ ] . to be included in this overview, at least % of studies within each sr had to fulfill all inclusion criteria. in four srs, % of included studies fulfilled all inclusion criteria [ , , , ] , although brendel and colleagues [ ] only included one study in total. in another four srs, % to % of included studies fulfilled all the inclusion criteria [ , , , ] . in the remaining twelve srs, % to % of included studies fulfilled all the inclusion criteria [ , , [ ] [ ] [ ] [ ] , , , [ ] [ ] [ ] . all srs primarily examined studies on school-based, rather than school-linked interventions. the srs covered eight health areas: nine on mental health [ , , , , , [ ] [ ] [ ] ] , four on oral health [ , , , ] , two on asthma [ , ] , and one sr each on sleep [ ] ; obesity [ ] ; vision [ ] ; menstrual management [ ] ; and sexual and reproductive health (srh) [ ] . eleven srs included only cluster-and individually- randomized controlled trials [ ] [ ] [ ] , , , , , , ] , seven srs included other types of controlled and uncontrolled experimental studies in addition to rcts [ , , , , , , ] , and two srs included only qes [ ] or controlled clinical trials [ ] . table the corrected covered area (cca) was found to be , indicating only slight overlap between the srs. calculations for the cca can be found in s appendix. table presents the remaining three of the four items of ballard and montgomery's checklist for overviews of reviews: ( ) levels of confidence in results for each included sr, ( ) publication year, and ( ) match in scope to the overview. a majority of the studies ( %) were given low or critically low levels of confidence. only three srs [ , , ] were scored as having moderate levels of confidence and just one [ ] was given a high level of confidence. the details of the quality appraisal of primary studies included in the srs are given in s appendix. [ ] oral health moderate % bastounis [ ] mental health low % brendel [ ] mental health critically low % chung [ ] sleep low % cooper [ ] oral health high % evans [ ] vision moderate % geryk [ ] asthma critically low % gold [ ] mental [ ] obesity critically low % stein [ ] oral health low % sullivan [ ] mental health critically low % walter [ ] asthma low % werner-seidler [ ] mental health low % a the four items of the checklist include: overlap, amstar rating of confidence, up-to-date, and relevance of included studies b see s appendix for amstar rating information c i.e., percentage of studies within the systematic review that clearly fulfill all inclusion criteria srh = sexual and reproductive health none of the srs evaluated comprehensive, multi-component, or multi-health area school health services. two srs found strong evidence for the potential effectiveness of educational interventions for children and adolescents with asthma diagnoses (table a) [ , ] . geryk and colleagues found that education on correct use of an inhaler improved inhaler technique, regardless of deliverer, method, or duration of the intervention [ ] . however, they did not assess risk of bias or appraise the quality of included studies [ ] . walter and colleagues found that family asthma educational programs for children and their parents or caregivers improved quality of life for both caregivers and children, and decreased asthma exacerbations for children [ ] . while results from primary studies were statistically significant in both srs, heterogeneity of interventions precluded meta-analysis by walter and colleagues [ ] and no reason was given for why meta-analysis was not performed by geryk and colleagues [ ] . hennegan and montgomery assessed the effectiveness of "hardware" and "software" menstrual management interventions (table b ) [ ] . hardware interventions included provision of sanitary products and software interventions focused on menstrual management education. a metaanalysis of two studies on sanitary pad provision found a moderate but statistically non-significant effect on school attendance. however, it was unclear whether these studies involved a health provider [ ] . outcomes across studies differed, but the authors noted trends toward improvement in menstruation knowledge, management practices, psychosocial outcomes, and school attendance. hennegan and montgomery found a high level of heterogeneity and substantial risk of bias in the included studies overall, thus they were unable to make conclusions about the effectiveness of menstrual management interventions [ ] . the effectiveness of school-based mental health services was assessed in nine srs (table c ) [ , , , , , [ ] [ ] [ ] , ] . srs addressed various intervention types: universal interventions [ , , , , , ] ; targeted interventions for military-connected children [ ] , children and adolescents at risk for depression and/or anxiety [ , ] , refugee and war-traumatized youth [ ] , and children referred to therapy [ ] ; and indicated interventions for children and/or adolescents diagnosed with autism spectrum disorder [ ] , depression [ , , ] , or anxiety [ , , ] . prevention and treatment of mood disorders was assessed in five srs, all of which targeted children and adolescents using rcts of established programs. higgins and o'sullivan assessed the friends for life program, a manual-based cognitive behavioral anxiety prevention program comprised of ten sessions with developmentally-tailored programs for different age groups [ ] . they found statistically significant improvements in self-reported measures of anxiety for participants who completed the program as compared to those in the control group [ ] . a sr and meta-analysis by bastounis and colleagues on the educational and preventative penn resiliency program (prp) and its derivatives found small, non-significant effect sizes for the prevalence of both depression and anxiety, favoring the intervention in the former and the control in the latter [ ] . the remaining three srs also assessed friends and prp along with additional often-overlapping programs. neil and christensen analyzed unique anxiety prevention and early intervention programs and found reductions in anxiety symptoms in % of the included studies [ ] . kavanagh and colleagues examined depression and anxiety group counseling programs based on cognitive behavioral therapy (cbt) and found statistically significant reductions of depressive symptoms at both four weeks and three months follow-up [ , ] . finally, meta-analysis by werner-seidler and colleagues of rcts on the effectiveness of depression and anxiety prevention and group therapy programs found small yet statistically significant effect sizes in favor of the intervention groups for both depression and anxiety as compared to control groups [ ] . although the overall degree of overlap between all srs within this overview was slight, the overlap between just these five srs targeting mood disorders was high (cca = ). assessments of music [ ] and art therapy [ ] in two srs reported weak evidence of effectiveness. gold and colleagues assessed daily music therapy as an intervention to improve verbal and gestural communicative skills and reduce behavioral problems in children diagnosed with autism spectrum disorder [ ] . meta-analysis found small but statistically significant effect sizes in favor of music therapy for gestural communication, verbal communication, and behavioral problems [ ] . oppositional defiant disorder (odd), separation anxiety disorder (sad), moderate to severe behavior problems, or learning disorders [ ] . the authors found improvements in classroom behavior, symptoms of odd, and symptoms of sad [ ] . however, in the studies included in both gold and colleagues' and mcdonald and drey's srs, the numbers of participants per intervention group were very small: - and - per study, respectively, introducing possibility of bias [ , ] . mostly favorable evidence of effectiveness was found in a sr of social-emotional interventions for refugee and war-traumatized youth from countries [ ] . improvements in trauma-related symptoms and impairment were found through narrative assessment of creative expression interventions, cognitive behavioral interventions, and multifaceted interventions [ ] . in contrast with gold and colleagues [ ] , this sr by sullivan and simonson found negative effects from music therapy interventions [ ] . however, there was no risk of bias assessment in this sr and therefore the results must be interpreted cautiously. the final sr on mental health services examined well-being interventions for children with a parent in the military [ ] . only one quasi-experimental study from the united states in was included in the sr. the study assessed a group counseling intervention and found no statistically significant effects on the prevalence of anxiety, self-esteem, internalizing behavior or externalizing behavior [ ] . schroeder and colleagues reviewed the effectiveness of obesity treatment and prevention interventions that specifically involved a school nurse (table d ) [ ] . most interventions involved school-nurse-delivered nutrition counseling, nutrition and health education, and some parent involvement or physical activity. meta-analysis indicated small, yet statistically significant, reductions in body mass index (bmi), bmi z-score, and bmi percentile for both obesity treatment and prevention [ ] . srs on oral health interventions focused on prevention [ , ] , screening [ ] , and education [ , ] (table e ). strongest evidence in favor of oral health interventions emerged from a sr on universal topical application of fluoride gel for the prevention of dental caries [ ] . meta-analysis results indicated a statistically significant effect on the before-after change in caries prevalence [ ] . a universal educational intervention on oral hygiene and caries produced weaker evidence of effectiveness [ ] . small but statistically significant effect sizes were found in favor of the intervention for mean plaque levels and oral hygiene, but no statistical significance was found for change in gingivitis indices [ ] . two srs on dental health screening [ ] and behavioral interventions for caries prevention [ ] found limited evidence of effectiveness. arora and colleagues did not find any rcts that looked at the effectiveness of dental health screening versus no screening on improving oral health outcomes, but their search did locate six rcts from the united kingdom and india with dental care attendance as the outcome [ ] . the data was too heterogeneous to meta-analyze, and the authors determined that the certainty of the evidence of the benefit of dental screening in increasing dental attendance was very low [ ] . the other sr examined behavioral interventions in the form of education on tooth brushing and the use of fluoride toothpaste in brazil, italy, united kingdom, and iran [ ] . due to the diversity in outcome measures and intervention intensities, the authors felt unable to make any evidence-based recommendations [ ] . a sr on sexual health interventions for prevention of sexually transmitted infections (stis) and human immunodeficiency virus (hiv) in sub-saharan african countries found that educational interventions were successful in increasing knowledge and attitudes for participants (table f ) [ ] . however, the sr suggested that the studies were ineffective in changing self-reported risky behaviors, although follow-up was either immediate or short-term (less than months) [ ] . this sr did not discuss the quality or risk of bias of included studies [ ] . chung and colleagues systematically reviewed and meta-analyzed universal sleep education programs as compared to no additional sleep intervention from australia, new zealand, brazil, and hong kong (table g ) [ ] . five of the included studies examined the same weekly sleep education program from the australian centre for education in sleep. the sixth study assessed a -day program in brazil. meta-analysis of the six studies showed statistically significant short-term benefits for weekday sleep time, weekend sleep time, and mood [ ] . however, these results did not persist at follow-up [ ] . evans and colleagues reviewed seven rcts from china, india, and tanzania on vision screening for correctable visual acuity deficits at or before school entry (table h ) [ ] . through meta-analysis of two rcts, the authors found that school vision screening combined with provision of free spectacles resulted in a statistically significant % increase in the wearing of spectacles at - months follow-up as compared to vision screening combined with prescription for spectacles only [ ] . evans and colleagues found no statistically significant difference in the proportion of students wearing spectacles at - months follow-up between vision screening with provision of ready-made spectacles and vision screening with provision of custom-made spectacles in a meta-analysis of three rcts [ ] . education on the wearing of spectacles in addition to vision screening as compared to vision screening alone did not have a significant effect [ ] . no srs found eligible studies comparing vision screening with no vision screening. this overview found srs covering primary studies. the majority of srs assessed educational, counseling, or preventive interventions, most of which were special research interventions rather than routinely-delivered school health services. no sr examined comprehensive or multi-component school health services, despite the fact that comprehensive services may be more efficient, easier to implement, and more sustainable than single interventions [ ] . results from this overview suggest that certain interventions can be effective in improving child and adolescent health outcomes, and thus may be worthwhile for integration into school health programs. vision screening is one of the most common forms of school health services [ ] , although the majority of programs are concentrated in high-income countries (hic) [ ] . although prevalence of visual impairment varies widely by ethnic group and age [ ] , who estimates that at least million children below age are visually impaired [ ] . evans and colleagues found strong evidence from china, tanzania, and india that school vision screening for correctable visual acuity deficits increased wearing of spectacles when spectacles were provided at no cost [ ] . a recent guideline from the international agency for prevention of blindness (iapb) reiterates the importance of free spectacles and goes further to suggest that low-and middle-income countries (lmic) adopt comprehensive school eye health programs [ ] . vision screening linked with free provision of spectacles, as a component of a comprehensive school eye health program, is an example of a cost-effective form of school health services that may be implemented. five srs covered depression and/or anxiety prevention and early intervention programs, with the friends for life program (friends) and the penn resiliency program (prp) most common [ , , , , ] . given that friends has been endorsed by who [ ] and was found to be effective in decreasing anxiety symptoms in all four srs where it was mentioned in this overview [ , , , ] , policy makers and school health officials may consider incorporating this or similar programs into existing school health services. the four srs that included prp found mixed evidence [ ] or no evidence of effectiveness [ , , , ] , bringing the popularity of this intervention into question. finally, creative therapy interventions seem to be effective for indicated populations of school-age children, such as children with autism spectrum disorder [ , ] . however, this conclusion should be interpreted cautiously due to small effect sizes, small sample sizes, and conflicting evidence on the effectiveness of music therapy between sullivan and simonson [ ] and gold and colleagues [ ] . comprehensive school programs that promote healthy school environments, health and nutrition literacy, and physical activity are one of the six key areas for ending childhood obesity recommended by who [ ] . this overview found only one sr that assessed obesity treatment and prevention delivered by a health professional in schools, despite the fact that over million school-age children and adolescents were overweight or obese in [ ] . schroeder and colleagues found that school nurses are well positioned to deliver nutritional counseling, design and coordinate physical activity interventions, and educate parents, students, and staff on health, nutrition, and fitness [ ] . however, all included primary studies were delivered in hic [ ] . schools are considered to be an ideal platform for oral health promotion through education, services, and the school environment [ ] . the most promising evidence from a sr was on topical application of fluoride gel for the prevention of dental caries [ ] . educational interventions had mixed effects. a sr that focused on behavioral education, such as demonstrating how to correctly brush teeth, found no evidence for reduction in caries [ ] , whereas a sr on oral hygiene and caries education found evidence for decreased plaque and improved hygiene [ ] . more research should be done to identify the content and methods of deliver that make some oral health education interventions more effective than others. it is difficult to determine overall effectiveness of school health services from this overview because the included srs do not sufficiently cover the health areas most relevant for children and adolescents. in , the top five leading causes of death for - year olds were lower respiratory infections, diarrheal diseases, meningitis, drowning, and road injury [ ] . among - year olds, the leading causes of death were lower respiratory infections, drowning, road injury, diarrheal diseases, and meningitis [ ] . finally, for [ ] [ ] [ ] [ ] [ ] year olds, the leading causes of death were road injury, self-harm, interpersonal violence, diarrheal diseases, and lower respiratory infections [ ] . leading causes of disability-adjusted life years (dalys) for - year olds were iron deficiency anemia, road injury, depressive disorders, lower respiratory infections, and diarrheal diseases [ ] . this overview shows that the current sr literature does address mental health, specifically mood disorders. however, the causes of death and disability beyond self-harm and depressive disorders are currently not addressed. although mortality and morbidity statistics vary by region and country, it is clear that the health areas included in this overview reflect a small subset of the global burden of disease for children and adolescents. furthermore, this overview exposes a mismatch between the sr literature on effectiveness of school health services and the actual school health services that are most commonly delivered. vaccinations have been identified as the most common type of intervention in schools in at least countries or territories [ ] , and there is evidence of effectiveness from primary studies regarding feasibility of school-based vaccination programs [ , ] . yet no srs on vaccinations fulfilled the inclusion criteria for this overview, suggesting the need for these srs to be conducted. additionally, at least countries or territories include some form of school health services that are routinely delivered, as opposed to special research interventions [ ] . this overview primarily found evidence for special research interventions, suggesting a need for assessment of routinely-delivered school health services. one of the central questions of this overview was whether the school health services that are regularly delivered across the globe are evidence-based. the mismatch in the sr literature identified by this overview demonstrates that more research must be done before an answer to this question can be determined. another important gap that this overview reveals is a lack of research on interventions carried out in lmic and low-income countries (lic). only one of the srs included in this overview examined studies from a majority of lmic and lic [ , ] . this is problematic given that health disparities for children and adolescents are greater in lmic and lic than in higher income countries [ ] . additionally, resources differ by income level and therefore effective interventions in hic may need to be tailored or changed entirely in order to be feasible in lmic and lic. who reports densities of less than one physician per population in countries and less than three nurses or although three srs mentioned the cost of specialized professionals delivering interventions versus teachers or a school nurse [ , , ] , cost, let alone cost-effectiveness, was not closely analyzed in any of the included srs. for useful recommendations to be made regarding school health services, cost-effectiveness must be more closely examined by primary studies and srs. although overviews offer a comprehensive method for synthesizing evidence, they also come with important methodological limitations. first, an overview is unlikely to include the latest evidence if recent primary studies have not yet been included in srs. this lag may preclude the ability for an overview to truly reflect current knowledge [ ] . while this overview found significant gaps in the evidence for certain health areas, this does not necessarily mean that relevant high quality primary trials have not been conducted. second, the ability of overviews to make valid and accurate conclusions is dependent upon the accuracy, rigor, and inclusiveness of the srs themselves. % (n= ) of srs included in this overview were given ratings of low or critically low confidence using the amstar checklist, although this is not unusual given the stringency of the checklist. nonetheless, it is interesting to note that all four of the srs given moderate or high levels of confidence were cochrane reviews. the remaining cochrane review was given a critically low level of confidence, though this may be because it was published in and standards for both the methods and reporting of cochrane reviews have improved in recent years. third, the scopes of individual srs often differ from the scope of the overview, a problem that ballard and montgomery call a "scope mismatch" [ ] . in this overview, srs with at least % of included studies fulfilling all criteria were included after extensive discussion between the authors and experts in the field. this implies that a narrower range of srs would have been eligible if a stricter cut-off had been selected, and vice versa. it is important to take this into account when interpreting results. finally, overlap of primary trials between srs can bias results of an overview [ ] . there is no definitive guidance on how to correct for overlap, as both including or excluding overlapping srs presents potentially biased results [ , ] . this overview measured overlap using the corrected covered area (cca) [ ] and did not exclude overlapping studies. however, the degree of overlap across all srs was graded as being small (cca = ), while there was high overlap in srs on mood disorders (cca = ). cca values for all health areas and calculations are available in s appendix. a key limitation of this overview is that only publications self-titled "systematic reviews" were included. this decision was made because of the vast numbers of reviews available and the increased rigor associated with the term "systematic". a sensitivity analysis comparing "systematic*" with "systematic review" found that the number of search results increased almost three-fold, but did not reveal any new articles that would eventually have met the subsequent eligibility criteria. another limitation is that this overview only included randomized and non-randomized controlled trials, quasi-experimental studies and other controlled study designs where health professionals delivered the intervention. while this strengthens the rigor of included studies and improves decision-making ability, it also excludes potentially relevant literature. a strength of this overview is that it attempted to answer a question that has not yet been answered regarding the effectiveness of both comprehensive and specific school health services delivered by a health provider. while other pillars of health promoting schools have relevant guidance documents, guidance on school health services is limited and not explicitly evidence-based. given the wide reach of schools and the fact that school health services already exist in most countries, international guidelines are needed to clarify whether school health services can be effective, and if so, which interventions should and should not be included. this overview makes an important first step toward that guideline. this overview presents multiple effective interventions that may be offered as a part of school health services delivered by a health provider. however, it is difficult to formulate an overarching answer about the effectiveness of school health services for improving the health of school-age children and adolescents due to the heterogeneity of srs found and the evident gaps in the sr literature. more than half of included srs analyzed mental health and oral health interventions, and no srs were found that assessed other relevant health areas, such as vaccinations, communicable diseases, injuries, etc. further, no srs evaluated comprehensive or multi-component school health services. if school health services are to truly improve the health of children and adolescents, they must comprehensively address the most pressing problems of this population. in order for policy makers and leaders in school health to make evidence-based recommendations on which services should be available in schools, who should deliver them, and how should they be delivered, more srs must be done. these srs must assess routine, multi-component school health services and the characteristics that make them effective, with special attention to content, quality, intensity, method of delivery, and cost. the gaps in the sr literature identified by this overview will inform the commissioning of new srs by who to feed into evidence-based global recommendations. who | global school health initiative focusing resources on effective school health: a fresh start to enhancing the quality and equity of education who information series on school health: oral health promotion: an essential element of a health-promoting school. geneva: world health organization school-based deworming: a planner's guide to proposal development for national school-based deworming programs deworm the world basic guide for school directors, teachers, students, parents and administrators usaid hygiene improvement project malaria control in schools: a toolkit on effective education sector responses to malaria in africa world bank open data global overview of school health services: data from countries world health organization. global accelerated action for the health of adolescents (aa-ha!): guidance to support country implementation preferred reporting items for systematic reviews and meta-analyses: the prisma statement covidence systematic review software veritas health innovation risk of bias in overviews of reviews: a scoping review of methodological guidance and four-item checklist amstar : a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both. the bmj overviews of systematic reviews: great promise, greater challenge systematic review finds overlapping reviews were not mentioned in every other overview amstar : a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both school dental screening programmes for oral health. the cochrane library the effectiveness of the penn resiliency programme (prp) and its adapted versions in reducing depression and anxiety and improving explanatory style: a systematic review and meta-analysis school-based sleep education programs for short sleep duration in adolescents: a systematic review and meta-analysis vision screening for correctable visual acuity deficits in school-age children and adolescents. the cochrane library do menstrual hygiene management interventions improve education and psychosocial outcomes for women and girls in low and middle income countries? a systematic review music therapy for autistic spectrum disorder. the cochrane library school-based cognitivebehavioural interventions: a systematic review of effects and inequalities fluoride gels for preventing dental caries in children and adolescents. the cochrane library are school nurses an overlooked resource in reducing childhood obesity? a systematic review and meta-analysis effectiveness of oral health education on oral hygiene and dental caries in schoolchildren: systematic review and meta-analysis school-based depression and anxiety prevention programs for young people: a systematic review and meta-analysis effects of school-based interventions with u.s. military-connected children: a systematic review primary school-based behavioural interventions for preventing caries a systematic review of school-based interventions that include inhaler technique education what works": systematic review of the "friends for life" programme as a universal school-based intervention programme for the prevention of child and youth anxiety primary-school-based art therapy: a review of controlled studies efficacy and effectiveness of school-based prevention and early intervention programs for anxiety a systematic review of school-based sexual health interventions to prevent sti/hiv in sub-saharan africa a systematic review of school-based social-emotional interventions for refugee and war-traumatized youth effectiveness of school-based family asthma educational programs in quality of life and asthma exacerbations in asthmatic children aged five to : a systematic review world bank country and lending groups -world bank data help desk school-based cognitivebehavioural interventions: a systematic review of effects and inequalities global variations and time trends in the prevalence of childhood myopia, a systematic review and quantitative meta-analysis: implications for aetiology and early prevention vision impairment and blindness. in: world health organization standard school eye health guidelines for low and middle-income countries the international agency for the prevention of blindness prevention of mental disorders: effective interventions and policy options geneva: world health organization obesity and overweight. in: world health organization who [internet human papillomavirus vaccination in tanzanian schoolgirls: cluster-randomized trial comparing vaccine-delivery strategies feasibility of delivering hpv vaccine to girls aged to years in uganda world health statistics : monitoring health for the sdgs, sustainable development goals. geneva: world health organization ensure healthy lives and promote wellbeing for all at all ages we would like to thank tomas allen for his guidance and support in designing the search strategy. we are also grateful for advice from the who school health services guideline steering group and guideline development group members. key: cord- -uzgya k authors: strömmer, sofia; barrett, millie; woods-townsend, kathryn; baird, janis; farrell, david; lord, joanne; morrison, leanne; shaw, sarah; vogel, christina; lawrence, wendy; lovelock, donna; bagust, lisa; varkonyi-sepp, judit; coakley, patsy; campbell, lyall; anderson, ross; horsfall, tina; kalita, neelam; onyimadu, olu; clarke, john; cooper, cyrus; chase, debbie; lambrick, danielle; little, paul; hanson, mark; godfrey, keith; inskip, hazel; barker, mary title: engaging adolescents in changing behaviour (each-b): a study protocol for a cluster randomised controlled trial to improve dietary quality and physical activity date: - - journal: trials doi: . /s - - -w sha: doc_id: cord_uid: uzgya k background: poor diet and lack of physical activity are strongly linked to non-communicable disease risk, but modifying them is challenging. there is increasing recognition that adolescence is an important time to intervene; habits formed during this period tend to last, and physical and psychological changes during adolescence make it an important time to help individuals form healthier habits. improving adolescents’ health behaviours is important not only for their own health now and in adulthood, but also for the health of any future children. building on lifelab—an existing, purpose-built educational facility at the university of southampton—we have developed a multi-component intervention for secondary school students called engaging adolescents in changing behaviour (each-b) that aims to motivate and support adolescents to eat better and be more physically active. methods: a cluster randomised controlled trial is being conducted to evaluate the effectiveness of the each-b intervention. the primary outcomes of the intervention are self-reported dietary quality and objectively measured physical activity (pa) levels, both assessed at baseline and at -month follow-up. the each-b intervention consists of three linked elements: professional development for teachers including training in communication skills to support health behaviour change; the lifelab educational module comprising in-school teaching of nine science lessons linked to the english national curriculum and a practical day visit to the lifelab facility; and a personalised digital intervention that involves social support and game features that promote eating better and being more active. both the taught module and the lifelab day are designed with a focus on the science behind the messages about positive health behaviours, such as diet and pa, for the adolescents now, in adulthood and their future offspring, with the aim of promoting personal plans for change. the each-b research trial aims to recruit approximately secondary school students aged – years from schools (the clusters) from hampshire and neighbouring counties. participating schools will be randomised to either the control or intervention arm. the intervention will be run during two academic years, with continual recruitment of schools throughout the school year until the sample size is reached. the schools allocated to the control arm will receive normal schooling but will be offered the intervention after data collection for the trial is complete. an economic model will be developed to assess the cost-effectiveness of the each-b intervention compared with usual schooling. discussion: adolescents’ health needs are often ignored and they can be difficult to engage in behaviour change. building a cheap, sustainable way of engaging them in making healthier choices will benefit their long-term health and that of their future children. trial registration: isrctn . registered on august . each-b is a cluster randomised controlled trial, funded by the national institute for health research (rp-pg- - ). the nhs long term plan sets out a prevention agenda in the uk aimed at reducing the risk of developing noncommunicable diseases (ncds) such as cardiovascular conditions and type diabetes [ ] . insufficient exercise and poor dietary quality are common and are linked to increased risk of ncds. ncds place a heavy burden on society, hospitals and community health services, costing the nhs £ billion a year [ ] . uk adolescents have poorer diets than other age groups, and fewer than % meet physical activity guidelines [ , ] . intervening during adolescence to support better health habits can bring a triple benefit: to the immediate health and wellbeing of the young person, to their own health in adulthood and to the health of the next generation [ ] [ ] [ ] [ ] [ ] . it is well-established that improving the dietary quality and nutritional status of both young women and young men before conception improves pregnancy and birth outcomes and therefore the long-term health of the offspring [ ] [ ] [ ] . it has also been suggested that adolescence is a critical period during which optimal nutrition could mitigate the effects of poor fetal and infant nutrition [ , ] . as a critical period of both physical and social development, adolescence is the time during which the physiological, mental and behavioural foundations of long-term health are consolidated. peak muscle and bone mass as well as cardio-respiratory fitness are reached during adolescence, and these physiological processes are both nutritionally sensitive and predictive of later health [ ] [ ] [ ] . in addition, widespread brain re-modelling during adolescence leads to a large increase in cognitive ability [ ] . adolescence is also a key time for the development of executive function and the capacity to make independent choices, follow them through and achieve goals, as well as the ability to form healthy social networks. lifelong behaviour patterns are established in adolescence, including choices about diet and physical activity (pa) [ ] . adolescence is a challenging time to intervene to improve health behaviours for both psychological and physiological reasons. adolescents find it difficult to engage with the long-term consequences of their lifestyle choices. developmental changes in brain structure leave them sensitive to emotional and social influences and to prioritising the immediate over the long-term; brain pathways involved in decision-making processes do not mature fully until early adulthood [ , ] . systematic reviews suggest that motivated and engaged adolescents can improve their health behaviours [ ] . however, little is known about precisely how to motivate and engage adolescents in sustaining positive changes long term [ , ] . the latest research evidence strongly indicates that successful interventions with adolescents are as follows: (i) multi-component, (ii) involve schools, (iii) engage and motivate adolescents to change their health behaviours and (iv) involve social support from friends and parents [ , ] . in addition, digital platforms show potential as complementary features in complex interventions targeting health behaviour change and are particularly relevant to this age group. approximately % of - year olds owned smartphones in , and % spent an average of h a week online [ ] . key strategies for effective engagement with digital interventions are recognised to include co-designing interventions with adolescents, the personalisation of interventions and connectivity to peers and the user's wider social networks [ ] . it is increasingly recognised that interventions need to facilitate collaboration between different agencies such as schools, community and parent groups and not rely on one setting, such as the school or family. interventions to improve adolescents' diet and pa have been implemented with varying success; effective engagement with, and motivation of, adolescents remains a pertinent issue. gender-specific issues should not be overlooked, and positive effects post-intervention may not be apparent in the short-term, making medium and longer-term measures important [ , , ] . many interventions favour combining health and nutrition education with behavioural skills training, even though evidence suggests that adolescents are not ignorant about the health implications of their food choices and pa habits, nor are they motivated by health in the distant future [ , ] . recent research has suggested that interventions designed to support adolescent health may be more engaging and successful if they align health agendas with adolescents' own values and priorities [ , ] . each-b is designed using a person-based approach [ ] [ ] [ ] with extensive co-creation to maximise alignment with adolescents' own values in order to make the intervention both engaging and effective. the views and input of parents and adolescents in the age range of each-b's target population have been continually sought and incorporated into the trial design through extensive engagement work with local schools, youth groups, and through lifelab's young ambassadors scheme. in addition, two advisory groups have been set up to ensure the intervention design and delivery are acceptable to parents of young teenagers and to the young people themselves. a number of 'game jams' involving approximately adolescents have been run throughout the development phase, in order to ensure the app reflects the values and priorities of the intended user group [ ] . aligning an intervention design with adolescent values and using fun, engaging methods of delivering behaviour change support as part of a multi-component, school-based intervention improves diet and pa habits of secondary school students. the aim of this cluster randomised controlled trial is to evaluate whether each-b, a complex intervention designed to engage, motivate and support adolescents aged - years, improves their dietary quality and pa habits. each-b is a cluster randomised controlled trial using a : allocation within a superiority framework. the intervention consists of three-components: ( ) face-to-face support from teachers trained in skills to support behaviour change, ( ) engagement in the lifelab school-based education programme and ( ) a digital intervention with games as well as peer-and parent-support features. we propose to evaluate each-b through a cluster randomised controlled trial. we plan to recruit boys and girls of middle academic ability in year (aged - years) from state secondary schools/academies (approximately students) to take part in the trial. year is the second year of senior school in the uk and was chosen to take part in each-b in consultation with schools for two key reasons: schools are better able to deliver the intervention at this time, before students start their gcse curriculums in year and timetabling becomes more difficult; adolescents in year often have increased levels of independence in terms of food choices whilst travelling to and from school alone or with friends. schools in hampshire, uk, and the surrounding counties will be eligible to take part. hampshire is a large county (pop. . million) in the south of england with a wide range of socioeconomic profiles. some rural areas of hampshire are affluent, but the two major cities southampton and portsmouth are in the most deprived quintile of local authorities in the uk [ ] . schools will be recruited from both rural and urban settings in order to reflect the diversity of the population (see the 'randomisation/blind ing' section for more on randomisation procedures). each school will be randomly allocated to either 'control' or 'intervention' status. of the schools recruited, will therefore be intervention schools where two classes of year students will complete the lifelab module, be offered support from teachers trained in skills to support health behaviour change and receive the digital intervention. the other schools will form the control group and will receive normal schooling. all state secondary schools and academies in hampshire and surrounding areas are eligible to take part in the each-b intervention trial. independent and selective schools including special schools and single-gender schools are excluded from taking part because by nature of being selective their inclusion could bias the study findings. lifelab's young ambassadors scheme is a scheme whereby young people who visit lifelab with their school can sign up to be a young ambassador in order to support lifelab's aims and objectives by taking part in special activities including being consulted on new ideas as they are developed and worked up by the lifelab team. in late , a successful pilot trial was run with students from six schools in the southampton area, to test and modify the intervention. the intervention comprises: i) professional development for teachers including training in communication skills to support health behaviour change, known as 'healthy conversation skills' (hcs), explained in detail below ii) lifelab educational module comprising in-school teaching of nine science lessons linked to the english national curriculum and a hands-on practical day visit to lifelab, held part way through the module iii) a personalised digital intervention (the 'app') with social support and game features the each-b intervention includes professional development (pd) for all teachers involved in delivering the life-lab educational module. the -day pd training course takes place at lifelab and focuses on science education relevant to the implementation of the nine lessons in school. it offers access to online support materials which describe the underpinning science. teachers are trained in hcs [ , ] to engage with their students in making plans to improve their diet and/or activity levels via a personal 'lifelab pledge'. teachers are trained how to support their students to keep their pledges, and how to use the digital intervention, through asking open questions and listening rather than telling. an additional hcs training session will be offered to the whole staff body in intervention schools to enhance the opportunity for adolescents to be supported at school to improve their health behaviour. hcs training was developed in southampton to provide communication skills to support behaviour change. these skills were designed in the first instance for health and social care practitioners to use with their patients and clients, but have since been adapted to the training of teachers to enable them to better support behaviour change in their students. while health promotion is not seen as a core requirement of a teacher's role, we have seen high levels of engagement from teachers throughout the development work and each-b pilot trial. it is widely acknowledged both in the scientific literature and by schools that diet and pa behaviours are significant factors in both academic performance and student wellbeing and there is growing evidence that health and health behaviours have measurable consequences for attainment [ ] . being more physically active at age is associated with higher attainment at gcse, while being obese at age is associated with lower attainment [ , ] . children from more disadvantaged backgrounds are more likely to be overweight/obese, have poorer diets and be less physically active. being overweight/obese can reduce children's self-esteem, which may lead to lower educational attainment and behavioural problems [ ] . therefore, schools and teachers are keen to learn skills that enable them to support students to eat well and be more active. the use of hcs encourages people to reflect on behaviours that they would like to change, in many cases making the unconscious, habitual behaviours conscious and therefore amenable to deliberate change. hcs trains people to use five key skills: ( ) creating opportunities for having healthy conversations; ( ) asking open 'discovery' questions that lead people to explore and find their own solutions; ( ) listening more than talking and so empowering people to identify and take control of their own behaviour change; ( ) reflecting on practice in order to be more effective; and ( ) supporting goalsetting using smarter action planning, providing people with a sense of change and progress. these skills were originally developed in collaboration with local health service commissioners in southampton, whose needs-assessment found that their healthcare providers lacked confidence to support clients to improve their diets and lifestyles [ ] . hcs training recognises that skills to support behaviour change need to go beyond education and instead empower individuals to take control of their health behaviours and to problem-solve. as with motivational interviewing, the training offers an approach to supporting behaviour change that is based on the understanding that giving people information is insufficient to change their behaviour; they must also be motivated to change and have the tools to implement that change. hcs training is philosophically underpinned by bandura's social cognitive theory of the socio-environmental and personal determinants of health [ ] . self-efficacy is a central construct in this theory and describes an individual's belief that he or she is capable of carrying out a specific behaviour, which implies that he or she also has the knowledge and skills to do so. hcs are designed to increase self-efficacy through empowering problem-solving, and employ behaviour change techniques [ , ] intended to support small changes in behaviour, leading to acquisition of mastery skills which bandura proposes as a means of raising self-efficacy. training in hcs is designed to increase the self-efficacy and hence build the capacity of practitioners and clients and, in doing so, change the ethos of those practitioners and their organisations to one that empowers change. the each-b intervention is designed to operate both at the level of individual behaviour change and at the level of changing the culture of schools to trigger automatic as well as reflective processes underlying behaviour change [ , , , ] . the lifelab educational module aims to engage adolescents with the knowledge and understanding needed to enable them to make appropriate health choices-their health literacy-and to motivate them to change their dietary and pa behaviours. the theme of the module is 'me, my health & my children's health', and it is delivered in an interactive and highly engaging format which sets scientific knowledge into a relevant and accessible context for this age group [ ] . the educational module is designed to be delivered as four pre-and five post-lifelab visit lessons delivered in science classes during the school day. the materials used in the educational module are explicitly linked to the english national curriculum, embed the messages of the lifelab visit and have been updated specifically for the each-b trial. for example, an additional lesson focusing on the influences of the food environment on healthy lifestyle choices has been added, in order to encourage the adolescents to critically analyse their own food environments and the influence these may have on their dietary behaviours. health messages in the module are linked to both the hands-on practical activities the students will carry-out on the day visit to lifelab and to the school-based activities. this approach is intended to ensure that the adolescents understand the long-term implications of their current diet and pa on their future health, their children's health and on the risk of ncds for both. halfway through the lifelab educational module in school, the students and their science teacher have a day visit to the purpose-built laboratory facility, based in southampton general hospital. the visit combines a mixture of hands-on practical work, reflection on lifestyle choices and learning about the science behind health messages. activities include: experiencing a variety of ways to measure health including assessing carotid artery blood flow and structure using ultrasound, measuring body composition, performing lung function tests, training in cpr and testing grip strength and flexibility extracting their own dna and carrying out gel electrophoresis experiments that illustrate how a healthy diet can induce epigenetic changes that alter dna structure and are passed from parents to offspring, with implications for cardiovascular and lifelong health for themselves and their children small group discussion sessions with scientists based at the hospital, to introduce students to the range of career options in scientific disciplines. at the end of the lifelab visit, and with support from lifelab staff, students are encouraged to make a 'pledge' about a positive change for their own health. students also download the each-b app onto their personal devices during the day (see below). the digital intervention will be in the form of a mobile phone application (app) with game features. it has been developed using a person-based approach to intervention development, combined with user-centred design principles for digital game design and a participatory design process. the design of the game is underpinned by self-determination theory and employs a range of behaviour change techniques [ , , ] . during the lifelab visit, students will be asked to download the app onto their personal mobile devices (android or ios) and log in. any student without a personal device will be given instructions for downloading the app at home via a shared family device. the app will involve creating a character and choosing games, quizzes and challenges to complete. players can choose challenges and none are compulsory. the app will allow players to connect with each other if they wish. parents/carers of students in the intervention will also be offered a companion app to help them support their adolescent in making healthy lifestyle choices. the parent app includes information about the different elements of the app developed for the young people taking part in the intervention. it also contains ideas, suggestions and prompts as to how parent and adolescent can join forces to improve food choices and activity levels for the whole family. all outcomes will be measured twice, once at baseline and again months later at follow-up. the trial has co-primary outcomes for dietary quality and pa. dietary quality will be assessed by a -item food frequency questionnaire (ffq). this ffq has been developed specifically for use with adolescents using data for boys and girls aged - years who took part in the national diet and nutrition survey. principal component analysis was applied to these data in order to identify indicator foods which best describe better and poorer dietary quality of uk adolescents. this ffq has shown good comparison with important nutritional biomarkers including -hydroxy vitamin d, total carotenoids, serum folate and vitamin c. using geneactiv™ accelerometers pa will be assessed as minutes of daily low, moderate and vigorous physical activity (lmvpa), also described as total pa [ ] . at baseline and again at the -month follow-up, geneactivs will be worn for days and the output data will be averaged over this period, or the maximum period of valid data. secondary outcomes for dietary quality are as follows: usual portions in the past month of water, sugar sweetened beverages (ssbs), chips and crisps and usual portions of fruit and vegetables consumed in a typical day. the number of portions of fruit and vegetables are analysed separately to estimate daily fruit and vegetable consumption for each adolescent [ ] . categories of pa will also be assessed as secondary outcomes, namely average acceleration, intensity gradient, sedentary time, light pa, light to moderate pa, moderate to vigorous pa, lmvpa -min, lmpa -min and mvpa -min. the categories of ' -min' restricts to activity that has a minimum of -min bout duration. all pa outcomes (primary and secondary) will be analysed separately for activity at the following times: weekdays, weekends, during school hours and during out-of-school hours. additional secondary outcomes are as follows: bmi zscores, with and without adjustment for pubertal status as indicated by standing height, sitting height and weight [ ] ; self-reported frequency of pa from a modified version of the youth physical activity questionnaire (ypaq) validated for use in - year olds [ ] ; behavioural self-regulation and self-efficacy for healthy eating and pa; and quality of life and wellbeing measured by two age-appropriate tools: the child health utility d (chu d) [ ] and the cantril ladder [ ] . behavioural regulation and self-efficacy for pa will be assessed by the behavioural regulation for exercise questionnaire [ ] and the pa section of the self-efficacy for healthy eating and physical activity measure (se-hepa) [ ] . behavioural regulation and self-efficacy for diet will be assessed using the recently developed confidence and behavioural autonomy (cba) scale. this is age-specific and has been validated against the healthy eating scales of the se-hepa and the treatment self-regulation questionnaire (tsrq) [ ] . a schematic schedule of enrolment, interventions and assessments is shown in fig. . unpublished analysis of earlier data from lifelab indicates an intra-school (class) correlation coefficient of . . forty-six schools each sending two classes amounting to approximately students from each school and students in total will provide % power at a . % significance level (accounting for two primary outcomes) to detect a . sd difference in diet quality score or minutes of total pa in intervention and control schools. comparable effect sizes have been considered in other health interventions as meaningful in terms of change in health behaviours, and our level of . sds falls in the mid-range of effect sizes reported in a meta-synthesis of meta-analyses of behaviour change interventions in the general population [ ] . we will recruit two year classes ( - years) from schools allowing for drop-out, though in previous lifelab studies only one school has ever dropped out. to minimise bias from loss of students to follow-up, we will request class lists for each participating class, so that missing participants at each stage of the follow-up can be identified and included in secondary analyses and process evaluation assessing uptake of the intervention. recruitment for each-b began in september and will run for years, with data collection taking place at schools at baseline and again months later. the life-lab team has worked with schools for many years and good systems for recruiting schools have been developed, so recruitment difficulties are not anticipated. we appreciate that control schools will not want to miss out on the intervention and therefore all control schools will be offered a visit to lifelab the following year. the schedule of enrolment for the trial is shown in fig. . schools will be recruited through a range of methods including presentations at relevant local meetings such as the secondary heads of science forum meetings, and letters sent to head teachers and heads of science of eligible schools in the recruitment catchment area. the recruitment pack for each-b includes a cover letter and information sheet for the school, offering basic information about the trial and explaining how the experience will differ for control and intervention schools. schools are then offered a meeting with the each-b research team at which further details are discussed and any questions answered. this meeting will take place with the head of science and a member of the senior leadership team at the school, at a time to suit them. it is also an opportunity for the research team and the school staff to establish how the intervention will run in the school, if it is allocated to the intervention arm, as each school operates differently in terms of timetabling science classes. schools will be asked to allocate two middle ability year classes to participate in the trial totalling approximately students. the teaching programme is designed for students in this age group and of all ability levels; there are no exclusion criteria for students. for students who may require more input (those who have english as a second language, for example), we provide support for schools in planning delivery of the module. specifics are discussed at the teachers' pd day. schools will already have in place provision for these students, and so it is a matter of ensuring that the lifelab materials are accessible by all participating students. following the meeting at school, the head teacher is asked to sign a consent form confirming they wish to take part, that they understand the trial procedures and to name the two classes that will participate. after signed agreement from the schools has been obtained and the classes taking part have been identified, schools will be randomised to receive usual schooling (control) or the each-b intervention (lifelab programme, hcs training for teachers, and access to the digital intervention) (see fig. ). we will use a minimisation procedure developed by the southampton clinical trials unit (tenalea), which aims to achieve a balance of schools in the two arms based on the following three criteria: -the proportion of students in the school receiving free school meals (cut-off > %); -the proportion of students in the school achieving l gcse (equivalent to a high 'c' grade) in english and maths (cut-off > %); -whether or not the school already participated in the full lifelab programme in the previous years. the randomisation is administered through a webbased secure system to which the each-b team submit the details of schools who have consented to participate. these are sequentially numbered and the allocation to intervention or control is then reported to the investigators. blinding from this point onwards is not possible except of the statisticians who will be analysing the data. many schools we recruit will have previously been involved in lifelab. contamination of the intervention effect is unlikely to occur as the year students taking part in each-b will not have visited lifelab before, and except for siblings, they will generally have limited contact with older children in the school. it will not be possible to blind schools and their students to their allocation due to the nature of the intervention. primary and secondary outcome data will be collected at baseline and follow-up visits by research staff to schools, conducted during class time. standing and sitting height and weight will be collected by trained research nurses from the clinical research facility at the southampton nihr biomedical research centre (sbrc). these measures will be used to derive body mass index z-scores and biological maturity [ ] . all researchers and research nurses working on the each-b trial will be trained in trial-specific procedures and be required to complete appropriate safeguarding and eating-disorder training. questionnaire data will be collected through participant completion of questionnaires on ipads during the baseline and follow-up visits to schools. the class will be divided into small groups of - students working with one member of the research team who will act as a facilitator. before students begin completing the questionnaires, the facilitator will use a trial-specific standard operating procedure to explain key points about the questionnaires and will remain with the group throughout the session to answer any questions that might arise. geneactiv™ accelerometers will be distributed to participants by trained research staff during the data collection sessions at both time points. the devices will be programmed to automatically start measuring at midnight on the first day of data collection and stop measuring precisely days later, in order to capture both weekend and weekday activity. a sampling frequency of hz will be used. participants will be asked to keep the device on their non-dominant wrist for seven full days, preferably without taking it off at any point. seven days after the baseline data collection visit, schools will be asked to return the geneactivs to the research team via courier or another secure method. after data collection visits to schools, all data will be downloaded from the ipads via a secure sockets layer (ssl) that encrypts the data before sending it and storing it in the database. the database itself is kept on a university server. all questionnaire data will be kept in accordance with general data protection regulations (gdpr), university of southampton data protection policy and in accordance with the protocols of the mrc lifecourse epidemiology unit leu). the data will be stored in password-protected computers by the research team and only accessible by them. data will be stored in access databases and managed with support from the data management staff of the mrc leu, who have extensive data management expertise and manage data from more than studies. after the trial is complete, anonymised data will be available to other researchers under our data sharing protocols. identifying information will be collected about participants, purely for the purposes of matching baseline and follow-up questionnaires. all identifying information will be stripped from the rest of the data after linkage is complete and will be stored separately. it will only be kept in case a further follow-up is planned. data will be analysed using stata, spss and mplus. the primary analyses will be according to the intention-totreat principle, comparing dietary quality and pa levels in the intervention and control groups using mixed effects linear regression to account for clustering within schools. the main analysis will compare these outcomes at baseline and at months follow-up. although randomised at the level of schools using a minimisation algorithm, there may still be disparities between the intervention and control participants at baseline. these will be assessed prior to analysis and relevant confounders will be incorporated in the models; factors to be considered include gender, exact age at recruitment and household area of deprivation using the income of deprivation affecting children index (idaci) score [ ] . adjustment for baseline dietary quality and pa levels will be included in the relevant models to allow an assessment of change from baseline. sensitivity analyses will examine effects of missing data, and multiple imputation will be used where appropriate, accounting for the clustered nature of the data. comparisons for secondary outcomes will also be modelled using mixed effects linear regression, with the use of binary models for binary outcome variables. mixed effect logistic regression will be used for rare outcomes and mixed effect binary regression (or poisson regression with robust variance if the binary regression models fail to converge) will be used for outcomes that occur in more than % of students. we will then conduct a mediation analysis to examine the role of these secondary factors in mediating the effect of the intervention on primary outcomes. the main analyses will be conducted using the latest available version of stata. planned subgroup analyses will focus on whether there are different effects for boys and girls, differing ethnicities, seasonal variation, idaci score and estimated biological maturity in outcomes. we will also determine the effect of each-b on outcomes for those who fully engage with the digital intervention (per protocol analysis) and assess uptake of the digital intervention by gender, ethnicity and idaci score. complier average causal effect (cace) modelling techniques will be used to examine factors that predict engagement with intervention components and to examine intervention effects specifically for students who engaged with those components. as this is a cluster randomised controlled trial, many of the assumptions underlying this method are unlikely to be valid, most obviously the independence of the participants [ ] . these assumptions will be assessed and methods of analysis developed appropriately. the cace analysis will be performed using mplus. data monitoring will be the responsibility of the trial team at the mrc leu. the steering committee will receive regular data reports as part of their bi-annual meetings. due to the low risk nature of the trial, a separate data monitoring committee is not deemed necessary. we are not expecting the trial to give rise to any adverse events or harms. however, a risk assessment was completed and submitted as part of the ethical approval process. all staff involved in visiting schools have the enhanced level of dbs to work with children, are trained in safeguarding and awareness of eating disorders and have basic levels of awareness about dealing with someone who may become anxious for any reason during a data collection visit. using the mrc guidance on process evaluation of complex interventions [ ] , focusing on the programme logic model, we will use mixed-methods to examine (i) implementation, (ii) context and (iii) mechanisms of impact of the each-b intervention. i) implementation: we will examine how intervention delivery is achieved and what is delivered (fidelity, dose, adaptations and reach) by, for example, monitoring downloads of the digital intervention on lifelab day as a proportion of those eligible, frequency of access to the digital intervention, as well as conducting structured, qualitative observations of teacher/student interactions, and teacher/student interviews. ii) context: we will assess context at school level by interviewing relevant staff about other activities and factors that may affect how the intervention was implemented and how it worked. the wider policy context will also be assessed at local and national levels by considering relevant healthy living initiatives or campaigns and their potential influence. iii) mechanisms of impact: we will conduct interviews with students, teachers and parents to explore their experiences of and engagement with the intervention as a whole and use in-app telemetry to explore usage of the digital intervention. some interviews will be carried-out with specific subgroups of students to ensure that the intervention is not stigmatising. analysis of the qualitative data collected as part of the process evaluation will be conducted with a view to achieving a comprehensive understanding of the way in which an intervention like each-b is implemented and how that relates to the outcomes. thematic analyses of all qualitative data from the process evaluation will be undertaken. structured, qualitative observations of teacher/student interactions and interviews with students, teachers and parents to explore their experiences of and engagement with the intervention will be analysed using various forms of content analysis. for observations of teacher/student interactions, structured record forms will be designed to monitor use of hcs. interviews will be audio-recorded, transcribed verbatim and analysed using inductive thematic analysis and a standard methodology [ ] . initial codes will be discussed between coders to reach agreement on themes, and then discussed with the wider research team and ppi panels. broad themes will then be broken down to identify commonly expressed themes and unusual cases. approximately % of the data will be coded by two team members to check that the coding scheme is identifying all the themes and concepts and that there is a shared understanding of what they are. findings will be used to assist with interpretation of the trial outcomes and to illuminate mechanisms through which the intervention has its effect. a model will be developed to estimate the costeffectiveness of each-b compared to usual schooling. the model will extrapolate short-term observed effects on diet, pa and quality of life (chu d) to estimate future health impacts and societal costs. there are many risks associated with poor diet and low pa over the life course, including increased incidence of a range of ncds and adverse social, economic and well-being outcomes. we will focus on four key risks for which there is good evidence of a short to medium-term impact: incidence of type diabetes, mental health, low birth weight and future loss of earnings [ ] . health outcomes will be quantified using quality-adjusted life years (qalys), including direct effects of diet and pa on quality of life (chu d), as well as losses associated with type diabetes, depression and low-birth weight pregnancies. costs will be estimated from a societal perspective, including costs to schools, local authorities and the nhs and loss of earnings for individuals. costs and qalys will be estimated over a time horizon of years in the base case, discounted at uk recommended annual rates [ ] . a range of sensitivity and scenario analyses will be conducted to assess uncertainty of the model predictions. this will include alternative assumptions about the persistence of observed effects on diet quality, pa and quality of life from the trial. this trial will estimate the effectiveness of a complex intervention to improve diet and pa in adolescents, designed to have reach and affordability. if it proves effective, the intervention could be rapidly and inexpensively disseminated to all secondary school students attending lifelab from across the wessex region. potential for the intervention to be introduced widely across the uk will be explored. some elements of the intervention will be easier to translate than others. in areas where there is already an educational intervention providing initial engagement for adolescents in thinking about their health, educators could be trained in communication skills to support behaviour change. the supplementary digital intervention is low-cost and sustainable. if successful in supporting behaviour change, the intervention has potential for both immediate impact on adolescents' health and well-being and for improving the health of the nation for generations to come. the intervention is designed to deliver outcomes aligned to the local authority's sustainability and transformation plan. as such, it represents an attempt to meet the need to provide preventive methods that can easily be up-scaled and that deliver technological solutions for major health issues. trial findings that will have wide application and impact include improvements in understanding how best to intervene with maximal effectiveness and cost-effectiveness to improve adolescent health behaviours, and to engage, and sustain the engagement, of adolescents. sub-group analyses of data will allow tailoring of the intervention to specific groups, e.g. the most disadvantaged, hardest to reach, or boys as distinct from girls. the programme provides information about the value of, and best practice in, co-creation of initiatives with adolescents and our understanding of mechanisms of creating change with adolescents. recruitment for the rct initiated in september and was due to be completed by june . the trial was halted in march due to the closure of schools in response to the covid- global pandemic. the trial team plans to restart the trial in late autumn . the trial protocol is version date august . the study has been registered on the isrctn database (isrctn , registered august ). the research sponsor is the university hospital southampton nhs foundation trust. the each-b research management team (comprising sts, meb, jvs and hmi) and research theme leads (jb, kwt and the management team) meet regularly to monitor the trial, while the remaining co-applicants and programme steering committee provide oversight through bi-annual meetings. we do not anticipate any protocol amendments, but may have to make changes as a consequence of the covid- pandemic. in the instance of a protocol amendment being required, we would discuss this with the programme steering committee, the funder, the sponsor and ethics committee and any participants affected. the main output from this research will be a fully developed, replicable intervention to improve adolescents' dietary quality and pa levels. we will determine the success of co-creation processes with adolescents, short-, medium-and long-term health benefits from intervening in adolescence, intervention cost-effectiveness, including reach/affordability and feasibility of rapid/inexpensive roll-out into routine practice in schools. in line with funder requirements, at the end of the trial after all statistical analyses are completed, we plan to grant public access to the full trial protocol, the anonymised dataset and statistical code used. dissemination pathways include close collaboration with stakeholders, including young people, feedback of findings to teachers and students, interactive workshops with stakeholders; conference presentations and a series of papers in open access peer-reviewed journals; links with professional societies and policy-makers; and regular press releases. all participants' data will be stored anonymously following the university of southampton guidelines. confidentiality and linked anonymity will be assured. information will not be reported in a way that would allow an individual participant to be identified. participants will only be known by id number when the data are reported. participants' identifying information will be stored separately from the data collected, but need to be kept to ensure linkage between the baseline and follow-up data. all interview data will be kept in accordance with the data protection law (including gdpr) and university of southampton and uk medical research council policies. data will be stored on password-protected computers by the research team and only accessible to them through the data manager. ancillary and post-trial care there are no expected adverse events or harms associated with the trial. no specific post-trial care is planned, but in an event where a participant was in any way adversely affected, the study team would work closely with the school and parents of the participant to ensure appropriate support is offered. all authors took part in the design of the study. meb, hmi, kwt, sts, scs and mb carried out pilot work and were involved in the conception, development and delivery of the programme of work. meb and hmi are principal investigators and together have overall responsibility for the each-b trial and wrote the protocol for the study. lb, dml and kwt were involved in the design, development and delivery of the teaching programme of work, including the pd programme for teachers, and wtl was involved in the design and delivery of the pd programme for participating teachers and leads on the hcs components and evaluation. meb, hmi and jb were involved in the design of the cluster randomised trial and concurrent process evaluation. meb, sts, scs, hmi and cv were involved in the design and validation of the research questionnaires. hmi is the statistician on each-b. pc, mb, hmi and sts manage the data for this study. jc, cc, dc, pl, mh and kg are study collaborators and were involved in the conception and development of the study. dl has advised on the design and development of the physical activity measures and is responsible for the analysis of the physical activity data. jl, nk and oo are responsible for the health economic modelling and cost-effectiveness analysis of the interventions developed in this programme. all authors have contributed to the manuscript and approved the final submitted version. this research is funded by uk nihr programme grants for applied research (rp-pg- - ). the views expressed are those of the authors and not necessarily those of the nihr or the department of health and social care. researchers working on this trial are also supported by the following funding sources: uk medical research council (mc_uu_ / ), nihr southampton biomedical research centre, wessex heartbeat and public health england. lifelab has also received research funding from the british heart foundation, the wellcome trust, cancer research uk, research councils uk, the bupa foundation, the primary science teaching trust (formerly the astra zeneca science teaching trust) and the epsrc (via the uos pathways to impact funding scheme). study sponsor and funder have had no role in study design and will have no role in collection, management, analysis or interpretation of data; the writing up of a final report; and the decision to submit papers for publication, and they will not have ultimate authority over any of these activities. data sharing is not applicable to this article as it is a protocol of an ongoing study and no data is reported. all anonymised datasets from the study will be deposited in a publicly available repository after the trial has ended. ethics approval and consent to participate full ethical approval was granted on july from the university of southampton's faculty of medicine ethics committee (ethics id ). once the school has agreed to participate, the head teacher has signed the consent form, and the randomisation process is complete, the appropriate trial documents for parents and students are provided for the schools to disseminate. two versions of the letter for parents and the participant information sheets have been produced: one for intervention schools and one for control schools. in both cases, parents and students are provided with contact details of the research team in case they have questions about taking part in the research trial. for both intervention and control schools, consent will be opt-in and collected by the schools prior to any research data being collected. in keeping with good practice, the consent form for intervention schools requires parents to initial boxes for each part of the intervention to ensure they understand and consent to each part separately. in all cases, student assent is also required and it is made clear that taking part is voluntary and a student or parent can withdraw their assent/consent at any time without giving a reason. the consent documents are collected by the study team at the baseline visit to schools and are thereafter stored securely at the mrc leu. no details, images or videos relating to an individual person are included. model consent forms will be provided on request. competing interests kg has received reimbursement for speaking at conferences sponsored by nutrition companies and is part of an academic consortium that has received research funding from abbott nutrition, nestec and danone. the university of southampton has received an unrestricted donation from danone nutricia to support lifelab's work with schools. wendy lawrence has received funding from danone nutritia early life nutrition for training and presentations. cc has received lecture fees and honoraria from amgen, danone, eli lilly, gsk, kyowa kirin, medtronic, merck, nestlé, novartis, pfizer, roche, servier, shire, takeda and ucb outside of the submitted work. outside of the submitted work, cv has a non-financial research relationship with a food retail company and maintains independence in all evaluation activities. this article, however, is not related to this relationship. the nhs long term plan the economic burden of ill health due to diet, physical inactivity, smoking, alcohol and obesity in the uk: an update to - nhs costs ): a survey carried out on behalf of public health england and the food standards agency. london: public health england engaging teenagers in improving their health behaviours and increasing their interest in science (evaluation of lifelab southampton): study protocol for a cluster randomized controlled trial longitudinal tracking of adolescent smoking, physical activity, and food choice behaviors tracking of obesityrelated behaviours from childhood to adulthood: a systematic review the international federation of gynecology and obstetrics (figo) recommendations on adolescent, preconception, and maternal nutrition: "think nutrition first annual report of the chief medical officer, , the health of the %: women. london: department of health our future: a lancet commission on adolescent health and wellbeing improving women's diet quality preconceptionally and during gestation: effects on birth weight and prevalence of low birth weight-a randomized controlled efficacy trial in india (mumbai maternal nutrition project) effects of dietary interventions on pregnancy outcomes: a systematic review and meta-analysis effects of dietary interventions on neonatal and infant outcomes: a systematic review and meta-analysis mother's education and the risk of preterm and small for gestational age birth: a drivers meta-analysis of european cohorts is adolescence a sensitive period for sociocultural processing? factors that affect bone mineral accrual in the adolescent growth spurt muscular strength and adiposity as predictors of adulthood cancer mortality in men pubertal timing and bone phenotype in early old age: findings from a british birth cohort study developmental trends in eating self-regulation and dietary intake in adolescents the developing social brain: implications for education parental support and adolescent motivation for dieting: the self-determination theory perspective school-based physical activity programs for promoting physical activity and fitness in children and adolescents aged to the effectiveness of interventions to increase physical activity among adolescent girls: a meta-analysis controlling childhood obesity: a systematic review on strategies and challenges delivering in-school interventions to improve dietary behaviours amongst -to -year-olds: a systematic review children and parents: media use and attitudes report strategies to engage adolescents in digital health interventions for obesity prevention and management. healthcare (basel) a systematic review of digital interventions for improving the diet and physical activity behaviors of adolescents a meta-analytic review of obesity prevention programs for children and adolescents: the skinny on interventions that work autonomy and control: the coconstruction of adolescent food choice harnessing adolescent values to motivate healthier eating a values-alignment intervention protects adolescents from the effects of food marketing intervention planning for a digital intervention for self-management of hypertension: a theory-, evidence-and person-based approach the person-based approach to enhancing the acceptability and feasibility of interventions the person-based approach to intervention development: application to digital health-related behavior change interventions idea jamming with teenagers. southampton: university of southampton london: ministry of housing, communities & local government healthy conversation skills: increasing competence and confidence in front-line staff making every contact count': evaluation of the impact of an intervention to train health and social care practitioners in skills to support health behaviour change the link between pupil health and wellbeing and attainment: a briefing for head teachers, governors and staff in education settings. london: public health england associations between objectively measured physical activity and academic attainment in adolescents from a uk cohort obesity impairs academic attainment in adolescence: findings from alspac, a uk cohort a mixedmethods investigation to explore how women living in disadvantaged areas might be supported to improve their diets health promotion from the perspective of social cognitive theory. psychol health the behavior change technique taxonomy (v ) of hierarchically clustered techniques: building an international consensus for the reporting of behavior change interventions from theory to intervention: mapping theoretically derived behavioural determinants to behaviour change techniques the effect of a behaviour change intervention on the diets and physical activity levels of women attending sure start children's centres: results from a complex public health intervention the southampton initiative for health: a complex intervention to improve the diets and increase the physical activity levels of women from disadvantaged communities estimation of fruit and vegetable intake using a two-item dietary questionnaire: a potential tool for primary health care workers an assessment of maturity from anthropometric measurements the validity of the youth physical activity questionnaire in - -year-old scottish adolescents the development of a paediatric health related quality of life measure for use in economic evaluation: the child health utility d (chu d). sheffield: the university of sheffield reliability and validity of an adapted version of the cantril ladder for use with adolescent samples a modification to the behavioural regulation in exercise questionnaire to include an assessment of amotivation reliability and validity of the se-hepa: examining physical activity-and healthy eating-specific self-efficacy among a sample of preadolescents self-determination, smoking, diet and health meta-synthesis of health behavior change meta-analyses london: local government association cluster randomized trials with treatment noncompliance process evaluation of complex interventions: medical research council guidance using thematic analysis in psychology tackling of unhealthy diets, physical inactivity, and obesity: health effects and cost-effectiveness the green book: appraisal and evaluation in central government we acknowledge sarah jenner, sara simao, daniel penn-newman, daniella watson and taylor morris who helped us collect preliminary data and develop research materials. kris tsenova created the artwork for the digital intervention and the education module. we are grateful to our ppi members ros horlock, paula twynham and rosie mackay for their advice and support. mrc lifecourse epidemiology unit, southampton general hospital, university of southampton, southampton, uk. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - jz tox authors: bayham, jude; fenichel, eli p title: the impact of school closure for covid- on the us healthcare workforce and the net mortality effects date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: jz tox background covid- is leading to the implementation of social distancing policies around the world and in the united states, including school closures. the evidence that mandatory school closures reduce cases and ultimately mortality mostly comes from experience with influenza or from models that do not include the impact of school closure on the healthcare labor supply or the role of the healthcare labor force in reducing the per infection mortality from the pathogen. there is considerable uncertainty of the incremental effect of school closures on transmission and lives saved from school closures. the likely, but uncertain, benefits from school closure need to be weighed against uncertain, and seldom quantified, costs of healthcare worker absenteeism associated with additional child care obligations. methods we analyze data from the us current population survey to measure the potential child care obligations for us healthcare workers that will need to be addressed if school closures are employed as a social distancing measure. we account for the occupation within the healthcare sector, state, and household structure to identify the segments of the healthcare labor force that are most exposed to child care obligations from school closures. we use these estimates to identify the critical level for the importance of healthcare labor supply in increasing a patient's covid- survival probability that would undo the benefits of school closures and ultimately increase cumulative mortality. findings the us healthcare sector has some of the highest child care obligations in the united states. % of healthcare provider households must provide care for children - . assuming non-working adults or a sibling years old or older can provide child care, leaves % of healthcare provider households in need of childcare during a school closure, while % of healthcare households are single-parent households. we document the substantial variation within the healthcare system. for example, % of medical assistants and % of nursing, psychiatric, and home health aide households have child care obligations, while only % of emergency medical personnel have childcare obligations. child care obligations can vary between states by over percentage points. a % decline in the healthcare labor force, combined with reasonable parameters for covid- such as a % case reduction from school closings and % baseline mortality rate implies that a % loss in the healthcare labor force must decrease the survival probability per percent healthcare worker lost by . % for a school closure to increase cumulative mortality. this means that the per infection mortality rate cannot increase from % to . % when the healthcare workforce declines by %; otherwise, school closures will lead to a greater number of deaths than they prevent. for school closures to unambiguously provide a net reduction in covid- mortality with these parameters, the school closures must reduce cases by over %. conclusion school closures come with many tradeoffs. setting aside economic costs, school closures implemented to reduce covid- spread create unintended childcare obligations, which are particularly large in healthcare occupations. detailed data are provided to help public health officials make informed decisions about the tradeoffs associated with closing schools. the results suggest that it is unclear if the potential contagion prevention from school closures justifies the potential loss of healthcare workers from the standpoint of reducing cummulative mortality. contagion prevention from school closures justifies the potential loss of healthcare workers from the standpoint of reducing cummulative mortality. no external funding. covid- ; coronavirus; sars-cov- ; outbreak; school closure; social distancing; non-pharmaceutical interventions; healthcare; labor supply; doctors; nurses. evidence before this study covid- is affecting many countries around the world. multiple countries, states, and school districts are employing school closures as a non-pharmaceutical social distancing strategy. support for school closures mostly comes from models and experience with influenza. few studies explicitly consider the tradeoff between case reduction and disease burden with the potential loss of healthcare workers to child care obligations. we found only two studies that attempted to quantify the potential child care burden of school closures for healthcare workers. these analyses were coarse, and no studies have explicitly considered the tradeoff between reduced transmission and healthcare labor's role in cumulative mortality. using current detailed data from the us current population survey, we quantify the exposure of the us healthcare sector, occupations within the healthcare sector, and individual us states to unmet child care obligations for us healthcare workers in the event of a school closure. these obligations could compromise the ability of the us healthcare system to respond to covid- . we identify the parameter space where school closure could lead to a greater number of deaths from covid- through healthcare labor force reductions. we find that the current best estimates of healthcare worker likely absenteeism to provide child care in the event of school closures imply great uncertainty to whether school closures will ultimately reduce covid- mortality. targeted pharmaceutical interventions for covid- are likely months away. however, supportive measures by healthcare providers are important. social distancing, including school closures, can limit covid- cases. however, the evidence that the potential transmission reduction benefits of mandatory school closures exceed the costs of potentially imposing greater child care obligations on healthcare workers, thereby reducing the healthcare workforce, is not overwhelming. we provide the first explicit analysis of the school closure tradeoff between case reduction and labor force impact on patient survival probability. a % decline in the healthcare labor force, combined with reasonable parameters for covid- such as a % case reduction from school closings and % baseline mortality rate implies that a % loss in the healthcare labor force must decrease the survival probability per percent healthcare worker loss by . % for school closures to increase cumulative mortality. this means that the per infection mortality rate cannot increase from % to . %; otherwise, school closures will lead to a greater number of deaths than they prevent. even if the only objective is to save lives, there is still a tradeoff associated with closing schools due to potential losses in healthcare labor force capacity. covid- 's global spread is triggering a range of public health responses. school closures are some of the highest-profile social distancing measures employed to slow the spread of an infectious disease. japan recently instituted a nationwide school closure, which drew a swift response from families scrambling to make alternative childcare arrangements. south korea, italy, and some us school districts have closed schools. school closures prevent contacts among children and reduce cases. but, there is a downside to closing schools, even if the only goal is saving lives during an epidemic. closing schools can inadvertently cause child care shortages that strain the healthcare system. lempel et al. estimated that the child care obligations associated with school closure could reduce key medical personnel by - %. here, we use the recent years ( - ) of the us current population survey to estimate the school closure induced child care obligations for the us healthcare labor force. approximately % of us households with a healthcare worker have at least one school-aged child age - , who requires child care. child care obligations are especially high and relatively inflexible in households where a parent is a nurse or medical assistant. understanding these tradeoffs are important in planning the public health response to covid- , because if the survival of infected patients is sufficiently sensitive to declines in the healthcare labor force, then school closures could increase deaths. the benefit from closing schools during an epidemic is to reduce transmission and new cases. cauchemez et al. estimated that extended school closures in france could reduce h n influenza cases by - %. bayham et al. show that schools are likely places for transmission among us children. however, bayham et al. also show voluntary behavioral changes, without mandatory shutdowns, appeared to reduce cases of the h n influenza on the order of - %. in a systematic review, focused on influenza and school closures, jackson et al. find some evidence that school closures are effective, but that the empirical evidence does not resolve how or when to close schools. furthermore, they find school closure mostly reduces infection in school children. koh et al. do not find strong evidence that school closures prevent the spread of hand, foot, and mouth disease. adda finds that while school closures do reduce incidence in france, the economic costs are large. the benefits of school closure are often estimated relative to a baseline of no voluntary changes in behavior, but it is likely that the correct baseline for forcasting the school closure effect on covid- includes other voluntary behavioral changes. the potential benefits of school closures must be balanced with the costs. smith et al., lempel, epstein, and hammond, and adda analyze the economic impacts of school closures. , , schooling is one of the most important investments we make in our children's futures, and we do not have good estimates of how prolonged school closures influence drop-out rates and future earnings. this uncertainty makes a holistic assessment of tradeoffs challenging. setting aside the economic costs and focusing on reducing mortality, school closures still create a tradeoff. many healthcare workers must reduce time spent providing patient care, running diagnostic tests, and tracing contacts, and increase time caring for their own children. this tradeoff should not be ignored because the capacity to care for infected individuals and trace contacts directly influences the development of an epidemic, the survival of infected patients, and ultimately cummulative mortality from the pathogen. first, we highlight two pathways school closure can effect pathogen induced mortality. school closures, , can affect mortality via ( ) reduction in cases and ( ) a reduction in the healthcare labor force that treats sick patients and prevents mortality. while no pharmaceutical treatment is available, supportive measures are still important for patient survival. define cumulative mortality as all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. where is the baseline mortality fraction for cases, / is the percent increase in the case mortality rate through the reduction in healthcare labor force from a school closure ( ) , and / is the percent decrease in cumulative cases from the school closure. if there are no school closures, , then ⁄ . this implies mortality is . this model highlights the tradeoff between the case-reducing effect of school closures and the cost in terms of lost healthcare labor supply. analyzing the net effect of school closures on mortality requires estimation of three factors that are not part of canonical epidemiological models. , the first term is , which is the effect of school closure on the healthcare labor force and is between zero and one. the second term is , which is a first-order approximation of the life-saving (mortality-reducing) effect of healthcare providers on the probability of a patient dying from disease or disease-related complications. the third term is / , which is the reduction in cases associated with a school closure. the first term, , is rarely calculated. lempel, epstein, and hammond provide a preliminary estimate for school closures in the us of a - % reduction in the healthcare workforce. to provide detailed estimates, we use data from the us current population survey (cps) to quantify the impact of school closures on healthcare labor supply, . the cps is an ongoing monthly survey of approximately , us households jointly administered by the us census bureau and bureau of labor statistics. we access the data via the integrated public use microdata. we use the recent dataset, which includes information on just over million individuals spread across . million households between january - . the detailed data in the cps allows us to characterize the family structure and likely withinhousehold childcare options for healthcare workers. it also enables us to describe the exposure to child care obligations for specific occupations within healthcare and across states. we calculate the share of healthcare provider households that are single parent, where a parent is defined to include head of household, opposite or same-sex spouse, partner or roommate, and an opposite or same-sex unmarried partner. we use the personal cps sample weights for all calculations to ensure that the estimates are nationally representative. the second term, , is calculated in development contexts and in emergency medicine, but to our knowledge has not been measured for accute infectious diseases (e.g., influenza or covid- ) or included explicitly in epidemiological models. it is useful to know the critical value of where school closure stops saving lives and starts increasing mortality, which is defined by the condition imposing condition ( ) as a strict equality and rearranging yields , when exceeds this value, then more lives are lost from school closures than saved. is the maximum percent increase in the mortality rate that does not reverse lives saved from school closures. is a more inutive quantity, and it also provides a number that is invariant to . the epidemiological literature has focused on the final term, / . this value is usually determined by adjusting the conditional infectivity, either parametrically or through a behavioral model within an epidemiological model to account for a school closure. models are required because there is little unconfounded experience with school closures during an epidemic, and few analyses of any behavior all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint change are empirical. one of the most detailed studies is cauchemez et al., which estimates that prolonged school closures would reduce cumulative influenza cases by - % in france, which implies ⁄ is between . and . . we focus on the midpoint of this estimate, . , but consider cumlative case reductions from school closures between and %. twenty-nine percent of healthcare worker households have an obligation to care for a child between and years of age. focusing on households without a non-working adult or sibling years or older who could potentially care for children ages - (https://www.redcross.org/take-aclass/babysitting/babysitting-child-care-training/babysitting-certification), we find that % or slightly more than in , healthcare worker households have child care obligations. this may be optimistic because non-working adults may not be able to provide care, e.g., they may require care themselves. on the other hand, it is possible that family members outside the household, neighbors, or friends could care for children, though no data are available in the cps on these possibilities. we find that % of healthcare households are single-parent households, which is greater than all other major industry classifications. within the healthcare sector, there are critical professions that are even more exposed to child care obligations (table ) . these include households with a nurse, psychiatric, and home health care aids, nurse practitioner, medical assistant, nurse anesthetist, diagnostics technologist, physician's assistant, or physician and surgeon. assuming that non-working adults or siblings or older can meet the child care obligations, % of nurse practitioners, % of physician's assistants, % of diagnostic technicians, % of medical assistants, % of physicians and surgeons, and % of nursing, psychiatric, and home healthcare aids would still have unmet child care obligations. this is just over % of the healthcare workforce. school closures may be especially challenging for single parents. . % of nursing, psychiatric, and home health aides are single parents. medical assistants ( . %) and licensed practical and licensed vocational nurses ( . %) are also more likely to be single parents than the healthcare average. together this is % of the healthcare workforce, and is the segment most likely to be providing infection control for the elderly in nursing homes and other facilities. registered nursing is the most common profession in the healthcare field and accounts for . % of the healthcare labor force, followed by nursing, psychiatric, and home health care aides ( . %). % of registered nurses have child care obligations, but perhaps % of registered nurses can meet these obligations with a non-working adult or older sibling, leaving % with unmet child care obligations. five percent of registered nurses are single parents, but this varies from state to state (figure ). the exposure of the healthcare labor force to school closures is not homogeneous throughout the us (appendix table ). how a policy trade-off plays out in one place may be different than another even if two locations experience a similar set of covid- cases. that is, different places may have similar projected benefits from school closures, but different costs. the greatest share of the healthcare labor force with child care obligations is in utah ( %), louisiana ( %), and missouri ( %), whereas the healthcare labor force in washington dc ( %), vermont ( %), and massachusetts ( %) have the least child care obligations. however, household structures also vary. therefore, if childcare obligations can be met by a non-working adult or older sibling, then south dakota ( %), oregon ( %), and missouri ( %) are the most exposed states to school closure induced healthcare worker shortages. washington dc ( %), new mexico ( %), and new jersey ( %) may be most able to cover their child care obligations. louisiana ( %), mississippi ( %), and pennsylvania ( %) have the greatest fraction of single-parent healthcare worker households. these differences are likely an interaction of variations in state-level healthcare regulation, cultural, and demographic differences. this requires state and local health officials all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. to consider the exposure of their own state (data available at https://github.com/jbayham/us_childcare_obligations) or region. using the data on child care obligations provides a first estimate of potential reductions in the healthcare labor force during a school closure. this estimate can be combined with projections of the case reductions from school closures to identify the parameter space where estimates of are important to inform whether school closures would reduce net mortality (figure ). if the is large, then the increase percent increase in the mortality rate is below , and closing schools saves lives. this is the case when school closures lead to many avoided cases, and few healthcare workforce effects. conversely, if is near zero, then then it is likely that the percent increase in mortality exceeds and school closure increases the cumulative mortality. this is the case if the school closure reduces the labor force substantially but provides a relatively small reduction in cumulative cases. there is a band where ≪ ≪ ∞, where whether or not closing schools saves lives depends on the value of , which is unknown. for the united states, and for most states within the us, the is not sufficently higher or lower to know which way a school closure will turnout without more information on . consider an example that uses cauchemez et al.'s ( ) estimate of a reduciton of cases of % from a school closure and assume baseline mortality to covid- of %. assume an epidemiological forcast without a school closure predicts , cases (about a / th of the cases currently reported in china), which implies deaths (italy has already reported deaths with , cases). the school closure reduces cases to , . the for this scenario is . , with an associate . . therefore, the mortality rate after the closure must rise to at least . % as a result of the % loss in the healthcare workforce in order to undo the benefits of school closures. this implies the elasticity of mortality with respect to the healthcare workforce must be less than - . in aboult value, which would imply doubling the healthcare workforce must not save more than . % more patients. what we know about social distancing policies is based largely on models of influenza, , where children are a vulnerable group. however, preliminary data on covid- suggests that children are a small fraction of cases and may be less vulnerable than older adults. if these early results hold-up, then the already uncertain transmission reduction benefits from school closures will be reduced relative to influenza. conversely, school closures may be implemented earlier in covid- outbreaks, which may lead to greater levels of prevented cases. furthermore, school closures may lead to other adults staying home, which could reduce cases. these are all important questions when considering school closures. school closures do expose the healthcare labor force to increased child care obligations, likely reducing support for infected individuals, which is critical. we also do not know how much a reduction in the healthcare labor force, and in what occupations within healthcare, decreases the probability of covid- patient survival. we do know that the segment of the healthcare work force most responsible for infection control in nursing homes is likely to be among the most highly impacted by school closure induced child care obligations. given our reasonable estimates of case reductions from school closures, a measure of the increased mortality risk to covid- patients from healthcare absenteeism to care for children is a critical, and to date unknown parameter. this analysis does not include non-covid- mortality that could occur from other conditions if the us healthcare work force is reduced, but risk to these patients should also be considered when deciding about school closings. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. minimizing the impact of the covid- and saving lives requires clear thinking and weighing tradeoffs. there will likely be cases when closing schools is sensible. however, policymakers and the healthcare experts advising them need to understand that closing schools may have severe knock-on effects on the healthcare system and that there is substantial uncertainty about the effectiveness of school closures for preventing infection beyond school children and on the impact of reduction in the healthcare workforce on patient survival. in the united states, the healthcare system appears disproportionately exposed to school closure induced labor shortages, and the segment of that system that provides infection control in nursing homes even more so. such potential healthcare workforce labor shortages should be a first-order consideration when considering the potential benefits and costs of school closures. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint figure . the map depicts the fraction of the healthcare workforce with possible childcare obligations under various adaptation assumptions: a) healthcare workers in households with at least one child age - , b) heathcare workers in households with at least one child age - and without a non-working adult or child over that might provide childcare, and c) healthcare workers in single-parent households. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint figure . critical level of the percent increase in mortality resulting from healthcare work force absenteeism associated with school closure induced child care obligations , , that would offset the mortality reduction achieved by school closures through case reductions (color scale). the actual percent increase in mortality must be below to justify closing schools. the red point, . , indicates the best national estimates of cases avoided due to school closures ( % [ %, %]) and the mean estimates of unmet healthcare workforce childcare obligations, %. this estimate accounts for the potential for other non-working adults or older siblings in the household to provide childcare. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint economic cost and health care workforce effects of school closures in the u.s estimating the impact of school closure on influenza transmission from sentinel data measured voluntary avoidance behaviour during the a/h n epidemic school closures and influenza: systematic review of epidemiological studies the epidemiology of hand, foot and mouth disease in asia economic activity and the spread of viral diseases: evidence from high frequency data the economy-wide impact of pandemic influenza on the uk: a computable general equilibrium modelling experiment the basic reproduction number of infectious diseases: computation and estimation using compartmental epidemic models integrated public use microdata series adaptive human behavior in epidemiological models behavioural change models for infectious disease transmission: a systematic review the effects of school closures on influenza outbreaks and pandemics: systematic review of simulation studies both authors worked closely to design and write the manuscript. jb lead the data management and analysis. we declare no competing interests the us current population survey is publically available, see flood et al. code to access and organize the data are hosted on bayham's github page, https://github.com/jbayham/us_childcare_obligations. key: cord- -iwdusvrw authors: gandolfi, alberto title: planning of school teaching during covid- date: - - journal: physica d doi: . /j.physd. . sha: doc_id: cord_uid: iwdusvrw learning and education are two of the biggest world issues of the current pandemic. unfortunately, it is seen in this work that, due to the length of the incubation period of covid- , full opening of schools in the fall of seems to be impractical unless the spread of the virus is completely under control in the surrounding region (e.g. with fewer than active cases every million people). in order to support the possibility of some in-person learning, we model the diffusion of the epidemic within each single school by a seair model with an external source of infection and a suitable loss function, and then evaluate sustainable opening plans. it turns out that blended models, with almost periodic alternations of in-class and remote teaching days or weeks, are generally (close to) optimal. in a prototypical example, the optimal strategy prescribes a school opening of days out of with the number of covid- cases among the individuals related to the school increasing by about %, instead of the about % increase that would have been a consequence of full opening. as clinical fraction is low in children, these solutions could lead to very few or no symptomatic cases within the school during the whole school year. using the density of active cases as a proxy for the number of pre- and asymptomatic, we get an indication for each country of whether either full opening, or blended opening with frequent testing, or no school opening at all, are advisable. more than one billion students are out of school because of covid- [ ] ; most of them are now using remote learning practices that present several drawbacks [ ] and, in most cases, have been hurriedly arranged. in addition, in most schools, school districts, and countries there is uncertainty on how to plan school activities for the - school year [ ] . as a result, learning and education are two of the biggest world issues of the current pandemic [ , ] . as we see below, the characteristics of covid- , and in particular the length of its incubation period of about days [ , ] , are such that cases within each single school tend to undergo a multifold increase should the school be completely open in the upcoming school year. this makes full opening of schools impractical unless the epidemic in completely under control in a region, or till a vaccine is available: difficulties have been already experienced by the first reopening attempts [ , ] . on the other hand, remote teaching has been put together in an emergency situation, and it appears to have badly affected most students [ ] [ ] [ ] , especially those from less advantaged contexts [ ] . at the same time, it has imposed, and it likely to impose again, an additional burden on many families. therefore, the hypothesis of a fully remote - school year is also impractical for different reasons. to help reconcile such conflicting circumstances, there is thus a need for studies and methods that suggest sustainable opening strategies; they should be aimed at supporting the possibility of some in-person learning while simultaneously containing extra infection transmissions, both at the level of each single school, and in a broader context. in this first work, we focus on a single school. what each school needs is a flexible instrument that incorporates some of the specific details of the school, the students, the personnel, and the surrounding area, and provides indications of the outcome of in-person school activities in terms of the potential spread of covid- within the school. some of the difficulties in this process are that it is hard to estimate the transmission rate within schools; that the individuals involved in the school are subject to potential external infectious transmissions; that asymptomatics are hard to detect; and that policy makers have to establish a relative importance of the two targets (in-person teaching and covid- containment). in this paper, we provide a model whose set-up and parameters incorporate several features of covid- dynamics related to a single school, and then outline the optimization process. we then study optimal solutions for a wide variety of plausible values of the parameters of the model, obtaining both an adaptable procedure that can be conformed to specific situations, and general policy indications. blended solutions appear to be extremely efficient in reducing the potential spread, and together with some detection of asymptomatic cases could offer the possibility of a sustainable planning for the whole year. besides containing the covid- diffusion, these solutions could be pedagogically acceptable, and could also become a driving model for the society at large. in this work we focus on a single school. to model the diffusion of covid- among the personnel and the students of the school, we set-up a suitable system of ordinary differential equations. in particular, we consider seair, a version of the sir model [ ] with an external source of infection [ , ] , and a control. the population is divided into: susceptible (s), pre-symptomatic or, equivalently, exposed (e), asymptomatic (a), infected (i) and recovered (r). variables are normalized so that s + e + a + i + r = . we assume that susceptible individuals might become pre-symptomatic or exposed (e) at transmission, entering a latency period in which they have contracted the virus and are contagious, without showing symptoms. the contagion can be caused by contact with other individuals with viral load, at a rate βc(t) (c(t) is the control, as described below); or, alternatively, it is caused by an infectious contact outside the school taking place at rate α. the asymptomatic members of the school community present a challenge, as they cannot be easily detected while still being infectious for a relatively long time: we assume accessibility to some type of fast screening with sufficient sensitivity [ ] : the contacts with asymptomatics within the school are then reduced by a parameter η, the false negative rate of the screening procedure. exposed individuals either develop symptoms at a constant rate δ, becoming infected, or progress into being asymptomatic with rate γ. the viral load is carried by exposed (e), asymptomatic (a) or infectious (i) individuals; however, infectious individuals are assumed to be isolated, while a large fraction of asymptomatic (a) is supposed to be detected by the screening procedure. both asymptomatic and infected individuals recover at rate ρ. as covid- mortality is particularly low in a mostly young population [ ] , we disregard mortality. the key differences with the usual seir model [ ] are: an external source of infection [ ] , [ ] , the possibility of transmission limited to hours per working day; a control indicating from the start whether, for each day, schools are open or closed; and the presence of asymptomatic individuals. with these assumptions, the system of ode modeling infectious transmissions of concern for the school is: exposed: infected: recovered: j o u r n a l p r e -p r o o f for a certain time interval [ , t ]. we consider weeks, and count time in hours, so that t = . the initial population at the beginning of the school year might be partly immune due to previous infections, but to avoid issues about efficacy and duration of the immunity, we assume that the initial population consists primarily of susceptible, s( ) ≈ , and a small fraction of exposed, so that s( ) + e( ) = . the function c(t) describes the control variable, and c(t) = for all times when the school is closed; these include all hours except from am to pm of those working days in which it has been decided that the classes are in person. for each of the working days, c(t) can be again if remote activities have been decided for that day, or if teaching is in person. formally, with t measured in hours, let m indicate the class of controls c(t) such that for some c i,j ∈ { , }, i = , . . . , , j = , . . . , , and ≤ t ≤ . lemma . . for each solution of ( )-( ), the total population s +e +a+i +r is preserved. in addition, ≤ s, e, a, i, r ≤ proof. let φ(t) = s(t) + e(t) + a(t) + i(t) + r(t); we have that φ( ) = and dφ dt = for all t from ( )-( ); it follows that φ ≡ by uniqueness of solutions of linear differential equations. in addition, r( ) ≥ and r ≥ , so that r(t) ≥ for all t. the other functions have to take the value before becoming negative by continuity. if i(t) = for some t, then i (t) > unless e(t) = , and the same applies to a(t), therefore let's consider the case that e(t) = . but then e (t) > unless s(t) = . in this last case, ( ) implies that all derivatives d n s(t) dt n = , in which case s(t) ≡ ; on the other hand, the system ( )-( ) with s = and nonnegative initial condition has an explicit, unique solution, which is seen to be non negative. proof. let x(t) = (s(t), e(t), a(t), i(t), r(t)) and express ( ) where is the number of hours in a week. take k = . extending c(t) = c for t ≤ , the conditions of peano's theorem are satisfied ( [ ] , ch. , th. ), so that there is a solution to the initial boundary value problem ( )-( ) for some positive t's. by considering r as above, one can see that the solution can be continued to the boundary of r ( [ ] , ch. , th. ), which is t = . in addition, as each component of x(t) satisfies x i ≤ by lemma . , f (c, x) is seen to be lipschitz in [ , ] × { x : x i ≤ for i = , . . . , }. hence, the solution is unique (see [ ] , ch. , th. ). the same reasoning can now be repeated from t = and with initial conditions given by the limit t → of the unique solution determined in the previous time interval. as c(t) has only jump discontinuities, the same procedure can be repeated in each interval, (see [ ] ), to show that there always exists a unique solution for all times t of the system ( )-( ). we are, however, interested in a finite time interval t , and hence in the transient solution up to time t . in the first part of the analysis, we will explore the effect of the opening policies on the number of remote teaching days and infection spreading. for this, we need an appropriate parameter selection. we make a first choice based on current observations, and present results in section , and then evaluate the modifications for a wide range of possible values in the sensitivity analysis section. as time is counted in hours, we need to scale all available parameter estimates, usually expressed for time counted in days, by a factor of . estimates of the infection rate β are typically around . - . [ ] ; however, as the school is likely to elicit more frequent contacts, we adopt the somewhat higher value of β = . / ≈ . × − . the value of β can also be computed from contact matrices and susceptibility. [ ] has contact matrices in school for most countries: averaging the contacts for class ages − with a factor for the number of classes, one gets an average number of daily contacts varying between (germany) and (italy); the contacts need to be revised by the recent containment measures, and related awareness of the population; a reasonable figure would be a factor of / . covid susceptibility (probability of infection upon contact) in school is estimated around . - . , see [ ] figure (b) as our equations are written for the school population only, which is about / of the total population, one needs to use a scale factor ; in addition, one needs to divide by to have the hourly contact number during school hours. in the end, estimates of hourly β range from β m ≈ . × / × × / ≈ . × − to β m ≈ . × / × × / ≈ . × − (this is a range of . - . for the daily β), in line with our choice above. in the stability analysis we consider the other values in this range. the duration of the latency period after infection and before symptoms are developed has been estimated at about days (see for example [ ] and [ ] ), so that γ + δ ≈ / × / = . / ; the clinical fraction, which is the fraction of symptomatic cases, depends on many factors [ ] , and it is substantially lower for younger individuals [ ] . we take a ratio of about , which is γ = . / ≈ . × − , and δ = . / ≈ . × − similarly, the average recovery period is about days, for mild cases [ ] , suggesting ρ = . / ≈ . × − ; more severe cases (i) are excluded from contacts, so their recovery rate is irrelevant: we use the same value of ρ. the parameter η describes how asymptomatic individuals are excluded from contacts with the other individuals; such a separation depends on the availability of detecting tests: we assume that a sufficiently reliable test is available, with a % test sensitivity, so that η = . . this assumption is subject to parameter sensitivity analysis in section below, where we see that a test sensitivity of at least % is needed for our calculations to make sense. we finally consider the external rate of infection. this is due to encounters of infectious individuals from the schools with others outside of the school; it is primarily based on the number of presymptomatic and asymptomatic individuals in the region around the school. this number is clearly unavailable, but an approximation can be obtained from the number of active cases, which is a well established figure [ ] : since about half of the infected shows symptoms, and the symptomatic period lasts longer than (perhaps times as long as) the asymptomatic, which itself lasts as the presymptomatic period of everyone infected, the fraction of presymptomatic and asymptomatic together is about the same as the fraction of active cases; the asymptomatics are not assumed to be detected outside of the school, so we take the density of active cases as a proxy for the density of infectious individuals that can be met outside of the school. a survey of the current situation in about countries shows that the fraction of active cases to the population ranges between . × − and . × − (see [ ] and supplementary material). this number must then be multiplied by the average number of contacts, similar to what done above to estimate β. as external contacts are, for the most part, between students and other individuals outside schools, we use the corresponding matrices in [ ] , with entries averaged over the various age classes: the averages vary between . for germany and . for italy; we then add a factor / for reduced mobility and awareness. susceptibility is taken again at . - . , and the scale factor is . plausible values of α are then in the range from . × . we take an intermediate external infection rate α ≈ . × − . with this value, in the observation period of weeks there is a moderate, but non negligible number of cases even if the school is totally closed; with our selection of parameters the individuals infected outside of the school will be about . % of the total. the parameter α will be carefully monitored in the sensitivity analysis, where it is seen that our optimality results are insensitive to the specific j o u r n a l p r e -p r o o f value of α provided that this lies in a specific range. to aid readability, one can consider an approximate conversion factor of . (to be adapted to local characteristics) between α and the density of active cases: α ≈ . × − corresponds to an overall density of active cases of approximately . × − / . ≈ × − active cases, or active case in inhabitants. this conversion factor and the above range of α will also be used to give a tentative indication of which countries can reopen schools and which cannot. table : recap of the model parameters and their estimated values. as initial condition, we consider two possibilities. in the first one, we assume that there are a few (about %) exposed individuals already present at the start of the school year: we start from e( ) = . . all others are susceptible, so s( ) = . . in the second, we assume that a careful screening is carried out at the beginning of the school, so we take s( ) = . in the second part of the analysis, we introduce a loss function which incorporates the aim of the planner to maximize the number of days with in-class vs. remote teaching, and simultaneously minimize the number of extra infectious transmissions due the opening of the school. for this, it is essential that the planner determines the relative weight τ of these two objectives. once a value of τ is fixed, the aim of the planner becomes that of minimizing a functional that combines these two objectives by the relative weight. to achieve her/his targets, the planner can fix a school calendar, deciding ahead of time which days are in-class and which are restricted to remote teaching; for simplicity, we consider here a plan for the whole year, but monitoring and en route adjustments can also be incorporated. we remain agnostic as to the value of the relative weight τ , and develop tools to analyze all possible scenarios. let s (t) be the fraction of susceptibles at time t in the hypothesis that teaching is completely remote, that is with c ≡ ; s ( )−s (t ) is then the fraction of individuals who receive an infectious transmission even if the school never opens; then, let s(t) = s c (t) be the fraction of susceptibles with opening plan c(t); we is the fraction of extra infectious transmissions due to the opening of the school j o u r n a l p r e -p r o o f journal pre-proof according to plan c(t). in addition, let n (c) = − t c(t)dt/ be the number of remote teaching days (the factor / is due to the number of hours that the school is open in a regular day). the loss function combining the two effects is then where τ is the relative weight of a day of in-class teaching to infectious transmissions: τ / can be interpreted as the number of days of in-class teaching that the planner considers equivalent to a % increase in the infectious transmissions in the school. the planner's objective becomes then with m as in ( ). it is easy to see that using the system ( )-( ) the optimization problem can be cast in a more standard form [ , ] , in which where e (t) is the exposed component of the solution of the system ( )- ( ) for the case c ≡ ; however, as we optimize over the very restricted class of functions ( ) , which are piecewise constant and depend on the finite number of parameters c i,j 's, the general theory of control optimization is not needed here: ( ) becomes a discrete optimization problem. once the parameters have been selected, for instance with the values given in table , we have optimized for each fixed value of τ ∈ [ . , ] by simulated annealing [ ] . for simplicity, we assume here that the decision about remote or in-class teaching is taken for each week, so the control c i = c i,j depends only on i = , . . . , . this still includes possible policies. for a fixed value of τ , we take vanishing "temperatures" k = e −k with ∈ ( , ], and a random initial configuration c ( ) (τ ) = {c , . . . , c } ∈ { , } . at iteration k = , . . . , , a random position m between and is selected and c m is changed intoc m = − c m , whilec m = c m for all other m 's. the system is solved using the opening rulec, and the loss function is computed in the solution. if the loss decreases, then c (k+ ) (τ ) =c; else, we set c (k+ ) (τ ) =c with probability k and c (k+ ) (τ ) = c (k) (τ ) with probability − k . we then repeat by drawing a new random integer m. this is repeated times for each initial configuration. the sequencec (k) (τ ) tends to a (local) minimum of the loss functional; the outputc(τ ) =c ( ) (τ ) of iterations is retained as a candidate (close to) optimal strategies for the given value of τ . in addition to the above, all the configurations c (k) (τ ), k = , . . . , , not only the (close to) optimal ones, are recorded for several values of τ , to get an j o u r n a l p r e -p r o o f idea of the possible scenarios. figure reports remote teaching days vs. fraction of infected in school at the end of the school year for each c (k) (τ ), k = , . . . , and for various values of τ . as benchmark cases we consider the full closure and the complete opening of the school, which is to say, the two most extreme choices of a fully remote teaching or regular hours a day in-class activities for the whole year. as we have assumed that there is a constant source of external infection in the region surrounding the school, even if the school is completely closed, which is c ≡ , the simulations based on ( )-( ) determine a number of cases. we have simulated the fraction of cases at school year end, and the number of cases in a school of individuals in the first two months. the parameters are those selected in the section . , except that we consider a variety of possible rates for the external source of infection. for example, for α = × − , we have that with the school completely closed there would be about . % cases (a+i). in a school with individuals this means that about individuals of the school would be infected (with or without symptoms) in the course of the year from contacts outside of the school. at day the number of infected individuals showing symptoms is about , and we assume that this is in line with the rate of infection in the overall community. if the school is completely open, on the other hand, simulations with c ≡ show a fraction of infected of about . % at the end of the school year, with already % of new cases, infected or asymptomatic, in the first days. in a school of , one would observe about cases, with perhaps a several symptomatic, in the first days, vs. the expected and very likely no symptomatic; a deviation which would not pass unnoticed. table compares the percentage of cases at the end of the school year, and the number of likely cases in the first two months, in the two cases of completely remote and fully in-class teaching. the values of α can be either determined from open cases in the area, contact matrix and susceptibility, as done in section . , or from the fraction of infected (a+i) that it causes at the end of the observation period. this last fraction is called seropositivity, and it is discussed in [ ] and [ ] ). seropositivity at the end of the school year, assuming the school is closed. is in table , column . the range of α covers all cases of interest. in table we assume that there are no active cases in the school at the start of the school year due to a sufficiently accurate initial testing. there is not much difference if we assume a % active cases for the largest values of α, but this last assumption would not be appropriate for small values of α and hide the possibility of opening the schools with almost no restrictions should one can see that complete opening of the school determines about a four fold increase in the number of cases, except for the extreme cases. if the virus is completely under control, which is less than active cases per million, or α < . × × − ≈ − , then the number of cases is so limited that schools can be safely reopen completely (with an initial screening and maintaining the physical distancing measures that are controlling the spread of the virus). if, on the other hand, there are more than about active cases per million in the surrounding region, or α > − then opening the school would not particularly aggravate the situation; but the number of cases is already so high that drastic measures need to be taken, such as keeping all schools closed. the conclusion is that, except in the extreme situations of almost complete elimination of covid- , or uncontrollable outbreak, the number of cases registered in a fully open school is likely to raise concerns from authorities before the end of the second month of school opening in any reasonable scenario; this is very likely to lead to the application of containment measures to the school, such as a forced, unplanned remote teaching. for these reasons, since this situation is likely to occur in most schools and in almost every external evolution of the epidemic, simulations indicate that it is advisable to attempt a sustainable, partial opening. this is the question faced here below. journal pre-proof with the reference parameters identified in section . , we have explored various opening strategies; for each policy c we have recorded the total fraction of infected, measured by the marginal decrease in susceptible s c ( ) − s c (t ), and the total number of remote teaching days n (c). recall that we consider policies with opening or closure applied for entire weeks. one can notice that, due to the form of the loss functional, some policies c are uniformly better than others regardless of the value of τ : define c as strictly preferable to c if s c ( ) − s c (t ) ≤ s c ( ) − s c (t ) and n (c ) ≤ n (c ) with at least one inequality strict; if a policy admits another one which is strictly preferable, the first policy can clearly be disregarded. we say that a policy is perfect with respect to the loss functional if there does not exist a strictly preferable one. on the other hand, once a value of τ is fixed, an optimal policy for τ is a policy minimizing the functional in ( ) . clearly, an optimal policy for some τ is perfect with respect to the loss functional, and, vice versa, a perfect policy with respect to the loss functional can be optimal for an interval of values of τ . in figure we plot remote teaching days vs. fraction of infected in school at the end of the school year for each c (k) (τ ), k = , . . . , and for various values of τ ∈ [ . , ]. such range corresponds to between . and days of in-class teaching rated equivalent to a % increase in the number of infected. notice, though, that figure does not allow one to retrieve the value of τ . one of the (likely) perfect opening strategies is represented by the red dot: it is very likely optimal for τ around . notice, finally, that the figure reports some, but very few of the strategies that are far from perfect or optimal. there are many others that our runs of the simulated annealing algorithm never visited. figure : fraction of extra infected, next to the . %, vs days of remote teaching, for several opening strategies. the potentially perfect, and hence optimal ones for some values of τ , are close to the continuous curve. one special possibly perfect and hence optimal solution corresponds to the red dot before turning to the exploration of specific optimal strategies, let us discuss j o u r n a l p r e -p r o o f the solid line in figure . experimentally, we have noticed that the outcomes of the perfect policies seem to lie close to a specific curve c, which corresponds approximately to where x is the percentage of covid- cases in the school population, and y is the number of remote teaching days. the curve c can be thought of as parametrized by τ , the relative importance of in-class teaching and covid- cases; in an abstract sense, each point on the curve c corresponds to one value of τ , although, as mentioned above, by the discrete nature of the opening strategies, more values of τ give the same perfect strategy. as indicated before, the red dot corresponds to a perfect strategy which is optimal for τ ≈ (see below). this curve c is of both theoretical and practical importance. in fact, . turns out to be a characteristic exponent of the comparison of opening days vs. extra infected; theoretically, it is quite interesting to explore the analytical reasons behind the appearance of such a curve; its role in the analysis of the solutions of the differential system ( )-( ); and the functional dependence of the value of the exponent on the values of the parameters in the system. in practice, if one was able to have some information about the dependency of the exponents from the parameters of the model different from τ , this would give an explicit benchmark for perfect strategies: for instance, if one fixes the number of opening days, then the curve indicates the best result that can be ideally achieved in the containment of the infection, or vice versa, without the need of an explicit numerical minimization. in this section we show one example of (nearly) optimal policy. as mentioned, a choice has been required by the policy maker: such a choice could have consisted of the minimal number of opening weeks, or of the maximal fraction of cases which is allowed by the circumstances. however, we adopted the less intuitive, but more intelligible possibility of using the relative weight of remote teaching vs. percentage of cases, as incorporated by the parameter τ (see section . ). we consider the parameters identified in section . and τ = , which means that days of remote teaching are considered equivalent to a % increase in the number of cases. the aim of the planner becomes then to solve the optimization problem ( ) , with the functional l defined in ( ). this can be achieved by a discrete optimization in { , } . we use here simulated annealing with iterations from a random seed, repeated for random initial conditions. the (possibly) perfect opening strategy indicated by the algorithm is ( , , , , , , , , , , , , , , , , , , , , , , , , , , , , ( ) , , , , , , , , , , , ) where indicates a week of remote, and a week of in-class activities; notice that there is an initial period of closure, before progressing to a regular alternation j o u r n a l p r e -p r o o f journal pre-proof of openings and closures. this is due to the assumed presence of % of cases at the start of the school year, and could be avoided with a very efficient screening before the start of the school year. the solution in ( ) has days of in-class teaching, with the additional infectious transmissions contained to . %, which is an increase of . % of the cases with respect to remote only activity. in a school of individuals this corresponds to creating an extra cases of covid- in the school year, in addition to the who would have been infected in any case. the administrators can decide to realize a stricter or more relaxed containment of the extra cases by assigning more or less weight to infectious transmissions. notice that the solution in ( ) is quite periodic, with alternated weeks of in-class and remote activities; figure shows the evolution of the epidemic functions under the policy in ( ) . table summarizes the change in infectious table : performance of optimal and random solutions with days of in-class teaching. first columns: total number of infectious transmissions; last columns: percentage increase with respect to remote only solution. "optimal" refers to the policy in ( ) identified by simulated annealing. we have also explored the possibility of selecting at random which weeks to open or close, with the constraint of having days of in-class activities, which is to say open out of the weeks. a simulation of the distribution gives that the increment in the number of cases is % on average, with a standard deviation of %, hence the solution in ( ) is more than sd's below what the average random selection would provide; in addition, it is also not subject to possible adverse fluctuations, so it is definitely preferable to a random, unjustified selection. it is, however, the case, that adopting a blended model with alternated open and closed weeks does provide in any case, even without optimization, a sizeable containment of the infection within the school. in section we carry out a sensitivity analysis, which shows that the optimal planning is pretty stable in case of some extra opening or remote days, and is only moderately affected by errors in parameter selection, within certain ranges. the optimal solutions turn out to be blended models, with approximately one week of in person teaching, and one week of remote learning. the pedagogical aspects of such models have been examined in various researches, for instance [ , ] it turns out that there are several advantages in blended learning: • in the current context, it seems to allow a sustainable programming of school operations; • it permits to take advantage of both in person and remote teaching, limiting drawbacks of both; one can, for instance, concentrate in person tests and labs for the in-class weeks, and less participative moments for remote teaching; • in general, it allows the use of a variety of learning modes and methods; • it can provide a pedagogical experience that can be replicated and expanded in the future in order to exploit new technologies while keeping regular personal contacts. of course, there are challenges, in particular concerning the ability of teachers and students to master the needed technologies and the alternating formats [ , ] . in addition, as schools could attempt planning in advance, especially if this is coordinated for most schools in some location, the regular economic and social activities could be somewhat adapted to the school calendar; for instance, more working hours could be planned during the weeks in which schools are open. clearly, there are several difficulties in implementing blended strategies, but the alternative, which is already unfolding in many situations, seems to be chaotic schedules, with schools attempting full openings, and then suddenly going back to closure as the first covid cases surface. we provide a sensitivity analysis in four parts. the first part concerns errors in the determination of the optimal solution. if one of the weeks for which remote teaching is planned by the optimal solution is instead changed to an in-class week, then the increment in the number of cases due to school opening ranges from an extra % to an extra %, compared to the extra % of the optimal solution. if one week is moved remote from inclass, then the increment in the number of cases is reduced to about %. this suggests that altering the number of weeks of opening is not optimal, but does not substantially change the final outcome: therefore, once an optimal policy has been determined, extra openings or closures can be adopted if needed without major changes in the final outcome. in addition, once the flexible blended model has been adopted, one can quickly and easily adapt to major changes in the external conditions, such as a sudden outbreak or a vaccine. the second part of the sensitivity analysis concerns each of the parameters of the model used to determine the optimal solution in ( ). we assume that the optimal policy has been determined, and observe deviations from the expected performance due to possible errors in the parameter determination. for each possible value of the parameters, we compute the fraction of cases in the cases of complete closure f cc , full opening f f o , and optimal solution f opt . then compare the percentage increase of optimal solution with respect to complete closure (f opt − f cc )/f cc with the analogous increment for the full opening (f f o − f cc )/f cc . such comparison is reported in table for the largest and the smallest values of (f opt − f cc )/f cc in the given range of the parameter. in addition, we indicate of each parameter the range of values for which the optimal solution achieves a substantial reduction with respect to complete opening, but limited to determining at most an % increase in the number of cases with respect to school closure. wee see that the most critical parameters are α, β and η. the range of α in which the optimization process has a change of being effective is − - − , which corresponds to about to active cases per million in the area around the school. for the infection rate β, the range in which optimization is effective is . - × − ; this is a very critical parameter, as observations in different countries lead to a range of . × − - × − . so, each region or country should monitor this rate and introduce strict measures to keep β within the above limits. finally, the fraction of undetected asymptomatic η must not exceed %: we assume availability of an easy, rapid and cheap test with a rate of false negative not exceeding %, such as crispr [ ] . the third part is about the selection of τ . in fact, the sensitivity to τ has already been discussed in section . : figure shows that the optimal strategies lie approximately on the curve c with equation ( ); even if the equation is not exact and the dependency on τ is not known analitycally, it nonetheless gives an idea of the variability of the optimal solutions as function of τ . the data plotted in figure has been obtained with τ ranging from to , which corresponds to a range between and days of in-class teaching equivalent to a % of extra cases. there is at most doubling of the cases for τ ≥ , and, correspondingly, for at least of remote teaching. this seems to indicate that next school year will allow no more than days of in-class teaching without pharmaceutical progress; recall that possibly one of two extra weeks can be gained with an efficient initial screening. the last part of the sensitivity analysis is about the weekly schedule. a possible alternative is to schedule opening or closure of the schools for each single day (still with a calendar fixed in advance). the optimization is now over possible policies. optimization by simulated annealing in this case determines a plot similar to that of figure . for instance, the optimal policy for τ = gives an increase of . % in the number of cases, vs. the . % increase of the weekly schedule; the daily schedule consists of of in-class teaching, instead of the which are considered by the weekly schedule. in summary, the daily schedule turns out to offer little advantage with respect to j o u r n a l p r e -p r o o f journal pre-proof the weekly option, but a more complicated implementation process. we have considered the issue of planning in-class activities for the school year ' -' . education is, in fact, one of the areas in which the measures to contain the current covid- pandemic have had a negative impact, with many countries and local authorities struggling to find acceptable plans for the next academic year. in this work we have focused on a single school. to aid the planning of teaching for the school, we have modeled the evolution of the covid- epidemic in the school by an extension of the sir model, with asymptomatic; an external, constant, source of infection; interactions within the school happening only during the hours of opening during week days; a reduced rate of encounter with asymptomatic due to a somewhat efficient screening system applied weekly. we have then determined realistic values for the remaining parameters of the model. next, we have set up an optimization problem, based on a preliminary assessment by the planner of the relative importance of ensuring in-class teaching vs. containing the epidemic spread. for each value of such relative importance and of the other parameters, we have carried out a discrete optimization by simulated annealing to obtain a (close to) optimal strategy within the space of all possible weekly opening policies. modeling of epidemic outbreaks by differential equations dates back to the original paper by kermack and mckendrick [ ] , with an enormous number of recent works on the current covid- outbreak. among these, [ ] discusses the impact of reopening various combinations of schools in england vs. keeping schools closed; results in [ ] are expressed in terms of the reproduction number r , and show that, in many cases, opening of all schools would bring the reproduction number above the critical value of . our model has many fewer categories and variables, and it is focused on one single school. in any case, our results also confirm that opening all schools is not advisable in uk, even with extra testing and blended models (see supplementary material). as far as optimization is concerned, methods similar to those used in this paper have appeared in many other contexts. in particular, several recent studies have considered optimal planning strategies in response to general epidemics [ , ] or, more specifically, to the covid- pandemic [ , ] ; to our knowledge, however, none has considered the current optimization problem for school planning. limitations. there are several limitations to our study. we use a variation of the sir model, in which we have averaged encounter rates and susceptibilities over several age classes and different countries to obtain reasonable parameters. it could be that local deviations from the values we have used might lead to sensibly different outcomes. the model includes no randomness, which, however, is an essential feature of both human interactions and virus spreading; randomness would also allow us to assess the variability of the predictions. as a result, there are no error bars in our predictions, except for the case of a random selection of the weeks of in-person teaching. the external infection rate is taken to be constant, as focusing on a single school does not allow us to model the evolution of the epidemic in the overall area. this rate, however, is not likely to be constant over a long period of time, but is subject to many variations due to expansion or containment of the outbreak. in addition, our results are only a first indication of a modeling methodology for the search of an optimal trade off between in-class teaching and containment of infectious transmissions. even if parameters are carefully and realistically selected, the values are based on information known at the time of this study; when a parameter has a wide range of variability we settle for one representative value; in addition, the worked out examples refer to an ideal school: for each particular concrete situation, one needs to adapt the model to the specific case. we also did not consider other alternatives, such as reducing the number of in-class students for each lesson. many possible alternatives could be conveniently incorporated in a more elaborate model. conclusions. first, we have seen in section . that the regular opening of a typical school would cause a multifold increase in the number of cases among the individuals involved with the school itself, both at days and at school year end. the only exception would be if the virus is under tight control in the region around the school, which is to say with less than active cases per million, in which case one can safely plan a complete reopening. if there are more than about active cases per million in the surrounding region, then the opening of the school would not particularly aggravate the situation; in that case it would, however, not be advisable to carry out any in person activity as the number of cases would be out of control (at least % of the school population in the first days of opening). the second conclusion, discussed in section . , is that careful planning of the opening weeks is capable of making a drastic reduction on the number of extra cases induced by opening the school. optimal or close to optimal solutions consist of blended models, with an alternation of weeks of remote and in-class j o u r n a l p r e -p r o o f activities; with about half of the weeks in class, for instance, one can reduce the number of cases by a factor of ; we have also seen that schedules with a choice between in-class and remote on a daily basis would not improve the reduction much. the likely cause of the need for the weekly alternation is the duration of covid- incubation period. the third result, in section . , is that to identify an optimal opening strategy suitable for a specific situation, planners are first required to explicitly incorporate into their objective the relative value of in-person teaching vs. reduction of covid- spread; once this is done, optimal or close to optimal solutions can be numerically estimated to produce one of the optimal or close to optimal strategies mentioned above. a random choice of the weeks of in-class activities would not reduce the number of new cases as effectively as an optimal choice, but would nonetheless achieve a substantial reduction with respect to full opening. in section we have also seen that the achieved reduction is preserved by small changes in the weeks of in-person activities. as such, one school could identify the number of in-class days or weeks, and slightly adjust the calendar to other needs. finally, in section , we have carried out a sensitivity analysis of the results. these have turned out to be rather stable for small errors in the selection of the model parameters. the more critical parameters are • the contact rate during school opening, a parameter which should be very carefully monitored and controlled via physical distancing and other measures; • the fraction of detected asymptomatic, for which schools should adopt some frequent and possibly not very expensive testing procedure; there is no need to have an exceptionally high reliability of the test itself, and we have shown viable opening policies based on reliability above %; • the external risk rate: as mentioned, one can achieve some control on the spread of the virus in the school only if there are no more than about active cases per million in surrounding area. based on the above broad indications, and pending adaptation to specific situations, we have determined for each country whether full opening is feasible, or whether a blended opening of schools for about half of the weeks, with systematic quick testing of all students and personnel, and physical distancing within the schools, would be sustainable. or whether, finally, school opening is not advisable in the current situation (see supplementary material). covid- educational disruption and response adverse consequences of school closures what will a return to school during the covid- pandemic look like? what parents need to know about school reopening in the age of coronavirus prepping to reopen, california schools desperate for guidance, money. calmatters what is a blended model of learning? by catherine lyst bbc scotland early transmission dynamics in wuhan, china, of novel coronavirus -infected pneumonia the temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study brief: covid- outbreak leads city of gttingen to shut schools through cbsnews: coronavirus flare-ups force france to re-close some schools retrieved the results are in for remote learning: it didn't work research shows students falling months behind during virus disruptions projecting the potential impacts of covid- school closures on academic achievement edworkingpapers jimmy sarakatsannis, and ellen viruleg covid- and student learning in the united states: the hurt could last a lifetime mckinsey and company, working paper mathematical and statistical estimation approaches in epidemiology modeling and analysis of an svirs epidemic model involving external sources of disease childs: an introduction to compartmental modeling for the budding infectious disease modeler saliva is more sensitive for sars-cov- detection in covid- patients than nasopharyngeal swabs. medrxiv age-dependent effects in the transmission and control of covid- epidemics s: epidemic thresholds with external agents gian-carlo rota -ordinary differential equations ordinary differential equations susceptible-infected-recovered (sir) dynamics of covid- and economic impact arxiv preprint projecting social contact matrices in countries using contact surveys and demographic data estimation of the asymptomatic ratio of novel coronavirus infections (covid- ) inferred duration of infectious period of sars-cov- : rapid scoping review and analysis of available evidence for asymptomatic and symptomatic covid- cases optimal quarantine strategies for covid- control models arxiv preprint deterministic and stochastic optimal control optimization by simulated annealing lessons from a rapid systematic review of early sars-cov- serosurveys blended learning guide to blended learning crispr/cas gene editing technology and its application to the coronavirus disease (covid- ), a review a contribution to the mathematical theory of epidemics the impact of school reopening on the spread of covid- in england optimal mitigation policies in a pandemic a simple planning problem for covid- lockdown key: cord- -nle axad authors: vlachos, j.; hertegard, e.; svaleryd, h. b. title: school closures and sars-cov- . evidence from sweden's partial school closure date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: nle axad to reduce the transmission of sars-cov- most countries closed schools, despite uncertainty if school closures are an effective containment measure. at the onset of the pandemic, swedish upper secondary schools moved to online instruction while lower secondary school remained open. this allows for a comparison of parents and teachers differently exposed to open and closed schools, but otherwise facing similar conditions. leveraging rich swedish register data, we connect all students and teachers in sweden to their families and study the impact of moving to online instruction on the incidence of sars-cov- and covid- . we find that among parents, exposure to open rather than closed schools resulted in a small increase in pcr-confirmed infections [or . ; ci . - . ]. among lower secondary teachers the infection rate doubled relative to upper secondary teachers [or . ; ci . - . ]. this spilled over to the partners of lower secondary teachers who had a higher infection rate than their upper secondary counterparts [or . ; ci . - . ]. when analyzing covid- diagnoses from healthcare visits and the incidence of severe health outcomes, results are similar for teachers but somewhat weaker for parents and teachers' partners. the results for parents indicate that keeping lower secondary schools open had minor consequences for the transmission of sars-cov- in society. the results for teachers suggest that measures to protect teachers could be considered. in the effort to contain the spread of sars-cov- most countries closed schools during the ongoing pandemic. an estimated . billion students in countries were affected by school closures in mid-april (unesco, ) . these closures are likely to have a negative impact on student learning and well-being, especially for students from disadvantaged backgrounds (dorn, hancock, sarakatsannis & viruleg, ; guessoum et al., ) . school closures also affect labor supply, not least among healthcare workers, hence reducing healthcare capacity (bayham & fenichel, ) . while the costs associated with school closures are high, modelling studies question their effectiveness in reducing the transmission of sars-cov- and direct evidence is largely missing (viner et al., ) . the absence of direct evidence is because school closures were usually implemented early, universally, and in close proximity to a raft of non-pharmaceutical interventions (npis) that have been documented and modelled to bring about large reductions in the basic reproduction number (hsiang et al., ; kraemer et al., ; pan et al., ; tian et al., ; maier & brockmann, ; auger et al., ) . this renders it difficult, if not impossible, to disentangle the effects of each specific intervention. sweden was an exception to the norm of universal school closures. on march , , one week after the first reported death from covid- , upper secondary schools moved to online instruction while schools for younger students remained open until the end of the school year in mid-june. while other npis were also implemented (public health agency of sweden, c), this partial school closure allows for a comparison of individuals and households who were differently exposed to open and closed schools, but otherwise faced similar conditions throughout the period of widespread contagion illustrated in figure . in this study, we link detailed register data from statistics sweden on the entire swedish population to all pcr (polymerase chain reaction)-identified cases of sars-cov- reported to the swedish public health agency and covid- cases requiring medical treatment reported to the national board of health and welfare between the time of school closure to the end of the school year. to study the broad impact of school closures on the transmission of the virus, we estimate differences in infection rates between parents exposed to lower and upper secondary students. we further analyze differences in infection rates between lower and upper secondary teachers as well as their household partners. for school closures to affect the transmission they must affect behavior and contact patterns. while absenteeism among lower secondary students increased during the spring, student absences were not dramatically higher that usual (see online supplement). the impact of school closures on the transmission of sars-cov- further depends on how the virus spreads between students, from students to adults, and among adults in school and at home. current reviews of the evidence suggest that while children and adolescents do get infected, they usually develop mild or no symptoms (ecdc, ) . the susceptibility to infection appears to be lower among the young, but there is some uncertainty regarding this as a large number of cases probably go undetected. children and adolescents with mild or no symptoms may still carry and spread the infection, but the evidence available indicates that infectiousness, just as the severity of symptoms, is increasing in age. outbreaks have been reported in connection to school openings and overnight summer camps (stein-zamir et al., ; szablewski, ) , but transmission within schools prior to their closure at the onset of the pandemic appears to have been limited (heavey, casey, kelly, kelly & mcdarby, ; macartney et al., ) . a general caveat concerning the available evidence is that most studies on the susceptibility and infectiousness of children and adolescents have been conducted when schools were closed and other npis were in place. differences between groups can be attributed to school closures if the groups are behaviorally and biologically similar in all other respects that affect the probability to get infected and tested. as lower secondary school (school years - , typical age - ) is compulsory and attendance to upper secondary school (school years - , typical age - ) is close to universal, the main selection concern regards the age of parents and students. parental characteristics (age, sex, income, occupation, region of origin and of residence) are controlled for, but the susceptibility and infectiousness are likely to increase in student age and general behavior may differ between younger and older students. we therefore focus our attention to parents exposed to students in the final year of lower secondary and first year of upper secondary school. the main concern regarding differences between upper and lower secondary teachers and their partners refers to partner characteristics that, as for parents, are adjusted for. given these restrictions and adjustments, the estimated differences can plausibly be attributed to the exposure . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . to open and closed schools and the study thus offers credible direct evidence on the impact of school closures on the sars-cov- pandemic. models predict that school closures can be effective if they actually reduce the number of contacts, the basic reproduction number is low (r < ), and the attack rate is higher in children than in adults (jackson, mangtani, hawker, olowokure & vynnycky, ) . the basic reproduction number for sars-cov- is above (c.-c. lai, shih, ko, tang & hsueh, ) and the attack rate in students is likely to be low relative to adults (ecdc, ). the theoretical prior is therefore that the impact of school closures on the transmission of sars-cov- among parents is low (viner et al., ) . for teachers and their partners, a more substantive impact can be expected. teachers at open schools were not only exposed to students but also to other adults, both at work and during their commute. upper secondary teachers, on the other hand, partly worked from school, but a substantive fraction did their teaching from home (see online supplement). we estimate differences in infections among parents, teachers, and teachers' partners who were differently exposed to lower (open) and upper (online) secondary schools using linear probability models (ols) and logistic regressions. descriptive statistics are shown in table . the preferred outcome is pcr-confirmed sars-cov- which has the highest incidence ( . cases/ among lower and upper secondary parents and . / among teachers). if we exclude healthcare workers who were targeted for testing, the incidence drops to . / among parents. one potential drawback of this outcome is that unbiased results rely on compared groups having equal propensity to get tested. in particular, it could be that those directly or indirectly exposed to open schools were more prone to get tested which would exaggerate the impact of school closures. the risk of such bias is alleviated by the limited testing capacity that forced testing to be targeted towards those with severe symptoms and care workers throughout most of the relevant period (see online supporting information). however, we also analyze covid- diagnoses from healthcare visits which is less likely to suffer from bias due to behavioral differences. healthcare coverage in sweden is universal and fees for doctor or hospital visits are low, assuring individuals in need of care will seek care. this is particularly true for hospitalizations since admittance to hospital is determined strictly on medical grounds. as receiving a covid- diagnosis is a less frequent event ( . / among parents; . / among teachers) these estimations have lower statistical power. low incidence is an even larger problem for severe cases (hospitalization or deaths) which has an incidence of . / among parents and . / among teachers. nevertheless, results for severe cases reported in the online supporting information are similar to those found for healthcare. the data covers the entire relevant swedish population and contains all reported cases as well as detailed . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . information on covariates (see materials and methods and the online supporting information for details). upper secondary schools moved online on march . allowing for an incubation period from infection to symptoms of about a week (lauer et al., ) , the cut-off date is set to march for teachers and april for parents and teachers' partners. the school year ends during the second week of june and the end date is therefore set to june for pcr tests and june for diagnoses through healthcare contacts. parental school exposure is defined by the school year that the youngest child in the household attends. in order to attribute estimated differences to school closures, households must be similar in all aspects that affects the likelihood of getting infected or tested, except for their exposure to open and closed schools. by narrowing the comparison to parents with the youngest child in the final year of lower secondary (year ) and first year of upper secondary school (year ), we reduce the risk of introducing biases due to confounding factors. figure shows the estimated odds ratios for pcr-confirmed sars-cov- parents from logistic regressions for where we adjust for age, sex, occupation, educational attainment, income, regions of residence and of origin (see method and materials). results for parents by school years - show there is a tendency to a positive age gradient, potentially indicating a higher parental risk of infection when exposed to older children. the most relevant comparison is therefore between school years and (reference category) for which we in table next, we analyze differences between lower and upper secondary teachers and their partners. upper secondary teachers constitute a relevant counterfactual to the work situation that lower secondary teachers had been in if their schools had moved to online instruction. the groups are also similar with respect to educational attainment and geographic dispersion. as there may still be differences in the household composition between the groups, wein addition to the controls used for parents -adjust for the occupation and educational attainment of teachers' partners, the number of children in separate age groups linked to the household, and whether or not the teacher is single. cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . fig. : sars-cov- odds ratios for parents by school year of the youngest child in the household. odds ratios estimated using logistic regression. the reference category is school year and % confidence intervals are indicated. in order to gauge the magnitude of the estimated effects for teachers, figure shows the incidence of detected sars-cov- in three-digit occupations with at least employees in ages - (healthcare workers excluded). upper secondary teachers in our sample are at the median while lower secondary teachers constitute the th most affected occupation. taxi drivers are the at the top of the distribution while driving instructors have the same level of infections as lower secondary teachers. as another comparison, table shows that the incidence of sars-cov- is similar among lower secondary teachers ( . / ) and the parents to the students that they teach ( . / , excluding healthcare workers). this is also the case for covid- diagnoses and more severe health outcomes. the higher incidence of infections among lower secondary teachers spilled over to their partners who have a higher incidence of positive pcr tests than their upper secondary counterparts [or . ; ci . - . ]. this is evidence of within household transmission from teachers to their partners. however, the estimates for covid- diagnoses for teachers' partners are not statistically distinguishable from zero [or . ; ci . - . ] and the same applies for severe cases [or . ; ci . - . ]. the relatively imprecise estimates for these outcomes . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . note: ***p < . , **p < . , *p < . . results are estimated using linear probability models (ols) in panel a, and logistic regressions (logit) in panel b. ci are shown in brackets. standard errors in parenthesis are clustered at the school level for teachers and their partners, and at the household level for parents. "open" indicates exposure to (open) lower secondary schools. "m. dep" refers to mean dependent variable for the reference category exposed to (online) upper secondary schools. the outcome "pcr" refers to positive pcr tests and "diag." refers to covid- diagnoses. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . also renders them statistically indistinguishable from the estimates for pcr-confirmed sars-cov- . we check the robustness of the main results in several ways. first, media searches reveal that some lower secondary schools closed spontaneously and preemptively, albeit usually for a brief period in time (see the online supplement). as privately run independent schools were over-represented in this group, we exclude individuals connected to such schools. this results in marginally larger estimates for parents and teachers' partners, and marginally smaller estimates for teachers. second, schools were allowed to let small groups of students return to school to complete practical assignments and such practices may have been more common among vocational students. we therefore exclude parents connected to students in vocational upper secondary programs. this makes the groups exposed to open and closed schools substantially less comparable and it leads to a reduction in the point estimate (from . to . cases/ ) and an increase in the standard error. third, rather than controlling for employment in the healthcare sectors, we drop teacher households where the partner is a healthcare employee. as expected, the results remain unchanged. fourth, we derive a slightly different measure of parental exposure to lower secondary schools and use a more restrictive population of parents but the results remain unchanged. these results can be found in tables s and s in the online supplement. how school closures affect the transmission of the virus depend on how they reduce contact between those potentially infected. this may differ depending on contextual factors and we analyze two types of heterogeneity. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . first, we allow the estimates for exposure to lower secondary schools to differ by population density in the district of residence. second, we let them vary by the regional rate of infections prior to school closure. the results presented in table s in the online supplement reveal interaction terms with large standard errors not allowing a clear interpretation. on march , , upper secondary schools in sweden moved to online instruction while lower secondary schools continued instruction as normal. this partial school closure provides a rare opportunity to study the impact on the transmission of sars-cov- during a period of widespread contagion. the impact of school closures on the transmission of the virus in society is best captured by the results for parents. we find that parental exposure to open rather than closed schools is associated with a somewhat higher rate of pcr-confirmed sars-cov- infections the positive association for pcr-confirmed cases could partly reflect other behavioral differences between households with slightly younger and older children, but if treated as a causal the estimates indicate that a hypothetical closure of lower secondary schools in sweden would have resulted in fewer detected cases among the parents in our sample. this can be related to detected cases in this group until mid-june ( cases when excluding healthcare workers). due to the sample restrictions made, the actual number of parents exposed to lower secondary schools is higher and if we extrapolate to a total of exposed parents, the estimates suggest fewer detected cases. this can be related to the country total of detected cases until mid-june ( excluding healthcare workers). the relatively low impact of school closures on the transmission of the virus cannot be attributed to particularly harsh precautionary measures within the schools that remained open. rather, the measures recommended (public health agency of sweden, b) are best described as mild in an international comparison (guthrie et al., ) . in particular, there were no imposed class size reductions and no recommendations concerning the use of face masks. face masks are also rarely used in sweden (yougov, ). are at the median, the lower secondary teachers in our sample constitute the th most affected group. other occupations with high infection rates (e.g. taxi drivers, driving instructors, social assistants, therapists, police . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . according to our estimates, fewer cases of sars-cov- had been detected among lower secondary teachers and their partners if lower secondary schools had closed. to this we can add the estimated fewer cases among parents. most transmission is within households so even if fewer detected cases is a lower bound, this can be seen as relatively low compared to the country total. based on an age-specific case fatality rate (cfr) of . % (see table s in the online supplement), this corresponds to approximately fewer deaths, . among parents and . among teachers and their partners. this counterfactual inference regarding mortality is highly uncertain, however, and in our sample we count a total of covid- related deaths at the lower secondary level ( parents, teacher, partner). the corresponding number at the upper secondary level is (all parents). as for severe health outcomes, we find such cases (including one death) among the lower secondary teachers in our sample. according to our estimates, this number had been down to if lower secondary schools had been closed. school closure is a costly measure with potential long-run detrimental effects for students. the results presented are broadly in line with theoretical work indicating that closing the schools is not a particularly effective way to contain sars-cov- (viner et al., ), at least not when facing as high a level of contagion as sweden did during the spring of . regardless of the limited overall impact on the virus transmission, keeping lower secondary schools open had a quite substantial impact on teachers. this should be taken into account and precautionary measures could be considered. it should, however, be noted that the incidence of sars-cov- and covid- among lower secondary teachers is on par with the incidence among the parents of the students they teach. finally, it should be mentioned that we do not study the impact of school closures on students. because of age related differences in access to testing (see online supplement), the severity of symptoms, risk behavior and patterns of socialization, results for students based on pcr-testing are likely to be biased and difficult to interpret. the rate of severe cases among children and adolescents is also too low to render a statistical analysis meaningful; such cases are recorded among the . million students in compulsory school age ( - ) and among the youths in ages - that comprise the bulk of upper secondary students. it can also noted there were zero covid- related deaths in age groups - recorded in sweden until late july. there might be other health implications among students, but analyzing these are beyond the scope of this study. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint we construct estimation samples for parents, teachers and their partners using registers held by statistics sweden. table reports descriptive statistics for parents, teachers and teachers' partners, starting with the incidence of positive pcr tests of sars-cov- as of june . since healthcare workers were prioritized for testing, we also present the incidence among parents and teachers' partners excluding those working in healthcare separately. we use ordinary least squares (ols) and logistic regressions to empirically analyze if sars-cov- infection . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint can be attributed to being exposed to open or closed schools. we estimate the following ols regression model for the three populations: parents, teachers and teachers' partners: the outcome y i is an indicator variable for a positive sars-cov- pcr test or being diagnosed with covid- by a doctor in outpatient care or at a hospital. y i , prior is an indicator for sars-cov- /covid- before the cut-off date (march for teachers, april for parents and partners). open is an indicator variable taking the value ( ) if individual i is exposed to (open) lower secondary schools. parents with the youngest child in lower secondary school is defined as exposed and parents with the youngest child in upper secondary school is defined as unexposed. lower secondary teachers and their partners are defined as exposed and their upper secondary counterparts as unexposed. x is vector of individual and household characteristics. when estimating the model for teachers the vector includes: indicators for age categories, sex, indicators for categories of educational attainment, indicators of categories of partners' occupation, region indicators of country of origin for those not born in sweden, log of household income, indicators of municipality of residence and household exposure to the number of children in age groups - , - , - , and + who reside in the same region as the teacher. the municipality fixed effects are exchanged for region fixed effects when estimating the logistic model. the equivalent vector of variables is used for teachers' partners, with the exception of own occupation instead of partner occupation. the vector of controls for parents include a similar set of variables as for teachers: age categories, sex, municipality of residence, educational attainment, occupational categories (own and partners'), region of origin for those not born in sweden, the log of disposable family income and indicators for missing data on any of these variables. standard errors are clustered at the school level when estimating the model for teachers and teachers' partners and at the household level when studying parents. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . jonas vlachos, edvin hertegård and helena svaleryd s . context enhanced cleaning of heavily exposed areas and keyboards/tablets. compared to school opening policies in other countries, the precautionary measures in sweden are best described as mild (guthrie et al., ) . in particular, there were no imposed reductions of class size and no recommendations concerning the use of face masks. on march , upper secondary schools and institutions of higher education moved to online instruction. upper secondary schools thus closed for normal instructions just as the number of deaths and icu admissions began to increase (see figure in the main text). although upper secondary school moved to online teaching, some teachers were still teaching online from the school premises. according to a survey conducted by a large teachers' union during the last week of april and first week of may, percent taught from the school, percent partly from home, and percent only from home (national union of teachers, ). as expected, compulsory school teachers mainly taught from school; merely percent of the teachers in compulsory schools had been partly teaching online from home and percent had only been teaching from home. there have also been media reports of substantial student absenteeism in compulsory schools. again there are no official reports but according to the same survey, percent of compulsory students were absent on a typical day. in a survey of compulsory schools conducted by the national board of education during late april, schools reported that absenteeism among compulsory school students was about normal, that there was an increase in absenteeism of between and percent, and stated an increase of more than percent (swedish national agency for education, b). based on these surveys, we draw the conclusion that student absenteeism at the lower secondary level was not dramatically high, even though there are local exceptions. the sample of parents is constructed as follows. we define household adults who are exposed to their own children (biological or adopted) or a new partner's children from a previous relationship as parents. for separated parents, . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . we use the household identifier in lisa to identify any current partner. this enables us to identify new couples who are either married or have common children. households consisting of unmarried cohabitant couples without common children cannot be identified and will be categorized as single households. the study population consists of parents who have children in school years - in the household, or biological children in these school years living in the same region. because parents are less likely to interact regularly with children living at a distance, only children residing in the same region are considered in the analysis. there are regions in sweden and they are thus relatively large geographical areas. there were recommendations against leaving the region of residence during most of the spring . we sort parents by the age of the youngest child connected to the parents in the household or through biological links. parents are considered exposed to lower secondary schools if their youngest child is enrolled in school years - . unexposed parents are defined by their youngest child being enrolled in upper secondary school. in the analysis we will focus on parents with the youngest child in school years and since they are likely to be the most similar in all other aspects, except for parents with their youngest child in the th school year being exposed to an open school. this analysis population consists of adults. the teacher sample consists of teachers working in lower and upper secondary schools according to the teacher register. teachers with children born in are excluded as they are likely to be on parental leave during the spring of . we also exclude those recorded as being on leave of absence during the fall of . the final sample consists of teachers. for the sample of teachers' partners, we connect partners to teachers using the household identifier from lisa. this enables us to identify partners who are either married to or have common children with the teacher. the resulting sample consists of partners. our main outcome variable is positive pcr test reported to the public health agency but we also analyse the incidence of covid- diagnoses from healthcare visits and severe cases of covid- (hospitalizations and deaths) reported to the national board of health and welfare. the first case of sars-cov- in sweden was reported on january, , and the disease was classified as a danger to public health and to society on the following day (public health agency of sweden, a). among other things, this classification means that all documented cases of active infection have to be reported to the public health agency. testing capacity was slow to expand and from march (week ), testing was directed towards healthcare employees and individuals with symptoms of covid- in need of healthcare. as can be seen in fig. s , testing increased substantially from early june (week ). healthcare is the responsibility of sweden's healthcare regions as is testing for sars-cov- . thus, there are regional differences in testing capacity as well as rules and recommendation regarding testing. some regions have recommended not to test children under (for example västra götaland and uppsala) and some have not had any age restrictions (for example skåne). the number of detected cases does therefore not well reflect the actual rate of infections and the rate of positive tests remained high throughout june (week ). by . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . june , a total of pcr tests had been performed ( per inhabitants) (public health agency of sweden, d). as discussed in the main text, a concern is that the propensity to get tested for sars-cov- is affected by being connected to open and closed schools, regardless of health status. this is less of a concern for covid- diagnoses made by the healthcare sector, especially severe cases which requires hospital care or cause death. as a robustness test we report results for severe cases defined as admittance to hospital or death due to covid- . the estimates presented in table s are similar to those found for healthcare diagnoses that are presented in the main text. despite school closures not being imposed by the government, some lower secondary schools spontaneously moved to online instruction. thus, some lower secondary school may be classified as having on-site instruction when they in fact conducted the teaching online. no official records on such closures exist but based on searches in media archives, they were rare and usually short-lived. there are approximately lower secondary schools in sweden and we find media reported closures. out of these, were reactions to cases among students or staff while were proactive. privately managed independent schools ( percent of all lower secondary schools) are vastly over-represented in reports on such proactive closures ( cases). see details on media searches in the next section. to explore this we re-estimate the ols model excluding individuals with their youngest child exposed to independent lower secondary schools. we find that this only marginally affects the results for teachers and their partners (table s ) but increases the estimates somewhat for parents (table s ) . we investigate if the estimated effects on teachers and teacher's partners are due to lower secondary teachers being more exposed to the healthcare sector through the teacher's partners occupation. this should be adjusted with the indicators for occupational categories included in the model. however, to further explore whether this is a concern we exclude teachers whose partners work in healthcare (occupational codes , , and ) and re-estimate the model. this change does not have any significant effect on the estimates (table s ). at the upper secondary level, schools were allowed to let small groups of students complete practical elements of education and assignments, provided that schools deemed that this could be done safely (swedish national agency for education, a)). this mainly concerned students at vocational programs since practical skills taught at vocational programs are difficult to teach online. students at vocational programs were also generally allowed to continue apprenticeships, provided that this was practically feasible. we investigate whether this affects our results by excluding parents with the youngest child exposed to upper secondary vocational programs. the estimates reported in table s show a small decrease in the estimates relative to the baseline. finally, we estimate the main model using an alternative measure of exposure to lower secondary school for . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . parents. instead of defining a parent as exposed if they have the youngest child in school year and not exposed if they have the youngest child in school year , we define a parent as exposed if they have a child in the household, or a biological or adopted child residing in the same region, in lower secondary school. we remove families with children not in secondary school. since we want to compare parents with lower secondary children to parents with children of a similar age we control for having a child in school year and . thus, the estimate presented in the last column of table s shows the effect of being exposed to a child in lower secondary school compared to being exposed to a child in upper secondary school year . the effect is of similar magnitude as in the main specification. the expected impact of school closures on virus transmission depends mainly on the magnitude of contact reduction. two factors that may be of importance for the effect is population density and how widely spread the virus was prior to schools closing. a study of us districts show that transmission of sars-cov- increases with population density (korevaar et al., ) . this suggests that when people live close, each person interact with more people and hence are more likely to transmit the virus. the consequences for the effectiveness of school closures is, however, unclear. on the one hand, school closures may have a larger impact in densely populated areas if isolation lead to larger reductions of the number of contacts. on the other hand, if a larger share of the interactions take place outside of school in densely populated areas, the expected effect should be smaller. to investigate this matter we implement a heterogeneity analysis by district population density, categorizing districts with a population density above the th percentile as high density districts. timing of the interventions has been shown to be important for the effectiveness of npis (s. . this suggests a larger impact of closing school when the spread within society is low. therefore we investigate whether the impact of school closures depends on the spread of the virus prior to schools were closed. regions with above the populated weighted median spread of cases per are categorized as high spread regions, i.e. the regions (cases per in parenthesis): stockholm ( ), uppsala ( ),Östergötland ( ), skåne ( ), sörmland ( ) and jönköping ( ). the econometric model is modified by adding interaction terms between indicators for high population density, respective high initial contagion, and exposure to lower secondary school as well as interactions with all control variables except for the municipality indicators. the results from the analysis is reported in table s . as the estimated standard errors are large, it is not possible to draw any clear conclusions. the interaction effect with high population density is negative or close to zero in all models but the estimates are not statistically significant. the interaction with initial level of contagion is also not statistically significant in any model. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint in order to get information on spontaneous closures of lower secondary schools, media searches were conducted using the service mediearkivet/retriver and on sveriges radio's site (public service radio with substantial local presence). search terms were permutations of "school closure" (skolstängning/skola stängd), "distance education" (distansundervisning), "online education" (onlineundervisning), "corona" and "covid". results for individual schools were followed by web searches to find more information on each particular case. spontaneous closures were recorded as proactive if they did not occur as a result of cases detected at the school and reactive otherwise. provided that information is available, a closure is labelled as brief if the duration was less than a week. in total, reports on closures were found ( among privately managed independent schools). of these were proactive ( among independent schools) while were reactive ( among independent schools). spontaneous closures thus appear to have been rare and independent schools are vastly over-represented among those that closed proactively. two of the reactive closures were on advice from the local disease protection officer and they both occurred late in the school year (june and ). information on the duration was usually not available, but of the reports from which the duration can be judged, were brief. several of the closures were also partial, meaning that school days were cut short, rolling schedules introduced, or that instruction partially moved online. details on each specific report are available from the authors. to extrapolate the expected effect of school closure on the number of deaths in sweden we derive the case fatality ratio (cfr) for different age groups. cfr is calculated by dividing the number of deaths with the number of cases and hence crucially depend on the testing regime. table s shows the incidence of detected sars-cov- in different age groups until june , , and the number of deaths among these cases reported until july . the numbers are shown both including and excluding healthcare workers, for which testing was more accessible. the cfr increases with age, except for the higher value for the youngest age group due to one dead child. this child was younger than one years old and thus not directly exposed to schools. the average age among teachers is , their partners and parents years old. based on the cfr distribution in table s we calculate the expected effect on mortality among lower secondary parents using a cfr of . percent. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . note: ***p < . , **p < . , *p < . . standard errors in parenthesis clustered the at the school level. "open school" is defined as having the youngest child in school year relative to school year . the results are estimated using linear probability models (ols). note: ***p < . , **p < . , *p < . . standard errors in parenthesis clustered at the household level. "open school" is defined as having the youngest child in school year relative to school year . "open school * " is an indicator for living in a household with a child in lower secondary school (see online supplement for details on sample restriction). in this specification we control for having a child in year or . the results are estimated using linear probability models (ols). note: ***p < . , **p < . , *p < . . standard errors in parenthesis are clustered at the school level for teachers and their partners, at the household level for parents. "open school" is defined as having the youngest child in school year relative to school year . the results are estimated using linear probability models (ols). . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . association between statewide school closure and covid- incidence and mortality in the us impact of school closures for covid- on the us health-care workforce and net mortality: a modelling study covid- and student learning in the united states: the hurt could last a lifetime covid- in children and the role of school settings in covid- transmission adolescent psychiatric disorders during the covid- pandemic and lockdown. psychiatry research, . re summary of school re-opening models and implementation approaches during the covid university of washington, dept of global health no evidence of secondary transmission of covid- from children attending school in ireland the effect of large-scale anti-contagion policies on the covid- pandemic. nature, - the effects of school closures on influenza outbreaks and pandemics: systematic review of simulation studies quantifying the impact of us state non-pharmaceutical interventions on covid- transmission the effect of human mobility and control measures on the covid- epidemic in china severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges effect of non-pharmaceutical interventions to contain covid- in china the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application transmission of sars-cov- in australian educational settings: a prospective cohort study. the lancet child & adolescent health effective containment explains subexponential growth in recent confirmed covid- cases in china coronapandemin och undervisningens genomförande association of public health interventions with the epidemiology of the covid- outbreak in wuhan, china standard errors in parenthesis clustered the at the household level for parents and school level for teachers and partners. severe cases include covid-diagnoses registered at hospital or at death. the effects are estimated using linear probability models (ols) and logistic regressions key: cord- -foucabzy authors: skedsmo, guri title: assessment and evaluation with clarifying purposes for policy and practice date: - - journal: educ assess eval account doi: . /s - - -x sha: doc_id: cord_uid: foucabzy nan in the first article, ysenbaert, van houtte and van avermaet report on their case study research exploring schools' assessment policies and teachers' assessment practices in six schools in the flemish education context in belgium. across the cases, several tensions are identified, such as finding the balance between responding to the challenges of diversity in the classroom and aligning the assessments with the requirements of centrally set attainment targets. the authors find a need for schools to use their autonomy to develop assessment policies on the school level that are adapted to the needs of diverse classrooms. moreover, these assessment policies need to be developed along with a schoolwide teaching and learning vision that also embraces diversity among students. in the second article, hofflinger and hippel explore test-based accountability in chile. in particular, they focus on school responses in terms of an inflation practice wherein low-performing students are made to miss high-stakes tests to prevent the school from being categorised as 'inadequate' and avoid the negative consequences of such a categorisation, such as reduced enrolment, funding and autonomy. the authors find that schools are somewhat successful in this effort to evade accountability. between and % of schools that would have been classified as 'inadequate' if all students took the test were instead classified as 'intermediate' or better. furthermore, more pressure is placed on schools serving disadvantaged students, given that the same thresholds are used for all schools regardless of the socioeconomic characteristics of the student body. the authors suggest adjusting accountability targets for these schools as a possible means of addressing this issue. in the third article, tuytens, devos and vanblaere review empirical qualitative and quantitative studies on teacher evaluation. while the quantitative studies focus more on the effects of teacher evaluation on student outcomes, the authors observe that qualitative studies more often consider teacher outcomes, such as behaviour, ability and motivation. based on the review, they identify various core elements and purposes embedded in evaluation practices which they argue to be part of a value chain. here, they expand a previous framework and present a more comprehensive model that aligns intended and perceived purposes, individual and organisational outcomes, and individual and organisational resources and context variables. the paper contributes to the existing knowledge based on teacher evaluation by providing insights on how such routines and practices can be embedded in human resource management (hrm). in the fourth article, geiger, amrein-beardsley and holloway use cases on teacher lawsuits that occurred throughout the usa as a point of departure for their analysis. the cases were presented in education week in and pertain to the use of student test scores to evaluate teachers. the researchers then purposefully selected the most high profile and controversial of the cases to better understand the measurement and pragmatic issues at play across the cases. the authors find that vam scores were particularly problematic with respect to reliability and validity. in addition, they found evidence of possible teacher-and school-level bias, bias across the various measures (e.g. observations and pp measures) used and lack of transparency with respect to vam measures. the selected case occurred in new mexico, where consequences were attached to teachers' vam scores, including the flagging of teachers' professional files if determined to be not 'value-adding'. this 'ineffective' classification ultimately prevented the teachers from moving teaching positions within the state. another issue was the teacher termination policies attached to new mexico teachers' vam scores. in the fifth article, huber and helm present and discuss the school barometer, a fast survey that was conducted in germany, austria and switzerland during the early weeks of the school lockdown to assess and evaluate the current school situation caused by covid- . the aim of the survey was to gather, analyse and present data in an exploratory way to inform policy and practice as a basis for reflection and decision-making in a time of crisis as well as for further research. the article presents some exemplary first findings and possible implications for policy, practice and research. the authors reflect on the strengths and limitations of such surveys as well as methodological options for data collection and analysis when used as a short monitoring survey approach. specifically, they discuss the methodological challenges related to hypothesis testing, the testing of causal effects and approaches for ensuring reliability and validity. the school barometer represents an example of how assessment and evaluation can contribute in a current crisis while also being part of future long-term crisis management when linked to further research. in the first two articles in this issue, the authors demonstrate and discuss ways by which assessment and evaluation form a basis for and can be integrated with learning and development. in the first article, ysenbaert et al. argue for the need for school policies, and incentives for schools to develop such policies, to develop integrated models of assessment which align assessment with teaching while providing flexibility for addressing individual needs. in the third article, tuytens et al. present a more comprehensive model of hrm that aligns the intended and perceived purposes of teacher evaluation, individual and organisational outcomes, and individual and organisational resources and context variables. two other articles demonstrate assessment practices where pressure and consequences, in terms of labelling of schools and school actors if the measured performance is below the threshold, do not foster learning. rather, they lead to unfairness and create distrust in assessments (they may not provide accurate information) and the authorities in charge of the systems. in the fifth article, huber and helm present an evaluation approach which makes it possible to provide information quickly to inform decision-making in times of crisis. some of these articles present approaches where assessment and evaluation have what popkewitz refers to as a 'policy clarification purpose ' ( , p. ) . he emphasises that policies and reform programmes aim to respond to perceived issues and problems that are not clearly defined and do not have linear outcomes. assessments and evaluations do not tell us what policy is most efficient or useful, but they can help illuminate the tensions, contradictions and ambiguities that underlie policies and educational practices. '[…] of the deepest value to the public debates around which schooling in a democracy is positioned (and of importance not only to policy makers) is an understanding of the strains and tensions found in the relations in school arenas […]' (popkewitz, , p. ) . following this, assessment and evaluation can potentially contribute to an increased understanding which can inform policy and improve practice. looking through the rearview mirror the swedish schoolhouse: a case study in transnational influences in education at the s world's fairs. peadagica historica some problems and problematics in the production of evaluation reformulating reform. curriculum history revisited. thesis for the doctor philos degree key: cord- -urpjr dz authors: combe, laurie g. title: school nurses: living the framework during covid- date: - - journal: nasn sch nurse doi: . / x sha: doc_id: cord_uid: urpjr dz nan as i write this letter to you, it is late april and we are in the midst of an unprecedented event. while it has been just months since we first learned of covid- (coronavirus disease ), i feel like i have been taking this walk with you for a much longer time. over the course of these months we have seen travel bans, physical distancing, rapid acceleration of case counts into the millions both worldwide and in the united states (see figure ), deaths in the hundreds of thousands (world health organization [who] , a), and brave healthcare providers on the frontline, often without the protection they need (american nurses association, ). forty-three states have ordered school closures, affecting . million students (education week, ) . the nasn conference is "going virtual" in order to keep attendees and conference production staff safe and healthy. the pace of change in the covid- response has been nothing short of frenetic (national broadcasting company news, ; who, b) . i regularly monitor the nasn all member forum school nurse net community and notice that nasn member katie morton ( ) sounded the first warning of the novel coronavirus on january seeking guidance about travel from china into her community (see figure ). since that time, all the school nurse net communities have logged almost covid- -related posts and the conversation quickly turned to school reentry planning. by tracking the expressed needs of school nurses surrounding covid- , the nasn staff has worked tirelessly to build coronavirus disease resources, including return to school guidance (nasn, a). while schools were still open, early discussions in the school nurse community focused on the need for school nurses to access the same level of personal protective equipment (ppe) as that recommended for other healthcare providers (centers for disease control and prevention [cdc], a [cdc], , b . school nurses discussed the evolving evidence surrounding covid- , considering how we could safely care for and isolate presumptive cases, manage aerosolizing procedures for ill or potentially asymptomatic students, and still provide safe care for the well population in our schools. with schools closed, we are learning new ways to engage students in maintaining their health, while practicing within the constructs of ethical, legal, and professional nursing standards. school systems are relying on the expertise of school nurses to provide factual information about covid- ; using their knowledge to develop plans that keep staff, students, and families healthy. as nurses always do, we are looking ahead, anticipating the needs of students and school communities for that time when schools reopen again. school nurses are keeping pace with the evolving evidence, understanding that forecast model assumptions are based on yet to be determined factors such as the long-term viability of this novel coronavirus, policy decisions of political leaders, and citizen behavior (michaud et al., ) . school nurses know that many children face disparities in healthcare access, nutrition, safe housing, transportation, and more. cindy begley's staff is making telephone calls to check on students with known health conditions, making sure that families have the resources they need to maintain health. collaboration with school counselors provides cindy's staff with a resource list to meet needs that families are expressing. school nurse amy ponce is making sure that distribution of instructional materials is done in manner that protects the health of students, families, and staff ( figure ). because their schools will be closed for the remainder of this school year, angela pesche and kindra schutt are staffing a medication pickup to return supplies to families, making sure they have enough medication at home (figure ). paulette abbey works with her school food service partners to ensure that students dependent on school nutrition continue to have their needs met (figure ). schools nurses are experts at adapting our nursing practice to the school environment. as schools flipped to remote learning we hopped on board. school nurses are dropping by teacher's remote classrooms to connect with students and collaborate with teachers to identify students at risk for chronic absenteeism. jenna palmisano ( ) produced a health education video, we are nurses!, addressing student fears about nurses and other healthcare providers in ppe (see figure ). mary coldwell and craig matthes are encouraging their students through social media, letting them know they are many of our colleagues have answered the call to serve the larger community, working on the front lines. andrea ferguson is working in the emergency room of her local hospital ( figure ). barbara maher and anthony torres are staffing a drive through covid- test site ( figure ). some school nurses, employed by hospital systems or health departments, are redeployed to other practice areas, necessitating selfevaluation of competence for the assignment and collaboration with employers to establish safe patient care environments. eileen gavin, in collaboration with her health department, is training fellow school nurses to conduct contact tracing, a natural fit for school nurse public health practitioners ( figure ). while working with one new mom, eileen was able to arrange delivery of infant formula, saving the mother and baby a risky trip into the community. this new paradigm of working from home presents possibilities and challenges. there can be time to research unique student health conditions, collaborate with pediatricians and other healthcare providers, and update individualized healthcare plans without the interruptions so common in a school health office. perhaps you have found space in your day to carefully analyze your health office data, preparing a presentation for your administrator about your work both before and during school closing. because many schools are relying heavily on school nurse expertise to help them navigate this crisis, more than a few school nurses administrators have expressed that they are working longer hours than ever to meet the needs of their school community. we have learned that managing work and family responsibilities within the same space and time can be stressful. you may have children at home who are used to a schedule, while your need to balance work and childcare make a schedule difficult to achieve. are you like me, juggling multiple curbside grocery pick-up services, trying to stock up on the essentials and favored items on a schedule that keeps your diet healthy and your family happy? you may be trying to safely care for older parents, or an immunocompromised family member and are fearful for their health when you must venture out into the community. perhaps you have made the decision to brave the frontline of this pandemic and isolate yourself from family because you are uncertain of your covid- status. others of you may be experiencing job loss and uncertain futures. a seat at the table: paving the way in speaking with imo jean douglas (personal conversation, april , ) about these opportunities and challenges, she shares that this pandemic has garnered her a seat at the executive team table. yet, imo jean is clear that it was the work she and her staff did prior to this crisis that paved the way. she looked to health data and the stories of students to demonstrate the necessity of school nurses to budget conscious administrators. she alerted her administrators early and often that covid- was coming and that they must be prepared. she continues to compile data and stories from this season of pandemic to advocate that she retain her seat at the table. nasn is paving the way for students access to school nurses. in late april, i sent a letter to president trump (nasn b), alerting him that it is imperative that students have access to a school nurse when schools reopen. this letter outlines for the president the essential role school nurses have in addressing the ongoing impact of the covid- pandemic and informing plans to reopen the nation's schools. we are asking that the white house fund , school nurses to conduct infectious disease surveillance, identification, and intervention for student physical and mental health concerns, health screenings, school-located vaccinations, and immunization compliance efforts. we know that keeping students in school will have tremendous implications for the economy, workforce, and families. our call for stakeholders to sign on to a petition to the president has garnered almost , signatures in days. you can help pave the way by contributing to nasn's ability to fund school health and school nurse research, research that can better define the value we bring to student health and learning. once again i want to ask that you support the doubledown for data president's endowment fund challenge ( figure ) i wonder what the course of covid- will be by the time you read this message. i wonder how our lives and the lives of students and families will be changed. will the expectation of increased mental health concerns in the school population be a reality? are schools and the larger community equipped to meet these increased needs? the promise of these events is that we are learning news ways to communicate with students and families-ways that i think will be good to continue beyond covid- . nasn and its affiliate organizations are certainly learning new ways to conference, communicate, advocate, and tap into the expertise of members. i want to encourage you to focus on self-care, remembering that you must be well to bring others to wellness. you have made me proud to be a school nurse alongside you and i look forward to the remainder of our journey. remember that we are #inittogether. with gratitude, ■ laurie figure . doubledown for data framework for st century school nursing practice: framing professional development what , nurses want you to know about treating covid- guidance for schools and child care programs before and during and outbreak strategies to optimize the supply of ppe and equipment map: coronavirus and school closures-update using the framework for st century school nursing practice in daily practice covid- models: can they tell us what we want to know? kaiser family foundation global health policy national association of school nurses school nurse net all member forum framework for st century school nursing practice™ shaping your practice and changing mindsets: framework for st century school nursing practice™ nasn calls on the president to include school nurses in proposals to re-open the economy and schools we are nurses! a new framework for school nurse self-reflection and evaluation covid- ) dashboard who timeline-covid- key: cord- -ehp q ry authors: haber, michael j.; shay, davis k.; davis, xiaohong m.; patel, rajan; jin, xiaoping; weintraub, eric; orenstein, evan; thompson, william w. title: effectiveness of interventions to reduce contact rates during a simulated influenza pandemic date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: ehp q ry measures to decrease contact between persons during an influenza pandemic have been included in pandemic response plans. we used stochastic simulation models to explore the effects of school closings, voluntary confinements of ill persons and their household contacts, and reductions in contacts among long-term care facility (ltcf) residents on pandemic-related illness and deaths. our findings suggest that school closings would not have a substantial effect on pandemic-related outcomes in the absence of measures to reduce out-of-school contacts. however, if persons with influenzalike symptoms and their household contacts were encouraged to stay home, then rates of illness and death might be reduced by ≈ %. by preventing ill ltcf residents from making contact with other residents, illness and deaths in this vulnerable population might be reduced by ≈ %. restricting the activities of infected persons early in a pandemic could decrease negative health impact. measures to decrease contact between persons during an influenza pandemic have been included in pandemic response plans. we used stochastic simulation models to explore the effects of school closings, voluntary confinements of ill persons and their household contacts, and reductions in contacts among long-term care facility (ltcf) residents on pandemic-related illness and deaths. our findings suggest that school closings would not have a substantial effect on pandemic-related outcomes in the absence of measures to reduce out-of-school contacts. however, if persons with influenzalike symptoms and their household contacts were encouraged to stay home, then rates of illness and death might be reduced by ≈ %. by preventing ill ltcf residents from making contact with other residents, illness and deaths in this vulnerable population might be reduced by ≈ %. restricting the activities of infected persons early in a pandemic could decrease the pandemic's health effects. t hree influenza pandemics have occurred during the th century (in , , and ) , and another pandemic is inevitable ( ) . the requirements for a pandemic virus include the existence of a new influenza a hemagglutinin for which there is little immunity, the ability of this strain to infect humans efficiently, and person-toperson transmission. such viruses are likely to arise in densely populated agricultural communities where contact between humans and birds or pigs are close and persistent ( ) . in , a highly pathogenic avian influenza a (h n ) virus was transmitted from live poultry to humans in hong kong special administrative region, people's republic of china, killing of infected persons ( ) . from december through june , , the world health organization confirmed human cases and deaths associated with influenza a (h n ) infections in humans ( ) , and in october , influenza a (h n ) infections among birds were identified for the first time in europe. currently circulating influenza a (h n ) viruses appear to infrequently infect humans, and person-to-person transmission, if it occurs, is certainly not efficient. however, international health officials are concerned that, as human exposure to such viruses increases, so does the possibility that a pandemic virus might appear. the next influenza pandemic in the united states could result in , to , deaths, , to , hospitalizations, and to million outpatient visits, with a direct economic effect between us $ and $ billion, according to set of estimates ( ). others have described the possible effects of vaccine and antiviral interventions. one study estimated that vaccinating % of the population would be necessary to achieve optimal cost benefits, assuming that development and mass production of a vaccine would require - months after the pandemic virus was characterized ( ). longini et al. ( ) estimated the effectiveness of rapid targeted antiviral prophylaxis of persons early in a pandemic by using epidemic stochastic simulations. they found that if the next pandemic virus had a similar virulence to that of the - pandemic virus, then delivering prophylaxis to % of exposed persons for up to weeks could reduce attack rates by %- % and death rates by . - . / , persons. however, such a strategy would require a stockpile of . billion doses of antiviral agents, which exceeds the current production capacity for these drugs for at least the next years. in the absence of adequate supplies of vaccines and antiviral agents, at least during the first wave of an influen-effectiveness of interventions to reduce contact rates during a simulated influenza pandemic za pandemic, public health officials should consider using interventions designed to reduce the number of contacts between infected or exposed persons and susceptible persons. the us department of health and human services influenza pandemic plan discusses several possible containment strategies, including those directed to single persons or entire communities ( ) . we used new stochastic simulation models to estimate the effects of several interventions of this kind. these models represented the spread of a pandemic in an urban us community, allowing for contacts in different settings (or mixing groups), including households, daycare centers, schools, workplaces and long-term care facilities (ltcfs). by using the age distribution of the us population ( ), we placed each person in the community in a stratum, defined by age group and (if > years of age) by residence in the community or in an ltcf. person-to-person transmission probabilities depended on the daily duration of contacts. contact rates and their duration varied by each person's stratum and mixing groups. by using these models to simulate an influenza pandemic, we estimated the effects of school closings, home confinement of ill persons (i.e., isolation) or their household contacts (i.e., quarantine), and reduction of contacts among residents of ltcfs on overall illness attack rates, hospitalization rates, and mortality rates. we simulated an influenza outbreak in a small urban us community. the simulation model used data from the asian influenza a (h n ) pandemic in - ( ) and from studies on us influenza-related excess rates of hospitalizations and death ( ) ( ) ( ) . the simulation process begins with the generation of a community of households, where the distributions of sizes of the households and ages of the household members follow the us census. every person in the community belongs to of age-dependent strata: preschool children (ages birth- years), schoolchildren (ages - years), adults (ages - years), seniors (ages > years) living at home, and seniors (ages > years) living in an ltcf. in addition, each person belongs to > mixing groups, according to his or her stratum: households, daycare centers, schools, workplaces, ltcfs, and the community. the mixing matrix is presented as table . on any given day, a susceptible person, a, makes contacts with other persons that may lead him or her to become infected. these contacts take place in each of a's mixing groups. the probability that person a becomes infected depends on the following input parameters: ) the number of different persons with whom person a has contact in each mixing group, ) the total duration, in minutes, of all the contacts with each of these persons, and ) the per-minute rates of infection transmission if the contacted person is infectious. the number and duration of contacts may be different on weekdays and weekend days. the values of the parameters that were used in this study are presented in the online supplemental materials appendix (available at http://www.cdc.gov/eid/content/ / / . htm). once person a becomes infected, he or she undergoes a latent period, followed by a period in which he or she is infectious. the mean length of the latent and infectious periods are input parameters. this model has new features that are not shared by the commonly used simulation models (such as the model in [ ] ) for transmission of influenza: ) the probability of transmission depends on the total duration of all contacts between persons, rather than on the number of times they make a contact, ) the transmission parameters do not depend on the population size, and ) different contact parameters can be specified for weekdays and weekend days. technical details of the simulation model are presented in the supplemental materials appendix. the basic reproductive number (r ) for this model is . . this value is within the range ( . - . ) estimated by mills et al. ( ) for the influenza pandemic. the interventions we examined in this simulation study were school closings, confinement of ill persons and their household contacts to their homes, and reduction in contact rates among residents of ltcfs. interventions were implemented at the start of the outbreak. when this intervention was implemented, schools closed when the prevalence of illness among children in the school exceeded a predetermined threshold, set to %, %, or % in the simulations. a school remained closed for a predetermined period ( , , or days). on weekdays, household and community contact parameters of children whose school was closed were assigned their weekend levels; their contacts with other children who continued to attend school and with working adults did not change. when this intervention was implemented, a given fraction of households were assumed to comply. if a household complied, then all of its members followed the confinement rules unless they had been previously ill and had recovered. we considered types of confinement: ill persons only, and ill persons and all the members of the same household. confinement began after a given number of days of illness ( , , or days) and did not depend on the severity of illness. if symptoms were severe, then the person reduced his or her duration of contacts with other household members by %. when a person was confined on a weekday (because of his or her illness or illness of another household member) and did not withdraw due to severe symptoms, then the duration of contacts with household members who continued to go to school or work did not change. durations of contacts with household members who stayed at home and were not withdrawn were the same as on a weekend day. when ill persons were confined, they returned to school or work day after their illness ended. when ill persons and other household members were confined, a person returned to school or to work day after his or her illness ended (even if other ill persons remained in the household). a person who did not become ill returned to school or work on the third day after the last day of illness of any household member (because the length of the latent/incubation period was assumed to be days). we examined the effects of interventions on ltcf residents: reduction in duration of contacts with other residents who were ill, and reduction in duration of contacts with visiting family members. contacts with ltcf staff did not change. we first ran a set of simulations using the baseline settings for all the parameters, without any interventions (online supplemental materials appendix). the average rates for the outcomes of interest-overall illness rate, hospitalization rate, and death rate-were calculated for simulations and used as baseline rates. for each intervention, we ran a set of simulations and used the aver-ages of these simulations as estimates of the expected rates under this intervention. the effectiveness of each intervention was defined as follows: effectiveness = [(baseline rate) -(rate with intervention)]/baseline rate we performed a sensitivity analysis to assess the robustness of our findings regarding the effectiveness of the modeled interventions. in common with all simulation studies, our findings depended on several parameters for which we have estimated values that we believe are reasonable starting points. these values included baseline contact rates, the probability of illness given infection, the relative infectiousness of an infected person without influenza symptoms, the probability of withdrawal to home because of severe symptoms, and the reduction in contact rates due to severe symptoms. we varied the values of these parameters and examined the effects of these changes on estimates of the effectiveness of school closings and confining ill persons to their homes. based on the simulations conducted with the baseline values of the pandemic parameters, the baseline rate of illness was . %, ( % confidence interval [ci] . %- . %), the baseline rate of hospitalization was . / , ( % ci . - . ) and the baseline rate of death was . / , ( % ci . - . ). these results were based on the assumption that the illness rates would be similar to their values in the influenza pandemic. two parameters affected the effectiveness of school closings: the percentage of ill schoolchildren required to close a school and the number of days the school remained closed. the effectiveness of the intervention varied as a function of the percentage of ill persons required for closing a school and the duration of the closure (figure ). for example, if each school were closed for days when the proportion of ill children exceeded %, then the overall illness rate was . ( % ci . - . ). the baseline illness rate was . ; therefore, the effectiveness of this intervention was ( . - . )/ . = . ( % ci . - . ). as expected, effectiveness usually decreased as the percentage of ill children required to close a school increased. the effect of the length of closure was less clear (figure ). when schools were closed, transmission in households and in the community increased; thus, school closings could increase death and illness rates in some groups. for example, when the illness rate required for school closing was %, then closing schools for days had the largest effect on hospitalization rates, compared with closings of or days. however, when the rate for closing was %, then closing schools for days had a smaller effect on hospitalization rates than closing for or days. in our models, confinement to home took place after a person showed symptoms of influenza. a delay of , , or days occurred between onset of symptoms (which coincided with the onset of infection) and the beginning of the confinement period. this delay and the proportion of households that complied with the confinement rules affected the effectiveness of the intervention. figure presents the effectiveness of these interventions as a function of the percentage of households that comply (between zero and %) for a delay of days. as expected, effectiveness usually increased with the compliance percentage. confining the ill persons and their household members was more effective than confining the ill persons only. for example, given a delay of days and % compliance, the effectiveness of these interventions on illness rates was . for confining the ill only and . for confining ill persons and their household members. effectiveness decreased when the length of the delay was increased. reducing contacts with ill residents of ltcfs decreased the rates of illness, hospitalization, and death for ltcf residents by > % (table ). reducing contacts also decreased the rates of hospitalization and death in the general population by up to % and %, respectively. figure presents the dynamics of the pandemic (a) without any intervention, (b) when schools are closed for days as the proportion of ill children exceed %, and (c) when ill persons and all their household contacts are confined after the second day of illness of the index case-patient and compliance is %. we see that these interventions do not affect the time to the peak of the pandemic (around week ). the rate of decline following the peak does not change under confinement to home, while it slightly decreases under school closing. the value of the basic reproductive number (r ) for the baseline setting of our parameters is . . because this value is higher than values used in recent simulation studies ( , ) , we evaluated the effectiveness of the interventions under smaller values of r . we found that reducing r resulted in an increase in the effectiveness of confinement to home and a decrease in the effectiveness of school closings. thus, our findings regarding the effectiveness of confinement and the lack of effectiveness of school closings remain valid for smaller values of r . the results of additional sensitivity analyses were as follows. emerging infectious diseases • www.cdc.gov/eid • vol. the most important parameters related to the effectiveness of school closings are those that underlie the contacts between children while they are in school. in our simulations we assumed that on a school day each child makes contact with other schoolchildren, each contact lasting minutes (see section d. .a in the online supplemental materials appendix). some of these contacts may be concurrent. to examine the effect of changing each child's exposure to other schoolchildren on the effectiveness of school closures, we increased and decreased the baseline duration of minutes by %. table shows the effectiveness of closing schools for days for the baseline values of duration of school contact. as we see, longer or shorter durations of contact while schools are open do not result in substantial changes in the effectiveness of school closings. we varied the values of several parameters in the baseline model and examined the effects these changes had on estimates of the effectiveness of confinement of ill persons to their homes (table ). we assumed that % of ill persons without severe symptoms were confined to home within days of symptom onset. when the fraction of infected persons who developed symptoms was increased from . to . , then the illness rate without an intervention (i.e., at the baseline level) changed only from . to . , while implementation of the intervention changed this rate from . to . . thus, the effectiveness of this intervention increased from . to . . the alternative values we used in table modeled a more severe pandemic than the pandemic modeled with the baseline initial values. the continuing epizootic of influenza a (h n ) among birds in asia and europe has raised concerns that the likelihood of an influenza pandemic may be increasing. shortages in the supply of neuraminidase inhibitors, the antiviral agents most likely to be effective against a pandemic influenza strain, and the months needed from the isolation of a pandemic strain until the availability of vaccine suggest that reducing contact rates between infected and uninfected persons will represent one of the few sets of interventions that can be rapidly implemented. we used a stochastic simulation model to estimate the effectiveness of several interventions that could reduce contact rates on pandemic-related outcomes. the use of individual-level (e.g., isolation and quarantine) and community-level (e.g., school closings) containment measures. our study considered possible interventions of both kinds, including early identification and confinement of case-patients and their household contacts, limiting visits to ltcfs, and closing of schools. our findings suggest that closing schools would result in relatively small reductions in morbidity and mortality rates during a pandemic. for example, when schools were closed when > % of children had influenza symptoms and remained closed for days, the rates of illness, hospitalization, and death decreased from the baseline rates of . %, / , , and / , to . %, / , , and / , , respectively. thus, the effectiveness of school closings was ≈ %- %. when we increased the threshold of illness incidence required for school closing to %, then these rates were . %, / , , and / , , respectively. these mild decreases in the rates of illness and death after school closures are explained by the fact that in our models, children whose schools were closed were more likely to increase their contacts with other groups. the attack rate of % that we used for school-age children may be considered high. however, if the attack rate were reduced, school closings would have an even smaller effect. our results do not contradict recent findings that vaccination of schoolchildren could be effective in controlling transmission during a seasonal influenza epidemic ( ) . vaccination of children reduces their chances of infection and of transmitting infection to household and community contacts, whereas closing schools may not decrease the likelihood of infection substantially and could increase the probability that an infected child will infect household and community contacts ( ) . the effect of school closings on overall illness rates in an influenza pandemic has been estimated in other recent simulation studies. germann et al. ( ) modeled the effect of a pandemic on the entire us population. they found that for r > . , closing of schools without any additional interventions had limited effectiveness. on the other hand, for r < . , school closings reduced the extent of illness. carrat et al. ( ) , by using a simulation model for the spread of influenza in a community, found school closings to be effective. we believe that these inconsistencies in the reported effects of school closings depend on the details of the various simulation models, especially on the way the community is affected by school closing in terms of increased contact rates of schoolchildren when their school is closed. our simulations predict that it might be possible to decrease illness and death rates by as much as % by reducing the contact rates of all ill persons. however, achieving this level of effectiveness would require persuading % of those with symptoms to withdraw to their homes and confine themselves. simulation studies by longini et al. ( ) and ferguson et al. ( ) found that quarantine, when used in conjunction with vaccines and antiviral agents, would be effective in containing an influenza pandemic in southeast asia. one should remember that the effectiveness of any behavioral/social intervention may vary across cultures. residents of ltcfs are likely to be at high risk for serious pandemic-related illness and death. we found that by limiting contacts of ill residents, illness and death may be reduced among other residents. these are notable findings, as this vulnerable population responds poorly to seasonal influenza vaccination, and they are unlikely to receive the limited quantities of pandemic vaccine when it first becomes available. the effectiveness of any particular intervention designed to reduce contact rates depends on the initial values selected for the parameters affecting influenza transmission (e.g., contact durations, probability of withdrawal due to severe symptoms), and a limitation of our study is that few data exist on which to base these values. studies designed to obtain reliable estimates of these parameters during seasonal, interpandemic influenza outbreaks should be a high priority. however, the major findings of this study seem to be robust, given a range of realistic values for the parameters we used. the target attack rates we used to calibrate the contact parameters (provided in the supplemental materials appendix) are high, but lowering these attack rates should not have a major effect on our findings, because both the pre-and postintervention incidence rates would decrease concomitantly. we did not make formal estimates of the economic costs and benefits of the interventions we examined. however, some likely consequences of school closings may be considered, given current childcare practices. obviously, the longer the duration of school closure, the more costly the consequences as working parents either have to take time off work to supervise children or pay for somebody else to care for them. if a large number of school days are lost, school districts might consider extending the school year, which would incur additional costs, although the conditions would be expected to vary greatly between school districts. these increased costs would have to be weighed against the limited predicted effectiveness of this intervention. encouraging the voluntary withdrawal of ill persons appears to be a more effective strategy than school closings in reducing the impact of a pandemic, and it may represent a relatively inexpensive intervention. however, researchers have found that us workers routinely miss < day of work after reporting onset of influenzalike illness ( ) . encouraging longer durations of work loss could decrease compliance with self-isolation and increase the economic cost per case avoided. home quarantine of the immediate family members of an ill person would likely increase the costs per case averted. for example, during the quarantine efforts related to the severe acute respiratory syndrome outbreak in toronto ( ) , many families found it too expensive to rigidly comply with a household-level quarantine of ≥ days. our stochastic simulation model has several strengths. the model considers the length of time persons are in contact, in addition to the total number of contacts. the model parameters we used are not related to the size of the simulated population, unlike previous models ( ) . we repeated the simulations conducted for this study with a population twice as large as the original population and the same input parameters. the resulting rates were almost unchanged, so the differences can be attributed to the random effects associated with these simulations. the weaknesses of our present model are that it requires many input parameters and that it does not include the effects of antiviral medications. our model allows for estimating vaccine effects for susceptibility and infectiousness; however, this option was not used in the present study. on during a severe pandemic, including isolation of persons with confirmed or probable influenza, voluntary home quarantine of members of households with confirmed cases, dismissal of students from schools and school-based activities, and closure of childcare programs. during a pandemic with a severity index of or (defined as a case fatality rate of > %), this new guidance recommends not only school dismissals of ≤ weeks but also measures to protect children from being exposed or exposing others to the pandemic virus via reduction of their out-of-school social contacts and community mixing. in this article, we assessed the effectiveness of school closures of - weeks duration after school absenteeism rates reached high levels. we assumed that children dismissed from schools would increase their out-of-school contacts. these assumptions reduced the effectiveness of school closures in our model. in future work, we will explore the effectiveness of early dismissal of students from schools, together with changes in out-of-school contacts, and other interventions using our model. in summary, if persons who suspect they are infected with pandemic influenza virus were to withdraw to their homes quickly, the rates of illness and death associated with a pandemic may be substantially reduced. the withdrawal of all household contacts may further reduce rates of illness and death, but this additional intervention is likely to be relatively costly and difficult to implement. restricting the movement of ill ltcf residents will be beneficial in reducing their adverse health outcomes. before early and rapid implementation of such interventions during a pandemic is feasible, the public will need to be educated about the early symptoms of influenza and measures developed to increase the social acceptability of self-isolation when ill. influenza pandemic preparedness plan for the united states influenza pandemic planning casecontrol study of risk factors for avian influenza a (h n ) disease, hong kong confirmed human cases of avian influenza a (h n ) the economic impact of pandemic influenza in the united states containing pandemic influenza with antiviral agents united states department of health and human services pandemic influenza plan profiles of general demographic characteristics for the united states mortality associated with influenza and respiratory syncytial virus in the united states influenza-associated hospitalizations in the united states estimation of influenza-associated deaths and hospitalizations in the united states transmissibility of pandemic influenza containing pandemic influenza at the source strategies for containing an emerging influenza pandemic in southeast asia strategy for the distribution of influenza vaccine to high-risk groups and children mitigation strategies for pandemic influenza in the united states a 'small-world-like' model for comparing interventions aimed at preventing and controlling influenza pandemics effectiveness and cost-benefit of influenza vaccination of healthy working adults: a randomized controlled trial factors influencing compliance with quarantine in toronto during the sars outbreak interim pre-pandemic planning guidance: community strategy for pandemic influenza mitigation in the united states-early, targeted, layered use of nonpharmaceutical intervention we thank martin meltzer for his thoughts on the potential economic consequences associated with the interventions we modeled and keiji fukuda for his comments on early versions of the manuscript. m.j.h. was partially supported by contract ipa from the centers for disease control and prevention.dr haber is a professor of biostatistics at the rollins school of public health, emory university, atlanta. his research focuses on statistical models and methods for infectious disease epidemiology. key: cord- -h atwd authors: diotaiuti, pierluigi; mancone, stefania; bellizzi, fernando; valente, giuseppe title: the principal at risk: stress and organizing mindfulness in the school context date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: h atwd background: in recent years the role of school principals is becoming increasingly complex and responsible. methods: this study was voluntarily attended by italian school principals who were administered the psychological stress measurement (msp), mindfulness organizing scale (mos), polychronic-monochronic tendency scale (pmts), and the scale of emotions at work (sew). results: the study has produced a path analysis model in which the relationships between the main predictors of principals’ work discomfort were explained. the effect of depressive anxiety on perceived discomfort (ß = . ) found a protective mediator in the mindfulness component that recognizes the sharing as a fundamental operational tool (ß = − . ), while an increasing sense of effort and confusion could significantly amplify the experience of psychological discomfort associated with the exercise of school leadership (ß = . ). conclusions: the model developed in this study suggests that focusing on organizing mindfulness can be a valuable guideline for interventions. research on work-related stress has long identified the school as one of the risk environments, and various studies and publications deal with the stress and burnout of school workers. most of the work produced devotes attention to teachers, and there are few reflections on the psychophysical resistance of the principal, especially considering all the legislative and organizational changes that have taken place [ , ] . these continuous changes, necessary in a rapidly changing society (well described by the "liquid society" metaphor coined by zygmunt bauman) become a cause of work-related stress for all school staff, especially principals. the ongoing reform of the education system in italy involves various professionals and individuals who have a role in the complex world of school: principal (dg-dirigente scolastico in italian); director of general and administrative services (dsga-it. direttore dei servizi generali e amministrativi), all administrative, technical and auxiliary staff (ata-it. personale amministrativo, tecnico e ausiliario), teachers, students and their parents (or parental figures). each of these players must adapt to regulatory changes, both individually and collectively, with time dictated by political choices, which can be both national and supranational; the educational system, through the schools operating on the territory and the people who effectively act and put into practice the regulatory frameworks decided by the government institutions, must follow and pursue the changes in a short time period, often far from the actual physiological timing that the institution can implement. the principal is an intermediate figure and a link between the institutional organizational framework, represented by the state, the regions, and the municipalities, and the framework of school individuals, represented by the teaching staff, ata staff, parents and students. many of the skills and int. j. environ. res. public health , , of competences required of professional staff, but also students, are not part of the training acquired over time and especially put into practice and enriched by personal experimentation and experience, so that even people with a ten-year work experience are forced to become new students learning subjects and skills hitherto untested, and therefore they are unaware of how gifted, capable and effective they are in resolving tasks [ , ] . the role of principal requires new skills and competences to meet the requirements and objectives defined by the proliferation of school reforms, regulations, and cultural changes [ , , ] . extensive research in various countries has already focused on principal burnout [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as sari [ ] notes, there may be curiosity and desire to learn and experience new skills and abilities, but the stress related to possible administrative errors and criminal consequences of errors or omissions, or low performance in failing to achieve results, remains high, with emotional drain and possible burnout consequences. moreover, implicitly, the principal may also experience the feeling that his own cultural and cognitive skills, as well as tools and resources, have become obsolete or are no longer suitable for current needs, thus increasing personal insecurity [ ] . these elements are sources of elevated stress levels in principals, because they become the point of reference and the catalyst of the stress of all professional and non-professional figures. not only must the principal solve his/her own problems, but he/she must also deal with those of his/her subordinates, in most cases without having adequate leadership experience and training. some countries have experienced a lack of candidates for the role of principal or an increase in resignations, because the teachers know they are not actually capable of fulfilling all the requirements the role calls for [ ] [ ] [ ] . the principal experiences a drastic reduction in his/her educational and pedagogical function and must acquire technical, administrative, cognitive and what we could call management engineering skills: as a principal he/she becomes responsible for the safety of staff and students and a manager of economic resources, with budgetary obligations. to carry out these tasks, he/she must have medical-legal and managerial skills which the teacher who is promoted to the role of principal does not possess. moreover, the change in commitments, as well as responsibilities, can be a source of great stress and a health risk, since the individual might not be able to manage, but above all to identify, the point where he must ask for help and activate a course of treatment. this applies both at the individual level and as the head of the safety and health of subordinate staff, having to decide when it is appropriate to subject a member of staff to a medical examination, especially when the latter does not want to or cannot deal with personal problems. the principal is not alone and employs ata and dsga personnel, but as the person responsible for their work, he/she still must have the ability to evaluate and correct any errors. the above concerns only the legal-administrative part and the school is viewed as a business that must be run efficiently [ ] . in addition to this, however, there is the management of the team in the areas of human resources and coordination. principals can contribute to shaping the school climate as they should promote and support students and teachers [ ] . this involves managing aspects related to students, social change and the management of a multi-ethnic society in continuous evolution, with changes in customs and processes [ , ] . for example, the new migratory flows have led some educational institutions to deal with the task of managing adult "students", independent adults and adults with a "different" culture, who are normally obliged to attend school: however, this type of "student" requires cultural and relational skills and management skills that the school staff may not have. the aim of this research is to draw attention to the figure of the principal as a person at risk of burnout and work-related stress for both exploratory, diagnostic and preventive purposes. the principal has to focus on the limit between being engaged in school life and avoiding workaholism, in order to live in a state of health and well-being [ ] . chronic stress stimulates negative behaviors and thoughts and problems related to emotions, feelings and physical health that can hinder effective school administration. according to scholars as langer [ ] , davidson et al. [ ] , vogus and sutcliffe [ ] , weick and putnam [ ] , and weick and sutcliffe [ ] , many positive benefits can be associated with individual and organizing mindfulness, such as health improvement, stress reduction, increased of creativity, and less risk of burnout. more specifically hoy et al. [ ] define "school mindfulness" as the extent to which teachers and administrators in a school carefully and regularly look for problems, prevent problems from becoming crises, are reluctant to oversimplify events, focus on teaching and learning, are resilient to problems, and defer to expertise. therefore, we also hypothesized in this work that components of the organizing mindfulness could have a significant role for the school principal in increasing or limiting his/her perceived work discomfort. ( ) monitor the stress levels of a large sample of principals belonging to different levels of italian schools; ( ) verify the incidence of specific pathologies associated with high levels of school stress; ( ) evaluate the relationships between the perceived stress, work discomfort and dimensions of organizational mindfulness; ( ) test the fit of a general path model illustrating the influence of the predictors on principals' work discomfort; and ( ) identify the role of organizing mindfulness on principal's perceived work discomfort. the study was voluntarily attended by school principals ( males and females) with an average age of . (sd = . ). they were invited to participate by means of an email indicating the purpose of the study, declaring the guarantee of anonymity and the use of the data collected for scientific purposes only, and therefore were requested to fill in a questionnaire online. approximately contact e-mails were sent, extracting the addresses from a special national list. the sample size determination was made by setting a -alpha confidence level at %, therefore with z normal value at the confidence level of . . the following formula was applied: x o = z (p × q)/b , with p as the proportion to be estimated and q the proportion of complementary character and b the desired precision set at %. hence: . ( . × . )/ . = . the response rate recorded was : , compatible with the fixed sample size ( > ). the average completion time was about min. tools administration took place upon the release and signing of the form for an informed consent of participation in accordance with the declaration of helsinki. the study was approved by the institutional review board of the university of cassino and southern lazio. in order to collect the data necessary to carry out the study, a questionnaire was built up and articulated into the following sections: ( ) socio-demographic information: gender; age; type of degree obtained; ( ) school environment: (a) how long (in years) have you been a school principal? (b) how many students altogether attend the school(s) you currently manage? (c) how long (in years) have you been a principal at the current location? (d) type of school(s) related to the management (primary/secondary school, technical school, high school, etc.); ( ) information on the current health of the subjects: body mass index, high/low blood pressure, possible presence of diabetes, cholesterol, heart disease, respiratory problems (e.g., asthma, bronchitis), migraines, stomach problems, back, neck or joint pain; ( ) psychometric measurements: (a) the test m.s.p. (psychological stress measurement) [ , ] . the test measures the state of subjectively perceived stress, and consists of items with likert response scale - (from not at all to very much) on the individual's perception of his cognitive-affective, physiological, and behavioral state. the overall test score provides a global index of the psychological stress state. in addition to the overall score, it is possible to calculate six other values that correspond to six different articulations of the way one perceives himself as being stressed: loss of control and irritability (i.e., "i am irritable, my nerves are on edge, i lose patience with people and things."); psychophysiological sensations (i.e., "i feel tense or strained."); sense of effort and confusion (i.e., "i feel overwhelmed, overpowered and overloaded."); depressive anxiety (mixed depression and anxiety symptoms: i.e., "i review the same ideas several times, i brood, i have the same thoughts over and over again, i feel my head full of thoughts."); pain and physical problems (i.e., "i have physical pains: back pain, headache, neck pain, bellyache."); hyperactivity and acceleration (i.e., "i walk quickly"); (b) mindfulness organizing scale (mos) [ , ] . this is a self-report measure that investigates the safety of the organization, or rather how the worker perceives that safety. it is based on concrete behavior that reflects the employee's relationship with the organization and his colleagues. the measurement is carried out on a three-point likert scale (from not at all to very much). the dimensions included in the items are: concern about failure, reluctance to simplify interpretations, sensitivity to operations, commitment to resilience, and deference to expertise. the s , s , and s scales were used for this study. a total of items make up these three scales. the s scale measures the degree of application of group decision-making and comparison with colleagues (awareness of the value of shared problem analysis: i.e., "when a crisis occurs, we rapidly pool our collective expertise to attempt to resolve it"), the s scale is designed to assess the organization's reluctance to simplify in the face of critical issues (awareness of the value of a non-rigid climate: i.e., "when discussing emerging problems with co-workers, we usually discuss what to look out for"), the s scale is intended to assess the level of organizational awareness (awareness of the value of mutual knowledge: i.e., "we discuss our unique skills with each other so that we know who has relevant specialized skills and knowledge"); (c) scheda per la rilevazione funzionale delle aziende (functional survey module for companies) [ ] . for this study, within the module, the scale of emotions at work was used; it consists of items with true/false answers to assess the emotions that prevail during work. the direction of the scale is oriented to specifically assess the work discomfort perceived by the person; (d) polychronic-monochronic tendency scale (pmts) [ ] . this measures the subject's ability to perform several tasks during the same time interval (i.e., "i feel at ease when i do several activities at once"). the measurement is carried out on a five-point likert scale (from strongly disagree to strongly agree) including five items. the data were processed using the statistical software spss version (ibm corporation, armonk, ny, usa) and amos ibm version (ibm corporation, armonk, ny, usa). the main analyses performed were: descriptive statistics to illustrate socio-demographic information; pearson and spearman bivariate and partialized correlations for all main measures (psychological stress, organizing mindfulness, work discomfort, polychronic-monochronic tendency) significant at p < . and at p < . , -tailed); kendal's point-biserial correlations between msp, work discomfort and reported physical ailments; cronbach's alpha as scale reliability coefficient; t-test to explore significance in stress score and polychronic tendency relating to gender; anova univariate test with p < . to explore significances between work discomfort, stress and organizing mindfulness; hierarchical regression to identify the predictors of work discomfort and stress; cohen's d and eta squared as measures of effect size; sem analysis to explore predictors' effects on work discomfort. to test the adequacy of the model the following eight indices were considered: ( ) the chi-square; ( ) the relationship between the value of the chi-square and the degrees of freedom; ( ) gfi (goodness of fit index); ( ) agfi (adjusted goodness of fit index); ( ) rmsea (root-mean-square error of approximation); ( ) rmsr (root mean square residual); ( ) cfi (comparative fit index); ( ) nfi (normed fit index); ( ) rfi (relative fit index); ( ) pnfi (parsimony adjustment to nfi); ( ) pcfi (parsimony adjustment to cfi); ( ) pclose (testing the null hypothesis that the population rmsea is no greater than . ). the main characteristics of the sample are illustrated in table below, while table presents the bivariate correlations between the measures used in the study. the corresponding dataset is available as supplementary material s . it can be observed in table that stress resulted inversely correlated to age, strongly correlated to work discomfort and inversely correlated to organizational awareness. among the components of stress most associated with the perception of work discomfort were depressive anxiety ( . **), the sense of effort and confusion ( . **), and irritability ( . **). as the number of years of service increases, organizational awareness also improves ( **), while stress (− . **) and the perception of work discomfort (− . **) decrease; at the same time, awareness of the value of mutual knowledge increases ( . **) and the person's level of hyperactivity decreases (− . **). the level of discomfort and perceived stress was not associated with the number of students in the administered institution. as seniority increased, there was also an increase in the number of students and therefore in the size of the school administered ( . **). partialized correlations with the perception of work discomfort, showed a decrease in the association between the variables (determined by the control variables), which however remained statistically significant with depressive anxiety ( . **), the sense of effort and confusion ( . **), awareness of sharing analysis ( . **), but no longer with irritability ( . ), awareness of no-stiffness ( . ), awareness of mutual knowledge ( . ). partialized correlations with the general measure of stress (msp) showed a reduction of associations with age variables (− . **) and work discomfort ( . **), and non-significance with awareness of sharing analysis ( . ), awareness of no-stiffness ( . ), awareness of mutual knowledge (− . ). table shows the distribution of stress levels among the sample, after the transformation of the raw score into t points and the relative comparison with the italian percentile calibration values of the scale. it can be noted that . % had high levels of stress and almost % of the principals were affected by moderate and high levels of stress. figure reports the distribution of stress scores compared to the type of school managed. it can be observed that the regency of the comprehensive schools with multiple age levels ( - ) was accompanied by the highest level of stress for the principals, although the univariate anova and tukey's post-hoc comparisons did not show significant differences between this level of stress and those associated with the other four types of school (p = . ). figure reports the distribution of stress scores compared to the type of school managed. it can be observed that the regency of the comprehensive schools with multiple age levels ( - ) was accompanied by the highest level of stress for the principals, although the univariate anova and tukey's post-hoc comparisons did not show significant differences between this level of stress and those associated with the other four types of school (p = . ). univariate anova reported, as illustrated in table , a significant inverse association between organizational mindfulness and stress scores; in addition, there was a significant association of the two variables (stress and organizational mindfulness) with the measure of perceived work discomfort. univariate anova reported, as illustrated in table , a significant inverse association between organizational mindfulness and stress scores; in addition, there was a significant association of the two variables (stress and organizational mindfulness) with the measure of perceived work discomfort. table reports point-biserial correlations between msp, work discomfort and the physical ailments the principals claimed to have. it can be noted that the disorders most associated with stress and the perception of work discomfort are: migraine, stomach problems, back and/or cervical pain. stomach problems are sometimes also associated with respiratory problems (e.g., asthma, bronchitis). in order to identify among the components of stress and mindfulness the predictors influencing work discomfort perceived by principals, a hierarchical regression analysis was carried out. hierarchical multiple regressions were run to determine if the addition of the stress and mindfulness components improved the prediction of work discomfort. the preliminary verifications of the regression assumptions excluded the presence of multivariate outliers. mardia's multivariate kurtosis index ( . ) was in fact below the critical value [p (p + ) = ]; therefore, the relationship between the variables can be considered substantially linear. low co-linearity was indicated by the low vif values (variance inflation factor) < and high tolerance values > . . for verification of the assumptions on the residuals, the average between the standardized and raw residuals was equal to ; the durbin-watson test had a value of . and was therefore indicative of the absence of autocorrelation. influential predictors have been identified in depressive anxiety the model is displayed in figure , where it is shown that work discomfort was mainly affected by depressive anxiety (standardized estimate of the regression weight of . for p < . ). the second influential predictor turned out to be the sense of effort and confusion (standardized estimate of the regression weight of . for p < . ), which in turn receives a major influence precisely from depressive anxiety (standardized estimate of the regression weight of − . for p < . ). the model has identified the awareness of the value of shared problem analysis as a significant negative predictor of work discomfort (standardized estimates of the regression weights − . for p < . ), furthermore this was negatively affected by depressive anxiety (standardized estimate of the regression weight of . for p < . ). subsequently, a sem analysis was performed, combining into one explanatory model the variables that previously revealed significant association with work discomfort. the model showed overall good fit measurements: χ = . df = p = . ; cmin/df = . ; rmr = . ; gfi = . ; agfi = . . baseline comparisons nfi = . ; ifi = . ; cfi = . . parsimony-adjusted measures pnfi = . ; pcfi = . ; rmsea: . ; pclose: . ; rmsea % . - . . the model is displayed in figure , where it is shown that work discomfort was mainly affected by depressive anxiety (standardized estimate of the regression weight of . for p < . ). the second influential predictor turned out to be the sense of effort and confusion (standardized estimate of the regression weight of . for p < . ), which in turn receives a major influence precisely from depressive anxiety (standardized estimate of the regression weight of − . for p < . ). the model has identified the awareness of the value of shared problem analysis as a significant negative predictor of work discomfort (standardized estimates of the regression weights − . for p < . ), furthermore this was negatively affected by depressive anxiety (standardized estimate of the regression weight of . for p < . ). table below summarizes the maximum likelihood estimates and regression weights estimates. the corresponding sem with amos is available as supplementary material s . the state of chronic stress leads to an inability to manage events, both in the sense of not being able to solve the conditions of difficulty that arise and in the sense of inability to prevent them and even of unconscious tendency to intensify and proliferate obstacles and stressful events [ ] . numerous studies have demonstrated that chronic stress also directly produces innumerable illness conditions, influenced by the inability to manage and improve one's own health [ ] . studies on work-related stress are increasing, as are publications on the difficult condition of school workers [ , ] . while the focus is on teachers, there are few reflections on the psychophysical fitness of the school manager, especially in light of the latest legislative and organizational changes. school leaders are also exposed to health risks related to work-related stress, but there are different aspects of the problem with respect to teachers and employees in general [ , ] . this involves an institutional figure thrown into the continuous proliferation of reforms, legal norms, structural changes, conflict management, and radical changes in customs and processes [ ] . we are substantially dealing with a figure who is at risk and to whom more research attention should be paid for exploratory, diagnostic and preventive purposes. our study first of all showed that about half of the sample of principals who participated in the research had moderate to high stress levels. this immediately emphasizes how current and critical the problem is. the widespread tendency to merge different school cycles (elementary and junior high schools) in the so-called "istituti comprensivi" (comprehensive schools, ages - ), for administrative reasons, was associated with a greater load of tension and pressure for principals, who probably find it difficult to manage with a single approach the problems and differences that arise from educational orientations and professional profiles traditionally characterized by a plurality of visions, different approaches to teaching and evaluation of students, diversity in the level of involvement of families in school life, different propensity and habit of teachers of different cycles (primary and secondary) to work on shared projects. it would seem that the management of these differences, rather than the size of the school (in terms of number of students, and therefore of teachers), is a reason for greater pressure and tension at work [ , ] . women principals showed significantly higher levels of stress than their male colleagues. in this regard, according to the data, their greater tendency to engage simultaneously in the resolution of several tasks, could indicate a greater resistance to delegation, a strong sense of personal responsibility that would lead them to a total (psychological) involvement which over time can overload the person, limiting the time of physical and mental recovery. in line with these data, studies by kiral [ ] have shown that women principals have higher levels of stress than male principals, as women have to reconcile the responsibility for domestic work with the official and public work they do in school. the first analyses of our study have indicated that the general perception of the principal's working discomfort presents on the one hand an association with the level of stress, which contributes to increase the value of the discomfort, and on the other hand an equally significant association with organizing mindfulness, which can substantially limit the negative effects of stress on perceived discomfort. this measure of discomfort includes de-motivational aspects, negative mood, disappointment for the unreliability of the context, negative balance between efforts and results, weight in conducting mediations, pessimistic view of the future, perception of others' insensitivity, doubts about one's self-efficacy. an interesting reflection that emerged from the observation of the data was that relating to the age and period of service of the principals. both the stress level and the perceived general discomfort had an inverse correlation with age and years of service. this suggests that experience can play a significant role in the development of the management and coordination skills required to best perform the complex functions of school leadership. the critical aspect concerns the italian situation where in the last decade there has been a substantial turnover of principals. therefore, only a few years ago, a large number of principals started their service role, in a context of extensive administrative changes imposed by the ministry of education. it is not difficult to hypothesize that in this situation the youngest principal may feel the weight and responsibility of an assignment that no longer finds a frame of reference in past experience. the results of the study confirmed the association between high levels of stress and somatization, which was already evident in the literature on the health implications of a chronic occupational stress condition: migraine, stomach problems, back and/or cervical pains, respiratory problems (e.g., asthma, bronchitis). these data were in line with mariammal et al. [ ] who stated that stress manifests itself in the form of chronic disorders or diseases such as hypertension, stroke, headache, and diabetes, as well as regular physical pain. some principals experience symptoms such as suppression of the reproductive system, anxiety, aggression, indigestion, stomach-ache, pain, dizziness, and rapid heartbeat. in addition, chronic stress creates muscle tension, fatigue, constipation, and arthritis [ ] . principal stress has even been associated with severe problems such as ischemia and heart problems [ ] . further results of the study have identified the predictors of work-related discomfort with greater accuracy in the components of stress and organizational mindfulness. through a regression analysis and then through sem, the effects and influence relationships between the predictors were identified. among the components of the msp, it seemed that depressive anxiety had the main role of influence. the anxiety component is characterized by aspects of recurrent ruminative cognition that amplify the sense of isolation, incomprehension, discouragement, and worry [ ] . this negative interpretative framework activates another significant component: the sense of effort and confusion perceived by the person, who develops thoughts of inadequacy, the impression that everything involves a considerable effort and that everything falls on his shoulders. this attitude can naturally encourage a lack of clarity in ideas and decreased attention and concentration. within the model, one of the three components of mindfulness, the awareness of the importance of sharing problem analysis, found a significant place. the effect of the variable limited the dimension of the perceived discomfort. if, on the one hand, the increase in pressure and tension drives the person to intensify their efforts by closing and defensively stiffening themselves, on the other hand, the ability to recognize the value of sharing and involvement of other collaborators and colleagues in order to deepen the understanding of the problems and the identification of the most appropriate management methods, can help one to come out of isolation and discover the value of confrontation and the functional exercise of delegation, reducing the sense of oppression and distrust. the anxiety component that characterizes the principal's stress can be mitigated by training and refresher courses focused on a model of organizing mindfulness. acquiring awareness of the value of sharing practice is the main theme, but two other aspects to be investigated should not be underestimated and which in this study have however shown strong positive correlations with the practice of sharing, and which perhaps constitute the necessary operational preparatory basis: awareness of the value/advantage of a non-rigid climate and awareness of the value of mutual knowledge. a lack of awareness of one's own way of acting can lead to behaviors that are not functional to the work context, to the quality of the interaction with one's colleagues and to the nature of the task required at the time [ ] . by investing in one's mindfulness, one can expect to break the old automatisms in favor of new behaviors, effective even in difficult times, as indicated by weick and sutcliffe, who believe it is necessary to rely on a mindfulness-oriented approach when there is a need to make a quick and important decision, giving priority to one's competence (or that of one's co-workers), rather than relying on one's authority. the study by beausaert et al. [ ] emphasized the influence of social support in the containment of stress and the burnout of principals. our study points out as a priority, above all, the awareness to which the principal must be individually accountable, i.e., the indispensability of a practice of sharing and mutual recognition of specificities and competences. in this way they move on to a proactive attitude that promotes social support in the first person, before expecting it (in due form) from others. when mindfulness becomes a shared social practice in an organization and permeates the routines, processes and practices among people and teams, and thus affects the organization as a whole, the organization itself becomes more resilient and proactive. even the educational institution has a vital need to promote responsible leaders at all levels, capable of maintaining self-control, a sense of balance and self-determination, despite the informational overload they have to deal with today. data collection through self-reporting measures should be expanded with a methodological design in which judgments about principal stress could be provided also by the staff and teachers of the school, in order to have a more balanced representation by an outside perspective. the cross-sectional approach of the study involved collecting data at a single point in time; instead, the extension of the study could include repeated administration at different times of the school year (beginning, middle, end). the reference hypothesis is that the level of stress and the perception of discomfort could vary in relation to different significant institutional moments, such as the opening and closing of the school year. in this regard it could be useful the novel use of neuroscience-based approaches in education, namely neurodicatics, which is directed to address the educational and psychological well-being of students and staff involved in education as part of the education environment [ ] . a further important contribution could be a specific focus on how principals cope with emergencies and on the functionality of their strategies to manage individual and collective stress triggered by the exceptional nature of the problems that the situation entails (e.g., ensuring teaching activities in safe environments after the spread of the covid- virus). at the moment, there are also no longitudinal studies that have monitored the evolution of the principal's leadership ability over medium-long intervals. it would therefore be important to understand if and how more mature and functional patterns for the containment and management of pressure in moments of personal tension and discomfort are learned and modeled over the course of his/her career. the results of this study have shown that the levels of stress and work discomfort perceived by principals are high and require both empirical investigation and targeted support and prevention interventions. in fact, the stress experienced by a principal is associated with various physical disorders and serious health risks, such as ischemia and heart problems. the study has produced a path analysis model in which the joint effects between the main predictors of principals' work discomfort were explained. the effect of depressive anxiety on perceived discomfort found a protective mediator in the mindfulness component that recognizes sharing as a fundamental operational tool, while an increasing sense of effort and confusion could significantly amplify the experience of psychological discomfort associated with the practice of school leadership. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , file s : principals stress.sav (dataset); file s : model work discomfort.amw (model). author contributions: p.d., g.v., and s.m. designed the study. p.d., g.v., and s.m. analyzed the data and discussed the results. f.b. and s.m. drafted the manuscript. g.v. and f.b. revised the manuscript. all authors approved the final manuscript. finally, the authors have agreed to be accountable for all aspects of the manuscript in ensuring that questions related to the accuracy or integrity of any part of it are appropriately investigated and resolved. all authors have read and agreed to the published version of the manuscript. funding: this research received no external funding. symptômes et caractéristiques du burn out [symptoms and characteristics of burnout school burnout and heart rate variability: risk of cardiovascular disease and hypertension in young adult females il nuovo dirigente scolastico preparing leaders to work with emotions in culturally diverse educational communities manuale per dirigenti scolastici e staff di direzione principal burnout: the concept and its components an analysis of burnout and job satisfaction among turkish special school headteachers and teachers, and the factors effecting their burnout and job satisfaction burnout among iranian school principals burnout 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bavaria the influence of morningness-eveningness on anxiety and cardiovascular responses to stress principals and stress: few coping strategies for abundant stressors effects of support on stress and burnout in school principals considering the role of neurodidactics in medical education as inspired by learning studies and music education authors are grateful to all school principals who took part with this study. the authors declare no conflict of interest. key: cord- -r x awlw authors: stage, h. b.; shingleton, j.; ghosh, s.; scarabel, f.; pellis, l.; finnie, t. title: shut and re-open: the role of schools in the spread of covid- in europe date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: r x awlw we investigate the effect of school closure and subsequent reopening on the transmission of covid- , by considering denmark, norway, sweden, and german states as case studies. by comparing the growth rates in daily hospitalisations or confirmed cases under different interventions, we provide evidence that the effect of school closure is visible as a reduction in the growth rate approximately days after implementation. limited school attendance, such as older students sitting exams or the partial return of younger year groups, does not appear to significantly affect community transmission. a large-scale reopening of schools while controlling or suppressing the epidemic appears feasible in countries such as denmark or norway, where community transmission is generally low. however, school reopening can contribute to significant increases in the growth rate in countries like germany, where community transmission is relatively high. our findings underscore the need for a cautious evaluation of reopening strategies that ensure low classroom occupancy and a solid infrastructure to quickly identify and isolate new infections. throughout the course of the ongoing covid- pandemic, the role of young people and children in transmission has been a question of particular concern [ , ] . this question is not only motivated by the goal to protect the younger generations; it is also known from other respiratory diseases that, because younger people tend to have more prolonged and physical contacts among themselves [ ] , they pose a greater risk of infection to each other as well as being likely to introduce the infection to their respective households [ ] and can drive the epidemic [ , ] . consequently, school closure is often one of the first measures considered when non-pharmaceutical interventions are needed to curb the spread of an epidemic. however, it is often implemented concurrently with other measures, making it difficult to assess its individual impact. many of the challenges inherent in quantifying the impact of closure remain when policy-makers subsequently turn to the reopening of schools. reopening presents a myriad of further questions, such as the ages of those returning, the physical circumstances and timing of their return, and the necessary condition which must be met on a community level before a return can be deemed safe. some work has already addressed these questions from a theoretical perspective of scenario planning [ ] , a valuable means of quantifying the expected impact of various measures without unnecessarily exposing staff and students to an increased risk of infection. in this work we present a complementary data-based approach which focuses on the effects of school closure and reopening in denmark, germany, norway, and sweden. we hope that our contribution can serve as a series of lessons learned from the outcomes of nations who have already reopened schools. clearly, modelling is essential in informing future decisions when faced with numerous possible actions. however, we believe our work fills a clear knowledge gap in the literature by addressing the context of school interventions alongside other measures, and the de facto impact of schools in a broader framework of epidemiological interventions. school closure and reopening not only affect transmission occurring on the premises; they also affect (and are affected by) the community transmission, the transmission within households with young children, and the measures taken to monitor and curb the outbreak. it must be remembered that the observed effects of these interventions are a product of underlying testing, reporting, and isolation (or other physical or social distancing) measures. the aim of this work is to carry out a comparative analysis of school interventions, making use of the diversity of available data streams, the varying age groups of the affected school children, and the variability in the timings of these interventions. where possible, we wish to examine roles in transmission played by a) different age cohorts of students, b) the timing of the school interventions (closure and reopening), and c) the background or community incidence of each country. we distinguish between countries with high and low levels of community transmission on the basis of daily covid- cases, rather than cases per capita. this is motivated by the feasibility of testing, tracing, and isolating cases, which need not scale with population size. data have been selected with the intention of studying a broad range of interventions. however, care has been taken to ensure that the data are representative of the underlying epidemic, and that incidence numbers at the time of intervention are large enough so that proper conclusions can be drawn without any signal being dominated by stochastic effects. where possible, we consider hospital admissions as the primary data source. this is a reasonably practical incidence measure and, unlike confirmed cases, is not as susceptible to variable testing rates in the wider population. however, it should be noted that hospitalisation only tells us about the specific subset of the population which is hospitalised. there are likely biases in such data toward older and sicker individuals. this could lead to longer than expected delays from school closures to an observed reduction in new admissions, as hospitalisations are more likely caused by second or third generation infections from an initially infected student. confirmed community cases have been used as a metric only in situations where hospitalisation data were not available or insufficient to reliably infer the effect of interventions -particularly relevant in the case of school reopening, which has predominantly been recommended in communities with significantly reduced daily incidence counts. in this case, care has been taken to ensure that there is evidence of consistent and thorough testing. we document the numbers of tests carried out and comment further on the reliability of confirmed cases as representative of the community epidemic in the supplementary material. only german states with at least cases at the point of school closure, and at least days of consecutively increasing cases prior to closures, have been selected for analysis. the selection was guided by the availability of data, the relative timings and scope of interventions, and the similar demographic profiles allowing for a better comparison. the effect of school closure is estimated using hospitalisation data for denmark and norway, and daily confirmed cases for germany and sweden. in all countries, we restrict the analysis of school closures to fall before the peak in reported cases or hospital admissions. hospital admissions data for norway starts days prior to school closure, totaling just admissions. we expect the mean time until interventions would be observed in hospitalisation data to be days after school closures [ ] . as norwegian hospitalisation data was too sparse to reliably infer the effect of school reopening, daily confirmed cases were analysed instead. swedish confirmed cases saw two distinct growth phases in the early part of the epidemic, likely caused by different testing regimes. for consistency with later data, we chose to work with the latter phase. in germany, daily confirmed cases are reported specifically for students under in the schools, kindergartens, holiday camps, after school clubs, etc. as well as for the staff working in these facilities. we concern ourselves with these numbers for the analysis of school reopening, rather than the total population aggregates on the state or federal level. consistent test numbers for both germany and norway around the time of school reopening suggest that the confirmed number of cases is less prone to biases than earlier in the pandemic. we are therefore less concerned about using these data streams in a reopening context, though they are still likely to exhibit greater weekend effects than hospital admissions. denmark reopened schools quickly enough following mass quarantine that hospitalisation data could be still used to monitor its effect, though we also verify these findings by analysing confirmed cases. the expected delay to observation in the danish hospitalisation data is to days [ ] . the announced re-opening of swedish upper secondary and higher education in mid-june will likely not be observed in the data until late june, and is thus not included in this analysis. of the four countries selected for comparison, denmark and norway are considered countries with low incidence, whereas germany and sweden are considered countries of medium to high incidence [ ] . in order to assess the impact of school closures, we developed a method to project forward the trajectory of cumulative cases or hospital admissions, under the scenario of no intervention. the method couples an ode epidemic model with a poisson gaussian process (gp) regression model. first, a selection of sample trajectories are generated via approximate bayesian computation (abc) fitting the ode compartmental model from the first day of data until days after school closures. this is based on the assumption that any change in growth rate within these time windows is unlikely to be attributable to the closures, given a . day mean incubation period for covid- [ ] . the gp regression model is then trained on the sampled trajectories and the data and is used to generate an estimate of the trajectory of cases. the gp model uses the same assumed lag period of days as was used in the abc fitting process, allowing more data points to be used in training the model. motivated by recent approaches proposed in [ , ] , the gp model helps to account for some of the structural discrepancies between the observed data and the simulated trajectories produced via the abc fitting method. additionally, the use of the gp model ensures that the modelled data points immediately after school closures closely follow the observed data, thereby making changes in the growth rate slightly easier to identify. we identify the first day on which there is a clear and sustained deviation from the modelled data, referred to as the response date. more precisely, a change in growth rate is considered an effective response to school closures if (a) it occurs more than days from the intervention date, (b) the deviation persists for at least days, and (c) exceeds the th percentile of the modelled data. the time window from school closure to response date defines the lag time (table , column ), which runs from the date of closure (acting as the zeroth day) up to but not including the response date defined as the first day of deviation from the projection. the growth rates are estimated through a weighted negative binomial regression applied to: the data during the lag time window since school closure (table , column ) and both the modelled (table , column ) and the observed data (table , column ) during equally long time windows after the response date (which marks the first included date in this window). in the case of baden-württemberg, berlin, north rhine-westphalia, and rhineland palatinate, it was necessary to shorten the period over which the observed post-response growth rate was calculated to ensure that the window did not exceed the point of peak daily incidence. the relative changes in the estimated growth rates can be used to assess the impact of interventions. however, we stress that the computed values should be viewed as representative of general trends in the epidemic, rather than definitive growth rates. in the supplementary material. with the number of sequential changes in interventions and loosened restrictions on personal movement and the operation of businesses, it is misleading to estimate a constant growth rate in new cases before and after schools reopened. we therefore consider a different method whereby the growth rate can be quantified following successive loosened measures. a smoother ρ(t) is applied over time t, such that the instantaneous growth rate is ρ (t) (c.f. a constant value in a phase of pure exponential growth). it is assumed that the daily new confirmed cases (or daily new hospital admissions) c(t) grow or decay exponentially, with noise added to account for small case numbers, i.e. c(t) ∝ e ρ(t) . to estimate ρ (t) we adapt a general additive model from the r package mgcv, using a quasi-poisson family with canonical link [ ] . smoothing is achieved using default thin plate regression splines. we consider the date of school closure as the first day on which all schools in a state were closed as a response to state or national government intervention. in most cases, however, there were local school closures prior to enforced closures. furthermore, most primary schools continued to be open to both vulnerable children and the children of key workers after national and state closures. with the exception of baden-württemberg, all german states closed schools on the th of march . as this was a monday, we have assumed the effective date of school closures is saturday march th , under the assumption that school activity is significantly reduced on weekends. schools in baden-württemberg closed on tuesday march th . school closures in hesse and rhineland palatinate were only partially observed: final year high school students were permitted to take their exams as planned in late march. table provides an overview of the observed change in daily growth rate in the period after school closures. these growth rates are consistent with previous estimates [ ] . we show the lag times until the effect of an intervention can be seen in the data, the growth rates pre-and post-response, and the relative change between the modelled and observed post-response growth rate. for ease of reading, a corresponding similarly, a weekend effect is observed in north rhine-westphalia on the st and nd of march. if these data points are omitted from the fit, then the observed post-response growth rate is . ( . - . ). this yields a relative reduction in the post-intervention growth rate of %. all states in germany saw a reduction in growth rate after the closure of schools, typically after a delay of around days. it should be noted, however, that all states experienced further interventions around the same time as school closures, namely the closure of national borders with france, switzerland, austria, denmark and luxembourg on march th , and the declaration of a national state of emergency -leading to the closure of sports facilities and non-essential shops, and restrictions on restaurants -on march th . in all states, except baden-württemberg, these interventions came and days after the effective date of school closures, respectively. in baden-württemberg school closures occurred one day after the closure of borders and on the same day as the declaration of a state of emergency. these concurrent interventions make it difficult to attribute the fall in cases solely to the closure of schools, and it is likely that there is a combination of factors contributing to the observed decay in growth rate. however, attention should be paid to baden-württemberg, which saw a similar lag time as other states, despite having different school closure dates relative to other interventions. we compare this state to north rhine-westphalia which saw comparable case numbers, and account for the three day difference in the closing of schools. the two curves exhibit very similar growth following school closure, and yield similar lag times when accounting for the weekend effect (see supplementary material). this is indicative of school closures being at least partially responsible for the reduction in growth rate. the states of hesse and rhineland palatinate allowed students aged - to sit examinations in late march. in all cases the exams were taken in schools under strict isolation conditions. neither of the states permitting examinations saw any significant detrimental effect on growth rates, compared to states which had similar case numbers prior to school closure, but where exams did not take place during this time period (e.g. lower saxony). this can be seen from comparable reductions in the growth rate in all three states (when accounting for the errors in the rates). further, rhineland palatinate managed to reduce the post-intervention growth to a similar (observed post-response) value as the other two states while holding exams despite a higher pre-response rate. this result indicates that, under controlled conditions with limited social interaction, older students sitting exams / . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint in school were likely not a significant driver of epidemic growth. we include the detailed results from the highlighted german states below, with the remaining analysed states detailed in the supplementary material. the difference between the modelled and observed post-response growth rates serves as an indicator regarding the effectiveness of interventions. bavaria and lower saxony had comparable pre-response growth rates, but lower saxony achieved a significantly greater reduction in the rate post-response. we argue this difference might be due to lower saxony having, at the time of intervention, a lower daily (and cumulative) incidence. additionally, north rhine-westphalia (when correcting for the strong weekend effect), hesse, and rhineland palatinate reached similar post-response growth rates despite rhineland palatinate having a much greater pre-response growth rate. at the time of intervention, rhineland palatinate and hesse also had lower daily (and cumulative) incidences. this underscores the increased effectiveness of earlier interventions, capable of breaking many transmission chains when community transmission remains low. the state of baden-württemberg clearly saw a decrease in the growth rate following school interventions, but we do not directly compare the growth rates in this state to others. a necessary change in the training period for the gp on the data means there is an unequal basis of comparison relative to other states. baden-würtemberg saw a reduction in growth rates very quickly after school closures -this is likely a result of reduced weekend testing rather than any response to school interventions. it is likely that the genuine response is seen on march th , after which there is a clear and sustained reduction in the growth rate ( figure ). baden-württemberg saw the following interventions around the time of school closures: • / -banned gatherings of more than people. • / -shut borders with france (fr), switzerland (ch), austria (at), denmark (dk) and luxembourg (lu). • / -closed schools. • / -state of emergency: closed sports and leisure facilities, closed non-essential shops, restrictions imposed on restaurants. • / -banned gatherings of more than people. closed all restaurants. modelled and observed cases in baden-württemberg. note that the strong weekend effect and days after school closure lead to artificially deflated values. as a result, it was necessary to fit the gp model to days after closure. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . hesse saw a similar response to school closures as other german states with moderate incidence (figure ), despite schools in the state permitting older high school students to sit examinations towards the end of march. hesse saw the following interventions introduced around the same time as school closures: • / -banned gatherings of more than people. • / -school closures (effective date). • / -shut borders with fr, ch, at, dk and lu. • / -state of emergency: closed sports and leisure facilities, closed non-essential shops, restrictions imposed on restaurants. • / -banned gatherings of more than people. closed all restaurants. lower saxony had a similar number of cases to the two states which permitted students to sit examinations in march. despite this, there was little difference between the reductions in growth rates -indicating that the examinations did not have a significant impact on the epidemic ( figure ). lower saxony saw the following interventions introduced around the same time as school closures: • / -banned gatherings of more than people. • / -school closures (effective date). • / -shut borders with fr, ch, at, dk and lu. • / -state of emergency: closed sports and leisure facilities, closed non-essential shops, restrictions imposed on restaurants. • / -banned gatherings of more than people. closed all restaurants. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . final year high school students in rhineland palatinate were required to sit examinations in the last two weeks of march. this does not appear to have had any significant effect on case numbers. note that the drop in growth rate on march st and nd is again likely to be a result of fewer tests reported during the weekend ( figure ). rhineland palatinate saw the following interventions introduced around the same time as school closures: • / -banned gatherings of more than people. • / -school closures (effective date). • / -shut borders with fr, ch, at, dk and lu. • / -state of emergency: closed sports and leisure facilities, closed non-essential shops, restrictions imposed on restaurants. • / -banned gatherings of more than people. closed all restaurants. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint this section considers the effect of closing schools in denmark, norway and sweden. for norway and denmark we consider hospital admissions, while for sweden we consider confirmed cases. as with germany, we use the officially announced date of school closures as a reference. the official date of closures in denmark fell on monday march th , and as such we take the effective closure date as saturday march th . in all three countries there were provisions in place to allow key workers' children to continue attending school. despite no official nation-wide closing of primary or secondary schools in sweden, there were local closures in response to outbreaks within the community. due to the low incidence in denmark and norway, the data for these two countries does not lend itself to reliably determining the response date of interventions, and estimates for the growth rates are therefore not reported. however, a visual inspection of the daily hospital admissions data in both countries (see the supplementary material) clearly finds a fall in cases following interventions. however, school closures in both denmark and norway occurred in conjunction with other interventions, and as such it is difficult to attribute any effect solely to the closures themselves. for completeness, we include the fits to daily and cumulative hospital admissions using the gp method in the supplementary material. the response date in sweden was days after school closure, and days after a ban on large gatherings. it is therefore unlikely that this signal can be attributed to the closures themselves. it is notable, however, that the limited closures in sweden were imposed in the absence of large-scale social restrictions. this indicates that school closures are most effective when implemented concurrently with other interventions. sweden's school closures were less restrictive than other countries, with only educational establishments for students aged or over being required to close. the first sustained reduction growth rate occurs days after school closures, and days after the banning of mass gatherings ( figure ). the limited response to the closure of education establishments should be viewed in the context of looser social restrictions. the eventual response after the banning of large gatherings indicates that school closures affecting older students without more widespread social interventions are unlikely to be effective. it is notable that there was an increase in weekly testing between march th and april th , which may have contributed to the apparent limited reduction in growth rate during this time. sweden saw the following interventions introduced around the same time as school closures: • / -banned gatherings of more than people. • / -social distancing advised but not enforced. • / -closed all education for students aged or over. • / -advised that travelling within the country should be reduced. • / -banned gatherings of more than people. the following events are possible confounders in the data (on a national level), and general indicators for the key dates of schools reopening: • shops were allowed to reopen on april th ; museums, gardens and zoos on april th , and hairdressers on may th . • return of final year (exam) students on april th . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . • return of mixed years on may th or may th . due to the variability in policies across german states, the dates of school reopening, and the ages of students returning were variable. the above is a summary of the general national trend. . confirmed cases of covid- in staff (red) and students (blue) in schools, kindergartens, holiday camps, and other educational venues or institutions for under- s. the exact age distribution of those tested is not known. left shows daily new confirmed cases, and right shows the instantaneous growth rate (shaded regions are % confidence intervals). solid vertical lines indicate when students returned to school, and dashed lines indicate other loosened measures. in april and early may with small numbers of primary school or exam students returning, there was no notable difference between the incidence among students and staff. accounting for the detection delay, the incidence among students was higher than that of staff following the return of more (and older) students on may th . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . the spike in daily cases observed following may th (figure , left) is likely a result of increased presentation for testing following a national announcement of school reopening, or increased community transmission following reopening of other parts of society which was subsequently contained . overall, there is a downwards trend in the incidence of staff, which is supported by the growth rate among staff being negative. the incidence among students decreases, and subsequently increases with a predominantly positive growth rate from the end of may (figure , right). . reported daily hospital admissions in germany, excluding those working in education, front-line healthcare workers, carers, catering, and hospitality. these numbers indicate transmission in a general, averageexposure population. left shows daily admissions, and right shows the instantaneous growth rate (shaded regions are % confidence intervals). the large confidence intervals on the instantaneous growth rates do not allow one to conclude if, following the reopening of schools, the growth rate has continued to be negative, or whether it is approximately zero. this suggests that the return of younger (and exam) students did not significantly impact the general hospitalised population. the stable, low, values of the incidence and growth rate until the middle of may indicate that the return of final year exam students and year students ( year-olds) either a) does not significantly increase transmission in schools or the community, or b) can increase transmission, but this is prevented due to the increased distancing in e.g. more spacious classrooms, and an effective testing and tracing system. this observed effect is quite a strong signal as small case counts appear even across a background of increased community transmission from late april onward with the opening of shops. it is therefore reasonable to conclude that these age groups do not strongly increase transmission in a setting of effective social isolation. however, the impact of most students returning to school from late may is different. in this time period, the incidence among staff qualitatively agrees with the national trend in hospitalisations (figure ) , i.e. staff do not appear to become increasingly infected following the return of more students. in contrast, there is a clear increase in the growth rate of students following may th . given that staff incidence is unchanged, and there was little effect of the return of younger years on their own, this suggests that the increase is due to either at three points in this data set, the recorded cumulative cases (from which the above daily cases were obtained) were inconsistent. these values were imputed using cases reported on the days immediately before and after. the findings do not change significantly upon exclusion of these data points. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint a) increased transmission among older students, or b) the impossibility of adequately carrying out physical distancing in venues at full capacity. note that we examine the impact of reopening for a longer period in germany than other countries, due to the possibility of specifically understanding the impact of reopening on the young student population. we have not done so for other countries because an equivalent data set was not available. the following events are an inexhaustive list of possible confounders in the data, and key dates for the return to school: • small businesses were allowed to reopen on april th ; further shops on may th , and some shops, restaurants, and cafes on may th ; zoos, museums, cinemas and similar venues opened on may th . • return of years to on april th . • return of years to and exam students on may th . • return of secondary school students on may th . there is no significant observable increase in the growth rate of hospital admissions following school re-opening to younger years, even bearing in mind the subsequent reopening of some businesses (figure ) . the low growth rate and small relative number of admissions indicate that the return of younger years to school settings did not contribute significantly to transmission, subject to adherence to social distancing. there are only very few data points following the reopening stage on may th when accounting for the expected delay. however, the present data does not suggest this event to have had a significant effect on national hospitalisations. we . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . verify this observation by comparison with confirmed cases (as they are subject to a shorter delay, and are recorded in greater numbers) in the supplementary material. this finding is further supported by the observation that the proportion of adults testing positive for covid- is lower among those working with children aged to than those working with students aged or over (further details in the supplementary material) [ ] . however, these numbers alone do not distinguish between infection acquired from the students, and infection acquired elsewhere. the following events are possible confounders in the data, and key dates for the return to school: • small businesses were allowed to reopen on april th ; larger gatherings of up to people were allowed from may th . • return of kindergarten students on april th . • return of years to , final year students, vocational training, and higher education which requires physical presence on april th . • return of the remainder of primary and secondary school the week commencing may th . . reported daily confirmed cases in norway. new cases are reported left, and right shows the instantaneous growth rate (shaded regions are % confidence intervals). solid vertical lines indicate when students returned to school, and dashed lines indicate other loosened measures. these numbers are obtained over a time period of increased testing, so there is little reason to believe the case numbers to be higher than reported. norway has a significantly large testing rate per capita. there is no observed increase in the growth rate of confirmed cases following any school reopening, even bearing in mind the subsequent reopening of some businesses (figure ). the consistently low growth rate and small number of admissions indicate that the return of most students to school settings did not contribute significantly to transmission, subject to adherence to social distancing. however, we emphasise that this effect is subject to very high levels of testing, with very low community transmission. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint our analysis suggests that school closures in denmark, norway and germany had some impact on epidemic growth rates, compared to the modelled un-intervened data. interventions always reduced the growth rate compared to our modelled scenario with no intervention; however the extent of this reduction strongly varied with the level of community transmission. school closures in germany, when analysed on a state level, typically resulted in a reduction in epidemic growth rate days after the intervention. we find comparable results in the state of baden-württemberg, which closed all schools later than other states. this indicates that school closures are at least partially responsible for the reduction in growth rate. however, the decision of two german states (hesse and rhineland palatinate) to permit final year high school students to sit examinations in late march does not appear to have had a significant impact on state-level case numbers. sweden implemented partial school closures which affected students aged or above. however, there is no clear evidence to suggest that this intervention is the cause of a later decline in the growth rate. the evidence for the impact of school closures on growth rates in norway and denmark is more limited. while there was a clear and significant reduction in growth rate of hospitalised cases after school closures, it has not been possible to disentangle this response from other interventions occurring at the same time. the reopening of schools to younger year groups and exam students in germany, denmark and norway has not resulted in a significant increase in the growth rate. the return of all students (up to age in denmark) does not appear to have increased transmission in denmark and norway; countries with low community transmission. however, the added return of most (primarily older) students in germany has increased transmission among students, but not staff. it is unclear whether older students transmit more, or if physical distancing is practically unfeasible in classrooms at high capacity. we argue that the different impact of reopening schools in germany may be due to higher levels of community transmission. despite an established system of testing, tracing, and isolation, the growth rate of the national german epidemic has increased in the period following reopening and is now close to zero. decoupling the effect of school closures from other interventions is not straightforward. this work does not claim to have achieved this, however the consistency of the signal across regimes with different intervention timelines suggests a positive effect of school closings. the degree of this effect is correlated with other interventions introduced in parallel which aim to reduce community transmission by different means. one cannot assume that school closures are a "mirror-image" of school reopenings. the two can provide context to one another, however a positive result for school closures is not a justification or repudiation of school openings. school closures are often one of the first measures introduced in populations with high incidence, whereas school reopening has been staggered with other gradually eased restrictions; often with only a small cohort of students returning initially. consequently, due to the additional backdrop of other interventions, e.g. retail closure and reopening, our findings should not be interpreted as presenting a causal link between individual interventions and changes in national case numbers. however, as most countries leave mass quarantine, reopening schools will likely only be one of several relaxation measures. in light of this, we believe our findings are a realistic assessment of the effects of school reopening in their natural context of wider societal changes. the presence (or lack) of signals in the data following school interventions are limited by the reliability of the available data. efforts have been made to consider the most reliable data streams, which we argue is hospital admissions, while bearing in mind that hospitalisations only affect a subset of the infected population. where this data was unavailable, we have considered confirmed cases while monitoring the degree of testing in place to ensure such numbers were indicative. where the availability and nature of tests changed (e.g. nasal swabs or antibody blood tests), it is not automatically clear that data from these different regimes can be compared. this is also true as the availability of tests expands from front-line workers to the general population. hospitalisations are less prone to some of these biases, but are still affected by protocols in reporting, e.g. the hospital admission of an individual with urgent medical needs who also tested positive for covid- , albeit with mild symptoms. school closures occurred early in the outbreak, with potential variations in diagnostic protocols, testing availability, and the nature of the data being collected. later in the pandemic, the quality and diversity of the data is now increasing as we shift from emergency measures to an infrastructure of long-term monitoring. / . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint however, as case numbers have fallen due to mass quarantines and other severe measures, over time some data streams simply have too few cases to be used for analyses. this has been seen in norway with the steady decrease in hospital admissions requiring a shift to using testing data for analysis. efforts have been made to clearly present these biases, and to quantify their impacts. where possible, we have cross-validated our observations using several data streams. the simulation of an unmitigated epidemic used to analyse school closures has been built around an seir model. while the system of ode's underpinning this model are well justified, they are inevitably unable to capture some of the processes present in the early stages of the epidemic. early, highly localised interventionssuch as the quarantining of the first early cases -are likely to have had some impact on the transmission rate which is not able to be captured in the model. this is particularly evident in states in which there was an abrupt change in growth rate in the period prior to school closures. the gaussian process regression method allows one to account for differences between the simulated epidemic trajectories and the observed cases, however this process is not without its limitations. the fact that closures occurred very early on in the epidemic means that the gaussian process method often had to be trained on a limited number of data points (the short posterior predictive trajectories from the ode before the intervention). although a gp, itself being a probabilistic model, is tailored to handle small data with exact uncertainty quantification, the process of estimating its hyperparameters, using mcmc, becomes challenging due to less than optimal mixing of some of these parameters. since the instantaneous growth rate relies on the derivative of splines, it is subject to increased error at the boundaries of the data. however, the observed signals are qualitatively robust to this limitation. due to the presence of weekend effects and the noisiness of some data streams due to the relatively low incidence following mass quarantine, the values of the instantaneous growth rate should be taken as a quantification of the trend in incidence rather than the true value on any given day. with some exceptions, we have considered the effects of school closure and reopening on the national level without accounting for inevitable geographic variability, the age distribution of those studied, and their profession (i.e. likelihood of exposure to infected individuals). the analysis of germany, particularly the comparison of staff and student infections, warns against the reliability in using national-level data to understand the immediate effects or impact of a single population. however, this does not imply that national data cannot be used, much like the lack of a signal on the national level following school reopenings does not guarantee a limited national impact. instead, this impact may only become first visible in subsequent generations. we must therefore be concerned not only with the delay until a signal might first be seen in the studied population, e.g. days, but also with the following generation of infections. this puts the minimum time scale between intervention and impact (assuming a change can be inferred from consistent data over a week, and a constant day incubation period) from to days if using confirmed cases, or to days if using hospital admissions (using an estimated day delay from infection to admission). a statistically challenging yet positive result is that, in many countries, there is insufficient data to analyse the impact of various interventions on an age-stratified level due to low incidence numbers. this is similarly true if examining cases stratified by occupation. our analysis is constrained to settings with high monitoring and intervention efficacy (including but not limited to high testing, tracing, and adherence to isolation). concerning e.g. the return of younger years, continued low incidence following their return to school does not imply that such a measure is inherently safe. in many instances, the students were spread over more classrooms with greater levels of physical distancing from each other and teachers, conditions which are not always practically feasible. similar caution should be had regarding the small or manageable effect of the return of older students, in particular with regard to: the likely increased number of crowded classrooms, as well as their added impact to community transmission if the latter is already relatively high. while all three studied countries seem to be effectively managing transmission, the volatility of the german growth rate in hospital admissions warns that a failure in control, or a sudden spike in cases, will likely have a stronger effect in germany than it would have in denmark or norway. key to this observation is the aforementioned delay before which the ripple effects of school reopening will travel from students to the population at large. furthermore, we highlight that the tenuous / . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . balance (net zero growth) in germany exists despite a swift and robust test and trace infrastructure, and school-level stratified monitoring. we question the possibility of an equally effective reopening in countries with a monitoring process reliant on national-level incidence data. our findings generally underscore the precarious nature of transmission control as it relates to the reopening of schools, particularly in numbers whereby physical distancing is unfeasible. the safe return of most (or even large proportions of) students to school is conditional on the successful implementation of a complete system of monitoring and interventions, jointly with low daily incidence, as observed in denmark and norway. this correlation with community transmission can be seen particularly clearly in germany, with confirmed cases increasing among students, and the halted decay in hospital admissions on the national level. the most severe impact appears to occur following the return of most older students to schools when community transmission remains significant (if managed). great care should be taken when attempting to infer the impacts of school reopening in other nations by comparison with the presented subset. it is insufficient to compare the number of daily tests carried out (or similar monitoring metrics), or the daily incidence (be it total or per capita). the speed of decline in daily cases is also a key quantity, as it informs us about the effectiveness of tracing, individual or household isolation, and the adherence thereto. the swiftness and effectiveness of targeted interventions become increasingly crucial as the daily incidence increases, due to the correspondingly greater challenges presented in managing the myriad localised outbreaks across e.g. reopened schools. policy-makers should carefully consider their nations' respective capacities and associated effectiveness of infection management before considering a partial or full reopening of schools. our findings suggest a small, strategically chosen, proportion of students to return in the first instance, with dedicated testing and monitoring of cases among staff and students over time scales where the effect can be appropriately assessed. any significant return of students to schools, particularly in countries with a high incidence, should not be considered unless an infrastructure is in place which would be able to swiftly identify and isolate most new cases as they appear. such a strategy may not be feasible if the incidence is too high. js modelled school closure from early data. hs analysed school reopening from late data. js and hs drafted the manuscript. sg implemented the gaussian process discrepancy model. all authors contributed to the writing of the manuscript. • germany: daily (and weekly) tests, hospital admissions, and confirmed cases from the robert koch institute [ ] . • norway: daily tests and hospital admissions from the norwegian institute of public health [ ] . • sweden: weekly tests, and confirmed cases from the public health agency of sweden [ ] . the compartmental model used to generate sample trajectories is outlined in figure s . multiple compartments have been used for the exposed (e) populations to model an erlang-distributed incubation period compatible with available estimates of mean and standard deviation of its duration [ ] . the i compartment splits into both detected (i d ) and undetected (i u ) infectious populations. the same model is used for hospitalisations, with hospitalisations taking the place of i d , and non-hospitalised cases taking i u . a higher variability, possibly country-dependent, in the time from onset of symptoms to detection/hospitalisation [ ] and limited knowledge on duration of infection and non-modelled pathway of hospitalised cases suggest a single compartment (i.e. exponential holding time) for these states is a reasonable and parsimonious choice. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . figure s . the epidemic model used to simulate cases. the model uses multiple exposed compartments to account for an erlang-distributed incubation period. in order to provide more data points for the fitting process, the model has been fitted to days after school closure. this is based on the assumption that any change in growth rate within these time windows is unlikely to be attributable to the closures, given the . day mean incubation period for covid- [ ] . simulation of the seir model ( figure s ) is achieved through approximate bayesian computation (abc). this fitting process is used to estimate the parameter vector: the parameter α is not estimated in this method, and is instead taken from the mean incubation period [ ] to be α = / . days − . we employ a weighted negative binomial loss function to measure the distance between the observed data and the posterior predictive trajectories generated by abc fitting. over-representation of earlier data points in the cumulative data is accounted for by introducing a weighting, w i , such that for a data point, x i , we have: the abc method generates posterior predictive trajectories, from which we select evenly distributed samples to be used as covariates with which to train the poisson gaussian process (gp) machine learning model. this model attempts to use the trajectories obtained via abc to replicate the observed data. as with the seir model fit, the gp model is trained with data days after closure for confirmed cases ( days, in the case of baden-württemberg). care has been taken to ensure the reproducability and robustness of these results, including testing with different distributions of and covariates, and running the model with re-calculated abc posteriors. testing data for germany figure s shows the number of tests carried out per day in german medical laboratories, along with the positive test ratio over the same period. this is not equivalent to the total number of tests carried out in germany, as not all laboratories provided this type of data; however, it can be used as an indication of general testing trends. there is a weekend effect occurring in the testing data for germany, with lower relative testing occurring on march th - th , th - th and st - rd . no corresponding change is seen in the positive test ratio, indicating that case numbers were likely consistent across these periods. as such, any fall in confirmed cases over these periods can likely be attributed to reduced testing, rather than a response to intervention. ignoring the weekend effect, the number of tests carried out across the period between march th and th was fairly stable. as most school closures in germany occurred on march th , we can expect the confirmed cases over this period to provide a reasonable representation of the underlying epidemic. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . figure s . daily testing from a subset of german testing laboratories during march. weekends are highlighted in grey. there is a periodic drop in testing occurring on weekends, particularly evident on sundays. these drops do not coincide with any changes to the positive test ratio. daily testing data for germany is not available after march. as such, it will be necessary to consider the weekly testing totals, which are made available through the rki. figure s shows the weekly testing numbers for germany, along with the weekly positive test ratio. note that weekly testing data for germany are released every wednesday. . weekly testing in germany remained consistent from march th , however the weekend effect (see figure s ) was likely present across the entire period. there were no abrupt changes in the positive test ratio. both denmark and norway saw a similar weekend effect in testing numbers, with midweek testing figures roughly % higher than weekend figures in denmark, and almost three times higher in norway. the weekly testing figures for denmark and norway are shown in figure s and s respectively. denmark displays two clear increases in testing capacity between march rd and th and again between april th and th . the increase in late march, combined with a relatively high positive test ratio, indicates that confirmed cases during this period might not be a suitable metric. similarly in norway there was a large increase in testing in the week commencing march th , very close to the date of school closures. as such, for both norway and denmark it will be necessary to consider hospitalisations as a metric for assessing the dynamics of the epidemic. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . figure s . weekly testing in denmark was not consistent across the period of this investigation, and so confirmed cases up to april th cannot be relied upon to provide a reliable representation of the underlying epidemic. the weekly testing figures for sweden are highlighted in figure s , along with the positive test ratio for the same period. testing rates around the time of school closures (march th ) were generally increasing, with a large increase occurring during the week beginning march th . this increase was accompanied by an increase in positive test ratio, indicating an increasing capability to identify and test infected individuals. as a result, it will not be possible to attribute any increase in case numbers after march th solely to the effect of interventions. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . we present the equivalent of table in the manuscript, but expressed via the doubling time (ln( )/growth rate). lag in figure s we consider the cumulative cases in both baden-württemberg and north rhine-westphalia on account of the states being very similar. we shift the cases in baden-württemberg back in time by three days, so / . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint as to coincide the dates of effective school closure (march th and th ). further, to aid comparison we rescale the cases in baden-württemberg by a multiplicative constant (approximately . ), so that the cumulative cases in both states are identical on the day of school reopening. we note that the pure exponential growth rate is unchanged by these transformations. the profiles of the data can now be compared, with the results from the gp fit to north rhine-westphalia providing a baseline from which the lag times can be found. figure s . cumulative cases for baden-württemberg and north rhine-westphalia when corrected for the three-day shift in school closure between the two. the effective day of school closure in both states is shown in red, with the timings of other interventions which took place in north rhine-westphalia included for reference. there is very good agreement between the two data streams despite the time difference in the school closure, suggesting comparable underlying transmission in the two states following school closure. additionally, when considering the weekend effect occurring in baden-württemberg, the lag times are comparable between the two states. the two states appear to be comparable both in terms of the overall cases following school closure, as well as the time taken until a response from an intervention can be observed in the data. clearly it would be unrealistic to assume school closure to be wholly responsible for the observed fall in cases, but the above-detailed correlations are evidence to suggest that they may have partially contributed to the total effect. bavaria saw a small decrease in case numbers immediately after school closures, although this is likely a result of changes in testing rates over this period. the first sustained decrease in epidemic growth rate occurs days after school closures ( figure s ). bavaria saw the following interventions around the time of school closures: • / -banned gatherings of more than people. • / -school closures (effective date). • / -local elections went ahead, with a high turnout. a large number of votes were submitted by post. • / -shut borders with fr, ch, at, dk and lu. • / -state of emergency: closed sports and leisure facilities, closed non-essential shops, restrictions imposed on restaurants. • / -banned gatherings of more than people. closed all restaurants. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . berlin saw a limited response to school closures, with a small increase in growth rate occurring days after intervention. testing rates throughout this period were consistent, indicating that confirmed cases are likely a good indicator of general trends in the epidemic ( figure s ). berlin saw the following interventions around the time of school closures: • / -banned gatherings of more than people. • / -school closures (effective date). • / -shut borders with fr, ch, at, dk and lu. • / -state of emergency: closed sports and leisure facilities, closed non-essential shops, restrictions imposed on restaurants. • / -banned gatherings of more than people. closed all restaurants. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . figure s . modelled and observed cases in north rhine-westphalia. denmark saw a staged closing of schools, with primary school closing on friday th of march, and all other schools following on monday the th of march. the effective date of secondary school closures is taken to be saturday th of march. while there is a clear decrease in growth rate in the period immediately following school closures, it is not possible to identify a single day on which this shift occurs. the gp model was unable to adequately predict the trajectory of cumulative hospitalisations in this period (s ). furthermore, estimating growth rates over this period is problematic as the peak in incidence occurs soon after school closures (s ). denmark saw the following interventions introduced around the same time as school closures: • / -banned public gatherings of more than people. • / -closed non-essential businesses. • / -closed educational establishments for students aged or over. • / -closed all schools (effective date). • / -banned gatherings of more than people. closed shopping centres. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . figure s . modelled and observed cumulative hospitalisations in denmark. the gp model has been fitted days after school closures, but is unable to adequately predict the trajectory of cases. as such, it is not possible to estimate any lag period for the response. figure s . modelled and observed incidence of hospitalisations in denmark. the gp model incorrectly predicts the exponential growth seen in the period prior to closures to continue, making it difficult to identify a response to school closures. norway closed schools at the same time as introducing a range of other restrictions on social life. as such it is not possible to attribute the observed change in hospital admissions solely to school closures. it is notable, however, that the observed reduction in hospitalisations is comparable in denmark and norway; both countries which simultaneously targeted schools and non-essential businesses ( figure s ). for completeness, we fit the gp model to cumulative hospitalisations ( figure s ). however the short training period for the model resulted in a wide confidence interval for this prediction. furthermore, as with denmark, the proximity of school closures to the point of peak incidence makes it difficult to adequately assess either the lag time or the change in growth rate occurring after closures. as such, it is difficult to draw any firm conclusions from the data. norway saw the following interventions introduced around the same time as school closures: . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint • / -closed all schools. • / -closed non-essential businesses. sports and cultural events cancelled. restrictions on restaurants. • / -advised against foreign travel. • / -closed ports and airports to all repatriating citizens and imports. figure s . modelled and observed cumulative hospitalisations in norway. the model is able to reasonably predict the trend in cases for around days after school closures, however the confidence in this prediction is very low. figure s . modelled and observed hospitalisations in norway. the gp model is less effective when dealing with the incidence data, failing to account for any points after the assumed lag period of days. as such, it is impossible to estimate post response growth rates in norway. we here present results in support of the main results of the paper, but which are not essential to the exposition of our findings. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . figure s . reported daily confirmed cases in denmark. new confirmed cases are reported left, and right shows the instantaneous growth rate (shaded regions are % confidence intervals). solid vertical lines indicate when students returned to school, and dashed lines indicate other loosened measures. we present these numbers in support of the observations made for daily hospital admissions due to the larger numbers of cases recorded here. these results are not qualitatively different from those obtained from hospitalisation data, but support the conclusions which are harder to draw from that data set due to the longer delay from infection to hospitalisation. sentinel survey information indicates the following for staff in different educational settings. a smaller proportion of staff working with young children have tested positive compared to staff working with older students. however, these numbers alone do not distinguish between infection acquired from the students, and infection acquired elsewhere (table s ) . tested population (%) positive tests (%) tests table s . a comparison of tests carried out among staff working in different stages of the danish educational and childcare sector dated june nd . we indicate the proportion of tested staff relative to estimated employee numbers in each group, and the percentage of those tested who test positive. for reference, the absolute numbers of tests are also shown. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint are children less susceptible to covid- systematic review of covid- in children shows milder cases and a better prognosis than adults social contacts and mixing patterns relevant to the spread of infectious diseases burden of influenza in healthy children and their households (retraction in: archives of disease in childhood modelling the impact of local reactive school closures on critical care provision during an influenza pandemic estimating the impact of school closure on influenza transmission from sentinel data the impact of school reopening on the spread of covid- koronavirus-modellering ved fhi ekspertrapport af den european centre for disease prevention and control. covid- situation update for the eu/eea and the uk, as of learning about physical parameters: the importance of model discrepancy considering discrepancy when calibrating a mechanistic electrophysiology model mixed gam computation vehicle with automatic smoothness estimation inferring change points in the spread of covid- reveals the effectiveness of interventions danish national health data authority. covid- og ansatte indenfor institutioner og undervisning overvȧgning af covid- daily situation reports on covid- dagsrapport og statistikk om koronavirus og covid- bekräftade fall i sverige -daglig uppdatering we gratefully acknowledge thomas house and ian hall for useful discussions and contribution to the methodology. the data streams used to carry out the analysis of this work can be found at:• denmark: daily tests, hospital admissions, and confirmed cases from the national serum institute [ ] .sentinel survey among educational staff is from [ ] . north rhine-westphalia saw a drop in cases between march th and nd . this was likely a result of decreased testing or reporting in this period. when accounting for this effect, the first sustained drop in epidemic growth rate occurs days after school closures ( figure s ).• / -local school closures in heinsberg.• / -banned gatherings of more than people.• / -school closures (effective date).• / -shut borders with fr, ch, at, dk and lu.• / -state of emergency: closed sports and leisure facilities, closed non-essential shops, restrictions imposed on restaurants.• / -banned gatherings of more than people. closed all restaurants. key: cord- -prp hiz authors: beck, dennis; beasley, jennifer title: identifying the differentiation practices of virtual school teachers date: - - journal: educ inf technol (dordr) doi: . /s - - -y sha: doc_id: cord_uid: prp hiz despite a large increase in enrollments of students in online courses at the k- level, there is very little research on the use of differentiation in fully online (called “virtual”) schools. this study asked virtual teachers from two different types of schools to discuss their differentiation practices, and compared differentiation practices of teachers across these schools. nineteen focus groups consisting of teachers were conducted. data were analyzed using tomlinson’s differentiation framework. results showed that the large majority of teacher comments about differentiation definitions, assessments, curriculum, grouping and strategies fell in the novice category, and that newer virtual school teachers may struggle in developing skills in differentiation in an online environment. teachers use the principles of differentiation to make decisions that are responsive to the needs of all students (tomlinson, (tomlinson, , . unfortunately, there is very little research on the use of differentiation in fully online (called "virtual") schools. this is important, because districts employing online education courses have increased enrollments from % to %, with rural districts making the most growth (gemin and pape ) . in recent times, a global pandemic forced many educators to teach remotely. in addition, students accessing this technology are more diverse than ever. enrollment of english language learners (ell) was higher in - ( . %) than in the previous ten years (u.s. department of education, office of special education programs, individuals with disabilities education act (idea) ). this has resulted in larger online classrooms with more diversity of students. although there is much research on the effectiveness of differentiation in the brick and mortar k- classroom (baumgartner et al. ; doubet ; valiandes ) and on the best practices of how teachers define and enact differentiation (archambault et al. ; westberg and daoust ) , there are few studies found on the use of differentiation in virtual schools. one study found that teachers struggle to find ways to differentiate instruction (beasley & beak, ) , but beyond that, no research compares differentiation practices of teachers across different types of virtual schools. in order to begin to explore the differentiation practices of virtual teachers, this research will ask the following research questions: . how do virtual school (vs) teachers differentiate teaching practices in an online classroom? . how do the differentiation practices of vs teachers compare across diverse kinds of online schools? literature review a differentiated approach to instruction is informed by principles that include providing high quality curriculum, utilizing flexible grouping, and administering ongoing assessments within a community that respects learning activities that are challenging for all students (tomlinson, ; . differentiation has often been confused with the term "personalized learning" and for the purposes of this study, it is important to distinguish between the two. dewaters ( ) stated, "personalized learning occurs when teachers and students work together to develop a personalized pathway to student learning" (p. ). dewaters ( ) claimed personalized learning diverges with differentiation in its highlighting of student interests, needs, readiness, and motivation. this global definition can encompass many instructional methods. in practice, many virtual schools use this term to promote learning that is student selected and student guided. in contrast, differentiated instruction is facilitated by the teacher working in partnership with the student needs. at times, a differentiated lesson may take on the aspects of a personalized learning assignment, but ultimately the learning experiences are guided by learning goals with the teacher facilitating the process (tomlinson b) . the literature has defined differentiation in practice primarily through research in the traditional face-to-face classroom (tomlinson, ; turner and solis ) . when observing classrooms, teachers who exhibit fidelity to the model of differentiation a) gather information to tailor instruction to student interest and learning profile as well as readiness to learn the topic or state standard, b) foster classroom community, c) group students flexibly, and d) utilize formative assessment for learning (doubet ) . these noted classroom indicators are supported by research in cognitive learning (bransford et al. ) . a successful learning community is knowledge-centered (bransford et al. ; miller & hudson, ) , learner-centered (ainley, hidi, & berndorff, ; bransford et al. ; tomlinson et al. ) , as well as assessmentcentered (bransford et al. ; glaser, chudowsky, pellegrino, ) . differentiated instruction has been shown to have a positive impact on student achievement (baumgartner et al. ; doubet ) . the best practices of teachers who differentiate instruction involve (a) the collection of information about student interests, learning profiles, and student readiness; (b) cultivation of a community in the classroom, (c) distribution of students into groups that can be easily changed based on the activity, and (d) use of formative assessment for learning (doubet ) . in differentiated classrooms, community is strengthened through the use of formative assessments that teachers use to determine student readiness, interest and learning profile to access a student's knowledge, skill, and/or understanding during a learning experience. however, there is little research or literature on what differentiation looks like in the virtual school classroom. how do teachers in virtual schools differentiate their instruction, and what does it look like across different types of virtual schools? during the covid- crisis, many schools required teachers to transition to online instruction (borup, jensen, archambeault, short, & graham, ) . however, online education has been growing even before this recent crisis, making it important to uncover best practices of differentiation in virtual schools. key aspects of teaching in a face-to-face classroom may look different online, but it will always be important for teachers to meet the needs of all students (bransford et al. ) . in this section we will briefly discuss online education and best practices in online teaching. forty-four of states reported online learning opportunities for k- students. virtual schools have grown both in numbers and in scope of offerings (gemin and pape ) . a virtual school refers to k- online learning programs offered fully online by an educational organization where students can earn credit toward graduation or promotion to the next grade level. despite this growth, evidence for achievement gains in virtual schools is poor (woodworth, raymond, chirbas, gonzalez, negassi, snow, & van donge, ; cremata, davis, dickey, lawyer, negassi, raymond, & woodworth, ) . most research focusing on student achievement in virtual schools has found them to be significantly less effective than brick and mortar schools (minnesota office of the legislative evaluator, ; woodworth et al., ) , though at least two studies found greater academic gains than in brick and mortar peers (wang & woodworth, ) . with that said, low student achievement is very common in virtual schools. over four years, colorado online schools yielded three times more dropouts than graduates. additionally, the scores of virtual school students' on state tests were, on average, to percentage points below the state average in reading, writing and math (hubbard& mitchell, ) . researchers in ohio examined achievement trends by students' achievement in their prior, comprehensive public school (cps) and found that students in virtual schools are performing worse on standardized assessments than their peers in traditional charter and traditional public schools (ahn and mceachin ; also tucker et al. ) . technology may offer great promise for the future of education, but the data is not showing that it isn't necessarily benefiting virtual schools. virtual schools do have potential advantages, such as accommodating schedules and a broader span of courses than may be offered locally. virtual schools can also serve disabled students who have difficulties attending in-person classes on a regular basis (miron and gulosino ) . some of the reported benefits of virtual schools include offering convenient lessons that fit a learner's schedule, enabling students who have other obligations during the daytime hours to finish their educational goals, and providing additional time periods for learning. virtual schools may also act as a shelter for cps students who have experienced bullying. these advantages may be some of the reasons why virtual schools receive high levels of student, parent and teacher satisfaction. teaching with a student's needs in mind is at the center of the philosophy of differentiation, which is explained in the next section. in this study, three virtual schools located in the mid-southern united states participated in focus group interviews over the course of the - academic year. ninety-two teachers were interviewed using a focus group protocol that was designed to gather information specifically in five categories: ) definition; ) curriculum; ) assessments; ) grouping; and ) strategies. approximately students attended four virtual schools in this mid-southern u.s. state during the - school year. three of the schools chose to participate in this study -two charters and one district-based. teachers from these schools were interviewed in focus groups to find out more about their differentiation practices. table represents the schools included in the study, pseudonyms are used for schools and individual teachers. the research questions for this study were: how do the differentiation practices of vs teachers compare across diverse kinds of online schools? a total of virtual classroom teachers were interviewed from three virtual schools located in the mid-southern united states (table ) . of those teachers interviewed, % of the participants self-identified as female and % of the participants self-identified as male. % of the participants reported to have or less years of teaching experience, % reported to have - years of teaching experience and % reported having or more years of teaching experience. % reported to have less than years of experience teaching online. a total of focus groups were collected over the course of a year. participants were contacted through the virtual school administration in september , researchers contacted each virtual school and asked permission to interview teachers either face-to-face or online. after obtaining institutional permission, administrators from each school provided dates and times for focus group settings. both researchers conducted focus groups for all three schools, with a maximum of teachers per group. for this study, teachers from the three schools participated in the focus groups (table ) focus groups were held with teachers in each school according to their academic subject area or specialty in late february, . the focus group protocol consisted of questions based on results of a previous survey of virtual school teachers' beliefs about differentiation. researchers chose focus group protocol as a way to verify the results of the survey used in the previous research study. as opposed to an interview, in a focus group discussion, researchers adopt the role of a "facilitator" or a "moderator." this allows the researcher to facilitate or moderate a group discussion between participants and not between the researcher and the participants. unlike interviews, the researcher thereby takes a peripheral, rather than a center-stage role in a focus group (bloor et al. ( ) ; johnson ( ) . after transcription, researchers examined focus group transcripts and code them based on tomlinson's differentiation work ( a). the interview protocol consisted of questions based on the teachers' responses at the focus group. an interview protocol was created by the researchers in response to findings from a previous study (beasley & beak, ) . this research revealed that ) online teachers understood the importance of getting to know their learners, but their practice contradicted that belief, ) online teachers report that they need to differentiate content, product, and process, and ) there was little mention of using assessment to inform instruction. this study built upon the findings of the previous work by focusing on further defining what teachers are doing to differentiate in the classroom, and comparing these practices across types of virtual schools. questions were centered on the three findings of the previous research (table ). the questions were also aligned with the principles of differentiation as defined by the previous research and literature review. this alignment to previous research and literature were important in order to build upon the findings by both the authors and experts in the field. of particular interest was finding out how virtual teachers are using assessment in their online classroom. the questions were kept purposefully open ended in order to facilitate discussion. data analysis of focus group transcripts occurred in two phases. first, a grounded theory approach (strauss and corbin, ) was used to code the data set. developing an initial codebook included the code, a definition of the code, and guidelines for using the code. afterwards, the researchers used those codes as they analyzed for themes finding respectful tasks* *based upon the work of tomlinson ( a tomlinson ( , b from the participants' responses. the goal was to reach categorical saturation (lincoln & guba, ) . a confirmatory analysis was also conducted through the use of a second coder. we then adapted hedrick's "ascending levels of intellectual demand ( ) for differentiation as a coding scheme for understanding how novices transition to apprentice, practitioner, and expert levels. hedrick's ( ) original framework looked at ways that educators grow in their understanding and application of differentiation in brick and mortar classrooms and this was considered as we identified ways teachers spoke about their use of differentiation in virtual classrooms (fig. ) . due to the lack of literature when it comes to how differentiation is being applied and defined in k- vss, the researchers applied the ascending levels of intellectual demand (aid) to participant responses. for this study, novices were noted by their lack of clarity of definition and application of differentiation. they tended to teach as if the whole class were the same. apprentices were more likely to tolerate some ambiguity and begin to see groups of students with similar needs. practitioners were able to apply the principles of differentiation in multiple ways fig. ascending levels of intellectual demand: differentiation and begin to utilize assessment data to change assignments for varying groups of students. finally, experts were able to begin to see students individually and plan proactively on a consistent basis. further characteristics are discussed for each level (e.g. "unsettled by the ambiguous nature of differentiation" in novice level) and thus developed a coding scheme that described how each of these descriptions looked in a vs. focus group transcripts were coded using this scheme. results on research question : how do vs teachers differentiate teaching practices in an online classroom? the interviews resulted in findings that were categorized by levels of differentiation but also allowed researchers to hear about the practices of virtual teachers. salient quotes were identified to communicate the findings in the area of ) defining differentiation, ) curriculum, ) grouping, ) teaching strategies, and ) assessment. similar to the initial research, vs teachers' definitions were more novice in nature. vs teachers spoke about differentiation being "different for different students" (novice, item ). a quote by one teacher exemplified this, "...differentiation to me, i think it's about the root word...to be different. if you are differentiating you're cooking outdoors, then you make changes to your cooking that make it look different than cooking in a kitchen" (vs , creative school). a more expert level response was demonstrated by two vs teachers, "...differentiating is based a lot on the relationships you build with the students. we learn about their past educational experiences, what their goals are for the future, and what obstacles might be in their path so that you can modify and adjust the way that you present material and help them to engage" (vs , creative school). many of the responses to curriculum in a differentiated classroom sought solutions that were already a part of their existing repertoire (novice, item ). very few vs teachers made connections across disciplines or saw ways to modify content, process, or product. one teacher from filmore virtual school noted, "i differentiate my lesson by changing the number of examples i give students who are farther along in the lesson. i also reduce the number of answers in a multiple choice for struggling students." a more practitioner level response demonstrated, "i look at my curriculum and see where students are both content wise, but also engagement wise. i try to engage student personal interests -and that looks different in different disciplines" (vs , filmore school). in the area of grouping, there were fewer comments than other categories. of the distinct quotes examined as a part of this study, directly correlated to grouping. this may be that many of the school lms systems did not allow many types of grouping strategies, or it could have been due to the fewer number of students in the "live sessions." as with other categories, the majority of vs teachers situated themselves within the novice level, primarily item that focuses on challenges instead of benefits. one example was, "i don't necessarily group, but i do allow them to chat with one another through our social discussion groups" (vs , filmore school). another identified this as an area for improvement, "if they don't call in, they don't show up, so they're not part of any grouping. i think this is my downfall" (vs , creative school). teaching strategies noted by the teachers were typically categorized by what they did when they were brick and mortar teachers (novice, item ). an example of this was by a teacher from the valentine school, "...sometimes you just revert back to some of the tools that you had when you were in a face-to-face classroom -like i would put a math problem up on the whiteboard and discuss the answer." one vs teacher had a more expert level answer (one that models differentiation with flexibility and fluency): "i really set out to gamify my class and it has done a world of good. my students have made so many gains both in engagement and academically" (vs filmore school). the area of assessment was more informative than in our last research study (beasley & beak, ) , representing % of the quotes being analyzed. previously, we found that very few vs teachers spoke about how they used assessment in a differentiated classroom. however, during this study's focus groups, one teacher noted "...sometimes we have to dig a little deeper -it's not exactly what it says when you first look at data-you have to start to talk with kiddos to find out what they need in our classroom" (vs , creative school). another answer that identified at a more practitioner level (item ) was: "i would say that most of the time (assessment) helps me to understand that a student may have to show me their answer in more than one way -not just a written answer" (vs , creative school). results for research question : how do the differentiation practices of vs teachers compare across diverse kinds of online schools? the results of the comparison of differentiation practices using these levels of differentiation are presented in table . the numbers in each cell represent the number of times that teachers in each school mentioned something that was coded as a definition, assessment, grouping, or strategy, and at which level. for all three virtual schools, the vast majority of teacher comments about differentiation definitions, assessments, curriculum, grouping and strategies fell in the novice category. teachers who made these statements showed evidence of being new or inexperienced to differentiated instruction. also, all three schools show a reduced, but still robust presence of comments in the apprentice column. this means that these teachers shared comments that showed that they knew more about differentiation of instruction and practiced it more than did novices, but still fell short of regular practice and expertise. an interesting contrast is found when comparing filmore virtual (district-based charter school) teachers' responses to that of the other schools at the practitioner or expert level for definitions, assessments, grouping, and strategies. filmore teachers had % of their responses coded at the practitioner and expert levels, while create and valentine virtual schools had higher levels of differentiation. however, filmore virtual school did show evidence of differentiation in the curriculum category at the practitioner and expert levels. another item that stands out in the data is that valentine virtual school appears to have much more expertise in their definitions of differentiation, which you might expect from the oldest statewide virtual school. also of note is that create virtual school appeared to have the most evenly spread distribution of comments about differentiation across the apprentice, practitioner, and expert levels of differentiation in all of the categories of differentiation. comparing differentiated instruction practices across schools by category is helpful in terms of considering some of the larger impacts of different types of virtual schools, but it does little to provide information on the more granular differentiated instruction practices of virtual school teachers. discussion on research question : how do vs teachers differentiate teaching practices in an online classroom? as stated previously, the large majority of teacher comments about differentiation definitions, assessments, curriculum, grouping and strategies fell in the novice category. also, all three schools show a reduced, but still robust presence of comments in the apprentice column. this falls in line with current statistics showing that virtual schools have grown both in numbers and in scope of offerings (gemin and pape ) . such large growth would most likely result in a large number of teachers who are untrained in online instruction. this also confirms research by kennedy and archambeault ( ) about the lack of teacher training to teach online, as well as the lack of training in how to lead and administrate instruction online. as seen in this study, this lack of confidence to differentiate is also in line with literature in the field. the most recent u.s. survey on preparation (bowsher, sparks, and hoyer, ) reported that % of teachers in their first year of teaching felt unprepared to differentiate instruction in the classroom. this same report noted that early career teachers need common planning times and regular support in order to increase in expertise. unless time is given for virtual teachers to receive professional development in differentiation, movement towards expertise may be impeded. many of the virtual school teachers' responses to curriculum in a differentiated classroom sought solutions that were already a part of their existing repertoire and had difficulty flexibly grouping. this confirms research by dixon, yssel, mcconnell & hardin ( ) that when adopting new strategies and techniques, teachers need multiple experiences to build efficacy. this literature suggests that novice teachers start by adopting new routines before working on more high preparation strategies or adjusting curriculum. it also supports research from the diffusion of innovations field that discusses the sometimes lengthy process that most people go through before they fully adopt an innovation as paradigm-shifting as the differentiation process (rogers ) . flexibility of instruction is another obstacle that is prevalent in the literature on differentiation (doubet ; tomlinson ; tomlinson et al. ) . in this study, vs teachers struggled to think of grouping strategies within the confines of their classroom. this study provided a look into the use of assessment in the differentiated classroom. it was revealing here to focus on what the few teachers who identified at a practitioner or expert level said about assessment. they focused on finding multiple means to assess student understanding and digging deeper to determine student needs. this corroborates research by tomlinson and moon ( ) that stresses assessment as imperative to differentiation. without knowing where students are, it is impossible to chart a personalized course for each student to reach the lesson's goals. although vs teachers were not utilizing assessment in the same way, all identified it as fundamental to differentiating instruction. discussion on research question : how do the differentiation practices of vs teachers compare across diverse kinds of online schools? a result that was mentioned previously was the comparison of filmore virtual (districtbased charter school) teachers' responses to that of the other schools at the practitioner or expert level for definitions, assessments, grouping, and strategies. this confirms previous research with virtual school teachers in the area of assessment (beasley & beak, ) . filmore teachers had % of their responses coded at the practitioner and expert levels, while create and valentine virtual schools had higher levels of differentiation. recalling that filmore virtual's is a new virtual school may help interpret this data. as a new virtual school that is district based, filmore virtual has used its own brick and mortar teachers as online course instructors. as a result, the relative inexperience of filmore's teachers with online learning may be the reason that they showed no evidence of differentiation at the practitioner or expert levels. however, filmore virtual did show evidence of differentiation in the curriculum category at the practitioner and expert levels. this may be due to a widespread belief among virtual school administrators that when a school first begins to provide online courses, there is overreliance on the online courses to differentiate instruction. this may also be due to research in differentiation that indicates that the act of differentiating is a highly nuanced skill that requires embedded training. tomlinson ( ) states: "for all its promise … effective differentiation is complex to use and thus difficult to promote in schools. moving toward differentiation is a long-term change process" (p. ). as a result, filmore virtual school teachers may be experiencing this long-term change process that tomlinson describes. to help move novice teachers along the continuum towards expertise, teachers may need to learn more about the strengths of virtual schools in finding ways to respond to student needs. in the beginning, virtual school teachers will need to know about and help students to take advantage of such things as accommodating schedules and a broader span of courses than other schools can potentially increase student engagement by pairing students with courses that are a good match for them while allowing them to take them at their own pace. they can also equip disabled students who have frequent medical appointments to attend virtual school classes and complete assignments on time. as teachers grow in their knowledge of differentiation, they can begin to learn more about how the community of virtual schools can act as a shelter for cps students who have experienced bullying. virtual school teachers can begin to weave community building, curriculum and assessment with flexible instructional decisions to accommodate for student differences. these advantages may be some of the reasons why virtual schools receive high levels of student, parent and teacher satisfaction compared to cps. another result mentioned that valentine virtual school appears to have much more expertise in their definitions of differentiation, which you might expect from the oldest statewide virtual school. many of the larger virtual school providers have defined programs of professional development for their teachers, and valentine virtual is included in this program (gemin and pape ) . they have a professional development (pd) program for all teachers that includes their own approach to best practices for online teaching. this pd program appears to have been successful in helping teachers understand the definition of differentiated instruction. this confirms what tomlinson and allan ( ) have suggested, that an educational leader may first want to assist teachers in defining differentiation as a first step to understanding what it is and is not. as virtual teachers begin to grow, leaders need to ensure that a common definition is identified. however, according to our data, this experience and training has apparently only been implemented successfully in the grouping category, but not in assessments, curriculum or strategy. although it is promising that valentine virtual teachers possess expertise in defining differentiation and in grouping, it is concerning that it hasn't impacted assessments, curriculum, or strategy, particularly because of these areas' proven impact on student achievement (baumgartner, lipowski, & rush, ; doubet ) , and particularly on student performance on standardized assessments (ahn and mceachin ; also tucker et al. ) . in the study, create virtual school appeared to have the most evenly spread distribution of comments about differentiation across the apprentice, practitioner, and expert levels of differentiation in all of the categories of differentiation. there may be many reasons for this even distribution, but one could be due to the hiring practices at create virtual school, which appear to focus on hiring more experienced teachers regardless of whether they have online experience. this hiring practice may have resulted in create virtual school teachers who are experienced with differentiating instruction in brick and mortar environments and who are learning to apply similar principles in online learning environments. it could also point to the ability to transfer differentiation skills across different kinds of learning environments, something that could increase quality of instruction in online schools, which is sorely needed (woodworth et al., ; cremata et al., ) . comparing differentiated instruction practices across schools by category is helpful in terms of considering some of the larger impacts of different types of virtual schools, but it does little to provide information on the more granular differentiated instruction practices of virtual school teachers. in this study, there were limitations that may have impacted the results. the small sample size of virtual classroom teachers does not allow the researchers to generalize across all contexts. another limitation was the instrument used to identify novices to experts in differentiation. the subjectivity of the aid when analyzing participant responses may have resulted in errors. finally, the lack of research in the topic does not allow for corroboration with other experts in the field. increasing the number of participants in the future may enhance the generalizability of the findings. research on the practice of differentiation is ongoing, both in the brick and mortar school as well as the virtual school. this research helped to reveal the differentiation practices of teachers across different types of virtual schools. it also made clearer some of the differences between the differentiation practices of expert online teachers and those who are less than expert. this is significant because little research has been done to discover the expert differentiation practices of online teachers. discovery of these practices should help to create professional development for all online teachers that is focused on building teachers skills in differentiation specific to the online learning environment. continued research on professional development in differentiation can reveal the connection between differentiation and increased online teacher efficacy (dixon et al. ) . previous research in virtual schools has found that teachers struggle to find ways to differentiate instruction. for example, it has shown that online teachers report that they need to differentiate content, product, and process, and also comprehend the importance of learner profiles. however, their practice contradicts their beliefs. actual practice shows that they do not use learner profiles as part of differentiation, and there is little mention of the use of assessment to inform instruction. the current study showed that newer virtual schools may struggle to acquire teachers who differentiate instruction at the practitioner and expert level. this points to a need to further investigate the instructional practices of the teachers who were practitioners and experts in the different categories of differentiation. future research should explore these expert and practitioner practices and seek to develop professional development interventions that are effective in helping teachers to differentiate instruction at these higher levels. a follow up study is planned where teachers whose responses demonstrated practitioner or expert levels of differentiation (tomlinson a (tomlinson , b will be invited to participate in a follow up interview that focuses on better understanding their expertise in differentiating instruction in a virtual school. the current study also showed that even experienced virtual schools may be effective in one area of differentiated instruction yet fail to deliver in others. other research has shown that obstacles to differentiation may be technological, instructional, administrative, and organizational in nature (aldossari ; doubet ) . future research should investigate what kind of obstacles may be hindering application of differentiated instruction in the areas of assessment, curriculum, and strategies, as well as considering potential solutions to overcome these obstacles. if curricular or technological obstacles to differentiation of instruction exist, curriculum and software designers should consider how to remove those obstacles while maintaining other development priorities. additionally, future research can examine how schools can partner with designers and developers of online curriculum to allow for maximum differentiation. student enrollment patterns and achievement in ohio's online charter schools interest, learning, and the psychological processes that mediate their relationship the challenges of using the differentiated instruction strategy: a case study in the general education stages in saudi arabia regular classroom practices with gifted students: results of a national survey of classroom teachers increasing reading achievement of primary and middle school students through differentiated instruction defining differentiation in cyber schools: what online teachers say supporting students during covid- : developing and leveraging academic communities of engagement in a time of crisis society for information technology & teacher education preparation and support for teachers in public schools: reflections on the first year of teaching supporting students during covid- : developing and leveraging academic communities of engagement in a time of crisis how people learn: brain, mind, experience, and school national charter school study: research report getting personalization right. the reading teacher differentiated instruction, professional development, and teacher efficacy teacher fidelity and student response to a model of differentiation as implemented in one high school (order no. ) keeping pace with k- online learning knowing what students know. the science and design of educational assessment ascending levels of intellectual demand within and beyond the parallel curriculum model troubling questions about online education. colorado: ednews. education and information technologies designing and implementing mathematics instruction for students with diverse learning needs it is good to talk': the focus group and the sociological imagination minnesota office of the legislative auditor virtual schools report the diffusion of innovation leadership for differentiated classrooms how to differentiate instruction in academically diverse classrooms let's celebrate personalization: but not too fast how to differentiate instruction in mixed-ability classrooms an educator's guide to differentiating instruction differentiating instruction in response to student readiness, interest, and learning profile in academically diverse classrooms: a review of literature assessment and student success in a differentiated classroom ohio e-schools: learning from their experience the misnomers of differentiating instruction in large classes individuals with disabilities education act (idea) database evaluating the impact of differentiated instruction on literacy and reading in mixed ability classrooms: quality and equity dimensions of education effectiveness evaluation of rocketship education's use of dreambox learning's online mathematics program the results of the replication of the classroom practices survey in two states online charter school study key: cord- -w wrwj authors: rothstein, rachel; olympia, robert p. title: school nurses on the front lines of healthcare: the approach to maintaining student health and wellness during covid- school closures date: - - journal: nasn sch nurse doi: . / x sha: doc_id: cord_uid: w wrwj in response to the novel coronavirus disease (covid- ) pandemic, most states in the united states enacted statewide school closures, ranging in duration from month to the remainder of the academic year. the extended durations of these closures present unique challenges, as many families rely on the school as a source of physical activity, mental health services, psychosocial support, child care, and food security. while the school doors may be closed, the school nurse can still play a vital role in emergency management. this article discusses challenges and proposes solutions to maintaining student health and wellness during extended school closures due to the covid- pandemic. furthermore, it is inevitable that until a vaccine for coronavirus is developed and readily available, many schools will continue to see future closures, though likely for shorter periods of time, as they respond to local outbreaks. what is the purpose of the "school nurses on the front lines of healthcare" series? the "school nurses on the front lines of healthcare" series will present cases reflecting emergencies commonly encountered in the school setting, focusing on an evidence-based approach to the initial management, stabilization, and disposition of the ill or injured child. special features unique to each article are extra credit points and report cards. extra credit points are trivia questions or clinical pearls scattered throughout the article related to the topic at hand. report cards are concise tables summarizing key points in each article that you can photocopy and laminate or photograph and keep on your smart device for easy access. during an otherwise normal afternoon in early march, your superintendent urgently assembles the school nurses, n asxxx . school nurses on the front lines of healthcare the approach to maintaining student health and wellness during covid- school closures principals, and other key faculty members, as well as local public health officials, to discuss the growing threat of covid- . your team ultimately decides to close the schools for the next weeks, effective immediately. shortly thereafter, your state governor declares a state of emergency and announces statewide closure of schools for a minimum of weeks. parents in the community begin contacting your team with concerns. many parents are essential workers and cannot obtain child care. others are concerned about food security, in the absence of school meal programs. the list of student health and wellness concerns continues. your school district is fortunate to have an emergency procedure in place for infectious disease outbreaks, and the school closure provides you and the school staff time to review and update your emergency and disaster plan, addressing many aspects of wellness for your students and their families. while the term social distancing is likely new to many, the concept of school closures as a tool for reducing spread of infectious diseases originated long before the covid- pandemic. during the - influenza h n pandemic, over schools, housing nearly , students, closed across the united states (klaiman et al., ) . while the current durations of school closures exceed those of other infectious disease outbreaks in recent history, consistent are the challenges to maintaining student health and wellness in light of closures. the national association of school nurses (nasn) recognizes the school nurse as a "vital member of the school team, who collaborates with community agencies to develop comprehensive emergency response procedures" (nasn, , para. ). likewise, the school nurse provides expertise in school health as a "leader and integral partner with school staff and outside agencies in developing comprehensive school plans/procedures" (nasn, , para. ) . nasn is continually providing up-to-date resources on covid- for school nurses, which incorporate emerging information from the centers for disease control and prevention (cdc) and other groups (cdc, b; nasn, ) . furthermore, the uncertainty surrounding future "peaks" of covid- or other infectious disease outbreaks emphasizes the importance of emergency and disaster planning. as school districts enter this unfamiliar phase of emergency management, the school nurse plays a critical role in addressing student health and wellness during school closures. the document, "considerations for school nurses when providing virtual care," highlights the role of virtual care in addressing student needs, continuing case management, providing resources to families, and mitigating health disparities (nasn, ) . nasn also provides, "guidance for school nurses to safely send and receive resources between school and home during covid- ," to ensure safe transfer of both student and school property, including backpacks, student medication, school supplies, electronic devices, and ongoing food services to socially and medically disadvantaged families (nasn, ). finally, "ideas for school nurse activities during the covid- pandemic" provides a list of activities that school nurses can complete in light of closures (nasn, ) . regarding school reopenings, nasn provides template letters, addressing state leaders, local leaders, and school superintendents, advocating for the inclusion of school nurses in planning processes (nasn, currently, sectors of the workforce qualify as "critical infrastructures" and are essential to the country's continued functioning, potentially leaving . million children, who otherwise would be in school, without adequate supervision (bayham et al., ) . while there is no definitive age at which it is appropriate to leave a child home alone, many resources are available to guide parents in this decision. leaving children home alone may present risks to both physical health and mental well-being. on the national level, the families first coronavirus response act includes a provision that expands protection for employees that during a public health emergency are unable to work due to a need for leave to care for their child because the school or day care has been closed or the child care provider is unavailable (moss et al., ) . similarly, the coronavirus aid, relief and economic security act includes a $ . billion expansion in funding of the child care and development block grant, to help states establish child care assistance for essential workers ( johnson-staub, ). examples of such interventions include temporary pandemic child care centers in ohio and regional enrichment centers in new york city. to assist your community's essential workers in obtaining child care coverage, view your state's emergency child care provisions and actions in response to covid- on the hunt institute website in table . also, the khan academy websites listed in table offer free online video classes, including music, games, crafts, and educational materials, for children to follow along while parents work or accomplish tasks in the home. the cdc recommend that all children aged to years participate in hour of moderate to vigorous physical activity each day, with an emphasis on aerobic activities such as walking, running, or biking, and that all children to years old participate in hours of physical activity daily, through activities spread over the course of the day (cdc, c). in addition to maintaining a healthy body weight and fostering musculoskeletal, cardiovascular, and mental health, physical activity in children is crucial to "self-expression, building self-confidence, social interaction and integration" (world health organization, , para. ). schools help children incorporate physical activity into their daily routines through participation in physical education (pe), recess, sports, and other extracurricular activities. as a result of school closures, children lose access to these structured and supervised sources of physical activity. the extended duration of school closures resembles summer recess, a period during which physical activity levels decline and sedentary time and body mass index rise (brazendale et al., ) ; unlike summer recess, the pandemic limits children's access to alternative sources of physical activity in the community, due to closures of public spaces such as parks, sports fields, tracks, and gyms. many families lack resources and equipment in the home for children to continue their usual activities. finally, social distancing limits the social interaction and bonding that physical activity often affords children. prior to the covid- pandemic, nearly % of children in the united states were overweight or obese, and children spent an average of . hours in front of a screen per day (brazendale et al., ; cdc, ) . both statistics are likely to rise in light of the pandemic. families now have a critical opportunity to adopt new attitudes and strategies to make physical activity a priority. to provide families in your community with outdoor physical activities that follow social distancing guidelines, visit the healthy children website in table . the online pe network website in table provides a list of grade-specific physical activities for the home, as well as monthly and weekly calendars to make these activities a part of children's daily routines. finally, in place of traditional screen time, the gonoodle website in table provides free online physical activity and mindfulness videos for young children to follow. approximately . million children in the united states participate in the national school lunch program, which provides discounted or free school lunches to children of low-income households (u.s. department of agriculture [usda], ). similarly, the school breakfast program provides school breakfast to nearly . million children in the united states. (usda, ). food insecurity disproportionately affects children residing in rural and large urban areas, as well as singleparent, black and hispanic households (no kid hungry, ). childhood hunger can contribute to malnutrition, difficulty concentrating, impaired academic performance, stress, poor social skills, and other adverse physical and mental health outcomes (no kid hungry, ). as a result of school closures, children lose access to these routine daily sources of meals. as of may , , children in these programs have missed a collective one billion school meals (no kid hungry, ) . to locate resources to help families in your community maintain food security, visit the no kid hungry and usda websites in table . even children residing in food secure households may encounter difficulty in maintaining healthy eating during school closures. for many children, school lunches provide the most nutritious meals of the day, by ensuring inclusion of fruits, vegetables, and milk. during school closures for summer recess, children typically consume fewer fruits, vegetables, and milk and more added sugars, saturated fats, and sodium (brazendale et al., ) . unlike summer recess, the pandemic presents unique barriers to obtaining healthy foods, due to grocery store closures, delivery delays, and item shortages, while access to "fast foods," which typically contain more saturated fat, sugar, salt, and calories, remain readily available. in efforts to expand reach during the pandemic, food banks are creating drive-through food distribution centers, preparing and distributing meals to children, prematurely opening summer meal programs, and keeping existing afterschool meal programs accessible. to locate a food bank in your community and to learn more about your local food bank's response to covid- , visit the feeding america website in table . to provide your community with strategies to make nutrition a priority during the pandemic, visit the nutrition website in table . to provide families with healthy eating tips, visit the cdc's website in table . for a list of healthy recipes that children enjoy, visit the academy of nutrition and dietetics' eat right website in table . the eat right campaign also helps parents find safe opportunities to engage their children in meal planning and preparation, which may help children become more curious about trying new foods. schools represent the largest providers of mental health services for children. of the % of children in the united states receiving mental health services, over % do so in school, specifically through school counselors, nurses, psychologists, and social workers (centers for health and health care in schools, ). in addition to counselling, schools provide crucial behavior management and crisis intervention services. as a result of school closures, children lose access to these critical resources. this is particularly concerning for children of racial and ethnic minority groups, who historically face greater difficulty in accessing mental health services. school closures also disrupt the structured daily routines that are critical for all children, especially those with underlying mental health and behavioral disorders. finally, in social distancing from peers, children lose access to a critical social support network. social isolation can further negatively affect mental health outcomes. school nurses searching for new strategies to address student mental health during the pandemic may schedule a zoom meeting with the crisis management institute ( ). the pandemic also presents unique psychosocial stressors, which may negatively affect the well-being of children and exacerbate underlying mental health disorders. children may experience additional home stressors, as parents face unemployment, economic hardship, and difficulty balancing work with their caregiver responsibilities. essential workers may experience greater stress in the workplace and spend less time with their children, which can negatively affect both the child's well-being and the parent-child relationship. physical activity, healthy eating, mindfulness, and sleep hygiene may all reduce psychosocial distress and improve well-being in children. to assist families in improving sleep hygiene, visit the child mind institute website in table . in addition to physical activity videos, the gonoodle website in table also provides online meditation and mindfulness videos for young children to follow. over seven million cases of child abuse and neglect are reported in the united states each year, four children of which die each day (safe horizon, ). professionals expect these numbers to increase in light of the pandemic, as household stress is a significant predictor of child abuse and neglect. in addition, victims have limited ability to seek refuge, due to closure of shelters, religious venues, and other community resources, as well as fear of contracting covid- in the emergency department. victims may connect with an advocate by visiting the national hotline website in table , by calling - - - or by texting loveis to . children may experience the illness, hospitalization, and death of a loved one from covid- . these hardships may be difficult to understand based on developmental age and can have long-term psychiatric consequences. to provide families in your community with guidance on how to help children cope with covid- , visit the nasn website in table . to engage young children in these discussions, age-appropriate covid- activity workbooks and coloring books are available on the cdc websites in table . children may also discuss covid- concerns with a crisis counsellor by visiting the crisis text line website in table or by texting home to . school closures significantly disrupt children's daily routines. literature on the structured day hypothesis demonstrates that healthy behaviors are "beneficially regulated when children are exposed to a structured day (i.e., school weekday)" versus weekends and summer recess, through, "compulsory physical activity opportunities, restricting caloric intake, reducing screen time occasions, and regulating sleep schedules" (brazendale et al., , p. ) . several nationally recognized organizations emphasize the importance of adhering to a structured schedule despite school closures. as an example, table displays a potential schedule for a late elementary school student, which incorporates resources mentioned in table . the khan academy website listed in table provides more comprehensive daily schedules for students in preschool to morning : to : wake up, get dressed, wash up, eat breakfast : to : start assigned schoolwork : to : play outside or complete an online pe network physical activity (table ) second grade, third to fifth grades, sixth to ninth grades, and th to th grades, as well as academic lessons/assignments by subject for students who are not receiving ongoing remote education during school closures (khan academy, ). finally, gasol foundation suggests creating themes for each weekday, to reinforce healthy behaviors and foster family time. examples on this website include "workout wednesdays," when family members participate in a family workout together, or "feelings friday," when family members gather to discuss their emotions surrounding covid- (gasol foundation, ) . families can utilize these resources to create a schedule that optimizes health and wellness during the pandemic. the return to school introduces new challenges to maintaining student health and wellness. several resources are available on the cdc's, "child care, schools and youth programs-plan, prepare, and respond," to assist schools, youth programs, camps sports and child care programs with the process of safely reopening or with maintaining safety for those that have already reopened (cdc, a). you visit the hunt institute website (table ) to learn more about your state's emergency child care provisions in response to covid- . likewise, you visit the no kid hungry and feeding america websites (table ) to locate resources to assist your community in maintaining food security. you disseminate tables and , as well as these state-and community-specific resources, to parents in your community. you receive feedback from several families that these tools assisted them in adopting a new and healthy routine, despite school closures. families in your community feel better equipped should the governor extend the duration of school closures. moreover, you feel better prepared, should your school district encounter intermittent "peaks" of covid- until a vaccine is developed and readily available, or an entirely new infectious disease outbreak occurs in the near future. if you have a clinical question, send your question to dr. olympia (rolympia@ hmc.psu.edu). questions will be selected and discussed as part of the "school nurses on the front lines of medicine" series. ■ robert p. olympia https://orcid .org/ - - - child care for essential workers during coronavirus outbreak understanding differences between summer vs. school obesogenic behaviors of children: the structured days hypothesis children's mental health needs, disparities and school-based services: a fact sheet screen time vs. lean time childcare, schools, and youth programs coronavirus disease (covid- ) frequently asked questions how much physical activity covid- resources for school counselors coronavirus quarantine: how covid- affects childhood obesity, healthy habits and vulnerable populations covid- and state child care assistance programs: immediate considerations for state ccdf lead agencies daily schedule for school closures variability in school closure decisions in response to h n : a qualitative systems improvement analysis the families first coronavirus response act: summary of key provisions coronavirus disease home page safe horizon national school lunch program global strategy on diet key: cord- - c qobr authors: pelaez, martha; novak, gary title: returning to school: separation problems and anxiety in the age of pandemics date: - - journal: behav anal pract doi: . /s - - - sha: doc_id: cord_uid: c qobr the shift to the postpandemic school environment will cause dramatic changes and is likely to increase separation problems. in this article, we look at the anxiety problems that some parents and their children might experience when school reopens after the covid- lockdown. using a behavioral theory of development, we provide suggestions for how to handle the departure and separation problems that may emerge as parents drop their children off at school. many parents are unsure about how to handle anxiety or fear as their children return to school or have to visit other environments outside their homes. social distancing has caused families to develop stronger dependencies at home and to create new routines that vary, in many instances greatly, from their prepandemic routines. families are adjusting to the new “normal.” they are keeping their children busy with schoolwork as best they can. in particular, families have likely developed close attachment relationships. families have been struggling with an unprecedented lockdown, and for many parents and their children, this extended period of family confinement and severe restrictions has been especially stressful, and the timing for returning to school is uncertain. we emphasize here that parents can be responsive to their children’s needs, plan ahead, provide reassurance, and depart firmly without vacillating, and we provide other tips to avoid inadvertently shaping children’s negative or anxiety behaviors as they go back to school. we offer some specific advice for parents and teachers to follow to prevent the departure and separation problems that typically develop during challenging behavioral interactions in school settings. the covid- virus invasion and its impact on human behavior have been quick and unprecedented. it has abruptly produced a series of significant challenges and opportunities for those who study human behavior. in this article, we look at some special implications for understanding its impact on children and how we care for them. we also look specifically at one aspect of this situation: departure and separation protests in young children (although older children, adolescents, and parents themselves may also experience separation issues). we view separation anxiety from the perspective of behavioral developmental theory. further, we make suggestions for how to deal with the young child's protests after requirements for social distancing are over. many parents are unsure about how to handle their children's anxiety or fear as their children return to school or have to visit other environments outside their homes. social distancing has caused families to develop strict home routines as they have been struggling with an unprecedented lockdown. for many parents and their children, this extended period of family confinement and severe restrictions has been especially stressful. but some families have adapted by creating constructive routines and learning environments for their children, maintaining a positive attitude, and trying to balance work at home with family time. others have fallen into more problematic patterns of behavior, such as coercive interactions. editor's note this manuscript is being published as part of a series of emergency publications designed to help practitioners of applied behavior analysis take immediate action to adjust to and mitigate the covid- crisis. this article was submitted on april , , and received final acceptance on may , . the views and strategies suggested by the articles in this series do not represent the positions of the association for behavior analysis international or springer nature. in this article, we stay focused on the separation situation and its effects on parents and young children by using the lens of behavioral development theory (novak & pelaez, ) . our treatment of separation protests is uniquely based on behavior-analytic research (gewirtz & pelaez-nogueras, , , . we conclude by offering specific suggestions about how to handle the separation difficulties that will inevitably emerge in some families upon returning to school. the covid- crisis came on quickly in the us winter and in the spring of , right in the middle of the school year. one of the immediate effects was the sudden disruption of the dynamic interactions among children and their care providers. in many communities, total social distancing was invoked with little warning. existing relations with teachers and peers were completely shut down, and families were left to scramble to find new ways to care for and educate their children as a replacement. our behavioral systems theory (bst; novak, novak, , novak & pelaez, ) incorporates developmental dynamicsystems concepts into behavior analysis. bst treats both schools and families as behavioral systems. in the case of schools, the ongoing reciprocal interactions among children and teachers and peers in the classroom develop into functioning systems that are highly efficient in producing reinforcers (both positive and negative). the same is true for the behavioral systems that children participate in at home with parents, grandparents, and siblings. these dynamic transactions take time to develop but become well attuned to the contingencies over time. a sudden and drastic change of conditions, as that which has occurred with the shutdown of schools in response to the covid- outbreak, means these well-adapted behavioral systems are thrown into chaos. that means many previously reinforced behaviors are no longer reinforced, establishing operations are no longer in effect, and once available cues for guiding the child (and parents) are now missing. consequently, the two systems have been disrupted quite decidedly. one way to look at the impact on behavioral development is to look at the disruptions they produce in all four terms of the behavioral contingency chain. in most u.s. households, time spent in school or preschool is a significant part of a child's everyday life. typical patterns of interaction emerged in the hours the child spent transacting with teachers and fellow peers. some of these interactions are primarily academic in nature, and nearly all of them involve social interactions. likewise, parents spent a significant part of the day at work, where they develop social interactions with coworkers and perhaps the public. in both the school and work environmental systems, participants develop strongly reinforced patterns of interactions based on behavioral contingencies and social rules. the new social-distancing rules in this age of pandemic have eliminated some of these former contingencies and created fresh ones. the conditions produced by the covid- pandemic serve as establishing operations or motivating operations for the development of attachment problems such as separation protest and anxiety. that is, the pandemic presents environmental variables that alter the effectiveness of stimuli, objects, or events as a reinforcer. among these possible motivational or setting conditions are the hospitalization of a parent or other family member, the unfortunate death of a parent or other family member, the worrisome tv news about the virus, the reports on job losses and a bad economy, and the negative reactions of others to the news. fear and anxiety can increase by watching continual updates on the covid- pandemic, and information directed at adults can cause anxiety and confusion, particularly in young children. all these experiences work to increase the general anxiety and fear of separation, leaving home and returning to school, and returning to a school environment that parents consider still unpredictable and uncontrollable. we offer some recommendations to parents on how to work with schools and teachers to cope with some of these emerging problems. although parent-child interactions were a part of wellestablished previrus family systems before and after school, with social-distancing policies, online virtual interactions have become nearly the sole source of social interactions outside the family. parental roles have also changed. no longer is the parent's role to support the child's learning in school. now, parents are the school. no longer do parents have to supervise their children for only a few hours before and after school. now they have daylong supervision responsibility. also, in cases where both parents are still working in essential jobs, stress is added to the situation by the need to secure childcare or education, with schools closed for half of the - school year and possibly the - . furthermore, the pandemic has placed additional stress on families by eliciting fear of contracting the illness and producing job and huge economic losses. we predict that one initial effect of the changes the pandemic has triggered is increased variability in behaviors of individuals in the family and school systems. largely, this variability occurs when formerly reinforced patterns of behavior are no longer reinforced because old system participants (e.g., teachers and peers) are no longer present. because of physical-distancing rules, for example, a friend's touch or hug may no longer be available to reinforce as it had in the past. thus, increased emotional withdrawal and frustration are also likely to result from withholding some reinforcers (i.e., extinction), which in turn further fuels response intensity and even more variability. together, these withdrawing conditions and the loss of reinforcement may evoke bursts of novel child responses that produce parental reinforcement during coercive interactions. put simply, the child may start acting out in exaggerated ways to obtain reinforcement (e.g., proximity, attention). unfortunately, we have evidence that many of these aversive child responses can be shaped by inadvertent parental responses that wind up strengthening undesirable child-parent behavioral interactions through negative reinforcement traps, as detailed in patterson's coercive family process (patterson, debaryshe, & ramsey, ) . one of the hardest decisions parents are making is determining when is the right time for their children to return to the classroom (summer or fall). the postpandemic situation is unique due to its length. the family interactional systems that have been reorganized and will newly emerge will have had a far longer time for patterns to be established. the scope of daily interactions is much broader than summer break and encompasses new educational responsibilities in addition to supervisorial and recreational ones. attachment and dependency are likely to be even stronger than the previous norm. therefore, what we are likely to see is another significant move of virus disruption in a highly efficient family system in which both parents and children are strongly attached and will have more difficulty adapting to the "new normal" systems in a postvirus era in schools. in the remainder of this article, we will utilize a behavioral systems approach to address this important issue, as we anticipate increasing problems when children return to their school environments. we will describe some of the research on the variables leading to separation protests and suggest actions that may reduce the number and intensity of these. parents' behaviors are likely to reinforce their children's proximity, attention, and affection. when the child's proximityand security-regulating behaviors become organized around a parent or caregiver, we say the child has developed an attachment (bretherton, (bretherton, , p. . by "organized," we mean that more than just individual responses occur. child attachment is characterized by dynamic patterns of behaviors reinforced by proximity to the parent or main caregiver. parents' behaviors, too, are reinforced by proximity to their children, and parents may become "attached" to them. thus, behavioral systems are bidirectional and reciprocal. parents are also liable to have heightened feelings of attachment and, if severe, may need additional help to treat behaviors characteristic of separation anxiety disorder that are described in the diagnostic and statistical manual of mental disorders (american psychiatric association, ). however, the focus of this article is on the prevention and compassionate management of young children's separation protests in returning to school. visit nearly any childcare center or preschool at the beginning of a new academic year and you are certain to observe young children's separation protests. these young children's protests typically take the form of whining, crying, throwing tantrums, clinging to the parent when leaving, and refusing to detach. separation protests are commonly used to measure security or insecurity of attachment and separation anxiety (kagan, kearsley, & zelazo, ) . we have emphasized that there is a possibility that the child's separation protest might develop as learned responses during interactions with primary caregivers (gewirtz & pelaez-nogueras, , , . gewirtz and pelaez-nogueras ( , , conducted several intervention studies on how mother-infant interactions influence the development of separation protests and distress. they applied the results of those studies to provide guidance to parents. in light of the covid- crisis, when children return to school and separate from their parents after a months-long period of -hr-a-day interaction that occurred during a period of social distancing, we predict that high rates of protest and distress may emerge, as well as high rates of refusal to leave the parents. this is likely to produce heightened child separation-protest responses that can disrupt classroom settings the new physical distancing rules, and provoke anguish and anxiety in parents. thus, the way that the departures and separations are handled by parents and teachers may have positive or negative consequences for all-children, parents, and school settings. researchers have shown that contingent maternal responding to separation protests can shape children's behaviors, such as whining, crying, and throwing tantrums (gewirtz & pelaez-nogueras, ) . they have further shown that separation protests could be reduced if parents differentially reinforced behaviors other than protest behaviors, such as playing with toys. although the research demonstrates how the family is a system, and although the caregiver is likely to shape the child's behavior in separation protests, the interactions have reciprocal effects. that is, although mothers and teachers can "inadvertently" reinforce the child's separation protest, the caregiver's behavior is likely to be negatively reinforced by reducing or stopping (at least in the short run) the child's crying/protesting and anxious behavior. specifically, the caregiver's attachment behavior can be shaped by terminating or escaping the aversiveness of his or her child's separation protest; simultaneously, the child's protest behavior is being shaped by the contingencies (e.g., touching, hugging, vacillating, talking, and overreassuring the child) provided by the parent returning to the protesting child in the classroom. few parents know how to cope with the crying and tantrums of their children, especially in situations in which they are getting ready to leave their children at school for the first time or when they will return to school during or after the pandemic. this lack of knowledge is completely normal. at the beginning of school, children's protests and cries can delay their parents' departures and cause them to return to school after dropping their children off. the research we discussed earlier shows that, during departures and at separation, parents' responses to children's protests can encourage and reinforce more of this distressing behavior. even more problematic is that when parents intermittently reinforce protests, these protests may escalate and get worse. in this section, we offer some advice for parents to follow to prevent maladaptive behavior interactions. the behavior-analytic view is that parental responses in this situation can encourage and otherwise shape the child's behavior patterns. unfortunately, our responses as parents often make it more difficult for our children to separate in the long term. we often inadvertently provide "misplaced contingencies" under such commendable intentions as providing a secure base or loving attention to our children and their needs. let us provide observations of what happens when a parent provides the "goodbye cues." a child's separation protests are routinely cued by a parent's preparations to leave the child in school or other settings. the behavioral cues that signal parental departure and separation can involve a mother picking up her car keys and purse, placing her baby in the crib or playpen while saying goodbye, or the arrival of a babysitter or caregiver. these cues serve as discriminative stimuli for child protests, as well as stimuli that evoke emotional distress in both child and parent. a parent may fear that the young child may not be safe in the school in this era of covid- . in older children, the cues would differ, and the parents may reason and provide explanations and a series of new instructions to the child. these new physical distancing rules may disrupt the existing family routine. we described this earlier as disruption and chaos in the dynamic system. departure-cued protests can include fusses, whines, whimpers, cries, and screams. in children with more advanced verbal and motor skills, protests may also involve grabbing the parent's body or clothes, clinging, pleading, or throwing a loud temper tantrum and refusal to leave the parent. these behaviors often cut short a separation from the parent. they also may further increase the emotional upset in the home setting. the parent's anguished responses to the child's protests can include delaying a departure, returning to hug the child, engaging in extensive reasoning with the child, vacillating during the departure, or hesitating to separate. the parent's anxious behavior may also include returning to school to pick up the child after having left the classroom in response to the teacher's call about a child's protest. often, parents return to touch, pick up, or hug and kiss the protesting child. these well-intended parental behaviors provide attention contingent on the child's protests, therefore strengthening them. in turn, during the transaction the child simultaneously trains the parent to behave this way, by stopping the protests contingent on the parent delaying departures, cutting short separations, or taking the child back home. thus, parents' behavior problems and anxiety responses can result from children's responses during the transactions. the serious situation of covid- virus transmission exacerbates all these scenarios, mainly because all the establishing operations or setting conditions it produces increase fear and anxiety of separation and the child getting infected. we discussed that parents' and teachers' attending reactions to infant protests during departures and after separations actually make recurrences of protests more likely in future situations. we also emphasized that intermittent reinforcement of protests will likely make them more resistant to extinction. the child's response bursts and variability are more likely to occur. parents with separation anxiety may have more fears and may want to check more frequently on their children. these parents may demand to know where their children are at all times by texting, making the teacher's job very difficult. in general, parents may have difficulties with separation from their children at all times, most specifically in light of the virus's risks of infection. to minimize separation distress, parents could program with teachers controlled visits to the child's classroom when the child has not recently protested and has remained there for varyingly longer periods. on each of these occasions, after parents drop off the child at school or classroom, the situation would be less stressful if the parent departs from the setting in a straightforward manner and is reassured by school personnel that the situation is under control. at the beginning of the separation episode, the classroom teacher in charge should provide reassurance to children that their parents will come back to pick them up at the end of the day. the new classroom routines and rules during and after the covid- pandemic should be established clearly and discussed ahead of time with parents and their children before they come back to school. some of the preliminary training can occur via virtual technology like zoom or telehealth techniques showing the new classroom arrangements and rules (e.g., handwashing routines, wearing face covers, physical distancing). it is important to keep in mind that parents and teachers should contact a professional if any child exhibits significant changes in behavior that suggest something else is amiss with the child that requires intervention from a child psychologist. going back to school after a prolonged and intense period locked down at home will require many adaptations to new environments, of which school is only one. in order to facilitate the shift from home to school, we offer several recommendations that parents can use to make parting less painful and anxious for their children. learn the new rules and routines of the classroom, and plan ahead. develop a plan for dealing with separation and new classroom routines and rules. involve the teacher's rules and plans in your own planning. discuss these with your child ahead of time. as a parent, you could write a description of your strategy for responding to your child's protests, e-mail it to the teacher, and invite the teacher to give input about how it fits into the classroom setting and how he or she may help you. at a minimum, this will keep the teacher informed of your intentions and concerns. listen carefully to the child. children give off verbal and nonverbal cues about their anxiety level. this will alert you to the likelihood of problems and may allow you to be especially reassuring and prepared for them. for younger children, while at home and talking to your child about returning to school, you might ask your child to make a drawing about his or her thoughts or concerns, and respond with truth and reassurance. model calm. be calm and relaxed. anticipate child protests and plan how to react calmly. model calm behavior during difficult situations, and "catch" your child being calm and richly reinforce it (see resources). create a less stressful environment. before your departure, ensure that your child is safe, relaxed, and positively engaged in an activity. use this to establish the conditions for a less stressful and safe departure. have the teacher greet your child in a warm and welcoming manner. check with the teacher about quickly providing a positive routine, such as helping prepare a snack or playing with a good friend. announce your departure. as soon as possible, follows physical distancing rules after arriving at the classroom, you should make sure the child follows physical distancing rules is welcomed into an activity (e.g., playing) and announce your departure to your child in a straightforward manner (e.g., by saying, "i'll be back for you later. i love you."). make the explanation clear and short. for younger children, you could simply say, "mommy has to go now, but i'll be back soon. have fun," give a kiss, and leave. for older children, you might also explain the departure ahead of time and indicate when you plan to return. reassure children that they are okay. however, once at school, such an explanation should be very short. do not negotiate. do not respond to protests with additional explanation or negotiation. negotiating runs the risk of reinforcing the child's protests. instead, focus on the positive. follow your plan, and leave without hesitating. depart without vacillating. you should leave the setting directly without hesitation. there is no need to appear rushed, but hesitating will only give the wrong cues and make it more difficult for you and your child. do not return. once you have left, you should not come back to pick up or talk to a protesting child. remember, occasional reinforcement of some protests will likely make the protests worse and make it harder for your child in the long term. behave consistently. have a routine that you continue to follow despite any protests. this predictability will make it easier for your child to learn that everything is normal. this will help your child integrate into the new daily postpandemic classroom routine. inconsistent experiences are likely only to produce more emotional upset and intermittently reinforce undesired behaviors. we would like to close our recommendations with a quote from the national association of school psychologists website: it is very important to remember that children look to adults for guidance on how to react to stressful events. acknowledging some level of concern, without panicking, is appropriate and can result in taking the necessary actions that reduce the risk of illness. teaching children positive preventive measures, talking with them about their fears, and giving them a sense of some control over their risk of infection can help reduce anxiety. this is also a tremendous opportunity for adults to model for children problem-solving, flexibility, and compassion as we all work through adjusting daily schedules, balancing work and other activities, getting creative about how we spend time, processing new information from authorities, and connecting and supporting friends and family members in new ways (nasp, ) . funding information the present study was not funded by any relevant grants or other funding sources. conflict of interest the authors declare that no relevant conflicts of interest influenced the nature of the present research investigation. ethical approval all procedures performed in studies involving human participants that participated in the studies cited that were conducted by pelaez et al. were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. informed consent this is a brief report, and no data were collected. informed consent in the studies cited in this article (by gewirtz & pelaez-nogueras, , , , was obtained from the parents of the infants who participated in those studies. specifically, if the parent's child met the characteristics for participation in the study and the parent provided consent-for-participation forms for both him-or herself and his or her child (via parental and child consent forms), the child would then begin the assessment procedures. the primary investigator was available and willing to answer any questions that the parent may have had regarding the study and his or her child's participation throughout every session. further, the consent form stressed that the parent may withdraw his or her child's participation at any time and that all participation was purely voluntary in nature. • coping with stress during infectious disease outbreaks: https://store.samhsa.gov/product/coping-with-stress-during-infectious-disease-outbreaks/sma - diagnostic and statistical manual of mental disorder patterns of attachment new perspectives on attachment relations: security, communication, and internal working models social-conditioning theory applied to metaphors like "attachment": the conditioning of infant separation protests by mothers the attachment metaphor and the conditioning of infant separation protests infants' separation difficulties and distress due to misplaced maternal contingencies leaving without tears: parents inadvertently train their children to protest separation helping children cope with changes resulting from covid- developmental psychology: dynamical systems and behavior analysis behavioral systems theory child and adolescent development: a behavioral systems approach a developmental perspective on antisocial behavior publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -odct kt authors: mcloughlin, gabriella m.; mccarthy, julia a.; mcguirt, jared t.; singleton, chelsea r.; dunn, caroline g.; gadhoke, preety title: addressing food insecurity through a health equity lens: a case study of large urban school districts during the covid- pandemic date: - - journal: j urban health doi: . /s - - - sha: doc_id: cord_uid: odct kt reduced access to school meals during public health emergencies can accelerate food insecurity and nutritional status, particularly for low-income children in urban areas. to prevent the exacerbation of health disparities, there is a need to understand the implementation of meal distribution among large urban school districts during emergencies and to what degree these strategies provide equitable meal access. our case study of four large urban school districts during the covid- pandemic aims to address these knowledge gaps. guided by the getting to equity (gte) framework, we conducted a mixed-methods study evaluating emergency meal distribution and strategy implementation in four large urban school districts (chicago public schools, houston independent school district, los angeles unified school district, and new york city department of education). we gathered data from school district websites on ( ) meal service and delivery sites and ( ) district documents, policies, communication, and resources. using qualitative coding approaches, we identified unique and shared district strategies to address meal distribution and communications during the pandemic according to the four components of the gte framework: increase healthy options, reduce deterrents, build on community capacity, and increase social and economic resources. we matched district census tract boundaries to demographic data from the american community survey and united states department of agriculture food desert data, and used geographic information systems (gis) software to identify meal site locations relative to student population, areas of high poverty and high minority populations, and food deserts. we found that all districts developed strategies to optimize meal provision, which varied across case site. strategies to increase healthy options included serving adults and other members of the general public, providing timely information on meal site locations, and promoting consumption of a balanced diet. the quantity and frequency of meals served varied, and the degree to which districts promoted high-quality nutrition was limited. reducing deterrents related to using inclusive language and images and providing safety information on social distancing practices in multiple languages. districts built community capacity through partnering with first responder, relief, and other community organizations. increased social and economic resources were illustrated by providing technology assistance to families, childcare referrals for essential workers, and other wellness resources. geospatial analysis suggests that service locations across cities varied to some degree by demographics and food environment, with potential gaps in reach. this study identifies strategies that have the potential to increase equitable access to nutrition assistance programs. our findings can support ( ) ongoing efforts to address child food insecurity during the pandemic and ( ) future meal provision through programs like the summer food service program and seamless summer option. future research should further examine the rationale behind meal site placement and how site availability changed over time. the novel coronavirus (covid- ) pandemic has brought unprecedented challenges to schools that were forced to close to prevent further exacerbation of infection. however, students' needs pertaining to nutritional supplementation through school meals persisted despite not attending school in-person. ensuring access to food during these critical times is of paramount importance to meeting the public health needs of vulnerable populations at greatest risk for food insecurity. further, as schools contemplate reopening plans, a greater emphasis is placed on ways to maintain practices developed over the closure period, protecting access to supplemental nutrition. federal nutrition assistance programs like the school breakfast program (sbp) and national school lunch program (nslp) help address child food insecurity [ ] [ ] [ ] and improve access to healthy foods [ ] . nearly million students participate in sbp and nslp daily [ ] , and % of sbp and % of nslp participants receive free or reduced priced meals based on household income, meaning their families do not have adequate means to purchase food in absence of safety net programs. for many students, school meals make up onethird to one-half of their caloric intake in a day [ ] . regular consumption of school meals can improve dietary intake; students who eat school meals every day consume more fruits and vegetables, fiber, and whole grains compared with those who do not [ , ] . accordingly, school meals provide a unique and rich source of nutrition for youth and may offset risk for overweight and obesity through mitigating food insecurity [ ] . children who are food insecure are at increased risk for obesity and diabetes [ , ] , two of the most common comorbidities associated with covid- hospitalizations [ ] . before the pandemic, one in three children and adolescents were diagnosed with overweight or obesity [ ] ; current school closures could exacerbate risk of obesity in childhood and increase disparities [ ] . ensuring the continuity of school meal programs during a national emergency [ ] is critical. in response to school closures and the ensuing threat of child food insecurity, us congress authorized support for school meal providers in covid- federal relief packages in march of . the united states department of agriculture (usda) responded by granting nationwide waivers allowing school districts to ( ) provide grab-and-go and home-delivered meals [ ] , ( ) expand the hours when they serve food [ ] , ( ) deviate from nutrition standards in the case of a supply chain disruption [ ] , and ( ) allow guardians to pick up meals without children present [ ] . the agency also waived the "area eligibility" requirement for the summer food service program (sfsp)/seamless summer option (sso) for states including california [ ] , illinois [ ] , new york [ ] , and texas [ ] , enabling districts to serve meals to low-income students where poverty was not as concentrated. under normal circumstances, usda only allows such meal provision where more than % of students qualify for free or reduced-price meals. with these waivers properly implemented, large urban school districts were in a position to effectively distribute emergency meals given that their operations already required complex food distribution, communication, and technology systems, and economies of scale can drive down costs in higher density populations. however, large urban districts typically serve predominantly ethnically, economically diverse populations in dense geographic areas. this creates distinct challenges given current social distancing recommendations. access is an important factor in establishing healthy dietary patterns. lower income and racial/ethnic minority children, who are more likely to live in areas with limited food access, may be particularly vulnerable to changes in access during disasters, including the covid- pandemic. an equitable emergency school meal system considers critical factors including availability (adequacy of the healthy food supply), accessibility (location and ease of getting to the food supply), affordability (food prices and perceptions of worth relative to cost), acceptability (attitudes about attributes of the food environment), and accommodation (how well local food sources accept and adapt to residents' needs) [ ] . there is limited research of these factors when schools are closed-during emergencies or during summer meal service-and more research is therefore needed. furthermore, the pressure on schools to re-open in the fall of is increasing, adding to the precarious state of school nutrition programs and meal service during the pandemic. data regarding how school districts implemented emergency school meal programming will provide a better understanding of "gaps in the food safety net" [ ] and help address areas for future improvement for schools as they consider reopening or continuing their emergency food service programming. accordingly, we sought to address two aims in this case study: . investigate the emergency school meal service strategies adopted by four of the largest school districts in the usa at the beginning of the covid- pandemic. . evaluate the degree to which districts promoted equitable access to emergency nutrition programming during the pandemic through a health equity lens. this case study focused on four of the largest urban school districts in the usa [ ] : chicago public schools (cps), houston independent school district (hisd), los angeles unified school district (lausd), and new york city department of education (nycdoe). these sites were selected based on ( ) location of the investigators' institutions (nycdoe), ( ) size of the school district (cps, lausd, and nycdoe are the three largest districts in the country), and ( ) seeking district case sites from diverse geographic locations (northeast, midwest, west, south) in the usa. furthermore, our ongoing collaboration with the urban school food alliance yielded information that houston was undergoing a reconstruction of their emergency school food response, warranting further study and investigation. table illustrates the demographic characteristics of these case study sites. to direct our assessment of each school district's meal distribution system, we developed an evidence-based set of guidelines for establishing an equitable emergency meal distribution system during a pandemic. since the onset of covid- in the usa, entities such as no kid hungry [ , ] have published information on best practices for providing meal service during school closures. furthermore, prior research on how emergency food supplies are deployed to address food insecurity during natural disasters [ , ] offers insight into this important issue. to our knowledge, there are no guidelines for evaluating equity in an emergency meal distribution system during a pandemic. we endeavored to address this need by developing guidelines that can inform future actions of urban school districts. this study was grounded in the getting to equity (gte) in obesity prevention theoretical framework to inform guideline development [ ] . the gte framework was designed to provide practitioners and researchers with strategies to increase the equity impact of policy, system, and environmental (pse) change interventions that aim to decrease obesity. the framework identifies opportunities for four key opportunities for intervention and action research that emphasize equity: ( ) increase healthy options (e.g., increase access to healthy food retailers), ( ) reduce deterrents (e.g., address threats to personal safety), ( ) improve social and economic resources (e.g., offer nutrition assistance programs), and ( ) build on community capacity (e.g., build strategic partnerships). using the gte framework [ ] , relevant scientific literature [ , [ ] [ ] [ ] [ ] [ ] [ ] , and information from various health-focused organizations (e.g., no kid hungry, feeding america, cdc, fda) [ , , , ] , we drafted evidence-based guidelines for increasing the equity impact of an emergency meal distribution system during a pandemic. guidelines were organized into four areas of action, similarly to the gte framework (see fig. ), and were used to direct a detailed evaluation of documents and media that describe each school district's meal distribution strategies. for the increasing healthy options domain, it was important to understand how meals were being distributed and promoted as a means to facilitate participation [ , ] , providing information on menu items and prioritizing healthy foods as an integral part of service [ ] , and providing as many meals as possible/feasible per collection period (i.e., breakfast and lunch, several days of food) [ ] . further, ensuring health and safety of children, families, and food service staff is an important component to maintaining access to emergency school meals [ , ] . in relation to reducing deterrents, literature and guidance points to the need to address barriers to accessing meals such as discrimination/stigma, language barriers, and safe access [ ] [ ] [ ] ] . as such, we were interested in the degree to which districts empowered children and families through inclusivity and attempted to reduce fears of discrimination through communication and outreach. the third quadrant, building on community capacity, was of particular interest, and through consultation of the literature, we sought to understand how districts collaborated with other organizations and networks to facilitate reach to children and families and promoted emergency school meals through these networks [ , , , ] . finally, we sought to understand how social and economic resources may be leveraged to facilitate access to school meals for those in greater need, such as provision of financial and logistical support to families [ , ] . high poverty is linked with greater food insecurity [ , ] , thus providing greater resources to families may help offset other costs, potentially mitigating the effect of covid- on food insecurity. we recognize that the sso and sfsp initiatives are developed with the sole purpose to offset food insecurity and thus are grounded following a health equity approach. the covid- pandemic has presented additional challenges, however, which warrant further study and thus our findings can be used to inform current and ongoing meal distribution during the pandemic. for this evaluation, we completed a comprehensive document analysis to characterize each district's emergency meal distribution system and to identify promotion strategies. we gathered information about emergency meal provision practices from each district website and social media pages, including press releases, food service policies and guidance, meal site information, and images between mid-march and mid-may . through such a process, we also were able to gather the number of meals served by each district over this time period. all documents were stored in a shared google drive folder to facilitate open coding. to capture overarching themes from documents and images, initial analysis was inductive, which allowed us to develop emergent open codes that were overarching concepts related to emergency school meal service [ ] . four members of the research team open-coded school districts' documentation separately and then met as a team to review open codes and discuss convergence/divergence. we then agreed upon an iterative coding strategy where one member led the initial document analysis and three members provided thorough member checking. using open coding, four research team members independently identified health and nutrition information for each school district's documentation. based on this independent document analysis of all four urban school districts, a research team member developed a list of open codes across the four domains of the gte framework. then, team members met to review all open codes and all school district documentation and reviewed and revised this list of codes [ , ] . this list was finalized to codes among the four domains (see table ). this iterative, team-based process facilitated a nuanced understanding of the factors that may impact emergency school meal service, and the degree to which district-level strategies could ensure equitable access to school meals. this approach enhanced integrity of the coding process but facilitated clear synergies to be identified to gte framework. the aim of the geospatial analysis was to understand the availability and accessibility of meal sites by census tracts. we reviewed school district homepages for covid- -related district location information. all districts included dynamic, searchable data on meal sites on one of these two pages. three districts included a link to a publicly available, interactive online map (lausd, hisd, cps) and one (nycdoe) included a drop-down menu of site locations. we used python code to download publicly available data (e.g., site name, address, hours of operation) from each district daily between april and april . meal site locations were triangulated using district documentation (e.g., press releases). for the spatial analyses, we either transferred meal site locations from mapped datasets or processed address information with the google maps application programming interface (api) through the batchgeo website and geocoded sites (mapped location from a physical address) to the highest level of positional accuracy possible. we decided to use food deserts as an important metric of food access because children living in these areas may have greater problems accessing healthier foods compared with non-food desert dwellers, and have a greater need for access to safety net programs like school meal programs in order to meet dietary recommendations. we joined the usda food desert locator dataset to a census tract boundary map file for food desert data. usda defines "food deserts" in urban areas as low-income census tracts where a significant number or share of residents is more than / mile from the nearest supermarket [ ] . we joined the food desert polygon data to meal site location point data. to identify meal sites relative to high poverty areas, racial minorities, and youth population (age - ), we used the american community survey, - , year estimates at the census tract level. we extracted census tract-level variables of "percent minority" (all non-white, including hispanic) and "percent poverty level" (income in the past months below poverty level, divided by total households) and joined these data to census tract boundary map files. we extracted the total population of children ages - in each census tract and used school district boundary files to select only the census tracts that fell within the school district perimeter. we joined the census sociodemographic data to the meal site location data and determined the number of meal sites per census tract. finally, we used univariate statistics to determine the prevalence (count and proportion) of meal sites in census tracts above and below the median census tract measure for each characteristic described above (i.e., percent poverty, percent racial/ ethnic minority, population - years of age). normally, cps serves around , meals a day [ ] . in april, the district served between , and , meals a day ( - % of typical service). for the week of th april, the same week used for gis data collection, lausd served between , and , [ ] ( - % of typical service), and nycdoe served between , and , meals to children per day ( - % of typical numbers) from its food "hubs." hisd, which typically serves , meals a week [ ] , distributed between , and , pounds a week in food boxes [ ] . after piloting different service concepts and strategies before spring break, all districts committed to models that remained the rest of the scheduled school year (see table ). a visual representation of availability and accessibility for nycdoe can be found in fig. . site distribution across cities varied, with two distinct strategies emerging. hisd and lausd operated fewer overall sites, but appeared to strategically place central collection points throughout the district. nycdoe and cps emphasized more sites and more localized coverage across district areas. for example, in the first weeks of school closure, cps served days' worth of breakfast and lunches from all schools [ ] . during spring break, cps provided grab-and-go meals from school sites. starting april th, the district consolidated food service at the schools with the highest participation before spring break. families could pick up days' worth of meals every weekday from : a.m. to : p.m. [ ] . hisd originally distributed meals from school sites in partnership with the houston food bank school market program [ ] but halted operations when staff were exposed to covid- [ ] . the district relaunched food service on april th, after spring break, distributing -pound boxes of food [ ] instead of meals. hisd continued to distribute boxes, daily (including weekends), from four to five locations across the city, depending on the day of the week [ ] . distribution times varied from morning to afternoon, depending on the site. lausd initially proposed opening family resource centers where children could study and obtain warm meals [ ] , but abandoned the plan before implementation. instead, on th march, the district announced grab-and-go food centers, open monday through friday from : a.m. to : a.m. [ ] . (as of th april, centers were open from : a.m. to : a.m.). working with the red cross, volunteers, and school food staff, lausd provided breakfast and lunch to families at pick-up sites. for the first week of school closures in new york ( - march ), nycdoe provided grab-and-go breakfast and lunches from each of its plus schools. the following week, starting rd march, nycdoce began providing students grab-and-go breakfast, lunch, and dinner monday through friday from : a.m. to : p.m. outside schools deemed citywide "hubs." the district continued to provide hot meals at "regional enrichment centers" for children of essential workers [ ] and launched a partnership with doordash to deliver tailored meals to medically vulnerable students [ ] . nycdoe temporarily closed sites for cleaning when staff fell sick and permanently shuttered others due to low participation. however, the general trend was to open more service locations; nycdoe operated approximately sites at the beginning of may. according to the gte framework, the two domains for increasing health equity through policy and systems change interventions are: "increase healthy options" and "reduce deterrents" to health promotion and healthy behaviors [ ] . the two key domains for building individual, household, and community resources and capacity are: "increase social and economic resources" and "build community capacity." our findings for urban school districts' responses to the covid- pandemic appear in table matched to the gte framework and are depicted in an adapted gte framework in fig. [ ] . districts' efforts to increase healthy options during the covid- pandemic varied by addressing barriers to accessing meals; offering a breadth of geographic eligibility and distribution of free meals, the number and quality of meals offered, and choices to serve children and adults; emphasized "healthy, nutritious" meals; and providing menus and nutrition information. each district took advantage of federal waivers to offer free meals during school closures: cps and lausd offered two free meals, and nycdoe offered three free meals a day. in contrast, hisd offered a bag of free groceries a week, with no apparent limit on site visits for families. all districts specified that food was available to students, but only three of the four used language that made it clear adults could also pick up food. for example, when still providing grab-and-go meals, hisd restricted the program to adults with children present and did not provide menus for the food boxes. all districts offered social distancing guidelines, but lausd and nycdoe emphasized the topic with specific reference to social distancing and wearing masks, specifically for staff. to reduce deterrents to health promotion and health behaviors during the covid- pandemic, districts provided grab-and-go meal locators and maps, up-todate information on site closures and the number of active sites, links to partner sites (i.e., food banks), and meal options for special or restricted diets (e.g., religious restrictions), as a proxy for addressing the fear of discrimination. for example, hisd partnered with houston food bank to distribute meals from convenient community locations, and lausd offered free meal distribution at homeless shelters. while nycdoe promoted kosher, halal, and vegetarian options, hisd was the only district that did not offer special or restricted diet options; cps did not list partner sites or offer vegetarian and gluten-free options. one important distinction to draw from houston's approach to meal service is that, providing large boxes of food (i.e., pounds) may present a barrier/deterrent for individuals seeking emergency nutrition, particularly if they do not have access to a car or other convenient transportation. providing multilingual information and resources was another way district leadership reduced deterrents for the inclusivity of diverse households; again, cps was the exception. hisd offered resources and educational materials in spanish and english; lausd in spanish, vietnamese, and english; and nycdoe in arabic, bengali, chinese, english, french, haitian, creole, korean, russian, spanish, and urdu ( languages). between april and april, the four districts had between and meal sites open daily. figure illustrates geographic depictions of meal site locations across nycdoe, distributed across census tracts coded for each variable of interest. table shows the distribution of meal sites in each district above and below the median census tract measure for each variable of interest. meal sites appeared to be in areas with highminority populations, except for cps. districts also appeared to place most meal sites in areas above the median poverty level. hisd had the greatest percentage ( %) of sites in areas of higher poverty, and nycdoe had the greatest number ( ; %). the maps reflect the trends in table , except for hisd and nycdoe. in hisd, only one high poverty area ( . % and above) contained a meal site, and in nycdoe, a considerable number of high poverty tracts did not have meal sites. images on school district documents and social media platforms that evoked equity and empowerment were another strategy to reduce deterrents. lausd used cheerful cartoon images of ethnically diverse children dressed in colorful clothes holding fruits and vegetables. nycdoe displayed smiling children of diverse additional maps for cps, hisd, and lausd can be found in an online appendix (https://wustl.box.com/s/z bs saixs rm k z yjkfrmsh qdlug). all districts employed varying degrees of health equity and empowerment language. cps stated that "everyone deserves to lead a healthy life" [ ] ; hisd emphasized the importance of "nutritional services mobilizing food safely" [ ] ; lausd called attention to an "outpouring of community support"; and nycdoe stressed "commitment to health and safety." districts also employed empathetic language to acknowledge families' hardship during the pandemic. lausd used explicit phrases like "support families in need" [ ] and new york devoted a webpage to "messages to families," which included memos from the mayor discussing the difficult time, the need to "come together," and that "no one can be turned away" [ ] . together, these districts' images and language indicate a concerted effort to promote diversity and inclusion in communication. districts built on community capacity by building community partnerships [e.g., establishing programs to support first responders and their families, fundraising, partnering with community-based organizations, and distributing staple foods (hisd only)]. hisd partnered with the regional food bank and the police department to distribute free food in communities. in addition, lausd partnered with the red cross to help distribute meals, and promoted a list of local wellness centers/school clinics available on the resources website to enhance awareness of children/ families. nycdoe also provided information on local community resources, such as regional enrichment centers which were available specifically for children of first responders, healthcare workers, and vulnerable populations. cps also took this initiative and provided resources for parents who worked as first responders pertaining to childcare and emergency nutrition opportunities. all districts promoted healthy behaviors by placing a majority of meal sites in tracts with above-median juvenile populations. cps placed the highest proportion of meal sites in these areas (n = , %), whereas hisd had the lowest proportion with ( %) of their meal sites in census tracts above median juvenile population. placement of these sites has implications for reach and participation in emergency school food service programming. urban school districts offered a wide range of resources for families impacted by covid- , beyond nutritional cells represent count and (percent) of total meal sites either above or below the district median note: n, number of meal sites per district a corresponding median measure for each district supplementation. all clearly communicated weekday hours to access free meals, promoting visibility of the school meal programming. hisd also offered weekend hours for adults and nyc provided afternoon hours for adults who needed food. further, all school districts provided access to wellness programs or centers; wellness resources can reduce deterrents such as discrimination and social exclusion [ ] . moreover, although indirectly related to food service, all districts provided technology, such as ipads and chromebooks to students to facilitate distance learning, in addition to wi-fi through partnerships with local/national companies. this may therefore offset financial barriers experienced by families in the covid- pandemic and increase their ability to access up-to-date information about meal sites. in usda-defined food deserts, hisd placed the greatest proportion of meal sites (n = , . %), whereas nycdoe placed only five meal sites ( . %). meanwhile, both cps and lausd placed roughly a third of sites in food deserts. finally, other considerations for all school districts in light of global crises such as covid- are local policies and programs that can increase resources to low-income households, beyond the school system. these include the federal stimulus plan, access to food assistance programs, and local efforts such as raising the minimum wage. using an innovative mixed methods approach, this study investigated approaches to covid- pandemic school meal service among four of the nation's largest urban school districts, and how strategies could ensure equitable access to supplemental nutrition during the pandemic. nutrition assistance programs such as nslp and sbp can reduce food insecurity and help prevent child obesity [ ] . understanding strategies to assure program access during emergencies is critical to promoting equity. in relation to increasing healthy options, districts used multiple strategies to encourage participation, but meals reached only a portion of the normal student population. there were no clear patterns to suggest whether the number or kinds of meals served, location and number of distribution sites, or time of day affected participation. nycdoe had the lowest average participation rate, which may have more to do with covid- prevalence and larger structural barriers (a factor for future study), than with specific distribution practices, or both. moreover, it was the largest district and the most difficult to navigate without public transport compared with lausd or hisd where cars are more common. it is important to consider the nuanced challenges presented in the covid- pandemic that may have previously been considered as strengths, such as predominant use of public transport and dense populations of urban context. notably, all districts deviated from initial distribution plans, suggesting that districts have much to learn when it comes to meal distribution during school closures. all districts took steps to increase access to healthy options; they provided at least one meal per day for students, displayed food safety information, and advertised that all children could eat for free regardless of district enrollment. but there were differences in the number of free meals provided, populations served, and communication about menu options. during school closures, not all districts published menus, making nutritional quality of foods unclear. these differences may impact food security, given strong reliance on school food programs in urban contexts [ ] . to reduce deterrents to participating in emergency school meal programs, hunger and nutrition advocacy groups encourage districts to provide multiple days' meals, serve adults, and post menu information. given the strong links between food insecurity and child/ adolescent obesity [ , , ] , the importance of school meal nutritional quality cannot be understated. it is highly likely that some form of emergency meal service likely to continue in the - school year and districts should strive to provide nutritional information and promote consumption of high-quality foods. districts should also strive to provide more information in multiple languages representing all the relevant cultures and nationalities to reduce deterrents to health, particularly fear of discrimination and other inequities confronted by diverse households. all districts provided interactive maps to display meal locations and hours, potentially making it easier for families to access school meals. in regard to building community capacity and improving social and economic resources, all districts had some form of building community partnerships including collaborating with first responder programs and promoting relief fundraisers. developing an inclusive community culture and partnerships are methods national anti-hunger organizations such as share our strength no kid hungry campaign [ ] recommend to improve meal distribution and ensure equitable access to nutrition. partnerships not only build community capacity, they can also help improve social and economic resources. for example, hisd's partnership with houston food bank facilitated weekend meals and outreach efforts to families. weekend meals can increase children's and families' food security [ ] . given the economic crisis that resulted from the pandemic, additional weekend meals may be a more important factor than ever. moreover, due to the significant economic crises of the covid- pandemic, it would also be essential for researchers to consider factors beyond the school system, such as equitable access to federal stimulus checks and local policies to aid low-income households through raising the minimum wage, for instance. further research is needed to understand how these macro-level factors may play a greater role in health equity in the coming months, particularly as schools begin to re-open and changes to emergency meal distribution manifest. although our study is the first to look at the spatial distribution of pandemic school meal site locations in large urban areas during the covid- pandemic, our results could be interpreted in the context of previous work examining meal site placement during school closures. one previous study examined the placement of summer meal sites [ ] and found that urban summer meal sites were more prevalent in larger, higher poverty, and majority non-white high schools. we found similar patterns during the response covid- pandemic, with more sites in the larger, higher poverty and higher minority areas. unlike summer meal sites, which are required to operate in low-income areas, where at least half of resident families' income falls below % of the federal poverty level [ ] , meal sites opened and operated during the covid- pandemic were not required to meet this same criterion. in this case, we did expect to see some level of variation in meal sites across census tract income levels, compared with the placement of summer sites. meal site placement that remained consistent with placing sites in areas with higher economic need is promising and could be, at least partially, based on a site or school's previous experience as a summer meal site. future research should include evaluations of how districts used these variables of interest or other equitybased considerations in their decisions concerning meal site locations during covid- , especially as areas of high poverty appeared to be the most consistently prioritized across the four cities. such an approach for future studies could be based on the idea that lowincome populations often have lower access to healthy foods, which is supported by previous research [ ] . with regard to food access, we found that sites were more equitably distributed based on demographic characteristics rather than food desert status. focusing on demographic rather than food environment characteristics could leave children living in food deserts without adequate access to healthy food, so further consideration should be given to food assistance and food access data. in this research, we focused on the availability of meals sites, but other dimensions of food access may be influential [ ] and should be examined in future studies. this study uses a novel equity framework to classify elements of intervention design, delivery, and contextual factors influencing provision of emergency healthy meals during unplanned school closures. these districts represent multiple regions of the country: the east coast (nycdoe), midwest (cps), south (hisd), and west coast (lausd). this study also employs a rigorous mixed methodology for document analysis and geo-spatial mapping. it analyzes school districts' efforts while the usa is in the midst of an unprecedented pandemic. our findings are currently relevant for school meal and other nutrition assistance providers given the persistent threat of covid- . congress has mandated that states submit a report to usda describing districts' use of school meal waivers year after this emergency. paired with state reports, this study can inform future plans for public health emergencies and accelerate the implementation of school meal service. we must also acknowledge some limitations of this study. recommendations to social distance and the time constraints the public health emergency created for school meal providers limited data collection. we relied completely on publicly available data such as websites and social media, and did not recruit participants for primary data collection to avoid burdening food service management and other school staff and stakeholders during such a critical time. although we collected information over a prolonged period (i.e., weeks), we plan to conduct follow-up research in collaboration with other research teams to identify the ways in which school districts approached their emergency school meal programs and deviations from initial plans. finally, although we chose urban districts in each major region of the usa, findings are not generalizable to other diverse urban districts. instead, with the data available, we were able to identify and characterize each district's public efforts to provide meals during school closures. this case study highlights salient challenges and successes within four urban communities, but more work is needed to address school food practices. while this study focused on urban areas, suburban and rural districts' responses also warrant study. there is also a need to understand the current use of geospatial data to determine the location of meal sites, how the location of sites changed over time, and what opportunities there are to better integrate such data to improve reach of emergency school meal programming. future studies could also examine centralized versus more widespread site distribution; the way districts distributed sites suggests an intentional, although varying approach to address food access issues. due to how meal distributions differed (i.e., number of meals per day and days of food), we could not establish whether more sites led to better reach. therefore, it may be beneficial to study the differential impact on population accessibility to school meals, as well as the impact on pickup, district purchasing, and dietary patterns. our study focused on food deserts rather than other food environment conditions such as food swamps due to the potential exacerbation of food insecurity due to meal site location for vulnerable children living in areas of low access to healthy food, but the relation of meal sites to food swamps, particularly as a competition for school meal site usage, should be a focus of future work. finally, and most importantly, additional work should be conducted to contextualize stakeholder perspectives on the distribution of meal sites, both from the perspectives of decision makers (i.e., those individuals or groups responsible for identifying meal site locations and approaches, developing and disseminating information about sites, and evaluating the relative success or failure of approaches) and of community members, program recipients, program collaborators, and decisionmakers (e.g., anti-hunger organizations, youth empowerment organizations, and local economic policymaking). for urban school districts, one future direction may be to develop and maintain partnerships with community organizations to prevent food insecurity during school closures due not just to pandemics or natural disasters, but also weekend and holiday closures. research suggests community organizations like food banks are valuable stakeholders in preventing food insecurity and insecurity-related chronic disease [ , ] , so sustained partnerships between these two entities may help reduce food insecurity. as schools begin to reopen in the fall of following pressure from federal agencies [ ] , we strongly urge equity to be placed at the forefront of decisions pertaining to school emergency food services. given the detrimental impact of covid- on the economic landscape of the usa and subsequent surge in food insecurity prevalence [ ] , continuation of meal sites that serve meals to students and families may serve as a vital strategy to offset ongoing depravity. this will be especially important for families/children at a greater risk for contracting covid- who wish to remain at home and/or for parents who still cannot return to work through job loss or ongoing furlough. further, depending on the school schedule and whether schools opt for a rotation system to minimize attendance and asking students to be at school on certain days of the week, continued operation of walk-up and drive-up meal sites would facilitate equitable access to supplemental nutrition despite students not attending schools full-time. continuation of various waivers granted by the usda may therefore be necessary to combat rising food insecurity rates and ease the transition of going back to school amid the pandemic. the findings provide a unique perspective to promote equitable food access to students and families during a public health crisis. school meal, summer meal, and emergency food service providers can use the findings to promote participation, especially among nutritionally vulnerable populations. the need to address urban communities' food security challenges by providing equitable access to meals during a public health emergency cannot be understated, and our study is one of the first to address food security issues during covid- . findings provide valuable insights for future meal provision both during public health emergencies and ongoing federal interventions to combat food insecurity in children and households. household food security status of families with children attending schools that participate in the community eligibility provision (cep) and those with children attending schools that are cep-eligible, but not participating the relationship between the school breakfast program and food insecurity united states department of agriculture. food security in the us children's food security and usda child nutrition programs school nutrition and meal cost study, final report volume : school meal program operations and school nutrition environments schools and obesity prevention: creating school environments and policies to promote healthy eating and physical activity eating school meals daily is associated with healthier dietary intakes: the healthy communities study eat smart, move more in schools: a comprehensive program to improve the school food environment outside of the nslp developed through a multi-state collaboration impact of the healthy, hunger-free kids act on obesity trends the association between food insecurity and obesity in children-the national health and nutrition examination survey food insecurity is associated with diabetes mellitus: results from the national health examination and nutrition examination survey (nhanes) - presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the new york city area prevalence of childhood and adult obesity in the united states covid- -related school closings and risk of weight gain among children food security information network (fsin) nationwide waiver to allow non-congregate feeding in the child nutrition programs nationwide waiver to allow meal service time flexibility in the child nutrition programs nationwide waiver to allow meal pattern flexibility in the child nutrition programs mealpatternnationwidewaiver final.pdf &sa= d&ust= &usg=afqjcngcbxhd mpqpye v-arqkjp orw. accessed nationwide waiver to allow parents and guardians to pick up meals for children e a e l i g i b i l i t y -a p p r o v a l . p d f & s a = g = afqjcngrbocrlivjlbrg_c cgbwadyjcfw e a e l i g i b i l i t y -a p p r o v a l . p d f & s a = g = afqjcnfpyaxnlqug xkvvwqhotgf ars q o m / u r l ? q = h t t p s : / / f n s -p r o d e a e l i g i b i l i t y -a p p r o v a l . p d f & s a = g = afqjcnhaikuwghaksqcdjeakfimvdluzqg the local food environment and diet: a systematic review identifying gaps in the food security safety net: the characteristics and availability of summer nutrition programmes in california, usa. public health nutr top largest school districts by enrollment and per pupil current spending summer meals emerging strategies and tactics for meal service during school closures related to the coronavirus emergency food during natural disasters. today's dietician relevance and uses of the getting to equity in obesity prevention framework a framework for increasing equity impact in obesity prevention impact of covid- on clinical research and inclusion of diverse populations feeding low-income children during the covid- pandemic ensuring equitable access to school meals violent crime and park use in low-income urban neighborhoods longitudinal associations between change in neighborhood social disorder and change in food swamps in an urban setting complete eats: summer meals offered by the emergency department for food insecurity tisch center for food nutrition & and policy. serving students during the coronavirus: best practices best practices for retail food stores, restaurants, and food pick-up/delivery services during the covid- pandemic impact and implementation findings from an experimental evaluation of playworks: effects on school climate, academic learning, student social skills, and behavior severity of household food insecurity is sensitive to change in household income and employment status among low-income families document analysis as a qualitative research method demonstrating rigor using thematic analysis: a hybrid approach of inductive and deductive coding and theme development chicago public schools. cps stats and facts ashx?moduleinstanceid= &dataid= &filename= - factsfigures.pdf. accessed hisd to relaunch food distribution efforts on april with centralized packing hub all cps schools to offer free meals for pick up during closure beginning tuesday a special message from interim superintendent of schools grenita lathan community impact newspaper. houston isd abruptly halts food distribution program after suspected covid- case houston isd plans to restart food distribution at five campuses next week covid- : children, families & schools what to do while los angeles unified schools are closed in response to the coronavirus: grab & go food centers distribute free meals to students doordash partners with department of education to deliver food to medically homebound students. a m n e w y o r k new york city schools meal sites school closures during covid- : opportunities for innovation in meal service a reach, effectiveness, adoption, implementation, maintenance evaluation of weekend backpack food assistance programs united states department of agriculture neighborhood environments: disparities in access to healthy foods in the u.s operating school meal programs in rural districts: challenges and solut i o n s publications/ _the_journal_of_child_nutrition_and edsource. california food banks partner with schools to serve families of students where some of the country's biggest school districts stand on reopening schools country-driven' approach needed to limit covid- damage to food security acknowledgments we are grateful to both healthy eating research, a national program of the robert wood johnson foundation, and the united states department of health and human services centers for disease control and prevention-supported nutrition and obesity policy research and evaluation network (nopren) for their support of an ad hoc joint covid- school implications working group and to nopren for fellowship support (gm, cd). key: cord- -jrd hia authors: mccleary, daniel f.; clark, frankie j.; dawes, jillian; flowers, jaime m.; ellis-hervey, nina title: going digital to address the school psychologist shortage date: - - journal: contemp sch psychol doi: . /s - - - sha: doc_id: cord_uid: jrd hia given the shortage of school psychologists, especially in rural areas, school psychology programs are beginning to emerge that provide students with the opportunity to attend classes through distance education (de) methods (i.e., virtually). despite many school psychology students and faculty holding negative perceptions of de methods, the use of de is expanding within the field of school psychology. this article provides an overview of existing de programs and existing de research, specific to school psychology, followed by a brief history of a de option at stephen f. austin state university that began in fall . solutions to common issues encountered with de are also presented. the field of school psychology continues to experience a shortage of practitioners and trainers (brock ; castillo et al. ; fagan ; reeves ; savage ) . dixon ( ) began a series of articles in the communique asking other school psychology practitioners to discuss the role online education could play in addressing these shortages. institutions such as eastern washington university (ewu), the university of calgary, wayne state university (wsu), fort hays state university, and the university of houston-victoria (dixon et al. ; drefs et al. ) already provide instruction to school psychology candidates through a variety of predominately distance education (de) methods (e.g., online, hybrid/blended). the purpose of this article is to provide a brief history of de programs, a summary of published de research findings within the field of school psychology, information about a de option provided by stephen f. austin state university (sfa), and recommendations for common issues associated with de. although this paper was originally written for programs contemplating incorporating de into their program, searching for ways to address the school psychology shortage, or address low enrollment numbers the information is also useful for programs forced into providing de options due to terminology de and its different modalities (online, hybrid/blended) come with a slew of terms that may be unfamiliar or, at times, difficult to discern differences between. for an orientation to concepts and common terms related to de, readers are encouraged to familiarize themselves with the concise work of fischer et al. ( ) , moy et al. ( ) , and moy et al. ( ) . in this paper, the term "face-to-face in-person" is used to refer to instruction physically occurring in the same room. the term "face-to-face virtually" refers to instruction wherein the instructor and some students physically meet in the classroom while other students attend the same classroom through virtual means (e.g., zoom). the first known school psychology program to offer de was ewu, which began offering a certificate in before transitioning to an online ed.s. program in (ruby et al. ) . although an online program, ewu requires students to attend functions on campus to interact with faculty and other students (for a full description, see waldron-soler et al. ). in , canada's university of calgary began an m.ed. online school psychology program that also required students to be on campus for specific components of the program (for a full description, see drefs et al. ) . fort hays state university started a school psychology program in that only requires students to physically attend a five-day workshop (dixon et al. ; fort hays state university n.d.) . since at least , the university of houston-victoria has provided individuals an opportunity to earn a specialist in school psychology (ssp) degree by completing approximately % of the coursework in an online format and the other courses either face-to-face in-person or through interactive television (dixon et al. ; hendricker and viola ) . beginning in , wsu began offering a m.a. in school and community psychology online via synchronous classes that include on-campus students. like other de programs, wsu requires students to attend campus about one evening per semester (dixon et al. ; wayne state university n.d.) . hendricker et al. ( ) surveyed school psychology program directors about de perspectives. they reported - % of school psychology programs provide instruction through some form of de (i.e., online, hybrid/blended) despite it not being advertised in program handbooks or on websites. of those programs, % provided de in theory courses and % reported de occurred in courses contained within the program but taught by non-program faculty. furthermore, the overwhelming majority of programs containing some form of de reported that neither the quality of the courses nor the program outcomes (i.e., praxis scores, graduation rates, employment rates) decreased after switching to de content delivery methods. viola et al. ( ) surveyed school psychology graduate students regarding their perceptions of the use of de within school psychology graduate programs. in general, students expressed more favorable opinions on hybrid/blended courses than online courses. only % of the participants perceived predominately online courses to be as beneficial as traditional face-to-face classes, whereas % believed hybrid/blended courses to be as beneficial as traditional classes. participants who had taken online and hybrid/blended courses reported disadvantages to include lack of interaction with others, fewer mastered skills, and reduced quality of instruction. nonetheless, participants who had taken online and hybrid/ blended courses in school psychology reported appreciation for the flexibility and convenience remote instruction allows, which provided them the opportunity to reach their educational goals. more recently, fischer et al. ( ) surveyed school psychology program faculty and found approximately % of school psychology programs include some form of de instruction. this is a notable increase from the - % de instruction reported by hendricker et al. in . similar to the results found by hendricker et al. ( ), fischer et al. ( reported de courses were more likely to be theorybased than skill-based (e.g., assessment) and faculty were more favorable of hybrid/blended courses than online courses. it is also worth noting that approximately % of school psychology faculty reported having plans for additional online course instruction within the next four years. despite expressed concerns from program directors, faculty, and students, published data from the university of calgary, university of houston-victoria, and ewu indicate students and alumni of de programs are adequately trained and prepared for the workforce (drefs et al. ; hendricker and viola ; ruby et al. ; schroeder ; waldron-soler et al. ). the school psychology program at stephen f. austin state university (sfa) has both an m.a. and ph.d. program. sfa is a regional university that is located in nacogdoches, tx, near the border of louisiana (i.e., east texas, tx). nacogdoches is in a rural area with approximately , residents (suburban stats ). the city is miles from houston, tx, and miles from dallas, tx. sfa has an overall student population of approximately , . the school psychology program at sfa is unique and needed due to the rural location. there are a total of school psychology programs in the state of texas and the nearest to the sfa campus is approximately miles away (grad schools ). the location also has a shortage of clinical and school mental health professionals; however, many of the students admitted to the program express interest in either serving in the local community or neighboring rural communities. this allows the program to create opportunities for the community to expand the reach of mental health professionals, specifically qualified to work in school systems. the m.a. in school psychology program was authorized by the texas higher education coordinating board (thecb) in . it was first approved by the national association of school psychologists (nasp) in . the master's in school psychology program adheres to the state requirements for licensure as a licensed specialist in school psychology (lssp) and the national training standards for certification as a national certified school psychologist (ncsp). the training requires semester hours, which includes an internship. the mission of the program is to ensure students learn to be ethical, responsible, and competent school psychologists. the program approach and teaching are that of the practitionerscientist/scholar. success refers to accomplishment of not only immediate goals but also long-range goals such as maximizing personal potentials, social integration, meaningful work, and contributing to society (stoltenberg et al. ) . faculty have various backgrounds and credentials including those holding the lssp, ncsp, bcba, and/or licensure as psychologists. faculty are dedicated to ensuring candidates develop high levels of interpersonal and collaborative skills and apply cultural competence and sensitivity to diversity as they fulfill their professional, legal, ethical, and social roles in education and society. students also have access to, and some training in, clinics and centers associated with the school psychology program including the human neuroscience laboratory, the janice a. pattillo early childhood research center, and the school psychology assessment center. due to the program location and need for more school psychologists in the local community and neighboring rural communities, the sfa school psychology program created a de option in that is delivered via zoom. it was designed for students at least miles away from the sfa nacogdoches campus. applicants must meet the same admission criteria as face-to-face students in the m.a. program and have the ability to attend courses via zoom video conferencing (i.e., strong internet connection, webcam, microphone). students attend classes via synchronous de. classes are physically conducted on the sfa campus with face-to-face students and de students attending virtually through zoom. in our experience, having de courses within the traditional classroom with face-to-face students attending allows live discussion to occur as it traditionally would. in addition to the standard program admission requirements, applicants must meet the criteria to be eligible for admission. the first requirement is that applicants' permanent address must be a minimum of miles distance from the university campus. additionally, applicants agree that if accepted, they will be able to meet the following criteria: & students must attend courses in real-time, with face-toface virtual conferencing via zoom. & some courses and meetings may be required on campus. students will be notified at the start of each semester dates they will be required to be on the university campus. & some courses require assessment materials. for these courses, students must be able and willing to work with an education region service center in texas (texas education agency n.d.) or local schools for access to testing kits or travel to campus to check out and return the materials from the program. & students will be responsible for all course assignments and program requirements, including thesis, practica, and internship. & in the event of a technology issue of the instructor's/ university's fault, the instructor will work with the student to ensure appropriate instruction is provided. if a technology issue of the student's fault occurs, the course policy for class absences will be followed according to the syllabus. students are admitted to cohorts that include campus-based and distance learning students. during the course of the program, de students are required to attend select activities on campus. these requirements are intended to familiarize de students with program faculty, on-campus peers, and to provide face-to-face in-person experiences for activities that are difficult to complete remotely, consistent with established de programs in school psychology at other institutions. while the activities reported below have been required by the sfa school psychology program in the past, the program is moving or considering moving many of these activities to de during the fall semester due to the covid- pandemic. it is unclear what the effects of this change may be. the first of these requirements is the annual student orientation, held prior to the beginning of each fall semester. requiring on-campus attendance at orientation helps connect de and campus-based students in the program to each other and to program faculty. this assists de students in establishing relationships with faculty and peers so that they feel connected to the program (waldron-soler et al. ). an additional on-campus requirement is in the fall semester for first-year full-time students. the program holds an annual research event, where students in all research methods courses in the school psychology program present a poster. in addition to student posters, there is an invited paper presentation by university faculty or another professional in a relevant field. this event is highly publicized and well attended by students, faculty, and administrators within the university. requiring all students to present on campus provides an additional opportunity for faculty and peer connections, as well as professional development and networking experiences. while both of these events could be attended via de, program faculty decided to require some on-campus presence by de students to help support them in building relationships with students and faculty in the program. faculty believe this is important for students to gain a professional identity and establish a professional network. student feedback has been overwhelmingly positive in regard to these on-campus requirements. students are also required to attend campus for three days to practice and demonstrate skills related to assessment. these designated days are noted in the assessment course syllabus and students are informed when they will be required to attend campus prior to the semester starting. the university has purchased zoom accounts for all faculty and students. additionally, school psychology program faculty, as well as students enrolled in clinical experience courses each semester, have accounts that are upgraded to be health insurance portability and accountability act (hipaa; and family educational rights and privacy act ( u.s.c. § g) compliant. this allows additional encryption so that meetings are secure. additionally, several classrooms in the building that house the school psychology program are deemed "refreshed classrooms," as they have been updated with the technology needed to provide de instruction. the refreshed classrooms on campus have updated zoom technology, desktop computers (including mouse and keyboard), web cameras that have the ability to move and swivel, projection technology (overhead), external audio speakers, and microphones (at the instructor's presentation desk). in most campus buildings these classrooms offer the basic settings including lights, desks (some may be re-arranged), and a prominent desk at the head of the classroom for the course instructor. however, the campus also has some classrooms that are deemed "zoom classrooms" or "distance learning classrooms" that were created to help facilitate a seamless zoom experience. these classrooms have many of the same setup options as the refreshed classrooms; however, they also have a larger space at the head of the classroom designated for instructors, televisions that project lessons and course presentations, more sound-proofing to avoid distractions and less seating than other classrooms due to the expectation that teaching and learning will mostly occur by way of the online platform. the university has invested in distance learning technology and has made available training and troubleshooting resources for faculty and students. zoom technology is used for all class meetings with de students, as well as for additional virtual meetings between students and faculty (e.g., advising). during the covid- crisis, the university addressed concerns that many students and employees have limited access to the internet. wireless coverage was added in various outdoor areas of campus and extended in range to support safe options to utilizing the internet while continuing to attend distance courses. furthermore, the university continues to provide students with guidance on emergency grant funding, including the coronavirus aid, relief, and economic security (cares) act: higher education emergency relief fund ( ), which has awarded grants to students, to support them in gaining resources they need to continue their education. this includes computers and other materials that may assist in creating a more successful student experience with distance learning. there has also been an effort to ensure students have discounts for these resources through the university. all program courses are offered as both de and campusbased. a separate section is opened for each type of student, and then the sections are co-listed, so that campus-based and de students attend courses together as a cohort. all students are expected to attend classes in real-time and to participate in class discussions. course requirements are identical for all students, with the exception of the method of class attendance. additionally, students and faculty utilize zoom for other meetings, including group project work, virtual office hours, and more. one possible area of difficulty in designing and offering a de program is in courses that include use of assessment materials and those where the primary purpose is field-based work, such as practicum and internship. our program has addressed this with two options for students: using community resources or using on-campus resources. there is incredible community support from the texas education region service centers for de students admitted to our program. the program faculty have established relationships with many educational administrators that lead to our de students' ability to use resources of education region service centers in their area. these resources can be used in assessment courses so that students are able to access assessment materials to use in learning and practice and for assessment course requirements. for de students living in areas where the program does not have an ongoing relationship, the student has two options: work with program faculty to establish a relationship between the program and the education region service center and/or travel to campus to check out and return materials required for use in the courses. to date, all distance learning students have been able to work with their local education region service centers to use materials. practicum and internship requirements for campus-based and de students are identical. while most campus-based students complete practicum requirements at a site near the university, it is more common for students completing internship to obtain an internship at a site farther from campus. therefore, the program had already established de guidelines for the internship. these were then generalized to practicum courses for de students and include the following: & students attend all practicum/internship class meetings in real-time via zoom & students obtain at least the minimum face-to-face supervision required by nasp and the texas state board of examiners of psychologists from the field supervisor & students complete all required hours and activities/assignments, as required by the program & university supervisors utilize hipaa and ferpacompliant zoom, as well as university email, to communicate with the student and the field supervisor and to complete site visits and field supervisor interviews to date, we have had one de student complete practicum (this student was the first student admitted to the program, we have yet to experience attrition with de students). feedback from the student and field supervisors have indicated it was a positive experience and that the student was well prepared for the experience and to continue on to internship. this student was the only person admitted through the de option in the - academic year, the - cohort included three students, and the - cohort currently includes nine students. as so few students have matriculated through the program at this time, we feel uncomfortable sharing specific outcome data, as it could readily be identified. however, our experience has been that de students receive grades, comprehensive exam scores, and practicum evaluations that lead to the successful completion of requirements toward the degree. given the widespread shortage of lssps in texas (barbre ), we receive numerous emails and calls asking for current students and alumni to fill positions in the public schools each week. the region education service center (texas education agency n.d.) reached out to sfa four years ago offering to provide equipment, proctors, and rooms for us to provide de through their facility for school psychology training. they expressed concern over being understaffed, having many of their lssp's close to retirement, and being unable to bring new psychologists into their area. they report much of the problem to be the rural nature of their districts, with schools being many miles apart and having no colleges close enough for people interested in the field to attend. the region service center is more than two hours away from sfa and even further away from other lssp training programs. they stated they felt they needed to "grow our own," noting that often the only way to get applicants who want to be in their service center area is for the applicant to be from the area, wanting to come home, or wanting to stay close to their childhood homes. the region education service center in conjunction with sfa school psychology faculty and interns provided meetings in their facility and numerous flyers, brochures, and phone calls were used to invite people to attend. we had several attendees but no follow through with applicants. as a faculty, we explored options and decided that designing a program through zoom for the m.a. degree might be an avenue to address the practitioner shortage as well as the declining number of applicants to our existing program. this was advertised through the region education service centers in texas. after this, we were approached by region education service center about participating in a new project from the texas education agency, because of our online option (zoom) to get more evaluators in texas public schools. they wrote a small and rural schools grant funded by the texas education agency (https://tea avoswald.tea.state.tx.us/ grantopportunities/forms/grantprogramsearch.aspx). the region center expressed a desire to work together to provide partial and potentially full funding for individuals who work in a small or rural district and are willing to commit to staying in the district for at least three years. they presented their grant information along with their partners in a training at a regional education conference. de courses, whether online or hybrid/blended, can cause faculty numerous issues. online teaching is like learning a new sport; a new sport can be time-consuming and onerous. we strongly encourage faculty considering teaching a de course to work with the center of teaching and learning, or similar office, on their campus. the center for teaching and learning at sfa requires instructors to complete a semester-long online instructor certification program before teaching any online course. we took this class in preparation for working with face-to-face virtual students and frequently collaborate with each other to troubleshoot issues or share new tips and techniques we have learned. faculty also regularly attend the center for teaching and learning workshops on new instructional strategies that use technological services of benefit to face-to-face in-person and face-to-face virtual students. instructors should invest a significant amount of time becoming comfortable with virtual learning modalities and interfacing with other individuals on these platforms before engaging in virtual teaching. while such courses are vital, some situations are difficult to anticipate and are learned through lived experiences. we wish to present the common issues we have experienced in this process and possible solutions to these issues (see table ). one of the main complaints of many faculty teaching an online course is passive students (lieberman ) . it is difficult to deliver lecture material to the quiet static of muted students without video, not to mention engage in discussion. face-toface in-person instruction allows faculty to read the faces of their students. the quiet space of the online classroom can be daunting. zoom offers a solution to this issue. zoom ( ) allows real-time polling during instruction. other learning management systems offer similar options. faculty can poll students on material as a simple check for understanding or as an opinion poll to increase participation and discussion. the polling can be set to anonymous, which allows students a safe space to voice opinions. this type of active student responding during instruction has a positive relationship with achievement (ellis et al. ; greenwood et al. ; rosenshine ) . another strategy is requesting a check for understanding during lectures. ask students to verbally state or use the chat box "thumbs up" or "thumbs down" to indicate they either understand the material or need more review of the material. the instructor can provide them the option of private messaging if they do not wish to publicly state they do not understand. this technique can promote engagement during online lectures (rosenshine ). zoom ( ) also allows the instructor to use a whiteboard. here, the instructor can draw or write in real time to the student. the instructor can also give control of the whiteboard to a student to answer a problem or demonstrate a skill. besides zoom, there is other software to help increase engagement in the online classroom. kahoots ( ) and quizizz ( ) allow the instructor to make a game out of quiz questions, allowing students to compete against each other. these platforms have the added benefit of allowing students to replay the quizzes at a later time to help prepare for exams. flipgrid ( ) allows the instructor to ask a question and students answer with a video message, creating a social media feel within the classroom. in a course with both face-to-face in-person and face-toface virtual learners, you can utilize these practices for both groups. you can embed polling into your powerpoints for traditional learners and use zoom ( ) polling for distance learners, and you can have the traditional learners physically give a thumbs up or thumbs down during lecture while the face-to-face virtual learners use the chatbox. struggling to get students to engage in discussion boards and projects in an online classroom is common. many students take very little action until the end of the semester (lieberman ) . this can be time-consuming and frustrating for the instructor. the instructor can model the behavior they wish to see from the student. for discussion boards and projects, the instructor can provide an example of an exceptional student from a previous semester or complete the project themselves to show exactly what is expected. this will also help alleviate some of the questions about the task. another recommendation is to provide choice during these discussion boards and projects. this way the students can engage in self-directed learning and develop interests within the course content. abrami et al. ( ) found in de classrooms that providing students with personally meaningful work, which students had some sort of choice in created greater levels of intrinsic motivation. allowing students to personalize their learning experience will create a better distance experience. another potential concern with online classrooms is an increase in email communication with questions from students. one recommendation from garrison and vaughan ( ) is to view these emails as part of the instruction. if an instructor receives a question in an email such as "do i need the latest edition of the book?", they should assume many students are wondering this and post the answer as an announcement. another recommendation is to create a discussion thread with the title questions for the instructor. this way every student will be able to benefit from the instructor's answers to the questions, and it may reduce the number of emails asking similar questions. however, it is also important to clearly communicate to students that digital correspondence, particularly in the absence of face-to-face in-person interaction, is important and valued. it is difficult sometimes in face-to-face in-person classrooms to create collaboration, and it becomes even more difficult in the face-to-face virtual classroom. zoom ( ) has breakout rooms. the instructor can choose to place students into smaller groups. during these breakout sessions, the instructor can join different breakout groups to check-in and engage the subgroup in the discussion or activity. when time is up, the instructor is able to give the students a warning timer for when the breakout rooms will end and everyone will return to the larger classroom. this creates a similar environment to the face-to-face in-person classroom, allowing students to work together in small groups. at sfa, when students in the faceto-face in-person classroom were broken up for group work the instructor was able to place the face-to-face virtual students into a breakout room so they could have equivalent experience. depending on the number of face-to-face inperson and face-to-face virtual students present, creating groups the aforementioned way may be problematic and the instructor may desire to arrange people in groups based on other factors. in this case, face-to-face in-person students can use their phone or laptop to join the virtual breakout rooms. the virtual learning environment inevitably comes with technical difficulties. anything can happen from video not working to full wi-fi failure. a few tips to help with this is to hold a tech session prior to the start of class. the instructor and students can work out difficulties prior to the semester beginning. this also can alleviate the anxiety for less tech-savvy students. providing documents for students to troubleshoot technical issues is also helpful. often a university's technology office has already created one that can simply be linked to the course. online teaching requires much flexibility. during the first class meeting the instructor should discuss with the student how technical difficulties will be handled (i.e., what will happen if the instructor loses the internet during the allotted course time or what will happen if a student loses the internet and cannot attend). there also should be a document in the course content with an explanation of these possibilities (e.g., handbook, syllabus, learning management system). with a little planning and a few extra steps, instructors can make the online classroom engaging and easy to navigate for any student. despite being ranked as the th best job (u.s. news and world report n.d.), the field of school psychology continues to face a national shortage (castillo et al. ; fagan ) . one way to address the shortage is for school psychology programs to consider providing instruction via de (dixon ; dixon ; moy et al. ) . to address the national, state, and local shortage of school psychology practitioners and trainers the faculty at sfa began offering a de option to students outside a -mile radius from campus. although we started with only one student it has expanded with the assistance of regional service centers and district leaders interested in a "grow your own" model. although the sfa de option is too young to provide outcome data, there is evidence from existing de programs that students are adequately trained and prepared for the workforce (dixon et al. ; drefs et al. ; hendricker and viola ; ruby et al. ; schroeder ; waldron-soler et al. ) . this is in contrast to the majority perception held by school psychology faculty and graduate students that de training options are inadequate. program faculty at sfa were also skeptical of de courses, especially skills-based courses. however, anecdotally, we have not found the de students' knowledge or skills to be inferior to face-to-face students in our program. course grades and other outcomes, such as comprehensive exams, are also commensurate between de and campus-based students, although the sample size is small. while de has the potential to increase the recruitment reach of school psychology training programs, provides flexibility to students, and has evidenced similar outcomes between students in de and non-de courses (hendricker et al. ) , there are some limitations to consider. one limitation is the perspectives of students and applicants. some graduate students report a lack of interaction and reduced skill acquisition when completing de courses ). the decreased face-to-face in-person interaction also has the potential to impair the quality of mentorship that can be provided. in particular, it may be more difficult for the faculty member to write strong letters of recommendation when they have limited face-to-face in-person interaction with the person in order to judge their professionalism and ability to interact with others in a live setting. another potential limitation is faculty perspectives. luongo ( ) completed a survey with fulltime and part-time faculty teaching de courses at a university. results indicate that about % of respondents disagreed that they were as satisfied with teaching de courses as using other modalities and that about % of participants indicated their workload is higher with de courses than courses using other modalities. over % of respondents indicated they either disagreed or strongly disagreed that they missed face-to-face contact with students when teaching de courses. in addition, luongo ( ) discusses several factors related to faculty investment and performance in de, including a lack of administrative support, lack of compensation, and inaccessibility of training and resources. accessibility to resources is not limited to a faculty barrier. a number of resources are needed to make de viable as a course modality option. these resources include, but are not limited to, technology and training for faculty and students. access to equipment and technology can somewhat be overcome by the university providing equipment and hot spots to faculty and students; however, there can continue to be issues with equity of content delivery, particularly for those students living in areas with limited broadband speed capabilities who are not able to travel to provided hot spot locations. sisneros and sponsler ( ) report % of individuals living in rural areas have inadequate bandwidth speed, compared with % of individuals living in urban areas. additionally, they report that as household income decreases, so does the likelihood of individuals having a bandwidth subscription, with lower-income black and hispanic households being even less likely to purchase bandwidth subscriptions than white low-income households. these data indicate limitations of access and equity across region, income, and race and ethnicity that need to be considered when planning for a de instructional model. access to training and support for faculty and students also need to be considered. a survey by viola et al. ( ) indicated students in graduate school psychology courses report using a number of technologies in de courses, including learning management systems (blackboard, canvas, etc.), real-time video applications (skype, zoom, etc.), and shared drives. many of these technologies may not have been used by faculty or students prior to the de course experience, requiring adequate training for best use of technology by faculty and usability for students. universities can provide this training through professional development opportunities and support services. students in a "grow your own" environment often have restricted supervisor options, which is another limitation de programs may face when trying to identify and select qualified supervisors for de students. providing training to supervisors and developing strong bilateral communication with supervisors about course requirements and evaluation of student performance is also a challenge from a geospatial distance. while additional limitations will no doubt be experienced by programs transitioning to a de model, faculty and student perspectives, technology access and equity, and training and support appear to be most frequently cited in the current literature. while these limitations may result in barriers to effective de course delivery, many can be overcome with careful planning and resource allocation. programs considering de should work with university administration to prepare for and prevent these barriers to the extent possible prior to offering de courses. school psychology de courses and de programs have received increased attention in professional outlets (i.e., the communique, journal articles, special issues) recently. although we anticipated this trend to continue before the pandemic, we more firmly believe de options will become more prevalent as faculty and student have been provided opportunities to experience and become more comfortable with this format. nonetheless, much more research is warranted and the discussion of how best to prepare students in skill-based classes is needed. data availability not applicable. conflict of interest the authors are affiliated with the program featured in this paper. ethical approval not applicable. consent to participate not applicable. code availability not applicable. interaction in distance education and online learning: using evidence and theory to improve practice a report on the state of school psychology in texas schools: - school year. research and practice in the schools where are the school psychologists? personnel needs in school psychology: a -year follow-up study on predicted personnel shortages and economic security act: higher education emergency relief fund online education: let's start the conversation online school psychology: blueprint to higher education conversation extending the university walls: using technology to prepare future practitioners canadian innovation: a brief history of canada's first online school psychology graduate program effective teaching principle and the design of quality tools for educators school psychology's significant discrepancy: historical perspectives on personnel shortages online content instruction in school psychology training faculty perceptions of distance education in school psychology training online school psychology program blended learning in higher education: framework, principles, and guidelines school psychology programs and degrees in texas opportunity to respond and student academic performance using distance education with nontraditional school psychology students trends and perceptions of distance learning in school psychology kahoot. (version . . ) [computer software discussion boards: valuable? oversued? discuss. inside higher ed an examination of distance learning faculty satisfaction levels and self-perceived barriers opportunities and challenges for online instruction in school psychology programs key concepts and terminology in online instruction: a primer for school psychology programs quizizz. (version . ). [computer software principles of instruction: research-based strategies that all teachers should know quality distance learning to address the school psychology shortage: a model for online respecialization school psychology matters preparation for a premaster's internship in a blended school psychology program: a case study. training and education in professional psychology broadband access and implications for efforts to address equity gaps in postsecondary attainment training models in counseling psychology: scientist-practitioner versus practitioner-scholar current nacogdoches, texas population, demographics and stats in education service centers distance education in graduate training programs: lessons learned from school psychology students instructional technology in graduate psychology distance education: trends and student preferences learning from graduate training in related fields: a model online school psychology program master of art in school and community psychology zoom. (version . . ) [computer software publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations mccleary is an associate professor at stephen f. austin state university. his primary interests include math fluency and the scholarship of teaching and learning clark is a clinical professor at stephen f. austin state university. she has plus years in private practice in deep east texas. currently she is the director of the apa accredited sfa charter school psychology internship program dawes is now an assistant professor at the citadel. her interests include math fluency and behavior analysis flowers is an assistant professor at stephen f. austin state university. her interests include bullying and behavior analysis ellis-hervey is an associate professor at stephen f. austin state university. her interests include selfesteem, locus of control, and personal presentation difficulties encountered by women of color key: cord- -wvu t authors: daly, bradford d.; gardner, rachel a. title: a case study exploration into the benefits of teaching self-care to school psychology graduate students date: - - journal: contemp sch psychol doi: . /s - - - sha: doc_id: cord_uid: wvu t it has long been established that school psychology practitioners experience high levels of burnout. as a means of preventing burnout among future practitioners, school psychology training programs are frequently encouraged to teach and model self-care to students. this is particularly important as the current generation of graduate students experience high levels of anxiety and depression, but there have been very few examples in the research literature of how training programs should teach self-care and whether it is actually effective. the current study presents results from an exploratory case study, which integrated self-care instruction into graduate school psychology curriculum with a small sample (n = ) of first-year school psychology students across two separate cohorts. students created written plans with self-care strategies that they attempted to implement over the course of their first semester in graduate school. a qualitative review of their plans and written reflections revealed that students described many sources of stress upon entry into training, and most needed to revise their strategies for coping as stresses changed during the semester. overall, student reflections revealed that the self-care activities were helpful to meet the demands of their graduate education. with the field of school psychology facing shortages in the near-and long-term future, the profession can ill afford to lose practitioners or prospective practitioners due to attrition. an estimated % of school psychologists leaves the field each year for reasons other than retirement (castillo et al. ), a number that in most regions of the usa exceed the projected number of new school psychology graduates entering the field each year. while the exact number and reasons that school psychologists leave the field are unclear, one potential reason that has been proposed is the high level of susceptibility of practitioners to burnout. research of school psychology practitioners (e.g., wilczenski ; kaplan and wishner ; mills and huebner ; huebner and mills ; worrell et al. ) has consistently found that while school psychologists have high levels of job satisfaction, they nevertheless also experience high levels of emotional exhaustion, feelings of depersonalization, and reduced sense of personal achievement, which unmitigated may result in burnout. newman ( ) proposed that unaddressed burnout can lead to problems with professional competence and engagement in ethically questionable practices. application of self-care strategies has frequently been recommended as a means of preventing burnout and increasing meaning derived from work. in fact, engaging in self-care has been described as an ethical imperative by multiple authors (lopez ; barnett and cooper ) . newman ( ) defined self-care behaviors as ones that are used to "establish and promote physical and emotional wellness, and to allay stress" (p. ); self-care behaviors serve to both mitigate stress and promote personal wellness, thereby ensuring the optimal quality of services provided by practitioners. selfcare has been conceptualized as a multi-dimensional construct, with some authors proposing four domains (newman) and others proposing six domains (butler et al. ) . common elements of self-care definitions include behaviors designed to promote physical wellness (e.g., physical exercise and nutrition), enhance cognition (e.g., professional development, supervision), promote emotional well-being (e.g., seeking counseling, communication), and deriving meaning from work (e.g., mindfulness practices, religious and spiritual practices). there is some correlational research regarding the effectiveness of self-care strategies on the functioning of mental health professionals. in a study of clinical psychologists, stevanovic and rupert ( ) found that psychologists who engaged in a greater number of self-care behaviors experienced greater job satisfaction. a survey of school psychologists (bolnik and brock ) found that % believed that engaging in self-care practices was important, and more frequent strategy use was associated with fewer physical symptoms of stress. coster and schwebel ( ) used a mixed methods approach to identify that % of psychologists had experienced personal impairment, and that effective strategies to address impairment included attending to personal relationships, going on vacations, striking a work-life balance, and developing self-awareness. in summary, this research evidence seems to provide at least some support for the importance of school psychologists engaging in self-care behaviors. based on these findings, researchers writing about self-care for psychologists frequently recommend that graduate programs directly teach self-care to prepare students to use self-care strategies in their future professional careers (newman ; huebner et al. ; barnett et al. ; barnett and cooper ), but there is very little empirical research to guide these recommendations. there are a small number of studies that have examined self-care strategy use by graduate students in mental health professions. myers et al. ( ) examined stress and self-care behaviors among clinical psychology graduate students and found that sleep hygiene, social support, and mindful awareness were related to lower stress levels. turner et al. ( ) , in a survey of school psychology interns, identified a number of strategies used by interns to manage stress including self-awareness activities, social activities, exerting control over professional activities, and appreciating intrinsic professional rewards, all of which were identified by the interns as being effective strategies. these studies provide some correlational evidence for the importance of self-care for graduate training, but the question of whether self-care behaviors can be successfully modified by training programs remains largely unexplored. several authors (lopez ; barton et al. ) have described various ways that school psychology training programs could integrate self-care into their curriculum, but there remains little evidence as to the effectiveness of these recommendations. lewis and king ( ) described efforts to integrate a self-care unit into social work students' field experiences, and provided qualitative evidence attesting to the benefits to students, providing a potential model for school psychology training programs to use. it is particularly important to consider the self-care needs of graduate students in school psychology given the overall context of high mental health needs among graduate students in general. a recent study (evans et al. ) found that graduate students both in the usa and internationally have six times the rate of depression and anxiety in comparison to the general population. there is some reason to believe that these rates may be higher in school psychology programs due to lower rates of financial support than other disciplines (proctor and truscott ) and the nature of school psychology training that involves intensive coursework and fieldwork (barton et al. ) . a study of training programs in school psychology (gadke et al. ) found that in the - academic year, one student withdrew from their training program for every . graduates at the specialist level and for every . graduates at the doctoral level. based on this information, it seems reasonable to consider school psychology graduate students a population that is vulnerable to stress and anxiety, yet vital to the future health of the school psychology profession. training programs need to develop a deep understanding of the sources of stress for graduate students in school psychology and a means by which to assist students in coping with these stressors. with self-care strategies having emerged as a promising practice in mitigating stress and burnout, training programs should seek to design ways to integrate self-care into their curriculum and monitor the effectiveness of doing so. an important question to consider when attempting to train students to apply self-care strategies is an understanding of the nature of self-care. much of the correlational research into self-care practices by mental health professionals has at times defined self-care as engagement in a number of coping behaviors at a specific point of time (e.g., stevanovic and rupert ; bolnik and brock ) . this research tradition suggests that self-care might be best conceptualized by considering the volume and diversity of coping skills employed by individuals. on the other hand, the im-paact framework (brown ) defines self-care as a more fluid concept, comprised of four stages: (a) prioritizing self-care, setting goals, incorporating into schedule, (b) implementing self-care plan and refine as necessary, (c) connecting internally and with an external social support network, and (d) thrive by fully engaging in the self-care plan and regularly re-assessing and adjusting the plan as necessary. these same core elements are consistent with the self-care plans developed, implemented, and revised by graduate students in the current study. moreover, drawing from the related literature on coping strategies for managing stress, coping skills have been theorized to fall within two distinct categories (i.e., problem-focused or emotion-focused; mcnamara ). using active problemsolving is an example of problem-focused coping. in general, such problem-focused coping is associated with better functioning and management of stress compared to emotionfocused coping. arguably, proactively planning for self-care in the midst of school-and work-related stress can be considered a form of problem-focused coping. the theoretical orientation is important because trainers interested in incorporating self-care into school psychology curriculum may make different decisions about how and what to cover based on an understanding of the construct of self-care. the present study was designed with two primary goals in mind. first, it is important for school psychology trainers to better understand the nature of the construct of self-care and how it can meet the needs of graduate student trainees. second, it is important to understand whether an intervention aimed at self-care could be effective as a means of addressing stress experienced by trainees in graduate school. therefore, this study was designed to provide an in depth understanding of the self-care practices of two cohorts of graduate students following a sustained semester-long intervention to integrate self-care into the school psychology training curriculum through a systematic case study inquiry. case study research has a long history in many disciplines, and is especially appropriate in circumstances in which an in-depth description of a case may inform future theory, research, and practice (creswell and poth ) . case study research is particularly useful in situations in which the primary research questions involve understanding the how and why behind a construct and intervention, especially when conducted over a period of time (yin ) . in this study, an analytic approach called explanation building (yin) was undertaken to provide a detailed examination of the experiences of the graduate students while experiencing a self-care intervention, intended to help other researchers and trainers conceptualize the fluid nature of self-care and whether an intervention based on the im-paact model could improve self-care practices among graduate students. two separate cohorts of graduate students across years (n = ) enrolled in their first semester at a school psychology training program participated in the present study. within each cohort, % of enrolled students participated in the self-care intervention in the present study and all completed the study through the three measurements. the school psychology program is a combined specialist level and doctoral level (psy.d.) program and is located in a small private university in the northeastern usa. in the present study, of the participants were members of the specialist program and the remaining were enrolled in the doctoral program. all students participated in the intervention described in the present study as part of the required coursework for their field experience seminar. per the university's committee on ethical research with human subjects, all participants were given the option to exclude the use of their anonymized data for this project. due to the small sample size in the present study, detailed demographics on the participants could not be reported due to the risk of revealing their individual identities; however, statistics on the overall graduate program during the time the study was implemented are available to convey information about the characteristics from the population that the participants derived. as reported by gadke et al. ( ) , approximately to % of school psychology graduate students nationwide are male, and approximately % are students of minority status. at the time of the study, the program overall had a percentage of students of minority status of %, which is lower than the national average cited above. the percentage of male students enrolled in the program overall was %, which is in line with the national average cited above. because of the very small number of male and ethnic minority students who were participants in this study, in all responses, identifying information was masked to prevent revealing identities. in some cases, participant responses were altered slightly to remove or change information that could be identifiable, in order to mask their identities. the two cohorts participating in the study were enrolled in consecutive academic years. the first cohort (n = ) was larger than the second cohort (n = ), but the group composition was overall similar. the large majority of students were in their early twenties and were enrolled in graduate school within a year of receiving their undergraduate degree; however, it is not possible to provide detailed age data due to risks to breaking participant confidentiality. per yin's ( ) recommendations, case study research can provide support for the external validity of their claims through replication of its procedures; therefore, having two separate cohorts complete the self-care intervention, while the overall number of participants is small, helps to strengthen the validity of the conclusions. the school psychology training program at which participants were located is housed within a college of graduate studies in counseling, psychology, and education. the school psychology's specialist program is approved by the national association of school psychologists and the psy.d. program is accredited by the american psychological association. at the time of data collection, there were full-time faculty with primary appointments to the school psychology program. the training program uses a cohort model with both specialist and doctoral students taking the same coursework in the first semester. students are required to take a total of credit hours in their first semester. the first semester within the training program emphasizes acquisition and maintenance of personal and professional skills. therefore, students take foundational coursework in learning and cognition, psychometrics, individual norm-referenced assessment, interpersonal communication skills, cultural awareness, and perspectives in school psychology. in addition to coursework, students in the training program are required to enroll in a school-based practicum experience day per week for a total of h in the first semester. finally, students also are assigned a graduate assistantship, which provides partial tuition remission in exchange for h of work per week. one unique characteristic of the training program is its location, which is in a rural community over an hour's drive away from major population centers. this provides students with experiences in rural schools and populations, but does make it more difficult for students who are working full time to commute. therefore, many of the students live in apartments in fairly close proximity to the university during the academic year. the primary author served as the instructor for the field experience course and the facilitator for the self-care intervention. in this role, there was the advantage of being embedded within the system and milieu along with participants, as well as the drawback of potential lessened objectivity from serving in an evaluative role to the participants. the instructor was a white male in his late thirties, and was in his first years as a fulltime core faculty within the school psychology training program over the course of the intervention. prior to joining the program faculty, the instructor had years of experience as a practicing school psychologist, and drew heavily on these experiences in describing the importance of self-care throughout the intervention. both cohorts participated in the self-care intervention in their first semester field experience seminar. the overall goal of the intervention was to help participants reach the thriving state of self-care based on the im-paact model (brown ) for application of self-care. the transactional model of coping emphasizes active application of coping in response to appraisals of stressful events (lazarus and folkman ) , and intervention in coping skills emphasizes application of problem-focused strategies and reducing use of avoidancefocused strategies (mcnamara ). the thriving state of self-care suggested by im-paact model predicts that this coping approach can be developed through promoting the prioritization of self-care, developing and evaluating a plan, and connecting internally to self-care and externally to others (brown ) . a summary of the steps of the self-care intervention designed to adapt the im-paact (brown ) framework is included in table for quick reference. during the first week's seminar, participants were prepared for their practicum experience through discussions about expectations and requirements. the first step of the self-care intervention, a didactic discussion led by the instructor, was embedded into this initial seminar. this discussion began with a short lecture about the effects of stress on professionals in school psychology, and the progression of unhealthy stress that can lead to burnout and problems of professional competence, based on the models of stress proposed by newman ( ) and mcnamara ( ) . participants were prompted to propose and share potential sources of stresses in their lives that could potentially lead to this unhealthy progression. the instructor then described that problematic stress could therefore lead to professionals engaging in ineffective and unethical practices. finally, the instructor introduced the notion that self-care strategies are posited to be a potential buffer against this negative progression. next, participants engaged in self-study with the goal of creating a plan with strategies that they could apply for selfcare. creation of a written plan to address stress through selfcare that is revised over a period of time is consistent with theoretical models of self-care (brown ) and parallels coping skill training as proposed by mcnamara ( ) . a series of readings and websites were provided to participants (see table for a list) to help them to generate ideas about how to structure and design a self-care plan. the participants were not given specific criteria for what format or content to include in their plans, but were advised to create plans that were multidimensional, individualized, specific, and realistic, so that the plan would reduce stress without adding new stress (newman ; lewis and king ) . participants submitted their self-care plans and a reflective writing piece to the instructor prior to the second week of class. these responses were used to facilitate a group discussion in the following seminar session. participants had the opportunity to share stressors and strategies they attempted to incorporate into the plans, which also helped to serve as member checks to ensure the credibility of the instructor's interpretations of their written responses. during this discussion period, the instructor primarily engaged in reflective listening while encouraging participants to think about ways that they could ensure the effective implementation of their plans. participants' self-care plans and written reflections were assigned a pass/fail grade based on completion of the requirements, which all participants did successfully. a brief written response was provided by the instructor to each participant, commenting on content in the student's reflective writing and encouraging their use of self-care strategies. the following is an example of one of these feedback comments: i'm glad to see the honesty in your response about feeling stressed. it's important to recognize when your stress level goes up and down so that you can react accordingly. don't be afraid to reach out to others, it's not a sign of weakness! good luck with following your self care plan this semester and managing your stress. discussion about implementing self-care strategies was embedded throughout seminars over the next several weeks, which was integrated with discussions about their professional work behaviors, and the nature of school psychology service delivery. while participants were encouraged to adjust their self-care routines throughout the semester, they were formally asked to revise and reflect on their self-care plans at two points. seven weeks into the semester (time ), participants were required to submit an updated self-care plan and written reflection in which participants were asked to reflect upon how well their plans worked, and to describe how they adjusted their plans to better meet their needs. the instructor used their responses in the following seminar session to again facilitate a group discussion about stress and self-care. finally, this process was repeated at the fourteenth week of the semester (time ) as a means for participants to have a summative examination of their self-care and stresses in their first semester of graduate school. as before, their responses were used to lead a debriefing session in the last field experience seminar session for participants to reflect on the role of self-care in their graduate education and to plan for the future. qualitative data used for the study included the student's selfcare plans and written reflections submitted as part of the coursework requirements at three measurements: time , time , and time . reflections and plans were both de-identified with student names and identifying characteristics removed from responses prior to analysis. as sources of data for this project, the written reflections were useful in understanding participants' cognitive appraisals of stressors and their coping responses, making them amenable to interpretation based on the transactional model of coping (lazarus and folkman ) . yin ( ) described tactics that can be used in case study research to support construct validity of conclusions. the first such tactic is through the use of multiple sources of information. while participant interviews and seminar discussions were not transcribed for separate analysis, they did provide external verification of the contents of the participants' written reflections, helping to support that they represent an accurate account of their experiences. a second tactic described by yin is establishing a clear chain of evidence for case study data. to analyze the written documents in this study, de-identified student reflections were initially downloaded from the online course management software. these raw responses were compiled into a single document in microsoft word. after compilation, participant responses were read by the primary author twice to identify excerpts of the response that could be relevant to understanding the responses of the participants based on the stages proposed by the im-paact model (brown ) . these excerpts were then extracted into a separate research database in microsoft excel, thus providing a clear chain of evidence for the qualitative data to be analyzed. once the participant response database was compiled, the analysis strategy for these data sources was developed based on recommendations for case study research by yin ( ) and creswell and poth ( ) . in general, a pattern matching approach was used to analyze the participant responses based on the im-paact model (brown ) and transactional model of coping (lazarus and folkman ; mcnamara ) . yin described pattern matching as a technique in case study research involving searching for patterns in the data predicted by theory or prior research. a subset of this approach, explanation building, is particularly useful when step brief description in the first field experience seminar, students were introduced to the idea of self-care as a means to avoid burnout. . guided self-study students were assigned to research self-care through a collection of articles and websites. specific readings assigned included: • self-care: the missing link in best practice -part i (lopez ) • self-care: the missing link in best practice -part ii (lopez ) • developing a self-care plan (reachout.com, n.d.) • best practices in early career school psychology transitions (silva et al. ) • self-care starter kit (butler and mcclain-meeder ) . self-care plan design students were required to design their own self-care plan and submit it to the instructor prior to the second week of the semester. at time ( week into the semester), time ( weeks), and time ( weeks), students completed written reflection about their sources of stress and how their self-care plan was working or was not working to help to manage these stressors. attempting to understand how and why observed outcomes occur, based on a prior theoretical model. this approach was selected as the major goals included understanding how participants appraised and planned self-care strategies to cope, how participants adjusted these plans over the course of the semester, and how the participants evaluated the intervention and plan as being effective in helping them meet the stressors experienced during graduate school. based on yin's ( ) recommendations, the analysis of the qualitative data followed an iterative process. the authors started the process with a conceptual model for stress and self-care, specifically the transactional model of coping (lazarus and folkman ; mcnamara ) and the im-paact framework (brown ). the analysis started at a broad level through an initial reading of all responses in the response database, followed by a closer reading at the element level within responses to examine for information that matched patterns predicted by theory and might yield the explanatory information needed to respond to the research questions. these responses were then developed into codes through which themes in responses could be identified, and as yin recommended, these initial themes were then compared to the prediction based on the theoretical models. finally, the explanations were again compared to the data, and the second cohort provided an opportunity to test these interpretive explanations with a new data source. responses were found to match patterns that were extracted into three major themes upon which the findings section is organized. several efforts were undertaken to ensure the validity of these analysis procedures. first, and very important as identified by yin ( ) , the authors considered several alterative and rival interpretations at multiple stages of the analysis process. as one example of such an explanation, the authors were aware that participants could experience stressful life events outside of graduate school which could influence their implementation and selection of self-care activities; to help guard against this threat, all responses were examined for information about outside stressors that could potentially have affected the individual participants. other rival explanations considered during the analysis phase are noted in the findings section. another strategy used to support the validity of the analysis was through triangulation of multiple information sources (creswell and poth ) . member checking to ensure credibility of the conclusions happened throughout the course of the semester through in-class discussions of the themes the instructor identified in participants' responses. the instructor also debriefed with the participants as a group following the submission of final grades to help make sure their responses would be similar in theme to the written products used for analysis in the present study. prolonged engagement with participants over the course of the academic semester helped the author to have a relationship that encouraged honest and candid responses, and is considered to be another good practice in qualitative research (creswell & poth) . the reliability and credibility of the explanations were supported in the research process through a memoing notetaking procedure to document contemporaneous process notes (creswell and poth ) . a replication of the intervention and data analysis of the study with two separate cohorts gave an opportunity for initial explanations developed after the first year to be checked with new data in the second year of the study, and provided additional support for the findings (yin ) . finally, while it must be noted that the primary author was the course instructor and therefore may have been prone to bias, being embedded with the participants over the course of a semester also helped to lend credibility to the analysis (creswell & poth) . the im-paact model (brown ) described the first stage of thriving with self-care to be prioritization of self-care. to support the prioritization of self-care, and participant development of problem-focused coping strategies (mcnamara ), the selfcare intervention began providing participants with didactics about self-care and stress, and requiring them to make an appraisal of their current stressors. a summary of stressors identified by participants at time is presented in table . as noted in the table, the majority of participants reported stress in relation to the rigors and difficulty of their graduate coursework, and their ability to stay organized and manage time to effectively meet these challenges. many of the participants described the origin of this stress being internal, as exemplified by the following response from a male graduate student in his early twenties: the biggest stressors in my life are largely internal. they're preconceived notions about what i should be doing, or what i should be able to do "on my own." they're also issues surrounding a consistent drive to "accomplish" as much as possible, to the detriment of my own health and well-being. by requiring participants to engage in this self-appraisal, the goal of the intervention was to help them prioritize self-care and develop plans to help them meet their needs, thus adopting a problem-focused coping strategy (mcnamara ) . per the im-paact model, "the goal at the prioritize stage is to identify a variety of activities that seem most likely to bring fulfillment and are realistic for the particular individual. consideration for personal interests and resources is key" (brown , p. ) . that said, the research literature in self-care gives little guidance as to how professionals should organize, plan, and implement self-care strategies. through an analysis of participants' self-care plans developed for time , it appears that the strategies that participants included in their initial plans largely mirrored the stressors they reported at the start of graduate school in primarily addressing academic and social-related sources of stress. all participants organized their strategies into several domains, which was recommended in some of the readings they completed as part of the assignment (e.g., butler and mcclain-meeder ) . a summary of the most common domains included by participants, and examples of strategies that they included under those domains, can be found in table . as noted, it appears that participants developed plans that were tailored to their individual needs based on their selfappraisal of stressors. all participants included strategies that were intended to support their ability to keep up with coursework and manage their time. all participants included a domain for physical health that initially included some kind of strategy related to physical activity or exercise, ranging from planning on visiting the gym days a week for exercise to going for a walk regularly. a large majority of participants (n = ; . %) included spending time with a significant other (defined in the study as spouse, partner, girlfriend, boyfriend, fiancé) as part of their self-care plan. consistent with previous research with self-care (lewis and king ), the majority of participants (n = , . %) included a spiritual health domain, which included a large number (n = ; . %) who indicated that religious expression, including prayer or attending religious observances, was an important part of their self-care. as noted, it was important to the study's analytic procedures to search for information that did not fit predicted patterns. to mitigate the effects of a history threat on interpretations, the authors considered it important to note when participants described unusual discrete events that could impact their stress and coping response. three participants did report outside events which were coded to be in this category, for example, the death of a loved one and a serious health issue. responses for these participants were flagged and efforts were made to analyze their descriptions of stressors and coping separately, so as to avoid drawing conclusions about the idiosyncratic responses to these events. it is important, however, for trainers to note the potential impact of similar events on the stress and coping of graduate trainees. finally, one theme present in the responses of social work trainees in previous self-care research (lewis and king ) was that of compassion fatigue and secondary trauma due to stressful situations at their field placement. while these themes were expected in the present study, they did not feature in participant responses outside of two participants. the authors interpreted this finding as being related to the typical activities at the field placements in the first semester of the training program, which primarily involve shadowing, observing, and assessment, as opposed to providing mental health counseling. the intervention procedures included several features which were designed to support participants' implementation of selfcare strategies, assess their plan, connect with self-care both internally and externally, and adjust their plans accordingly. these included the cycles of reflections and revisions at time and time and the group discussions about self-care in the seminar. the im-paact model (brown ) predicts that these processes are important to successful application of selfcare in response to constantly shifting sources of stress. participants were asked at time and time to re-evaluate their sources of stress to provide an insight into their shifting appraisals of stress. the most prominent theme in participant responses regarding the evolution of their stresses was the need to find a balance between academic demands and other activities. there was a wide variance of responses to how participants evaluated their success at finding this balance at time and time , with participants describing the semester overall as causing very little stress and some conveying that they experienced extreme stressors. consistent with other work on self-care that has followed individuals over a length of time (e.g., lewis and king ), self-care practices for participants in this study evolved over the course of the semester in response to their shifting appraisals of stress, with participants experiencing some successes and challenges implementing strategies that they initially thought would be successful. all participants made adjustments to their plans at time , and all but two made further adjustments at time . a summary of the most common adjustments to plans can be found in table . as noted, most participants experienced difficulties in time management as a primary barrier for regular application of self-care strategies. many participants reported that they initially under-estimated the time that coursework, practica, and other responsibilities related to graduate school would take. one student, who had written at the start of the semester that graduate school had already threatened to become overwhelming, commented at the end of the semester, "i quickly realized that those stressors from undergrad and the ways i would cope with them, were not going to help this year." as noted in table , there were some participants for whom the cycle of revision and reflection on self-care plans provided impetus to seek out counseling support. all of these participants described the change as being very positive; for example, one female graduate student in her early twenties wrote on the benefits of seeking counseling: "i am so glad i started following this assignment because i have never felt so mentally healthy and fit in my entire life. i feel like a whole new person this semester because i am prioritizing my mental and physical health." one domain of self-care that participants found particularly difficult to implement was in the area of spiritual health. onethird of participants who originally included strategies regarding religious expression removed these strategies when revising their plan. this finding was an interesting difference from results reported in other research (lewis and king ), but overall is consistent with the im-paact model in terms of participants developing a plan that is adapted to their needs. theme : participants perceived that the intervention procedures supported their thriving with self-care brown ( ) proposed that the final stage in the implementation of self-care based on the im-paact model is characterized by thriving. reaching this stage does not mean that a professional's self-care plan is a finished product, but rather that the professional has fully engaged in a plan for self-care which is continually re-assessed and adjusted to meet changing needs. individuals at the thriving stage will have prioritized their wellbeing as a fundamental aspect of their professional effectiveness. in the present study, evidence that participants were in the thriving stage was evaluated through how they described their reactions to their self-care plan and their descriptions of their approach to self-care as part of their professional functioning following the semester-long intervention. importantly, a participant who is thriving could still struggle with implementing certain aspects of her plan, as long as self-care was clearly described as a priority and she provided evidence of committing to a continual cycle of assessment and adjustment of a self-care plan. based on the model, all participants in the study were characterized as being at least partially in the thriving stage of self-care at the end of the intervention. due to the richness of their responses, excerpts from three student reflections are presented below, which represented the continuum of responses to these questions (yin ) . the first such response, from a female participant, demonstrated the importance, predicted by the im-paact model (brown ) , of prioritizing self-care, and adjusting self-care behaviors to provide a balance of thriving in a way that enhanced productivity. the participant also described a commitment to continuing these practices in her graduate school and professional career, an important mark of thriving: overall, i think that having a self-care plan has been helpful this semester. if i had not thought about doing activities that i enjoy or that help me to relax, i think my only focus would have been school work. i am glad that through this assignment i was able to be reminded about the importance of taking time for myself and doing activities that i enjoy. i also think that while implementing my self-care plan took time away from completing school work, having that break made me more productive once i started school work again. i think that continuing to implement my self-care plan will be helpful as i continue through graduate school and begin working as a school psychologist. the second illustrative excerpt, from a male participant, exemplified how participants described the role of self-care as fundamental to their future professional functioning: i imagine that any professional career i choose will come with assorted ethical dilemmas and stressinducing deadlines. such troubles must not only be met with adequate clinical expertise, but an effective self-care regimen. i imagine that as i become more familiar with the program, and how best to formulate a self-care plan, i will be extremely prepared for these situations… my experiences will self-care throughout this semester, and the forthcoming, will be influential while rival explanations were actively considered in making interpretations of thriving in participant responses, such as whether some outside influence rather than the intervention procedures might have led participants to make these statements, it is important to point out that the design of the research made it impossible to rule out some alternate explanations for the results. one example is that of a maturation threat; in other words, that the experiences of the participants could simply be explained by normal changes that would have occurred over the first semester in graduate school even in absence of any intervention related to self-care. while some participants described outside stressful events or mitigating factors in implementation of their plans, all nevertheless attributed their commitment to self-care at least in part to the semester-long intervention. despite the focus in the field of school psychology in attracting and retaining professionals to meet the personnel shortage, and the long-recognized risks of burnout in the profession (e.g., huebner & mills, ) , there is little research on the application of self-care strategies as a means to prevent burnout and attrition. this is especially important for training programs, who obviously play a large role in developing new school psychology professionals to address personnel shortages. while there are many scholars (e.g., newman ; huebner et al. ; barnett et al. ; barnett and cooper ) who have recommended that training programs include self-care in their curriculum, most of the existing research into self-care is correlational, and therefore, it is unknown how training programs should best accomplish this among many other priorities, and whether interventions to teach self-care are effective. the present study attempted to address this shortage in the literature by developing an intervention for self-care that was implemented over the course of the semester and documenting outcomes of the intervention. as a case study example of a self-care intervention, this study is intended to guide the development of future research, as a proof of concept that a selfcare intervention developed based on the im-paact model and the transactional model of coping (lazarus and folkman ; mcnamara ) can be feasibly integrated into school psychology training. all participants who participated in this self-care intervention considered the creation of a self-care plan in the context of their field experience class, with check-ins to monitor and adjust the plan, to be a helpful exercise. several participants were quite enthusiastic, reporting that the stressors they experienced in graduate school were much more intense than anticipated. consistent with research documenting high levels of stress and anxiety among graduate students (evans et al. ) , participants in the present study described many and varied stressors at the start of their graduate education. the largest source of stress was graduate training itself, with students worrying about being able to manage the volume and difficulty of the material. students also frequently expressed anxiety regarding managing their personal and family relationships amid the demands on their time caused by coursework. students participating in this study designed self-care plans that appeared to be geared to meet the stresses they anticipated; however, many of the students found their initial plans to be inadequate to meet the demands of the training program. the im-paact model (brown ) described application of selfcare to be a process that best results in a plan that is individualized, continually assessed, and adjusted to changing stressors. previous research with self-care for students in mental health training programs (lewis and king ) also documented that self-care strategies for students evolved over the course of the semester and needed to be monitored and revised to be effective. there was evidence that the intervention procedures helped to support these behaviors among participants; future research should further explore the process of adopting and sustaining self-care behaviors, to inform efforts at instructing students and practitioners to engage in these behaviors. while the present study described a case study which should suggest several important hypotheses for future research to consider, there are some important limitations in the design of the study that must be taken into account in interpreting the results. first, the study's goal was to obtain an in-depth understanding of the stresses that school psychology trainees face and the strategies they use to cope with those stressors after a self-care intervention. as a result, the participants were all enrolled in a single graduate program, and were overall a more homogenous group in comparison to school psychology graduate students overall. this may be particularly important if considering applying the conclusions of this study to students who are members of a minority group. proctor and truscott ( ) for example found that themes of fitting in with other cohort members as being a paramount concern to students of minority status, but in the present study among largely white european american students this theme was much less prominent. future research should therefore consider using a larger and more heterogeneous sample to understand how the study's conclusions might extrapolate to students of minority status. the second important limitation was the lack of a comparison group to determine whether any described findings are causally related to the self-care intervention. without a control group, the design of this case study was such that it could not rule out whether, for example, the experiences described by students might simply represent normal maturation in response to starting a graduate program. finally, an important limitation for the present study was in nature of the measures used for data collection and analysis. while efforts were taken to ensure the dependability and credibility of the data and conclusions, students were submitting the artifacts for a grade in their course, and may have been incentivized to exaggerate the importance or value of the self-care plan. likewise, there were no external measures to estimate how often students were actually implementing strategies as they described in their reflections, nor any quantitative measures of stress and burnout. despite these limitations, the present study should provide researchers with a proof of concept model for embedding selfcare into school psychology training. given the research into graduate student mental health (e.g., evans, ) and especially when considering the covid- pandemic, for which there have already been documented negative effects on mental health in general (gao et al. ) , it is important to consider the mental health needs of students enrolled in school psychology training programs. furthermore, it is an important professional outcome of training for students to develop coping mechanisms while in training. it is vital for future research to extend the understanding of the construct of self-care and explore interventions that can help inform practice and training recommendations. creating a culture of self-care in pursuit of wellness: the self-care imperative 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psychologists' job satisfaction: a -year perspective in the usa case study research and applications acknowledgments the authors would like to thank jennifer daly and nicholas sanders for assistance with copyediting and manuscript preparation.data availability the datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request. conflict of interest the authors declare that they have no conflict of interest.ethics approval the research reported in this manuscript was classified as exempt by the institutional review board at a small, northeastern private university. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.dr. bradford daly is an assistant professor and program director in the school psychology program at alfred university. he has years of experience as a practicing school psychologist and continues to consult in public schools. he is interested in applications of positive psychology and professional issues of school psychologists, particularly at the secondary school level. recently he has contributed to a project involving the effects of social media on the wellbeing teenagers.dr. rachel gardner is an assistant professor of school psychology at alfred university. her primary professional interests include tiered school-based mental health services and promoting student' subjective well-being. her research interests include the impact of social media usage on adolescents' wellbeing, provision of evidence-based mental health services within an mtss framework, and positive psychology interventions applied to youth in school settings. key: cord- -iu dijsl authors: rosenstock, linda; helsing, karen; rimer, barbara k. title: public health education in the united states: then and now date: - - journal: public health rev doi: . /bf sha: doc_id: cord_uid: iu dijsl it was against a background of no formal career path for public health officers that, in , the seminal welch-rose report( ) outlined a system of public health education for the united states. the first schools of public health soon followed, but growth was slow, with only schools by . with organization and growing numbers, accreditation became an expectation. as the mission of public health has grown and achieved new urgency, schools have grown in number, depth and breadth. by mid- , there were accredited schools of public health, with more in the pipeline. while each has a unique character, they also must possess certain core characteristics to be accredited. over time, as schools developed, and concepts of public health expanded, so too did curricula and missions as well as types of people who were trained. in this review, we provide a brief summary of us public health education, with primary emphasis on professional public health schools. we also examine public health workforce needs and evaluate how education is evolving in the context of a growing maturity of the public health profession. we have not focused on programs (not schools) that offer public health degrees or on preventive medicine programs in schools of medicine, since schools of public health confer the majority of master’s and doctoral degrees. in the future, there likely will be even more inter-professional education, new disciplinary perspectives and changes in teaching and learning to meet the needs of millennial students. decades, tensions between the evolving fields of medicine and public health continued to be reflected in discussions about the future of public health. by the 's, growth in the number of sph had stalled (there were only by ), and economic challenges of schools were large, dominated by inadequate funding to pay faculty salaries, obtain necessary facilities and purchase needed equipment. schools increasingly turned to the national institutes of health (nih) for research funding. there was growing interest in building departments of preventive and community medicine within medical schools-many of these would prove forerunners of subsequent independent sph, but that future was uncertain and unplanned at the time. the first major government investment in public health education came in with the hill-rhodes bill which provided funds for training and project grants for public health. this was the beginning of a period of renewed interest in public health as applications to sph increased. schools began to thrive, with growth from sph in sph in to in . concomitant with the growth in independent public health schools were important changes in the numbers and composition of formally trained public health professionals. during the 's teaching methods changed, with greater attention to problem-based learning, especially in medical schools. support for public health professional education has been inconsistent over the decades, with a marked erosion of federal funding, beginning in the 's. this trend only reversed in the last few years but is again at risk in the wake of a serious recession. state government support also has been variable but significant; of the current schools are public institutions, with different levels of state assistance. most schools with state funding have seen that support eroded over the last few years, some very significantly. a recent article in the chronicle of higher education provided data about declines in state support for public universities. the average state cut was . percent, with at least four state cuts exceeding percent. today, sph train public health professionals at multiple levels, provide services to their local communities and beyond, and conduct research to prevent disease, disability and avoidable mortality at the individual, community and societal levels. schools also translate research into evidence-based policies and practices in communities, clinical care settings and governments, non-governmental organizations (ngos) and private organizations. research in sph ranges from basic laboratory research (e.g., to explain molecular signatures for particular viruses, cancers and other diseases) to applied research in communities as well as policy research. in fact, it is this continuum from basic research to translation of research into practice and policies that makes sph especially relevant and skilled in solving problems. public health researchers often collaborate with faculty in schools of medicine, pharmacy, dentistry, nursing, and others. they conduct bench and clinical research as well as communication research, comparative effectiveness studies, clinical effectiveness research and translational research, frequently with community-based research components. these varied roles reflect, in part, the fact that public health is not just a profession, but also a professional culture and commitment. sph educate undergraduate, master's, doctoral, postdoctoral, and certificate students. schools also provide continuing education to public health professionals within and beyond their geographic reach. the us centers for disease control and prevention (cdc) funds training centers within sph charged with developing leadership skills among certain groups of health professionals (e.g., those from underserved groups). similarly, the cdc has funded preparedness centers that focus on training particular kinds of professionals within assigned geographic regions. , this training and related concepts enabled schools to provide direct responses to training needs of first responders and health department personnel, in response to the events following september , and outbreaks such as severe acute respiratory syndrome (sars) and influenza a (h n ). since september , , public health students and many practitioners are trained to understand concepts and language of biosurveillance, health risk communication, and the critical roles government agencies and nongovernment partners play in responding to public health emergencies. the landmark institute of medicine (iom) report, the future of public health, criticized sph for being overly research intensive and disconnected from practice. in response, many schools made administrative and policy changes that institutionalized the means by which practice communities can access academic public health expertise and also increased opportunities for academicians to connect with communities. despite some successes in addressing acknowledged deficiencies in practice, there still are many challenges to create permeable boundaries between academic public health and practice. for example, the need to demonstrate publication productivity may cause many younger faculty members to choose professional focus areas that have quicker timelines to publication than those required to build relationships and consensus with practice communities. some schools have modified their appointments and promotion guidelines to reflect the importance of practice, but this varies from school to school. within sph, students pursue their education with an extraordinarily interdisciplinary range of faculty, including biomedical scientists, medical care professionals, behavioral and social scientists (e.g., economics, sociology, politics), epidemiologists, biostatisticians, information scientists, lawyers, health service researchers and health educators, among others. as a result, sph are well-positioned to be university leaders in collaborations with other schools, organizations and within the communities they serve. increasingly, there are collaborations with schools of journalism, social work, and regional and city planning. this reflects, in part, recognition of the complexity of health and healthcare and the forces that influence them. the association of schools of public health (asph) was founded in by a group of seven sph concerned about the growth of public health education programs. asph worked closely with apha to develop standards and definitions for sph. from to , apha conducted accreditation of graduate professional education in public health, at first centered almost exclusively in sph, but later including other college and university settings. in , the independent council on education for public health (ceph) was established by apha and asph. responsibility for evaluation of sph was transferred to ceph, which initially limited its focus to school accreditation. in the late s, ceph responded to requests from practitioners and educators to undertake accreditation of community health/ preventive medicine programs and to a request from apha to assume additional responsibility for community health education programs. in , these separate programmatic categories were combined into a single category of public health programs. ceph is the accrediting body for sph, but other organizations accredit particular programs within sph. these include the commission on accreditation for dietetics education (cade) and the commission on accreditation of health management education (cahme). asph started as an association "representing university faculties concerned with graduate education of professional personnel for service in public health; to promote and improve education and training of such personnel, and to do such other things as may improve the supply of trained personnel for all phases of public health activity." over time, asph became the national organization whose members are ceph-accredited sph, not just in the us but internationally with inclusion of an accredited school in mexico and an associate member school in france, which is in the process of accreditation. asph membership includes all cephaccredited member schools, in ( figure ), , which together, graduate over , students each year. growth of schools and students in the most recent period has been dramatic ( figure ). additionally, six associate member schools are scheduled to become fully accredited sph within the next two years, and others have indicated intent to become fully accredited. , growth of schools is expected to continue as states and private institutions recognize their value, and student interest grows. ceph accredits about public health programs in a variety of kinds of institutions, e.g., mph programs in medical schools. some programs are not ceph-accredited. estimates gathered from (association for prevention teaching and research; unpublished survey) indicate that less than , graduates/year come from ceph-accredited programs. the number of graduates from unaccredited schools and programs is unknown. several large, for-profit, online universities also offer public health programs and degrees. there is considerable concern about the growth and quality of these programs. in an effort to establish public health as a recognized, certified profession, asph, apha, the association for prevention teaching and research, the association of state and territorial health officials and the national association of county and city health officials established the national board of public health examiners (nbphe) in september . nbphe's purpose is to "ensure that students and graduates from schools and programs of public health accredited by ceph have mastered the knowledge and skills relevant to contemporary public health." nbphe is an active, independent organization that develops, administers and evaluates a voluntary certification exam once every year. graduates of cephaccredited schools and programs are eligible to take the exam. as of this writing, the number of examinees each year is small (about , ) but growing. it is not known what the ultimate effect of the exam will be on job availability, selection, salaries or on the quality of the public health workforce. ceph's focus is improvement of health through assurance of professional personnel who can identify, prevent and solve community health problems. the council has several objectives, including to: promote quality in public health education through a continuing process of self-evaluation by schools and programs that seek accreditation. assure the public that institutions offering graduate instruction in public health have been evaluated and judged to meet standards essential for the conduct of such educational programs. encourage-through periodic review, consultation, research, publications, and other means-improvements in the quality of education for public health. to achieve this mission, ceph reviews sph resources, structure and programs through its established criteria, which are updated periodically. accredited sph must offer coursework in at least the five core areas of knowledge basic to public health: biostatistics; epidemiology; environmental health sciences; health services administration; and social and behavioral sciences. the core, broad knowledge areas form the basis of how schools structure curricula. however, schools are not limited to these disciplinary areas. some schools have added departments of genetics, maternal and child health, nutrition and other areas. nothing precludes expansion of the five core areas, but all students must get sufficient exposure to core public health disciplines (table ) . over the last several years, asph has developed competencies in a number of areas, such as undergraduate education and master of public health programs, and identified cross-cutting areas, such as cultural competence, public health biology and health informatics which augment the disciplinary focus of the core areas. review of competencies shows the richness of subject matter area included under disciplinary areas, such as epidemiology. across schools, it is expected that students gain skills in a variety of areas and also emerge with understanding about the multiple determinants of health, using the kind of social ecologic model identified in the iom report, who will keep the public healthy? the accreditation process is based on peer review, in which a site visit team visits each school and evaluates their self-study and the processes behind it. according to the ceph website , site visitors must: be a senior academician (e.g., dean, associate dean, department chair or senior faculty member); or a senior public health practitioner (i.e., primarily employed by a public health department, non-profit organization, healthcare organization, etc. with preferably at least years of experience in public health); and have at least a master's degree (practitioners) or a doctoral degree (academicians); and possess strong writing, communication and analytical skills. ceph is responsible for selecting site visit teams, chairs and assuring that guidelines are followed throughout the accreditation process for each school (table ) . in , ceph amended and strengthened accreditation criteria for schools. sph now are required to have at least five full-time faculty members for each of the five core areas of study (minimum of faculty members) and must offer at least three doctoral degrees in three distinct programmatic areas. again, they are not restricted to this minimum, and most mature schools have many more programs. some also offer joint degrees with schools of social work, medicine, dentistry, nursing, city and regional planning, law, business, information and library sciences and other areas. accreditation requirements are a floor and not a ceiling. • environmental factors including biological, physical and chemical factors that affect the health of a community. • planning, organization, administration, management, evaluation and policy analysis of health and public health programs. • concepts and methods of social and behavioral sciences relevant to the identification and solution of public health problems. schools must be independent, with status similar to other professional schools at their universities. that aside, the perceived value of sph undoubtedly varies across universities and is likely to be affected by a school's rankings, success in obtaining grants and contracts and other issues. criteria for programs are similar to those for schools, with some differences. each degree program and area of specialization must have clearly stated competencies that guide development of educational programs. these define what a successful learner should know and be able to do upon completion of a particular program or course of study. asph developed master's degree core competencies in to serve as a resource and guide and continues to develop competencies in several other priority areas, such as preparedness. accreditation has both advantages and disadvantages. from the perspective of students and the field, accreditation assures a minimum level of quality in relation to established criteria. specifying core disciplines that must be represented and taught, identifying core competencies and clearly specifying relationships between goals, learning objectives and student outcomes is a strength of the process. but such a process also carries threats to innovation if criteria are interpreted too narrowly and do not permit new developments in format, methods and content of training programs. there also is more emphasis on teaching and service aspects of schools and less on research which, for research universities, is an important part of the mission. in addition, costs of accreditation, both direct and indirect, have grown as the complexity of the process has grown. lengthening the time period between reviews might be appropriate in view of this. fifty years ago, the profile of a public health student was a white physician or nurse who pursued an mph in order to practice at a health department or other similar setting. today, about eight percent of public health students have medical degrees. current public health students are younger, with less work experience, and more varied in the academic disciplines and the perspectives they bring to the profession. they also are more diverse in terms of ethnicity, race, age, socioeconomic backgrounds and culture and related characteristics. , students' and trainees' characteristics vary as much as diversity of the schools themselves. in , over , students were enrolled in accredited sph (table ) ; about one third of students were part-time, and many were trained in online programs with limited in-person classroom contact hours (distance education offered at schools). in , females represented percent of graduates. minorities (including asians) received percent of graduate degrees awarded to us students. sixty percent of graduates received mph degrees. doctoral degree recipients were dominated by phds, about -fold more often than doctor of public health graduates. international students, despite small dips in enrollment in recent years, continue to grow and now constitute percent of graduates. in , across all accredited sph, there were over , faculty members. overall, program areas with highest concentrations of graduates are health services administration ( %), epidemiology ( %) and health education/behavioral sciences ( %). "other" program areas included percent of graduates, despite efforts to categorize degree classifications into one of the ten categories in asph's annual survey. this may reflect diversity of offerings, as well as efforts to adapt to new priority areas and other emerging areas of focus, such as health equity, health systems modeling, public health preparedness, health implications of climate change, and chronic disease prevention. graduates from public health accredited schools and programs conduct research and teach in universities, international bodies and nonprofit organizations, manage healthcare and health insurance systems, work in the private sector and for foundations, are public health leaders in state, local and federal health agencies, and work globally and locally in many different roles. in the us, academic public health continues to grow in size and stature. the scope of public health education is expanding to new collaborations among health professions and other professional degree programs and includes college and even high school students. broadening public health education as a core body of knowledge for students, not just in other health professional schools but well beyond, was augured by the iom's report, who will keep the public healthy? specifically, the report called for a dramatic upsurge in master's level training in public health for medical professionals, citing the need to train as many as half of all medical school students at this level. inter-professional education extends far beyond more traditional medicine and public health training. for public health, it is seen when multiple professions' disciplines collaborate to advance the knowledge and skills of professionals and students. public health schools have a long history of collaboration with other schools and colleges within their own universities. these include formal dual degree opportunities. some of the most common joint degrees include mph/md degrees, but also degrees joint with law (mph/jd), dentistry (mph/dds), social work (mph/msw), nursing (mph/msn), business (mph/mba) and veterinary medicine (mph/dvm). several schools offer dual degree training with schools of communications, journalism, information and library science, public policy, city and regional planning, education and international affairs. these combinations allow students to integrate curricula towards their particular interests. there is no conceptual limit to potential joint and dual degree programs; they are likely to increase in the coming years. for many years, a small number of schools offered undergraduate study of public health including public health majors. recently, public health has emerged in a broad spectrum of undergraduate programs amidst growing interest in public health. in , the american association of colleges and universities surveyed their membership and found that institutions offered undergraduate majors, minors or concentrations in public health. universities with sph clearly dominate the playing field, with schools offering public health as a major area of concentration, and offering a minor concentration, accounting for nearly , under graduate students in . a recent front page washington post story captured this interest, in an article entitled "for a global generation, public health is a hot field." public health as a field has an increasingly wide appeal for students concerned with what the st century holds for the world's population, and some potential applicants would like to be able to enter the field with less time in school. additionally, there is also increased attention to opportunities at the community college level for public health education. applicants' interest in sph is growing at a remarkable rate, eclipsing other health professional fields, such as medicine. there was a percent growth in the number of applicants between and , from about , to , / year. despite annual healthcare costs in the neighborhood of $ trillion usd/ year, the us ranks th in life expectancy and nd in infant mortality among the world's nations. the us invests less than two percent of each heathcare dollar on prevention while spending percent of that dollar treating preventable diseases. such an imbalance defies peer-reviewed findings that show prevention activities in most instances are far more costeffective in improving health than medical treatment. , unlike medical schools, sph do not receive core federal funding for education [beyond a small pool of students], such as is received through medicare funding for medical residents or core federal funding for research and service/care available through the veterans administration for faculty effort. while the nih bench science model drives much of the highly valued research at sph, progress has been made in garnering nih and foundation support for applied research in epidemiology, behavioral sciences, health policy, and environmental health. limited fiscal resources, however, often make it difficult to mobilize and sustain research articulated by the practice sector and communities of need. funding for sph comes from a variety of sources, which include: tuition and federal sources: health resources and services administration (hrsa), cdc and nih funding of students; research supported by federal, state, city and not-for-profit organizations (~$ million in ), and foundation, corporate and philanthropic support. state and city universities and colleges often receive support from the relevant governmental level. the amount of this support varies, and has in general been significantly declining in recent years. for example at uc berkeley and ucla, core support has eroded to about percent. funding at sph ebbs and flows depending on current governmental priorities. as mcginnis and foege observed, "one of the most difficult challenges is that the urgent does not crowd out the important. in health, this challenge is especially difficult, because urgent matters can be so riveting…" examples of interventions with known major impacts on individual health include tobacco control and injury prevention activities. however, as colgrove et al. stated, "the current funding system for sph is piecemeal and largely reactive and constrains the ability of sph to meet essential societal needs. we argue that the federal government should invest significant and sustained financial support for this work through a dedicated funding stream." this would be a milestone for a field that lacks support to carry out its essential functions. several programs at sph contribute to the nation's health through provision of effective, up-to-date public health training to public health workers via a network of regional centers. to successfully carry out their charge, these centers have formed formal partnerships, particularly with local and state health agencies. in late , hrsa funded public health training centers (phtc), of which are located at sph, nearly doubling the previous network of training centers. phtcs aim to develop the existing public health workforce as a foundation for improving the infrastructure of the public health system. phtcs are based on collaborations with health departments and foster close advisory roles for academia and practice partners in their geographic areas. cdc-supported centers for public health preparedness (cphp) which began in and funded schools to prepare frontline public health workers to respond to bioterrorism and infectious disease outbreaks. in , these centers were redesigned, and new preparedness and emergency response learning centers (perlc) were funded at schools. these centers support workforce development needs by offering assistance to their state, local and tribal public health partners and are developing consistent curricula using public health workforce competencies. in , cdc funded nine schools to establish preparedness and emergency response research centers (perrcs). centers connect public health researchers with scientists involved in business, engineering, legal, and social sciences and conduct research that will evaluate the structure, capabilities, and performance of public health systems for preparedness and emergency response activities. the cdc prevention research centers (prc) program funds prevention centers, the majority of which are housed in sph. the prc program is an effective model for applied population-based prevention research. community and research partners collaborate to develop programming and identify successful aspects of research projects that can be disseminated to other communities. prcs play a leading role in translating bench and clinical research findings into practice in complex and diverse community settings. this kind of research, which adapts, refines, and demonstrates the effectiveness of community interventions, is contributing to understanding mechanisms for improving the health of populations. prcs are integrally related to public health education, not just through interactions with community public health professionals but also through opportunities for involvement of students. examples of other research and training centers in sph include: education and research centers (niosh), which conduct research and training and make recommendations for the prevention of work-related illnesses and injuries; centers of excellence in health statistics (nchs), which improve data collection systems to help develop and evaluate prevention programs; injury prevention centers (ncipc), which fund and monitor research in three phases of injury control: prevention, acute care, and rehabilitation; centers for genomics and public health (nceh), which study all elements of our human genome and how they relate to human health and disease; public health research and education centers (phrecs) within the veteran's administration, which conduct research, education and outreach on health promotion and disease prevention activities for veterans; and, centers of excellence in environmental health (nceh), which partner with state and local health departments, to develop state-of-the-art environmental health programs based on the essential public health services. in addition to these examples, there are many other centers and institutes within sph. public health professionals have been forced over an extended period of time to do more with fewer people, a problem greatly exacerbated by the recent global recession. "given the increasing complexity of public health science, meeting these challenges means training many more specialists in the many sub-disciplines of public health. as well, the availability and capacity of a global public health workforce needs to be significantly expanded." although for some time, there has been widespread recognition that the us has a shortage of well-trained public health professionals, no quantitative estimates of projected needs had been taken prior to . at that time, a taskforce of the asph set about quantifying public health workforce needs projected for . , , we summarize below the findings and implications of the workforce report and related subsequent efforts. as shown in table , "in , there were , fewer public health employees than in . , the workforce ratio in - public health workers for every , us residents-although a likely underestimate of need, was used as a benchmark. given population increases, a total of , (vs. the , available) would have been necessary in to maintain the workforce ratio that existed two decades earlier. in , a public health workforce of more than , would be needed to achieve the : , ratio. that creates a need for some , more workers than are available today." table public health workforce to us population ratios even that number is undoubtedly conservative, since public health departments across the us absorbed substantial personnel cuts during the recession of - . extrapolation of these data to projected shortages by state is demonstrated in figure . these estimates also do not take into account the large potential retirement effects of an aging worker cohort. although some retirements may be postponed due to the economic recession, by , more than , us public health workers in government- percent of an estimated , -person workforce-will be eligible to retire. in addition, the estimates are supply-based and do not attempt to quantify need or demand or the serious issue of geographic distribution and discipline-specific projects (e.g., laboratory workers vs. epidemiologists). several other organizations (e.g., apha , trust for america's health, association of academic health centers (aahc) ) and efforts have addressed specific disciplines. for example, the american association of medical colleges (aamc) has reported a shortage of , public health physicians, recommending a doubling of public health physicians currently in practice. moreover, there are demonstrated racial and ethnic disparities and significant geographic gaps in the public health workforce as the sullivan commission on diversity in the healthcare workforce concluded. "today's physicians, nurses, and dentists have too little resemblance to the diverse populations they serve, leaving many americans feeling excluded by a system that seems distant and uncaring. the fact that the nation's health professions have not kept pace with changing demographics may be an even greater cause of disparities in health access and outcomes than the persistent lack of health insurance for tens of millions of americans." sullivan commission on diversity in the healthcare workforce. public health workforce shortages are even more critical in much of the developing world. for example, sub-saharan africa has percent of the world's population and percent of the global burden of disease-yet it commands less than one percent of the world's health expenditures. the world health organization has said there is a "major mismatch" between population needs and the available public health workforce in terms of overall numbers, relevant training, practical competencies and sufficient diversity to serve all individuals and communities. , events and population health changes of the last few decades, have shown that countries do not exist in isolation and are increasingly interdependent. , , health professionals move from one country to another in a permeable manner. similarly, health conditions know no borders. , an epidemic that starts in the us, africa or thailand may become worldwide for non-communicable conditions as well as communicable diseases. tobacco companies found global markets after they became stymied in the us. a similar phenomenon is occurring with regard to availability of processed foods and obesity. the internet has made global communication instantaneous and accessible to more and more individuals regardless of country. burgeoning funding for aids through the us president's emergency fund for aids relief (pepfar) provided support for many public health researchers to conduct global research. older us sph have undertaken global activities for many years, although in the past, the area was referred to as international health. in dreaming of a time, korstad described the global travels and sanitation consultation of faculty in environmental sciences and engineering at the university of north carolina in the 's and 's. in other departments, faculty members and students traveled around the world as they worked on health projects. participation of americans in leading roles in international health, such as outlined in the preface to this edition by donald henderson, was not uncommon. however, except in a few schools with organized departments of international health (such as harvard and johns hopkins), systematic attention to international/global health was inconsistent. asph is leading an effort to develop global health competencies, and individual schools have been engaged in this effort as well. to many, global health and public health are indistinguishable. both global health and public health share many characteristics, including an emphasis on population-level policies, as well as individual approaches to health promotion. the commission on education of health professionals for the st century said that "maintaining a comparative global perspective can enrich existing curricula, thereby reducing the demand for extra time and space." the current focus on global health, separate from international health, is broader and not solely about developing countries. the us is approaching years of formalized public health professional education. some features present at the outset remain today, notably, recognition of a distinct field that is science-based across a broad spectrum of activities, from the laboratory to bedside to communities, both domestically and globally. there continues to be some tension about the relationship between medicine and public health, with some holding to distinct boundaries, and others claiming the need for better integration. however, with broad research collaborations across schools and growing numbers of medical students receiving public health training, old dichotomies between medicine and public health are breaking down. even the term has come under review, with an increasing number of cited references referring to population health as a better descriptor of the field conventionally known as public health. much has changed; with change has come evolution in the structure and functions of public health education. globalization has spared little, and certainly not the health arena. as recognition of the importance of global health has grown, and with it, attendant economic resources, the area of global health -which an increasing number would define as synonymous with global public health -has caught on with great interest, capturing the increasing attention of the medical education and care communities as well. , public health schools and training programs have responded to the growing interest of students and have flourished as they couple this interest with longstanding activities of their own faculties. perhaps most dramatic over the past years is growth in numbers of students and their diversity. this trend promises to continue despite economic challenges created by recession. our field is exciting and better understood than it has been throughout most of its history. the importance of public health education should continue to grow, not only as its own distinct field but in the context of increasing interprofessional education, team-based learning and increasing opportunities to link research and education to didactic learning and practice, in the us and globally. over the last few decades, there has been greater attention paid to building the evidence base for public health, adapting a model that originally was built for medicine and operationalized in the us preventive services task force (uspstf). the cdc's task force on community preventive services is the us body charged with assessing evidence for public health interventions. , focus on building the evidence base for public health is an important trend. this review has not focused on changes in healthcare delivery and payment that accompany the patient protection and affordable care act. expansion of health insurance coverage for millions of americans is accompanied by a number of central issues relevant to public health education, including a central emphasis on the importance of prevention and public health, with recognition of the importance of workforce development and funding. moreover, there is a large role for sph in conducting comparative effectiveness research to answer important questions about which public health and healthcare interventions are most effective in practice. we look forward to major opportunities to improve and innovate in public health education as a result of the passage of this historic legislation. as we enter a new decade, well-trained public health graduates are needed more than ever before. we face huge global threats, such as lack of safe water, emerging infections, wars, global income inequality, climate change, global obesity epidemic and changing demographic patterns associated with global aging. new technologies have potential to ameliorate some of the divide between rich and poor, developed and developing countries by providing access to information and tools to use information for improving the health of individuals and societies. as globalization makes the world smaller, public health graduates from the us and other countries are needed to strengthen health systems around the world. the complexity of these problems requires that students be trained, not in disciplinary silos but in interdisciplinary environments where they learn how to discover, find, synthesize and use information for health improvement. the commission on health professionals for a new century, an ambitious agenda for health professional training in the new century concluded, "the next generation of learners needs the capacity to discriminate vast amounts of information and extract and synthesize knowledge that is necessary for clinical and population-based decision making." new skills, like data mining and visualization, will become increasingly important as we face terabytes of data that require sense making. research synthesis and health informatics also are likely to be increasingly important. some schools have begun to integrate teaching of the core disciplines, on the assumption that most students will work in interdisciplinary settings, and that the silo approach to disciplines is no longer appropriate. over time, it is likely that there will be more integrated teaching and learning across disciplines, because the amount of content is growing at an enormous rate, beyond what can be absorbed into courses conducted in isolation. inter-professional training should become more frequent, as well. we look forward with great interest and enthusiasm to changes that are likely to come in sph as our students increasingly demonstrate that they learn and communicate very differently than their predecessors. the millennial generation of students and teachers is expanding conventional teaching with their use of new media and their skills for information search and synthesis. , we anticipate that, coupled with additional technological advances, these factors will drive significant changes in the way we educate future generations of public health professionals. for example, future classrooms are likely to offer global connections to facilitate hybrid learning, with students from different countries participating in discussions. students increasingly view themselves as global citizens, and that bodes well for the future of public health. need for stable core funding for schools of public health. changing patterns of teaching and learning for new generations of students. integrating global and domestic missions of public health. integrating academic and practice missions. accommodating the tremendous knowledge explosion within the context of accreditation expectations. dealing with new problems, (e.g., climate change), and new disciplinary areas, (e.g., neuroscience). need for more inter-professional education among health sciences schools. accredited schools of public health have grown and continue to grow in number, depth and breadth. despite growth of programs, there is widespread recognition that the us has a shortage of well-trained public health professionals. accredited sph train professionals at multiple levels, provide service to local and global communities, and conduct and translate research at the individual, community and societal levels. despite many successes in addressing public health practice contributions in academia, there remain many challenges (e.g., most schools lack consistent funding mechanisms that are not research-oriented). with globalization and increased complexity involved in strengthening health systems around the world, today's students must be trained, not in disciplinary silos but in interdisciplinary environments. institute of hygiene: a report to the general education board of rockefeller foundation professional education for public health in the united states evolution of public health and preventive medicine in the united states the future of public health. institute of medicine of the national academies the future of the public's health in the st century who will keep the public healthy? educating public health professionals for the st century. institute of medicine of the national academy of sciences disease and discovery: a history of the johns hopkins school of hygiene and public health medical education in the united states and canada: a report to the carnegie foundation for the advancement of teaching health professionals for a new century: transforming education to strengthen health systems in an interdependent world federal support of schools of public health state support for higher education continues to fall are schools of public health needed to address public health workforce development in canada for the st century? public health preparedness centers for public health preparedness program: from vision to reality asph: association of schools of public health asph: association of schools of public health. member schools map nbphe: national board of public health examiners changing demographics of public health graduates: potential implications for the public health work force association of schools of public health. asph annual data report asph policy brief -confronting the public health workforce crisis: executive summary liberal education and public health: surveying the landscape for a global generation, public health is a hot field. the washington post a call to action: public health and community college partnerships to educate the workforce and promote health equity association of schools of public health. asph policy brief -creating a culture of wellness: building health care reform on prevention and public health greater use of preventive services in the us health care could save lives at little or no cost the immediate vs. the important schools of public health: essential infrastructure of a responsible society and a st-century health system ga: the centers for disease control and prevention the public health workforce : new challenges for the health-care workforce, a critical prognosis. the chronicle of higher education public health personnel in the united states : second report to congress bureau of health professions. the public health workforce enumeration issue brief -the public health workforce shortage: left unchecked, will we be protected public health workforce shortages out of order, out of time: the state of the nation's public health workforce: a report by the association of academic health centers physician shortages to worsen without increases in residency training sullivan commission on diversity in the healthcare workforce. missing persons: minorities in the health professions taking stock: health worker shortages and the response to aids working together for health: world health report global health is public health towards a common definition of global health dreaming of a time: the school of public health: the university of north carolina at chapel hill, - . chapel hill: the university of north carolina at chapel hill on the eradication of smallpox and the beginning of a public health career developing competencies for a graduate school curriculum in international health patient protection and affordable care act committee on comparative effectiveness research prioritization board on health care services. initial national priorities for comparative effectiveness research the future of learning institutions in the digital age teaching digital natives key: cord- -ns ehm authors: xiang, mi; yamamoto, shohei; mizoue, tetsuya title: depressive symptoms in students during school closure due to covid‐ in shanghai date: - - journal: psychiatry clin neurosci doi: . /pcn. sha: doc_id: cord_uid: ns ehm nan since the first identification of a patient with covid- , this infection has spread rapidly worldwide, with a high case fatality rate among the elderly and individuals with chronic diseases. while this virus is less likely to cause serious illness in the younger generation, many affected countries have adopted school closure as a measure to prevent its spread among the community. concerns may be raised about the adverse effects of school closure not only on education but also on the health of children. the drastic changes in children's lifestyle during school closure may deteriorate their mental health. the present paper reports the changes in depressive symptoms among students in shanghai caused by school closure due to the covid- epidemic. the present study was originally designed to assess the health and health-associated factors using a web-based survey among schoolchildren (ages - years) and their parents from five schools across in shanghai, china. details of the study procedure are described in appendix s . first survey was conducted during january to , (before school closure) and the second survey was conducted from march to , (during school closure). depression was assessed using the children's depression inventory-short form (cdi-s); the total score ranges from zero to , with a higher score indicating more depressive symptomatology. we also asked positive aspects of lifestyle change during school closure. we calculated the mean and sd of cdi-s scores before and during school closure and tested the difference using paired t-test. a within-subjects anova was conducted to determine the interactions of time × subgroup (sex, school levels, and income levels) in terms of cdi-s score. we declare statistical significance when p < . for paired t-test and p< . for interaction test. of , students in the target schools, , ( %) participated in the first survey. of these, , participated in the second survey. overall, the mean cdi-s score significantly decreased between the two surveys: . before school closure and . during school closure (p< . ). in stratified analyses, children in middle school showed a greater decrease in cdi scores than those in primary school (p for interaction = . ), whereas a lower cdi-s score during school closure was consistently observed across genders and household income categories. as regards positive aspects of lifestyle change during school closure, %, %, and % of children were satisfied with having more time "at home," "with their parents," and "doing their own things," respectively. this article is protected by copyright. all rights reserved. school closure with staying-at-home policy should drastically decrease the opportunity of direct contact with persons other than family members, decrease physical activity, and increase the time of screen viewing, all of which may deteriorate mental health of school children. however, the present result is at variance with prior hypothesis; rather, we observed fewer depressive symptoms during school closure compared to those measured before the closure. the present finding based on pre-and post-observation without comparison group should be interpreted with caution. nevertheless, this unexpected finding could be attributed to relatively good educational environment at home. in shanghai, nearly all families own a personal computer or smartphone, which has been used for communication with school before the pandemic. soon after school closure, each school has started providing an online and tv program for education, and children have received interactive education at home from their teacher and communicated with classmates on a daily basis. such remote educational system might have contributed to better mental health during school closure. children, especially those under high academic pressure, might have less stress at home environment than at school setting. analysis by school type showed that middle school students, who this article is protected by copyright. all rights reserved. had a higher depressive score before school closure than primary school students, showed a greater decrease in depressive symptoms during school closure. this may partly reflect greater academic stress preparing for entrance examinations in middle school. as regards poisitive aspects of staying at home, most children reported being satisfied with having more time "at home," "with their parents," and "doing their own things." taken together, a well equiped home-schooling system, together with fewer stressors and positive aspects of staying at home, may have jointly outweighed the potential harm of school closure to children's mental health. in conclusion, the present longitudinal study in shanghai during the covid- does not show any evidence of increased depressive symptoms among students after a two-month school closure. additional study is required to assess long-term effect of school closure on mental health and educational attainment of children. ) . cdi-s, children's depression inventory-short version; cny, chinese yuan. *calculated using paired t-test. † calculated using within-subjects anova by the interaction effect of time × subgroup [sex, school levels (primary or middle school), or income levels (< , , , to , , > , to , , or > , cny)]. people who are at higher risk for severe illness united nations educational, scientific and cultural organization. covid- educational disruption and response mitigate the effects of home confinement on children during the covid- outbreak technical manual update. north tonawanda, this article is protected by copyright. all rights reserved. ny: multi-health systems academic stress in chinese schools and a proposed preventive intervention program this research was funded by the national natural science foundation of china (grant number ) for mi xiang. the study protocol was approved by the ethics committee of shanghai, jiao this article is protected by copyright. all rights reserved.tong university school of medicine (sjupn- ). all authors have indicated they have no financial relationships relevant to this article to disclose. this article is protected by copyright. all rights reserved. key: cord- -dqsg e authors: tang, suqin; xiang, mi; cheung, teris; xiang, yu-tao title: mental health and its correlates among children and adolescents during covid- school closure: the importance of parent-child discussion date: - - journal: j affect disord doi: . /j.jad. . . sha: doc_id: cord_uid: dqsg e background: : school closures due to the covid- outbreak have affected % of the world's students physically, socially, and psychologically, yet rigorous investigation into their mental health during this period is still lacking. methods: : a cross-sectional online survey of , primary and secondary school students from shanghai, china was conducted during march – , . besides demographic information, psychological distress (including depression, anxiety, and stress), life satisfaction, perceived impact of home quarantine, and parent-child discussions on covid- were assessed. results: : the three most prevalent symptoms were: anxiety ( . %), depression ( . %), and stress ( . %). participants were generally satisfied with life and . % became more satisfied with life during school closures. senior grades were positively correlated with psychological symptoms and negatively associated with life satisfaction, whereas the perceived benefit from home quarantine and parent-child discussions on covid- were negatively correlated with psychopathological symptoms and positively correlated with life satisfaction. among participants who perceived no benefit from home quarantine, those who had discussions with their parents about covid- experienced less depression, anxiety, and stress. limitations: limitations included the inability to infer the casual relationship, no parental report for mental health of children aged to , and the inadequate measurement of parent-child discussion. conclusions: : mental health problems and resilience co-existed in children and adolescents during the covid- outbreak. given the important role of parent-child discussions, open communication between parents and children about the pandemic should be encouraged to help children and adolescents cope with mental health problems in public health crisis. the outbreak of the coronavirus disease has brought global affects with over million cases and over million deaths as of september , (world health organization, a . most countries implemented social distancing measures to slow down the spread of the infection, the most universally used methods include home quarantines and national school closures. in shanghai, china, where this study was conducted, there were local cases and imported cases by march , , when the data collection ended (state council information office of the people's republic of china, ) . shanghai launched its highest-level emergency response against the covid- on january , , and the level emergency response had been active until march , . during the level emergency response period, shanghai had closed all public venues and canceled all large-scale public events, and required people from infected areas to stay at home or be quarantined in groups for days (cui, ) . due to school closures, online learning had been implemented to all . million primary and secondary students in shanghai (shanghaieye, ). as estimated by the united nations educational, scientific and cultural organization (unesco) on march , , school closures have affected % of the world's students (i.e., more than . million children and young people in countries) by restricting access to education (united nations educational, scientific and cultural organization, ) . besides the potential inequality caused by the digital divide and distance learning practices (united nations educational, scientific and cultural organization, ) , school closure may also cause disruptions in the physical activity, social interaction, and mental health of children and adolescents . restrictions on movement can have a substantial psychological impact. a systematic review of studies examined the psychological impact of pandemic-related quarantine on hospital staff, parents, children, and the general population (brooks et al., ) . they found that quarantined people had an elevated risk of developing acute and post-traumatic stress symptoms and disorders, and showed a higher prevalence of general psychological symptoms, emotional disturbances, depression, stress, low mood, irritability, and anxiety-induced insomnia. serious concerns about the mental health of children and adolescents during the pandemic-related quarantine and school closures have been raised (golberstein et al., ; wang et al., ) ; the debate whether the benefits of the mandatory measures outweigh the psychological costs continues viner et al., ). an earlier study found % of isolated or quarantined american children in areas severely impacted by h n met the criteria for posttraumatic stress disorder (ptsd) based on parental reports (sprang and silman, ) . current evidence related to covid- were all provided by chinese scholars. the prevalence of depressive symptoms were . % in primary school students (xie et al., ) and . % in secondary school students (tang and ying, ) . the prevalence of anxiety symptoms in china were . %, . %, and . % in primary students from hubei province (xie et al., ) , primary and secondary students from shaanxi province (li et al., ) , and secondary school students from sichuan province (tang and ying, ) , respectively. in addition to capturing children and adolescents' mental health status during the covid- pandemic, key risk and protective factors need to be identified to further understand who are in the at- risk subgroups and what could be done to protect them against mental health problems and to maintain their mental health status. perceptions on the epidemic and its possible impact matter in predicting mental health status. fear of being infected was another risk factor for depressive symptoms in primary school students (xie et al., ) and anxiety symptoms in children and adolescents during the covid- pandemic (li et al., ) . similarly, it is likely that for children and adolescents who are confined to their homes due to school closure during the covid- pandemic, their perception of the impact of home quarantine may play an important role in relation to their mental health. during home confinement, parents are usually the ones who interact the most with children and adolescents. the world health organization (who) advises parents to discuss covid- with their children in an honest and age-appropriate way that addresses children's concerns and eases their anxiety (world health organization, b) . in this way, close and open communication between parents and children may serve as a protective factor in children's mental health. previous studies on epidemics and pandemics, however, have not examined either the perceived impact on children and adolescents nor that of parent-child discussion. to address these gaps in the literature, we aimed to estimate the prevalence of depressive, anxiety, and stress symptoms, and levels of life satisfaction, among children and adolescents experiencing home quarantine and school closure in shanghai due to covid- . we also aimed to identify factors related to their mental health status, with a closer examination of the role of the perceived impact of home quarantine and parent-child discussion relating to mental health status during the covid- pandemic. we followed the cherries guideline for online surveys (see supplementary file ). this crosssectional online survey was conducted among , students in primary school (grades - ), junior secondary school (grades - ) and senior secondary school (grades - ). cluster sampling was adopted to randomly select one school from each of the six districts in shanghai, china, and all students in the six identified schools were recruited. depression, anxiety, the chinese version of dass- (wang et al., ) was used to measure psychological distress during home quarantine due to covid- . the chinese dass- is a self-report measure containing seven items in each subscale to measure the constructs of depression, anxiety, and stress. participants responded to each item by rating the frequency and/or severity of symptoms over the previous week using a -point likert scale ( = did not apply to me at all, = applied to me very much or most of the time). to yield equivalent scores to the full -item version of dass- , the total score of each subscale was multiplied by two for scores that range from - . the scale could be used to identify individuals who were at high risk of developing mental health problems with cutoffs, for depression, for anxiety, and for stress (lovibond and lovibond, ) . the dass- has previously been used to assess children and adolescents aged to in china (mellor et al., ; and populations after traumatic events such as the sichuan earthquake (chan et al., ; . in the current sample, the cronbach's alphas of depression, anxiety, and stress subscales were . , . , and . , respectively. life satisfaction was assessed using two parameters, namely, current life satisfaction and a change in life satisfaction since the pandemic. the former was assessed using a -point likert scale ( = extremely dissatisfied, = extremely satisfied), and the latter asked the question, how do you see your current life now compared to before? and rated on a -point likert scale (- = a lot worse, - = worse, = pretty much the same, = better, = a lot better). children and adolescents' perceived impact of home quarantine due to covid- was measured in terms of positive and negative impact using a self-constructed questionnaire. the positive impact consisted of five possible benefits and negative impact consisted of eight potential problems. participants were asked to rate how well each item applied to them on a -point likert scale ( = did not apply to me at all, = applied to me very much). the average scores of each dimension were calculated to represent the overall positive and negative impact. the differences between the two dimensions were recoded as perceived beneficial ( ) if the average score of the positive impact was higher than that of the negative impact, and recoded to perceived not beneficial ( ) if the average score of the positive impact equaled to or was lower than that of the negative impact. if participants rated "did not apply to me at all" for all items, their data would be excluded in analyses using this variable. in the current sample, the cronbach's alphas of the positive and negative impact dimensions were . and . , respectively. parent-child discussion on covid- was measured by asking participants whether their parents had discussed covid- with them or not. if the participants responded yes, they were asked about the frequency of discussion during the past month. answers were recoded as not at all ( ), at least once monthly ( ), at least once weekly ( ), or at least once daily ( ). the online survey was conducted during march - , (after approximately two months of the covid- outbreak). the survey was performed by wenjuanxing (wjx.cn). prior to distribution, we piloted the survey within several co-workers, postgraduate students, primary and secondary school students who did not study in the sampled schools and asked them to give feedback on content and functionality. a link to the survey was sent to six teachers in charge of student affairs in six schools. the teachers then forwarded the link to all classes' social network groups and asked parents to have their child completing the self-reported questionnaire. both the students' and parents' consent were obtained. for students, implied consent was adopted. the consent information page including the purpose of the study, voluntariness of participation, confidentiality, length of time of the study, data retention was presented before entering the formal survey. only when the students ticked the box "i understand the information described above and agree to participate in this study", they would enter the formal survey. otherwise, the webpage window would be closed automatically. for parents, as the schools agreed to participate in the study, passive consent was adopted. if they did not wish to give consent, they were requested to make this known to the school. to ensure quality responses, research assistants and teachers were assigned to each of the six schools to be on duty online. if the parents and students were confused about certain questions in the questionnaire, they can ask the research assistant and teachers immediately so that the questions could be explained and clarified on time. other than clearing the confusion, the research assistant and teachers were required not to provide help on answering questions. parents were instructed to assist primary school students. ethical approval was obtained prior to data collection from the ethics committee of shanghai jiaotong university school of medicine (sjupn- ). the characteristics of the sample were summarized using descriptive statistics. cronbach's alpha coefficients measured the internal reliability of each subscale of dass- and the self-constructed questionnaire for the perceived impact of home quarantine; a cut-off of . was used to indicate good internal reliability. comparative profiles of demographic and quarantine-related variables between screened and unscreened subgroups of depression, anxiety, and stress were examined using chi-square tests. independent sample t-tests and one-way analyses of variance (anova) were conducted to investigate group differences in symptom levels of depression, anxiety, and stress, and life satisfaction. binary logistic regression analyses and multiple linear regression analyses were conducted to examine the independent associations between demographic and quarantine-related characteristics and depression, anxiety, stress, and life satisfaction. demographic and quarantine-related variables were entered into the regression models as independent variables, and mental health variables were considered dependent variables. multivariate analyses of variance (manova) were used to test the interaction between significant quarantine-related variables in association with the severity of depression, anxiety, and stress, and simple effect tests were conducted following significant interactions. the associations were reported as odd ratios (ors) or unstandardized coefficient b and % confidence intervals (cis). p values <. were considered statistically significant (two-sided). analyses were performed using ibm spss statistics, version . the total of , students were approached, and , students completed the survey, with a response rate of . %. among the respondents, students did not fill out their demographic information and students responded in patterned ways. their responses were excluded, resulting in a valid sample of , students. post-survey interviews with teachers suggested that the two most frequently mentioned reasons for not submitting or not completing the survey were having forget to fill out the questionnaire during the given time frame and the lengthiness and tediousness of the survey. the mean age of the sample was . ± . years, ranging from - . boys accounted for % of the sample. the proportion of primary, junior secondary, and senior secondary school students were . %, . %, and . % respectively. over half ( . %) of the students experienced the impact of home quarantine as positive. the majority ( . %) had discussed the pandemic with their parents. table shows the detailed demographic and quarantine-related information. table provides the percentages that met threshold for depression, anxiety, and stress, as well as means and standard deviations of symptom scores in the total sample and subgroups. among the participants, . % had experienced symptoms of anxiety, followed by . % for depressive symptoms and . % for stress symptoms. five hundred participants ( . %) met thresholds of depression, anxiety, and stress at the same time. percentages of mild, moderate, severe, and extremely severe levels of these symptoms are listed in supplementary table . senior secondary school students yielded the highest prevalence and levels of depressive, anxiety, and stress symptoms, and primary school students the lowest. positive screens and symptom levels for depression, anxiety, and stress were higher among those who were more negative about the impact of home quarantine and who had no discussion about the covid- with their parents. boys and girls did not show any difference in the prevalence and symptom levels of all three conditions. table presents the means and standard deviations of overall and subgroups' levels of life satisfaction. participants were generally satisfied with their current status, with the mean score ( . ± . ) significantly higher than (satisfied) [t( ) = . , p < . ]. the median was (very satisfied) and the mode was (extremely satisfied), indicating that half of the participants were very satisfied and those who were extremely satisfied were the largest subgroup ( . %). most participants experienced no change in life satisfaction ( . %), and . % were more satisfied with their lives during the pandemic. bonferroni post hoc tests showed that senior secondary students were more satisfied with life than primary (p = . ) and junior secondary (p = . ) students. participants who perceived themselves as having benefited from the quarantine reported higher current life satisfaction. in addition, participants who had discussed the pandemic with their parents reported higher levels of current life satisfaction than those who had not. table shows the odds ratio for depression, anxiety, and stress from binary logistic regression analyses and the unstandardized coefficients for the symptom levels from multiple linear regression analyses. the rates of positive screens for depression, anxiety, and stress and the symptom levels varied based on grade, perceived impact of the quarantine, and parent-child discussion about the pandemic. junior secondary school students were at higher risk of developing anxiety and experienced more severe levels of all symptoms than primary school students, while senior secondary students were at higher risk of developing depression, anxiety, and stress than other grades. the presence of perceived benefit and parent-child discussion lowered the risk of meeting the threshold of depressive, anxiety, and stress symptoms. similarly, being senior secondary students and the absence of perceived benefit and parentchild discussion were associated with higher levels of all three types of symptoms. table presents demographic and quarantine-related variables relating to current and changed life satisfaction in two multiple linear regression models. taking all factors into consideration, participants who perceived themselves as having benefited from the home quarantine and had discussion on covid- with their parents reported significantly higher levels of current life satisfaction. besides, perceived beneficial was significantly associated with increased life satisfaction during the pandemic. interestingly, senior secondary school students were less satisfied with life than others, but their levels of life satisfaction had increased more dramatically than others during the pandemic. in order to further explore how children and adolescents' intrapersonal perception on the impact of quarantine, interpersonal interaction with parents regarding covid- , and the interaction effects between perceived beneficial and parent-child discussion in association with the severity of depression, anxiety and stress, as well as levels of life satisfaction, were examined. the (perceived beneficial) × (parent-child discussion) anova analysis indicated significant effect for interactions between perceived beneficial and parent-child discussion for depressive this study examined the mental health status of children and adolescents during school closure due to covid- in shanghai, china. primary and secondary schools started online education from march , and continued until schools started to reopen after april , . children and adolescents' most prevailing mental health problem was anxiety, with a prevalence rate of . %, which is slightly higher than reported in a study conducted one month earlier ( . %; li et al., ) mostly due to the elevated prevalence for secondary school students. in china, secondary school students face more academic pressure than primary school students as they need to prepare for the zhongkao (entrance examination for senior secondary school) and gaokao (the national higher education entrance examination). since our study was conducted in the first two weeks of online schooling, adjusting to new forms of learning, online classes, and studying alone at home without faceto-face interaction with teachers and classmates may have contributed to secondary school students' more elevated anxiety levels. the second most prevalent mental health problem was depression, with an incident rate of . %. the rate for primary school students ( . %) was very similar to the pooled prevalence of depressive symptoms in chinese primary school students reported in a recent systematic review ( . %; % ci: . %- . %; xu et al., ) but lower than reported in primary school students in wuhan and huangshi, in hubei province, who were also surveyed during the same period of time ( . %; xie et al., ) the latter is understandable as hubei was the most seriously affected area in china during the covid- pandemic. the prevalence rate of depression for secondary school students ( . %) was slightly lower than the pooled prevalence rate reported in a systematic review on depression among chinese secondary school students ( . %; % ci: . %- . %; tang et al., ) and lower than findings during the covid- pandemic (tang and ying, ) . the lower prevalence of depressive symptoms among secondary school student in our study could be explained by the choice of screening instrument. screening instruments can affect findings among adolescents (tang et al., ) but not children (xu et al., ) . the study's adapted dass- showed a relatively lower prevalence of depression than other measures (tang et al., ) . the prevalence of stress symptoms ( . %) was lower than anxiety and depressive symptoms. acute stress symptoms are usually observed within the first month of a stressful event (american psychiatric association, ). since the current data was collected more than one month after the outbreak of covid- , due to the strict measure implemented by the chinese government, the pandemic was under control by early march, so children and adolescents may have started to experience less stress symptoms. notwithstanding, researchers and clinicians should pay attention to the fact that three out of twenty children and adolescents suffered from stress symptoms and provide timely intervention before they develop ptsd. children and adolescents were generally satisfied with their current life status. over % thought their lives were either the same or better since the pandemic. this finding was not surprising, given the co-existence of psychological distress and resilience in children and adolescents when faced with change or adversity. the study conducted in wuhan and huangshi, hubei province, showed that . % of primary school students worried about being infected with covid- ; that said, . % of them were optimistic about the development of the pandemic (xie et al., ) , supporting the finding that negative and positive feelings could co-exist during a traumatic event such as the covid- pandemic. our study revealed a pattern that depression, anxiety, and stress among children and adolescents increased gradually and substantially from primary and junior secondary to senior secondary school. this is consistent with previous findings in systematic reviews (barker et al., ; tang et al., ; xu et al., ) . the increased psychological distress from early to late adolescence might be due to biological changes (especially hormonal changes during adolescence), increased adverse life events (e.g., academic pressure increasing from primary to secondary school, interpersonal challenges with parents and friends; schubert et al., ) . our study also found that as adolescents progress to higher grades, their life satisfaction decreased. this was not surprising given the negative correlation between life satisfaction and psychological distress in the current study (from -. to -. , all ps < . ). benefit finding was identified as one of the important protective factors against depression, anxiety, and stress. our study found that children and adolescents perceived home quarantine as more positive than negative, and this yielded less psychological distress and more life satisfaction. their perceived benefits of home quarantine included increased time available to spend with parents and on personal activities. benefit finding is defined as "the positive effects that result from a traumatic event" (helgeson et al., ) and is commonly observed among children and adolescents facing healthrelated threats such as cancer (rosenberg et al., ; wicks and mitchell, ) and diabetes (helgeson et al., ; tran et al., ) . a meta-analysis of studies showed that benefit finding in adults was related to less depression and more positive well-being, but was unrelated to anxiety (helgeson et al., ) . another meta-analysis of studies conducted on children and adolescents found a significant to marginally significant negative relation between post-traumatic growth (or benefit finding) and depression, anxiety, general symptoms of stress, and emotional distress (meyerson et al., ) . our findings also echo other studies conducted during the covid- pandemic. for instance, primary students who were not optimistic about the development of the pandemic were at an increased risk of depressive symptoms compared with those who were optimistic (xie et al., ) . in short, benefit finding can buffer the possible negative impact brought on by home quarantine and school closures due to pandemic. guiding children and adolescent to perceive the pandemic from a constructive angle and to discover positive aspects in difficult times could be an appropriate way to help them to cope with the pandemic. parent-child discussion on the pandemic was another important protective factor, as children and adolescents who discussed the pandemic with their parents were less likely to present symptoms of depression, anxiety, and stress. even in those who failed to find benefit from their current situation, parent-child discussion still prevented them from developing more severe symptoms of depression, anxiety, and stress. this highlights the crucial role of open communication between parents and children when coming to terms with stress and crisis. among all psychosocial factors associated with depression in chinese adolescents, parent-child communication had the largest effect size (r = . ; tang et al., ) in line with previous findings, the duration and frequency of communication alleviated the effects of stressors on depressive symptoms (guang et al., ) . our study also found that the frequency of discussion about the ongoing public health crisis mitigated depression, anxiety, and stress, and boosted life satisfaction. it is worth noting that chinese parents tend to use supportive behaviors rather than verbal expression to deliver care and love to their children compared with american parents (zhang and wills, ) , so chinese parents especially need to be encouraged to discuss life events more frequently with their children to facilitate their children's mental health during times of public health crisis. several limitations should be considered in interpreting the results. first, the cross-sectional design of this study was unable to illuminate the causal relationship between risk and protective factors, and mental health outcomes. a longitudinal study with follow-up surveys after the schools reopen is needed to clarify these relationships. second, although primary school students completed the survey with the assistance of their parents, self-reporting on dass- by primary school students, especially those who aged - years old, may not be as reliable as other age groups due to possible misinterpretation of some items of the mental health measures. future research should include both self-reporting and parental reports when assessing mental health outcomes among young children to guarantee the data accuracy. third, parent-child discussion was measured merely in terms of frequency. the way how parents communicate with children, the specific content of their discussion on the pandemic, the quality of parent-child relationship, and their role in contributing to children's mental health status, are worth exploring in future studies. finally, some psychosocial factors of children and adolescents' mental health (e.g., academic performance, academic pressure, peer relationship; tang et al., ) were not included in this study. yet, this study cannot eliminate the possibility of residual confounding caused by these unmeasured variables. moreover, we did not had the chance to explore the reasons for not participating in the survey from the % non-respondents, whom may be from disadvantaged groups such as who had less parents' supervision for online learning, were less adaptive to online learning like reading online materials, or suffered from more psychological distress. despite these limitations, this study recruited a large representative sample of children and adolescents, assessed their mental health status, and identified the risk and protective factors during covid- related school closures in shanghai, china. anxiety, depression, and stress were common among children and adolescents experiencing home quarantine and school closure due to the outbreak of covid- . however, students were generally satisfied with their current life status. secondary school students were at greater risk of developing depression, anxiety, and stress, whereas benefit finding and parent-child discussion on the pandemic were two protective factors. policy makers and mental health professionals should ensure that mental health interventions are available and tailored to help children and adolescents cope with public health a participants who rated "not applied to me at all" for all items of perceived impact of home quarantine were excluded in all analyses (n = ). * p < . , *** p < . . correlates diagnostic and statistical manual of mental disorders prevalence and incidence of anxiety and depression among children, adolescents, and young adults with life-limiting conditions: a systematic review and meta-analysis the psychological impact of quarantine and how to reduce it: rapid review of the evidence extending the utility of the depression anxiety stress scale by examining its psychometric properties in chinese settings cities launch emergency response to contain virus coronavirus disease (covid- ) and mental health for children and adolescents depressive symptoms and negative life events: what psycho-social factors protect or harm left-behind children in china? benefit finding among children and adolescents with diabetes. medical illness and positive life change: can crisis lead to personal transformation? a meta-analytic review of benefit finding and growth investigation on the influencing factors for anxiety related emotioanl disorders of children and adolescents with home quarantine during the prevalence of novel coronavirus pneumonia manual for the depression anxiety stress scales factorial invariance of the dass- among adolescents in four countries posttraumatic growth among children and adolescents: a systematic review hope and benefit finding: results from the prism randomized controlled trial the psychological effects of quarantining a city depressive symptom trajectories in late adolescence and early adulthood: a systematic review online classes as schools to stay closed posttraumatic stress disorder in parents and youth after health-related disasters the th press on prevention and control of the covid- held in shanghai investigation on mental health status and its influencing factors among secondary school students during the covid- epidemic benefit finding, affective reactions to diabetes stress, and diabetes management among early adolescents unesco rallies international organizations, civil society and private sector partners in a broad coalition to ensure #learningneverstops school closure and management practices during coronavirus outbreaks including covid- : a rapid systematic review. the lancet child & adolescent health mitigate the effects of home confinement on children during the covid- outbreak the adolescent cancer experience: loss of control and benefit finding world health organization, a. who coronavirus disease (covid- ) dashboard [www document helping children cope with stress during the -ncov outbreak mental health status among children in home confinement during the coronavirus disease prevalence of depressive symptoms in primary school students in china: a systematic review and meta-analysis application of the short version of depression-anxiety-stress scale to stress assessment on students after earthquake a u.s.-chinese comparison of affectionate communication in parent-child relationships ) depression = . ; χ ( ) anxiety = ) anxiety = . ; χ ( ) stress = . ) stress = the authors gratefully acknowledge the research members, families, children, and adolescents who took part in this research, without whom it would not have been possible. table means key: cord- - khael authors: munday, j. d.; sherratt, k.; meakin, s.; endo, a.; pearson, c. a. b.; hellewell, j.; abbott, s.; bosse, n.; cmmid covid- working group,; atkins, k. e.; wallinga, j.; edmunds, w. j.; van hoek, a. j.; funk, s. title: implications of the school-household network structure on sars-cov- transmission under different school reopening strategies in england date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: khael background school closures are a well-established non-pharmaceutical intervention in the event of infectious disease outbreaks, and have been implemented in many countries across the world, including the uk, to slow down the spread of sars-cov- . as governments begin to relax restrictions on public life there is a need to understand the potential impact that reopening schools may have on transmission. methods we used data provided by the uk department for education to construct a network of english schools, connected through pairs of pupils resident at the same address. we used the network to evaluate the potential for transmission between schools, and for long range propagation across the network, under different reopening scenarios. results amongst the options evaluated we found that reopening only reception, year and year ( - and - year olds) resulted in the lowest risk of transmission between schools, with outbreaks within a single school unlikely to result in outbreaks in adjacent schools in the network. the additional reopening of years and ( - and - year olds) resulted in an increase in the risk of transmission between schools comparable to reopening all primary school years ( - year olds). however, the majority of schools presented low risk of initiating widespread transmission through the school system. reopening all secondary school years ( - year olds) resulted in large potential outbreak clusters putting up to % of households connected to schools at risk of infection if sustained transmission within schools was possible. conclusions reopening secondary school years is likely to have a greater impact on community transmission than reopening primary schools in england. keeping transmission within schools limited is essential for reducing the risk of large outbreaks amongst school-aged children and their household members. school closures are one of many non-pharmaceutical interventions that can be employed during epidemics of droplet infections, such as influenza, to reduce transmission, and can be highly effective [ ] [ ] [ ] . however, there are substantial societal and economic costs associated with closing large numbers of schools, such as limiting children's access to education and requiring caregivers to stay at home (impacting on household income and on economic activity) [ ] [ ] [ ] [ ] [ ] , which can affect deprived households most . as with any public health intervention, it is important for policy makers to balance the public health benefits of school closures with the associated economic and social impact. to do this effectively, clear understanding of the relative benefit of closing schools and therefore the potential impact of reopening is required. school closures were introduced as a central component of the response to the covid- outbreak in many countries around the world . the uk closed all schools on rd march to all but the children of essential workers and the most vulnerable. schools in england remain closed to the majority of students until the beginning of the coming academic year (september ). although reported cases of covid- are low amongst school-aged children, the role of children in transmitting covid- remains unclear. [ ] [ ] [ ] [ ] [ ] it remains an open question whether reopening schools might increase transmission within the community and therefore lead to a resurgence of the disease in the population. notwithstanding the poorly quantified risk, there is growing concern regarding the potential impact of prolonged closures on the wellbeing of the population at large . a recent report from the royal society voices concerns that maintaining widespread closures does not just pose a risk to children's wellbeing in the immediate term but may also have long term consequences for the skill level of the future workforce and therefore economic growth of the uk . the potential impact of reopening schools on transmission is twofold: firstly, the number of potentially infectious contacts increases through children mixing in schools. secondly, transmission within schools can facilitate transmission between households, and households with multiple school-aged children may act as a route for transmission between schools. this second impact can be considered as a network of schools and households linked by pupils. while strict stay-at-home orders (so-called lockdowns) as implemented in many countries had the aim of removing the links of network such that chains of infection could not progress beyond individual households, reopening schools has the potential of reconnecting households with each other such that longer chains of infection can arise. here, we investigated the potential impact of reopening schools on the connectivity of the school and household network and, consequently, on the potential size of an outbreak among families with school-aged children, under the assumption that children are effective at transmitting the virus. we did so by using a large data set of household addresses of school children in england to quantify probability of transmission through pupils who reside in a common household as the edges on a network of schools. we used this framework to analyse the potential for these links between schools to form large networks of infectious contact and therefore large outbreak clusters within the school-age population and their household members. individual level de-identified data of pupils attending state funded schools in england was provided by the uk department for education (dfe) under a formal data sharing agreement. the use of this data was also reviewed and approved (ref: ) by the london school of hygiene & tropical medicine research ethics committee. the data includes an entry for each pupil for each institution they attend, unique reference number (urn) for the school, school postcode, pupil's postcode and pupil's address, collected between september and december . we combined the student's postcode and address to assign a household code for each group of pupils that were found to live at the same address, where we assume each individual address operates as a single household for social distancing purposes. using our generated household code, we were able to estimate the number of unique contacts between each pair of schools. for each pupil we have included only institutions coded as the pupil's current main school and have excluded pupils listed as boarders (those who are resident at their school during term time). more details of data cleaning are included in the supplementary information. typically, there are school years in the english school system (figure ), which each run from september to september. children enter reception aged and complete years of primary school leaving year aged . they transition to secondary school into year where all pupils are expected to complete years of secondary education (until the age of ) . at this point children are able to leave school or progress to further education (fe), which may be in the same institution as other secondary school years or a separate institution offering only fe courses. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint there are exceptions to this two/three institution framework, where some schools offer a different subset of school years (for example the first years of primary education). for this analysis all reopening scenarios are assumed to operate on a school year basis, hence assuming that all children from the appropriate years return regardless of the nature of their institution. we considered six reopening scenarios relevant to policy in england, illustrated in figure . in each scenario different combinations of year groups return to school: early-years education (reception and year , i.e. - year olds) and time-sensitive groups in transition e.g. through exam certifications or transitional years (years , i.e. - year olds, year , i.e. - year olds and year , i.e. - year olds) . we used the data to construct a network of schools linked through households. each edge on the network of schools is weighted by the number of unique contacts between schools that occur through shared households. for example, if in a given household, children attend school and children attend school , this corresponds to unique contacts between school and school . the total number of unique contacts between schools and , denoted by !" , is the sum of unique contacts over all households. (figure ). concretely, !" = #,! #," # where #,! is the number of children in household who attend school . from this network, we created a transmission probability network ( figure ) where we estimated the probability of transmission between schools i and j ( %&'(),!" ) we defined the definition of transmission between schools as an outbreak in one school leading to an outbreak in an adjacent school on the network. we simplify within-household transmission such that only direct transmission between contact pairs occurs (neglecting the potential for transmission through other members of a household) and hence approximate the transmission probability between schools through a single contact pair as. where *+ is the probability of an outbreak in school i given one infection, !(,," is the probability of a child in school being infected and is the probability of transmission between children in the same household. the probability of transmission between schools j and i through all contact pairs can be approximated as is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint the probability of an outbreak *+ in any given school with reproduction number was assumed to be, which reflects a geometric distributed contact rate within the school . we assumed homogeneous mixing within the school population. we then approximated the probability of a student in school being infected " . based on the expected final size of an outbreak with reproduction number r, where / is the final outbreak size as a proportion of the school population. we set , the per-contact probability of transmission between children in the same household to . (consistent with estimates of household secondary attack rate of sars-cov- ). we repeated the analysis for a range of r values between . and . , leading to outbreak size between and % of school children, broadly spanning the range of reported outbreak sizes of covid- in schools , . for each scenario, we assumed all pupils within the years specified attended school and contributed to transmission. we assumed that pupils outside of the specified years did not attend school and therefore did not contribute to transmission. to simulate this condition, we constructed a network using only data of pupils in the specified years. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint to summarise how the potential of transmitting to adjacent schools in the network varies with r (within school) and the reopening scenario we calculated the distribution of the weighted degree of the transmission network (the distribution of the expected number of schools infected through households by each school) for each scenario, where the weighted degree of school , ! was defined as: to summarise the potential spread of the virus across the network of schools, we sampled instances of binary outbreak networks, where transmission between each pair of schools either occurs (edge weight of ) or does not occur (edge weight ) ( figure ). since transmission probabilities are reciprocal, the eventual number of schools in any outbreak cluster can be defined as a connected component of the outbreak network (i.e. all schools are connected by edges equal to ). for a particular school , the schools in the same connected component are those that would be infected in an outbreak seeded at that school ( ). the same schools are those in which a seeded outbreak would eventually infect this school ( ). hence the distribution of the connected components gives an indication of expected outbreak size and therefore risk posed to and by individual schools in the network. schools vary in size considerably, with large differences between secondary and primary schools. to reflect the size of outbreaks in terms of the number of households at risk, we calculated the number of households with children attending schools within each connected component in the network. specifically, we calculated the number of unique households with children attending the schools in each component (in the appropriate years for each scenario). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint to summarise the risk of larger outbreak clusters, we present the distribution of the number of households associated with each connected component. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint the full contact network ( figure ) included , schools, with . million primary school children and . million secondary school children in attendance, living at . million unique addresses. including only pupils associated with the reopening scenarios reduces the network to containing between % and % of all schools and between % and % of all households (table ). networks of contact through households between , state funded schools in england plotted by location. a) network with all school years in attendance, b) network with only primary school years in attendance, c) network with only secondary school years in attendance. nodes show schools with size determined by the weighted degree of the node (number of unique contact pairs with any other school). edge widths that indicate the number of unique contact pairs between the schools the edge connects. red nodes show secondary schools (mean age >= years), blue nodes show primary schools (mean age < years). followed by degree distributions of the networks of contact through households. d) a histogram of the number of schools connected by at least one contact pair and e) a histogram of the number of unique contact pairs with all other schools in the network including all school years (i.e. that shown in panel a). for all schools (grey) dots, secondary schools (mean age >= years, red circles), and primary schools (mean age < years, blue, circles). f) a histogram of the number of schools connected by at least one contact pair and g) a histogram of the number of unique contact pairs with all other schools in the network including all school years (grey), the network including only secondary school years (blue) and the network including only primary school years (red). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint the mean unweighted degree of the schools in the network (average number of schools each school is connected to) was with a maximum of . the mean number of contact pairs to all other schools was with a maximum of . (figure ) . secondary schools were more connected to the network with higher mean degree, schools, and weighted degree, contact pairs. primary schools were less connected with mean degree and weighted degrees of schools and contact pairs, respectively and a maximum degree of schools and weighted degree of contact pairs. when we included only primary school years in the network (i.e. secondary years did not attend school) the mean degree reduced to schools and mean weighted degree to contact pairs. when only secondary school years were included the mean degree and weighted degree reduced to schools and contact pairs respectively. with all schools fully open, the mean weighted degree of the transmission probability network (i.e. the mean expected number of schools infected by any individual school) varied between . for r of . , to . for an r of . . the school with the highest weighted degree varied between . to . for r of . and . respectively. when the network was modified to only include pupils from certain years the mean degrees decreased ( figure is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint to that of fully opening only, primary schools (table ) . of these, scenario (reception and years , and ) had the lowest mean degree for each value of r, between . and . . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint table median, mean and maximum weighted degree on the transmission probability network (expected number of schools infected by each school) and median and range of largest component size, households and schools, over realisations of the binary outbreak networks, for each scenario for r values between . and . . the largest connected component of the realisations of the binary outbreak networks, that is, the number of schools in the largest connected part of the network, increased with r for each scenario, increasing the number of households at risk (figure , figure s (supplementary information)). for scenario (reception, year , year ) the median largest components simulated ranged between and schools or and , households across r values considered, and there were very few exceeding schools in each realisation (figure ) , these connected components typically represented fewer than , households in total. . cc-by-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint figure the number of households with children attending a school in each largest connected component of the binary transmission networks (estimated potential outbreak cluster size) generated from transmission probability networks for school reopening scenarios. vertical bars show the % credible intervals. the green dashed line shows the total number of households in the school system. adding either of secondary school years or to the network (scenarios and ) increased the largest connected component size considerably. the size of the largest component was comparable to scenario at a low r of . , with a median largest component size of less than schools for all scenarios. however, the largest connected components for realisations at r of . reached many tens of schools for scenarios and ( and respectively, compared to for scenario ) and thousands more households ( , and , compared to , for scenario ). adding both years and had similar largest component size to - at . ( schools and , households) however the largest component at . was much larger than the other scenarios affecting schools and , households. opening only primary school years (scenario ) resulted in comparable largest component sizes to scenario at lower values of r but at r of . resulted in a median largest component of less than a third of schools (median of ) and less than half as many households (median of , ). largest components were consistently larger when only secondary schools were included in the network, with a median of schools and , households with an r of . increasing to , schools and , , households at an r of . which accounts for % of the schools and % of households. despite the increase in largest component size at higher values of r, for scenarios - , the substantial majority of schools remained in small components of less than schools, even with r at . : , (> % of schools in the network), , ( %), , ( %), , ( %), , ( %) for scenarios - respectively. whereas for scenario , where all secondary school years return, only ( % of schools in the network) schools formed components of less than schools. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint our results suggest that reopening schools with a small selection of school years may only present a small risk of transmission between schools and, consequently, the households of school children. in particular, the analysis highlights the difference in risk posed by secondary schools relative to primary schools, where reopening even a small subset of secondary school years (years and ) increases the connectivity between schools considerably, whereas opening all primary schools resulted in lower connectivity in the network. furthermore, opening only secondary schools resulted in the highest connectivity of all the scenarios evaluated. recent studies showed that outbreaks in primary schools were smaller than in secondary schools in the same area and that older children might pose a greater risk of onwards transmission in households than younger ones . in combination, these studies suggest that primary schools pose a lower risk of increasing community infections than secondary schools and support the prioritisation of primary schools for reopening although if children in secondary schools were better able to practice physical distancing than primary schools, this could act to counterbalance the additional risk. under the assumption that primary school children transmit the virus less efficiently than older school children , the difference between the scenarios of reopening either primary or secondary schools would be expected to be greater than what we found. in the extreme case where primary school children were not able to transmit the virus at all, the scenario of reopening all years would be the same as reopening only secondary schools. our assumption is that transmission in school aged children is sufficient to sustain an outbreak within a school i.e. r > . although there is some evidence of transmission within schools , and that closing schools reduced the growth rate of the epidemic , other studies have shown that transmission in schools did not contribute greatly to the overall epidemic prior to closure , . scientific consensus on this matter remains elusive , and our results should therefore be considered in light of the most recent available evidence to the reader. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint although we found that varying the reproduction number within the schools, r, had a substantial impact on the number of households in the largest potential outbreak cluster (indicated by the largest component), there was little impact on the results for the vast majority of schools' component sizes, suggesting that particular parts of the network were more closely connected than the rest of the network. this could result in particular areas being disproportionately affected following the reopening of schools. increasing r also had some impact on the weighted degree distribution of the transmission probability network, suggesting that in that case the virus may spread more effectively across connected components even if the eventual outbreak cluster size remained similar. this may impact the effectiveness of targeted interventions, as identifying a school outbreak before an outbreak in an adjacent school has been seeded may become more challenging. this is analogous to challenges in contact tracing due to pre-symptomatic infection , . our network focusses on transmission in schools and households between school-aged children and aims to provide insight into the capacity for transmission within schools and households to develop into large outbreak clusters involving multiple schools. further, we cannot account for mixing among children from different schools or households occurring outside of school contexts . the data from which the network was constructed, included only state-funded schools in england with children coded as school years reception to year in official data, which accounts for % of children in this age bracket. the addition of independent schools would increase the size and possibly the connectivity of the network, however only % of children in england attend an independent school so the impact may be marginal. the way we quantified the probability of transmission between schools assumed that each school outbreak reached its unmitigated final size -this may be unlikely before interventions, such as targeted school or class closure are introduced. for example, closure of schools when a small number of cases are reported could be an effective means to curb transmission , early on, however, to the knowledge of the authors, the effectiveness of such reactive closures is yet to be quantified in the context of sars-cov- . this framework also implies a well-mixed contact network within each school, final sizes are likely to be smaller if schools implement social bubbles to introduce community structure in the contact network and therefore reduce the probability of a school wide outbreak . the reproduction number was assumed to be invariant between schools, this approach was chosen to maintain the parsimony of the approach, as modelling internal transmission dynamics of individual schools would increase complexity considerably. we assumed transmission between members of the same household to occur with probability q = . , which is consistent with estimates of the household secondary attack rate , . to assess the robustness of the results to this assumption, we re-ran the analysis with = . and = . (supplementary material), where although the sizes of the connected components changed, the relative impact of scenarios remained comparable to the main analysis. our analysis provides insight into the potential for school-based and household-based contacts between children to combine to create long chains of transmission which could result in infections within many thousands of households. we highlight that the impact of restarting school on transmission varies substantially between the tested scenarios. reintroducing primary school years had much lower risk of transmission between schools . cc-by-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint than secondary school years. we also highlight that maintaining restrictions on contact between children within schools to ensure a low within-school reproduction number may be highly influential, as the rate of transmission between schools increases rapidly with r on some parts of the network. furthermore, such restrictions will be essential for suppressing transmission in the event that all secondary schools are opened. further analysis using this network may provide more precise guidance, particularly on reactive school closure strategies in the event of detecting a school outbreak, where the network itself may serve as a tool to aid targeted interventions. our results are directly applicable to the school system in england. although the network properties of school systems around the world may vary, we anticipate these results would be qualitatively similar in other settings with broadly comparable education frameworks. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint school closure and mitigation of pandemic (h n ) , hong kong estimating the impact of school closure on influenza transmission from sentinel data the impact of unplanned school closure on children's social contact: rapid evidence review estimating the costs of school closure for mitigating an influenza pandemic mitigating pandemic influenza: the ethics of implementing a school closure policy economic evaluation of individual school closure strategies: the hong kong h n pandemic impact of school closures for covid- on the us healthcare workforce and net mortality: a modelling study school closure and management practices during coronavirus outbreaks including covid- : a rapid systematic 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risk in households: insights from primary schools in matsumoto city this research was partly funded by the national institute for health research (nihr) using uk aid from the uk government to support global health research. the views expressed in this publication are those of the author(s) and not necessarily those of the nihr or the uk department of health and social care (pr-od- - : wje). health protection research unit for immunisation nihr nakajima foundation (ae). dfid/wellcome trust (epidemic preparedness coronavirus research programme /z/ /z: cabp) the following funding sources are acknowledged as providing funding for the working group authors. alan turing institute (ae). bbsrc lidp (bb/m / : ds). this research was partly funded by the bill & melinda gates foundation bmgf (opp : ka) uk aid from the uk government to support global health research. the views expressed in this publication are those of the author(s) and not necessarily those of the nihr or the uk department of health and social care (kvz) health protection research unit for immunisation nihr : ngd; health protection research unit for modelling methodology rpa\ : pk). uk dhsc/uk aid/nihr (itcrz : hpg). uk mrc (lid dtp mr/n / authors of this research receive funding from uk public health rapid support team funded by the united kingdom department of health and social care (tj) the authors would like to thank the her majesty's government's department for education for extracting and arranging access to the appropriate pupil data. the study was conceived by all of the authors, and designed by jdm, srm, ks, wje, ajvh and sfunk. the data was cleaned and matched by jdm and ks. the general "school network" methodology was developed by jdm, kea, jw and ajvh. the analysis in this study was carried out by jdm. the results were interpreted and the manuscript was written by jdm, srm, ks and sfunk and edited by all authors. the authors have no competing interests key: cord- - rws authors: bonal, xavier; gonzález, sheila title: the impact of lockdown on the learning gap: family and school divisions in times of crisis date: - - journal: int rev educ doi: . /s - - -z sha: doc_id: cord_uid: rws the lockdown of schools in spain to confront the effects of covid- caused an enormous impact at both societal and educational levels. schools and families had to react rapidly to a new teaching and learning scenario without the benefit of previous planning or government guidelines. in this context, some schools were better able to adapt to the new circumstances than others. likewise, the structure and size of families’ economic, social and cultural capital produced significant differences in the learning opportunities for children from different backgrounds. this article assesses the impact of the school lockdown on the learning gap between children from different social backgrounds in catalonia. based on , responses to an online survey administered between and march to families with children aged between and , the authors’ analysis shows that learning opportunities varied significantly. middle-class families were able to maintain higher standards of education quality in a critical context, while children from socially disadvantaged families had few learning opportunities both in terms of time and learning experiences (schoolwork and maintenance of after-school activities). results differed by type of school (public/private) where students were enrolled, family economic, social and cultural capital, and family living conditions. in the final part of the article, the authors highlight the importance of the role of the school in ensuring learning opportunities for children from low socioeconomic backgrounds, and they discuss some policy implications of their findings. l'impact du confinement sur les écarts en matière d'apprentissage : disparités familiales et scolaires en période de crise -la fermeture des écoles en espagne durant le confinement pour faire face aux effets de la covid- a eu d'immenses répercussions sur les plans sociétal et éducatif. les écoles et les familles ont rapidement réagi au nouveau scénario de l'enseignement et de l'apprentissage sans pouvoir s'appuyer sur une planification préalable ou des directives gouvernementales. dans ce contexte, certaines écoles ont réussi mieux que d'autres à s'adapter à ces nouvelles circonstances. de même, la structure et la taille du capital économique, social et culturel des familles a montré qu'en fonction de leur milieu, les enfants avaient des possibilités d'apprendre très inégales. cet article évalue l'impact de la fermeture des écoles en espagne durant le confinement sur les écarts en matière d'apprentissage chez des enfants de différents milieux sociaux en catalogne. s'appuyant sur réponses à une enquête en ligne menée entre le et le mars auprès de familles avec des enfants âgés de trois à dix-huit ans, l'analyse des auteurs révèle de considérables disparités concernant les possibilités d'apprendre. les familles de la classe moyenne ont réussi à maintenir un niveau d'éducation élevé dans cette situation critique, tandis que dans les familles défavorisées sur le plan social, les possibilités des enfants étaient restreintes, tant en termes de temps que d'expériences éducatives (devoirs et maintien d'activités extrascolaires). les résultats étaient différents en fonction du type d'établissement (public/privé) où les élèves étaient inscrits, de la situation économique de la famille, du capital social et culturel de cette dernière et de ses conditions de vie. dans la dernière partie de l'article, les auteurs soulignent l'importance du rôle de l'école pour garantir la possibilité d'apprendre aux enfants de milieux socioéconomiquement faibles. ils abordent en outre un certain nombre de conséquences qu'entraînent leurs constatations pour les politiques en matière d'éducation. el impacto del cierre escolar en la brecha de aprendizaje: divisiones familiares y escolares en tiempos de crisis -el cierre de escuelas en españa para hacer frente a los efectos de la covid- causó un enorme impacto tanto a nivel social como educativo. escuelas y familias tuvieron que reaccionar rápidamente a un nuevo escenario de enseñanza y aprendizaje sin contar con planificación previa o con directrices gubernamentales. en este contexto, algunas escuelas fueron capaces de adaptarse mejor a las nuevas circunstancias que otras. asimismo, la estructura y el tamaño del capital económico, social y cultural de las familias produjeron diferencias significativas en las oportunidades de aprendizaje de los niños y niñas de diferentes orígenes. este artículo evalúa el impacto del cierre de las escuelas en la brecha de aprendizaje entre el alumnado de diferentes orígenes sociales en cataluña. sobre la base de . respuestas a una encuesta en línea realizada entre el y el de marzo de a familias con hijos e hijas de entre y años, el análisis muestra que las oportunidades de aprendizaje varían significativamente. las familias de clase media pudieron mantener niveles más altos de calidad educativa en un contexto crítico, mientras que los niños de familias socialmente desfavorecidas tuvieron pocas oportunidades de aprendizaje, tanto en términos de tiempo como de experiencias de aprendizaje (tareas reducing physical contact has been the most common strategy adopted by governments to reduce the spread of covid- . it has led most countries around the world to close their schools for periods of time. the lockdown of schools in spain, one day before the declaration of a state of alarm on march , has had an enormous impact at both societal and educational levels. schools and families had to rapidly adjust to a new teaching and learning scenario without the benefit of previous planning or guidelines from the spanish ministry of education or regional departments of education. in this context, some schools were better able to adapt to the new circumstances than others. likewise, the structure and size of families' economic, social and cultural capital (resources) produced significant differences in the learning opportunities for children from different socioeconomic backgrounds. while the covid- pandemic has no precedent in terms of effects on the economy and social life, schools have previously experienced periods of closure. for example, large-scale outbreaks other diseases (such as ebola and influenza), teacher strikes, natural disasters and violent conflicts have already forced schools to stop their activity in the past. researchers and international organisations have studied the effects of school closures on students' learning and found a measurable loss in the acquisition of basic skills, particularly for the most disadvantaged children (quinn et al. ; cattaneo et al. ) . it is highly likely that the current pandemic will have a dramatic long-term impact on students' competencies and increase existing education inequalities. in this article we reflect on how school closure produces unequal learning opportunities in terms of both formal and non-formal education for children and youth from different socioeconomic backgrounds attending different types of schools in catalonia. during the covid- catalan lockdown, the instructional time received by students from different social backgrounds has been unequal, as has the educational value of the activities developed at home in non-school time. we investigated what learning opportunities were available to students from different backgrounds by administering an online survey during the second week of the state of alarm (i.e. between and march ). hypothesising that exposure to learning (both formal and non-formal) among children from lower-income households was likely to be lower than for their peers from higher-income families, we expected this to irremediably increase the existing learning gap between them. based on the results of our survey, this article reflects on how students' social background and conditions of confinement were associated with their learning opportunities. these unequal opportunities, in turn, are likely to exacerbate existing inequalities in skills acquisition and academic performance. we begin our article with a review of existing evidence on learning losses and their unequal impact on different groups of students due to past periods of school shutdown. the next section provides information on the context in which we conducted our own survey and the methodology we used to analyse the data. we then present our key findings regarding inequalities in formal and non-formal education, while the final section draws conclusions and discusses some policy implications of our findings. although it has only been a few months since schools in many countries around the world closed due to the covid- pandemic, research on the educational effects from that closure has already been notably rich. obviously, most of these studies have measured the short-term effects of the lockdown. it is certainly too early to know whether these effects can be considered a learning loss or just a temporary effect as a result of disuse that can be easily regained with practice (coe et al. ) . beyond this recent evidence, several previous studies based on student absenteeeism and past school closures have focused on the impacts of being out of school on learning outcomes (abadzi ; eef ) . these research studies can help us understand the potential effects of current school closures on learning and the mechanisms by which educational inequalities occur. some of the reasons causing schools to reduce or even stop their activity in the past range from summer school holidays, student or teacher absenteeism and teacher strikes to violent conflicts. extant research evidence based on these experiences identifies the existence of a general loss of learning connected to school absence, which can be more or less severe depending on students' previous performance, family characteristics, age, and education pathway, among other factors. school closure, even when distance (remote) learning is offered, usually implies a reduction in instructional time and, by consequence, also a decline in learning time. past studies have consistently identified a positive relationship between learning time and student achievement -along with other educational outcomes (abadzi ; hanushek ; scheerens ) . therefore, some studies have used instructional time as a proxy for "opportunities to learn". variables that measure exposure to curriculum seem to show a greater effect on learning than variables related to teacher behaviour or school climate. students' prolonged and repeated exposure to stimuli and feedback has been identified as a key educational resource. even though evidence is not always conclusive (cattaneo et al. ) , most research suggests that students with a migrant background, and those who are socioeconomically disadvantaged, will be slower learners. thus, these students are likely to benefit more from an increase in learning time than their peers (dahmann ; gromada and shewbridge ; huebener et al. ). consequently, not going to school reduces learning opportunities for all, but particularly for students from low-income backgrounds and less-skilled children. student (or teacher) absenteeism can also be understood as a lack of instructional time. student absenteeism results in poorer academic achievement, gaps in skills development, abilities and behaviours necessary for educational success, and an increased likelihood of school dropout. effects go beyond the school environment: absence has also been connected to risky behaviours such as smoking, juvenile delinquency, alcoholism, drug use, risky sexual behaviours or unwanted pregnancies, as well as other effects in adulthood (e.g. unemployment, alcoholism) which diminish life opportunities for young absentees (abadzi ; coe et al. ; gonzález motos ) . in spain, as in other european countries, the school lockdown between march and june seamlessly merged with the beginning of the summer holidays, resulting in students' absence from school lasting six months. past studies comparing students' learning progress during the school year with the summer break have consistently shown that achievement tends to slow or decline over the summer holidays. although there is no agreement on the magnitude of the learning loss during holidays (von hippel ), it is clear that it is steeper for mathematics than reading, and it is especially acute for upper-grade students . moreover, research points out that this loss does not affect all students equally; during the summer period, educational inequalities between socially advantaged and disadvantaged children increase (alegre ) . beyond the evidence related to breaks in the ordinary school year (e.g. absenteeism, summer break, instructional time), other research studies have analysed the effects on students' learning caused by external factors. for example, the literature on weather-related school closures (e.g. as a result of heavy snowfall or hurricanes) also contributes to a better understanding of the potential unexpected consequences of disruptive closures. most of the evidence is based on small samples, is geographically specific and refers to short break periods. however, in all cases, studies have found a clear effect of each day of school cancellation on achievement, especially among students attending less-resourced schools or coming from low-income families (goodman ; marcotte and hemelt ) . teacher strikes have also forced a sudden cancellation of classes. strikes affect student learning outcomes (mainly in terms of achievement and grade repetition) by reducing the time that children attend school, but they also affect the quality of schooling and increase the likelihood that students may engage in risky behaviour (jaume and willén ). in addition, research has found heterogeneous (uneven) effects, with children from vulnerable families being most affected by school disruptions (jaume and willén ; belot and webbink ). the main difference between school closures examined by previous studies and the current lockdown is that this time schools have managed to maintain some contact with students, mostly through online instruction. however, despite the efforts to support remote learning, many students have not had access to it. existing data about the digital gap and surveys conducted during the lockdown indicate that teachers have been unable to contact a significant number of students, mainly because of lack of an internet connection or adequate devices to engage in distance learning ; van lancker and parolin ). even when students have been able to connect, remote learning during school closures seems to have widened the existing attainment gap between students from different socioeconomic backgrounds (coe et al. ) . for instance, a recent study undertaken by researchers from brown and harvard universities into the use of an online mathematics programme (zearn) before and during lockdown in the united states (chetty et al. ) shows a sharp decline of student progress in mathematics in classrooms located in low-income zip (postal) code areas, while in high-income zip code areass no changes were observed (goldstein ) . before the covid- pandemic, in-school learning had already been proven to be more effective than distance learning. furthermore, student outcomes resulting from online learning have been shown to be poorer, on average, than outcomes resulting from face-to-face instruction (heppen et al. ) . conditions for effective remote learning (good internet connection, and clear explanations, scaffolding and feedback from teachers) are not easy to accomplish. the combination of a digital gap with teacher inexperience in providing high-quality distance learning makes it difficult to improve students' learning opportunities . moreover, there is now a significant risk that vulnerable students have less access to quality teaching than their peers, widening the attainment gap due to the school lockdown (coe et al. ; kay et al. ) . if it has already been proven that students from low-income families experience more interruptions and disruptions of their instructional time under normal school conditions (abadzi ; alegre and benito ) , then poor distance learning can be understood as a new obstacle to effective learning. apart from school organisation or distance learning strategies developed by teachers, parental engagement in children's learning, practised in some families alongside school attendance, becomes more important when school is replaced by at-home instruction. educational studies have demonstrated that parental involvement and the quality of learning at home improve academic outcomes. research in this field has consistently observed greater academic achievement among students whose parents are actively involved in the educational process. it has also found a clear relationship between engagement in the learning process and parental background in terms of social class and ethnicity (oreopoulos et al. ) . unequal parental capacities to help children with their homework and different uses of family time have been extensively documented (meyer et al. ; mora and escardíbul ) . therefore, family reactions to school lockdown are likely to produce significant differences in the learning opportunities for children from different types of social background (burgess and sievertsen ) . regardless of their causes and mechanisms, previous crises have had a more intense and negative effect on student learning outcomes in contexts with higher proportions of disadvantaged families (borse et al. ; chen et al. ; iqbal et al. ; sadique et al. ; shores and steinberg ) . previous research also suggests that the learning loss can not only have a limited short-term effect, but may in fact result in cumulative losses (abadzi ). likewise, other researchers suggest that the current school lockdowns will widen the learning gap between vulnerable children and their peers, possibly even reversing the progress made during the last decade to narrow this gap (coe et al. ; van lancker and parolin ). on march , the catalan government ordered the closure of all schools and colleges (universities) due to the covid- pandemic. more than one million students in compulsory education were asked to stay at home. during the first two weeks of confinement, the catalan department of education advised schools not to provide new curriculum content, in an attempt to contain the adverse effects of the technological gap on educational inequalities. despite the department of education's order, some schools reacted rapidly to the new circumstances and switched to remote learning strategies. other schools stopped their activities entirely. between these two extremes, there were a range of reactions and responses. between and march, after two weeks of school closure and before the spanish government ordered a more restricted lockdown, we (the authors of this article) conducted an online survey with families whose children were aged between and . the survey was addressed to families with children enrolled in preschool ( - years old), primary school ( - years old), lower secondary education ( - years old) and post-secondary academic or vocational education ( - years old). organised into two main sets of questions, our survey included items, with a variable number of subquestions that branched out to adapt to respondents' particular contexts. the first group of items characterised the different social, spatial and technological conditions in which families had to respond to the school lockdown (such as the size of their home, the availability of outdoor spaces, access to the internet and to digital devices). the second set of questions was designed to assess children's learning opportunities in (a) formal education (contact with school teachers, frequency and types of school tasks, feedback provided by schools), (b) informal education (uses of time, family activities, support to carry out school tasks) and (c) non-formal education practices from home (participation in after-school activities, types of activities and forms of participation). in addition, our survey included variables to characterise respondents' social and economic conditions, such as family structure, parental education attainment, income, ethnic origin, gender and work status. to test the survey, we conducted a brief pilot. due to the exceptional circumstances, the pilot was implemented using an informal strategy: we asked families with children of different ages and on different school pathways to complete the survey. after the pilot was completed, we distributed the final version of the survey through various online communication channels (twitter, facebook and whats app). this strategy helped us to improve the survey's visibility among different social groups as well as reducing sampling bias linked to the use of social networks (blank ) . we also mobilised strategic contacts in the most deprived areas of catalonia to reach those families less likely to answer online surveys. in total, , people clicked the link to the survey, approximately , started to answer it and more than , families completed it. after excluding some respondents for reasons like incomplete answers, children's ages outside our survey's scope, families not living in catalonia, the final sample included information from , families (with a total of , children aged between and ). as expected, high-income and highly educated parents were over-represented. due to the lockdown and the urgent need for data collection, we were unable to complement our online survey with other strategies such as telephone calls or face-toface interviews. we opted instead to use non-response weighting to bring the sample closer to the population distribution. to do this, we weighted the sample by the level of parental education attainment, as a key variable of social and cultural differentiation, and as a reliable and accurate source of census data. table shows both the weighted and unweighted samples. of our survey respondents, % were female, . % lived in heterosexual biparental households, . % lived in single-parent households and . % were part of a same-sex couple. more than half of the households ( %) were composed of four members, three-member households represented % of the sample, and % of the households had five or more members. sample distribution according to school variables accurately reflects the distribution between the public and private school sectors. in catalonia, the proportion of children in compulsory schooling who attend public schools is % (this was . % in our sample), while % of students attend private subsidised schools ( . % in our sample), and only % are enrolled in private independent schools ( . % in our sample). our sample was also well balanced regarding children's ages, with a slight over-representation of younger students. for our data analysis, we split information across three databases according to different units of analysis. first, a "household database" included information about family members living together during the confinement. second, a "student database" collected information about the learning activities of children aged between and . third, the "after-school activities database" contained information about the extracurricular activities children were involved in before and after the lockdown. we also constructed some subsamples to carry out specific analyses. in this article, we present some data based on subsamples by children's age or educational level. in this section, we present and discuss the data from our survey regarding social differences in the learning conditions of catalan children and the impact of these conditions on learning opportunities during lockdown. inequalities in families' economic, social and cultural capital impacted on student learning opportunities by different means, including school responses to the lockdown, access to digital facilities and the level of parental learning support. this section reviews how these factors are drivers of inequalities in the three domains of learning: formal, non-formal and informal. learning conditions at home differ on the basis of a number of variables. for example, the amount of physical space and access to technological devices both have an impact on the learning conditions of children. our survey included questions about the size of the household and the outdoor spaces available, to serve as indicators of the physical conditions of confinement. the responses showed that most homes of confined families had a balcony or other outdoor space/s. however, there were significant differences among residents depending on the size of the municipality (families living in cities had less access to outdoor spaces) and other social indicators, such as parental education attainment. for instance, % of families with an adult who had completed compulsory education did not have outdoor spaces, while this was only the case for % of households in which at least one adult held a university degree. likewise, % of households in the poorest income quintile (q ) had less than square metres of space, reducing to % in the case of the richest income quintile (q ). students also had different internet connectivity conditions and unequal opportunities to access technological devices to carry out their schoolwork. since we could only implement an online survey, we were unable to grasp the full extent of the actual digital divide. data from the catalan department of education estimate that there are currently approximately , school children without access to an internet connection (vallespín ) , which would situate the digital gap at between % and % of all children in the catalan education system. our survey revealed that . % of our sample had only a mobile phone internet connection or no connection at all. most families in our survey had access to a high-speed internet connection. however, there were important differences regarding the "possibilities of use" of technology. for example, . % of respondents declared that they had access to a single device at home or only had access to a mobile phone. we calculated the number of devices per person and found that in % of cases there was less than one device per person. the digital divide can also be expressed in terms of access to devices based on the composition of the household. in our survey, % of households with two children had only one device available; % of households with three children had two or fewer devices available; and % of households with three children had three or fewer devices available. access to technology depends on the level of family income. while % of surveyed families in q (the lowest income quintile) had access to only one digital device, for families in q (the highest quintile), the corresponding figure was only %. furthermore, while % of families in q had access to four or more devices, this applied to % of families in q . taking into account the size of the household, % of families in q did not have access to one device per person. among families in q , the number of households with less than one device per person reduced to %. figure shows the proportion of households for each income quintile that had access to one or more device per person. differences are sorted by income, with an inequality factor of . between q and q . in addition, the unequal impact of the crisis on parents' working situation also altered the social and psychological conditions that ensured an adequate learning process. our survey revealed that before lockdown, % of adults were unemployed, while % were working full-time. when we asked about the impact of the covid- crisis on their working situation, these figures changed dramatically: % of respondents indicated that they had lost their job. of those who were still working, only . % were able to go to work "normally". the rest ( %) were working from home, either with the same schedule or with a more flexible schedule. of those still working, % considered it likely that they would lose their job. of those who were already or became unemployed, % knew that they were entitled to unemployment benefits, while the other % either knew that they were not entitled or did not know. our survey included questions regarding how much time children invested in schoolwork every day since the beginning of the school lockdown, how frequent the contact was with their school and teacher/s, how often they received online teaching lessons, whether they had to complete specific homework tasks, and how often these tasks were reviewed and returned to children. taking into account the intensity of all these tasks, we composed an index of opportunities to learn (otl). to compose the index, which ranged from to points, we normalised indicators and aggregated (combined) frequencies. we only used this index for a subsample of the older children aged between and , since assigned school tasks or online teaching were more unlikely for younger age groups. in terms of the index, . % of students had an otl equal to , meaning that they dedicated less than one hour a day to school tasks, had almost no communication with teachers and did not have homework to do or to be reviewed. at the other end of the index, . % of students had a maximum otl of , meaning that they dedicated more than four hours per day to schoolwork, had frequent contact with their teachers and received regular feedback for school assignments. the majority ( . %) of all surveyed students in this subsample had an otl score below points. interestingly, we found the otl score to be positively related to a number of variables. having greater access to digital devices, being enrolled in more advanced courses (older students had a higher otl), being native to spain or living in a higher-income household were all factors associated with higher otl scores. students enrolled in private schools, both independent and private subsidised ones, had significantly higher otl scores than those enrolled in public schools. there are explanations for this difference. for example, the catalan department of education announced that the first two weeks after the approval of the state of alarm would be a non-school period. therefore, a significant number figure opportunities to learn (otl) index, by school sector and educational level note: baccalaureate refers to two years of optional upper secondary education preparing students for tertiary level of public schools did not develop school tasks during these two weeks, waiting for new instructions from the department. despite this announcement, private subsidised and independent schools did not stop their teaching activity. one of the plausible explanations for this difference lies in the economic dependency of private schools on fees. they needed to keep providing a service to users despite the exceptional circumstances. figure shows the distribution of the otl index across school sectors for different educational levels. the bar chart reveals strong differences by school sector for students enrolled in the last years of primary education and for those in lower secondary education. the chart also shows how students in vocational education and training (vet) had the lowest otl of post-compulsory education. we also found the otl index to be clearly related to the level of parental education attainment and to family income. for instance, % of children in families from the richest quintile (q ) had an otl score of points or higher. this reduced to % in the case of the poorest quintile (q ). the absence of schooling increases the importance of families as teacher substitutes in the learning process. our survey included questions regarding whether adults in the family helped students in their school tasks during the relevant lockdown period. an initial remarkable result appeared in terms of gender: while % of female adults stated that they supported their children to do schoolwork, only % of male adults did. as expected, gender differences were clearly observed, and family support was higher in the case of younger children. figure shows that for children undertaking primary education, the support of mothers who had completed compulsory figure adult support for school tasks, by gender and highest parental education attainment education was comparable to that of mothers with higher educational (in bourdieu's terms) cultural capital. however, for students enrolled in lower secondary education, the differences increased dramatically: only % of mothers who had completed compulsory education helped with homework, while % of the most educated mothers did. providing support for school tasks to students in post-compulsory education declined to close to % for all groups. there are several reasons why support for schoolwork may not have been provided by adults in a student's household. these reasons are remarkably different depending on the level of parental education attainment. in those households in our survey with children enrolled in lower secondary education, % of the families with an adult who had a university degree and who did not provide support for schoolwork argued that the child did not need it. this reason was only argued by % of respondents from those households with adults who had completed only compulsory education. in this case, a lack of knowledge was proffered as a significant reason for not giving support (by % of respondents), which reduced to % for those parents with a university degree. differences in cultural capital are therefore reflected in the capacity and possibilities of families to help children with their school tasks. in addition, (and not just in times of school closure) families' cultural capital and everyday informal practices have effects on children's learning experiences and opportunities. interestingly, confinement, which maximises the interactive time between family members, provides ideal research conditions for assessing informal learning activities. figure reveals that in those households with children aged between and , there were three activities in which social differences were especially acute. first, accompanying children in reading was more frequent by far in families with high cultural capital: % of families with an adult who had completed university studies responded that their children aged between and read with an adult every day. this reduced to only % in the case of families with an adult who had completed compulsory education. second, differences were also visible in foreign language informal practices: % of families with an adult who had completed university studies responded that foreign language practice was done daily or several days a week, while this practice was carried out by only % of families with an adult who had completed compulsory education. third, sporting activities were practised several times a week by % of families with university degrees and only by % of families with compulsory education. by contrast, other activities were more frequent among families with lower cultural capital. the most relevant was playing video games: % of families with an adult who had completed compulsory education reported that their children of preschool age played video games every day or several times a week. this applied to only . % of families with parents who had completed university studies. the differences were also significant in the frequency of watching educational television programmes ( % of households with parents who had completed compulsory education and who had children enrolled in preschool education reported that their children did so every day, versus % of parents with a university degree), and in following and carrying out educational activities available via social media. in such an extreme situation of school absence, it appears that families with less cultural capital made more use of external resources to support their children's learning activities, while families with more cultural capital were more confident in their own abilities to respond to their children's learning needs. in the case of older children, the same differences were observed, although the gap increased in musical and other artistic activities and was less pronounced in playing video games. participation in after-school activities is a source of differential learning opportunities for children from different socioeconomic backgrounds (bradley and conway ; gonzález motos ; lauer et al. ; potter and morris ) . our survey compared whether children participated in one or more after-school activities before and after the beginning of the lockdown. we found that the effects of covid- on these kinds of activities have also increased the gap between socially advantaged and disadvantaged children. before lockdown, after-school activities were more frequent for students enrolled in primary education. this was particularly the case for children whose parent/s had completed a university degree ( %), compared to children from households with parent/s who had completed compulsory education ( %). this polarisation increased particularly for children enrolled in lower secondary education with at least one unversity-educated parent ( %) versus those whose parent/s had completed compulsory education ( %). we also found families' economic, social and cultural capital to be associated with the number of after-school activities in which children participated. for example, for families with two children in lower secondary education, the average number of after-school activities for highly educated households was . , compared to . for households with lower educational levels. sports, foreign languages and artistic practices were the most frequently reported after-school activities. by income level, students in families in the lowest quintile (q ) were more represented in sports and compensatory education activities, while children from the richest quintiles were over-represented in foreign language and musical after-school activities. as expected, we found that most after-school activities ( %) were interrupted during lockdown. however, economic and cultural inequalities were also visible in the probability of maintaining after-school activities despite the confinement. figure shows that children whose parent/s had lower educational levels already had lower participation in after-school activities before lockdown and were more likely to be unable to continue them after schools closed than children from families with higher educational levels. there are two main reasons for this difference in after-school activities after the closure of schools. first, families with higher economic, social and cultural capital participated in activities that were more likely to continue online (e.g artistic activities, foreign languages), compared to the activities more commonly practised by families with lower capital (e.g. sports). second, voluntary interruption of afterschool activities was also higher among families with lower ecoomic, social and cultural capital, due to difficulties in coping with the costs during times of crisis. for instance, compensatory education activities -which could be undertaken online -were voluntarily interrupted by % of families who had previously engaged in figure after-school activities before and after lockdown, by parental education attainment compensatory education, in this context, refers to those after-school classes that some students attend to reinforce their learning competencies. these classes are usually offered to students with marked learning difficulties. them. this voluntary interruption was particularly high among children whose parent/s attained compulsory education ( %), compared to those whose parent/s had a university degree ( %). despite schools' efforts to maintain learning activities during lockdown, our analysis reflects significant inequalities in exposure to school learning depending on family characteristics (income, level of educational attainment) and school characteristics (educational level, school sector). the absence of schooling neutralises the benefits of socialisation provided by early childhood education for the most vulnerable children. it also disrupts those processes of guidance and accompaniment which are especially important for adolescent students in their study, work and life transitions. in addition, the digital divide and visible differences in access to technological devices among students have left some children and young people without options to connect to learning for at least three months or, more probably, for six months. inequalities in being able to maintain school-based learning are not the only existing ones. our analysis shows that families with a lower level of parental education attainment have fewer resources and less knowledge to help their children with school tasks. these limitations become more acute when support from the school has been reduced and demands for autonomous work by students increase. likewise, family economic, social and cultural capital also influences the adoption of everyday informal learning practices that are more or less aligned with school logic, which readily increases differences in learning opportunities and familiarity with what is considered valid knowledge. after-school activities are also more likely to be maintained among children from wealthier families, which also increases the existing inequalities in this area. in sum, if the pre-covid- school system already had significant limitations in its ability to eliminate existing social inequalities, the closure of schools during this period of lockdown poses enormous challenges for developing effective policies to compensate for learning losses and learning inequalities. school lockdown has not affected all children in the same way, and significant material and human resources will be necessary to ensure that the most vulnerable children can catch up. an educational emergency plan with social and educational objectives will be needed to restore children's socio-emotional and cognitive skills. the individual and social costs of not intervening are just too high, unfair and unequal. it is the responsibility of states and countries to ensure the right to education in these difficult times. instructional time loss in developing countries: concepts, measurement, and implications are summer programs effective in improving learning and educational outcomes in students? barcelona: ivàlua-fundació jaume bofill climas y (sobre todo) culturas escolares: cómo se explican y qué permiten explicar familia y relaciones 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cultural organization) (n.d.-a). instructional time. ibe-unesco glossary of curriculum terminology learning time. ibe-unesco glossary of curriculum terminology population figures [online resource we should avoid flattening the curve in education: possible scenarios for learning loss during the school lockdowns. education for global development the long-run effects of teacher strikes: evidence from argentina remote learning: rapid evidence assessment. london/cambridge: education endowment foundation (eef)/edtechhub. retrieved projecting the potential impacts of covid- school closures on academic achievement the covid- slide: what summer learning loss can tell us about the potential impact of school closures on student academic achievement the effectiveness of out-of-school-time strategies in assisting low-achieving students in reading and mathematics: a research synthesis unscheduled school closings and student performance patterns of literacy learning in german primary schools over the summer and the influence of home literacy practices home environment and parental involvement in homework during adolescence in catalonia (spain) the intergenerational effects of compulsory schooling family and schooling experiences in racial/ethnic academic achievement gaps: a cumulative perspective seasonal dynamics of academic achievement inequality by socioeconomic status and race/ethnicity: updating and extending past research with new national data estimating the costs of school closure for mitigating an influenza pandemic effectiveness of time investments in education. springerbriefs in education series the impact of the great recession on student achievement: evidence from population data educació detecta . famílies sense ordinador o sense connexió per poder seguir les classes a distància covid- , school closures, and child poverty: a social crisis in the making is summer learning loss real? education next publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations he has been a member of the eu network of experts in social sciences and education (nesse) and is an editorial board member of several international journals on education policies and educational development. xavier has widely published in national and international journals and is the author of several books on the sociology of education, education policy and globalisation, and education and development. he has worked as a consultant for international organisations such as unesco, unicef, the european commission, and the council of europe she is a member of the institute of government and public policy (igop) and the globalisation, education and social policies (geps) research centre. her research interests include public policy analysis and social inequalities, with a special focus on education and migration. sheila has conducted several research projects in this field, and she has experience in policy analysis and policy evaluation the data that support the findings of this study are available from the corresponding author, upon reasonable request. authors' contributions: all authors contributed to the study conception and design. material preparation, data collection and analysis were performed by xavier bonal and sheila gonzález. all authors read and approved the final manuscript. key: cord- -wrxejwms authors: song, jungeun; kweon, yong-sil; hong, sung hee; kim, joonbeom; chun, ka hye; bahn, geon ho; yook, ki-hwan; shin, dongwon; hong, hyun ju title: characteristics of first visit pediatric patients with suicidal ideation and behavior: an -year retrospective chart review date: - - journal: soa chongsonyon chongsin uihak doi: . /jkacap. sha: doc_id: cord_uid: wrxejwms objectives: our study aimed to analyze the demographic and clinical characteristics of children and adolescents during their first visit to psychiatric outpatient departments for the management of suicidal ideation and behavior, and to compare the changes before and in or later. methods: this multicenter study was conducted at five university hospitals in a metropolitan area in south korea. medical records of patients aged – years were retrospectively reviewed from january to december . patients were analyzed by classifying them into suicidal and non-suicidal groups based on their visit to the hospital for management of suicidal ideation or suicide attempt and other mental problems, respectively. results: there were differences in the year of visit, diagnosis, education level, and referral sources between patients in the suicidal and non-suicidal groups. multiple regression analysis was conducted based on the sex, education level, referral by school, and diagnosis of depression in patients in the suicidal group, which revealed significant association. conclusion: suicide-related problems were significantly associated with the sex, education level, referral by school, and a diagnosis of depression in the patients. a well-connected referral system would be necessary for professional mental health management of high-risk children and adolescents. ation and behavior occurring in adolescence tend to persist in adulthood [ ] , and are predictive factors of adult psychiatric disease. such thoughts and behavior have been associated with functional impairment in work, education, and social relationships [ ] . therefore, characterization and early detection of adolescents at high risk of suicide and initiation of interventions are crucial to prevent suicidal behaviors and decreased function in adulthood, and to improve the quality of life of the individuals. although early detection and treatment is important to address the issue of suicide among adolescents, the proportion of youths availing mental health services has been reported to be low. among korean youths who attempted suicide within the last year, only . % reported receiving treatment after the attempt in [ ] , and a study based on health insurance data from south korea also reported that the treatment rate for psychiatric disorders in youths aged ≤ years was . % [ ] . the factors affecting the treatment rate are diverse, including social, healthcare system-related, and personal factors. social stigma is an important social factor that affects the use of healthcare services, especially mental health services, and younger individuals have been reported to be more sensitive to stigma [ ] . the aforementioned studies signify that sufficient expert therapeutic interventions are not being provided to young individuals at high risk of committing suicide. the role of school-based mental health services has been further emphasized due to the growing need for suitable interventions to address the issue of suicide among adolescents. school-based health services have been reported to be effective at improving mental health and preventing psychiatric disorders in adolescents [ ] . furthermore, school-based prevention and intervention programs have been initiated to deal with suicide-related issues. in particular, such screening programs have been successful at detecting at-risk youths and linking them with expert treatment services. in south korea, a system for proactive management of the mental health of students was established in , which annually tested the emotional and behavioral characteristics of students at schools throughout the country. the system screens high-risk students and refers them to external institutions, if required, after counseling at school [ ] . in addition, since , support projects have been initiated at regional educational offices to construct a regional collaborative model for students' mental health, in order to prepare a system for interventions in the high-risk group. furthermore, since , projects have been implemented to provide support to unconnected interest groups, wherein mental health experts visit schools, thereby enabling management of high-risk groups within the school [ ] . however, studies have not been con-ducted to investigate the actual changes in the number of adolescents visiting mental health departments, sources of referral, or clinical characteristics after initiation of these schoolbased public mental health projects. the present report is of a large-scale study based on data obtained from pediatric mental health outpatient departments. we analyzed the trends and changes before and in or later, following initiation of school-based mental health projects, and compared the demographic and clinical features and sources of referral of patients visiting the mental health department for management of suicidal ideation or behavior and other chief complaints. in this manner, we aimed to provide evidence to help establish effective youth suicide prevention and management policies. we retrospectively reviewed the medical records of patients aged - years who visited the pediatric mental health department for the first time at one of five university hospitals in the capital region between january and december . a total of patients met the criteria. we investigated the date (day, month, and year) of first visit, age, sex, education level (elementary, middle, and high), school year, chief complaint, duration of disease, referral source, final route of referral before hospital visit, and provisional diagnosis at first visit of all patients. diagnosis was based on first visit records or treatment records within weeks of the first visit, and described according to the th revision of the international classification of diseases (icd- ) [ ] . data was collected via an online report form and data input sheets, and students at special schools or in special classes at regular schools, preschool children, and high school graduates were excluded. this study was approved by the institutional review board of hallym university sacred heart hospital (irb no.: - ). the chief complaints of the patients were classified as emotional, behavioral, suicide-, family-, school-, thought-related problems and others. patients who visited hospitals with a complaint of suicide ideation or behavior were classified as the "suicidal group" and those who visited for other reasons were classified as the "non-suicidal group." multiple inputs were permitted for patients with more than one referral source, chief complaint, or diagnosis. cross-tabulation analysis was performed to compare sociodemographic characteristics, year of visit, referral source, and diagnosis between the two groups. the patients were divided into two groups to analyze the year of visit as those who visited in or later, when the students' emotional and behavioral screening test and school-based mental health projects became more prevalent, and those who visited before . furthermore, additional cross-tabulation analysis was performed on the number of patients who visited the hospital in each group. referral source was categorized as "via school" or "not via school," and diagnosis was categorized as "depression" or "not depression" for further cross-tabulation analysis. finally, multivariate logistic regression analysis was performed with sex, education level, year of visit, referral source, and diagnosis as independent variables, to investigate their correlation with inclusion in the suicidal group. stata . (statacorp lp, college station, tx, usa) was used for all analyses. a total of patients were analyzed, including male ( . %) and female ( . %) patients. the most frequent education level was elementary school ( patients, . %). there were patients ( . %) in the suicidal group and patients ( . %) in the non-suicidal group. the non-suicidal group included patients ( . %) with behavioral, ( . %) with emotional, ( . %) with school-related, ( . %) with thought-related, and ( . %) with family-related problems. there were ( . %) and ( . %) female patients in the suicidal and nonsuicidal groups, respectively, which represented a significantly higher ratio of female patients in the former group (p< . ). furthermore, there was a significant difference in the education level between patients in the two groups. there were elementary school ( . %), middle school ( . %), and high school ( . %) students, and dropouts ( . %) in the suicidal group, and elementary school ( . %), middle school ( . %), high school ( . %) students, and dropouts ( . %) in the non-suicidal group (p< . ). there was no significant difference between the two groups with respect to the years of visits (p= . ); however, in the total sample, the highest number of visits was in the year ( patients, . %), and the lowest was in the year ( patients, . %) ( table ) . overall, the number of patients showed a generally decreasing trend and the ratio of patients in the suicidal group showed minimal changes. however, there were no major changes until , when the ratio was the highest, and patients in the suicidal group accounted for % of all cases ( fig. ) . comparison of the number of patients in the suicidal group revealed that there were patients before , accounting for . % of all cases, and patients in or after , accounting for . % of all cases. the ratio of patients in the suicidal group was significantly higher in or later (p= . ). fig. . annual trends in visits in total, suicidal and non-suicidal groups. overall, the number of patients showed a generally decreasing trend and the ratio of patients in the suicidal group showed minimal changes. the dataset included patients with multiple routes of referral. in both groups, the most common referral source was observation by a caregiver. significant differences were observed in the routes of referral between the two groups on cross-tabulation analysis (p< . ) ( table ). based on categorization of the referral source, a significantly higher ratio of patients in the suicidal group ( patients, . %) were referred via their schools compared to those in the non-suicidal group ( patients, . %; p= . ). multiple inputs were accepted in the analysis of diagnoses. the most common diagnosis of patients in the suicidal group was depression ( patients, . %), whereas that in the nonsuicidal group was hyperactivity disorder ( patients, . %). cross-tabulation analysis revealed significant differences in the diagnoses of patients between the two groups (p< . ) ( table ) . likewise, a cross-tabulation analysis after categorizing the diagnoses as "depression" or "not depression" also showed a significant difference between patients in the two groups (p< . ). among male patients, the ratio of patients increased grad- gender differences by education level in the suicidal group. among male patients, the ratio of patients increased gradually from elementary to middle to high school levels. among female patients, there was a dramatic increase in the ratio at the middle school level, which was maintained at the high school level. we included the cases with one or more referral sources. *p＜ . we included the cases with one or more diagnoses. *p＜ . ually from elementary to middle to high school levels. among female patients, there was a dramatic increase in the ratio at the middle school level, which was maintained at the high school level (fig. ) . cross-tabulation analysis revealed significant sex-related differences based on education levels (p= . ); however, there were no significant differences in the rates of referrals via school (p= . ), diagnosis of depression (p= . ), or the year of visit (before or in or later; p= . ) ( table ). in regression analysis of patient data in the suicidal group, sex, education level, rate of referrals via school, and rate of diagnosis of depression showed significant results, while the year of visit (before or in or later) was not significant (pseudo r = . ) ( table ) . female patients showed . times higher relative risk (p< . ). analysis based on the education level revealed that, compared to elementary school students, the relative risk of suicide increased with the education level from middle school to high school, and dropouts showed the highest risk ( . -fold; p< . ). the relative risk of suicide was higher among patients referred via school (p< . ) and among those with a diagnosis of depression (p< . ). this study was a chart review comparing the characteristics of pediatric patients who visited a mental health department for the first time between and for the management of suicidal ideation or behavior with those who visited for management of other complaints. although studies have been conducted previously on youth suicide in various groups, this is the first -year multicenter study in south korea investigating all first visit patients. the most common chief complaint of the patients was behavioral problems ( . %), and the most common diagnosis values are presented as n (%) unless otherwise indicated. *p＜ . was hyperkinetic disorder ( . %). this could be related to the disease prevalence, but could also be attributed to different treatment rates depending on the chief complaint or diagnosis. a study conducted in the united states on the treatment of adolescents in the mental health department revealed rates of . % and . % for attention-deficit/hyperactivity disorder (adhd) and behavioral disorders, respectively, while lower treatment rates were observed in patients with anxiety disorders, eating disorders, and substance abuse [ ] . thus, it is essential to perform studies on trends in the prevalence and treatment rates of psychiatric disorders among adolescents in south korea in order to elucidate differences in the frequency of chief complaints. a general decreasing trend was observed in the number of patients when we analyzed the annual trends in the total group of first visit patients. this could be related to the gradual decrease in the size of the adolescent population in south korea. the total number of patients was lowest in , which could be due to the middle east respiratory syndrome (mers) outbreak, wherein people avoided visiting medical institutions. notably, the total number of patients showed minimal changes from , and although there was a generally decreasing trend, the ratio of patients visiting hospitals for management of suicide-related problems showed no significant changes until , when the ratio was at its highest. the korean youth risk behavior survey reported that the proportion of youths who attempted suicide was . % in . subsequently, although there were minimal changes, a continually decreasing trend to . % was observed in , and the rate of suicidal thoughts also showed a decreasing trend from . % in to . % in [ ] . moreover, the mortality due to suicide among the - -year-old population also decreased from . deaths per population in to . deaths in [ ] . thus, our findings were not possibly due to an increase in the ratio of adolescents with suicide-related problems, but rather due to an increase in early detection, linkage with medical services, and treatment. in this study, we compared the data of patients before and in or later, when school-based mental health projects became more prevalent, and found a significant increase in the rate of patients with suicide-related problems in and after . the year of hospital visit (before or in or later) did not show significant effect in the regression analysis. this suggests that, rather than a direct effect of the specific time period (in or later) on suicide-related problems, an indirect effect exists via the relationships with other variables, such as sex, education level, referral via schools, or depression. analysis of the referral sources revealed that patients in the suicidal group were referred at a significantly higher rate through schools than other routes. this may be because schools consider problems related to suicide more serious than other problems, which would mean an increased likelihood of referral of their students to psychiatric departments. externalizing behaviors are more readily detected by parents and are linked to mental health services based on parental demands; however, problems such as depression are less frequently recognized by parents [ ] . in particular, youths with suicide-related problems demonstrate additional difficulties in seeking help from others or receiving treatment due to the associated social stigma [ ] . this emphasizes the role of schools in dealing with the issue of suicide among adolescents. the effects of school-based programs may differ depending on the presentation of mental health problems, and it has been reported to be more effective in cases of internalizing than externalizing behavioral problems [ ] . therefore, referral to a professional treatment center via school can play a crucial role in cases of internalizing problems, and schools should adopt measures to enable early recognition of such problems in young individuals. furthermore, we believe that the referral and linkage efficiency could be improved further through projects to support the treatment costs of patients being referred to a treatment center via schools. such projects are already being implemented in some regional education offices in south korea. the rate of suicidal group was found to increase with the education level from elementary to middle to high school. the overall ratio of female patients was higher in the suicidal group. although male patients did not show major differences in the rate of suicide-related problems at the elementary, middle, or high school levels, a significant increase in the rate was observed among female patients at the middle school level. these results are consistent with a previous study, which reported that the overall rate of self-harm among adolescents was higher in female patients [ ] . the study also reported that while female patients showed a peak in midadolescence, male patients showed a gradually increasing trend up to late adolescence. this could be because females start puberty earlier than males do, and this period of biological changes is associated with a higher risk of suicide [ ] . these sex-related differences tend to disappear in early adulthood, and it has been reported that adolescent suicide attempts are predictive of the same in adulthood among female but not male patients [ ] . among individuals with suicide-related problems, female patients are known to avail psychiatric treatment and general mental health services at a higher rate than male patients [ ] . this could be related to our finding of more female patients in the suicidal group. another significant finding of our study was that the ratio of youths who had dropped out of school was higher in the suicidal group. problems such as depression and anxiety are known to increase the risk of dropout from school [ ] , and more than half of the students who drop out of middle school have been reported to suffer from psychiatric disorders [ ] . in addition, young individuals with suicide-related problems who quit school are more likely to have the same issues in adulthood [ ] . this demonstrates the need for efforts to detect high-risk youths outside of the education system and initiate therapeutic interventions. a study that assessed the relationship between hospital schools and return-to-school rates in adolescents hospitalized in a closed psychiatry ward revealed that youths who were admitted in hospital schools showed higher return-to-school rates [ ] . based on these results, we believe that development of hospital schools could decrease the dropout rate of youths with psychiatric disorders, thereby ensuring a positive effect on their prognosis. psychiatric disorders are known to be a strong risk factor for suicide among young individuals. in particular, mood disorders, substance abuse, and disruptive behavior disorders have been shown to be closely associated with youth suicides [ ] . likewise, in our study, depression was the most frequent diagnosis among patients in the suicidal group ( . %), while that in the non-suicidal group was adhd ( . %). similarly, in the regression analysis, the diagnosis of depression was associated with . times higher relative risk of suicide-related problems compared to other diagnoses. adolescents with depression who attempt suicide have been reported to demonstrate stronger intention to commit the act compared to those without depression, and suffer more serious physical injuries due to suicidal behaviors [ ] . moreover, several symptoms of depression, such as non-suicidal self-harm, sleep disorders, and cognitive symptoms have been reported to be associated with suicide-related problems [ ] . this demonstrates the need for detailed assessments to diagnose depression and identify high-risk symptoms in adolescents who visit hospitals with suicide-related problems. studies have shown certain differences in the relationship between suicide-related problems and psychiatric disorders in young individuals depending on the patients' sex and age. a study that compared - -year-old and - -year-old pediatric suicide victims reported that adhd was more strongly associated with suicide-related problems in the former group compared to depression [ ] . in a finnish study based on psychological autopsy results, among those who committed suicide, adolescent girls showed a higher rate of depression than boys did, while boys showed a higher rate of substance abuse [ ] . in the present study, depression was the most common diagnosis among patients in the suicidal group, irrespective of sex. thus, it is essential to consider age and sex when dealing with psychiatric disorders related to suicide. this was a multicenter study and, in particular, the first to analyze the change in patient trends since the implementation of school-based public mental health projects. the merit of this study lies in the fact that it demonstrates the importance of school-based mental health services and cooperation between systems. this study also has several limitations. first, there are many variables (other than demographic variables and referral source) that can affect hospital visits among youths with suicide-related problems [ ] ; however, we could not control these variables. second, we depended on the diagnoses assigned by clinicians, rather than through objective structured assessment tools. third, because this was a retrospective study, we were unable to follow-up on the response to treatment and maintenance of outcomes after the first visit. fourth, there were certain inconsistencies between the medical records collected from the five different hospitals. in the future, a prospective study should be performed on the factors affecting clinical characteristics and hospital treatment, and differences depending on referral source in youths with suicide-related problems. this study was an -year retrospective chart review of patient data, wherein we analyzed the characteristics of adolescents who visited hospitals for the first time for management of suicide-related problems. female sex, higher education level, dropping out of school, referral via school, and a diagnosis of depression were significantly associated with suicide-related problems. korean centers for disease control & prevention. statistics of surveillance of health behavior in adolescents in prevalence, correlates, and comorbidities of dsm-iv psychiatric disorders in children in seoul, korea prevalence, correlates, and treatment of lifetime suicidal behavior among adolescents: results from the national comorbidity survey replication adolescent supplement characteristics of adolescents who visit the emergency department following suicide attempts: comparison study between adolescents and adults adult associations of childhood suicidal thoughts and behaviors: a prospective, longitudinal analysis adolescent suicidal ideation as predictive of psychopathology, suicidal behavior, and compromised functioning at age korean centers for disease control & prevention. statistics of surveillance of health behavior in adolescent in prevalence of treated psychiatric disorders in korean child and adolescent patients in program evaluation activities: outcomes related to treatment for adolescents receiving school-based mental health services screening for depression and suicidality in adolescents: the korean annual nationwide survey analysis on effectiveness of mental health professional's school visit service for intervention world health organization. the icd- classification of mental and behavioral disorders: clinical descriptions and diagnostic guidelines. geneva: world health organization service utilization for lifetime mental disorders in u.s. adolescents: results of the national comorbidity survey-adolescent supplement (ncs-a) depressive and disruptive disorders and mental health service utilization in children and adolescents predictors of help-seeking for suicidal ideation in the community: risks and opportunities for public suicide prevention campaigns the effectiveness of school-based mental health services for elementary-aged children: a meta-analysis sex differences in developmental trends of suicide ideation, plans, and attempts among european american adolescents pubertal transition, stressful life events, and the emergence of gender differences in adolescent depressive symptoms sex differences in suicides among children and youth: the potential impact of misclassification service use and unmet needs in youth suicide: a study of trajectories mental disorders and termination of education in high-income and low-and middle-income countries: epidemiological study what proportion of failure to complete secondary school in the us population is attributable to adolescent psychiatric disorder? adolescent predictors of incidence and persistence of suicide-related outcomes in young adulthood: a longitudinal study of mexican youth successful schooling rate and satisfaction of the inpatient hospital school participants among the child and adolescent psychiatric inpatients in the closed ward psychiatric contacts among youths aged through years who have made serious suicide attempts depressive disorders and suicidal intent in adolescent suicide attempters specific depressive symptoms and disorders as associates and predictors of suicidal acts across adolescence suicide in elementary school-aged children and early adolescents antisocial behaviour in adolescent suicide this work was supported by the ministry of education of the republic of korea and the national research foundation of korea (nrf- s a b a ). the authors have no potential conflicts of interest to disclose. jungeun song https://orcid.org/ - - - yong-sil kweon https://orcid.org/ - - - key: cord- -shog bx authors: larcher, vic; dittborn, mariana; linthicum, james; sutton, amy; brierley, joe; payne, christopher; hardy, hannah title: young people’s views on their role in the covid- pandemic and society’s recovery from it date: - - journal: arch dis child doi: . /archdischild- - sha: doc_id: cord_uid: shog bx objective: there has been little formal exploration of how young people see their role in the covid- pandemic. design/setting: focus-group discussion with children’s hospital young people’s forum members ( / ) to explore their perspective on the impact of covid- on both their lives and those of their community, on school closures, and the role they wished to play in society’s recovery from the pandemic. audio recordings were transcribed verbatim using nvivo software and analysed using an inductive thematic analysis approach. outcome: four major themes identified: ( ) awareness of pandemic’s impact on others: participants showed mature awareness of the effects on broader society, especially the elderly, socially disadvantaged and parents. ( ) perceived impact on their own lives: principal concerns were the educational and practical repercussions of school closures and social isolation, including effects on educational prospects. ( ) views about school reopening: young people understood the broader rationale for school reopening and were generally positive about it, but expressed concerned about their safety and that of others. ( ) communication issues: a need for clear, concise, understandable information readily accessible for young people was expressed. up to now, they felt passive recipients rather than participants. conclusion: young people were concerned about their future, their family and broader society, consistent with a high level of moral development. they want to be active participants in social recovery, including concepts around return to school but require appropriate information and a means by which their voices can be heard. the alternative suggested roles as pawns or pathfinders were discounted. in the uk and elsewhere, the response to the covid- pandemic has been 'guided by the science'. but the implication that there is one form of science whose facts should guide policy is erroneous, since medical, social and economic disciplines are all involved. moreover, science is not value-free, since inevitable disparities in scientific advice need to be resolved by judgements that are value-driven and which need to take into account the views of those involved if ethical principles of equal concern and respect are to be satisfied. the wider psychosocial and economic consequences of the pandemic are fast emerging. although young people (yp) have suffered less as a direct result of covid- disease than other vulnerable groups, for example, the elderly and bame (black, asian and minority ethnic) community, - the indirect impact on them has been marked. perhaps of more significant concern is the educational and psychosocial effects of physical distancing, social isolation and school closures on children's welfare. reopening of schools is, therefore, a key element in the recovery process, because of the benefits it brings, but it remains a matter of dispute. the broader role yp might play in the recovery process needs to be explored since they will, as adults, bear the economic and psychosocial consequences of the pandemic. yet there has been little formal exploration of how they view their roles in this process or of how their voices might be heard. to begin to address this, we explored the views of yp on the impact of the pandemic on both their lives and those of their community, on school closures and of the role they wished to play in recovery. ► sars-cov- has not severely clinically impacted children compared with other groups, yet they have been profoundly affected by control measures, for example, social distancing and school closures. ► the views of young people on the effects of the pandemic on society, and their role in recovery for it have been little explored. ► roles as partners, pawn or pathfinders in society's recovery from the pandemic have been suggested for young people; it is unknown which they think suitable. ► the main concerns of young people about the pandemic are its effects on others (known to them; the abstract vulnerable) and on their own education/future. ► young people do not consider roles as pawns or pathfinders in society's emergence from the pandemic as suitable, preferring to be partners. ► young people felt recipients rather than participants in the pandemic process; they need appropriate accessible information and means by which their voices can be heard. we conducted an audio-recorded focus-group discussion with available members ( - audio recordings were transcribed verbatim using nvivo software and analysed using an inductive thematic analysis approach. four major themes with subthemes were identified: yp comment: boxes and . . awareness of the impact on others: participants showed clear recognition of the effects of the pandemic and the imposed control measures on broader society, focusing mainly on three groups: the elderly, socially disadvantaged and parents. i. elderly and communication and technology challenges: participants were all technology proficient, being able to join a video-conference forum, but had experienced communication challenges with their more elderly relatives who often lacked skills to use online communication means: (p ). participants recognised this as a broader problem, including for those organisations that would typically support older people who were themselves now facing communication problems. ii. socially disadvantaged: participants were aware of challenges posed by social inequalities on those from underprivileged backgrounds. notably, they mentioned that students who lacked the required devices for homeschooling needed support and the provision of appropriate, updated and dynamic materials: (p ). iii. challenges for parents: participants discussed how competing responsibilities posed by school closure and working from home was challenging for their parents, who often lacked teaching skills and the time needed to support children with homework: (p ). i. school closure: many felt that home-schooling and online learning had been an overwhelming experience produced by such factors as the new format, the lack of peer interaction for discussion and education and lack of proper support from their teachers. participants expressed how support received was inadequate and slow regarding submission deadlines: (p ). ii. suspended grades: for those preparing for a-levels, with the need to obtain specific grades to progress to university, cancellation produced feelings of disappointment, impotence and uncertainty about their future, for which they had been working hard and looking forward. lack of clear guidance and information about how this will be managed contributed to concerns: (p ). iii. social isolation: regarding social isolation, participants mentioned difficulties with being unable to socialise faceto-face with their peers, with whom they would normally share their personal struggles, as expressed by this participant: (p ). participants also expressed challenges produced by the interruption of their usual coping mechanisms, such as sport or music activities. while some of them were adapting readily to new online formats, there were concerns about the significant amount of screen time to which they were now exposed. . yps' views about school reopening box synthesis of themes and subthemes from young people's forum focus-group discussion (i) "so, all of my grandparents are very social, but i think i found staying in quite hard, especially because they don't understand technology as well […] . so, none of them would be able to face time." (p ). ii. socially disadvantaged "they kind of came out and asked who had the provision to actually access the internet to do work. and i think for the people who didn't, they were given textbooks. and to me, that seems a little bit outdated and something that maybe isn't too useful because that's not a constant stream of information." (p ) iii. challenges for parents "but obviously, our parents don't know everything about things. so, then we try, and you google. but then when google doesn't give you the answer, then you rely on your parents. but your parents can't really teach you anything. so, you kind of feel bad because you can't do anything because you don't understand it, but they don't understand your work either." (p ) iv. schools' staff: this is included under school reopening concerns . the perceived impacts on their own lives i. school closure "we have been almost overwhelmed. i know i have. with the amount of work that's been set all at one time. and a lot of the teachers don't respond when you ask them for assistance with things we might be stuck on." (p ) ii. suspended grades "oh, i was going to have my a-levels this year, but obviously they got cancelled and it, kind of feels like the sort of rite of passage that kind of got them. yeah. for me that you don't get to go through, like because i want to do medicine. i've done so many things for the past two years. just as often leads up to and it's like you've built up all this momentum. but the end result and like thought of going through exams or just disappear so you don't have that place of relief for this sort of energy and everything that's been building up." (p ) iii. social isolation "so, it might be very hard for some people because we can't see our friends anymore. and i know that i haven't got much family, so my friends are more like my family and i depend on them. it's not being able to have that human contact in person is really hard." (p ) i. rationale: views toward school reopening were variable. some thought that this was necessary to allow parents to go back to work. in contrast, others considered this unsafe as children would be the first in society to have close contact with other people: (p ) ii. concerns: various anxieties were expressed about school reopening related to impracticable infection control measures, including social distancing steps, that would lead to risks for staff and any students' family members who might be at high risk of severe disease: (p ). iii. age groups' particular issues: most comments were concerned with year - returnees, rather than younger children, but a view concerning the impact on university students was also expressed: (p ). i. right to appropriately tailored information: participants agreed that yp had the right to receive information about the pandemic, but acknowledged the challenges associated with adapting it to recipients' needs and different age groups. however, they felt that comprehensive details should be provided while avoiding complicated wording and lengthy and confusing content: (p ). ii. problems related to available information: participants mentioned several issues with how information was being presented, which they felt was not explicitly aimed at a yp audience. many knowledge resources provided overwhelming and confusing amounts of data, with inconsistencies between government/institutions' official reports and what they saw shared on social media. however, they also identified the risk of fake news on some platforms, which concerned them due to the considerable uncertainty it could generate among yp. iii. linear communication model-yp as passive recipients: information was reportedly received from different channels, especially from schools: "[…] we've also been receiving weekly updates from the college." however, only a few mentioned that they had opportunities to express their questions and opinions. most participants agreed with the feeling of not having a voice during the pandemic or during the recovery process: (p ). iv. yp's participation in the recovery process: many expressed the view they should be part of this process, as they are and will be directly affected by the pandemic in multiple aspects of their current and future lives: (p ). they suggested various ways in which yp' voices could be heard: from looking at what yp are currently discussing and their opinions on social media and other platforms, in collaborative or national surveys by active representation and participation in the youth parliament, and in events like the current forum: (p ). we asked the yp to write a paragraph about being one of two yp authors on the paper, to reflect their contribution to the paper-judging only two proved incredibly hard, therefore we have elected to share these as an online supplementary file. (yp paragraphs) the discussion held in this focus group with yp provided remarkable insight on their awareness of moral issues concerning themselves, their families and the wider community, consistent with higher levels of moral development. they demonstrated knowledge of themselves over time in respect of future ambitions and need for education. additionally, participants expressed willingness to participate in the response and recovery process by actively looking for both information sources and means to voice their views. however, most acknowledged the lack of opportunities to express their opinions and actively participate in the process. the un convention on the rights of the child (uncrc) and the uk children act provide for the rights of children to express their views on matters that concern them, and for those views to be given due weight in accordance with their age, maturity and understanding. professional bodies such as the general medical council and royal college of paediatrics and child health have sought to involve yp in discussions about policy and other matters. the reason for yp's voices not being considered in the pandemic response and regarding recovery measures is unclear. it might be due to persistent notions of incomplete moral development and agency in this group, even though some children have shown the capacity for self-directed acts of box synthesis of themes and subthemes from young people's forum focus-group discussion (ii) "i don't think until mps are sitting together in the house of commons, i don't think we should be sitting together in school just because we're younger and we aren't at risk of having a more serious infection, doesn't mean we should be the first to have to fully go back and reintegrate. i don't think that is a really safe thing to do because that is a blanket." (p ) ii. concerns "there's obviously a lot of concerns about how the risk of infection will be strictly managed all day long because i know like at some schools there's key workers kids who all go in, and it's really good the social distancing. all-day long. but at the break and lunchtimes, there's no social distancing at all. so, all the kids go back together and then at pick up. all the parents are together as well." (p ) iii. age groups' particular issues "i was kind of a forgotten group of uni students in the sense that all unis have different policies about what they're doing. some are holding their exams; some aren't. and then the question of the future, they sort of forget the fact that we're all racking up nine thousand pounds every year, even if we're not allowed to go back." (p ) i. right to information-tailored "so, even if it's presented in a different way, you're entitled to the same information in life as an adult should be." (p ) ii. threats related to available information iii. linear communication model-yps as recipients only "they're not listening to children, young people much at the moment." (p ) iv. yp's participation in the decision-making process "i think that's a general view at the moment that it's going to affect this generation most with all the debt and everything that's building up out of this." (p ) "yeah. i was thinking maybe one way that children and young people could get their words heard was maybe a schools or youth group sent out like a q and a. and then the answers and their opinions were collaborative and maybe sent to mps or somebody higher up." (p ) kindness and altruism. participants in this focus group showed great ability to understand complex issues and, more importantly, expressed the need and willingness to actively participate, supporting the social and legal commitments to them represented in the above international and national standards/laws. although the participants were sophisticated in terms of their medical knowledge, gleaned from past experiences and participation in the ypf, they felt that the information that they had received was complicated, confusing and in some instances, contradictory. if this was the case for this group, it seems highly likely that it would apply to an even greater extent to those who lacked their experience and knowledge. again, this group demonstrated concern for others by suggesting strategies to alleviate this problem. this issue requires urgent attention with lack of transparent, appropriately presented and complete information impeding yp's participation in this process. initially, national debates about the covid- pandemic were primarily concerned with discussions about the medical science including the direct effect of the virus on vulnerable individuals and maintenance of critical care capacity, so little involved yp who were not perceived likely to be significantly affected. however, this is no longer the case given the recent identification of the paediatric multisystem inflammatory syndrome temporally associated with covid- , and the highlighting of the socioeconomic consequences of physical distancing, social isolation and school closures, and of now a recovery to the 'new normal'. children's issues, including how we must mitigate the profound detrimental effects on them as a social priority, is finally becoming clear. return to school has been an essential element in this because of the significant interruption of education that has occurred, the vital part played by schools in the psychosocial support and protection of vulnerable children, and the freeing of parents to contribute to the economy. participants in the focus group were aware and concerned that yp and younger children have been and will continue to be indirectly affected by the pandemic and the control measures imposed, notably social isolation, school closure and for older participants, the impact of suspended grades. after reading the jme paper about their potential role as pawns, pathfinders or partners in the recovery process, the comments made by the yp provided a clear indication of the position that they would wish to play in response to the pandemic. comments about the limited provision of relevant and accessible information, together with the almost complete lack of consultation of children throughout were indicative of passive 'pawn' role insofar as others made decisions on their behalf in which they had no active part. concerns expressed about their safety and that of others, together with the view that others should be taking similar risks suggested that they did not regard the 'pathfinder' role, even with safeguards, an attractive one. in contrast, general support for a more active partnership role was clear; this is undoubtedly consistent with pre-existing standards, especially the uncrc. to be clear, the need to make urgent difficult decisions in the national interest is a crucial role of government and we do not seek some plebiscite of those most affected (ie, teachers, yp and parents regarding school closures) before urgent decisions are taken. but we do argue that consultation of such groups ought to occur as soon as possible after any urgent action, and indeed before if there is time. we wish to highlight a few strengths and limitations to this study. there was a broad range of participants, all actively involved, which allowed the ypf to eloquently contribute to a little explored, hot topic affecting them. the presence of the authors of the provided paper might have made it more difficult to voice disagreement, though this was not the author's experience. although the aim was not to look for representativeness, this is a particularly educated and informed group in terms of prior involvement in, and experience of, child health issues. therefore, it is unclear how generalisable to other yp our findings are. there is a lack of information concerning the role of children and yp in the covid- pandemic response. from this limited study, it seems clear that yp have relevant concerns about themselves (especially their future), their family and broader society. they feel that they should have an active part to play in both return to school and the recovery process, though they have had little mechanism as yet to do so. they do not see themselves as either pathfinders or pawns, but rather partners in society's emergence from the pandemic. twitter vic larcher @viclarcher ethical road map through the covid- pandemic guidance: responding to covid- : the ethical framework for adult social care mitigating the wider health effects of covid- pandemic response risks to children and young people during covid- pandemic covid- ) related deaths by ethnic group sars-cov- infection in children coronavirus disease in children -united states the effects of social deprivation on adolescent development and mental health school closure and management practices during coronavirus outbreaks including covid- : a rapid systematic review children of covid- : pawns, pathfinders or partners? using thematic analysis in psychology united nations general assembly. convention on the rights of the child uk public general acts not just a phase. a guide to the participation of children and young people in health services clinical characteristics of children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov- paediatric inflammatory multisystem syndrome: temporally associated with sars-cov- (pims-ts): cardiac features, management and short-term outcomes at a uk tertiary paediatric hospital contributors jb conceived the project after email exchange with the editor in chief. vl and jb wrote the jme paper and blog which the ypf read before the focus group. as leads the yp, and with md and jl organised the focus group and contributed to the paper. md and vl transcripted and jb, vl and md analysed the data. all yp were invited to comment on why they might be authors, and we think they are all so good we include as an addendum. all authors approved the final manuscript.funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.competing interests none declared. provenance and peer review not commissioned; externally peer reviewed.data availability statement data are available on reasonable request. the data consist of the full transcripts, the audio recording has been destroyed.this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid- pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. joe brierley http:// orcid. org/ - - - key: cord- - wsn t r authors: zhang, caiyun; ye, maolin; fu, yunwei; yang, minyi; luo, fen; yuan, jinhua; tao, qian title: the psychological impact of the covid- pandemic on teenagers in china date: - - journal: j adolesc health doi: . /j.jadohealth. . . sha: doc_id: cord_uid: wsn t r purpose: the covid- pandemic poses a challenge to adolescent psychological health. the aim of this study was to survey junior high and high school students in china to better understand the psychological consequences, such as anxiety, depression, and stress, of the covid- pandemic. methods: a cross-sectional online survey using structural questionnaires was conducted from april , , to april , . demographic information and general information related to the pandemic were collected. psychological consequences were assessed by the impact of event scale-revised and the depression, anxiety and stress scale. influencing factors were assessed by the brief resilience scale and coping style questionnaire. results: our sample comprised junior high school students (male = , mean age = . years) and high school students (male = , mean age = . years). resilience and positive coping were protective factors for the occurrence of depressive, anxiety, and stress symptoms in junior high and high school students (p < . ). positive coping was a protective factor for trauma-related distress in junior high school students (p < . ). negative coping is a risk factor for depression, anxiety, stress symptoms, and trauma-related distress in junior high and high school students (p < . ). conclusions: during the covid- pandemic in china, more than one fifth of junior high and high school students' mental health was affected. our findings suggested that resilience and positive coping lead to better psychological and mental health status among students. in contrast, negative coping is a risk factor for mental health. the psychological impact of the covid- pandemic on teenagers and adolescents is substantial, causing more than one fifth of chinese junior high and high school students to experience symptoms of depression, anxiety, and stress. when addressing these symptoms, resilience and positive coping are protective factors, whereas negative coping is a risk factor. not only the risk of death from the viral infection but also psychological consequences among people, particularly because of the long-term nature of the pandemic, which is still developing. previous research has revealed a profound and wide range of psychological impacts of infectious outbreaks on survivors, family members of infected patients, medical staff, and the general public [ , ] . the psychological consequences of infectious diseases have been reported to include depressed mood, anxiety, poor sleep, and increased fear and stress levels [ , ] , with posttraumatic stress disorder (ptsd) and depressive disorders being the most prevalent long-term psychological conditions [ ] . the psychological impacts of the covid- pandemic on teenagers and adolescents seem to be far greater than the impact on adults because they are more vulnerable to the negative effects of stress [ ] . following the outbreak, national school closures had been implemented, and students were required to stay at home. reduced social interaction, stay-athome restrictions, difficulties in schoolwork, substantial changes to daily routine, fear of becoming sick, and boredom can create dramatic psychological effects on teenagers and adolescents. developmental motivations and hormonal changes make teenagers and adolescents highly attuned to peer groups, making it challenging to isolate at home. for instance, during the severe acute respiratory syndrome (sars) epidemic, a cross-sectional study revealed that psychiatric morbidities in a general population were associated with younger age [ ] . the psychological impact of covid- on teenagers is a serious concern during the outbreak and thereafter. the present study sought to examine depressive, anxiety, stress, and trauma-related distress symptoms in a sample of junior high and high school students. the covid- pandemic can be regarded as an acute, largescale, and uncontrollable stressor that will have a significant effect on individuals' mental health. however, little is known about how teenagers cope with acute large-scale stressors such as the covid- pandemic. accumulated evidence has indicated that different coping strategies are associated with different adjustment outcomes after trauma [ , ] . generally, there are two types of coping strategies: active and passive coping. active coping involves actively doing something to reduce stress, such as problem-solving, planning, and cognitive restructuring, whereas passive coping involves ignoring and avoiding sources of stress, such as denial and substance use [ ] . we expected teenagers with positive coping to be associated with better mental health outcomes during the covid- pandemic. another potential factor influencing teenagers' psychological outcome in the event of the covid- pandemic is resilience. the construct of resilience refers to an ability to maintain positive mental health in the face of adversity or stress [ ] . a high level of resilience provides protection from various mental health conditions. for instance, higher resilience in adolescents aged e years was associated with a lower level of depression, stress, and anxiety [ ] . when dealing with stress induced by the covid- pandemic, teenagers with a high level of resilience are expected to have a positive mental health status. a web-based cross-sectional study was performed to assess the psychological impacts of the covid- pandemic on junior high and high school students. we hypothesize that positive coping and a high level of resilience are protective factors for the mental health consequences of the covid- pandemic. a cross-sectional online survey using structural questionnaires was conducted from april , , to april , . participants, stratified by age and gender, were recruited from guangzhou no. middle school and longchuan no. one middle school in guangdong, china. the study was approved by the ethics committee of jinan university, and all participants provided written consent before they answered the questionnaires. demographic and general information. demographic information was collected, including age, gender, grade, and education levels of parents. in addition, information related to the covid- pandemic was collected, such as residence location during the pandemic, whether their parents were frontline personnel during the pandemic, whether they were confirmed cases, whether their family or friends were confirmed cases, and whether they had peers around during the pandemic. brief resilience scale. the brief resilience scale (brs) was developed as a reliable measure of a unitary construct to measure the most basic and original meaning of resilience, that is, "an individual's ability to bounce back or recover from stress" [ ] . it represents a factor related to resilience resources and health outcomes. the participants were asked to indicate the extent to which they agreed with a statement according to a -point scale, ranging from (strongly disagree) to (strongly agree). the brs has been validated in a variety of populations under different levels of stress and has demonstrated good psychometric properties. the chinese version of the brs has demonstrated acceptable internal consistency with cronbach's alpha values of . and . in hong kong and mainland samples, respectively [ ] . to the best of our knowledge, the brs has not been validated in a sample of teenagers. we conducted psychometric analyses on the data set. results of exploratory factor analysis suggested extraction of two factors (positive and negative items) that account for . % of total variance. the cronbach's alpha coefficient was . , suggesting good reliability of the chinese version of the brs. coping style questionnaire. the coping style questionnaire (csq) was developed by xie [ ] on the basis of both chinese and foreign scales [ ] . it consists of items measuring the use of coping strategies with two dimensions, active coping ( items) and passive coping (eight items). a higher score represents a greater active/passive coping tendency. participants rate the frequency with which they adopt the strategy in the face of stress on a -point scale, ranging from (never) to (very often). xie validated the csq in a chinese sample aged e years. its internal consistency was . , with two subscales and whole scale's cronbach's alpha were . , . , and . , respectively. a previous study investigating a sample aged e years indicated that the internal consistency measured by cronbach's alpha were . , . , and . for the subscales and the whole scale, respectively [ ] . nevertheless, the csq has been used in several studies with teenagers as the subjects [ , ] . the instrument also demonstrated good reliability and validity in chinese samples [ ] . we conducted psychometric analyses on the data set. results of exploratory factor analysis suggested extraction of two factors (positive coping and negative coping) that account for . % of total variance. the cronbach's alpha coefficients for the two subscales were . (active coping) and . (passive coping), suggesting good reliability of the chinese version of the scale. impact of event scale-revised. the original impact of event scale (ies) is a -item measure of psychological responses to trauma. there are three subscales, namely, the intrusive subscale, avoidance subscale, and hyperarousal subscale. participants rate each item on a -point scale, ranging from (no problems) to (frequent problems). the mean score for ptsd is , and a cutoff score of has been used to estimate the prevalence of ptsd symptoms [ , ] . the ies-revised (ies-r) can be anchored to any specific event, such as sars or covid- infection. the ies has been validated in a sample of adolescents in taiwan who had experienced floods and mudslides caused by typhoon morakot [ ] . the results suggested that the ies was a reliable and valid instrument with cronbach's alpha of . and three factors of intrusion, hyperarousal, and avoidance accounted for . % of the variance. another study validated the ies in a sample of urban high school students who experienced the september , , terrorist attack [ ] . the results yielded three factors (intrusion, hyperarousal, and avoidance) and found a moderate correlation between the ies and my worst experience scale as a measure of posttraumatic stress reactions in individuals exposed to a specific traumatic stressor [ ] . the chinese version of the ies-r has demonstrated good reliability and validity with cronbach's alpha of . in a student sample [ ] , and cronbach's alpha for the three subscales were . (intrusion), . (avoidance), and . (hyperarousal) in a patient sample from the accident and emergency department [ ] . in our sample, cronbach's alpha coefficients for the three subscales were . (intrusion), . (avoidance), and . (hyperarousal), suggesting good reliability of the chinese version of the scale. twenty-one-item depression anxiety stress scale. the -item depression anxiety stress scale (dass- ) is a well-developed instrument evaluating levels of depression, anxiety, and stress, with seven items in each subscale. it asks whether the described situation is applicable and requires participants to rate the applicability on a -point scale, ranging from (never) to (almost always). the total scores of the full scale and subscales were obtained, and a higher score indicates a more severe level of depression, anxiety, or stress. the depression subscale score was divided into normal ( e ), mild depression ( e ), moderate depression ( e ), severe depression ( e ), and extremely severe depression ( e ). the anxiety subscale score was divided into normal ( e ), mild anxiety ( e ), moderate anxiety ( e ), severe anxiety ( e ), and extremely severe anxiety ( e ). the stress subscale score was divided into normal ( e ), mild stress ( e ), moderate stress ( e ), severe stress ( e ), and extremely severe stress ( e ). the dass- has been demonstrated to be a reliable and valid measure in assessing mental health in the chinese population [ ] . the dass was previously used in research related to sars [ ] . the dass has been widely used in children and adolescents between the ages of and years [ e ]. we conducted psychometric analyses on the data set. the results of exploratory factor analysis the suggested extraction of three factors (depression, anxiety, and stress) that account for . % of total variance. the cronbach's alpha coefficients for the three subscales were . (depression), . (anxiety), and . (stress), suggesting good reliability of the chinese version of the scale. statistical analysis was performed using spss version . (ibm, chicago, il). first, descriptive analyses were conducted to describe the demographic characteristics of chinese junior high and high school students. all results of quantitative variables are reported either as the mean ae standard deviation (sd) or frequency (percentage). second, to investigate the variables contributing to coping and resilience, multivariate regression analysis was used to assess the association between demographic variables and three dependent variables, including positive coping, negative coping, and resilience. third, the prevalence of depressive, anxiety, stress, and trauma-related distress symptoms were calculated for the junior high and high school students. finally, multivariate logistic regression models were performed to explore potential influencing factors of depressive, anxiety, stress, and trauma-related distress symptoms during the covid- pandemic. the variables of age, gender, grade, and other general information related to the pandemic were included as covariates. adjusted odds ratios, and % confidence intervals ( % cis) were obtained from logistic regression models. each hypothesis was tested using a two-tailed analysis at the a ¼ . level of significance. the questionnaires were administered to junior high school students, and completed the questionnaires with a response rate of . %. of the junior high school students (male ¼ , mean age ¼ . years), ( . %) had siblings, ( . %) had peer partners, and ( . %) stayed in mainland china (places other than hubei) during the covid- pandemic. the questionnaires were administered to high school students, and completed the questionnaires with a response rate of . %. of the high school students (male ¼ , mean age ¼ . years), ( . %) had siblings, ( . %) had peer partners, and ( . %) stayed in mainland china (places other than hubei) during the covid- pandemic. the demographics and general information are summarized in table . the results are summarized in tables and . only a few demographic variables had a significant association with the dependent variables. for junior high school students, higher resilience was significantly associated with male gender (p < . ) and father's college education (p ¼ . ), whereas negative coping was significantly associated with covid- patients (p ¼ . ). for high school students, higher resilience was significantly associated with male gender (p < . ), mother's college education (p ¼ . ), and siblings (p ¼ . ). the mean scores for the dass- were . (sd ¼ . ) in the junior high school students and . (sd ¼ . ) in the high school students, with a significant between-group difference (p ¼ . ). the prevalence of psychological symptoms is summarized in table . moderate depressive symptoms were found in . % of junior high school students and . % of high school students, and severe-to-extremely severe depressive symptoms were found in . % of junior high school students and . % of high school students, with no significant between-group difference (c ¼ . ; p ¼ . ). moderate anxiety symptoms were found in . % of junior high school students and . % of high school students, and severe-to-extremely severe anxiety symptoms were found in . % of junior high school students and . % of high school students, with a significant betweengroup difference (c ¼ . ; p ¼ . ). moderate stress symptoms were found in . % of junior high school students and . % of high school students, and severe-to-extremely severe stress symptoms were found in . % of junior high school students and . % of high school students, with significant between-group difference (c ¼ . ; p ¼ . ). trauma-related distress was found in . % of junior high school students and . % of high school students, with no significant between-group difference (c ¼ . ; p ¼ . ). the regression results are presented in to the best of our knowledge, this study is the first to investigate the psychological consequences of the covid- pandemic and its influencing factors on junior high and high school students. this study highlights several important findings. first, the results revealed significant rates of psychological consequences of the covid- pandemic. second, similar and different patterns were found between junior and high school students. the junior high and high school students had a similar prevalence of depression and trauma-related distress symptoms, and high school students had a higher prevalence of anxiety and stress symptoms than junior high school students. third, positive coping and resilience were protective factors for depression, anxiety, and stress symptoms in both junior high and high school students, whereas negative coping was a risk factor for depression, anxiety, and stress symptoms in both samples. for trauma-related distress, resilience was a protective factor, negative coping was a risk factor in both samples, and positive coping was a protective factor for junior high school students but not for high school students. the rates of depression symptoms ( . % for junior high school students and . % for high school students) and anxiety symptoms ( . % for junior high school students and . % for high school students) were higher than the rates reported by recent studies that did not involve any epidemic or pandemic. for instance, a recent study was conducted with a sample of , children aged e years old from provinces in china, and the results revealed that the rates of depressive symptoms are significantly lower in urban areas ( %) than in rural areas ( %) [ ] . another study surveyed , junior high school students aged e years in shang hai city (an urban area in china), and the results showed that the rates of anxious symptoms and depressive symptoms were . %and . %, respectively [ ] . in contrast, the rates reported in this study were comparable with those found in previous studies in similar epidemic or pandemic contexts [ , , , ] . a study suggested that nearly one in five participants had depressive symptoms in the covid- epidemic, indicating that the uncertainty of epidemic progression would cause greater psychological pressure on the public [ ] . a recent cross-sectional study examined the impact of the covid- pandemic on mental health in local chinese residents, table the psychological outcomes of the participants all, n (%)/mean (sd) junior school students, n (%)/mean (sd) senior high school students, n (%)/mean (sd) and the results indicated a mild stressful impact [ ] . previous studies have shown that children and adolescents who are exposed to traumatic experiences during disasters may suffer from high levels of posttraumatic stress [ ] . regarding anxious symptoms, a study evaluated the psychological condition of college students during the covid- epidemic, and the results indicated that . % of college students were afflicted with anxiety [ ] . common sources of anxiety during the epidemic include the increasing number of confirmed cases and suspected cases, the increasing number of provinces and countries affected by the outbreak, the shortage of masks and disinfectants, and the overwhelming and sensational news headlines [ ] . furthermore, we found that high school students ( . %) had a higher prevalence of anxiety symptoms than junior high school students ( . %). during the data collection period, the schools were still shut down, and the students were staying at home. all junior high and high schools in china were postponing classes and using distant remote learning methods. troublesome anxiety can be exacerbated during the senior high school years as students face extreme transitions and social and academic pressures [ , ] . this added stress has been shown to increase with age and be related to increased levels of anxiety [ ] . according to muris and mayer [ ] , as children age, their brain development continues and allows them to link their physical symptoms to an anxiety-provoking situation. broeren and muris [ ] maintained that as adolescents gain cognitive abilities, they would have more negative thoughts and interpretations of events, leading to increased anxiety. as part of the increased responsibility and independence students receive as they get older, emotional support often decreases in secondary school. it has been found that anxiety symptoms can escalate when emotional support decreases [ ] . the results showed that positive coping is a protective factor for anxiety, depression, and stress symptoms in junior high and high school students, whereas a negative coping style is a risk factor for anxiety, depression, and stress symptoms in both groups. in our study, active coping style involved positive appraisal and thinking, distancing, problem-solving and helpseeking. these active coping strategies can enhance mental health by promoting an individual's sense of control over a chaotic environment and creating opportunities for satisfying relationships with a support network [ , ] . accumulating evidence has suggested that positive coping is a protective factor for mental health. for example, it has been found that the use of a positive coping style could promote university students' academic adjustment and reduce displays of maladaptive behaviors [ ] . negative coping includes behaviors of avoidance, such as keeping feelings to one's self, avoiding the situation, and staying away from people. it is suggested that prominent use of negative coping strategies tends to impede adaptation and psychological health [ , ] and to exacerbate the negative effects of stress, perhaps leading to feelings of loss of control and helplessness [ ] . a negative coping strategy is employed when an individual's perceived resilience and social support is inadequate and when stressors are perceived as uncontrollable [ , ] . as the covid- pandemic is threatening and unpredictable, it is likely to be perceived as a stressor out of control. the current results showed that resilience is a protective factor for anxiety, depression, and stress symptoms in junior high and high school students. this could be indicative of the role that resilience plays in safeguarding the mental health of teenagers during the covid- pandemic. similar findings were reported in previous studies [ , ] . resilience refers to the ability to bounce back from stress, and the brs was developed to measure this ability [ , ] . although the brs measures a person's belief in how well he or she can bounce back from stress rather than his or her success in doing so, this belief may serve as a prerequisite of the individual's actual ability to bounce back [ ] . the current result suggesting that male students have higher resilience is consistent with the literature. on the one hand, previous studies reported higher resilience level in male than female in a university sample [ ] and in a community sample [ ] . on the other hand, the moderation effect of resilience on health outcomes was more prominent in males than females who exposed to earthquake [ ] . similarly, the moderation effect of resilience on psychological distress was more prominent in males than females in a university sample [ ] . the results extended previous findings by demonstrating that higher parental education level was associated with children's higher resilience. this is reasonable because parents with high educational levels are more likely to provide fulfilling educational, employment, and interpersonal futures for their children compared with parents with low educational levels, thus foster more positive self-image, greater life satisfaction, and more available support. nevertheless, there are several limitations to the present study. first, the cross-sectional study design precluded the ability to establish a causal relationship between psychological symptoms and coping/resilience, and causal inferences must be drawn with caution. future work may wish to follow up with young adults and to establish a predictive role of coping style and resilience for the occurrence of psychological symptoms. second, the online survey method relied on the self-selection of respondents and may lead to biased estimates, particularly when the response rate is low. finally, all the constructs were assessed by self-report, and the estimated relations among psychological outcomes, coping, and resilience might be biased by the reporter effect. future work may wish to use a multimethod approach for assessment. during the covid- pandemic in china, more than one fifth of junior high and high school students' mental health was affected. our findings suggested that resilience and positive coping lead to better psychological and mental health status in students. in contrast, negative coping is a risk factor for psychological and mental health. this study can be used to formulate psychological interventions to improve the mental health of junior high and high school students during the covid- pandemic. the psychological impact of the covid- epidemic on college students in china stress and psychological distress among sars survivors year after the outbreak immediate and sustained psychological impact of an emerging infectious disease outbreak on health care workers prevalence of psychiatric morbidity and psychological adaptation of the nurses in a structured sars caring unit during outbreak: a prospective and periodic assessment study in taiwan generalized anxiety disorder, depressive symptoms and sleep quality during covid- outbreak in china: a web-based crosssectional survey long-term psychiatric 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and social supports. wiley series on personality processes coping strategies as mediators in relation to resilience and posttraumatic stress disorder trauma reactivity, avoidant coping, and ptsd symptoms: a moderating relationship? resilience as the ability to bounce back from stress: a neglected personal resource? university students' resilience level: the effect of gender and faculty demographic and childhood environmental predictors of resilience in a community sample resilience in adolescence: gender differences two years after the earthquake of l'aquila prevalence of psychological distress and the effects of resilience and perceived social support among chinese college students: does gender make a difference? the authors thank the local school in guangdong province and students for participating in this study. this study was supported by research grants from the science and technology program of guangdong, china ( b ). key: cord- -xhyqg u authors: keeling, m. j.; tildesley, m. j.; atkins, b. d.; penman, b.; southall, e.; guyver-fletcher, g.; holmes, a.; mckimm, h.; gorsich, e. e.; hill, e. m.; dyson, l. title: the impact of school reopening on the spread of covid- in england date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: xhyqg u background: in the uk, cases of covid- have been declining since mid-april and there is good evidence to suggest that the effective reproduction number has dropped below , leading to a multi-phase relaxation plan for the country to emerge from lockdown. as part of this staggered process, primary schools are scheduled to partially reopen on st june. evidence from a range of sources suggests that children are, in general, only mildly affected by the disease and have low mortality rates, though there is less certainty regarding children's role in transmission. therefore, there is wide discussion on the impact of reopening schools. methods: we compare eight strategies for reopening primary and secondary schools in england from st june, focusing on the return of particular year groups and the associated epidemic consequences. this is assessed through model simulation, modifying a previously developed dynamic transmission model for sars-cov- . we quantify how the process of reopening schools affected contact patterns and anticipated secondary infections, the relative change in r according to the extent of school reopening, and determine the public health impact via estimated change in clinical cases and its sensitivity to decreases in adherence post strict lockdown. findings: whilst reopening schools, in any form, results in more mixing between children, an increase in r and hence transmission of the disease, the magnitude of that increase can be low dependent upon the age-groups that return to school and the behaviour of the remaining population. we predict that reopening schools in a way that allows half class sizes or that is focused on younger children is unlikely to push r above one, although there is noticeable variation between the regions of the country. given that older children have a greater number of social contacts and hence a greater potential for transmission, our findings suggest reopening secondary schools results in larger increases in case burden than only reopening primary schools; reopening both generates the largest increase and could push r above one in some regions. the impact of less social-distancing in the rest of the population, generally has far larger effects than reopening schools and exacerbates the impacts of reopening. discussion: our work indicates that any reopening of schools will result in increased mixing and infection amongst children and the wider population, although the opening of schools alone is unlikely to push the value of r above one. however, impacts of other recent relaxations of lockdown measures are yet to be quantified, suggesting some regions may be closer to the critical threshold that would lead to a growth in cases. given the uncertainties, in part due to limited data on covid- in children, school reopening should be carefully monitored. ultimately, the decision about reopening classrooms is a difficult trade-off between increased epidemiological consequences and the emotional, educational and developmental needs of children. the emergence of a novel strain of coronavirus, now named sars-cov- , in wuhan city, china, in late , has resulted in a global pandemic that spread to every region in the world. when the sars- cov- virus infects humans it can result in covid- disease, with symptoms including a fever, a continuous dry cough, a shortness of breath and a loss of sense of taste and smell [ ] . in severe cases, the symptoms can require hospitalisation and admission to intensive care, with ventilation required in the most severe cases in order to assist with breathing. as the number of confirmed cases increased both nationally and globally, there was a concern that hospital and intensive care capacities would be rapidly overwhelmed without the introduction of in- terventions to curb the spread of infection. with this in mind, many countries introduced a range of social distancing measures, such as the closing of workplaces, pubs and restaurants, the restriction of leisure activities and the closing of schools. in the uk, the introduction of many of these measures was announced during the week of th march, with schools, along with the hospitality sector, closing on friday th march. full lockdown measures were subsequently introduced three days later, on the evening of monday rd march. at the time of writing, over , people in the uk have been confirmed to have been infected with covid- , with over , confirmed deaths of individuals who have tested positive for infection. the decision to close schools is a balance between the risk associated with transmission in the school environment and the educational and welfare impact upon children of shutting down education es- tablishments. evidence from a range of sources suggests that children are, in general, only mildly affected by the disease and have low mortality rates [ , ] . this is reflected in the fact that by th may there had been , covid- associated deaths in hospitals in england, but only of those were in the - year age group [ ] . in a retrospective study of , paediatric covid- cases in china [ ] , . % of children had mild or moderate disease while . % were severe or critical; similarly low levels of severe disease are reported in other regions [ , ] . the health risks of school attendance for any individual child is therefore thought to be low. however, there is less certainty regarding children's role in the transmission of sars-cov- [ , ] . this can be broken down into two key questions: (i) how likely are children to become infected, and (ii) once infected, are children likely to transmit infection? a recent meta-analysis concludes that children and young people under the age of may be less likely to become infected: the odds ratio for becoming infected upon contact with an index case compared to adults (> years old) is . (ci . , . ) [ ] . this conclusion is based on pooling the results of contact tracing and population-screening studies, most of which find evidence that the attack rate in children may be lower than in adults [ , ] , but one does not (bi et al. [ ] ). all contact tracing studies are hampered by the problem that symptom-based surveillance is likely to systematically under detect cases in children [ ] . seroprevalence surveys so far do not find any significant effect of age on the probability of possessing antibodies against covid- , although those under the age of five are not always included in surveys [ ] [ ] [ ] . two cross sectional pcr studies hint at lower susceptibility in children, since they found no sars-cov- pcr positive children under the age of [ , ] , but a pcr-based survey by the uk office for national statistics found no difference in the probability of infection between age classes [ ] . further, large-scale seroprevalence studies which fully sample all age groups will be necessary to fully resolve these questions. overall the balance of evidence cautiously suggests that children may have a lower inherent susceptibility. if it exists, such lower susceptibility could be physiological [ ] or could be due to cross reactive immune responses from other childhood infections, with cross-protection between other human coronaviruses and sars-cov- hinted at by recent studies [ , ] . there is little evidence from contact tracing and clinical investigations about the relative infectiousness of children. children hospitalised with covid- readily shed the virus above the likely transmission threshold [ ] [ ] [ ] , with detection of virus in nasopharyngeal (nasal) swabs, oropharyngeal (throat) swabs, sputum, or faeces [ , ] . however, in their review of contact tracing and population-screening studies, viner et al. [ ] found just one relevant study comparing infectiousness by age: zhu et al. [ ] , which shows that children make up a low proportion of index cases in households. as pointed out by viner et al., this particular result could be explained by children being less likely to get infected in the first place rather than children being less infectious once they have actually contracted the virus. there is also evidence suggesting that mild cases in adults could be less infectious than severe or critical cases [ ] , but it remains unknown whether this result extends to asymptomatic or mild cases in children. thus, children with severe symptoms are likely infectious, but it is harder to determine how transmissible the virus may be from children with few or no symptoms. we are aware of three reported studies of sars-cov- infection within the school environment. a retrospective serology study of individuals with links to a school-based outbreak in oise, france, showed that the infection spread readily within and outside the school to reach students, teachers, staff, and families [ ] . in contrast, an australian government study of cases in schools in western australia [ ] identified nine children and nine adults who tested positive for sars-cov- (located across different schools), but found only two secondary cases when testing a third of the close contacts of these cases ( samples). in ireland, six sars-cov- cases were identified who had attended or taught in schools. none of school related child contacts or school related adult contacts showed any symptoms, but asymptomatic cases could have been missed [ ] . the australian school cases were identified between th march and rd april, and the first irish school case was identified at the beginning of march. the first oise school cases, by contrast, were identified on the nd february . the greater awareness of covid- by march, during which the who declared covid- as a global pandemic, likely helped to control the australian and irish school-based outbreaks sooner than in oise. in the uk, at the time of writing (late may ), cases of covid- are declining and there is strong evidence to suggest that the effective reproduction number has dropped below across the country. a multi-phase relaxation plan for the country to emerge from lockdown began on th may, with a greater emphasis on returning to work if practical. as part of this staged process, primary schools in england are scheduled to partially reopen on st june; reception, year and year children are initially due to return to the classroom, with an emphasis on maintaining social distancing measures where possible. in this paper we investigate the epidemiological impacts of reopening schools in england, focusing on different combinations of year groups. we extend a previously developed dynamic transmission model for sars-cov- , which is fit (on a regional basis for the uk) to real-time data on confirmed cases requiring hospital care and mortality. we compare and contrast multiple possible strategies for reopening both primary and secondary schools, focusing upon determining the effect of given year groups returning to school upon future epidemic behaviour. by elucidating the risks associated with particular age groups returning to school, we seek to contribute to the evidence base on the likely role . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint of schools in the containment and control of this outbreak. transmission model in order to perform the analysis of school reopening, we extended a previously-developed determin- istic, age-structured compartmental sars-cov- transmission model [ ] . this model stratifies the population according to current disease status, following a susceptible-exposed-infectious-recovered (seir) paradigm (fig. ) . we assumed the latent period to be erlang distributed, modelled within the compartmental framework via division of the latent state into three stages. infectious cases were partitioned by presence of symptoms, meaning we tracked symptomatic and asymptomatic individuals separately. additional layers of complexity included differentiating by isolation and household status. we provide a listing of model parameters in table , with a description of the model equations given in supporting text s . we use the predicted number of symptomatic individuals to estimate the number of hospital admissions, icu admissions and deaths, by estimating the proportion of symp- tomatic individuals requiring hospitalisation, icu admission and the proportion that eventually die, and the distribution of times through each of these states. for hospital admissions and cases requiring treatment in icu, the proportions going through each state and the distribution of times taken were drawn from the covid- hospitalisation in england surveillance system (chess) data set that collects detailed data on patients infected with covid- [ ] . the risk of death was also captured with an age-dependent probability, while the distribution of delays between hospital admission and death was assumed to be age-independent, with both these two quantities determined from the public health england (phe) death records. with the inclusion of age-structure, transmission was governed through age-dependent mixing ma- trices, based on uk social mixing patterns [ , ] , and age-dependent susceptibility. to capture the effects of social distancing measures that were introduced in the uk to reduce transmission, we scaled down the mixing matrices associated with schools, work and other activities while increasing the within household transmission matrix (see supporting text s ). in a refinement to the base model, we imposed an amended age-stratification of the population. whilst in previous work the population was stratified into five year age brackets, for this study we separated those aged between and years old into single year cohorts, with the remainder of the population stratified into five year age brackets as before ( - yrs, - yrs and so on). the final age category corresponded to those aged years or above. modelling school reopening scenarios we used this model framework to evaluate eight strategies for reopening schools from st june. the eight school reopening options we considered assumed that, from the st june, the following school year groups would return to school: cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . we stratified the population into susceptible, exposed, detectable infectious, undetectable infectious, and removed states. solid lines correspond to disease state transitions, with dashed lines representing mapping from detectable cases to severe clinical cases that require hospital treatment, critical care (icu), or result in death. we separated those aged between and years old into single years, with the remainder of the population stratified into five year age brackets. see table for a listing of model parameters. note, we have not included quarantining or household status on this depiction of the system. for clarity, in all the strategies considered here we assumed that children of key workers continued to attend school at the currently observed level. we assessed the school reopening scenarios at a regional scale, modelling the population of england aggregated to seven regions (east of england, london, midlands, north east and yorkshire, north west england, south east england, south west england). this involved the use of region-specific posterior parameters obtained in our prior work, where we fit our base transmission model on a region- by-region basis, using a monte carlo markov chain (mcmc) fitting scheme, to four timeseries: (i) new hospitalisations; (ii) hospital bed occupancy; (iii) icu bed occupancy; (iv) daily deaths (using data on the recorded date of death, wherever possible) [ ] . the inference was performed from epidemiological data until th may . our assessment of school reopening strategies comprised three strands. firstly, we quantified how the process of opening schools and year groups affected contact patterns and anticipated secondary infections. secondly, we related the scale of school opening to the relative change in r, assuming the same transmission patterns in the rest of the population as during the strict lockdown phase. finally, we gauged the estimated change in clinical case and its sensitivity to changes in community transmission following the easing of lockdown measures on th may. we outline each item in further detail below. contacts and secondary infections any school reopening plan will inherently alter age-group contact patterns compared to contact struc- tures observed during the lockdown. we attempted to resolve how these alterations in social in- teractions propagated into the transmission dynamics by tracking secondary infections arising from . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint figure s ) h r household quarantine proportion − can be varied according to scenario n r a population size of a given age by region ons symptomatic index cases and infected index cases (either symptomatic or asymptomatic), respec- tively. specific to this aspect of the analysis we focused on a single region, namely the midlands and the posterior parameter set with the maximum likelihood. we first assess the contact structure and transmission under two distinct lockdown assumptions ('strict closure' and our default assumption of 'weaker closure'). the 'strict closure' scenario assumed that there was no additional mixing between school-age groups during the lockdown period. 'weaker closure' assumed there was more limited adherence, leading to higher mixing between school-age groups compared to the 'strict closure' setting. we also consider six of the eight reopening strategies (omitting those with half class sizes as these are bounded above by the full-class strategy). for each we show the age-mixing matrix between age-groups; . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . the transmission matrix from a symptomatic infectious individual; the transmission matrix from an average infectious individual (recognising the many will be asymptomatic or in household quarantine); and the expected number of secondary cases an average infectious individual of a particular age-group will generate. reproductive number analysis the reproductive ratio or number (r) has become a universally recognised quantity in the description of covid- dynamics; it is defined as the average number of secondary cases from an average index case -where the second average is important as it samples across all infectious states including asymptomatics and those currently under household isolation. to prevent the occurrence of a second phase of exponential growth in infection, it is crucial that relaxation of social distancing measures do not result in the value of r rising above . on these grounds, there is interest in predicting the magnitude of a rise in r that could result from the reopening of schools, and our confidence in this result. we considered all eight school reopening scenarios and examined the increase in r per region under each of the eight strategies. to compute r, we used the contact matrices associated with the given choice of school reopening and accounting for the regional population structure, whilst assuming the same level of mixing in the rest of the population as during the strict lockdown; therefore any changes in r are driven by changes in school-age mixing. we calculated means and intervals from simulation replicates with parameter sets sampled from the posterior parameter distributions. clinical case impact the prior methods focused on the reproductive ratio r, which is both an instantaneous measure (r can be calculated at any or every time point) and a long-term calculation (as it utilises an eigenvalue approach to generate the asymptotic r). calculation of quantities of public health interest requires the simulation of the full temporal dynamics from the start of the outbreak to the closing of schools for the summer holidays on nd july. in addition, we considered the sensitivity of reopening schools to other potential changes in population mixing patterns (and hence different values of r) driven by other changes to the lockdown since th may. these changes to population mixing were generated by reducing the adherence with lockdown measures, bringing the mixing matrices closer to the pre- pandemic norm. we performed these simulations, using the full dynamic model to generate estimates of the symp- tomatic cases, deaths and icu admissions between st june and nd july, for each of the eight school-opening strategies. we compared these measures, aggregated over this -day period, to a scenario where school closures remain in place beyond the st june. for each reopening strategy and each region, we performed a total of replicates. in each replicate we sampled parameter values randomly from all posterior parameter distributions, with the exception of the adherence level. the potential reduction in adherence values, from th may, inevitably gen- erates different r values at the point of school reopening (measured by the observed growth rate of the outbreak in the model simulation). as a consequence, for comparative purposes we segregated the estimated increases in epidemiological quantities (comparing different school opening strategies for fixed underlying parameters) into three categories according to the r value before school reopening: below . , between . and , or between and . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint choice of reopening strategy influences contact structure and secondary infection risk we first investigated the impact of alternate strategies for reopening schools upon contact patterns be- tween individuals and the effect of this upon transmission of sars-cov- and occurrences of covid- infection. our results for the midlands, and the posterior parameter set that maximises the likelihood, are summarised in fig. . for all scenarios investigated we observe several common trends. contact is most common between individuals of the same, or similar ages (fig. , first row) . there was also greater contact between children and adults between the ages of and , reflecting interactions between children and their parents, as well as between elderly people. this increased likelihood of contact within and between those age groups is reflected in the risk of secondary infections occurring (fig. , second and third rows) . the second row accounts for age-dependent susceptiblity, and shows the expected number of secondary infections in each age (y-axis) from a symptomatic index case of a particular age (x-axis). the third row incorporates the likely state of an index infection (symptomatic, asymptomatic or in household quarantine -as predicted by the underlying odes) thereby reducing the potential transmission from particular age-groups (supporting figure s ). if schools remain closed, with a high level of adherence to the lockdown within this younger age-group (fig. , first column) we observe that contact between children, and therefore the risk of secondary infection occurring, is extremely low. should adherence to lockdown be weaker (fig. , second column) , we observe a higher rate of mixing between children and a slight increase in risk of secondary infections occurring. for both of these scenarios the average number of secondary infections per index infection is below for all age groups and the value of r remains significantly below . we now investigate the impact of various strategies for school reopenings. we first investigate the scenario of reception, year and year children returning to school -the policy that is scheduled to be implemented on st june in england (fig. , third column) . in this scenario, we observe a slight increase in contacts compared to the "weaker closure" scenario, with increased transmission between individuals in these age groups. however, crucially, even within these age groups, the total number of secondary infections per index case remains below one (third column, final row, red bars) and the overall reproduction number value of r was only observed to have slightly increased from the scenarios in which schools remain closed. a slight increase in mixing, and hence r, was again observed when all primary schools are opened (fig. , fourth column) , but we predict that r remains below . to conclude this segment of the analysis, we investigated the impact of school reopening strategies that involved some, or all, secondary school children returning to the classroom. if children from key years of and return to school (in addition to some or all primary school children), a significant increase in mixing was observed within those age groups; the number of secondary infections as a result of index infections in secondary schools was predicted to be above one (fig. , fifth and sixth columns). however, this expected number of cases is distributed across multiple age-groups thereby dissipating the worst effects. in general, we found secondary schools to represent a higher risk of increased transmission potential than primary schools. this could lead to higher values of r when all secondary schools are opened; but for all scenarios investigated, even the scenario in which all schools are opened, we found strong support for r remaining below in the midlands (fig. , final column) assuming that all other transmission patterns remain unchanged. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint effect of school reopening on reproductive number next, we sought to estimate changes in r that may result from school reopenings alone -assuming the transmission patterns in the rest of the population are maintained at strict lockdown phase levels. in contrast to the the first part of the analysis, which focused on a single set of parameters and a single region (fig. ) , here we explore the full parameter uncertainty and compare different parts of the country. for all school opening scenarios, within the seven regions of england, we observe an increase in r compared to what we predict for keeping schools closed until the end of this academic year (fig. and supporting figure s ). this is to be expected, given the increase in contact between children that such reopening scenarios would allow. however, the magnitude of increase is predicted to be relatively low, depending on the age-groups that return to school. in general, the more year groups allowed to return to school at one time, the greater the effect on r, with the return of secondary school children having the greatest impact. the impact of allowing multiple year groups to return to school can still be small: opening a fraction of the age-cohorts in each school generally leads to a moderate (less than . ) increase in r, especially if children can be taught in smaller class sizes which is assumed to lead to a proportionate reduction in within school transmission. there is however considerable variation between the regions and here we focus on four exemplars. for london and north east england & yorkshire, the increase in r is considerably less than that for east of england and the midlands across all reopening scenarios. for the former, even allowing all age groups to return to school (while maintaining tight control in other age-groups) is highly unlikely to increase r above , with both means and % prediction intervals falling well below this threshold (figs. (a) and (b) , the % prediction intervals, as the name suggests, contain % of all predicted values across the entire posterior distribution of parameters). this low r value is especially true for london, which saw the most abrupt rise and subsequent decline in cases. however, this is not the case for the east of england (fig. (c) ) and the midlands (fig. (d) ). in these regions, allowing schools to fully reopen could increase r above , with such an occurrence lying within the % prediction intervals. we attribute these regional differences to both heterogeneity in the observed rate of epidemic decline and the differential proportion of school age school in each region; the midlands has the highest proportion of older teenagers in the country. quantifying clinical case impact stemming from the re-opening of schools our final piece of analysis examined the extent to which each of the eight school reopening strategies may contribute to clinical case outcomes, using the full dynamic model. we also considered the sensitivity of reopening schools to other potential changes in population mixing patterns (and hence different values of r) driven by other changes to the lockdown since th may. in each scenario, reopening schools increased the absolute number of cases, icu admissions and deaths as a result of increased transmission (fig. ) . note that these increases will not be restricted to the children that return to school, since the greater transmission will lead to increased cases in other age groups. echoing our earlier findings, strategies in which a larger number of children return to school generally resulted in larger increases. in addition, older children had a greater effect, so that reopening secondary schools results in larger increases than only reopening primary schools. the opening of schools on st june, is just one of a number of changes that began on th may. in the previous sections we focused on school reopening, assuming that mixing (and hence transmission) within the wider population has remained unchanged. here we allow the relaxation of lockdown measures to precipitate an increase of r within the community and calculate the additional change . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . from the opening of schools. we consistently find that school reopening has a larger impact when r in the community is high, leading to a greater increase in cases, icu admissions and deaths. however, by far the largest increase in any of these key quantities is driven by the underlying change in r due to relaxations other than the reopening of schools (figs. (b) , (d) and (f)). we also give the % prediction intervals. solid red lines identify the absolute increase required to raise r above , within each region, alongside % and % intervals (shaded red areas). means and intervals are calculated from replicates sampled from the posterior parameter distributions. all scenarios are implemented on st june and continued until nd july. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint in this paper, we have used a mathematical model to consider the implications of various potential strategies for reopening schools in england. we have compared the different strategies by presenting mixing matrices and discussing their implication for onward transmission, and by analysing the increase in the reproduction number and absolute number of cases, icu admissions and deaths compared to those predicted if schools remain closed. in the absence of other changes, the complete opening of all schools is not predicted to raise the reproductive ratio above one. it must be noted that even in the case where r remains below one, the increased transmission resulting from school reopening will likely lead to a small increase in the absolute number of cases, icu admissions and deaths. reopening schools, in any form, is going to result in more mixing between children, an increase in r and thus, equivalently, more transmission of the disease. however, we can constrain and potentially minimise the extent of this increase by selecting a subset of year groups to return to school. in doing so, we restrict the increase in r to very low levels and, crucially, avoid the possibility of increasing r above . these findings are in agreement with studies applied to other nations showing an apparent non-primary role of school settings as a driver of sars-cov- transmission. a statistical study in us counties looking at the relationship between the reduction in growth rate and the timing of different state and local government social distancing interventions found school closures to not be statistically significant [ ] . further, in terms of suppressing spread of sars-cov- , a mechanistic transmission model judging the impact of non-pharmaceutical interventions in switzerland, by their potential to reduce r below at a national level, predicted school closures alone would typically be insufficient [ ] . in choosing a specific reopening policy, one must weigh up the benefits to both children and parents that are gained from allowing more year groups to return to school, with the risks associated with increased transmission. in light of the variation in effects on r between regions, reopening policies may benefit from heterogeneity across the country, in order to allow the most children possible to return to school without threatening a resurgence of disease prevalence. our results also highlight the benefit to be gained from small class sizes and hence maintaining such measures of social distancing. we also discerned a higher risk of increased transmission potential for reopening secondary schools versus the reopening of primary schools. such a relationship may be partly attributed to the observed larger number of contacts of secondary school children compared with primary school children [ ] . on the other hand, other contributory factors include differences between age classes in susceptibility and, if infected, displaying symptoms [ , ] . these may consequently lead to secondary school children having a larger contribution to overall transmission throughout the population. increasing levels of contacts between school children also inevitably leads to greater absolute numbers of infections, detected cases, icu admissions and, regrettably, deaths, even if the reproductive ratio is not raised above one. for this reason, we also estimate the increase in the numbers of these outcomes as a result of reopening schools using the different strategies. the ranking of the different strategies for these outcomes mirrors the ranking in terms of increases in r. the impact of reopening schools also depends on the behaviour of the wider population. if there is more mixing within the adult (and elderly) population, the effect of reopening schools will be exacerbated by the generally higher infection levels and contacts in the community. reopening schools will then lead to greater increases in case numbers over and above the increases due to greater mixing. in general we find that even small changes in r due to the behaviour of the general population swamp the impacts of reopening schools. we would stress that, such increases must be viewed in the context of the restrictions currently placed on pupils and parents. ultimately, it is a societal decision to balance the benefits to pupils' welfare and education against the epidemiological consequences. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . to consider the effects of specific school years returning, this work made some simplifying assump- tions, and our results therefore have limitations. in particular, in this paper we consider only an england-specific context. the devolved administrations employ a different school system from eng- land, including different school term dates, which may affect the outcome of reopening schools on specific dates. future work could incorporate such differences, some of the epidemic variability be- tween nations will be captured by the model parameter fits that are already performed for all the devolved nations. as we have shown, the context in which school reopening happens will also have an impact on its effect. while we consider different population level mixing patterns, this exploration is necessarily constrained; for example it may also be the case that the opening of schools allows more parents to return to work, increasing their risk of infection. indeed, a recent surge in cases in seoul, south korea linked to a distribution centre, has identified at least one sars-cov- positive high-school student, whose family member worked at the centre. this was followed by the re-implementation of localised lockdown and social distancing measures, including the closure of schools, days after their phased reopening [ ] . it will also be important to consider the impact of school re-openings in combination with other concurrent measures, such as the recently rolled out nhs test and trace system in england (beginning on th may) [ ] , which aims to trace close recent contacts of anyone who tests positive for sars-cov- and, if necessary, notify them to self-isolate at home to prevent onward transmission. effective contact tracing breaks transmission chains, but may also subject school classes to tracing and isolation. even without national-scale relaxation in the lockdown measures, the behaviour of the general population is likely to change over time, in ways that are difficult to predict. beyond these considerations, we have also neglected the many possible side effects of reopening schools, such as parents interacting at the school gates, teachers' exposure while travelling to school (or in the staff room), or the effects of school reopening on children mixing outside of school. the decision to gradually reopen schools in england, starting on st june with reception, year and year , has caused significant debate amongst scientists, policy makers, teachers and parents regarding the risks associated with such a change in policy. our work provides evidence suggesting that the reopening of primary schools will result in only a small change to the value of r, indicating that the risk associated with this policy change is low. as more children return to school, we would expect the value of r to rise further, although our forecasts indicate that school reopening alone is unlikely to push r above . however, given the number of other changes that have occurred over the past month and are scheduled for the coming months, there is the potential for a gradual increase in r; it is therefore vital that all epidemiological indicators are monitored closely during this relaxation phase and measures reinstated should there be a significant rise in cases in the future. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . the data were supplied from the chess database after anonymisation under strict data protection protocols agreed between the university of warwick and public health england. the ethics of the use of these data for these purposes was agreed by public health england with the government's spi-m(o) / sage committees. data availability data on cases were obtained from the covid- hospitalisation in england surveillance system (chess) data set that collects detailed data on patients infected with covid- . data on covid- deaths were obtained from public health england. these data contain confidential information, with public data deposition non-permissible for socioeconomic reasons. the chess data resides with the national health service (www.nhs.gov.uk) whilst the death data are available from public health england (www.phe.gov.uk). . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint supporting text s description of the complete system of model equations. details on the mechanisms underpinning social distancing measures within the model framework supporting figure s posterior distributions of key model parameters from fitting to date until st june. the left-hand graphs show how the probability of symptoms (d a ) and susceptibility (σ a ) varies with age; given the low value of alpha most of the age-dependence is in the displaying of symptoms. the right-hand graph shows the relative adherence with lockdown measures in each region; high values correspond to a dramatic reduction in the mixing matrix, while an adherence of zero returns the matrix to pre-lockdown levels. this figure supplements the information in table . bars show the % credible intervals from the posterior distribution. distribution of household symptomatic, asymptomatic and isolated cases in each age group on st june. used in conjunction with fig. . bottom segments (blue shading) represent symptomatic infection. middle segments (orange shading) represent asymptomatic infection. top segments (yellow shading) represent those in isolation. filled dots specify the fraction of the population within that age bracket. increase in reproductive number, r, under eight school reopening scenarios for three regions in england. estimates are depicted for the following three regions: (a) north west, (b) south east, (c) east of england. for each scenario, bars represent the mean absolute increase in r, compared to what we would observe if schools remained closed. we also give the % prediction intervals. solid red lines identify the absolute increase required to raise r above , within each region, alongside % credible intervals (dashed red lines). means and intervals are calculated from replicates sampled from the posterior parameter distributions. all scenarios are implemented on st june and continued until nd july. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint covid- patients' clinical characteristics, discharge rate, and fatality rate of meta-analysis systematic review of covid- in children shows milder cases and a better prognosis than adults covid- infection in children: estimating pediatric morbidity and mortality. medrxiv preprint pages covid- total announced deaths epidemiology of covid- among children in china evolving epidemiology and impact of nonpharmaceutical interventions on the outbreak of coronavirus disease susceptibility to and transmission of covid- amongst children and adolescents compared with adults: a systematic review and meta-analysis school closure and management practices during coronavirus outbreaks including covid- : a rapid systematic review changes in contact patterns shape the dynamics of the covid- outbreak in china modes of contact and risk of transmission in covid- among close contacts epidemiology and transmission of covid- in cases and of their close contacts in shenzhen, china: a retrospective cohort study infection fatality rate of sars-cov- infection in a german community with a super-spreading event repeated seroprevalence of anti-sars-cov- igg antibodies in a population-based sample from seroprevalence of covid- virus infection in guilan province suppression of covid- outbreak in the municipality of early spread of sars-cov- in the icelandic population nasal gene expression of angiotensin-converting enzyme in children and adults pre-existing and de novo humoral immunity to sars-cov- in humans journal pre-proof targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals virological assessment of hospitalized patients with covid- an analysis of sars-cov- viral load by patient age a critical assessment of some recent work on characteristics of pediatric sars-cov- infection and potential evidence for persistent fecal viral shedding prolonged viral shedding in feces of pediatric patients with coronavirus disease a novel coronavirus from patients with pneumonia in china cluster of covid- in northern france: a retrospective closed cohort study covid- in schools -the experience of nsw no evidence of secondary transmission of covid- from children attending school in ireland predictions of covid- dynamics in the uk: short-term forecasting and analysis of potential exit strategies covid- hospitalisation in england surveillance system (chess) -daily reporting social contacts and mixing patterns relevant to the spread of infectious diseases projecting social contact matrices in countries using contact surveys and demographic data strong social distancing measures in the united states reduced the covid- growth rate. health aff assessing the impact of nonpharmaceutical interventions on sars-cov- transmission in switzerland the korea times. calls grow to delay school opening as virus fears persist department of health and social care all authors declare that they have no competing interests. key: cord- -jisp l authors: meyers, keith; thomasson, melissa a. title: can pandemics affect educational attainment? evidence from the polio epidemic of date: - - journal: cliometrica (berl) doi: . /s - - - sha: doc_id: cord_uid: jisp l we leverage the largest polio outbreak in us history, the polio epidemic, to study how epidemic-related school interruptions affect educational attainment. using polio morbidity as a proxy for epidemic exposure, we find that children aged and under, and school-aged children of legal working age with greater exposure to the epidemic experienced reduced educational attainment compared to their slightly older peers. these reductions in observed educational attainment persist even after accounting for the influenza epidemic of . the global covid- pandemic in early led to severe disruptions in nearly all aspects of life. stay-at-home orders closed schools and non-essential businesses, generating widespread unemployment and large declines in economic activity that cannot yet be fully assessed. the social and economic costs of this crisis have been widely felt. a united nations report suggests that global gdp could decline by % (united nations ), while the managing director of the international monetary fund stated that she anticipates that the pandemic will generate "...the worst economic fallout since the great depression," with global gdp contracting by % (gopinath ) . according to unesco, over . billion students, almost % of all learners, have had their schooling disrupted by this crisis. at least countries have closed schools in response to the covid- threat, which may interrupt human capital accumulation in children and contributes to the social costs of the pandemic (unesco ). while most people alive today have never witnessed this scale of the pandemic, research on other epidemics may provide insights into the possible magnitude of the disruption. in this paper, we examine the polio epidemic to measure how it impacted the educational attainment of children during the outbreak. the epidemic is unique in that it was the first and largest major polio epidemic in the usa and was one of the first major public health crises many nascent public health departments faced. the epidemic proved to be the first of many outbreaks that would continue until the advent of the salk vaccine in , although trevelyan et al. ( ) suggest that the epidemic was more intense than subsequent outbreaks. while smaller in magnitude than the more famous - spanish flu, the polio epidemic started in new york city and new jersey, but then spread across the usa, resulting in mass quarantines and prolonged school closures at the start of the - academic school year. the polio epidemic may have affected educational attainment in two ways. first, infected children may have missed school during the period of their illness and recovery. second, even children who did not have symptoms missed school during periods of closure or because their parents refused to send them to school. thus, we leverage geographic variation in the severity of the epidemic, as well as the fact that polio differentially affected children of different ages, to measure how the disease affected the educational attainment reported by these children as adults in the census. children living in areas with high polio morbidity were more likely to be infected and more likely to endure school closures than children in less-affected areas. children under were more susceptible to polio, yet children aged and older may have been more likely to quit school during a prolonged closure and join the labor force. during the ebola outbreak, five million children in africa faced school closures (sifferlin ) . schools in sierra leone were closed for nine months, and schools in guinea and liberia were closed for six months (sifferlin ; paye-layleh ) . in nigeria, schools that were supposed to open in august remained closed until october (bbc news ). studies examining the impact of the sars outbreak on the chinese economy show that it resulted in temporary negative shocks to tourism, retail sales and personal consumption, as well as a . % reduction in gdp (siu and wong ; hanna and huang ) . more recent studies of the impact of ebola on the countries of guinea, liberia and sierra leone suggest that "aversion behavior"-the actions taken by people to avoid illness, and the actions taken by investors as they anticipate these behaviors-led to a loss of $ . billion in , or about % of their combined gdp (thomas et al. ) . prior to , most of the us population had limited experience with the poliovirus. while minor and relatively isolated outbreaks occurred previously in the usa, the outbreak affected more than three times the number of people relative to previous years. in , . cases of polio were reported per , people, compared to the - rate of . per , population (paul ; nathanson and kew ) . the outbreak had greater rates of morbidity than later epidemics, and there is evidence that the epidemic may also have been under-reported (nathanson and kew ). our results show that children born in states with more reported polio cases had lower educational attainment compared to slightly older birth cohorts who would have already completed schooling before the - school year and that the decline in educational attainment varied depending on their age during the outbreak. we find that a one-standard-deviation increase in polio intensity reduced years of completed schooling by . years for children between the ages of and in . for children who were and under during the epidemic (and who were most susceptible to polio), a one-standard-deviation increase led to a . year reduction in years of completed education. these results are robust to controlling for - influenza intensity. polio is caused by the poliovirus, an enterovirus transmitted fecal-orally. evidence on egyptian stele (depicting an adult with a withered leg and crutch) dating from - bc suggests that polio has existed for thousands of years (nathanson and kew ) . although the existence of polio dates to antiquity, it was not until the late th century that epidemics began occurring in the usa and europe. the most plausible explanation for this pattern is that before the late th and early twentieth centuries, most people were probably exposed to polio during infancy when infants had circulating maternal antibodies that made the infection much less severe. over the next years, improved public sanitation delayed exposure to the virus, thus increasing both the age of primary infection and the severity of the illness, since maternal antibodies were no longer circulating. as a result, epidemics that became increasingly severe were reported each summer and fall, and the average age of persons infected increased (nathanson and kew ; centers for disease control and prevention ) . in , there were over deaths and , more documented infections, numbers that eclipsed those of previous (and subsequent) epidemics (lavinder et al. ; trevelyan et al. ) . table details the number of reported polio cases and deaths by state during the epidemic. nearly every state reported polio cases, with massachusetts, new york, new jersey, and connecticut reporting the highest numbers of reported polio infections. while much of the epidemic was in the northeastern usa, there were also sizable outbreaks in minnesota, montana and mississippi. it is important to note that while cities may be more heavily impacted by modern epidemics, polio was unusual in that it was precisely better hygiene that predisposed victims to a later, and more severe, onset. in places with poor sanitation, children would have been exposed at much younger ages (often infancy, without obvious symptoms) and be less likely to suffer a severe case. while many cities had improved sewer and sanitation (thus leading to the large flare of the epidemic in ), rural areas had traditionally been "healthier" places to live from a sanitation standpoint. lavinder et al. ( ) note that the epidemic was more prevalent and intense in small towns and the country than in large cities, and "...not due to mere chance but to some fundamental law" (p. ). the outbreak generated substantial fear. commonly known as "infantile paralysis," polio often caused mild (or no) symptoms but could attack the nervous system and cause permanent paralysis. little was known about how the disease was spread, and no vaccine for polio would be available for another forty years. the disease transcended class lines and affected the poor and the rich alike. in new york city, for example, a large share of cases occurred among native-born children living in single-family dwellings with clean surroundings and indoor toilets (lavinder et al. ). actions taken to curb the spread of polio public health officials struggled to contain the outbreak, which began in june and lasted until november. as the disease spread, officials became increasingly desperate. to prevent the spread of the epidemic and allay public fear, officials implemented various non-pharmaceutical interventions, such as strict quarantines. placards were also placed in front of homes of infected individuals to notify the community of the public health risk. in at least one case, a child suffering from polio was forcibly removed from his home to a hospital, against the wishes of his parents (emerson ) . in new york city, children ages and under were prohibited from leaving new york city for travel unless they produced "... a certificate that the premises occupied by them were free from poliomyelitis, and had been free from this disease since january , ." this was supplemented by a medical examination of such travelers at the point of departure (emerson ) . in new york city and philadelphia, health officials ordered the city streets to be washed with millions of gallons of water each day (offit ; rogers ) . government policy also called for a ban on public gatherings in areas where numerous cases were reported (emerson ) . in the summer of , new york public health officials barred children from movie theaters and libraries, closed sunday schools and banned picnics (the new york times c, e; rogers ) . fears that domesticated animals could spread the disease led to the extermination of , cats and dogs (the new york times b). even though drastic measures were undertaken to control the spread of the disease, hundreds of new infections were reported every week. as autumn approached, fears that afflicted youths would infect their peers and a belief that quarantines could limit the virus' spread led many school districts to delay the start of the school year until the epidemic waned. as the new york department of health noted, "the unprecedented virulence and extent of the existing epidemic, and unfamiliarity with the disease, has engendered in the public such a state of mind that concession to public alarm seemed advisable" (emerson ) . the extent and nature of these closures varied across political and geographic divisions of the usa due to the decentralized structure of the nation's public health (the new york times d) . newspaper accounts show numerous other cities nationwide joined new york in postponing the start of school. philadelphia delayed opening schools until september th (and then to october nd), and both washington, d.c., and fort wayne schools were not reopened until october nd (the washington times company ; evening public ledger a, b) . schools closed across the nation even in states with small numbers of cases. for example, some schools closed in seattle, with only five cases reported (the tacoma times ) . but, even when schools may not have been officially closed, there is evidence that parents worried about sending their children to school. for example, even after the delayed reopening in new york city, worried parents withheld their children from school; up to , students were absent the first few weeks after schools opened, and the district announced "leniency" for parents who failed to send their children to school (the new york times a). to gain insight into the extent of school closures nationally and when they occurred, we searched newspaper records contained in the chronicling america newspaper archive (library of congress ). cities in the most heavily affected areas are most frequently mentioned, but newspaper accounts demonstrate that school closures tended to occur wherever an area experienced a breakout of polio. the epidemic started in june, accelerated in july and persisted through the start of the academic school year. these data most likely represent a subset of school closures, since not all newspapers are covered in the library of congress archive. moreover, our search procedure may have failed to reveal all closures. many newspapers referred to school closures in different cities, or even in major cities in other states. although we found some articles in which a city expressly stated schools would remain open (including chicago, detroit and milwaukee), for most cities we could not ascertain with certainty that they did not close. in fig. , we plot the frequency of school delay announcements in the chronicling america newspaper archive for all newspapers from july , , to november , the search used the entire chronicling america newspaper archive for the usa during the active epidemic. the search required either the word(s) "polio" or "poliomyelitis" or "infantile" or "paralysis" to be referenced on the same page as the words of "school" and "postpone". we allowed the words "school" and "postpone" to be within five words of each other. many articles even referred to the disease as the "baby plague" or "plague". searches for "school closures" also yielded similar results, but with a flatter distribution and wider tails because they often referred to sunday school closures. . school postponement notifications in us newspapers began to increase in the weeks preceding the start of the academic school year (normally around september or september ), peaked during the two first weeks of september and gradually decreased until the first week of october. the latest public school start date we observe in the chronicling america newspaper archive (library of congress ) was october , , although some new england preparatory schools advertised that they postponed their start dates into the middle of october. the newspaper articles also reveal that the persistence of the epidemic caused public health officials and school boards to repeatedly push back school start dates. washington, dc initially planned to start on time, then pushed back the start two weeks to september , and then finally postponed until october . boston, ma did the same. the entire state of pennsylvania also postponed school starts multiple times until the first week of october. to dig deeper into the relationship between city-level school closures and polio outbreaks, we further searched for announcements of school closures in cities for which we have polio morbidity data digitized by van panhuis et al. ( ) . these were cities that voluntarily participated in the disease reporting system conducted by the us public health service, which published this information in its weekly bulletins. we were able to find newspaper accounts of school postponement for of these cities. table in the appendix provides more detailed information on the city names, the dates of postponement, and the name of the newspaper in which we found the information. of these cities, % opened over weeks late, with . % opening on october , . only three (chicago, detroit, and milwaukee) opened on time. figure presents the relationship between weekly polio morbidity in these cities and the timing of school postponements using data from project tycho. we highlight the relationship between the epidemic timing and school postponement by separating this information by the timing of the school starts: schools that started on time, schools that delayed their start dates to a later date in september, and schools that delayed school starts into october. a mass of polio outbreaks in august and september coincided with delayed school starts in many affected cities. polio outbreaks continued in these cities throughout october, and the epidemic ended in november. in fig. , we focus in on a subset of the reported data for cases under per week. for many cities with schools that opened in october there large numbers of reported weekly polio cases throughout september and well into october. schools that postponed only a few weeks into september generally had fewer reported cases of acute polio than either the schools that started in october. in the few schools that we confirmed opened on time, most city week observations report low numbers of polio infections. the exception to this pattern is the city of chicago, which opened schools on time even while polio still spread throughout the community. there is a broad and well-documented literature in economics and in economic history about the impact of early childhood health and economic shocks on later life outcomes (almond et al. ) . within this literature, there are a number of papers that demonstrate the long-run consequences of infectious disease shocks in childhood. beach et al. ( ) show that reduced early-life exposure to typhoid improved later life educational attainment, perhaps by improving cognitive functioning. bhalotra and venkataramani ( ) also find that reduced infant exposure to waterborne disease is associated with improved test scores for girls and improved heights for boys. there is also evidence that individuals who do not contract infectious diseases during epidemics can also be affected. for example, parman ( ) examines the impact of the influenza pandemic on the siblings of those children born during the outbreak and finds that older siblings received an additional three months of education, while younger siblings received slightly less education relative to children who did not have a sibling born during the pandemic. to our knowledge, no one has examined the case of polio in the usa, which is unique in that educational attainment may have been affected even despite the fact that relatively small numbers of people contracted polio. the polio epidemic may have reduced educational attainment through two channels. children who contracted the disease may have missed school while ill, thus reducing educational attainment, although this might depend on whether they contracted a mild form of the disease or paralytic polio. gensowski et al. ( ) find that children who contracted paralytic polio in denmark in were more likely to earn a university degree than children who did not, suggesting that these children shifted investment into education as their comparative advantage moved from jobs requiring certain physical abilities to those requiring more cognitive skills. other children may have experienced reduced educational attainment if they missed school for short periods of time due to school closures or avoidance, or if they delayed entry into formal schooling (for example if the parents of a wouldbe kindergartner decided to wait until the fall of to send the child to school). younger children who have their schooling disrupted even for short periods of time may suffer negative effects. for example, marcotte and hemelt ( ) find that unscheduled school closings due to weather negatively affect student performance on third-grade state assessment examinations. middle-grade students may have lower educational attainment if their learning is disrupted during key periods of development (lloyd ; hernandez ) . whether these effects persist in the long run is less clear. card and krueger ( ) find that men educated in states with higher-quality schools (measured by term length, student-teacher ratio and teacher wage) have higher returns from their schooling. pischke ( ) uses data from a german school district that had a one-time shortening of the school year and finds that the short school year had no negative effect on earnings and employment later in life, although it did lead to more students repeating a grade and fewer students entering higher secondary school tracks. other research does suggest that interruptions in schooling at crucial points of development may have long-run impacts. hernandez ( ) suggests that children in third-grade experience a pedagogical shift from "learning to read" toward "reading to learn." a failure to develop this proficiency in reading on time appears to inhibit human capital accumulation of students and limits the potential to keep pace with their peers. lloyd ( ) suggests that students who perform at a lower level than their peers may be more likely to drop out of school, with third-grade academic performance accurately predicting whether an individual would drop out of school nearly % of the time. in addition to the effect on younger children of missing school, prolonged school closures may have caused older children of legal working age to exit school and enter the labor force. a sizable literature on the effects of compulsory schooling laws suggests that educational attainment is affected by increases in the legal age of school exit, increases in the age at which children can legally work and decreases in the maximum age of school entrance. in , states had legislated as the minimum age for employment in manufacturing (moehling ) . around . % of males and . % of females between the ages of and were active participants of the workforce in , suggesting that many school-age individuals in would have possessed workforce alternatives to education (carter and sutch ) . together, this evidence implies that a notably large portion of youth ages and older had viable and legal employment alternatives available to them in . moreover, if worried parents delayed their youngest children's entry into school for a year, research by deming and dynarski ( ) suggests that these "redshirted" children may have less educational attainment than their non-redshirted peers since they reached a legal working age having acquired fewer years of formal schooling. using the polio epidemic as a natural experiment, this paper examines the effect of epidemics on the educational attainment of children. our primary identification strategy relies on interacting different age cohorts with polio morbidity rates in an individual's birth state. data on polio morbidity at the state level come from lavinder et al. ( ) , who provide comprehensive information pertaining to statelevel polio morbidity covering most of the continental usa. we use the data from lavinder et al because the us public health reports do not include areas with only a few cases (us surgeon general and us public health service ). in all specifications, we interact data on polio morbidity with dummy variables for an individual's age in , since children of different ages may have been differently affected by polio. given that nearly % of children infected by polio before were under the age of , they may have been more likely to experience the effects of polio directly than children of older ages (nathanson and kew ) . on the other hand, children who were and over may have been more likely to leave school for the workforce than children of younger ages. a priori, we expect children under and children ages or older to have their educational attainment impacted at a greater rate than those between the ages of and . we use polio morbidity as our primary data for two reasons. first, complete data on school closures are not available, and state-level data on attendance is not reported for the - school year. second, polio morbidity data enable us to capture both the direct and indirect impacts of polio on children. polio may have directly affected school attendance for the children who contracted the disease. since do we not have information on whether specific individuals contracted polio, we instead rely on the polio case (notification) rate in an individual's birth state. in addition, the epidemic may have indirectly affected children who missed school either because schools closed, or because fearful parents kept them at home. schools in areas with higher rates of polio were more likely to close, and parents were more reluctant to send their children to school even when schools were open. relying on school closure data may fail to capture some of these "avoidance" behaviors. for example, data on average daily attendance for five boroughs in new york city prior to , information on enrollment, attendance and length of the school year was reported in the annual report of the us commissioner of education. however, in , the office switched to reporting biennially, and the information was not reported in / (u.s. bureau of education ). to try to disentangle the school closure effect from other effects, we did run regressions using a sample of of the cities we found specific closure dates for in our search of newspapers that also were big enough to be listed as a place of residence in the census. while statistically significant in one specification, results (available upon request) show the same general pattern we describe below. (available only between and ) indicate that average daily school attendance in new york city fell in the - school year, as shown in fig. . we measure polio epidemic exposure by reported infections at the statelevel so that our main identifying assumption is that people who were school age in would be exposed in their state of birth. this assumption would be violated if children and adolescents migrated between states prior to the epidemic. while we can only speculate as to the number of children who moved across states during childhood, we do know that in , % of people were residing in the same state in which they were born. further, migration would bias the treatment effect toward zero if migration prior to the epidemic was not systematically correlated with polio morbidity. using data from the us census, we find that the difference between state of birth and state of residence in is less of a concern and that assigning polio virulence to state of birth is a reasonable proxy for exposure to the epidemic. in fig. , we present the share of persons who were not observed in their state of birth in the decennial census. on average, % of people ages to in in states that reported polio still resided in their state of birth. to test whether the epidemic influenced the educational attainment of exposed cohorts, we match a sample of white males born between and with the polio morbidity rate in their state of birth, and the years of education they report having in the us census (ruggles et al. ). we restrict the sample to males since they entered the workforce as permanent participants. we also exclude blacks, since margo ( ) finds that the highest grade of school reported in the census does not accurately represent years of schooling received for african americans. table reports summary statistics for the primary sample. equation ( ) represents the reduced form empirical regression used to study the effects of the epidemic on educational attainment: s,a denotes years of completed education for individual i, born in state b, residing in state s in , and born in birth year cohort a. to compare the relationship between polio morbidity in and educational attainment, we compare the educational attainment of persons born between and (who were between the ages of and at the time of the epidemic) to those who were born between and (who were between the ages of and ). this allows us to compare the educational attainment of persons who plausibly could have been in school with those who would have already completed school. in order to test our hypothesis that children of different ages may have been deferentially impacted by the polio epidemic based on their labor market alternatives, we bin them into four age groups: ages - (born - ), ages - (born - ) , and children under age (born - ) . we interact polio morbidity per population ( polio b ) in an individual's birth state with these age-specific cohort groups ( agebin a,j ). the omitted cohort is individuals aged to (born - ). the variables b , s , and a denote state of birth, state of residence in , and age cohort fixed effects. the identification of polio morbidity's effect on educational attainment comes from comparing different age cohorts from the same birth state while controlling for current state of residence, and national shocks common across birth cohorts. state of birth fixed effects control for factors common across persons born in the same state, and state of residence fixed effects control for factors that are shared among persons residing in the same enumeration state. common shocks shared across birth year cohorts, such as wwi, are controlled for using birth year fixed effects. in the full specification, we also include state-level economic and demographic controls for by state of birth, and control for schooling laws that applied to each birth year cohort from each state. these controls are denoted by x b and include doctors per capita in , education expenditures per capita in , the natural log of manufacturing wages per earner in , and the natural log of population in . interacting these controls with age cohort fixed effects allows the effect of these state-level characteristics on educational attainment to vary across different age cohorts. these interactions allow the state-level treatment effect of the epidemic to vary across birth year cohorts. the schooling laws, constructed by lleras-muney ( ), denote the ages of mandatory school entry, age of school exit and age at which children could obtain work permits for each birth year cohort from each state of birth. these laws proxy for idiosyncratic changes in schooling regulations for each state of birth by birth year cohort. finally, i,b,s,a denotes a heteroskedastic error term clustered by state of birth. in our analysis, we run three different regressions, reported in table . columns ( )-( ) report results from estimating equation ( ) with the years of schooling as the dependent variable. column ( ) includes fixed effects for state of residence in , state of birth, and birth year. column ( ) also includes the full set of fixed effects and birth state by age-cohort trends to control for potential underlying geographic trends common across cohorts born in different areas of the country, and column ( ) we thank adriana lleras-muney for providing these data. the mandatory schooling law data start in and continue until . we assign the cohort-specific law by subtracting the year the law was enacted from the age students can leave school. this identifies the birth year cohort to which the law plausibly applied. the laws are assigned for each year of birth by state of birth. in cases where there might be more than one applicable law, we assign the minimum age implied by the laws. in the case of missing values, we linearly interpolate if the missing value is between two birth years. in cases where we cannot linearly interpolate, we assign the schooling law from the closest birth year without a missing value in the state of birth, e.g., if values for birth years and are missing and we have values for , the values for are assigned to the earlier cohorts. this process works for all states except for wyoming where we cannot assign variable values for individuals. inclusion or exclusion of the schooling laws has little effect on estimated regression coefficients. replaces the trend variables with flexible state of birth economic and demographic controls for interacted with age cohort dummies, and adds schooling law controls. all coefficient results discussed refer to our preferred and most restrictive specification, ( ), unless otherwise noted. results presented in table confirm our hypothesis that the polio epidemic of had different effects on educational attainment for children of different ages. schoolaged children who were old enough to have labor market alternatives (i.e., those who were between ages and in ) and those who were living in areas more affected by the epidemic had lower educational attainment than similarly-aged children living in areas with lower polio morbidity rates. this result is robust even in our models with the most restrictive controls. a one-standard-deviation increase in the polio morbidity rate per persons (reported as . in table ) results in persons ages - having around . fewer years of educational attainment on average. the reference cohort of persons ages - had, on average, nine years of education. a back-of-the-envelope calculation suggests that this effect is equivalent to every fifteenth individual completing one less year of education. there is also a statistically significant and negative relationship between polio morbidity and educational attainment for children and under. the estimated coefficient suggests that a one-standard-deviation increase in polio morbidity decreases educational attainment by . years-the equivalent of one in ten people of the cohort completing one less year of education. in contrast, state-level polio morbidity in does not have a statistically significant relationship with educational attainment in individuals who were over , but too young to legally work in most states. the estimated coefficients on the interaction between polio morbidity and persons ages to in are negative, although the standard errors are large and they are not precise zeros. relative to individuals born before , the educational attainment of people born in those years do not appear to be strongly affected by polio morbidity rates. we test whether our results are robust to using two-year age groupings. results (reported in , / and / . the reference birth cohort is / . standard errors are clustered by state of birth and are in parentheses. all specifications include state of birth fixed effects, fixed effects for state of residence in , and birth year fe. economic controls are controls interacted with age cohort dummies. these variables include state-level doctors per capita, education expenditures per capita, log manufacturing wages per earner and log population. cohort schooling laws included proxies for the age of school entry, age of school exit and age of work permit that varies by state of birth and by year of birth * p < . , * * p < . , * * * p < . the appendix) are robust and confirm our main findings: that students with the ability to work legally, and children younger than experienced reduced educational attainment relative to children in the middle grades. in addition to state-level data, we have county-level data on polio morbidity from lavinder et al. ( ) for a select set of states in the eastern usa, as shown in fig. . these county data cover the northeastern part of the usa, which experienced much higher rates of polio cases and deaths than other regions. while we do not have data on county of birth, we can connect this county-level measure of polio intensity to county of residence in and restrict the sample to persons residing in the same state they were born in. essentially, we assume that people living in a particular county in (as long as it is in their birth state) were also born in this county. if we assume that migration within the usa between and is not systematically correlated with polio morbidity in , then migration bias would just introduce classical measurement error in county-level exposure to the epidemic. the results using the county-level polio morbidity in table mirror the results using state-level data. while the estimated coefficients are smaller, the relationship between education and polio is negative and statistically significant for all cohorts ages to in . these results suggest that children living in counties with greater polio cases in , on average, had lower levels of educational attainment than slightly older persons. a one-standard-deviation increase in county-level polio cases reduced the educational attainment of persons born between ages to by . years relative to the reference cohort born a few years earlier. similarly, persons aged - also experienced reduced educational attainment; a one-standard-deviation increase in polio morbidity in this age group reduced educational attainment by . years. children under during the polio epidemic had the largest reductions in educational attainment, with a one-standard-deviation increase in polio morbidity associated with a . year reduction in average educational attainment. measurement error could occur, however, if people move between high-morbidity and low-morbidity areas for reasons related to education-for example, if people born in rural counties moved to more urban counties for jobs. in this case, we may be overstating the impact of polio morbidity on educational attainment at the county level. for this reason, we have more confidence in the state-level results that aggregate rural and urban counties and include state of residence in fixed effects that further control for migration. an alternative method would be to link individuals observed between the and censuses and assume that if they reside in the same county, they were likely born in that county. this could further reduce measurement error, but requires significant resource investment and is still not entirely free of measurement error. we also examine whether our results are robust to two alternative specifications. first, the polio outbreak primarily affected young children under the age of five. as sanitation improved, epidemics in later decades tended to infect older children more frequently. using age-specific infection rates for the polio epidemic from a study by dauer ( ) in boston, we substitute our interaction of the polio notification rate variable with age category bins with a new variable created by interacting the polio notification rate for and the age-specific polio infection rate from dauer ( ) . we assign infection risks of % for persons under age four in , % for persons ages to , % for persons ages to , and % for anyone above the age of . in table , we present the main regressions specifications using this alternative measure of polio epidemic exposure. the negative and statistically significant coefficients in specifications ( ) and ( ) are consistent with our main findings and are larger in magnitude. according to specification ( ), for persons aged four and under, an increase in polio cases by one per people decreased educational attainment by . years. for persons between ages five and nine, the reduction in educational attainment was . years, while educational attainment fell by . years for people aged to during the epidemic. it is also plausible that persons exposed to the polio epidemic were also affected by the influenza pandemic, which killed over , people in the usa. the flu came in three waves. the first, with low mortality, came in the spring of . a second wave associated with higher mortality came in fall/winter , with a third wave arriving in early . we include the - flu mortality rates from garrett ( ), the united states bureau of the census ( ), and the united states bureau of the census ( ) into our analysis, since public health departments were not required to report influenza cases. table provides state-level data on influenza deaths by state in and . to control for the intensity of the influenza epidemic, we include the state-level influenza mortality rate interacted with age bins in our regression, even though the disease was most virulent and lethal in older populations than the children we focus on (i.e., adults of prime-working age). this exercise addresses potential concerns that the polio epidemic is spuriously correlated with influenza intensity. we study the effects of influenza intensity on observed education in for a subset of states used in the main analysis ( of ). results reported in table show that including the influenza death rate and its interactions with age groups does not affect our finding that children of legal working age in states with greater numbers of polio cases had less educational attainment. it attenuates the statistical significance of the epidemic for children ages to in in specifications ( ) and ( ) but strengthens it in specification ( ). the effect of influenza mortality itself on educational attainment is statistically insignificant in specifications ( ) and ( ). in specification ( ), we find influenza mortality increases educational attainment for persons born between and . this result is consistent with the findings in parman ( ) , who shows that families results reported are for white males. sample is restricted to individuals residing in the same state as their state of birth. years of education are top coded at years. age cohorts interacted with polio correspond to birth years / , / , and / adjusted educational investments in children in response to reduced human capital caused by prenatal exposure to the flu: slightly older children with siblings who were prenatally exposed to the flu pandemic experienced increased educational attainment. to further substantiate the main results, we also perform a placebo test comparing men ages to in to reference cohort of those ages to . results, reported in table , indicate that there is not a consistent pattern between reported polio morbidity and educational attainment. there is a small, negative and statistically significant relationship between polio intensity and the level of education observed for the age to cohort at the % level in specification ( ). however, this relationship is not consistent across all specifications, suggesting that polio morbidity at the state level is relatively uncorrelated with unobserved factors that could affect observed educational attainment in . in the appendix, we include an additional specification test using two-year birth cohorts, which do not substantively change our results. our primary results focus on men because they had more workforce opportunities and may have responded differently to polio than women. table reports results from our primary specification on a sample of women in . we find that the polio epidemic affected girls under age in a similar way to men. girls aged - also reported reduced educational attainment in high-polio areas, although not in our most saturated model. in our male sample, we find that boys aged - living in areas with higher polio did not have a statistically significant lower educational attainment than boys living in areas less affected by polio, although the standard errors are large and the results are an imprecise zero. for girls aged - , the adj r . . . estimated coefficients are negative and statistically significant. we are not sure why. in general, males and females had different returns to education, and it might have been that girls, for whatever reason, did not return to school in the same numbers as their male counterparts. the first major polio epidemic in the united states struck in the summer of and persisted into the fall. with over , cases of polio diagnosed, the epidemic tested the nascent system of public health departments. officials engaged in a variety of measures to stem the outbreak, including quarantines, washing streets, and closing public schools. in this paper, we focus on the effect of the epidemic on years of schooling reported by individuals in the us census. educational attainment could have been reduced for infected children, or for children who spent less time in school because their schools were closed or because their parents refused to send them. while we do not have data on school closures, we leverage geographical variation in polio morbidity as a proxy and focus on whether children of differential ages were differently affected. younger children were more likely to have the virus, and older children may have opted to join the workforce instead of stay in school. our results show that children of legal working age living in areas with higher rates of polio infection had diminished educational attainment than similarly aged children living in states with lower infection rates. this result, which is robust and consistent across specifications, does not hold for age groups who were not of legal working age, nor does it hold for slightly older children who had already completed their secondary schooling. we find stronger negative educational effects for children who were most susceptible to the virus and who were age and under during the epidemic. table women: effect of polio notification rate per on educational attainment of age cohorts specifications to are the same specifications presented in the main analysis in table , except these results are for white women. years of education are top coded at years. age cohorts interacted with polio correspond to birth years / , / , and / our results suggest that a one-standard-deviation increase in polio intensity during the epidemic reduced educational attainment by . years for the age - cohort and . years for the cohort age and younger. based on the average reported days in the school year, these numbers suggest that annual attendance declined by roughly days in the age - group and days among children et al. ( ) aged and under. these numbers are not trivial; they are within the range of lleras-muney's ( ) finding that compulsory attendance and child labor laws increased educational attainment by about days. another way of looking at the impact is to look at the aggregate effect in the cohort. for the cohort of children aged - during the epidemic, these numbers suggest that on average, about out of of them had one year less of educational attainment in their lifetime. the impact is even larger among younger children, with one out of every children attaining one less year of education. these cohort-wide reductions in educational attainment suggest that public quarantines, school closures, and parental fear magnified the social cost of the poliomyelitis epidemic, and may shed light on how epidemics can affect even people who do not contract the illness. clearly, school closures, even for short times, may reduce educational attainment among affected children. today, with virtual instruction, it is likely that these effects may be smaller. it is also likely that the impact on high-school completion will be mitigated because many jobs require high school diplomas. nevertheless, greater numbers of students are now enrolled in college. these results suggest that if these students leave school (either because they do not like virtual learning, or have a negative financial shock), they may be less likely to return and suffer from lower overall educational attainment as a result. , / , / , / , / , / , / , / , and / , / , / , / , / , / , / , / , and / we test sensitivity of our main analysis using alternative reference cohorts. the main analysis uses persons who would have been between and during the polio epidemic. we prefer using multiple birth year age bins study the polio epidemic. this is because our age variable is constructed from year of birth, there is some imprecision in lining up school attendance and age in . in particular, some persons who reach the age of in may have started the school year as year olds. we find a relatively negative and consistent relationship between polio virulence and the age someone was in . we redo the main regressions using persons aged - , - , , - , - , and as reference cohorts. we include an additional interaction between polio and the age cohorts removed from the reference cohort as a "placebo cohort." we find results consistent with our main analysis. using single year reference or falsification cohort in a specification using multiple year bins can alter the statistical significance of our results. potential issues arise from using either year olds alone as a reference cohort or using year olds alone as a falsification cohort. our preferred specifications using economic controls attenuate these effects, the regressions with these controls present negative and statistically significant results consistent with the main regression specification (tables , ) . table alternative reference cohort specification with falsification, part specifications to are the specifications presented in the main analysis and use the reference birth cohort is ages to in (birth years / ). specifications to use a reference cohort ages to and persons aged in are in the "placebo cohort." results reported are for white males. years of education are top coded at years. age cohorts interacted with polio correspond to birth years / , / , and / . standard errors are clustered by state of birth and are in parentheses. economic controls are controls interacted with age cohort dummies. these variables include state level doctors per capita, education expenditures per capita, log manufacturing wages per earner, and log population. cohort schooling laws included proxies for the age of school entry, age of school exit, and age of work permit that varies by state of birth and by year of birth. * p < . , * * p < . , * * * p < . table alternative reference cohort specification with falsification, part specifications to are the specifications that use ages to in as the reference birth cohort and persons aged to are in the "placebo cohort." specifications to use a reference cohort age and persons aged to in are in the "placebo cohort." results reported are for white males. years of education are top coded at years. age cohorts interacted with polio correspond to birth years / , / , and / . standard errors are clustered by state of birth and are in parentheses. economic controls are controls interacted with age cohort dummies. these variables include state level doctors per capita, education expenditures per capita, log manufacturing wages per earner, and log population. cohort schooling laws included proxies for the age of school entry, age of school exit, and age of work permit that varies by state of birth and by year of birth. * p < . , * * p < . , * * * p < . childhood circumstances and adult outcomes: act ii is the influenza pandemic over? long-term effects of in utero influenza exposure in the post- u.s. population the effect of age at school entry on educational attainment: an application of instrumental variables with moments from two samples cognitive development and infectious disease: gender differences in investments and outcomes does school quality matter? returns to education and the characteristics of public schools in the united states fixing the facts: editing of the u.s. census of occupations with implications for long-term trends and the sociology of official statistics early life health and cognitive function in old age epidemiology and prevention of vaccine-preventable diseases public reaction to a severe polio outbreak in three massachusetts communities studies on the epidemiology of poliomyelitis symposia: investment in children: the lengthening of childhood a monograph on the epidemic of poliomyelitis evening public ledger, p evening public ledger ( b) school opening postponed childhood health shocks, comparative advantage, and long-term outcomes: evidence from the last danish polio epidemic human capital and social capital: the rise of secondary schooling in america, - the great lockdown: worst economic downturn since the great depression the impact of sars on asian economies published: the annie e casey foundation lavinder ch, freeman aw, frost wh ( ) epidemiologic studies of poliomyelitis in new york city and the northeastern united states during the year chronicling america: historic american newspapers were compulsory education and child labor laws effective? an analysis from to in the u.s prediction of school failure from third-grade data unscheduled school closings and student performance race, educational attainment, and the census compulsory schooling legislation and school attendance in turn-of-the century america: a 'natural experiment' approach state child labor laws and the decline of child labor from emergence to eradication: the epidemiology of poliomyelitis deconstructed the cutter incident: how america's first polio vaccine led to the growing vaccine crisis childhood health and sibling outcomes: nurture reinforcing nature during the influenza pandemic the impact of length of the school year on student performance and earnings: evidence from the german short school years weekly reports for dirt and disease: polio before fdr million kids aren't in school because of ebola acknowledgements thomasson acknowledges support from the julian lange professorship. we thank see tables , and . the economic impact of ebola on sub-saharan africa: updated estimates for . technical report. world bank group trevelyan b, raynor-smallman m, cliff ad ( ) key: cord- - hprecor authors: cohen, j. a.; mistry, d.; kerr, c. c.; klein, d. j. title: schools are not islands: balancing covid- risk and educational benefits using structural and temporal countermeasures date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: hprecor background: school closures around the world contributed to reducing the transmission of covid- . in the face of significant uncertainty around the epidemic impact of in-person schooling, policymakers, parents, and teachers are weighing the risks and benefits of returning to in-person education. in this context, we examined the impact of different school reopening scenarios on transmission within and outside of schools and on the share of school days that would need to be spent learning at a distance. methods: we used an agent-based mathematical model of covid- transmission and interventions to quantify the impact of school reopening on disease transmission and the extent to which school-based interventions could mitigate epidemic spread within and outside schools. we compared seven school reopening strategies that vary the degree of countermeasures within schools to mitigate covid- transmission, including the use of face masks, physical distancing, classroom cohorting, screening, testing, and contact tracing, as well as schedule changes to reduce the number of students in school. we considered three scenarios for the size of the epidemic in the two weeks prior to school reopening: , , or detected cases per , individuals and assumed the epidemic was slowly declining with full school closures. for each scenario, we calculated the percentage of schools that would have at least one person arriving at school with an active covid- infection on the first day of school; the percentage of in-person school days that would be lost due to scheduled distance learning, symptomatic screening or quarantine; the cumulative infection rate for students, staff and teachers over the first three months of school; and the effective reproduction number averaged over the first three months of school within the community. findings: in-person schooling poses significant risks to students, teachers, and staff. on the first day of school, - % of schools would have at least one person arrive at school with active covid- , depending on the incidence of covid in the community and the school type. however, reducing class sizes via a/b school scheduling, combined with an incremental approach that returns elementary schools in person and keeps all other students remote, can mitigate covid transmission. in the absence of any countermeasures in schools, we expect - % of teaching and non-teaching staff and - % of students to be infected with covid in the first three months of school, depending upon the case detection rate. schools can lower this risk to as low as . % for staff and . % for students by returning elementary schools with a hybrid schedule while all other grades continue learning remotely. however, this approach would require - % of all school days to be spent at home. despite the significant risks to the school population, reopening schools would not significantly increase community-wide transmission, provided sufficient countermeasures are implemented in schools. interpretation: without extensive countermeasures, school reopening may lead to an increase in infections and a significant number of re-closures as cases are identified among staff and students. returning elementary schools only with a/b scheduling is the lowest risk school reopening strategy that includes some in-person learning. that would be lost due to scheduled distance learning, symptomatic screening or quarantine; the cumulative infection rate for students, staff and teachers over the first three months of school; and the effective reproduction number averaged over the first three months of school within the community. findings in-person schooling poses significant risks to students, teachers, and staff. on the first day of school, - % of schools would have at least one person arrive at school with active covid- , depending on the incidence of covid in the community and the school type. however, reducing class sizes via a/b school scheduling, combined with an incremental approach that returns elementary schools in person and keeps all other students remote, can mitigate covid transmission. in the absence of any countermeasures in schools, we expect - % of teaching and non-teaching staff and - % of students to be infected with covid in the first three months of school, depending upon the case detection rate. schools can lower this risk to as low as . % for staff and . % for students by returning elementary schools with a hybrid schedule while all other grades continue learning remotely. however, this approach would require - % of all school days to be spent at home. despite the significant risks to the school population, reopening schools would not significantly increase community-wide transmission, provided sufficient countermeasures are implemented in schools. interpretation without extensive countermeasures, school reopening may lead to an increase in infections and a significant number of re-closures as cases are identified among staff and students. returning elementary schools only with a/b scheduling is the lowest risk school reopening strategy that includes some in-person learning. evidence before this study scientific evidence on covid- transmission has been evolving rapidly. we searched pubmed on september for studies using the phrase ("covid- " or "sars-cov- ") and ("model" or "modeling" or "modelling") and ("schools") and ("interventions"). this returned studies, of which were retained after screening. a wide variety of impacts from school closures were reported: from - % of deaths at the lower end to reducing peak numbers of infections by - % at the upper end. drivers of this variability include (a) different epidemic contexts when school closure scenarios are enacted, (b) different timeframes and endpoints, and (c) different model structures and parameterizations. thus, considerable variation in predicted impacts of school closures has been reported. added value of this study to our knowledge, this is the first modeling study that explores the trade-offs between increased risk of covid- transmission and school days lost, taking into account detailed data on school demographics and contact patterns, a set of classroom countermeasures based on proposed policies, and applies them to range of community transmission levels. if rates of community transmission are high, school reopening will accelerate the epidemic, but will not change its overall course. however, even if rates of community transmission are low, complete school reopening risks returning to exponential epidemic growth. staged school reopening coupled with aggressive countermeasures is the safest strategy, but even so, reactive school closures will likely be necessary to prevent epidemic spread. implications of all the available evidence the impact of school reopening on the covid- epidemic depends on the transmission context and specific countermeasures used, and no reopening strategies are zero risk. however, by layering multiple types of countermeasures and responding quickly to increases in new infections, the risks of school reopening can be minimized. keywords: covid- , agent-based modeling, disease transmission, school reopening primary and secondary schools around the world closed due to covid- transmission in march and april . a recent observational study [ ] estimated that school closures were associated with a % decline in weekly covid- incidence and a % decline in weekly covid- mortality; some modeling studies have also found large impacts of school closures [ , ] . however, several other studies [ , , ] have estimated that closing schools has only a relatively small impact (e.g., - % reduction in deaths). attention is now focused on the risks and benefits of reopening schools. in-person learning has educational, social, and physical benefits; schools provide a safe space for educational instruction, support the development of social and emotional skills, and address nutritional needs, especially for families of lower socioeconomic status. for these reasons and more, the american academy of pediatrics issued a statement [ ] on july advocating for bringing students back to the classroom for in-person learning. yet much remains uncertain about the role schools, and school-age students in particular, play in covid- transmission, as well as how effective school-based interventions will be in preventing transmission. while children may be both less susceptible and less likely to develop severe infection, nearly a quarter of teachers are at a greater risk of serious illness if infected with covid- [ ] . in an npr/ipsos poll of teachers across the us conducted in july, % of teachers indicated that they are worried about risking their own health in returning to in-person education and % would prefer primarily remote, distance learning [ ] . much can be learned from the experience of school reopening across europe and asia; some countries successfully reopened without a significant increase in covid- cases [ ] , while others were forced to quickly re-close after outbreaks were observed in multiple schools [ , ] . in general, countries that reopened schools tended to do so when the case detection rate over the most recent day period was below per , -nearly times lower than the case detection rate in the united states as of august [ ] , with significant heterogeneity both across and within states. additionally, many of these settings returned younger students in-person first with some degree of staggering the start, stop, and break times within schools. in this analysis, we used an agent-based model of covid- transmission to evaluate the health and educational outcomes associated with various school reopening strategies. specifically we compared behavioral interventions such as mask usage, physical distancing and hand hygiene; case detection interventions, including symptomatic screening, follow-up diagnostic testing and contact tracing; and structural interventions, including classroom cohorting, schedule changes, and combinations of remote and in-person learning. we used covasim, an agent-based model of covid- transmission and interventions [ , ] , to estimate the impact of school reopening on disease transmission and the extent to which school-based interventions could . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . mitigate epidemic spread within and outside schools. covasim includes demographic information on age structure and population size; realistic transmission networks in different social layers, including households, schools, workplaces, long-term care facilities and communities; age-specific disease outcomes; and within-and between-host variations in infectivity to capture sub-and super-spreading and front-loaded infectivity. we modeled schools to match age mixing patterns between students within elementary schools, middle schools, and high schools [ ] (see appendix a for more details on the school network structures). key inputs and assumptions of our modeling approach have been documented in our methodology papers [ , ] . the covasim model is fully open source and is available for download via github and the python package index; more information is available at https://covasim.org. we compared seven alternative school reopening strategies that vary the degree of school-based transmission mitigation. countermeasures were nonpharmaceutical interventions (npis), which implicitly include face masks, six foot separation, and hand washing, which together are assumed to reduce transmission by % [ , , ] ; class cohorting, in which students and teachers have minimal contact outside their own classroom (see figure ); and symptomatic screening, with % follow-up diagnostic testing and % follow-up contact tracing. a/b scheduling splits classrooms into group a students (who attend two days per week, e.g. mon-tue) and group b (who attend two different days per week, e.g. thu-fri). . all in-person with no countermeasures . all in-person with countermeasures . all in-person with countermeasures and a/b scheduling . elementary and middle in-person with countermeasures, high school remote . elementary in-person with countermeasures, middle and high school remote . elementary in-person with countermeasures and a/b scheduling, middle and high school remote . all remote . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint figure : a schematic diagram of the in-person school contact networks of students, teachers and additional school staff under different cohorting strategies. normal, pre-covid- patterns allow for mixing of students and teachers across grades and classrooms. a cohorted strategy places elementary and middle school students into classrooms with their own teachers, preventing contact between students in different classrooms in these schools. high schools remain mixed due to the highly individualized schedules of students in many u.s. based high schools, including those in king county, washington. teachers and additional school staff have contacts with each other to reflect their use of common staff paces such as a teachers' lounge, front office, and other rooms for preparation. we applied our interventions to elementary, middle, and high schools and assumed that preschools and universities will remain closed. we assumed that high schools would not be able to implement classroom cohorting, as it would be too challenging to coordinate the highly variable schedules of students at this level (see figure for a schematic of the school network differences). we simulated the first three months of the school term (sept. we used data from king county, washington to define the population and contact network structure, including the average class size and the distribution of students enrolled per school [ ] . the effective reproduction number in the absence of school reopening and the case detection rate in the two weeks prior to school reopening served as signals of both the direction of cases as well as the size of the epidemic. results presented in this work assume that epidemic is declining slowly when schools are fully closed (i.e., r e = . ). in conjunction with our assumption around epidemic control with full school closures, we considered three scenarios for the size of the epidemic . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . in the two weeks prior to school reopening: , , or diagnosed cases per , individuals. these numbers fit within the low, medium and high case detection bands defined by states across the united states for school re-opening guidance. in-person schooling, even with sufficient countermeasures, poses significant risks to students, teachers, and staff. on the first day of school, - % of schools would have at least one person arrive at school with active covid- (including all students, teachers, and staff), depending on the covid case detection rate in the community and the school type ( figure ). these infections may show few symptoms and go undetected, especially if they are in younger children. symptomatic individuals may be screened and sent home immediately upon arrival. active covid- infections also may not lead to onward transmission within schools, depending on per-contact infectivity. this highlights the importance of procedures within schools to minimize risk of transmission, detect and isolate cases, and contact and quarantine any known contacts. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . closely examining infections present in school, our model shows that in the absence of any countermeasures and at a case detection rate of per , in the two weeks prior to school reopening, nearly a quarter of teachers and staff and over % of students would visit school while infectious with covid- in the first three months of school ( figure ). this risk can be reduced . -fold by reopening schools when the case detection rate is per , ; when there is more covid in the population, there will inevitably be more covid in schools. we find that implementing countermeasures that limit transmission and detect, trace, and quarantine cases within schools would lead to an even larger reduction in the cumulative covid- infection rate at each of the three case detection rates we considered. the inclusion of face mask usage, physical distancing, classroom cohorting, symptomatic screening, testing and tracing in schools would lower the risk to students, teachers, and staff over -fold at a case detection rate of per , . this risk can be lowered further with the addition of scheduling efforts that reduces the number of students present at school. an a/b school scheduling approach that returns elementary schools in-person and keeps all other students remote would minimize the presence of covid within (and outside of) schools. the predicted cumulative covid- infection rate for people in schools could be as low as between . and . % for teachers and staff and to between . and . % for students, depending on the case detection rate. this represents at least a -fold reduction in the risk of covid- for teachers and staff in schools relative to a strategy that returns all individuals in-person with no countermeasures. within the king county population of approximately . million, we estimate that this approach would require an additional - , tests for students, teachers and staff over the first three months of school, depending on the case detection rate in the two weeks prior to school. we find that an a/b scheduling approach, in which classrooms are split into two groups that attend school two days a week on different days, reduces covid- transmission in schools to . - . % for teachers and staff and . - . % for students, depending upon the covid case detection rate. this represents about double the risk compared to an elementary-only approach, but gives all k- students some time for in-person learning, whereas the elementary-only approach restricts in-person learning to elementary school students, at least initially. these strategies come at a significant educational cost, requiring up to % of school days to be spent at home, due to either planned distance . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . learning or related to detected covid- infection (figure ) . provided sufficient countermeasures are implemented within schools, the covid- infection rate in the population prior to school reopening has more influence on the infection rate within schools than the specific schooling strategy. we find an over seven times reduction in the infection rate for people in schools if schools are reopened when the case detection rate in the community is at per , compared to per , . at any given case detection rate, additional countermeasures will have a marginal impact on the rate of infection within schools at a large cost of missed in-person school days. we find that reopening schools will not significantly increase communitywide transmission, provided sufficient school-based interventions are implemented ( figure ). if community transmission is decreasing in the absence of in-person schooling, a return to in-person learning with appropriate countermeasures is unlikely to add significantly to community transmission. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint figure : trade-offs between within-school infection rate and missed in-person school days (due to either scheduled distance learning, quarantine or infection. due to considerable uncertainty in the roles k- students, teachers, and staff play in covid- transmission, we performed several sensitivity analyses to see if results were robust to a range of reasonable assumptions. while our baseline assumed that infected k- children are as infectious as adults, consistent with findings from pcr cycle threshold counts [ ] , a recent study using data from south korea suggests that students under the age of might be half as infectious as older students and adults [ ] . implementing this variation in the model leads to similar conclusions ( figure ) . a second sensitivity analysis concerns the susceptibility of school-age children to covid- infection. in our base case, we assume that - year-olds have a % reduced susceptibility to infection and - year-olds have a . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . % reduced susceptibility to infection [ ] . however, a recent study among children in australia challenges this broadly held assumption, arguing that children are similarly vulnerable to the virus [ ] . we find that without any countermeasures in schools, this assumption has a significant impact on the infection rate for people in schools ( figure ) as well as the effective reproduction number in the community ( figure ). with sufficient countermeasures, this assumption does not change our general policy conclusions. any return to in-person learning will pose covid- infection risk for students, staff and teachers. however, modeling results suggest that, depending on the case detection rate of covid- in the community, a carefully organized incremental approach that returns the youngest students first . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint with a reduced schedule would minimize the risk of infection within schools and provide important benefits to the neediest children. while this analysis is based upon school enrollment and population demographic data from king county, washington, lessons can be applied to other settings to inform decision-making around school reopening. for example, as of august , , king county had a -day case detection rate of compared to in new york city per , people. this would translate to nearly twice as many schools in the region with at least one person arriving at school with an active covid- infection on september st compared to new york city, depending on the size of the school. it may also indicate a different optimal strategy for school reopening in these cities. in fact, seattle public schools, citing a case detection rate in the city of well over per , in the last two weeks of august, announced they would be starting the fall with completely remote learning [ ] , while new york public schools announced they . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . would start with an a/b hybrid scheduling approach [ ] . schools in the san francisco region have also mostly opted for remote learning, in line with recent model-based recommendations finding a high risk of school reopening [ ] . we find that the solution with the lowest health risk also has the highest educational cost, in terms of days of distance learning required. we note that days of distance learning are not experienced equally by students and the benefit gained varies considerably based on both age and other factors such as socioeconomic status and resources available to their families [ ] . decision makers must therefore balance the benefits of in-person education with the safety of teachers and staff, while continuing efforts to decrease the incidence of covid- outside of schools. regardless of the approach taken, it will be critical to monitor schools with symptomatic screening and react quickly to the emergence of cases in schools with testing and contact tracing. our results support the strategy of returning elementary school students to school either full-time or with an a/b schedule while keeping all other students remote for the first three months of school to best minimize the risk of covid- infection in schools. returning elementary schools in-person first is both relatively lower-risk and higher-benefit; elementary school aged children are less susceptible to infection [ ] and potentially less likely to transmit infection [ , ] . additionally, they may benefit more from in-person learning and pose more of a burden on family members. these results have been put into practice by some countries, which have returned elementary students to school first and older students at a later stage after observing an absence of significant school-based transmission [ , ] . however, these countries had a significantly lower covid- incidence rate prior to reopening schools than many parts of the us, including washington state, are experiencing today. while agent-based modeling is able to capture many details of populations and disease transmission, our work has important limitations and assumptions that could impact our findings. there is still a high degree of uncertainty around the susceptibility, symptomiticity/severity, and infectivity of covid- in children, particularly since schools in most locations shut down early in the epidemic. our analysis is based on the most recent scientific literature for each of these parameters. we assumed individuals under had a - % reduced risk of developing symptoms [ ] and - % . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . reduced risk of acquiring infection [ ] . we varied this in sensitivity analysis so that individuals under are equally as susceptible to infection as individuals age to . we assume that an infectious individual is times more likely per day to transmit to a household contact than a school contact, based on estimated numbers of hours spent in each setting per week. we assume all individuals infected with covid- are equally likely to transmit infection per contact, and varied this assumption in sensitivity analysis so that individuals under years old are half as likely. after being diagnosed, all individuals are assumed to reduce their daily infectivity by % for home contacts, % for community contacts, and % for school and work contacts. additionally, the household contacts of these individuals may be traced, notified, and school contacts removed from school for a full -day quarantine period. while we anticipate that schools will be able to help identify contacts of diagnosed students or staff, the large number of contacts within schools may place additional burden on local or state contact tracing efforts and our analysis does not represent this. we assume that household transmission is not reduced while children are attending school in person, nor that it increases when students are learning remotely. in terms of structural choices, we made several assumptions about the implementation and impact of school reopening strategies. we chose to not model increased transmission associated with parents/guardians returning to work following a return to in-person learning. we also assume students who participate in remote learning are not in contact with anyone from school. on days that students are learning remotely, we do not account for any potential interaction students may have in a congregate care setting, such as an alternative after-school care program. we have assumed that, if implemented, all elementary and middle schools will be able to enforce classroom cohorting, whereby students are grouped into a classroom and are only in contact with other students and teachers in that classroom. we note that cohorting may be difficult to implement given the complexity of class scheduling for student bodies with multiple academic tracks, elective classes, and degree requirements. therefore, we may be underestimating the transmission impact of school reopening, and further caution should be taken in any decisions to return to in-person learning. we assumed that, if implemented, symptomatic screening would occur daily in schools and students or teachers presenting with covid-like symptoms would be sent home. those who are symptomatic may be asked to take a diagnostic test, which we assumed returns a result within two days. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . students who received a negative test result return to school the next day, and students who received a positive test result are isolated at home for days. our results do not depend on school staff administering the diagnostic tests. we are not explicitly modeling after-school care, which many working parents depend upon to cover the gap between school hours and working hours. families who use these services may also be more likely to be essential workers. we also do not model transportation to and from school, which may be an important source of transmission and which also depends on school resources. schools around the world are grappling with the challenge of returning to in-person learning in the covid era. much remains unknown about the role children play in covid- transmission within schools and in the broader community, but the latest science suggests that younger children are less susceptible and show fewer symptoms if infected. from schools that never closed in sweden to reopening examples in europe and asia, lessons on how the us might resume in-person learning are abundant and diverse. our computational modeling synthesizes this evidence, and the latest results give reason for optimism. yet reopening schools is not a zero-risk activity. symptom screening is imperfect, and covid- will be present in the respiratory system of students, teachers, and staff on day one. additionally, a return to in-person learning would allow parents and guardians return to work, which could be accompanied by an associated increase in transmission outside schools. but the solution with the lowest health risk has the highest educational cost, the majority of which lands on those families most marginalized and underresourced: those without access to technology and private tutors, and whose parents or guardians work in the essential economy. schools must open; the question is when and how, so as to balance the benefits of in-person education with the safety of teachers and staff, all while realizing that covid is not just a school problem. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint in order to model realistic school reopening scenarios, we equipped the model to generate networks within schools that reflect proposed cohorting by age and grade. we modeled schools to approximate age mixing patterns [ ] between students within preschool, elementary schools, middle schools, high schools, and universities. using the county-level school enrollment data [ ] , we simulate contacts within schools, mixing between students and teachers, and clustering of students into cohorts. mixing of students and teachers can be thought of as following three main patterns: ( ) students sorted in classroom cohorts of the same grade with one or two teachers, ( ) students mixing with random contacts mostly within the same grade and at least one teacher, and ( ) students mixing with random contacts across the entire school and at least one teacher. the first mixing pattern resembles the contact structures commonly found in pre-school and elementary schools, where students are generally taught by one teacher and stay with the same classroom of contacts throughout the day. the second pattern reflects mixing patterns often found in middle schools and high schools where students have individualized schedules and mostly interact with other students in the same grade. the third mixing pattern reflects university settings where student interaction occurs in classes, dorms, and in other spaces on campus. student mixing in these institutions display less age assortativity because of the high variability of age when students enroll, use of common spaces such as libraries and dining halls, and other aspects of on-campus life. in addition to students and teachers, schools also include additional staff members such as principals, counselors, nurses, maintenance, and cleaning staff. using information on the estimated ratio of students to all staff members, we model the number of additional non teaching staff expected for each school and the contacts for them as random contacts across the entire school. this reflects the overall more varied contact patterns of non-teaching staff with students, teachers, and other staff members. we used an optimization procedure to find a set of parameters that resulted in our desired combinations of effective reproduction number in the absence of school reopenings (specifically, r e = . ) and cumulative diagnosed cases in the two weeks prior to the beginning of the school year (specifically, , , or diagnosed cases per , people). the parameters used . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint for the optimization were the number of seed infections at the start of the simulation, and the reduction of workplace and community contacts. we calibrated these model parameters using the tree-structured parzen estimator sampler in optuna, a python-based optimization library. the sampler finds regions of the parameter space that minimize a scalar objective function by learning the relationship between the parameters and the objective, as well as the overall probability of various objective values. we defined the objective function to be the sum of squared differences between the target values (i.e., average effective reproduction number from september st to december st; cumulative covid- cases in the two weeks prior to september st) and the corresponding model outputs. in all cases, we found a roughly % reduction in workplace and community contacts resulted in r e = . . initializing the simulation with roughly , , and seed infections resulted in scenarios of , , or diagnosed cases per , people over a two-week period. we ran the analyses with the top best-fitting parameter sets to each of these three scenarios. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint association between statewide school closure and covid- incidence and mortality in the us covid- and inequalities sex by school enrollment by type of school by age for the population years and over american community survey -year estimates hand hygiene, mask-wearing behaviors and its associated factors during the covid- epidemic: a cross-sectional study among primary school students in wuhan physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov- and covid- : a systematic review and meta-analysis how many teachers are at risk of serious illness if infected with coronavirus? school openings across globe suggest ways to keep coronavirus at bay, despite outbreaks age-dependent effects in the transmission and control of covid- epidemics school closure during the coronavirus disease (covid- ) pandemic: an effective intervention at the global level? social contact networks and mixing among students in k- schools in pittsburgh, pa the effect of school closures and reopening strategies on covid- infection dynamics in the san francisco bay area: a cross-sectional survey and modeling analysis age-related differences in nasopharyngeal severe acute respiratory syndrome coronavirus (sars-cov- ) levels in patients with mild to moderate coronavirus disease (covid- ) no evidence of secondary transmission of covid- from children attending school in ireland covid- , children, and schools: overlooked and at risk most teachers worried about reopening schools, coronavirus poll finds". in: npr controlling covid- via test-trace-quarantine". in: medrxiv covasim: an agent-based model of covid- dynamics and interventions". in: medrxiv interventions to mitigate early spread of sars-cov- in singapore: a modelling study community use of face masks and covid- : evidence from a natural experiment of state mandates in the us what do we know about children and coronavirus transmission? implications of the school-household network structure on sars-cov- transmission under different school reopening strategies in england projected effects of nonpharmaceutical public health interventions to prevent resurgence of sars-cov- transmission in canada determining the optimal strategy for reopening schools, the impact of test and trace interventions, and the risk of occurrence of a second covid- epidemic wave in the uk: a modelling study early release -contact tracing during coronavirus disease outbreak educators and superintendents urge a safe return to school this fall seattle public schools announces plan for remote learning this fall reopening the world: reopening schools-insights from denmark and finland school closure and management practices during coronavirus outbreaks including covid- : a rapid systematic review new york schools can reopen changes in contact patterns shape the dynamics of the covid- outbreak in china schools network structure we simulated a representative sample of the . million king county, washington residents in covasim. we use student enrollment data for king county available from the american community survey [ ]. this data gives an estimate of the enrollment rates by age for students attending any educational institution at the county level key: cord- - n j jo authors: stein-zamir, chen; abramson, nitza; shoob, hanna; libal, erez; bitan, menachem; cardash, tanya; cayam, refael; miskin, ian title: a large covid- outbreak in a high school days after schools’ reopening, israel, may date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: n j jo on march , israel’s government declared closure of all schools. schools fully reopened on may . ten days later, a major outbreak of coronavirus disease (covid- ) occurred in a high school. the first case was registered on may, the second on may. they were not epidemiologically linked. testing of the complete school community revealed students (attack rate: . %) and staff members (attack rate: . %) who were covid- positive. on march , israel's government declared closure of all schools. schools fully reopened on may . ten days later, a major outbreak of coronavirus disease (covid- ) occurred in a high school. the first case was registered on may, the second on may. they were not epidemiologically linked. testing of the complete school community revealed students (attack rate: . %) and staff members (attack rate: . %) who were covid- positive. as part of the coronavirus disease (covid- ) pandemic containment measures, israel's government declared complete closure of all educational facilities on march . limited schools reopening (kindergartens, grades - and - ) only in small groups was approved on may . subsequently, all school classes reopened on may , with requirement for daily health reports, hygiene, facemasks, social distancing and minimal interaction between classes. ten days later, the first major covid- school outbreak in israel emerged in a high school. the first case was registered on may and the second on may. the two cases were not epidemiologically linked. testing of the complete school community revealed students (attack rate: . %) and staff members (attack rate: . %) who were covid- positive. overall, some persons were infected (students, staff members, relatives and friends). in this report, we aim to describe the investigation and epidemiological characteristics of the school's outbreak. school is a regional public school; students arrive from suburbs and neighbourhoods, by public or school bus. it contains , students aged - years (grades - ) and staff members. the school reopened after months' closure on monday, may . students returned to their previous classrooms and received instructions on preventive procedures. on - may (tuesday to thursday), an extreme heatwave occurred. hence, the ministry of health exempted schoolchildren from facemasks for these days. the first covid- case (student a) was notified on may . the source of infection was unknown. close contacts from household (n = ), students (n = ) and teachers (n = ) were instructed to self-isolate. the second case (student b) was notified on may . according to the epidemiological investigation, both students attended school during the days of - may and reported mild symptoms (anosmia, ageusia, fever and headache). they were from different grades and were not epidemiologically linked. with the emergence of two unrelated cases within days, the district health office declared an 'outbreak status' including school closure, isolation instructions and testing of the school community. during that long weekend (a jewish holiday, - may ), mass covid- testing was conducted as a joint effort of the school leadership and community, the four health funds, magen david adom (national emergency services organisation), the local municipality and the district health office. ten teachers and students who had not attended school since reopening were excluded. most of the remaining school community was tested, of staff members and , of , students. overall, students and staff members were confirmed as covid- -positive. the data from the epidemiological investigation are shown in the table. the covid- rates differed between groups. male cases were slightly overrepresented. the rate of cases reporting symptoms, upon meticulous questioning, was % ( / ) among students and % ( / ) among staff. the leading symptoms reported were cough, headache, fever, sore throat and myalgia. one emergency room visit was recorded and no hospitalisations. covid- rates were higher in junior grades ( - ) than in high grades ( - ) ( figure ). the peak rates were observed in the th grade ( cases in one class and cases in two other classes) and the th grade ( cases in one class). of the cases in teachers, four taught all these four classes, two taught three of the four classes and one taught two of these four classes. an environmental school inspection reported crowded classes: - students per class, class area - m², allowing . - . m² per student (below the . m² standard). distancing among students and between students and teachers was not possible. furthermore, during the extreme heatwave, air-conditioning functioned continuously in all classes. the air-conditioning system was separate for each class. the junior grades ( - ) and the high grades ( ) ( ) ( ) are situated in one large building, yet in separate wings, and share the schoolyard and public spaces. according to the school schedule, students study days (sunday to friday) for - h weekly ( . - . h daily on average). daily travel time to school depends on distance and traffic conditions and lasts - min. most students also participate in extracurricular activities such as sports teams or dance classes for an average of - h per week. as at june , of ( . %) students and of ( %) staff members have recovered (with two negative pcr results). evaluating the recovery period revealed that % of asymptomatic cases recovered within days vs only % of symptomatic cases. by mid-june , additional confirmed covid- cases had occurred among close contacts of the first school's cases. these included siblings attending other schools, friends and participants in sports and dancing afternoon classes, students' parents and family members of school staff. the large school outbreak led us to evaluate the age distribution of covid- cases before and after schools' reopening. from week to week in , , confirmed covid- cases were reported in the jerusalem district. as schools reopened on may , the evaluation point selected was week later, on may (week ). the evaluation showed that before may , the proportion of the - years-olds (representing schoolchildren), was . % ( / , ) of cases in weeks - , increasing to . % ( / ) after may , in weeks - ( figure ). from week to week in , , confirmed covid- cases were reported nationally, , cases in the jerusalem district and , cases in all the other districts in israel, excluding jerusalem. the age pyramid of confirmed covid- cases in the jerusalem district vs nationally (excluding jerusalem) showed a prominence of the - years-olds in jerusalem, . % vs. . % in all the other districts ( figure ). on january , israel's health minister declared covid- infection a notifiable disease requiring immediate reporting. by june , some , confirmed covid- cases had been reported with fatalities [ ] . israel's population is . million (median age: years) [ ] . like other countries, israel implemented diverse containment measures including quarantine. nationally, there are . million schoolchildren, , kindergarten children and , teachers and staff [ ] . (median age: . years), characterised by moderate to low socioeconomic status and large households [ ] . the high school outbreak in jerusalem displayed mass covid- transmission upon school reopening. the circumstances promoting infection spread involved return of teenage students to their regular classes after a -month closure (on may) and an extreme heatwave (on may) with temperatures rising to °c and above [ ] that involved exemption from facemasks and continuous air-conditioning. classes in the first affected school had more than students. israel's secondary school classes are crowded (average: students in public schools) compared with the organisation for economic cooperation and development (oecd) average ( students) [ ] . covid- in a school necessitates a prompt response. classmates and teachers should be considered close contacts (particularly in crowded classes), as should students in groups mixing several classes, extra-curricular activities and school buses. temporary school closure is prudent (especially in large regional schools) pending investigation results. most student cases presented with mild symptoms or were asymptomatic. severe acute respiratory syndrome coronavirus (sars-cov- ) infection in children and adolescents is considered mild compared with adults. a review of studies ( , hospitalised paediatric patients) presented overall good prognosis for that age group [ ] . a chinese study of paediatric cases infected with sars-cov- reported main signs of fever, cough and pharyngitis, % were asymptomatic [ ] . in a european multicentre study ( children), covid- was usually mild, a small fraction developed severe disease and mortality was rare [ ] . in a study in new york state, kawasaki-like disease and myocarditis have been linked to covid- infection, with the condition termed multisystem inflammatory syndrome (mis-c) in children [ ] . french paediatric surveillance data also support linkage between sars-cov- infection and mis-c [ ] . the role of children and adolescents in covid- spread is equivocal; epidemiological data imply insignificance of children in transmission [ ] . school closure is a public health tool in influenza pandemic preparedness plans, based on high infectiousness and susceptibility in schoolchildren and high contact rates [ ] . school reopening policy after the covid- lockdown varies considerably between nations and therefore requires ongoing assessment [ ] . covid- prevention in schools involves studying in small groups and minimising student mixing in activities and transportation. teachers and parents should lead by wearing facemasks, hand hygiene, keeping physical distance etc. school attendance should be avoided at any sign of illness. learning from home may also reduce the need for class attendance. outdoors classes should also be considered. covid- prevention encompasses avoiding the 'three cs': closed spaces with poor ventilation, crowded places and close-contact settings [ ] . the european centre for disease prevention and control's report on air-conditioning and ventilation systems and covid- recommends increasing air exchange rate and outdoor air use and decreasing air recirculation, aiming to reduce spread in indoor spaces [ ] . finally, appropriate planning of covid- prevention for the next school year is essential. israel ministry of health (moh) population of israel on the eve of population of israel on the eve of school closure and management practices during coronavirus outbreaks including covid- : a rapid systematic review timeliness and completeness of routine childhood vaccinations in young children residing in a district with recurrent vaccine-preventable disease outbreaks organisation for economic cooperation and development (oecd) severe acute respiratory syndrome coronavirus (sars-cov- ) infection in children and adolescents: a systematic review sars-cov- infection in children covid- in children and adolescents in europe: a multinational, multicentre cohort study multisystem inflammatory syndrome in children in new york state sars-cov- -related paediatric inflammatory multisystem syndrome, an epidemiological study reopening schools after the covid- lockdown closure of schools during an influenza pandemic clusters of coronavirus disease in communities heating, ventilation and air-conditioning systems in the context of covid- the authors wish to acknowledge mr. danniel leibovitch, the school's headmaster, the school's teachers and staff, the students and families for their cooperation and compliance. the authors wish to thank the jerusalem municipality and mayor mr. moshe lion, for their support and leadership. the authors wish to thank the jerusalem branch of mda (national emergency services organization) for the rapid mass testing. finally, the authors would like to thank the devoted public health teams in the district health office and the community clinics teams of the four heath funds in jerusalem. none declared. chen stein-zamir, nitza abramson and hanna shoob collected data, performed the investigation and data analysis and wrote the manuscript. erez libal, menachem bitan, tanya cardash, refael cayam and ian miskin performed the patients' follow-up, provided data and reviewed the manuscript. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord- - r authors: uscher-pines, lori; schwartz, heather l.; ahmed, faruque; zheteyeva, yenlik; tamargo leschitz, jennifer; pillemer, francesca; faherty, laura; uzicanin, amra title: feasibility of social distancing practices in us schools to reduce influenza transmission during a pandemic date: - - journal: j public health manag pract doi: . /phh. sha: doc_id: cord_uid: r schools are socially dense environments, and school-based outbreaks often predate and fuel community-wide transmission of seasonal and pandemic influenza. while preemptive school closures can effectively reduce influenza transmission, they are disruptive and currently recommended only for pandemics. we assessed the feasibility of implementing other social distancing practices in k- schools as a first step in seeking an alternative to preemptive school closures. methods: we conducted focus groups with education and public health officials across the united states. we identified and characterized themes and compared feasibility of practices by primary versus secondary school and region of the united states. results: participants discussed school practices ( within-school practices implemented as part of the school day and reduced-schedule practices that impact school hours). participants reported that elementary schools commonly implement several within-school practices as part of routine operations such as homeroom stay, restriction of hall movement, and staggering of recess times. because of routine implementation and limited use of individualized schedules within elementary schools, within-school practices were generally felt to be more feasible for elementary schools than secondary schools. of reduced-schedule practices, shortening the school week and the school day was considered the most feasible; however, reduced-schedule practices were generally perceived to be less feasible than within-school practices for all grade levels. conclusions: our findings suggest that schools have many options to increase social distance other than closing. future research should evaluate which of these seemingly feasible practices are effective in reducing influenza transmission in schools and surrounding communities. they may also recommend other social distancing measures in other community congregate settings (eg, workplaces, mass gatherings) to slow the spread of disease and thereby relieve pressure on overburdened health care and public health systems. [ ] [ ] [ ] schools are important settings for social distancing. because public schools are socially dense environments where more than million students congregate across the united states each day, schools can fuel community-wide disease transmission. [ ] [ ] [ ] in addition to promoting respiratory etiquette and hand hygiene and engaging in frequent environmental cleaning, schools can implement practices that promote social distancing to potentially protect large numbers of vulnerable children, as well as limit secondary transmission to adults within their households and communities. over the past decade, research and guidance on social distancing in us schools have mainly focused on school closure as the most impactful, albeit disruptive, social distancing practice. while a substantive evidence base documents that school closure can mitigate influenza pandemics, there may be potentially less disruptive opportunities to increase social distance among students who remain in school. [ ] [ ] [ ] nonetheless, feasibility, acceptability, and effectiveness of the full range of school social distancing practices have not been explored. [ ] [ ] [ ] to address this gap, the us centers for disease control and prevention and rand health conducted a large qualitative field study to examine the feasibility of social distancing practices other than school closures in k- schools. through focus groups with senior education administrators across the united states, we aimed to identify potential social distancing practices beyond school closure, describe barriers to and facilitators of implementing these practices for at least weeks to decrease the spread of influenza before many students become ill, and rank practices by feasibility. in the summer of , we conducted focus groups with education and public health officials via webinar. participants were selected from all us department of health & human services (hhs) regions to explore perspectives that might differ by region of the country. we also recruited participants representing both primary and secondary schools to compare how barriers and facilitators might vary by grade level. we recruited superintendents, principals, teachers, school nurses, state school nurse consultants, district transportation directors, state health pandemic planners, and school safety representatives. for each hhs region, we assembled a purposive sample of participants by searching professional association web sites and lexisnexis and conducting snowball sampling with focus group attendees. within professional association web sites, we searched for lists of members, conference attendees, and association leaders. in lexisnexis, we looked for names of school leaders who were quoted in the media about emergency preparedness. a total of participants, representing all regions, participated in a total of focus groups. the number of participants per focus group ranged from to , with a mean of . each participant participated in a single focus group. a team of moderators trained in qualitative research conducted the focus groups via webinar. most of the groups consisted of participants from a single hhs region, but groups included a mix of several hhs regions to accommodate scheduling preferences. participants were contacted via e-mail and offered a $ gift card as an incentive. all focus group discussions were recorded and then transcribed. each group followed a semistructured protocol, and participants were asked to identify, mention experience with, and discuss the feasibility of practices within of categories: practices that could be implemented within a normal school schedule ("within-school practices") and those that would require an altered school schedule ("reduced-schedule practices"). to limit focus group duration to minutes, groups discussed within-school practices ( focused on elementary schools, focused on secondary schools, and discussed both elementary and secondary schools) and discussed reducedschedule practices (both elementary and secondary schools). in each group, participants were first presented with a list of practices assembled by the study team and asked to brainstorm any additional practices that could be implemented in k- schools. second, they discussed any direct experiences with listed practices as well as implementation barriers and facilitators of each individual practice. finally, they selected the most and least feasible practices from among the full list of practices. feasibility was defined as "ease of implementation" in this context. this study was approved by rand's institutional review board, and all participants gave oral consent to participate at the start of each focus group. standard qualitative analysis techniques were used to identify and characterize instances of themes arising from the various topics covered in focus group protocols (eg, each identified practice) as well any unanticipated themes that emerged. two of the authors read each transcript and independently coded. to ensure that different coders were interpreting the data as similarly as possible, we ( ) developed descriptive and precise codebooks that gave each code a clear definition and meaning; ( ) performed intercoder agreement checks prior to analyses where all analysts read the same text, coded independently, and discussed areas of disagreement; and ( ) performed supervisory reviews of the analysis at regular intervals. we compared themes by hhs region as well as by secondary versus elementary school settings, and we reported any differences identified. dedoose qualitative research software was used to facilitate data handling, coding, and thematic analyses. for ranking, participants in within-school practices focus groups could vote for practices they perceived as the most feasible and perceived as the least feasible. for the focus groups on reduced-schedule practices, participants voted for the single most feasible and single least feasible practice. individual votes were summed. we first present the full menu of social distancing practices (other than school closure) discussed by focus group participants as well as high-level summaries of perceived barriers, facilitators, and variation by region and/or grade level. we then present details on the barriers and facilitators for the within-school and reduced-schedule practices that ( ) were deemed most feasible by participants and ( ) could be implemented continuously over at least weeks. participants discussed a total of school practices ( within-school practices and reduced-schedule practices). while practices were identified by the study team in advance through policy and literature reviews, additional within-school practices were identified by focus group participants. additional practices included limiting group work, limiting congregation at arrival and dismissal, encouraging solo physical activity, canceling cross-school transfer for special programs such as dual enrollment, reducing congestion in the school health office, and educating students and family members to maintain their distance. practices with which participants reported prior experience as part of routine operations or in response to the h n pandemic are shown in table . for elementary schools, the more common within-school practices that some schools use as part of routine operations included homeroom stay, restricting hall movement (walking single file, a foot apart), segregating recess area by class, staggering recess times, segregating the cafeteria by class, and staggering lunch periods. common within-school practices implemented for the first time in response to the h n influenza pandemic included canceling field trips, canceling assemblies, limiting visitors, and reducing congestion in the health office. several participants also reported experience with reduced-schedule practices as part of routine operations and in response to emergencies, including operating on a -day week because of budget constraints, and shortening the school day (delayed start or early dismissal for severe weather). the perceived barriers and facilitators for each practice, as well as variability by grade level, are shown in table . within-school practices were generally perceived to be less feasible for secondary schools than elementary schools for a variety of reasons (eg, lack of homeroom; individualized and complex class schedules, including off-campus education activities; classes have students from multiple grades; need to use lockers to retrieve textbooks and other belongings). reduced-schedule practices, such as shortening the school week or the school day, were perceived to be less feasible than withinschool practices in both elementary and secondary schools because of complexities related to scheduling, transportation, staff work hours, communication to families, food preparation and provision, and a variety of regulatory issues (eg, required in-person instructional hours, union rules requiring duty-free periods for teachers) listed in table . the need to arrange childcare was especially challenging for the parents of elementary school students. as shown in tables and , the within-school practices most frequently perceived as feasible in both primary and secondary settings included canceling field trips ( votes), canceling assemblies ( votes), rearranging desks to increase space between students in the classroom ( votes), restricting hall movement (elementary only) ( votes), and limiting nonessential staff and visitors ( votes). the practices most frequently identified as least feasible included moving class outdoors ( votes), staggering class start and dismissal times ( votes), separating classes into smaller groups ( votes), and shortening and staggering lunch times ( votes). the reduced-schedule practice considered the most feasible was a shortened school week affecting the entire school ( votes), and the least feasible was selective dismissal of one class or one grade in a school ( votes). we did not identify keeping one cohort of students together throughout the day is the norm in many elementary schools, though students often leave the classroom for specialty classes such as physical education, music, art. rearrange desks to increase space schools experiment with a range of seating configurations depending on grade level, students' needs, subject area, and available space and furniture. one participant discussed experience with this practice during the h n pandemic, noting that he configured students' seats so that they were no longer facing each other in a pod formation. hallway and bathroom practices restrict hall movement several participants stated walking single file, about a foot or an arm's length apart, is common in elementary schools and leads to orderly transitions between classes. it has also been used successfully during fire drills. limit bathroom use to reduce congestion in the bathroom bathroom use is highly grade level-specific. elementary school students are more likely to have common bathroom breaks when students use the bathroom in groups, while middle and high school students typically use bathroom passes. limit congestion during arrival and dismissal several participants reported limiting congestion by requiring students to report directly to their first class upon arrival and to remain in their last class until just before departing the campus. stagger class start and dismissal times to reduce load in hall a few participants reported experience with staggering the start and end time of the school day, and with staggering the class start and end times throughout the day without changing the overall length of the day (mainly for schoolwide activities such as assemblies). for instance, a participant reported modifying class start and dismissal times to separate the grades, reduce congestion in the halls, and protect the smaller and younger students. schoolyard and recess practices segregate recess area by class some elementary schools segregate the recess area by class (eg, one class is on the field and one is using the playground equipment). it is also common in smaller schools for each class to have its own time slot in the recess area. shortening the school day several participants described experience with this practice, often due to severe weather ( -or -h delay or early dismissal), or for professional development activities for staff. dismissal of one grade several participants described having used this practice, such as dismissing an entire grade for a field trip or for testing purposes. none any substantive variation in perceived feasibility by us region; however, moving class outdoors was considered more feasible in locations with mild climates. moving desks at least -ft apart to increase the physical distance between students is a social distancing measure that could be implemented in some classrooms. one participant discussed experience with this practice during the h n pandemic, noting that he configured students' seats so that they were no longer facing each other in a pod formation and indicated that this required some substantial "geometry." barriers to this practice did not vary by location in the united states; however, there was variation by grade level because different types of seating arrangements are used for different ages. barriers to implementing this practice included insufficient classroom space to spread desks out, inflexible seating arrangements and furniture, and negative impacts on students with special needs (eg, those who need to be located in the front row during instruction). participants indicated that flexible seating arrangements, such as desks and chairs that can be moved separately, would facilitate reconfiguring a classroom to create additional space between students during a pandemic. also, a region participant discussed how schools with limited space could rearrange students (eg, have all students face front, limiting face-to-face contact) without necessarily rearranging furniture. finally, a region participant discussed encouraging elementary-age students to fully utilize all the spaces in the classroom (eg, rug on the floor, rocking chair) rather than be limited to using desks and chairs at all times. he explained, they like gathering in a carpet area or … like a rocker or pillows. so, when they're doing their work, they can move around the room and find a different spot that's not at their table, and so that's a little bit more of a common practice at the elementary level. with a shortened school week, students do not attend days in a row. this practice can apply to required instructional time states typically require a minimum number of instructional hours or school days as a condition for funding. if these requirements are not met, schools, or particular classes, must make up missed time by meeting on holidays or weekends or extending the school day. required physical activity (including recess and physical education) participants discussed some legal and regulatory challenges in decreasing or eliminating recess time or physical education class as part of a within-school practice or as a result of reduced-schedule practices, including the fact that some communities have requirements about the number of physical activity minutes students must receive. required duty-free or planning time for staff participants cautioned against asking teachers or other staff to supervise students at times that were previously designated as "duty-free" or reserved as planning periods. to extend duty hours, schools would either need to hire substitutes to supervise students or ask existing staff to assume the extra responsibilities, in some cases violating union terms. required time for certification a variety of practices can harm students' ability to earn credit or fulfill requirements for certification. for example, certain career and technical programs require students to complete a certain number of hours to meet the criteria for certification. legal obligation to adhere to individualized education plans (iep) a number of practices (eg, movement restriction in class, cancellation of cross-school transfer, reducing school hours) can conflict with the ieps of special needs students. federal law obliges schools to adhere to ieps. mandated standardized testing a number of practices (eg, shut down computer lab, reduce school hours) can interfere with students' preparation for or the actual delivery of mandated standardized testing. many communities require that teachers have certain qualifications (eg, be certified, pass a background check) as a condition of employment. such requirements can be barriers to hiring additional staff in an emergency. the entire school (eg, all students attend monday through wednesday only), or the school can alternate so that some students, for example, attend monday and tuesday and the others attend thursday and friday. several focus group participants noted that their districts were already operating on a -day week because of budget constraints. the leading barriers to implementation of this practice include burden on parents to find childcare, impact on students who rely on schools for meals, need to make up missed instructional hours, challenging communication with parents about complex school schedules especially with alternating days, lower educational quality, and burden on staff. these barriers did not vary significantly by region of the country or grade level, with the exception of childcare needs being more pronounced for elementary-aged children. participants identified several facilitators to this practice. many felt that a shortened school week would be less of a burden than shortened days on parents and on the bus transportation system because the disruption would be limited to to days per week rather than all school days. participants recommended having the whole district or at least all schools within the same feeder cluster operate on the same schedule to reduce some of the complexities for families with multiple children. factors that could increase the feasibility of this practice include giving parents plenty of advance notice, engaging key stake-holders to explain why and how the new schedule will work, obtaining a waiver from the state so that district funding is not reduced, putting a distance learning plan in place to continue instruction when students are at home, and offering meal programs off the campus for students not attending school on a given day. through focus groups in all hhs regions, we identified and reviewed social distancing practices that schools could implement in an influenza pandemic while continuing to operate. participants reported prior experience with several within-school practices in elementary schools as a part of routine (nonemergency) operations. these practices included homeroom stay, restriction of hall movement, segregation of recess area by class, staggering of recess times, segregation of cafeteria by class, and staggering of lunch periods. within-school practices were generally perceived to be more feasible for elementary schools than secondary schools. reduced-schedule practices, such as shortening the school week or the school day, were perceived to be less feasible than within-school practices in both settings. the practices considered the most feasible were those that involved isolated events like field trips and all-school assemblies or furniture rearrangement that had little to no staffing, curricular, or cost table ). c considered feasible for elementary schools only. implications. in general, the practices considered the least feasible were those that required smaller classes, more staff, more square footage, or staggered start and end times. these were considered less feasible because they require hiring more staff on short notice, rewriting lesson plans, altering parent work schedules on a daily and prolonged basis, or finding more space in already crowded buildings. within-school practices that do not change the academic calendar in any way are easier to implement than reduced-schedule practices. in addition, reduced-schedule practices that affect the entire school rather than subgroups of students are easier to implement because an alternating schedule presents additional challenges (eg, need for additional bus routes, extra burden on parents with students on different schedules, and need to explain complex scheduling with clear, consistent messages). state and district policies on required instructional time, required physical activity hours, duty-free periods for teachers, professional development hours for teachers, and/or teacher qualifications were frequently mentioned as key barriers to making significant changes to school schedules and supervision of students. schools can surmount some of these regulatory legal and regulatory barriers by seeking waivers and building flexibility into staff contracts. our findings indicate that several within-school practices could be implemented in elementary schools to reduce the transmission of influenza (eg, keeping students in their homerooms for the entire school day, restricting hall movement, segregating recess area by class, staggering recess times, segregating the cafeteria by class, staggering lunch periods). these practices might be more effective in reducing disease transmission across classrooms than within a classroom. practices such as canceling assemblies or field trips might be feasible but might not have a sustained impact on disease transmission because they are onetime events rather than practices that alter the nature of social interactions at school. implementation of some practices, such as rearranging desks to increase space, might be feasible but might not keep students from mixing, as it would be difficult to limit students' movement in class (eg, require students to stay seated at all times). students are likely to continue moving within the room and interacting with their peers. among the reduced-schedule practices, shortening the school week for the entire school was perceived as the most feasible. shortening the school week might be an alternative to prolonged school closure. to our knowledge, our study represents the first one to comprehensively assess the feasibility of social distancing practices in schools. this study builds on our previous review of the literature, which found that neither the peer-reviewed and gray literature nor the pandemic influenza guidance and plans included details on the range of potential practices or on the barriers schools would likely encounter in implementing social distancing practices. therefore, public health and school leaders had limited evidence to inform decisions about social distancing in schools. our previous literature review identified epidemiological and modeling studies that assessed the effect of selected school practices on reducing influenza transmission. in addition to school closures, these studies considered limiting use of congregation spaces such as the cafeteria as well as a number of social distancing practices that our study identified as infeasible (eg, class and grade dismissal, classroom movement restrictions, and staggered classroom schedules). an additional modeling study reported that shortening school week may be effective in reducing virus transmission. hence, further epidemiologic and modeling efforts are needed to explore the range of effects of social distancing practices that our study identified as feasible according to educators. this study has a number of limitations. first, we did not include parents because we chose to focus on educators' implementation challenges in school settings. while parents are crucial to ensuring compliance, they do not make the decision to implement social distancing measures and are not tasked with enforcing them in schools. nonetheless, many focus group participants are also parents or report to them, and they commented on how practices would be received by parents. second, although we engaged numerous stakeholders and achieved thematic saturation, findings represent the perspectives of focus group participants and might not be generalizable. third, we focused on the united states and did not engage school leaders from other countries, nor assess the implications of this work for an international audience. future research should explore the feasibility of school social distancing practices in a wide range of developed and developing countries. fourth, due to time constraints, some focus groups discussed within-school practices and others discussed reducedschedule practices. fifth, we explored perceptions of feasibility. more research is needed on the effectiveness of identified practices on reducing disease ■ schools and public health officials can jointly consider multiple practices to reduce influenza transmission during a pandemic as an alternative to closing. ■ these practices can also be considered in other outbreaks of infectious disease that affect school settings. ■ practices vary with respect to feasibility, with practices that can be implemented as part of the school day (vs those that affect school hours) considered the most feasible. transmission. before such evaluations are completed, infectious disease transmission experts can consider our data to determine which of the top-rated practices may be helpful in reducing school-based transmission of the disease(s) of interest. the study's strengths included the following: the study was based on a large number of focus groups, included participants from all hhs regions, and assessed feasibility by primary versus secondary school. in summary, our findings suggest that schools have options to increase social distance during an influenza pandemic or other infectious disease outbreak as alternatives to closing. given that influenza pandemics occur in waves and can last for months, it is critical to identify and consider alternatives to extended school closure, which is burdensome to students, parents, and employers. future research should evaluate the effects and optimal timing and duration of a set of seemingly feasible practices on influenza transmission in schools, given that selecting effective social distancing practices is the ultimate goal of policy makers and practitioners. in addition, feasibility and acceptability of the most promising practices will eventually need to be evaluated among other audiences, including parents and students. community mitigation guidelines to prevent pandemic influenza-united states the effects of school closures on influenza outbreaks and pandemics: systematic review of simulation studies school closures and influenza: systematic review of epidemiological studies evidence compendium and advice on social distancing and other related measures for response to an influenza pandemic school opening dates predict pandemic influenza a(h n ) outbreaks in the united states spatial transmission of pandemic influenza in the us emergency preparedness and response: school dismissals to reduce transmission of pandemic influenza school practices to promote social distancing in k- schools: review of influenza pandemic policies and practices weekends as social distancing and their effect on the spread of influenza key: cord- -ivv x authors: odongo, david otieno; wakhungu, w. j.; stanley, omuterema title: causes of variability in prevalence rates of communicable diseases among secondary school students in kisumu county, kenya date: - - journal: z gesundh wiss doi: . /s - - - sha: doc_id: cord_uid: ivv x purpose: to determine causes of variability in communicable disease prevalence rates among students in secondary schools to inform policy formulation in the public health sector. methods: a representative cluster sample size for students was estimated using fisher et al.’s formula while schools, sub-counties and education zones were clustered and sample size was calculated based on coefficient of variation by school type. data were collected by questionnaire, medical examination using standard procedures, and focus group discussion, and descriptive analysis was performed on the completed questions. comparisons between risk factors were made by chi-square and anova analysis using spss for windows (version . ; chicago, il) software. a p value of < . was considered statistically significant. results: there was significant variation between communicable disease prevalence rates and age (x( ) ( , . ) = . ), school size (x( ) ( , . ) = . ), gender (x( ) ( , . ) = . ) and class of students (x( ) ( , . ) = . ), and bed and desk spacing (p < . at % ci). however, there was no significant association in prevalence rates between both locality and type of school. there was strong evidence that student age has an effect on prevalence rates. the prevalence rate of malaria was higher in male ( . %) than female students ( . %) compared to prevalence of diarrhea, which was higher in female ( . %) than male students. conclusion: this study has revealed that the prevalences of diarrhea, tuberculosis, pneumonia and other respiratory tract infections are lower among female secondary school students than males and that the prevalence of malaria is higher in males than females. age of secondary school students is a significant vulnerability factor for malaria, diarrhea, tuberculosis and pneumonia, which were the important communicable diseases most prevalent among secondary school students in kisumu county, kenya. the majority of kenyans continue to seek treatment in health care facilities for ailments that can be controlled through preventive and promotive measures (who ) , with malaria as the leading cause of morbidity at % followed by respiratory diseases ( . %). tuberculosis control has been more challenging, with high tuberculosis (tb) prevalence of per , , tb/hiv co-infection prevalence of % and a growing threat of multidrug resistance/extra-drug resistance (mdr/xdr) tb (who ) . overcrowding and intermittent use of antibiotics are some of the challenges facing tb control. kisumu county suffers from a high burden of communicable diseases as well as emerging threats. according to the kenya demographic and health survey by the government of kenya ( ) , the county has one of the highest hiv/aids prevalence rates at % higher than the nyanza region rate of . % and national rate of . %. these results have been replicated by a demographic and health survey in kisumu west (kwhdss ), which found high levels of hiv/aids, diarrhea, malaria, multi-drug resistant tb (mdr-tb) and other communicable diseases with more than half of the population relying on surface water as the main source of drinking water while % of the households share toilets and % have no toilets. being a highly disaster-prone county, floods and drought affect different geographical zones annually with a varying degree of damage to the health infrastructure and people's health, causing interruption of the access to safe water and collapse of the sanitation infrastructure (mournie ) . these facts indicate that many people in kisumu county, students included, are at risk of contracting communicable diseases such as diarrhea, typhoid, intestinal parasite infections, trachoma and schistosomiasis, among others, which account for millions of school days lost (cdc ) . a major contributing factor to this burden of communicable disease is inadequate access to safe water and sanitation infrastructures (boschi-pinto et al. ) . the introduction of free schooling in public schools in kenya has witnessed increased enrollment in secondary schools with the number rising from . million in to . million in (gok a, b), leading to increased student membership in the existing hostels and other social amenities in the schools. at present, there is no national guideline to provide a framework for the transformation of school health service into an integrated county health service, while strategies and interventions to work with other ministries have not been spelled out in kenya's health sector strategic plan, - (gok a . integration of school health services into national and county health services will ensure timely surveillance, prevention and treatment of communicable diseases in schools. high school students are a neglected group in which very little research has been done concerning the health challenges they face in schools (muna ) . there is a need to determine the causes of variability in communicable disease prevalence rates among students to provide baseline information that can be used by policy makers to develop viable intervention programs to address the communicable disease burden in schools. the study applied mixed methods to collect and analyze public health-related risk factors using a correlational research design. the first three sub-counties out of the six were purposively selected based on the decreasing value of the coefficient of variation by student gender and type of school. a representative cluster sample size (n = ) for , students was estimated using fisher et al.'s formula, while the sample size per sub-county was based on the coefficient of variation by student gender. thirty percent of schools, out of , were selected based on the coefficient of variation by school type. students in sampled schools were clustered and selected by cluster sampling. key informants and observation units were purposively sampled, while focus group discussions were sampled by quota. a series of data collection tools were used. these included: ( ) use of a questionnaire to collect data on the locality of the school, type of school, size/enrollment of the school, demographic information and morbidity of students. bed spacing in hostels and desk spacing in classrooms were observed using an observation checklist. ( ) focus group discussion guide used to listen and gather information from different people to corroborate data from the field. thematic issues included groups' perceptions of skill-based education and a communicable disease surveillance system. an in-depth interview guide was used for index cases. students who did not self-report clinically confirmed illnesses in the questionnaire were taken to the nearest health facility for medical examination. the observed medical examination results of interest were: (a) blood slides testing positive for the malaria parasite; (b) a positive culture for mycrobacterium tuberculosis confirming tuberculosis infection. an acceptable sputum specimen had more than leukocytes and fewer than epithelial cells per lower field. the most common pathogens detected were bacteria such as streptococcus pneumoniae, staphylococcus aureus and klebsiella species. sensitivity testing was done for positive results; (c) positive rapid urine antigen testing for streptococcus pneumonia; (d) positive stool tests for clostridium difficile for respondents having diarrhea or watery stools. other tests done were an antigen test for notavirus, ova and parasite examinations and antigen tests specific for the parasites g i a r d i a l a m b i a , e n t a m o e b a , h i s t o l y c a , crystosporidium and parvum. ( ) a descriptive analysis was performed on completed questions. comparisons between risk factors were made by chi-square and anova analysis using sas version . . (carry, nc, usa). a p value < . was considered statistically significant. we ensured the study was conducted within the international standard procedures for medical examinations. as a result medical examinations were carried out in health facilities in the neighborhood of the schools and performed by medical professionals. verbal consent from participants was obtained before any data were collected. participants were informed about the intention to utilize data collected for the dissertation and then intervention. research authorization was sought from the school of graduate studies at masinde muliro university of science and technology and the administration of kisumu county, kenya. there were ( %) respondents, male and female students sampled in schools. characteristics of respondents, including age, gender and class showed significant variation, p < . ; however, there was no significant variation in the religion of respondents. the most predominant student age bracket was - years, forming . % of the sampled population. we observed wide geographical variations in prevalence rates of communicable diseases among schools with the highest being malaria at . % and the lowest being pneumonia at . % (fig. ) . there was significant variation in the prevalence of communicable diseases among secondary schools in kisumu county. this study found a significant association between the communicable disease prevalence rates and age (x , . = . , fig. ) of students, school size (x , . = . , fig. )/enrollment, gender (x , . = . , fig. ) and class (x , . = . , fig. )/form of student, bed spacing in hostels (x , . = . , fig. ) and desk spacing in classrooms. however, for both the locality and type of school there was no significant association with prevalence rates of communicable diseases. further analysis using anova showed strong evidence that age has an effect on prevalence rates of communicable diseases among secondary school students. the prevalence rates of tuberculosis and pneumonia were . and . % in schools where standard bed spacing in hostels was not observed and % for other observations. a significant association between bed spacing and prevalence rates of tuberculosis and pneumonia was observed among students; however, there was no strong evidence that the intervention had an effect. prevalence rates of tuberculosis and pneumonia were high at . and . %, respectively, in schools where standard desk spacing in classrooms was not observed and almost zero percent in schools that observed standard bed spacing. desk spacing in classrooms and prevalence rates of tuberculosis and level of education of respondent p < . ; x , . = . p = . at %confidence interval; df = . prevalence rate (%) n= -malaria -diarrhea -tuberculosis -pneumonia -other respiratory tract infecƟons -pregnancy related -other illnesses fig. prevalence of communicable diseases in secondary schools in kisumu county, kenya pneumonia had a significant association (x , . = . , fig. ) but there was no strong evidence that the intervention had an effect. the prevalence of malaria varies widely from area to area, as has been shown by several studies in uganda (nankabirwa et al. ; pullan et al. ; kabatereine et al. ). the findings of these studies, which showed that in uganda - % of school-age children have parasitemia at any one time, concurs with the determined prevalence rate of malaria that we observed ( fig. ; x , . = . ). this also concurs with the results of a study on the prevalence of malaria parasitema by gitonga and others ( ) in kenyan schools between september and march , which found an overall prevalence rate of %; however, this ranged from to %. it also agrees with the findings of a study by dai et al. ( ) ; oduro et al. ( ) and others for senegal, the gambia, and mauritania in which the prevalence of malaria infection in school-age children ranged from to %. it is very concerning that malaria prevalence among secondary school students may interfere with their educational development. the effect of malaria infection on school absenteeism has been confirmed in several studies (leighton and foster ; brooker et al. ) showing that it contributes to between and % of school absenteeism per year. studies by lengeler ( ) and lim et al. ( ) have shown there is strong evidence that, at the individual level, regular use of an insecticide-treated net (itn) or long-lasting insecticide-treated nets (llin) substantially lowers the risk of malaria infection. this may be attributed to the fact that as children become older and more independent, parents have less control over the time when they go to bed, where they sleep and whether they use a net, frequently resulting in low fig. prevalence rates related to student gender net coverage in children in this age group. however, evidence is needed to concretize the reasoning. this study has revealed that student age is a predisposing factor to infectious diseases (fig. ) . the same results were observed in a study by baker et al. ( ) on infectious diseases attributable to household crowding in new zealand, which showed that children less than years old exposed to greater household crowding had . times the odds of pneumonia as children exposed to the least crowding. the findings of this study showed that the prevalence of tuberculosis and pneumonia is high among students where there is overcrowding in hostels and classrooms (figs. and ) . it also revealed that students in the - -year age bracket have a higher incidence of tuberculosis and pneumonia than those in the years and greater age bracket . findings by the cdc ( ) also confirm these results. the world health organization (who ) , while addressing sex and gender in epidemic-prone infectious diseases, found that differences between males and females arise because gender-based roles, behavior and power. for most infectious diseases (fig. ) , the difference in prevalence rates between males and females is more likely to be due to differences in exposure than to differences in immunity. for example, in many societies females spend more time at home than males during the day and therefore experience greater daytime household exposure to infections, for instance caring for the sick and changing baby diapers, than males. male students, in their normal lives, spend more time outside the households in the evening than female students, so they are more exposed to mosquito bites than females. this study has revealed that the prevalences of malaria, tuberculosis, pneumonia and other respiratory tract infections are lower among female than male secondary students. for reasons that are not well understood, a study by who ( ) found that females had lower mortality rates from severe acute respiratory syndrome than males, a pattern that was maintained after adjusting for age. despite the scarcity of information, there are strong indications that sex and gender are necessary for the transmission and control of epidemic-prone diseases. age of secondary school students is a significant vulnerability factor for malaria, diarrhea, tuberculosis and pneumonia, which were the important communicable diseases most prevalent among secondary school students in kisumu county, kenya. acknowledgments this work was not supported by any organization. its contents are solely the responsibility of the authors. all authors report no conflicts of interest. the authors would like to thank the members of the centre for disaster management and humanitarian assistance of masinde muliro university of science and technology for their continued guidance during the research. funding this research was fully funded by the corresponding author who is a phd student at masinde muliro university of science and technology. the research was in pursuit of an academic degree and therefore the other two authors were supervisors. ethical approval research authorization was granted by the school of graduate studies of masinde muliro university of science and technology and the administrators of kisumu county, kenya. all procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional research committee and with the helsinki declaration and its later amendments or comparable ethical standards. as a result, medical procedures were carried out in health facilities in the neighborhood of the schools and performed by medical professionals. informed consent verbal consent from participants was obtained prior to the commencement of focus group discussion, in-depth interviews, use of the questionnaire, school transect walking during the environmental health observation and medical examination. permission to capture some of the research sessions on tape was obtained prior to the event. participants were informed of the intention to utilize data collected for the dissertation. infectious diseases attributable to household crowding in new zealand: a systematic review and burden of disease estimate estimating child mortality due to diarrhea in developing countries situation analysis of malaria in school-age children in kenya-what can be done? summary health statistics for us children: national health interview survey bionomics of malaria vectors and relationship with malaria transmission and epidemiology in three physiographic zones in senegal river basin transforming health; accelerating attainment of health goals government of kenya (gok) ( ) population and housing census. ministry of planning. government printer kisumu west health and demographic surveillance system economic impacts of malaria in kenya and nigeria, report submitted to the office of health. usaid, washington lengeler c ( ) insecticide treated bed nets and curtains for preventing malaria net benefits: a multicounty analysis of observational data examining associations between insecticides treated mosquito nets and health outcomes health needs assessment for kisumu effect of zinc supplementation on morbidity due to diarrhea in infants and children in elsabeen hospital in sana'a, yemen. who regional office for eastern seroepidemiological and parasitological evaluation of the heterogeneity of malaria infection in the gambia plasmodium infection and its risk factors in eastern uganda kenya: epidemiological country profile on hiv and aids. world health organization and unaids communicable diseases epidemiological profile for horn of africa. world health organization, geneva world health organization the authors have not received any grants from any companies. all authors report no conflicts of interest.open access this article is distributed under the terms of the creative comm ons attribution . international license (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord- -y ufxwhq authors: bahn, geon ho title: coronavirus disease , school closures, and children’s mental health date: - - journal: soa chongsonyon chongsin uihak doi: . /jkacap. sha: doc_id: cord_uid: y ufxwhq coronavirus disease (covid- ), which was first identified in wuhan, china, in late december of is rapidly spreading across the globe. the south korean government has ordered the closure of all schools, as part of its attempts to use social distancing measures to prevent the spread of covid- . the effects of the school closures on reducing contagion are generally positive; however, the measure is controversial because of the socioeconomic ripple effect that accompanies it. the author briefly reviewed the existing literature on the mental health aspects of disasters and presents the issues related to school closures due to pandemics, from medical and socioeconomic perspectives and in terms of children’s mental health. the results of this review suggest that research on children’s mental health in relation to the adoption of school closures as a pandemic mitigation strategy is urgently needed. a cluster of pneumonia cases of unknown origin from wuhan, hubei province, was reported to the china national health commission on december , [ ] . one week later, chinese scientists had isolated a novel coronavirus from these patients. the report of a novel coronavirus outbreak was published online by the lancet on january , . according to the report, new patients from countries other than china, such as thailand, korea, and japan, had been confirmed as having coronavirus disease (covid- ) [ ] . the number of confirmed cases has increased exponentially, and new cases have been reported worldwide [ ] . based on past research on the severe acute respiratory syndrome (sars) epidemic of [ ] and the middle east respiratory syndrome coronavirus (mers-cov) epidemic of to [ ] , which were both types of coronavirus, it can be surmised that co-vid- affects not only physical disabilities but also mental health [ ] . while there have been several reports on the mental health problems faced by medical workers in wuhan (such as stress, anxiety, depressive symptoms, insomnia, denial, anger, and fear) [ ] , the mental health problems of the patients with covid- have yet to be thoroughly investigated [ ] . furthermore, countermeasures employed by american [ ] , chinese [ ] , and korean psychiatrists [ ] have been found to be insufficient to predict or resolve these mental health problems, especially those of infants, children, and adolescents. school closure, a social distancing strategy, is considered to effectively reduce community transmission [ ] . as the cov-id- outbreak enters its third month, the ministry of education in korea has postponed the new school year from the first of march to weeks later for kindergarten, elementary, middle, and high schools across the country. all colleges and universities have also decided to postpone the beginning of the new semester for weeks or more. most childcare centers nationwide have been closed indefinitely for several weeks. with schools and institutions shutdown, the author, as one of the professionals in the field of children's mental health, is not only deeply concerned about the covid- outbreak, but also about the mental health of our children. how will the prolonged epidemic and school closures affect children's mental health? how should mental health professionals prepare and for what? what should be our focus if the disaster continues for longer than we expect? although an examination of all obstacles and possible solutions may not be possible at the moment, this study reviewed the existing literature for methods (even temporary or incomplete ones) to address children's mental health issues that are related to the covid- outbreak and resultant school closures. the disasters and stressors experienced by humans include life/developmental stressors, such as parental divorce or vehicle accidents, human-generated acts of violence, such as rape or war, and natural disasters, such as earthquakes or pandemics [ ] . studying previous disasters can help us prepare for new ones, even in the types of disasters are different from one another. the / attacks (an example of human-generated acts of violence) were a one-time planned attack that was traumatizing to many people, both directly and indirectly. in a child psychopathology study with students, conducted months after / [ ] , it was found that children of parents who lost their jobs or were restricted in their travel after / , were likely to experience posttraumatic stress disorder or other anxiety disorders. experiences of disaster-related adversity extend beyond traumatic exposure and include the long-term ripple effects of post-disaster life issues and economic difficulties. therefore, mental health services that focus on the impact of disasters on students' lives are needed. one study conducted to years after the / attacks, on adolescents and family members, found that minority, low-income, and single-parent adolescents were the most affected groups [ ] . if both the parents and their children are likely to develop post-disaster mental health symptoms, the official in charge should be particularly concerned. during the sars outbreak, mild to serious psychiatric symptoms were reported: patients diagnosed with sars experienced anxiety, loneliness, boredom, anger, and worry about the effects of quarantine and contagion on family members and friends; medical staff experienced fear of contagion and of infecting others; and uncertainty and stigmatization was experienced by both staff and patients [ ] . the human immunodeficiency virus/acquired immunodeficiency syndrome (hiv/aids) pandemic evolved to become the second leading cause of disability and the fourth leading cause of mortality worldwide by the end of the th century [ ] . the results from a review of published papers on hiv/aids suggest that mental health professionals are in a unique position to adapt and apply the strategies that they use to treat psychiatric symptoms to prevent engagement in hiv transmission behaviors and, therefore, play a critical role in curbing the epidemic [ ] . mental health professionals in china have developed co-vid- outbreak countermeasures for patients, their families, and medical staff based on methods used in the sars outbreak [ ] : first, mental health support should be provided by multidisciplinary mental health teams that have been es-tablished by health authorities at regional and national levels. second, there must be clear communication and regular and accurate updates about the covid- outbreak, in order to reduce the sense of uncertainty and fear. third, services to provide psychological counselling using secure electronic devices and applications must be available. fourth, patients who have experienced disasters should be screened regularly for depression, anxiety, and suicidality. if necessary, personalized psychiatric treatment that has been specifically tailored for the individual patient, including psychotherapy and psychotropic medication, should be provided [ ] . phone counselling should be employed because face-to-face therapy and interviews with play therapy are to be avoided due to the possibility of infection. therefore, it is necessary to develop programs that use the internet and smartphones, and social workers, family therapists, pediatricians, and community-related agencies, as well as psychiatrists, should be required to undergo training on these programs [ ] . when pandemic infectious diseases occur, school closures are a social distancing measure that is often conducted to reduce their impact [ ] . however, there is little data on the short-and long-term consequences. the effects may vary depending on the timing of the closure decision and regional consistency. non-pharmacological interventions, including school closures, church closures, the banning of mass gatherings, mandated mask wearing, isolation, and disinfection and hygiene measures were implemented during the spanish influenza of to in the united states. these interventions were determined in the early stages of the epidemic and implemented for a long time [ ] . they had a moderate effect on the total mortality rate (perhaps reducing mortality by - %), and contributed greatly to the peak mortality reduction (around % in some cities) [ ] . public health measures, including school closures, implemented in sydney, australia, in might have reduced cumulative attack rates during the influenza pandemic by up to % [ ] . during the - asian flu pandemic, closing schools and keeping children and teenagers at home contributed to a reduction of the attack rate by over % [ ] . in the case of sars in hong kong, in , government mandates and public concern resulted in considerably lower levels of confirmed influenza compared with the preceding five years [ ] . interventions such as school closures, banned mass gatherings, mask wearing in public, and staying at home as much as possible worked well. a large-scale survey of eight different european countries conducted to determine the basic reproduction number on weekdays versus weekends and during regular versus holiday periods, found that school closures can have a substantial impact on the spread of a newly emerging infectious disease that is transmitted via non-sexual close contacts [ ] . with the first case of h n flu on may , , the governor of osaka, japan, decided to close all high schools and junior high schools in osaka prefecture from monday, may , to sunday, may , following the weekend days of may and observed at most schools [ ] . the prefecture-wide school closure strategy might have had an effect on the reduction of virus transmission and elimination of successive large outbreaks. in addition, it resulted in greater public awareness about the need for preventive measures. in alberta, canada, school closure reduced transmission of pandemic influenza (ph n ) among students by more than %, and this was a key factor in interrupting transmission in countries with social contact networks similar to those in canada [ ] . results of a household survey conducted in china on the socio-economic impact and inconveniences of school closures due to the influenza a (h n ) pandemic indicated that the majority ( %) of families supported the policy despite economic inconvenience [ ] . in a small, rural community in north carolina in the united states, % of households were in favor of school closures as a community mitigation strategy against the outbreak of influenza b [ ] . in contrast, late or delayed decisions to close schools can be ineffective, as was the case in france during the pandemic [ ] . at that time, public health officials were concerned that early school closures could increase public anxiety and create a sense of crisis. the decision was delayed until after - % of children had fallen ill. rodriguez et al. [ ] examined the hypothesis that school closures during an influenza outbreak would result in lower absenteeism after the return to school. in , among schools in king county, washington, in the united states, closed during a seasonal influenza outbreak, while remained in session. there was no significant difference in post-break absenteeism between the schools that had closed and those that remained in session. one study in singapore [ ] investigated the ) public holiday effect, ) school vacation effect, and ) school closure during outbreaks effect on hand, foot, and mouth disease (hfmd) transmission with a large sample, for several years. while the results of that study provided evidence that school closure reduces hfmd transmission [ ] , the effect was small. given the mild and self-limiting cases of hfmd and large burden following a school closure, the evidence from these reports suggests that policy makers should weigh very carefully whether the effect (particularly, the economic burden on families) of school closures for the mitigation of a pandemic is tolerable before deciding to implement them. are not yet known [ ] , one study reports that the appropriate control measures for the disease include a quarantine and an observation period of days for suspected cases, based on a mean incubation period of . days, with the th percentile of the distribution at . days [ ] . one of the questions associated with school shutdown is when to reopen the schools. deciding on whether or not to close schools, one of the main factors to consider is the economic impact on the households involved and on society [ ] . when schools are closed, parents who have to go to work should quickly find people or facilities to take care of their children. otherwise, it is inevitable that the child stays at home alone [ ] . generally, institution closings are a more serious and urgent issue for parents of toddlers and kindergarteners, than they are for parents of elementary, middle, and high school students [ ] . kids attending such programs are too young to help themselves, and, therefore, one of the parents has to work less or even has to quit his or her job when the institution closes, which will most likely result in the family experiencing economic difficulties [ ] . after the closing of childcare centers, parents who work need different sources of care for their children. studies suggest that respiratory infection rates are lower in childcare groups with less than six children [ ] . it is very difficult to find such facilities at a time when the institutions are shut down because of a pandemic. in response of covid- outbreak, the korean government has developed nationwide emergency child care programs for parents who need child care support because of extended closures at child care centers, kindergarten schools, and elementary schools [ ] . in addition, for low-income households or for families in which the parents' marriage is unstable, long school closure durations can lead to more difficulty in learning for the child [ ] . in the longer term, the more likely the child will be to drop out. also, students who rely on the free lunch served at school can face related problems [ ] . closing childcare programs or kindergarten schools could cause serious financial implications for these institutions and their employees [ ] . many childcare programs are small business operations or are run by faith-based institutions. closing these programs because of a pandemic could lead to longterm interruption in the provision of such services. although there is evidence that school closures can mitigate the scope of an epidemic, they have substantial impact on the economy of a society, including the enormous cost of lost productivity due to parental absence from work [ ] . for parents who cannot quit their jobs, this situation can cause distress. the impact of school closures, thus, will vary depending on socioeconomic factors and the home environment. if the school closure were to last months, % of low-income households would be faced with serious financial problems as opposed to % of high-income households [ ] . this proportion drops to % and %, respectively, if people stay home for month. one uk study found that about % of the uk gross domestic product could be affected by a -week school closure [ ] . using a stochastic individual-level model to simulate severe pandemic influenza in the united states, it was found that total costs of strategies involving school closures for weeks was approximately to times more compared with the single intervention strategies of prevaccination or full-targeted antiviral post-exposure prophylaxis [ ] . this finding indicates that strategies that involve school closures can reduce the us gross domestic product by . %. through the assessment of national-level preparedness for epidemic and pandemic outbreaks, researchers developed an epidemic preparedness index (epi). the epi consists of five sub-indices measuring the economic resources, public health communications, infrastructure, public health systems, and institutional capacity of countries [ ] . although the epi provides global comparative standards, it does not consider pandemic-specific and age-specific factors. there is very little mental health data on the school closures that can occur during a pandemic, compared with disasters caused by terrorism or traffic accidents. to the best of the author's knowledge, this is the first review on children's mental health to examine school closures during pandemics. three topics regarding children's mental health and school closures were examined in order to support children's development during and after the covid- outbreak: fear management, misinformation control, and strengthening resilience (fig. ) . infectious diseases can cause fear and anxiety among children. parents can relieve their children's fears and anxieties by explaining covid- to them in terms they can understand [ ] . stress can build up when children have to stay at home and playing outside is banned because of a situation such as a pandemic [ ] . indoor physical activities that allow families to relax together are helpful. parents should help children to maintain a regular daily life with activities they can perform indoors. if a family member or a relative is diagnosed with a pandemic disease, the stress in the family increases. if a child or a parent is hospitalized and confirmed as having covid- , anxiety can increase because of family isolation. depending on the child's situation, counseling or short-term psychotherapy may be helpful [ ] . assessment for acute stress disorder or posttraumatic stress disorder may be required in cases of severe fear or anxiety. guidelines provided by government agencies and expert organizations offer useful information for treating anxiety [ , ] . the internet has a considerable amount of misinformation about covid- , including fake news. children will be vulnerable to such exaggerated news and misinformation because they do not have enough knowledge or the capacity to differentiate between fact and fiction [ ] . if contents from the media, including the social networking service (sns), are not managed properly, the risk for chaos and the likelihood of a surge of fear among the population regarding medical care facilities will be heightened. misinformation and fear can lead to discrimination and stigmatization. this may lead to children rejecting or avoiding proper medical interventions. parents should evaluate the information their children are receiving about covid- on social media and internet websites [ ] . mental health professionals frequently update co-vid- information through the sns or the websites of expert groups to minimize damage due to misinformation. the purpose of the school closures is to prevent the spread of the pandemic and to protect children from the virus. however, school closings can hinder the healthy development of children [ ] . closing schools deprives children of schoolbased peer interactions and their daily routine. that is, in ad- enhance post-disaster growth dition to being psychologically and physically vulnerable during the pandemic, the children will also experience disruptions in their development or delays in their educational progress [ ] . parents should help children who have to stay at home to create a daily routine at home, and also allow the use of sns or smartphones so that children with limited outdoor activities can communicate with their friends [ ] . children need resilience to restore pre-disaster levels of function and psychological equilibrium [ ] . improvements in access to resources, the encouragement of participation, and equitable treatment are required in order to promote children's resilience to disasters [ ] . support and cooperation from families and communities are also needed. as a method of promoting strength, parents should spend more leisure time with their children than usual. strengthening resilience can promote post-disaster growth [ ] . future research should examine the factors mediating the potential for post-disaster growth that are related to covid- and school closures, especially during children's developmental period. the members of the korean academy of child and adolescent psychiatry have gained much experience in the field of disaster psychiatry through several serious incidents, including a single incident of death witnessed by fourth graders at an elementary school in [ ] , suicidal death among elementary school students from to [ ] , and the sewol ferry disaster of [ ] . the academy organized a new committee for disaster and trauma, and the committee created guidelines on the covid- outbreak, including "coping with the stress of covid- epidemic: tips for families getting through school closing period" [ ] and "tips for adolescents: coping with the stress of covid- epidemic" [ ] . the guidelines were published in korean and english for use by the general public immediately after the co-vid- outbreak. infants and children are vulnerable to diseases and, therefore, society must develop measures to protect them in the event of an epidemic such as covid- . infectious disease management personnel should establish a system to cooperate with educational institutions, hospitals, medical personnel, nurseries for infants and toddlers, and mental health service providers. unexpectedly, the covid- outbreak has lasted for months and has extended to many people and many countries, prompting an unprecedented nationwide order of daycare centers; elementary, middle, and high schools; and colleges to close. mental health professionals should prepare for patients with mental health problems related to long school closures and the reopening of schools. they should also pre-dict and take countermeasures against any problems that may arise in the near future and in the long run. a novel coronavirus outbreak of global health concern what we know so far: covid- current clinical knowledge and research long-term psychiatric morbidities among sars survivors psychological impact of the mers outbreak on hospital workers and quarantined hemodialysis patients timely mental health care for the novel coronavirus outbreak is urgently needed the mental health of medical workers in wuhan, china dealing with the novel coronavirus mental health care measures in response to the novel coronavirus outbreak in korea covid- ) mental health guideline for the psychological protection from covid- school closures during the influenza pandemic: national and local experiences care of children exposed to the traumatic effects of disaster attack-related life disruption and child psychopathology in new york city public schoolchildren -months post- / adolescent behavior and ptsd - years after the world trade center terrorist attacks of the immediate psychological and occupational impact of the sars outbreak in a teaching hospital youths and hiv/aids: psychiatry's role in a changing epidemic online mental health services in china during the covid- outbreak estimating the impact of school closure on influenza transmission from sentinel data quantifying social distancing arising from pandemic influenza targeted social distancing design for pandemic influenza respiratory infections during sars outbreak estimating the impact of school closure on social mixing behaviour and the transmission of close contact infections in eight european countries influenza (h n ) outbreak and school closure, osaka prefecture effects of school closure on incidence of pandemic influenza in alberta, canada social and economic impact of school closure resulting from pandemic influenza a/h n household responses to school closure resulting from outbreak of influenza b, north carolina association between school closure and subsequent absenteeism during a seasonal influenza epidemic the effect of school closure on hand, foot, and mouth disease transmission in singapore: a modeling approach korean society of epidemiology -ncov task force team. an interim review of the epidemiological characteristics of novel coronavirus early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia dynamic modelling of costs and health consequences of school closure during an influenza pandemic pandemic influenza planning: addressing the needs of children national institute of child health and human development (nichd) early child care research network. child care and common communicable illnesses in children aged to months government to extend child care hours as virus outbreak continues. the korea herald public response to community mitigation measures for pandemic influenza estimating the costs of school closure for mitigating an influenza pandemic economic evaluation of influenza pandemic mitigation strategies in the united states using a stochastic microsimulation transmission model assessing global preparedness for the next pandemic: development and application of an epidemic preparedness index children and disasters: understanding vulnerability, developing capacities, and promoting resilience-an introduction a -month prospective follow-up study of psychological symptoms, psychiatric diagnoses, and their effects on quality of life in children witnessing a single incident of death at school suicidal deaths in elementary school students in korea none. the author has no potential conflicts of interest to disclose. geon ho bahn https://orcid.org/ - - - key: cord- -xuurjaz authors: vanhems, philippe title: sars-cov infection and primary school closure date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: xuurjaz nan to the editor: the recent review of brooks et al. on the impact of unplanned school closure on children's social contact [ ] for controlling outbreaks brings interesting information that could apply to coronavirus disease . in addition, a recent case in france of a -year-old child infected by sars-cov- [ ] raises the issue of risk assessment for other children at a same school and/or in a same classroom. identification of contacts between classmates is of high importance for appropriate screening and implementation of preventive measures at a primary school level but also at a family level. it has been reported that the patterns of contacts strongly differ according to age and school grade. for example, based on radiofrequency identification devices (rfid) technology, it was reported that young french children (age years) in a primary school [ ] had a median of contacts per school day and a median of minutes of cumulated contact per day. older children (age - years) had a median of contacts per day and a median of minutes of cumulated contact per day. an aggregate analysis emphasised that young children interacted with many schoolmates of the same or similar age (age - years) while older children restricted their contacts mostly to their own age stratum, like in england [ ] . the practical application of such an observation would therefore be to help public health authorities identify the children at higher risk of exposure. the decision to close a school totally or partially according to the age of an index case should be discussed. however, in an emergency context such as the covid- pandemic where scientific knowledge regarding the virus is still lacking, total closure of a school was reasonable and reassuring for parents. management of such an event raises two issues in public health decisions. on the one hand, an understandable precautious public health decision for total school closure, and on the other hand, a detailed risk assessment with a potentially different decision. although sars-cov- is not influenza or a respiratory syncytial virus, previous studies have identified the major impact of different social contacts of children by age which could have an impact on the spread of respiratory viral infections in schools [ ] . attack rates would differ according to grade or age, which determine the different contact patterns between children and would make it possible to adapt infection control measures [ ] . however, a more discriminant risk estimation by age at onset of a public health emergency would appear not to be useful but might be helpful regarding strategies of re-opening schools with sequential access to courses. nevertheless, at least retrospectively, detailed analysis of inter-individual contact remains a key determinant with viral characteristics in order to understand the dynamic of viral transmission in close environments such as primary schools. the impact of unplanned school closure on children's social contact: rapid evidence review covid- , france et monde. [infections with novel coronavirus (sars-cov- ), covid- , france and worldwide high-resolution measurements of face-to-face contact patterns in a primary school measuring social networks in british primary schools through scientific engagement estimating within school contact networks to understand influenza transmission mitigation of infectious disease at school: targeted class closure vs school closure none declared.this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord- -bbw m authors: caridade, sónia maria martins; sousa, hélder fernando pedrosa e; pimenta dinis, maria alzira title: the mediating effect of parental involvement on school climate and behavior problems: school personnel perceptions date: - - journal: behav sci (basel) doi: . /bs sha: doc_id: cord_uid: bbw m as a reflection of the culture and norms of the school community, the school climate (sc) is a potential factor connected to students’ major behavior problems (bp). parental involvement (pi) is considered as an essential factor for sc, contributing to promote good students’ educational results, as well as better social functioning. the present study aimed to analyze the mediating effect of pi on the relationship between sc and bp, taking into consideration the school personnel perceptions. a total of school personnel (teachers versus no teachers) aged between and (m = . , sd = . ), mainly female ( %), were integrated in the sample. based on the perception of the school personnel, the results indicate moderate level of pi and sc, as well as the existence of different bp in the school context. the mediating effect of pi in the relationship between the sc and bp has been demonstrated. these results suggest that, if the sc and pi are improved, it could be an effective strategy to enhance the social functioning of students in the school context. this study thus contributes to a comprehensive empirical analysis of how pi can improve the relationship between the sc and the bp of portuguese students. different definitions about school climate (sc) concept (e.g., [ ] ) can be found in literature. for the purpose of this study, sc was defined as a wide term involving the quality and character of school life in terms of norms and values, interpersonal relationships, social interactions, and organizational processes, structures, and culture [ ] . considered a multifaceted concept, sc includes observable characteristics of schools, organizational behavior of school personnel, and shared values among students and school personnel [ ] . in this sense, sc has been conceptualized as a critical factor in school life because it establishes socially acceptable behavior at school, which is able to influence and shape interactions between all school members (i.e., students, teachers, and parents) as well as their development at various levels [ ] . many different sc models have been proposed (e.g., [ ] [ ] [ ] ). sc has been operationalized into four main dimensions, with different elements: (i) safety, that could involve physical and emotional safety, rules and norms [ ] ; (ii) teaching and learning encompassing, for example, support for learning, quality instructional, social, emotional, and ethical learning (e.g., [ ] ); (iii) relationships, which involve respect for the community, school diversity school and collaboration, morale and connectedness (e.g., [ ] ); (iv) environmental-structural, which includes physical characteristics, aesthetics and space size, and resources [ ] , school connectedness/engagement, and physical layout and surroundings [ , ] . in the present study, only environmental-structural (e.g., access, adequate space and materials; adequate aesthetic quality and size of school; extracurricular offerings) and (ii) and relationships (e.g., mutual support and ongoing communication; school-community involvement) dimensions are considered. these two selected sc dimensions to this study are supported in the context of the literature framework. in fact, many of the sc-centered studies only address two of the above-mentioned dimensions (e.g., [ , ] ) or even a single construct [ ] . in addition, the literature proved the existence of a relationship between the institutional environment, interpersonal relationships, and bp, which has not been observed in other sc dimensions, that is, safety, teaching, and learning, since they were not equally considered in the analysis of the various studies [ ] . the multidimensionality of the sc is very important, as it will allow one to better assess the changes to be made in the school context [ ] , as well as to identify the main bp disturbing the good school functioning, able to interfere with the students' performance [ ] , all decisive factors to prevent the development of a delinquent trajectory [ ] . for the purpose of this study, the bp concept has a broader meaning, which includes a combination of disruptive, antisocial, delinquent, deviant, and risk or externalizing behaviors [ , ] , that could have serious negative consequences for adolescents or general society. absenteeism and incivilities practiced in the school context are also considered [ ] . school-centered research has recognized the existence of several bp, such as violence and indiscipline, which can be easily identified in the school context, capable of contributing to the breakdown of social attachment through the use of force and aggression [ , ] , drug abuse [ , ] , delinquent, antisocial behaviors, and incivilities occurring in the school or in the space surrounding schools [ , ] or even absenteeism [ ] . while members of the school community, parents play an important role in the learning environment, in defining school policies and practices, as well as in the academic and social performance of students [ ] . considered as a multidimensional concept [ ] , pi comprises several behavioral practices of parents (e.g., parents' style of life, parental expectations and aspirations, domestic rules and parental supervision, helping with homework, or communicating with teachers) [ ] , which support students' educational progress. however, the multiple definitions of pi [ ] in this study are conceptualized as participation in school-related activities, and communication is both an involvement and a means of improving pi [ ] . joyce epstein [ ] , focused on creating and strengthening the bonds between school, family, and community, developing the most widely cited and researched pi model in school. in order to foster the growth of students' success in school in different domains (for example, school, social, relational and behavioral), epstein's [ ] model established six types of pi: (i) parenting, which consists of taking care of the health and safety of children, developing good parenting skills in training and preparing children for school and providing a peaceful situation at home, allowing children to focus on their learning activities and complete their studies and making their homework; (ii) communicating, which involves establishing effective ways of communicating from school to home and from home to school about school programs and progress in children's learning (e.g., sending messages or letters, phone calls, parents visiting the school, sending news by teachers and directors); (iii) volunteering, which requires the involvement of family members, with available skills to support students in their learning process, inside and outside school; (iv) learning at home, with regards to the relationship between teachers and parents to help students to learn better at home; (v) decision-making, encouraging parents to participate in decision-making to increase student academic performance (e.g., parent participation in the parent-teacher association meeting) and (vi) community collaboration, which involves connections, relationships, and activities able to promote school-family-community collaboration in developing student learning. according to this model, parents, school, and community have spheres of influence shared in the students' learning process. the wide and diverse sc-centered research identifies sc as an important factor for increasing the results and improving the school environment. a positive sc will allow students to learn through cooperative learning, team cohesion, and mutual trust [ ] . other sc benefits on the prevention of school violence have also been consistently demonstrated, such as promoting healthy relationships, school connectedness, dropout prevention [ ] , lower levels of absenteeism (e.g., [ ] ), and less aggression and violence [ ] . an association between sc and bp has also been demonstrated [ ] , as well the link between sc and school violence [ , ] . in line with this, the research has shown that a decline in sc is related to higher bp at school [ ] , which include telling lies and breaking rules [ ] and externalizing behaviors [ ] , or even in the increase of absenteeism [ ] . research has shown that inadequate characteristics of the physical environment (e.g., inadequate lighting, hiding places, gang-related or hate-related graffiti, inadequate supervision of corridors, and poor maintenance) can create opportunities for conflict and violence and foster tolerance for aggressive behavior [ ] . a meta-analytic review developed by reaves et al. [ ] concluded by a significant relationship between students' perceptions dimensions of sc and bp over time. more specifically, a relationship between the institutional environment, interpersonal relationships and bp was found; the same not occurring in the other dimensions of the sc, i.e., safety, teaching, and learning, as they were not equally considered in the analyses of the many studies. furthermore, aldridge, mcchesney, and afari [ ] tried to analyze how sc influenced the prevalence of bullying and delinquent behavior, demonstrating the importance dimensions, such as connection to the school, the existence and clarity of the rules, and the support the teacher, have in preventing victimization experiences and delinquent behavior in the school context. in turn, a longitudinal analysis by dorio et al. [ ] focused on middle school students' perceptions of sc factors that are associated with bullying participant behaviors in the traditional and cyber contexts found that students' observations of delinquency and illegal behaviors on school grounds were positively associated with engagement in bullying and outsider behaviors. absenteeism is another significant chronic problem in schools that is also related to sc factors. then, a large study by van eck et al. [ ] in the u.s. with , middle and high school students from schools, using multilevel latent profile analysis, found that students who consider the sc to be more negative are more likely to have higher rates of absenteeism. as the proportion of students who perceive the sc as "moderate" or "negative" increases, the rates of chronic absence also increase. other studies intend to analyze the link between the sc dimensions and psychological and behavioral adjustment. for example, a study by way, reddy, and rhodes [ ] involving young adolescents found that a decline in all dimensions of the assessed sc (e.g., teacher and peer support, opportunities for students' autonomy in the classroom, and clarity and consistency of school rules) was associated with declines over time in psychological and behavioral adjustment. the benefits of increasing sc dimensions in the reduction of certain behavioral problems among peers, such as bullying, have also been documented. in this way, it has been shown that students are less likely to be bullied when they feel a sense of belonging to the school they attend to, thus being more confident, and when they are constantly involved in the classroom [ ] . crime prevention through environmental design (cpted) has been identified as a promising approach in redesigning schools, making them safer, more welcoming, and comfortable [ ] . based on an architectural philosophy that aims to prevent criminal or anti-social behavior through the climate, cpted primarily focused on natural surveillance, access control, and territorial reinforcement [ ] , considering four main mechanisms: space design, space use, circulation and circulation patterns, and territorial characteristics and space deterioration. the benefits of cpted in reducing the incidence of bp and school violence have been largely demonstrated (e.g., [ , ] ). when analyzing the associations between the physical attributes of schools and violence-related behaviors, vagi et al. [ ] , found an association between better scores on the cpted and a lesser probability of students missing school due to safety issues. school-wide positive behavior support (swpbs) constitutes another proactive, quite popular approach that tries to improve the academic and behavioral outcomes for students by targeting the school's organizational and social culture through different strategies: (a) defining clear expectations for all students at the school; (b) disseminate and promote these expectations to all students; (c) encourage the practice of these expectations; (d) positively reinforce the desired behavior; (e) defining clear consequences for problematic behavior; (f) extending expectations to the whole school; and (g) data collection and use for ongoing decision making [ ] . a longitudinal study developed by wienen et al. [ ] with teachers from elementary schools that agreed to participate and started swpbs implementation found a slight increase in the perception of the prosocial behavior and a decrease in the behavior problems with peers, reporting also different effects in children, teachers, and schools. despite swpbs being considered a very promising approach, given its applied nature, it is not without limitations, requiring the development of more studies based on a more robust and rigorous methodology [ ] . the pi in students' schooling has been perceived as having multiple and important benefits [ ] , including in managing bp initiated in school environments. pi has been successfully associated with increased social and emotional health and to a reduced dropout and substance use [ ] ), as well as better social functioning [ ] . the positive relationship between pi and students' social functioning is based on several empirical evidences. it has been proven that the greater involvement of parents in the education of their children promotes communication with school personnel about the adjustment and school behavior of children [ ] . it allows a better understanding of children's social difficulties at school, addressing and reinforcing positive behaviors at home, and it makes possible to mediate students' behavioral difficulties and problems, such as situations of violence and indiscipline in school [ ] . the study developed by el nokali, bachman, and votruba-drzal [ ] concluded that pi is associated with a decrease in bp and improvements in children's social skills. children with highly involved parents showed better social functioning and less bp, something that was attributed to the fact that communication between parents and teachers focused essentially on the management of students' social and bp. the research conducted by thompson et al. [ ] on teachers' perceptions about pi found that teachers rating low pi also reported worse student behavioral indicators, recognizing more externalizing behaviors, fewer social skills, more symptoms of attention deficit, and disturbing behaviors in relation to adults and colleagues. a portuguese study by caridade, azevedo, dinis, sani, and nunes [ ] conducted with school personnel participants also found a significant association between pi and the perception of students' general behavior, with % of the professionals rating student's general behavior as bad, also rating pi as poor. the literature also shows that a positive sc is more likely to increase pi in children's school activities. as an example, some studies (e.g., [ , ] ) found a strong and positive correlation between sc and pi, concluding that the improvements observed in sc, enhance the involvement of parents in the schooling process. when parents are highly involved in the school community, the sc is more vigilant and favorable [ ] . overall, the existing international literature indicates that there are important associations between sc, pi, and bp. however, the majority of the international (e.g., [ , , ] ) and portuguese [ ] research relating these variables under study is focused on students, and the number of studies focusing on parents and teachers [ ] or school staff (e.g., [ , ] ) is scarce. furthermore, portuguese studies that have analyzed the mediating effect of pi on the relationship between sc and bp are unknown. therefore, this area of research is still opened for further investigation, in order to support the need to improve school instructional and relational practices, identified as predictive of positive academic and social performance [ ] . in addition, the systematic and continuous collection of data about sc perception is essential to establish data-driven interventions for safer and healthier schools [ ] . therefore, this study addresses a valuable contribution to the portuguese literature by comprehensively assessing sc, pi, and bp, investigating whether pi mediates the relationships between sc and bp, considering the school personnel perceptions. more specifically, this study intends to: (i) identify the level of sc, pi, and bp; (ii) determine the relationship between pi, total sc, and its dimensions (i.e., environmental-structural and relationships) and bp; and (iii) investigate the mediation effect of pi on the relationship between total sc and its dimensions (i.e., environmental-structural and relationships) and bp. the following hypotheses are investigated: a positive correlation between the total sc and its dimensions (i.e., environmental-structural and relationships) and pi. a negative correlation between total sc and its dimensions (i.e., environmental-structural and relationships) and bp. low levels/scores of total sc and its dimensions (i.e., environmental-structural and relationships) and pi are expected to be predictors of high levels/scores of bp. pi is expected to mediate the relationship between sc (i.e., environmental-structural and relationships) and bp. the convenience sample integrated school personnel participants, aged between and years (m = . , sd = . ), mainly female ( %, n = ), and with higher education, . % (n = ). a total of . % (n = ) have secondary education and . % (n = ) have basic education. a substantial percentage of the sample ( . %, n = ) are teachers, with a significant percentage, . %, having more than years and . % under years of service (cf. table ). table ). the option to focus these three different school geographic locations was related to the fact that this study follows a research project conducted in the city of porto (lookcrim project). in addition, lisbon and porto constitute the two main portuguese cities which have the highest crime rates. sociodemographic information. school personnel were asked to provide background and demographic information including age, sex, marital status, education, school personnel function, school geographic location, and years of professional experience. school climate (sc) [ ] . participants were asked about environmental-structural dimension, through three variables: school surrounding physical environment, organization/quality of school spaces, and school infrastructural conditions considering the number of students. similarly, the relationships dimension also included three other variables: dynamics of extracurricular activities developed at school, dynamics of extracurricular activities involving the school and other institutions, and school engagement with the community. for the six variables, school personnel were asked to rate perception using a five-point likert-type scale (ranging from -very poor to -very good). participants were then asked to justify the given answer, selected from a predefined checklist. all five-point likert scales were recoded in a three-point likert scale, joining the lowest, i.e., responses and , and the highest, i.e., responses and , values, to create three distinct groups: poor vs. fair vs. good. the cronbach's alpha (α) was calculated for each dimension to verify the internal reliability of sc, considering that it is more suited to likert scales [ ] as it is the case in this study. in this sense, the original ratings were added to estimate total sc (α = . ) and their main two dimensions: environmental-structural (α = . ) and relationships (α = . ). the factor analysis reveals the adequacy of the sample through the means of the kaiser-meyer-olkin (kmo) measure of sampling adequacy [ , ] ( . > . ) and through the bartlett's test [ ] (χ = . ; p < . ). the factor loads are above . , varying between . and . , indicating an adequate level of validity of the selected items. students' behavior problems (bp) [ ] . participants were asked about students' behavior, considering three variables: absenteeism, disruptive behavior, and incivilities. concerning absenteeism, school personnel were requested to rate the perception using a five-point likert-type scale (ranging from -very low to -very high). in this study, this variable was dichotomized based on occurrence, i.e., yes for ratings and vs. no for all other ratings. then, a list of seven disruptive behaviors (e.g., widespread disrespect; disrespect for teachers; disrespect for school personnel; disrespect between students; manifestation of aggressive behavior; tobacco/drug abuse; alcohol consumption) were presented to the participants, who were then asked to identify those behaviors that happened in their school, through yes vs. no responses. an index of disruptive behavior (idb) was created through the sum of yes responses by participants. a similar strategy was applied regarding incivilities. participants were asked to select yes vs. no responses upon a predefined list of four items, i.e., scatter/throw trash around the school; destroy/damage equipment; disturbing school functioning; use of inappropriate language. an index of incivilities was then constructed, adding the yes responses. finally, a global index of behavior problems was established summing the positive responses across all assessed variables, i.e., absenteeism, disruptive behavior, and incivilities. parental involvement (pi) [ ] . school personnel were asked to rate the perception about the pi in the school activities, using a five-point likert-type scale (ranging from -very low to -very high), with one item only. data collection was accomplished during the school years of / and / , through an internet-administered survey. recruitment of participants occurred through several means, including direct contact with each school principal by email or phone and a request to forward the questionnaire survey link to the entire school personnel. the questionnaire was developed as part of the lookcrim research project in which this study is included, based on the reference literature in this area. the instrument was subjected to two validation procedures. first, the questionnaire was analyzed and revised by two senior researchers in this field, who made important contributions, both in terms of the content of the items and in terms of the order in which they are presented to the participants. subsequently, the questionnaire was subjected to a pre-test accompanied by spoken reflection with a sample of potential participants ( teachers and non-teachers). the results showed that the instrument proved to be adequate in terms of language and understanding of the items, which did not raise difficulties for the participants to respond. all subjects gave their informed consent for inclusion before participating in the study. respondents were given a narrative preamble explaining the study and informing them of the voluntary nature of the participation, prior to completing the questionnaires items. the study was conducted in accordance with the declaration of helsinki, and the protocol was approved by the ethics committee of university fernando pessoa (ufp) porto, portugal, project "diagnosis of the school climate", april , no specific reference assigned, date acting as reference id. the research project was also submitted and approved by the portuguese ministry of education (ref. no. ). the software ibm statistical package for social sciences (ibm spss for windows, version . , ibm corp., armonk, ny, usa) [ ] was used to data analysis. allowing the characterization of the sample and the identification of the sc, pi, and bp levels (objective i), descriptive univariate analyses were employed. a pearson correlation analysis was performed to explore the relationship between the variables under study, i.e., sc, pi, and bp (objective ii). aiming to explore a possible mediating effect between sc, pi, and bp (objective iii), hierarchical regression analyses were also carried out. in order to test mediation between variables, the model of spss command process v . , was used, a more robust way to deal with mediation, according to recent literature [ ] . table presents data relating sc, bp, and pi in school based on school personnel perception. the mean for total sc was . (sd = . , range = - ), which is in a moderate level. environmental-structural (m = . , sd = . ) is the higher rated highest sc dimensions. regarding the different dimensions of bp, the mean of disruptive behavior is the highest (m = . , sd = . ), followed by absenteeism (m = . , sd = . ) and incivilities (m = . , sd = . ). the correlations between the main variables of the study are presented in table . except for the relationship between absenteeism and environmental-structural sc., it can be verified that all variables are statistically correlated with each other. table . pearson's correlation results for the variables under study (n = ). using the stepwise method, a multilinear regression was produced with all variables, pi, total sc, and its dimensions, i.e., environmental-structural and relationships, as predictors variables, and bp as the dependent variable. no significant model was found. a significant multilinear model with a nearly significant environmental-structural sc (β = − . ; p = . ) variable included and pi (β = − . ; p < . ), explaining . % of bp variance, was found. the environmental-structural sc was not a pi predictor, but it was marginally significant. table in order to test mediating effects between total sc and its dimensions, i.e., environmental-structural and relationships and bp, the model of spss command process v . [ ] the present study has as main purpose to analyze the mediating effect of pi on the relationships between sc and bp, taking into consideration the school personnel perceptions, a topic that has not been previously studied in the portuguese context. similarly, and despite the existence of several international studies focused on these variables proving the existence of a positive relationship sc and bp (e.g., [ , , , , ] ) and between sc and pi (e.g., [ , ] ), demonstrating that the educational and relational practices of school are important predictors of successful results [ ] or even that pi increase social functioning [ ] and could yet reduce other problems behaviors (i.e., dropout and substance use) [ ] , research about mediators is sparce. this study is expected to represent a relevant contribution to the understanding of this phenomenon and to contribute to the development of other studies in this area. moreover, clarifying the relationship between the variables under study is, therefore, extremely important to implement and improve the level of these variables, sc, pi, and bp, in schools, namely the communication and interaction between family and school and the social functioning of students. based on the perception of school personnel, the results of this study allow to conclude by an moderate level of pi and sc, as well as the existence of different bp in the school context (i.e., absenteeism, disruptive behaviors and incivilities), also found in other portuguese studies [ , ] , which could impact in students' academic achievement [ ] , as well their social and emotional health [ ] . it has been shown that a greater pi in the education of children promotes communication with school personnel [ ] , allowing for mediation of students' difficulties and behavioral problems detected in schools, such as situations of violence and indiscipline [ ] . in turn, a positive sc has been associated with healthy relationships, a greater school connection, and the prevention of school dropout [ ] , with the reduction of lower levels of absenteeism [ ] and less aggression and violence [ ] . it should be highlighted that, with the exception to the relation between absenteeism and environmental-structural sc, all variables under study, i.e., sc, pi, and bp, are statistically correlated with each other. more specifically, pi has a positive important relationship with sc and its dimensions (i.e., environmental-structural and relationships) and a negative significant association with the different bp considered (i.e., disruptive behavior, absenteeism and incivilities). this means that if parents are more involved in school, it is possible to promote sc and decrease bp and, in this way, overall school improvement within the school context performance will be obtained. these results document and reinforce the important role of parents in their children's school process, whose involvement needs to be widely promoted [ ] . in addition, total sc and relationships sc are the present study has as main purpose to analyze the mediating effect of pi on the relationships between sc and bp, taking into consideration the school personnel perceptions, a topic that has not been previously studied in the portuguese context. similarly, and despite the existence of several international studies focused on these variables proving the existence of a positive relationship sc and bp (e.g., [ , , , , ] ) and between sc and pi (e.g., [ , ] ), demonstrating that the educational and relational practices of school are important predictors of successful results [ ] or even that pi increase social functioning [ ] and could yet reduce other problems behaviors (i.e., dropout and substance use) [ ] , research about mediators is sparce. this study is expected to represent a relevant contribution to the understanding of this phenomenon and to contribute to the development of other studies in this area. moreover, clarifying the relationship between the variables under study is, therefore, extremely important to implement and improve the level of these variables, sc, pi, and bp, in schools, namely the communication and interaction between family and school and the social functioning of students. based on the perception of school personnel, the results of this study allow to conclude by an moderate level of pi and sc, as well as the existence of different bp in the school context (i.e., absenteeism, disruptive behaviors and incivilities), also found in other portuguese studies [ , ] , which could impact in students' academic achievement [ ] , as well their social and emotional health [ ] . it has been shown that a greater pi in the education of children promotes communication with school personnel [ ] , allowing for mediation of students' difficulties and behavioral problems detected in schools, such as situations of violence and indiscipline [ ] . in turn, a positive sc has been associated with healthy relationships, a greater school connection, and the prevention of school dropout [ ] , with the reduction of lower levels of absenteeism [ ] and less aggression and violence [ ] . it should be highlighted that, with the exception to the relation between absenteeism and environmental-structural sc, all variables under study, i.e., sc, pi, and bp, are statistically correlated with each other. more specifically, pi has a positive important relationship with sc and its dimensions (i.e., environmental-structural and relationships) and a negative significant association with the different bp considered (i.e., disruptive behavior, absenteeism and incivilities). this means that if parents are more involved in school, it is possible to promote sc and decrease bp and, in this way, overall school improvement within the school context performance will be obtained. these results document and reinforce the important role of parents in their children's school process, whose involvement needs to be widely promoted [ ] . in addition, total sc and relationships sc are negatively associated with all variables of bp under study, which means that if the sc is further improved, it will be possible to promote positive changes in student's behavior [ ] , also reducing disciplinary and violence problems, such as those demonstrated by other previous evidence through cpted [ ] . in this study, pi emerges as the main predictor of bp, explaining . % of the variance. total sc and its two dimensions assessed in this study (i.e., environmental-structural and relationships) also emerged as predictive factors for bp, but with low weights ( . %, . %, and . %, respectively). this result is particularly important in terms of the changes to be implemented in the school context, specifically in terms of the characteristics of physical spaces and the type of relationships promoted at school. an important finding of this research is related to the mediating effect of pi on the relationship of total sc and its dimensions (i.e., environmental-structural and relationships) and bp, concluding that the sc alone does not explain the improvement of behavior, which is verified only when there is pi. it is clear, therefore, that focusing only on improving school conditions as a strategy to reduce bp in school, may be ineffective in itself. actions should be combined with promoting pi in their children's school process more than ever [ ] . the present study has some limitations that should be addressed. it is an exploratory study with a cross-sectional nature, focused on school personnel perception comprised by participants which are mainly teachers ( %), and attending only the reports about student's bp. in future studies, it would be extremely relevant to consider the perceptions of other important actors in the school context, such as parents, students, community members, or even social workers [ ] and analyzing records on specific issues about the sc and students' behavior. two sc dimensions are being considered in this study. however, because sc is a broader multidimensional construct, future studies should consider other dimensions (e.g., safety, teaching, and learning) to better intervene in the school context and guide reform efforts [ ] . the variable pi was only constituted by a generic item, limiting a more detailed exploration of this dimension, something to be addressed in future studies. in the same way, this study only attended to the mediating role of the pi, and future studies should also consider its potential moderating role. given the importance that the pi has in the students' school life, future longitudinal studies are necessary in order to better identify the predictors involved. finally, low alpha values constitute another limitation that should be considered and further explored in future investigations with this questionnaire. the pandemic problem generated presently by coronavirus covid- that required the implementation of remote learning, highlighted and reinforced the important role of pi in the students' education, something that should be reflected in further studies analyzing this important particular and extremely relevant period of time. the present study allowed us to highlight the important role that the sc, particularly translated in terms of pi fundamental relevance, has in the decrease of students' bp, considering the perceptions of the studied school personnel. the focus on teaching professionals (i.e., teachers and non-teachers) constitutes an added value of this paper, since most of the international research has focused mainly on students and, sometimes, on teachers and parents. it was possible to conclude that the influence of the improvement of sc in the management and reduction of bp is particularly visible in the face of the increase in pi. this is an important result that should be consequently considered extremely valuable, influencing the design of policies and strategies to improve the social functioning of students in the portuguese school context. it is important, therefore, to favor a more ecological approach, focusing on pi, in the context of sc, as a single and combined force to promote interaction between school and family. the lack of time reported by parents has been pointed out as one of the main reasons for reducing pi. the use of social media and online platforms to reach parents and involve them in their children's school life should be increased. as an example, the use of webinars and discussion forums could be important and empowering tools to achieve this. encouraging and energizing families to get involved in school activities, as well as carrying out joint activities with children, inside and outside the school context, can improve students' behavior, with important beneficial implications outside the school context. in addition, the development of support parent networks is another useful strategy for fostering effective participation and decision-making across the school, contributing 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comparison and connections between school climate, school safety and adolescents' antisocial behavior across three types of schools da indisciplina ao clima de escola: a voz dos alunos school social workers' perception of school climate: an ecological system perspective discovering statistics using ibm spss statistic the essentials of factor analysis ibm corporation released ibm spss statistics for windows, version ; ibm corp introduction to mediation, moderation, and conditional process analysis: a regression-based approach a meta-analysis: the relationship between parental involvement and african american school outcomes this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -fio cjj authors: nan title: peripheral nerve society meeting july – , sitges, barcelona, spain date: - - journal: j peripher nerv syst doi: . /jns. sha: doc_id: cord_uid: fio cjj nan the peripheral nerve society was founded in from two groups of academic investigators, peripheral nerve study group and peripheral neuropathy association of america, interested in the basic biology and function of the peripheral nervous system and its application to the clinic. their invite only biennial meetings involved - attendees in cloistered settings organized by shoestring and local initiative. from this, we have grown remarkably. we now have an annual meeting of over people including meetings within the meeting for the special interest groups in inflammatory, diabetic and hereditary neuropathy. with this substantial growth and the success of jpns, the journal of the peripheral nervous system the society continues to flourish. has proven to be a year full of exciting changes for the society. pns has transitioned from a biannual, to an annual meeting. next year, the meeting will be taking place at the renaissance baltimore harborplace hotel from - july in baltimore, maryland. the development of a new website has been completed, please visit www.pnsociety.com to see the new face of the society. finally, pns has adopted new executive staff. with their guidance and the leadership of an active and diverse board of prominent professionals in the field the peripheral nerve society continues to grow and anticipates more exciting changes in the year to come. the peripheral nerve society provides annual meetings, teaching courses, guidelines, and other resources to aid in the education of members. becoming a member of pns means collaborating with prominent global professionals in the field to develop and provide the best treatments for people with peripheral nerve diseases and setting standards of care within the field. please participate in our future by joining the pns, volunteering for a project aligned with your interests and sending your ideas for the future to the executive office, or board member. peles e . department of molecular cell biology, rehovot, israel. two schwann cell-dependent mechanisms control the presence of na + channels at the nodes of ranvier: i. clustering of the nodal complex by glia-derived proteins and ii. restriction of nodal proteins within the nodal gap by the paranodal junctions. these mechanisms depend on specific cell adhesion molecules that mediate the contact between myelinating glia and their underlying axons at the forming nodes and the paranodal junction. during myelination, na + channels initially clustered at heminodes that border each myelin segment. this process requires gliomedin, nrcam and neurofascin (nf ), three cell adhesion molecules (cams) that mediate the interaction between schwann cell microvilli and the axon. na + channels clustering activity of gliomedin is tightly regulated by two distinct and functionally opposing proteolytic events. while the clustering activity of gliomedin is enhanced by its shedding from the surface of schwann cells by a furin protease, its activity is negatively regulated by bone morphogenetic protein /tolloid-like (bmp /tld), and tolloid-like (tll ) metalloproteinase. cleavage by these enzymes restricts the activity of gliomedin to the nodal area and prevents the formation of ectopic clusters along axons that are devoid of myelin segments, as well as below the myelin internodes. hence, proteolytic processing of gliomedin facilitates, yet limits, the clustering of na + channels to specific sites along the axon in a timely manner. furthermore, axon-glial contact mediated by gliomedin and nf at the nodes, not only plays a role in na + channel clustering during development, but also contributes to the long-term maintenance of na + channels at nodes of ranvier. in addition to clustering by gliomedin, the distribution of na + channels is restricted between two growing myelin segments by the flanking paranodal junction. at this site, axon-glia contact is mediated by a distinct set of cell adhesion molecules (i.e., caspr, nf and contactin) that also delineate the underlying axonal and glial cytoskeleton. this paranodal junction-dependent restriction of na + channels to the nodes is mediated by the spectrin-based paranodal axonal cytoskeleton. illa i . neuromuscular unit, neurology department, hospital santa creu i sant pau, universitat autònoma de barcelona, barcelona, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune disorder of the peripheral nerves with clinical and immunological heterogeneity. currently, the diagnosis of cidp is based on clinical and electrophysiological criteria and does not take into consideration the presence of immune biomarkers. several autoantibodies against proteins of the node of ranvier in patients with cidp have now been described. these antibodies define specific cidp subtypes sometimes referred to as nodopathies and can have diagnostic and prognostic implications. anti-contactin (cntn ) antibodies. we have described the presence of antibodies to cntn in a small subset of patients with cidp. these patients shared a phenotype and have poor response to ivig. the anti-cntn antibodies are predominantly igg . pathological studies from skin and sural nerve biopsies of patients show morphological changes in the paranodes. experimental data supporting the pathogenicity of anti-cntn igg antibodies include: a) demonstration in vitro that the antibodies disrupt the binding of the cntn -caspr complex to neurofascin- (nf ); b) intraneural injections of antibodies progressively and specifically disrupt the paranodal axo-glial junction; and c) chronic infusion of antibodies induced clinical and electrophysiological worsening in animals with experimental autoimmune neuritis (ean). anti-nf antibodies. antibodies to neurofascins were first reported in patients with guillain-barré (gbs) and cidp and subsequently, antibodies specific to the nf isoform were found in a small group (< %) of patients with cidp. studies by us and confirmed by others have demonstrated that patients with cidp and anti-nf antibodies have a distinct phenotype that often includes a low-frequency tremor and poor responses to ivig. the autoantibodies are predominantly of the igg subtype. the passive transfer of monoclonal anti-neurofascin antibodies (which recognize all neurofascin isoforms) to mice with ean strongly exacerbated the severity of the pathology, but no studies have yet demonstrated that patient-derived anti-nf igg antibodies are pathogenic. a pathogenic role of the antibodies is however supported by sural nerve biopsies from patients with cidp and anti-nf antibodies that showed paranodal demyelination in the absence of inflammation, the loss of septate-like junctions and, the interposition of cellular processes between the paranodal loops and the axolemma. these alterations are reminiscent of those found in nfasc-null mice suggesting that anti-nf antibodies may specifically disrupt the nf -cntn -caspr complex at the paranodes. antibodies to other nodal proteins. recently neurofascin- and neurofascin- were reported as the main targets of autoantibodies in five patients with igg reactivity against the nodes of ranvier; the antibodies were predominantly igg . these patients presented with clinical features distinct from those in patients with anti-nf igg antibodies. four of these patients had subacute onset of sensory ataxia without tremor. the presence of anti-caspr antibodies has been reported in two patients with inflammatory neuropathies, one classified as cidp, the other as gbs. both patients had intense neuropathic pain. the skin biopsy from both patients showed paranodal disruption. some patients whose sera show nodal or paranodal reactivity in teased nerve fiber preparations have antibodies against other nodal proteins, such as gliomedin or neuronal cell adhesion molecule (nrcam) . the skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. indeed some mechanoreceptors can detect even nanometer-scale movements. movement is transformed into electrical signals via the gating of mechanically-activated ion channels at sensory endings in the skin. the sensitivity of piezo mechanically-gated ion channels are controlled by stomatin-like protein- (stoml ), which is required for normal mechanoreceptor function. under pathophysiological conditions following nerve injury or diabetic neuropathy the slightest touch can produce pain. it is at present unclear whether peripheral changes in sensory mechanotransduction may underlie hypersensitivity associated with neuropathic pain. here we have examined the role of the stoml modulation of piezo channels in mechanoreceptors and nociceptors to under pathophysiological conditions. we recently developed small molecules that act as inhibitors of stoml function. peripheral application of stoml inhibitors can alleviate hypersensitivity in models of neuropathic pain. our data strongly suggest that tactile evoked pain in models of peripheral neuropathy may be at least partly driven by sensitization of sensory mechanotransduction driven by stoml . coleman m . john van geest centre for brain repair, cambridge, uk. axons are lost early in many neurodegenerative disorders of peripheral and central nervous system. the degeneration of transected axons (wallerian degeneration) can be slowed tenfold by overexpression of a variety of nad-synthesizing enzymes, such as isoforms of nmnat or the related mutant fusion protein, wld s . wallerian degeneration is also delayed by deletion of tlr adapter protein sarm , a protein recently reported to promote nad degradation. it is important to understand fully the mechanism of wallerian degeneration because related mechanisms contribute to axon loss in a number of disease models, including models of peripheral neuropathies, parkinson's disease, multiple sclerosis and glaucoma. new data also suggest a role in hereditary spastic paraplegia. while depletion of nad is an attractive hypothesis for the mechanism of wallerian degeneration, especially as nad can be increased by dietary methods, it cannot explain a number of key observations. fk , an inhibitor of nampt, blocks the nad salvage pathway and strongly depletes nad, including within axons. however, instead of killing axons as the nad hypothesis would predict, it does precisely the opposite: it phenocopies the protective effect of wld s . moreover, ectopic expression of the bacterial enzyme nmn deamidase, a protein absent in mammals, protects injured axons both in transgenic mice and in primary neuronal cultures, but it has no effect on nad levels either under basal conditions or in degenerating nerves. these observations fit better with a proposed toxic role for the nad synthesis intermediate nmn, a model that can also explain the protective effect of wld s . a full understanding of the pathway should identify a number of points where intervention could be a treatment for multiple axonopathies. as with any medical discipline, expansoin of knowledge about the fundamental science behing a disorder of the human nervous system comes part and parcel with a change in our understanding of the epidemiology of any given disorder or groups of disorders. recent advances in our fundamental understanding of inflammatory neuropathies of the peripheral nervous system have been accompanied by drastic changes in our understanding of the neuroepidemiology of these disorders -the specific populationss affected by peripheral neuropathies, as well as the varying importance / contributions of select peripheral neuropathies to the overall burden of peripheral nervous system (pns) disease, and how this shift in epidemiological understanding influences the clinical approach to diagnosis and management of patients with pns disease. the past few decades have witnessed a paradigm shift in many aspects of pns disease diagnosis and treatment; from the association of human immunodeficiency virus (hiv)-associated neuropathies; to the increassing recognition of hereditary / familial peripheral neuropathies; to the increased recognition of specific neuropathies such as multifocal motor neuropathy with conduction block. in addition, timely events such as the recent, and increasingly irrefutalbe evidence for a link between zika virus and a guillain-barré syndrome, and the rather unexpected resurgence of peripheral neuropathies due to previously 'exotic' etiologies such as lepromatous neuropathy require prompt clinical attention. this plenary session aims to describe the evolving neuroepidemiology of peripheral nervous system disorders, and how these changes may influnece the clinical approach to the diagnosis, prognostication, and treatment of otherwise 'unusual' periphal nerve diseases. oxaliplatin chemotherapy for colorectal cancer is seriously limited by neurotoxic side effects which are not fully understood. oxaliplatin-induced peripheral neurotoxicity (oipn) comprises an acute syndrome and a chronic sensory neuropathy. the acute symptoms, notably cold hyperalgesia, have been attributed to transient ion channel dysfunction, and the worse they are the more severe the chronic neuropathy that ensues. we designed a combined in vitro and in vivo project, using neurophysiology to better understand the pathogenesis of oipn. in the in vitro study, differentiated f cells (rat drg neurons x mouse neuroblastoma n tg- cell line) were incubated for and hours in . m oxaliplatin, and their electrophysiological properties studied by patch-clamp. the treated f cells showed relatively depolarized resting membrane potentials, significantly decreased firing frequencies, and increased sodium current densities. moreover, a decrease in erg (ether-à-go-go-related gene) potassium current was also evident. in the in vivo study, we applied nerve excitability testing (net) to a wistar rat model of oipn. to investigate the acute syndrome, we compared behavioural and neurophysiological data of animal cohorts (controls and oipn rats, n= each) before and after oxaliplatin administration ( mg/kg, iv). twenty-four hours after the injection we observed differences between the groups in behaviour (cold plate test, p= . ) and in superxcitability of motor axons (p= . ). to investigate the chronic neuropathy, we compare a control group (n= ) with a treated group (n= , oxaliplatin mg/kg twice weekly x weeks, iv). both groups are studied with behavioural, neurophysiological (sensory and motor nerve conduction studies, net), and pathological (caudal and sciatic nerve, skin biopsy, drgs) methods. data are collected at baseline, end of treatment and weeks after treatment; to obtain a full net profile of all significant changes. in this highly translational approach to oipn, the in vivo net changes in the acute and chronic rat models can be matched on the one hand to findings from in vitro experiments, and on the other to clinical data, since net is also easily applied in humans. of these results. in denmark there is a unique situation to conduct epidemiological studies facilitated by the danish civil registration and the danish national hospital registry (dnhr). this enables us to identify all gbs patients in denmark in a given period. from the same period as the igos cohort was included we have identified all gbs patients admitted to or seen in outpatient's clinics of hospitals in denmark (september st to december st ). records from the population based danish cohort will be reviewed for demographic and clinical data and compared to the patients included in igos from denmark, as well as with the igos europe/america cohort. during this period patients from denmark have been included in igos. the danish group is comparable to the europe/america group not counting the danish patients (n= ) of the igos cohort in regard to sex and age at entry, gbs disability score at nadir, and percentage of patients needing mechanical ventilation. in the danish igos group % are males, the median age is ( - ) years, the mean(sd) gbs disability score at nadir . ( . ), and % of the danish group needed mechanical ventilation. in the europe/america group % are males, the median age is ( - ), mean(sd) gbs disability score at nadir . ( . ) and % of the group needed mechanical ventilation. at the meeting we will compare and present data from the danish population based cohort as well as epidemiological data. chronic inflammatory demyelinating polyneuropathy (cidp) is a common autoimmune disease of the peripheral nervous system (pns) that causes sensorimotor impairment. mice with a dominant autoimmune regulator gene (aire) g w mutation on the non-obese diabetic (nod) background (nod.aire gw/+ mice) develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. in sapp, demyelination is caused primarily by th t cells; however, the contributions of nerve-resident cells such as schwann cells are poorly understood. we identified a population of non-hematopoietic, integrin alpha + (itga +) cells in the pns that increases in frequency and number during sapp. these itga + cells coexpress numerous schwann cell markers including sox , p , s b, myelin protein zero, and peripheral myelin protein , suggesting that itga + cells are schwann cell-like. additionally, during sapp, these itga + cells upregulate the extracellular matrix protein periostin (postn), which has recently been shown to promote macrophage recruitment and activation in inflammatory disease and cancer. our data indicate that macrophages are pathogenic during sapp. therefore, we hypothesized that itg a+ cells promote macrophage recruitment during sapp via postn production. to test this hypothesis, we performed in vitro chemotaxis assays. conditioned media from nod.aire gw/+ nerve promoted significantly more macrophage chemotaxis than conditioned media from postn −/− nerve. furthermore, postn recombinant protein was sufficient to induce macrophage chemotaxis in vitro. our findings show that itg a+ schwann cell-like cells mediate macrophage chemotaxis by upregulating postn during sapp and suggests postn as a novel target for the treatment of cidp. "wear-off" frequency will be analyzed by assessing the proportion of subjects with any given degree of gs and rods intracycle fluctuation and the proportion of cycles in which gs and r-ods fluctuation occurs. to determine the extent of "wear-off" the degree of difference between maximum and minimum gs, r-ods, tugs, onls, and vas scores will be analyzed. currently subjects from different sites have been enrolled ( sites eligible for enrollment). this interim study report will provide preliminary representative data, demonstrating ivig "wear-off" effects on gs and other outcome measures. by better understanding the frequency and extent of ivig treatment-related fluctuations we expect that these results will help facilitate development of cidp treatment optimization strategies. we also expect that this information will be important in forming hypotheses to be tested in future studies (for example, comparing different dosage intervals, optimal ivig taper guidelines, or assessing the long-term outcome of short-term cycle to cycle clinical fluctuations). neuropathic pain is a frequent feature of peripheral neuropathy causing a significant impact on patients' quality of life and health care costs. resolving the genetic architecture of painful neuropathy will lead to better disease management strategies, risk stratification, and counselling. therefore, we aim to develop a reliable technique to rapidly and accurately re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. whole exome sequencing of thousands of samples remains expensive for clinical use. several targeted enrichment approaches are currently available to selectively enrich for genomic regions of interest. in this study, we compared the sensitivity, specificity, targeting efficiency, reproducibility of performance and cost effectiveness of truseq ® custom amplicon-next generation sequencing (tsca-ngs) and molecular inversion probes-next generation sequencing (mips-ngs) methods. for both methods, we constructed a targeted enrichment kit to capture the coding and exon-flanking intron sequences of nine sodium channel genes (scn a, scn a-scn a, and scn b- b) expressed in nociceptive neurons. probes were designed for the two methods using their respective informatics pipelines. in total, patients with diabetic and idiopathic neuropathy were tested by both methods. among the patients, patients were tested previously by sanger sequencing for scn a-scn a. approximately kb was captured and sequenced. % of the targeted regions showed an average coverage of ≥ x in tsca-ngs, and % in mips-ngs. we managed to identify potential pathogenic mutations and polymorphism variants by mips-ngs and tsca-ngs. moreover, we observed a perfect agreement ( %) between sanger sequencing data and those obtained using mips-ngs and tsca-ngs. both ngs approaches showed user-friendly software to design probes and exhibited a similar on-target efficiency. although the overall coverage per region varied across different dna samples, it was sufficient to detect any variant in these regions. mips-ngs has more versatile assay design, demonstrated a high degree of flexibility with probes re-placement and > x cheaper than tsca-ngs. mips-ngs is a reliable, flexible, and inexpensive method to detect genetic variations in thousands of patients. in our centers, this technology is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients. alonso-jiménez a , , belvis-nieto r , diaz-manera j , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; centro para la investigación biomédica en red para enfermedades raras, ciberer, madrid, spain; neurology department, hospital universitario dexeus, barcelona, spain. most acute demyelinating polyneuropathies have an immune-mediated pathogenesis and are included within the guillain-barré syndrome spectrum. occasionally, other mechanisms such as metabolic, infectious or toxic may lead to gbs-like presentations. thermatrim ® and pura alegria ® are different brands of the same illegal slimming product that is sold through online vendors and in which the exact composition is unknown. here we present a patient with an acute demyelinating polyneuropathy secondary to the intake of the slimming product "pura alegría". a year-old woman with no remarkable medical history reported days history of distal numbness in her feet that progressed in one week to her knees and her left hand. she had had an upper respiratory tract infection ten days prior to these symptoms. the neurological examination showed absent distal vibratory and arthrokinetic sensations and arreflexia in lower limbs, decreased vibratory sensation in her hands and a ataxic gate. the lumbar puncture showed . g/l of proteins with no cells. the emg fulfilled diagnostic criteria for acquired demyelination. intravenous immunoglobulin therapy was started but the symptoms kept worsening and corticosteroids were started. the patient mentioned then the slimming product. a brain mri showed diffuse leukoencephalopathy that was asymptomatic. steroids and the pura alegria slimming products were withdrawn and the patient recovered completely after one year of follow-up. "pura alegría", "thermatrim" and "thermatrim plus" are slimming products that were forbidden in spain after several cases of acute leukoencephalopathy and acute polyneuropathy. exact composition is unknown although the spanish drug agency detected the pesticide malonoben, a tyrosin kinase inhibitor, among the components. since nine cases of pura alegria/thermatrim neurological toxicity, including leukoencephalopathy and polyneuropathy have been described. all cases had good outcomes after treatment withdrawal, although recovery is slow and may be incomplete. our case highlights the need to carefully consider drug toxicity, including dietary supplements, in the differential diagnosis of gbs specially when evolution does not follow typical patterns. alvarez s , klein d , martini r , moldovan m , , krarup c , . center for neuroscience, university of copenhagen, denmark; department of neurology, developmental neurobiology, university hospital würzburg, germany; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. charcot-marie-tooth neuropathy type a (cmt a) resulting from peripheral myelin protein kda (pmp ) overexpression is the most common hereditary motor and sensory neuropathy in humans. the transgenic pmp (pmp tg) mouse line c carrying copies of the human pmp gene, has a slowly progressing neuropathy phenotypically like cmt a with thin and abnormally thick myelin profiles and supernumerary schwann cells. in addition, pmp tg nerves showed activated macrophages leading to axon-myelin compartment disruption and maldistribution of k+ channels (kohl b et al, am j pathol. ; : ) . the aim of the present study was to investigate the motor axon excitability in pmp tg versus wt littermates. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle and "threshold-tracking" the plantar compound muscle action potential (cmap). at age months, when the post-developmental maturation was nearly complete in the wt, the pmp tg cmap showed an increase in latency by %. the cmap amplitude was decreased by %, although the mean motor unit size (mscan method) appeared unchanged indicating a lack of collateral sprouting. furthermore, pmp tg showed abnormalities in both passive cable properties and voltage dependent parameters. at age month, the cmap latency of pmp tg was increased by % as compared to wt. in contrast to this marked conduction slowing along the tibial nerve from to months of age, the progression of excitability changes localized at ankle appeared modest. nevertheless, when pooling data from to months, the increase in pmp tg latency was correlated (spearman p< . ) with an increase in accommodation half-time during depolarizing electrotonus (+ % of threshold) from to ms and a reduction of the late subexcitable period of the recovery cycle from to % of threshold, both changes consistent with a redistribution of k+ currents consistent with the maldistribution of k+ channels. our data suggest that in the pmp tg cmt a model, a functional, thus potentially reversible abnormality in k+ channel distribution, accumulates along the nerve and aggravates the conduction impairment due to impaired myelin formation and maintenance. alvarez s , krarup c , , moldovan m , . center for neuroscience, university of copenhagen, denmark; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. mice heterozygously deficient of myelin protein p gene (p +/−) show a mild progressive dysmyelinating neuropathy, with conduction slowing and impaired excitability, phenotypically similar with charcot-marie-tooth disease type b (cmt b). we found that in p +/− the accumulating myelin abnormalities were paralleled by progressive changes in voltage-dependent motor axon function resulting in neurotoxic membrane depolarization (rosberg mr, et. al. neurobiol dis. : ) . the aim of this study was to investigate the relationship between demyelination and motor axon function in p +/−. demyelination of the right sciatic nerve by topic lysophosphatidylcholine (lpc) application was carried out in p +/− and wild-type (wt) mice, in year (mature) and years (aged) groups. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle (distal to lpc demyelination) and "threshold-tracking" the plantar cmap responses. live imaging studies by cellvizio (mauna kea technologies, paris, france) confocal laser endomicroscopy were carried out in transgenic mice expressing the fluorescent reporter yfp in peripheral nerve axons under the thy promoter. in mature wt the sciatic morphological and electrophysiological demyelinating features following lpc could be readily observed at hours but disappeared by weeks. no morphological changes could be observed at the tibial level. consistently, no conduction or excitability changes could be observed at the right tibial neve level as compared to the left tibial nerve in wt. in contrast, in p +/− the motor axon function was impaired at the tibial nerve level at weeks after sciatic lpc demyelination. in mature p +/−, although the cmap amplitude appeared preserved, the distal motor latency was prolonged whereas the excitability measures showed reduced deviations during threshold electrotonus and increased refractoriness at the expense of superexcitability of the recovery cycle, both consistent with membrane depolarization. furthermore, in aged p +/− the delayed tibial conduction was associated with a drop in cmap amplitude and a prolongation of the strength-duration time constant. taken together these data suggest that focal demyelination aggravates membrane dysfunction along the entire motor axon in p +/− providing a novel experimental model to explore the link between demyelination and axonal membrane dysfunction in cmt b. amass l , li h , gundapaneni b , schwartz j , keohane d . pfizer inc., new york, ny, usa; inventiv health inc., burlington, ma, usa. a number of factors can influence disease progression in transthyretin familial amyloid polyneuropathy (ttr-fap), a rare, fatal, hereditary amyloidosis. this analysis evaluated the specific role of baseline neurologic severity on neurologic disease progression in ttr-fap. a predictive model was created based on longitudinal data from val met patients who participated in the tafamidis (a selective ttr stabilizer) clinical development program. data from the intent-to-treat population of the double-blind, placebo-controlled registration study (tafamidis group, n= ; placebo group n= ) and its two consecutive open-label extension studies in which all patients received tafamidis were used. the second extension study is ongoing, but a formal, prospectively-planned interim analysis was conducted with the cut-off date of december , . this analysis focused on the first months of treatment for the overall study cohort analyzed. the neuropathy impairment score-lower limbs (nis-ll) was used to assess neurologic functioning at baseline and at subsequent study visits. a linear mixed-effects model for repeated-measures (mmrm) analysis, with baseline nis-ll, treatment, and their interactions with time as fixed effects, was used, and the slope and intercept for each patient were included as random effects. patients were primarily caucasian with early-stage neurologic disease (baseline nis-ll mean [standard deviation]: tafamidis, . [ . ]; placebo, . [ . ] ). across both groups, disease progression increased with increasing levels of baseline severity (nis-ll) (p< . ). however, the predicted magnitude of change from baseline to month for tafamidis was consistently less than that for placebo across a range of observed baseline nis-ll values, suggesting a disease-modifying effect of tafamidis. similar findings were observed for the nis-ll muscle weakness subscale. this mmrm analysis in patients with val met ttr-fap demonstrates that disease progression strongly depends on baseline neurologic impairment and highlights the disease-modifying effect of tafamidis across a range of baseline levels of neurologic severity. clinicaltrials.gov identifiers: nct , nct , nct . amino h , misawa s , sekiguchi y , shibuya k , watanabe k , suichi t , kuwabara s . department of neurology, chiba university, chiba, japan. guillain-barré syndrome (gbs) is a potential life threatening neurological disorder and respiratory insufficiency is one of the critical complications. eramus gbs respiratory insufficiency score (egris) is a method for predicting the chance of respiratory insufficiency in gbs. however, clinical characteristics and courses can vary for subtypes of gbs, whose occurrences differ for each region: acute inflammatory demyelinating polyneuropathy (aidp) is very common in european countries, whereas acute motor axonal neuropathy (aman) is frequently seen in asian countries. the aim of this study is to investigate the usefulness of egris in japan, where aman is more common than in the netherlands. clinical and electrophysiological profiles of consecutive gbs cases, who visited our hospital within days after symptoms onset between and , were reviewed. of the gbs patients, % were classified as aidp and % as aman according to the electrodiagnosis criteria by ho and colleagues. higher egris scores correspond to higher risk of respiratory insufficiency in total of the gbs patients, as well as in aidp patients. however, in patients with aman, egris scores did not always match the chances of respiratory insufficiency: up to % of the patients with low risk of egris showed respiratory failure, whereas only % of the patients with high risk of egris needs intubation/mechanical ventilation. in aman, associations with mechanical ventilation were seen for rapid progression (shorter duration between onset and hospital admission), more decreased vital capacity, and more frequent autonomic involvement. egris is useful also for japanese gbs patients. however, for aman patients, it should be used with discretion. another score to predict respiratory insufficiency might be required in asian countries. anandan c , litchy wj , laughlin rs , leep hunderfund an , naddaf e . mayo clinic, rochester, usa. in patients with suspected ulnar neuropathy, nerve conduction studies (ncs) are commonly requested to help with diagnosis and localization. however, routine ncs are often normal or not localizing. ulnar ncs recording from the first dorsal interosseous muscle (ncs-fdi) is thought to increase the diagnostic yield of electrodiagnostic testing, although not commonly considered. we developed a quality improvement strategy to routinely perform ulnar ncs recording from the abductor digiti minimi muscle (ncs-adm) as well as ulnar ncs-fdi in all patients referred for suspected ulnar neuropathy. we utilized the dmaic (define, measure, analyze, improve, control) model of process improvement to define our problem and create a map of the current process for ulnar neuropathy diagnosis in our electromyography laboratory. we determined baseline performance via review of distal sensorimotor polyneuropathy (dpn) is the most common complication of diabetes and risk factors beyond hyperglycemia have proven important particularly in type diabetes (t dm). only few prospective studies from early-stage t dm exist. we aimed to study the development of dpn during the first years after a screening-based diagnosis of t dm. from the addition-denmark study participants were eligible for this study. dpn was assessed by the michigan neuropathy screening instrument questionnaire (mnsi) at four time-points during follow-up. dpn was defined by a mnsi score ≥ . participants ( %) were positive in mnsi at baseline and thus excluded from this study. by kaplan-meier plot we evaluated the cumulative incidence of dpn and in cox proportional hazard models we calculated hazard ratios (hr) for the intervention groups in the addition trial and for various covariates proposed to influence the development of dpn. models were adjusted in steps for intervention group, age, sex, baseline mnsi, lipid-lowering and anti-hypertensive treatment. this study cohort consists of participants ( % men) with a median age of . years (p ;p : . ; . ) and median baseline hba c of . (p ;p : . ; . ). a cumulative incidence of % was seen during years of diabetes. there was no statistically significant difference in hr between the intervention groups or by sex but a significantly higher hr of . ( %ci: . ; . ) was seen for age (per year). the highest hr was found for a history of cardiovascular disease (myocardial infarction or stroke) up to ten years prior to the diabetes diagnosis with a hr of . ( . ; . ). weight, waist circumference, body-mass index and methylglyoxal (log transformed) showed modest but statistically significant associations with incident dpn with standardized hrs of . ( %ci: . ; . ), . ( %ci: . ; . ), . ( %ci: . ; . ) and . ( %ci: . ; . ) respectively. this study demonstrates a fairly low cumulative incidence of dpn in people with screen-detected t dm and provides evidence that macrovascular disease, obesity and oxidative stress are important risk factors for dpn even at the earliest stages of t dm. andermann syndrome, also known as agenesis of the corpus callosum and peripheral neuropathy (accpn), is an autosomal recessive disorder with a broad spectrum of mild to severe neuromuscular and psychiatric consequences. the gene variants causing disease were first identified in french-canadian families. in the present study, we intended to phenotype and genotype a series of non-french-canadian familial cases presenting with charcot marie tooth disorder associated with agenesis/dysgenesis of the corpus callosum. for this purpose, seven families, of consanguineous marriage, were studied. patients were clinically and para-clinically investigated using mri and electrophysiology (mncvs). for some, a sural nerve biopsy was taken. microsatellite markers around the accpn locus were used in two large families; followed by sanger sequencing of all the exons and intron-exons boundaries of the gene, in one patient from each of the families. the age at onset of the disease was at birth in the patients from the largest consanguineous family ( affected individuals). the biopsy from one patient showed a severe demyelinating neuropathy with many hypomyelinated fibres and mostly secondary axonal changes. these finding were compatible with electrophsiological data where the mncvs are of demyelinating range. dysgenesis of the corpus callosum in one patient and agenesis in another sib were revealed by mri. we identified one homozygous truncating mutation in this family. interestingly, no causative variant was found in a patient from another family and showing homozygous haplotype. two different heterozygous variants were identified at one hit in two patients from two non-consanguineous families. genetic investigations will be continued to identify the possible second hit. in the remaining families, no variant was found. the negative family cases will be subjected to ngs. at this stage, it is tempting to speculate on the genetic heterogeneity of accpn in our series. baba m , suzuki c , ogawa c , tomiyama m . department of neurology; diabetes center, aomori prefectural central hospital, aomori, japan. in we introduced a staging system of severity of diabetic polyneuropathy (dpn) by nerve conduction study (ncs) of the lower limb: sensory ncs of the sural nerve and motor ncs of the tibial nerve. the system consists of five stages; ncs- (normal): no abnormalities, ncs- (mildly abnormal): presence of delay of mcv, scv, minimal f-wave latency, or positive a-wave, ncs- (moderately abnormal): decrease in sural snap less than uv, ncs- (severely abnormal): decrease in plantar muscle-cmap to - mv, ncs- (ultimately abnormal): plantar muscle-cmap lost or less than mv with trace of sural-snap. to examine validity of the system, we conducted -year prospective observation on development of diabetic foot (df) by the ncs staging system. in addition, occurrence of ischemic heart disease (ihd) and stroke (is), and death of neuro-vascular events were also counted. n - , we carried out ncs in diabetics, and categorized them by the ncs staging system: % was ncs- , % was ncs- , another % was ncs- , % was ncs , and % was ncs- . we then followed them and prospectively counted the occurrence of df, ihd and is in patients (mean age ys). the occurrence of df during the following years was; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- : %. occurrence of any of df, ihd and/or is was as follows; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- %. there was no death from nsc- , − , and − groups (n= ), while two from ncs- group (n= ) were found dead in a bed or on a driver's seat, and other two from ncs- group (n= ) died of sudden cardiac arrest or infection after foot amputation. in summary, the present ncs grading system seems to work satisfactory not only for diagnosis of severity of the current dpn, but also for prognostic prediction of the dpn-related foot and vascular events. bacon c , feely sme , shy me . university of iowa hospital, iowa city, ia, usa. for patients with charcot-marie-tooth disease, also known as hereditary motor and sensory neuropathy, the rasch modified cmtnsv is a validated measurement of symptoms and impairment. the score is comprised of nine parameters of a clinical examination, including nerve conduction studies (ncs) . each parameter has an individual score ranging from zero to four, with the composite score having a maximum of prior to rasch modification. for patients who do not complete a ncs, the cmt exam score (cmtesv ) is used, with a maximum score of before rasch modification. one parameter of the cmtesv is the vibration sense. using a rydel tuning fork, a care-giver measures vibration sense in a patient's feet, ankles, and knees and a score is determined by the severity of reduced vibration sense. in cmt a patients, % received a score of out of , noted by a reduced vibration sense at the knee. in order to capture a more sensitive vibration measurement, we tested ways of taking the total raw score of the rydel tuning fork at each point of vibration sense, the toe, ankle, and knee bilaterally. scores range from zero to , with reflecting full vibration sense. in this modified measurement, vibration sense scores varied in wider distribution. for cmt a patients captured in this studied, a modified vibration sense score results in a potentially more sensitive total cmtesv score. bae dw , an jy . st. vincent's hospital, the catholic university of korea, suwon, korea. diabetic lumbosacral radiculoplexus neuropathies (dlrpn) are usually subacute painful, monophasic, asymmetrical lower limb neuropathies with incomplete recovery due to ischaemic injury and microvasculitis. the diagnosis relies mostly on clinical suspicion and characteristic electromyographic findings. however, in acute phase, neuroimaging has more important diagnostic significance than electrophysiological studies. here we describe mri and ultrasound findings in a -year-old woman with dlrpn who was diagnosed with diabetes three years ago, but has not received any other treatment. she had a -day history of acute-onset severe pain with weakness of muscles innervated by left femoral and obturator nerve and decreased sensation in left l -l dermatome. nerve conduction studies showed reduced amplitude in left femoral nerve and electromyography showed only increased insertional activity in left iliopsoas muscle and no volitional activity in muscles innervated by left femoral and obturator nerve. ultrasound revealed increased cross-sectional area (csa) of left femoral and lateral femoral cutaneous nerve. mri showed enhancement in left l , nerve roots and proximal femoral nerve and increased signal intensity in left iliacus and iliopsoas. we diagnosed her with dlrpn and started corticosteroid. nerve ultrasound has not been previously reported in a patient with cabin air in commercial airliners originates from aircraft engines or auxiliary power units. this bleed air may occasionally be contaminated with hydraulic fluids and engine oil that contains a number of potentially hazardous chemicals including tricresyl phosphate (tcp). over the last years reports are emerging about aircrew members that experience symptoms such as tingling or burning of extremities in addition to headache, and vertigo. this "aerotoxic syndrome" is controversially discussed in the literature and has been attributed to exposure to organophosphate contaminated cabin air. since tcp has been associated with peripheral neuropathy we aimed to determine the frequency of peripheral neuropathy among frequent flyers. civilian air crew members and frequent flying passengers (m:f = : , median age years, median exposition time in aircrafts of . hours) were examined at the university hospital of cologne or at the frankfurt airport (iata code fra) by a detailed questionnaire of past medical conditions, a standardized neurological examination and nerve conduction studies of sural, tibial, and ulnar nerves. we identified subjects with clinical and electrophysiological evidence for large fiber peripheral neuropathy. incidence of peripheral neuropathy was not correlated to exposition time in aircrafts. in addition subjects showed signs of ulnar neuropathy, subjects reported abnormal vibration sensation, subjects suffered from gait imbalance and individuals reported tingling of extremities. our study shows a . % prevalence of large fiber peripheral neuropathy among frequent flyers. comparison of these data with prevalence rate in an age-matched control group will reveal a possible association of chronic exposure to cabin air and risk for peripheral neuropathy. the high incidence of the symptom tingling in our cohort warrants further studies to determine the risk for small fiber neuropathy in this condition. intravenous immunoglobulin (ivig) are human igg derived from plasma pools of healthy donors. although there are studies in literature evaluating their effectiveness in different pathological animal models, there are no data about their possible role on bortezomib (btz)-induced peripheral neuropathies. female wistar rats were treated following a preventive schedule (btz and ivig co-treatment for and weeks) and a therapeutic schedule ( weeks of btz treatment followed by a -week ivig-btz co-treatment). caudal nerve conduction velocity (ncv), plantar and dynamic tests were performed at different time points. animals were sacrificed after ws (acute phase) or ws (chronic phase) and tissue samples (dorsal root ganglias -drg-, sciatic nerve, caudal nerve, skin) were collected for morphological, morphometrical and immunohistological analysis.in the preventive schedule, ivig was not able to rescue caudal ncv reduction caused by btz neither after nor after weeks of co-treatment. same results were observed in the therapeutic schedule. on the other hand, the evaluation of mechanical allodynia and cold hyperalgesia showed that ivig injection protected from btz effect in both treatment schedules. morphometric analysis evidenced that, even if not statistically significant only the preventive schedule has a tendency to protect the caudal nerve from btz damage. this result is consistent with the morphological evaluation of the nerve. also, intra-epidermal nerve fibers density was preserved in the preventive schedule but not in the therapeutic one. finally, sciatic nerve and drg macrophage infiltration levels tended to be reduced in the therapeutic schedule and were brought back to ctrl (rats not treated or injected with ivig alone) levels in the preventive one. in conclusion, we were able to demonstrate for the first time that ivig treatment especially used as preventive treatment option may reduce btz-induced neuropathic painful pointing out the possible role of inflammation in the pathogenesis of this invalidating pathology. this work was supported by kedrion spa. we studied the prevalence, the molecular cause and clinical presentation of hereditary motor neuropathies in a large cohort of patients from the north of england. detailed neurological and electrophysiological assessments and next generation panel testing or whole exome sequencing were performed in patients with clinical symptoms of distal hereditary motor neuropathy (dhmn, patients), axonal motor neuropathy (motor cmt , patients) or complex neurological disease predominantly affecting the motor nerves (hmn plus, patients) . the prevalence of dhmn is . affected individuals per . inhabitants ( % confidence interval: . - . ) in the north of england. causative mutations were identified in out of index patients ( . %). the diagnostic rate in the dhmn subgroup was . %, which is higher than previously reported ( %). we detected a defect of neuromuscular transmission in cases and identified potentially causative mutations in patients with demyelinating multifocal motor neuropathy. many of the genes were shared between dhmn and motor cmt , indicating identical disease mechanisms therefore we suggest changing the classification and include dhmn also as a subcategory of cmt. abnormal neuromuscular transmission in some genetic forms provides a treatable target to develop therapies. barohn rj , gajewski b , pasnoor m , brown l , herbelin l , kimminau k , jawdat o , parks c , shlemon p , dimachkie mm and the pain-controls study team . the university of kansas medical center, kansas city, ks, usa. cryptogenic sensory polyneuropathy (cspn) is a common slowly progressive neuropathy that affects adults and presents with significant neuropathic pain for which multiple medications have been tried including antiepileptics, antidepressants, topicals and narcotics. a web based survey among neuromuscular experts suggested pregabalin as being more effective than other medications, however there are presently no comparative studies to assess the most effective medication. the objective of this study was to determine which of the pharmaceutical therapies (pregabalin, duloxetine, nortriptyline or mexiletine) is most effective for neuropathic pain and best tolerated in cspn. to achieve this objective we performed a prospective randomized open labelled comparative effectiveness adaptive design study of cspn patients through the patient centered outcomes research institute (pcori). cspn patients who fulfilled the inclusion and exclusion criteria were enrolled into this study. patients underwent a baseline neurological evaluation and randomly assigned to one of the neuropathic medications for months. the primary outcome is the change in likert-like pain scale. the secondary outcomes included nih pain interference scale, nih fatigue interference scale, nih sleep disturbance scale, sf- and adverse events. the outcome measures are performed at baseline, month , and . statistical analysis using bayesian adaptive design developed by berry consultant software will be performed to determine winner and losers (winner = greater than point improvement in pain). total number of patients to be enrolled is . recruitment has been challenging and a number of recruitment techniques have been used. to date, there have been patients screened, patients randomized from us sites. anticipated completion of enrollment by june and end of final patient assessment by september . interim analysis performed after first patients completed their months as part of bayesian adaptive design analysis and occurs every weeks. the distribution of randomization of patients to the medications at last adaptive design randomization was . % to medication , % to medication , . % to medication and . % to the th medication. this study may give physicians and patients evidence for future management of cspn patients. transthyretin amyloidosis (attr), which encompasses a group of disorders with significant clinical variability, is caused by transthyretin (ttr) derived amyloid deposition. the clinical aspects of autonomic nervous system involvement in attr are only partially known. the ongoing, multinational, longitudinal, observational transthyretin amyloidosis outcomes survey (thaos) provides the opportunity to expand our understanding of dysautonomia in attr. data from all symptomatic subjects enrolled in the thaos registry with a diagnosis of attr (cut-off date: january , ) were assessed for the presence and temporal course of autonomic symptoms, genotype and phenotype associations, and clinical burden according to the frequency and severity of symptoms. of symptomatic subjects enrolled in thaos, ( . %) had autonomic symptoms at enrollment including: gastrointestinal ( subjects, . %), urinary ( , . %), erectile dysfunction ( , . %), orthostatic hypotension ( , . %), xerophthalmia ( , . %) and dyshydrosis ( , . %). subjects with autonomic manifestations, compared with those without, were younger (mean age [standard deviation, sd] of . [ . ] vs . [ . ] years), with a longer duration of attr symptoms ( . [ . ] vs . [ . ] years). autonomic dysfunction was less common with wild-type attr ( of subjects, . %) than in mutation groups: val met ( / , . %); non-val met/non-cardiac ( / , . %); and "cardiac mutations" (val ile, leu met, thr ala, or ile leu mutations; / , . %). similarly, time (mean [sd] , years) from first attr symptoms to onset of autonomic symptoms, was longest for wild-type attr ( . [ . ] ) followed by "cardiac mutations" ( . [ . ]), non-val met/non-cardiac ( . [ . ]), and val met ( . [ . ] ). autonomic symptoms were present at disease onset in over a third of subjects ( , . %) . autonomic dysfunction was less frequent in subjects with cardiac phenotype ( of subjects, . %), than with mixed ( / , . %) or neurologic ( / , . %) phenotypes. the burden of autonomic symptoms (mean [sd] ) varied by genotype, val met ( . [ . ] , non-val met/non-cardiac ( . [ . ], "cardiac mutations" ( . [ . ]), wild-type attr ( . [ . ]), and by phenotype, mixed ( . [ . ]), neurologic ( . [ . ]), cardiac ( . [ . ]). dysautonomia is common, and a significant burden, in subjects with hereditary forms of attr. its prevalence is higher in val met than in other genotypes, and in the neurologic or mixed phenotypes. the objective of this study is to assess the usefulness of motor unit number index (munix) technique in charcot-marie-tooth type a (cmt a) disease and to test correlation between munix and clinical impairment. munix technique was performed in abductor pollicis brevis (apb), abductor digiti minimi (adm) and tibialis anterior (ta) muscles in the non-dominant side. a munix sum score was calculated by adding munix of these muscles. muscle strength was measured using the mrc (medical research council) scale. disability was evaluated with several functional scales including cmt neuropathy score version (cmtnsv ) and overall neuropathy limitation scale (onls). cmt a patients with known pmp gene duplication were enrolled. the munix of the adm, apb and ta muscles were correlated with the mrc of the corresponding muscle (p< . ). munix sum score was correlated with clinical scales: cmtnsv (r=− . , p< . ), onls (r=− . , p< . ). in conclusion, munix correlates with muscle strength and clinical measurements of disability in cmt a patients. the munix technique evaluates motor axonal loss and correlates with disability. the munix sum score may be a useful outcome measure of disease progression in cmt a. the objective of this study was to assess the usefulness of mri in charcot-marie-tooth type a (cmt a) disease and to test correlation between muscle fat fraction and clinical impairment. mri was performed in the non-dominant lower limb of cmt a patients and healthy controls. fat fraction of tibialis anterior muscle, cross section area and volume of sciatic nerve were determined. muscle strength of dorsiflexion was measured using a dynamometer. disability was evaluated with cmt neuropathy score version (cmtnsv ). cmt a patients with known pmp gene duplication were enrolled. fat fraction of tibialis anterior muscle was significantly increased in patients compared to healthy controls. it was correlated with muscle strength (r=− . , p< . ) and cmtnsv score (r=− . , p< . ). cross section area and volume of sciatic nerve were significantly increased in patients compared to healthy controls. in conclusion mri fat fraction correlates with muscle strength and clinical measurement of disability in cmt a patients. it may thus be a useful outcome measure of disease progression in cmt a. basta i , peric s , cobeljic m , bjelica b , bozovic i , kacar a , nikolic a , rakocevic stojanovic v , stevic z , lavrnic d . neurology clinic, clinical center of serbia, school of medicine, university of belgrade, belgrade, serbia. there is a complete lack of data about epidemiological and clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) in serbia and surrounding countries. furthermore, there is a striking scarcity of information about quality of life (qol) in cidp patients and all qol studies have been conducted in countries with high standards of health care. in august we have designed the iness (inflammatory neuropathy study of serbia) in order to comprise as many patients with cidp from serbia, republic of srpska (bosnia and herzegovina) and montenegro covering population of more than nine million people. our first aim is to analyze overall impact of cidp on physical, mental and social areas of life measured with generic, symptom specific and disease specific questionnaires -sf- , inqol and cap-pri, respectively. furthermore, we aim to analyze influence of the disease on patients' working status and presence of depressive mood measured by beck's inventory. following features of patients are included: sociodemographic data, clinical aspects of the disease, level of disability, severity of sensory symptoms, presence of comorbidities, electrophysiological characteristics, as well as fatigue, autonomic symptoms and neuropathic pain. we intend to define the most significant predictors of decreased qol in order to focus on patients with the highest risk and to improve care of cidp. we also want to see if cidp patients in complete remission as per clinical findings still have reduced quality of life. we have recruited patient so far and we expect to include around subjects overall. we will present the first data of the study at the pns meeting . beaudonnet g , prud'hon s , cauquil c , labeyrie c , not a , bouilleret v , adams d . neurophysiology chu bicêtre, le kremlin bicêtre, france; neurology chu bicêtre, le kremlin bicêtre, france. familial amyloid neuropathy (fap) is a life-threatening disease of autosomal dominant inheritance due to transthyretin (ttr) gene mutation, a liver-produced protein. current treatments slow down its natural course and are indicated from the very first objective symptoms. we aimed to evaluate two neurophysiological markers: sympathetic skin response (ssr) and heart rate variability (hrv) in the early detection of sympathetic damages due to fap. ssr and hrv were assessed in ttr gene mutated patients with neither clinic nor electroneuromyographic abnormalities and controls matched on gender and age. cases were recruited consecutively from current care in the french reference center for rare diseases of bicetre university hospital. ssr was recorded on the two palms and on the sole of the left foot with to stimulations between and ma. hrv was registered during three conditions of seconds each: normal breathing, deep breathing ( cycles of seconds of inspiration and seconds of expiration) and valsalva manoeuver during seconds. valsalva ratio, defined by the ratio between the longest and shortest rr intervals, was significantly higher in the control groups after bonferroni correction (means of . and . , respectively, p< . ). there was no significant difference between the two groups for any ssr parameter, although means of amplitudes were systematically higher in controls than among cases. our results confirm that autonomic nervous fibers are damaged early in both clinical and electroneuromyographic asymptomatic patients mutated on the ttr gene. valsalva ratio seemed to be the most discriminative marker. long-term follow-up with test repetition and confrontation with cardiologic assessment will help to precise how these tests could be used in current care. they might help to identify high risk patients to propose them an appropriate early treatment and could be used to follow treatment efficacy. familial amyloidotic polyneuropathy (fap) was originally characterized by andrade as an axonal neuropathy which subsequently was found to be associated with a number of mutations in the plasma protein transthyretin (previously named prealbumin). it is now recognized that cardiomyopathy may be a significant factor in a majority of patients with the hereditary form of transthyretin amyloidosis (fap) and many of the transthyretin (ttr) mutations are associated with cardiomyopathy with no or minimal signs of peripheral neuropathy. attr-wt also called senile cardiac amyloidosis and senile systemic amyloidosis is recognized as late-onset, usually in the th or th decade of life, and the fact that the majority of patients are males. transthyretin neuropathy proven by nerve biopsy has been rarely reported in this population. here we report our experience with patients having attr-wt characterized by cardiomyopathy but also with varying degrees of peripheral neuropathy. clinically, the neuropathy appears as typical axonal or mixed axonal/demyelinating neuropathy as is seen in fap. pathologically, two types of ttr deposition have been found, ( ) intraneural ttr amyloid deposits as seen in fap are present in some patients and ( ) other patients have extensive vascular deposition of amyloid in both perineural arteries and veins without deposits within nerve trunks. in conclusion, peripheral neuropathy may definitely be a part of the attr-wt clinical presentation and with the increase in numbers of attr-wt cardiomyopathy patients being identified, it is important to ascertain whether any evidence of peripheral neuropathy is due to the amyloidosis and not to compounding syndromes such as diabetes mellitus type ii. besora s , santos c , izquierdo c , martinez-villacampa m , simó m , bruna j , , velasco r , . neuro-oncology unit, hospital universitari de bellvitge-institut català d́oncologia, ĺhospitalet, barcelona, spain; medical oncology department, institut català d́oncologia, ĺhospitalet, barcelona, spain; department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. oxaliplatin (oxa) is the first-line chemotherapy agent in the treatment of colorectal cancer (crc). oxa-induced peripheral neuropathy is the most frequent long-term side-effect. retreatment with oxa is frequently considered in patients as salvage treatment. patients receiving oxa-based chemotherapy regimen at least twice at our institution between and were reviewed. the aim of this study was to investigate whether retreatment with oxa increases the risk of developing or worsening previous oxa-induced peripheral neuropathy. the severity of neuropathy was measured by national cancer institute-common toxicity criteria (nci), total neuropathy score (tns) © and nerve conduction studies. one hundred twenty-five crc patients were included. median age was years. after first-line oxa-based chemotherapy, . % of patients developed neuropathy according nci, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. severity of neuropathy was grade ( . %), grade ( %), and grade ( . %). median time to retreatment with oxa was months. frequencies of neuropathy before retreatment were as follows: . % grade , . % grade , and . % grade . after retreatment, severities of neuropathy were . % grade and . % grade . no patient developed grade . . % of patients did not develop neuropathy. peripheral neuropathy was the reason for stopping prematurely treatment after first-line and retreatment in . % and . % of patients, respectively. worsening of previous nci score was observed in . % of patients. the great majority of patients ( . %) remained within the same nci score than before retreatment after median [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients that did not develop neuropathy after first treatment (n= ), only and patients developed grade and , respectively, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients who initially developed grade and neuropathy, no differences in tnsc © scores just before and after finishing retreatment with oxa were identified ( [ - ] vs [ - ], p= . ). retreatment with oxa in crc patients is a feasible option even in patients who developed moderate or severe neuropathy previously. lack of worsening of previous neuropathy is observed in the great majority of patients. neurological monitoring of patients candidates to retreatment with oxa should be considered. bessaguet f , sturtz f , magy l , , desmouliere a , bourthoumieu s , demiot c . ea -myelin maintenance & peripheral neuropathy, faculties of medicine and pharmacy, university of limoges, limoges, france; department of neurology, reference center for rare peripheral neuropathies, university hospital of limoges, limoges, france. prolonged pressure and the resulting local ischemia are widely accepted as the primary etiology of skin pressure ulcers (pus) but precise mechanisms of their formation remain unclear. in this study, we wanted to study the potential role of sensory small nerve fibers in regulation of inflammation during pus formation. to achieve this goal, we developed a mouse model of a purely sensory neuropathy and this was induced by resiniferatoxin (rtx). in this model, seven days after a single injection of rtx ( g/kg; ip), mice present a thermal and mechanical hypoalgesia associated with large substance p (sp) and calcitonin gene-related peptide (cgrp) depletion without neurodegeneration. this model mimic quite well what is observed in early stages of sensory nerve fiber defect. studies have shown that sp and cgrp are involved in cutaneous inflammation regulation. in fact, these neuropeptides are released by sensory fibers and are pro-inflammatory mainly through recruitment of immune cells and vasodilation. thus, we studied gene expression of pro and anti-inflammatory cytokines by a rna array approach during pus formation in control and rtx mice. seven days after a single injection of rtx, epidermis, dermis and subcutaneous tissue layer were pinched with magnetic plates during hours. pressure induced a stage pus. gene expression was evaluated in each compressed area h after pressure. results showed mainly a down-regulation of gene expression in pus of rtx mice compared to control mice. a decrease of cgrp/sp in skin sensory small fiber increased pus formation associated with an increase of interleukins (il)- , il- , il- and il- expression and a decrease of il- expression in rtx mice. supplementary experiments with rt-qpcr for each cytokine will be necessary to confirm these preliminary results. these observations suggest a cgrp/sp role in regulation of cytokines expression during pus formation. the new inflammatory profile exhibited in this study might help in the design of new treatments improving the quality of life of neuropathic patients prone to developing bedsores. bhattacharyya bj , jayaraj nd , belmadani a , ren d , rathwell ca , hackelberg s , miller rj , menichella dm . department of neurology, northwestern university, chicago, il, usa; department of pharmacology, northwestern university, chicago, il, usa. painful diabetic neuropathy (pdn) is one of the most common and intractable symptoms of diabetes, affecting % of diabetic patients. the hallmarks of pdn are neuropathic pain and small fiber degeneration, manifested by the loss of dorsal root ganglion (drg) nociceptor axons. neuropathic pain is associated with nociceptor hyper-excitability in the absence of physiologically appropriate stimuli. in states of neuropathic pain, drg nociceptors become increasingly responsive to a variety of excitatory influences, including inflammatory cytokines. in particular, we have shown that stromal cell derived factor- (sdf- ) and its receptor cxcr are necessary for the generation of neuropathic pain in mouse models of pdn. however, the molecular mechanisms leading to the hyper-excitability of drg nociceptors in pdn are unknown, as are the mechanisms leading to small fiber degeneration. this fundamental gap in our knowledge represents a critical barrier to progress in developing novel therapeutic approaches for pdn. the objective of this study is to identify the molecular cascade linking cxcr /sdf- chemokine signaling to drg nociceptor hyper-excitability, neuropathic pain, and small fiber degeneration in pdn. drg nociceptors can be identified by a series of molecular markers, including expression of the sodium channel na v . . indeed, > % of na v . -expressing drg neurons are nociceptors. feeding mice a high fat diet (hfd) for several weeks induces glucose intolerance, obesity, and mechanical allodynia, a particular pain hypersensitivity associated with pdn. using the hfd model combined with dreadd receptor technology, we have shown that reducing excitability of na v . -expressing neurons prevents and reverse neuropathic pain, neuronal calcium overload, mitochondrial dysfunction, and small fiber degeneration. furthermore, we have shown that cxcr receptors are necessary for neuropathic pain and small fiber degeneration in pdn. taken together these data demonstrate that na v . nociceptor hyperexcitability in pdn is driven through the activation of cxcr receptors. inhibition of hyperexcitability can prevent and reverse the development of pdn. furthermore, these observations will advance our understanding as to how changes in excitability, calcium influx, and mitochondrial dysfunction in nociceptors contribute to neuropathic pain and small fiber degeneration in pdn, which is a critical barrier to progression for effective and disease modifying treatment for pdn. bianco m , terenghi f , gallia f , nozza a , scarale a , fayoumi mz , giannotta c , morenghi e , nobile-orazio e . poems (polyneuropathy, organomegaly, endocrinopathy, m protein and skin changes) syndrome is an unusual multisystem disease with neurological disability due to a severe disabling polyneuropathy, with high mortality by multiorgan failure. peripheral blood stem cell transplantation (pbsct) is considered the treatment of choice for poems while lenalidomide is the most promising therapy for patients not eligible for pbsct. the aim of the present study was to compare the long-term effects on clinical, biological and neurophysiologic parameters in patients with poems treated with lenalidomide or pbsct. the clinical, biological and neurophysiologic data were reviewed in poems patients treated with pbsct (n: ) or lenalidomide (n: ). the mrc sumscore on muscles, onls scale, vegf serum levels and nerve conduction studies were assessed before (t ) and after (t ) and years (t ) of treatment and the differences were compared using anova. combining the two groups of patients, there was a significant improvement after treatment in the mean mrc sumscore (t = ± ; t = ± ; t = ± ; p = . ), in the mean onls score (t = . + . ; t = . + . ; t = . + ; p = . ), in the ulnar mean distal motor latency (t = . ± . msec; t = . ± . msec; t = . ± . msec; p = . ), distal compound muscle action potentials amplitude (t = . ± . mv; t = . ± . mv; t = . ± . mv; p = . ), motor conduction velocity (t = . ± . m/sec; t = ± m/sec; t = . ± . m/sec; p= . ) and serum vegf levels (t vs t : p = . ; t vs t : p = . ). the difference was also significant when we separately analyzed patients treated with lenalidomide and pbsct and there was no difference between the two groups in any of the analyzed parameters. treatment with pbsct and lenalidomide significantly and similarly improved clinical, biological and neurophysiologic parameters in patients with poems syndrome up to two years. since pbsct may not be suitable for all patients, lenalidomide may represent an effective and a valuable alternative in these patients or in those relapsing after pbsct inducing a prolonged clinical, biological and neurophysiologic improvement. bis d , tao f , abreu l , sleiman p , hakonarson h , zuchner s and inherited neuropathy consortium. dr. j.t. macdonald department for human genetics, hussman institute for human genomics, university of miami, miami, fl, usa; center for applied genomics, the children's hospital of philadelphia, philadelphia, pa, usa. inherited peripheral neuropathies are clinically and genetically heterogeneous diseases that can cause distal muscular atrophy and sensory loss. alleles in over one hundred different genes have been shown to cause peripheral neuropathies; yet, greater than % of axonal neuropathy patients do not receive a genetic diagnosis. large scale exome studies are now beginning to be sufficiently powered to perform mutational burden analysis. this approach compares damaging allele frequencies of cmt cases with a control group to identify additional causes for neuropathies. this approach will also identify genes that require an oligogenic inheritance to cause a phenotype. in a deviation from the classic linkage-based and heuristic variant filtering approaches to gene identification, we are performing burden analyses in a large cohort of cmt families. in known neuropathy genes, we saw that neuropathy cases carried on average . rare, non-synonymous variants, while unrelated non-neuropathy controls harbored . variants (p= . e- , mann-whiney u-test). enrichment of rare, non-synonymous variants in cmt disease genes within inherited peripheral neuropathy cases suggests the presence of multiple weaker alleles in individual patients. we also performed an unbiased exome-wide gene-based burden analysis and ranked genes after multiple testing correction. several new candidate genes were identified that need further follow up conformational studies. a number of known cmt and related genes were observed in the list of top candidates. we are currently analyzing additional aspects of this sample and are actively seeking more cmt exomes to enlarge our study. in summary, statistical methods traditionally reserved for more 'common' phenotypes' increasingly are becoming available for rare disease genetics. c bl/ mice were treated with bortezomib alone or in combination with monastrol. neuropathic changes were assessed by nerve conduction studies and histological analysis. analysis of axonal morphology was performed with light and electron microscopy. anti-neoplastic properties of monastrol alone and in combination with bortezomib were assessed in different blood cancer cell lines. prolonged treatment with bortezomib induced a sensory neuropathy in mice. significant changes in axonal morphology correlated with reduced function of peripheral nerves. the administration of monastrol substantially ameliorated morphological features of axonal alterations and sensory neuropathy. cytotoxicity studies in blood cancer cell lines showed no interference of monastrol with the cytostatic effects of bortezomib. our data indicate that monastrol may alleviate bortezomib induced neuropathy. the favorable cytotoxic profile of monastrol makes it an interesting candidate as neuroprotective agent to prevent bortezomib-induced neuropathy. boczonadi v , meyer k , gonczarowska-jorge h , , bartsakoulia m , roos a , , bansagi b , zahedi rp , talim b , bruni f , kaspar b , , lochmüller h , boycott km , müller js , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; rinch, columbus, oh, usa; leibniz-institute für analytische wissenschaften-isas-e.v., dortmund, germany; capes foundation, brazil; department of pediatrics, hacettepe university children's hospital, ankara, turkey; dbbb,university of bari aldo moro, bari, italy; department of neuroscience, the ohio state university, columbus, oh, usa; department of genetics, cheo, university of ottawa, ottawa, canada. while autosomal dominant mutations in gars, encoding the glycyl-trna synthetase, have been identified in patients with charcot-marie-tooth peripheral neuropathy (cmt d) and distal spinal muscular atrophy type v (dsma-v), autosomal recessive mutations cause mitochondrial disease affecting skeletal muscle and heart. gars is a bi-functional enzyme and it is responsible for normal protein translation both in mitochondria and the cytoplasm. in this study we have focused on the mitochondrial function of the gars by investigating a mouse model (gars c r) , human fibroblasts and induced neuronal progenitor cell lines (inpcs). mild mitochondrial abnormalities were detected in skeletal muscle of the gars c r mice while no other tissues were affected. control and patient fibroblasts harboring gars mutation were directly converted into inpcs. we identified tissue specific impairment of mitochondrial function in neuronal cells carrying not only recessive but also dominant gars mutations, suggesting neuron-specific effects of mitochondrial alterations.comparative proteomic analysis of inpcs showed significant changes in mitochondrial proteins. furthermore, the reduction of the vesicle-associated membrane protein-associated protein b (vapb) and its downstream pathways in gars-deficient inpcs suggests that altered mitochondria-associated endoplasmic reticulum (er) membranes (mam) may also contribute to the motor neuropathy. boczonadi v , king ms , bansagi b , roos a , , eyassu f , borchers c , lane m , ramesh v , lochmüller h , pyle a , griffin h , smith ac , chinnery pf , , alan j robinson aj , edmund rs kunji ers , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; mitochondrial biology unit, medical research council, cambridge; leibniz institute of analytic sciences (isas), dortmund, germany; uvic-genome bc proteomics centre, vancouver, canada; paediatric neurology, royal victoria infirmary, newcastle upon tyne foundation hospitals nhs trust, newcastle upon tyne, uk. members of the mitochondrial carrier family (slc ) transport nucleotides, keto acids, amino acids, fatty acids, co-factors and inorganic ions across the mitochondrial inner membrane. several inherited diseases with very variable clinical presentations are associated with dysfunctional mitochondrial carriers. we report a patient with childhood-onset spinal muscular atrophy and mitochondrial myopathy caused by a homozygous mutation in slc a , encoding the mitochondrial oxodicarboxylate carrier (odc). the mutation renders the carrier dysfunctional and, consequently, -oxoadipate cannot be imported into the mitochondrial matrix. computer modelling of the metabolic defect caused by the mutation predicted that the impaired transport leads to accumulation of -oxoadipate, pipecolic acid and the known neurotoxin quinolinic acid, which were precisely confirmed by targeted metabolomics in serum and urine. exposure of -oxoadipate and quinolinic acid reduced the level of mitochondrial complexes in sh-sy y cells in-vitro suggesting a possible pathomechanism. here we demonstrate that -oxoadipate and quinolinic acid are toxic for spinal motor neurons and their increased levels may contribute to neuropathy. and families have been reported. interestingly, most mutations target the same amino acid residue (k e, k m, k n, k t) in the highly conserved -crystallin domain of the hspb protein. the spectrum of diseases caused by mutations in the hspb gene was recently expanded to distal myopathy. hspb is ubiquitously expressed, but is highly expressed in motor neurons and muscles. the hspb is a chaperone that participates in clearing misfolded poly-q containing proteins such as mutant huntingtin and ataxin- involved in respectively huntington's disease and spino-cerebellar ataxia. hspb directly interacts with the co-chaperone bag and their role in chaperone-assisted selective autophagy is well described. to delineate the molecular deficits and functional consequences of hspb mutations we generated a knock-in (ki) mouse model for the k n missense mutation mimicking the neuropathy phenotype. we observed that homozygous mutant mice (hspb k n/k n ) develop a progressive axonopathy, with decreased compound motor action potential amplitudes, and loss of large and medium myelinated axons. this results in locomotor deficits with an impaired performance at the rotarod and grip strength tests. at the ultrastructural level, the hspb -ki model displays severe signs of axon degeneration and a clear myofibrillar myopathy, as observed in some patients with hspb mutations. interestingly, hspb positive aggregates were found in the sciatic nerve and gastrocnemius muscle of our mutant mice. additionally, our model allowed us to generate hspb knock-out (ko) mice using the same targeting vector. strikingly, the homozygous hspb -ko animals do not show any sign of axonopathy and display a much milder myopathy than the hspb -ki animals. these data suggest that part of the pathomechanisms is due to toxic gain-of-function of the mutant protein. boutahar n , reynaud e , nasser y , camdessanché jp , antoine jc . university hospital, saint-etienne, france. dysimmune sensory neuronopathies (snn) depend on neuron cell death induced by an inflammatory reaction in dorsal root ganglia. we have recently identified the intracellular tyrosine kinase (trk) domain of the fibroblast growth factor receptor- (fgfr ) as the target of antibodies in a subset of patients with non paraneoplastic snn. fgfr is one of the four fgfrs and has been involved in sensory neurons maintenance during development and cell death induction after axotomy. fgfrs ligand fixation results in the activation of several intracellular pathways through adaptator protein interactions with the trk domain. in particular ras activation may lead to cell proliferation or apoptosis through erk / or p map kinase signaling. the p mapk pathway is also involved in neuronal cell death induced by nmda and ampa receptor activation. as fgfr is a cell surface protein, human antibodies may interfere with the receptor functioning as a growing number of evidence has showed with other cell surface antibodies in neurological diseases. to test this hypothesis we developed an in vitro model using fvbn mice cortical neurons culture exposed to a rabbit polyclonal antibody reacting with the trk domain of fgfr . comparatively to normal rabbit iggs, the fgfr antibody induced neuron cell death in a dose dependent manner. neuron cells were exposed to fgfr antibody concentration leading to - % cell death in absence or presence of the p mapk inhibitor sb and the expression of fgfr , glur subunit of ampa receptors and nr subunit of nmda receptor was measured by quantitative rt-qpcr. the fgfr antibody induced an upregulation of fgfr while the glur and nr subunits were modulated. these changes were prevented in presence of sb . these preliminary results indicate that anti-fgfr iggs may interfere with the functioning of the intracellular domain of the protein and the expression of nmda and ampa receptors through the p map kinase pathway. this model may be used to test the effect of human anti-fgfr iggs in vitro. braathen gj , tveten k , holla Øl , busk Øl , hilmarsen ht , svendsen m , høyer h . section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. next-generation sequencing (ngs) has during the last years entered the clinical diagnostics. ngs has proven to be very efficient in the diagnostics of disorders where multiple genes can be involved. our ngs-based targeted gene panel consists of hereditary neuropathy genes, i.e. mostly charcot-marie-tooth genes. this study is a retrospective study of clinic samples received between may and february . we describe the identified novel likely pathogenic sequence variants, according to international guidelines. in this period we identified novel, not previously described, likely pathogenic sequence variants in the following genes: aars, fgd , gan, hint , litaf, lrsam , mme, mpz, nefl, pmp , sbf , sh tc and yars. there is now a large range of genes causing hereditary peripheral neuropathies and many likely pathogenic sequence variants. likely pathogenic sequence variants are not only identified in old well established neuropathy genes but also in the newer genes like mme. modelling brown-vialetto-van laere syndrome in c. elegans mutations in slc a and slc a , which encode the riboflavin transporters rfvt and rfvt . patients with rfvt deficiency exhibit proximal and distal limb weakness, sensory ataxia, diaphragmatic paralysis, optic atrophy, sensorineural deafness and bulbar palsy. riboflavin is critical for the biosynthesis of flavin mononucleotide and flavin adenine dinucleotide, essential cofactors for carbohydrate, amino-acid and lipid metabolism. mutations in slc a reduce or abolish rfvt expression resulting in impaired riboflavin uptake into sensory and motor neurons. high-dose riboflavin treatment can improve or stabilise a patient's condition, however the optimum dose and long term effects of riboflavin treatment, and disease pathomechanisms remains poorly understood. to further understand the pathophysiological consequences of slc a mutations, we propose developing an animal model for bvvl. caenorhabditis elegans (c. elegans) are small round transparent nematodes extensively used for studying the genetics and molecular biology of neurodegenerative diseases. there are two c. elegans riboflavin transporter genes, rft- and rft- . based on protein sequence homologies and expression profiles for both genes, rft- is the ortholog of rfvt . the expression of rft- is regulated by riboflavin availability and knock-down of the rft- gene by sirna perturbs c. elegans development. our aim is to develop a knock-in c. elegans model of bvvl. human rfvt and c. elegans rft- protein sequences were aligned with clustal omega to identify conserved amino acid residues associated with bvvl mutations. the amino acid involving the l p rfvt mutation is conserved in rft- (residue l ). to create our model, we will introduce the l p rft- mutation into the rft- locus in c. elegans genomic dna using crispr/cas- technology. this bvvl c. elegans model will allow us to explore the pathogenic consequences of rfvt deficiency underlying motor nerve degeneration and to evaluate drug therapy regimes by determining the optimal riboflavin dose and treatment initiation, and trialing other compounds that may improve benefits seen with riboflavin supplementation. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune-mediated inflammatory disease of the peripheral nervous system. cidp can present chronic progressive or relapsing-remitting courses and can predominantly affect motor but also sensory nerve fibers causing weakness of proximal and distal muscles. it represents the most common chronic autoimmune neuropathy and is pathologically characterized by focal inflammatory-mediated demyelination followed by axonal degeneration. recently, we have developed a new animal model for cidp, the chronic-ean, induced in lewis rats by active immunization with s-palmitoylated p ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) peptide. this model fulfills electrophysiological criteria of demyelination with axonal degeneration, a result confirmed by immunohistopathology. the late phase of the chronic disease is characterized by an accumulation of il- + cells and macrophages in sciatic nerves and as well as high serum il- levels. it is a reliable and reproducible animal model for cidp that can now be used for translational drug studies for chronic human autoimmune-mediated inflammatory diseases of the peripheral nervous system, particularly cidp, for which, there is a real need for new immunotherapies. the aim of this study was to test the therapeutic efficacy of ivig and a recombinant fc fragment (fcrec) in this new cidp animal model. treatments with ivig and fcrec proved effective in preventing further progression of cidp in rats. the therapeutic treatments not only decreased the maximal clinical scores of the cidp rats compared to albumin treatment but also abolished the disease chronicity. interestingly, a better efficacy of fcrec treatment compared to ivig was demonstrated at histological level, with the myelinated fibers well preserved and the greatly reduced accumulation of macrophages and il- + cells in sciatic nerves. ivig and fcrec therapeutic activities in this model can also be followed by measurement of antibodies in the serum and could therefore be used as biological markers. the current study provides for the first time direct evidence that ivig is effective in the treatment of cidp rats and suggests that a novel fcrec compound is more effective than ivig. it will contribute to the development of more effective and safer drugs for the treatment of autoimmune peripheral neuropathies like cidp. each time, one measurement with supramaximal stimulation according to the conventional ncs and another measurement with % enhanced stimulation intensity were conducted. we analyzed the results with special consideration of minimal and maximal fiber velocities and of the velocity with maximal fiber proportion. as a result, we detected statistically significant differences between cidp patients and controls in all these parameters. compared with controls, cidp patients had slower minimal and maximal fiber velocities and also the spectrum of motor conduction velocities was definitively shifted to slower velocities. slower minimal conduction velocities could be detected in some cidp patients by using the enhanced stimulation intensity. interestingly, in some cidp patients the conventional ulnar ncs were normal, but the collision technique showed fibers with a conduction velocity of less than m/s, indicating demyelination. to our knowledge, this is the first study using hopf's collision technique systematically in cidp patients. significant differences between the variation of ulnar motor nerve conduction velocity of cidp patients and controls could be detected. this method also showed signs of demyelination in some cidp patients with normal ulnar ncv. by enhancing stimulation intensity above the threshold of supramaximal stimulation in conventional ncs, the collision technique may be even more valuable. the clinical course of guillain-barré syndrome (gbs) is highly variable and some patients may develop treatment-related fluctuations (trfs) as an indication of ongoing disease activity and temporary treatment effect. other patients present as gbs, but subsequently develop repetitive relapses as an indication of acute-onset chronic inflammatory demyelinating polyneuropathy (a-cidp). this distinction is important because treatment may differ. we determined the frequency and clinical presentation of patients with gbs-trf and a-cidp in the first patients included in the international gbs outcome study (igos) with a follow-up of at least months. thirty-eight patients ( %) were excluded because of alternative diagnoses. of the remaining patients, ( %) had at least one trf, ( %) had a-cidp. preliminary analysis showed no significant differences between the groups (total-gbs, gbs-trf and a-cidp) for sex, age, sensory symptoms, cerebrospinal fluid results and mechanical ventilation. a-cidp patients had a median age of years (iqr - ), % was male, and all patients were treated in the acute phase of the disease (intravenous immunoglobulins (ivig) ( ), plasma-exchange (pe) ( ) or methylprednisolone (mp) ( )). gbs-trf patients has a median age of years (iqr - ), % was male and all patients received treatment (ivig ( ) and pe ( ). gbs-trf patients showed more antecedent events ( % versus %, p= . ), a higher gbs disability score (≥ ) at nadir ( % versus %, p= . ) and less frequently developed ataxia ( % versus %, p= . ) than patients with a-cidp. onset to nadir was longer in a-cidp than in gbs-trf ( days (iqr - ) versus days (iqr - ), p= . ) and the total gbs group ( days (iqr - ), p= . ). the time until the first clinical deterioration tends to be longer in the a-cidp patients (median days (iqr - ) versus median days in the gbs-trf group, not significant). the diagnosis a-cidp was made after a median of days . in conclusion, this first analysis identified distinctive characteristics of gbs-trf and a-cidp in support of a different pathogenesis that may help with early identification of these disorders in clinical practice. additional results will be presented at the conference. chronic inflammatory demyelinating polyneuropathy (cidp) is a disorder with a highly diverse clinical presentation, electrophysiological phenotype, response to treatment and outcome. this heterogeneity may indicate the presence of distinct subtypes of cidp, which may have a different pathogenesis and require more personalized treatment. the international cidp outcome study (icos) is a prospective, observational, international multi-center study that aims to describe this variation in clinical and electrophysiological subtypes and to define the clinical and biological determinants of these subtypes, disease activity, treatment response and outcome. in addition icos aims to provide an infrastructure for conducting new (therapeutic) studies in cidp, similar to the international gbs outcome study (igos). all patients fulfilling the efns/pns ( ) diagnostic criteria for cidp can be included in icos, independent of age, duration and severity of disease or treatment. we collect information on neurological deficits, diagnostic characteristics, various validated clinical outcome measures, previous and current treatment and we collect biomaterials (dna, cerebrospinal fluid, nerve biopsies and repeated serum samples). icos was started as a pilot study in dutch university centers. by february , patients were included in icos, patients recently diagnosed with cidp and previously diagnosed patients. included were ( %) males and ( %) females with a median age of years (iqr - ). the current cohort consists of classic (sensory-motor) cidp, madsam and pure motor variants. of the patients diagnosed in the past, patients ( %) were treated (intravenous immunoglobulins (ivig) ( ), prednisolone ( ), subcutaneous immunoglobulins (scig) ( ), dexamethasone ( ), plasma-exchange ( ) and ivig with methylprednisolone (mp) ( )). the recently diagnosed patients all received treatment (ivig ( ), dexamethasone ( ), plasma-exchange ( ) and patients were treated in a pilot study with ivig with methylprednisolone (mp)). the protocol has been evaluated and adjusted and will be shared with other researchers via the inflammatory neuropathy consortium and igos consortium. our aim is to include at least cidp patients worldwide with a minimum follow-up period of years. dominant mutations in glycyl trna synthetase (gars) cause inherited axonal neuropathy (charcot-marie-tooth type d). mutations in the mouse gars gene cause a similar phenotype, and represent valid disease models. we routinely use two mouse strains, one with a severe neuropathy, and one with a milder, later onset neuropathy. using these mouse models, we are exploring the mechanisms through which gars mutations cause peripheral axon degeneration. efforts include ribosome tagging to isolate ribosome-associated mrnas specifically from motor neurons, and non-canonical amino acid tagging to visualize and isolate newly synthesized proteins. taking advantage of the anatomy of motor neurons, we are able to analyze cell bodies separately from peripheral axons in both of these approaches. in addition, we are using these mouse models for preclinical studies testing gene therapy approaches to treat cmt d. consistent with our previous genetic studies in mice, knockdown of the mutant transcript, while preserving sufficient levels of the wild type, is a very successful approach when administered before the onset of neuropathy. we are now testing this approach in mice after the onset of symptoms, and in mice carrying a mutant gars allele associated with human disease. these studies are potentially translational, and also address mechanistic questions such as the timing and cell autonomy of the pathophysiology. burnor e , yang l , hao z , patterson k , quinn c , scherer ss , lancaster e . the university of pennsylvania, philadelphia, pa, usa; the second xiangya hospital, central south university, hunan, china. autoantibodies to two isoforms of neurofascin (nf and nf ) have been reported in patients with guillain-barre syndrome (gbs) or chronic inflammatory demyelinating neuropathy (cidp). it is not clear which of these responses reliably distinguish autoimmune neuropathies from other severe neuropathies, which responses are transient versus persistent, or which responses may target multiple isoforms of neurofascin. in addition, approximately % of neuropathy patients have no known cause, and it is unknown whether a subset of these patients may have autoantibodies to neurofascins. we have studied cohorts of patients with autoimmune neuropathy (n= ), genetic neuropathy (n= ), and idiopathic neuropathy (n= ) for igg and igm responses to neurofascins. neurofascin antibodies were found in ( %) of patients with autoimmune neuropathy, and ( . %) idiopathic neuropathy patients, but only % ( of ) in patients with genetic neuropathy. follow-up serum samples were available for positive cases. persistent responses were associated with chronic neuropathy while transient responses were seen in gbs or with remission of cidp. most patients had responses specific to either nf of nf . however, a particularly severe, treatment-resistant form of cidp, approaching a locked-in state, was seen in a patient with a unique response to all three isoforms of neurofascin (nf , nf , nf ). treatment of this patient with rituximab resulted in clinical improvement and resolution of the neurofascin antibody response. in conclusion, autoantibodies to neurofascins distinguish autoimmune neuropathies from severe genetic neuropathies, but the clinical phenotype may depend on the persistence and isoform specificity of the immune response. antibodies to the common domains shared by nf and nf may portend a severe but treatable neuropathy. a subset of idiopathic neuropathy patients may have an autoimmune mechanism. burns j , , sman ad , cornett kmd , wojciechowski e , , walker t , menezes mp , , mandarakas mr , rose kj , , bray p , , sampaio h , farrar m , , refshauge km , raymond j and the fast study group. university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia; university of new south wales, sydney, australia. exercise has undisputed benefits for human health and potentially as a treatment for neuromuscular disease. but there is also a risk of harm due to overwork weakness. we report the results of a -month randomised, double-blind, sham-controlled trial evaluating progressive resistance exercise of foot dorsiflexor muscles in pediatric cmt. sixty patients ( cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx ) aged - years were randomly assigned to undergo -weeks ( sessions) of moderate-intensity progressive resistance exercise or sham exercise. the primary endpoint was change in isometric dorsiflexion strength between groups assessed by hand-held dynamometry (expressed as a z-score based on age-and sex-matched normative reference values, positive values indicate an improvement in strength). the primary safety endpoint was change in muscle and fat volume of the muscles responsible for dorsiflexion by mri between groups. secondary outcomes were function (balance, long jump, -min walk test), walking ability ( d gait analysis), self-reported ankle instability, parent-reported quality of life (physical summary, psychosocial summary and global impression of change scores), and adverse events. fifty-five ( %) children completed the trial. adherence was comparable between exercise ( %) and sham ( %) groups. while patients experienced muscle soreness during training in the exercise group, adverse events did not differ between groups. the mean z-score for dorsiflexion strength increased in the exercise group by % at -months (from − . ± . to − . ± . ) and decreased in the sham group by %, mirroring the natural history of cmt (from − . ± . to − . ± . ). between-group ancova-adjusted difference at -months was . ( %ci, . to . ; p= . ). the mean scaled scores for mri muscle volume and fat volume were comparable between groups at months (p> . ). the global impression of change scores favoured the exercise group at -months (p= . ) and -months (p= . ). there was no other measurable effect of exercise. pre-specified subgroup analyses according to age ( - years vs. - years) showed a larger treatment effect with exercise in adolescents. targeted progressive resistance exercise was effective at halting progression of dorsiflexion weakness without detrimental effect on muscle morphology or other signs of overwork weakness in children with cmt. byung-nam y , yoon-ho h , suk-won a , seok-jin c , jung-joon s . inha university hospital, incheon, south korea; seoul metropolitan government boramae medical center, seoul national university, seoul, south korea; joong ang university hospital, seoul, south korea; seoul national university hospital, seoul, south korea. a years old female patient visited the hospital with weakness in both upper limbs from months under the suspicion of motor neuron disease with the finding of extensive denervation changes on electromyography. whole spine mri showed ventral nerve rootlet enhancement in the c and t bundles. the emg had confirmed active denervation changes in the muscles innervated by bilateral c ∼c roots. the cerebrospinal fluid culture test showed a protein (csf) elevation to mg / dl. she got a steroid pulse therapy. she had the symptoms of dry mouth and dry eyes during the recent years. a salivary gland scan test was performed for the possibility of sjögren's syndrome, and as a result, absorption of the contrast agent in both parotid and submandibular glands was decreased. the shammer test showed mm on the right side and mm on the left side. she was diagnosed of sjögren's syndrome. after weeks, overall strength improvement was observed, and bilateral shoulder abduction was improved to mrc grade or higher. the follow-up spine mri also showed that the initially seen ventral nerve root enhancement was disappeared. the case had visited the hospital with major symptoms of weakness and atrophy of the muscles, showing similar pattern to motor neuron disease, and was diagnosed as inflammatory polyradiculopathy and confirmed as primary ss during differential diagnosis. this case suggests that primary ss may induce inflammatory polyradiculopathy, which shows motor symptoms as major symptoms rather than sensory symptoms, and that a fast and accurate diagnosis is needed in terms that it can be treated with steroids and appropriate immune suppressive agents. traumatic injuries to peripheral nerves are frequent. however, effective pharmacological treatments are lacking. curcumin, a polyphenol found in rhizomes of curcuma longa, has been shown to develop antioxidant, anti-inflammatory, and neuroprotective properties. however, due to its poor hydrosolubility and its extensive metabolism, the use of large curcumin doses is required for therapeutic purpose. the aim of the present study was to investigate the effects of a local infusion of a low curcumin dose on nerve regeneration and functional recovery after sciatic nerve injury in rats. the experiments were conducted in g sd male rats submitted to unilateral sciatic nerve crush at d . curcumin was solubilized in polyethylene glycol and continuously administered using osmotic pumps ( . mg/day until d ) with a catheter delivering the drug near the lesion site. functional analyses using von frey, beam walking, static sciatic index (ssi) and grip strength tests were carried out at d (reference test) and every week after injury (d , d , d , d and d ). in addition, an evoked electromyogram was performed at d and d . after euthanasia (d ), nerve and muscle samples were collected and analyzed by light and electron microscopy. functionally, a significant improvement of the mechanical sensitivity (+ %) was observed at d in the curcumin-treated group (n= ) vs. vehicle group (n= ). in curcumin-treated group, skillful walking and finger spacing of the ipsilateral paw (ssi) were fully restored respectively at d and d contrary to vehicle group. furthermore, curcumin treatment improved the grip strength recovery (+ % at d ). the electrophysiological results indicated a full recovery of motor nerve conduction velocities (mncv) after days of curcumin treatment, while mncv remained altered in vehicle group ( % of the mncv at d ). morphometric analysis of nerve sections using g-ratio showed an improvement in the thickness of the myelin sheath in curcumin treated animals (+ % vs. vehicle group). histological investigation of gastrocnemius muscle indicated decreased neurogenic lesions in curcumin group. proteomic analysis is currently under investigation to understand the mechanisms involved in curcumin effects. our data could lead to the development of new therapeutic strategies in peripheral nerve regeneration using low doses of curcumin. previous studies suggest that the metabolic syndrome (mets) is associated with distal symmetrical polyneuropathy (dsp), and that diabetes, pre-diabetes, and obesity are the main metabolic drivers. the aim of this study is to investigate the association of mets components with retinal and cognitive function in a bariatric surgery cohort prior to surgery. patients were recruited from the adult bariatric surgery clinic at the university of michigan and lean controls from a research website (no mets components based on ncep/atpiii definition). participants underwent extensive metabolic phenotyping including a glucose tolerance test and fasting lipid profile. dsp was defined using the toronto consensus definition of probable clinical neuropathy. retinal function was measured with frequency doubling technology perimetry (average mean deviation), and cognitive function with the nih toolbox (composite score). univariate linear regression models were used to evaluate the association between mets components and retinal and cognitive function. to date, we have recruited bariatric surgery participants and lean controls. in the bariatric population, the mean (sd) age was ( . ) with % female compared with a mean age of . ( . ) with % female in the lean group. in the bariatric group, . % had diabetes, . % pre-diabetes, and . % normoglycemia. the dsp prevalence was % in lean controls, . % in normoglycemic, . % in pre-diabetic, and . % in diabetic bariatric participants (p< . for trend). retinal function was . ( . ), − . ( . ), − . ( . ), and − . ( . ) (p= . for trend), and cognitive function was . ( . ), . ( . ), . ( . ), . ( . ) (p= . for trend) in these same groups for lean controls, normoglycemics, pre-diabetics and diabetics, respectfully. pre-diabetes (− . , %ci: − . ,- . ) was the only mets component associated with retinal function, and waist circumference was the only one associated with cognitive function (− . , %ci − . ,- . ). dsp and retinal function, but not cognitive function decline with worsening glycemic status. similar to previous data for dsp, pre-diabetes and obesity are associated with retinal and cognitive function respectively. interestingly, while clinical dsp is common in this population, clinical retinopathy and dementia are not, indicating that dsp may be the first metabolic complication in the morbidly obese. and anti-cntn antibody-positive cases were confirmed by cba and were of igg subclass in half of them. by cba we identified additional / ( %) anti-caspr seropositive patients, whose isotype is currently being tested. sera of anti-nfasc and anti-cntn igg seropositive patients and patients with anti-caspr antibodies stained paranodes by indirect immunofluorescence on mouse teased nerve fibers. of note, seronegative patients for known antibodies showed reactivity against node and/or paranodes. compared to other seronegative cipd patients, seropositive patients had more frequently subacute onset of the neuropathy and a younger age at onset, particularly for nfasc or caspr antibodies. weakness was more severe and was often associated with proprioceptive loss, sensory ataxia and tremor. neuropathic pain was not a feature of caspr -seropositive patients. frequent findings were increased distal motor latencies and temporal dispersions on nerve conduction study and a higher protein level in csf. finally seropositive patients tended to have a higher disability and showed worst response to ivig. rituximab was effective in one patient with anti-nfasc antibodies and two patients with anti-cntn antibodies showed good and persistent recovery after cyclophosphamide. prevalence of antibodies was % in italian cidp patients and their presence was associated with distinctive clinical features. their determination, followed by characterization of igg subclass in positive cases, has clear clinical impact, by helping to guide therapeutic choices. the reactivity against nodal and paranodal components in sera from patients without known antibodies suggests that other targets could play a role in the autoimmune response in cidp and they still need to be identified. mutations in amynoacyl trna synthetases (arss), enzymes that catalyse the covalent attachment of amino acids to their cognate trna, are responsible for autosomal dominant cmt , intermediate cmt (cmt-i) and dhmn. we report the case of a male of italian ancestry who first presented with bilateral ankle clonus at three months, followed by toe walking and ankle instability. the ankle clonus subsided during adolescence. in the third decade he developed progressive walking difficulties followed by distal sensory loss. neurological examination at the age of revealed a steppage gait, distal lower limb weakness, decreased pinprick to the ankles, and reduced vibration sensation at the knees. reflexes were brisk in the upper limbs, reduced at the knees and absent at the ankles. muscle tone was increased in the lower limbs and plantar responses were extensor. nerve conduction studies revealed an axonal neuropathy. brain and spinal cord mri were normal. sanger sequencing of pmp , gjb , mpz , gdap and mfn were negative. sureselect focused exome sequencing (agilent technologies, santa clara ca, usa) demonstrated a c. g>a, p.arg his mutation in aars. the p.arg his mutation in aars has previously been reported in families with intermediate or axonal motor-sensory neuropathy, and in one case was associated with sensory-neuronal deafness. cns involvement has not previously been described with this mutation. mutations in aars have been associated with a range of phenotypes including cmti, cmt and dhmn with variable age on onset ranging from to years (mean years). of note, the aars p.gly arg mutation has been reported in a family with cmt and pyramidal signs. this study provides further evidence that pyramidal tract involvement can be an early feature of cmt n due to mutations in aars, further expanding the spectrum of arss-associated phenotypes. small fibres in the skin are vulnerable to damage in metabolic or toxic conditions such as diabetes mellitus or chemotherapy resulting in small fibre neuropathy and associated neuropathic pain (np). whether injury to the most distal portion of sensory small fibres due to a primary dermatological disorder can cause np is still unclear. recessive dystrophic epidermolysis bullosa, (rdeb) is a rare condition in which mutations of proteins of the dermo-epidermal junction lead to cycles of blistering followed by regeneration of the skin. damage is exclusive to the skin and mucous membranes, with no known direct compromise of the nervous system. it is increasingly recognised that most rdeb patients experience daily pain, the aetiology of which is unclear but may include inflammation (in the wounds), musculoskeletal (due to atrophy and retraction scars limiting movement) or np. in this study we investigated the incidence of np and examined the presence of nerve dysfunction in rdeb patients. around three quarters of patients presented with pain of neuropathic characteristics which had a length dependent distribution. quantitative sensory testing of the foot revealed striking impairments in thermal detection thresholds combined with an increased mechanical pain sensitivity and wind up ratio (temporal summation of noxious mechanical stimuli). nerve conduction studies showed normal large fibre sensory and motor nerve conduction however skin biopsy showed a significant decrease in intraepidermal nerve fibre density. autonomic nervous system testing revealed no abnormalities in heart rate and blood pressure variability however the sympathetic skin response of the foot was impaired and sweat gland innervation was reduced. we conclude that chronic cutaneous injury can lead to injury and dysfunction of the most distal part of small sensory fibres in a length dependant distribution resulting in disabling np. these neuropathic pain (np) following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons which begins to normalise over time. we investigated the composition and distribution of shaker type potassium channels (kv channels) within the nodal complex of myelinated axons following injury. at the neuroma that forms after damage, expression of kv . and . (normally localised to the juxtaparanode) was markedly decreased. in contrast kv . and . , which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in the rat and in humans. within the dorsal root (a site remote from injury) we also noted a redistribution of kv channels towards the paranode. blockade of kv channels with dtx after injury reinstated hyperexcitability of a-fibre axons and enhanced mechanosensitivity. changes in the molecular composition and distribution of axonal kv channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury. members of the francophone anti-mag cohort group are listed in "appendix". appendix polyneuropathy with anti-mag igm antibodies is classically progressive, predominantly sensory, and distal with ataxia and sometimes postural-intention tremor. we assessed clinical, biological, electrophysiological, and histopathological features in patients with igm gammopathy and anti-mag antibody titres higher than btu. we focused on characteristics of patients according to the anti-mag antibody titres at diagnosis. we retrospectively and prospectively analysed standardized report forms of patients from fourteen french-speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset and last follow-up was . years ( . - . ). about . % of patients presented with a "variant" clinical phenotype independently of anti-mag titres (< or ≥ btu). this included acute or chronic sensorimotor polyradiculoneuropathies, paucisymptomatic sensory polyneuropathy and multifocal neuropathy. at the most severe disease stage, . % of patients were significantly disabled. anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in respectively , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. sixteen percent of patients did not meet the electrodiagnostic criteria of definite demyelinating neuropathy, independently of anti-mag titres (< or ≥ btu). nerve biopsy, performed in nineteen patients, provided support to the diagnosis of anti-mag neuropathy in some particular issues (low titres of anti-mag, unusual clinical or electrophysiological phenotype). we assessed the degree of probability (probable, possible or uncertain) that patient neuropathies are directly related to anti-mag antibodies, according to anti-mag titre, electrophysiological data and nerve biopsy characteristics if available. it appears uncertain in patients with low anti-mag titres ( . % of the whole population). the clinical phenotype didn't appear to be different according to anti-mag antibody titre. many of the patients with low anti-mag titres presented "genuine" anti-mag neuropathy as demonstrated by edx studies, clinical presentation and sometimes nerve biopsies. for a small proportion of these patients, a direct relation between neuropathy and anti-mag antibodies is uncertain due to atypical clinical presentation, axonal neuropathy pattern or nerve biopsies, and positivity of antigangliosides antibodies. we assessed therapeutic management, response to immunotherapies and adverse events in a cohort of patients presenting with igm gammopathy and anti-mag antibody titres higher than btu. we retrospectively and prospectively analysed standardized report forms of patients from french speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset to diagnosis and last follow-up were respectively . ( - ) and . years ( . - . ). anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. seventy eight percent (n = ) of patients received immunotherapies. transient response to ivig or plasma exchanges at six month was observed in less than and % of patients respectively. chemoimmunotherapies and rituximab were more frequently administered in the group of patients with malignant hemopathies (n = ) compared to mgus (n = ) (mean lines of therapy = . , range - , sd . versus . , range - , sd . , p = . ). more than % of patients (n = ) received rituximab monotherapy. clinical worsening, mostly transient and reversible, was observed in / patients after rituximab. clinical response to rituximab at months and/or during - months follow-up period was observed in . % of patients, and correlated with anti-mag titre ≥ btu ( / responder versus / non-responder patients, p = . ). at - months follow-up, responder patients presented shorter symptom duration compared to non-responders, though not significant after logistic regression ( . years, range . - . , sd . in responder patients versus . years, range . - . , sd . , in non responder patients, p = . ). in some cases, electrodiagnostic studies were recorded during rituximab treatment follow-up and showed in responder patients a clear improvement of motor conduction velocities. these data may support another clinical trial to study benefice of rituximab in anti-mag neuropathies early in the disease. it raises issues about the value of incorporating electrodiagnostic parameters as end-points. schwann cells (scs) are essential for axon integrity and myelination in the uninjured pns. after pns injury, scs function as "first responders" , undergoing phenotypic re-programming and orchestrating many processes that lead to functional nerve repair. receptors in scs that contribute to sc repair programs remain incompletely understood. we identified a member of the ionotropic glutamate receptor (igr) family, n-methyl-d-aspartate receptor (nmda-r), in scs that is upregulated after nerve injury and acts as a co-receptor with ldl-receptor related protein (lrp ). lrp is a well-known regulator of the sc response to pns injury. herein, we used pcr to profile igr expression in cultured rat scs. obligate receptor subunits required for assembly of nmda-rs, ampa-rs, and kainate receptors were identified. treatment of rat scs with - microm glutamate or . - . microm nmda robustly activated akt and erk / . the response was transient and bimodal; glutamate concentrations greater than microm failed to activate cell-signaling. phosphoprotein profiling demonstrated other cell-signaling and transcription factors regulated by glutamate in rat and human scs, including p s -kinase, glycogen synthase kinase- , ribosomal s kinase, c-jun, and creb. activation of cell-signaling by glutamate in scs was blocked by eliminating calcium from the medium, by the selective nmda-r antagonist, mk , and by genetic silencing expression of the obligate nmda-r nr subunit. phosphoinositide -kinase/pi k functioned as an essential upstream activator of both akt and erk / in glutamate-treated scs. by activating pi k and erk / , glutamate promoted sc migration. glutamate ( microm) or nmda ( microm) injected into crush-injured sciatic nerves robustly activated p-erk / in both myelinating and non-myelinating scs in vivo. these results identify igrs as potentially important cell-signaling receptors in scs that may promote axon-glial interactions. understanding the function of sc nmda-r is important given current efforts to develop nmda-r-targeting drugs for patients with pain, depression, and alzheimer's disease. while frequently overlooked in a therapeutic context, scs are extremely important in the pathogenesis of chronic neuropathic pain. if these drugs modulate the activity of sc nmda-r and sc physiology, the response to pns injury may be altered and the possibility that neuropathic pain develops increased. candayan a , atkinson d , durmus tekce h , parman y , jordanova a , battaloglu e . department of molecular biology and genetics, bogazici university, istanbul, turkey; center for molecular neurology, antwerp university, antwerp, belgium; istanbul medical school, istanbul university, istanbul, turkey. charcot-marie-tooth (cmt) disease is a group of inherited peripheral neuropathies affecting one in individuals worldwide. the disease presents both clinical and genetic heterogeneity. so far, mutations in genes and loci are associated with cmt with autosomal dominant, autosomal recessive, x-linked and mitochondrial inheritance. despite the advances in genetic testing, approximately % of all cmt patients worldwide remain without a molecular diagnosis. we have investigated unrelated cmt patients of turkish descent, in all of which pmp duplication has been excluded previously. we used multiplex amplification of specific targets for resequencing (mastr) assay to sequence exonic regions of common cmt genes. recurrent mutations were identified in cases in mfn , gjb , mpz and hint genes. we have also identified novel variants in cases in mfn , pmp , gars, aars, ighmbp and gdap genes, all of which are very rare or not present in the variation databases and are predicted to be pathogenic by in silico tools. familial segregation analyses are ongoing for novel variations. mfn and gjb genes were the most commonly mutated causative genes in this cohort. cases without molecular diagnosis after the mastr testing are candidates for further analyses such as whole exome sequencing or whole genome sequencing. outcomes of the current study and our previous experience with turkish cmt patients suggest a high genetic heterogeneity. our insight is that different genetic strategies or larger panels are essential to determine the causes underlying cmt especially in regions where rare recessive types of the disease can be observed due to high frequency of consanguineous marriages. capoccitti g , giannini f , ginanneschi f , casali s , insana l , rossi a . university of siena, siena, italy. several efforts have been made to elaborate new electrophysiological criteria for early diagnosis of guillain-barré syndrome (gbs) and to differentiate demyelinating (aidp) from axonal (aman/amsan) forms. the aims were to verify the diagnostic power of total cmap (tcmap) duration, firstly applied in gbs field. this parameter was compared with commonly used neurophysiological measures, including negative phase cmap duration (ncmap), and was added to modified rajabally criteria. we reviewed the clinical and electrophysiological data of patients with gbs (level or of brighton clinical criteria). each patient underwent at least two neurophysiological studies, the first within weeks, the second between and weeks from symptom onset. at least four motor and three sensory nerve conduction studies were recorded for each test. regarding early diagnosis, the binary logistic regression model with multiple variables, including ncmap duration, showed that the features of predictive model presenting greater significance (p < . ) were tcmap duration, sural sparing and a-waves. among these, tcmap duration showed greater significance (p = . ). the tcmap was diffusely prolonged in aidp compared to aman/amsan, already in first examination and confirmed in the second one. roc analysis for tcmap duration in aidp vs. aman/amsan showed: cut-off . ms, auc . , ppv . %. we propose the tcmap duration as a new useful electrophysiological measure for early diagnosis of "generic" gbs and for early differentiation between aidp and aman/amsan. moreover, the prolongation of tcmap, the presence of a-waves and sural sparing represent a strongly diagnostic predictive triad of aidp. aifm (apoptosis-inducing factor, mitochondrionassociated- ) has captured great attention from biomedical researchers due its critical role in the regulation of cell apoptosis. this flavoprotein is typically located in the mitochondrial intermembrane space where it is associated with respiratory chain complex-i. upon a cell-death insult, aifm is cleaved into a kd protein that is released into the cytosol. the kd peptide may enter the nucleus to trigger chromosome condensation and fragmentation, initiating a caspase-independent pathway of apoptosis. however, this nuclear translocation may be blocked by cytosolic heat-shock protein- (hsp ) that binds with the fad domain (aa - ) of aifm . mutations in aifm gene have resulted in several clinical phenotypes, including a family with cmtx (glu val). clinical deficits in these patients usually involve multiple organs. in this study however, we identified a family with a novel missense mutation (phe leu) in aifm that developed a late-onset sensory motor axonal polyneuropathy by nerve conduction criteria. the proband and affected siblings exhibited distal muscle weakness and atrophy with normal cognitive and cranial nerve functions. there was no obvious phenotype from other organs. interestingly, this phe leu mutation affects a highly conserved amino acid at the center of the fad domain. we hypothesize that this mutation impairs the binding between aifm and hsp , leading to an enhancement of cell-death signaling. this family therefore provides a unique opportunity to explore how altered apoptotic signaling affects peripheral nerve system. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr chronic inflammatory demyelinating polyneuropathy (cidp) can be the key symptom leading to diagnosis of associated lymphoma. patients with diagnosis of cidp according to efns/pns criteria associated with b cell lymphoproliferative disorders (bld), in one center, between november and november were included. demographical, clinical, nerve conduction, immunological, histological data and response to treatment were recorded retrospectively. eight patients ( men), median age yo were included. onset of polyradiculoneuropathy was either chronic (n= ) or acute (n= ). neurological condition led to diagnosis of bld in all but one case, because of onset (n= ) or worsening of neuropathy (n= ). clinical presentation was that of cidp in patients or pseudo-canomad in and plexopathy in one. lymphoma type was: lymphoplasmacytic (n= ), diffuse large b cell (n= ), small lymphocytic (n= ), marginal zone (n= ), unclassified small b cell (n= ). only patients presented with lymphadenopathies. bld was diagnosed in all patients on myelogram or bone marrow biopsy, performed because of cytopenia (n= ), atypical (n= ) or severe (n= ) neuropathy. monoclonal gammapathy was identified in / patients (igm n= , igg n= ). neuromuscular biopsy was performed in patients and disclosed endoneural infiltration in . anti-neuronal or antigangliosides antibodies were positive in patients. none of the patients presented a systemic autoimmune disease, hemolytic anemia associated with bld (n= ). immunomodulating treatment was administered in all patients (ivig n= , plasma exchange n= , steroids n= ) and immunosuppressants (n= ). immunochemotherapy for lymphoma was initiated because of lymphoma type or severity in cases, in cases because of the associated neuropathy. median follow-up was of months after treatment initiation. four out of patients treated within months of neurological onset improved as well as one out of patients whose preexisting neurological condition had worsened. two patients presented neurological relapse during progression of lymphoma. two patients died. unusual presentation of cidp -i.e., rapid progression or treatment failure -should lead to further testing for associated lymphoma. because general symptoms and lymphadenopathy often lack, diagnosis requires analysis of bone marrow with lymphocyte phenotyping. early treatment with immunochemotherapy was associated with better prognosis in our series. cardiac scintigraphy is a useful tool for the diagnosis, prognosis and pre-symptomatic early detection of familial amyloidosis-associated neuropathies three main types of familiar amyloidosis: transthyretin (ttr), apolipoprotein a and gelsolin. cardiac involvement is a leading cause of morbidity and mortality; one new described mutation strongly related with isolated cardiac amyloidosis is the ttr val ile. the discovery of tests that allow early diagnosis of cardiac involvement in amyloidosis and to infer about the etiology of the disease is of major importance. in a cohort of patients with different types of familiar ttr amyloidosis, we aimed to assess the role of mtc- , -diphosphono- , -propanodicarboxylic acid ( mtc-dpd) in detecting myocardial amyloid infiltration. we enrolled four patients diagnosed with late familiar amyloidosis, which mutations were documented at deoxyribonucleic acid analysis: three patients with ttr val met mutation and one patient with ttr val ile mutation. three patients were asymptomatic for cardiac involvement and one patient (val ile mutation) had a previous diagnosis of heart failure. myocardial uptake of mtc-dpd scintigraphy was semiquantitatively and visually assessed at five minutes and three hours.the uptake of mtc-dpd highly demonstrated amyloid in cardiac area in two out the three cases of ttr val met and in ttr val ile. ttr val ile case presented the highest uptake due to the exclusive deposition of amyloid in cardiac area resulting in severe heart failure. in hereditary transthyretin-related amyloidosis, including the mutations ttr val met and val ile, mtc-dpd cardiac scintigraphy can identify infiltration even in asymptomatic individuals, allowing an early diagnosis of cardiac compromise in this group of diseases. we can consider that this non-invasive test would be a tool with potential importance in the diagnosis, prognosis and pre-symptomatic early detection of cardiac amyloidosis, giving emphasis on its applicability in familial forms of amyloidosis. cervellini i , galino j , zhu n , birchmeier c , bennett dl . ndcn university of oxford, oxford, uk; max-delbrück-center for molecular medicine, berlin, germany. erk/mapk pathway has a critical role in pns development since its involvement in many physiological processes. sustained erk / mapk activation in schwann cells enhances myelin growth during development and overcomes signals ending myelination leading to a continuous myelin production. however, strong activation of erk has also been shown to cause schwann cells dedifferentiation and demyelination in vivo. our aim was to investigate whether a mild activation of this signalling pathway in adult schwann cells (scs), by expression of gain of function mek dd allele, could have a beneficial role in remyelination and regeneration after injury. erk/mapk activation in adult scs in plpcreer t ;mek dd mutant mice, did not affect myelination during development. following sciatic nerve injury, wallerian degeneration was enhanced in mutants pushing towards a dedifferentiation stage of scs as previously described. however, mapk activation was detrimental during regeneration with a delay in functional recovery and a negative impact in both myelinated and non-myelinated fibres compared to controls. one month after injury the total number of axons in mutant sciatic nerves was half of the controls. although no differences in g-ratio have been found in the two groups, mutants presented a higher number of myelinated axons showing myelin disruption with start of myelin decompaction, lack of cajal bands, abundant sc processes surrounding axons and a shorter sc elongation, as seen by decreased internodal distance. in addition, we found a negative effect of mapk activation also in small diameter axons with the presence of abnormal remak bundle structures, reduced number of c-fibres/remak bundle and a significant decrease in intra epidermal nerve fibres density in the skin. we concluded that mild mapk activation has a different role in development and remyelination where negatively affects axon survival, myelin stability, remak bundle formation and small fibres regeneration. cervellini i , galino j , zhu n , bao lu , bennett dl . ndcn, university of oxford, oxford, uk; harvard medical school, boston, ma, usa. neprilysin (nep) is an endopeptidase which has been of interest due to its potential role in neurodegeneration and pain as a consequence of its ability to degrade amyloid and substance-p respectively. nep expression is not limited to cns and it has been reported to be expressed in schwann cells, nodes and schmidt-lanterman incisures. our interest in this gene was related to recent findings that have associated homozygous and heterozygous nep mutations with charcot-marie-tooth type- . in old mice lacking nep subtle morphological changes have been reported. our aim was to determine whether nep expression was modulated by nerve injury and to investigate its role in axon regeneration and re-myelination. we find that nep gene expression was decreased after nerve crush and furthermore was dependent on the growth factor (and pro-myelin signal) neuregulin- . in control mice nep expression was transiently reduced and returned to baseline at day after injury, in neuregulin- knock-out (ko) mice, in which re-myelination was impaired, the expression was still decreased at day . in assessing behavioural measures of locomotor and sensory function one month after sciatic nerve crush, nep ko mice showed a functional regeneration comparable to wt, as seen by sciatic functional index measurement, beam and toe spreading tests. the only significant difference we observed between wt and ko was in the sensorial test, showing ko mice recovering faster in the pinch test by days after crush. the results for all the tests at baseline did not differ between the two groups. detailed histological analysis of nerve repair was undertaken using electron microscopy. there was no difference between wt and ko in total axon number, g-ratio, axon diameters and other myelin features one month post crush. in summary, although nep expression is regulated by nerve injury in a neuregulin- dependent fashion this endopeptidase is dispensable for axon regeneration and re-myelination after nerve injury in the rodent. diabetes neuropathy is a common complication of diabetes, and neuropathic pain has a detrimental impact on quality of life. this study investigated sensory nerve excitability properties to elucidate the axonal changes of diabetic neuropathy. a total of diabetes patients ( type ii, and type i) were enrolled in this study. clinical assessment, nerve conduction studies, and nerve excitability testing data were analyzed to determine axonal dysfunction in diabetic neuropathy. among those patients, seventeen subjects had complained of spontaneous painful sensation over feet or hands (painful cohort), and seventy-eight patients had no sensory symptoms or decreased the sensation over foot (non-painful cohort). sensory nerve excitability of the painful cohort showed reduced late subexcitability (p= . ), increased superexcitability (p= . ) in compared to the non-painful cohort. there is no difference in disease duration, blood glucose levels (hba c) between these two cohorts. these findings suggested the possible pathogenesis of painful sensory axons might be hyperpolarized or slow potassium channels dysfunction. these insights our further understanding of painful diabetic neuropathy, and may provide a basis for neuroprotective or therapeutic approaches for painful polyneuropathy. the main purpose of this study was to assess the clinical feasibility of diffusion tensor imaging (dti) for the diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp). between march and december , we prospectively enrolled patients with definite cidp according to the efns criteria and two control groups: healthy volunteers matched on age and sex and patients with cmt- a. using a -t magnetic resonance imaging scanner, we obtained dti scans of brachial plexus of these groups and prepared fractional anisotropy (fa) maps, and compared these values between groups. adc values and cervical nerve roots diameters on stir sequences were evaluated too. two neuroradiologists, blinded to clinical informations, reviewed mri studies independently. in all patients with cidp, we also performed clinical evaluation and electroneuromyography. significantly decreased fa values (p< . ) and increased adc values were observed in cidp patients compared with healthy subjects. there is no significant difference between cidp and cmt group. inter-observer concordance was excellent for fa values ( c= . ; p< . ) and moderate for adc values ( c= . ; p< . ) and cervical nerve root diameters ( c= . ; p< . ). there is a significant correlation between fa and disease duration (r = − . , p < . ), inclusion mrc score (r = . , p < . ) and between fa mesured on c c and incat score at inclusion (r = − . , p < . ). no significant correlation is observed between fa and electrophysiological indices. compared with healthy subjects, cervical nerve root diameters were significantly increased (p< . ) in patients with cmt and cidp. contrary to fa values, moderate level of concordance was found between inter-observers measurements of diameters (cclin = . ). our preliminary data prove the clinical feasibility and reproductibility of dti for the evaluation of plexus and cervical nerve roots in patients with cidp. cheng yj , teng a , goh ejh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. the sural-sparing pattern of the sensory nerve action potentials (snap) of guillain-barré syndrome (gbs) has been attributed to greater immunological injury of the blood-nerve barrier at its most vulnerable regions. we asked if entrapment sites, such as median nerve at the wrist, are more predisposed than the distal nerve endings to such injury. we compared the median snap with radial snap measured antidromically at digit in gbs patients whose nerve conduction study showed the sural-sparing pattern. the terminal nerves at digit are of similar length, but those of median nerve are prone to compression, often subclinically, at the carpal tunnel while those of radial nerve are not. we defined the sural-sparing pattern as a greater decrease in median and or ulnar snap than that of the sural, compared to age and height-matched normal controls. a total of gbs and miller fisher patients from our institution's database were studied. patients had the sural-sparing pattern, of whom had pre-existing carpal tunnel syndrome. of the remaining patients with sural-sparing, had abnormal median snap at digit , while had both abnormal median and radial snaps at digit . none had isolated abnormality of the radial digit snap. among the cases that had abnormal median and radial snaps at digit , the mean percentage decrease when compared to age and height matched norms was greater in median nerve compared to radial nerve ( % and % respectively). of the patients without sural-sparing pattern, had normal snaps; patient had inexcitable sensory nerves while the other had a length-dependent decrease in snap. in the latter patient, unlike those with sural-sparing, there was no differential decrease of median snap over radial snap at digit . our findings suggest that the disruption of blood nerve barrier at entrapment sites rather than the distal nerve endings may underlie the pathophysiology of the sural-sparing pattern seen in gbs. chiba a , uchibori a , gyohda a . kyorin university, tokyo, japan. serum igg anti-gq b antibody is the most specific biomarker for fisher syndrome and its related disorders (fs-rd), but approximately - % of the patients are seronegative for it in conventional assays (gq b-seronegative). some molecules need ca + cation to interact with their ligands, and antibodies with such a property (ca + -dependent antibodies) are reported. we have found that such a ca + -dependency is also present in igg anti-gq b antibody, and majority of gq b-seronegative patients with fs-rd have this type of antibodies. in patients with final clinical diagnoses as fs-rd (fisher syndrome, guillain-barré syndrome with ophthalmoplegia, bickerstaff brainstem encephalitis, and acute ophthalmoplegia), were seropositive for igg antibodies against gq b-related antigens (isolated gq b in , and gq b-conatinig complexes in two) in conventional elisa using phosphate-buffered saline. in the remaining patients, eight ( %) turned positive for igg antibody against gq b-related antigens (isolated gq b in seven and gq b-conatinig complexes in one) in elisa using ca + -added tris-buffered saline. the reaction strengths increased depending on ca + concentration, and reached to nearly maximum level in the physiological concentration. all the patients with the ca + -dependent antibodies were also positive for igg antibody against gt a-related antigens, suggesting that the terminal disialo residue common to both the gangliosides would be important as an epitope also for the ca + -dependent antibodies. in the patients with ca + -non-dependent antibodies, only two showed increased titers of igg anti-gq b antibody by adding ca + , and showed significantly decreased titers. this difference in the effect of ca + -addition between ca + -dependent and ca + -non-dependent antibodies suggests that ca + would not be just an enhancer of the antigen-antibody reaction. there are four single bonds between the two pyranose rings in the terminal disialo, and those rotatable bonds make it possible for the disialo structure to take various conformations. a molecular model shows that the distance between two minus-charged carboxy groups in the disialo could vary from nearly zero to approximately , pm and that the disialo would take specific conformations, if divalent ca + cation, which size is approximately pm in diameter, interacts with these two minus-charged groups. the ca + -dependent antibodies might recognize such particular conformations of gq b. charcot-marie-tooth disease (cmt) is a genetically and clinically heterogeneous disorder with variable inheritance modes. it is characterized by loss of muscle tissue and touch sensation, predominantly in the feet and legs but also in the hands and arms in the advanced stages of disease. as several molecules have been reported to have therapeutic effects on cmt, depending on the underlying genetic causes, exact genetic diagnostics have become important for executing personalized therapy. aminoacyl-trna synthetase (arss) genes encode enzymes responsible for charging trna with corresponding amino acids. arss are ubiquitously expressed, essential enzymes responsible for performing the first step of protein synthesis. specifically, arss attach amino acids to their cognate trna molecules in the cytoplasm and mitochondria. recent studies have demonstrated that mutations in genes encoding arss can result in neurodegeneration, raising many questions about the role of these enzymes in neuronal function. mutations in six cytoplasmic ars genes have been reported as the cause of cmt. this study was performed the whole exome sequencing to identify genetic defects in korean cmt patients from unrelated families. variants were sorted with cmt gene list that includes almost genes were related cmt neuropathy, and additionally sorted wes data as ars genes. capillary sequencing for family members and more than controls revealed five novel mutations, c. g>a (p.d n), c. c>t (p.s f), and c. c>a (p.p h) in gars; c. c>a (p.p t) in mars; _ ga>at (p.d i) in yars gene in each family. the mutation sites were well conserved between different species and each mutation were located in the well-conserved catalytic domain or between two catalytic domains or anticodon-binding domain. in silico analysis predicted all mutations may affect protein function. clinical features were similar to those reported in other countries, but differed in terms of age at onset and degree of disability. we believe that those novel ars mutations are the underlying causes of the each family. a -year-old man presented with a -year-history of weakness in biltearal upper limbs. he was complaining of intermittent fasciculation of upper and lower limbs with gradually worsening of paresthesia for years. dysphagia and dysarthria were also presented years ago. there was no patient affected muscle weakness and bulbar symptoms in his family members. in neurological examination, the patient had weakness in biltereal upper and lower limbs (mrc grade ) and prominent distal sensory loss were combined in length dependant pattern. deep tendon reflexes were absent on bilataral biceps and knee joints. in nerve conduction study, there was consistent with demyelinating sensorimotor polyneuropathy. molecular diagnostic analyses those spinobulbar muscular atrophy (sbma) and mutation related to peripheral myelin protein (pmp ) gene were performed and confirmed expansion of expansion of a polymorphic cag in androgen-receptor (ar) gene and deletion of pmp gene. smba, also known as kennedy disease, is an adult-onset, x-linked recessive trinucleotide, polyglutamine (poly-g) disorder caused by expansion of a polymorphic cag tandem-repeat in exon of ar gene on chromosome xq - . charcot-marie-tooth disease (cmt) is the most common hereditary neuropathies and cmt cases with motor conduction velocities(mcvs) of upper limb below m/s are defined as demyelinating (cmt ) and those with mcvs above m/s are defined as axonal (cmt ). most families with cmt linked to duplication of pmp gene on the short arm of chromosome ( p . ), called cmt a. the reciprocal deletion of pmp gene is a responsible genetic defect in % of hereditary neuropathy with liability to pressure palsy (hnpp). these "classical" phenotypes of cmt a and hnpp have been considered which are determined by different mutation mechanism of the same gene. however, an overlap of cmt a and hnpp due to pmp gene deletion was reported that suggestion the phenotype of hereditary neuropathies may differ variably. herein, we report a patient who simultaneously presented clinical and electrophysiologic features of smba and cmt a with genetical confirmation of cag expasion and deletion of pmp gene. charcot-marie-tooth type a disease (cmt a) is an inherited peripheral neuropathy stemming from overexpression of pmp protein in schwann cells due to the duplication of the pmp gene. this leads to abnormal schwann cell differentiation and dysmyelination, eventually leading to axonal loss and muscle wasting. no approved treatment is currently available for cmt a. we conducted a systems biology level analysis of the signaling network putatively underlying the processes driving cmt a pathology. based upon this, we identified and tested three repurposed drugs -baclofen, naltrexone and sorbitol -alone and in combination to determine their ability to rescue aberrant myelination in cultures derived from cmt a transgenic rats overexpressing pmp gene. to this end, we studied a validated in vitro co-culture model of sensory neurons and schwann cells adapted to -well culture plates. this model allows measurement of the appearance of myelin proteins as an index of the physiological process of in vivo myelination. total myelin length was quantified with an automatic image analyzer following pmp immunostaining. we first determined the full dose-response curves of single drugs, emphasizing their promyelinating activities. we then tested binary combinations of very low and inactive doses of each drug and compared these to the activity of the combination of the three, namely pxt . whereas combination of any two drugs was not significantly active at the doses tested, combination of all three produced a synergistic improvement in myelination. these findings clearly demonstrate the necessity of using pxt over its single components and highlight the value of pleiotropic combinational repurposing of drugs at low doses as a novel approach for rapid drug development in cmt a and other disorders. autonomic dysfunction is frequently observed in guillain-barré syndrome (gbs) and affects approximately % of the patients. it has been shown that the sweating function can be impaired in gbs. the aim of the present investigation is to summarize the current knowledge on sweating disturbances in gbs patients. we have used appropriate terms to systematically search for references published until and indexed in the following databases: medline, embase, lilacs and cochrane. the inclusion criteria were a diagnosis of gbs and a description of the methods used to test the sudomotor function. the search was limited to the english language. relevant information about study design, methods of assessing the deficit of sweating, patient's characteristics and main results were collected. we selected original references for the final analyses. the majority of the studies were cross-sectional in nature and there were two longitudinal studies. the severity of sweating impairment varied according to the applied method, ranging from normal to almost sympathetic nervous system failure. in seven research papers, the sympathetic skin response was used to evaluate the sudomotor function in patients, and approximately % demonstrated abnormal results. however, researchers used different stimulation protocols and parameters to interpret their results. regarding whole-body sweating test, four research papers applied the thermoregulatory sweating test in patients and they showed areas of anhidrosis on the lower limbs in all of them. eight patients presented sweating impairment on the upper limbs and abdominal wall. results on the sudomotor axon reflex test suggested a length-dependent pattern of sweating loss according to one case report. in another study, eight gbs patients were tested only on the distal leg and foot dorsum and the authors proposed an association between post-ganglionic sudomotor function and antiganglioside antibodies. the present literature review showed that the studies of sweating disturbances in gbs patients included only small cohorts. future studies with larger patients sample sizes are necessary to investigate the patterns of sweating loss in gbs and their changes along the follow up. funding: grants # / - and # / - , são paulo research foundation (fapesp). sensory neuronopathy (sn) represents a rare subgroup of peripheral neuropathies characterized by degeneration of primary sensory neurons at the dorsal root ganglia on the spinal cord. depending on the neuronal population affected, its clinical presentation may manifest as gait ataxia, proprioceptive sensory loss and positive and negative sensory symptoms. although a few reports have mentioned areas of anhidrosis in sn, we were not been able to find previous case series studies on the sweating function in sensory neuronopathies. the aim of the present investigation was to study the whole-body distribution of sweating on both anterior and posterior surfaces in patients diagnosed with sn. quantitative sensory testing for cold and warm sensation threshold (method of levels) was performed on the dorsum of the hands and feet in a randomized order. we tested the thermoregulatory sweating using a sweat chamber ( to ∘ c air temperature and % relative humidity). the oral and skin temperature was monitored and the test time did not exceed min. in order to study the sudomotor axon reflex we employed the q-sweat device on standardized body sites. the test was performed on both sides, simultaneously. we included seven patients (three male; mean age . years) with a mean disease duration of . months (range - ) and a confirmed diagnose of sn. patients presented an asymmetrical loss of cold and warm threshold on hands or feet compared with healthy control (p< , ). regarding the tst results, we found a striking variation of sweating disturbances, ranging from small areas of anhidrosis on the trunk to complete failure of the sympathetic nervous system. two patients underwent the axon reflex test and there was an asymmetrical and mostly distal pattern of sweating loss in one of them and a distal-symmetrical on the second one. our findings indicate a great variability of sweating losses in sn, not overlapped to the sensory loss areas. currently, we are testing more patients in order to confirm our results. funding: grants # / - , # / - and # / - , são paulo research foundation (fapesp). diet, exercise, and inflammation are established modulators of peripheral nervous system function, including pain. prior work examining exercise consistently demonstrates a benefit on heightened pain from a number of acute and chronic pain models. in the present work, we investigated several parameters of peripheral nerve function relevant to pain in rats bred for high (high capacity runners, hcr) or low running capacity (low capacity runners, lcr). the longtime selective breeding of these rat substrains has created divergent intrinsic aerobic capacities and predisposition of metabolic conditions between lcr and hcr rats. examination of the role of sex in the development of chronic pain has established key differences in males and females. to understand gender specific differences, this study focused on female rats to understand the role of metabolic status and peripheral nerve function in females. our analysis identified numerous parameters of peripheral nerve function relevant to pain and neuropathy that are different among lcr and hcr female rats. lcr female rats display reduced hind paw mechanical sensitivity, increased hind paw intraepidermal nerve fiber density and trka-positive epidermal axons, increased numbers of langerhans and mast cells in the hind paw dermis, and increased overall fat mass relative to body weight compared to female hcr rats. examination of sensory and motor nerve conduction velocities, thermal sensitivity, and mrna expression of selected genes relevant to peripheral sensation found no differences between hcr and lcr females. together these results suggest that a genetic component of aerobic capacity and metabolic status can influence sensory sensitivity and specific aspects of inflammation and immune responses in peripheral tissues, which may lead modify the animal's responses to tissue damage and painful stimuli. the lcr and hcr rat model will provide a useful model in the future to assess the involvement of metabolic status in the development of pain. ivig is often considered treatment of first choice in chronic inflammatory demyelinating polyradiculoneuropathy (cidp) because of its rapid onset of action and its relatively safe long-term adverse event profile. clinical trials published so far focused on a loading dose of . g/kg ivig and/or a standard maintenance dosage of . g/kg ivig once every weeks, but have not investigated different dosing options. this study is a prospective, double-blind, randomized, parallel group, multi-center phase iii efficacy study and will be conducted in centres in canada, eu, russia, ukraine and australia. adult patients with definite or probable cidp according to the efns/pns criteria will be enrolled and randomized : : to receive either . g/kg or . g/kg or . g/kg ivig (panzyga ® ) for seven maintenance infusions at -week intervals during the dose-evaluation phase. the starting loading dose will be . g/kg ivig (panzyga ® ) for all patients. primary objective: efficacy measured as percentage of responders (decrease in adjusted incat score by at least point) in the . g/kg ivig (panzyga ® ) arm (given every weeks) at week as this should corroborate the existing and published evidence on efficacy of ivig in cidp. secondary outcome: percentage of responders at week in the . g/kg and . g/kg ivig (panzyga ® ) arms relative to baseline and compared to the . g/kg arm. the procid study aims to confirm published clinical results obtained with the . g/kg standard dose and will in addition evaluate one higher and one lower maintenance dose, with the aim to offer cidp patients a more adequately dosed and effective treatment policy. * este medicamento no se encuentra comercializado en españa. understanding the rate of disease progression in patients with charcot-marie-tooth disease (cmt), both within and between subtypes is important for clinical prognosis and is crucial for clinical trial design. due to the progressive nature of cmt, intervening at the earliest stages of the disease is a priority. measuring progression of a disease with both motor and sensory deficits requires a multi-item composite scale. the cmt pediatric scale (cmtpeds) is a well-tolerated psychometrically robust -item scale measuring fine and gross motor function, strength, sensation and balance for children and adolescents aged - years with cmt. the aim of this study was to determine the rate of disease progression of children and adolescents within and between genetic subtypes of cmt. ( female) participants aged - years enrolled in the inherited neuropathies consortium were included in this study. demographic, anthropometric and diagnostic information were collected at baseline and -year follow-up. disease progression was measured with the cmtpeds. on average cmtpeds scores progressed at a rate of . points over -years ( % change from baseline, p< . ). there was no difference in rate of disease progression between males and females. of the most common genetic subtypes, participants with cmt a/pmp duplication progressed by . points ( % change from baseline, p< . ), nine participants with cmt b/mpz mutation progressed by . points ( % change), six participants with cmt a/mfn mutation progressed by . points ( % change) and seven participants with cmt c/sh tc mutation progressed by . points ( % change). participants with cmt a progressed faster than those with cmt a (p= . ). children with cmt a progressed consistently during childhood and adolescence while children with cmt b and cmt a progressed faster during childhood than adolescence. overall, children with cmt progress at a significant rate over -years according to the cmtpeds. understanding the rate at which affected children deteriorate is essential for adequately powering clinical trials of disease-modifying interventions. queen square, london, uk; department of neurology, university of iowa carver college of medicine, iowa city, ia, usa; department of neurogenetics, the national hospital for neurology and neurosurgery, ucl institute of neurology, london, uk; department of molecular neuroscience, ucl institute of neurology, london, uk and national hospital for neurology and neurosurgery, queen square, london, uk. in recent years, targeted ngs panels have changed the diagnostic work-up in patients with inherited neuropathies. however, there is limited data on the impact of targeted ngs panels on the diagnosis of cmt patients in everyday practice. the aim of this study was to investigate the impact of targeted ngs panels on the diagnosis of cmt across two tertiary referral centres in the united kingdom (london) and united states (iowa). in london, patients with a diagnosis of cmt (previous pmp duplication and common cmt genes excluded in appropriate cases) underwent targeted ngs panel sequencing covering genes associated with cmt and additional genes associated with hsp or als. a variable number of genes, ranging from to , were analysed depending on the clinical phenotype of the patients. a definite molecular diagnosis was achieved in cases ( %) including pathogenic and likely pathogenic mutations in sh tc ( cases), gjb ( cases, including cases with mutations in the promoter and 'utr regions), gdap ( cases), fgd ( cases), aars ( cases), ighmbp ( cases), mpz ( cases), nefl ( cases). vus were further identified in patients. the diagnostic rate was higher in demyelinating cmt cases ( / , %), compared to cases with axonal cmt ( / %), dhmn ( / , %) and hsn ( / , %). in iowa, patients were investigated by ngs panels covering to genes associated with cmt. a molecular diagnosis was reached in / ( %), and in particular / ( %) demyelinating and / ( %) axonal cmt cases. the most frequent genes identified were gjb ( cases), mfn ( cases), sh tc ( cases) and ighmbp ( cases). vus were identified in patients, including cases with novel variants in aars, warranting additional testing such as segregation of the variant in the family or functional validation studies. in clinical practise, targeted ngs panels represent an effective approach for the diagnosis of cmt. the lower diagnostic rate in london is likely to be due to prior sanger sequencing and exclusion of mutations in common cmt genes in this patient population. more than genes are known to cause cmt and an even larger number are known to cause peripheral neuropathy as part of a more complex neurological disorder. despite the use of custom panels, a significant proportion of patients with inherited neuropathy have no molecular diagnosis. the aim of this study was to investigate the diagnostic yield of a disease-associated gene exome (sureselect focused exome, agilent technologies, santa clara ca, usa) in the diagnosis of cmt and in cases with complex neurological syndromes associated with neuropathy. thirty-one patients with molecularly undiagnosed inherited neuropathy were analysed with sureselect focused exome sequencing. six patients had a more complex phenotype including learning difficulties, cerebral white matter changes, ataxia and pyramidal tract involvement. a genetic diagnosis was achieved in / ( %) of cases by detecting a mutation in cmt-associated genes mpz ( cases), aars, nefl, bscl , bicd and trpv . of note, six cases had mutations in genes which are not covered by currently available diagnostic targeted ngs panels, including kif a, polg, mme ( cases), dnajb , and a novel candidate gene. the average coverage was higher compared to the usual coverage of whole-exome sequencing; % of the targets were covered at x or more, and % of the targets were covered at x or more. this study provides evidence that the sureselect focused exome is a useful tool for the diagnosis of cmt and complex neurological disorders and provides further insight into the phenotypic spectrum of genes associated with inherited neuropathy. changes in the cis-regulatory sequences of a gene's untranslated regions (utr) are increasingly recognised as a significant cause of inherited disease in humans. for example, variants in the non-coding region of gjb account for % of all mutations in our cohort of cmtx patients. one of the biggest challenges in analysing the large number of non-coding variants in a gene is identifying those that are disease-causing and those which are polymorphisms. the aim of this study was to implement a reliable method for the in-vitro functional validation of non-coding variants in the promoter and 'utr regions of gjb . in our cohort of cmtx patients we have previously identified seven mutations (c.- _ inst, c.- g>a, c.- t>g, c.- t>c, c. t>c, c.- g>c, c.- c>t) in the promoter region and one novel mutation in the 'utr (c. c>t), which were considered likely to be pathogenic based on the clinical phenotype, segregation in affected family members and absence in control databases. we have now generated a luciferase-based reporter system and optimised it in a hela cell line. mutations in the promoter region were generated by site-directed mutagenesis using a commercially available gjb promoter clone (genecopoeia). our preliminary results show a reduction of luciferase activity for the c.- t>c and c.- _ inst mutations compared to the wild-type promoter. this difference was increased when transcription factors sox or egr were co-transfected with the t>c and c.- _ inst mutations respectively. validation of other variants is currently ongoing. if successful, our study will provide a useful tool for the validation of mutations in non-coding regions of gjb . moreover, it will constitute a proof-of-principle approach to the functional validation of non-coding variants in other cmt genes known to cause disease by a loss of function. recessive and dominant mutations in leucin-rich repeat and sterile alpha-motif-containing (lrsam ) have been associated with cmt p. lrsam is a ubiquitin e ligase containing a ring domain in its c-terminal, which is crucial for correct protein folding and ubiquitination activity. to date, the majority of dominant mutations reported have resulted in a frame shift disrupting a major portion of the ring-domain, although point mutations in this domain have also been described. the aim of this study was to report the prevalence, clinical features and genetic findings of patients with cmt p in our centre. we performed targeted next-generation sequencing in genetically undiagnosed cmt patients and identified cases with heterozygous mutations in lrsam ( . %) from unrelated families. the mutations identified included frameshift insertions and deletions, a non-frameshift deletion and non-sense and missense point mutations. all of the mutations were novel and were located in or flanking the ring domain. the average age of disease onset was in the rd decade but an earlier onset was reported in two cases. four had a positive family history in keeping with autosomal dominant inheritance. symptoms at presentation were heterogeneous and encompassed distal numbness, unsteadiness, distal weakness of upper or lower limbs and foot deformities. positive sensory symptoms, including tingling and shooting pains, and cramps were also frequently reported. neurological examination showed mild to moderate distal atrophy and weakness, with early ankle plantar flexion involvement in three patients. loss of vibration and reduced joint position sense were often prominent in the lower limbs and appeared to be disproportionate to the degree of weakness and impairment of pinprick sensation. ankle jerks were absent but knee and upper limb reflexes could be normal or brisk. after an average disease duration of years, all but one patient was able to walk independently. nerve conduction studies showed a sensory and motor axonal neuropathy with normal conduction velocities. our study highlights that mutations in lrsam are a relevant cause of cmt and are associated with prominent large fibre sensory loss. in recent years, the implementation of ngs panels for the molecular diagnosis of cmt has increased the number of patients with a genetic diagnosis. nevertheless the interpretation of a particular variant as disease causing can be challenging especially when multiple variants are identified in a single patient. we report two illustrative cases of such challenges. the first index case was born of non-consanguineous healthy parents. he presented with falls in early childhood. over the years he developed foot deformities and progressive length dependent weakness. he had multiple orthopaedic operations to his feet. the past history was also notable for kyphoscoliosis, sensorineural deafness from the age of and bilateral cataracts. nerve conduction studies at the age of revealed a demyelinating neuropathy consistent with the clinical phenotype of cmt . his younger brother had a similar, although more severe phenotype. a litaf (c. c>t,p.pro ser) mutation was identified by sanger sequencing and was present in both affected brothers but also in the unaffected sister. ngs for cmt -associated genes was therefore performed and identified two compound heterozygous pathogenic mutations detected in sh tc (c. c>t, p.arg *; delg, p.arg serfs* ), which segregated with the disease in the family. the second case describes a brother and sister with early onset demyelinating cmt associated with scoliosis and cranial nerve involvement. the male proband underwent ngs and a single previously reported pathogenic intronic splice-site mutation in sh tc (c. - a>c) was found. relative read-depth analysis of ngs was performed to look for possible copy number variants in sh tc , thus identifying a deletion of exon , which was confirmed by long pcr. cruz-velásquez gj , miramar-gallart md , alarcia-alejos r , roche-bueno jc , rodríguez-valle a , capablo-liesa jl . neurology service, university hospital miguel servet, zaragoza, spain; genetics unit, clinical biochemistry service, university hospital miguel servet, zaragoza, spain. charcot-marie-tooth disease (cmtd) defines a clinical and genetically heterogeneous group of inherited peripheral neuropathies characterized by chronic motor and sensory impairment. the type cmtd- also known as cowchock syndrome, is the product of the mutation in the apoptosis inducing factor mitochondria associated gene (aifm ). it is a slowly progressive, recessive, x-linked disease characterized by axonal neuropathy, deafness, and cognitive impairment. our purpose is to describe new cases, brothers, children of non-consanguineous parents, with a characteristic phenotype and a new mutation in the aifm gene. both siblings present from childhood, progressive weakness in lower limbs with diffuse amyotrophies. needing tenotomy before the year due to equinovarus foot. likewise they develops sensory deafness and one of them requires unilateral support at and the other wheelchair at , this one need a pacemaker for an atrioventricular block at . with brain functions and normal language, sensorial deafness, proximal and distal weakness in the four limbs with intense amyotrophies, predominantly distal. tactile and painful sensitivity decreased in glove and sock pattern. an extensive metabolic and biochemical study was normal. the electroneurography demonstrates an axonal neuropathy without response in most of the nerves explored. the electromyography shown a myogenic pattern with distal predominance. brain mri was normal in both cases. through a genetic study by exoma targeting genes associated with cmt and inherited related neuropathies was identified the homicigosis mutation in the aifm gene (p.glu lys), located in the chromosomal region xq . (cmtx ). cowchock syndrome is a rare entity, with few cases described in the literature. the in silico analysis indicates in of the predictors used (provean, sift, polyphen , lrt, mutationtaster, mutationassessor and condel) , that it is a deleterious variant. we audited all patients with peripheral neuropathy caused by a paraprotein, who received treatment and attended king's college hospital peripheral nerve service between - . patients were identified retrospectively from our database of patients attending the peripheral nerve outpatient clinic. clinical information was obtained retrospectively from hospital electronic patient records. we excluded patients with poems syndrome or in whom the neuropathy was not felt to be caused by the paraprotein. we identified patients with a diagnosis of paraproteinaemic neuropathy. we excluded four who did not fulfill the diagnostic criteria and eleven who had received no treatment or were under diagnostic study. we included patients in the final audit. ( %) had igm paraprotein. the haematological diagnosis was monoclonal gammopathy of undetermined significance (mgus) in %, waldenstroḿs macroglobulinaemia %, and lymphoma or plasmacytoma %. after treatment, overall ( %) patients improved neurologically, ( %) stabilised, and ( %) worsened. in the patients who received more than one type of treatment, we analysed outcomes according to the most powerful treatment received. patients received rituximab alone of which ( %) improved, ( %) stabilised and ( %) worsened. nine patients received rituximab combined with cyclophosphamide or bendamustine, of which ( %) improved, ( %) stabilized and ( %) worsened. eight patients received intravenous immunoglobulin, of which ( %) improved, ( %) stabilized and ( %) worsened. four patients received other chemotherapy, of which improved, stabilised, worsened. two patients received corticosteroids and both worsened. there was improvement in / ( %) with mag antibodies and / ( %) without. there was improvement in / ( %) with mgus and / ( %) with haematological malignancy. there was improvement in / ( %) with kappa light chains and / ( %) with lambda. factors associated with better outcome (by univariate analysis) were negative mag antibodies, kappa light chain, and haematological malignancy. there was no significant difference between treatments in the proportions who improved. cumberbatch m , cox a . addenbrooke's hospital, cambridge, uk. treatment of patients with autoimmune neuropathies such as chronic inflammatory demyelinating polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) has centred on the use of intravenous immunoglobulin (ivig). however, ivig therapy is associated with systemic side-effects, treatment wear-off effects and regular hospital attendance. subcutaneous immunoglobulin (scig) is an efficacious alternative that can be flexibly dosed, self-administered at home and avoids the 'peaks' and 'troughs' observed with ivig. these factors may combine to improve patient satisfaction and alleviate hospital capacity issues. here, we report on clinical and patient experience of switching from hospital-based ivig to home-based manual push scig for the treatment of cidp and mmn. this was a clinical case series of patients ( cidp, mmn; mean age . years) who were clinically stable on ivig and wished to switch to manual push scig. starting scig dose was equivalent to the final ivig dose for each patient (mean . g/kg week). clinical efficacy (medical research council sum score, -m walk, modified inflammatory neuropathy cause and treatment score, overall neuropathy limitations scale, romberg test) and patient-reported outcomes ( -item short form health survey , life quality index [lqi] ) were assessed at baseline and at regular intervals until the final visit ( - months after switching). at baseline, patients cited 'convenience' as their primary reason for switching to scig. eight patients completed the full assessment period and successfully undertook administrations at home (via hospital-at-home service in cases). dose adjustments, based on clinical need, were required in patients. treatment efficacy and patient quality of life, measured by sf- , were maintained after switching to scig; overall patient satisfaction, measured by lqi, increased from % to %. in the lqi, 'convenience', 'travel time/cost' and 'interference-work' were significantly improved (p< . ) after switching to scig therapy. adverse events included mild erythema and localised swelling, as expected for a ml subcutaneous injection. these findings suggest that manual push scig therapy is a viable alternative to ivig for patients with cidp and mmn, as it maintains disease stability, is more convenient for patients and may help ease hospital capacity concerns. cumberbatch m , soares regua d , cox a . addenbrooke's hospital, cambridge, uk. intravenous immunoglobulin (ivig) is used to treat a number of chronic autoimmune neurological diseases. in most centres, infusions are given at slow rates as there is a perception that this reduces the risk of adverse events (aes). this results in longer in-patient admissions, or frequent day case attendances, impacting on both the patients' quality of life and hospital capacity. however, there is little evidence to suggest that slow infusion rates are required. we used the manufacturer recommendations to optimise infusion rates and reduce the time patients spend in hospital. we report a retrospective audit which describes the impact of different ivig infusion rates on patients' clinical condition. the audit comprised three -month assessment periods: january-june (cohort ; infusion rates of . ± . ml/kg/hr, n = ); january-june (cohort ; . ± . ml/kg/hr, n = ) and july-december (cohort ; . ± . ml/kg/hr, n = ). clinical data were reviewed to determine: patient demographics, duration of infusion; time spent in hospital; and incidence of aes. the three cohorts were well matched in terms of patient demographics ( patients were treated in all treatment periods). cohorts and had significantly shorter treatment episodes than cohort ( . and . vs. . hours, p< . ), spent less time on the unit over the month period ( . and . vs. . hours, p< . ) and had fewer admissions/patient ( . and . vs. . , p< . ). the overall incidence of confirmed aes (mainly headaches) was similar across the cohorts (cohort : %; cohort : %; cohort : %). these findings indicate that increases in ivig infusion rate are well tolerated and significantly reduce treatment time, which benefits patients and offers potential cost savings and reduced pressures on hospital capacity for healthcare providers. cunningham me , yao d , meehan gr , barrie ja , willison hj . university of glasgow, glasgow, uk. one mechanism of injury in the acute motor axonal neuropathy (aman) form of guillain-barré syndrome (gbs) is the attack of peripheral nerve axons by anti-ganglioside antibodies (agabs). rodent models have demonstrated that that binding of these antibodies activates the complement cascade, resulting in the insertion of the terminal component, membrane attack complex (mac) into the axonal membrane. complement activation also results in the release of anaphylatoxins, which are known to recruit phagocytic immune cells to the site of injury. our current in vivo mouse model of agab and complement-mediated injury are acute and severe, resulting in respiratory distress over several hours of such magnitude to warrant termination of experimental procedures. to observe and potentially target immune cell infiltration following agab and complement-mediated injury, a subacute model extending over days is required. here, we demonstrate the development of such a model. to compare differences in immune cell infiltration subacutely under control and injury conditions, mice with endogenous expression of egfp in monocytes and macrophages underwent a modified agab and complement-mediated injury, resulting in a less severe phenotype than previously published models. six days following injury, immune cells in the diaphragm were compared by immunofluorescence and flow cytometry. flow cytometry found overall presence of neutrophils was significantly increased in the diaphragm. macrophages were also increased in injured mice, although did not achieve statistical significance at this timepoint. these results were reflected in immunofluorescent staining of the diaphragm where egfp+ macrophages were quantified surrounding the neuromuscular junction (the primary injury target in this model). the development of an extended mouse model of agab and complement-mediated injury is important, since acute models do not take into consideration either the late-term effects of complement-mediated activation at the nerve membrane, or the recovery phase. future studies will look at the effect of inhibiting complement activation on the presence of immune cells in distal motor nerves. family- is a large australian family with an autosomal dominant form of dhmn (dhmn : omim % ) -a group of length-dependent neurodegenerative disorders affecting the lower motor neurons leading to chronic disability. we recently reported a novel . mb chromosomal insertion within the dhmn locus which we hypothesise is likely to cause disease by dysregulating the expression of one or more nearby genes. studying gene dysregulation in peripheral nerve disease is challenging as the relevant tissues (spinal cord and peripheral nerve) are not easily accessible in patients. therefore, alternative strategies are needed to elucidate the disease mechanisms and pathways involved in peripheral nerve degeneration. to address this problem, we have devised a two-tiered strategy to assess dysregulation of candidate genes using patient lymphoblast and fibroblast cell lines. these cell lines can be easily established, are minimally invasive to obtain, and will harbour the natural mutation and genetic background of patients. our strategy firstly uses lymphoblast gene expression profiles as an initial screening tool to prioritise candidate genes for assessing altered expression. differentially expressed genes will then be modelled in c.elegans where behavioural and nerve morphology can be assessed. using rt-pcr, we have screened dhmn candidate genes in patient and control lymphoblast cell lines. eighteen candidate genes were expressed in lymphoblasts. twelve of the eighteen genes were prioritized for further analysis based on expression in both lymphoblast and neural tissues. quantitative analysis using qrt-pcr taqman assays revealed that ube c, was differentially expressed between patients and controls. it is important that patterns of differential expression can be recapitulated in neural cell-specific models. as part of our second strategy, we have generated patient and control induced pluripotent stem cell derived motor neurons (ipsc-mns) from reprogrammed fibroblasts. using this model, we will perform rna-seq and qpcr experiments to examine disease-relevant alterations in gene expression in neural tissue. we predict that utilization of these two strategies will shed light on the pathogenic mechanisms underlying the dhmn insertion and provide useful insights of pathways leading to peripheral nerve degeneration. the outcome of guillain-barré syndrome remains unchanged since plasma exchange and intravenous immunoglobulin were introduced over years ago. pathogenesis studies on gbs have identified the terminal component of complement cascade, the membrane attack complex, as a key disease mediator and thus a therapeutic target. the inhibition of complement in guillain-barré syndrome (ica-gbs) trial looked at the first use of c pathway inhibition with eculizumab in humans with gbs in a randomised, double-blind, placebo-controlled trial. its primary outcome was to look at safety and tolerability of administration concomitantly with ivig and in the context of severe (gbs disability score or greater) disease. participants were recruited for a month period, with regular follow up. subjects were screened, with ( %) being randomised. the two main causes for failure to proceed were participant concerns around eculizumab side effect profile, specifically the meningitis risk, and also intercurrent infection precluding treatment. five received eculizumab for four weeks, alongside standard intravenous immunoglobulin treatment, with receiving placebo. the safety outcomes, monitored via adverse events capture at each trial visit, showed eculizumab to be well tolerated and safe when administered in conjunction with ivig. the most common adverse events were mild derangement in transaminases or infection. there were no infusion reactions. primary and secondary efficacy outcomes were captured via gbs disability scores, mrc sum scores, rasch overall disability scores and overall neuropathy limitation scores. for the primary efficacy outcome at weeks after recruitment, of placebo and of eculizumab-treated subjects had improved by or more grades on the gbs disability score. all patients had improvements in other measured parameters. this trial highlights the challenges in recruiting acutely unwell patients, due to time constraints and intercurrent infection. although the small sample size precludes a statistically meaningful analysis, these pilot data indicate further studies on complement inhibition in gbs are warranted. charcot-marie-tooth disease (cmt) affects about one in . people. currently more than genes have been identified, with the most different phenotypes. the majority of cases in western countries are autosomal dominant and classified as demyelinating and axonal according to electroneuromyography (enmg). the clinical condition is characterized by weakness and predominant sensory changes in the feet and hands. sometimes there are different phenotypes. recently, variants in heterozygotes in the hars gene (histidil-trna synthetase) have been described associated with cmt called type w.to report a case of cmt-sensitive phenotype with a probable new mutation in hars gene (p.leu arg).a male patient, adopted son, caucasian, drug addict, for three years suffered pain in lower limbs, of great intensity, refractory to drug treatment. the examination showed retrognathism, abolition of patellar and achilleas reflexes, painful and thermal anaesthesia and apalesthesia in the feet.the enmg showed reduced sensory action potentials in sural and superficial fibular nerves. laboratory investigations for painful polyneuropathy of thick and fine fibres was normal. sural nerve biopsy revealed axonal predominance neuropathy. exome sequencing revealed a mutation in the hars gene with a pathogenic variant in heterozygosity, with replacement of the amino acid leucine at position by arginine. our patient, although we did not know the antecedents, presented painful polyneuropathy, whose genetic research, although not unequivocal, indicated a variant called cmt w. few cases of this variant were described, with several mutations. our case revealed mutation hitherto unknown (p.leu arg). we conclude by the importance of a thorough genetic evaluation, in cases of sensory polyneuropathy of unknown cause. small fiber neuropathy (sfn) is a condition that affects the small a -and c-fibers, leading to severe neuropathic pain and autonomic dysfunction. several sodium channel gene mutations have been found in patients with sfn, with scn a-gene mutations being the most frequent. because current available sodium channel blockers are not selective for na v . , these treatments often result in numerous side effects. lacosamide is an anticonvulsant that targets specific sodium channels with a slow-inactivation state, while sparing those with normal activity. several mutations of the scn a-gene with an impaired slow-inactivation of na v . have been found in patients with sfn. therefore, a positive effect of lacosamide on pain reduction in these patients is expected. the primary objective of this study was to determine the effect of lacosamide versus placebo on pain in subjects with scn a-associated sfn. secondary objectives were to determine the effect of lacosamide on autonomic symptoms, sleep interference, and quality of life, and to examine the safety and tolerability. the lacosamide-efficacy-'n'-safety in sfn (lenss) study was a randomized, placebo-controlled, double-blind, crossover-design study. subjects were randomized to start with lacosamide and end with placebo or vice versa. during both of the two phases of the study, the subjects were treated for a period of eight weeks of mg bid, preceded by a titration period, and ended by a tapering period. patients filled in a pain diary twice daily and scored a set of validated questionnaires on autonomic symptoms, sleep interference, and quality of life at multiple study visits. in total patients with scn a-associated sfn were included between november and february . the subjects had a median age of years, ranging from to years. sixty percent of the patients were female. the final results of the study, including the primary and secondary outcomes, will be presented. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces must be in intimate contact with nerve fibres to have a proper function, but it has been shown that this is compromised due to the foreign body reaction (fbr). this fbr is the first response of the nonspecific immune system against an implanted device and is characterized by a first inflammatory phase followed by a second antiinflammatory and fibrotic phase. this process results in the formation of a tissue capsule around the interface causing function loss due to the physical separation between the active sites of the electrode and nerve axons. taking this into account, here we have tested several anti-inflammatory drugs such as dexamethasone, ibuprophen and maraviroc to reduce macrophage activation as well as clodronate liposomes to reduce monocyte/macrophage infiltration. moreover, sildenafil have been administered as an antifibrotic drug to reduce collagen deposition in a fbr model with longitudinal parylene c-based intraneural devices implanted in rat sciatic nerve. briefly, animals were systemically treated with dexamethasone, ibuprophen, sildenafil, maraviroc or clodronate liposomes for two weeks, and nerve damage, inflammatory reaction and matrix thickness around the implant were assessed. treatment with dexamethasone, ibuprophen or clodronate liposomes significantly reduced the inflammatory response in the nerve in comparison to saline group while sildenafil or maraviroc had no effect on iba positive cells infiltration in the nerve. however, only dexamethasone was able to significantly reduce the matrix deposition around the implant after two weeks of treatment. these results support the idea that inflammation triggers the foreign body response in peripheral nerves and a potent anti-inflammatory treatment with dexamethasone could have a beneficial effect on lengthening intraneural interfaces lifespan. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces functionality decreases over time, among other factors, due to the foreign body response (fbr), which encapsulates the implanted devices and physically separates the active sites from the nervous tissue. here we have studied the fbr to parylene c or polyimide thin devices implanted in rat sciatic nerves, assessing thickness of the tissue capsule, signs of inflammation and nerve damage. we have characterized the responsible cells of this response and several molecular mediators over months of implant to find differences between the fbr to both materials. after weeks of implant, the inflammatory response due to the surgery was already decreased, whereas in the implanted nerves it reached its highest levels to then decrease at chronic time points. besides, the amount of foreign body giant cells (fbgc), as a result of macrophage fusion, found in the tissue capsule around the implant also increases progressively to reach a maximum after weeks. on the other hand, molecular analysis of the environment revealed a peak of inflammatory cytokines during the first day of implant to return to standard levels thereafter. however, an increase on ccls molecules was found at later time-points for both materials. with regard to the capsule thickness, all the devices were surrounded by a tissue deposition which appeared soon after the implantation. however, in the case of polyimide devices, the tissue capsule showed a peak weeks after the implant and signs of remodeling thereafter, while the parylene c devices showed a second increase from to weeks in comparison to polyimide devices. immunohistochemical and electron microscopy analysis revealed two different cell types implicated in the fbr in nerve to both materials: macrophages, in close contact with the interface, and fibroblasts which appear after weeks surrounding the tissue capsule. although further analyses are needed to elucidate the differences in the fbr to parylene c and polyimide polymers, these results can help to determine therapeutic targets in order to reduce this response and to improve the intraneural interfaces lifespan. delmont e , antoine jc , paul s , boucraut j , attarian s . referral centre for als and neuromuscular diseases, marseille, france; referral centre for neuromuscular diseases, saint etienne, france; immunology laboratory, saint etienne, france; immunology laboratory, marseille, france. peripheral neuropathies with antibodies against myelin associated glycoprotein (mag) are chronic sensory neuropathies characterized by the presence of an igm monoclonal gammopathy and high levels of anti-mag antibodies. these antibodies recognize a specific epitope called human natural killer (hnk ) shared by nk lymphocytes and several components of the peripheral nerve (mag, p , pmp , sgpg, phosphocan). recently an elisa test has been developed to detect antibodies against hnk epitope. our objectives were to determine the sensitivity and the specificity of anti-hnk antibodies in the diagnosis of anti-mag neuropathy and to know if these antibodies were correlated with the severity of the disease. anti-hnk antibodies were assessed in anti-mag neuropathies and in negative controls: chronic inflammatory demyelinating polyradiculoneuropathies (cidp), miller fisher syndromes, sensory neuronopathies, length-dependant axonal sensory polyneuropathies, healthy controls. in anti-mag neuropathies, were recorded age, disease duration, incat sensory sum score (iss), overall neuropathy limitation scale (onls), rasch-built overall disability scale (rods), mrc sum score, anti-mag antibodies titer, peak dosage of the igm monoclonal gammopathy. anti-hnk antibodies were measured with gangliocombi™ mag elisa test and anti-mag antibodies with anti-mag autoantibodies elisa test both from buhlmann company. anti-hnk antibodies were positive in / anti-mag neuropathies, and in / controls (sensitivity %, specificity %). in anti-mag neuropathies, anti-hnk titer was correlated with sensory deficiency evaluated with the iss score (r= . , p= . ) and with disability evaluated with the rods (r= − . , p= . ) and onls scales (r= . , p= . ). anti-hnk titers were not related to age, disease duration, mrc sum score, anti-mag antibodies titer, peak dosage of the paraproteinemia. anti-mag antibodies titers were associated with none of the characteristics of the patients with anti-mag neuropathy. anti-hnk antibodies have good sensitivity and specificity in the diagnosis of anti-mag neuropathy. compared to anti-mag antibodies, their value is that their titers are related to the disease severity. these results need to be confirmed in a larger prospective cohort. chronic inflammatory demyelinating polyradiculoneuropathy (cidp)is a heterogeneous and treatable immune-mediated disorder that critically lacks biomarkers to support diagnosis. recent evidences indicate that paranodal proteins (contactin- , contactin-associated protein- , and neurofascin- ) are the targets of autoantibodies in a subset of patients with cidp showing distinct clinical presentations. particularly, these biomarkers appear to have clinical relevance and help to orientate therapeutic choice. here, we examined five patients presenting an igg reactivity against the nodes of ranvier and the axon initial segment. using a proteomic approach, cell-based assays and elisa, we identified neurofascin- (nfasc ) and neurofascin- (nfasc ) as the main targets of autoantibodies at the nodes of ranvier. four patients displayed predominantly antibodies of the igg subclass, whereas one patient presented igg antibodies that activated the complement pathway in vitro. these antibodies recognized different epitopes than the previously described anti-neurofascin- igg suggesting different pathogenic functions. accordingly, patients with anti-nfasc / igg showed a distinctive clinical presentation. most patients had a severe phenotype associated with conduction block or decreased distal motor amplitude. tremors or neuropathic pain were not observed. four patients presented with a subacute-onset and sensory ataxia. of interest, the neuropathy occurred concomitantly with nephrotic syndromes in two patients and with an igg -related retroperitoneal fibrosis in one patient. this suggested that autoantibodies could be responsible for the occurrence of both disorders. intravenous immunoglobulin and corticosteroids were effective in three patients, and one patient improved following cyclophosphamide and rituximab treatment. clinical remission was found to correlate with the depletion of anti-nfasc / antibodies and the loss of igg reactivity toward the nodes of ranvier. in addition, recovery of conduction block and of distal motor amplitude were observed following remission and suggested a nodo-paranodopathy. our data demonstrate that nodal antigens are the target of autoantibodies in a subgroup of patients with cidp. this emphasizes that the pathogenic mechanisms involved in chronic immune-mediated demyelinating neuropathies are broad and may include dysfunctions of the nodes of ranvier. mutations in the neurofilament heavy (nefh) gene have been recently identified as a rare cause of autosomal dominant, axonal charcot-marie-tooth disease (cmt ). the clinical spectrum of this condition remains to be delineated. we report two french families with an axonal, predominantly motor, dominantly inherited form of cmt caused by two previously unreported mutations in the nefh gene. twelve patients belonging to two different families were included in the study. they displayed an axonal motor and sensory neuropathy, with no mutations in known axonal cmt genes. a remarkable feature in all patients was the early involvement of proximal muscles of the lower limbs, occurring approximately to years after the onset of motor deficit. proximal weakness affected predominantly the iliopsoas muscle, whereas quadriceps and hamstring muscles were relatively preserved. muscle weakness and muscle wasting progressed rapidly, with most of the patients requiring walking assistance after years of disease evolution. three patients in family had brisk reflexes. nerve-conduction velocity studies displayed evidence of a motor and sensory axonal neuropathy predominantly affecting the lower limbs. original deletions of nucleotides near the end of the coding sequence of nefh were identified: in family , c. _ del (p.lys argfs* ), and in family c. _ del (p.lys glyfs* ) causing a frameshift. interestingly, this frameshift leads to the loss of the terminating codon and to the translation of additional amino acids encoding a cryptic amyloidogenic element, suggesting that this type of mutations could induce protein aggregation. consistently, we showed that overexpression of the mutated forms of nefh in a human neuroblastoma cells induced the formation of protein aggregates. we also observed that it triggered caspase activation and apoptosis. using electroporation of chick embryo spinal cord, we confirmed in vivo that mutated nefh formed aggregates and triggered apoptosis of spinal cord neurons. altogether, this suggests that these mutations in nefh cause protein aggregation and neurotoxicity in neurons expressing nefh. progressive loss of such neurons would explain the early motor involvement and the pyramidal signs observed in some patients. our results provide a physiological explanation to the presence of cmt and als clinical features in affected patients. del valle j , , delgado-martínez i , righi m , santos d , , cutrone a , bossi s , d'amico s , micera s , , navarro x , . neuroprosthetic devices that are aimed to restore sensorimotor limb function of amputee patients require highly selective electrodes designed to establish a tight relationship with the nerve, allowing the bidirectional transduction of signals between nerve fibres and the interface and enabling close-loop control from the user. differently from extra-or intraneural interfaces, regenerative nerve electrodes are designed to enable electrical interface with regrowing axonal bundles of injured nerves, aiming to achieve high selectivity for recording and stimulation. however, most of the developed designs pose an obstacle to the regrowth mechanisms due to low transparency and cause an impairment of the nerve regeneration. in this work, we present a novel double-aisle planar regenerative electrode, a new type of highly transparent, non-obstructive regenerative electrode, which allows the selective stimulation and recording of separated nerve fascicles. the design consists of a thin and flexible double-sided electrode longitudinally inserted across a conduit thus creating two separated aisles in which regenerating fascicles can independently regrow after nerve transection. electrodes implanted in acutely transected nerves of rats showed the capability of selectively stimulating and recording different fascicles inserted in the aisles. moreover, chronic implantation of the electrode in a nerve gap of mm after sciatic nerve section allowed for fascicle regeneration and reinnervation of distal muscles as confirmed by the high number of myelinated axons inside each aisle, good biocompatibility, and adequate nerve conduction. in addition, three and six months after implantation, independent stimulation and recording of each separately regenerated fascicle were possible. our results demonstrate the potential contribution of the doubled-aisle regenerative electrode to selectively interface different fascicles of an injured nerve with no deleterious effects on nerve regeneration. therefore, this multi-aisle regenerative electrode may be suitable for neuroprosthetic applications, such as prostheses for the restoration of hand function after amputation or severe nerve injuries. demichelis c , garnero m , franciotta d , cortese a , callegari i , mancardi gl , schenone a , leonardi a , benedetti l . department of neuroscience, rehabilitation, ophthalmology, genetics, maternal and child health, university of genoa and irccs aou san martino-ist, genoa, italy; laboratory of neuroimmunology, irccs, "c. mondino" national neurological institute, university of pavia, pavia, italy; u.o. neurology, asl imperiese, imperia, italy. querol et al. showed that neurofascin (nf ) antibodies identify a chronic inflammatory demyelinating polyradiculoneuropathy (cidp) phenotype characterized by severe polyradiculoneuropathy, poor response to intravenous immunoglobulins (ivig), and disabling tremor. neurological improvement after therapy with rituximab has been previously reported in three patients with cidp with igg anti-nf antibodies. herein we describe the acute-onset of a case of cidp positive for nf igg antibodies resistant to conventional therapies and responsive to rituximab. the patient is a year-old woman who presented acute onset ataxia and gait disturbances; her symptoms progressed over two weeks and distal weakness, numbness and paresthesias appeared too. the nerve conduction study was suggestive for a motor-sensory polyradiculoneuropathy mainly demyelinating. the cerebrospinal fluid analysis showed elevated protein level and normal cellular count. the patient was initially diagnosed with guillain-barré syndrome (gbs) and treated with plasma exchange without improvement. an ivig cycle was started with a partial relief but at the time of admission to the rehabilitation center the patient still had a marked weakness in all four limbs. after six months she presented a further clinical deterioration and she was restricted to wheelchair. there was no response to additional treatment with ivig, while pulse corticosteroid treatment determined a significant clinical improvement. during the next months, despite the maintenance of steroid therapy, the patient presented a progressive deterioration and she was again restricted to wheelchair. postural and intention tremor appeared at upper limbs and became progressively more disabling. anti-nf ab dosage resulted positive. rituximab was administered at a dosage of mg/m /weekly for weeks. after three months the tremor improved, allowing her to eat independently and the patient was able to walk with bilateral support. antibodies anti-nf were negative. after six months she walked without support and she was able to stitch crochet. as previously reported, in this case a cidp positive for igg nf developed severe polyradiculoneuropathy with predominant distal weakness, ataxia, disabling tremor and resistance to conventional therapies. interestingly the onset was gbs-like. the correct identification of these cidp subtypes has diagnostic, prognostic and therapeutic implications. rituximab con be useful in these patients. demir Ö , yazıcı t . department of neurosurgery, university of gaziosmanpaşa school of medicine, tokat, turkey; department of neurosurgery, kent hospital, giresun, turkey. it is still challenging problem to maintain motor and sensory functions of peripheral nerve after nerve transection. after the nerve injury, calcium concentration in the damaged area increases. then the calcium ions act like cytotoxic agents in the damaged area. nifedipine is calcium channel blocker. we aimed to investigate the effects of nifedipine on nerve regeneration by modulating calcium in the damaged area. twenty-four swiss albino male rats were divided into two groups. left sciatic nerve transection surgery was performed to the all rats in both groups. then the all transected nerves were sutured primarily with epineural interfascicular method. in the experimental group, the anastomosis sites were wrapped with a piece of gel foam soaked into diluted nifedipine solution. in the control group, the anastomosis sites were wrapped with a piece of gel foam soaked into saline solution. we evaluated the effect of nifedipine by using functional, electro-physiological and histopathological studies after the surgeries. in the postoperative second week, walking test was performed and sciatic function index was calculated. in the postoperative third week electroneuronography (enog) was performed. there are significant differences between two groups. nifedipine improved nerve recovery functionally (p< . ) and electro-physiologically (p< . ). in the postoperative fourth week, we performed histopathological examination. in the experimental group with nifedipine there were more organized axons that reached the aim. we conclude from these results that nifedipine is an effective nerve protective agent when used locally at the anastomosis site after the transection of the nerve. the lack of effective, disease-modifying therapies for cmt highlights the need for novel preclinical models suitable for drug discovery. studies in rodent models of cmt tend to be time-consuming, and findings so far have translated poorly into clinical trials. primary and induced pluripotent stem cell (ipsc)-derived neuronal cultures are an established model of neurological diseases. however, due to the random distribution of neuronal bodies and neurites that happen when plating these cells, this system is not ideal to investigate axonal, length-dependent processes like peripheral neuropathies and particularly cmt. to optimize this well-established model system, we developed a robust human platform to study axonal morphology and physiology based on motor neuron neurospheres. we differentiated motor neurons from human induced pluripotent stem cells, purified them by magnetic sorting and cultured them in suspension until they formed neurospheres. floating neurospheres can be maintained in agitation for months as a reliable source of motor neurons. after neurospheres are platted, axons rapidly grow out of them in a radial fashion, resembling dorsal root ganglia cultures. this configuration allows for a better visualization of axons in imaging studies and for continued axonal growth over at least a -day period. axons grew at an average rate of micrometers/day and reached up to cm in length. neurospheres can be fixed and stained allowing for morphological analysis and investigation of protein distribution in axons. this system is also ideal for time-lapse imaging to study axonal transport of organelles and neurofilament kinetics. lastly, our motor neuron neurosphere system lends itself well for high content screening platform. neurospheres can be plated in -well plates where multiple compounds can be tested and the axons easily imaged by a high content screening microscope. in summary, we developed a new platform to investigate motor axons in vitro, which are particularly useful to study length-dependent processes such as inherited peripheral neuropathies and may facilitate the identification of new therapeutic compounds using high content screening systems. mutations in the neurofilament light chain (nfl) gene cause autosomal dominant axonal charcot-marie-tooth neuropathy (cmt e). nfl is a major component of the neuronal cytoskeleton, and is believed to function in conjunction with nfm and nfh to provide structural support for the axon and regulate axon diameter. despite the significant advances in understanding its biological basis, there is still no effective, disease-modifying therapy for cmt e, in part due to the paucity of preclinical models suitable for drug discovery. the development of novel preclinical platforms that can faithfully mimic mechanisms of axonal degeneration in vitro would be an essential and valuable resource to better understand the biology of cmt e and identify potential targets for therapy development. to address this, we generated control and cmt e patient-derived motor neurons and cultured them in suspension until they formed neurospheres. immunostaining of cmt e neurospheres with nfl and tubb antibodies revealed numerous areas of nfl accumulation in n s cmt e axons, resembling the accumulations of mutant nfl protein seen in the processes of catecholaminergic neuronal cell line cad overexpressing several nfl mutants. further analysis demonstrated that areas of nfl accumulation were also immunopositive for nfh, pnfh and nfm and that at least nfl and nfm co-localized in the same areas of deposits. taken together, these results demonstrate that abnormal axonal neurofilament distribution is a feature of cmt e ipsc-derived motor neurons and involve all three neurofilament subunits. we also developed an image analysis routine to allow for automatized quantification of neurofilament distribution. preliminary quantification of nfl signal intensity revealed that axons from patients have a weaker nfl signal compared to control axons, but present several signal peaks above the range observed in controls, which related to the areas of nfl accumulation. these results suggest that nfl accumulates in certain regions of cmt e axons but is reduced in the areas with no accumulation. these findings can be readily adapted into a high content screening platform and will be used to identify compounds able to reverse this axonal phenotype. in summary, we identified a strong axonal phenotype in human cmt e motor neurons with potential as a screening platform for drug discovery. nodal and paranodal proteins have been identified as antigens in peripheral inflammatory neuropathies, however the frequency and clinical relevance of antibody responses against these targets remain poorly investigated in gbs. patients with acute onset inflammatory neuropathies were identified by exploration of the local databases of the departments of neurology and the institute of neurology of the medical universities in innsbruck and vienna. patient data, electrophysiological classification and presence of anti-gangliosid antibodies were retrospectively retrieved by review of patient records. only patients with typical clinical presentation and electrophysiological results consistent with one of the subtypes of gbs were included in the study. among forty-nine patients, thirty-five were classified as aidp, six aman, three amsan, three mfs, and two pharyngo-cervico-brachial gbs. of the included patients had anti-ganglioside-antibodies. ten patients with the initial suspicion of aidp had a disease duration of more than months and were reclassified as cidp. all patient and twenty sera of control patients with non inflammatory polyneuropathy were screened by an optimized tissue based assay using rat brains for immune responses against surface antigens, and by cell-based assays with transfected hek cells for antibodies against contactin (cntn ), contactin (cntn ), contactin-associated-protein (caspr ) and neurofascin- (nf ). in the tissue based assay some of the patients showed a light neuropil staining. none of gbs patient's sera had antibody reaction to cntn , cntn , caspr or nf in cell-based assays. among the cidp patients, two patients demonstrated reactivity against cntn with similar clinical presentation as previously described. none of the control patients had any antibody reaction to the performed tests. our results suggest that antibody responses to cntn , cntn , caspr or nf are absent in austrian gbs patients, although more patients will be screened to substantiate these preliminary results. furthermore, it remains to be established whether antibodies against cntn may predict a chronic course in acute onset inflammatory neuropathies. a few variants of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described with a frequency of - %. their relation and possible evolution into typical-cidp remain unclear, as is their treatment response possibly because of differences in diagnostic criteria. we used the data from a web-based database on italian patients with cidp to determine the frequency and characteristic of these variants, the possible evolution into typical-cidp, and their treatment response. all the patients were assessed at study entry and the disease course before inclusion was analyzed. by february- , we included patients ( men, women), aged - years (median years) with a mean disease duration of . years (range . - years) and complete data available from . based on the clinical data and our revised diagnostic criteria, patients ( %) were classified to have atypical cidp at onset and for the following two years including with dads ( %), with motor cidp ( %), with sensory cidp and cisp ( . %), with lewis-sumner syndrome ( . %), and with recurrent cranial neuropathy. at study entry, patients ( %) had progressed into typical cidp after - years (median years) while ( %, % of total) still had atypical cidp after . - years (median years) with a similar proportion of progression ( - %) within each group. the diagnosis of atypical cidp at entry fulfilled efns/pns criteria in ( %). csf studies were diagnostic in / ( %) patients, nerve biopsy in / ( %), and nerve imaging in / ( %) tested patients. similarly to typical cidp, % of treated patients with atypical cidp improved after treatment with a proportion of response varying from % to % in the different forms. most patients with sensory or motor cidp had however an unsatisfactory response to steroids. this study shows that he proportion of patients with atypical cidp varies during the course of the disease with almost % of the patients evolving into typical cidp within years from onset. in addition, response to treatment is frequent in atypical cidp even if not all the forms respond to the same therapies. only few studies investigated the frequency of antecedent events and comorbidities in patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and little is known on the role of possible predisposing factors, dietary, and lifestyle habits, on the onset and progression of the disease. we used the data from a web-based database on italian patients with cidp to determine the frequency of antecedent events and comorbidities and the possible role of predisposing factors including lifestyle and dietary habits and exposure to toxic agents, using a structured questionnaire. partners of patients served as controls. impairment was evaluated using the mrc sumscore and disability with incat and r-ods scales. logistic regression was used to calculate odds ratio (or) with % confidence interval (ci) for the risk of cidp. sex and disease-duration were included as covariates. by february- , patients were enrolled, with complete data on patients for antecedent events and comorbidities and patients and controls for lifestyle habits. ninety-two patients ( %) reported an antecedent event, mostly infection or vaccination ( %). one or more comorbidity were present in % of the patients including hypertension ( . %), thyroid disorders ( %) and diabetes ( . %) and in % influenced the choice of initial therapy. exposure to toxic environmental agents (odds ratio [or] = . ; % ci, . - . ), cigarette smoke (or = . ; % ci, . - . ), and dietary supplements (or = . ; % ci, . - . ) were associated with a higher risk of cidp while rice consumption was associated with a reduced risk (or = . ; % ci, . - . ). concerning disease severity, more severely affected patients more frequently consumed raw-meat (or = . ; % ci, . - . ) and white meat (or = . ; % ci, . - . ), while rice (or = . ; % ci, . - . ) and soft drink consumption (or = . ; % ci, . - . ) and physical activity were associated with lower disability (or = . ; % ci, . - . ). this study confirms that comorbidities are frequent in patients with cidp and often influence the choice of initial therapy. in addition preliminary data show that toxic exposure and some lifestyle and dietary habits may influence the onset and progression of cidp. doppler k , schuster y , weishaupt a , sommer c . department of neurology, university hospital würzburg, würzburg, germany. autoantibodies against the paranodal protein contactin- have recently been described in patients with cidp. in most patients, autoantibodies of the igg subclass are predominant and are supposed to be pathogenic. the role of igg anti-contactin- is so far unclear. in the present study, igg of three different patients, one with igg anti-contactin- , one with a low titer of igg anti-contactin- and one with a high titer of igg anti-contactin- , and of controls were injected into the sciatic nerves of lewis rats. nerve conduction studies of the injected nerve and motor and sensory testing were performed before and after injection. conduction blocks and motor deficits were detectable in the two patients with high titers of igg and igg , not in the patient with low titers. the percentage of conduction blocks was . % in rats injected with igg of the igg patient and % in those injected with igg . motor deficits were detectable in both patients with conduction blocks but were most apparent in the patient injected with igg of the igg patient. no differences in sensory testing were observed. conduction blocks and motor deficits improved after five days and were normal after seven to eight days. our data give the first evidence of pathogenicity of igg anti-contactin- autoantibodies, not only igg . igg of the igg patient induced a more severe clinical and electrophysiological phenotype compared to the igg patient. remarkably, this reflected the clinical phenotype of the patients, as the igg patients showed an acute-onset of sensorimotor symptoms at the time of blood withdrawal whereas the igg patient presented with a more chronic course of disease. doppler k , frank f , koschker a-c , reiners k , sommer c . department of neurology, university hospital würzburg, würzburg, germany; endocrinology and diabetes unit, department of medicine i, university hospital würzburg, würzburg, germany. axoglial dysjunction and paranodal demyelination have been discussed as potential mechanisms of nerve fiber damage in diabetic neuropathy. studies on human tissue are limited, as nerve biopsies are invasive and only rarely performed in patients with confirmed diabetic neuropathy. skin biopsy has recently been suggested as a good tool to analyze paranodal and nodal changes of myelinated fibers. in the present study, we analyzed the paranodal and nodal region in myelinated fibers of skin biopsies of patients with diabetic neuropathy, patients with diabetes mellitus without neuropathy, and normal controls. immunofluorescence of skin sections with antibodies against caspr, neurofascin, sodium channels and myelin basic protein was performed to assess paranodal/nodal architecture, segmental demyelination and myelinated nerve fibers. staining with antibodies against protein gene product . was used to quantify unmyelinated nerve fibers. we found an increase of elongated ranvier nodes and a dispersion of neurofascin at the distal leg in patients with diabetes mellitus with and without neuropathy and at the finger in patients with diabetic neuropathy. an increased dispersion of caspr was only found in biopsies of the finger in patients with diabetic neuropathy. our data show that skin biopsy is an appropriate tool to analyze nodes of ranvier in patients with diabetes mellitus. structural nodal changes are detectable in diabetic neuropathy, and even in diabetic patients without neuropathy. dourado me , fernandes u , vital al , ramos e , urbano jc , sena a , queiroz jw , jeronimo smb . federal university of rio grande do norte, natal, brazil. the erasmus gbs outcome score (egos) is a validated prognostic model that uses acute phase and easy-to-obtain clinical characteristics to determine outcome at months in patients with gbs. this study aims to assess the validity of egos in rio grande do norte, brazil, and to compare with another european study. data collected prospectively from a cohort of patients with gbs of rio grande do norte, brazil, between june and august , was assessed. ninety patients were excluded for missing data or diagnoses of miller fisher syndrome and atypical forms of gbs. to calculate the egos, the gbs disability score was assessed in the second week of disease and at months. to compare this study with the european one in independent group proportions, we used the student's t-test, being considered statistically significant p< , . the patients included were divided in four groups based on egos. thus, patients had egos between and ; had egos between . and . ; had egos and had egos between . and . in the first, second, third and fourth group, ( %), ( . %), ( . %) and ( . %) of the patients were unable to walk independently after six months of the disease, respectively. overall, of the patients analyzed, ( . %) had poor outcomes in this study. in the european paper, based on the same group division, of ( . %), of ( %), of ( %) and of ( %) were unable to walk independently. comparing both studies, the patients of this study were younger, more seriously ill in the first weeks and with more sensitive deficits. there were no difference relative to sex, cranial nerves deficits and presence of anti-gangliosides antibodies. using the student's t-test for ability to walk after months according to egos stratification, we achieve in the first group p= . ; in the second p= . ; in the third p= . ; and in the fourth p< . .the egos did not have a good capacity to predict the ability to walk after months of gbs in rio grande do norte, brazil. historically, guillain-barré syndrome (gbs) epidemics are rarely seen. between and , we treated and followed cases of gbs in the state of rio grande do norte with a yearly incidence of . / , . no seasonality was observed. the mean age of the patients was years (range, - ), with % of the cases younger than years. demyelinating variant was the most frequent subtype of gbs. in march , the first report of autochthonous transmission of zika virus (zikv) was determined in natal, brazil. later that month, we documented an increase in incidence of gbs in natal, brazil. the incidence in was of . / . . of the cases of gbs diagnosed in , were diagnosed from march through may, which coincided with the outbreak of zikv in natal, brazil. eighteen patients ( % of the cases) had a history of rash and fever prior to onset of gbs symptoms, with the median age of years (range, - ) . the electroneuromyography studies of these patients indicated that ( . %) had acute inflammatory demyelinating polyneuropathy, ( . %) had acute motor axonal neuropathy, and ( . %) was inconclusive. the mean time from onset of zikv infection symptoms to onset of the gbs were days (range, - ). the mean time of nadir was days (ranged, - ) . cranial neuropathies were present in patients ( . %). nine patients were bedridden ( %) and ( . %) required mechanical ventilation. the mean protein content of the central spinal fluid was . g/l, with the white blood cell count below /mm in all patients. they were all treated with intravenous igev. they all improved quickly. anti-gm was negative in all patients. rt-pcr was negative for dengue, chikungunya and zika. serum mac-elisa igm for zika and dengue was made in patients and it had % of positivity. prnt for zika and dengue had % positivity. in summary, we report a geographically and temporally defined cluster of gbs associated with an outbreak of acute rash in the state of rio grande do norte, brazil. as the prevalence of diabetes mellitus continues to increase worldwide, diabetic complications represent a growing burden to patients and society. distal symmetrical polyneuropathy (dsp) is a common complication that affects up to % of diabetic patients. dsp reduces patient quality of life due to chronic pain, ulcerations, and may lead to lower extremity amputations. despite its high prevalence, the mechanisms underlying diabetic dsp are poorly understood and several mechanisms are believed to play a role. we hypothesize that diabetic dsp arises from microvascular complications characteristic to diabetes. specifically, capillary dysfunction -disturbances in capillary flow patterns -is a likely candidate to explain development of dsp, as it can limit oxygen and nutrient delivery to nervous tissue, causing nerve dysfunction and damage, and thus development of dsp. we will study this hypothesis utilizing the state-of-the-art blood flow imaging techniques to visualize and quantify endoneureal blood flow and then link these findings with measures of dsp (e.g. nerve conduction velocity; intra-epidermal nerve fibre density) in animal models. we will include several animal models of diabetic dsp caused by either type or type diabetes. two photon microscopy and optical coherence tomography allow visualisation and quantification of capillary transit times and blood flow within peripheral nerves at high resolution. we hypothesise that changes in blood flow patterns and subsequent impairment of nutrient and oxygen delivery to nervous tissue precede the onset of diabetic dsp. if our experiments support this prediction, we will attempt to develop interventions that improve capillary blood flow to prevent or delay the development of dsp. duman o , saracoglu m , haspolat s , bozkurt o . department of child neurology, akdeniz university hospital, antalya, turkey. axonal polyneuropathies are very heterogeneous group of diseases which are very rarely seen during infantile age. some of them may be accompanied by developmental retardation, severe muscle weakness and progressive course. we aimed to present two cases of axonal sensorymotor neuropathy with infantile onset and atypical course. our -year-old boy patient was admitted to our clinic for progressive gait loss since one month. he was the first offspring of consanguineous parents with normal prenatal and natal history. electromyography revealed axonal sensorymotor polyneuropathy. metabolic and cerebrospinal fluid (csf) examinations for etiology were all normal. brain and spinal magnetic resonance imaging (mri) were normal. he had partial benefit from oral steroid treatment. in the course of disease along with four neuropathy attacks he had significant benefit from serial intravenous immunoglobulin treatments in two years clinical course. a month-old girl who is the first offspring of nonconsanguineous parents was admitted to the clinic for acute tetraparesis. axial sensorymotor polyneuropathy was detected in the electromyography. metabolic and cerebrospinal fluid (csf) examinations were normal. she had three more acute polyneuropathy attacks during ivig cessation period. both patients revealed with serial immunoglobulin treatments but unresponsive to riboflavine treatment. we aimed to discuss our rarely seen and the pathogenesis is not completely understood cases' course. serial ivig treatment may be helpfull for such patients' treatment. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominantly inherited disorder caused by mutations of the transthyretin (ttr) gene. the mutant amyloidogenic transthyretin protein causes the systemic accumulation of amyloid fibrils that result in organ dysfunction. ttr-associated fap is a progressive and fatal disease, if left untreated, and should be considered in the differential diagnosis of any person presenting with a progressive polyneuropathy, particularly with accompanying autonomic involvement. the clinical, electrophysiological, histopathological, and genetic characteristics of patients from turkey ( female, male) from eleven families with polyneuropathy and mutations in ttr were evaluated. two patients had no family history of ttr-fap and were considered as sporadic cases, and the remainders were familial cases displaying an autosomal dominant inheritance pattern. sequence analysis of the ttr gene revealed five mutations (p.val met, p.glu gln, p.gly glu, p.glu gly and p.gly glu). most common mutation was p.val met (in unrelated families). mean age at disease onset was . ± . years (range - years). the most commonly reported initial complaint was paresthesia in the feet (asymmetric in three patients). four patients ( male) with the p.glu gln mutation presented with carpal tunnel syndrome. two patients with the p.gly glu mutation showed episodes of dysarthria and hemiparesis, consistent with this genotype. seven patients died during the follow-up period as a result of systemic involvement. this study suggests that our cohort of ttr-fap patients from turkey exhibits clinical and genetic heterogeneity. ebenezer gj , liu y , judge dp , cunningham k , truelove s , carter nd , sebastian b , byrnes k , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; division of cardiology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. effect of amyloid deposition on cutaneous nerves was assessed in subjects with pathogenic ttr variants and control subjects. three groups of subjects each including ttr-fap patients, age/gender-matched healthy subjects and disease controls as well as ttr mutation carriers without neuropathy (ttr-nopn) and with al-amyloid underwent neurological examination and mm skin biopsies. micron sections were stained with anti-pgp . , anti-ttr and congo red. amyloid burden with imagej, intraepidermal (ienfd) sweat gland (sgnfd) and pilomotor densities (pmnfd) measured and correlations between amyloid burden, fiber subtype, neuropathy impairment score-ll (nis-ll) and nis sensory subscore were evaluated. ienfd, sgnfd, and pmnfd were all significantly reduced in ttr-fap patients vs. healthy controls while mutation carriers had intermediate reductions. lower nerve fiber densities were associated with nis-ll (p< . ). congo red staining revealed brilliant red amyloid deposits with apple-green birefringence within dermal collagen, sweat glands, and arrector pili muscles. amyloid infiltration was observed in the endoneurium and perineurium of small fiber sensory and autonomic nerves that innervate sweat glands and arrector pili muscles. cutaneous amyloid deposition was detected in % of ttr-fap and not in healthy or disease controls subjects. both al and / ttr-nopn subjects were congo red positive. amyloid burden was inversely correlated with ienf (p< . , r=− . ) sgnf (p< . , r=− . ), pmnf (p= . , r=− . ) distal leg densities, and correlated with nis-ll (p= . , r= . ) and nis sensory subscore (p= . , r= . ). wild-type ttr staining was less prominent in pathogenic ttr carriers. the diagnostic sensitivity and specificity to detect amyloid in skin were % and % in ttr-fap. the repeat measurement of the amyloid burden from the same section with imagej was r = . , p< . and different sections from the same biopsy was r = . and p< . . we conclude that endoneurial amyloid contributes to sensory and autonomic nerve injury. amyloid burden correlated strongly with sensory/autonomic axon densities and nis-ll. skin punches offer a convenient alternative to establishing a tissue diagnosis. amyloid burden is an attractive biomarker marker for ttr-fap and treatment effect. the study was supported through a grant from pfizer. ebenezer gj , truelove s , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. we investigated differences of unmyelinated sensory nerve fibers in the distal limb among healthy-weight subjects (bmi < kg/m ) and overweight/obese subjects (bmi ≥ kg/m ), aged - years. subjects underwent neurological examination and mm skin punches from distal leg (dl), thigh (dt), and proximal thigh (pt) sites, from which micron sections were stained with anti-pgp . antibody; intraepidermal nerve fiber density (ienfd; fibers/mm) and epidermal thickness were assessed. a second dl biopsy was processed for electron microscopic examination and both thick and thin sections were examined for ultrastructural changes. multivariable linear regression models were used to assess the effect of age, gender, height and weight. after controlling for height, age, and obesity, females were found to have lower distal leg ienfd (− . ; p= . ). increasing age and height were significantly associated with decreasing dl ienfd, with decreases of − . fibers/mm per years (p<. ) and − . fibers/mm per cm (p< . ), respectively. even after controlling for height, being overweight/obese was associated with reduced dl ienfd, with . fibers/mm lower dl enfd than healthy-weight individuals (p=. ). these findings remained consistent across distal thigh and proximal thigh enfd, though not all associations remained significant. the epidermis was thicker in obese subjects across the lower limb, most pronounced at the distal leg (μm, mean± sd, healthy-dl: ± . , dt: . ± . , pt: . ± . , obese-dl: . ± . , dt: ± . , pt: . ± . ). under em very few intact dermal nerve bundles were identified at the proximal thigh sites. the atrophic and degenerating axons were seen with perineurial infiltration by dense collagen in obsess/overweight subjects but not age/gender matched controls. obesity further accelerates attenuation of epidermal nerve fibers across the lower limb even after controlling for other associated factors. echaniz-laguna a , geuens t , petiot p , péréon y , adriaenssens e , haidar m , capponi s , maisonobe t , fournier e , dubourg o , degos b , salachas f , lenglet t , eymard b , delmont e , pouget j , juntas morales r , goizet c , latour p , timmerman v , stojkovic t . strasbourg university hospital, strasbourg, france; peripheral neuropathy group, vib department of molecular genetics and institute born bunge, university of antwerp, antwerpen, belgium; lyon university hospital, lyon, france; nantes university hospital, nantes, france; hôpital de la pitié-salpétrière, paris, france; nice university hospital, nice, france; marseille university hospital, marseille, france; montpellier university hospital, montpellier, france; bordeaux university hospital, bordeaux, france. in this study, we describe the phenotypic spectrum of distal hereditary motor neuropathy caused by mutations in the small heat shock proteins hspb and hspb and investigate the functional consequences of newly discovered variants. among unrelated patients with distal motor neuropathy, we identified mutations in hspb ( index patients/ ; . %) and hspb ( index patients/ ; . %) genes. patients have slowly progressive distal ( %) and proximal ( %) weakness in lower limbs, mild lower limbs sensory involvement ( %), foot deformities ( %), progressive distal upper limb weakness ( %), mildly raised serum creatine kinase levels ( %) and central nervous system involvement ( %). we found a broad range of disease onset with some patients presenting with foot drop at the age of years, and others presenting symptoms only after years. disease progression was slow in all patients, and even with a disease duration of more than years patients were still able to walk. none of our patients were wheelchair dependent. muscle pathology, nerve pathology and electrophysiology showed in all cases a slowly progressive, mostly symmetrical and predominantly distal motor axonal neuropathy. mild sensory involvement was observed upon nerve conduction studies, mostly the lower limbs, in % of cases. we identified hspb and hspb mutations, including respectively and not previously reported. transmission was either dominant ( %), recessive ( %) or de novo ( %). three missense mutations in hspb (pro ser, gly asp, gln arg) cause hyperphosphorylation of neurofilaments, while the c-terminal mutant ser leu triggers protein aggregation. two frameshift mutations (leu fs, ala fs) create a premature stop codon leading to proteasomal degradation. two mutations in hspb (lys met/asn) exhibited increased binding to bag . we demonstrate that hspb and hspb mutations are a major cause of inherited motor axonal neuropathy. mutations lead to diverse functional outcomes further demonstrating the pleotropic character of small heat shock proteins. eftimov f , querol l , rajabally ya and on behalf of all participants of the st enmc workshop. academic medical center, amsterdam, the netherlands; hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; aston university, birmingham, uk. although chronic inflammatory demyelinating polyneuropathy (cidp) is a treatable neuropathy further research is urgently needed to define the diagnostic clinical and electrophysiological boundaries of cidp and its subtypes, and to define the role of biomarkers in supporting the diagnosis, monitoring disease activity and predicting response to treatment and outcome. in recent years, several national registries and biobanks have been developed to enable systematic data collection in cidp. an international registry with large number of patients is needed to allow answering many important questions and develop validated prognostic models to predict outcome in individual patients with cidp. at the inflammatory neuropathy consortium (inc) meeting in , the inc members agreed that a european neuromuscular center (enmc) workshop would be the ideal setting to reach a consensus on the infrastructure of database and biobanks. the st enmc workshop will take place on may - , with participants representing different countries. primary objective of the workshop is to reach a consensus on inclusion and exclusion criteria, core sets and recommended sets of clinical data, diagnostic data and follow-up points and a manual of operations for collection of biomaterials. a secondary objective is to construct an infrastructure to allow sharing data between different databases and biomaterials. conclusions of the consensus meeting and outline of further perspectives will be presented at the peripheral nerve society meeting in . eichinger kj , burns j , cornett k , bacon c , shepherd m , mountain j , sowden j , shy r , shy me , herrmann dn . clinical outcome assessments that measure functional ability are important endpoints for clinical trials. dr. burns has led the development/validation of a functional outcome assessment (cmtpeds) for individuals with charcot marie tooth disease (cmt) ages - years in the inc rdcrn. the cmtpeds is reliable and sensitive to change. however a validated functional outcome measure (fom) for adults with cmt is needed. our data in - year-olds indicated that the cmtpeds could be modified for adult use. however, some items of the cmtpeds (e.g. balance beam and jumping) have floor effects in adults with cmt. we have developed an adult cmt-fom modeled on the cmtpeds, and refined based on literature review, patient interviews, a large-scale cmt patient survey and expert opinion. the cmt-fom is a performance-based scale comprising items that are combined to form a composite score to quantify functional ability of adults with cmt. the cmt-fom shares items with the cmtpeds. four items were added to measure functional abilities relevant to adults (sit to stand, meter walk/run, stair climb, and timed up and go test). the cmt-fom scoring mirrors the cmtpeds. to generate a score ranging from - , raw item scores are converted to z scores, based on age-and sex-matched normative reference values form the norms project and categorized to a - likert along a continuum of impairment levels. we have conducted a pilot study of the cmt-fom in adults with cmt a ( male, female, age . ± . yrs) of differing severity (cmt exam score (cmtes) range - ). the cmt-fom is feasible, individuals were able to complete all items, and takes minutes to perform. the mean cmt-fom score was . ± . (range - ). concurrent validity of the cmt-fom is supported by an association with the cmtes (r = . ). the overall score did not demonstrate floor or ceiling effects. in summary the adult cmt-fom is well-tolerated and captures upper and lower limb strength, dexterity, balance, speed, ambulation and endurance. the cmt-fom requires validation in a large longitudinal cohort, prior to application in clinical trials. estilow t , glanzman am , burns j , cornett kmd , menezes mp , shy r , moroni i , foscan m , pagliano e , pareyson d , laurà m , bhandari t , muntoni f , reilly mm , finkel rs , sowden j , eichinger k , herrmann dn , shy me , yum sw , ramchandren s and on behalf of the inherited neuropathies consortium. cmt is associated with progressive impairment of the hands. reducing this impairment by treating children who are in the early stages of the disease is crucial. studies assessing measures of cmt hand function in children and their associations with patient-reported outcomes are lacking. we analyzed the upper extremity items from the cmt pediatric scale (cmtpeds) and pediatric cmt quality of life scale (pcmt-qol) in children ages - years enrolled in the inherited neuropathies consortium, to explore the relationships between measures of hand function (impairment, activity, and activities of daily living), and patient-reported outcomes. weak grasp ( %), hand pain ( %), and tremor ( %) were prevalent impairments. performance on activity level tasks, the nine hole peg test ( hpt) and functional dexterity test (fdt), were impaired in % and % of the cases, respectively. patients reported difficulty "sometimes" to "always" in opening a jar/lid ( %), zipping/buttoning ( %), writing ( %), carrying a plate without spilling food ( %) and putting on shoes ( %). patients reporting tremor showed significant differences on the hpt (p=. ). grip strength was shown to have a moderately significant correlation with performance on the fdt (r=. ; p<. ). stepwise multiple linear regression showed that grip strength (beta=−. ; p<. ), hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to open a jar (adjusted r =. ; p<. . grip strength (beta=−. ; p<. ), and fdt (beta . ; p<. ) were predictive of ability to carry plate without spillage (adjusted r =. ; p<. ). grip strength (beta=−. ; p<. ), hpt (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to put on shoes (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to zip/button (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and hpt (beta . ; p<. ) were predictive of ability to use a pen/pencil (adjusted r =. ; p<. ). children with cmt present with frequent limitations in adl performance impacting qol. the upper limb measures of the cmtpeds are associated with hand performance and interventions to improve grip strength and reduce pain should be investigated further with respect to their impact on improving function, and ultimately qol. evans me , morrow jm , wastling s , sinclair cdj , fischmann a , shah s , emira ak , hanna mg , yousry ta , thornton js , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; neuroradiological academic unit, ucl institute of neurology, london, uk; university of basel hospital, basel, switzerland. responsive outcome measures are needed in charcot-marie-tooth disease (cmt) to allow adequately powered clinical trials to test novel therapeutics. we have shown high responsiveness of mri quantified intramuscular fat accumulation in calf muscles of cmt a patients over months. the aim of the present study was to assess the responsiveness and longitudinal validity of quantitative mri over a year follow-up period. we undertook two further sets of quantitative mri, myometric and clinical assessments in the original mrc centre cmt a quantitative mri cohort. mri sequences included fat quantification using the point dixon fat-water separation method, t quantification and magnetisation transfer imaging. of the patients with genetically confirmed cmt a were assessed at baseline ( male, mean age . ± . years), underwent repeat assessments a median on months (data already published), underwent repeat assessments at a median of months, and underwent a final assessment at a median of months. the primary outcome measure currently being analysed is mean calf muscle fat fraction at a single axial slice a fixed distance distal to the knee joint. results of this analysis and correlation with clinical measures will be presented at the peripheral nerve society meeting. fainmesser y , dori a , , drory ve , . department of neurology and neuromuscular service, tel-aviv medical center, tel-aviv, israel; department of neurology, sheba medical center, ramat gan, israel; sackler faculty of medicine, tel aviv university, israel. the use of anabolic drugs by those who wish to increase lean body mass is widespread and not well supervised medically. a years old man was referred for evaluation of a slowly progressive sensory and motor disturbance in the distribution of the left ulnar nerve. his symptoms began months after repeated self-injections of anabolic steroids and vitamin e into the biceps and triceps brachii muscles bilaterally. his examination showed increased muscle mass of the injected muscles with a hard-rubbery consistency, atrophy and weakness of left interossei, mild weakness of the bilateral biceps brachii and sensory loss in an ulnar nerve distribution. nerve conduction studies showed a left ulnar neuropathy with reduced motor and sensory response amplitudes and denervation in the left first dorsal interosseus, as well as mild myopathic changes with significantly reduced insertional activity in both biceps muscles. mri of the soft tissues of the arms showed massive fibrosis and infiltration of fat in the arm muscles compressing the ulnar and median nerves on the left. neurolysis of the left ulnar nerve was performed, and the ulnar nerve was found enclosed in a fibrotic mass throughout the entire length of the upper arm. the brachial artery and the median nerve were similarly enclosed in fibrotic tissue and were released. a biopsy of the affected muscles showed muscle necrosis and fibrosis. the patient was treated with physiotherapy and losartan with only mild improvement in the consistency of the muscles, but without clinical improvement in ulnar nerve function, and worsening of nerve conduction. this case illustrates severe peripheral nerve damage due to entrapment in massive muscle fibrosis following improper intramuscular injection of anabolic steroids for cosmetic purposes. fabrizi gm , testi s , høyer h , braathen gj , squintani g , bertolasi l , ferrarini m , taioli f , cabrini i , pancheri e , cavallaro t , tonin p . department of neuroscience, biomedicine and movement, university of verona and department of neuroscience, aoui verona, italy; section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. inherited diseases of nerve and muscle may overlap phenotypically or coexists as facets of the same disorder. two families whose probands were initially diagnosed with a lower motor-neuron (lmn) syndrome and a hereditary distal motor neuropathy (dhmn) turned out to represent a vcp (valosin-containing protein)-related syndrome and a gne (udp-n-acetylglucosamine -epimerase/n-acetylmannosamine kinase)-related "distal myopathy" . both conditions shared the presence of rimmed vacuoles (rv) in muscle biopsies. in family , the year-old male proband had a lmn syndrome manifesting at age years (wasting/weakness, cramps, fasciculations of thigh muscles, later involving the distal upper limbs). his year-old brother had lower limb weakness and diffuse pain in bones/joints since age . the father was diagnosed with a "muscular dystrophy" thirteen years before dying a years; by age years he had developed a behavioural frontotemporal dementia (ftd). electrodiagnosis (edx) disclosed myopathic changes with ongoing denervation together with a mild sensory-motor axonal polyneuropathy in the proband, and a chronic sensory-motor axonal polyneuropathy in the brother. mri showed fatty replacement of weakened muscles in both siblings and diffuse changes of bones consistent with paget disease (pd) in the elder sibling. in family , a year-old african woman was affected by weakness and wasting starting at the distal lower-limb muscles at age years and soon progressed proximally; parents were not consanguineous and two sisters out of six siblings, deceased in their thirties for post-partum complications, had a similar disease. edx mainly disclosed a neurogenic process with ongoing denervation. muscle biopsies from the three family- patients and from the family- proband showed a myopathy with rv. next-generation sequencing demonstrated a heterozygous c. c>t change of vcp leading to a known pathogenic p.arg cys substitution in all family- patients, and two compound heterozygous c. g>a and c. g>a changes of gne in the family- proband leading to known p.ala thr and p.ala thr substitutions. vcp is known to cause autosomal dominant amyotrophic lateral sclerosis- , charcot-marie-tooth disease type y or inclusion body myopathy (ibm) with pd and ftd (ibmpfd); gne causes the autosomal recessive nonaka myopathy (alias ibm ). both cases emphasize the clinical and neurophysiological heterogeneity of those disorders. fargeot g , vandendries c , labeyrie c , viala k , theaudin m , adams d . neurologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neuroradiologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neurologie, groupe hospitalier universitaire pitié salpêtrière, paris, france. the diagnosis of cidp is often challenging, especially when electrophysiological signs of demyelination are lacking. plexus mri has documented nerve abnormalities in small series of typical cidp but its contribution in patients with no electrophysiological signs of demyelination remains to be proved. we report the results of plexus mri in a serie of patients suspect of having cidp without efns pns definite electrophysiological criteria. we did a retrospective study of patients consulting in kremlin bicetre and pitié salpétrière hospital. lumbar or brachial plexus mri (or both) were performed and we assessed nerve trophicity, t -stir signal intensity and gadolinium enhancement as well as the topography of abnormalities. a consensus diagnosis was made by a group of experts (based on clinical data and other supportive criteria) and allowed us to classified patients in "cidp" or "other diagnosis". the practical contribution of plexus mri to the diagnostic algorithm has been studied. diagnosis of cidp was made in patients. mri was abnormal in % of cidp patients and showed nerve roots hypersignal/hypertrophy/enhancement in respectively . / . / . % of patients. the pattern of abnormalities was often asymetrical ( . %), diffuse ( . %) or multifocal ( . %). after unblinding, the mri confirmed the diagnosis of experts in . % of patients and changed the diagnosis in % of patients. plexus mri has shown to be useful in our serie to confirm the diagnosis of experts or to modify it in patients ( %) . the "classical" pattern described in definite cidp (diffuse nerve root hypertrophy and hypersignal) was documented in % of our cidp patients whereas less typical pattern (focal or multifocal abnormalities, hypersignal without hypertrophy) was found in %. plexus mri seems usefull when facing patients suspected of having cidp when electrophysiological criteria are not met: both symetrical diffuse or asymetrical multifocal patterns can be found and should be always correlated to the clinical examination and other supportive criteria. the specificity of such abnormalities remains to be studied. feely sme , rebelo a , abreu l , tao f , bacon c , zuchner s , shy me . university of iowa, iowa city, ia, usa; dr. john t. macdonald department of human genetics and hussman institute for human genetics, miller school of medicine, university of miami, miami, fl, usa. mutations in bcle-associated athanogene (bag ) have been shown to cause a distal myofibrillar myopathy and cardiomyopathy that can severely affect children or only affect adults depending upon the particular mutation. children with severe cardiomyopathy and myopathy have also developed axonal peripheral neuropathy, consistent with the known localization of bag in neurons as well as in muscle. we have identified two large autosomal dominant families with adult onset charcot-marie-tooth disease (cmt ) with the identical novel missense mutation pro ser, a codon previously shown to cause severe or mild myopathy depending on the amino acid substitution. these families expand the phenotypes caused by mutations in bag to include cmt and provide an additional example of adult onset cmt that may previously have been diagnosed as chronic idiopathic axonal neuropathy (ciap). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. myelin protein zero (mpz), expressed only by myelinating schwann cells, has sequence alterations that have previously been reported to cause demyelinating, intermediate, and axonal forms of charcot marie tooth (cmt) disease. we describe a rare duplication in mpz which is causing an early onset, demyelinating form of cmt in our patient. patient was a product of a normal pregnancy and delivery. early milestones were delayed. she began walking at months of age. she was not able to keep up with her peers and was the slowest runner. she started wearing smos at years of age and afos by years of age. she was diagnosed with scoliosis at years of age and started wearing a brace at years. her examination at years of age showed that her fdi, apb, and adm were all / bilaterally. weakness in her lower extremities included / foot eversion and / great toe dorsi flexion. she had tight heel cords bilaterally. pinprick sensation was reduced throughout in her upper and lower extremities and absent at her toes bilaterally. vibration sensation with a rydel tuning fork was absent at her toes and ankles and reduced at her knees and fingers. nerve conduction studies were performed and revealed no responses in all sensory nerves tested with the exception of the radial nerve which had normal latency and mildly slowed conduction velocity ( m/s). prolonged latencies, demyelinating range slowing (between - m/s), and low cmap amplitudes in almost all segments of the median and ulnar motor nerves were also observed. these findings were consistent with a hereditary sensorimotor demyelinating polyneuropathy. she was diffusely areflexic and her total cmt pediatric score (cmtpeds) was / which is in the severe range. her cmt neuropathy score (cmtns) was / in the moderate range. the duplication of mpz has previously been identified as a rare cause of cmt b. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. mutations in egr cause a severe, demyelinating form of cmt, cmt d. we describe a novel mutation in egr which led to extreme variability in severity in a family. proband was a year old girl who was the product of a normal pregnancy and delivery. early milestones were on time. problems with walking started at years of age. at . she was unable to use stairs, run, or jump. at she was wearing bilateral afos. by she was using a wheelchair and started breathing assist at night. she lost arm lift but could still hold a pen. by she could not ambulate independently and breathing assist was required day/night. she lost the ability to write and developed a head drop. by years she could not sit up. on exam her head and neck muscles were / . upper limbs, deltoids, biceps, wrist ext/flex, finger ext, fdi, and adm were / bilaterally. triceps were / ; finger flexors and apb were / bilaterally. lower limbs were / and she had contractures on the right. sensory examination was normal. she was diffusely areflexic. ncvs were absent. both the cmtns and cmt pediatric score were severe at / and / . exome sequencing revealed a r l variant in egr which was likely pathogenic. the probands' mother also had this mutation. she had no reported symptoms at the age of with strength at / throughout with the exception of left foot eversion which was +/ and great toe dorsi flexion which was -/ bilaterally. sensory exam showed a decrease of vibration and pinprick sensation at left toe. she had a normal gait, tandem gait and could toe walk, but not heel walk. she was diffusely areflexic. ncvs showed absent sensory responses. motor ncvs showed her latencies were prolonged and velocities reduced ( - m/s). cmtns was mild ( / ). additional family members who had the r l mutation were evaluated including maternal grandmother who was moderately impaired ( / ) and maternal aunt who was severe ( / ). no other mutations that could cause another known neuropathy or myopathy were identified and mitochondrial sequencing was normal. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). fisgun a , luan x , hoke a . johns hopkins university, baltimore, md, usa. molecular mechanisms that underlie slow distal axonal degeneration seen in chemotherapy induced peripheral neuropathy (cipn) are unclear. however, several identified molecular targets suggest shared mechanisms with wallerian degeneration. since spontaneous mutation in wlds mice and genetic deletion of sarm gene lead to slow wallerian degeneration, we asked if wlds or sarm knockout (ko) mice are resistant to distal axonal degeneration induced by several chemotherapy agents. we chose chemotherapeutic drugs from different classes of agents, paclitaxel (taxane), cisplatinum (platin-based drugs) and bertozomib (proteasome inhibitor), to model cipn in mice. primary outcome measure was evaluation of epidermal nerve fibers in the hind paw plantar footpads. secondary outcome measures included thermal sensation and nerve conduction studies. sarm ko mice were almost % protected against development of sensory neuropathy but the protection in wlds mice was partial. this study confirms the pivotal role sarm plays in mediating axonal degeneration and identifies inhibition of sarm activity as a potential therapeutic target for prevention of cipn. florio f , scapin c , ferri c , feltri ml , wrabetz l , d'antonio m . myelin biology unit, san raffaele scientific institute, milan, italy; hjkri-university of buffalo, ny, usa. myelin protein zero is the most abundant structural protein in myelin of the pns. in humans, more than mutations in p are associated with hereditary neuropathies. deletion of serine causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in mice (wrabetz et al., ) . p s del protein is misfolded and is retained in the er where it gives rise to a dose-dependent unfolded protein response (upr) (pennuto et al., ) . the upr results in the activation of transcriptional and translation control programs that reduce protein synthesis and increase the folding and degradative capacity of the cell. usually, when this first response is not sufficient the cells may activate apoptosis resulting in cell death. however, in p s del schwann cells there is no cell death suggesting that these cells may respond differently to chronic stress. transcriptomic analysis performed on p s del nerves showed increased expression of transcription factors normally present only in the early phases of differentiation such as c-jun, sox and id (d' antonio et al. ) . in order to understand the role of the expression of these factors in the peripheral nerve myelination we used ex vivo and in vivo approaches and we showed that sox and id act as negative regulators of myelination. these results suggest that the expression of sox and id may contribute to the hypomyelination observed in p s del mice. as such, we reasoned that their ablation could ameliorate the phenotype. surprisingly, the ablation of these factors in the p s del mouse severely worsens the neuropathy bursting schwann cell differentiation and increasing the expression of both p wild type and mutant allele with concomitant exacerbation of the upr. this suggests that the overexpression of early differentiation factors in schwann cell under chronic er-stress is an adaptive mechanism that limits differentiation and reduces the expression of toxic proteins. intravenous immunoglobulin (ivig) is the treatment of choice for the guillain-barré syndrome (gbs). the working mechanism of ivig in gbs is undefined, but most likely all potential effects are dose dependent. the pharmacokinetics (pk) and pharmacodynamics (pd) of ivig in gbs are highly variable between patients and a rapid consumption or clearance of ivig is associated with poor recovery. in the current study we developed a model to predict the pk of a standard dosage of ivig ( . g/kg for consecutive days) in individual patients with gbs. non-linear mixed-effects modelling (nonmem) was used to construct a model based on a cohort of gbs patients, with a total of sequential serum igg levels. the final model accurately describes the day to day increment in igg levels during the -day course and the initial rapid fall and graduate decline to steady-state levels thereafter. we explored several potential covariates that improved the predictive capabilities and decreased the between-subject variation in the model. the model including these covariates were evaluated successfully (bootstrap analysis) and through numerous simulation studies each based on (simulated) gbs patients. in conclusion, a first accurate and robust nonmem model for the pk/pd of standard ivig treatment in gbs was developed. the model can be used to predict the pk in individual patients applying a few simple baseline characteristics. in addition, the effect of different treatment regimens of ivig in gbs on a population pk/pd level can be simulated. this modeling technique is a new tool to optimize the pk in individual patients and the study design for new trials with ivig in gbs. forese mg , pellegatta m , rivellini c , podini p , quattrini a , previtali sc , taveggia c . axonal neuregulin (nrg ) type iii is an essential instructive signal for peripheral nervous system (pns) myelination, as its expression determines whether axons are myelinated as well as the thickness of the myelin sheath. we recently demonstrated that gamma-secretase cleavage of nrg type iii generates an axonal intracellular fragment, which translocates in the nucleus to upregulate the expression of the prostaglandin d synthase (l-pgds) gene in neurons. l-pgds catalyzes the conversion of prostaglandin h into prostaglandin d (pgd ). we also showed that specific inhibition of l-pgds activity impairs in vitro myelination. accordingly, myelin in l-pgds null mice is noticeably thinner, thus indicating that l-pgds is a new modulator of developmental pns myelination. previous studies have shown that prostaglandins are involved in the process of wallerian degeneration (wd) and axonal regeneration after injury. thus, to determine whether l-pgds and pgd could be important in pns regeneration and remyelination, we performed sciatic nerve crush injury in months old l-pgds null and wild type control mice and we analysed nerves by morphologic, biochemical, histological and molecular approaches at different time points (t) after crush. we focused on three phases: degeneration (t -t ), axon regeneration (t -t ) and remyelination (t ). our results indicate that in l-pgds null mice the amount of myelin proteins synthesized after crush as well as the number of remyelinated fibers do not change, suggesting that l-pgds might be dispensable for remyelination. however, we observed an increased number of macrophages in null nerves during regeneration (t ), possibly as a consequence of an increase in the blood-nerve barrier (bnb) permeability, indicating potential alteration in the regeneration process in l-pgds null mice. these results suggest that l-pgds could have a different role in developmental pns myelination and after injury. whether other prostaglandins and synthases might compensate for l-pgds activity is currently under investigation. fornasari be , , raimondo s , , ronchi g , , crosio a , budau ca , el soury m , muratori l , , tos p , battiston b , geuna s , , gambarotta g . department of clinical and biological sciences, university of turin, italy; neuroscience institute cavalieri ottolenghi, turin, italy; microsurgery unit, health and science city, cto, turin, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. to repair nerve gaps following severe peripheral nerve injuries, chitosan tubes were proved to give good results, comparable with those obtained with nerve autografts, the gold standard technique. to further improve peripheral nerve regeneration using chitosan tubes, a conduit enrichment strategy was developed using longitudinal skeletal muscle fibres, which have been previously shown to be good fillers in the "muscle in vein" experimental paradigm, where they played a trophic and a structural role. to this aim, rat median nerve gaps were repaired using two different conduits: mm chitosan tubes filled with a longitudinal piece of pectoralis major muscle ("muscle in tube") and hollow chitosan tubes. samples were harvested at early time points ( , , , days) for biomolecular and morphological analysis, and later ( months) for stereological analysis. autologous nerve grafts were used as gold standard positive control in the early time points. biomolecular analysis carried out on in vitro degenerating muscle and on "muscle in tube" at early time points show that the muscle produces high levels of soluble isoforms of neuregulin , a key factor for schwann cell survival and activity, usually released by schwann cells after nerve injury. functional assay and stereological analysis carried out on the distal part of regenerated nerve months after nerve repair, show no significant differences in the regeneration outcome between hollow chitosan tube and "muscle in tube" groups. therefore, we conclude that for short gaps (≤ mm), both hollow chitosan tube and "muscle in tube" are good techniques to repair nerve defects and we suggest that the "muscle in tube", which spontaneously releases neureg-ulin , might be a promising strategy to promote regeneration when the gap is longer or the repair is delayed in time. foucquier j , bertrand v , jouve e , truillet r , mandel j , laffaire j , blin o , magy l , lehert p , , hajj r , guedj m , cohen d , attarian s . pharnext, issy-les-moulineaux, france; aix marseille université, aphm, marseille, france; hôpital dupuytren, limoges, france; university of melbourne, melbourne, vic , australia; faculty of economics, louvain, belgium. charcot-marie-tooth disease type a (cmt a) is a rare disease belonging to a group of inherited, progressive, chronic motor and sensory peripheral neuropathies. the charcot-marie-tooth neuropathy score (cmtns) (shy et al., ) and the overall neuropathy limitations scale (onls) (graham & hughes, ) are considered as the main clinical scales for evaluating progression of disability associated with cmt. as cmt a is a slowly progressive neurodegenerative disease, the choice of endpoints and their ability to monitor small changes over time remain a major concern for clinical drug development. with this in mind, we studied a cohort of french cmt a patients with a follow-up ranging from months to . years resulting from the merge of two multicentre clinical trials (micallef et al., ; attarian et al., ) and a non-interventional study (unpublished) . the sensitivity to change of both cmtns and onls were assessed using a mixed effect model estimating annual progression with time in years, cmtns or onls baseline value as covariates, study centre as a fixed factor and patients as a random effect to account for the repeated measures. disease progression was estimated to be + . points per year on the cmtns (p = . ) and + . points per year on the onls (p = . x − ), both corresponding to a deterioration of impairment and disability. while both endpoints have similar and favourable properties, our set of observations led us to conclude that the onls could be more promising to monitor disease progression in cmt a. frasquet m , , lupo v , mas f , , vílchez r , chumillas mj , , espinós c , sevilla t , , . hospital universitari i politècnic la fe, valencia, spain; instituto de investigación sanitaria la fe, valencia, spain; centro de investigación príncipe felipe, valencia, spain; eresa, valencia, spain; centro de investigación biomédica en enfermedades raras (ciberer), valencia, spain; departamento medicina, universitat de valencia, valencia, spain. mutations in the bicd gene are a cause of dominant spinal muscular atrophy, lower extremity predominant (smaled). we report six patients belonging to three spanish families who carry three different novel mutations in the bicd gene. we describe clinical, electrophysiological and magnetic resonance imaging (mri) data. we provide results of muscle biopsy of one patient and skin biopsy for the study of epidermal nerve fiber density (enfd) of other two patients. genetic diagnosed was reached using a gene panel for genetic testing of cmt and dhmn. three novel mutations in the bicd gene that segregated with the disease were detected: p.val gly; p.tyr his and p.s l. the most frequent clinical phenotype consisted of mild weakness in proximal muscles of lower limbs combined with foot deformities. one patient had prominent sensory symptoms and abnormalities on sensory examination. other two patients had minor sensory abnormalities on examination. in one patient sensory and motor nerve action potentials were reduced, in the rest of patients electrophysiological studies showed normal motor and sensory nerve responses, with chronic denervation predominantly in muscles of lower limbs. mri studies at the level of tight and calf were performed in all patients. the most affected muscles were rectus femoris, vastus lateralis and medial gastrocnemius. mri studies at the level of feet were obtained from five patients and showed that there was not fatty infiltration in intrinsic foot muscles. mri at the level of pelvis muscles performed in four patients showed marked fatty infiltration of gluteus medius muscle in two of them. muscle biopsy performed in one patient showed myopathic features. skin biopsy was performed in two patients of the same family. in the older patient, who had minor sensory abnormalities on examination, there was a marked reduction of enfd that followed a length-dependent pattern. in conclusion, we report three new pathogenic mutations in the bicd gene. in our study we include mri findings at the level of pelvis and feet, which allow us to better define the pattern of muscle involvement related with this gene. our results also raise the subject of a possible sensory involvement in the disease. hereditary neuropathy with liability to pressure palsies (hnpp) is an autosomal dominant disorder that usually results from deletions in the pmp- gene. the neuropathy is unique in that it manifests with recurrent mono-neuropathies at common sites of compression. while spontaneous recovery from episodes of nerve injury usually occurs, it is often incomplete, and over time patients may develop a length dependent polyneuropathy. given the relapsing/remitting nature of hnpp symptoms, standard clinical scores, such as the cmt neuropathy score, are not effective at capturing the severity or progression of the disease. a specific tool is therefore needed for measuring clinical severity of hnpp in preparation for emerging clinical trials. in the current study, we evaluate a new pilot measure, the hnpp severity score (hnpps). the score is composed of patient reported questions addressing current and prior sensory and motor symptoms, and the impact of symptoms on quality of life, followed by items based on a motor examination. total scores vary from - , with higher scores indicating increased disease severity. in this study, the hnpps was administered to patients with genetically confirmed hnpp at the ucl institute of neurology. subjects included males and females with a mean age of years (+/− , range - ). the mean hnpps was . points (+/− . , range - ) and the data did not demonstrate major skew. the cronbach alpha for the hnpps was . , and items based on the physical examination showed the least variability. a modest correlation was observed between the hnpps and the cmt examination scores (pearson correlation . , ci . - . ). we conclude that the hnpps may be a useful measure of clinical severity in hnpp, and should be refined in larger patient cohorts. fridman v , sillau s and on behalf of the inherited neuropathies consortium (inc) . university of colorado hospital, aurora, co, usa; university of iowa hospitals and clinics, iowa city, ia, usa. the most common of the hereditary neuropathies (hn) is cmt a, an autosomal dominant demyelinating neuropathy that results from duplications in the pmp- gene. recent advances in defining the pathomechanisms of the disease have led to an increasing number of potential therapies; however, the absence of reliable natural history data and the paucity of sensitive clinical outcome measures have been barriers to effective clinical trials. the charcot marie tooth neuropathy score (cmtns) was developed to quantify impairment and measure progression in hn. it was observed that while the score discriminates well between mildly and severely impaired patients, it tends to cluster together patients in the middle range of severity. to improve the score's sensitivity, rasch analysis-based weighted category responses were developed. we report a longitudinal study of weighted cmtns and cmt examination scores (cmtes) over a three-year time frame in patients with cmt a. baseline, one year, two year and three year wcmtnsv /wcmtesv scores were available for / , / , / and / patients respectively. mean wcmtns (sd)/wcmtes (sd) scores were as follows: . ( . )/ . ( . ) at baseline, . ( . )/ . ( . ) at one year, . ( . )/ . ( . ) at two years and . ( . )/ . ( . ) at three years. a mixed regression model showed significant change in wcmtns and wcmtes at years (mean change from baseline at years was . points (p=. ) for wcmtns and . points (p=. ) for wcmtes. significant change as compared to baseline was also seen at years (mean change from baseline . points (p= . ) for wcmtns and . points (p= . ) for wcmtes. we conclude that weighted cmtnsv scores show change over the first three years of the inherited neuropathies consortium natural history study and are a helpful measure of progression in cmt a. fridman v , novak p , david w , macklin ea , mckenna-yasek d , walsh k, oaklander al , brown r , hornemann t , eichler f . massachusetts general hospital, boston, ma, usa; university of massachusetts medical school, worcester, usa; university hospital zurich, zurich, switzerland. hsan is an autosomal dominant, severe sensory motor polyneuropathy caused by mutations to serine palmitoyl-coa transferase. mutations shift the substrate preference from serine to alanine leading to formation of neurotoxic -deoxysphingolipids ( -deoxysl). treatment with high-dose l-serine has been shown to reduce -deoxysl accumulation and improve neuropathy in transgenic hsan mice. we report a two-year, delayed-start, placebo-controlled clinical trial evaluating the safety and efficacy of oral l-serine in hsan . eighteen hsan subjects were equally randomized to l-serine ( mg/kg/d) or placebo for year. at -weeks, the placebo group crossed-over and all participants took open-label l-serine for one additional year. sixteen subjects completed their -week visit, and no serious adverse events related to l-serine were reported. participants randomized to l-serine experienced a decline in charcot marie tooth neuropathy scores (cmtns) over year relative to placebo (− . units, % ci − . to − . , p = . ). both groups improved in the second year of the study, with a diminished difference in cmtns at weeks (− . units, % ci − . to . , p= . ). skin biopsies from the distal leg site were largely devoid of intra-epidermal nerve fibers (ienf), but at year, a greater increase in ienf density was seen in participants on l-serine versus those on placebo (median change of vs. fibers/μm , p= . ). -deoxysl levels declined among participants on l-serine versus those on placebo after one year of treatment ( % decrease in deoxysphinganine vs. % increase on placebo, p < . ), and placebo participants experienced similar declines in -deoxysl levels after crossing over to l-serine. we conclude that l-serine is a safe and potentially efficacious treatment for hsan . fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the relationship between guillain-barré syndrome (gbs) and malignancy is uncertain. under the diagnostic criteria of paraneoplastic neurological syndrome (pns) by euronetwork, , neuropathy with no definite onconeural antibodies identified due to gbs has been classified as "non-classical" paraneoplastic disorder. we retrospectively analyzed data of consecutive patients admitted with gbs from birmingham, u.k. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) to calculate the relative cancer risk using different definitions and determined characteristics of malignancy-associated gbs. patients were classified according to definitions: ) all cases of malignancies excluding preceding diagnoses > year before gbs and with no evidence of malignant disease activity at the time of gbs diagnosis, and ) malignancies diagnosed > years post-gbs onset as per pns criteria. total number of gbs patients with malignancy was ( . %). a total of patients ( . %) fulfilled requirements for inclusion as malignancy-associated gbs as per existing criteria for pns. associated malignancy consisted of angioimmunoblastic t-cell lymphoma ( ), poorly differentiated squamous cell carcinoma of nasal septum ( ), gastric adenocarcinoma ( ), hepatocellular carcinoma due to hepatitis b ( ), rectal carcinoma with liver metastasis ( ) and myelodysplastic syndrome ( ). malignancy was globally commoner in our gbs cohort compared to the general population (odds ratio: . ; ci: . - . ; p = . ). however, this was unconfirmed if paraneoplastic criteria were applied. gbs patients with cancer were significantly more likely to be older (p = . ), hyponatremic (p = . ) and demonstrate more electrophysiological axonal loss (p < . ). cerebrospinal fluid (csf) protein levels were lower in the malignancy group (p = . ) and neurological improvement was less likely (p = . ). in-patient mortality was significantly higher in patients with malignancy (p < . ). none of the patients in the malignancy group had positive anti-ganglioside antibodies or anti-neuronal antibodies (anti-hu, yo, ri, crmp ). we conclude global cancer risk is higher in gbs than in the general population, although definition-dependent. application of the strict published criteria for paraneoplastic syndrome reduced the number of cases and suggested absence of a link. malignancy requires consideration in elderly, hyponatremic subjects with normal csf protein, severe electrophysiological axonal loss who fail to improve post-treatment. fu liong h , aude-marie g , anne-catherine an , emilien d , julien c , shahram a , yusuf r . anti-myelin associated glycoprotein antibody (anti-mag) neuropathy is a slowly progressive neuropathy resulting in disability through distal sensory more than motor deficits and tremor. there are currently no evidence-based treatments for anti-mag neuropathy and the effect of the disease on quality of life (qol) in this patient population is unknown. our objective was to assess determinants of qol in patients with anti-mag neuropathy. the sf- questionnaire was assessed in patients, from marseille, angers (france) and birmingham (united kingdom). routine clinical evaluations included mrc sum score, incat sensory score, inflammatory rasch built overall disability score (i-rods), ataxia score, jamar grip dynamometry, timed meter-walk, neuropathic pain symptom inventory (npsi) score, and fatigue severity score (fss). there were males and females. mean age was . years (sd: . years). mean disease duration was . years (s.d.: . years). there were no significant differences between the french and u.k. cohorts in terms of gender distribution, age, disease duration, anti-mag antibody titre. all physical assessments, including mrc sum score (p= . ), jamar grip dynamometry (p= . ), onls (p= . ), irods (p= . ), incat sensory score (p= . ), -meter timed walk (p= . ), ataxia score (p= . ), tremor score (p= . ), were comparable. prevalence and/or severity of pain (p= . ), fatigue (p= . ), restless legs syndrome (p= ) and cramps (p= . ) were also similar. physical component summary (pcs) and mental component summary (mcs) of the sf questionnaire were significantly lower than in reported normal subjects of both countries (p< . ). all sf- qol domains correlated with i-rods, except mcs for which significance was however approached (p= . ). pcs correlated with mrc sum score, ataxia score, timed m-walk, tremor, jamar grip dynamometry, npsi pain score, fss and level of social support. mcs correlated exclusively with fss and level of social support. in multivariate regression, pcs was associated independently with i-rods (p< . ) and npsi pain score (p= . ), whereas mcs was associated independently with fss (p= . ). qol is accurately predicted in anti-mag neuropathy by the i-rods and fss, lending support to their use in clinical and research settings. effective measures to improve qol should include tremor and neuropathic pain treatment, fatigue management and improved social support. fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the electrophysiological diagnosis of guillain-barré syndrome (gbs) is dependent on a number of abnormalities affecting different parameters in peripheral nerves on nerve conduction studies (ncs). diagnostic sensitivity varies with different electrodiagnostic criteria, practices as well as extensiveness of nerve study. however, the most efficient method of performing electrophysiology for guillain-barré syndrome (gbs) is unknown. we retrospectively analyzed electrophysiological data of consecutive gbs patients from birmingham, uk ( - ), studied ≤ weeks post-onset. we first identify abnormal nerves from various regions which produced gbs electrodiagnosis using the recently described criteria by rajabally et al. we subsequently used pre-established hypothetical nerve conduction study protocols to determine the potential optimal method of achieving electrodiagnosis. we found the sensitivity of electrophysiology for each gbs subtype was dependent on the upper and lower limb nerves tested. in acute inflammatory demyelinating polyneuropathy (aidp), abnormalities were predominant in the arms, whereas leg abnormalities predominated in axonal gbs. in aidp, the most common abnormal parameters were distal motor latency ( . %) and conduction block ( . %), and the most frequently affected nerve was the median ( . %). prolonged f-waves were present in % and f-waves were absent in %. mcv was the least frequently abnormal ( . %) with demyelinating range slowing, significantly lower than cb or dml prolongation (p< . in both cases). in axonal gbs, reduced motor amplitudes ( . %) and conduction block ( . %) were the most common parameters, and the most frequently abnormal nerve was the tibial ( . %). f-waves were absent in . %. . % of all motor nerves were unexcitable, significantly more common in lower limb nerves compared to upper limb nerves (p= . ). on comparison of different hypothetical ncs protocols ( unilateral protocols with nerves, with nerves as well as the protocols with -nerves and with exclusive bilateral upper limb and lower limb testing), unilateral -nerve (median, ulnar, common peroneal and tibial) testing produced the highest diagnostic sensitivity for both aidp ( . %) and axonal ( . %) gbs. electrodiagnostic sensitivity in gbs is thus dependent on nerves tested and parameters considered. each subtype preferentially involves specific nerves and parameters. these findings may help per-procedure interpretation, improve electrodiagnostic sensitivity, and reduce patient discomfort. funakoshi k , nagashima t , kokubun n , hirata k , yuki n . department of neurology, dokkyo medical university, tochigi, japan; department of neurology, mishima hospital, nagaoka, japan. recently, igg anti-ganglioside complex (gscs) antibodies have been reported in patients with guillain-barré and miller fisher syndrome. some researchers (nobile-orazio et al., ) reported igm anti-gscs antibodies in multifocal motor neuropathy (mmn) and other chronic immune-mediated neuropathies. in ten of eleven mmn patients with anti-gm antibody, there was a decreased reactivity to gm /gd a compared to single antigen gm . similarly, in one of two chronic inflammatory degenerative polyradiculoneuropathy (cidp) patient with anti-gm antibody, there was a decreased reactivity to gm /gm , gm /gd a, gm /gd b, gm /gt b compared to gm . these relationships were defined negative interaction. in one cidp with anti-gd b antibody, anti-gm /gt b and -gm /gt b reactivity increased although gm and gm were negative (positive interaction). in japan, on the other hand, this correlation remains unclear. sera were investigated from one mmn patient ( y/o male) with anti-gm , and anti-gd a antibody, one cidp patient ( y/o female) with anti-gm , and one subacute sensory motor polyradiculoneuropathy ( y/o male) with anti-gd b, and anti-gq b. igm antibodies to gscs gm /gd a were tested with a mixture of gm and gd a (each pmol/well) as antigen. anti-gm /gd a antibodies were judged to have positive interactions when the optical density was . greater than the sum of the antibodies against individual gm and gd a, and negative interactions when the optical density was % or less compared to the antibodies against individual gm or gd a. antibodies to at least one combination of two of the six gangliosides (gm , gm , gd a, gd b, gt b, and gq b) were similarly tested and judged for positive or negative interactions. in one mmn, anti-gm /gd a had negative interactions. in one cidp, anti-gm /gd a, and anti-gm /gq b had negative interactions. in one subacute sensory motor polyradiculoneuropathy, various anti-gscs antibodies including gd b or gq b had negative interactions. in the cidp patient mentioned above, anti-gm /gt b or anti-gm /gt b had no positive interactions. the relationship between this attenuated reactivity presumably driven by adjacent gangliosides and the mechanism of chronic immune-mediated neuropathies needs to be clarified. galino j , cervellini i , zhu n , stöberl n , fricker fr , lee g , hütte m , lalli g , bennett dl . the nuffield department of clinical neurosciences, john radcliffe hospital, university of oxford, oxford, uk; wolfson card, king's college london, guy's campus, london, uk. schwann cells (scs) are the myelinating cells in peripheral nerve system and are important for normal peripheral nerve development and repair. rala and ralb are small gtpases that have been implicated in neural tube closure, neurite branching and have been described as upstream effectors of proteins involved in cell migration and membrane dynamics. due to their potential role in sc function here we investigated, by genetic ablation in transgenic animals of one or both gtpases, their importance in sc function in adulthood and in nerve repair. we showed that ral gtpases are dispensable for sc function in the naïve state. however ral signalling (provided by rala or ralb) is required for effective remyelination of axons following nerve injury. moreover, absent ral signalling produced defects in axon reinnervation of distal organs and a delay in motor function recovery after nerve injury. we also studied the ral dependent cellular mechanisms that may be responsible for impaired sc remyelination and noted abnormal sc lamellipodia formation that prevent normal axial and radial axon remyelination. this work demonstrates for the first time a novel mechanism for ralgtpases that controls sc lamellipodia formation and their importance in normal sc function during peripheral nerve repair. garcía-lareu b , , , ariza l , cobianchi s , , chillón m , , , , navarro x , , , bosch a , , . tumor necrosis factor (tnf) alpha has been implicated in the pathogenesis of diabetic peripheral neuropathy (dpn), among other inflammatory demyelinating diseases and neuropathic pain. tnf alpha is a pro-inflammatory cytokine that can act at several stages in the demyelination process. it is produced by schwann cells (scs) in the peripheral nervous system (pns) after nerve injury and released into the local environment to attract and activate macrophages at the site of injury, contributing to wallerian degeneration. in vivo studies demonstrated a local inflammation in the sciatic nerve of rats after injection of tnf alpha, followed by demyelination and axonal degeneration. furthermore, the application of tnf alpha resulted in acute mechanical hiperalgesia, a main characteristic of neuropathic pain and therefore tnf alpha is postulated as a biomarker for painful changes after nerve injury. with the aim to characterize tnf alpha effects in chronic neuropathic pain and in diabetic neuropathy, a transgenic mouse model overexpressing tnf alpha cdna under the peripheral myelin protein p promoter was generated. here we characterized the overexpression of tnf alpha in myelinated scs at different stages of myelination (postnatal days , and ) showing that high levels of tnf alpha in sciatic nerve leads to the downregulation of the major pns myelin proteins (p , mbp, pmp , mag) compared to wild type mice, correlating with the loss of structured myelin and an increase in p ntr, a marker for immature and non-myelinated scs in the sciatic nerve. iba staining showed high levels of macrophage infiltration in both sciatic nerve and spinal cord tissues, compared with wild type animals. stress conditions were induced by sciatic nerve crush surgery after which recovery and subsequent remyelination were delayed in the transgenic mice, as evaluated by the sciatic functional index (sfi) and electrophysiological tests. on the other hand, mechanical and thermal nociception seemed to be unaltered, with or without lesion. this model could be helpful in the characterization of the role tnf alpha in pain development, injury and dpn as well as in developing efficient therapeutic strategies to modulate such pathological conditions. garcía-sobrino t , , blanco-arias patricia , , vidal-lijó mp , quintáns bea , , sobrido mj , , pardo j , . department of neurology, hospital clínico, santiago de compostela, spain; neurogenetics research group, instituto de investigaciones sanitarias (idis), santiago de compostela, spain; genomic medicine group (u ), centre for biomedical network research on rare diseases (ciberer), spain; department of neurophysiology, hospital clínico, santiago de compostela, spain. charcot-marie-tooth (cmt) disease is a genetically heterogeneous group of hereditary motor and sensory neuropathies. more than genes were involved in the disease pathogenesis. the objective of this study was to assess the genetic distribution of cmt disease in galicia (northwestern spain). patients were diagnosed as cmt if they had a consistent neurological and/or neurophysiological examination or if they had sensory motor neuropathy with a positive family history. a total of cmt adult patients ( % females) were evaluated with a median age of [ - ] years. the molecular diagnosis was achieved in patients ( %) with a higher success in cmt ( %) than cmt ( %). globally, pmp duplication was the most frequent finding ( %), followed by mutations in mpz ( %), mfn ( %), gjb ( %) and gdap ( %). in cmt , with expception of the pmp duplication, pathogenic variants in egr , nefl and mpz gene were most common. pathogenic variants in mfn and gdap accounted for % of cmt patients. several patients referred to our institution as cmt were diagnosed as hereditary motor neuropathy (hmn) and pathogenic variants in the bscl gene were the most frequent. pathogenic variants not previously related to cmt were identified in mpz, mfn , gjb , egr , nefl and sh tc genes. sporadic or autosomal recessive (ar) cmt accounted for the % of all diagnoses. the genetic epidemiology of cmt in galicia follows a similar pattern to other populations, although some remarkable features are axonal gdap and demyalinating egr pathogenic variants as well a seemingly elevated proportion of ar cases. mutations in mpz gene are associated to a wide range of phenotypes. the r c, in particular, is associated to a severe and early onset disease. some cmt patients respond to immunosuppressive or immunomodulatory treatment, some of them harbouring mpz mutations. we present the case of a -year-old brazilian female with a severe cmt b (r c) that presented an acute episode of worsening after a febrile exanthematic disease. she complained of tingling, stopped walking without support, and could not raise her arms. her emg showed an asymmetrical reduction of motor cv, reduced amplitudes, severe temporal dispersion and possibly conduction block. csf protein was mg%, with normal cell count. diagnosed as gbs, she received ivig. curiously, she markedly improved, regaining the functional status of youth, recovering functions that she had long lost. on emg, motor amplitudes improved significantly, but cv remained the same. reviewing carefully her past history, we identified some motor fluctuations along her adult life. at age , while pregnant, she developed positive sensory symptoms, became unable to get up from the chair and could not walk without assistance. after delivery, she improved, but did not return to baseline. one year later, she presented a new transitory functional worsening after an influenza illness. her parents were healthy but two out her children have a severe cmt. her first daughter never walked and died at the age of six. six months after treatment with ivig, she faced a new relapse. we pulsed with corticosteroids, with great response. she remains stable to date, four months after treatment. the episodic fluctuations and the evident response to treatment clearly suggest an associated immunomediated process. the fast improvement of the amplitude with maintenance of cv suggests that another mechanism in addition to demyelination and axonal degeneration is involved. we propose the existence of an associated channelopathy. funded by cnpq, faepa, pronas (ministry of healthy) gbs is an acute, immune-mediated neuropathy that comprises three major subtypes: aidp, aman, and amsan. on clinical grounds, this distinction is based on nerve conduction studies (ncs): aidp is a demyelinating neuropathy, aman is an axonal motor neuropathy, and amsan, an axonal sensory and motor neuropathy. recently, it has been demonstrated that axonal gbs, in addition to axonal degeneration, may present conduction block (cb) that can progress to axonal regeneration or revert, characterising a reversible conduction failure (rcf), a finding usually associated to good prognosis. patients presenting axonal cb are frequently diagnosed as having aidp, a mistake that can be avoided with two sets of ncs, one as early as possible and the second after three weeks of disease. following these recommendations, we retrospectively classified a brazilian group with gbs followed in our institution that had been submitted to two ncs sets. from september to january , patients fulfilled clinical criteria for gbs at clinical hospital of ribeirão preto and had more than one ncs accomplished. at least four motor nerves (median, ulnar, peroneal, and posterior tibial) and five sensory nerves (medial, ulnar, radial, sural, and peroneal superficial) were evaluated in each examination, according to routine procedures. first ncs revealed patients with aidp ( %), cases of axonal gbs ( aman, amsan, %), and patients with equivocal results ( %). at follow-up study, patients ( %) had their classification changed. the main shifts were from aidp to axonal group and from equivocal results to aidp. aidp increased to % (n = ), axonal gbs increased to % (n = , aman, amsan), and there were no more equivocal patients. although the majority of studies have shown that aidp is more frequent in western countries while axonal gbs predominates in eastern countries, we found a different pattern of distribution in our population, with predominance of the axonal subtypes. the considerable increase of axonal gbs at follow-up studies reinforces that serial emg are mandatory for accurate diagnosis of gbs subtypes. prospective studies are now being carried out in order to confirm these results. in amyloid light chain (al) amyloidosis, misfolded light chain (lc) accumulates and causes progressive peripheral neuropathy (pn) and progressive failure of critical organs such as the heart and kidneys. progressive ascending sensorimotor neuropathy is often a related clinical finding. the deposition of toxic lc amyloid in peripheral nerves is associated with paresthesias, pain, muscle weaknesss, orthostatic hypotension, and diarrhea or constipation in approximately % of patients with al amyloidosis. there are no approved treatments for al amyloidosis. current therapeutic approaches for al amyloidosis are intended to limit lc production but do not directly target misfolded lc deposited as amyloid in organs. we report phase / trial results of neod , an investigational monoclonal antibody that targets misfolded lc and may neutralize circulating lc aggregates and clear insoluble deposits. trial inclusion criteria were one or more plasma cell-directed treatment completed before enrollment, partial or greater hematologic response to any previous therapy, and persistent organ dysfunction. neod was administered intravenously every days during a dose-escalation phase ( patients; . , , , , , , or mg/kg in a + study design) and an expansion phase ( patients; mg/kg). we assessed safety, tolerability, pharmacokinetics, immunogenicity, organ responses based on consensus criteria, and pn responses using the neuropathy impairment score-lower limb (nis-ll). neod treatment was not associated with dose-limiting toxicities or serious adverse events, drug-related discontinuations, or antidrug antibody development in any patient (n = ). of patients with measurable pn at baseline (n = ), % achieved pn response based on the nis-ll score after months of treatment, which resulted in a median % nis-ll score reduction (mean baseline nis-ll, . ). in a best response analysis, % of cardiac-evaluable patients (n = ) and % of renal-evaluable patients (n = ) met respective criteria for organ response. improvements in neuropathy have not been previously shown in al amyloidosis. these results demonstrated that monthly neod infusions were safe, well tolerated, and associated with responses across three different organ systems. gervais cbl , ross ma , goodman bp , khoury ja , muzyka i , smith be . mayo clinic in arizona, scottsdale, az, usa. this work describes clinical, examination and electrophysiologic findings in a cohort of patients with length dependent sensorimotor peripheral neuropathy (ldsmpn) with ventral abdominal sensory loss. ldsmpn affects the longest nerve fibers, namely those innervating structures in the feet and hands. what is less well appreciated is that length dependent involvement of sensory nerve fibers in ldsmpn from the thoracic segments gives rise to ventral abdominal sensory loss. consecutive patients seen for ldsmpn (n= ) were evaluated for the presence or absence of ventral abdominal sensory loss. demographic variables, symptoms and quantitative neurologic findings (neuropathy impairment score [nis]) were examined using descriptive statistics. final diagnoses were noted. ventral abdominal sensory loss to pinprick (which was asymptomatic in all patients tested) was documented in / ldsmpn patients ( . %), mean age was . years (range - ), m:f gender ratio was . ( : ), mean nis was . (range - ), ncs/emg abnormalities were found in / patients (the remaining showing objective evidence of small fiber sensory involvement). ldsmpn patients without ventral abdominal sensory loss (n= ) had a mean age of . (range - ), m:f of . ( : ), and mean nis of . (range - ). no patient ( / ) had dorsal torso sensory loss between the shoulder and buttock levels on either side. diagnoses of the ldsmpn patients with vs. without ventral abdominal sensory loss included charcot marie tooth or other hereditary neuropathy (n= vs. ), abnormal carbohydrate metabolism (n= vs. ), idiopathic (n= vs. ), hypothyroidism (n= vs. ), inflammatory (n= vs. ) and other (n= vs. ). ventral abdominal sensory loss appears to be common in patients diagnosed with ldsmpn of a variety of causes including inherited neuropathies; in addition to those innervating distal limb territories, distal sensory fibers from the thoracic region represent another category of length dependent involvement in ldsmpn; ) the clinical examination of ldsmpn should include the ventral abdomen. gibbons c , garcia j , casasola m , freeman r . beth israel deaconess medical center, harvard medical school, boston, usa. objective: to determine the relationship between measures of autonomic function, electrochemical sweat conductance (esc) and intra-epidermal (ienfd) and sudomotor nerve fiber density (sgnfd). background: structural and functional measures of small fiber neuropathy have been studied in patients with diabetes, but little information comparing these techniques exists. design/methods: we studied patients with diabetes (ages ± yrs, gender f) and healthy control subjects (age ± yrs, gender f). subjects underwent examination scores (nis-ll), quantitative sensory testing, autonomic testing (heart rate variability, valsalva maneuver, tilt test), esc, and millimeter punch skin biopsies at the distal leg and distal thigh for ienfd & sgnfd. results: there were strong correlations between exam scores (nis-ll) and biopsy ienfd (r=− . , p< . distal leg; r=− . , p< . distal thigh), and sgnfd (r=− . , p< . distal leg; r=− . , p< . distal thigh). moderate correlations were noted between exam scores and qst (r values . - . , p< . ), ienfd and qst (r= . - . ). modest correlations were noted between esc and parasympathetic function (r= . - . , p< . ). modest correlations were noted between esc and ienfd (r= . , p< . , but only at the distal leg) and esc and nis-ll (− . , p< . ). no correlations between exam scores and autonomic function were noted. no correlations were detected between esc and sgnfd (r=− . - . ) or esc and sympathetic adrenergic function (r= . ). conclusions: differences between tests are expected based on our understanding of the pathophysiology and natural history of diabetic neuropathy. exam scores, qst results and biopsy results do correlate, and are consistent with prior studies. in contrast, there was little relationship between tests of autonomic function and exam scores, similar to prior studies and our understanding of autonomic function and differences in p. esc had little correlation with either exam scores or biopsy ienfd or sgnfd. there was a modest correlation between esc and parasympathetic function. the exact relationship between esc and diabetic peripheral neuropathy is not clear, and further research studies are needed to determine the role this technique has in clinical practice. objective: to determine the optimal tissue thickness of skin biopsy sections for studies of cutaneous nerve fibers. background: although analysis of intra-epidermal nerve fiber density (ienfd) is routinely reported using μm thick tissue sections, many recent studies of peripheral alpha-synuclein deposition use or μm frozen sections, or μm paraffin embedded tissue sections. design/methods: we compared the results of biopsies from patients with parkinson's disease (pd), using tissue sections each of , and μm thickness. tissues were stained with pgp . and stained for phosphorylated alpha-synuclein (p-syn). the total number of dermal structures (hair follicles, sweat glands, pilomotor muscles) were quantified, nerve densities analyzed, and the frequency of p-syn positive results. we also studied μm paraffin embedded tissue samples from patients with pd. results: in the biopsies of patients with pd there were no differences in the number of sweat glands, hair follicles or pilomotor muscles in , or μm sections. there were significantly fewer blood vessels noted in and μm sections compared to μm sections (p< . ). ienfd and sgnfd declined with tissue thickness (p. . , all) and there was increased variability in results in thinner tissue sections. there was a highly significant reduction in p-syn positive sections in thinner tissue sections (p< . , all tissues compared to μm sections). paraffin embedded tissue sections had significantly lower nerve densities and positive p-syn results (p< . ) compared to all frozen tissue sections of , and μm thickness. thinner tissue sections carried a greater risk of false positive result or indeterminate results due to difficulty interpreting overlap with pgp . . conclusions: tissue sample thickness plays a critical role in interpretation of skin biopsy results. thinner tissue sections, or paraffin embedded tissue sections, do not provide equivalent data and significantly underestimate nerve densities and positive alpha-synuclein results with increase false positive results despite similar numbers of dermal tissue structures. ace- is an investigational protein therapeutic that acts as a localized ligand trap for myostatin and other negative regulators of muscle growth. local injection of ace- into the gastrocnemius muscle of wild-type, mdx, and sod mice produced dose-dependent increases in muscle mass and force without systemic effects. in a phase single-center, double-blind, placebo-controlled dose escalation study in post-menopausal women, unilateral injections of ace- into the rectus femoris (rf) or tibialis anterior (ta) muscle were generally safe and well tolerated. mean percent changes from baseline in muscle volume of the injected muscle were + . % in the rf and + . % in the ta at the highest dose administered with minimal changes observed in the contralateral side and placebo-treated subjects. frequent related aes (≥ %) included injection site pain, pain in extremity, injection site discomfort, and muscle twitching, with similar incidence in ace- and placebo-treated groups. all aes were grade - and reversible. together, these preclinical and clinical results support further studies of ace- in myogenic and/or neurogenic diseases with focal loss of muscle strength and function, including cmt. a phase study is ongoing in facioscapulohumeral muscular dystrophy (fshd). study a - is an ongoing multicenter, two-part, phase study to evaluate the safety, tolerability, pharmacodynamics, efficacy, and pharmacokinetics of ace- in patients with cmt and cmtx. part is open-label and will enroll up to dose-escalating cohorts ( patients per cohort); part is randomized, double-blind, and placebo-controlled, and will enroll an additional patients. ace- will be administered bilaterally to the ta muscle once every three weeks for up to five doses. a safety review team will meet periodically throughout the study to review safety data and make dosing recommendations, including the recommended dose level for part . eligible patients must have genetically confirmed cmt or cmtx with mild-moderate weakness in ankle dorsiflexion. safety and tolerability will serve as the primary outcome for part , muscle volume evaluated by mri for part . additional outcome measures of interest include strength by quantitative muscle testing, function by motor tests, and quality of life by the cmt-health index questionnaire. our study was aimed to evaluate the functional and morphological consequences of cellular and humoral responses in chronic inflammatory demyelinating neuropathy (cidp), using extensive standardized high-resolution sonography (hrus) and nerve conduction study (ncs) protocols in incident treatment-naive patients. we enrolled consecutive, newly diagnosed, treatment naive patients with cidp. in addition to all relevant clinical examinations, all patients underwent a standardized ncs and extensive hrus protocol, of median, ulnar, tibial, fibular and sural nerves. we assessed standard nerve and fascicle size, and echogenicity. we found focal sonographic enlargements in multiple nerves and nerve segments with and without ncs abnormalities. the degree of nerve hypertrophy was not associated with presence of ncs features of demyelination, i.e. / ( %) of median nerve segments showed enlargement without strong decrease in motor conduction velocity and / ( %) hypertrophic median nerve segments revealed no conduction block. a lower distal cmap of median nerve was related with lower mrc sums-scores (p < . ). we found no correlation between age, disease duration or mrc sum-score and nerve size. cellular and humoral responses in cidp may lead to nerve enlargement along the length of nerves, that can be detected by hrus, whereas ncs allows identification of its' specific focal disruption in nerve function. the most common complication of diabetes is peripheral neuropathy, which has prevalence as high as % and is characterized by damage to neurons, schwann cells and blood vessels within the nerve. the concept of schwannopathy as an integral factor in the pathogenesis of diabetic neuropathy is re-emerging, and it is now known that schwann cells cultured in hyperglycemic environments underproduce neurotrophins and exhibit loss of axonal associations, further indicating a non-optimal glial cell activation and function. furthermore, the increased expression of p ntr in myelin sheaths around fibers that are susceptible to axonal degeneration in diabetic neuropathy suggest an important role for this molecule in disease progression. with this project, it is our main goal to evaluate how disruption of p ntr signaling in the schwann cells affects the pathophysiology of diabetic neuropathy. by using a fluorescent live/death cell viability assay, our preliminary data indicate that wild-type schwann cell cultures present increased cell death rate h after stimulation with high levels of glucose. the p ntr has a highly recognized role in the activation of death signals and when absent, we observed that schwann cells are significantly more resistant to apoptosis in hyperglycemic conditions. the role of p ntr receptor signaling in neuron-schwann cell communication and myelination under in vitro diabetic conditions was investigated with primary schwann cell-sensory neuron co-cultures. after eight days of ascorbic acid stimulation, both under euglycemic and hyperglycemic conditions, myelination was assessed by confocal microscopy using specific markers for neurons and myelin. results highlight a compromised ability of wild-type schwann cells to myelinate axons when exposed to a hyperglycemic environment, which was even intensified in co-cultures with schwann cells lacking the p ntr . to complement the in vitro studies, we are modeling type diabetes in a p ntr schwann cell conditional ko mouse model and plan to investigate nerve mrna expression profile to disclose genetic regulation depending on this receptor signaling and its modulatory role in endoneurial hypoxia and neuroinflammation. the results from this project will provide an integrated vision of how impaired schwann cell activity guides neuropathy progression. gondim faa , barreira aa , cruz mw , cunha fmb , de freitas m , frança mc jr , marques w jr , nascimento ojm , oliveira asb , pereira rc , pupe c , rotta ft , schestatsky p . panelists on behalf of the scientific department of peripheral neuropathy, brazilian academy of neurology, brazil. neuropathy is one of the most common neurological manifestations of several diseases and sfn has been progressively receiving more attention in the medical literature. the aim of this study is to generate a set of recommendations to define and diagnose sfn in brazil. a group of neurologists, members of the scientific department of peripheral neuropathy from the brazilian academy of neurology reviewed a preliminary draft prepared by the first author that was distributed by email. the panelists got together on . . at the city of fortaleza, brazil, to discuss and finish the text for the first submission of the manuscript. sfn can be defined as a subtype of neuropathy characterized by selective involvement of unmyelinated or thinly myelinated sensory (sometimes also autonomic) fibers. it is usually characterized by sensory (pain/dysesthesias/pruritus) or combined sensory and autonomic complaints, associated with an almost entirely normal neurological examination (except for sensory changes). electromyography is normal. a growing list of medical conditions has been linked to sfn, although there is no evidence-based literature to support the use of any specific set of screening tests to diagnose the etiology of sfn (the panelists will suggest a basic screening panel). sfn may also serve as a fallacious but useful terminology to uncover discrepancies in the normal values from different neurophysiology laboratories. in brazil, skin biopsy is not usually performed and initial forms of leprosy may have predominant small fiber involvement. there are several tests to demonstrate involvement of small sensory and autonomic fibers. skin wrinkling test, sympathetic skin responses & heart rate variability (conducted on emg machines) and thermoregulatory sweat test may be low-cost screening alternatives. after the final meeting on . . , we finished the first draft for submission to arquivos de neuropsiquiatria (together with a translation to portuguese as supplementary material), the official journal of the brazilian academy of neurology to serve as a source for the definition and diagnosis of sfn in brazil. the final draft will be submitted after presentation at the pns meeting in barcelona on . . the lower cranial nerves (lcn) include the paired glossopharyngeal, the vagal, the accessory and the hypoglossal nerves. these are involved in the execution of swallowing, speech, phonation, tasting, as well as sensory and autonomic functions. lcn can be affected in central and peripheral nervous system diseases. this study investigates the use of ultrasound (us) to detect lesions of the peripheral course of lcn. in addition mri can be used in unilateral lcn local lesions to demonstrate indirect signs of nerve lesions such as muscle atrophy. the patients were examined in supine position with the neck in maximum extension with either a regular or a portable us system (logiq e and logiq e ge healthcare, milwaukee, wisconsin) and high frequency transducers ( to mhz). according to the detected pathology longitudinal or transversal images were recorded. a series of exemplary observations are demonstrated. in one case a paraganglioma in the glossopharyngeal nerve at the carotid sinus was found. in another patient an us of the thyroid gland revealed a nerve tumor, which was later identified to be a schwannoma. in another case multiple neurofibroma were identified in the vagal nerve during a routine neurofibromatosis screening. us has become also important in investigations of lesions of the accessory nerve, following damage by surgery. nerve continuity and scar formation can be distinguished. lesions of the hypoglossal nerve can be caused by tumor infiltration. in one patient an infiltration of the nerve by a squamous cell carcinoma, and in another case by a low grade sarcoma were detected. in addition mri demonstrated an atrophy of the tongue. identification and assessment of the lcn from the point of exit of the skull to their endpoint can be pursued by us techniques, which have been described for the individual nerves. us is easily applicable and -in addition to the "static" image of the mri -allows assessing nerve positions during different movements, as well as detecting muscle movements as fasciculations. mri can also be used to detect not only more proximal lesions, but also changes of the skeletal muscle by denervation. grümme l , hattenhauer st , wolffram k , kleinschnitz c , mausberg ak , stettner m . university hospital essen, essen, germany; heinrich-heine-university, düsseldorf, germany. in chronic inflammatory demyelinating polyneuropathy (cidp), t cells are suspected to play a crucial role in myelin destruction. cd and cd t cells contribute to the inflammatory process, while the exact mechanism of myelin damage is still under debate. to elucidate the molecular interaction of t cells and myelin sheets, we compared neuritogenic and control cells in a live imaging in vitro model. the myelinated fibres of rat dorsal root ganglia (drg) served as model for the peripheral nervous system. drgs of embryonic (e ) lewis rats were cultured; myelination was initiated after one week in vitro and continued for two additional weeks. lewis rats immunized with p and neuritogenic t cells of the lymph nodes were obtained ten days after immunization. control t cells were prepared from healthy rats. for vital tracking, neuritogenic t cells were stained with orange cell tracker, while the control cells were labelled with cfse. myelin detection in vital cultures was assessed by incorporation of c fatty acids conjugated with a fluorophore into the myelin layer. experiments were performed in a conditioned microscope chamber, the two t cell populations were added simultaneously to the drg culture and migration was tracked using live cell imaging. we observed differing migration patterns for neuritogenic and control t cells. the velocity as well as the directionality was altered. after initial contact, the non-neuriotgenic t cells in close proximity to myelin subsequently decreased over time, while the numbers of neuritogenic t cells close to myelin remained stable. long term incubation with neuritogenic t cells affected the myelin integrity in regard to the intermodal length as well as the myelin ratio. further experiments will elucidate the specific effects of neuritogenic t cells to decipher the role of t cells during inflammation in the pns, which could be useful in developing targeted therapies. gundapaneni b , sultan mb , keohane dj , schwartz j . inventiv health inc., burlington, ma, usa; pfizer inc., new york, ny, usa. transthyretin familial amyloid polyneuropathy (ttr-fap) is a rare, life-threatening disorder caused by protein destabilizing ttr gene mutations, broadly classified as val met (the most common mutation worldwide) and non-val met genotypes. tafamidis, a highly-specific ttr-stabilizer, is the only medicine approved to delay neurologic progression in ttr-fap. the objective of this post-hoc analysis was to compare the effects of tafamidis on neuropathy progression in patients with val met and non-val met genotypes. val met patients were participants in a randomized, double-blind, placebo-controlled clinical trial of tafamidis, while non-val met patients were participants in an open-label tafamidis study. patients were grouped into three cohorts: val met tafamidis (n= ); val met placebo (n= ); and non-val met tafamidis (n= ). baseline disease severity and change in disease severity from baseline to month was assessed using the neuropathy impairment score-lower limbs (nis-ll). the effect of tafamidis in the val met and non-val met cohorts versus the val met placebo cohort was determined using a mixed-effects model for repeated measures (mmrm). at baseline, patients in the non-val met cohort were older, had longer symptom duration, and more advanced neurologic impairment than the val met cohorts. at month , the baseline-adjusted mean ± standard error change in nis-ll was comparable between the val met tafamidis and non-val met tafamidis cohorts ( . ± . and . ± . , respectively). these changes were smaller than that observed in the val met placebo cohort ( . ± . ; p= . vs val met and p= . vs non-val met) indicating less disease progression. based on predicted values from the mmrm analysis, the size of the change in nis-ll across the full range of baseline nis-ll scores was remarkably similar in the val met tafamidis and non-val met tafamidis cohorts and was consistently smaller than that observed in the val met placebo cohort. moreover, in all three cohorts, as baseline nis-ll increased, the predicted level of disease progression also increased. in conclusion, while controlling for baseline disease severity, tafamidis delayed disease progression to a comparable extent in val met and non-val met patients. the similar trajectories of disease progression across val met and non-val met patients suggest that these two genotype groups may be more similar than previously considered. clin-icaltrials.gov identifiers: nct ; nct . gutmann l , shy m . university of iowa, iowa city, ia, usa. post tetanic potentiation (ptp) is a physiological phenonmenon seen with disorders of the neuromuscular junction (nmj). it is caused by the influx of ca++ into the terminal axon during the tetanus resulting in an increased number of acetylcholine (ach) vescicles released by each axonal action potential. in myasthenic syndromes it results in improved nmj function by increasing the probability of achieving a large enough end plate potential to generate a muscle action potential. ptp has different appearances depending on whether the nmj defect is pre-synaptic or post-synaptic. in pre-synaptic defects (the most common being lambert-eaton syndrome) there is an increased amplitude of the muscle action potential after a tetanus that persists up to minutes. prolonged ptp has now been reported in disorders, one on a genetically determined neuropathy/myasthenic basis and the other on an acquired toxic origin. prolonged ptp up to minutes was reported in families with a motor neuropathy (having leg weakness and foot deformites) and a congenital myasthenic syndrome caused by a heterozygous mutation of the synaptotamin ii (syt ) gene (c t>g p.pro [p.asp ala] and c g>a [p.pro leu]). electrophysiological testing showed features of a presynaptic defect with prolonged ptp persisting up to minutes. the same phenomenon has been noted in the acquired pre-synaptic defect caused by botululinum toxin. the ptp continued up to minutes. syt is the synaptic vescicle calcium sensor in the terminal axon, allowing for fusion of ach containing vescicles with the presynaptic membrane and the synchronous release of ach. the fusion requires a complex assembly process involving snare proteins. both the syt gene mutation and botulinum toxin affect normal syt function, the mutation by altering amino acids in the calcium-binding domain and the toxin by binding to syt as well as gangliosides gd a and gt b on the neural membrane. the mechanism for the ptp prolongation remains unknown. prolonged ptp appears to be a unique physiological abnormality resulting from altered syt . this phenomenon has been decribed to occur in syt mutations causing congenital motor neuropathy/myasthenic syndrome and botulism. the abnormality may represent a physiological marker for a presynaptic nmj defect involving altered syt . hachisuka a , , senger j , curran m , chan km , . division of physical medicine and rehabilitation, university of alberta, edmonton, canada; department of rehabilitation and medicine, university of occupational and environmental health, kitakyushu, japan; division of plastic surgery, university of alberta, edmonton, canada. background: motor unit number estimation (mune) techniques are valuable tools in neuromuscular disease. among them, the multiple point stimulation (mps) is one of the most common used. contamination by distant single motor unit potentials (smups) generated by neighboring muscles is a potential confounding factor. this is particularly problematic in ulnar neuropathy, one of the most common neuropathies in humans. reason being that the ulnar nerve innervates the majority of hand muscles. the goals of this study are to test the hypotheses that ) distant smups all have an initial positive deflection and ) elimination of smups generated by distal muscles will significantly lower the mune results in the hypothenar muscles. methods: to address the first hypothesis, we tested subjects by stimulating their median nerve while recording smups simultaneously over the hypothenar and thenar muscles. for the second hypothesis, we carried out mps mune of the hypothenar muscles using multi-channel recordings placed over ulnar innervated intrinsic muscles across the hand. when a smup with an initial positive deflection was detected at the hypothenar electrodes, its original was systemically tracked through all the recording channels. results: in the first series of experiments, in accordance with the dipole theory, all smups recorded at the hypothenar recording electrodes had an initial positive polarity. in the second series of experiments, of the studies carried out in subjects, distant smups generated by muscles other than those in the hypothenar eminence represented ± . % (mean±sd) of the overall sample. mune calculated using only smups generated by the hypothenar muscles was ± , compared to ± if all smups were included (p < . ). the extent of increase in mune was highly correlated with the proportion of distant smups found in each study (r = . , p < . ). conclusion: in contrary to some studies suggesting that smups from distant muscles could have an initial negative deflection, we found all smups from distant muscles had a positive deflection. exclusion of those smups from the sample had a significant impact on the mune results. hagiwara w , konno s , kihara inoue m , fujioka t . toho university, tokyo, japan. matrix metalloproteinase (mmp) plays crucial roles in developing immune-mediated neuritis as guillain-barré syndrome (gbs) and its animal model experimental autoimmune neuritis (ean). to investigate the intraneural expression of mmps during ean and the effect of a phosphodiesterase- inhibitor cilostazol (clz) on it, ean rats were treated with either mg/kg/day of clz or vehicle from one day post immunization (dpi). to induce ean female lewis rats were immunized with synthetic peptide from bovine p protein. cauda equina (ce) were removed in several time points, total rna was extracted and reverse-transcribed to obtain cdna that was subjected to real-time pcr analysis for expression of mmp- , mmp- and tissue inhibitor of matrix metalloproteinase- (timp- ) messages. mmp- and mmp- messages peaked at dpi, that is presymptomatic phase of ean. all rats developed motor paralysis at dpi, mmp- and mmp- messages subsided at this moment. however, timp- message reciprocally increased at dpi, persisted through dpi. treatment of clz suppressed motor paralysis of ean significantly. mmp- message peaked at dpi and mmp- message peaked at dpi. on the other hand, both messages at dpi were suppressed compared to untreated ean rats. timp- message in clz treated rats peaked at dpi coincided with motor paralysis peak. both mmp- and mmp- messages might result in subsequent upregulation of timp- that finally downregulates mmps activity and inflammatory process. clz treatment suppressed and delayed expression of mmps and facilitate timp- expression, resulting suppression of ean. the precise mechanism of expression of mmps and timp- remain unclear, however, that mmp- and mmp- messages peaked at dpi suggests involvement of pro-inflammatory cytokines such as tumor necrosis factor-alpha or interleukin- . clz might suppress these cytokines resulted in mmps down regulation. mmp- less affected by clz and thus stimulated timp- expression at dpi. clz might rational treatment for immune-mediated neuropathy via mmps modulation although further investigation especially in-vivo study is needed. haidar m , de winter v , asselbergh b , bouhy d , timmerman v . peripheral neuropathy research group, vib, university of antwerp, antwerp, belgium. the small heat shock protein hspb (hsp ) gene is ubiquitously expressed and encodes for a chaperone protein with essential cellular functions. our lab was the first to identify missense mutations in hspb responsible for axonal charcot-marie-tooth neuropathy (cmt f). since then we became interested in understanding the physiological functions of hspb and its association with cmt neuropathies. we demonstrated the involvement of hspb in microtubule stability. because of the link between autophagosome formation and its intracellular transport, and microtubules stability, we believed that the macro-autophagy process could be regulated by hspb . macro-autophagy is a cellular housekeeping process during which autophagosomes target, envelop and degrade aberrant protein aggregates and damaged organelles. there is strong evidence for an essential role for autophagy in the maintenance of neuronal homeostasis; hence its impairment can lead to a neuropathic condition. our data indicate that macro-autophagy is disrupted by hspb cmt-causing mutations. combining novel microscopy and interactomics techniques we unravelled the way different cmt-causing mutations in hspb impair the autophagic pathway. our data present the impairment of autophagy as a possible pathomechanism for cmt-causing hspb mutations. hajjar h , gautier b , berthelot j , gonzalez e , gess b , young p , tricaud n . institute of neurosciences of montpelllier, inserm, university of montpellier, montpellier, france; universitätsklinikum münster, klinik für schlafmedizin und neuromuskuläre erkrankungen, münster, germany. cmt a, the most common of charcot-marie-tooth diseases, results from the duplication of peripheral myelin protein (pmp ) gene. this gene encodes for a small protein of kda, pmp , mainly produced by schwann cells and the excess of pmp leads to demyelination. there is no cure for this disease but one approach for a treatment is gene therapy. a transgenic rat model exists for cmt a, which possesses copies of the mouse pmp gene. our goal is to provide a proof of principle for gene therapy in peripheral nerves using this rat model of cmt a. our strategy is to reduce the overexpression of mouse pmp protein in rats schwann cells using short hairpin rnas (shrnas). shrnas are small non-coding rnas that specifically bind to targeted mrnas resulting in their degradation. we tested for the efficiency of several shrnas targeting mouse pmp in vitro to find two shrnas that reduce pmp levels. the shrnas have been cloned in an adeno-associated serotype (aav ) viral vector together with green fluorescent protein in order to detect infected cells. aav was selected for its high transduction rate of myelinating schwann cells, for its good diffusion and low immunogenicity. we plan bilateral injections in the sciatic nerve of control and diseased rats. the efficiency of this gene therapy will be checked by assessing muscle strength (grip test), way of walking (catwalk), mobility (rotarod) and nerve conduction velocity of treated cmt a rats versus non-treated. the process of myelination and myelin maintenance in schwann cells will be analyzed by biochemistry and electron microscopy. biochemical tests include western blot for pmp protein expression in sciatic nerve, immunohistochemistry for pmp protein expression in myelinating schwann cells and pcr for mrna pmp expression. if the therapy is successful in rats, it could possibly be later on used in clinical trials. ultrasound is a widely-used tool in diagnosing carpal tunnel syndrome (cts). several different methods for sonographical evaluation of median nerve damage exist, such as calculating the ratio of cross sectional areas (csa) of median nerves in various sites. this enables detection of actual nerve swelling proximal to the carpal tunnel as an expression of median nerve damage inside the carpal tunnel (as seen in cts). in comparison to other diagnostic methods no data exist about the prevalence of cts like changes in an unselected population. a series of non-selected fresh cadavers were examined. arms of fresh, non-embalmed whole body cadavers were examined. the medical record did not allow to obtain conclusive information on the peripheral nerves. using a regular ultrasound system with a mhz transducer, median nerves were identified and tracked along their course in the forearms. csa measurements of the median nerves were performed at two sites in each arm: .) halfway between the elbow joint and wrist, .) directly proximal to the carpal tunnel. csa ratio was calculated with the following formula: csa ratio = csa wrist (cm ) csa forearm (cm ) csa ratio < . was found in ( . %) arms, csa ratio ≥ . in ( . %) arms and csa ratio ≥ in ( %) arms. csa ratio ≥ . was detected in . % of women and % of men. the overall mean (± sd) age was . ± . years. men ( . ± . ) were significantly younger than women ( . ± . ; p = . ). a weak but significant correlation between age and csa ratio was found in women (spearman -− . ; p= . ), but not in men (p= . ). the mean bmi for csa ratio ≥ . was . ± sd . . based on a csa ratio ≥ . as a criterion for cts, the present ultrasound results are consistent with the average cts prevalence reported in previous studies, which were obtained with electrophysiological methods. this study on a large unselected series of cadavers confirms the comparability of both methods. instrumental activities of daily living (badl and iadl), falls and gait patterns in the large, prospective, population based rotterdam study. in total, participants of this study (mean age years, % women) underwent a polyneuropathy screening involving a symptom questionnaire, neurological examination and nerve conduction studies. screening yielded four groups: no, possible, probable and definite polyneuropathy. participants were interviewed about badl (stanford health assessment questionnaire), iadl (instrumental activities of daily living scale) and frequency of falling in the previous year. in a random subset of participants, gait was assessed with an electronic walkway (gaitrite). associations of polyneuropathy with badl and iadl were analyzed continuously with linear regression, and dichotomously with logistic regression. history of falling was evaluated with logistic regression and gait changes were evaluated with linear regression. we found that participants with definite polyneuropathy had more difficulty in performing badl and iadl than participants without polyneuropathy. polyneuropathy related to worse scores of all badl (especially walking) and three iadl components (housekeeping, traveling, and shopping). participants with definite polyneuropathy were two times more likely to fall, and these falls more often resulted in injury. participants with polyneuropathy had worse gait parameters on the walkway, including lower walking speed and cadence, and more errors in tandem walking. in summary, chronic polyneuropathy is strongly associated with significant impairment in daily life. recognition of polyneuropathy and related disability is very important in order to inform, support and possibly treat patients, and to prevent future falls and dependence in daily functioning. patients with polyneuropathy often suffer from tingling sensations, numbness, weakness and pain. these symptoms are used in several screening questionnaires, most of which were developed for high-risk patient groups, such as individuals with diabetes mellitus. in most tools equal weights are applied to all symptoms, while some might be more informative than others. we evaluated the diagnostic value and frequency of occurrence of individual symptoms of chronic polyneuropathy and constructed and validated a simple screening questionnaire that can reliably help to diagnose polyneuropathy in low-risk patient groups. in a multi-step procedure, we initially compiled a twelve-item questionnaire concerning symptoms of polyneuropathy. the questionnaire was completed by polyneuropathy patients and controls (headache, transient ischemic attack, multiple sclerosis). we calculated the sensitivity, specificity and likelihood ratios of each individual symptom. next, stepwise multivariable logistic regression was used to create a compact model, able to discriminate cases from controls using only the most informative symptoms. a simple scoring system was subsequently developed based on the regression coefficients of this reduced model. external validation was subsequently conducted in a population of cases with chronic idiopathic axonal polyneuropathy and controls without polyneuropathy. performance was assessed with discrimination (area under the curve, auc), and calibration. numbness and tingling feet were most frequently reported by polyneuropathy patients and had the highest sensitivity. feeling as if walking on cotton wool and allodynia of the feet had the highest specificity. multivariable logistic regression yielded a model that contained these four symptoms, complemented with balance problems and tingling hands. based on this regression analysis, the erasmus polyneuropathy symptom score (e-pss) was created, a score ranging from to . this polyneuropathy symptom score had a good performance (auc . ) in de derivation set and proved to be valid in the external population (auc . ). in this study, we created a simple, validated polyneuropathy symptom score (e-pss) that takes both the individual value of only six different symptoms and its frequency into account. this tool can be helpful as screening instrument in clinical practice and for future studies on polyneuropathy. multiple sclerosis (ms) is traditionally viewed as a central nervous system disease. to date, there is no unequivocal evidence implicating involvement of the peripheral nervous system (pns). this study aims to prove whether the pns is additionally affected and if so, to detect, localize and quantify these peripheral nerve lesions in patients with multiple sclerosis (ms) by applying high-resolution mr-neurography (mrn) with large anatomical coverage in combination with standard electrophysiological and neurological tests. we prospectively enrolled patients with confirmed ms (> years), two patients with clinically isolated syndrome (cis), and age-/sex-matched healthy volunteers. any other potential causes for a concomitant polyneuropathy were excluded. all ms patients underwent detailed neurological and electrophysiological testing. tesla mrn with large anatomical coverage from lumbar plexus and spinal nerves down to ankle level was performed in all participants by using fat-saturated, t -weighted turbo-spin-echo (tse) sequences (tr/te / ms) and a dual echo tse sequence for t -relaxometry (tr ms; te /te / ms). a d t -weighted, fat-saturated space sequence (tr ms; effective te ms) was used for imaging of the lumbar plexus. manual segmentation of spinal/sciatic/tibial/peroneal nerves was performed on a total of , axial slices. besides evaluation of nerve t w-signal, detailed quantification of nerve lesions by analyzing morphometric (nerve caliber) and microstructural markers (proton-spin-density and t -relaxation-time) was conducted. mean lesion load at thigh level was higher in ms ( . ± . ) vs. controls ( . ± . ;p< . ). nerve proton-spin-density was also higher in ms (tibial/peroneal: . ± . / . ± . ) vs. controls (tibial/peroneal: . ± . / . ± . ;p< . ). in contrast, t -relaxation time was significantly higher in controls (tibial/peroneal: . ± . / . ± . ) vs. ms (tibial/peroneal: . ± . / . ± . ;p< . ). proximal tibial and fibular nerve caliber was also significantly higher in ms (tibial: p< . ; fibular: p= . ). for the first time, pns lesions in ms patients could be visualized and quantified in vivo by high-resolution mrn. lesions are indicated by an increase of proton-spin-density and a decrease of t -relaxation-time. nerve caliber as a morphometric criterion also significantly increased. this proof-of-concept study may offer new insights into the pathomechanism of ms and might have future implications on therapeutic approaches. immunoglobulin g (igg) fc-gammars confer diverse effector functions by linking the cellular and humoral arms of the immune system that has been involved in the pathogenesis of guillain-barré syndrome (gbs). in the post-polio era, the polymorphisms of fc-gammar and their relevant knowledge have become one of the main targets for new therapeutic strategies for the treatment of gbs patients. differences in severity and frequency of gbs subtypes found between south-asian and western populations can be attributed to their genetic susceptibility. therefore, we aimed to determine fc-gammar polymorphic alleles (fc-gammariia: h /r ; fc-gammariiia: v /f ; fc-gammariiib: na /na ) and their possible link with gbs on the currently available large gbs cohort in bangladesh. fc-gamma r polymorphisms of gbs patients and healthy controls were genotyped using sequence-specific pcr. for validation, we carried out the sequencing of some samples for fc-gammariia and fc-gammariiia alleles. no significant differences were found regarding the distribution of fc-gammar genotypes and allele frequencies in gbs patients and controls. fc-gammar-h/h- genotype was significantly predominant in patients with severe disease compared to patients with mild disease (p= . , or, . ; % ci, . - . ). no other significant associations were found in gbs patients for candidate alleles and disease severity. fc-gammariiia-f/f- was found to be significantly predominant in anti-gm antibody positive gbs patients compared to anti-gm antibody negative patients (p= . , or, . , % ci, . - . ). fc-gammariiia-v alleles were significantly higher in patients with poor prognosis when compared to patients with good outcome (p= . , or, . , % ci, . - . ). no significant association of fc-gammariiib genotypes and alleles were found with gbs patients, disease severity and disease outcome. extensive subgroup analysis revealed no significant association in genotype and allele frequencies between aman and aidp subtype. in conclusion, igg fc-gammar polymorphisms do not constitute significant risk markers for susceptibility to gbs, however homozygous fc-gammariia-h might be involved in the severe form of gbs. in addition, fc-gammariiia-f/f- might play an important role in the molecular mimicry against nerve gangliosides in gbs. further studies that enroll a large number of patients (e,g. igos) are required to confirm the present findings from different geographical areas. hayes jm , o'brien pd , backus c , feldman el . department of neurology, university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common complication observed in patients with impaired glucose tolerance and type diabetes. male mice fed a high fat diet (hfd) develop metabolic impairments and pn serving as an appropriate animal model to study pn development and progression. it is well documented that female mice fed a hfd display a degree of protection against hfd-induced metabolic changes with mice retaining relatively normal insulin sensitivity. this protection is attributed to differences in fat accumulation and to the anti-diabetic effects of estrogen. based on these sex-dimorphisms we hypothesized that hfd-fed female mice would also exhibit resistance to developing pn. in the present study male and female c bl /j mice were fed either a standard diet ( % kcal fat; sd) or a high fat diet ( % kcal fat; hfd) from wk. at wk, wk and wk, neuropathy phenotyping was performed on all groups complemented with longitudinal metabolic assessments including insulin tolerance testing (itt). neuropathy phenotyping consisted of hindpaw latency to heat stimulus, motor and sensory nerve conduction velocities (ncvs), and terminal intraepidermal nerve fiber (ienf) counts. assessment of insulin resistance through itt demonstrated that during early hfd feeding, female hfd-fed mice exhibited relatively normal insulin responsiveness, while male hfd mice exhibited insulin resistance. despite this finding, wk female hfd mice displayed a similar pattern of pn to that of their male counterparts, with similar fold-changes in hindpaw latency and sensory and motor ncvs. therefore, although female hfd-fed mice exhibit resistance to hfd-induced metabolic changes, they display a pn comparable to male hfd-fed mice suggesting that systemic insulin resistance does not mediate pn. further studies are underway investigating the role of insulin signaling in the peripheral nerves of female hfd-fed mice. anti-mag (myelin-associated glycoprotein) neuropathy is a disabling autoimmune peripheral neuropathy caused by monoclonal immunoglobulin m (igm) autoantibodies that recognize the carbohydrate epitope hnk- (human natural killer- ). this glycoepitope is highly expressed on adhesion molecules, such as mag, present in myelinated nerve fibers. since the pathogenicity and demyelinating properties of anti-mag autoantibodies are well established, current treatments aim at a reduction of autoantibody levels. however, the therapies applied so far are primarily immunosuppressive and lack selectivity and efficacy. we therefore hypothesized that a significant improvement of the disease condition could be achieved by selectively neutralizing the pathogenic anti-mag antibodies with carbohydrate-based ligands mimicking the natural hnk- glycoepitope. in an inhibition assay, a mimetic (mimhnk- ) of the natural hnk- epitope inhibited mag-binding by pathogenic igm antibodies from patient sera, however only with micromolar affinity. therefore, considering the multivalent nature of the mag-igm interaction, polylysine polymers of different sizes were substituted with the mimetic. with the most promising polylysine glycopolymer pl (mimhnk- ) the inhibitory effect on patient sera was improved by a factor of up to , per epitope, consequently leading to a low nanomolar inhibitory potency. since clinical studies indicate a correlation between the reduction of anti-mag igm levels and clinical improvement, an immunological surrogate mouse model for anti-mag neuropathy, producing high levels of anti-mag igm, was developed. the observed efficient removal of these antibodies with the glycopolymer pl (mimhnk- ) represents a first step towards an antigen-specific therapy for anti-mag neuropathy. hinder lm , mendelson f , backus c , feldman el . university of michigan, ann arbor, mi, usa. in the united states % of children and young adults are obese and at risk of developing prediabetes. prediabetic patients largely develop the same macro-and microvascular complications as patients with type diabetes, including peripheral neuropathy (pn). moreover, recent clinical data suggest normoglycemic obese patients develop pn. central obesity, characterized by excess fat storage in visceral white adipose tissue, leads to systemic metabolic dysfunction largely due to an imbalance between pro-inflammatory/anti-inflammatory adipokine production. subcutaneous adipose tissue is considered 'benign', but adopts a visceral-like phenotype in response to metabolic stress, with reduced thermogenicity, reduced brown adipose identity, and increased pro-inflammatory gene expression. the popliteal adipose tissue (pat) depot, corresponding to subcutaneous adipose, is adjacent to the peripheral nerve affected in pn, contains the lymph node for lymphatic drainage of the hind limb, and expands following local, sterile hind paw inflammation. the aim of the current study was to characterize pat changes in the high fat diet (hfd) mouse model of obesity and pn, and consider its contribution to peripheral nerve dysfunction. we previously reported c bl /j mice fed % hfd from - wk develop obesity, 'prediabetes' and pn; and switching mice back to a standard diet from - wk improves metabolic and pn phenotypes. at and wk pat was bilaterally dissected and the lymph node removed. the left pat was processed for histomorphometry, and the right pat for rt-qpcr. at wk hfd was associated with a significant shift in adipocyte size-frequency distribution, with a greater number of larger adipocytes. switching the hfd mice back to standard chow from - wk restored the size-frequency distribution towards age-matched controls. rt-qpcr was performed to assess changes in thermogenicity (ucp ), brown adipose identify (cidea) and sterile inflammation (saa ). at and wk hfd pat had reduced thermogenicity and brown adipose identify, and increased sterile inflammation. this switch towards a visceral-like phenotype was reversed in the hfd mice switched back to standard chow. in summary, hfd-induced changes in pat histomorphometry and adipose identity closely associate with pn phenotype. these preliminary data suggest a potential role for pat-nerve signaling in pn. hinder lm , backus c , hayes jm , feldman el . university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common and debilitating complication of obesity and diabetes that triggers pain and loss of sensation. substantial nerve damage occurs in many patients prior to noticeable symptoms and no treatments are currently available; therefore, there is a critical need to identify treatment strategies that impact the underlying disease pathogenesis. our in vivo fluxomics data in the bks-db/db mouse model of type diabetes (t dm) and pn suggest that 'metabolic reprogramming' occurs in the t dm nerve to downregulate mitochondrial oxidative phosphorylation of substrates derived from glycolysis and fatty acid beta-oxidation. therefore, we hypothesize that distinct systemic metabolic alterations occur in obesity and diabetes which induce tissue-specific metabolic reprogramming within the peripheral nerve, altering fuel utilization and ultimately leading to tissue dysfunction. we contend that identifying conserved bioenergetic profiles across mouse models of pn will provide insight into key pn mechanisms. the current study utilized two mouse models of pn: the % high fat diet (hfd) mouse model of obesity and prediabetes at wk of age ( wk hfd), and the leptin receptor-deficient bks-db/db model of t dm at wk of age. mitochondrial function was determined in primary dorsal root ganglia (drg) neurons and sural nerve tissue from both models using the seahorse xf analyzer. resting mitochondrial oxidative metabolism was upregulated in drg neurons from mice with pn, with increased resting atp production and maintained mitochondrial coupling. in contrast, resting atp generation was decreased in sural nerve from mice with pn, with decreased coupling efficiency. relative spare respiratory capacity was attenuated in both drg neurons and sural nerve from mice with pn, indicating that mitochondria were less able to increase respiration in response to an energetic challenge. moreover, mitochondrial copy number was unchanged in drg neurons, but decreased in sural nerve tissue of mice with pn compared with respective controls. these data suggest a change in absolute number and function of sural nerve mitochondria, and a conserved cross-model proximal-distal bioenergetic profile in pn. we are currently exploring the relationship between these changes and pn pathogenesis. hoang ttn , umapathi t . hospital, ho chi minh city, vietnam; national neuroscience institute, singapore. ho chi minh city (hcmc) is the biggest metropolitan city in southern vietnam. its population is more than ten million. adult patients with neurological disorders are seen at six city public hospitals, including , hospital. there has been no systematic study of guillain barré syndrome (gbs) at hcmc or in vietnam in general. we are in the process of starting a prospective gbs database that we hope to expand to the other public hospitals at hcmc. here we describe our experience from . we saw gbs patients at , hospital. most of the cases were admitted in the rainy season, from late april to november, when mosquito-borne flavivirus infections are more common. patients were seen in the first week of illness and reported antecedent fever. two patients had diarrhea. diagnoses were made largely on clinical features, cerebrospinal fluid analysis and nerve conduction study. clinical findings include limb weakness, numbness and vii cranial nerve palsy. extraocular eye movements were affected in one patient. none had respiratory involvement severe enough to require artificial ventilation or intensive care. there were no pure miller-fisher syndrome cases; we suspect this might be related to the mild deficits that did not prompt hospitalization. nerve conduction studies showed typical features such as loss of f waves and abnormal blink reflex. the electrophysiology of patients' was dominated by demyelinating changes, one case was largely axonal and the remaining patient had normal electrodiagnostic study. repeat nerve conduction studies were not feasible because of limited resources. neurologists use corticosteroids as the main treatment. intravenous immunoglobulin and plasma exchange are costly and not reimbursed by medical insurance. we are currently preparing to systematically study gbs in southern vietnam, specifically with regards to possible role of antecedent flavivirus infections. we are also exploring the possibility of using low volume plasma exchange as a feasible cost-effective therapeutic modality. about % of patients with guillain-barré syndrome (gbs) treated with intravenous immunoglobulin (ivig) or plasma exchange deteriorate after initial improvement or stabilization -a phenomenon that is termed treatment-related fluctuation (trf). it is important to distinguish acute onset cidp (a-cidp) from gbs-trf during early course of the disease, because their therapeutic strategies and prognoses are different. herein, we describe a patient with gbs-trf, but with an extended progression phase that exceeds weeks. a -year-old woman was admitted due to acute onset progressive leg weakness and diplopia that had developed weeks prior (onset, d ). on neurological examination, right facial palsy was also observed. lower extremity weakness was moderate in proximal and distal muscles (mrc grade iv) with absent knee and ankle jerks. sensory examination revealed no abnormality in all modalities. she denied any recent diarrhea or upper respiratory infection, and vaccination. albumino-cytologic dissociation was noted in csf analysis; white blood cell count of / l and protein level of . mg/dl. nerve conduction study revealed demyelinating sensorimotor polyneuropathy with prolonged distal latency and conduction blocks. anti-ganglioside antibodies (gm igm/g, gd b igm/g, and gq b igm/g) were all negative. following ivig treatment, she was discharged with considerable improvement (d ). days later, she was re-admitted due to deterioration of leg weakness and hand clumsiness (d ). after another ivig treatment, she was discharged with clinical improvement (d ). days later, however, she was admitted again (d ) due to another considerable deterioration with four extremity weakness being worst at this time (mrc grade ii-iv in upper extremity, grade ii in lower extremity). a-cidp was considered given the progression phase exceeding weeks, but, we decided to give another treatment with ivig instead of a switch to corticosteroids because of uncertainty regarding distinction between a-cidp and gbs-trf. she was significantly improved following ivig treatment, and finally discharged (d ). thereafter, there has been no further deterioration during long-term follow-up of year. conclusively, this is a rare case of gbs with extended progression phase and trf. we propose that this could be referred to as subacute inflammatory demyelinating polyradiculoneuropathy (sidp) with trf. hsieh s , chao c . national taiwan university hospital, taipei, taiwan. transthyretin (ttr)-related familial amyloid polyneuropathy (fap) constitutes a major etiology of adult-onset hereditary neuropathies worldwide, in particular, a mutant ttr of ala ser (ttr-a s) in taiwan, the most common cause of acquired genetic neuropathy with adult onset (> years of age) of taiwanese patients. fap is a pan-modality neuropathy involving motor, sensory, and autonomic components of the peripheral nervous system with early involvement of small fibers as a major symptom. the early symptoms of fap are sometimes minimal and difficult to ascertain, mainly related to the fact that conventional electrophysiological examinations were not sensitive enough to detect small fiber neuropathy. skin biopsy with quantification of intraepidermal nerve fibers (ienf) has become one of the standard approaches to diagnose small fiber sensory neuropathy based on pathological documentation of nociceptive nerve degeneration. to explore the issue of early biomarkers in fap, we performed skin biopsy and compared ienf density with parameters of nerve conduction studies (ncs) and quantitative sensory testing (qst) on subjects ( men, aged . ± . years) with genetic confirmation of ttr-a s: patients and carriers. the ienf densities were significantly reduced compared to the age-and gender-matched controls in carriers ( . ± . vs. . ± . fibers/mm, p = . ) and patients ( . ± . vs. . ± . fibers/mm, p = . ). the latter was consistent with our previous report (neurology, : - , ) . the abnormal rate of ienf density was significantly higher than that of ncs and qst, respectively. in conclusion, there was significant skin nerve degeneration in carriers with ttr-a s. compared with qst and ncs, ienf density assessment had the highest abnormal rate and highest sensitivity to detect neuropathic changes in the early stage of fap. charcot-marie-tooth type p (cmt p) has been associated with frame-shift mutations in the ring domain of lrsam (an e ligase). this study describes families with a novel missense mutation of lrsam gene and explores pathogenic mechanisms of cmt p. this american family with dominantly inherited axonal polyneuropathy reveals a phenotype similar to those in previously reported non-us families. the affected members in our family co-segregated with a novel missense mutation cys arg that alters a highly conserved cysteine in the ring domain. this mutation leads to axonal degeneration in the in vitro neuronal cell-line. moreover, using protein mass spectrometry, we identified a group of rna binding proteins (including fus, a protein critically involved in motor neuron degeneration) that interacted with lrsam . the interactions were disrupted by the cys arg mutation, which resulted in reduction of intranuclear rna-binding proteins. a knockin mouse of cys arg has been created for further explorations of cmt p mechanisms and therapeutic development. together, our findings suggest that the mutant lrsam may aberrantly affect the formation of transcription machinery. given a similar mechanism has been reported in motor neuron degeneration of amyotrophic lateral sclerosis, abnormalities of rna/rna-binding protein complex may play a role in the neuronal degeneration of cmt p. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr ). this double-blind, multicenter, parallel-group trial randomized ( : ) adult participants (n= ) with chronic inflammatory demyelinating polyradiculoneuropathy being treated with intravenous immunoglobulin (ivig) or corticosteroids to . mg fingolimod (n= ) or placebo (n= ) once-daily. previous treatment was discontinued (ivig) or tapered (corticosteroids). in the total trial population, there was no significant difference between the groups in the primary outcome, time-to-first confirmed worsening (≥ -point increase on the adjusted inflammatory neuropathy cause and treatment [incat] scale vs. baseline), or time-to-first any worsening (confirmed on incat assessment or unconfirmed). no significant difference between the two treatment groups was shown on secondary outcomes; change from baseline in grip strength and rasch-built overall disability scale (r-ods) at six months or trial end. analyses of pre-specified subgroups were performed for primary and secondary outcomes. this trial evaluated the efficacy and safety of fingolimod in chronic inflammatory demyelinating polyradiculoneuropathy (cidp). corticosteroids, intravenous immunoglobulin (ivig) and plasma exchange are recognized treatment options but no other immunomodulators demonstrated efficacy in a controlled trial. fingolimod has been shown to be efficacious and is approved for the treatment of relapsing multiple sclerosis. results from experimental autoimmune neuritis in rats suggested that it might show an effect in cidp. in this double-blind, multicenter, parallel-group trial, cidp participants receiving ivig or corticosteroids were randomized to once-daily fingolimod . mg or placebo ( : ). participants were stratified by inflammatory neuropathy cause and treatment disability (incat) scores and prior treatment. previous ivig treatment was discontinued after one final course before randomization. previous corticosteroid treatment was tapered over weeks. the primary outcome was time-to-first confirmed worsening (≥ -point increase on the adjusted incat score versus baseline). secondary outcomes included change in grip strength and rasch-built overall disability scale (r-ods) score from baseline to month and at trial end. the trial was stopped for futility by an independent data monitoring committee after a pre-planned interim analysis based on pre-specified criteria. in all, participants received fingolimod (ivig: , corticosteroids: ; age: . ± . years [mean±standard deviation]; male: . %); received placebo (ivig: , corticosteroids: ; age: . ± . years; male: . %). the percentage ( % confidence interval) of participants free from confirmed worsening at the trial end was not significantly different between fingolimod ( . % [ . %- . %]) and placebo ( . % ( . %- . %); p= . ). in the first days, approximately % participants experienced worsening. at trial end, approximately % participants had no worsening. there was no significant difference after six months or at the trial end (whichever occurred earlier) in the secondary endpoints. adverse events were reported in / and / participants in the fingolimod and placebo group, respectively. there were no deaths. nine participants in the fingolimod group and in the placebo group had serious adverse events. adverse events leading to trial drug discontinuation occurred in ( %) participants on fingolimod and none on placebo. no new safety signals emerged in this trial. acknowledgment: the authors consulted for or were employed by the study sponsor novartis pharma ag, basel, switzerland. myelin sheath enwraps non-nociceptive mechanoselective abeta-afferents transmitting touch/vibration sense. a prominent reduction in the mechanical stimulus required to evoke a withdrawal response in rodents, a phenomena interpreted as mechanical allodynia, arises due to peripheral nerve/myelin damage. evidence has emerged that nerve injury-induced mechanical allodynia depends on the adaptive immune/t cell activity in female but not male rodents. having previously demonstrated both the release of the cryptic - peptide regions of myelin basic protein (mbp - ) following sciatic nerve chronic constriction injury (cci) and the direct, robust and t cell-dependent ability of the pure mbp - peptides to induce mechanical allodynia after injection into the intact sciatic nerve, we hypothesized that mbp - contributes to sexual dimorphism in mechanical allodynia. the pure mbp - wild-type (wt), its histidine (his) mutant or scramble peptides were administered into an intact sciatic nerve fascicle in male and female rats or mice, followed by von frey testing. intra-sciatic mbp - -wt peptide induced robust and lasting allodynia in females. in contrast, males responded with a brief and mild decline in mechanical sensitivity for one day post-injection of both wildtype and control peptides. the algesic ability of mbp - -wt was diminished in the his mutant. we here present the molecular changes in the sciatic nerve, drg and the spinal cord after the intra-sciatic mbp - injection in male and female animals. in addition, using the biotin-labeled mbp - peptide and the hrp-labeled goat anti-rat igg/igm antibodies, we developed an elisa to quantitatively assess seropositivity for the specific anti-mbp - peptide igm/igg autoantibodies in female and male rats post-cci. human serum from female patients with multiple sclerosis was used for control. our work corroborates the findings of sexual dimorphism of mechanical hyperpathia and suggests its potentially autoimmune nature in females. iijima m , nishi r , ikeda s , kawagashira y , koike h , sobue g , katsuno m . nagoya university, nagoya, japan. non-obesity diabetic (nod) b - knockout (ko) mice are characterized by chronic and progressive neuritis and expected as models of immune-mediated neuropathies, especially cidp. hindlimb-predominant weakness due to inflammatory demyelination followed by axonal degeneration begins from around twenty week-age in all female mice until thirty week-age. to clarify the efficacy of immunoglobulins as immune-regulating therapeutics and the similarity of pathogenesis of human cidp, we injected intraperitoneally human-derived immunoglobulins (ipig, mg/mg bw/week) and saline as a control to totally forty female mice. clinical and pathological estimations in sciatic nerves were performed in time series. as a result, the ipig-treated group was protected from weight loss which could be related to axon loss followed by muscle atrophy as well as inflammatory demyelination between twenty-five week-age and thirty week-age compared to the control. in addition, the pathological findings in sciatic nerves showed that ipig apparently suppressed inflammatory infiltrates. about the subsets of inflammatory infiltrates, while macrophages (cd +) and lymphocytes (cd +) highly existed and suggested to play a main role in the neuritis until thirty week-age, only macrophages naturally disappeared after thirty week-age without any therapeutic induction. immunoglobulins effectively suppressed only macrophages although that did not suppress cd + lymphocytes. in conclusion, nod b - ko mice respond to immunoglobulins in a similar manner to human cidp and this efficacy is due to the suppression of macrophage-dominant pathogenesis. therefore, macrophage-derived pathogenesis is for the main target of immunoglobulin therapy and we should focus on the lymphocyte-derived pathogenesis which might plays an important role in non-responders to immunoglobulins. ikeda s , nishi r , kawagashira y , iijima m , koike h , katsuno m , sobue g , . department of neurology, nagoya university graduate school of medicine, nagoya, japan; research division of dementia and neurodegenerative disease, nagoya university graduate school of medicine, nagoya, japan. chronic inflammatory demyelinating polyneuropathy (cidp) is an acquired immune-mediated polyradiculoneuropathy that is characterized by heterogeneous clinical manifestations. typical cidp is defined as neuropathy manifesting in a progressive manner, stepwise manner, or with recurrent symmetrical proximal and distal weakness and sensory impairment in all four limbs. although they occur at a lower proportion than the so-called typical cidp, atypical forms, such as multifocal acquired demyelinating sensory and motor (madsam), distal acquired demyelinating symmetric (dads), pure sensory, pure motor, and focal, are considered to cidp subtypes. thus far, pathological features characterizing each clinical subtype have not been fully elucidated. we analyzed clinical and pathological correlations in consecutive cidp patients who underwent sural nerve biopsy and fulfilled the definite or probable efns/pns criteria. there were male and female patients. the age at biopsy was . ± . (mean ± sd) years, and the duration from the onset of neuropathy to biopsy was ± months. fifty-five percent (n = ) of the patients were classified as having typical cidp. regarding atypical cidp, madsam (n = , %), dads (n = , %), and pure sensory (n = , %) subtypes were the major subtypes, while pure motor (n= , %) and focal (n= , %) subtypes were rare. no significant difference was found among these subtypes in terms of sex, age at biopsy, and disease duration. sural nerve biopsy specimens revealed that the densities of large myelinated fibers significantly decreased in the madsam subtype than in the other subtypes (p = . ). in addition, the variation in nerve fibers among fascicles was more conspicuous in the madsam subtype than in typical cidp (p= . ). patients with the dads subtype tended to show the formation of onion-bulbs. in conclusion, pathological findings of sural nerve biopsy specimens were different among the cidp subtypes. further studies are needed to clarify mechanisms leading to different pathological features. small volume plasma exchange (svpe) can be an affordable and potentially effective alternative form of plasma exchange. svpe is the repeated removal of small volumes of supernatant plasma over several days via sedimentation of patient whole blood. the aim of this study is to assess the clinical feasibility and safety of svpe in patients with gbs in low-income countries. twenty adult patients with gbs diagnosed as per the criteria for gbs of the national institute of neurological and communicative disorders and stroke (ninds) were enrolled for svpe at a centre in bangladesh. serious adverse events (sae) were defined as the number of patients developing severe sepsis associated with the central venous catheter (cvc) or deep venous thrombosis in the limb where the cvc is placed for svpe. the svpe procedure was considered safe if less than of svpe-treated gbs patients have a sae, and feasible if eight litres of plasma could be removed in at least of svpe-treated gbs patients. among the cases who received svpe, ( %) patients were male and the age range between to yrs. all the patients were quadriplegic and bedbound at enrolment for svpe with a median mrc score of (iqr, - ). cranial nerve involvement, autonomic dysfunction and requirement for assisted ventilation were observed in ( %), ( %) and ( %) patients respectively. electro physiologically ( %) patients were motor axonal and ( %) patients were sensory-motor demyelinating type. during the svpe none of our patients experienced sae and one patient experienced central line associated blood stream infection. common adverse effects were transient intravenous fluid responsive hypotension during the svpe sessions in ( %), cv catheter insertion site hemorrhage in ( %) and hypersensitivity reaction to fresh frozen plasma in ( %) patients. there was no hypo-albuminemia, anemia or electrolyte imbalance observed in most patients ( %) treated with svpe. improvement in one or more grade of the gbs disability score at four weeks after the onset of svpe was observed in ( %) patients. in conclusion svpe can be a safe, feasible and cost effective alternative to standard pe in the developing countries. guillain-barré syndrome (gbs) is a descriptive disease entity defined by a set of clinical, electrophysiological and laboratory criteria. various clinical phenotypes exist that may be triggered by different antecedent infectious events. although the disease appears to affect primarily the elderly in developed countries, but, scenario is different in developing countries. bangladesh has made an impressive progress towards the eradication of poliomyelitis, and no new cases have been reported since . gbs, an acute polyradiculoneuropathy, is the most frequent cause of acute flaccid paralyis. the crude incidence rate of gbs in < years of age reported here appears to be . × to × higher than that reported in the literature. we conducted a hospital based observational study including patients fulfilling the national institute of neurological disorders and stroke (ninds) criteria for gbs patients between and in dhaka medical college hospital, dhaka, bangladesh. detailed clinical, electrophysiological, serologic and microbiological data were obtained. gbs affected predominantly in young adults males (m/f= : ) living in rural areas. antecedent events were recorded in > % of patients; frequent events being gastroenteritis (> %) and upper respiratory tract infection ( %). more than % of the patients were bed-bound (gbs disability score ) at entry and about % patients required mechanical ventilator. about % patients did not receive specific treatment either intravenous immunoglobulin (ivig) or plasmapheresis due to high expensive treatment cost. % patients had died during hospitalization. % of patients had an axonal variant of gbs and evidence for a recent c. jejuni infection ( %). c. jejuni infection was significantly associated with serum antibodies to the gangliosides gm and gd a, axonal neuropathy, and greater disability. in conclusion, the majority of the patients do not receive standard treatment with ivig in view of its high price. therefore, we developed low-cost treatment strategies and conducted a safety and feasibility trials for small volume plasma exchange (svpe) on gbs patients in bangladesh. in future, it is essential to conduct a phase ii clinical trial to assess the efficacy of svpe for low-in-come countries. prognosis of guillain-barré syndrome (gbs) has not improved in last two decades. current therapies (intravenous immunoglobulin, ivig and plasma exchange, pe) had been proved to be effective on two third of patients in developed world. unpredictable and poorly understood clinical course of gbs hamper treatment development. in bangladesh, most patients affected by gbs cannot afford specific treatments with ivig or pe instead most of them receive only supportive care. therefore, we aimed to compare the outcome of ivig treated patients with supportive care patients in improvement of gbs disability score and mrc sum score by using world's largest gbs cohort in bangladesh. we conducted a prospective observational study enrolling gbs patients between and from dhaka medical college hospital and national institute of neuroscience and hospital, dhaka, bangladesh. only gbs patients ( %) received standard ivig treatment. in current analysis, ivig treated patients and age, sex and severity matched controls (supportive care only) were considered. outcome of both groups were compared using fisher's exact or chi square test and survival analysis were performed by kaplan meier method using log rank test. among patients (cases and controls), male/female ( / ), median age years, % patients were bed-bound, one-fourth patients required mechanical ventilation and % were axonal. we did not found any significant differences of treatment outcome in both cases and control groups in gbs disability score (week : p= . , months: p= . ) and mrc sum score (week : p= . , months: p= . ). survival analysis revealed, the differences of time required for independent locomotion, improvement of one gbs disability score and improvement of mrc score were not statistically significant between treatments (ivig) and supportive care patients. in conclusion, our analysis showed that standard dose of ivig use has no considerable advantage to improve specific outcome measures among gbs patients in bangladesh. as the phenotype of gbs in bangladesh is different from developed world; therefore, an efficacy trial for ivig is needed for developing countries like bangladesh or new targeted therapeutic strategies can append beneficial effects for gbs patients. isose s , , watanabe k , omori s , sekiguchi y , beppu m , shibuya k , amino h , suichi t , misawa s , kuwabara s . graduate school of medicine, chiba university, chiba, japan; national hospital organization chiba east national hospital, chiba, japan. diabetic neuropathy is a frequent cause of neuropathic pain, suggesting the small-fiber involvement. additionally, persistent peripheral pain-related inputs could cause neuronal hyperexcitability and complex interactions of the nociceptive pathways, i.e., central sensitization. to investigate the pathophysiology of neuropathic pain in diabetic neuropathy, we studied pain-related evoked potentials (preps) after selective intraepidermal electrical stimulation (ies) to a-deltaand c-fibers in diabetes patients with neuropathic pain (n= ) and without neuropathic pain (n= ). we also conducted a longitudinal study to assess changes in preps and pain profiles in patients with neuropathic pain months after the start of treatment with duloxetine. this study is registered with the umin clinical trials registry, umin . ies was applied in the hand and foot, and preps were recorded from the cz electrode referenced to the linked earlobes. we evaluated prep latencies, amplitudes, and amplitude ratios of preps after c/a-delta -fiber stimulation. in the conventional nerve conduction studies, patients with neuropathic pain significantly showed conduction slowing and decreased snap amplitudes in the median and sural nerves compared with those in patients without neuropathic pain. in pain-related sep studies, there were no significant differences in prep amplitudes and latencies after a-delta -or cfiber stimulation between the patients with neuropathic pain and without it. prep amplitude ratios after c/a-delta -fiber stimulation tended to increase in patients with neuropathic pain compared to patients without pain. after the treatment with duloxetine, c/a-delta -prep amplitude ratios were significantly decreased after both hand and foot stimulation, and as for numerical rating scale (nrs) scores as the intensity of pain. patients with less pain relief showed the tendency of higher c/a-delta prep amplitude ratios before treatment compared to patients with better pain relief. the correlation between reduction of c/a-delta prep amplitude ratios and nrs reduction did not reach statistical significance. this pain-related sep study demonstrated that abnormal cortical response in patients with neuropathic pain could improve after the treatment with duloxetine, this might reflect the cortical hyperexcitability as a central sensitization. this study is funded by shionogi & co., ltd. the sponsors played no role in the design and management of the study, collection and analysis of data, interpretation of the results, or the writing of the writing of the report. peripheral nerve injury is commonly associated with traumatic injury which is often amenable to surgery. despite improved methods in surgical repair, functional recovery remains a challenging clinical problem that often leads to significant morbidity in patients. alternative therapies that could augment surgical repair may be beneficial in functional outcomes. neuroinflammation is a complex pathway with different cellular components and cytokines that are activated following peripheral nerve injury. macrophages are responsible for the breakdown of debris following injury as well as promotion of regenerative signals. macrophage polarization is the process by which macrophages take on phenotypically distinct functions based on the local environment and signaling cues. exercise has been shown to drive macrophage polarization from a pro-inflammatory m phenotype towards an anti-inflammatory m phenotype in numerous tissues, but remains uninvestigated in the peripheral nervous system. the purpose of our study was to identify how exercise affects macrophage polarization, motor and sensory function, and neuroregeneration following sciatic nerve crush. c bl/ mice underwent sciatic nerve crush injury and were then given access to running wheels (exercised) or not given access to running wheels (sedentary) for weeks. exercised mice ran an average of . km per night. injured exercised mice were protected from the development of thermal hyperalgesia when compared to injured sedentary mice. exercised mice had fewer paw slips on beam walk testing compared to sedentary mice. no differences were measured in mechanical sensitivity or motor coordination and balance assessed by rotarod. while motor nerve conduction velocities were significantly reduced for injured mice compared to uninjured controls, motor nerve conduction velocities from injured exercised animals were significantly higher than injured sedentary animals suggesting improved nerve recovery with exercise. injured sciatic nerves from exercised mice demonstrated increased m macrophages compared to sciatic nerves from injured sedentary mice. the behavioral changes and altered macrophage polarization correlated with increased epidermal nerve fiber density, improved myelination, and increased in vitro neurite outgrowth from injured exercised animals. therefore, exercise alters macrophage polarization towards an anti-inflammatory phenotype which improves repair and recovery of the injured peripheral nerve. jack mm , shah k , everist b , reyna j , hylton p . university of kansas medical center, kansas city, ks, usa. diffusion tensor imaging (dti) has long been used to evaluate the location and integrity of white matter tracts in the brain. dti uses quantitative data and directionality of water diffusion to determine axonal connectivity of the nervous system. the technology has only recently been utilized in limited settings in the peripheral nervous system due to challenging technical factors and lack of widespread availability. magnetic resonance (mr) neurography or peripheral neurography is a technique which uses diffusion to differentiate between intraneural and perineural tissues. it allows for fascicle patterns to be visualized particularly in the setting of peripheral nerve sheath tumors. peripheral nerve sheath tumors of various pathologies cause surrounding nerves to be involved or displaced in a range of directions. this technique helps determine the anatomic location of these nerve fibers in relation to the mass, which is particularly helpful at distinguishing neuromas from schwannomas. this data is invaluable to the surgeon to ensure a safe and low morbidity operation. while this technology has benefit particularly with surgical planning, it has been underutilized due to the challenges of requiring complex software to produce fiber tracks and the inability to translate these images into the operating room. here, we utilized brainlab software that is commonly available and utilized in surgical suites to produce images of the radial nerve fiber tracts with an associated peripheral nerve sheath tumor prior to surgical resection. while the software is commonly used in the central nervous system, it has not been reported to have been used in the peripheral nervous system. this software offers a high usability and produces anatomically correct and reliable fiber tracts. this technique overcomes the reliance on highly specialized software and extensive training required for use that most other tractography software has. utilizing peripheral neurography in this case allowed for complete surgical resection without postoperative deficits. this data offers clinicians an option to investigate peripheral nerve fibers in various pathologic states, to plan appropriate operative trajectories to peripheral nerve pathology, and to improve surgical outcomes for patients with peripheral nerve sheath tumors. jacobsen b , parry g , allen j , walk d , muley s , ortega e . barrow neurological institute, phoenix, az, usa; university of minnesota, minneapolis, mn, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is an immune mediated neuropathy that is responsive to immunomodulatory agents such as glucocorticoids, intravenous immunoglobulin (ivig) and plasma exchange (pe). the specific immunopathogenic mechanisms of cidp remain unclear but there is increasing interest in nodal proteins as a site of the immune attack. even though the majority of patients respond to one of the aforementioned immunomodulatory agents there are some who are unresponsive or incompletely responsive to these first line agents and other more aggressive treatments may be necessary. cyclophosphamide and stem cell transplantation may be effective but are associated with considerable morbidity. anecdotal reports suggest that rituximab may be beneficial for some patients that fail first-line therapy, especially if they have antibodies to nodal proteins. we present four patients with intractable cidp who responded to rituximab. one of the three patients had diabetes. disease duration prior to starting rituxan was short ( months) in two patients and longer and months in the other two. all patients had failed treatment with glucocorticoids and ivig, and in two, plasma exchange and ivig-pe were also ineffective. two of the four patients were quadriparetic and non-ambulatory. two gm doses two weeks apart of intravenous rituximab were instituted in all patients. all patient tolerated the treatments well without adverse effects. all patients responded within four weeks and continued to improve at six months. other immunomodulatory agents were successfully tapered but not totally discontinued. it remains unclear whether antibodies to nodal proteins were present in these patients. in conclusion, although rituximab efficacy remains uncertain on the basis of randomized controlled clinical trials, it may be beneficial in selected patients otherwise intractable to first-line treatments. further studies are necessary to better understand which patients may benefit most from rituximab and where in the treatment algorithm rituximab should be applied. neuropathic pain is a chronic condition seen in patients suffering a direct injury to the peripheral or central nervous system or an indirect injury due to, e.g., diabetes or multiple sclerosis. current treatment options fall short of preventing or completely relieving patients of their pain. for years, research has focused on understanding the role of neurons in neuropathic pain pathogenesis while overlooking the role of supportive cells in general and satellite glial cells (sgcs) in the dorsal root ganglion in particular. these cells not only buffer the neuronal microenvironment they are also involved in controlling the electrical activity flowing through the neurons and in neuropathic pain pathogenesis. the aim of this project is to understand the role of sgcs in neuropathic pain development and thereby aid the identification of new drug targets. to purify the sgcs from adult mice we optimized a fluorescently activated cell sorting (facs) protocol. the success of our purification method was confirmed using qrt-pcr and visual inspection of the sorted cells. finally, we are running rna sequencing on sgcs after peripheral nerve injury to compare their transcriptome to that of uninjured cells at different time points. the results from our study are likely to deepen our understanding of how sgcs contribute to the development and maintenance of neuropathic pain. guillain-barrésyndrome (gbs) is an immunemediated disorder in the peripheral nervous system (pns) triggered by molecular mimicry against nerve gangliosides. one of the cell surface receptors (fas)-ligand (fasl) interaction transmits apoptotic signal to eliminate the auto-reactive b and t-cells, which generates cross-reactive antibody against nerve cells. host genetic polymorphism of fas and fasl may alter their expression and induce aberrant apoptotic response to develop gbs. therefore, we determined the single nucleotide polymorphisms (snps) of both fas receptor (− g/a and - a/g) and fasl ligand (− c/t) in gbs patients (n= ) as well as healthy controls (n= ) using the lightcycler technique. serum level of soluble form of fas and fasl was measured using commercially available sandwich elisa kit. comparison of genotype, allele and haplotype frequencies was done with the gbs subgroups based on the clinical and serological data. ag heterozygote (p= . , or= . , % ci= . - . ) and polymorphic g-allele (p= . , or= . , % ci= . - . ) of fas receptor - a/g promoter snps were significantly associated with anti-ganglioside (gm ) antibody positive gbs patients. in addition, − g-allele (p= . , or= . , % ci= . - . ) and - g/- g haplotype (p= . , or= . , % ci= . - . ) were predominantly associated with the axonal variant of gbs patients. serum soluble form of sfas (median levels pg/ml vs. pg/ml, p= . ) and sfasl (median levels pg/ml vs. pg/ml, p= . ) were found to be elevated in anti-gm antibody positive gbs patients compared to anti-gm negative patients. no significant association was found in genotypic distribution between gbs patients and healthy controls. in conclusion, fas/fasl promoter snps are not a susceptible factor for gbs but could be one of the influencing factors to develop cross-reactive anti-ganglioside antibodies in gbs patients in bangladesh. furthermore, functional studies with a larger sample size (using cohort like international gbs outcome studies-igos) are required to explain the immune pathogenic role of these snps for gbs patients. jang sy , yoon ba , shin yk , yun sh , jo yr , park ji , shin kj , kim jk , park ht . dong-a university, busan, south korea; inje university, busan, south korea. myelination is essential for the proper function of the nervous system. schwann cells, which form the peripheral myelin sheath, have the unique ability to dedifferentiate and to destroy the myelin sheath under various demyelination conditions. during schwann cell dedifferentiation-associated demyelination in wallerian degeneration after axonal injury, schwann cells exhibit myelin and junctional instability, down-regulation of myelin gene expression and autophagic myelin decomposition. however, in inflammatory demyelinating neuropathy, it is still unclear how schwann cells react and contribute to segmental demyelination before myelin scavengers, macrophages, are activated for myelin clearance. here, we show that schwann cell dedifferentiation-associated demyelination is a mechanism involved in the initial demyelination observed in a mouse model of inflammatory demyelinating neuropathy using ultrastructural, biochemical and microarray analyses. myelin uncompaction and myelin membrane instability generated by dedifferentiated schwann cells lead to autophagolysosome-dependent cytoplasmic amputation between the axon-containing myelin sheath and the schwann cell body, resulting in the formation of the "myelin corpse", thereby allowing macrophages to phagocytose the myelin corpse in the end stage of segmental demyelination. we found myelin corpse formation in inflammatory demyelination to be a process similar to the myelin rejection during wallerian degeneration, which appeared to be dependent on schwann cell autophagolysosome activation since schwann cell-specific atg knockout mice exhibited delayed myelin rejection following nerve injury. finally, lysosome inhibition in schwann cells not only prevented segmental demyelination but also delayed the progression of clinical stages by suppressing the myelin corpse formation in inflammatory demyelinating neuropathy. thus, our findings indicate that demyelination by schwann cells and macrophages might be part of a process that includes sequential divisions of labor with respect to myelin rejection and digestion, respectively. in conjunction with previous studies showing schwann cell dedifferentiation and autophagy in toxic and hereditary neuropathies, the concept of "schwann cell dedifferentiation-associated demyelination" provides insight into the development of possible therapeutic strategies to prevent schwann cell demyelination in peripheral demyelinating neuropathies. methods: electrophysiological data, the charcot marie tooth exam score (cmtes), and bmi from patients with known cmt a were obtained and analyzed. results: when controlled for age, bmi does not affect studies of ulnar motor nerve conduction in ctm a patients, but rather specific components of the cmt exam scores (cmtes, loss of pinprick sensation and motor strength in the lower extremities). discussion: bmi and clinical components of the cmtes are correlated, but it is uncertain which is the primary effect -i.e., whether the reductions in pinprick sensation and motor strength in the lower extremities lead to a higher bmi, or higher bmi results in these signs. introduction: charcot-marie-tooth disease type c (cmt c) is a rare, dominantly inherited neuropathy caused by mutations in the lipopolysaccharide-induced tumor necrosis factor (litaf) or small integral membrane protein of the lysosome/late endosome (simple) gene. methods: we present a case series comprised of patients in whom cmt c is caused by a gly ser substitution in the encoded protein. we focus on clinical presentation, electrodiagnostic analyses, and our findings in the context of previously described cases. results: the gly ser mutation causing cmt c is a mild form of cmt, as patients walked on time, had less weakness than those with charcot-marie-tooth disease type a (cmt a), had a charcot marie tooth neuropathy score (cmtns) indicative of mild disease, and had faster ulnar and median motor nerve conduction velocities compared to those with cmt a. discussion: the g s mutation in litaf seems to be clinically indistinguishable from a mild presentation of cmt a. jha mk , russell k , lee y , rothstein jd , morrison bm . johns hopkins university, baltimore, md, usa. peripheral nerves are highly dependent on metabolic energy to maintain both basic cellular functions such as axon transport, na+/k+ ion gradients, and myelination, as well as to support regeneration following injury. though glucose certainly provides some metabolic support, our recent studies have shown that monocarboxylates, such as lactate, pyruvate, and ketone bodies, also contribute to recovery from peripheral nerve injury. monocarboxylate transporters, particularly mct , are the predominate transporters for monocarboxylates in the peripheral nerve. in a recent publication, we found that mct heterozygous null mice, which express % less mct in all cells, have slowed nerve regeneration and reduced myelination following sciatic nerve crush. this study was limited by the global reduction of mct , which is widely expressed in schwann cells (sc), dorsal root ganglia (drg) neurons, endothelial cells, macrophages, and perineurial cells within the regenerating peripheral nerve. to better understand the mechanism by which mct contributes to normal nerve function and nerve regeneration, we produced and validated conditional mct null (mct loxp) mice that allow selective deletion of mct from scs, drg neurons, endothelial cells, or macrophages through mating to cell-specific cre lines. we are currently quantifying peripheral nerve development, aging, and regeneration in each of these mouse lines. following sc-, but not drg-, specific mct knockdown, sensory peripheral nerves develop demyelination by months of age, manifest by reduced myelin thickness, increased g-ratio, and reduced conduction velocity. studies are ongoing in cultured scs to determine the mechanism for demyelination. neither sc nor drg knockdown of mct impairs nerve regeneration following sciatic nerve crush. these results suggest that sc-specific mct is critical for maintaining myelin in sensory, but not motor, peripheral nerves as they age. they also suggest that mct expression in peripheral nerve cell types, other than sc and drg, is important for nerve regeneration. ongoing studies are determining the contribution of mct in other peripheral nerve cell types, particularly endothelial cells and macrophages, to normal development and regeneration following injury. our results will clarify the role of lactate and its transporter, mct , in peripheral nerve function, potentially suggesting novel targets for demyelinating neuropathies or nerve injuries. small fiber neuropathy (sfn) is a condition in which the smallest nerve fibers are affected, characterized by neuropathic pain and autonomic dysfunction. according to international criteria, sfn diagnosis is based on clinical symptoms in combination with abnormal temperature threshold testing (ttt) and/or reduced intraepidermal nerve fiber density (ienfd) in skin biopsy. skin biopsy is moderately sensitive, invasive and the process is time consuming and expensive. ttt is a widely available diagnostic tool, but lacks specificity. previous studies introduced stimulated skin wrinkling (ssw) as an objective, non-invasive diagnostic tool to detect sympathetic nerve dysfunction in sfn by means of a categorical assessment. however, our unpublished data has shown that inter-observer reliability of categorically assessed ssw is quite low. in this current study we will use a new digital method for ssw quantification: the digit wrinkle scan© (dws©). the primary study objective is to define normative values for dws© expressed as total wrinkle length per fingertip surface (mm/mm ). subsequently we investigate the applicability of dws© in patients with definite sfn, based on abnormal iefnd and/or ttt, determining the dws© sensitivity and specificity, as well as its validity. for this cross-sectional study, we will include healthy participants and patients diagnosed with sfn. eligibility is based on meeting the inclusion and exclusion criteria and providing written informed consent. skin wrinkling is induced by emla (eutectic mixture of local anesthetics) cream© application and captured by taking pictures. the primary outcome measure is total length of wrinkles per mm as shown on the photographs, which will be calculated by a new software program. patients are stratified according to age and gender. based on the results of healthy participants, normative values will be defined. inter-and intra-observer reliability will be determined. in the sfn group, additional correlation analysis will be conducted to determine the correlation between dws© and different outcome measures (sfn-symptom inventory questionnaire, visual analogue pain scale, neuropathic pain scale, sfn-rasch-built overall disability scale, ienfd and ttt). we expect to provide digitally quantified ssw (dssw©) normative values that can be used in clinical practice in the diagnostic workup for sfn. cmt , characterized by axonal degeneration, is an inherited motor and sensory neuropathy accounting for about % of total cmt patients. the cmt subtype shows on its own, a vast genetic heterogeneity with more than mutations in known genes rendering the identification of relevant drug targets and therapies very challenging. so far, only hdac inhibitors have shown promising results in mouse models for hspb mediated axonal cmt (cmt f), albeit such a single gene approach may have a limited relevance at clinical levels owing to the limited number of patients per genotype. in this study, we investigated common causative molecular players of cmt associated axonal degeneration. for this, an itraq based proteome analysis was performed on five patient's derived lymphoblasts bearing different cmt causal mutations alongwith respective age and gender matched unaffected controls. software-assisted interpretations of the obtained data led us to identify two proteins which were significantly downregulated in cmt patients compared to controls. these two proteins were then validated using western blotting and qpcr on patient derived lymphoblasts and fibroblasts. our results prompted us to unveil whether these two proteins can be used as potential biomarkers for identifying cmt patients. therefore, through a europe-wide collaboration, we constructed a cohort of cmt patients and healthy controls. these two proteins exhibited significant downregulation in this cohort suggesting a potential new role of these proteins as cmt biomarkers. remarkably, we were also able to validate the significant decrease in ineurons (neurons differentiated from patient derived ipscs) strengthening the importance of our finding and also suggesting the relevance of these proteins in the pathogensis of axonal cmt. this will be the first study involving multiple cmt causal genes at once, thereby holding the potential to offer new drug targets and potential biomarkers with wider application both clinically and pharmaceutically. mesenchymal stem cells (mscs) represents a valuable source of stem cell therapy, can differentiate into various cell types. we investigated of the neuromuscular potential of human tonsil-derived mscs (t-mscs) for neuromuscular regeneration in trembler-j mice that is considered to be a model for charcot-marie-tooth disease type a (cmt a diseases), which is involving hereditary motor and sensory peripheral neuropathies. the t-mscs differentiated toward skeletal myocytes, as evidenced by increased expression of skeletal muscle-related markers (including troponin i type , and myogenin) and the formation of myotubes in vitro. in situ transplantation of t-msc-derived myocytes (t-myocytes) into gastrocnemius in trembler-j mice, a mouse model of cmt a, enhanced motor function, as identified with recovery by a compound muscle action potential (cmap) amplitude. and the regenerated shape of the sciatic nerve and skeletal muscle by immunochemistry, without the formation of teratomas. furthermore, the expression levels of nerve growth factor (ngf) and glial cell line-derived neurotrophic factor (gdnf) were significantly increased in t-myocyte compared with t-mscs in vitro. these results indicate that the transplantation of t-myocyte can be a therapeutic option of cell therapy for the neuromuscular regeneration in hereditary peripheral neuropathy, comprising cmt a disease. kagiava a , karaiskos c , richter j , tryfonos c , lapathitis g , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory, the cyprus institute of neurology and genetics, nicosia, cyprus; department of molecular virology, the cyprus institute of neurology and genetics, nicosia, cyprus; neurology clinics, cyprus school of molecular medicine, the cyprus institute of neurology and genetics, nicosia, cyprus. x-linked charcot-marie-tooth disease (cmt x) is a common form of inherited demyelinating peripheral neuropathy resulting from mutations affecting the gap junction protein connexin (cx ). using a cx knockout (ko) mouse model of the disease, we have shown that targeted expression of virally delivered cx results in morphological and functional improvement. since patients with cmt x express mutant forms of cx in schwann cells, that could potentially interact with virally delivered wild type (wt) cx through dominant-negative effects, we also treated mutant mice expressing the t i, r w and n d mutations associated with cmt x on a cx ko background. all three mutants were localized in the perinuclear compartment of myelinating schwann cells consistent with retention in the er (t i) or golgi (r w, n d) with loss of physiological expression in non-compact myelin areas. following intrathecal delivery of the human gjb gene we could detect the virally delivered wt cx correctly localized in the non-compact myelin areas only in t i/cx ko mutant mice, but not in the other two mutants, suggesting dominant effects of the r w and n d mutant but not of the t i mutant. gjb treated t i/cx ko mice showed improved motor performance, along with lower ratios of abnormally myelinated fibers and reduced numbers of inflammatory cells in all tissues examined compared to mock-treated animals. in contrast, gjb treated r w and n d mutant mice showed only slight but not statistically significant improvement. this study provides additional proof of principle for a clinically translatable gene therapy to treat cmt x even in the presence of endogenously expressed cx mutants, since at least one er-retained cx mutant did not interfere with the expression of virally delivered cx allowing a therapeutic benefit similar to cx ko mice. however, golgi-retained mutants may interfere with virally delivered wt cx and other approaches besides gene addition may be needed for effective treatment. funding: muscular dystrophy association (grant mda to kak). kaida k , kadoya m , koike h , iijima m , takazaki h , ogata h , moriguchi k , shimizu j , nagata e , takizawa s , chiba a , yamasaki r , kira j-i , sobue g , ikewaki k . national defense medical college, tokorozawa, japan; nagoya university graduate school of medicine, nagoya, japan; kyushu university, fukuoka, japan; university of tokyo, tokyo, japan; tokai university school of medicine, isehara, japan; kyorin university, tokyo, japan. antibodies to a glial protein, neurofascin (nf) have recently been identified in approximately % of patients with chronic inflammatory demyelinating polyneuropathy (cidp), which are igg -predominant. igg anti-nf -associated cidp may be a distinct subtype from typical cidp in terms of clinical features and response to immunotherapy. however, a diagnostic criterion of anti-nf -associated cidp has not established yet. to develop optimal criteria and design the best treatment plan for the anti-nf -associated cidp, procedures for determining anti-nf antibodies should be simplified, prevalent, and reproducible, as well as being accurate. cell-based assay (cba) has hitherto been utilized for determining antibodies to nf in sera from patients, results of which have been confirmed by immunohistochemistry (ihc) using teased nerve fibers from rodents. these methods are the most reliable techniques, while not necessarily easy-to-use and easy to maintain in most laboratories. in the present study, we aimed to validate the diagnostic utility of a conventional enzyme-linked immunosorbent assay (elisa) for determination of anti-nf antibodies and the igg subclass. sera from patients with efns/pns criteria-met cidp were examined with elisa using human recombinant nf . to verify elisa results, ihc on rat sciatic nerves, western blot (wb) and cba using nf -transfected and naive hek cells were conducted. the human nf -based elisa clearly distinguished between anti-nf antibody-positive and -negative sera. fifteen cidp patients ( %) were igg anti-nf antibody-positive, which were confirmed by wb, ihc and cba studies. none of disease controls or healthy subjects had positive results. twenty-five sera randomly selected from anti-nf -negative cidp sera were also negative on cba. the anti-nf activities on elisa were significantly positively-correlated with those on cba (p < . ). analyses of clinical and laboratory findings showed that anti-nf -associated cidp was characterized by younger onset, distal dominant phenotype, tremor, sensory ataxia, higher protein levels in cerebrospinal fluid, and poor response to ivig, which were consistent with those in previous studies. this elisa combined with determination of the igg subclass is a simple and reliable method for initial screening for anti-nf antibodies. the genetic abnormality responsible for x-linked charcot-marie-toothy neuropathy subtype cmtx was recently identified by whole genome sequencing to be a kb insertion into chromosome xq . . the clinical profile of cmtx in childhood is not well described. we reviewed the clinical characteristics, neurophysiological profile and cmt pediatric scale (cmtpeds) assessments of children with genetically confirmed cmtx . cmtx was characterized by early onset, and early and progressive hand weakness. most affected children were symptomatic within the first two years of life. the most common presentation was with equinovarus foot deformity in the first year of life. cmtpeds analysis in these children revealed that cmtx progressed more rapidly ( . ± . points/ year, n= ) than cmt a and cmtx . grip strength in the second decade of life in most affected males was two standard deviations below age-and sex-matched normative reference values. the most severely affected individual was wheelchair bound at years of age and two individuals had no movement in the small muscles of the hand in the second decade of life. there was only a single symptomatic female identified and she had mild signs. nerve conduction studies showed a demyelinating sensorimotor neuropathy with motor conduction velocity in eight children while one child had a length-dependent sensorimotor axonal neuropathy. understanding the unique phenotype of cmtx is essential for directing genetic testing, as the cmtx insertion will not be detected on the snp microarrays, multi-gene panels or whole-exome sequencing currently used for the diagnosis of cmt. the early onset of disease coupled with rapid progression means that many children with cmtx will have severe disability within the first two decades of life and hence early diagnosis is needed for early commencement of rehabilitation. kapoor m , catania s , sarri-gonzales s , lunn mp , manji h , reilly mm , carr as . centre for neuromuscular diseases, national hospital for neurology and neurosurgery, london, uk; department of neurophysiology, national hospital for neurology and neurosurgery, london, uk. the conventional dosing of ivig in cidp and mmn is based on treatment trials that used bolus and maintenance dosing of ivig between - . g/kg. there are rare published articles reporting the efficacy of higher maintenance ivig doses. we present three cases of inflammatory neuropathies, who are currently stabilized on ivig doses of mg- mg/kg of ivig per month, refractory to standard dose ivig and other immunosuppressants. the first case is a year-old-lady with cidp who presented with episodes of ascending sensory disturbance, weakness, and diplopia. she had activity related fluctuations and pre-dose deterioration on g/kg/month ivig. she then had an acute deterioration with mrc sum score dropping from to even with additional plasma exchange. her bilateral foot drop (mrc grade - ) and fluctuations persisted with an increase of ivig to . g/kg/month. she is now clinically stable (ankle dorsiflexion mrc grade - , mrc sum score ) on mycophenolate and g ivig weekly ( . g/kg/month). case is a -year-old male fitness instructor with mmn and sjogren's syndrome. he presented with recurrent proximal and distal weakness that responded to g/kg of ivig and deteriorated with iv methylprednisolone. he had peri-dose fluctuations, intermitted proximal weakness, and persistent foot drop (ankle dorsiflexion mrc grade - ) at . mg/kg/month, worsening to mrc grade - on . g/kg/month and fluctuating between mrc grade - on . g/kg/month. an increase of ivig to g weekly ( . g/kg/month), has resulted in mmn rods scores of / , improved distal power and return to full capacity at work. case is a -year-old man with predominantly upper limb cidp. he received g/kg ivig without any benefit, had no response to doses of plasma exchange, doses of cyclophosphamide or dose of rituximab between and . since , he has received g/kg/month ivig with improvement of mrc sum score from to . these cases highlight that some patients require a much higher than conventionally prescribed dose of ivig, and that these doses are tolerated over years without serious adverse events. idiopathic rapid eye movement sleep behaviour disorder (irbd) has been identified as a precursor of alpha-synucleinopathies, such as parkinson's disease, dementia with lewy bodies, multiple system atrophy. several studies linked changes in cutaneous innervation with central nervous system pathology in neurodegenerative disorders. recently small fiber neuropathy and alpha-synuclein deposition in the skin found to be a potential biomarker in parkinson's disease. we evaluated the epidermal innervation of irbd patients and age and sex-matched controls from skin punch biopsies from the distal leg using pgp . immunohistochemistry. furthermore, a battery of clinical examinations were performed on patients and controls alike, including structured interviews, clinical motor and non-motor questionnaires and rating scales (e.g. unified parkinson's disease rating scale [updrs], non-motor symptoms questionnaire [nms-quest] and beck depression inventory, epworth sleepiness scale, evaluation of cognitive and olfactory functioning as well as blood samples. irbd patients, compared to controls, showed a significant reduction in intraepidermal nerve fiber density (p = . ), whereas the axon swelling ratio, did not differ between groups. patients with irbd reported non-motor symptoms more frequently than controls (updrs i, nms-quest). olfaction and daytime sleepiness differed between both groups, whereas there were no differences regarding cognition. these in vivo findings demonstrate small fiber neuropathy in irbd patients that are associated with non-motor symptoms indicating that peripheral abnormalities may occur early in irbd. they warrant larger scale longitudinal studies in order to investigate their prognostic value. katz j , lewis r , spatafora d . california pacific med center, san francisco, usa; cedars-sinai medical center, los angeles, usa; neuropathy action foundation (naf), santa ana, usa. multifocal motor neuropathy (mmn) is a rare condition that affects . in every , individuals worldwide and is associated with motor dysfunction and moderate to severe disability. the neuropathy action foundation conducted a global survey to determine the impact of mmn on patient quality of life (qol) and gaps in patient/provider educational needs. the first global mmn qol survey was an item internet questionnaire available between january and july , . the survey focused on three primary areas: timely and accurate diagnosis, the efficacy of treatment, and the impact of the disease on patients qol. the survey was completed by patients from countries. the majority of respondents said they were diagnosed between the ages of and years ( . %), more than % reported that it took more than one year to be diagnosed and more than % reported that it took - years or longer to be accurately diagnosed. with respect to treatment options : . % reported receiving intravenous immune globulin and . % reported receiving subcutaneous immune globulin therapy. other therapies being used to treat mmn were gabapentin ( . %), and pregabalin ( %). almost half ( . %) said that mmn often impacts their overall schedule. half of the participants reported that mmn often or always interferes with their employment; % had difficulty typing on a computer or using a telephone, . % had trouble concentrating, and . % said they had to work really hard to pay attention or else they would make a mistake. this is the first assessment of mmn from a patient's perspective. the survey highlighted critical issues relating to the diagnosis, management, and impact on the qol of individuals with mmn. the data also identified gaps and insights in provider education relating to proper diagnoses and management of the condition from a patient's perspective. katz j , levine t , dimachke m , barohn r . forbes norris center, san francisco, ca, usa; phoenix neurological institute, phoenix, az, usa; kansas university medical center, kansas city, ks, usa. in the united states, cidp cases are submitted to insurance companies to determine whether ivig therapy is appropriate. this is done using specified diagnostic criteria, which reduce diagnosis to a boolean analysis, where a disease can only be present or absent. this leaves no room for uncertainty, even when it truly exists. boolean criteria are useful for clinical trials, but fall short where real decisions are made under uncertainty and based on perceived cost/benefit analysis. this project attempts to elucidate root causes of uncertainty and to find solutions to this dilemma. we asked cidp experts to select a single diagnosis in cases where ivig was approved using the submitted case records. while there was agreement on many cases, in the five most "uncertain" cases no more than reviewers agreed on a single condition, who chose up to four separate entities. among these, at least three reviewers diagnosed an immune neuropathy in all five. the root cause of the disagreement, to a large degree was unclear documentation (aunts) which consisted of pasted, missing, and disorganized data. reviewers missed useful information, admitting it was too difficult to fully parse records. to resolve uncertainty, reviewers admitted to discounting certain reported datum to help fit the entity they suspected, such as reported therapeutic responses or certain electrodiagnostic/exam findings. other disagreement, however, reflected the complexity of neuropathy diagnosis, such as knowing if improvement was due to natural history or treatment, unawareness of rare presentations, or analyzing a true uncle (complex case). reviewers used bayesian (select most likely diagnoses from a list) and fuzzy logic (compare best fits to base cases). when the "best" diagnosis did not fit the base case, they had to re-interpret the data. improving review procedures requires eliminating aunts by collecting all key information and simplifying how records are presented. it also needs more advanced data methods to analyze common and rare borderline presentations (uncles like mama v pma, cidp v cspn, etc..), developing diagnostic algorithms that address real uncertainty, educating prescribers and patients on process, and creating systems that measure outcomes longitudinally after induction or tapering of therapy. keisuke y , miyuki m , motoi k , susumu k . department of neurology, faculty of medicine, kinki university, osaka, japan. anti-ganglioside antibodies are closely associated with clinical phenotype and specific symptoms in acute immune-mediated neuropathies. igg anti-gq b antibodies are specifically associated with miller fisher syndrome (mfs), bickerstaff brainstem encephalitis (bbe) and guillain-barré syndrome (gbs) with opthalmoplegia (gbs-op). in addition, ganglioside complexes (gscs) containing gq b also can be targets in such diseases, and might be associated with the clinical features. however, factors regulating clinical phenotype in those gq b-associated antibodies-positive diseases have not yet been known. for investigating the differences of antibody reactivities among those diseases, we examined, using combinatorial glycoarray, igg antibodies to ten individual glycolipids [gm , gm , gm , gd a, gd b, gq b, galactocerebroside (gal-c), lactosylceramide (laccer), ga , sulfatide] and glycolipid complexes consisting of two of the glycolipids listed above in sera from patients with gbs-op who were positive for anti-gq b antibody by elisa (gbs-op-gq b), patients with mfs with the clinical triad (opthalmoplegia, ataxia, and areflexia), and patients with bbe. by combinatorial glycoarray, overall sensitivity of antibodies to gq b and gscs containing gq b was . % ( / ) in gbs-op-gq b, . % ( / ) in mfs, and . % ( / ) in bbe, respectively. there were no significant differences in antibody reactivities between mfs and bbe. it is notable that antibodies to gscs containing gd b were more frequently found in gbs-op-gq b patients than in mfs or bbe patients (e.g., gd b/sulfatide: p= . and p< . , respectively). presence of the antibody reactivities to gscs containing gd b may possibly be related with clinical features of gbs-op-gq b, including frequent need of artificial ventilation. kennedy r , , , carroll k , , paterson k , ryan mm , , , mcginley jl , . royal children's hospital, parkville, australia; university of melbourne, parkville, australia; murdoch childrens research institute, parkville, australia. problems with walking and footwear fit are often reported by children and adolescents with charcot-marie-tooth disease (cmt). a cross-sectional, case controlled study of gait was conducted in children with cmt and typically developing (td) children. gait was assessed barefoot and in two types of the participants' own typical footwear; optimal (e.g. athletic shoes) and suboptimal (e.g. slip-on footwear). the aims were to determine differences in spatio-temporal (s-t) gait variables between children with cmt and td children; and to investigate the effect of footwear choices. twenty-nine independently ambulant children aged - years with confirmed genetic or clinical diagnoses of cmt, and age and gender matched td children participated (mean age . years; males). exclusion criteria included developmental disorders, other neuromuscular conditions or musculo-skeletal diseases that could affect gait, and lower limb injury or surgery in the preceding months. assessment included s-t gait patterns, footwear characteristics, metre run, and cmtpeds. gait was assessed at self-selected speed with an electronic walkway (gaitrite™), with trials for each condition. the primary gait variable assessed was speed; other variables included step length, step time, cadence, base of support width and step-to-step gait variability. across all footwear conditions children with cmt walked more slowly ( regeneration of cutaneous unmyelinated axons is known to be slowed in dm-patients and after -months, the density of intraepidermal nerve fibers (ienf) does not return to baseline levels after chemical or mechanical axotomy. however, the long-term outcome of regeneration in dm or control subjects is not known. additionally, it is not clear if the regeneration of sensory distal axons is length-dependent. here we measured the rate of axonal regeneration -months after chemical denervation using a capsaicin model in dm patients (n= / dm /dm ) without neuropathy, and controls. dm skin punches were performed at distal thigh at baseline, -hours post-capsaicin, and at , , and days. blood glucose and hgba c were measured at baseline, , and days. healthy controls had skin punches at both distal leg and proximal thigh at baseline, after capsaicin chemical axotomy, and days , , and . regeneration rate was significantly higher at the thigh in healthy controls ( . fibers/mm/day ( % ci: . - . fibers/mm/day) compared to dm (p= . ), but no difference between dm ( . fibers/mm/day % ci: . - . fibers/mm/day) or dm ( . fibers/mm/day % ci: . - . fibers/mm/day) (p= . ). comparing regeneration rate at different time intervals, showed that regeneration was significantly slowed between day and dm patients, while it continued with the same rate in controls. blood glucose or hga c had no effect on regeneration rate. ienfd returned to baseline in controls by -months ( % of baseline) while it is did not in dm subjects, %/ % (dm /dm ) of ienfd baseline, (p= . dm vs. controls). there was no difference in regeneration rate ienfd %-baseline by -months at distal leg and proximal thigh in controls (p= . ). these results suggest that the rate and outcome of regeneration is independent of the length of the axon. additionally diabetic patient have incomplete nerve regeneration after months regardless of diabetes type or the level of glycemic control. regeneration of axons slowed down over time in patients with dm and reached a plateau after days. kiessling p , lledo-garcia r , watanabe s , langdon g , tran d , bari m , christodoulou l , price g , smith b , byrnes w , brock m , jolles s . ucb pharma, monheim, germany; ucb pharma, slough, uk; ucb pharma, braine-l' alleud, belgium; ptx solutions ltd, london, uk; ucb pharma, raleigh, durham, usa; university hospital of wales, cardiff, uk. ucb is a humanised high-affinity monoclonal igg antibody developed to bind human neonatal fc receptor (fcrn), selectively inhibiting igg salvage and recycling. conditions such as myasthenia gravis (mg) are characterised by pathogenic igg autoantibodies; inhibition of fcrn may provide a suitable therapeutic approach. this phase i, double-blind, dose-escalating, first-in-human study (nct ) evaluated the safety and pharmacology of ucb . forty-nine healthy adults (mean age years, range - ) were randomised and received a single dose of intravenous (iv) or subcutaneous (sc) placebo (n= and n= , respectively), or a single dose of iv or sc ucb ( , or mg/kg; n= per dose, per administration). subjects were followed up until day . one placebo iv subject did not complete the study. twenty-seven of subjects ( %) receiving ucb , and / ( %) receiving placebo, reported ≥ treatment-emergent adverse event (teae) of mild/moderate intensity. severe teaes occurred in four subjects, all in the ucb mg/kg iv group (headache [n= ], back pain [n= ]). no serious aes occurred. incidence of infections was similar with ucb and placebo. the most frequently reported infection was nasopharyngitis. treatment-related teaes were reported by % of subjects receiving ucb and % receiving placebo: the most common in the ucb -treated groups were headache ( / ; %) and vomiting ( / ; %); these occurred more frequently with the iv than sc route. non-linear increases in ucb plasma concentration-time profile with increasing dose were observed with ucb . serum igg was reduced in a dose-depended manner with ucb iv and sc: decreases from baseline to day with ucb iv were . %, . % and . % for , and mg/kg doses, respectively, and . %, . % and . %, with ucb sc doses, respectively. these data indicated that the fcrn inhibitor ucb effectively reduced serum igg, with sc administration generally better tolerated than iv. further to these observations, the efficacy, safety and tolerability of ucb sc for chronic-intermittent treatment of moderate-to-severe mg are being evaluated in an ongoing phase ii, multi-centre, randomised, double-blind, placebo-controlled study (eudract - - middle east respiratory syndrome (mers) has a high mortality rate and pandemic potential. however, very little information has become available on this syndrome since it first erupted in . this study aimed to evaluate the frequency of neurological complications and their clinical presentations in mers. we reviewed the medical records of all patients who were diagnosed with laboratory-confirmed mers coronavirus (cov) infections and subsequently admitted to a single reference center for mers treatment during the outbreak in korea. in total, patients ( . %) reported neurological symptoms during or after mers-cov infection. the potential diagnoses in these cases included bickerstaff's encephalitis overlapping with guillain-barré syndrome, critical illness polyneuropathy or other toxic or infectious neuropathies. neurological complications did not appear concomitantly with respiratory symptoms, but were instead delayed by - weeks. neuromuscular complications were not rare in mers-cov-infected patients, and they may have previously been underdiagnosed. understanding neurological manifestations is important in an infectious disease like mers, because evaluation is frequently limited during treatment, but it can interfere with prognosis and sometimes require modification of treatment. kim hj , hyun jk , kim tu . dankook university, cheonan, south korea. the diagnosis of carpal tunnel syndrome (cts) is based on clinical symptoms, physical examinations and supported by nerve conduction study (ncs). ultrasonographic examinations can be performed to assess peripheral nerves with less discomfort and the surrounding anatomic structures. while the usefulness of ultrasonography (usg) in the cts has been reported, no study to date has compared the diagnostic utility of various usg findings for cts. we investigated the correlation of various usg findings to the clinical symptoms/signs and ncs findings in patients with cts. twenty-eight hands ( patients) with cts based on electrodiagnostic criteria and clinical symptoms such as tingling sensation or pain in the first to third fingers, burning sensation, paresthesia and weakness of hand grip power. all subjects were examined with usg. cross-sectional area (csa) and flattening ratio (fr) of the median nerve was calculated at level of radio-ulnar joint, pisiform and hamate. swelling ratio of the median nerve and palmar displacement of the flexor retinaculum was also calculated. clinical assessment was conducted using the boston carpal tunnel questionnaire (bctq) scale and historical-objective (hi-ob) scale. the analysis of correlation between usg findings and clinical symptom scales/ncs findings was performed using correlation analysis. the csa of the median nerve at level of radio-ulnar joint was significant correlated with bctq scale, hi-ob scale, distal motor latency, and conduction velocity (cv). the csa of the median nerve at level of pisiform was significantly correlated with hi-ob scale, distal motor latency, and cv. the fr of the median nerve at level of radio-ulnar joint was significantly correlated with bctq scale, hi-ob scale, distal motor latency, and cv. the swelling ratio of the median nerve was also significantly correlated with distal motor latency and cv. in patients with cts, csa of the median nerve at level of radio-ulnar joint was most closely related to ncs findings and clinical symptoms. so, csa of the median nerve at radio-ulnar joint might be a complementary tool for the diagnosis of cts. kitaoji t , tsuji y , ashida s , yamada t , ishii r , tanaka a , mizuno t . department of neurology, graduate school of medical science, kyoto prefectural university of medicine, kyoto, japan. the case was a -year-old woman. first, she noticed paresthesia in the right plantar eight months before admission and in the left plantar four months before admission. three months before admission, she developed muscle weakness in her feet. the muscle weakness and paresthesia extended to the lower legs in a few months. twenty days before admission, she experience difficulty in walking. on admission, the muscle weakness was observed in the legs, especially in the right tibial anterior muscle (ta). there was severe sensory disturbance and loss of deep tendon reflex in the legs. she had trouble walking due to the weakness and sensory aphasia. in nerve conduction study (ncs), conduction block was observed between the ankle and popliteal in both tibial nerves. the blood level of angiotensin converting enzyme (ace) was elevated. cerebrospinal fluid analysis was normal. there was no enhancement in the lumbar nerve roots shown on mri. gallium- scintigraphy showed hot spots on bilateral hilar lymph nodes and mediastinal nodes and biopsy of mediastinal nodes showed non-caseating epithelioid granuloma. therefore, we diagnosed her with sarcoid peripheral neuropathy by sarcoidosis. by using the nerve ultrasound, partial spindle-shaped nerve enlargement was observed at the part of conduction block in the left tibial nerve. we started the treatment with methyl prednisolone ( mg, days) and oral prednisolone therapy ( mg/kg/day). after treatment, the paresthesia and muscle weakness in the legs had gradually improved. the partial enlargement in the left tibial nerve also improved on the -hospital day. in ncs, the conduction block improved, however, the compound muscle nerve potential of tibial nerve decreased because of axonal damage. this partial spindle-shaped nerve enlargement by using ultrasound has never been reported in sarcoid peripheral neuropathy before. the nerve ultrasound may be useful for evaluation of therapeutic effect of sarcoid peripheral neuropathy. peripheral nerve injuries are still debating problems in the world because of poor recovery. there is absolutely a need for new therapeutic agents to improve outcome by altering nerve regeneration. there are some studies in the literature about some therapeutic agents that used in the cases of peripheral nerve injuries. despite these studies, an agent with clinical use has not been presented yet. in this experimental study, we aimed to analyze the effects of curcumin (cur) in the cases of peripheral nerve injuries. forty rats were randomly and equally divided into four groups. the first group was control group. rats in this group were not operated. right sciatic nerve injuries were performed to the other groups. the second group was operation group with no therapeutic agent. the third group was operation and local cur applied group. the fourth group was operation and systemic cur applied group. electrophysiological evaluations were performed with electroneurography (enog) before and after the surgeries. systemic use of cur although caused improvement in the enog values but could not make a positive contribution to the nerve regeneration statistically. additionally local use of cur made negative effect to the nerve regeneration statistically. according to our statistical results we could not recommend cur as a nerve protective agent. klein cj , wu y , jentoft me , mer g , spinner rj , dyck pjb , dyck pj , mauermann ml . mayo clinic, rochester, usa. intraneural perineurioma is a hypertrophic peripheral nerve tumor having immunoreactivity to epithelial membrane-antigen, negative for s- . the origin of perineurial cells is debated to be similar to meningeal cells. ip does not metastasize, but motor deficits accumulate over time from tumor growth in nerve and plexus. after schwannomas and neurofibromas, perineuriomas are the most common nerve tumor of young adults. a chromosome q deletion has been reported in one patient. we identified ip cases from our previously published clinical cohort with available flash frozen ip tissue for dna isolation. wes with cnv analysis and cgh microarray analysis (agilent x k superprintg ) were performed on extracted dna; had available germline dna (lymphocytes and buccal tissue). we compared the exome data against online and in-house control data (∼ , ) examining variants less than . frequencies, predicted damaging or nonsynonymous. wes identified three novel, heterozygous, damaging mutations in tumor necrosis factor receptor-associated factor (traf ) in of ( %) cases; p.l p (n= ), p.h r (n= ) and p.s r (n= ). mutations were within the wd domain, p.l p, p.h r within exon and p.s r within exon , and mapped to a limited region of traf with protein structure modeling. two of cases ( . %) showed macroduplications/deletions on multiple chromosomes, including chromosome , confirmed with cgh microarray analysis and cnv results from exome data analysis. four of ( %) had no discovered mutation. age of onset or severity did not correlate with type of mutations. this study provides strong evidence that traf is a specific tumor driver of ip. mutations in traf are also linked to benign intracranial meningiomas suggesting a shared pathogenesis and close origins of perineurial and meningeal cells. study supported by: mayo foundation and the mayo center of individualized medicine. klein i , , bobylev i , , lehmann hc , . university hospital cologne, cologne, germany; center for molecular medicine cologne (cmmc), cologne, germany. peripheral neuropathy is a common side effect of paclitaxel. clinical evidence suggests that the delivery mechanism of paclitaxel formulations influence time course and severity of paclitaxel induced peripheral neuropathy. in a preclinical model we studied access, distribution and toxicity of two paclitaxel formulations (nanoparticle albumin-bound paclitaxel (nab) and solvent-based paclitaxel) in the peripheral nervous system (pns). c bl/ mice were treated with mg/kg or mg/kg of nab-paclitaxel or solvent based paclitaxel. kinetics of paclitaxel in neurons was assessed by a newly established immunostaining technique. neurotoxicity was evaluated by functional assays and nerve morphology. paclitaxel accumulated mostly in dorsal root ganglia, whereas distal nerve segments showed only low uptake of paclitaxel. treatment of mice with the two paclitaxel formulations resulted in paclitaxel uptake mostly in nf + larger fiber neurons. in ib +, and cgrp+ small fiber neurons, paclitaxel was less frequently detected. nab-paclitaxel was incorporated more rapidly compared to solvent-based paclitaxel but neurons also showed a faster clearance of nab-paclitaxel compared to solvent based paclitaxel. functional assays and nerve conduction studies indicated that nab-paclitaxel was less neurotoxic compared to solvent-based paclitaxel. this is the first study that characterizes in detail the access of nab-paclitaxel and solvent based paclitaxel into the pns. our findings have important implications to understand the pathomechanisms of paclitaxel induced neurotoxicity and to develop neuroprotective strategies by preventing access of paclitaxel to the pns. the aim of our study was to investigate the etiology of neuropathy in patients with rheumatoid arthritis (ra). subjects were neuropathy patients with ra admitted to our department. laboratory investigations, nerve conduction studies (ncs) and sural nerve biopsy were performed. mean patient age was . years (range, - years), and mean disease duration was . years (range, - years). clinical diagnosis for neuropathy was rheumatoid vasculitis (rv) in patients, rv with acute motor axonal neuropathy (rv-aman) in and chronic inflammatory demyelinating polyneuropathy with ra (ra-cidp) in . rheumatoid factor ( / ) and rheumatoid arthritis particle agglutination ( / ) was high and c tended to be lower in the rv group (rv and rv-aman). anti-ganglioside antibodies were examined in patients, with positive results in . an rv-aman case was diagnosed with motor-dominant clinical presentation and the presence of anti-galnac-gd a immunoglobulin (ig)g antibody. no other cases with rv were examined for anti-ganglioside antibodies. positive results for anti-gm and gm igg antibody were seen in one ra-cidp patient. we evaluated sural/median (s/m) ratio) for sensory nerve action potential (snap). s/m ratio was low in rv cases ( / ) and high ( / ) in immune-mediated cases, suggesting a so-called normal sural abnormal median pattern in immune-mediated neuropathies. the rv-aman case showed a moderate value in s/m ratio. nerve biopsy revealed thinly myelinated nerve fibers in ra-cidp cases compatible with demyelination, while the rv group showed the typical pathology for necrotizing vasculitis. rv cases were treated with prednisolone (psl), intravenous methylprednisolone, intravenous cyclophosphamide and increased psl dose. ra-cidp and rv-aman were treated with intravenous ig. in conclusion, neuropathy in ra can be divided into vasculitic and immune-mediated groups. ncs, and the s/m ratio of snap with some laboratory parameters in particular, may be of use in differential diagnosis and deciding treatment strategies. koh s , wong shj , loh kw , chng ysk , pawa c , ei ma , lee bjh , subramaniam t , umapathi t . national neuroscience institute, singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university singapore, singapore; tan tock seng hospital, singapore; khoo teck puat hospital, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful autonomic neuropathy that develops with abrupt improvement in glycaemic control. typically, type or dm patients on insulin or oral hypoglycaemic agents (ohga), present with painful neuropathy and autonomic dysfunction within weeks of rapid improvement in glucose control. current emphasis to achieve good glycaemic control rapidly may inadvertently increase incidence of tind, hence the impetus to understand risk of over-zealous glycaemic control. we therefore set out to study the occurrence of tind in a dm cohort of a tertiary hospital. we screened all patients who had two hba c measurements between and . during this period, approximately patients were seen per year. we found patient-encounters that showed hba c decrease of ≥ % over months or ≥ % over months. we then used a structured checklist of tind symptoms to shortlist cases. these case-encounters were scrutinised and classified as; 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or uncertain temporal relationship to decrease in hba c; unlikely tind: alternative explanation exists for symptoms. only one case was deemed 'probable tind'-a middle-aged man with newly diagnosed type dm who presented to emergency department with palpitations and worsening 'frozen feet' sensation that disturbed sleep. his hba c decreased by . % in weeks. his symptoms improved within a month with neuropathic pain medications and resolved months later. he also developed maculopathy and proliferative retinopathy. ten months later, he developed significant proteinuria. four other cases were classified as 'possible tind' while the remaining were unlikely tind. our study is limited by retrospective design and reliance on hospital records. nevertheless, our findings suggest that tind is uncommon in a general cohort of dm patients. on the other hand, the number of patients with painful neuropathy and acute dysautonomia symptoms contemporaneous with rapid decline in hba c raises the intriguing possibility that forme fruste of tind exists and one should interrogate the rate of hba c decline in dm patients with these symptoms. koike h , kadoya m , kaida k , ikeda s , kawagashira y , iijima m , kato d , ogata h , yamasaki r , matsukawa n , kira ji , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; national defense medical college, tokorozawa, japan; nagoya city university graduate school of medical sciences, nagoya, japan; kyushu university, fukuoka, japan. we investigated the morphological features of chronic inflammatory demyelinating polyneuropathy (cidp) with autoantibodies directed against paranodal junctional molecules, particularly focusing on the fine structures of the paranodes. sural nerve biopsy specimens obtained from cidp patients with anti-neurofascin antibodies and patient with anti-contactin antibodies were assessed. these antibodies were examined using sera obtained from patients with cidp who fulfilled the criteria of the european federation of neurological societies/peripheral nerve society. thirteen cidp patients without these antibodies were also examined to compare pathological findings. characteristic light and electron microscopy findings in transverse sections from patients with anti-neurofascin and anti-contactin antibodies indicated a slight reduction in myelinated fiber density, with scattered myelin ovoids, and the absence of macrophage-mediated demyelination or onion bulbs. teased-fiber preparations revealed that segmental demyelination tended to be found in patients with relatively high frequencies of axonal degeneration and was tandemly found at consecutive nodes of ranvier in a single fiber. assessment of longitudinal sections by electron microscopy revealed that detachment of terminal myelin loops from the axolemma was frequently found at the paranode in both anti-neurofascin and anti-contactin antibody-positive cidp patients compared with antibody-negative cidp patients. patients with anti-neurofascin antibodies showed a positive correlation between the frequencies of axo-glial detachment at the paranode and axonal degeneration, as assessed by teased-fiber preparations (p < . ). in conclusion, paranodal dissection without classical macrophage-mediated demyelination is the characteristic feature of patients with cidp with autoantibodies to paranodal axo-glial junctional molecules. koike h , ikeda s , takahashi m , kawagashira y , iijima m , misumi y , ando y , ikeda si , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; kumamoto university, kumamoto, japan; shinshu university hospital, matsumoto, japan. peripheral neuropathy is the cardinal feature of familial amyloid polyneuropathy (fap), but its mechanism has not been fully elucidated. we used electron microscopy to examine schwann cells and endoneurial microvessels. sural nerve biopsy specimens from fap patients with transthyretin val met mutation were assessed. patients were consisted of early onset cases from endemic foci and late onset cases from non-endemic areas. loss of nerve fibers with or without neighboring amyloid deposition was a common feature. the amount of amyloid deposition was greater relative to the extent of nerve fiber loss in early onset cases than in late onset cases. the atrophy of schwann cells, particularly nonmyelinating cells, that were apposed to amyloid fibrils was more conspicuous in early onset cases than in late onset cases. the numbers of endothelial cell nuclei, endothelial cell profiles, and occluded microvessels were significantly increased in the fap patients compared with patients with nutritional/alcoholic neuropathies (p < . , . , and . , respectively). findings suggestive of the disruption of blood-nerve barriers, such as the loss of tight junctions and the fenestration of endothelial cells, were also more frequently found in the fap patients (p < . ), irrespective of the presence or absence of amyloid deposition. in conclusion, these findings suggest that direct insult of amyloid fibrils causes schwann cell damage resulting in the predominant loss of small-fiber axons characteristic of early onset cases. in addition, vasculopathy may also participate in the pathogenesis of neuropathy, particularly in late onset cases. kolb n , smith ag , singleton jr , beck s , howard d , dittus k , karafiath s , mooney k . university of vermont, burlington, vt, usa; university of utah health, slc, ut, usa. chemotherapy induced peripheral neuropathy (cipn) is a major cause of morbidity due to numbness, pain, and gait instability. this prospective study compares the current standard care for cipn symptom management to a new care delivery model which utilizes an automated symptom tracking program paired with a nurse practitioner led intervention triggered by moderate to severe symptoms. all participants beginning taxane or platin based chemotherapy called a telephone based automated symptom tracking program daily (symptom care at home -sch) to report chemotherapy related numbness and tingling. sch tracked the presence and severity of neuropathic symptoms and their interference with activities of daily living (adls) on a - scale. participants were randomized to two groups. the usual care (uc) group was advised to call their oncology provider for recommendations on symptom management. in the nurse practitioner (np) group, when symptom severity was ≥ participants received automated self care strategies and a call from a nurse practitioner to provide treatment recommendations based on consensus guidelines. patients participated in the study. mean duration of follow up was . ± . days with . ± . calls. the np group had fewer days with any neuropathic symptom ( . % ± . vs. . % ± . , p= . ), with moderate to severe neuropathic symptoms ( . % ± . vs. . % ± . , p< . ) or days of distress from neuropathic symptoms ( . % ± . vs. . % ± . , p= . ). on days with moderate to severe symptoms participants also reported burning ( . ± . %), weakness ( . ± . %), balance problems, ( . ± . %), and tripping ( . ± . %). there was no significant difference between groups in the interference in adls (np . ± . vs. uc . ± . , p= . ). overall the automated telephone system effectively identified neuropathy symptoms and their severity. compared to usual care in which patients must independently reach out to their care team for symptom management, sch is effective in decreasing symptom prevalence, severity and distress. kouton l , kremer l , tard c , morales r , kuntzer t , attarian s , boucraut j , delmont e . referral centre for als and neuromuscular diseases, marseille, france; neurology department, strasbourg, france; neurology department, lille, france; neurology department, montpellier, france; neurology department, lausanne, switzerland; immunology laboratory, marseille, france. antibodies against proteins of the node of ranvier have been recently described in severe chronic inflammatory demyelinating polyradiculoneuropathies (cidp). they target paranodal proteins, namely contactin (cntn ) and neurofascin (nf ). cell-based assay and elisa are available in research, but no gold standard technic is admitted for the detection in routine of these antibodies. our objective was to evaluate if flow cytometry analysis is an efficient technic to detect antibodies against ctn and nf in a large cohort of cidp patients. flow cytometry analysis were performed on a bd facs-diva. human embryonic kidney (hek) cells were transfected either with nf or cntn . sera were diluted / . antibodies anti-cntn or nf were revealed using fitc conjugated anti human igg antibodies. delta mfi (mean fluorescence intensity) was calculated as mfi of transfected cells less mfi of non-transfected cells. measures were normalized using positive controls and negative controls from healthy blood donors. sera of cidp patients from different french neuromuscular referral centres were analysed with flow cytometry. respective delta mfi were (standard deviation ) and (standard deviation ) for antibodies against nf and cntn in cidp antibodies negative patients. antibodies against nf were found in patients (respective mfi , , ) and against cntn in two other patients (respective mfi and ). isotype of these antibodies was igg in patients and igg and igg in the remaining patient. all the patient had severe cidp. four patients had poor response to intravenous immunoglobulins (ivig) and have been treated with immunosuppressive drugs. as usually reported, the patient with anti-nf antibodies had postural tremor. flow cytometry seems effective to detect antibodies against nf and cntn . compared to other assays, benefits of flow cytometry are: to analyse a large number of sera in the same time and to give objective numerical results expressed in mfi that can be compared to the results of other samples. further studies are needed to confirm that flow cytometry can be the best test to assess antibodies against cntn and nf in routine. the mechanism by which intravenous immunoglobulins (ivig) improves peripheral nerve function in multifocal motor neuropathy (mmn) is unknown. the rapid clinical improvement following ivig could be related to blocking complement deposition on gm epitopes, change in ion-channel properties of affected motor axons, or both. the present study investigated median nerve motor excitability parameters at ∘ c just before ivig administration as well as at the peak of clinical improvement in patients with mmn. the investigated nerves were characterized either by conduction block (n= ), demyelinative slowing without block (n= ), or motor axon loss (n= ). the results of motor excitability testing in mmn showed no difference between pre-and post ivig recordings. clinical assessment of apb muscle showed increase in mrc score in patients and decrease in patient after ivig administration. in patients mrc score of the apb remained the same. those findings indicate that clinical changes following ivig administration are not related to excitability parameters of affected motor axons in mmn. where impulse conduction is blocked or markedly slowed in motor axons but is normal in sensory axons. sensory symptoms or signs are usually absent but have occasionally been reported in skin areas innervated by nerves with prominent motor axon loss. although the mechanism of selective motor involvement in mmn is unresolved, it may be related to differences in antigenic properties between motor and sensory axons or differences in biophysical properties. the objective of the present study was to compare ion-channel activity in both motor and sensory axons of nerves affected by mmn. affected nerves had to have motor conduction block, demyelinative slowing on motor ncs, or motor axon loss, whereas sensory ncs had to be normal. we performed excitability tests of motor and sensory axons in affected median nerves of mmn patients and healthy controls at ∘ c. conditioning and test stimuli were delivered at the median nerve at the wrist; cmaps were recorded from the thenar muscle and snaps from the rd digit. results of motor excitability testing in mmn showed fanning-out of threshold electrotonus, decreased i/v slope, and increased superexcitability, all compatible with persistent hyperpolarization of resting membrane potential in motor axons. sensory excitability testing in mmn showed decreased subexcitability but was otherwise normal. this may indicate minimal involvement of sensory axons in mmn. krarup c , , moldovan m , , alvarez s , ciano c , pisciotta c , pareyson d . university of copenhagen, copenhagen, denmark; rigshospitalet, copenhagen, denmark; fondazione irccs istituto neurologico c. besta, (incb), milan, italy. mutations in the gene coding for myelin protein zero (mpz, p ) are associated with different forms of charcot-marie-tooth (cmt) disease. we describe a family harboring a frameshift mutation (c. dela / p.asp thrfster ) in the p gene, predicted to result in a nonfunctional p truncated very early in the extracellular domain. this offered the rare opportunity to assess the consequences p deficiency in absence of the potential gain-of-function effects of the mutations itself. conventional conduction studies and multiple measures of nerve excitability by "threshold tracking" were carried out in heterozygote parents (aged and ) and their two homozygote sons (aged and ). in the homozygous patients, all distal limb cmaps and snaps were absent. for neurophysiological assessment, the spinal accessory nerve was stimulated at the neck and cmap was recorded over the upper trapezius muscle. eight normal subjects, mean age , were used as control. the two sibs showed a severe phenotype with early onset, severe scoliosis, complete loss of distal movements and relevant proximal weakness, cmt examination score (cmtes) - / ; both heterozygous parents had very mild adult-onset neuropathy with cmtes < / . control subjects had a trapezius cmap with a latency of . ms and an amplitude of . mv. heterozygotes had a mild cmt type b phenotype, with a cmap latency of . ms and an amplitude of . mv whereas the homozygotes had a severe neuropathy with a cmap latency of . ms and an amplitude of . mv. consistently, the homozygotes had a more severe impairment in excitability with a rheobase of . ma as compared to . ma in the heterozygote and . ma in controls. deviations in excitability measures were similar to our previous reports in p +/− and p −/− mice. mathematical modeling, indicated both altered passive cable properties due to dysmyelination and depolarizing features with increased na+ currents. our data suggest that p deficiency is associated with impaired axonal na+ channel function, arguing for the translational value of na+ channel blocker treatments as found in p null mouse models. krishnarajah s , divino v , mallick r , dekoven m . csl behring, king of prussia, pa, usa; quintilesims, fairfax, va, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is a rare neurological disorder of the peripheral nervous system. the objectives of this retrospective real-world study were to compare demographic and clinical characteristics among cidp cases and matched controls and to assess cidp treatment utilization. adults newly diagnosed with cidp between / / and / / were identified in the quintilesims pharmetrics plus health plan claims database (first diagnosis date termed the index date). eligibility requirements were: confirmation of cidp (second cidp diagnosis or initiation of cidp therapy) within year of initial diagnosis, continuous health plan enrollment in the months prior to diagnosis (the pre-index) and the years following diagnosis (the follow-up), and no cidp diagnosis or use of cidp therapy in the pre-index. a total of , cidp cases met the study eligibility criteria. cases were direct-matched to controls based on age, gender, region, health plan, and payer type at index, and pre-index charlson comorbidity index score. the final sample consisted of cases matched to controls (both: mean [sd] age . [ . ]; . % male; . % commercially-insured). alternative pre-index diagnoses among cases included inherited neuropathies ( . %) and chronic acquired polyneuropathies ( . %). in the pre-index, neuropathic pain ( . % vs. . %), back pain ( . % vs. . %), and use of opioids ( . % vs. . %) and anti-convulsants ( . % vs. . %) were significantly higher among cases compared to controls (p< . for all). median total pre-index healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . ). over the follow-up, median total healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . [mean $ , and $ , ] ). cidp-related therapy costs accounted for . % of total healthcare costs for cases. the majority of cases ( . %) initiated cidp therapy over the follow-up, in a mean of . ( . ) days from initial diagnosis. half ( . %) of cases initiated treatment with corticosteroids only, while . % initiated ivig only. over the follow-up, . % of cases used any corticosteroid, while . % used any ivig. our findings suggest a substantial clinical and economic burden of cidp compared to matched controls. corticosteroids and ivig were most commonly used to treat cidp. kronlage m , baeumer p, pitarokoili k , schwarz d , schwehr v , godel t , heiland s , gold r , bendszus m , yoon ms . department of neuroradiology, heidelberg university hospital, germany; department of neurology, st. josef hospital, ruhr university of bochum, germany. objective: to evaluate large coverage magnetic resonance neurography (mrn) in chronic inflammatory demyelinating polyneuropathy (cidp). methods: in this prospective study patients with cidp and healthy controls were examined by a standardized mrn protocol at tesla. lumbosacral plexus was imaged by a t -weighted d-sequence ( mm isotropic voxel size); peripheral nerves of the upper and lower extremity by axial t -weighted turbo-spin-echo sequences ( . x . mm in-plane resolution). lesions were characterized by nerve cross sectional area (csa) and t -weighted signal (nt ). additionally, t -relaxometry of the sciatic nerve was performed using a multi-spin-echo sequence. all patients received a complementary electrophysiological exam. results: patients with cidp exhibited increased nerve csa and nt compared to controls (p < . ) in a proximally predominating pattern. roc analysis revealed best diagnostic accuracy for csa of the lumbosacral plexus (auc = . ) and nt of the sciatic nerve (auc = . ). csa correlated with multiple electrophysiological parameters of demyelinating neuropathy (f-wave latency, nerve conduction velocity) of sciatic and median nerve, while nt only correlated with f-wave latency of sciatic and not median nerve. t -relaxometry indicated that mr-signal increase in cidp was due to increase in proton-spin-density (p < . ), and not increase in t -relaxation time. conclusion: both nt and csa might aid in diagnosis of cidp, but csa correlates more robustly with electrophysiological parameters. since best diagnostic accuracy was shown for proximal nerve locations, mrn may be a useful complementary tool in select cidp cases. kühnemund j , wetzel c , bégay v , moshourab r , lewin gr . mdc & bih, berlin, germany; mdc, berlin, germany; charité, berlin, germany. damage of peripheral sensory nerves due to diabetes, herpes zoster infection, chemotherapy or trauma can cause chronic neuropathic pain. common symptoms include increased pain sensation (hyperalgesia), touch-induced pain (allodynia), paresthesia and spontaneous pain. we currently have poor understanding about the underlying molecular mechanisms at the peripheral level and treatment of patients suffering from neuropathic pain is inadequate. recent meta-analysis studies show that common first-line medications only yield nnts (numbers needed to treat) between . to . . it is still unclear to what extent allodynia can be attributed to changes in the physiological properties of intact sensory afferents. in this study, we aimed to elucidate whether changes occur in intact sensory afferents that innervate the plantar skin of the hind-paw following induction of neuropathic symptoms. this question was of particular interest considering that blocking mechanotransduction in the skin can alleviate mechanical hypersensitivity in neuropathic pain models (wetzel et al nature neuroscience ( ): - ). we used the chronic constriction injury (cci) model in mice which is behaviourally characterized by robust mechanical hypersensitivity. we made electrophysiological recordings from primary afferent neurons which had intact axons passing through the constriction to innervate the plantar skin using an ex-vivo skin-nerve preparation. we were able to record from myelinated afferent fibers and unmyelinated c-fibers with receptive fields in the control uninjured plantar skin as well as plantar skin of cci mice. we will present evidence that changes in the mechanosensitivity of sensory fibers innervating the glabrous skin may contribute to the symptoms of neuropathic pain. neuropathology, national hospital for neurology and neurosurgery, london, uk; irccs foundation "carlo besta" neurological institute, milan, italy; neurogenetics unit, national hospital for neurology and neurosurgery, london, uk; nuffield department of clinical neurosciences, oxford, uk; department of clinical neurophysiology, norfolk and norwich university hospital, uk. hsn secondary to sptlc / is a rare slowly progressive neuropathy resulting in marked sensory loss, especially nociception and significant motor deficit. despite most of the patients having the same c w mutation in sptlc , there is marked heterogeneity in the phenotype. l-serine oral supplementation has been suggested as potential therapeutic candidate however the lack of outcome measures is a major limiting factor in the initiation of a clinical trial. we undertook a natural history study to identify outcome measures that are responsive enough to be used in a clinical trial. the assessments used were cmt neuropathy score (cmtns version and cmtns version rasch modified), mri of calves and thighs, computerised myometry, quantitative sensory testing (qst), comprehensive neurophysiological assessment, proximal thigh skin biopsy for intra-epidermal nerve fibre density (ienfd), plasma dsl levels and patient based questionnaires (neuropathic pain symptom inventory and sf- v ). standardised response mean, srm (mean change/standard deviation of change) was used to compare responsiveness between tests. patients were recruited: with sptlc (c w) and with sptlc mutations. when analysed as a whole cohort, proximal calf mri fat fractions showed the most significant change over months. ienfd, plasma dsl levels, npsi and sf- v showed minimal change or the change was not in the clinically expected direction. for subsets of the remaining assessments which showed the highest responsiveness, the cohort was sub-divided into mild-moderate (cmtns ≤ ) and severe (cmtns> ) subgroups. in the mild-moderate subgroup, the greatest improvement in responsiveness was seen in computerised myometry (ankle plantarflexion: srm=− . and ankle eversion: srm=− . ). in the severe subgroup, qst (vibration detection thresholds on hands: srm=− . and face: srm=− . and pressure pain threshold on the face: srm= . ) and proximal calf mri fat fractions (srm range= . - . ) showed the greatest improvement. focusing on subgroups classified according to disease severity improved the responsiveness of some tests into the highly responsive range with mri still being the best outcome measure. this will reduce the number of participants required to power a clinical trial and might be a possible solution for designing a clinical trial for a rare, slowly progressive disease with a heterogeneous phenotype. kugathasan u , clark aj , suriyanarayanan s , laurá m , wilson e , , kalmar b , greensmith l , , hornemann t , reilly mm * , bennett dlh * . hsn secondary to sptlc / mutation is a slowly progressive sensory motor neuropathy leading to profound sensory loss with variable but often severe motor deficit. the genes sptlc and encode for the essential enzyme serine palmitoyltransferase (spt) which catalyses the rate limiting step in the sphingolipid de-novo biosynthesis. mutations in these two genes alter the substrate specificity of spt leading to the synthesis and accumulation of atypical metabolites called -deoxysphingolipids ( -deoxysl). plasma levels of -deoxysl are raised in hsn patients. deoxysphingolipids have been shown to be toxic in avian and by our group, in mammalian primary drg and motor neuron cultures. firstly, this study looked at the effects of -deoxysl on the survival and neurite integrity of human ipsc derived sensory neurons following exposure to different concentrations of deoxysphingolipids. later, we determined if there was autonomous -deoxysl production in hsn patient ipsc derived sensory neurons. sensory neurons were differentiated from human ipscs using a combination of small molecular inhibitors. deoxysphingolipids were found to be neurotoxic in this model after hours of treatment. in control lines, there is a significant reduction in neuronal survival following treatment with both -deoxysphinganine ( -deoxysa) and -deoxymethylsphinganine ( -doxmethsa). a clear dose-dependent increase in the expression of the axonal injury marker, atf , is seen with both -deoxysphngoid bases. in both instances, -deoxysa is more neurotoxic than -doxmethsa, which is similar to the findings in avian and mammalian primary neuronal cultures. autonomous -deoxysl production is seen in ipsc derived neurons obtained from three different hsn patients with the levels being significantly greater than that seen in multiple control lines. this is the first study to demonstrate that human ipsc derived sensory neurons can be used as an in-vitro model for hsn , providing a great opportunity both to probe the pathomechanisms mediating deoxysphingolipid toxicity and to test potential therapeutic agents. recent studies have demonstrated an association between autoantibodies directed against antineurofascin- (anti-nfasc ) and a subpopulation of cidp patients characterized by sensory ataxia, tremor and poor response to ivig treatment. here we report on the clinical features of three patients who developed acute changes in their phenotype during the course of their neuropathy, a potential clue to recognize a neuropathy with predominantly humoral dysimmunity. case reports: our index patient had developed sensory changes over weeks, followed by irregular locomotion, and muscle weakness with general areflexia. a first run of ivig improved the patient in a week, but he relapsed within days. a second ivig course with prednisone again normalized deficits within days. ivig runs and rituximab were still necessary to treat a nd , and then a rd relapse before obtaining complete improvement. the course of the neuropathy was months. two other patients were encountered with more chronic courses but in whom periods of worsening or improvement suggested a relapsing-remitting neuropathy, either spontaneously or following immunomodulating treatments. in all three, extensive work-ups were negative, anti-nfasc igg were positive, and detailed repeat nerve conduction studies demonstrated fluctuating conduction blocks. discussion: our report underscores that in chronic cidp patients a relapsing-remitting course could be encountered as a key feature of anti-nfasc neuropathy. this not yet described characteristic course could be of value when deciding using rituximab instead of immunomodulating treatments. kusunoki s , morikawa m , kuwahara m , ueno r , samukawa m , hamada y . faculty of medicine, kindai university, osaka-sayama, japan. anti-glycolipid antibodies are often detected in sera from patients with autoimmune neuropathies, such as guillain-barré syndrome (gbs), chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and multifocal motor neuropathy (mmn). not only individual glycolipid antigens but also mixtures of two different glycolipids (glycolipid complexes) are sometimes recognized by serum antibodies. to investigate antibody activities against large number of glycolipid complexes in serum samples from patients with gbs, mmn, and cidp, we examined igm and igg antibodies against glycolipids [gm , gm , gd a, gd b, gq b, galnac-gd a, lm , galactocerebroside (gal-c), asialo-gm (ga ), and sulfatide] and glycolipid complexes consisting of two different glycolipids listed above, by using combinatorial glycoarray. serum was obtained from patients with gbs, patients with cidp and patients with mmn, all in the acute or relapsing phase. serum was also obtained from healthy controls and patients with other neurological diseases. we investigated the relationships between the clinical features and presence of those antibodies. high titers of igg antibodies were detected almost exclusively in gbs patients. in contrast, igm antibodies were frequently present in mmn and gbs. among the anti-glycolipid complex antibodies in gbs, anti-gm /sulfatide, anti-ga /sulfatide, anti-gm /gd a, and anti-gq b/sulfatide igg antibodies were common ( , , , and patients, respectively). igg antibodies against antigens containing gm were significantly correlated with pure motor gbs (p < . ) and those against antigens containing gq b were significantly correlated with gbs with ophthalmoplegia (p < . ). in seven of the patients with anti-gq b/sulfatide complex antibodies, the antibodies were specific to the gq b/sulfatide complex rather than the individual gq b and suldfatide antigens. moreover, four patients did not have antibodies other than those to the anti-gq b/sulfatide complex. in patients with mmn, igm antibodies to antigens containing gm or galnac-gd a were present in % and . %, respectively. glycoarray is efficient for detecting antibodies against numerous glycolipid complexes in immune-mediated neuropathies. we need further investigations on other immune-mediated diseases using larger number of antigens. kuwabara s , misawa s , sekiguchi y , susumu kusunoki and the jet-gbs study group . department of neurology, chiba university, chiba, japan; department of neurology, kindai university, osaka, japan; japanese eculizumab trial for guillain-barré syndrome, chiba, japan. guillain-barré syndrome is a monophasic immune-mediated neuropathy, but a substantial number of patients with severe disease have poor recovery, even if treated with immunoglobulin. recent studies suggest that complement activation plays a pivotal role in gbs-associated axonal degeneration, and eculizumab is a monoclonal antibody that specifically binds to complement component and inhibits complement activation. jet-gbs is an investigator-led, phase , randomized, placebo-controlled trial conducted in hospitals, this trial aims to investigate the safety and efficacy of eculizumab for treatment of severe gbs. patients were randomly assigned ( : ) to treatment with immunoglobulin plus either eculizumab ( mg/day; n= ) or placebo (n= ) once weekly for weeks. the primary outcome measures are safety and efficacy (the proportion of subjects who regain their ability to walk independently at week ). the secondary outcome measures included the proportion of subjects who were able to walk independently at week , and other measures such as mrc sum scores, nerve conduction parameters. enrollment for the trial began in august , and follow-up of the last patient was completed in october . analyses will be made in april , and the results will be presented at this meeting. this trial is registered with clinicaltrials.gov identifier: nct , and funded by the japanese agency for medical research and development, and alexion pharmaceuticals inc. neurofascin , a paranodal protein in peripheral nerve, is a target antigen for autoantibodies in a subset of chronic inflammatory demyelinating polyneuropathy (cidp). anti-neurofascin antibody-positive cidp is characterized by onset at younger age, tremor, and refractoriness to ivig. we have treated four patients with anti-neurofascin antibody-positive cidp at kindai university hospital. anti-neurofascin antibody was detected by both elisa and cell-based assay in those patients. igg subclass was predominantly igg in all four cases. the median age of the patients at admission was . years [range: - years]. among the four patients, three had tremor, and two had severe cerebellar ataxia. cerebrospinal fluid protein levels were remarkably increased [median: . mg/dl, range: - mg/dl]. although ivig treatment was administered in all four patients, the responses were poor or partial. in contrast, plasma exchange (pe) was performed in all four patients and the clinical symptoms dramatically improved in two of them. corticosteroids were also effective in those two patients. sural nerve biopsy was performed in all four patients. although sensory nerve action potentials of the sural nerves from those patients were not evoked, the transverse semithin sections of sural nerves from three patients revealed only slight or mild loss of myelinated fibers. partial paranodal demyelination was observed in teased-nerve fibers from three patients. in addition, abnormal paranodal lesions such as loss of the transvers bands were observed by electron microscope in all four patients. those electron microscopic findings were not observed in control patients with anti-neurofascin antibody-negative cidp. anti-neurofascin igg antibody-positive cidp shows distinctive clinical and pathological features. labeyrie c , besson f , vandendries c , cauquil c , beaudonnet g , not a , durand e , adams d . neurologie adulte, chu bicêtre, le kremlin bicêtre, france; médecine nucléaire, chu bicêtre, le kremlin bicêtre, france; clinique bizet, paris, france; unité de neurophysiologie clinique, chu bicêtre, le kremlin bicêtre, france. study of the proximal portion of the peripheral nervous system (pns) is difficult because less accessible to electrophysiological exploration and biopsy. the indications of mri are increasing in proximal neuropathies to analyse morphology of the roots, plexus and proximal nerves: integrity of the nerve bundle, inflammation or infiltration of these structures. pet-computed tomography (pet-ct) can detect infiltration of the proximal segments of the pns, with hypermetabolism of roots, plexuses and large nerve trunks. pet-ct is also useful in detection of solid neoplasm which can infiltrates pns and primary nerve sheath tumors. however, spatial resolution of pet -ct scan is limited to explore the roots and plexuses for moderate hypermetabolisms. we believe that a mild hypermetabolism could be highlighted in inflammatory neuropathies or in mild tumor infiltrations. we performed a fusion of whole body pet (performed because of suspected neoplasia) and plexus mri (pet-mri) images in ten patients with a peripheral neuropathy for which we suspected proximal involvement. the pet-ct and mri were first read separately, then with merge of the images by a nuclear physician and a neuroradiologist. five out of ten patients presented with hypermetabolism of pns (hmpn+) on pet-mri: root (n= ), and/or dorsal root ganglia (n= ). median sul of lesions was . . among the hmpn+ patients: hypermetabolism was already apparent on pet-ct in cases, the merge invalidated abnormalities seen on pet-ct in patients and mri detected additional lesions, not visible on pet-ct, in one patient. all hmpn+ patients had hypersignal and or hypertrophy of pns on mri either diffuse (n= ), or multifocal (n= ). among hmpn-(no hypermetabolism) out of ( %) had abnormal mri ( multifocal and diffuse). gadolinium enhancement was found in all patients receiving gadolinium in the hmpn+ (n= ) and only in / patients in the hmpngroup. final diagnoses of hmpn+ patients were neurolymphoma in one ( %), and idiopathic cidp in the others ( %) with histological proof on nervous root biopsy in one of them. final diagnosis in hmpn-patients was cidp in out of and sequelae of neuropathy in relation to lymphoma without relapse for the last one. cidp was more disabling (onls≥ ) in hmpn+ than in hmpn-group % vs. %. pet-mri could be helpful to detect a proximal inflammation of the pns, and to plan further testing. further studies are needed to evaluate the prognostic value of hmpn+. lancaster e , li j , liem r , scherer ss . perelman school of medicine, university of pennsylvania, philadelphia, pa, usa; columbia university school of medicine, new york, ny, usa. heterozygous nefl n s/+ mutant mice are the first animal model of a charcot-marie-tooth disease e (cmt e). people with this mutation have a severe, early onset axonal neuropathy. axons in the mutant mice have reduced number of neurofilaments and decreased diameters. they also show early onset of tremor and abnormal hindlimb clasping behavior. we measured the compound action potentials (caps) from tails every weeks from the same cohort of nefl n s/+ mutant mice and their wt littermates from to weeks. even at the age of weeks, the amplitude of mutant caps was only ∼ % of wt caps ( ± . microv, n= vs ± . microv, n= ), and the caps stayed substantially smaller than those in wt mice for weeks ( ± . microv, n= vs ± . microv, n= ). the conduction velocity and the duration of caps in mutants were slower and wider compared to those of wt. separate cohorts of mice (n= mutants and n= wt) were sacrificed at different time points for analysis by light microscopy. in caudal nerve of mutant mice, the number of axons was significantly reduced compared to that of wt at weeks and, by weeks, it was reduced more than %. this model system may be useful for preclinical studies of treatments for cmt e since the animals show progressive neuropathy over weeks, which can be objectively measured electrophysiologically and anatomically. despite more than cmt genes identified today, at least % of cmt patients do not carry a mutation in any of these genes. progress in gene identification in recent years suggests that there are still many more cmt disease genes to be discovered; however, it has become rare to gather support from multiple large families that allow for conclusive linkage analysis. we have studied multiple extended dominant cmt families with linkage support for a gene at chromosome p . . originally a czech family yielded a two point lod score of . at this locus and a family from southern italy showed a lod score of . . whole exome sequencing of multiple family members identified missense mutations in the gene atpase na+/k+ transporting subunit alpha (atp a ). atp a has not been associated with human diseases thus far. in expression studies on teased fiber preparations we already confirmed predominant expression in the nodes of ranvier of peripheral nerves. through collaborative efforts in the inherited neuropathy consortium and beyond we identified five additional multigenerational families via exome or sanger sequencing resulting in a total of seven unique segregating missense changes: leu arg, ile thr, ala thr, asp phe, pro ala, pro thr and asp ala. five of these mutations fall into a remarkably narrow motif associated with the sodium binding structure of atp a , flanking the flexible hinge motif. functional studies in different model systems (mammalian cell lines, xenopus oocytes, patient fibroblast lines) are underway to determine whether a loss or a dominant gain-of-function represents the disease mechanism. taken together, we show strong support for a major new dominant cmt gene, atp a . this finding represents a new pathway and an attractive new target for therapy development in axonal cmt. laurá m , ramdharry g , , singh d , kozyra d , skorupinska m , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; school of rehabilitation sciences, st george's university of london/kingston university, uk; royal national orthopaedic hospital, stanmore, uk. charcot-marie-tooth (cmt) disease is the most common inherited peripheral neuropathy. foot deformities are frequent complications and orthopaedic surgery is often required. however there are no evidence based guidelines on the type or timing of the surgery. only few studies have described the long-term results of surgical procedures and evidence regarding optimal surgical management of these patients is lacking. we prospectively studied surgical management of cmt patients attending our centre. we collected data and assessed cmt patients before and after surgery. data included: history of ankle instability, pain, skin condition, details of physiotherapy and orthotic management, assessment of lower limb strength, charcot-marie-tooth examination score (cmtes), foot posture index, ankle dorsiflexion range of movement and specific questionnaires (foot index and manchester-oxford foot questionnaire, modified fatigue severity scale and modified falls efficacy scale), details of surgical procedures. patients were assessed yearly after surgery. so far patients ( males and females, age range - ) have been evaluated prior to surgery. all patients but one had genetically confirmed cmt ( cmt a, cmtx, cmt a). patients have been assessed after year, patients after years, patients after years and patient after years from surgery. a wide range of surgical procedures were performed by one dedicated orthopaedic surgeon. preliminary results showed reduction of number of falls in / ( %) patients and improvement of callosities in / ( %) patients at year follow up. there was also significant improvement of alignment of the operated foot (p= . ) and pain (p= . ). there were no significant changes in measures of strength and ankle range of motion. further analysis on a larger number of patients will be important to determine the long-term outcome of surgery. data acquired from this study will help develop orthopaedic intervention guidelines and identify areas for further research. lavigne-moreira c , oliveira mf , marques vd , onofre ptbn , dos santos acj , nascimento ojm , barreira a , marques w jr . division of neuromuscular diseases and neurogenetics, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; department of neurology, fluminense federal university, rio de janeiro, brazil. fap associated to ttr mutations is defined as a length-dependent axonal sensory and motor polyneuropathy that at early stages affects mainly the small nerve fibers, associated to autonomic and thermo-algesic sensations. the expected emg pattern was that of an axonal sensory and motor polyneuropathy, but in fact several unexpected patterns may be found. in this study we present the results found in a brazilian population with ttr mutation. patients were divided in three groups: ttr-met of early onset, ttr-met of late onset and ttr non-met . in the first group (ttr-met of early onset), ( . %) examinations were normal, ( . %) were axonal, were demyelinating fulfilling pidc criteria, one suggested a predominately motor polyneuropathy and the final one presented a lower motor neuron disease pattern. in the second group (ttr-met of late onset), ( . %) had an axonal pattern, ( . %) had an intermediate cv, ( . %) had a demyelinating pattern, had a lumbossacral pattern and the final one had no definite pattern. among the non-ttr met , two patients had the ttr -asp tyr, one presented an axonal pattern, while the second presented initially an axonal pattern, that changed to a demyelinating pattern in the second examination. patients with ile val mutation presented an axonal neuropathy associated to cts. most patients with demyelinating or intermediate pattern were treated with corticosteroids or ivig, with no satisfactory results. this small series of patients shows clearly that fap-ttr is associated to several emg patterns in addition to the expected sensory and motor axonal polyneuropathy. this variability is present in the same family and in the same patient in different occasions. clinicians should be alert to these possibilities to do not delay diagnosis and treatment. identifying the mechanisms involved in this variability could improve our knowledge of this intriguing disease. lavin tm . greater manchester neurosciences centre, salford royal hospital, manchester, uk. subcutaneous immunoglobulin (scig) has evidence from small trials for its use following intravenous igg (ivig) loading in chronic inflammatory demyelinating polyneuropathy (cidp). potential advantages of scig include stable igg levels and reduced complication rates. scig de novo can be considered as igg levels gradually rise with subcutaneous delivery and maybe beneficial for patients for whom complications are a concern. we present cases of cidp who were initiated on subcutaneous immunoglobulin g ( g/kg/month, % scig, hizentra, csl behring) following corticosteroids; without intravenous igg loading. patient is year old with a history of ihd with a progressive lower limb sensory changes and sensory ataxia over years. neurophysiology confirmed sensory demyelinating changes. he did not respond to corticosteroids. concerns were raised about the potential risk orf adverse cardiac events with iv immunoglobulin. scig therapy was introduced at g weekly. initial therapy has been well tolerated and objective measures (rods-cidp score, jamar grip strength, hole peg) have remained stable through initiation, and at months. patient is yr old male with a past history of branch retinal vein thrombosis. he developed a sensorimotor polyneuropathy over years with neurophysiology fitting efns criteria for cidp. given concerns regarding previous thrombosis, a trial of scig was given at g/week, with resolution of sensory ataxia and improvement in objective markers (jamar grip strength, rods-cidp score, hole peg) at months. unfortunately an adverse event occurred of an urticarial skin reaction. patient is a yr old male who presented with motor predominant cidp. there was no response to corticosteroids but improvement following plasma exchange. due to a past history of transient ischaemic attack, scig was started. there has been a positive response after months with improvement in walking distances and stable objective markers (rods-cidp score, grip and pinch strength). our cohort remained neurologically stable during initiation of scig, with adverse reaction. our experience would support further trials in this area regarding the efficacy of scig compared to ivig in both the short and long term. diabetic peripheral neuropathy is the most common and debilitating complication of diabetes and it is associated to neuropathic pain and non-traumatic amputations. despite the economic burden and human costs, nowadays there is not a specific treatment to cure diabetic peripheral neuropathy. hyperglycaemia and dyslipidemia-mediated oxidative and endoplasmic reticulum stress has been linked to diabetic peripheral neuropathy, among other altered pathways. in order to study in more detail the molecular mechanisms that lead to the development of diabetic peripheral neuropathy we set up in vitro models of the disease using nsc- (motoneurons) and med . (sensory neurons) cell lines or primary cultures of dorsal root ganglia and motoneurons exposed to saturated fatty acids. it has been reported that palmitic acid is able to induce endoplasmic reticulum stress and oxidative stress in multiple cell types including schwann cells and myenteric neurons, resulting in a good model of dyslipidemia-mediated stress. here we found that palmitate induces upregulation of the molecular chaperone bip/grp and the ccaat-enhancer-binding protein homologous protein (chop) mrnas and a dose-dependent downregulation of the apoptosis marker bcl- in primary cultures. moreover, palmitic acid induces loss of neuron dendrites and cell death at high doses and upregulation of heme oxygenase (ho- ) and chop at lower doses in the nsc- cell line. we are currently characterizing multiple molecules implicated in oxidative stress, endoplasmic reticulum stress and inflammation pathways in these cell types to find new therapeutic targets to treat type diabetic sensorimotor polyneuropathy, that will be subsequently validated in the db/db mouse model by pharmacological or gene therapy strategies. lee bjh , ohnmar o , wong j , koh sj , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful peripheral neuropathy and autonomic dysfunction that occurs within weeks of tight glycaemic control. therapy with either insulin or oral hypoglycaemic agents (ohga) may result in tind. the quantum and rate of decline in hba c predicts development and severity of tind. both type i and ii dm patients are prone to this iatrogenic complication. besides the expected morbidity associated with painful somatic and autonomic neuropathy, tind patients also develop life-threatening eye complications such as maculopathy. with greater awareness, we picked up at least typical cases of tind in recent few months. however, there was a fourth possible tind case, whom we feel deserves special attention. the circumstances surrounding this case are common and may go under-recognised in acute hospitals. a year-old man with -year history of poorly controlled type ii dm was admitted with a partial left middle cerebral artery stroke. one month before admission, his hba c was . %. his hospitalization was prolonged because of his considerable disability that required rehabilitation. he stayed weeks. his blood sugar was difficult to control with hyperglycaemic episodes requiring ohgas and insulin. the highest capillary blood glucose recorded was mmol/l. he also had episodes of hypoglycaemia. three weeks into hospitalization, he developed severe orthostatic hypotension. he came to our attention two months later when he was sent for autonomic screening tests for severe postural hypotension, in spite of therapy with fludrocortisone and midodrine. his hba c was . %. he had marked orthostatic hypotension suggestive of sympathetic dysfunction. we were not able to discern if he had a painful neuropathy because of his aphasia. there was no recent ophthalmology review. we suggested a diagnosis of possible tind. he was discharged with lower doses of ohga. however, day after discharge, he attended the emergency department for a syncopal episode. this case raises intriguing questions on the safety of glucose control paradigms commonly employed in patients with acute stroke and myocardial infarction as well as the possible role of major fluctuations in blood glucose in the development of tind. lee hs , kim sm . presbyterian medical center, jeonju, korea; yonsei university college of medicine, seoul, korea. the occurrence of peripheral neuropathy by a tumor within or compressing a nerve in neurofibromatosis (nf) type and is relatively well known. however, nf presenting with demyelinating polyneuropathy unrelated to tumor masses is rarely reported. we report a rare presentation of an nf patient with demyelinating polyneuropathy of subacute onset. a -year-old woman was referred to our hospital with paresthesia on both limbs months before. she was healthy and had no family history. the symptoms worsened over the next months, tremor and weakness in both limbs occurred. although she treated as a chronic inflammatory demyelinating polyneuropathy (cidp) with intravenous steroid and immunosuppressant in another hospital, symptoms were getting worse. bifacial numbness and left eyeball pain occurred weeks ago and she visited our hospital. on neurologic examination, bilateral facial sensory hypoesthesia and symmetrical both limbs weakness (mrc grade iv+) were observed. decreased touch and pinprick sensation on both limbs were observed like stocking-glove distribution. the reflexes were sluggish. on nerve conduction study, sensorimotor demyelinating polyneuropathy with conduction blocks and temporal dispersions was observed. bilateral r , r responses were prolongation on facial blink test. mri of cervical spine and brain revealed contrast enhancing tumor-like enlargement of multiple nerve roots and cranial nerves. nerve biopsy was performed on right supraorbital nerve, neurofibromatous changes were observed. the symptom deterioration was stopped without treatment. gene test dose not revealed nf type and . on review of the literature, polyneuropathy in nf patient can result from tumor masses within the proximal nerve roots, or along the peripheral nerve, or in the extramedullary lesion affecting neighboring nerve roots. thus, axonal type polyneuropathy and focal amyotrophy have been reported in nf. demyelinating polyneuropathy has not been reported before in our knowledge. although the etiology of demyelinating polyneuropathy in nf requires further clarification, some authors claim that unknown local toxic or metabolic influences of the endoneurial pathological cells on adjacent nerve fibers. in this patient, atypical symptoms were observed in cidp such as cranial nerve involvement and stocking-glove sensory distribution. in this case, a nerve biopsy can be helpful the accurate diagnosis. lee jy , yoo jh , kang dk , bae js . department of neurology hallym university, seoul, korea. acute disseminated encephalomyelitis (adem) is an uncommon post-infectious inflammatory demyelinating disorder of central nerve system, while guillain-barre syndrome (gbs) is a prototype of acute post-infectious peripheral neuropathy. previous reports regarding the coexistence of these relatively rare diseases suggest that certain immunogenicity within central and peripheral nerves may share a common autoimmune process during the disease course. a previous healthy years old man was admitted because of fever, headache, nausea and myalgia in department of infectious disease. two weeks before admission, he suffered from watery diarrhea for days and spontaneously recovered. at initial presentation, he had high fever( ∘ c), headache and myalgia and intermittent horizontal diplopia. a few days after admission, he began to complain of drowsy mentality, bilateral extremities weakness, especially lower limbs, dysarthria, bilateral facial paralysis, urinary retention, dyspnea. on neurologic examination, he had mildly drowsy mentality, symmetric muscle weakness scoring of on bilateral hip, knee flexion and finger extension, but he had no sensory symptoms. he showed gazed evoked nystagmus with no extraocular muscle palsies. deep tendon reflexes were not present. pulmonary function test revealed a severe restrictive pattern. csf studies disclosed a dissociative increase of protein contents ( mg/dl) without pleocytosis. anti ganlioside antibody assay identified an anti-gt a igg positivity in his serum. nerve conduction study (ncs) showed prolonged motor terminal latencies and slow motor conduction velocity on multiple nerves. in contrast, sensory ncs revealed no abnormal findings. imaging studies unexpectedly revealed apparently symmetrical lesions across bilateral brainstem and basal ganglia suggesting a diagnosis of adem. after both ivig and high dose steroid treatment, he remarkably recovered from disturbed mental state and motor weakness. about month after the symptom onset, he could walk with assistant aid and discharge to other hospital for rehabilitation. our case suggest that certain component of autoimmunity simultaneously result in both cns and pns inflammation. specific immunological mechanism is remained to be elucidated. although we could not conclude whether cellular component or humoral component is dominant for our case, the presence of anti gt a antibody suggest a role of humoral mechanisms. the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. leitao amf , , araújo df , marques h , pamplona l , , souza mh , , nbraga ll , , gondim faa , . the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. the classic guillain barré syndrome (gbs) is characterized by motor weakness, hyporreflexia, but limited sensory deficits. sensory variants involving either small or large fibers or both are unusual and represent a diagnostic challenge. we described patients presenting with the sensory variant of gbs and retrospectively analyzed the clinical and electrophysiological findings of patients fulfilling the criteria for sensory gbs according to oh et al. criteria. six patients were identified (mean age years: range - years). four had a previous infection. they all consulted due to distal painful paresthesias and allodynia. on examination the patients presented normal strength and normal cranial nerves through the course of the disease with reduced knee and ankle reflexes in patients. distal hyperesthesia to pinprick was identified in and one of them additionally had hyperhidrosis and constipation. two additional patients presented hypoesthesia to pinprick and temperature. one patient had distal proprioceptive sensory loss with sensory ataxia. csf albumin cytological dissociation was present in patients. nerve conduction studies (ncs) identified a sensory motor demyelinating neuropathy in patients. among the with normal ncs, had abnormal cold and warm threshold in their qst evaluation. all patients received symptomatic treatment for the neuropathic pain and only two ivig therapies. longstanding pain, fatigue or both were persistent findings in patients after a mean follow up of months. in conclusion the sensory variant of gbs is both an infrequent presentation and a diagnostic challenge. longstanding pain and fatigue are common persisting findings. the epidemic of zika virus (zikv) throughout the americas and asia, and the subsequent rise in reported cases of guillain-barré syndrome (gbs) caused worldwide concern. as of january , countries have reported evidence of mosquito-borne zikv transmission and in of these countries, a sudden increase of gbs has been reported. moreover, case studies and a case-control study further indicate that zikv may trigger gbs. however, accurate diagnosis of both zikv and gbs in many of these studies is disputed, and a comprehensive description of the clinical phenotype of gbs related to zikv is lacking. the international gbs outcome study (igos) is a prospective observational study on the factors determining the onset, clinical course and outcome of gbs. at present, a research consortium of centers from countries has included patients in igos. our aim is to investigate zikv-related gbs in igos as is already occurring in colombia. in igos-zika, data on clinical features and ancillary investigations will be collected in zikv endemic areas according to the igos protocol with some modifications. first, igos-zika has a case-controlled study design to define the association between gbs and zikv and other arboviruses. second, urine samples will be collected and additional questions on preceding events will be asked, focusing on arbovirus infections. third, a more limited follow-up is required. our aim is to recruit additional centers via the inflammatory neuropathy consortium (inc) and centers in all arbovirus endemic regions that are willing to participate. the focus of igos-zika will be on the accuracy of the diagnosis of both gbs and zikv and on defining the associated clinical phenotype, course and outcome. igos-zika provides the opportunity to combine data and biobanks from various geographical regions using a standardized protocol and to compare these data with data and biosamples already collected in igos. studying these cases will help to optimize diagnostics and care for gbs patients in arbovirus endemic countries and provides a unique opportunity to further understand the pathogenesis of gbs. moreover, this study design and network can be used to adequately respond to other future viral epidemics related to gbs. lerat j , cintas p , dzugan h , , magdelaine c , , sturtz f , , lia as , . service de biochimie et génétique moléculaire -chu de limoges, limoges, france; service de neurologie et d'explorations fonctionnelles -chu de toulouse, toulouse, france; ea -université de limoges, limoges, france. pharc syndrome is an autosomal recessive neurodegenerative pathology leading to demyelinating polyneuropathy, hearing loss, cerebellar ataxia, retinis pigmentosa and early-onset cataract. these various symptoms can occur at different ages, so that pharc syndrome can be a differential diagnosis of charcot-marie-tooth disease (cmt) associated with deafness. only abhd mutations have been reported in patients. we described the th mutation and compared our results to the literature data. we analysed by next generation sequencing (ngs) strategy using a targeted cmt and associated neuropathies -gene panel the dna of a -year old male who has suffered from demyelinating sensory and motor polyneuropathy and ataxia since the age of . bilateral sensorineural deafness was diagnosed at the age of five. bilateral congenital cataracts were operated on at the age of . a new large complex homozygous mutation, with one deletion of seven base pairs and one insertion of base pairs, was detected. by analyzing our patient data and those of the literature, we evaluated that, in pharc syndrome, sensorineural deafness always occurs as the first feature in late teens. the ophthalmological symptoms are cataracts that occur at a mean age of yo and then retinis pigmentosa at a mean age of . demyelinating sensory-motor polyneuropathy is the most variable characteristics, which occurs in the thirties. we report the first large complex homozygous mutation in pharc syndrome, which is certainly under-diagnosed. therefore, it seems interesting to include abhd in the panels of the five symptoms, especially deafness ones. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a chronic disabling disease that often improves with immune therapy. to date, the most reliable diagnostic criteria for cidp are the efns/pns revised criteria, with a reported sensitivity of % and specificity of %. we implemented a web-based database to collect data from patients with cidp followed by italian centers with expertise on cidp to determine the frequency and characteristic of cidp and it variants, the diagnostic criteria used for their diagnosis, the possible evolution into typical cidp, the association with specific anti-nerve antibodies, and their response to therapy. all the patients were evaluated at the time of inclusion and will be followed for two years to monitor their outcome and response to therapy. by february we included patients with cidp and variants ( men, women), aged - years (median ) with a mean disease duration of . years (range . - years). based on clinical symptoms, cidp was defined as typical in % and atypical in %. the diagnosis of typical cidp fulfilled efns/pns criteria in % of the patients while nerve conduction studies were not diagnostic in % (grouped as clinical cidp) or not available in %. we analyzed the frequency of supportive criteria for the diagnosis of cidp in patients with clinical cidp and found that increased csf proteins, demyelination or cell infiltration on nerve biopsy and imaging abnormalities consisting with cidp on us or nmr were present in %, % and % of the patients, respectively. a relapsing course was present in % of patients with clinical cidp, increasing the reliability of the diagnosis for cidp. in addition an improvement after one or more therapies was reported by % of the patients, with a positive response to ivig in %, steroids in % and plasma exchange in %, similarly to what observed in patients fulfilling efns/pns criteria. in % of the patients with clinical cidp two or more supplementary criteria for cidp were present. this study on a large population of patients is providing useful information that may help to revise the current diagnostic criteria for cidp. li j , cannell m , suragani r , pearsall r , kumar r . acceleron pharma inc, cambridge, usa. charcot-marie-tooth (cmt) is the most common hereditary peripheral neuropathy and is characterized by demyelination and/or axonal damage of peripheral nerves and muscle weakness. foot drop, steppage gait, and foot deformities are a typically seen in cmt patients. consequently, falls are commonly reported in these patients. improvement of dorsiflexor muscle function to prevent falls may improve quality of life and activities of daily living in patients with cmt. ace- , a locally-acting ligand trap that binds growth and differentiation factors (gdfs) and activins, has previously been shown to increase muscle mass and force in both duchene muscle dystrophy (dmd) and amyotrophic lateral sclerosis (als) mouse models. in the current study, we evaluated the therapeutic effects of ace- to improve muscle strength in the trembler (tr-j) mouse model of cmt a. these mice harbor a mutation in the peripheral myelin protein (pmp ) known to cause cmt a. seven-month old (b .d -pmp tr-j /j) mice were administered ace- ( g, twice weekly) intramuscularly to one of the unilaterally tibialis anterior (ta) muscle for weeks. the contractility of the ta muscle was evaluated during isometric contraction. all data were compared to the uninjected contralateral control hind-limb. after weeks of ace- treatment, ta muscle mass was increased by % (p< . ) and its physiological cross-sectional area was increased by % (p< . ). the increase in muscle mass correlated with an increase in strength, with maximum tetanic force and twitch force improved by % (p< . ) and % (p< . ), respectively. in addition, temporal properties during isometric contraction, such as maximum rate of contraction and relaxation, were accelerated by % and % respectively (p< . ) in the ace- -treated ta muscle compared to its contralateral hind-limb. pathological and biochemical assessment of ace- -treated mice showed enlarged myocyte area (+ %, p< . ) and reduced atrogin- mrna expression (− %, p< . ). together, these results demonstrated that ace- attenuates the degree of muscle atrophy and also improves muscle function in a mouse model of cmt a. the current study provides proof of concept for the use of ace- as a therapy for cmt to improve dorsiflexor muscle function and alleviate foot drop. we have generated a rat model of charcot-marie-tooth disease a (cmt a) harboring the p.arg trp mfn mutation, whose human counterpart results in a severe, early-onset axonal neuropathy. the mutation was made using zinc finger nuclease-mediated genome editing in fertilized rat eggs. a large cohort of mutant and wt littermates were characterized behaviorally and found to develop multiple motor deficits that worsened over time. nerve conductions of the tail (caudal nerve) was performed on a separate cohort of mutant (n= ) and wt littermates (n= ) every weeks from to weeks. mutant rats showed a progressively decline in the amplitude of the compound action potential after weeks, whereas the amplitude progressively increased in their wt littermates. separate cohorts of rats were sacrificed at , , and weeks and analyzed by light microscopy. in mutant rats, there was a reduced density of myelinated axons and active axonal degeneration in distal but not proximal nerves, and in the fasciculus gracilis of the cervical spinal cord at and weeks. these findings were not present in the -week-old cohort of mutant rats, or in wt rats at or weeks. a genetically authentic animal model of cmt a that develops a progressive, length-dependent axonal neuropathy will be a valuable tool for examining the pathogenesis and treatment of cmt a. lindborg ja , niemi, jp , defrancesco a , zigmond re . case western reserve university, cleveland, usa. traditionally the role of immune cells in nerve degeneration and regeneration has focused on the infiltration of inflammatory monocytes into the distal nerve after nerve injury and the phagocytosis by the resulting macrophages of myelin and axonal debris, thereby clearing a path for regenerating axons. therefore, it was surprising when we discovered that in ccr knockout (ko) animals, in which the entry of these inflammatory monocytes does not occur, that wallerian degeneration precedes normally. we now report that the reason for this is that neutrophils and schwann cells compensate for the decrease in macrophage accumulation. furthermore, nearly complete depletion of circulating neutrophils by systemic injection of an antibody to ly g leads to an inhibition of myelin clearance both in ccr ko and in wild type animals. on the other hand, we have demonstrated a second site of macrophage accumulation in wild type animals, namely around axotomized sensory neurons in dorsal root ganglia (drgs). blockade of that accumulation, for example as occurs in ccr ko animals, leads to a dramatic impairment of nerve regeneration. to examine the relationship between macrophages and regeneration further the monocyte chemokine ccl was overexpressed in drg neurons in intact animals by viral infection using an aav containing the ccl coding sequence. the resulting overexpression of ccl led to the accumulation of macrophages in drgs even though no injury had taken place and subsequently to an increase in the intrinsic growth capacity of the sensory neurons. examination of changes in gene expression in the drgs in these animals revealed increased expression of the cytokine leukemia inhibitory factor and an increase in its downstream signaling pathway that involves the phosphorylation and nuclear translocation of stat . strikingly, pharmacological blockade of stat activation inhibited the increase in the neurons' growth capacity produced by the virus. these results reveal unexpected interactions between immune cells and neurons facilitating nerve degeneration and regeneration and could lead to therapies to improve regeneration after injury or in disease. lin j , , qiao k , , huang j , , zhao cb , , lu jh , . institute of neurology, fudan university, shanghai, china; department of neurology, huashan hospital, fudan university, shanghai, china. we used terminal latency index (tli) as a tool in differentiation between poems syndrome and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). comparison of median and ulnar nerve conduction studies including motor conduction velocity (mcv), distal motor latency (dml) and terminal latency index (tli) were studied in poems patients, matched cidp patients and normal controls. in this cohort, the average age at evaluation was . ± . years old in poems group and that of cidp patients was . ± . years old. except the ulnar terminal latency index in cidp group, poems and cidp patients demonstrated prolonged distal latencies, low conduction velocities and increased terminal latency indexes compared with the normal group. reduced conduction velocities and higher terminal latency indexes in poems group than in cidp group was found. increased tli was demonstrated in . %(median nerve) and . %(ulnar nerve) poems and that in cidp patients was . %(median nerve) and . %(ulnar nerve). decreased tli was found in . %(median) and . %(ulnar) cidp patients and none in poems. temporal dispersion (td) and conduction block (cb) were more often seen in cidp patients with increased tli than that in poems. compared with cidp and poems showed greater slowing of the intermediate nerve segments and relatively more uniform demyelination. about % cidp demonstrated more distal conduction slowing and more td and cb especially in those with increased tli. terminal latency index combined with td and cb may be helpful in differentiating poems from cidp. lin y , sung j , chang t , jowy t . department of neurology, taipei municipal wanfang hospital, taipei, taiwan. the purpose of our study is to exam whether electrophysiology changes could be detected in prediabetes patients and to discover the possible mechanism of nerve injury in prediabetes stage. we analysis and compare the nerve excitability test data between prediabetic patients and age-matched normal control subjects. prediabetes is defined by american diabetes association (ada) as one of the three following: hba c . % to . %, fasting glucose mg/dl to mg/dl, and hour oral glucose tolerance test to mg/dl. patients with radiculopathy, myelopathy, entrapment neuropathy such as carpel tunnel syndrome, and polyneuropathy were excluded. the strength-duration time constant (sdtc) and superexcitability showed significant difference (p< . ) between two groups. we also find increased threshold electrotonus in depolarization (ted) and reduced relative refractory period (rrp) and refractoriness in . msec. these early changes in prediabetic patient are similar in nerve excitability feature of diabetic patients. however, the above changes are not found in motor axonal excitability test. our data supports that nerve excitability test may be a useful, non-invasive, and less time dependent tool to detect peripheral nerve injury in prediabetic stage. the sensory axons are more vulnerable than motor axons. superexcitability is the most sensitive parameter in prediabetes. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominant disorder caused by mutations of ttr gene and is associated with variable penetrance. ttr-fap is rare, except for endemic areas. this is a retrospective study of ttr-fap patients diagnosed at our center between - . we identified four families with different ttr mutations. in one family with v a mutation, nine family members over four generations diagnosed with ttr-fap was followed since . in the other families the index cases with different mutations were identified between - . affected family members with v a mutation developed severe progressive polyneuropathy with cachexia, with onset of the disease between ages and . three patients presented with marked visual symptoms (one patient underwent vitrectomy). nine patients died to years after disease onset. two patients (sisters) underwent liver transplantation -one died after years of disease at age , second is years old and wheelchair-bound as her symptoms continue to progress. the ttr mutations diagnosed in the index cases of three other families are: d v, f l and v m. they all presented with similar clinical picture of late-onset ttr-fap with predominant progressive axonal sensory, motor and autonomic polyneuropathy. all three index cases were men, the onset of symptoms was between - years with numbness and paresthesia in the feet followed by weakness and autonomic dysfunction. all had excessive weight loss resulting in cachexia and were diagnosed with cardiomyopathy. no patient suffered from visual symptoms. all three patients progressed to stage ii of ttr-fap -walking with assistance. time to diagnosis was . - years. due to advanced stages of their disease these patients were not suitable for therapy with tafamidis or the liver transplantation. the ttr d v (p.d v) was confirmed as a de novo mutation, which is uncommon in ttr-fap. in the remaining families carriers of ttr mutations were identified and are followed up. pedigree analysis of the family with f l mutation revealed affected members who with high probability died from ttr-fap. our study suggests that patients with ttr-fap in poland exhibits clinical and genetic heterogeneity. liu x , fan d . department of neurology, peking university third hospital, beijing, china. objective: to identify the gene mutation of chinese charcot-marie-tooth pedigrees and investigate the correlation among the clinical manifestation, electrophysiology and mechanism of different genotype. methods: we included pedigrees with cmt enrolled in our hospital from january, to december . we recorded clinical features, cmtns and electrophysiological data at diagnosis. the patients underwent mutation analysis of pmp , cx , mpz, mfn , hspb , hspb using mlpa, dhplc and sanger gene sequencing. results: we found pmp duplication pedigrees ( . %), cx pedigrees ( . %), mfn pedigrees ( . %), mpz pedigrees ( . %) conclusions: in chinese han population, the proportion of pmp duplication is relatively low, the majority of clinical manifestation is classical cmt. axonal cmt can show isolated lower extremity injury, with central nervous system involvement. hmn may be an underestimated clinical types, the identification should be done with caution in differential diagnosis. liu y , liu b , sebastian b , wozniak km , wu y , slusher b , polydefkis m . johns hopkins school of medicine, baltimore, usa. chemotherapy-induced peripheral neuropathy (cipn) is a common dose-limiting toxicity in the treatment of many cancers. most cipn studies preferentially focus on sensory fiber loss and dysfunction. here, we compared the structural and functional recovery of autonomic fibers in sweat glands (sweat gland nerve fiber density, sgnfd) and sensory fibers (intra-epidermal fiber density, ienfd) in mouse footpads after exposure to a maximum tolerated dose (mtd) of several common chemotherapy agents. additionally, we assessed footpad sweat production as a functional correlate to sgnfd reductions. female balb-c mice ( -animals/group) were treated with a mtd of four anti-tubulin drugs: paclitaxel (pca, mg/kg), ixabepilone (ixa, mg/kg), eribuline (erib, . mg/kg), vinoelbine (vino, mg/kg), or corresponding placebo given intravenously, mwf for two weeks. recovery was assessed at -hours, , , , , and weeks following the last dose. footpads were processed to visualize epidermal nerve fibers using pgp . and autonomic nerve fibers with tyrosine hydroxylate and pgp . . ixabepilone-treated mice experienced significant reductions in sgnfd at hrs, while ienfd nadir occurred at a later time point, -weeks. the recovery to baseline levels occurred more quickly for ienfd ( -weeks) than sgnfd ( -weeks). in contrast, vinorelbine and eribuline treated mice experienced a maximum deficit in sgnfd and ienfd at hrs and sgnfd recovery was slower ( -weeks) compared to ienfd ( -weeks). pca-treated animals showed more severe ienfd and sgnfd deficits compared to the other agents with both ienfd and sgnfd not recovering completely until -months. reductions in th-sgnfd were comparable or more pronounced to decreases in pgp . -sgnfd for all agents and timepoints. pca-treated animals demonstrated reductions in footpad sweat droplet number thereby providing a functional correlate. together, these data indicate that in mouse models of cipn, autonomic nerve fibers are affected more severely than sensory nerve fibers, and also recover more slowly than intraepidermal nerve fibers. autonomic dysfunction may be an important and under-appreciated consequence of chemotherapy exposure. the pi -kinase vps (pik c ) synthesizes phosphatidylinositol -phosphate (pi p), a lipid critical for both endosomal membrane traffic and macroautophagy. human genetics have implicated pi p dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating charcot-marie-tooth (cmt) peripheral neuropathy. here, we investigated the role of vps , and pi p, in mouse schwann cells by selectively deleting vps in this cell type. vps -schwann cell knockout (vps scko ) mice show severe hypomyelination in peripheral nerves. vps −/− schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. upon reaching the promyelinating stage, vps −/− schwann cells are significantly impaired in the elaboration of myelin. nerves from vps scko mice contain elevated levels of the lc and p proteins, indicating impaired autophagy. however, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in vps scko mice is caused by impaired autophagy. endosomal membrane traffic is also disturbed in vps −/− schwann cells. we investigated the activation of the erbb / receptor tyrosine kinases in vps scko nerves, as these proteins, which play essential roles in schwann cell myelination, are known to traffic though endosomes. in vps scko nerves, erbb was hyperphosphorylated on a tyrosine known to be phosphorylated in response to nrg exposure. the overall level of erbb was also decreased during myelination. our findings suggest that the loss of vps alters the trafficking of erbb / through endosomes. abnormal erbb / signaling may contribute to the hypomyelination observed in vps scko mice. lombardi r , devaux j , cortese a , dacci p , benedetti l , demichelis c , lauria g . irccs foundation "carlo besta" neurological institute, milan, italy; aix-marseille université, marseille, france; irccs c. mondino national neurological institute, pavia, italy; university of genoa and irccs aou san martino-ist, genoa, italy. the recent identification of igg anti-neurofascin (nfascin) antibodies in a group of patients has widened the spectrum of presentation for chronic inflammatory demyelinating polyradiculoneuropathy (cidp). these patients can be distinguished by disabling tremor, poor response to intravenous immunoglobulin and distal and sensory disturbances. cell-adhesion molecule nfasc and cntn are expressed on the paranodal junction (pnj) of nodes of ranvier, and play key roles on sodium channel clustering and glia-axon interactions. quantification of unmyelinated intraepidermal nerve fibers (ienf) is a useful parameter employed in small nerve fiber pathology diagnosis. in addition the immunohistochemistry evaluation of dermal nerve fibers allows to examine morphological changes of myelin sheath and ranvier nodes structure. we performed immunofluorescent colocalization studies using antibodies to visualize axons (protein-gene-product . , neurofilament, tubulin), sheath of myelin (myelin-basic-protein) and specifically nodal/paranodal/juxtaparanodal structures (pannfascin, nfascin , nfascin , caspr, cntn , potassium and sodium channels) in skin tissues from seronegative and seropositive cidp patients. we analyzed axon and myelin sheath damage, abnormal nodal-paranodal-juxtaparanodal architecture and morphometric parameter as internodal length of ranvier nodes. our results on skin biopsies from three igg nfasc -positive cidp patients revealed complete loss of nfasc staining at the paranodes, asymmetrical paranodes and widening of the nodes of dermal myelinated nerve fibers. one igg cntn -positive cidp patient showed abnormal nodal/paranodal immunostaining with different features as compared with igg nfasc -positive patients suggesting specific changes. however, such alterations were not found in four seronegative cidp patients. our data support the hypothesis that examining specific axonal and myelin markers could provide diagnostic and prognostic clues on nodo-paranodopathies. the goal of this study is attempt a possible correlation between the presence of serum autoantibodies and structural changes in nodal/paranodal regions of dermal nerve fibers in cidp patients. knowledge of autoantibodies expression in the peripheral myelinated nerves of cidp patients could serve for stratifying patients and potentially guiding personalized treatments. lunati a , lerat j , dzugan h , , rego m , magdelaine c , , bieth e , calvas p , cintas p , gilbert-dussardier b , goizet c , journel h , magy l , toutain a , urtizberea j , sturtz f , , lia as , . charcot-marie-tooth disease is one of the most frequent inherited peripheral neuropathies ( / ). so far, mutations in more than genes have been identified causing either the demyelinating form (type ) or the axonal form (type ). duplication of pmp gene is the most frequent cause of autosomal dominant demyelinating form. autosomal recessive demyelinating form is often due to sh tc gene mutations. patients suffer then from early severe neuropathy starting in the first decade. scoliosis and deafness are often observed. we analysed patients suffering from peripheral neuropathy, by multiplex-ligation-dependant-probe-amplification (mlpa), followed by targeted next-generation-sequencing (ngs) using a -gene custom panel designed for the diagnosis of charcot-marie-tooth and associated neuropathies. mutations of interest were verified by sanger sequencing. diagnosis was positive for patients. as expected, the most frequent mutation was the pmp duplication detected in patients. deletion of pmp was observed in patients and pathogenic point mutations were detected in patients. sh tc gene appeared to be the most frequently mutated with nine patients diagnosed. associated with known mutations, four new mutations have been identified: two nonsense mutations and two missense mutations. all these patients presented deafness and/or scoliosis. sh tc appears to be an important gene involved in charcot-marie-tooth disease, often associated with deafness and /or scoliosis. it is important to pay attention to these associated symptoms in charcot-marie-tooth patients in order to guide their diagnosis and to improve their medical care. lupo v , , frasquet m , , sánchez-monteagudo a , , barreiro m , alberti ma , casasnovas c , quintáns b , , , camacho a , domínguez c , sedano mj , pelayo al , pardo j , sobrino t , sobrido mj , , , sevilla t , , espinós c , . mme (membrane metalloendopeptidase) mutations, inherited in an autosomal recessive fashion, have been recently identified in japanese probands (higuchi et al. ) . thus, mme has been included in the list of cmt genes as a new autosomal recessive axonal form, cmt t (mim ), and moreover, it is considered a strong candidate for the genetic diagnosis of unsolved late-onset cmt cases. in fact, few months later auer-grumbach et al. ( ) reported european probands with autosomal dominant late-onset cmt and mutations in mme. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy using an updated version of our custom gene panel, which includes mme. in this study, we report probands with cmt , intermediate cmt or dhmn/cmt and homozygous or compound heterozygous mutations in mme, and proband with cmt and a heterozygous mutation in mme. we have identified different type of mutations: novel splice donor variant, frameshift, nonsense and missense mutations. the two nonsense changes, p.trp * and p.arg *, and the splice donor variant c. + g>a, were detected in homozygous or compound heterozygous state in patients, while the frameshift mutation, p.pro leufs* , was detected in homozygous or heterozygous in the remaining patients. strikingly, the two nonsense and the frameshift mutations had been previously reported as causative for autosomal-dominant cmt t (auer-grumbach et al. ) . out of three missense mutations, one is novel (p.his tyr), and two are reported in control databases (p.asn lys and p.arg trp). this study shows that the autosomal recessive cmt t is common in spanish population, and moreover, it suggests that screening of mme using gene panel testing could help to improve diagnosis of unclarified inherited peripheral neuropathies cases. funds: isciii (pi / , pi / ); fundació per amor a l' art. inherited peripheral neuropathies (ipns) encompass a group of disorders highly heterogeneous, clinically and genetically. charcot-marie-tooth (cmt) disease is closely related to distal hereditary motor neuropathy (dhmn) or distal spinal muscular atrophy (dsma), and some patients show additional signs associated with amyotrophic lateral sclerosis (als). targeted gene panel and exome sequencing are considered to be powerful and cost-effective tools for diagnosis of these disorders. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy: families were investigated by exome sequencing and, cases were tested using different updated versions of a gene panel . each version comprises , or ipn genes, respectively and it shows a high coverage performance: percentage of analyzable target base with > coverage was , %. both exome and gene panel capture libraries were based on sureselect capture technologies (agilent technologies), and sequencing was performed in miseq or hiseq illumina equipment. we have identified novel genes and novel mutations in known genes, broadening the phenotypical spectrum associated with ipns. exome sequencing has allowed us to identify causative gene in % of familiar or sporadic cases: morc , aars, bscl , kif a, gars, egr , fig , dnajb , drp , ighmbp , dao, sod , fig . gene panel testing has been mostly performed in sporadic cases, and it has allowed us to identify either disease-causing or candidate mutations in % of cases: kif a and bicd were the most common genes mutated. update gene panels neuro and neuro have revealed novel mutations in genes recently associated to cmt and cmt disease: mme and pmp , respectively. in sum, both strategies have helped us to achieve a more accurate clinical and genetic reclassification of these disorders, an impossible challenge using conventional sequencing methods. our study expands the clinical phenotype previously associated to known ipn causing-gene, and emphasizes that gene panels should be considered as a first diagnosis method for unclarified ipn patients. funds: isciii (pi / , pi / , pi / ); fundació per amor a l' art. magy l , mathis s , goizet s , tazir m , vallat j-m . department and laboratory of neurology, national reference center for rare peripheral neuropathies, chu limoges, france; department of neurology, chu bordeaux, france; department of medical genetics, chu bordeaux, france; department of neurology, chu algiers, algeria. charcot-marie-tooth (cmt) disease is a hereditary neuropathy with a relatively homogeneous phenotype but is genetically heterogeneous. moreover, nerve conduction studies distinguish different forms, adding another level of complexity. the current classification of cmt being difficult to understand for physicians, scientists and patients, we presented and published a proposal for updating this classification, based on inheritance, nerve conduction findings and gene/mutation involved. inputs from colleagues prompted us to conduct a survey in order to try to reach some consensus about our proposals. we conducted an internet survey between october and december . the link to complete the survey was sent several times by email with an introduction to more than people. participants were contacted through the emailing list from the last cmt meeting in venice (september ) and additional physicians and scientists who are involved in cmt care and research were contacted as well. one hundred seven people from various countries (mainly france, italy and the usa) answered the survey. most ( %) of the participants were between and years of age, % being physicians and % being scientists. the vast majority ( %) considered the proposal constituted an improvement over the historical classification whereas % wanted to keep the old one. about the order of information, % of participants thought the mode of inheritance should come first, whereas % felt the phenotype should be placed at the beginning. ninety-one percent of people thought cmt should be kept as a generic name for hereditary sensory and motor neuropathy. for pure sensory neuropathy, % favoured hsn over hsan although % thought the opposite and % of participants felt dhmn should be kept for distal motor neuropathy. about nerve conduction findings, % of participants thought the intermediate phenotype has to be kept and % favoured our proposal to replace " " by "de" (for demyelinating) and " " by "ax" (for axonal). finally, % of responders thought that genetic information should be included in the classification of cmt. overall, our proposal of a new classification received a very good appreciation from physicians and scientists implicated in the care of patients with hereditary neuropathy. mallik r , hubsch a , gaida a , barnes d . csl behring, kop, usa; csl behring, bern, switzerland; csl behring, ottawa, canada. the risk of hemolytic events (hes) with intravenous immunoglobulin (ivig) therapy appears to be linked to the isoagglutinin (anti-a and anti-b) level of the specific ivig product. using published anti-a and anti-b titers for seven ivig products and corresponding he rates reported to the eudravigilance database, we developed a mathematical model to predict the risk of he to patients receiving ivig products of given anti-a and anti-b levels. modeling was performed separately for the risk to patients with blood groups a, b, ab and o and the overall population risk was estimated assuming a blood group distribution of % a, % b, % ab and % o. applying the prediction model, we calculated the he risk for an ivig product produced via a chromatographic process (privigen ® , csl behring) a) without any isoagglutinin reduction measures ( ) ( ) ( ) ( ) ( ) ( ) ( ) , b) with an anti-a donor screening program eliminating approximately % of donors with high anti-a titers ( ) ( ) ( ) , c) incorporating an anti-a/anti-b specific immunoaffinity chromatography (iac, igisolo tm ) step in the manufacturing process (since ) and d) with both measures (b and c) combined; as well as for an ivig product produced with a cohn-like cold ethanol fractionation process (carimune ® nf/sandoglobulin ® , csl behring). isoagglutinin titers in ivig products, measured by european pharmacopoeia direct assay, were provided by dr c bellac, swissmedic, bern, switzerland. the predicted risk was highest with the chromatographically purified ivig without isoagglutinin reduction ( . cases expected per kg ivig used). anti-a donor screening reduced the predicted risk to . cases/ kg. a greater risk reduction was predicted with the iac isoagglutinin reduction step ( . cases/ kg). the combination of both methods produced little benefit ( . cases/ kg) versus iac alone. the predicted hemolytic risk with ivig produced by cohn-like ethanol fractionation was low ( . cases/ kg). an observational cohort study to confirm these hemolytic risk reductions is in progress. at present, the observed hemolytic risk for anti-a donor screening appears consistent with the prediction calculated by the model; results for iac isoagglutinin reduction are expected in . the cmt infant scale (cmtinfs) is an outcome measure of functional ability for young infants and children aged < years. cmtinfs aligns with the cmt pediatric scale and cmt neuropathy score to measure disease severity across the lifespan. to measure gross motor and fine motor function, cmtinfs comprises of two subscales: gross motor (e.g. head control, crawling, walking, jumping and hopping) and fine motor function items (e.g. grasping, reaching, tearing paper and buttoning). overall and subscale-specific function is expressed as a z-score based on normative reference values (positive z-scores indicate poorer function). a total of controls aged - months (mean age , sd m) have been assessed across australia (n= ), thailand (n= ) and usa (n= ). total cmtinfs z-scores did not differ significantly between sites (australia vs thailand) (p= . ) or gender (p= . ). data collection is ongoing and infants aged < years are eligible for inclusion. to date, infants ( % male) aged - months (mean age , sd m) with a range of cmt subtypes ( cmt a, cmt d, cmt c, cmt x and unidentified gene) have been assessed with cmtinfs. mean total z-score for infants with cmt ( . , sd . , range: − . - . ) was significantly higher than controls ( . , sd . , range: − . - . , t=− . , p= . ). differences between affected infants and controls were larger in infants older than months. infants with cmt a (cmtinfs z-score . , sd . ) and cmt c (z-score . ) were less affected than cmtx (z-score . ) and cmt d (z-score . ). the gross motor function subscale differed significantly between cmt cases and controls ( . , sd . vs . , sd . ; p=. ) and a significant difference was also observed for the fine motor function subscale ( . , sd . vs . , sd . ; p= . ). reliability, factor and rasch analysis of the cmt-infs is underway to assess validity. initial results support the sensitivity of cmtinfs in distinguishing between infants with and without cmt. preliminary analyses also suggest the scale is sensitive to genetic subtype. with increased power, cmtinfs promises to become a useful outcome measure of disease severity and function in infants with cmt. manso c , querol l , mekaouche m , illa i , devaux j . aix-marseille université, marseille, france; universitat autónoma de barcelona, barcelona, spain. contactin- , contactin-associated-protein- (caspr ), and neurofascin- (nfasc ) are essential for the formation of paranodal axoglial junctions. igg autoantibodies to contactin- , caspr , and nfasc are associated with subsets of patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp) presenting with common clinical features. anti-contactin- igg autoantibodies have been shown to be pathogenic and to affect the paranodal axoglial junctions in vivo and in vitro. by contrast, the pathogenic effect of anti-nfasc igg have not been demonstrated. here, we purified anti-nfasc igg from cidp patients' plasma and investigated their effects after passive transfer. to determine whether these antibodies can pass the paranodal barrier, we performed intraneural injections of anti-nfasc igg autoantibody. by contrast to anti-contactin- igg , anti-nfasc did not penetrate the paranodal regions after intraneural injections, but bound to the surface of the schwann cell. to perform chronic exposure, lewis rats were implanted with intrathecal catheter and anti-nfasc igg were administrated in a daily manner during three weeks. igg to nfasc , but not control igg , induced progressive clinical deteriorations characterized by gait ataxia and hindlimb paraparesis. these deteriorations were associated with nerve activity loss in motor spinal nerves and with a selective loss of the paranodal specialization characterized by the disappearance of the caspr /contactin- /nfasc complex at paranodes. the passive transfer of anti-nfasc igg thus seem to induce similar pathogenic effects as the anti-contactin- igg . however, the pathogenic mechanisms leading to paranode disappearance appear different. our findings indicate that igg directed against nfasc are pathogenic and further show that these antibodies are reliable biomarkers of a specific subset of cidp patients. better fit the needs of the consortium and improve patient experience. these enhancements include creating mobile friendly webpages, updating enrollment form content, access to a customized dashboard, and the ability of registrants to explore their data in comparison to other registrants. we will review these enhancements in depth, and demonstrate their impact on the growth and development of the rdcrn inc contact registry. martinez c , hubsch a , watson dj , shebl a , wallenhorst c , simon tl . institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl bering ag, bern, switzerland; csl behring llc, king of prussia, usa; csl bering gmbh, marburg, germany. hemolytic anemia (ha) is a complication of intravenous immunoglobulin (ivig) treatment, particularly in patients receiving high dose ivig for immune modulation, such as guillain-barré syndrome or chronic inflammatory demyelinating polyneuropathy. the primary mechanism for the increased risk is believed to be passive acquisition of anti-blood group a and b antibodies (isoagglutinins) from the ivig product. to reduce the quantity of isoagglutinins, an anti-a donor screening was implemented for the ivig privigen ® from - and donors with high titers were excluded from contribution to pooled plasma. anti-a donor screening was replaced since with an immunoaffinity chromatography step, which decreases isoagglutinins to a greater extent, but no data are available to test its clinical effectiveness. to test the effectiveness of the donor screening, two cohorts of patients treated with privigen ® before and after start of donor screening were identified from a hospital-based administrative database of us hospitals with in-and outpatient discharge diagnoses, procedures, drug utilization and laboratory tests between / and / (period ) and between / and / (period ). privigen ® dose per kg body weight was estimated from the daily quantity administered and age-and sex-specific us population body weight estimates. ha within days of privigen ® use was assessed from manual records review and the incidence rate of ha in the two periods compared. incidence rate ratios (irr) of ha were adjusted for sex, age, treatment setting, indication and dose per kg body weight using period as reference. the incidence rate of ha was . / , person-days ( % confidence interval: . - . ) in period ( has in , person-days) and . ( . - . ) in period ( has in , person-days). the adjusted irr was . ( . - . ). significantly less ha risk was found with high dose (≥ . g/kg body weight) privigen ® , irr . ( . - . , p= . ). we conclude that anti-a donor screening and exclusion of donors with high anti-a titers from plasma pools is associated with a decreased risk of ha with ivig. matsumoto a . department of neurology, keijinkai jozanki hospital, sapporo, japan. subacute myelo-optico neuropathy (smon) is the intoxication of clinoquinol with main clinical symptoms of paresthesia and spasticity of legs. these symptoms have been considered to be elicited by the disturbance of spinal cord and peripheral nerve as the intoxication of clinoquinol. however, as to the patients with smon who have been still living after the onset of disease, the examination of nerve conduction velocities are in normal ranges and the clinical symptoms of peripheral neuropathy are not observed now. in order to investigate whether the peripheral neuropathy were observed in the early stage of smon, we investigated the longtidunal changes of electrophysiological results in patients who could examine the nerve conduction studies from early stage of smon until the present time. as to the disturbance of pyramidal tract functions (myelopathy) in smon patients, the central motor conduction times were calculated by transmagnetic stimulation of motor cortex, cervical roots and lumbar roots. the peripheral nerve conduction velocities of sensory nerve were examined with the sural nerves. as the results, in patients with smon who could examine the electrophysiological examination from the early stages of smon until to years later, the central motor conduction times of leg muscles from motor cortex to lumbar roots were prolonged in the smon patients compared to the normal cases. these results suggest the presence of disturbances of conduction velocities of spinal cord. conduction velocities of sensory nerve velicities (sncvs) showed the delayed sncvs of sural nerves( - m/sec) at the first examination from the onset of - years. however from to years later, sncvs of these cases were covered to - m/sec. from these electrophysilogical examinations, it was suggested that the presenting main symptoms of smon were the disturbance of myelopathy, and the disturbance of peripheral nerve function had been recovered after onset of smon being elapsed a long time, natural killer (nk) cells are part of our innate immune system with regulatory and effector functions. they comprise the first line of defence in the recognition and destruction of virus-infected and pathologically altered cells. different studies suggest that the treatment with intravenous immunoglobulins (ivig) has an immunomodulatory effect on nk cells. ivig is a first-line treatment for various autoimmune diseases in particular in chronic inflammatory demyelinating polyneuropathy (cidp). the lack of a predictive marker for ivig responsiveness in cidp avoids the early preservation of non-responding patients. to better understand the effect of ivig in patients with cidp, we tested whether ivig treatment altered the nk cell status. additionally, we analysed if the alteration in the populations may serve as a surrogate marker in predicting the outcome of ivig treatment. using semi-quantitative pcr and flow cytometry in the peripheral blood of patients with cidp, we analysed the effects of ivig on the nk cell population before treatment initiation and h after first dose and correlated the changes with the reponsiveness to ivig. ivig administrations induced a reduction in the expression of several typical nk cell genes. interestingly, this ivig-induced reduction of nk cells was reversible four weeks after the ivig treatment. flow cytometry data revealed that ivig reduced the cytotoxic cd dim nk cell population, while regulatory cd bright nk cells remained almost unaffected or were even increased. interestingly, we found that the observed effects on nk cells almost exclusively occurred in cidp patients who responded to ivig therapy. correlation between the changes in the nk cell population and treatment efficiency suggests a crucial role for nk cells in the immunomodulatory mechanism of ivig. further studies are warranted to investigate whether the differences in the nk cell status of patients with cidp represent a reliable surrogate marker in predicting the outcome of ivig therapy. mccray b , sullivan j , woolums b , aisenberg w , lloyd t , sumner c . johns hopkins university, baltimore, usa. mutations in transient receptor potential vanilloid (trpv ), a calcium-permeable non-selective ion channel, cause charcot-marie-tooth disease type c (cmt c). trpv is unique in that it represents the only membrane-expressed ion channel in cmt and thus a potential therapeutic target. previous work has suggested that trpv mutations lead to gain of channel function and toxicity in cultured cells. neuropathy-causing mutations of trpv largely cluster in the cytosolic ankyrin repeat domain (ard) that is known to mediate protein-protein interactions, suggesting that pathogenesis may be related to disruption of such interactions. in order to identify trpv -interacting proteins, we performed two unbiased proteomics screens and identified multiple cytoskeletal-modifying proteins including syndapin- , a neuronal protein known to promote axonal outgrowth by influencing the actin cytoskeleton. in cultured cells, we have shown that trpv and syndapin- co-localize to highly dynamic actin-rich cellular processes and together stimulate robust neurite extension, but this facilitation of neuritogenesis is impaired by disease-causing mutations in trpv . we have also shown that over-expression of syndapin reduces trpv -mediated calcium influx in cultured cells and rescues toxicity of mutant trpv . in addition, syndapin over-expression suppresses mutant trpv phenotypes in a drosophila model of trpv -related neuropathy. further, we have demonstrated that treatment of drosophila with a specific trpv channel antagonist ameliorates trpv mutant toxicity. together, our data highlight the importance of trpv interaction with cytoskeletal proteins such as syndapin- in the pathogenesis of cmt c. specifically, our results suggest that mutations in trpv disrupt the normal role of trpv in regulation of cytoskeletal dynamics and that interactions with the cytoskeleton reciprocally modulate trpv channel function and influence toxicity of mutant trpv . mcgonigal r , yao d , barrie ja , crawford c , willison hj . university of glasgow, glasgow, uk. guillain-barré syndrome (gbs) is in part mediated by anti-gm ganglioside antibodies induced by preceding infections. anti-gm antibodies target plasma membrane gm that is extensively distributed in both glial and axonal membranes, particularly at the node of ranvier. antibodies deposited at this site in models of gbs are associated with complement deposition, conduction block, structural disruption of ion channels and macrophage infiltration. the wide distribution of the gm ganglioside target leads to unwanted complexity in ascribing pathological outcomes to injury of cell-specific membranes, in particular unravelling the consequence of paranodal schwann cell membrane injury on axonal function, and vice versa. to overcome this impasse, we have generated transgenic mice through glycosyltransferase manipulation that express gm exclusively in neurons or glia, thus allowing us to very specifically target and injure axonal or glial membranes with a single anti-gm ganglioside antibody. through this route we can create mouse models of both the axonal and demyelinating forms of gbs, induced by a single anti-gm antibody, thus creating otherwise highly comparable conditions. here, we show anti-gm antibody binding is restricted to the nodal axolemma in galnact −/− -tg(neuronal) mice and conversely to paranodal loops in galnact −/− -tg(glial) mice. when anti-gm antibody and a source of complement is added to a nerve-muscle ex vivo injury paradigm, there is a loss of axonal integrity (i.e. loss of neurofilament immunolabeling) when the neuronal membrane is targeted in galnact −/− -tg(neuronal). conversely, axonal integrity is maintained when the paranodal membranes are decorated by antibody and complement products ex vivo in galnact −/− -tg(glial) mice. in a passive immunisation model in vivo, galnact −/− -tg(neuronal) mice acutely develop weakness, respiratory dysfunction, associated complement deposition, and degenerative pathology in distal axons. in contrast, galnact −/− -tg(glial) mice have significantly fewer abnormalities under the same acute conditions. these data indicate the high vulnerability of axonal membranes to acute injury and underline the importance of developing specific axonal protection strategies. in summary, targeting the nodal axolemmal or glial membranes allows us to study associated nodal pathology, and determine the downstream consequences on function and axon fate, currently a major area in gbs clinical research. memon a , madani s , schultz l , grover k , arcila-londono x , sripathi n , ahmad bk . neuromuscular division, department of neurology, henry ford hospital, detroit, michigan, usa. objective: to differentiate sensory electrophysiology, tli and treatment response in patients with paraproteinemic cidp. background: low tli has been reported as a useful electrophysiological marker for mag-cidp. to our knowledge comparison of sensory electrophysiology and tli of paraproteinemic cidp subgroups have not been previously reported. methods: retrospective review(january -december ) of patients with cidp fulfilling electrophysiological criteria(aan ad hoc subcommittee and albers and colleagues).cidp patients with diabetes(n= ) were excluded. patients were divided into idiopathic (n= ) and paraproteinemic cidp(n= ). paraproteinemic cidp sub-groups: mag( ), non-mag( ) and igg( ) were compared to idiopathic cidp( ). these groups were compared for demographics, history of cancer, csf protein, sensory conductions, tli measurements and response to treatment using chi-square tests for binary and categorical variables and t-tests for continuous measures. results: there was a higher proportion of females in idiopathic-cidp compared to non-mag-cidp ( % vs %). idiopathic group having a higher proportion of patients on monotherapy( % vs %) and combination therapy( % vs %) compared to non-mag. higher mean csf protein compared to mag-cidp(p= . ) was seen in the idiopathic. the difference between idiopathic and igg-cidp was significant for overall rx response(p= . ) and rx response in patients with follow-up(p= . ). for both variables, patients in the idiopathic group had a higher proportion of patients on combination therapy and lower proportion of no treatment offered compared to patients in the igg-cidp. % of non-mag-cidp patients had a history of cancer vs % of mag-cidp. none of the other differences were significant. there were no group differences in sensory electrophysiology and tli. conclusions: sensory electrophysiology and tli may have no value in differentiating paraproteinemic cidp. csf protein is higher in idiopathic cidp compared to mag-cidp. idiopathic-cidp has a higher proportion of females compared to non-mag-cidp and a higher proportion of patients on combination therapy compared to igg-cidp. cancer screening should be considered in patients with non-mag-cidp. memon a , madani s , ahmad bk , schultz l , grover , arcila-londono x , sripathi n . department of neurology, henry ford hospital, detroit, michigan, usa. introduction: sensory electrophysiology and terminal latency index (tli) differences have been described in various cidp sub-groups. objective: evaluate electrophysiology, tli and treatment response in idiopathic and diabetic cidp. methods: retrospective review of patients with cidp who underwent electrodiagnostic evaluation (january -december . patients fulfilled electrophysiological criteria described by ad hoc subcommittee of american academy of neurology (aan) and albers et al. we excluded patients( ) with acute inflammatory demyelinating neuropathy, hereditary sensorimotor neuropathy, vasculitis and polyneuropathy with paraproteinemia. patients were divided into idiopathic( ) and diabetic( ) groups. these groups were compared for age, sex, history of cancer, csf protein, response to treatment, sensory response abnormalities and tli measurements using chi-square tests for binary and categorical variables and t-tests for continuous measures. all testing was at the alpha= . level. results: group differences for age, sex, history of cancer, csf protein and treatment response were not significant. comparing tli values in measurable responses, the difference between the two groups for tibial tli was significant (p= . ), with idiopathic group having a lower mean as compared to the diabetic. tli values differences for median, ulnar and peroneal nerves were not significant. the difference in abnormal rates of sensory responses was significant for the sural nerve with the idiopathic group having a lower rate compared to the diabetic group ( % vs %, p< . ). no differences were noted for the ulnar, median and radial nerves. tibial tli and sural sensory responses have some value in differentiating the two groups. larger prospective studies are needed to confirm our findings. ivig is an important treatment option for cidp. although recommended by treatment guidelines, little is known about the consequences of temporary ivig withdrawal to assess ongoing immunoglobulin need. path is a randomized, double-blind trial of the subcutaneous immunoglobulin (scig) igpro (hizentra ® , csl behring) in cidp. before scig randomization, subjects underwent periods of ivig withdrawal ( weeks or until pre-determined indications of clinical deterioration) and ivig restabilization (igpro ; privigen ® , csl behring). subjects not showing deterioration during withdrawal period were withdrawn from study. to proceed to scig randomization, subjects had to achieve "cidp stability" (no relevant change in incat score at last two restabilization visits and at least the same total score as at screening). subjects entered the ivig withdrawal period. ( %) of these qualified for igpro restabilization; subjects ( %) were not ivig dependent, and ( %) were withdrawn for other reasons. one subject withdrew consent before igpro dosing. at the end of the igpro restabilization period, cidp stability was achieved in % of subjects ( did not reach stability, were withdrawn for other reasons). post-study follow-up information was available for / subjects who did not reach stability: ( %) had improved to baseline clinical status and had not, meaning at least % of the subjects improved to their pre-study status. during the restabilization period, / subjects ( %) improved in at least one of the predefined outcome measures. on average, subjects improved by . points in incat total score, . points in i-rods centile score, kpa in mean grip strength (dominant hand), and . points in mrc sum score. improvement occurred with a median of days after the first igpro dose in one or more efficacy outcome measures and in % of cases after the third igpro maintenance infusion. headache and nasopharyngitis were the most frequently reported adverse events (aes) during restabilization. aes deemed causally related were mostly mild or moderate. no unexpected aes or laboratory or vital sign findings associated with igpro occurred during the study. in summary, igpro reversed neuromuscular disability and improved activity/participation after previous clinical deterioration during an ivig withdrawal period. pmp duplication is the most frequent cause of charcot-marie-tooth disease (cmt). since it discovery, more than genes have been identified to be potentially responsible for cmt disease. however only single nucleotide variations (snvs) or small indels have been described. this could be due to the new sequencing strategy (ngs), especially ngs by amplicon sequencing, for whose few convenient tools are available and easily usable to detect cnvs responsible for inherited disease. to overcome this problem, we designed "cov'cop", a user-friendly tool able to detect cnvs among amplicons sequencing data. using the run's coverage file provided by the sequencer, "cov'cop" simultaneously analyzes all the patients of the run using a two-stages algorithm containing correction and normalization levels and provides an easily understandable output, showing with various colors, potentially deleted and duplicated amplicons. we validated our method on several datasets, including those of our targeted ngs panel screening genes known to be involved in cmt and close pathologies. cov'cop detected easily pmp duplication and deletion in our patients, confirmed by mlpa. in addition, cov'cop permitted the detection of new cnvs different from the pmp duplication, in cmt patients. we confirmed these cnvs by quantitative pcr and cgh array. we present here one of these cnvs: the duplication of aars gene detected in cmt patients and we discuss the pathogenicity of this new cnv. additional cnvs responsible for cmt disease are probably still to be discovered and we believe that cov'cop will help molecular geneticists to rapidly identify them. poems (polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscraisia and vascular endotherial (vegf) overproduction. although number of therapeutic interventions for plasma cell dysorders have been applied to poems syndrome, there have been no randomized clinical trials. this phase / double blind, randomized, placebo comtrolled trial was performed to investigate the safety and efficacy of thalidomide for patients with poems syndrome who are not eligible for stem-cell transplantation. the primary endpoint was the reduction rate of serum vegf concentrations at weeks in intention to treat analysis. additional outcomes of long-term extension study included progression-free survival. twenty-five poems patients were randomly assigned to either thalidomide plus dexamethasone or placebo plus dexamethasone from nov , , to july , . one patient in the placebo group was excluded from analyses because of a protocol violation. the adjusted mean serum vegf reduction rate at weeks was . (sd, . ) in the thalidomide group compared with − . ( . ) in the placebo group (p= . ). the kaplan-meier rate of progression-free survival at months was . in the thalidomide group, as compared with . in the placebo group (hr, . ; % ci, . to . ). in the randomized study period, mild sinus bradycardia was more frequent in the thalidomide group than in the placebo group ( % vs %; p= . ). thalidomide suppresses serum vegf concentrations and lengthened pregression-free survival in poems patients who are ineligible for stem cell transplantation. although thalidomide treatment has a risk of bradycardia, the benefits would exceed the risk. this study is registered with the umin clinical trials registry, umin . montes-chinea ni , coutts m , vidal c , courel s , rebelo a , abreu l , zuchner s , saporta, ma , . department of neurology, university of miami, miami, usa; department of human genetics, university of miami, miami, usa. autosomal dominant mutations in synaptotagmin- (syt ), a synaptic vesicle protein that functions as a calcium sensor for neurotransmission, have been previously linked to presynaptic neuromuscular junction (nmj) dysfunction and motor neuropathy in two families. both pathogenic mutations (asp ala and pro leu) were located in the c b domain of syt , which is essential for neurotransmitter release at the nmjs. we report a family with a new missense mutation in the c b domain of syt and a similar phenotype characterized by a slowly progressive, predominantly motor neuropathy and evidence of presynaptic nmj dysfunction on nerve conduction studies. the index case is a year-old woman with gradually progressive weakness of her extremities. she had normal developmental milestones, but was found to have bilateral high arched feet and hammertoes and occasional falls around the age of . she gradually developed progressive leg weakness, worsening bilateral hand cramping, weak handgrip, and only mild paresthesias on distal extremities. family history is remarkable for similar symptoms reported by her maternal grandfather, two maternal uncles, her mother and a younger sister. her neurological exam revealed inability to walk on heels or toes, significant distal lower extremity weakness and absent ankle deep tendon reflexes. cranial nerve examination and coordination were normal and there were only non-specific sensory changes in the lower extremities. emg/ncs revealed normal sensory responses throughout; however, motor nerve evaluation demonstrated globally reduced amplitudes with a > % increment after brief isometric contraction. further electrophysiological evaluation with slow ( hz) repetitive nerve stimulation of the right ulnar motor nerve revealed a % decremental response in amplitude and a > % increase in amplitude immediately after a one-minute period of sustained muscle contraction, which rapidly extinguished after one minute. voltage-gated calcium channel (vgcc) antibodies and a chest ct were normal. targeted sanger sequencing revealed an ile lys mutation in syt , which is located in the c b domain and is predicted to impair protein function. syt -related neuropathy is a rare disease, but should be suspected in patients presenting with a combination of pre-synaptic nmj dysfunction (resembling lambert-eaton myasthenic syndrome) and a predominantly motor neuropathy, especially in the context of a positive family history. charcot-marie-tooth (cmt) disease affects roughly in , individuals and is described as an inherited peripheral neuropathy primarily affecting distal muscles. limited studies detail patient-reported impact of muscle weakness on functional activities. this anonymous survey was developed with input from clinical experts and patient interviews and aimed to better understand the prevalence and impact of various cmt clinical manifestations on patients' lives. the survey was administered online to the hereditary neuropathy foundation's (hnf) patient contact database and is ongoing through june . here we present preliminary data on patient characteristics and disease impact for cmt patients collected february - , . respondents were mostly female ( %) and mostly from the us ( %). median age (range) at symptom onset was years ( - years), at diagnosis was years ( - years), and at present was years ( - years). the sample was representative of all cmt types (cmt , , , ,and x). the most common physical and clinical manifestations of cmt were problems with balance ( %), ankle weakness/foot drop ( %), loss of feeling or abnormal sensation in the lower leg/foot ( %), and hand muscle weakness ( %). maintaining balance, walking long distances, and climbing up and down stairs were key challenges associated with ankle weakness/foot drop. foot drop was considered by % to be the primary factor contributing to falls, which averaged . falls to ground per month. of those with foot drop, % had bilateral weakness. a majority of respondents ( %) used some form of assistive device for mobility, including ankle-foot orthotics/below-the-knee leg braces ( %), canes/walking sticks ( %), and custom foot orthotics/inserts ( %). the most common drug therapy included pain and anti-inflammatory medications ( %). foot surgery was the most common surgical procedure received ( %) and toe surgery was the most common surgery considered ( %). key symptoms that affected quality-of-life "very much" included problems with balance ( %), ankle weakness (foot drop) ( %), and fatigue ( %). these data suggest a high prevalence of lower leg muscle weakness; therefore, therapies aimed at improving ankle weakness and the resulting foot drop and imbalance may be beneficial to patients' daily functioning and quality of life. morano m , , gambarotta g , ronchi g , , cillino m , fornasari be , , fregnan f , , tos p , cordova a , moschella f , geuna s , , raimondo s , . department of clinical and biological sciences, university of turin, orbassano (to), italy; neuroscience institute of the "cavalieri ottolenghi" foundation (nico), university of turin, orbassano (to), italy; plastic and reconstructive surgery. department of surgical, oncological and oral sciences, university of palermo, palermo, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. nerve fiber regeneration and complete functional recovery after peripheral nerve injury do not always occur and can be influenced by many factors including patient age, gender, lesion site, injury severity, size of the gap between damaged nerve stumps and time interval that elapses before performing surgical repair. the poor outcome occurring after a long delay can be due to loss of the neuron ability to regenerate, loss of the schwann cell ability to support regeneration and, of course, progressive muscle atrophy. the aim of this study was to investigate the nerve regeneration after delayed repair and to study the degenerative processes of the denervated distal nerve stump and denervated muscle. in particular, the analyses were focused on the role of nrg/erbb system, that is expressed both in nerve and in muscle tissue, during degenerating and regenerating processes. functional recovery analysis performed after nerve repair showed that only the group repaired immediately and not the groups repaired with a delay of or months, recovered partially. nevertheless, morphological analyses demonstrated that, despite the delay, the nerve fibers are still able to regenerate, even if they are fewer and smaller than the immediate repaired group. moreover, the analysis of the nrg /erbb system showed a significant decrease of soluble nrg in both degenerating and delayed-repaired nerves. the poor outcome after delayed nerve regeneration might be explained by schwann cell impairment and the consequent ineffective support for nerve regeneration. as regards denervated muscle analysis, results showed that erbb receptors expression is related to the innervated state of the muscle, with an upregulation of erbb clearly associated with denervation state. interestingly, nrg isoforms are differently regulated depending on the type of nerve injury. future experiments will be needed to address the in vivo efficacy of different isoforms of nrg both in injured nerve and denervated muscle. we planned a multicenter, prospective, randomized, single blind, controlled study to evaluate the efficacy and safety of an innovative rehabilitation protocol based on the use of treadmill training in a cmt a population. the protocol required that subjects were blindly randomized into two treatment groups, spe (three months of respiratory, proprioceptive and stretching exercises) or trespe (the same treatment plus aerobic training at the treadmill). subjects were evaluated at baseline (t ), after three month of treatment (t ) and further three months of follow up free of therapy (t ). the full assessment included: -mwt (primary outcome measure), -mwt, walk- , short physical performance battery (sppb); lower limbs dynamometric strength evaluation; berg balance scale (bbs); cmt neuropathy score; medical outcomes study short form (sf ). a total of subjects (mean age of . ± . years) were recruited. at t we found a significant improvement in both groups in the -mwt (p< . ), -mwt (p< . ), bbs (p< . ) and sppb (p< . ), while at t only the -mwt was still significantly improved (p< . ) in the spe group. no significant differences between groups were observed for any of the outcome measures. performances on walk did not significantly change during follow-up (p= . ). concerning the sf , we did not observe consistent changes during follow-up or consistent differences comparing the two treatments. in conclusion, this multicenter, prospective, randomized, single blind, controlled study shows that the combination of respiratory, proprioceptive and stretching exercises has a positive impact on the performance of cmt patients, especially regarding walking tests. the aerobic exercise at the treadmill, is well tolerated but apparently does not add any further improvement to the conventional treatment. we speculate that the relatively low clinical severity of the patients, due to the selection criteria, may have prevented a positive effect of treadmill exercise. müller m , dohrn m , romanzetti s , reetz k , gess b . university rwth aachen, department of neurology, aachen, germany. muscle mri is increasingly used in neuromuscular patients to detect changes in muscle volume, muscle fat infiltration and edema. muscle mri are mostly analyzed by qualitative means as quantitative analysis is time-consuming and not well established. here, we developed a novel method for semi-automated segmentation of muscle mri data sets. based on axial t -weighted dixon mri stacks, muscle volumes were quantified by an adapted water-shed algorithm. muscle volumes of thighs and calves were determined separately and the ratio of thigh/calf was calculated. myopathy, neuropathy patients and healthy controls were included in the study. muscle volumes determined by semi-automated segmentation were very similar to manually segmented data sets, differences being < %. this was the case for patients as well as healthy controls. the time-saving effect of automated segmentation was very strong ( vs min. per patient). muscle volumes of the thigh and also of the calf of myopathy patients showed a highly significant difference (p< . ) compared to healthy subjects. in neuropathy patients there was a just significant difference (p< . ) of muscle volumes compared to healthy patients that did not sustain in bonferroni's multiple comparison test. the ratio of thigh/calf muscle volume was significantly different comparing patients with myopathy and neuropathy (p< . ). subgroup analyses of different groups of myopathy patients showed highly significant differences (p< . ) in myositis, limb-girdle-muscular dystrophy and metabolic myopathy, compared to healthy patients, but no significant differences in-between these groups. taken together, the data shows that automated segmentation of muscle mri allows for exact and fast quantification of muscle volumes in neuromuscular patients. higher patient numbers are necessary to test differences between specific disease groups. further studies should also address the possible use as a marker of disease progress for clinical studies or therapy monitoring. murakami t , nishimura h , nagai t , hemmi s , kutoku y , sunada y . department of neurology, kawasaki medical school, kurashiki, japan; department of pathology, kawasaki medical school, kurashiki, japan. familial amyloidotic polyneuropathy (fap) is an autosomal dominant hereditary systemic amyloidosis caused by mutation of transthyretin (ttr) gene, and usually shows sensory dominant polyneuropathy and autonomic neuropathy at the initial stage. the pathogenesis of neuropathy is not well understood, and explained by several mechanisms, including such as mechanical compression, vessel occlusion, ttr toxicity and schwann cell dysfunction. we describe a sporadic patient with late-onset fap due to ttr e k. she noticed dysesthesia first in the foot at age . the symptoms were slowly progressive, and abnormal sensations were extended up to the both upper arms and the both knees at age . distal muscle weakness and atrophy was also observed in the extremities. she noticed difficulties in walking and frequent diarrhea. echocardiogram revealed diffuse left ventricular hypertrophy, suggesting cardiac amyloidosis. amyloid deposits were not detected in the endoneurim or perineurium of the sural nerve years after the onset of the disease, but a marked loss of myelinaed and unmyelinated nerve fibers was observed in it. ttr-derived amyloid deposits were confirmed in the peroneous brevis muscle, salivary gland and heart tissue. dna analysis revealed the heterozygote mutation, p.e k (e k)/c. g>a, of ttr gene, and she was diagnosis as fap. these findings suggest that the proximal parts of peripheral nervous system might be strongly involved by ttr aggregates or amyloid fibrils. blood-nerve barrier in the distal part of peripheral nerves could be preserved until later in the patient. several biopsy sites other than nerve may be helpful and necessary for diagnosis of ttr amyloidosis in mild or late-onset fap as our case. amyloidgenic element (cae) encoded by the ' utr. this study also identified a de novo c. _ dup frameshift mutation predicting p.lys glnfs* in nefh from a cmt family with atypical clinical symptom of proximal dominant weakness. this mutation is located near the previously reported frameshift mutations, suggesting a mutational hot spot. these relatively frequent deletion/duplication events with this resign might be caused by the putative hairpin structure. patient's lower limb mri revealed a marked hyperintense signal changes in the hip muscles than those in the thigh or lower leg muscles. this study also observed an anticipation pattern of earlier onset ( yrs old for mother to yrs old for daughter) and more severe symptoms in later generation. therefore, this study suggests that the stop loss and translational elongations by the ' utr of the nefh mutations may be relatively a frequent genetic cause of axonal peripheral neuropathy with the specific characteristics of proximal dominant weakness and an anticipation pattern. neil j , haghi ashtiani b , choumet v , musset l , léger jm . department of immunology, pitié-salpêtrière hospital (ap-hp), paris, france; department of neurology, national referral center for rare neuromuscular diseases, pitié-salpêtrière hospital (ap-hp), paris, france; institut pasteur, emerging diseases epidemiology unit, paris, france. the myelin-associated glycoprotein (mag) is a transmembrane glycoprotein localized in periaxonal shwann cells and oligodendroglial membranes of myelin sheaths. mag contains a carbohydrate epitope (hnk- ) that is a target antigen in autoimmune peripheral neuropathy associated with monoclonal igm gammopathy. in these neuropathies, numerous studies report the absence of correlation between the titers of anti-mag antibodies and the disease course. anti-mag titers and igm level at diagnosis are not always associated with disease severity and there is not good correlation between pre-and post-treatment anti-mag titers in patients who respond clinically to immunomodulators. mag belongs to siglec- a family and the linkage of sialic acid to the underlying sugars is an important determinant of siglec binding. mag shows high affinity for alpha- , -linked sialic acid ( , -sa).moreover, human monoclonal igm possesses heavy chain glycosylation sites at asn , , , and with sialylated olidgosaccharides and high-mannose type oligosaccharides. igm may bind to mag via these glycan epitopes as an alternative and additional route of antigen binding other than through the fab v regions. this mag-glycans igm interaction may be clinically neutral but could lead to an overvaluation of the biological results. in this study, we analyzed sera from patients with igm reactivity against mag: of them had an anti-mag neuropathy with various degrees of severity, and the last one had igm monoclonal gammopathy, strong serum anti-mag reactivity but no neurological disease. igm were extracted and purified from these sera by affinity chromatography. for each batch, an aliquot was digested by jack bean alpha-mannosidase and anti-mag reactivity was performed by elisa and indirect immunofluorescence (iif), before and after demannosylation. these extracts, tested in an iso quantitative way with regard to the original serum, showed a decrease of activity (elisa) and intensity (iif) after demannosylation. furthermore, elisa anti-mag was carried out in sera from patients with igm monoclonal gammopathy without neurological impairment: of them ( . %) showed a significant biological response. taking into account the fact that anti-mag antibodies are pathogenic (in animals models), these results support the hypothesis of neutral intermolecular interactions between igm and mag. ng cjb , ng jph , tay lb, t , umapathi . yong loo lin school of medicine, national university of singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. patients with postural orthostatic tachycardia syndrome (pots) have marked tachycardia with no associated decrease in pbp. we asked if the reflex tachycardia, which corresponds to postural dizziness in these patients, could be a result of a decrease in casp. two male patients, and years of age, with clinical features typical of pots went through a complete battery of autonomic screening tests. the sympathetic and parasympathetic responses were normal other than a heart rate increase of and beats per minute, respectively, on standing. there was no significant decrease or increase in pbp on standing for and minutes. casp was measured non-invasively by a device bpro r that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure waveform from which a numerical casp value is derived. the casp measurements for both patients did not decrease on standing for and minutes. our preliminary observation suggests that the basis of tachycardia in pots patients may not be a decrease in central blood pressure. we are proceeding to systematically study more pots patients to corroborate the above observation. we are also trying to compare the difference between pbp and casp in pots and age, gender-matched normal controls. the major limitation of the study is the model-based mathematical derivation, rather than direct measurement, of casp. we are proceeding to systematically stud y more pots patients to corroborate the above findings. ng jph , ng cjb , t umapathi . lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university of singapore, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. orthostatic hypotension (oh) is a prominent component of autonomic dysfunction (ad), the consequent hypoperfusion of vital organs responsible for considerable morbidity and mortality. these structures are exposed to casp rather than pbp. we sought to understand the relationship of casp to peripheral blood pressure (pbp) in patients with autonomic dysfunction exposed to orthostatic stress. we reviewed autonomic function tests of patients tested at our laboratory over a -year period. the patients were divided into cohorts: ( ) no ad and no oh ( ) no ad, with oh ( ) mixed ad ( ) parasympathetic dysfunction ( ) sympathetic dysfunction. casp was measured non-invasively by a device, bpro r , that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure-wave form from which a numerical casp value is derived. the difference and ratio of casp to pbp was recorded at rest and after minutes of standing. out of patients had complete data and a definitive final diagnosis. cohorts - had , , , , patients respectively. mean casp-pbp difference in cohorts - were − . , − . , − . , − . , − . respectively at minutes, and − . , − . , − . , − . , − . respectively at minutes. mean casp/pbp ratio in cohorts - were . , . , . , . , . , . respectively at minutes, and . , . , . , . , . respectively at minutes. there was no significant difference in the response of casp and pbp to orthostatic stress across abnormal cohorts to and in comparison with the "normal" cohort (p= . to . ). there was also no relationship to symptoms, namely postural dizziness. in conclusion, autonomic dysfunction does not seem to affect the casp-pbp relationship as measured by non-invasive means. the absence of true normal controls, the exclusion of significant number of patients because of incomplete data and the model-based mathematical derivation rather than direct measurement of casp are limitations that we aim to address in follow-on studies. niu jw , guan hz , cui ly , guan yz , liu ms . the department of neurology, peking union medical college hospital, chinese academy of medical sciences, beijing, china. we aimed to explore the correlation between afterdischarges in motor nerve conduction studies and clinical motor hyperexcitability in patients with voltage-gated potassium channels (vgkc) antibodies. six patients with positive serum antibodies to contactin-associated protein-like (caspr ) or/and leucine-rich glioma-inactivated protein (lgi ) were recruited, including with autoimmune encephalitis, and with cramp-fasciculation syndrome. electromyography (emg), nerve conduction studies (ncs) and f waves were performed, and afterdischarges were assessed. one patient was followed up. five patients had clinical evidence of peripheral motor nerve hyperexcitability (myokymia or cramp), and four of them had abnormal spontaneous firing in concentric needle electromyography. prolonged afterdischarges following normal m waves were present in all six patients, including the two patients who had no emg evidence of peripheral nerve hyperexcitability (pnh). in the patient who was followed up, afterdischarges disappeared after treatment with intravenous immunoglobulin (ivig). afterdischarges in motor nerve conduction study might be more sensitive than needle electromyography for detecting peripheral motor nerve hyperexcitability in patients with vgkc antibodies, and could disappear gradually in accordance with clinical improvement and reduction of antibodies. in our research, multiple sites measurement of cross sectional areas (csa) by ultrasound was performed to differentiate charcot-marie-tooth type a (cmt a) and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). twenty-eight patients with cidp, patients with cmt a, and healthy controls (hc) were recruited prospectively. consecutive ultrasonography scanning was performed from wrist to axilla on median and ulnar nerves. csas were measured at predetermined sites of each nerve. cmt a had significantly larger csas at all sites of median and ulnar nerves (all p< . ). in cmt a, csas increased gradually and homogeneously from distal to proximal along the nerve, except potential entrapment sites. cidp displayed three different morphological patterns, including mild enlargement in patients, prominent segmental enlargement in , and slight enlargement in one, among which different treatment responses were observed. all patients with mild nerve enlargement treated with intravenous immunoglobulin (ivig) were responsive ( / ), while less than half of those with prominent segmental enlargement ( / ) were responsive (p< . ). the patterns of csa enlargement were different in cmt a and cidp patients. consecutive scan along the nerve and multiple sites measurement by ultrasound could supply more detailed morphological feature of the nerve and help to differentiate cidp from cmt a. guillain-barre syndrome (gbs) mainly consists of acute inflammatory demyelinating polyradiculoneuropathy (aidp) and acute motor axonal neuropathy (aman). in aman, conduction block (cb) could be reversible or followed by axonal degeneration. we aimed to identify the correlation between existence of cb and the functional outcome for patients with gbs. gbs patients were prospectively recruited for serial electrophysiological tests and disability evaluation. all patients received treatment of intravenous immunoglobulin (ivig), and their disabilities were evaluated on the hughes functional grading scale before and month after treatment. patients were classified into aidp, aman, equivocal or normal according to electrodiagnostic criteria described by rajabally et al. aman patients who had follow-up nerve conduction studies were further classified into three groups. group was typical aman without conduction block, group had reversible conduction block, group had conduction block and subsequential axon degeneration. electrophysiological study results showed aidp, aman, equivocal and normal. probable or definite conduction block was observed in aidp patients and aman patients. aman with cb had higher reduction of hughes grade at one month ( . ± . vs . ± . ,p= . ), and lower percentage of patients with slow recovery (unable to walk independently at six months) ( % vs %, p= . ) compared with aman without cb. there were no significant differences between aidp with cb and without cb, in the reduction of hughes grade at one month. among the aman patients who were followed up, were typical aman without cb (type ), had reversible cb (type ), had cb and subsequential axon degeneration (type ). hughes grades at nadir were similar, while patients with reversible cb (type ) had the largest hughes grade reduction at one month (type - . vs type - . vs type - . ). none of the patients with axon degeneration (type ) showed rapid recovery, while % of those with reversible cb (type ) had rapid recovery (improvement by two or more hughes grades within four weeks after onset). electrodiagnosis of aman with conduction block, especially reversible conduction block, might be a marker of good recovery. we report a follow-up study of nerve ultrasound in a patient with primary neurolymphomatosis. a -year-old female presented with -months history of asymmetric limb pain, paresthesia, and weakness. electrodiagnostic studies and spinal cord mri showed an axonal neuropathy involving cervical and lumbosacral root, brachial plexus and left median nerve. detection of malignant b lymphocytes by cytology and flow cytometry of cerebral spinal fluid confirmed the diagnosis of b-cell non-hodgkin lymphoma. nerve ultrasound showed dramatic enlargement of upper, middle and lower trunks of left brachial plexus (cross sectional area-csas mm , mm , mm respectively), middle trunk of right brachial plexus (csa mm ), and proximal part of left median nerve (csa - mm ). five months later, after five chemotherapy of rituximab and high-dose methotrexate, and intrathecal injection of cytosine arabinoside and dexamethasone, the patient had clinical improvement. nerve ultrasound also showed alleviation of nerve enlargement. the csas of upper, middle and lower trunks of left brachial plexus were mm , mm , mm respectively; the csa of middle trunk of right brachial plexus was mm ; the csa of proximal part of left median nerve was - mm . peripheral nerve ultrasound could help locate the distribution of nerve involvement, and reveals disease progression. nolano m , provitera v , stancanelli a , caporaso g , saltalamacchia am , borreca i , lullo f , califano f , lanzillo b , iodice r , manganelli f , barone p , santoro l . irccs "salvatore maugeri" foundation, institute of telese terme (bn), italy; "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; center for neurodegenerative diseases (cemand), department of medicine and surgery, neuroscience section, university of salerno, italy. a peripheral nerve involvement has been demonstrated in pd with the evidence of a small fiber pathology as possible intrinsic feature of the disease and a higher occurrence of large fiber neuropathy in patients longtime treated with l-dopa. however the role that disease itself and ldopa have on small and large fiber pathology in pd is still debated. we studied morphology and function of cutaneous innervation, in idiopathic pd patients ( male, aged . ± . ), including naïve and l-dopa treated subjects without electrophysiological signs of neuropathy, with the aim to assess and characterize small and large fiber involvement and the effect of l-dopa on it. all patients underwent a screening to rule out potentially neurotoxic conditions such as glucose intolerance, dysendocrinopathies, vitamin e, b and folic acid deficiency, hepatic or renal failure, hiv or connective tissue disorders. skin biopsies were obtained from thigh, leg and fingertip from the more affected side and bilaterally from thigh and leg in patients. samples were processed with indirect immunofluorescence technique using primary antibodies to mark different sensory and autonomic fiber populations. density of intrapapillary myelinated endings (ime), meissner's corpuscles (mc) and epidermal nerve fibers (enfs) was obtained as well as a semi-quantitative assessment of sudomotor, pilomotor and vasomotor innervation. further evaluation included sympathetic skin response, quantitative sensory testing and dynamic sweat test. morphological and functional findings were compared with data extracted from our age and sex stratified normative dataset. ienf, ime, mc densities were lower (p< . ) compared to controls in both naïve and l-dopa treated patients without differences between them except for mc density that was lower in l-dopa treated subjects ( . ± . vs . ± . /mm ). a loss of autonomic nerves was also found in both groups compared to controls. significant abnormalities (p< . ) of thermal sensory thresholds, tactile thresholds, mechanical pain perception and reduced sweating output were present and similar in both groups. our work confirms in pd an intrinsic peripheral nerve pathology involving both small and large fibers. small fiber pathology isn't affected by l-dopa treatment while sensory large fibers involvement, already present in naïve patients worsens with ldopa treatment. noto y , garg n , li t , timmins hc , park sb , shibuya k , kiernan mc . brain and mind centre, the university of sydney, sydney, australia. the diagnosis of amyotrophic lateral sclerosis (als), a progressive, fatal neurodegenerative disorder defined by combined upper and lower motor neuron involvement, remains clinically based. the purpose of this study was to determine the ultrasound appearance of peripheral nerves in als patients, and to investigate whether parameters such as distal/proximal ratios of nerve cross-sectional areas (csas) may effectively differentiate disease mimics from als. nerve ultrasound of the median, ulnar, and tibial nerves was performed in als patients compared to mimic patients ( patients with peripheral nerve hyperexcitability syndromes (pnhs) and patients with multifocal motor neuropathy (mmn)). comparison of nerve and the distal/proximal ratios was undertaken by ultrasound and compared across clinical and neurophysiological parameters.compared to normal controls, csa of the median nerve at the upper arm was decreased in als (p < . ). in comparison to als mimic disorders, csa at the proximal site of the median, ulnar and tibial nerve and the forearm/upper arm ratio of the median and ulnar nerves had diagnostic values. in addition to csa of the median, ulnar, and tibial nerves, the median and ulnar nerve forearm/upper ratios may provide a useful marker in for the diagnosis of als. approximately in , individuals are diagnosed with charcot marietooth (cmt) disease, making it the most common hereditary peripheral neuropathy. there is no documented cure for cmt, however, many of those affected, report difficulty with mobility, imbalance, and weakness of the feet and hands. in the general population, patients reporting difficulty walking, falls and/or fear of falling, and poor strength are often referred to physical (pt) and occupational therapists (ot) to skillfully address the impairments and help restore function and quality of life (qol). for patients diagnosed with cmt this is unfortunately not the norm, often leaving patients without any skilled guidance on managing their functional impairments. patients ( males) with a mean age of . years ( - ), went through a progressive and skilled pt intervention over the course of months. the program included: therapeutic activities and exercise, neuromuscular reeducation, and manual techniques to address the documented deficits. patients were progressed through the program based on the borg scale. each patient was assessed prior to and post the commencement of the program with the berg balance scale, minute walk test, timed up and go, sit to stand in seconds, foot self selected and fast gait speed, the lower extremity functional scale, activities balance confidence form, upper extremity functional index, oswestry, and the sf- qol measure. the patients were seen by the same skilled pt - a week for weeks. all but patients improved in all measures taken, indicating an improvement in function and overall quality of life. participants reported a total of falls in the months prior to the initiation of the study and only fall was reported during the month pt intervention. this study makes a strong case for the utilization of skilled pt to address deficits in patients with cmt. additionally, the utilization of objective, valid and reliable outcome measures in this population may help healthcare practitioners establish baseline function and response to change. a large randomized control trial is recommended to study the effects of a specific pt intervention on outcome measures in patients with cmt. ogata h , fujita a , yamasaki r , matsushita t , kira ji . department of neurology, neurological institute, graduate school of medical sciences, kyushu university, fukuoka, japan. clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) patients with autoantibodies against neurofascin (nf) , one of the paranodal proteins, have been elucidated while the relation between anti-nf antibody levels and long-term clinical course in these patients still remains elusive. we retrospectively collected clinical, electrophysiological and immunological data of three anti-nf antibody-positive cidp patients. they were all males and their ages at onset were , , and years old. their clinical severity was evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale. anti-nf antibody levels were measured by flow cytometry using hek cell lines stably expressing human nf . after immunotherapies of various combinations, including intravenous immunoglobulin, plasmapheresis, corticosteroids and other immunosuppressants, were introduced, their clinical parameters were gradually improved. decreased or absent dtrs were normalized and grip strength was increased by more than kg. hughes functional scale scores were decreased by at least one point compared with those at nadir. ncs findings of all three patients also showed obvious amelioration. for example, their f wave latencies in the right ulnar nerve were improved from to ms, from to ms, and from to ms, respectively. anti-nf antibody levels after treatment were decreased in two patients whose pre-and posttreatment sera were available. when dose of oral prednisolone was being tapered, they experienced re-exacerbation of clinical parameters, especially dtrs and grip strength. their ncs findings and serum anti-nf antibody levels were also deteriorated. exacerbation of these laboratory data in one patient preceded his clinical fluctuation, which suggests that ncs and serum anti-nf antibody levels could be used as early disease activity markers. in this case series, not only clinical but also laboratory data support a notion that anti-nf antibody-positive cidp patients were reactive to combined immunotherapies including corticosteroids. even though various treatments were administered to them, efficacy of oral corticosteroids seemed to be dose-dependent. optimal disease activity markers and immunotherapies for long-term maintenance of remission in anti-nf antibody-positive cidp should be identified. evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale, after starting immunotherapies, ohnmar o , kamyw , ng lfp , t umapathi . university of medicine , yangon, myanmar; singapore immunology network, a*star, singapore; national neuroscience institute, singapore. singapore's zika virus (zikv) outbreak started in late august . over a period of months, we studied patients enrolled into our institution's prospective guillain-barré syndrome (gbs) database for relationship to zikv infection. we also studied gbs controls that were seen before the established outbreak and non-gbs controls. the index cases tested negative for zikv pcr in blood and urine. we proceeded to test zikv igg, igm, dengue virus (denv) igg and igm, and neutralization assays against zikv and denv. one patient with anti-gq b igg positive miller fisher syndrome had detectable zikv igm and zikv igg. the serum showed low titre denv igm and denv igg. follow-up serum at about months showed increase in zikv igg. we believe this patient has zikv-gbs. one patient with acute motor sensory axonal neuropathy and another with acute inflammatory demyelinating polyneuropathy had high zikv igm but low denv igm and igg. another patient with mfs showed high levels of zikv and denv igm but low igg. the latter two patients had gbs before the zikv outbreak in singapore. we suspect these patients could have zikv-gbs, but are awaiting convalescent sera for confirmation. two patients seen during the outbreak had detectable levels of zikv igg but serial testing showed a decline after a period of - months. the initial and follow-up sera showed raised denv igm and igg levels in one and raised igg levels in the other. in addition, both had stronger neutralizing capacity against denv than zikv suggesting that the initially detectable zikv igg levels was due to cross reactivity with previous denv infection. four patients, gbs control and non-gbs controls also showed serological response consistent with previous exposure to denv. one normal control showed nil exposure to both viruses. in summary, using various overlapping serological methods we diagnosed definite and suspect zikv gbs cases. our findings highlight ) insensitivity of blood and urine pcr to diagnose zikv-gbs ) the problems of interpreting zikv serology from cross-reaction with denv ) serial serology increases diagnostic accuracy. okar sv , ergunay k , bekircan-kurt ce , , erdem-ozdamar s , , tan e , . department of neurology, hacettepe university, ankara, turkey; virology unit, department of medical microbiology, hacettepe university, ankara, turkey; neuromuscular disease research laboratory hacettepe university, ankara turkey. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated polyradiculoneuropathy. it is believed that acute inflammatory demyelinating poliradiculoneuropathy is caused by t-cell mediated autoimmune response targeting peripheral nerve myelin. molecular mimicry plays a role in the pathogenesis of some gbs cases. this mechanism has been well demonstrated in the acute motor axonal neuropathy (aman) variant, in which autoantibodies to camphylobacter jejuni share epitopes with peripheral nerve gangliosides. this molecular mimicry mechanism can be attributed to some cases with atypical triggers such as zika virus or west nile virus infections. moreover there are accumulating clinical data for vector borne viral infections triggering gbs. we evaluated vector-borne viral infections in our gbs and aman patients. eight patients with gbs, two patients with aman and as a control group, seven patients with normal pressure hydrocephalus were included. gbs and aman was diagnosed with clinical, electroneuromyograpic and cerebrospinal fluid (csf) findings. cerebrospinal fluid serum and urine samples were examined for vector borne viral infections via generic flavivirus and phlebovirus pcr. we also documented our patients prognostic scores such as modified erasmus gbs outcome score (megos) and modified erasmus gbs respiratory insufficiency score (megris). the mean age of the patients was , ( - years) , six of them were female. all csf, serum and urine samples of our patients and control patients were negative for flavivirus and phlebovirus families. the preliminary results of our study in this our small cohort did not show any correlation between the vector-borne viral infections and gbs. further studies with broad number of patients are needed for more suggestive results. neuromyotonia (nmt) consists of spontaneous motor unit activity that reflects increased peripheral nerve excitability, leading to involuntary, persistent muscle activity, visible as muscle twitching at rest, with generalized, easily provoked cramps. since the first electrophysiological (emg) description of nmt by denny-brown and foley ( ) , there has been discussion about the origin of the abnormal electrical activity recorded in needle emg studies. we studied two patients. patient 's nmt is aggravated by cold, and he has an associated non-progressive mild polyneuropathy with demyelinating features. he is now -year-old and has been followed in our centre for years. he is negative for anti-vgkc antibodies. firstly, he was treated with carbamazepine and phenylhydantoin with poor response, but he has shown major improvement on intravenous immunoglobulin (ivig) during the last years. patient is a year-old man with nmt, followed for year, with high titters of anti-vgkc antibodies ( pmol/l; normal< ). he improved on ivig during the last months. screening for neoplasia was unremarkable in both patients (negative anti-neuronal antibodies, in particular anti-hu, anti-yo and anti-ri antibodies; normal computerized tomography scan of the chest and abdomen). in addition to routine studies, we tested synchronicity to spontaneous discharges in different motor units in simultaneous recordings made with two needle electrodes in the first dorsal interosseus muscle. time-locked fasciculations in these double recordings would represent abnormal ectopic activity initiated in a nerve trunk with ephaptic stimulation of a nearby axon. in patient , this research protocol was applied once, years after regular ivig treatment. patient was investigated before and year after ivig. both patients improved after ivig, mirrored by a striking decrease in the amount of spontaneous activity on emg. moreover, our technique did not detect synchronous spontaneous activity (time-locked fasciculations) on the second assessment, although this was predominant before treatment in patient . in nmt, abnormal discharges originate both in distal axonal branches and in more proximal segments. it appears that ivig is more effective in blocking antibody activity in proximal axonal segments, perhaps related to factors such as blood-nerve barrier, temperature or differing ion channel distributions. celiac disease (cd) is a chronic, multisystem and immune-mediated disorder characterized by small-bowel sensitivity to dietary gluten in genetically predisposed individuals. neurological manifestations may occur in about % of patients, including peripheral nerve involvement. recent growing evidence strongly suggests that peripheral neuropathy in cd may be autoimmune and associated with anti-ganglioside antibodies. a -year-old woman presented with slowly progressive weakness of her right hand and fingers extensors. medical and family history were unremarkable. on examination, muscle strength (medical research council-mrc) was scored for right wrist and finger extensors, for right abductor pollicis longus and for right ankle dorsiflexion. right triceps brachii and brachioradialis reflexes were weak, but normal elsewhere. the remainder of the neurological examination was normal. neuroaxis magnetic resonance was unremarkable, specifically with no gadolinium-enhancing lesions. motor and sensory nerve conduction studies were normal. no conduction block or abnormal temporal dispersion was found. needle electromyography showed severe neurogenic changes with abnormal spontaneous activity in right radial-innervated muscles, and chronic neurogenic changes in homolateral tibialis anterior, peroneus longus and extensor digitorum brevis. ganglioside antibody testing was positive to igg anti-gm antibody, but negative to anti-gm and other anti-ganglioside antibodies. additional blood tests were unremarkable, in particular cryoglobulin testing was negative. intravenous immunoglobulin improved weakness, as right extensors of the wrist and fingers scored (mrc). a monthly treatment was initiated, which after months was changed to every weeks to preserve function. a diagnosis of mama was established. later, cd was diagnosed in her daughter due to chronic diarrhoea. our patient underwent anti-tissue transglutaminase antibodies determination and small-bowel biopsy after years disease' duration, and a diagnosis of cd was made. gluten-free diet was started, but her neurological picture did not change after six months. in our case the presence of igg anti-gm antibody may support a causal link between mama and biopsy-confirmed cd, and the lack of response to gluten-free diet may be explained by chronic axonal injury induced by memory t-cells. this case broadens our knowledge about neurological manifestations in cd, raising a probable association with purely axonal multifocal motor neuropathies and anti-ganglioside antibodies. Õunpuu s , , pogemiller k , acsadi g , pierz k , , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa. orthopaedic surgical intervention of the foot and ankle is performed in persons with charcot-marie-tooth (cmt) for improving foot pain, ankle instability, orthosis fitting/comfort issues and shoe wear. the impact of these surgeries on ankle function during gait is not known. therefore, the goal of this study was to measure gait changes in ankle function following surgical intervention by means of computerized motion analysis. fourteen patients with cmt ( ± years pre and ± years post-operative) with bilateral orthopaedic surgery were included. all patients had a plantar fascia release plus some combination of other soft-tissue (posterior tibialis and tendo-achilles lengthenings; extensor hallucis longus, peroneus longus and anterior tibialis transfers) and bony procedures (metatarsal and cuboid osteotomies). all patients completed two gait analyses (pre and on average . years post-surgery) during barefoot walking using a standardized d motion analysis protocol. the changes in ankle kinematics and kinetics and temporal-spatial parameters were analyzed in reference to a control group of patients with cmt without intervening surgeries with . years between gait analyses as well as normal reference data. the surgical group showed a significant increase in height between the pre and post-operative analyses and no changes in walking velocity (pre: ± , post: ± , normal: ± cm/sec). similarly, the ankle kinematics and kinetics showed no changes as a result of surgery. however, there was a trend for increased peak ankle dorsiflexion (pre: ± , post: ± , normal: ± degrees). as in the surgical group, the control group showed an increase in height but no simultaneous increase in walking velocity (test : ± , test : ± cm/sec). the control group also showed no changes in ankle kinematics and kinetics suggesting that the impact of the disease is, for the most part, not noticeable over . years. the results show that surgical intervention that primarily addresses foot alignment does not negatively impact ankle kinematics and kinetics during gait. the comparison with the control group supports this finding. however, there are some other findings such as the increase in peak dorsiflexion during stance in some patients that will need to be examined further in larger cohorts. Õunpuu s , , pogemiller k , pierz k , , , acsadi g , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa. ankle-foot-orthoses (afos) are commonly prescribed for patients with charcot-marie-tooth (cmt) to improve gait and reduce ankle instability and falling. there are no studies that examine bracing outcomes using objective evaluations. the purpose of this study was to examine the impact of various afo designs on gait parameters in children and adolescents with cmt. we predicted that the afos would improve excessive and delayed peak dorsiflexion in stance and equinus in swing. fifteen patients (mean age ± years) were analyzed barefoot and with their prescribed afos by means of a standardized motion analysis protocol. a full clinical examination was also completed including strength and passive range of motion measures. the afos included solid, hinged and posterior leaf spring (tapered, less supportive) designs. sagittal plane ankle kinematics and kinetics and temporal-spatial parameters were analyzed in comparison to normal controls. walking velocity improved from ± to ± cm/sec demonstrating the functional benefits of afos. ankle plantar flexion angle at initial contact improved from − ± to − ± degrees demonstrating improvement in drop foot in swing and at initial contact reducing the risk of tripping. however, the degree of peak ankle dorsiflexion in stance remained the same and excessive at ± degrees barefoot and ± degrees with afos suggesting that in many patients the afo design was not sufficiently supportive. peak ankle plantar flexor moment showed improvement from . ± . to . ± . nm/kg highlighting the improved base of support provided by the afos that compensate for ankle plantar flexor weakness. however, peak power generation was reduced from . ± . to . ± . w/kg indicating that some of the available ankle strength was impeded with the afos. the results of this study suggest that orthoses can provide improved gait outcomes which will improve overall function. however, there are individual differences in patient impairment (strength, range of motion and bony deformity) and associated gait presentation that need to be accounted for in afo design. afos do not always function as intended due to the complex interaction between patient impairment, orthosis stiffness and orthosis design. motion analysis can assist in identifying the specific afo needs for an individual with cmt. painous c , lópez-pérez ma , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, barcelona, spain; hospital san pedro, logroño, la rioja, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune neuropathy with a heterogeneous clinical spectrum. specific autoantibodies define different clinical phenotypes. cidp with nf antibodies constitutes a specific cidp subset in which a high incidence of postural and intention tremor has been described and that responds poorly to immunoglobulins. we report a patient with an anti-nf positive cidp that presented head, voice and limb tremor that improved with immunotherapy. a -year-old man with unremarkable medical history, presented at the age of with progressive distal weakness and paraesthesia. numbness, gait ataxia and action tremor involving voice, head and limbs appeared sequentially. the emg showed features of acquired demyelination fulfilling cidp diagnostic criteria. a first course of intravenous immunoglobulins was ineffective. the weakness, ataxia and tremor worsened significantly, needing two walking aids first and becoming wheelchair bound later on. he received six plasmapheresis cycles that were also ineffective and oral corticosteroids ( mg/kg) were started with mild improvement. after corticosteroid tapering the patient developed a severe relapse and was referred to our centre. five plasma exchange cycles followed by rituximab ( mg/m , doses) were added to the corticosteroids. three months later the weakness, ataxia and tremor, including the head and voice tremor, started to improve significantly. six months later the patient presented a significant improvement and was able to walk unaided. the anti-nf antibody titres fell from / pre-rituximab to be undetectable. limb tremor is known to occur in patients with inflammatory neuropathies and, specifically in anti-nf + cidp patients but, to our knowledge, this is the first report of a cidp patient presenting with head and voice tremor ever reported. the improvement of tremor with immunotherapy strongly suggest that anti-nf autoantibodies are involved in its pathogenesis, expanding the phenotype of anti-nf specific clinical features. palaima p , peeters k , l. pelayo-negro a , garcía a , gallardo e , garcía-barredo r , de vriendt e , infante j , berciano j , jordanova a . vib and university of antwerp, center for molecular neurology, molecular neurogenomics group, antwerp, belgium; university hospital "marqués de valdecilla", santander, spain. charcot-marie-tooth disease type g is an autosomal dominant and slowly progressing inherited neuropathy which was first described over years ago. it has been attributed to a single spanish family consisting of individuals with affected members spanning four generation. initially, the genetic defect was linked to a . mb region in q -q . . however, extensive sequence and structural variant analyses using whole genome sequences (wgs) of three affected individuals did not reveal any known or novel genetic causes within the region. over the last decade, serial clinical re-evaluation of the entire pedigree was performed leading to changes to the affection status of seven individuals redefining the disease-linked region to chr q . -q . (z max = . at theta = ). additional family members were then submitted for whole exome sequencing. this has led to the identification of a novel missense variant in the e ubiquitin-protein ligase lrsam (p.cys tyr) that was previously not covered by wgs. the variants co-segregated with disease and were absent from controls. other mutations that are known to disrupt the ring domain of lrsam have been previously reported to cause both autosomal dominant and recessive cmt type p (cmt p). unlike prior reports, we demonstrated that the mutation does not influence overall protein levels of lrsam , nor of its ubiquitylation target tsg in patient derived lymphoblasts. transcriptomics analysis identified significant upregulation of another e ubiquitin-protein ligase (nedd l) and of a key regulator of axonal degeneration (tnfrsf ). notably, magnetic resonance imaging of lower-limb musculature systematically showed fatty atrophy in both clinical and subclinical mutation carriers emphasizing its use for the identification of mildly affected members. our findings demonstrate that the isolated genetic entity cmt g is caused by a missense mutation in lrsam and should be reclassified as cmt p. moreover, we reveal novel molecular players associated with lrsam dysfunction, and highlight pathways and therapeutic targets shared with amyotrophic lateral sclerosis and alzheimer disease. damage to peripheral nerves is a prerequisite for neuropathic pain. however, it remains unclear why some neuropathy patients have pain, but others with identical nerve conduction, qst and ienfd do not. we propose to examine the distribution of trpv and its co-localization with cgrp and sp in epidermal nerve fibers in patients with and without neuropathic pain. we aimed to define the expression pattern of trpv in normal healthy subjects first, and study the co-localization of trpv with cgrp and sp in normal controls and patients with painful neuropathy. skin biopsies from neuropathy patients and normal subjects were utilized. anti-trpv antibody generated in our university with support from ninds-funded dept. of neuroscience monoclonal core (ns ) was used. combined immunohistochemistry were performed to identify co-expression of trpv with cgrp, sp and pgp . . the distribution of trpv in controls revealed a proximal to distal gradient similar to that observed for ienf labeled by pgp . . trpv staining was more intense in nerve terminals in the epidermis. combined immunostaining revealed that % of pgp . labeled fibers in the epidermis were trpv +, while % of cgrp+ fibers trpv +. patients with pain had a higher density of trpv + fibers compared to that of patients with numbness. a greater proportion of cgrp+ fibers ( %) in the painful patients were trpv +. expression of trpv in controls exhibits a distal to proximal gradient. trpv expression and co-localization with cgrp were altered in neuropathic pain patients, suggesting that this receptor plays an important role in pathological states. activation of the nop-receptor (nop-r) by its endogenous ligand nociceptin/orphanin fq or non-peptide agonists modulates nociception and analgesia in neuropathic pain models. the wide distribution of nop-r and its endogenous ligand represents an attractive treatment target. since pain is the most prominent feature in pc, we defined expression of the nop-r in plantar skin biopsies and assessed whether alterations exist in pc-affected vs pc-unaffected and anatomically matched control skin. skin biopsies from k a pc and control subjects were immunohistochemically stained for nop-r. combined immunostaining for nop-r with pgp . , neurofilament h (nfh) and cgrp was used to define nop receptor expression in the epidermis and upper dermis. robust nop-r was detected in epidermal keratinocytes and a subset of pgp . + fibers in both epidermis and dermis. staining was inhibited through pre-incubation with a nop-r blocking peptide and western blot analysis using homogenized human skin tissue demonstrated a band at ∼ kd consistent with nop-r molecular weight. nop-r expression occurred in dermal nfh+ a beta-fibers in all groups though no cgrp+ fibers co-expressed nop-r. pc-affected skin had slightly lower nop-r expression than in pc-unaffected skin and a similar pattern in anatomically matched locations from healthy control subjects was observed. nop-r is expressed in human plantar skin epidermal keratinocytes as well as a subset of epidermal and dermal nerve fibers. these fibers are pgp . +, cgrp-and many co-express nfh. nop receptor is a viable pharmaceutical analgesic target in pc patients irrespective of its slight down-regulation as compared to pc-unaffected skin. this work was supported by grünenthal gmbh. pareyson d , stojkovic t , leonard-louis s , reilly mm , laurà m , parman y , battaloglu e , tazir m , bellatache m , bonello-palot n , sacconi s , guimarães-costa r , attarian s , latour p , megarbane a , schenone a , ursino g , sabatelli m , luigetti m , santoro l , manganelli f , quattrone a , valentino p , murakami t , scherer ss , dankwa l , shy me , bacon cj , herrmann dn , pisciotta c , previtali s , bolino a . milan, italy; paris, france; london, united kingdom; istanbul, turkey; algiers, algeria; marseille, france; nice, france; lyon, france; genoa, italy; rome, italy; naples, italy; catanzaro, italy; kurashiki, japan; philadelphia, usa; iowa city, usa; rochester, usa. cmt b and b are characterized by recessive inheritance, early onset, severe course, slowed nerve conduction, myelin outfoldings, association with loss-of-function mutations in myotubularin-related protein- and − (mtmr , mtmr /sbf ), respectively, involved in the metabolism of ptdins p and ptsins( , )p phosphoinositides, key regulators of membrane trafficking. in a multicentre retrospective study to better characterise cmt b, we collected a minimal dataset of information including cmtes/cmtns on cmt b patients, cmt b ( unrelated families) and cmt b ( families). cmt b patients were younger and with earlier onset than cmt b : last visit was performed at a mean age of years (sd . ; range - ) for cmt b and years ( . ; - ) for cmt b ; disease onset occurred at a mean age of . years ( . ; - ) in cmt b as compared to . years ( . ; - ) in cmt b ; delay in motor milestones occurred in / cmt b and / cmt b subjects. eleven cmt b patients became chair-bound, whereas all cmt b subjects but one are still ambulant, although with afos for patients and requiring unilateral support in two cases. both disease types are characterised by vocal cord involvement ( / cmt b and / cmt b ); respiratory involvement was seen almost exclusively in cmt b patients (n= , four requiring non-invasive ventilation and one tracheostomy, as compared to one cmt b patient on niv at age ); two cmt b subjects and an affected cmt b relative not included in the present study died of respiratory complications. glaucoma (n= ) and buphthalmos (n= ) occurred only in cmt b . cmtes/ cmtns scores were higher in cmt b patients in spite of their younger age, indicating more severe disease: cmt b = cmtes mean . (n= ; sd . ; range - / ), cmtns mean . (n= ; sd . ; range - / ,; cmt b = cmtes mean (n= ; sd . ; range - / ), cmtns mean (n= ; sd . ; range - / ). our data confirm that cmt b is more severe than cmt b . interestingly, mtmr interacts with mtmr . mtmr is a catalytically active phosphatase, whereas mtmr is a catalytically inactive protein, known to increase mtmr enzymatic activity. thus, in cmt b nerves a residual enzymatic activity of mtmr may result in a less severe clinical phenotype as compared to cmt b . the charcot-marie-tooth disease (cmt) national registry is fully operative (https:www.registronmd.it) with collection of clinical/genetic information (minimal dataset) and reporting of clinical scales; cmt patients have registered thus far and have chosen one of the reference centers; information collected during the ad hoc visit have been entered in the registry for of them. registered patients have the chance to participate to a study that requires filling online self-reported questionnaires related to five important issues: disease course and complications during pregnancy; use, efficacy and tolerability of orthotics and assistive devices; outcome of surgery for skeletal deformities; safety of anesthesia; occurrence of sleep disorders (including evaluation of fatigue, anxiety and depression). by february , patients and relatives/friends (as healthy controls) have filled the questionnaires. we are performing a first explorative analysis of results, but data collection on all questionnaires will be prolonged until novemebr , , to obtain a larger sample. pregnancy: / cmt women had at least one pregnancy; complications ranging from mild to severe occurred in / pregnancies vs / in controls. cmt worsened in pregnancies ( patients) with no recovery in instances. prenatal diagnosis was performed in / pregnancies. satisfaction related to surgical procedures for foot deformities, assessed with vas (score - ), was . ± . (n = ). repeat surgery was required in / instances. sleep: the epworth sleepiness scale questionnaire revealed abnormalities of sleep in / cmt patients ( %) and in / controls ( %). the vast majority of cmt subjects ( / ; mean . ± . ), but also of controls ( / ; . ± . ) were not good sleepers according to the pittsburgh sleep quality index (psqi) (range - , good sleep). fatigue: scores of modified-fatigue impact scale (range - , no fatigue) were higher for cmt (mean ± . ) than controls (mean . ± . ). hospital scale for anxiety and depression: / cmt subjects had mild-to-severe anxiety and / mild-to-severe depression as compared to / and / controls, respectively. data analysis on orthotics and anesthesia is ongoing. in conclusion, the first data analyses confirm that there are problems related to all the five domains explored, that will need to be specifically addressed in patients' care. supported by telethon-uildm grant gup . charcot-marie-tooth disease (cmt) is the most frequent motor and sensory peripheral neuropathy and is known to have the uniform demyelination. the traditional definition of conduction block is the reduction of negative peak of compound muscle action potential (cmap) of proximal segment relative to adjacent distal segment more than %. in this study, we tried to find the frequency of conduction block in cmt a patients and to investigate the differences of the clinical manifestation. we enrolled unrelated cmt a patients ( males and females) with pmp duplication and undertook nerve conduction studies from to . stimulation sites were wrist, elbow and axilla for median nerve study. eleven patients ( . % of all enrolled patients) had ncs features suggestive of conduction block. compared to cmt a patients without conduction block, functional disability scale was significantly higher in cmt a patients with conduction block (p < . ). however, onset age and disease duration were not different between cmt a patients with and without conduction block. in addition, conduction block was more frequently observed in distal segments than proximal segments. we suggest that the frequency of conduction block in cmt a patients is not low, and there is some heterogeneity of demyelination in cmt a patients. also, in cmt a patients, the conduction block might have relationship with clinical disability. park jg , park ms . yeungnam university college of medicine, daegu, korea. the subacute combined degeneration (scd) is diagnosed by a characteristic clinical manifestation, spinal mri, and decreased serum vitamin b levels. we report a case of scd with elevated homocysteine and methlymalonic acid level in the situation of spurious elevation of vitamin b concentration. an years old man presented with progressive gait disturbances and sensory disturbances. about a year ago, he felt both hands and feet paresthesia and gait disturbances. four months before the visit, nerve conduction tests were performed elsewhere due to symptoms. polyneuropathy was found. the laboratory test showed megaloblastic anemia. however, serum vitamin b concentration was increased to pg / ml (normal: - pg / ml). his symptoms progressed gradually. at presented, neurological examination showed spasticity of the lower limb without weakness, and vibration and position sensation were decreased in both lower limbs, and showed ataxic gait. spinal cord mri showed a lesion with a long t high signal intensity from c to t in the posterior column of the spinal cord. further laboratory test were added, then while folate was normal level but homocysteine at . umol/l (normal: - umol/l), and methylmalonic acid at . um / l (normal: - . um / l) were increased. finally, scd caused by vitamin b deficiency was diagnosed. in addition, gastric endoscopy was performed to find the cause of vitamin b deficiency and chronic atrophic gastritis was found. after cobalamine treatment for months, hemoglobin level was improved and his symptoms were all improving with a little gait disturbances. false-elevation of vitamin b level could lead to delayed diagnosis and cause irreversible changes in the nervous systems. one of the most commonly used methods to measure vitamin b concentrations is competitive-binding assay, which may result in normal levels even with vitamin b deficiency due to the effect of anti-intrinsic factor antibodies. when vitamin b deficiency is suspected, even if the vitamin b concentration is normal, additional tests of homocysteine and methylmalonic acid should be considered. parman y , durmus h , deymeer f , oflazer-serdaroglu p , battaloglu e . istanbul university, istanbul faculty of medicine, department of neurology, istanbul, turkey; bogazici university, istanbul, turkey. hereditary motor and sensory neuropathies, also called cmt neuropathies, are the most common group among the hereditary neuropathies. cmt subtypes are grouped by axonal, demyelinating or intermediate phenotype, or by autosomal-dominant (ad), autosomal-recessive (ar) or x-chromosomal inheritance. cmt- is considered axonal and characterized by distal muscle wasting, mild sensory loss and normal or near-normal nerve conduction velocities. mutations in more than genes are known to be able to cause autosomal dominant (ad) or recessive (ar) cmt- to date. the genetic heterogeneity in charcot-marie-tooth (cmt) is a challenge for genetic diagnostics. clinical clues and frequencies of mutations in cmt genes from large cohorts may help to develop strategies for efficient genetic testing. here, we present the clinical, electrophysiological and genetic features of patients from turkey, diagnosed as genetically confirmed cmt- in the department of neurology, istanbul faculty of medicine our clinic between and . genetic testing was performed for gdap by dna sequencing and samples from patients were exome sequenced (wes). twenty-one male and ten female patients from unrelated families were investigated. segregation was ar in eight, ad in six families and two were isolated cases. intraand interfamilial variability in the age of onset with a range of - (mean . ± . years) and disease progression rate were striking. slowly progressive weakness and atrophy of distal muscles in the feet and/or hands were the most common presenting symptoms. mfn was found in four unrelated ad kinship and in unrelated ar kinships and was most common gene mutated among whole cohort. gdap was the most commonly mutated gene among ar families ( / ). wes revealed further mutations in aars, nefl, kif b and hspb , however, the causative mutation could not be identified in known cmt genes in about % of patients. our data indicates the marked intra-and inter-familiar phenotype variability in cmt- as previously described in literature. many more genes causing arcmt- remain to be discovered. pasnoor m , veerapaneni k , murphy r , statland jm , kimple d , hamasaki a , glenn md , herbelin l , barohn rj , jawdat o , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. electrodiagnostic (edx) studies involving electromyography/nerve conduction study (emg/ncs) are useful for diagnosis of neuromuscular disorders. although these are safe procedures, emg and ncs often evoke anticipated pain and anxiety. there are no published research studies to support the notion that providing detailed information to patients prior to these procedures would reduce procedural pain and ameliorate anxiety levels afterwards. the objective of this study was to perform a prospective survey to assess anticipated pain and anxiety in patients presenting for edx studies, design a detailed patient education form, and assess the change in perceived pain and anxiety pre and post procedure in the standard of care (soc) education versus the printed detailed education instrument group. after completing a brief pain/anxiety questionnaire, patients were randomly assigned to either soc verbal education group or to read the detailed education form. another brief questionnaire was completed post procedure. seventy-eight patients were enrolled at the time of abstract submission, in intervention and in soc groups. mean age of patients enrolled was ± years. pain was anticipated by % patients as a visual analog scale (vas) score mean of . ± . with no significant difference between both groups (p= . ). post-procedural pain was reported in % of patients with mean pain score of . ± . and no significant difference between the two groups (p= . ). anxiety pre-procedure was reported by % patients with a mean likert-like score of . ± . score in all cases (p= . between both groups). post-procedural anxiety frequency was reduced to % with mean level of . ± . (p= . between both groups). most patients reported pain with both emg and ncs ( %). we found that edx testing evokes moderate pain in most and in some cases moderate anxiety level. the detailed education intervention did not attenuate the frequency or severity of pain. post-procedural anxiety was expectedly reduced in severity and frequency in both groups with a suggestion that anxiety severity is lesser in the detailed education group but numbers were small. further studies with more detailed questionnaire and perhaps web-based education in larger population may be useful to reduce pain/anxiety in patients presenting for edx studies. pasnoor m , roach c , barohn rj , statland j , jawdat o , dick a , glenn m , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. diagnostic criteria for chronic inflammatory demyelinating polyneuropathy (cidp) have been designed for use in clinical trials. there is limited data on the comparative diagnostic utility of these criteria in the clinic setting and it is unknown if some of these criteria are better for predicting treatment response. the objective of this study is to compare the sensitivity of various diagnostic criteria and review treatment response. after obtaining local irb approval, we performed a retrospective chart review of cidp patients seen at the university of kansas medical center between and . we abstracted the clinical, electrodiagnostic, and treatment information. we determined the frequency of patients fulfilling each of the following criteria: efns , aan, saperstein and incat. we defined treatment response based on treating physician's impression of change, patient-reported functional improvement or one point grade change in the mrc grade. fifty-three cidp patients charts were reviewed, were excluded due to missing data. mean age was . , and ( %) were female. elevated csf protein was seen in / ( %). twenty-eight ( %) fulfilled efns definite criteria, ( . %) efns probable, ( . %) aan, ( . %) incat, and ( . %) the saperstein criteria. treatment response in patients who fulfilled efns definite/probable criteria included / to ivig, / to iv or oral corticosteroids, / to mycophenolate mofetil; among patients who fulfilled aan criteria, these were respectively / , / and / ; among those meeting incat criteria / , / and / ; and for saperstein criteria it was / , / and / . half to two-thirds of patients responded to plex based on different criteria. while all cidp criteria can similarly predict ivig treatment response, the efns criteria are most sensitive for the clinical diagnosis of cidp. pellegatta m , canevazzi p , forese mg , podini p , quattrini a , taveggia c . division of neuroscience and inspe, axo-glia interaction unit, san raffaele scientific institute, milan, italy; division of neuroscience and inspe, experimental neuropathology unit, san raffaele scientific institute, milan, italy. axonal nrg type iii is an essential instructive signal for pns myelination, since its expression determines whether axons are myelinated and also the thickness of the myelin sheath. previous studies have shown that secretases' cleavage controls nrg type iii activity at post-transcriptional level. specifically, the beta-secretase bace- cleavage of nrg type iii promotes myelination, whereas the alpha-secretase tace inhibits pns myelination by inactivating nrg type iii. after a damage, the axon located distally to the site of injury degenerates and loses the myelin sheath, following a process termed as wallerian degeneration. unlike the cns, axons in the pns can regenerate and regrow. several studies have shown that regeneration is favored by the trans-differentiation of schwann cells, which start to create a permissive environment for axonal regrowth. in addition, it has been shown that nrg regulates the early stages of the dedifferentiation process and is essential for creating a permissive environment to regeneration. similarly, the beta secretase bace- promotes pns regeneration and remyelination. in the present study, we analyzed the role of the alpha-secretase tace during wallerian degeneration. our data indicate that tace is upregulated in sciatic nerves after injury, distally to the site of damage. since tace is expressed in schwann cells and axons, we analyzed the role of both glial and neuronal tace during degeneration and regeneration processes. thus, we performed crush injury in sciatic nerves of two different transgenic lines carrying a conditional deletion of tace either in schwann cells (p -cre//tace flox/flox ) or in neurons (chat-cre//tace flox/flox ). the analyses of the different phases of the wallerian degeneration in these animals suggest a role of glial tace during the early stage of the process. in fact, we observed an increased number of myelinated axons only in p -cre//tace flox/flox mice, while we did not detect substantial differences in mutant chatcre//tace flox/flox mice. these results suggest that even during regeneration tace has an inhibitory role, as deletion of tace in schwann cell likely induces the activation of a neuro-protective program that we are currently investigating. péréon y , nizon m , küry s , besnard t , quinquis d , boisseau p , magot a , mussini jp , mayrargue e , barbarot s , bézieau s , isidor b . reference centre for neuromuscular disorders; dept. of genetics; dept. of paediatric surgery; dept. of dermatology, university hospital, nantes, france. the hereditary sensory and autonomic neuropathies (hsan) constitute a clinically and genetically heterogeneous group. hsan are associated with sensory dysfunction, altered pain and temperature perception with varying degrees of autonomic dysfunction, and abnormal small fibers neurodevelopment. more than ten genes have been described so far in the hsan group. original classification encompassed four entities, but additional subgroups continue to be described. hereditary sensory neuropathies (hsan) type ii are characterized by autosomal recessive inheritance, onset at birth and self-mutilating behavior. until now, one homozygous frameshift variant, c.[ _ del], p.(lys phefs* ), was reported in arl ip (adprribosylation-like factor -interacting protein ) in a consanguineous family. patients presented with spastic paraplegia, diffuse sensory and motor polyneuropathy and acromutilation. the disorder was classified as autosomal recessive spastic paraplegia spg . here, we described a new patient with congenital insensitivity to pain, sensory neuropathy, self-mutilation, and spastic paraplegia. whole exome sequencing showed a homozygous frameshift variant c. [ _ del], p.(lys phefs* ) in arl ip . the protein harbours reticulon-like short hairpin transmembrane domains and has a role in endoplasmic reticulum shaping. the variant causes an additional c-terminus hydrophobic domain which could alter its function. arl ip interacts with atlastin- responsible for spg a and hsan type d. this report highlights the role of arl ip in pathophysiology of insensitivity to pain and spastic paraplegia. péréon y , nguyen a-l , leclair-visonneau l , fayet g , nguyen j-m , magot a . laboratoire d'explorations fonctionnelles, hôtel-dieu, nantes, france; département de statistiques médicales, hôtel-dieu, nantes, france. carpal tunnel syndrome (cts) is responsible for sensory and/or motor symptoms that may be increased by wrist flexion or extension (phalen sign). previous studies have shown that short duration (e.g. min or less) flexion or extension was not responsible for significant changes of median nerve conduction parameters. the objective is the study was to determine how prolonged extension (reverse phalen's test) affects median and ulnar nerve conduction parameters in normal subjects. after providing informed consent, normal subjects ( females, age rank - years) underwent motor and sensory testing of both median and ulnar nerves from the dominant side. edx testing was performed using standard techniques with wrist in neutral position, then during passive wrist extension ( ∘ ) for minutes, with sequential recording of both motor action potential (cmap) and sensory action potential (snap) latencies and amplitudes every minutes after extension onset. ulnar nerve conduction parameters remained unchanged in all the subjects. regarding median nerve, groups of subjects could be individualized: subjects with no changes; subjects with only significant decrease of snap amplitude (− %); subjects with both snap amplitude and velocity decrease; subjects with both cmap and snap significant changes. when present, changes did not appear before min, with snap decrease being the earliest observed abnormality. prolonged wrist extension was responsible for median nerve edx parameter modifications in / normal subjects. sensory fibers were firstly affected, corresponding to the chronology clinically encountered in cts. based upon these results, the same protocol will be used in patients presenting with mild clinical cts, in order to try and identify an additional edx technique being useful in the diagnosis of cts. one could also wonder whether the subjects with the most important changes would be more prone to present cts in the future. perez-siles g , , drew a , , ellis m , kidambi m , takata r i , speck-martins c e , hagerman k a , siskind c e , day j w , ginzberg m , nicholson g , , , kennerson m l , , . northcott neuroscience laboratory, anzac research institute, sydney, australia; sydney medical school, university of sydney, sydney, australia; molecular medicine laboratory, concord repatriation general hospital, sydney, australia; sarah network rehabilitation hospitals, brasilia, df, brazil; department of neurology, stanford health care, stanford, ca, usa; pediatric neuromuscular unit, wolfson medical center, holon, israel. mutations in the atp a gene cause of x-linked hereditary distal motor neuropathy (dhmnx). to date, missense mutations (t i and p s) in this copper transporting atpase are the only confirmed mutations causing dhmnx in two independent families. next generation sequencing (ngs), including whole exome and whole genome sequencing, is increasing the rapid detection of variants in genes known to cause disease, however the absence and size of additional families, make it challenging to determine the pathogenic or benign status of variants identified. we have recently identified new variants in atp a in patients with progressive peripheral neuropathy, suggesting further genetic heterogeneity of dhmnx. two of these new variants, pe v and pm v, are located at highly conserved amino acids within domains of atp a (a-domain and p-domain, respectively) that are critical for the catalytic cycle of the copper transporter. we have also identified a third variant (c. - a>g) located bases upstream exon that is predicted to abolish a conserved branch-site, a consensus intronic sequence necessary for the processing of immature rna during splicing. our recent investigations using both patient fibroblasts and an atp a conditional knock in mouse model for dhmnx expressing atp a t i , the orthologue of the human atp a t i mutation, showed reduced atp a protein levels and defective retrograde trafficking of atp a, as pathological hallmarks of dhmnx atp a causative mutations. to elucidate if the newly identified pe v, pm v and c. - a>g atp a variants are pathogenic, we have systematically assessed patient fibroblasts harbouring these mutations for altered parameters previously found in the pathogenic t i atp a mutation. we predict that functional characterisation of atp a using patient fibroblasts harbouring newly identified variants will provide the evidence to ascertain whether these variants are disease causing. establishing a systematic functional readout for atp a variants will improve accuracy of genetic counselling and patient management of dhmnx in those cases where genetic evidence is limited. this study represents a complementary and necessary approach to the use of ngs, for validating the pathogenic status of variants identified and for expanding the genetic heterogeneity of dhmnx. month of birth (mob) have been defined as a risk factor for more than diseases. for instance, susceptibility for multiple sclerosis (ms) seems to be associated with being born in may, while lower risk of ms is observed in those born in november. there are no studies that assessed potential association between mob and a risk for guillain-barré syndrome (gbs). we sought to determine if the risk of gbs is associated with the mob. study included gbs patients diagnosed in serbia, montenegro and republic of srpska in the period from january , until december , . mob of patients and of general population were compared. patients with gbs had tendency to be born in october ( % increase compared to general population, p= . ) and were less likely to be born in june ( % decrease, p= . ). when consider specific seasons, gbs patients were more likely to be born in winter months ( % increase, p< . ). no associations were found between month/season of birth and disease severity. results of this pilot study showed that gbs patients are more likely to be born in cold months, and less likely to be born in june. this might be explained by higher exposure to different pathogens during pregnancy in cold months. in accordance with this, it is well known that early exposure to infective agents reduces risk of allergic and autoimmune diseases. further studies are needed to test our findings in different cohorts and in regions with different climate. these results may shed new light on the disease pathogenesis. majority of patients with guillain-barré syndrome (gbs) tend to have a successful recovery, but in number of them significant long-term consequences may negatively affect their quality of life. aim of this research was to analyze the outcome of the disease one year after the acute episode of gbs. among patients diagnosed with gbs in seven tertiary centers in serbia, montenegro and republic of srpska during , subjects were retested after one year ( % males, mean age ± years). functional disability of patients was estimated based on the gbs disability scale (gds). severe form of the disease (gds score - ) was registered in % of patients at admission, % at nadir, and % on discharge. after one year follow-up period, % of patients had no symptoms of gbs, % had mild symptoms, % was able to walk but not to run, % needed unilateral support during walking, while % was wheelchair bound or bedridden. lethal outcome was registered in three patients during acute phase of gbs and in four more during one-year follow-up. paresthesias were present in % of gbs patients, musculoskeletal pain in %, and fatigue in % one year after acute phase of the disease. factors associated with the worse functional outcome (gds grade above ) after one year were: age above , preceding respiratory infection, and worse gds on discharge. one year after the onset of the disease, significant number of our gbs patients had neurological impairments including sensory symptoms, pain, fatigue and muscle weakness which may significantly affect patients' everyday functioning. phetteplace je , saade d , bacon c , shy me . university of iowa hospitals and clinics, iowa city, ia, usa. charcot-marie-tooth (cmt) disease, also known as hereditary motor and sensory neuropathy, is a heritable peripheral neuropathy caused by genetic mutations in many different genes. cmt type a is the most common form of cmt. individuals affected with cmt a commonly have distal muscle atrophy, foot deformities, abnormal gait, and reduced nerve conduction velocities caused by demyelination of the peripheral nerves. cmt a is known to be caused by a duplication of the pmp gene at p . . the proposed disease mechanism of cmt a is believed to be increased gene dosage of the pmp gene, although the exact function of pmp is not known. there have been previous reports in the literature of individuals who carry a homozygous duplication of pmp , therefore having four copies of the gene. however, many of these reports have focused on adult patients who symptoms may not vary significantly from individuals who harbor only a duplication of pmp . a month old male presented to the university of iowa hospital due to a family history of cmt a and concern for delay in independent ambulation. he was known to have both a maternal and paternal family history of cmt a, although the families were believed to be unrelated. on exam he was unable to walk independently, but could take a few steps when his hands were held. per report he began crawling and scooting at months of age. he was not found to have any structural abnormalities or atrophy in his upper or lower extremities. electrodiagnostic testing revealed a demyelinating neuropathy. only one nerve was tested, as the patient was unable to tolerate additional shocks. the peroneal nerve was found to have a nerve conduction velocity of m/s. based on the clinical presentation, electrodiagnostic examination and family history deletion and duplication testing for pmp was performed. this testing revealed a homozygous duplication of pmp , which has been previously reported to cause cmt a. this case supports the gene dosage disease mechanism, as our patient appears to be more severely affected than a typical infant or toddler affected with cmt a. piscosquito g , saveri p , provitera v , stancanelli a , ciano c , magri s , taroni mpz mutations are associated with the demyelinating early-onset charcot-marie-tooth (cmt) type b and with the axonal later onset cmt i/cmt j. paresthesias/dysesthesias and pain have been already reported in some cmt types and also in cmt i/j. we report patients ( from families, sporadic cases,) carrying the mpz c. dela mutation (p.asp thrfster ). this is predicted to be a loss-of-function mutation, leading to p haploinsufficiency. most of the subjects have genetic ancestry from puglia region (southern italy). two patients carried the mutation in homozygosity, showing a severe phenotype: onset at birth, severe scoliosis, proximal weakness, distal limb plegia, no autonomous walking, cmt examination score (cmtes) - / . all the other heterozygous patients had mild adult-onset (mean age , range - ) neuropathy, walked without aids or ankle-foot orthoses, had cmtes < / . five showed mild foot deformities, / had foot and / hand weakness (mrc never less than / ), / sensory loss in lower limbs (ll). paresthesias/dysesthesias (mostly tingling and burning in type) localized in hands and ll were reported by / subjects, neuropathic pain by / . in the homozygous patients, all cmaps and saps were absent. in all other subjects, motor conduction velocities (cv) were > m/s (range - m/s) and sensory cv > m/s (range - m/s). cmaps as well as saps were normal or moderately reduced in amplitude, indicating only slight axonal loss. we confirm that heterozygosity is associated with very mild cmt, whereas homozygosity causes a very severe neuropathy. our data suggest that paresthesias and neuropathic pain are very common and should be considered as part of the phenotype. although the clinical picture suggests a small fiber neuropathy, no such evidence is provided in the current literature. in the attempt to further investigate this field, we found in a -year-old male patient of our series that all thresholds were increased at quantitative sensory testing, and several abnormalities of small nerve fibers were present in skin biopsy. apparently, p aneuplody causes neuropathy and small fiber involvement through a mechanism different from other mpz missense mutations. poitras t , chandrasekhar a , singh v , martinez j , zochodne dw . neuroscience mental health institute, and division of neurology, department of medicine, university of alberta, edmonton, alberta, canada. a microarray mrna analysis of dorsal root ganglia examined in long-term diabetic mice yielded an unexpected upregulation of a unique set of proteins known as major urinary proteins (mups & ). originally, this family of barrel shaped proteins was described as including mediators of pheromone secretion in the urine of rodents, apparently unconnected to other functions. we identified its unanticipated expression in sensory neurons of adult mice and rats and explored its potential impact on the properties of these neurons. analysis of rat and mouse sensory neurons showed widespread and pan-neuronal cytosolic mup expression, not only in the cell body, but also in distal sensory projections located in the dermis of the footpad. to assess the role that mup might offer in the growth behaviour of adult neurons, we examined sirna-induced knockdown of mup on pre-injured dissociated drg primary cultures. mup knockdown in both species showed a significant increase in neurite outgrowth following mup sirna treatment when compared to a scrambled sequence control. mice treated with streptozotocin (stz) model features of human type diabetic polyneuropathy, including decreases in multi-fiber motor and sensory measurements, and changes in thermal sensation in the extremities. to explore the potential role of mup upregulation specifically in diabetes, we examined the impact of non-viral intranasal injection of mup sirna on indices of neuropathy in chronic type diabetic mice. following treatment, mice treated with mup sirna showed improvement in both motor and sensory nerve conduction velocities compared to scrambled controls. taken together, this data suggests that mup plays a growth-suppressive role in both diabetic and non-diabetic neurons and may also contribute to the electrophysiological and behavioral abnormalities associated with diabetic polyneuropathy. the repurposing of mups in sensory neurons identifies an interesting role for a supposed pheromone protein. [supported by cihr, cda and adi] enhancement of their outgrowth properties (singh et al, ) . here we asked whether similar activation, using low dose capsaicin, might similarly ramp up the regenerative activity of sensory neurons. using rat adult primary sensory neurons, we identified a dose dependent relationship between capsaicin exposure and their outgrowth with enhancement at lower doses and expected neurotoxicity at higher doses. doses that enhanced outgrowth were nonetheless associated with rises in intraneuronal calcium. we next tested whether a similar impact of trpv activation in vivo might exist. we used a high sciatic crush injury mouse model to assess the impact of capsaicin at low ( um) or high ( mm) doses applied directly at the site of axon outgrowth after injury. by days following the single application of capsaicin, low doses of capsaicin showed more rapid recovery of thermal, but not mechanical sensation, whereas high dose capsaicin showed no significant difference compared to vehicle treated mice. in parallel with the neurotoxicity observed in vitro, the high dose capsaicin resulted in a decrease in sensory nerve conduction velocity, while the low dose showed no significant difference when compared to the vehicle treated animals. there was no impact on motor axon recovery. taken together, subtoxic trpv activation with intraneuronal calcium mobilization, as applied to regenerating sensory axons, enhances their outgrowth after injury, and improves behavioural recovery similar to that following extracellular electrical stimulation. [supported by cihr] hereditary transthyretin (hattr) amyloidosis is a rapidly progressive, life-threatening disease caused by ttr gene variants, resulting in ttr misfolding and amyloid deposition in multiple organs, including peripheral nerves. hattr amyloid polyneuropathy has prominent small fiber involvement characterized by neuropathic pain and dysautonomia. measurement of intraepidermal and sweat gland nerve fiber density (ienfd, sgnfd) in skin biopsies has been useful in identifying small fiber abnormalities in hattr. we report the effect of patisiran, an investigational rnai therapeutic for the treatment of hattr, over a -month period on ienfd, sgnfd and dermal amyloid content from the phase open-label extension (ole) study. skin biopsies (distal leg, distal thigh) were obtained every months for up to years within the phase ole study (patisiran . mg/kg iv, q w, nct ) and analyzed in a blinded manner by a central laboratory. all analyses are exploratory; nominal p-values are reported without adjustment for multiple testing. twenty-seven patients enrolled; preliminary data indicated patisiran was generally well tolerated; resulted in sustained mean serum ttr lowering of ∼ % for > months and a mean . -point decrease in mnis+ (n= ). twenty-four patients underwent serial skin punch biopsies. at baseline, mean ienfd was . and . fibers/mm, while mean sgnfd was . and . m/mm for distal thigh and distal leg, respectively. amyloid was detected in ∼ % of skin biopsies, mean baseline dermal amyloid content . % and . % for distal thigh and distal leg, respectively, which inversely correlated with ienfd and sgnfd. overall, ienfd was stable throughout the -year treatment. sgnfd increased over time relative to baseline, reaching statistical significance at , , and months for distal thigh (p< . , all time points) and at months for distal leg (p= . ). dermal amyloid content decreased over time and reached statistical significance at , and months for distal thigh (p< . , all time points) and at , , , and months for distal leg (p< . , all time points). in summary, improvements in sgnfd and dermal amyloid content observed over a -year period suggest that these parameters have the potential to serve as biomarkers of response to patisiran treatment. polydefkis m , neuhaus s , doherty l , ebenezer gj . department of neurology, johns hopkins university, baltimore, md, usa. misdiagnosis and delayed identification of an etiology for sensory neuropathies hampers adequate management and delays initiation of therapy. we present several cases that illustrate variable presentations of amyloid neuropathy (an) and an complicated by amyloid arthropathy. a -year-old former college athlete developed painless difficulty walking. he was unable to keep up with family members during routine outings and subsequently was unable to navigate uneven ground during a hike, something he had previously excelled at. balance decreased, making bicycling difficult. neurological evaluation -months after symptom onset led to a diagnosis of peripheral neuropathy. a reversible neuropathy panel was normal though a c was elevated ( . %). over several months, his strength/sensation deteriorated despite diet improvement, losing lbs and a c improvement ( . %). upon referral, emg revealed a sensorimotor neuropathy with markedly reduced/absent sural, peroneal and tibial motor amplitudes. there was mild orthostasis. nis was . skin biopsies revealed severe ienfd, sgnfd reductions and positive congo red staining, confirmed with birefringence. a sural nerve biopsy confirmed amyloid deposits. genetic testing revealed the ttr-fap variant leu his. there was no history or suspicion for a family history of amyloid. a -year-old male with a history of controlled diabetes x years developed abrupt onset of rls months after a lymphoma diagnosis. treatment with rituximab led to radiographic resolution of the lymphoma though the patient developed progressive peripheral neuropathy confirmed by ncv/emg. new-onset early satiety, mild orthostasis and ed emerged. skin biopsies demonstrated a lichenoid pattern of amyloid deposition consistent with aa-amyloid. a -year old woman with leu his ttr-fap since developed progressive leg weakness, reduced ability to walk and low back pain. emg demonstrated progression of her sensorimotor neuropathy and superimposed multilevel radiculopathy. spine mri demonstrated severe lumbar stenosis requiring surgical decompression. postoperatively, she regained strength, walking more easily. congo red staining of vertebral bone demonstrated widespread positive staining with birefringence consistent with amyloid arthropathy. these cases illustrate to wide sprectrum of amyloid neuropathy presentation, an example of amyloid arthropathy-induced spinal stenosis and the utility of skin biopsy to diagnose and manage amyloid neuropathy. the differentiation between hereditary neuropathy and chronic inflammatory demyelinating polyneuropathy (cidp) in children is especially significant because of completely different treatment possibilities and prognosis in these conditions. the aim of the study was to compare electrophysiological abnormalities in a group of children and young adults with demyelinating neuropathy of chronic or subacute onset. retrospective analysis of clinical and nerve conduction study (ncs) data included children and young adults with charcot-marie-tooth neuropathy x type (cmtx ), children with charcot-marie-tooth neuropathy type a (cmt a) and children with cidp. in our study / cmtx had both axonal and demyelinating changes in ncs study. the aan and efns electrophysiological cidp criteria were fulfilled in / cmtx , / cmt a and / cidp patients. additionally patients with cmtx are classified with efns criteria as "probable/possible cidp". a distal compound muscle action potential (dcmap) was > ms in all cmt a and / cidp patients but none with cmtx . abnormal median/normal sural snap (amns) parameter was observed in // cidp and / ctmx patients but not any cmt a patient while abnormal sural/normal median snap (asnm) parameter was found in / patients with cidp. a difference between conduction velocities (cv) of two corresponding nerves > m/s was seen in / cmtx and / cidp patients but no one with ctm a. one patient with cmtx had especially conspicuous difference between nerve conduction in lower limbs, with axonal neuropathy and demyelinating features, and upper limbs with no changes. one -years-old patient with genetically confirmed cmtx had no abnormalities in ncs. electrophysiological data of cmtx , cmt a and cidp indicate diffuse demyelination, however in cmtx axonal changes coexist, seen in / patients, and asymmetrical abnormalities between corresponding upper and lower limb were observed in / patients. our study has showed that duration of dcmap is useful not only in diagnosing an inflammatory neuropathy but also in differentiating cmtx from cidp. however presence of the not homogenous abnormalities in ncs in patients with cmtx may mimic inflammatory neuropathy. prada v , mori l , francini l , accogli s , ursino g , gemelli c , schizzi s , grandis m , bellone e , mandich p , schenone a . department of neurosciences, rehabilitation, ophthalmology, genetics and maternal and child health, university of genoa, genoa, italy. the overwork weakness (ow) problem in cmt disease has been debated for long time. it has been reported that the non-dominant hand (ndh) of patients with cmt disease is stronger than the dominant hand (dh) as a result of ow and some authors verified this hypothesis using mrc on different muscles (van pomeren, ). more recently, piscosquito et al. ( ) found that the ow phenomenon does not exist using a dynamometer and the hole peg test, a dexterity test. we recruited cmt patients and healthy controls. we evaluated the strength of the -pinch and of the hand-grip with a dynamometer, the opposition ability with the thumb opposition test (tot) and applied an instrumental testing of hand function using the sensor engineered glove test (segt), which previously demonstrated its sensitiveness to measure severity of hands dysfunction in cmt patients. tot was significantly higher in the ndh compared to dh (ndh= , ± , ; dh= , ± , , p< . ) in cmt patients, instead there was no difference in dh and ndh in healthy controls. in the evaluation of -pinch and of the hand-grip, ndh performed slightly but not significantly better than the dh ( -pinch: dh= , ± , n; ndh= , ± , n; ± , n; ndh= , ± , n, p:ns) . in normal controls we confirmed the % rule (noguchi & demura, ). finally, segt results were similar between the ndh and dh in cmt patients but in normal controls there was a better performance in the dh. in conclusion, this study supports the existence of the overwork weakness in cmt, evident in every measurements. dexterity and overall ability of the hands impaired in the dh, probably because of compensating movements, compared with the healthy controls in the weaker hand. finally, our results support the importance of avoiding supramaximal exercises and educating the cmt patients to prevent incorrect movements, which may overload the hand muscles. prasad k , ohnmar o , teng a , cheng yj , umapathi t . national neuroscience institute, singapore; yangon general hospital, yangon, myanmar; yong loo lin school of medicine, national university of singapore, singapore. antecedent infections that have been linked to guillain-barré syndrome (gbs) include campylobacter jejuni, haemophilus influenza, epstein-barr virus, cytomegalovirus, mycoplasma pneumoniae and influenza a virus. in south-east asia, the burden of mosquito-borne flavivirus infections is considerable. even in a small country like singapore, with a population of only . million and in spite of excellent sanitation and public health measures, dengue virus (denv) infections can exceed , a year. both symptomatic and asymptomatic denv infections are associated with gbs. zika virus (zikv) infections were detected in singapore in late august . doctors working in south-east asia anecdotally report that gbs cases increase after rainy season when mosquitos breed. we are currently planning studies to estimate the hitherto unknown burden of gbs associated with denv, zikv and other mosquito-borne viruses in this region. as a preliminary step, we reviewed the gbs cases seen at our institution in for evidence of denv and zikv. three out of the tested cases were positive for denv serology. one of these was also positive for denv pcr. all these patients had symptomatic dengue. two had acute inflammatory demyelinating polyneuropathy (aidp) and acute motor axonal neuropathy (aman) subtype of gbs. none the patients tested were positive for zikv pcr in serum and urine. one patient, with miller fisher syndrome (mfs), had serological evidence of recent zikv. he did not have clinical zikv symptoms. in mfs cases, the initial detectable zikv igg was later proven to be due to cross reactivity with previous denv infection. in cases ( amsan, aidp, mfs) the initial zikv igm was raised. we await analysis of convalescent sera to decide if they indeed had zikv or the results were related to previous and co-infection with denv. our preliminary findings indicate that flavivirus infections may account for at least some of the gbs cases in singapore. the lack of symptoms in some cases and the interactions between zikv and denv antibodies make accurate diagnoses challenging. prior r , , benoy v , , vanden berghe p , van den bosch l , . ku leuven-university of leuven, department of neurosciences, experimental neurology and leuven research institute for neuroscience and disease (lind), leuven, belgium; vib-center for brain & disease research, laboratory of neurobiology, leuven, belgium; laboratory for enteric neuroscience, translational research center for gastrointestinal disorders, k.u. leuven, leuven, belgium. charcot-marie-tooth disease (cmt) is the most common inherited neurodegenerative disorder of the peripheral nervous system. it is divided into two main subtypes, a demyelinating subtype (cmt type ) and an axonal form (cmt type ). cmt is a length-dependent disease, in that it effects the longest neurons in the body, thus the muscles in the peripheral regions are affected first and foremost. moreover, the majority of cmt patients share a classical phenotype with shared pathological hallmarks, such as distal muscular atrophy, reduction in nerve conductions, etc. but also molecular pathological hallmarks, like the breakdown in the transport of organelles and vesicles in neurons in a process called axonal transport. currently, there is no cure or effective treatment available to cmt patients. histone deacetylase (hdac ) has been shown to be a key regulator in axonal transport. moreover, inhibiting hdac has been demonstrated to stabilize microtubules, which act as molecular tracks for motor proteins and facilitates axonal transport of cargos. our labs current focus is on cmt type a, the main cause of cmt, which is caused by a duplication of a segment of chromosome p . containing the gene encoding peripheral myelin protein (pmp ). pmp is mainly expressed by schwann cells, the cells that myelinate neurons in the peripheral nervous system. in the work presented, the commercial mouse schwann cell-line, sw cells, was transfected to overexpress pmp using a lentivirus vector system to mimic that of cmt a patient schwann cells. the overexpression of pmp was confirmed using immunofluorescence and western blot techniques. these transfected sw schwann cells were then co-cultured with primary mouse dorsal root ganglion neurons (drgs) isolated from adult mice. these co-cultures where then analysed after weeks in vitro for axonal transport. the investigated groups were: a co-culture of drgs + sw cells, a co-culture of drgs + sw cells transfected with pmp , and a co-culture of drgs + a sw cell-line transfected with green fluorescent protein using a lentivirus vector system, and a monoculture of drgs. furthermore, this work demonstrates that the overexpression of pmp in schwann cells can impair axonal transport in co-cultured drgs in comparison to the other co-culture groups. moreover, these axonal transport defects were able to be rescued by the treatment of a hdac inhibitor, tubstatin a. to conclude, selective hdac inhibitors have been shown to be a beneficial treatment for a number of cmt subtypes in preclinical studies in our lab and offer as a viable treatment for a currently, untreatable debilitating disease. provitera v , caporaso g , stancanelli a , piscosquito g , di caprio g , saltalamacchia am , santoro l , nolano m . "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; department of neurosciences, reproductive and odontostomatological sciences, university 'federico ii' of naples, naples, italy. the observation of rate and patterns of cutaneous nerves regrowth after mechanical or pharmacological distal axonotmesis, has proven to be an excellent tool to study human nerve regeneration in normal and pathological conditions. most of the observations, however, are referred to small fibers, possibly due to the availability of excellent models of reversible distal cutaneous small fiber axonopathy. no such model is available to study the regeneration of myelinated fibers in human. we studied regrowth of cutaneous large fibers in fingertip of patients with carpal tunnel syndrome after surgical decompression. we recruited patients (m/f / ; age . ± . years) with carpal tunnel syndrome candidate to surgery. patients underwent clinical and electrophysiological evaluation, quantitative evaluation of discriminative threshold at rd fingertip. in the same site, patients also underwent mm punch skin biopsy. skin sections were stained by immunohistochemical techniques and cutaneous innervation was analyzed by confocal microscopy. meissner corpuscles and their myelinated afferences were assessed following previously published procedures. twelve months after surgery, patients repeated functional evaluation and underwent a second skin biopsy two mm apart from the first one. mean density of mcs/mm was . ± . at time and . ± . at follow-up, mean density of myelinated endings was . ± . at time and . ± . at follow-up. however, not in all patients regeneration occurred. based on the variation of mc density between time and follow-up we were able to identify patients in which active regeneration had occurred. in this subgroup mean density of mcs/mm was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . in the same group, mean density of myelinated endings was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . we describe morphological patterns associated to nerve regrowth in the biopsies of patients in which nerve regeneration occurs. we propose an in vivo model to study regeneration of large myelinated fiber endings in human skin. in addition to the count of nerve endings, the identification of patterns associated to nerve regeneration can increase the diagnostic yield of skin biopsy. the most common type of charcot-marie-tooth disease is caused by a duplication of pmp , leading to dysmyelination, axonal loss and progressive muscle weakness (cmt a). currently, no approved treatment for cmt a patients is available. among others, preclinical therapeutic approaches aim to correct the pmp overexpression in order to ameliorate axonal loss and muscle weakness. we previously reported that the novel polytherapeutic drug pxt , a low-dose combination of baclofen, naltrexone and sorbitol, improved dysmyelination and axonal loss after long-term application in adult pmp transgenic rats, a known animal model of cmt a. interestingly, after short-term application in young cmt a rats we observed a long-lasting prevention of muscle weakness as well but without obvious effects on dysmyelination and axonal loss. improved distal motor latencies in the electrophysiological recordings and a shift in the axonal diameter distribution raised the hypothesis that therapy may improve the neuromuscular endplate pathology as another therapeutic target for cmt a. here, we report a preclinical trial in adult cmt a rats treated from postnatal week for consecutive weeks as the most effective regimen in order to facilitate a deeper understanding of the mode-of-action of pxt . long-term therapy effects were confirmed by an ameliorated behavioral phenotype, improved distal motor latencies and also nerve conduction velocity in the electrophysiological recordings. histological analysis revealed an increased number of neuromuscular endplates, which were lowered to wildtype level after pxt treatment. further investigations include detailed analysis of peripheral nerve myelin thickness, internodal and nodal length, terminal axonal sprouting at the neuromuscular endplate and muscle fiber subtyping in treated cmt a rats. charcot-marie-tooth disease (cmt) is a rare hereditary neuropathy for which novel treatments are being developed and urgently needed. only few clinical trials and natural history studies have been performed. evaluation of intervention efficacy is hampered by slow progression and lack of sensitive outcome measures. the interindividual disease variability is high and prognostic disease measures are not established for cmt patients. previously, we have identified skin-derived disease and progression biomarkers in a large european and us-based cmt a cohort. within the german charcot-marie-tooth disease network (cmt-net, www.cmt-net.de) we aim to establish easier accessible biomarkers in blood of cmt a patients. blood analysis is less invasive and can be repeatedly performed during clinical trials and routine patient care. we have started clinically assessing approximately young, adolescent and adult cmt a patients at clinical centres (münster, münchen, göttingen) including a large set of clinical outcome measures (cmtnsv , onls, mwt, mwt, walk , hpt, sf- , fss, psqi, ess, bdi-ii). we will sample blood and skin tissue once a year at , and months for an observational period of years. tissue samples will be analyzed on the transcriptional, translational and epigenetic level. we envision that the diagnostic and progression biomarkers may be used to measure therapeutic effects within clinical trials and later in clinical routine monitoring. this is the first trial testing "circulating" biomarkers from blood in a prospective observational trial in cmt a patients. igm peripheral neuropathy is a slowly progressive and heterogeneous disease with symptoms ranging from mild foot numbness and minor imbalance to severe neuropathic pain and sensory and motor dysfunction. international consensus regarding assessment and treatment of patients with igm peripheral neuropathy is lacking. this might be caused by the repeated use of suboptimal outcome measures, the small trial sizes with low numbers of treated patients, the indolent disease course needing a longer follow-up period to capture relevant changes, or the possibility that administered treatments were not aggressive enough. the imagine study is an international, multi-centre, prospective, observational cohort study, which will result in a unique collection of a large number of prospectively collected and highly standardized clinical data, and a biobank from a large population of well-defined patients with igm peripheral neuropathy. the main objective is to describe in detail the variation in clinical subtypes, clinical disease course, past and current practice of treatment, antibody titres, and clinical picture at the various levels of assessing outcome. patients being at least years, and fulfilling the international criteria for igm peripheral neuropathy are eligible. exclusion is primarily based on concomitant diseases influencing peripheral nerve function. several study parameters measuring weakness, sensation, activity and participation, ataxia, pain, and quality of life are of interest. in february an enmc kick of meeting is organized, bringing together an igm peripheral neuropathy study group. the meeting will focus on the development of a core set of outcome measures to be used in future studies in igm peripheral neuropathy. the imagine study started in the netherlands in september , and to date patients were included. the imagine study starts in the united kingdom, italy, and france in the beginning of . centres recruiting at least patients with igm peripheral neuropathy are eligible to participate in the study. during the pns global recommendations from the enmc meeting, as well as the first results of the imagine study, will be presented. puget s , paolantonacci p , burlot l lfb biomédicaments, les ulis, france; lfb biotechnologies, les ulis, france. in dysimmune neuropathies patients (mmn, cidp, gbs), ivig have become the gold standard due to their efficacy and tolerability. acute renal impairment, a rare but serious adverse event, can be induced by all ivig. its incidence is not determined precisely for each ivig. it mostly occurs in patients at risk, with pre-existing conditions (prior renal insufficiency, diabetes, mellitus, arterial hypertension, elderly > years old, dehydratation, hypervolemia, sepsis, paraproteinemia or concomitant use of other nephrotoxic drugs) and treated by immunomodulatory doses. as the number of these patients increases over the years, the choice of ivig in association with the precautionary measures will be decisive to reduce or avoid the occurrence of this adverse event. from s to s, the nephrotoxicity of ivig has been explained by tubular toxicity related to osmotic nephrosis. sucrose, a stabilizer of some ivig, has been implicated as one of the causes of this. even if several new ivig without sucrose are used, renal impairment cases have been described and studies have highlighted that sucrose is not the only ivig stabilizer associated with tubular damage. however, in a recent study, renal impairment cases ( / ) have occurred in cidp elderly patients treated by free-sucrose ivig. the occurrence of renal failure seemed to be low (only / ) for patients treated with sucrose-ivig in association with precautionary measures (hydratation, adaptation of dosage according body mass index, rate infusion). otherwise, since s, cases of renal impairment secondary to hemolysis have been increased with some ivig but would be rare for full ethanolic fractionated products as they have been identified to generate very low occurrence of hemolysis. the occurrence of renal impairment related to ivig seems to be multifactorial, need to find out all causes (mechanisms of different stabilizers, hemolysis, and concentration of ivig). registries would be helpful for physicians to define the incidence, relative risk of ivig-related renal impairment and the right ivig in association with precautionary measures in these patients at risk. the published ultrasound (us) studies on chronic inflammatory demyelinating polyradiculoneuropathy (cidp) report diffusely increased cross-sectional area (csa) of nerves. these data have not been correlated with histological patterns. to date, no further information about us ultrastructural nerve modification has been provided because of limited frequency range ( - mhz) of the us probe. the aim of this preliminary study is to evaluate the correlation between histological findings from sural nerve biopsies and us patterns found with uhfus ( mhz) from sural nerve at the ankle. four patients with cidp underwent uhfus nerve evaluation of clinically affected sural nerve before undergoing a sural nerve biopsy. us findings included: cross-sectional area of the nerve, connective tissue depth and changes in echogenicity of fascicles. those data were then compared to the histological findings obtained in transversal and longitudinal sections. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened and in two of the four patients hyperechogenicity of fascicles was observed. also, thanks to the ultra-high-resolution of the probe, a direct correlation of the histological and us images was possible, so as a direct measurement of internal microstructure of the nerve. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of sural nerve can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue, as confirmed by the histological study. in particular, we have observed a hyperechogenicity of fascicles in most severe patients; in those cases, histology confirmed an increase of endoneurial depth. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed and will be presented during the meeting. puma a , cambieri c , panicucci e , desnuelle c , raffaelli c , sacconi s . high-resolution ultrasound (hfus, mhz) of peripheral nerves is a valuable non-invasive, painless complement to neurophysiology, especially in the workup of cidp. nevertheless, the current spatial resolution of echographic images doesn't allow a detailed study of the nerves. ultra-high frequency ultrasound (uhfus, mhz) is a new tool with a - times higher spatial resolution than traditional hfus. the aim of this preliminary study is to evaluate sensory and motor nerves structural characteristics found with uhfus in patients with definite cidp. seven patients with cidp underwent uhfus nerve evaluation of median, ulnar, peroneal and sural nerves, bilaterally. us findings included: cross-sectional area of the nerve, connective tissue depth, nerve vascularization and changes in echogenicity of fascicles. patients also underwent electroneurography (eng) and plexus mri. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened. two of the seven patients presented an epineural hypervascularization, observed at the doppler evaluation; none of them was treated by iv immunoglobulines. echographic changes were present even in the absence of mri abnormalities (root hypertrophy). eng characteristics correlated with the us patterns. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of nerves can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue. in particular, uhfus allows a more detailed study of nerves, demonstrating that the structural abnormalities hit connective tissue, while -conversely to previous studies -fascicles anatomy seems to be spared. we did not show nerve vascularization except in non-treated patients. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed. purcell l , , wojciechowski e , , gibbons p , , jamil k , , menezes, m , , burns j , . sydney children's hospitals network (randwick and westmead), new south wales, australia; university of sydney, new south wales, australia; universiti kebangsaan, kuala lumpur, malaysia. - % of children with cmt are reported to have hip dysplasia. we aimed to investigate the relationship between radiological hip dysplasia indicators and walking pattern in children with cmt. thirty children ( female; . ± . yrs; ± . cm; ± . kg cmt a, cmtx , cmtx , cmt f, cmt a) underwent d gait analysis ( dga), and had an anterior-posterior pelvis radiograph within months of assessment. radiographs were reviewed by two orthopaedic surgeons, and the reliability of measures of dominant limb hip health via radiograph was assessed. of the measures, measures had an intraclass correlation coefficient > . between raters, and the more experienced surgeon's measures were used for further analysis. the measures of acetabular index (ai), centre edge angle (cea), neck shaft angle (nsa), medial joint space, head width, lateral uncoverage, migration percentage and triradiate were used to investigate correlations with kinematic and kinetic dga variables of the pelvis and dominant limb hip, knee and ankle in planes, temporal spatial parameters and gait profile score. dga data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model. gait data were compared to typically developing children ( female; . ± . yrs, ± . cm, ± . kg). five of affected children had a migration percentage > ∘ , representing % of our sample. maximum hip abductor moment in terminal stance was significantly lower than normative reference values, and was moderately correlated with of the radiographic measures (ai r=− . , p= . ; nsa r=− . , p= . and medial joint space r=− . , p= . ). walking speed (normalised to leg length) was correlated with medial joint space (r= . , p= . ) head width (r=− . , p= . ) and triradiate (r=− . , p= . the ganglionopathies are a unique group of disorders with a varied etiology which includes autoimmune disorders, paraneoplastic syndromes and toxin induced causes. it has been observed that despite extensive investigations the cause of a sensory neuronopathy is often idiopathic in approximately - % cases. electrodiagnostic criteria to diagnose a possible ganglionopathy requires at least one sensory action potential absent or three sensory action potentials < % of the lower limit of normal in the upper limbs plus less than two nerves with abnormal motor nerve conduction study in the lower limbs. ulnar sensory-motor amplitude ratio values (usmar) lower than . is useful in differentiating ganglionopathy from axonal length-dependent neuropathy. we performed a retrospective analysis of patients who were either admitted or evaluated in the outpatient department in department of neurology, nizam's institute of medical sciences, with a possible ganglionopathy as per the diagnostic criteria proposed by camdessanchéet al. consecutive patients who sought treatment during a year period with a clinical pattern of sensory neuronopathy were analysed retrospectively. we reviewed the electrodiagnostic studies of patients and calculated the ulnar sensory-motor amplitude ratio values in comparison with patients with a clinical and electrophysiological diagnosis of diabetic axonal sensorimotor neuropathy. the clinical profile of a ganglionopathy from the indian subcontinent comprised of a prominent sensory ataxia as the initial presenting clinical manifestation with a predilection towards involvement of large-fiber sensory modalities. electrophysiologically a pattern of absent snaps was characteristically encountered with a significant value of usmar less than . , enabling differentiation from axonal length-dependent neuropathy. rahman im , jahan it , nahar s , khalid mm , jahan i , hayat s , , , islam z . guillain-barré syndrome (gbs) is a post-infectious autoimmune polyradiculoneuropathy where immune response is triggered by molecular mimicry between glycolipid antigens expressed by an infective agent such as campylobacter jejuni (c. jejuni) and human peripheral nerve gangliosides. cd molecules are mhc-like structures specialized in capturing and presenting a variety of glycolipids to antigen-specific t lymphocytes. the objective of this study was to investigate the possible role of coding region polymorphisms of cd genes in the pathogenesis of gbs in bangladeshi population. single nucleotide polymorphisms (snps) of exon of cd a (* /* ) and cd e (* /* ) in well defined gbs patients and healthy controls were studied to delineate their effect in developing gbs. we genotyped exon of both cd a (cys/tryp) and cd e (gln/arg) genes through pcr-rflp. to validate these findings, direct sequencing of pcr product was performed for at least samples for each position. we found no differences in genotypes and allele frequencies of both genes in gbs patients compared to controls. however, compared to control individuals with cd a* /cd e* haplotype were . times more likely to develop gbs, whereas individuals with cd a* /cd e* haplotype had a reduced relative risk by . times. a positive association of cd a* /cd e* haplotype was observed only in axonal form of gbs ( . % vs. . %, p = < . ). haplotype cd a* /cd e* was prevalent among anti-gm antibody positive gbs patient compared to anti-gm antibody negative patients ( . % vs. . %) though it was not statistically significant. snps in cd a and cd e were not associated with antecedent c. jejuni infection, disease severity and disease outcome at months of follow up. in conclusion, cd polymorphisms are not a susceptibility or disease causing factor in gbs. conversely, increasing knowledge of this field may offer new dimension for the research to elucidate better answer for disease pathogenesis and also contribute to conduct high power meta-analysis. extensive genetic testing was performed on a unique patient, who received national media attention due to her severe cmt phenotype. the index patient was never able to walk independently, had difficulty breathing and swallowing and demonstrated aspiration by a barium swallow study. nerve conduction velocity testing revealed velocities of m/s in her upper extremity. she required continuous positive airway pressure (cpap) for an upper respiratory infection and developed bilateral vocal cord paralysis. we used the combined brief assessment of motor function (bamf) scale to evaluate her disability. her fine motor scale was a / , her upper extremity gross motor scale was a / and her lower extremity gross motor scale was / . unfortunately, she died at age in her sleep, presumably from respiratory arrest. the proband's father was asymptomatic, however, his neurological exam showed pes cavus bilaterally and mild atrophy of the first dorsal interossei muscles in his hands. his nerve conduction velocities were slowed to m/s in the upper extremities and his cmt neuropathy score (v ) was / . we performed whole exome and subsequently whole genome sequencing in the trio. comprehensive structural variant analysis for copy number variations, large deletions, and recombinations was completed by a combination of software tools. known cmt genes were excluded as the underlying cause and the only viable candidate gene remaining was sh tc . we showed expression of sh tc in peripheral nerve and schwann cells. sh tc is a paralogue to sh tc , which causes the recessively inherited dysmyelinating form cmt c. sanger sequencing confirmed the variants in our family, p.v m (c. g>a, chr : ) and p. a p (c. g>c, chr : ) , and showed the segregation of the heterozygous variations. we hypothesize that the heterozygous paternal allele had a minimal effect and let to a very mild or subclinical form of peripheral neuropathy. of interest, the recessive sh tc gene has also been shown to cause mild aberrant forms of peripheral nerve degeneration in carriers of heterozygous disease alleles. in summary, this paper will present genetic and molecular evidence from an extensive n= study proposing a novel cmt gene. charcot-marie-tooth (cmt) is an important cause of morbidity worldwide. it is a heterogeneous disease manifesting as progressive weakness, wasting and loss of feeling in a length-dependent pattern. there are an increasing number of cmt-related genes in the literature. more recently, recessive mutations in the hint gene have been reported as causative of predominant motor axonal neuropathy associated with neuromyotonic discharges on emg. one of the characteristics of autosomal recessive cmt is it varying frequency in different populations and ethnic groups. we sought to evaluate the frequency of mutations in the hint gene in a brazilian cohort with axonal hereditary neuropathy. all patients included in this study born within south east area of brazil. the group consists of consecutive patients with axonal neuropathy screened for recessive or sporadic axonal neuropathy in the neurogenetics laboratory of clinical hospital of ribeirão preto. direct sequencing of the coding region of hint gene was done. among patients screened, were suspected of having recessive axonal neuropathy without neuromyothonic discharges, and two have axonal neuropathy associated with neuromyothonic discharges on emg. we did not find any disease causing mutations among our patients. some previously reported studies reported a high frequency of mutations in the hint gene among recessive axonal neuropathies in some european countries. our results demonstrated that frequencies of mutation underlying genetic hereditary neuropathies are different between different ethnic groups and have implications for the organization of services management of cmt, for genetic counseling, and for gene flow in different world populations. funded by cnpq, fapesp, faepa, pronas (ministry of health). rodriguez-menendez v , ballarini e , malacrida a , , ceresa c , semperboni s , , meregalli c , cavaletti g , nicolini g . school of medicine and surgery, experimental neurology unit, university of milano-bicocca, monza, italy; phd program in neuroscience, university of milano-bicocca, monza, italy. bortezomib (btz) is a proteasome inhibitor widely used for multiple myeloma treatment. btz induced peripheral neuropathy (bipn) is the most frequent adverse effect. bipn in humans, is a dose dependent painful sensory neuropathy characterized by nerve axonopathy and a tendency to recover in the follow-up period after btz withdrawal. bipn affects principally dorsal root ganglia (drg) and different rodent models have shown alterations in sensory neurons, small unmyelinated axons, large myelinated axons, axonal mitochondria and schwann cells. in this work, we evaluated the effects of btz in vitro on drg neurons isolated from adult mice. our interest focused on dystonin, a protein able to interact with all the three components of cytoskeleton (microtubules, microfilaments and intermediate filaments) and able to bind map b (a mt-associated protein) through which can influence golgi apparatus morphology. there are different neuronal isoforms of dystonin and in particular dystonin-a is able to modulate tubulin acetylation and stability through interaction with map b. it is noteworthy to underline that map b expression in the mature nervous system is restricted to sensory neurons. western blot analysis demonstrated that a treatment with nm btz induces a statistically significant decrease in tubulin acetylation. this result does not go along with our previous study where we observed a tubulin polymerization increase after btz treatment. therefore, we have decided to focus our interest on soma organelle organization that is well known to be dependent from cytoskeleton structure. immunofluorescence images showed an altered distribution of acetylated tubulin in soma cytoplasm after hour treatment. moreover, confocal analysis of cis golgi (gm ) demonstrated that in btz treated neurons the normal golgi structure is lost showing a spot-like non perinuclear labeling distribution. additionally, dystonin distribution seemed comparable to that of cis golgi apparatus suggesting that btz could induce a change in dystonin localization which in turn affects golgi organization, probably through map b.these data suggest that the cytoskeleton alterations, induced by btz, could probably cause wrong maturation and trafficking alteration of golgi vesicles consequently impairing the correct sensorial function. romão tt , aleixo bfl , wedemann d , herculano fgn , prado h , cal h , pupe c , bittar c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. acute motor and sensory axonal neuropathy (amsan) is a clinical variant of guillain-barré syndrome (gbs) which has rarely been reported in association with human immunodeficiency virus (hiv) and neurosyphilis. we describe a case of a -year-old woman who started with muscle weakness in the left leg followed by weakness in the right leg and in upper limbs in one month. motor symptoms were followed by urinary and fecal incontinence. no preceding symptoms, as diarrhea or fever were referred. neurological examination showed global areflexia with impairment of vibratory and tactile modalities, as well as loss of proprioception. pinprick sensation was moderately compromised. no impairment of higher mental functions was observed. other causes of neuropathies were ruled out. cerebrospinal fluid (csf) examination showed cell/mm , increased protein level, normal glucose level and negative vdrl test. blood analysis showed hiv and positive serology tests, vdrl : and positive t. pallidum hemagglutination assay. igm cytomegalovirus antibody was negative, cd was in normal range and hiv load was undetectable. cranial and spinal cord magnetic resonance imaging revealed extensive involvement of the posterior spinal cord tracts which supports the diagnosis of neurosyphilis. nerve conduction studies showed a significant reduction in compound motor action potentials amplitudes, particularly at lower limbs, and absent sural nerve responses. electromyography revealed positive waves and fibrillation at lower limbs. treatment with methylprednisolone, human intravenous immunoglobulin and ceftriaxone has been settled, resulting in improvement of upper limbs paresis and sphincters dysfunction. antiretroviral therapy was initiated. this amsan case is associated with central nervous system involvement, encompassing an encephalomyeloradiculopathyrelated to hiv/t. pallidum co-infection. c.jejuni is the main etiological agent associated with amsan, but there are some reports of hiv/cmv co-infected patients who presented this gbs variant. facing a patient with amsan, mainly when cns involvement is present, it is of crucial importance to undergo investigation of an ongoing infectious process, such as hiv and syphilis, considering the impact of early treatment on prognosis. roodbol j , , yiu e , doets a , , de wit mcy , monges ms , van nes s , catsman-berrevoets ce , van den berg b , , jacobs bc , and on behalf of the igos-kids consortium. only a few prospective cohort studies so far have investigated the clinical presentation and course of guillain-barré syndrome (gbs) in children using age-adjusted outcome scores. in we started a study based on the igos protocol but adjusted for children with gbs (igos-kids). (pediatric)neurologists from argentina, australia and the netherlands joined forces to determine the clinical and biological determinants of disease progression and recovery in gbs in children from different geographical areas. for each age category specific pain scales are used including the comfort score for children < years old, pain faces revised age - years old and numerical rating scale in children ≥ years old and outcome measures. a new strength score was developed: gbs kids score. this score will be used and validated in addition to the mrc-sum score. the onls, r-ods and fss are also be used in igos-kids. age-dependent quality of life questionnaires were added to the igos-kids protocol, namely the pedsql and the pedsql multidimensional fatigue scale. these scales are available for children ≥ years old. an activity score validated for neuromuscular disorders for children ≥ years old was also added. currently there are children included in igos-kids, eight children from australia and five from the netherlands. additional clinicians and researchers interested in gbs in children are most welcome to participate in igos-kids. prognostic models have been developed to predict the highly variable clinical course of guillain-barré syndrome (gbs) in adults. the clinical course of gbs is equally variable in children, but the current prognostic models have not been validated in children. in this study, we aimed to identify in children with gbs the characteristics at hospital admission that predict the clinical severity and outcome. the study was conducted in two patient cohorts from europe: one (largely) german cohort of children and one dutch cohort of children. clinical information was obtained regarding preceding infection, first symptoms, neurological deficits at admission and nadir, results of additional investigations (cerebrospinal fluid and nerve conduction studies). clinical severity, course and outcome was defined by the gbs disability score, especially the ability to walk unaided, at a follow-up of month, months, months and months after onset of symptoms. univariate and multivariate regression analyses were performed initially on the two separate cohorts. combined the cohorts consisted of children (age median years, range - years) including boys ( %). the median duration between onset of symptoms and hospital admission was days (range ). pain at admission was remarkably frequent and present in ( %) children. in the combined cohorts, children ( . %) developed respiratory failure and one child died. multivariate regression analysis showed that in both cohorts strong predictors of respiratory failure available at hospital admission were cranial nerve involvement, a higher gbs disability score and a shorter period in days between onset of symptoms and admission. this information was used to develop and validate a prognostic model for children with gbs that will be presented at the conference. in conclusion, based on the analysis of two independent cohorts of patients the predictors of respiratory failure and clinical recovery were identified and validated. similar factors were identified for adult patients, although the prognosis in children in general is better than in adults. this information will be used to develop a simple prognostic model for current clinical practice to predict the chance of respiratory failure and outcome in children with gbs. the negative trials of vitamin c in cmt a have highlighted the lack of sensitive outcome measures in charcot-marie-tooth disease (cmt). neurofilaments are abundant neuronal cytoskeletal proteins and their concentration in blood is likely to reflect axonal breakdown. we therefore examined plasma neurofilament light chain (nfl) concentration as a potential biomarker in cmt. blood samples were collected from patients with cmt and age matched healthy controls over a -year period. disease severity was measured using the weighted cmt examination and neuropathy scores. plasma nfl concentration was determined using an in house developed simoa assay based on the nfl antibodies from the nf-l light elisa kit (umandiagnostics). plasma nfl concentrations were significantly higher in cmt patients (median: . pg/ml; iqr: . - . ) than in controls (median: . pg/ml; iqr . - . , p< . ) and correlated with disease severity as estimated by the weighted cmt examination (n= , r= . , p< . ) and neuropathy scores (n= , r= . , p= . ) . concentrations were also significantly higher when subdividing cmt patients by genetic subtype; cmt a (n= , p< . ), sptlc (n= , p< . ) and gjb (n= , p= . ) or into demyelinating, cmt (n= , p< . ) or axonal, cmt (n= , p< . ) forms compared to healthy controls. there was no significant difference in the plasma nfl concentration after year in patients with cmt (n= , mean difference − . pg/ml, % confidence interval − . - . ) or healthy volunteers (n= , mean difference − . pg/ml, % confidence interval . - . ) which is unsurprising as cmt is a slowly progressive disease in which the rates of axonal degeneration are likely to be constant and elevated. in summary, we have shown that plasma nfl levels are significantly raised in patients with cmt and that they correlate with disease severity. this is of relevance not only for the field of cmt but for peripheral neuropathies in general, and suggests that plasma nfl holds promise as a biomarker of peripheral neuropathy in both routine practice and clinical trials. neurolymphomatosis (infiltration of the peripheral nervous system by lymphoma cells) is a rare and usually devastating condition belonging to the spectrum of lymphoma-associated neuropathies. cerebrospinal fluid examination with cytologic examination, flow cytometry and clonality testing by pcr may show malignant cells especially when nerve root involvement is prominent. however, nerve biopsy remains a useful tool to confirm the presence of malignant cells invading the nerve. neuropathological features are important and pcr-based immunoglobulin or t-cell receptor clonality testing on nerve fragments may add notable value for the diagnosis of neurolymphomatosis, although this has not been systematically investigated. we retrospectively studied clinicopathological data and clonality results of nerve biopsy samples in patients with nl from centres, performed between and . among patients with nl, we found % of b-cell lymphoma and one t-cell lymphoma. nl was the first manifestation in , % of patients. the main clinical pattern was symmetrical progressive sensorimotor polyneuropathy in % of patients and pain was a prominent feature in %. clonality testing showed a monoclonal rearrangement in ( %) cases, oligoclonal rearrangement and no amplification in ( %). the main histological pattern was perivascular infiltration of predominant b-cells in the epineurium, without signs of vasculitis. the extent of axon loss was highly variable between patients. various chemotherapeutic regimens were used and the median overall survival was months. only one case showed a monoclonal pattern among control nerve samples from patients with other types of neuropathy including vasculitis and cidp. overall, we found that clonality testing on nerve biopsy specimens may provide decisive information on the presence of neurolymphomatosis, with a sensitivity of . %, a specificity of . %, a positive predictive value of . % and a negative predictive value of . %. we confirm the utility of nerve biopsy for the diagnosis of nl and show the great yield of pcr-based clonality testing to assess the malignant feature of peripheral nervous system lymphoid infiltrates. despite an accurate diagnosis, neurolymphomatosis still remains a devastating disease with an overall poor prognosis. in the last years, cumulative data have shown that patients with amyotrophic lateral sclerosis (als) and mouse models of the disease present loss of small epidermal and dermal nerve fibers and sensory dysfunctions, in addition to the classical motor symptoms. our objective is to characterize this small fiber neuropathy and to clarify if axonal loss involves all sort of fibers equally, or if there is some specificity. for this purpose, we performed an immunohistochemical characterization of total intraepidermal nerve endings (protein gene product; pgp . ), peptidergic (calcitonin gene-related peptide; cgrp) and nonpeptidergical nerve epidermal endings (isolectin b ; ib ) of the sod g a mouse at different stages: presymptomatic stage ( weeks), disease onset ( weeks) and in symptomatic stage ( weeks). the sympathetic sweat gland innervation was immunolabeled for vasoactive intestinal peptide (vip) from very early stage ( weeks) to disease onset ( weeks). the results showed a marked reduction of the intraepidermal nerve fibers already in the presymptomatic stage compared to the wildtype littermates (p< . ). this axonal loss affected more markedly the nonpeptidergic axons from the disease onset stage ( % axonal loss, p< . ), whereas no significant differences were found in the cgrp positive fibers ( . % axonal loss). a reduction of the sympathetic innervation of the sweat glands was also found from the disease onset stage ( % axonal loss, p< . ). in summary, we have found that nonpeptidergic and sympathetic innervation of the skin are predominantly affected in the sod g a mouse model. these findings suggest that this specificity could be used as an accessible biomarker for the disease. hereditary and inflammatory neuropathies are peripheral nerve disorders of different pathophysiology whose identification is crucial for therapeutic approach. diagnosis of cmt is easy when there is a family history, disease course is slowly progressive, neurophysiological findings are homogeneously abnormal. cases of cmt patients with inflammatory-like phenotypes leading to a misdiagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described. the presence of "red flag signs", such as slowly progressive decline, mild sensory symptoms, minimal electrophysiological progression and no response to therapy should prompt re-evaluation of the diagnosis of cidp. the introduction of nerve neuroimaging has contributed to the diagnostic work-up. we describe cmt patients ( men, women, mean age . ± . yrs, mean neuropathy duration . ± . yrs) with genetically confirmed cmt, who were initially diagnosed with cidp. neurophysiology showed demyelinating features in patients, the remaining patients had axonal features. csf analysis showed albumin-cytological dissociation in patients, oligoclonal igg bands were present in patient. nerve ultrasound in patients with demyelinating neuropathy showed diffuse increased cross-sectional area. mr-neurography in one patient confirmed diffuse nerve hypertrophy and increased signal intensity, supporting the hypothesis of a overlap syndrome. all patients were treated with immunomodulatory therapies. among patients with demyelinating features, patients underwent ivig for months, without benefit; the remaining were treated with steroids, showing temporary improvement. the patients with axonal neuropathy complaining of a progressive history, underwent ex adjuvantibus plasma exchange and ivig, without benefit. given the lack of benefit from therapies, screening for hereditary neuropathies was performed. among patients with demyelinating neuropathy, had cmt a, cmt b, cmt d. among the patients with axonal neuropathy, one was diagnosed with cmt k. in patients an overlap syndrome was present. several clinical and laboratory features (csf protein elevation), might contribute to the misdiagnosis of cidp in cmt patients. refractoriness to immune-modulatory treatment should rise the suspicion of a hereditary neuropathy. an overlap cmt/cidp syndrome may be considered, in front of acute/subacute deterioration and/or proximal muscles involvement. two patient with an overlap syndrome showed benefit after either steroids or ivig. ruiz m , tripodi sm , campagnolo m , zara g , salvalaggio a , ruggero s , toffanin e , anglani m , briani c . neurology, department of neuroscience, university of padua, padua, italy; neuroradiology, padua hospital, padua, italy. facial diplegia is a rare regional subtype of guillain-barrè syndrome characterized by rapidly progressive bilateral facial palsy in absence of other cranial neuropathies, ataxia or limb weakness. the diagnosis is based on history, clinical examination, laboratory data. the outcome of this variant appears to be better than that of classical gbs. although about % of gbs patients clinically exhibit facial nerve involvement, there are only few reports that demonstrate mri gadolinium facial nerves enhancement. a -year-old previously healthy man sought neurological advice for the acute onset of right perioral paresthesias and hyposthenia at the extensor hallucis longus bilaterally. in the previous month, during a trip to china and japan, he had suffered from gastroenteritis. at neurological examination he presented with bilateral extensor hallucis longus hyposthenia. ankle jerk reflexes were absent. one week later, he developed bilateral facial palsy. neurophysiology disclosed absence of activity in the facial nerves innervated muscles bilaterally and signs of mild partial denervation at right l metamer. csf analysis showed increased protein levels ( mg%) and . leucocytes/ l. serological tests for infectious agents were negative and serum levels of angiotensin converting enzyme were normal. antibodies to monosialoganglioside gm of igg isotype were positive ( / ). t brain mri showed, after gadolinium administration, marked bilateral enhancement of the facial nerves in their extra-and intra-canalicular segments. t lumbosacral mri scan ruled out the presence of disc diseases as well as signal modifications within conus medullaris and cauda equine nerve roots. the patient was treated with iv immunoglobulins ( . g/kg/die for days) with benefit on facial weakness. at follow-up examination months later, the patient presented a further improvement of the facial diplegia and neurophysiology disclosed a partial recovery of activity in the facial nerves, with persistent axonopatic damage. bilateral extensor hallucis longus hyposthenia persisted. in conclusion, we report on a regional subtype of guillain-barrè syndrome with the curious association of facial diplegia and bilateral extensor hallucis longus hyposthenia, and igg anti-ganglioside antibodies, that are not commonly described in facial diplegia. moreover, we provide t brain mri evidence of facial nerve involvement. rumora ae , tabbey ma , lograsso g , dolkowski j , haidar j , lentz si , feldman el . department of neurology, university of michigan, ann arbor, usa; department of internal medicine, division on metabolism, endocrinology and diabetes, university of michigan, ann arbor, usa. diabetic neuropathy (dn) is the most common complication of diabetes affecting up to % of diabetic patients. the pathogenesis of dn in type diabetes is directly related to the metabolic syndrome associated with the western diet composed of elevated levels of long chain saturated fatty acids (sfas) and low levels of medium chain sfas. long chain sfas are associated with insulin resistance and dyslipidemia while medium chain sfas have been associated with decreased lipid accumulation and improved mitochondrial function. since dn is primarily a disorder of the sensory dorsal root ganglion (drg) neurons, we sought to evaluate the impact of sfa hydrocarbon chain length on mitochondrial trafficking mechanisms that are critical for distributing atp throughout drg axon to provide energy for synaptic transmission. we hypothesize that sfas with longer hydrocarbon chains will impair mitochondrial trafficking whereas medium length sfas will not impact mitochondrial movement along the drg axon. in this study, we examined the impact of sfa hydrocarbon chain length on mitochondrial trafficking, directionality and velocity in primary mouse drg neurons. drg neurons were treated with increasing concentrations of long chain sfas, stearate and palmitate, or medium chain sfa, laurate, ranging from . to micromolar for twenty-four hours. drg neurons treated with long-chain sfas, palmitate and stearate, showed a significant decrease in the percentage of motile mitochondria whereas medium chain sfa, laurate, does not alter mitochondrial motility. we next assessed whether motile mitochondria in drg neurons treated with palmitate or stearate exhibited altered directionality or velocity of mitochondrial trafficking. palmitate and stearate treatments resulted in a trending decrease in the number of mitochondria trafficking in both anterograde and retrograde directions. furthermore, . to micromolar palmitate and stearate induced a significant decrease in mitochondrial velocity. laurate treatment, on the other hand, retained directionality and velocity of mitochondrial trafficking. these results suggest that hydrocarbon chain length of saturated fatty acids plays an important role in regulating mitochondrial trafficking mechanisms in dorsal root ganglion neurons. impaired mitochondrial trafficking in drg neurons exposed to elevated levels of long-chain sfas may play a critical role in the progression of dn. dyslipidemia is a critical factor that contributes to the development of diabetic neuropathy (dn). the progressive nerve damage associated with dn correlates with the dyslipidemic state characterized by elevated circulating levels of harmful saturated fatty acids (fas) and low levels of beneficial unsaturated fas. in dn, primary sensory dorsal root ganglion (drg) neurons exhibit energy dyshomeostasis and mitochondrial dysfunction, however, little is known about the differential impact of saturated and unsaturated fas on mitochondrial mechanisms in drg neurons. mitochondrial trafficking is an essential mechanism for transporting mitochondria throughout drg axons to provide cellular atp for neuronal function and neurotransmission. since mitochondrial trafficking is regulated by metabolic flux, we sought to determine whether saturated fa, palmitate, and unsaturated fa, oleate, have differential effects on mitochondrial trafficking in drg neurons. we evaluated mitochondrial trafficking patterns and the mitochondrial membrane potential (mmp) in primary drg neurons treated with physiologically relevant concentrations of palmitate, oleate, and combinations of both fas. primary drg neurons treated with . to micromolar palmitate induced a significant and dose-dependent reduction in the percentage of motile mitochondria. these palmitate treatments also induced a dose-dependent reduction in mitochondrial velocity but did not impact the directionality of mitochondrial movement. alternatively, . to micromolar oleate treatments did not impair the percent of motile mitochondria, the direction of mitochondrial movement, or mitochondrial velocity. since palmitate and oleate have differential effects on mitochondrial motility, we next assessed whether oleate could counter the inhibitory effect of palmitate on mitochondrial trafficking. surprisingly, oleate/palmitate mixtures at ratios of : or : prevented palmitate-induced impairment of mitochondrial trafficking. we assessed the impact of palmitate and oleate on mmp by staining palmitate and oleate-treated drg neurons with tetramethylrhodamine methyl ester. drg neurons treated with micromolar palmitate exhibited an increase in the percentage of depolarized mitochondria while mitochondria in oleate-treated drg neurons retained mmp. interestingly, drg neurons treated with : oleate/palmitate mixtures also maintained polarized mitochondria. these results suggest that saturated and unsaturated fas have a distinct impact on mitochondrial trafficking mechanisms in drg neurons and that equimolar ratios of oleate/palmitate can prevent impairment of mitochondrial trafficking. of g alpha- in m r overexpressed sensory neurons significantly reversed the m r-induced reduction in relative levels of total neurite outgrowth (mean±sd: . ± . (control) vs . ± . (g alpha- knockdown), p< . , n= neurons). further, treatment of m r overexpressing neurons with mt or pirenzepine sequestered g alpha- / proteins at the m r and significantly reversed the impaired cytoskeleton-mediated reduction in total neurite outgrowth (mean±sd: . ± . (control), ± (+ nm mt ), ± . (+ micromolar pirenzepine); n= , and neurons, respectively; for treated groups p value= . vs control by one-way anova). mt and pirenzepine also significantly restored mitochondrial trafficking and abundance of mitochondria in the distal neurites. our findings suggest a novel mechanism in which modulation of m r influences tubulin polymerization, mitochondrial distribution in nerve terminals and controls axonal outgrowth and regeneration. funded by cihr and nih. muscarinic receptors are a group of five g-protein coupled receptors (gpcrs) that are targeted by drugs for the treatment of several human pathophysiological conditions. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) antagonists of the muscarinic acetylcholine type receptor (m r) induced elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt et al., ) . one of the major cellular effectors activated by gpcrs is extracellular signal-regulated kinase (erk). the erk signaling cascade regulates a variety of cellular processes including growth and proliferation. both g protein and beta-arrestin mediated signaling pathways can lead to erk activation by phosphorylation through different kinases. activated erk in turn can phosphorylate about cellular substrates, thereby mediating diverse functions. in this study, we have analyzed beta-arrestin recruitment, as part of the receptor internalization process induced by agonist/antagonist binding. in addition, we studied phosphorylation of erk by mt and pirenzepine using isoelectric focusing with phospho-erk specific antibodies and a variety of cell-based assays including beta-arrestin and g protein (gnas/gnasl/gnaq/gna /gna /gna ) knockout cell lines. our study revealed that beta-arrestin is recruited to the m r upon mt and pirenzepine treatment. treatment with nm mt and micromolar pirenzepine significantly increased the dual phosphorylation of tyr and tyr residues of erk / in primary rat sensory neurons (p< . ) in comparison to muscarinic agonist carbachol ( micromolar) and general antagonist atropine ( micromolar). we have identified multiple distinct phosphorylation events on the m r by isoelectric focusing that are specific to mt and pirenzepine induction. further, we have shown that mt and pirenzepine-mediated erk phosphorylation is dependent on both g protein and beta-arrestin recruitment to m r. finally, we reveal that increased erk phosphorylation by mt and pirenzepine significantly (p= . and . , respectively) increased phosphorylation of camp responsive element binding protein (creb) at ser . these results show for the first time that antagonists of the m r can activate the erk signaling pathway and possibly drive phenotypic change in adult sensory neurons. funded by cihr # mop- . charcot-marie-tooth disease (cmt) is representative of inherited neuropathies affecting an estimated in people. the immense advances in gene discovery gained from next-generation sequencing (ngs) projects have revealed the extent of cmt's genetic heterogeneity, with over loci already identified. this knowledge is rapidly translated into clinical comprehensive gene testing panels, often containing over genes. such a large genomic space will invariantly yield variants of uncertain clinical significance (vus) in nearly any person tested. this rise of the number of vus creates major challenges for genetic counseling. in addition, less individual mutations in already known genes are being published as the academic merit is decreasing, and most such testing now happens in clinical laboratories. we propose to capture more of this data in the cmt field to gain a more complete collection of alleles in cmt genes, ideally in conjunction with detailed phenotypic data. this represents a rational approach to eventually reduce the number of vus. thus, we have created a unique, community-driven variant database for cmt researchers and clinicians. the inherited neuropathy variant browser provides simple, user-friendly access to currently reported cmt variation, including patient-level genotypic and phenotypic information. we have also designed an interactive rating system of genetic variation to assist the community with interpretation of vus. for the initial release, we have collected genetic variation, along with genotypic and phenotypic data when available, from published literature, clinical lab reports, and our in-house database. we highly encourage new submissions of not only observed pathogenic variation, but also variation of unknown significance. the goal is to provide a platform for the cmt community to store, share, and discuss genetic data in order to resolve variation of uncertain significance as a joint-effort. with active participation, we aim to provide the community with a more complete mutational spectrum in cmt genes to assist allelic interpretation and patient diagnosis. neurofilaments are strictly neuron-specific intermediate filaments crucial for maintaining axonal architecture. pathogenic mutations in nefl, which encodes the light chain of neurofilament, cause dominantly and recessively inherited charcot-marie-tooth neuropathy (cmt). the nefl-associated neuropathy can be either axonal (type e) or demyelinating (type f). most pathogenic nefl variants are dominantly inherited missense mutations, which are thought to cause disease by inducing nefl aggregation, leading to the disruption of axonal transport. however, investigation of disease mechanisms caused by the mutations has been complicated by the neuronal specificity of nefl. we identified a homozygous nefl variant c. c>t predicting a nonsense change p.arg * in a patient with early-onset cmt. to elucidate the disease mechanism, we used pluripotent stem cells, reprogrammed from patient's skin fibroblasts, to differentiate patient-specific spinal motor neurons. the motor neurons revealed a near complete loss of nefl mrna, and absence of nefl protein. our results establish that nefl is not essential for the development of human nervous system but its absence causes progressive axonal neuropathy. we currently profile the transcriptomic alterations of the motor neurons lacking nefl using single cell rna sequencing to identify compensatory pathways. friedreich's ataxia (frda) is the most common autosomic recessive ataxia, due to a trinucleotide expansion within the frataxin gene. within the wide phenotype, frda patients present also with an axonal neuropathy whose pathological mechanisms are not completely known. eight patients ( women, mean age . yrs, range - ) with genetically confirmed frda underwent neurophysiological and nerve ultrasound evaluation at four limbs bilaterally. echogenicity and cross-sectional area (csa) of median, ulnar, radial, peroneal, tibial, and sural nerves were recorded. mr neurography and diffusion tensor imaging (dti) analysis were performed in one patient; fractional anisotropy (fa), radial (rd) and axial (ad) diffusivity of median, radial and ulnar nerve were calculated at proximal, intermediate and distal sites. all patients presented with sensory axonal neuropathy. seven patients ( %) presented with increased csa of median and ulnar nerves at arm and axilla. mean median nerve csa at mid-upper arm was . mm (normal values < mm ), mean ulnar nerve csa at mid-upper arm was . mm (normal values < mm ). mean median nerve csa at axilla was . mm (normal values < mm ), mean ulnar nerve csa at axilla was . mm (normal values < mm ). mr neurography (performed in one patient) confirmed diffuse swelling and signal hyperintensity of median and ulnar nerves at the arm and dti analysis showed abnormal values of fa, ad and rd along the whole course of evaluated nerves thus suggesting a wide alteration of nerves structure. frda patients presented with an axonal neuropathy characterized, at ultrasound, by a nerve enlargement strictly limited to mid-upper limbs in all patients, findings that cannot be solely explained by a dying-back axonopathy, as suggested by several authors. neither a dorsal root ganglia neural loss could explain by itself our findings, because a diffuse csa reduction would have been expected. on the whole, these findings represent a peculiar feature in frda, but its pathophysiologic meaning remains unclear. inherited peripheral neuropathies are an important health concern for which there is currently no disease-modifying therapy. dogs are affected by a variety of peripheral neuropathies that are breed-specific, indicating a strong genetic component. the use of in-bred populations, such as pure-bred dogs, is advantageous for genetic dissection of disease. acquired peripheral neuropathy (apn) is an inherited late-onset generalized polyneuropathy with high prevalence in labrador retrievers. the most prominent features of apn, laryngeal paralysis and pelvic limb weakness, are associated with the longest peripheral motor nerves in the dog. the pathologic features of apn are similar to human peripheral neuropathy. our aim is to understand the genetic and pathologic features of apn for development of this condition as a naturally occurring large animal model for human disease. we performed a genome-wide association study (gwas) and short-read high-depth whole genome sequencing (wgs) to investigate the genetic underpinning of apn in the labrador. our gwas data indicates that apn is an autosomal dominant disease. the initial analysis from the wgs study resulted in a potential causal variant with an autosomal dominant pattern; this variant is associated with an axonal gene. the neuropathologic progression and histologic features of apn are poorly defined. using genetic markers from our gwas study, we are able to confidently identify labradors with pre-clinical apn, from which nerve biopsies can be obtained. preliminary analysis of biopsies from labradors suggest apn is an axonopathy. further histologic data from preclinical and symptomatic dogs is being obtained to further define the pathogenesis in this model. mutations in morc lead to an axonal form of neuropathy (cmt z). to date, nine families have been published with mutations in the morc gene, showing that this gene is frequently involved in cmt. while the recent genetic data clearly established the causative role of morc in cmt z, its phenotypic consequences in patients and role in neuronal biology remains to be clarified. therefore, we aim to look for altered genetic and biochemical pathways with a transcriptomic approach in order to investigate the role of morc in hereditary peripheral neuropathy. we have performed transcriptomic analysis using a human gene expression microarray (v x k, agilent technologies), in three hek- t cell lines: control, morc knock-down (kd) and the overexpression of the most common morc mutation, the p.r w (np_ ) (kr). differential gene expression assessment was carried out using limma moderated t-statistics. standard analysis techniques perform one test for each gene. thus, for each gene, a t-test statistic is reported together with its corresponding p-value. in this analysis we have used the conventional multiple testing p-value correction procedures proposed by benjamini hochberg to derive adjusted p-values. the preliminary results reveals that kd shows up-regulated genes involved in transmission of nerve impulse and cilium metabolism, suggesting that morc might act at this level in the peripheral nervous system. otherwise, kr shows a major alteration of main axonal metabolic pathways, including the overexpression of genes related to the generation of neuronal action potential, transport through the axon and its targeting in synapse formation. kr also shows a marked alteration of gene expression related to organization, assembly and cilium movement and with the axonemal dynein complex assembly. this study provides an important step towards understanding the pathomechanism underlying to morc p.r w and its role in cmt z. aifm encodes a mitochondria associated apoptosis inducing factor. mutations in aifm lead to a wide spectrum of neurodegenerative disorders: cowchock syndrome; a combined oxidative phosphorylation deficiency (coxpd ) with severe encephalomyopathy; x-linked deafness with peripheral sensory neuropathy; spondyloepimetaphyseal dysplasia (semd) with mental retardation; and an infantile motor neuron disease. previous studies showed severe defects in mitochondrial metabolism, related to redox function, mitochondrial fragmentation, and respiratory deficiencies. in addition, some mutations impair the protein expression of aifm and cause an increase in caspase-independent apoptosis. by targeted next-generation sequencing, we detected the aifm c. c>t (p.phe ser), in a year-boy. this mutation was confirmed in his year-old affected brother. electromyography and nerve conduction velocities studies revealed an axonal polyneuropathy with exclusive involvement of motor fibers, with an early childhood-onset. both children currently show normal cognitive and cranial nerves functions. the in silico structural modeling of human aifm showed that the mutation of a phenylalanine to serine at position disrupts the hydrophobic interaction between phe and pro , and consequently, it destabilizes an alpha-loop domain. cartoon of protein superposition between two different human aifm structures suggest that a lack of constraints in this region could affect the interaction between - helix and the - -hairpin regions, a very important stage for the functional activity of the aifm protein. patient-derived fibroblasts were used to investigate the pathological effect of the p.phe ser mutation: fibroblasts from patients show a similar mrna but different protein expression of aifm compared to healthy control fibroblasts. however, they have an aberrant morphology, from fibroblastic to polygonal shape, and they are larger than control fibroblast; mitochondria from mutant fibroblasts are markedly fragmented compared to controls; the viability of the patient's fibroblasts is lower, but it does not correlate with an increase in apoptosis. instead, it seems to be caused by an increase in the expression of genes activating the senescent program, like p and p . our study confirms that variable effect of different mutations on the protein function may contribute to the clinical variability observed in aifm patients. funds: isciii (pi / ); fundació per amor a l' art. sango k , takaku s , niimi n , yako h . diabetic neuropathy project, tokyo metropolitan institute of medical science, tokyo, japan. coculture models of neurons and schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained from the primary culture with embryonic or neonatal animals. because it is recognized that some biological properties of both neurons and schwann cells change with maturation and aging, culture systems of adult animal cells appear to mimic peripheral nerve degeneration and regeneration better than those of immature animal cells. we have established spontaneously immortalized schwann cell lines from long-term cultures of adult fischer rat peripheral nerves. one of these cell lines, designated ifrs , has been shown to retain distinct schwann cell phenotypes, such as spindle-shaped morphology with expression of glial cell markers, synthesis and secretion of neurotrophic factors and cytokines, and fundamental ability to myelinate neurites in cocultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed pc cells. our current investigation focuses on the establishment of the coculture system of ifrs cells and nsc- motor neuron-like cells. nsc- cells were seeded at a low density ( x /ml) and maintained for a week in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (bdnf, ng/ml). after overnight exposure to mitomycin c (mmc, micro g/ml), nsc- cells were cocultured with ifrs cells ( x /ml) and maintained in serum-containing medium supplemented with bdnf ( ng/ml), ciliary neurotrophic factor (cntf, ng/ml) and coenzyme q ( micro m). under this culture condition, overgrowth of nsc- cells was prevented and gradual movement of ifrs cells toward the neurites emerging from nsc- cell bodies was observed. double-immunofluorescence staining carried out at day of the coculture showed myelin protein zero-immunoreactive ifrs cells surrounding the beta iii tubulin-immunoreactive neurites. this coculture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g. amyotrophic lateral sclerosis, charcot-marie-tooth diseases and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them. santos pp , torezani gs , maciero l , pagliarini lfd , romão tt , abunahman ms , ferreira is , bittar c , pupe c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. zika virus (zikv) is a flavivirus related to dengue, yellow fever and west nile viruses, and has been recently associated to the occurrence of neurological complications in children and adults. previous studies have linked zikv to the development of guillain-barré syndrome (gbs), myelitis, meningoencephalitis and ophthalmological manifestations in adults. guillain-barré syndrome (gbs) encompasses a spectrum of post-infectious neuropathies characterized by different distributions of weakness and sensory impairment. serum anti-ganglioside antibodies are often found and are related to different clinical patterns. recently we have encountered patients in rio de janeiro, brazil, with distal edema in lower limbs, acute weakness, pain and sensory disturbances during the acute stage of an acute febrile exanthematous illness. the symptoms persist for up to days, with complete resolution afterwards, without specific therapy. blood concentrations of muscle enzymes show normal values, and electromyography and nerve conduction studies (emg/ncs) are usually unremarkable. of note, all cases have had positive rt-pcr for zika virus, indicating an illness that occurred during the viremic phase of this arbovirus infection, and complete recovery within the expected timeframe for the resolution of the systemic viremia. there is a subset of patients who developed acute weakness very early after the initial viral symptoms, with clinical, laboratorial and electrophysiological findings that substantially differ from gbs. we describe three cases with similar features suggestive of an acute infective polyneuritis (aipn). one might hypothesize that zikv might lead to a direct neurotropic injury, significant enough to cause weakness and sensory complaints, but not severe enough to cause permanent damage, resolving in conjunction with decreasing blood viremia. we consider that these patients differ from classical gbs because their illness begins during the acute febrile stage of an infective illness and the clinical course is more rapid leading to complete resolution in a few days. savransky a , mozzoni j , massaro sanchez mp , reisin r , monges ms . department of neurology, hospital de pediatria j.p. garrahan, buenos aires, argentine; department of physical therapy, hospital de pediatria j. p garrahan, buenos aires, argentine; department of neurology, britain hospital, buenos aires, argentine. our objective is to describe a series of children with guillain barré syndrome (gbs) included in the igos protocol. as part of the igos multicenter protocol, pediatric patients meeting gbs diagnostic criteria, who consulted within the first weeks of symptom onset and had parental consent to participate in the study were included. patients were also offered to participate in the extended -year protocol. all patients were evaluated according to the igos protocol. patients were recruited between october and june . twenty-four patients, eight girls, participated. ages ranged from months to years (mean . years). all patients agreed to participate in the extended protocol, which was completed by of them. eight patients completed the -year follow-up and are still under evaluation. five patients were lost to follow-up.twenty-two had the classic variant of gbs and two miller fisher. radicular pain in the back or lower limbs was reported by %. twenty-three patients underwent lumbar puncture and albumin-cytological dissociation was found in . in all cases, csf was stored for proteomic studies. all patients underwent emg showing aidp in , and amsan and aman in each. one patient with aidp developed to cidp. in patients full-spine mri was performed and cauda equina enhancement was found in every case. three patients required ucip, two with invasive and one non-invasive ventilation. all patients were treated with gammaglobulin, with a second dose at weeks in cases with a poor response. all patients who followed the protocol were evaluated with the gbs disability score. median score was at baseline and between and at the -year assessment. we describe a series of children with gbs as a part of an international protocol including patients of different ages. pain was a frequent and early symptom and could be determined despite the young age of our patients. most patients fully recovered. we were invited to participate in igos kids to better assess this age group. saysavath k , somchit v , t. umapathi . mittaphab hospital, vientiane, lao pdr; setthathilath hospital vientiane; national neuroscience institute, singapore, singapore. laos people democratic republic is a country of . million people in south east asia. largely agricultural, its capital and metropolis is vientiane. adult neurological services are concentrated at mittaphab hospital, vientiane serviced by three neurologists. on the average about six cases of guillain-barré syndrome (gbs) are seen per year. it is believed that most patients do not seek medical attention. cases appear to cluster during the rainy season. most patients present late, often at the second week of illness when recovery is unapparent after seeking treatment from traditional medicine doctors and at district hospitals. a substantial number of patients seek treatment at hospitals across the border, in neighboring provinces of thailand. common antecedent symptoms are viral prodrome and diarrhea. miller fisher syndrome appears to be rare, possibly because of the mild deficits that do not prompt patients to seek medical attention. diagnosis is made largely from clinical features and from spinal fluid analysis. nerve conduction studies are not available. patients are often treated with steroids by internists. intravenous immunoglobulin and plasma exchange are not available. common complications include pneumonia, autonomic dysfunction (fluctuating blood pressure), pressure sores and depression. patients who develop respiratory failure are nursed at a twelve-bedded intensive care units. plans are afoot to set up a prospective gbs database, systematically study antecedent infections, including of flaviviruses, and develop low-volume plasma exchange as a feasible therapeutic modality. a gene therapy approach for treating cmt c neuropathy schiza n , markoullis k , richter j , tryfonos c , kagiava a , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory; department of molecular virology; neurology clinics, cyprus institute of neurology and genetics and cyprus school of molecular medicine, nicosia, cyprus. charcot-marie-tooth type c (cmt c) is the most frequent form among recessively inherited demyelinating neuropathies and results from mutations in the sh tc /kiaa gene. sh tc mutations cause loss of function of the sh tc protein suggesting that gene replacement therapy may be useful for treating cmt c. sh tc −/− mice develop all major aspects of the human pathology including early onset progressive peripheral neuropathy with hypo-and demyelination along with decreased motor and sensory nerve conduction velocities, offering a relevant model for testing treatments for cmt c. in order to develop a gene replacement strategy for cmt c, we generated a novel lentiviral vector, lv-mpz-sh tc .myc, to drive expression of the human sh tc cdna under the control of the myelin protein zero (mpz/ p ) promoter specifically in myelinating schwann cells. a c-terminus myc tag was included to facilitate expression analysis. a control vector (mock) was also produced in which the sh tc cdna was replaced by the egfp reporter gene. we first confirmed expression of hsh tc in hela cells transfected with the pcdna -cmv-sh tc .myc vector. immunofluorescence analysis confirmed a strong expression of sh tc specifically at the plasma membrane with additional localization in a dotted pattern intracellularly. for in vivo gene delivery we used both intraneural and intrathecal injections of the lv-mpz-sh tc .myc vector in -week to -month old sh tc −/− mice. expression of virally delivered hsh tc was assessed weeks after injection. immunofluorescence analysis showed hsh tc immunoreactivity in perinuclear schwann cell cytoplasm in sciatic nerve teased fibers of sh tc −/− mice following both intraneural and intrathecal delivery, while lumbar intrathecal gene delivery resulted additionally in expression of hsh tc in the lumbar roots. real time pcr analysis confirmed hsh tc mrna expression in both lumbar roots and sciatic nerves. thus, we have developed a novel lentiviral vector for schwann cell targeted gene delivery to treat cmt c and for testing possible therapeutic effects in the mouse model of the disease. hereditary neuropathies are a group of disorders which are characterised by the systemic impairment of peripheral nerves. more than neuropathies are associated with causative gene defects [ ]. charcot marie tooth (cmt a) neuropathy is the most frequent hereditary neuropathy, triggered by a mutation in the peripheral myelin protein gene (pmp ). cmt a leads to a primary loss of myelin sheath and afterwards to a degeneration of axons [ ] . symptoms appear with the degeneration of axons, whereas demyelination is thought to be largely asymptomatic. for that reason, we investigate in the mechanisms of axonal degeneration. for our analysis, we used purified axoplasma without detectable myelin proteins of the sciatic nerves of weeks old pmp -c mice. in this early stage of the axonal degeneration sarm was significantly increased. the nad + concentration in the axoplasma was dramatically reduced. this correlates to the previous finding that sarm promotes axonal degeneration by cleavage of nad + . additional studies showed an increase of the nad + -dependent axonal protective factor sirt in pmp -c sciatic nerves. nmnat- , known as the active component of the wallerian degeneration slow gene, was unaffected in axoplasma of pmp -c sciatic nerves. summarised, these results indicate that the pathway of sarm , nad + and sirt may play a critical role in axonal degeneration in neuropathy. we report two unrelated czech patients with cmt b , both sporadic cases in the family. patient is a year old man with congenital glaucoma after ophthalmological surgeries. his early motor development was normal. at the age of years parents noted gait problems, first neurological examination was at the age of years, when emg showed diffuse motor and sensory neuropathy with severely decreased ncv ( - m/s). he developed foot deformities (pes cavovarus) and underwent corrective orthopedic surgeries at the age of years. after several dna tests for demyelinating cmt the sfb gene was sanger sequenced and a novel missense mutation p.ile asn was found in homozygous state in the patient and in heterozygous state in both parents. the patient was later tested also by ngs of a panel of all genes to be causal for hereditary neuropathies and no other potentially causal variants were detected. patient is years old man, with normal early motor development. at the age of years parents noted gait problems with distal leg weakness which progressed into distal leg plegia at the age of years. hand weakness was noticed since the age of years. he has severe atrophies of distal muscles of all extremities, is self ambulant. at the age of , emg showed unrecordable responses from nerves of lower limbs and ncv was measurable only on ulnar nerve and was m/s. at the age of years increased intraocular pressure was diagnosed and he uses anti glaucoma eye drops. after many single gene tests for demyelinating cmt, we used ngs of a panel of genes known to be causal for hereditary neuropathies and two novel heterozygous, probably pathogenic variants affecting invariant splice sites were detected: c. - a>g and c. - _ del, both confirmed by sanger sequencing. the first variant is also in the father, but the second is probably de-novo (not detected in parents, despite correct parentity). sekiguchi y , kikuchi si , konno si , sekiguchi m . department of orthopaedic surgery, fukushima medical university school of medicine, fukushima, japan. carpal tunnel syndrome (cts) is the most common entrapment neuropathy. ultrasonography can be used to detect anatomic changes in cts. more recently, it has been shown that doppler ultrasonography can detect increased intraneural blood flow in cts. the purpose of this study was to determine the most suitable finding of pre-and postoperation in cts by ultrasonography. a total of wrists of patients with nerve conduction study (ncs) proven cts were evaluated with ultrasonography. we measured the median nerve's cross-sectional area (csa) and intraneural blood flow of median nerve by ultrasonography. the correlation between these ultrasonographic measurements, ncs severity and duration of clinical cts symptoms was analyzed. the csa (mean, mm ) was no significantly reduction after successful carpal tunnel release. morphologic median nerve changes may persist for a longer period regardless of successful surgery and clinical improvement. however, intraneural blood flow is increasing after successful carpal tunnel release. we conclude that doppler ultrasonography results strongly correlate with post operated cts improvement. hence doppler ultrasonography is a useful method for functional improvement of pre-and post cts operation. senger jl , chan km , olson jl , webber ca . university of alberta, edmonton, canada. the beneficial effects of a preinjury crush conditioning lesion (cl) on peripheral nerve regeneration is well-documented in animal models. no human studies have been attempted to date, given the ethical dilemma of deliberately injuring an intact nerve, and the difficulty in predicting the timing of a nerve injury. recent studies demonstrate that hour of electrical stimulation (es) produces effects similar to cl in neuronal cultures. this, coupled with a surgical environment favoring nerve transfers, in which an intact nerve is deliberately cut to reinnervated a denervated muscle, means that es may be clinically translatable to enhance regeneration. this study hypothesizes that es prior to nerve injury will enhance nerve regeneration. twelve sprague-dawley rats were divided into four groups based on conditioning-type to the mid-common peroneal (cp) nerve: es ( ), crush ( ), sham-es ( ), and naïve ( ). one week following conditioning, they underwent a cut/coaptation of the cp nerve at the sciatic trifurcation. post-cut day , nerves and dorsal root ganglia (drgs) were collected. axonal counts of nerves stained with nf- revealed similar regeneration between es and crush ( . vs. . mm, p= . ) that was superior to sham-stimulation ( . mm) or no-conditioning ( . mm, p< . ). a greater number of axons at the distal tip were present in animals that received either type of conditioning compared to the unconditioned cohorts. drgs were stained with neuronal injury marker growth associated factor- (gap- ), and satellite cell glial cells with glial fibrillary acidic protein (gfap). significant increase in gap- expression at three days was observed in es and crush cohorts compared to sham or naïve (p< . ) cohorts. the satellite glial cells of es and crush conditioning showed a significant increase in gfap expression ( . % and . % respectively) compared to sham ( . %) and naïve ( . %) drgs. by demonstrating similar improvements in axon regeneration, this proof of principle project suggests that es conditioning may produce regenerative outcomes comparable to the classical crush injury model. in turn, this suggests that es may be a promising method for delivering conditioning lesions in clinical trials for conditioning nerves prior to surgical intervention. we report a unique case of newly developed waldenstrom's macroglobulinemia (wm) in a patient with chronic inflammatory demyelinating polyneuropathy (cidp) with antibodies against myelin-associated glycoprotein (mag) and sulfatide who was undergoing treatment with intravenous immunoglobulines (iv-ig). subsequent rituximab infusions did not have a positive impact. patients with wm can develop demyelinating and axonal polyneuropathies and few patients have anti-mag and/or anti-sulfatide antibodies. anti-mag antibodies ( % of wm) are associated with sensorimotor axon loss and demyelination and anti-sulfatide ( % of wm) with sensory axonal loss. rarely, both antibodies can be present, with a more severe clinical phenotype. cidp associated with anti-mag and anti-sulfatide antibodies can represent independent entities, not associated with wm. there are no reports to date of patients with cidp associated with anti-mag and anti-sulfatide antibodies that developed wm during immunomodulatory therapy with iv-ig. in addition, subsequent rituximab infusions after the iv-ig were stopped have not been proven beneficial, as has been previously reported for anti-mag cidp patients. seventy-six year old right-handed gentleman presented with persistent numbness in his left foot, three months following artificial disc placement in his lumbar spine. gradually he developed sensory ataxia. no radicular signs were present on exam or impingement on serial spine mri's. ncs/emg studies were consistent with a cidp variant with severely prolonged distal motor latencies. serum anti-mag and anti-sulfatide antibodies were elevated. chronic therapy with iv-ig was able to partially stabilize the symptoms; however, six years later he newly developed wm. subsequent infusions with rituximab, after iv-ig was stopped, did not improve the clinical picture or the ncs/emg findings. wm can newly develop in an autoimmune setting, such as cidp associated with anti-mag and anti-sulfatide antibodies. in this particular case, there was an ongoing immunomodulatory therapy for our cidp patient, as he had monthly iv-ig infusions. this may reflect a possible induction of pathological b cell clone proliferation during the iv-ig treatment. subsequent rituximab infusions, after the iv-ig was stopped, did not improve the symptoms or the demyelination features on ncs/emg. he continues to be symptomatic despite efforts. charcot-marie-tooth disease (cmt) is an inherited neuropathy without known cure (prevalance: : ). duplication of the gene encoding the peripheral myelin protein of kda (pmp ) underlies the most common subtype cmt a. severely affected cmt patients suffer from sensory and motor symptoms with wheelchair-boundness. the clinical phenotype is highly variable and is determined by the amount of axonal loss, but the molecular mechanisms of the disturbed neuron-glia interaction are poorly understood. risk factors have not been investigated. therefore, cmt-net, a german network funded by the german ministry of education and research (bmbf, bonn, germany) includes interdisciplinary expertise from molecular biology, neurology, neuropathology and human genetics in order to identify genetic and non-genetic risk factors of disease severity of cmt by: (i) examining the mechanisms of the disturbed axon-glia-interaction and neuronal vulnerability, (ii) identification of genetic modifiers and (iii) novel therapeutic targets, (iv) validating outcome measures in children and adults, (v) establishing a biobank and (vi) exploring the disease burden via an internationally harmonised patient registry. cmt-net includes three service structures cmt-net will focus on cmt a, but also includes rarer subforms. we will provide the scientific basis for the development of translational approaches to therapy in patients. our approach bridges cutting edge molecular screening techniques, transgenic animal models of altered axon-glia interactions (fly, chick, mouse, rat), state-of-the-art genomic technologies and human patient trials in order to understand and treat the disease aggravation in cmt. sezer g , tekol y , sezer z , . erciyes university, betül ziya eren genome and stem cell centre, kayseri, turkey; erciyes university, school of medicine, pharmacology department, kayseri, turkey; erciyes university, good clinical practice and research centre, kayseri, turkey. analgesic effects of antidepressant drugs are well known for a long time, however, their systemic side effects limit their usage as an analgesics. venlafaxine is an antidepressant drug that has different structure. our purpose was to investigate whether systemic analgesic effect has been proved drug, venlafaxine, has local peripheral antinociceptive action. we applied vanlafaxine ( μl , , , μg) to male, sprague-dawley rats' paws by intraplantar injection and also by intraperitoneal route ( , , , mg/kg) in formalin test, a model for acute and tonic pain. we also pretreated another groups of rats with mg/kg naloxone (opioid receptor antagonist), mg/kg cpt (adenosine a receptor antagonist) or saline (ip.) before μg/paw venlafaxine injection. to check the effect is local or not, we determined the blood levels of venlafaxine in at different time points after both the local and systemic applications by gc-ms method. datas were expressed as number of flinches and total time for biting/licking of the injected paw over phase ( - min) or phase ( - min) and analyzed using the student's t-test. venlafaxine induced antinociception at , and μg concentrations by the local peripheral application and at , mg/kg doses by the systemic application in formalin test and the effects were comparable. pretreatment with naloxone diminished the effect of venlafaxine in the both phases, however, it was not statistically significant. pretreatment with cpt decreased venlafaxine induced antinociception only in phase . neither naloxone nor cpt changed formalin induced nociceptive behaviors alone. this is the first that determines the peripheral antinociceptive actions of venlafaxine in rat formalin test. with roles of opioid and adenosine a receptors in this action. our results suggest that venlafaxine has local peripheral antinociceptive effect and such an activity may led to trials for to use this drug as a cream-gel formulation for analgesia in clinics in the future. topical application might permit the attainment of higher and more efficacious concentrations in the region of the sensory nerve terminal, with limited systemic side effects. shah a , hoffman em , klein cj , staff np . mayo clinic, rochester, usa. cipn is a common dose-limiting complication for patients with cancer. the long-term disease burden of cipn is compounded by increasing cancer survivorship, yet there are minimal data on long term outcomes following onset of cipn, especially in population-based studies. we utilized the rochester epidemiology project to examine incidence and disease burden of cipn among individuals of olmsted county, minnesota with neurotoxic chemotherapy exposure between and . clinical records were queried for the presence of neuropathic signs, symptoms and icd- diagnostic codes as well as for patient provided information on impairment with activities of daily living and use of pain medications. a total of individuals with incident exposure to neurotoxic chemotherapy agents between and were identified. based on aan criteria for identifying peripheral neuropathy, ( . %) of these individuals were determined to have cipn, while ( . %) controls did not. the median time from incident exposure to reported symptom onset was days (iqr . - ). patients with cipn received a neuropathy icd- diagnosis in merely cases ( . %). median survival following incident chemotherapy exposure among all cases and controls was . years with a significantly longer mean survival in cases with cipn as compared to that of controls ( . years vs. . years, p< . ). in addition to acute effects in cipn, individuals surviving greater than years following exposure to neurotoxic chemotherapy continue to self-report increased symptoms of numbness (or . , % ci . - . ) and pain (or . , % ci . . . ) of the extremities. through utilization of patient provided information, our study was able to collect data on long-term impairment associated with previous history of exposure to neurotoxic chemotherapy. our results are consistent with previous reports of the high incidence of cipn in the first two years following incident exposure. additionally, our results provide evidence of high incidence of cipn independent of individual chemotherapeutic agent used. additionally, our results indicate icd- -cm diagnostic code attribution may dramatically underestimate the magnitude of cipn. increased survival following exposure to neurotoxic chemotherapy and its long-term disease burden necessitates further study of among survivors. the utility of quantitative muscle ultrasound as a marker of disease severity in charcot-marie-tooth (cmt) disease subtypes was investigated. muscle ultrasound was prospectively performed on individual muscles from cmt patients ( cmt a, cmtx , cmt a) and compared to muscles from age and gender-matched controls. muscle ultrasound recorded echogenicity and thickness in representative muscles including first dorsal interosseus (fdi) and tibialis anterior (ta charcot-marie-tooth (cmt) disease is the most frequent inherited peripheral neuropathy, and there is currently no available cure. the most common subtype of cmt, cmt a, is completely associated with duplication of the pmp gene, which encodes peripheral myelin protein of schwann cells. previous studies of cmt a mainly relied on rodent models, and it is not yet clear how pmp overexpression leads to the phenotype in patients. based on induced pluripotent stem cell (ipsc) technology, we herein developed a brand new in vitro cell model of cmt a, called cmt a-hipscs, in the hopes of simulating the developmental progress of the disease and gaining new insights into its pathogenesis. here, we efficiently derived ncscs from cmt a-ipscs and assessed the potential of the isolated cmt a-neural crest stem cells (ncscs) to differentiate into peripheral neurons and schwann cells using defined media. we found that, unlike normal control ncscs, cmt a-ncscs rarely generated schwann cells. instead, cmt a-ncscs produced numerous endoneurial fibroblasts in the schwann cell differentiation system. we further established a pmp -overexpressing ipsc model, and obtained similar results when pmp -ncscs were subjected to schwann cell differentiation. these results suggest that the development of schwann cells in cmt a patients is interrupted by the duplication of pmp . with the exception of the demyelination-remyelination process, developmental disabilities of schwann cells should be considered as an underlying cause of cmt a. shimoi t , yamada t . international university of health and welfare, tochigi, japan, cmt japan, tokyo, japan. charcot-marie-tooth (cmt) disease is the most common hereditary motor and sensory neuropathy. our preliminary report suggests that a certain cmt patient has the recruitment disorder of motor units during muscle fatigue and this disorder may be a factor of "super fatigability" in motor neuropathy patients. if the "super fatigability" occurs, we would expect patients with this characteristic to become slower in recovery from muscle fatigue than patients without this characteristic. in order to verify this hypothesis, we measured characteristic of recovery from muscle fatigue in charcot-marie-tooth patients with electromyographic study. twenty three participants were asked to maintain their % of maximal voluntary isometric contraction (mvc) of elbow flexor until exhaustion as the fatigue exercise. in addition, the participants asked to perform s of their % of mvc at , , , , , , , , , s after the fatigue exercise as recovery tasks. the surface emg (semg) signals of biceps brachii muscle were determined during the exercise and tasks. muscle force, median power frequency (mdpf) and the root mean square of semg amplitude (rms) were used as objective parameters of muscle fatigue. borg scale was used as a subjective parameter of muscle fatigue. six of twenty three participants showed significant decrease of rms during the fatigue exercise. in consideration of this result, we compared alteration of mdpf in recovery task between six participants with decrease of rms (abnormal group) and seventeen participants with increase of rms (normal group). as the result, the abnormal group had at least s as the recovery time from muscle fatigue in contrast with s of normal group. the recovery time from muscle fatigue in subjective parameter was shorter than the time in objective parameters in each group. our data support the "super fatigability" hypothesis. and that hypothesis may induce "hidden muscle fatigue". comparison between complex regional pain syndrome type and based on electrophysiologic, imaging and clinical findings shin jy , moon jy , sung jj . seoul national university hospital, seoul, republic of korea. complex regional pain syndrome (crps) is a constant regional neuropathic pain characterized by various kinds of motor, sensory, and autonomic changes. conventionally, the crps is divided into type i and ii according to the absence and presence of nerve injury. but the pathogenesis of crps is not fully understood yet. and there is still no systematic comparative study between crps type i and ii. we compared between crps type i and ii using multimodal approaches including electrophysiologic, imaging, and clinical findings. the patients ( type i and type ii) diagnosed with crps using the international association for the study of pain (iasp) diagnostic criteria were included. type i and ii were divided by electromyography and nerve conduction study. we obtained clinical information such as continuing pain, allodynia, hyperalgesia, edema, temperature, skin color, sweating, trophic change from patients. all patients were evaluated by bone scan, thermography, quantitative sudomotor axon reflex test (qsart), quantitative somatosensory test (qst). the ratio of qsart and temperature threshold abnormality in type ii was higher compared to type i ( . % vs . %, . % vs . % respectively, p = . and . ), among clinical symptoms, sweating change significantly high in type ii compared to type i ( . % vs . %, p = . ). other electrophysiologic and imaging, clinical findings were not significantly different in both type. in this study, we identified that crps type i and ii are distinguished not only by the nerve injury but also by the sudomotor function, and qsart can serve as a good technique to differentiate between crps type i and ii. it is estimated that there are two distinct pathogenesis in crps. our results may be helpful to diagnose crps correctly and understand the pathogenesis of crps. sjogren syndrome (ss) is an autoimmune inflammatory disorder of exocrine glands resulting in xerophthalmia and xerostomia. ross syndrome is a rare entity characterized by tonic pupil, hyporeflexia, and segmental anhidrosis. we present a -year-old hispanic woman with debilitating sensory and autonomic neuropathies, and persistently elevated anti-ss-a and anti-ss-b antibodies, without the classic sicca complex. she initially developed diarrhea and an ear infection, then felt toe and later leg numbness, which eventually spread to her cheeks and tongue, over few months. four years later, her thumbs and index fingers started tingling. also, she developed orthostatic lightheadedness, tachycardia, segmental hypohidrosis of the right abdomen, and hyperhidrosis of the remaining trunk, intermittent erythema, chronic diarrhea, and a -pound weight loss. her exam demonstrated orthostatic hypotension, bilateral tonic pupils and light-near dissociation, sectoral palsy of the right iris sphincter, stocking-distribution diminished sensation to all modalities, pseudoathetosis, areflexia, dysmetria, intention tremor, romberg, and sensory gait ataxia. mri of neuraxis demonstrated t -weighted hyperintensity in the dorsal spinal cord from c to the lower thoracic level, with mild atrophy. electrodiagnostic testing was consistent with moderate-to-severe, length-dependent, asymmetric, sensory polyganglionopathies. csf showed oligoclonal bands. serology showed elevated antinuclear antibody ( : , reference < : ), ss-a ( , reference < u), ss-b ( , reference < u), and rheumatoid factor ( , reference < iu/ml) titers. the remaining workup was negative for infection (syphilis, hiv, htlv, hepatitis, lyme disease), paraneoplastic syndrome (anti-hu and ganglionic nicotinic acetylcholine receptor antibodies), pyridoxine intoxication, malignancy (chest/abdomen/pelvis ct, breast ultrasound, axillary lymph node flow cytometry, colon and esophagus biopsy), celiac disease, vitamin deficiency, autoimmune disease (anti-aquaporin and anti-gq b antibodies), and adrenoleukodystrophy. the patient received ivig and steroid with some gait improvement and currently takes mycophenolate. midodrine and fludrocortisone resolved her dizziness. this case highlights an important, treatable sensory ganglionopathy and systemic autonomic neuropathy due to sjogren syndrome, and illustrates the overlapping clinical triad of ross syndrome, which may guide future management. siles am , , assylbekova d , , diaz-manera j , , rojas-garcia r , , cortes e , , gallardo e , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, univeristat autònoma de barcelona, barcelona, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. inflammatory neuropathies are a heterogeneous group of peripheral nerve diseases that respond to immune-therapies. chronic inflammatory polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) are two chronic inflammatory neuropathies responding to intravenous immunoglobulins (ivig). b lymphocytes are involved in their pathogenesis. while widely used in clinical practice, ivig's mechanism(s) remain not completely understood. ivig are reported to lead to b-cell anergy and to increase regulatory t-cell function and frequency. regulatory b cells (bregs) are a rare subset of b lymphocytes that suppress immunopathology acting upon several target cells in the immune system. impaired breg yields have been described in a plethora of autoimmune conditions. the presence of regulatory b cells in inflammatory neuropathies and the effect of ivig therapy on their frequencies has not been studied. the aim of this study is to describe the frequencies of bregs in cidp and mmn and the effect of ivig on their frequencies. patients fulfilling diagnostic criteria for cidp or mmn and matching controls were included. pbmcs were obtained by gradient centrifugation before ivig infusion and one week after treatment. b-cells were isolated with negative selection magnetic beads, cultured and activated with the tlr agonist odn and anti-human igg+iga+igm. il- secretion capacity was assessed by flow-cytometry. twenty-eight patients were included of whom where cidp and mmn. of all patients included, received ivig and were suitable for pre and post ivig breg frequency comparisons. breg frequencies did not differ in patients (before ivig treatment) and controls (p= . , mann whitney test, two-tailed). however, the frequencies of bregs significantly increased one-week after treatment with ivig (p= . , wilcoxon matched pairs test, two-tailed). when stratifying by disease subtype, breg frequencies increased in cidp patients after ivig (p= . , wilcoxon matched pairs test, two-tailed) and in mmn (p= . , wilcoxon matched pairs test, two-tailed) although results did not reach statistical significance in mmn. this is the first study that studies the breg frequencies in cidp and the first study that addresses the effect of ivig on breg frequencies. our study provides the proof of principle that bregs could become a biomarker for response to ivig but this would need a larger and prospective study. siles am , , martínez-hernández e , diaz-manera j , , rojas-garcia r , , gallardo e , , illa i , , graus f , querol l , . paraneoplastic neuropathies (pn) are rare, immune-mediated disorders of the peripheral nerve with important prognostic implications. ectopical expression of neural antigens in the tumor leads to the development of onconeural antibodies. several autoantibodies associate to pn, including anti-hu, anti-caspr or anti-cv antibodies but a significant proportion of pn lack identifiable antigens. adhesion molecules that are autoantigens in other neuropathies, like contactin- , are present in several types of tumors. our study proposes a systematic screening of autoantibodies against neural cell-adhesion molecules and neural structures to detect novel antigenic reactivies in pn. thirty-five patients followed in our centre and at the neuroimmunology-multiple sclerosis unit at hospital clínic de barcelona, with pn fulfilling diagnostic criteria of possible (n= ; . %) and definite (n= ; . %) paraneoplastic disease were included. serum samples were obtained and tested by immunocytochemistry against contactin- (cntnt ), neurofascin (nf ) and the cntn /caspr complex. primary cultures of dorsal-root ganglia (drg) and rat schwann cells were incubated with patients' sera to detect antibodies targeting neural structures. ten individuals ( . %) presented with a tumor and a neuropathy involving both sensory and motor symptoms. the remaining patients ( . %) presented with a tumor and a classical sensory neuronopathy without anti-hu or any other onconeuronal antibody. among the latter, ( . %) patients were diagnosed with small-cell lung carcinoma. the rest of the individuals ( . %) associated diverse malignancies. we did not detect any sera reacting against cntn , nf or the cntn /caspr complex. in igg antibody screening experiments, patients ( . %) reacted against drg neurons, of them ( . %) reacting strongly, and patients ( . %) reacted mildly against rat schwann cells. in igm experiments, patients ( . %) reacted slightly against drg neurons and patients ( . %) against rat schwann cells, of them ( . %) featuring strong staining. experiments screening antibodies against motor neurons and immunoprecipitation assays are ongoing. overall, % of patients reacted strongly against either neurons or schwann cells. our study did not detect antibodies against the neural adhesion molecules cntn , nf and the cntn /caspr complex in patients with pn. however, a significant proportion of pn patients harbour antibodies targeting neural structures, which suggests that novel neoplasm-associated antigens remain to be discovered. simmons m , tao f , abreu l , zuchner s , li j . department of neurology, vanderbilt university school of medicine, nashville, tennessee, usa; hussman institute for human genomics, university of miami, miami, florida, usa. objective: despite of a shared genetic mutation of the trisomy of chromosome p (c p ), patients with charcot-marie-tooth type- a (cmt a) present with a high variability of their disease severities. the underlying cause for the variability is still unclear. in this study, we tested a hypothesis whether a second genetic mutation known to damage the nervous system is also present in cmt a patients with early onset and severe phenotypes. methods: from a cohort of patients with cmt a mutation (chromosome p duplication), we identified patients with an early onset (< or = years of age) of the disease. four of the eleven also had dna testing for a panel of known cmt-related genes and sequencing of mitochondrial dna in addition to the dna testing for c p duplication. results: besides the c p duplication, we identified three additional mutations in the four patients with early onset. the mutations were a missense mutation of arg his in mpz gene, an a g mutation in mitochondrial trna for glycin and a homozygous mutation of c p duplication. three of the four had symptomatic onset at birth. one showed symptoms at years of age. conduction velocities were severely reduced in all four patients (from to m/s). interpretation: traditional approaches to identify genetic modifiers, including snp association, assume that those modifiers are clustered in a small region of human genome and shared by the studied patients. however, our study suggests that genetic modifiers in cmt a may be highly diverse and scattered throughout the genome, which could make the conventional approach via the genetic variants association difficult. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr sjogren's syndrome(ss) is a systemic auto-immune disease that apart from exocrine glands may affect any organ. involvement of peripheral nervous system results in wide spectrum of neuropathic manifestations. the aim of our study was to evaluate the clinico-electrophysiological patterns and pathological characteristics of neuropathy in sjogren's syndrome (ss) patients presented to neuromuscular clinic in a tertiary hospital from south india. this is a retrospective study from the departments of neurology, rheumatology and pathology from nizam's institute of medical sciences. twenty one patients with diagnosis of ss and peripheral neuropathy between to were analysed. clinical records, conventional nerve conduction studies, lip and nerve biopsy reports were collected.in patients with ss associated neuropathy,male to female ratio was : . in ( . %) neuropathy was the initial manifestation,while in ( %)exocrinopathy preceded neuropathy. the patterns of neuropathy included mononeuropathy multiplex(mnm) in patients ( %),ganglionopathy in ( %),length dependant, trigeminal, autonomic neuropathy and cidp in ( %)and cranial neuropathy in ( %).eighteen( %) were seropositive..schirmer's test was positive in ( . %).nerve biopsy showed vasculitis in patients, demyelinating and axonopathy in patients each.we conclude that neuropathy is frequently the initial presentation of ss.mnm is the common pattern followed by ganglionopathy. pattern of neuropathy helps in arriving at the diagnosis of ss. confirmation of ss is not by mere serology. schirmer's test and lip biopsy are equally essential for the diagnosis especially in seronegative patients when clinical index of suspicion is high. siskind ce , tesi rocha c . stanford health care, palo alto, ca, usa; stanford university, stanford, ca, usa. here, we report the first case of a human found to have a homozygous, presumed disease causing variant in the arl ip gene, causing charcot marie tooth disease (cmt) with central nervous system findings. the proband was born at term with apgars of and at and minutes. he was found to have iugr and was hospitalized for days for weight gain. he developed respiratory distress during the admission and was intubated. due to inability to extubate, he was transferred to stanford children's hospital, where he remained for three months. noted during admission was hypotonia, areflexia, minimal voluntary movment, sinus tachycardia, and dysautonomia. brain mri found polymicrogyria and cerebral underdevelopment with generally normal-appearing brainstem, with moderate ventriculomegaly. ncs found length dependent polyneuropathy with axonal degeneration. follow up muscle and nerve biopsy found immature muscle and amyelinating neuropathy the patient had normal plasma amino acid, acylcarnitine, lactate, pyruvate, urine organic acid testing, opththalmology exam, newborn screen, and normal array cgh. genetics ordered whole exome sequencing through baylor genetics laboratory (houston, tx, usa), which found a homozygous nonsense variant in arl ip : c. c>t, p.r x. a second child had been identified by baylor with two variants in this gene. that child had hypotonia, respiratory distress and seizures, and a muscle biopsy consistent with sma. the parents of that child chose to withdraw care at months of age. our patient's parents continued with aggressive therapies, including tracheostomy and g-tube for feedings. he had several subsequent hospitalizations for respiratory distress, possible seizure activity, and buldging anterior fontenelle, but now, at two years of age, has made developmental progress and is living at home with his family. he is able to smile, reaching for toys and swatting objects. he has little voluntary movement, and no longer responds to light touch stimuli. overall, this is the first picture of a child affected with a severe amyelinating form of cmt that causes weakness, hypotonia, and possible seizures, with the main concerning feature being the severe respiratory distress that may be life threatening, but can be managed with extreme care. charcot marie tooth (cmt) disease is the most common inherited peripheral neuropathy. patients frequently ask whether pregnancy will affect their cmt, whether cmt will affect their pregnancy, the optimal delivery and whether they or their child will have a higher risk of complications during pregnancy or delivery. so far few studies address these questions. currently no guidelines exist for the management of pregnancy, delivery and postnatal care in cmt patients. the aim of the study is assess the impact of pregnancy on cmt and assess how cmt affects pregnancy, delivery and care of the new born baby. we designed a retrospective questionnaire with expert help from an obstetrician with a special interest in pregnancy in patients with medical conditions. the questionnaire is divided into four parts (prior, during, after pregnancy and delivery) and includes questions on impairment, falls, pain, fatigue and respiratory complications during those periods; type of delivery, possible complications, details of anaesthesia and difficulties looking after the baby in the first months postpartum. so far women ( pregnancies) with cmt and related disorders have answered the questionnaire. % of patients had cmt a, the remaining had various subtypes of cmt and related disorders. patients reported deterioration of cmt symptoms during pregnancy in % of pregnancies with resolution of symptoms after pregnancy in % of pregnancies. of symptoms questioned walking ( %), balance ( %), and hand function ( %) deteriorated the most. there was an increased use of orthoses and walking aids during pregnancy. the majority of women ( %) had natural delivery, % were assisted and % had caesarian sections which was similar to the uk population ( %). no complications with anaesthesia were reported. the survey is currently ongoing. we plan to survey consecutive patients. data acquired from this survey will provide valuable information on current practice and will inform future guidelines and standard of care in charcot marie tooth disease. multifocal motor neuropathy (mmn) is a slowly progressive disorder in adults, characterized by asymmetrical limb weakness, mainly affecting the arms. despite beneficial effect of immunoglobulins, weakness gradually progresses. a major determinant of muscle weakness is the degeneration of affected motor axons. treatments aiming to reduce loss of motor axons require objective tools to quantify such an effect. therefore, we applied the compound muscle action potential (cmap) scan, which is an electrophysiological method that, with increasing transcutaneous stimulus-currents, successively activates all motor units (mus) in a muscle. it captures the contribution of enlarged mus due to reinnervation by the presence of relative large discontinuities in the scan. the aim of the present study was to identify pathophysiological changes of mu-loss and reinnervation in mmn patients by means of the cmap-scan. recordings were obtained from mmn patients. cmap-scan recordings were performed in the median nerve at the wrist where motor responses were recorded from the thenar muscle. we determined the number of largest cmap-scan discontinuities by means of a novel marker, d , where a low number is indicative of mu-loss and enlarged mus. furthermore, we applied the recently developed method of professor hugh bostock for obtaining a mu-number estimate from the cmap-scan. the median peak cmap amplitude was . mv (range . - . mv) and median d was (range - ). in three mmn patients with a normal maximum cmap amplitude (> mv) a reduced d (< ) was found indicative of mu-loss and enlarged mus. furthermore, d and the estimate of mu number were significantly related (r = . , p < . , n = ). the findings suggest that the cmap-scan is a sensitive tool in detecting the underlying pathological changes of reinnervation and mu-loss in mmn, more so than standard maximum cmap amplitude. it is quick and easy to perform and has the potential to be useful for follow-up studies. smith ag , thurgood b , revere c , hauer p , aperghis a , singleton jr . university of utah, salt lake city, utah, usa. corneal confocal microscopy (ccm) directly and quantitatively assesses corneal innervation including nerve fiber length (nfl) and density (nfd). ccm has shown promise as a diagnostic test. we have previously demonstrated that ccm has a diagnostic performance for diabetic neuropathy (dpn) similar to skin biopsy with assessment of intraepidermal nerve fiber density (ienfd) and nerve conduction studies (ncs). the responsiveness of these surrogate measures to dpn progression and their relation to clinically meaningful outcomes has not been well explored. diabetic patients undergoing annual retinopathy examination were recruited. each underwent ccm, ienfd, ncs including sural sensory and peroneal motor responses, the utah early neuropathy score (uens), the norfolk quality of life -diabetic neuropathy (nqol-dn, a validated neuropathy specific qol scale), and a minute walk test ( mwt). with dpn based on symptoms ( %) or signs underwent repeat testing at months and at months. at baseline, nqol-dn correlated with sural sensory amplitude (ssa) (− . , p< . ), peroneal motor conduction velocity (pcv) (− . , p< . ) and ienfd (− . , p< . ). no ccm metric was related to qol. mwt distance correlated with ssa ( . , p< . ), nfl ( . , p< . ) and nfd ( . , p< . ). over months, there was a significant worsening in dpn signs assessed by the uens (increase . +/− . , p< . ). ssa declined . uv (p< . ) and ienfd . fibers/mm (p< . ). there was no change in any ccm metric, pcv or nqol-dn. these findings suggest measures of distal axonal integrity are most sensitive to neuropathy progression, with ienfd having the greatest responsiveness. in contrast, ccm was not responsive to dpn progression. both ncs and ienfd (but not ccm) were significantly correlated with neuropathy-specific qol, whereas ncs and ccm measures correlated with physical functioning. the responsiveness of ienfd and ssa, and their relationship to qol support their selection as endpoints in dpn clinical trials. chronic inflammatory demyelination polyradiculoneuropathy (cidp) affects in , people, and is marked by chronic autoimmune infiltration of peripheral nerves and destruction of the myelin sheath. with current therapies, only % of cidp patients achieve complete remission. to produce more effective, mechanism-based therapies, we study mice with a partial loss of function g w substitution in the autoimmune regulator (aire) gene on the non-obese diabetic (nod) background (nod.aire gw/+ ) that develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. autoimmunity can result from defective immunosuppression. the potent, immunosuppressive cytokine interleukin (il- ) is increased in the peripheral blood mononuclear cells (pbmcs) of active phase cidp patients relative to remission phase patients. further, pbmcs from cidp patients produce il- in response to the myelin protein p . despite these findings, whether il- is important for cidp pathogenesis is not known. thus, we sought to determine the role of il- in sapp. il- was highly upregulated in sciatic nerves of nod.aire gw/+ mice with sapp, suggesting it may play an immunosuppressive role in pathogenesis. however, genetic ablation of il- in nod.aire gw/+ mice lead to a paradoxical delay in disease development. age-matched il- -deficient nod.aire gw/+ mice exhibited no sciatic nerve infiltrate and no reduction in nerve conduction during electrodiagnostic studies. interestingly, the delay in sapp was specific, since the incidences of five other autoimmune manifestations in il- -deficient nod.aire gw/+ mice were unchanged relative to il- -sufficient nod.aire gw/+ controls. importantly, il- -deficient nod.aire gw/+ mice did not have colitis, which is consistent with previous studies of il- deficiency on the nod background. il- is known to perform effector functions in autoimmunity by promoting b cell secretion of immunoglobulins. however, genetic ablation of b cells did not affect neuropathy development in nod.aire gw/+ mice, suggesting b cells are dispensable for pathogenesis and unlikely to mediate the protective effect of il- deficiency. il- -deficient nod.aire gw/+ cd + t cells, which are sufficient to transfer sapp, exhibited increased activation, increased interferon gamma secretion, and preserved nerve-specific t cell activation. these data suggest t cell activation and priming are unperturbed and not the mechanism of protection. in summary, our data showed that il- was paradoxically an effector cytokine in sapp. long exercise test (let) has been used especially in myotonic syndromes and muscle channelopathies. marked decrement in compound muscle action potential (cmap) amplitude after prolonged exercise was previously reported in patients with paramyotonia congenita, hyperkalemic or hypokalemic periodic paralysis. we describe a patient with secondary hypokalemic paralysis who showed abnormal let results. a -year-old man presented with ascending flaccid paralysis which evolved in a hyperacute fashion. the patient became quadriplegic after two hours. initial laboratory evaluation revealed severe hypokalemia, with normal thyroid function. we performed electrodiagnostic studies including long exercise test as proposed by mcmanis et al. nerve conduction study was normal, but marked decrement in cmap amplitude (up to % decrease after minutes) was noted after prolonged exercise. despite oral and intravenous potassium replacement, serum potassium level was not corrected as expected. the unusual clinical course prompted for evaluation of secondary etiologies. abdomen computed tomography scan revealed a . x . cm-sized mass in the left adrenal gland. aldosterone to renin ratio was elevated, suggestive of primary hyperaldosteronism. genetic study for cacna s mutation turned negative. after receiving laparoscopic adrenalectomy, the patient experienced no further attacks, and also was able to stop his antihypertensive medication. let may show abnormal results in condition with reduced membrane excitability, even without true channelopathy. according to international criteria, the diagnosis small fiber neuropathy (sfn) is based on clinical symptoms in combination with a reduced intraepidermal nerve fiber density (ienfd) in skin biopsy and/or abnormal temperature threshold testing (ttt). the sensitivity of skin biopsy is moderate to good, although ienfd is normal in about % of patients with sfn complaints. furthermore, ttt is a widely available diagnostic tool, but lacks specificity. corneal confocal microscopy (ccm) has been described and is used in clinical practice as an objective, non-invasive diagnostic tool to detect small nerve fiber damage in patients with diabetes mellitus. this study examines the applicability of ccm in patients with sfn, and the value of ccm as an additional diagnostic tool in sfn. we will include healthy participants to compare the results with the recently published ccm normative values, and patients referred to the sfn center maastricht with the clinical picture of sfn. corneal nerve fiber density (cnfd), branch density (cnbd), fiber length (cnfl), and the tortuosity coefficient (cnft) will be determined in all participants. the results will be compared with the ienfd and ttt. preliminary results will be presented. southanalinh k , university of health sciences, vientiane capital, lao p.d.r. located in south east asia, lao pdr is a landlocked country with a population of about . million inhabitants. the health indicators are among the lowest in south east asia. the total health caregivers in consisted of , persons corresponding to a ratio of . health workers per inhabitants. the main network for health care service provision remains the public system. its health care facilities consist of four central teaching and referral hospitals; five regional hospitals, including one teaching hospital; provincial hospitals; district hospitals, and about health centers. only one in seven sick people receives modern health care treatment. most people rely on self-medication and/or reliance on self-healing. neurological care is a very new field. knowledge of common neurological disorders among both the lao population and medical staff is only beginning to be spread. there are three neurologists in the country. six neurology residents are currently being trained in a three-year program supported by the association pour la promotion des neuro-sciences au laos (association for the promotion of neuro-sciences in laos) and the asean neurological association. indeed, resources are scarce. in the peripheral nerve diseases domain for example, we have only one electromyography machine that was only temporary used when emg experts from france and from singapore came to teach residents. a significant mismatch between the provision of specialized neurologic services and the needs for them exists, especially in rural areas. also, health insurance is not available for the majority. as a consequence, patients have to bear the costs themselves, which constitutes a limit to the access of available healthcare facilities. neurologic training centers, laboratory facilities and equipments are limited. optimizing available human resources, integrating primary, secondary, and tertiary healthcare tiers and making medical treatment more affordable are need to improve neurologic care in the developing world. in certain low-income countries with limited human and financial resources, it may be difficult for governments to apply some of these recommendations on their own. in these circumstances, it is suggested that countries work with international agencies, nongovernmental organizations or other partners to put their plans into practice. spina e , topa a , iodice r , tozza s , dubbioso r , ruggiero l , santoro l , manganelli f . department of neuroscience, odontostomatological and reproductive sciences, university of naples "federico ii", naples, italy. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a disabling disease and about % of patients may become persistently disabled over time. our aim was identify clinical prognostic factors of long-term disability in a large series of cidp patients. we collected data from cidp patients with definite diagnosis according efns/pns criteria and positive response to first-line therapies (immunoglobulin or corticosteroids) including sex, age of onset, phenotype, disease duration, course of disease (monophasic/relapsing-remitting or chronic progressive) and disability at the time of diagnosis assessed using the modified rankin scale (baseline mrs). all patients had clinical assessment of disability through mrs within the last months (last mrs). ordinal logistic regression model was applied to evaluate the relationship among the clinical parameters and last mrs, considered as ordinal outcome ( - ). anova test for repeated measures was applied to test the overall effects of different course on disability accumulation while t-test was performed to evaluate inter-group differences for parametric variables. we found a significant relationship between last mrs and the course of disease [p< . , z= . , or: . ]. disability accumulation was greater in patients with chronic progressive course than those with monophasic/relapsing-remitting course of disease [p= . ]. moreover, patients with progressive course were older [p= . ]. our data suggest that chronic progressive course of disease may be a major negative prognostic factor for long-term disability in cidp patients. to note that a chronic progressive course of disease is also associated with an older age from the beginning and a more pronounced worsening over the course of disease. sprenger a , lichtenstein t , henning t , lehmann hc . department of neurology, university hospital of cologne, cologne, germany; institute of diagnostic and interventional radiology, university hospital of cologne, cologne, germany; department of neurology, university hospital of cologne, cologne, germany. an unresolved problem in the treatment of inflammatory neuropathies is the lack of valid and reliable diagnostic biomarkers to evaluate axonal damage. we investigated if "diffusion tensor imaging" (dti) and mri t w multi echo dixon imaging are eligible methods to determine proximal nerve injury in chronic inflammatory demyelinating polyneuropathy (cidp). in this prospective observational cohort study the sciatic nerve of cidp patients and age matched healthy controls was investigated. all subjects underwent multimodal mri imaging to determine fractional anisotropy (fa) and muscle fat fraction of the biceps femoris and quadriceps femoris muscle. patients were evaluated by mri, clinical examination and nerve conduction studies at baseline and after six months. the mean fractional anisotropy (fa) value was significantly lower in the sciatic nerve from cidp patients compared to controls. fat fraction of the biceps femoris and quadriceps femoris muscle were significantly higher in cidp patients compared to controls. mri outcome parameters remained unchanged after six months. our study demonstrates the utility of mri imaging to differentiate between "healthy" and functional constricted proximal nerve segments. we postulate that dti and dixon mri might be eligible methods to assess proximal nerve damage in cidp. the presence of peripheral myelin protein (pmp ) has been known for decades, but its functional role was uncovered only recently. recent characterization of pmp -deficient mice revealed a role of pmp in the lipid homeostasis of myelinating schwann cells. in this study, we analyzed the functional impact of pmp on myelination. to decipher the role of pmp , experimental demyelination was performed in myelinating dorsal root ganglia cultures, and in vivo re-myelination was assessed after experimental peripheral nerve damage. we used the myelinating dorsal root ganglia (drg) model in pmp -deficient schwann cell cultures, combined with an established de-and remyelinating protocol in order to analyze myelination in vitro. we also performed experimental nerve crush in pmp -deficient mice. morphometric parameters were defined for the in-vitro experiments and functional parameters such as nerve conduction velocity and the clinical score were additionally measured for the in vivo experiments. structural analyses of the drg cultures revealed fibers expressing myelin basic protein (mbp) and pmp , as well as fibers positive for mbp alone. in contrast to our previous in vivo data, we were also able to detect myelin segments that stained positive for pmp , but were negative for mbp. pmp -deficient drg-cultures demonstrated slightly greater nodal lengths than the control cultures. this trend was significantly augmented after in vitro de-and remyelination, which also resulted in decreased internodal lengths only now, while conserving an intact myelin structure. concomitantly, in vivo nerve crush gives rise to a more severe phenotype in pmp -deficient mice than in wild-type controls. consistent with this, nerve conduction studies showed a delay in remyelination, and analysis of semi-thin sections demonstrated an altered fiber structure in the peripheral nerve biopsies. together, these data suggest that in addition to its role in glial cell lipid homeostasis, pmp also plays a role in remyelination of the injured peripheral nervous system. anti-mag neuropathy remains a difficult diagnosis to treat given its limited therapeutic options. of all interventions, rituximab has emerged as the most effective, although its effect has been with mixed results, especially in patients with advanced axonal loss. lenalidomide is another promising immune modulating therapy, whose effect has been well demonstrated in neuropathy associated with poems (polyneuropathy, endocrinopathy, organomegaly, m-spike protein, and skin changes) syndrome, a condition that has several striking parallels to anti-mag neuropathy. the use of lenalidomide has not been previously described in anti-mag neuropathy. herein, we describe a case of lenalidomide-responsive anti-mag neuropathy in a patient with advanced axonal loss. suichi t , misawa s , sato y , beppu m , sekiguchi y , shibuya k , watanabe k , amino h , kuwabara s . department of neurology, graduate school of medicine, chiba university, chiba, japan; clinical research center, chiba university hospital, chiba, japan. polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes (poems) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscrasia and vegf overproduction. several diagnostic criteria for the disorder have been published, but sensitivity/specificity analyses, and their validation have never been performed. the aim of this study is to establish valid diagnostic criteria for poems syndrome. consecutive poems patients, seen at chiba university hospital since , were screened. of these, we have set a gold standard group of poems syndrome, based on treatment response and exclusion criteria during -year follow-up, and patients was diagnosed as having definite poems syndrome. we also collected patients with cidp (demyelinating neuropathy control) and with multiple myeloma, primary amyloidosis, or mgus (m-protein control). criteria for poems syndrome was defined as having two of the three major criteria (polyneuropathy, m-protein, and elevated serum vegf level) and at least two of the four minor criteria (extravascular volume overload, skin changes, sclerotic bone lesions, and thrombocytosis) which were determined by logistic regression analyses. according to the criteria the sensitivity was %, and the specificity was %. our results indicate that the proposed criteria have an excellent diagnostic accuracy, and are useful in clinical practice, presumably leading to early diagnosis and treatment. intraepidermal electrical stimulation (ies) is a new technique to that assesses the function of a-delta fibers in the epidermis. using this technique, we previously reported that the epidermal pain threshold was two-hold in asymptomatic diabetic patients than in normal subjects (muscle nerve : - , ). subsequently, we reported that the elevated pain threshold negatively correlated with intraepidermal nerve fiver density (jpns : s , ). empirically, it is known that lowering the skin temperature makes it less likely to feel pain. therefore, it is necessary to investigate whether the results of ies are affected by skin temperature. the aim of this study was to investigate the influence of a low skin temperature on pain threshold. we recruited subjects with a mean age of . years. for nociceptive stimulation, we used an ies method with a concentric micro-needle electrode that was developed specifically for the selective stimulation of cutaneous a-delta fibers. we placed the ies electrode onto the extensor digitorum brevis and began stimulation with intensity strong enough for the subject to feel a pricking sensation, then reduced the current in steps of . ma until no sensation was felt. we defined pain threshold as the minimum electrical intensity at which a subject felt a pricking sensation. firstly, we measured pain threshold at skin temperature above degrees celsius. then, we put an ice pack on the extensor digitorum brevis for min to lower the skin temperature, and measured pain threshold at skin temperatures below degrees. mean pain threshold values above degrees and below degrees of skin temperature were . and . ma (p< . ), respectively. our data indicated an elevated pain threshold in epidermis with a low skin temperature. one of most common methods for nociceptive stimulation is painful co laser stimulation. some co laser stimulation studies reported pain threshold increased with a low skin temperature. our result is similar to that of co laser stimulation. pain threshold using ies is very easy and non-invasive technique. it may be useful for the evaluation of small fiber neuropathy. svačina mkr , röth p , bobylev i , sprenger a , zhang g , sheikh ka , lehmann hc . department of neurology, university hospital cologne, cologne, germany; department of neurology, university of texas, houston, usa. intravenous immunoglobulins (ivig) are an effective treatment in guillain-barré-syndrome (gbs). in most patients, the optimal ivig dose and regime is unknown. in serum and ivig preparations, immunoglobulin (ig) g form igg dimers, which are assumed to consist of idiotypic/anti-idiotypic antibody pairs. however, data about kinetics of igg dimer formation in gbs are lacking. to study igg dimer formation, c bl/ mice were injected with ivig and anti-gd b antibody or pbs. blood sera were collected h, h and week post injection. a third cohort received an anti-gd a/gt b antibody and blood was collected h post injection. igg was extracted and subtyped into polymeric, dimeric and monomeric fractions using the Äkta fplc system. dialysed dimeric and monomeric igg fractions were examined for the presence of anti-ganglioside antibodies by anti-ganglioside antibody elisa. further, blood samples from gbs patients were collected before (pre-ivig) and after treatment with ivig (post ivig). serum samples were examined for igg dimers and monomers using the Äkta fplc system. in the mouse model, a maximum peak of igg dimer formation was observed h post injection. in gbs patients' samples, igg serum levels and igg dimer content was significantly higher after treatment with ivig. we demonstrate here the feasibility to assess igg dimer formation in an animal model and in gbs patients' samples after treatment with ivig. h after ivig treatment appears to be the optimal time point to assess igg dimer formation. further studies are warranted to determine the utility of igg dimer formation as surrogate marker for treatment response in gbs. svaren j , moran jj , wu x , gutmann l , shy m . university of wisconsin-madison, madison, wisconsin, usa; university of iowa, iowa city, iowa, usa. development of outcome measures for clinical trials in cmt a is a major challenge given the slowly progressive nature of the disease. outcome measures can be used to measure a) target engagement for a given therapy, as well as b) disease process and c) disease burden. several candidate therapies have been shown to reduce pmp levels in cmt a rodent models and thereby ameliorate the symptoms of pmp overexpression. measuring pmp mrna reduction in human trials has so far been limited to analysis of skin biopsies by qrt-pcr, which did not demonstrate clear elevations of pmp mrna during, nor a reduction following, ascorbic acid trials. the analysis of skin biopsies is hampered by variable amounts of schwann cells (sc) in skin biopsies, as well as the variable amount of pmp in sc as previously established by immuno-em in cmt a skin biopsies (katona et al., ) . therefore, it is important to develop optimal normalization criteria to address the variability inherent in skin biopsy analysis. ideally this will employ normalization to sc-specific genes that are not altered by cmt a status. to optimize normalization, we have performed rna-seq analysis of skin biopsies from patient and control skin biopsy samples. analysis of these data after normalization to read depth indicated that pmp levels were . fold higher in cmt a patient samples compared to control skin biopsies. however, there was significant variability in pmp levels particularly in cmt a samples, which may be due to variable amounts of schwann cells in cmt a skin. using a combination of sc-specific genes for normalization, we were able to reduce the apparent variability and optimize the differential levels between cmt a and control skin biopsy samples. we also identified other sc-specific genes that were apparently induced in cmt a skin biopsies relative to control. these studies provide a new framework for gene expression analysis in skin biopsies, enabling more precise evaluation of pmp levels in clinical trials for cmt a as a measure of target engagement. in addition, the normalization framework may also be applicable to other types of cmt. chronic inflammatory demyelinating polyneuropathy (cidp) is the most common chronic autoimmune neuropathy, with an estimated prevalence of between and per , people. it can cause temporary disability in the affected individuals and may eventually lead to permanent disability or death. cidp is commonly treated with intravenous immunoglobulin (ivig) therapy or corticosteroids. octagam ® % is licensed for cidp in france, while octagam ® % is licensed for cidp in germany and belgium. this analysis presents data from three open, multicenter, non-interventional, single-arm, non-controlled studies of a post-authorisation safety surveillance (pass) program for the subset of patients receiving octagam ® % or % for neurological indications, focusing on patients with cidp. briefly, data from in-and out-patients in austria, france, germany, and uk treated with octagam ® for neurological disorders were collected by physicians and analyzed to assess safety and tolerability of the treatment. of patients included in the three studies, patients ( . %) received octagam ® for neurological indications, of which patients ( . %; mean age . years [range - ]) had cidp. the mean dose of octagam ® per course was . g/kg bw for patients with cidp; for the other neurologic indications, the dose ranged from . (for multiple sclerosis) to . g/kg bw (for guillain-barré syndrome). premedication was not needed in . % of these patients. the development of clinical appearance since last observation (mean: every . months) was assessed for of the cidp patients by their treating physicians. the majority of observations ( . %) assessed the patients as stable and . % showed even an improved clinical appearance. only . % of the observation periods resulted in deteriorations. adverse drug reactions were rare: of the infusions received by patients with neurological disorders, . % of infusions were associated with an adr ( . % of infusions in cidp patients). overall, treatment with octagam ® was effective and well-tolerated in patients with cidp. these results are consistent with data for the overall patient population (including patients with primary and secondary immunodeficiencies, dermatological and other diseases). szepanowski f , szepanowski lp , kleinschnitz c , kieseier bc , stettner m . department of neurology, medical faculty, university duisburg-essen, essen, germany; department of neurology, medical faculty, heinrich-heine-university, düsseldorf, germany. lysophosphatidic acid (lpa) is a pleiotropic signaling lipid that acts as ligand for at least six specific g protein coupled receptors. schwann cells (sc) are known to mainly express the lpa receptor subtype. an emerging body of in vivo evidence has linked lpa with injury induced peripheral nerve demyelination as well as neuropathic pain. however, the molecular mechanism underlying its demyelinating effect has remained largely unclear. myelinated dorsal root ganglia (drg) cultures were treated either with lpa, lpa + am (lpa antagonist) or vehicle. we assessed myelin basic protein, tumor necrosis factor alpha (tnf-alpha) as well as the sc differentiation marker sox by immunocytochemistry. additionally, myelin was investigated by sudan black staining. to better understand the relevance of lpa signaling for demyelination in vivo, we performed sciatic nerve crush in c bl/ mice treated with am at mg/kg in order to study schwann cell expression of tnf-alpha, sox and sox , a marker for sc dedifferentiation, by immunohistochemistry. in drg cultures, lpa caused a significant reduction of myelin as demonstrated by both sudan black staining and immunocytochemical analysis of myelin basic protein. demyelination was paralleled by an upregulation of tnf-alpha as well as downregulation of sox . lpa mediated effects were found to be blocked by addition of the lpa receptor antagonist am . in c bl/ mice, am treatment prior to crush injury increased sox expression in scs in the distal nerve stump while reducing the number of cells expressing sox . these data indicate that lpa may be a critical factor to shift scs towards an injury-associated phenotype and contribute to the onset of wallerian degeneration. szepanowski lp , szepanowski f , kleinschnitz c , stettner m . department of neurology, university hospital essen, essen, germany. glyphosate-based formulations comprise the world's most commonly used herbicides. in non-resistant plants, glyphosate exerts toxic effects most likely via inhibition of aromatic amino acid synthesis by interfering with the shikimate pathway. while glyphosate is the active ingredient, herbicidal formulations contain several adjuvants, including polyethoxlated alkylamines (poeas). although glyphosate has long been considered safe for use in humans and animals, several studies have implicated glyphosate and/or the commonly used adjuvants in cytotoxicity, carcinogenicity and endocrine disruption. furthermore, glyphosate-based herbicide has been reported to mediate neurotoxicity in immature rat hippocampus involving glutamate excitotoxicity. however, it remains unclear whether glyphosate alone or in combination with its adjuvants may have detrimental effects on myelin integrity in the peripheral nervous system. myelinated dorsal root ganglia (drg) cultures were treated over the course of ten days with either pure glyphosate or a glyphosate-based herbicide at concentrations of . %, . % and . %. the concentration of the glyphosate-based herbicide was matched with regard to glyphosate content ( %). controls were treated with equal amounts of vehicle adjusted for the ph. subsequently, cultures were stained with sudan black and myelin content was assessed by determining the number of internodes per neurons. while glyphosate, regardless of its concentration, did not show any effect on myelin content, the glyphosate-based herbicide caused significant demyelination in a concentration-dependent manner. notably, at . %, drg cultures were completely devoid of myelin and appeared severely necrotic. these data raise the possibility that not glyphosate itself, but rather the adjuvants in glyphosate-based herbicide formulations may cause demyelination. the open question whether demyelination is a direct effect of the adjuvants or a consequence of increased cellular glyphosate uptake due to permeabilization warrants further investigation. tan cy , tan mp , yeoh ky , goh kj , shahrizaila n . department of medicine, university of malaya, kuala lumpur, malaysia. in guillain-barré syndrome (gbs), autonomic dysfunction is common and accounts for significant morbidity and mortality. there have been many studies investigating the electrodiagnosis of gbs but few have studied autonomic dysfunction in gbs. the current study comprehensively investigates quantitative autonomic function in patients with gbs and its variant. ten gbs patients were prospectively recruited and the results were compared to age-and gender-matched healthy controls. a series of autonomic function tests including computational (power spectrum analysis of heart rate variability (hrv) and baroreflex sensitivity (brs) at rest) and challenge tests (deep breathing, eyeball compression, active standing, valsalva manoeuvre, isometric exercise and ice-water hand immersion) were performed. parasympathetic function was represented by high frequency (hf) hrv, heart rate responses to deep breathing, eyeball compression, valsalva manoeuvre and active standing. sympathetic function was represented by low frequency (lf) hrv, blood pressure responses to active standing, sustained handgrip and ice-water hand immersion. in the frequency domain analysis of hrv, low frequency (lf: . ± . vs . ± . ; p= . ), high frequency (hf: . ± . vs . ± . ; p= . ) and total power spectral densities (psd: . ± . vs . ± . ; p= . ) were significantly reduced in patients compared to controls. the mean up slope ( . ± . vs . ± . ; p= . ), down slope ( . ± . vs . ± . ; p= . ) and total brs slope ( . ± . vs . ± . ; p= . ) were significantly lower in the gbs group. the diastolic rise in blood pressure upon ice-water hand immersion was significantly lower in gbs group compared to controls ( . ± . vs . ± . ; p= . ). our findings suggest that computation dependent tests (hrv and brs) were sensitive at detecting autonomic dysfunction and baroreceptor reflex insensitivity in gbs patients. in contrast, ice-water hand immersion was the only reliable challenge test making it useful as a bedside measure of autonomic function in gbs patients. sjögren's syndrome (ss) is an autoimmune disease that affects both east and west. nevertheless, we still have limited knowledge of how autoantibodies in ss affects the peripheral nervous system. in this study, we investigated the peripheral neuropathy in ss and sicca complex using the nerve excitability test, to elucidate how peripheral nerves are affected. we have enrolled a total of patients with ss or sicca complex. of these, two patients were excluded due to co-morbid carpal tunnel syndrome. each patient received clinical evaluation, examination for ssa/ssb antibodies titer, the nerve excitability test, conventional thermal quantitative sensory test, and conventional nerve conduction study. compared to normal control subjects, motor nerve excitability test of ss patients with positive ssa or ssb antibodies (n = ) were found to have increased rheobase (p< . ), increased relative refractory period (rrp) (p< . ), increased refractoriness at . ms (p< . ), increased accommodation toward depolarizing current in threshold electrotonus (te) (p< . ), and decreased superexcitability (p< . ). the sensory axonal study in seropositive ss also revealed increased rrp (p< . ), increased refractoriness at . ms (p< . ), and increased accommodation toward hyperpolarizing current in threshold electrotonus (te) (p< . ). meanwhile, in seronegative ss and sicca complex (n = ), we found no significant axonal properties changes. the present study revealed that peripheral nerves are affected differently in seropositive ss and in seronegative ss/sicca complex. in seropositive ss, motor axons tended to be depolarized, and both sensory and motor axons have increased refractoriness. the findings suggested that ssa and ssb antibodies might play a role in the inactivation of transient sodium channels. the effects of the antibodies on transient sodium channels might be the basis of peripheral neuropathies and even cardiac arrhythmias and heart block in ss. charcot-marie-tooth disease type a (cmt a), caused by the pmp duplication on chromosome p . , is the most common subtype of inherited peripheral neuropathies and affects in , individuals worldwide. while sharing the same genetic cause, cmt a patients often present great variability in their phenotypic presentation and disease severity. the cause of the phenotypic variability is largely unclear. in this study, we performed genome-wide association study (gwas) to identify novel genetic modifiers of various phenotypes in cmt a. dna samples from cmt a patients were genotyped on illumina omniexpress platform. after standard quality control, the dataset includes k markers in individuals ( individuals from european ancestry, and individuals from asian ancestry). we focused our analyses on the european population. logistic regression in plink was used to analyze the association between the clinical outcomes and patients' genotypes in an additive model. for cmt neuropathy score (cmtns), the analysis was performed using linear regression in plink, adjusting for patients' age. the analyses yielded several suggestive association signals. an association peak on chromosome was identified in difficulty with eating utensils (lead snp rs , chr : , p= . e- , odds ratio= . ). the peak is located within a non-coding gene linc . hearing loss showed an association peak on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ), located in an intergenic region near the megf gene. in foot plantar flexion, an association signal was identified in the dscam gene on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ). cmtns showed an association signal on chromosome (lead snp rs , chr : , p= . e- , beta= . ), located within an intergenic region close to dffb, c orf , and linc . while these suggestive signals require further validation, our study provides novel insights into the genetic architecture of cmt a. novel genetic modifiers may serve as potential targets for therapeutic interventions in the future. teng a , ohnmar , kalpana p , chai yh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. we present an intriguing diagnostic puzzle, that was eventually cracked serendipitously. a -year-old man was seen for bilateral ptosis. evaluation for myasthenia gravis was negative. nevertheless, a diagnosis of ocular myasthenia gravis was made and he was put on pyridostigmine. he did not respond. the ptosis progressively worsened. he sought a second opinion. at this evaluation, he was noted to have complex ophthalmoplegia without diplopia, bilateral facial weakness and mild bulbar weakness. he had no sensory complaints. the limb examination was remarkable for slightly reduced reflexes, normal strength, and other than increased vibration threshold at the toes, intact sensory examination. repeat serological and electrodiagnostic work-up for myasthenia gravis was negative. a myopathic disorder such as chronic progressive external ophthalmoplegia was considered. serum creatine kinase and lactate were normal. he underwent biceps muscle biopsy which showed increased cox-negative and sdh positive fibers, supporting the then clinical impression of a mitochondrial cytopathy. at this point, he underwent blepharoplasty to improve his vision. routine histological examination of the levator palpebrae muscle showed amyloid deposits. this prompted a review of the earlier biceps biopsy, which revealed amyloid deposits that were not appreciated before. at this point, the significance of the patient and his son's history of lattice corneal dystrophy became apparent. he also reported that his late mother had similar facial appearance as his. the patient's nerve conduction study showed length-dependent sensory axonal polyneuropathy, right carpal tunnel syndrome and bilateral facial neuropathy. he had no definite symptoms of autonomic neuropathy. cardiac evaluation was unremarkable. the final diagnosis of familial gelsolin amyloid polyneuropathy was made. genetic confirmation for the patient and his family is being planned. we highlight the key clinical features of gelsolin neuropathy. the symmetric cranial neuropathy can resemble a muscle or neuromuscular junction disorder and the relative sparing of the cardiac muscle, somatic and autonomic nerves contrasts with transthyretin-related amyloid polyneuropathy. neuropathy is one of the most common long-term complications of diabetes. furthermore, % to % of diabetic neuropathy patients will develop neuropathic pain. the pathophysiology of neuropathic pain in diabetic peripheral neuropathy is complex and not fully understood. a potential mechanism is a change in voltage gated sodium channels, such as nav . . loss of function mutations in this channel cause insensitivity to pain, whereas gain of function mutations have been linked with different pain syndromes including small fiber neuropathy. in a cohort of patients with diabetic peripheral neuropathy we investigated whether mutations in nav . were associated with diabetic neuropathic pain. twelve nav . variants were identified in nine participants all within a cohort of participants with painful diabetic peripheral neuropathy. five of these variants were previously associated with pain disorders: v l, m l; w r, r h, l v. among the other variants two of them met the criteria of potential pathogenicity based on predictive algorithms and were further studied. functional analysis by whole cell patch clamp showed that one of these variants (m t) drastically impairs the inactivation of the channel by shifting the steady-state fast-inactivation towards more depolarizing potentials. there were no phenotypic difference between those participants with pathogenic variants and those participants without pathogenic variants. no rare nav . variants were found in participants with painless diabetic peripheral neuropathy. these observations suggest that mutations in nav . may contribute to painful diabetic peripheral neuropathy. tholance y , rosier c , f bouhour , psimaras d , kuntzer t , taieb g , créange a , delmont e , camdessanché jp , antoine jc . university hospital, saint-etienne, france; university hospital, lyon, france; university hospital, paris, france; university hospital, lausanne, switzerland; university hospital, montpellier, france; university hospital, creteil, france; university hospital, marseille, france. dysimmune sensory neuronopathies (snn) encompass paraneoplastic snn and snn associated with systemic autoimmune diseases such as sjögren syndrome, lupus or inflammatory bowel or rheumatic diseases but also a number of apparently idiopathic cases. biomarker antibodies are well-known in paraneoplastic snn but are lacking in non paraneoplastic cases. from a mono-center retrospective study we identified in anti-fgfr antibody as a potential biomarker of dysimmunity in patients with idiopathic or systemic autoimmune disease associated sensory neuropathy. the identified patients were more frequently women and had a non lenthg dependent neuropathy suggestive of snn. anti-fgfr antibody was the only immunological marker in / of cases at initial work-up although / of patients eventually developed with time systemic autoimmune disease. to confirm the incidence and the clinical pattern of patients with anti-fgfr antibodies we launched a prospective multicenter french study including patients with a sensory neuropathy suspected to be a snn of no paraneoplastic, genetic or metabolic origin. we present here the results on the first included patients compared to healthy blood donors. anti-fgcr antibodies were searched by elisa using the trk intracellular domain of the protein (invitrogen©). we found patients positive for anti-fgfr antibody ( . %). these patients were women and men aged . years as a mean ( - ). the neuropathy was acute and subacute in one patient respectively and progressive in the others. six patients fulfilled the diagnosis criteria of snn and the last one had a sensory neuropathy in the lower limb with abnormal sensory action potentials in the four limbs suggesting snn without reaching the requested criteria. one patient developed uveitis which is a new symptom with anti-fgfr ab. an unclassified dysimmune context was present at the initial work up in patients and one patient developed sjögren syndrome with follow-up. as a whole the clinical pattern of these patients is consistent with that of the initially published series. the lower prevalence of positive sera may be due to more stringent criteria used for elisa but needs to be confirmed on the complete prospective series. peripheral neuropathy research registry (pnrr) neurological assessment was scored using the total neuropathy score clinical version (tnsc), comprising pinprick and vibration sensibility, deep tendon reflexes, strength and patient symptom report. compound sensory action potential (csap) amplitudes were recorded antidromically at the lateral malleolus, stimulating the sural nerve at the mid-calf. of the total sample, % reported lower limb neuropathy, with % of patients reporting 'quite a bit' or 'very much' severity of tingling and numbness in their feet. the average sural csap amplitude was . ± . v and % of patients had sural amplitudes below the lower limit of normal for age. the total tnsc score correlated with the pro fact-gog ntx score (r = −. , p<. ) and sural amplitude (r = −. , p <. ). vibration sensibility correlated with the overall fact-gog ntx score (r =−. , p <. ), and sural amplitude (r = −. , p <. ). sural amplitude correlated with patient reported severity of numbness and tingling in the lower limbs (r =−. , p <. ) but not with the overall fact-gog ntx score. patient reports of neuropathic symptoms in the lower limb correlate with both objective neurophysiological and clinical measures of neuropathy severity. identifying links between objective neurophysiological markers and patient reported outcomes are critical to assess the impact of clinical interventions. tomaselli pj , gouvea sp , nyshyama kfs , nicolau n jr , lourenço cm , marques w jr , . division of neuromuscular diseases, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; neurogenetics, department of neurosciences and behaviour sciences, university of são paulo, ribeirão preto, brazil. mutations in the gab junction beta -protein gene (gjb ) are the second most frequent cause of charcot-marie-tooth disease (cmt), accounting for approximately % of cmt cases worldwide. the gjb codes for connexin protein (cx ). in the peripheral nervous system, the cx is expressed in the schwann cells and allows intercellular traffic of ions and small molecules between opposed cells. we analysed retrospectively detailed clinical and neurophysiological data of five families carrying novel gjb mutation submitted for testing at our neurogenetics laboratory. mutations were identified by bidirectional sanger sequence analysis of gjb coding region. we identified a total of subjects from five different kindreds with novel mutations (p.a v, p.l w, p.l q, p.f s, p.r l). these five novel mutations segregate with phenotype, are located in highly conserved amino acids among gjb and other gab junction protein sequences and among different species, are not present in any public database (exac, dbsnp and genome database), and were not found in normal brazilian controls. in silico analysis, predict these variants to be pathogenic, there was no male-to-male transmission; males were more severely affected than females. four out seven female have subclinical neuropathy and were only identified after clinical and electrophysiological evaluation. the conduction velocities were in the intermediated range in the males patients and higher in the females included in this study. we describe five new pathogenic mutations causing cmtx in a brazilian population and expand the number of causative mutations in the gjb gene. funded pure neural leprosy (pnl) is a slowly progressive, predominantly sensory patchy neuropathy presenting with positive and/or negative sensory symptoms, which are usually followed over time by distal asymmetrical weakness. despite rare, monomelic involvement in leprosy has already been reported. we sought to describe the clinical and electrophysiological patterns of an unusual leprosy neuropathy presentation. clinical data were retrospectively collected from nine patients who had monomelic involvement and were referred for further investigation to the emg lab. seven out nine patients were male. four patients had a brachial plexus like presentation and five have a lumbosacral plexus like presentation. the initial complaint was hypoesthesia in four patients, tingling in two patients and hypoesthesia with tingling in two patients. severe pain was observed in just one patient. all individuals from the group of patients with lumbosacral plexus-like presentation and three with brachial plexus-like presentation had no sensory nerve action potentials (snaps) for all nerves tested in the affected limb with or without motor involvement. one patient with brachial plexus-like presentation had focal slowing of conduction velocity with temporal dispersion of both median and ulnar nerves in the affected limb. one patient with plexus-like presentation had snaps with low amplitude of all nerves in the affect limb. the diagnosis of leprosy was confirmed by nerve biopsy findings, anti-pgl antibody, and positive response to specific treatment. nerve biopsy was performed in four patients, and the bacillus was found in two. the anti-pgl antibody was positive in four patients. plexus mri was performed in two patients and was normal. we found the distribution of motor and sensory symptoms were restricted to on limb in this group of patients. as a typically patchy disorder pnl may affect any nerve, although the reason why damage are restrict to only one limb has to be elucidated. the description of these cases increases the clinical spectrum of leprosy neuropathy. this possibility should be considered in the differential diagnosis of patients with plexopathy from endemic areas after excluding other causes. funded ataxia with oculomotor apraxia type (aoa ) is a very complex disorder characterized by an early-onset progressive cerebellar ataxia with cerebellar atrophy and peripheral neuropathy and it is caused by recessive mutations in the aprataxin gene (aptx ). when the neuropathy is present, it has been described in % of cases as primarily axonal. we describe a case of aoa due compound heterozygous mutations in aptx associated with demyelinating neuropathy. the patient was born from healthy and non-consanguineous parents and presented in the first decade with progressive cerebellar ataxia, multidirectional ophtalmoparesia, oculomotor apraxia, choreiform movements of limbs and peripheral neuropathy. he had normal cognition and stopped walking at age of . blood tests were unremarkable with normal levels of leucocytes, serum proteins, immunoglobulin, cholesterol, vitamin e, and alfa-feto protein. brain mri showed severe cerebellar atrophy. the motor conduction velocity in the upper limbs was slow with preserved amplitudes. the distal latencies and the minimal f wave latencies were prolonged. there was no evidence for superimposed acquired demyelinating neuropathy. direct sequencing of the aptx gene revealed two variants, c. - a>g, p? and c. g>a; p.w x. the first variant is novel and affects a highly conserved acceptor splice site of exon . the other variant is the most common portuguese variant, the nonsense mutation w x is located in exon . parental dna was tested and confirmed the variants were in different alleles. the presence of a demyelinating neuropathy in aoa suggests that phenotypic variability in this condition may be larger than previously considered. at the same time, it increases the differential diagnosis of inherited conditions with cerebellar ataxia and demyelinating neuropathy. finally, this finding opens the functional effects of the aptx gene. funded by: cnpq, fapesp, faepa, pronas (ministry of health). topa a , spina e , iodice r , tozza s , ruggiero l , dubbioso r , esposito m , santoro l , manganelli f . university of naples "federico ii", naples, italy. we report our -year experience of subcutaneous immunoglobulin (scig) in a cohort of patients with chronic inflammatory demyelinating polyneuropathy (cidp) from a tertiary care neuromuscular center. we analyzed data from cidp patients ( males and females, mean age: ± . years; mean age at onset: . ± . years; disease duration: . ± . years) treated with scig and with a follow-up period of months. all patients were previously responders to intravenous immunoglobulin (ivig). eight patients had a typical cidp and five patients had an atypical variant of cidp. five patients switched to weekly maintenance scig therapy (continuous regimen) because of short-lasting response to ivig therapy. eight patients with a longer lasting response to ivig received scig with a pulsed regimen similar to that used for ivig (from to cycles per year); seven of them because of difficulty in hospitalization and one for allergic reaction to ivig. changes in clinical status were assessed over the period of follow-up by using clinical evaluation of muscle strength, modified rankin scale, overall neuropathy scale and inflammatory neuropathy cause and treatmentsum score. in patients we evaluated also six minute walking test, hole-peg-test and meter walking test. all the five patients treated with a continuous regimen of scig remained clinically stable throughout the follow-up period. among the patients receiving pulsed scig treatment, out of ( %) responded to scig similarly to ivig, while three patients ( . %) worsened and needed to be treated again with ivig and the other one ( . %) stopped any therapy. subcutaneously administered immunoglobulin were well tolerated and no patients complained of adverse events. in conclusion, our findings confirm that continuous scig therapy is efficacious in maintaining clinical stability in patients with short-lasting response to ivig. moreover, our data suggest that pulsed therapy with scig may represent an alternative therapeutic option for the treatment of a subset of cidp patients. touvier t , ferri c , mastrangelo r , glimcher l , , wrabetz l , , , d'antonio m . myelin biology unit, division of genetics and cell biology, san raffaele scientific institute, dibit, milan, italy; department of cancer immunology and virology, dana-farber cancer institute, boston, usa; department of medicine, harvard medical school, boston, usa; hunter james kelly research institute, university at buffalo, buffalo, usa; department of biochemistry, university at buffalo, buffalo, usa; neurology, jacobs school of medicine and biomedical sciences, university at buffalo, buffalo, usa. mpz glycoprotein is an abundant product of terminal differentiation in myelinating schwann cells. the mutant mpzs del causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in transgenic mice. mpzs del protein is retained in the endoplasmic reticulum (er) of schwann cells and induces an unfolded protein response (upr) characterized by activation of perk, atf and ire /xbp pathways. we have previously reported that activation of chop and gadd , two downstream mediators of perk, is pathogenetic in mpzs del mice (pennuto, ; d' antonio, ) but the role of the other upr branches remains to be investigated. in this study, we investigated the role of the er stress sensor enzyme ire and of xbp -a transcription factor specifically activated by ire -in mpzs del pathogenesis. we generated a new mouse model with schwann cells-specific ablation of xbp and in parallel we exploited mpzs del dorsal root ganglia (drg) explant cultures in which xbp signaling is modulated by gain/loss of function approaches. we observed that absence of xbp dramatically worsens hypomyelination and electrophysiological/locomotor parameters in young and adult mpzs del neuropathic animals. interestingly we observed that perk, atf and ire -mediated ridd signalings are upregulated in neuropathic animals lacking xbp . this suggests that activation of xbp targets have an essential role in limiting mpzs del toxicity, which cannot be compensated by other stress responses. moreover, we demonstrated in mpzs del drg cultures that inhibition of xbp splicing by u c (cross, ) decreases myelination whereas activation of xbp splicing by quercetin (wiseman, ) slightly ameliorates myelination. altogether, these data demonstrate that xbp pathway has a critical adaptive role in mpzs del neuropathy and suggest that activation of this pathway may be beneficial for cmt b and perhaps for a broad range of neuropathies characterized by upr activation. tozza s , bruzzese d , iodice r , esposito m , dubbioso r , ruggiero l , topa a , spina e , santoro l , manganelli f . department of neuroscience, reproductive sciences and odontostomatology, university of naples "federico ii", naples, italy; department of public health, university federico ii of naples, naples, italy. in cmt a patients, the clinical impairment progressively increases over time and correlates with the axonal loss. evidence has suggested that the decline of physical performance in cmt a patients may reflect a process of normal ageing. the aim of our study was to describe, by a case-control cross-sectional design, the progression of physical impairment with ageing in cmt a patients. we enrolled cmt a patients ( m; range - years) and sex-and age-matched healthy controls. to assess physical performance, all patients and controls underwent -meter walk test ( mwt), -minute walk test ( mwt) and -hole peg test ( hpt) of their dominant (d) and non-dominant (nd) sides. moreover, to assess clinical disability, impairment and quality of life in the cmt a group we used the charcot-marie-tooth neuropathy score (cmtns), the mrc sum score and the short form- (sf- ) questionnaire. the linear regression model was used to evaluate the changes over time of clinical measures in patients and controls. the chow test was used to determine whether the ageing had a different impact on clinical measures for the two groups. physical performance worsened with ageing in both patients and controls, but with a greater slope for cmt a patients [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. the rate of deterioration of physical performance was not different between patients and controls until the th year of age. after the th year of age the rate of deterioration became greater in cmt a group [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. moreover, in cmt a patients also cmtns, mrc sum score and sf- worsened with ageing and with a greater rate of deterioration after the th year of age. our study demonstrates that clinical decline in cmt a patients goes parallel to the normal ageing process until the th year of age, whereupon the clinical deterioration accelerates. tsouni p , devic p , moura b , planque e , bédat-millet al , devaux j , steck aj , delmont e , hottinger af , kuntzer t . dcn, chuv, lausanne, switzerland; centre de référence maladies neuromusculaires, hospices civils de lyon, lyon, france; cabinet médical, epinal, france; département de neurologie, chu de rouen, rouen, france; cnrs, crn m-umr , université aix-marseille, marseille, france; centre de référence maladies neuromusculaires et sla, hôpital la timone, marseille, france. new therapeutic options in immuno-oncology have allowed significant progress in the management of melanomas. treatment usually consists of a combination of two monoclonal antibodies targeting cytotoxic lymphocyte-associated protein and programmed cell death- . as a result, the immunologic barrier protecting tumor cells is overcome allowing an antitumor response. we report cancer patients with immune checkpoint inhibitor-induced neuropathies as a complication of this immunomodulating oncologic treatment. case reports: our index patient developed severe myalgia days after introduction of ipilimumab-nivolumab followed by painful paresthesias of the face and extremities day after the nd cycle of treatment. generalized areflexic quadriparesis (mrc ) with gowers'sign and distal loss of vibratory sensation were found. a -day course of ivig plus corticosteroids (cs) had no effect. three monthly ivig courses were necessary to improve the deficits at months. a survey of sfnp members revealed other patients with similar acute courses but with phenotypes varying from sensorimotor deficits with areflexia and myalgia to purely sensory ataxic forms following immunomodulating treatment. work-up including anti-nodal antibodies were negative in patients. from the other , had abnormal csf and had necrotizing myopathy. detailed repeat ncs demonstrated signs of nerve hyperexcitability and of demyelination or conduction blocks. evolution was slowly favorable following ivig and cs. discussion: our report underscores that atypical acute generalized demyelinating neuropathies are induced by these novel treatments. they may be associated with severe myalgia or other systemic toxic effects. discontinuation of the oncologic treatment depends on severity of symptoms. outcome was slowly favorable following ivig or cs, albeit slower than in the case of primary inflammatory neuropathies, probably given the long half-life of the monoclonal antibodies. gap junctions (gjs) are membrane channels found in most tissues connecting adjacent cells or different cell compartments as in schwann cells. they are involved in electrical connectivity and metabolic homeostasis allowing the passage of small molecules such as ions, second messengers, nucleotides and peptides. an important functional role of peripheral nerve connexins is suggested by their involvement in x-linked inherited neuropathy as well as in acquired neuropathy caused by oxaliplatin. although gjs play a role in electrical connectivity their specific role in the formation of the sciatic nerve compound action potential (cap) remains unclear. the aim of this study was to investigate the role of peripheral nerve connexins in the electrical responses of the mouse sciatic nerve under normal and stress conditions. for this purpose we used sciatic nerves of three different mouse models, the cx knockout (ko), cx ko and the cx /cx double knockout (dko) mice. using our ex vivo model for extracellular recordings we exposed sciatic nerves from different genotypes to three different gj blockers: octanol, -beta-glycyrrhetinic acid (gra) and octanoic acid (oa) and recorded the cap. amplitude and duration of the cap were used as an indication for the effects of the different blockers on the cap formation. all gj blockers caused a gradual decrease of the cap without any changes in the duration of the cap in all genotypes, suggesting progressive disturbance of axonal membrane excitability in the absence of one or two gj proteins. comparison of the three genotypes showed that cx may play a dominant role in the maintenance of the cap formation since nerves from cx ko mice proved to be more sensitive to the gj blockers compared to the cx ko nerves showing a faster decline of the cap amplitude. moreover the effect of gj blockers was similar in cx ko and dko nerves. finally, the effect of gj blockers on the dko nerves implies the presence of another gj protein. in conclusion, our results confirm the direct functional involvement of cx gj channels and cx hemichannels in the cap formation and indicate the existence of at least one more connexin in peripheral nerve. ca( +)-dependent anti-ganglioside antibody in seronegative guillain-barrÉ syndrome uchibori a , gyohda a , chiba a . kyorin university, tokyo, japan. we have reported ca + -dependent igg anti-ganglioside gq b antibodies in gq b-seronegative patients with fisher syndrome and its related disorders (fs-rd). in patients with fs-rd who were gq b-seronegative in conventional assays using phosphate-buffered saline without ca + , % turned seropositive for gq b-related antigens in assays using ca + -added tris-buffered saline. objective: we investigated whether ca + -dependent anti-ganglioside antibodies was present also in ganglioside-seronegative patients with other clinical disease types of guillain-barré syndrome (gbs) other than fs-rd. methods: the subjects were the following: patients with final clinical diagnosis as gbs (acute motor axonal neuropathy [aman], n = , and acute inflammatory demyelinating polyradiculoneuropathy [aidp], n = ), and patients with final clinical diagnosis as sensory ataxic neuropathy (san), n = . all subjects were ganglioside-seronegative in the conventional assays. we assayed serum igg antibodies against various gangliosides (including asialo-gm ) in elisa using tris-buffered saline as a basal buffer in both ca + -added and -non-added conditions. increase of the optical density (od) more than . in ca + -added condition compared with ca + -non-added one was taken significant, i.e. positive for ca + -dependent antibody. results: ca + -dependent antibody was negative in all aman and san patients. in aidp, the antibody titers (ods) against gainac-gd a were significantly increased in patients, and those against asialo-gm were increased in other patients in ca + -added condition. however, the titiers of those ca + -dependent antibodies were all at low level. conclusions: in clinical disease types of gbs other than fs-rd, ca + -dependent igg antibodies against ganglioside were detected in a few patients with aidp, but the positive rate and the antibody titers were low compared with case of gq b-seronegative fs-rd. ca + -dependent antibodies against ganglioside are considered to be more specific for gq b. the aim of the study was to investigate the relationship between median sensory conduction of median nerve and ulnar nerve in patients diagnosed with carpal tunnel syndrome. two hundred and eighty-six hands with carpal tunnel syndrome and hands in control group were investigated. patients were staged clinically and electrophysiologically. diagnosis of carpal tunnel syndrome was made according to the presence of paresthesia, pain in the innervation area of the median nerve, weakness and atrophy in the median nerve innervated muscles, positive phalen and tinel tests. median motor and sensorial nerve conduction study, including first, second, third finger and palm, and ulnar motor and sensorial nerve conduction of fifth finger studies were performed to all patients and control group. the ratio of distal latency and velocity of nerve conduction of first, second, third and palmar branches to fifth finger was calculated. distal latency of first, second, third finger and palm of patients with cts are longer and velocity is more slowly than controls. in addition to these findings, the velocity of fifth finger is also slower and distal latency of this one is longer than healthy subjects. the most sensitive method of classifying the carpal tunnel syndrome as normal, mild and moderate is the ratio of distal latency and velocity of second finger (p< . ). carpal tunnel syndrome is the most common encountered neuropathy. in nerve conduction studies can be used the ratio of distal latency and velocity of second finger as determine the degree of carpal tunnel syndrome. the most surprising finding of this report is the nerve conduction studies of fifth finger. the subclinical susceptibility of fifth finger can be explained by overusing of wrist. ursino g , gemelli c , grandis m , reni l , bellone e , geroldi a , gotta f , mandich p , ferrara m , schenone a dinogmi university of genoa, genoa, italy; irccs-aou san martino hospital genoa, genoa, italy. charcot-marie-tooth (cmt) neuropathy represents a clinically and genetically heterogeneous group of hereditary peripheral neuropathies characterized by chronic motor and sensory impairment. to date mutations in up to genes may cause cmt. the aim of this study is to describe our large population of cmt patients, and, within this, highlight specific phenotypes. the cmt clinic in genova, started in . during the years, patients underwent complete neurological, rehabilitative, neurophysiological examinations and genetic testing. the patients were routinely tested for common genes (as pmp , gjb , mpz, mfn ), while in specific cases we followed the candidate gene approach testing single genes based on the genotype-phenotype correlation. however, the ngs techniques were used when routine genetic testing was negative and a clear genotype-phenotype correlation could not be identified. cases are present to date in our database of cmt patients. in ( . %) patients, in spite of a clinical diagnosis of cmt, a genetic diagnosis is still lacking; ( . %) patients had alternative diagnosis (i.e hereditary spastic paraparesis etc.). instead, in patients ( . %) a defined genetic diagnosis was reached, of them being females ( . %) and males ( . %). among these, except for the more common cmt a, hnpp and cmt x phenotypes, we frequently observed patients affected by cmt b and cmt f. according to most literature data, we observed ( . %) patients with cmt b and patients ( . %) affected by cmt f. at the first visit, the cmt b phenotype was clearly length-dependent: . % patients showed impairment of the lower limbs and saving of the upper limbs; in terms of severity of the neuropathy, the mean cmtns was . and the mean age was . years. similarly, % of patients affected by cmt f, present with the same phenotype; the mean cmtns at the first visit was . and the mean age was . years. in conclusion, based on the experience of the genova cmt clinic, we describe a large population of cmt patients and a specific phenotype in cmt b and f patients, characterized by involvement of the lower limbs and selective sparing of the upper ones, which may help in addressing the diagnostic algorithm. non-freezing cold injury (nfci) develops following sustained exposure to cold temperatures, resulting in tissue cooling but not freezing. this can result in persistent sensory disturbance of the hands and feet including numbness, paraesthesia and chronic pain. both vascular and neurological aetiologies of this pain have been suggested but remain unproven. we prospectively approached patients referred for clinical assessment of chronic pain following non-freezing cold injury between february and november . of patients approached consented to undergo detailed neurological evaluations including: questionnaires to detail pain location and characteristics, structured neurological examination, quantitative sensory testing, nerve conduction studies and skin biopsy for intra-epidermal fibre assessment. of the study participants all had experienced nfci whilst serving in the united kingdom armed services and the majority were of african descent ( . %) and male ( . %). many patients reported multiple exposures to cold. the median time between initial injury and referral was . years. pain was principally localised to the hands and the feet, neuropathic in nature and in all study participants associated with cold hypersensitivity. clinical examination and quantitative sensory testing were consistent with a sensory neuropathy. in all cases large fibre nerve conduction studies were normal. the intra-epidermal nerve fibre density, however, was markedly reduced; ⋅ % of subjects having a count at or below the ⋅ centile of published normative controls. using the neuropathic pain special interest group (neupsig) of the international association for the study of pain (iasp) grading for neuropathic pain % had probable and ⋅ % definite neuropathic pain. chronic non-freezing cold injury is a disabling neuropathic pain disorder due to a sensory neuropathy. why some individuals develop an acute painful sensory neuropathy on sustained cold exposure is not yet known but individuals of african descent appear vulnerable. screening tools, such as the dn questionnaire, and treatment algorithms for neuropathic pain should now be used in the management of these patients. funded by the wellcome trust and the uk ministry of defence guillain-barré syndrome (gbs) is highly heterogeneous regarding clinical presentation, course, electrophysiological subtype and outcome. in part this variety is associated with differences between geographical regions, although this had not been investigated in a single comparative study. one aim of igos is to define the influence of the geographical origin on the heterogeneity of gbs. in igos all gbs patients within weeks of onset can participate, independent of age, variant, severity and treatment. in february , patients were included from countries. the first inclusions in igos were used in this analysis. seventy-five patients ( %) were excluded because of alternative diagnoses (n= , including a-cidp), protocol violations (n= ) or missing data (n= ). of the remaining patients, % were males and % female with a median age of years (iqr - ). at entry % presented with tetraparesis and % with paraparesis. during follow-up % needed mechanical ventilation and % died. of all gbs patients % received treatment ( % ivig, % pe). the remaining % received supportive care only (mild gbs or low social-economic status). antecedent events were reported in % of patients, including upper respiratory tract infection ( %) and gastro-enteritis ( %) as the most frequent events. the pure motor form was the predominant subtype in patients from bangladesh ( %). in europe/americas and asia (without bangladesh) the predominant subtype was the sensorimotor form ( % in europe/americas and % in asia). in asia (without bangladesh) there was a relatively larger proportion of patients with mfs/mfs-overlap syndrome ( %) than in other regions ( - %, p< . ). the proportion of patients able to walk unaided at months after follow-up was % in europe/americas, % in bangladesh and % in asia (without bangladesh). kaplan-meier analysis comparing electrophysiological subtypes of gbs (as reported by neurologist) showed that patients with inexcitable nerves or axonal neuropathy needed more time to regain the ability to walk unaided than the patients with demyelinating or equivocal result (log-rank test, p < . ). these findings demonstrate the extensive geographical differences in gbs. future igos studies will investigate the role of genetic and environmental factors that additionally could explain these differences. van den bergh pyk , attarian s , grapperon am , nicolas g , cassereau j , rajabally ya , delmont e , woodard jl , piéret f and the university of louvain gbs electrodiagnosis study group * . corticosteroids are considered as one of the first line treatments for chronic inflammatory demyelinating polyneuropathy (cidp). different types of corticosteroids are used and there are no comparative studies assessing the improvement rates, remission rates, tolerability and the side effect profiles of these treatment regimens. in addition, there are currently no reliable predictors of favorable treatment response to steroids, which would greatly ease the choice of first line treatment. in this retrospective study we will compare efficacy, tolerability and safety of three different corticosteroid regimens used as first line treatment in three large cidp centers in netherlands, serbia and italy. treatment naïve cidp patients who received either pulsed dexamethasone, pulsed methylprednisolone or daily prednisone will be included in the study. data will be extracted from patient charts. the primary outcome is the percentage of treatment responders at months after start of first treatment, in which treatment response is defined as subjects who improved after treatment and are either without treatment after six months or are still being treated with the first chosen therapy. secondary endpoints include the remission rates and in case of a relapse, the mean duration of remission to relapse; the discontinuation rate within months of treatment due to inefficacy, adverse events or intolerance; and the frequency of adverse events and serious adverse events (sae) during treatment or within month after stopping treatment. furthermore, we will explore the value of previously reported potential predictors of treatment response. results will be presented at the peripheral nerve society meeting . van lieverloo g , , musters a , adrichem m , esveldt r , doorenspleet m , klarenbeek p , van schaik i , de vries n , eftimov f . academic medical center, department of neurology, amsterdam, the netherlands; academic medical center/university of amsterdam, department of clinical rheumatology and immunology, amsterdam, the netherlands. following reports that pathogenic antibodies are present in a minority of patients with chronic demyelinating inflammatory neuropathy (cidp), we studied whether oligoclonal expansions of b-cell clones are present in patients with cidp. recently, we developed a new method for b-cell receptor (bcr) repertoire landscaping based on high throughput sequencing (hts) of rna extracted from blood. bcr repertoire was analyzed in patients with cidp: patients with active disease and starting treatment (group ), patients with stable disease using intravenous immunoglobulin (ivig) treatment in which treatment withdrawal was attempted (group ), and patients in remission (i.e. no treatment in the last months, group ). clinical parameters and sampling was performed at baseline (group , and ), at months after start of treatment (group ), at months or earlier in case of relapse in group and at baseline only in group . most cidp patients had highly expanded bcr clones, regardless of disease activity and response to treatment. however, in group , the most expanded b-cell clones at baseline showed no overlap with the expanded bcr clones after improvement. based on these preliminary data expanded bcr clones are observed in the peripheral blood of most cidp patients, regardless of disease activity (active, stable disease or remission/cure). functional characterization of these expanded clones remains to be performed. van rijs w , fokkink wjr , tio-gillen ap , brem md , jacobs bc , huizinga r . erasmus mc, university medical centre, rotterdam, the netherlands. myeloid cells, including monocytes, macrophages and dendritic cells, are critically involved in the induction of adaptive immune responses, clearance of pathogens and in the initiation of tissue repair. in the guillain-barré syndrome (gbs), macrophages are present in the peripheral nerve, where they phagocytose (damaged) myelin and axons. dendritic cells (dc) are increased in the cerebrospinal fluid of patients with gbs. however, the composition and phenotype of monocytes and dc subsets in the peripheral blood is unclear and it is unknown if these cells can be used as biomarker to monitor disease activity or response to treatment. here we investigated the frequency and phenotype of six myeloid subsets in the peripheral blood mononuclear cells (pbmc) using advanced -color flow cytometry. pbmc were isolated from patients with gbs, before and after immunomodulatory treatment, and age and gender-matched, healthy controls. the frequency of total monocytes, determined as percentage of cd + cells, was increased in gbs patients compared to controls (p< . ). the monocyte population was skewed towards more intermediate (cd +cd +; p< . ) and less non-classical (cd -cd +; p< . ) monocytes. classical (cd +cd -) and intermediate monocytes as well as cd c+ dc expressed significantly higher levels of cd compared to healthy controls. in contrast, the expression of cd and siglec- was significantly higher in the non-classical monocytes of gbs patients compared to controls. no differences were observed in the expression of cd , cd , cd and siglec- . immunomodulatory treatment strongly reduced the frequency of non-classical monocytes and all dc populations in cd + pbmc. the expression of cd , cd c and hla-dr was reduced in classical monocytes after treatment. in addition, siglec- expression was reduced in several monocyte and dc populations after treatment. in summary, our data identify significant changes in the monocyte compartment in gbs. the decrease in non-classical monocytes may suggest that these cells have migrated to peripheral tissues, promoting the differentiation of classical monocytes into intermediate monocytes. further analysis should reveal whether these changes are related to preceding infections, disease severity and response to treatment. in mme. the two mutations were absent from control databases (e.g. exac), affected highly conserved aminoacids and were predicted to have deleterious effects by in silico analysis. unfortunately, both parents were deceased and we were therefore unable to prove that the two mutations reside on separate alleles. mme encodes the metalloprotease neprilysin whose role in peripheral nervous system is still unclear. higuchi and colleagues have described japanese cmt families with late-onset sensory-motor axonal neuropathy and recessive loss-of-function mutations in mme. we report two novel missense mutations in mme in a case of late-onset cmt . we hypothesize an autosomal-recessive mode of inheritance as most likely given the clinical phenotype and the absence of a family history. anti-contactin- (cntn ) antibodies were recently identified in a subgroup of patients with chronic inflammatory demyelinating polyneuropathy (cidp) showing acute/subacute onset of severe sensory-motor neuropathy and poor response to intravenous immunoglobulin (ivig) and corticosteroids. these antibodies belong to the igg isotype and interact with cntn -neurofascin (nf ) complex at paranodes leading to loss of nodal integrity. a -year-old man presented with acute onset of distal weakness in the lower limbs, four limbs paraesthesias and sensory ataxia. at clinical examination ankle swelling was also observed. nerve conduction study showed a demyelinating polyneuropathy and cerebrospinal-fluid examination revealed cyto-albuminologic dissociation. sural nerve biopsy disclosed diffuse loss of myelinated fibres. at routine blood test serum albumin was reduced and proteinuria was gr/ hours, thus leading to the diagnosis of nephrotic syndrome. kidney biopsy showed changes consistent with membranous glomerulonephritis, together with sub-epithelial deposits of immune complexes and complement deposition. treatment with ivig and corticosteroids did not improve neurological status, while membranous glomerulonephritis showed moderate response to ivig. a six-month course of cyclophosphamide was started leading to normalization of renal function and muscle strength and partial improvement of sensory ataxia. the patient did not require any further treatment and after years his condition remains stable. cntn antibodies were tested on a recently collected patient's serum and resulted positive on both elisa and cell-based assay. the patient here reported showed the typical clinical features of anti cntn -associated cidp including older age, acute onset, severe motor impairment and sensory ataxia. the contemporary occurrence of membranous glomerulonephritis was reported in only one other case. contactin- is expressed at low levels in the kidney and a direct damage of anti-cntn antibodies could be hypothesized. alternatively, renal damage might have been secondary to unspecific immune complexes deposition. a good response to anti-cd rituximab was recently reported in patients with cidp associated with anti-cntn and anti-nf antibodies. notwithstanding this single-case observation, our report suggests that also cyclophosphamide may be considered an effective therapy in anti-cntn antibodies-associated cidp and membranous glomerulonephritis, leading to persistent clinical remission. velasco r , , besora s , santos c , sala r , izquierdo c , simó m , gil-gil m , , jiménez l , pardo b , calvo m , palmero r , clapés v , bruna j duloxetine is the only agent demonstrated effective in treating pain related with chemotherapy-induced peripheral neuropathy (cipn). patients with symptomatic cipn treated with duloxetine were retrospectively collected in a single-institution. aim of the study was to evaluate the drug's efficacy and rate of compliance. only patients with cipn with distressing positive symptoms (pain, numbness and/or paresthesia), and non-progressive disease were included. cipn was graded employing the total neuropathy score (tns © ) and national cancer institute-common toxicity criteria. response to duloxetine was assessed with patient global impression of change (pigc) scale ( : no benefit; : excellent response). consecutive first one-hundred cipn patients treated with duloxetine were analyzed. median age was ( - ). , , and received platinum, taxane, bortezomib and vincristine-based regimen, respectively. median tnsc © was ( - ). severity of neuropathy was grade ( %), grade ( %), and grade ( %). sixteen patients were on treatment with other analgesic agents. median time from finishing chemotherapy to duloxetine initiation was months [ - ]. median pigc score was [ - ]. among responders, . % and . % scored low ( - ) and high ( - ) benefit, respectively. fifty-seven ( %) patients discontinued early duloxetine due to intolerable side effects (n= ) the goal of this study was to determine whether predicted fork stalling and template switching (fostes) during mitosis deletes exon in peripheral myelin protein kd (pmp ) and causes a gain-of-function mutation associated with peripheral neuropathy in a family with charcot marie tooth disease type e. two siblings previously reported to have genomic re-arrangements predicted to involve exon of pmp were evaluated clinically and by electrophysiology. skin biopsies from the proband were studied by rt-pcr to determine the effects of the exon re-arrangements on exon mrna expression in myelinating schwann cells. transient transfection studies with wild type and mutant pmp were performed in cos and rt cells to determine the fate of the resultant mutant protein. both affected siblings had a length-dependent demyelinating neuropathy with severely slow nerve conduction velocities (< m/sec). rt-pcr studies of schwann cell rna from one of the siblings demonstrated a complete in frame deletion of pmp exon (pmp delta ). transfection studies demonstrated that pmp delta protein is retained within the endoplasmic reticulum and not transported to the plasma membrane. our results confirm that that fostes mediated genomic rearrangement produced a deletion of exon of pmp , resulting in expression of both pmp mrna and protein lacking this sequence. in addition, we provide direct experimental evidence for endoplasmic reticulum retention of the mutant protein suggesting a gain-of-function mutational mechanism consistent with the observed cmt e in this family. pmp delta is another example of a mutated myelin protein that is misfolded and thus likely to contribute to the pathogenesis of the neuropathy. in poems and cidp, distal limb nerve conduction studies are limited in identifying demyelination and detecting treatment effects in severely affected patients. blink reflex r latency may help to not only identify demyelination but also provide a meaningful treatment outcome measure especially in severely affected patients. poems and cidp patients having undergone routine nerve conductions and blink reflex testing were identified. correlation between r latency, limb nerve conduction studies and neuropathy impairment scores (nis) was calculated with treatment. blink reflexes were performed in patients ( poems, cidp; nis range: - points). overall, r latency prolongation occurred in . % of patients ( . % poems, . % cidp). patients with r prolongation (> ms) had more severely affected nerve conductions in both poems (ulnar cmap . mv vs . mv, p= . ) and cidp (ulnar cmap . mv vs . mv, p< . ). r latency correlated with nis severity in poems better than cidp (r = . vs . , p=< . vs. . ). follow-up nis and r latency evaluations after treatment were available in patients ( poems, cidp). the r latency changes were concordant with the nis changes in % of poems and % of cidp patients. in severely affected patients [ulnar cmap amplitude ≤ . mv ( . %: / )] except for one, all had prolonged r (> ms), allowing for treatment follow up and initial diagnosis. blink reflex r latencies are valuable in defining demyelination in severely affected poems and cidp patients, but also provide a sensitive, early treatment outcome measure among those same severely affected patients. watson dj , martinez c , wallenhorst c , hubsch a , shebl a , simon tl . csl behring llc, king of prussia, usa; institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl behring ag, bern, switzerland; csl behring gmbh, marburg, germany. chronic inflammatory demyelinating polyneuropathy (cidp) rarely occurs in children. clinical trials in pediatric patients have not been performed and there are little data on therapy with intravenous immunoglobulin (ivig). we performed an observational trial to investigate the risk of adverse events of special interest (aesi) with ivig, i.e. hemolytic anemia, aseptic meningitis, acute renal failure, thromboembolic events and anaphylactic reactions. the study cohort was derived from the us premier perspective database and consisted of patients < years with a diagnosis of cidp (icd- cm diagnosis code . ) treated with the ivig privigen ® (csl behring, bern switzerland) between jan and dec . we identified pediatric cidp patients: preschool children (age - years at first treatment for cidp), children ( - years) and adolescents ( - years); females and males; white, one black and two allocated to "other race". six patients had a history of other ivig use for guillain-barré syndrome, one patient for myasthenia gravis and one for immunodeficiency before the diagnosis of cidp. the mean privigen ® dose calculated from the cumulative quantity of privigen ® per treatment episode and the corresponding age-and gender specific median of the us population body weight estimate was . g/kg body weight. the number of recorded treatment episodes per patient ranged from to . using an at-risk period of days for hemolytic anemia, and days for other aesi after each privigen ® administration, no aesi were observed in the patients with cidp with a total of person-days at-risk for ha and person-days at-risk for other aesi. this observational study shows that ivig (privigen ® ) is used for treatment of cidp in pediatric patients with or without concomitant conditions and revealed no particular safety issues in this patient group. hsn- is a peripheral neuropathy most frequently caused by missense mutations in the sptlc or sptlc genes, which code for two subunits of the enzyme serine palmitoyltransferase (spt). spt catalyzes the first and rate limiting step of de novo sphingolipid synthesis. it has been shown that mutations in spt cause a change in enzyme substrate specificity which results in the production of two atypical sphinganines, deoxysphinganine (dsp) and deoxymethylsphinganine (dmsp), rather than the normal enzyme product, sphinganine (sp). levels of deoxysphingolipids are elevated in the blood of hsn- patients and this has been shown to cause the peripheral nerve damage characteristic of the disease, which affects both sensory as well as motor axons. however, the underlying pathomechanism of how deoxysphingolipids damage neurons remains elusive. here, dsp and dmsp-mediated neurotoxicity was examined in primary mouse motor and sensory neurons, by assessing cell survival and neurite outgrowth following exposure to different concentrations of sp, dsp or dmsp. the abnormal enzyme products were found to have a rapid and dose-dependent neurotoxic effect in primary neurons. we also explored the potential mechanisms that underlie deoxysphingolipid neurotoxicity, by characterizing mitochondrial function and changes in calcium handling. we found that mitochondrial dysfunction and calcium handling deficits may be key mediators of abnormal sphingolipid neurotoxicity, in both motor and sensory cell models. specifically, we revealed mitochondrial abnormalities, signs of endoplasmic reticulum stress and dysfunction of store-operated calcium channels. we propose that early deficits in mitochondria and calcium handling may underlie deoxysphingolipid neurotoxicity and thus present potential therapeutic targets for hsn- . months. one patient was excluded because the decrease in hba c was not contemporaneous with weight loss. the mean and median decrease in bmi per month was . and . respectively. the mean and median interval between surgery and decrease in hba c was . and . days respectively. records of these patients were scrutinized and classified as: 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or temporal relationship to decrease in hba c is uncertain; 'unlikely tind': when an alternative explanation exists for symptoms. only one patient was classified as 'possible tind': a -year-old woman who developed neuropathic pain in both lower limbs month after a rapid hba c decline of . % and about months after bariatric surgery. she had no documented autonomic symptoms. our study is limited by small cohort size, retrospective design and reliance on hospital records. nonetheless, it demonstrates that besides nutritional neuropathies, tind should also be considered in dm patients who develop peripheral neuropathy after bariatric surgery. in another study, we found tind is uncommon in a general cohort of diabetic patients. the occurrence of 'possible tind' in only dm patients corroborates earlier data that weight loss may act in tandem to increase the risk of tind. woolums bm , tabuchi m , sung h , sullivan jm , mamah c , yang m , blum id , wu mn , sumner cj , lloyd te . johns hopkins university, baltimore, usa. dominant missense mutations in the gene encoding the cation channel transient receptor vanilloid, family member (trpv ) cause inherited neuropathies including charcot-marie-tooth disease c (cmt c). in vitro, mutations in trpv that cause cmt c cause a gain of trpv channel function and increased intracellular calcium which subsequently leads to cellular toxicity. however, the mechanisms by which cmt c mutations in trpv lead to neuronal dysfunction in vivo remain poorly understood. we generated transgenic drosophila that express either wild-type or a cmt c causing trpv mutant (trpv r c ) to assess the effect of trpv r c on neuron function in vivo. selective expression of trpv r c in drosophila ccap neurons (n ccap ) results in a failure of drosophila wing expansion that is blocked by genetically inactivating the channel pore, demonstrating the requirement of channel function in mediating this phenotype. perforated patch clamp analysis of n ccap reveals that trpv r c causes a calcium-dependent increase in n ccap neuronal excitability. this hyperexcitability is restored to control levels by application of a trpv selective antagonist. high level expression of trpv r c causes synaptic and dendritic degeneration, both of which are rescued genetically by inactivating the channel pore or pharmacologically by feeding larvae a trpv selective antagonist. we conducted a genetic screen in n ccap and found that camkii knockdown potently suppresses the trpv r c mediated wing expansion phenotype and selectively rescues degeneration of synapses but not dendrites. we also find that trpv r c , but not controls, disrupts mitochondrial transport in axons by increasing the number of stationary mitochondria. our data demonstrate that trpv r c elevates neuronal intracellular calcium which disrupts mitochondrial transport and mediates neurodegeneration through compartment-specific calcium-mediated signaling pathways, and supports the further investigation of trpv antagonists as potential therapeutics for the treatment of cmt c. mood disorders, including anxiety and depression, are commonly observed among chronic pain patients with prevalence estimates ranging from to %. comorbidity between mood and chronic pain disorders has been linked to altered limbic regulation of the hypothalamic-pituitary-adrenal (hpa) axis. stress activates the hpa axis and can initiate and/or exacerbate symptoms related to both chronic pain and mood disorders. previous studies from our laboratory have investigated the influence of early life stress on mechanical pain hypersensitivity, visceral hypersensitivity and behavioral evidence of mood disorder later in life. here, we are testing the hypothesis that chronic stress exposure in adulthood can increase somatic and visceral sensitivity and anhedonic behaviors in a mouse strain with a genetic predisposition to anxiety. adult, female a/j mice were exposed to repeated foot shock stress for continuous days and tested for alterations in mechanical sensitivity, sucrose preference, visceromotor response (vmr) during urinary bladder distension, and serum corticosterone levels. mice that underwent shock stress had a significantly decreased mechanical withdrawal threshold in the hind paw compared to their baseline and sham group measurements. sucrose preference was measured prior to shock exposure and throughout the shock paradigm as an indicator of anhedonic behavior. mice that underwent shock stress displayed a trend toward decreased sucrose preference, indicating anhedonia, in comparison to mice in the sham group that did not display anhedonia. mice that underwent shock stress displayed significant increases in vmr during bladder distension compared to sham mice. finally, serum corticosterone levels were significantly higher in the mice that underwent shock stress compared to sham mice, indicating a stress-induced increase in hpa axis output. together these data suggest that chronic stress exposure can induce mechanical allodynia, visceral hypersensitivity, and depression-like behaviors in an anxiety-prone mouse strain. future studies will incorporate gene expression in the hypothalamus, amygdala, and hippocampus, as well as investigation of possible downstream peripheral neuroimmune modulation and neuronal morphology changes. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated neuropathy. as prognostic markers of gbs, modified erasmus gbs outcome score (megos), erasmus gbs respiratory insufficiency score (egris), and Δigg have been reported. however, the proportions of subtypes of gbs are known to be different between the western countries and japan, it remains to be elucidated whether those markers can also be applied to gbs in japan or not. we here investigated retrospectively gbs patients and determined the megos and the egris of those cases. among them, Δigg could be obtained in cases. we evaluated the prognosis using gbs outcome score (functional grade: fg) at months; we called good prognosis when fg at months was less than and poor prognosis when that was or more. as a result, in cases with higher score than at megos on admission cases ( %) had poor prognosis and in cases with higher score than at megos at day of admission cases had poor prognosis. in cases with higher score than on egris cases ( %) needed the mechanical ventilator. patients with good prognosis had higher Δigg(average: mg/dl) than patients with poor prognosis(average: mg/dl). we calculated the cut-off value of Δigg in patients, which was mg/dl. patients with higher Δigg than mg/dl could significantly walk independently at six months (p< . ). patients ( %) had poor prognosis in patients with lower Δigg than mg/dl. ( %) of patients were treated with the single cycle of intravenous immunoglobulin (ivig). other patients ( %) were treated with the combined therapies, such as intra venous methylprednisolone and/or plasmapheresis or the second cycle of ivig in addition to ivig. although there was no difference in prognosis between patients with the single cycle of intravenous immunoglobulin (ivig) and patients with the combined therapies, in the patients who had fg> and megos > on admission, the combined therapies made better prognosis than the single course of ivig (p< . ). we found that megos, egris and Δigg were also available in japan. the efficacy of the combined therapies in severe gbs patients should be investigated in the future large scale prospective studies. yiu em , , , burns j , , , menezes mp , , ryan mm , , and for the paediatric cmt best practice guidelines consortium. royal children's hospital melbourne, melbourne, victoria, australia; murdoch childrens research institute, melbourne, victoria, australia; university of melbourne, melbourne, victoria, australia; university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia. charcot-marie-tooth disease (cmt) often presents during childhood. common symptoms include weakness, limb pain and cramps, foot deformity, and balance impairment. guidelines for the optimal management of common problems experienced by children with cmt do not currently exist. development of these guidelines will provide an evidence base for the management and monitoring of children with cmt. a series of systematic literature reviews utilising the grade (grades of recommendation, assessment, development, and evaluation) approach were conducted to answer pre-specified key clinical questions related to the management of paediatric cmt. these included treatment recommendations for symptoms such as weakness, pain, balance impairment, joint deformity, and impaired upper limb function, and anticipatory monitoring for associated complications such as hip dysplasia. this yielded minimal to no evidence for the pre-specified clinical questions, and evidence-based management recommendations could not be made. consensus-based statements will therefore be formulated via a three-round delphi process, to be conducted in . the delphi panel will consist of local and international medical and allied health professionals who have experience in the management of children with cmt. efficacy of pxt in the treatment of adult patients cmt a (n= ) was shown in a multicenter, randomized, double-blind, placebo-controlled phase ii study (attarian et al. ) . pxt taken x/day, orally, for consecutive months was well tolerated and safe. significant improvement of disability was observed for the highest tested dose, thought indicative for an early, meaningful change in disease course (meta-analysis by mandel et al., ) . this formed the rationale to initiate a multicenter, randomized, double-blind, placebo-controlled pivotal phase iii study (clin-icaltrials.gov: nct ) of pxt in mildly to moderately affected cmt a patients in december . the primary objective is to assess the efficacy of doses of pxt compared to placebo on disability as measured by the mean change from baseline overall neurology limitations scale (onls) score at month and . furthermore, efficacy on the proportion of responders (i.e. improvement of onls), impairment (cmtns-v ), functional tests ( -mwt, qmt, -hpt), electrophysiological parameters (cmap, snap and ncv) and quality of life (eq- d, vas) are secondary endpoints. pursuant this study, patients will be eligible for a -month extension study, in which pxt assigned patients will continue with the previously assigned dose, whereas placebo patients will be randomized to one of the two pxt doses. the study is conducted in investigational sites in countries (eu, canada and us). in december patient randomization was completed (n= ). the screen failure rate was %, as expected ( patients were screened). the independent dsmb recommended to continue the study as planned following a safety analysis on the first patients treated for > months. preliminary baseline characteristics are based on patients (data not cleaned). the study population had a mean age of . ± . years (range - ; male . %) of which . % had a confirmed genetic diagnosis of cmt a. the mean cmtns-v was . ± . and the mean motor nerve conduction of the ulnar nerve was . ± . m/s. ten patients withdrew from the study, due to adverse events unrelated to study treatment. the last patient completing the study is expected in march . zhang g , ghosh p , lin j , ghauri a , sheikh ka . department of neurology, university of texas health science center at houston, houston, tx, usa. assessment of epidermal nerve fiber density and its structure integrity is critical for the diagnosis and evaluating the effectiveness of potential therapies in small fiber neuropathies. currently, skin biopsies, at multiple sites, are most commonly used to assess these diseases. these studies are expensive and time consuming due to cumbersome processing and quantification techniques and serial biopsies over time are often not feasible due to costs and patient acceptance. moreover, vast majority of normative data for skin biopsies in humans are available only for few distal sites and a significant proportion of patients with small fiber neuropathies have focal or regional symptoms not involving the commonly biopsied sites in the leg. live imaging could overcome these limitations and provide a noninvasive real time assessment of epidermal nerve fibers all over the body. we previously found that anti-ganglioside antibody (aga) is an effective neuronal delivery vector for transport of various cargos, such as fluorescent dyes, to peripheral nerves. in the current proof of concept study, we examined whether non-invasive multiphoton microscopy can be used to probe/image the epidermal nerve fibers in living animals after systemic and/or local delivery of fluorescently conjugated aga. we found that the individual nerve endings in skin and cornea are distinctly labeled, and visualized under two-photon microscope. the epidermal nerve fiber labelling by fluorescent-tagged aga was further validated using transgenic mice selectively expressing yellow fluorescent protein in their nervous system. in-vivo multiphoton imaging provide a tool with potential for dynamic longitudinal evaluation of small fiber neuropathies, including nerve degeneration and regeneration, without tissue removal. thus, the use of multiphoton microscopy in conjunction with fluorescently labeled aga as neuronal vector can have many research and clinical applications, such as labeling and live visualization of epidermal nerve fibers to assess small sensory nerve fibers in health and disease. zhou y , tavori h , lee s , al salihi m , fazio s , notterpek l . mcknight brain institute, university of florida, gainesville, florida, usa; knight cardiovascular institute, oregon health and science university, portland, oregon, usa. the majority of hereditary neuropathies are due to abnormalities in peripheral myelin protein (pmp ), whose genetic variants include increased expression (gene duplication), haploinsufficiency (gene deletion), or point mutations. phenotypic heterogeneity in clinical presentation is common among hereditary neuropathy patients even within the same family, the cause of which has not been determined. to investigate the role of pmp in the pathogenesis of the neuropathies we have generated and characterized pmp null (pmp −/− ) mice (amici et al., ) whose peripheral nerves show alterations in lipid metabolism (lee et al., ) . to examine the molecular changes underlying these abnormalities we determined the expression of cholesterol synthesis (srebp pathway), and cholesterol uptake, transport and efflux genes (lxr pathway). in affected nerves, we found the cholesterol synthesis pathway inhibited, while the lxr pathway, and particularly apoe and abca , upregulated at the mrna and protein levels. since pmp is expressed at low levels in the liver, the central organ for the regulation of cholesterol in the body, we studied liver tissue form pmp −/− mice. liver from pmp −/− mice showed significant hepatomegaly, clear features of microvesicular steatosis, as well as marked increase in lxr pathway genes and proteins (abca and apoe), as compared to wt. ultrastructural studies identified lipid droplets and significantly enlarged mitochondria in the liver of male pmp −/− mice, which is not due to mitochondria fusion, as the levels of mfn and remained similar to wt. as disturbed hepatic cholesterol homeostasis induces the activation of kupffer and stellate cells, we determined the extent of inflammation in nerve and liver tissues from pmp −/− mice with leukocyte (cd b) and macrophage markers (cd ). in nerve sections, we detected an increase in the number of cd b+ cells, which was confirmed by western blots. in the liver of pmp −/− mice we found a significant increase in cd -reactive kupffer cells and elevated levels of tnf-alpha. the severe dysregulation of cholesterol metabolism in nerve and liver, including neuroand hepatic inflammation in the absence of pmp − suggest that dysregulated cholesterol metabolism and inflammation may act as a disease modifier in pmp -dependent neuropathies. our aim was to ascertain frequency of and risk factors for tee, arterial (ate) and venous thromboembolic events (vte) in neuromuscular patients receiving regular ivig. we performed a retrospective case-note review of inflammatory neuropathy patients receiving regular ivig treatment. we collected the following data over a month study period this analysis suggests tee incidence is higher in ivig treated patients than comparable population-based rates. examination of tee occurrence in age and vascular risk factor matched ivig-treated and ivig-naïve individuals is required to appreciate the excess risk associated with ivig treatment. référence des maladies neuromusculaires et la sla, hôpital de la timone ha) and whether temporal dispersion (td) parameters are helpful. fifty-eight patients diagnosed according to asbury and cornblath ( ) were prospectively included between january and september . edx and were performed - (mean= ) and - days (mean= ) after disease onset, respectively. there were no differences in classification consistency between ho and ha at edx (p= . ) and edx (p= . ), but more patients were classified as aman when comparing ra with ho and ha at edx (p< . ). at edx , ra classified more patients as equivocal with ho (p< . ) and as aman with ha (p< . ) ) or edx (ho, fe p= ; ra, fe p= . ; ra with td, fe p= . ). gm , gd a, gd b and gq b igg antibodies were tested (willison at edx , only ho showed maybe more antibodies with aman compared to aidp (fe p= . , phi=− . ) and with aman compared to equivocal cases since correlation with factors associated with axonal gbs, in casu rcf and antibodies, is far from exclusive, the usefulness of edx subtype classification using specific criteria sets, remains doubtful. the frequency of rcf indicates that nodal/paranodal alterations may represent the main pathophysiology in more gbs patients than currently thought ruiz m , lessi f , cacciavillani m , riva m , salvalaggio a , campagnolo m , briani c . neurology, department of neuroscience, university of padua, padua, italy; hematology and clinical immunology unit, department of medicine, university of padua, padua, italy; cemes, data medica group, padua, italy.arsenic trioxide (ato) is highly effective in treatment of acute promyelocytic leukemia (apl). it is licensed in italy for treatment of relapsed-refractory apl and for first line chemotherapy in low risk patients. ato most frequent side effects are well described, but less is known on ato induced. we describe apl patients who were treated with all-trans retinoic acid (atra)/ato as first line therapy. the characteristics of ato induced neuropathy was prospectically analyzed by neurological evaluation using both the total neuropathy score, clinical version (tnsc) (a validated scale for chemotherapy induced peripheral neuropathy) and neurophysiological assessment. patients have been evaluated at baseline, at the end of the induction phase, at the end of ato/atra treatment and year after discontinuation of treatment. baseline neurophysiology was performed at the end of induction phase. both patients were men, respectively and -yr-old. none of the patients had previous history of neuropathy. baseline tnsc was (no clinical signs of neuropathy) in both patients. neurophysiological evaluation performed after the end of induction cycle did not reveal signs of peripheral neuropathy in both patients. patient received mg of ato during induction, total , mg. patient received mg of ato during induction, total , . both patients developed leg numbness during consolidation cycles and patient also hand numbness. tnsc at the end of therapy was in patient and in patient . neurophysiology at the end of therapy detected signs of sensitive axonal neuropathy in both patients. they received full doses of ato consolidation ( . mg/kg/day for days/week, on alternate months for total months and tretinoin weeks on weeks off). during the first year of follow-up both tnsc and neurophysiology year after the end of consolidation cycle were consistent with full recovery. our patients developed sensory axonal neuropathy during ato therapy, that clinically manifested during consolidation cycles and improved up to complete recovery during follow-up. published case reports show that outcomes may not be as good as in our patients. a multicenter prospective study evaluating the characteristics of ato-induced neuropathy in apl is ongoing. ruiz m , campagnolo m , salvalaggio a , cacciavillani m , taioli f , fabrizi gm , briani c . department of neuroscience, neurology unit, university of padua, padua, italy; data medica group, emg unit, cemes, padua, italy;mutations in the mitochondrial copper-binding protein sco , cytochrome c oxidase assembly protein, have been reported in several cases of fatal infantile cardioencephalomyopathy with cox deficiency. we identified compound heterozygous variants in sco in two unrelated patients with isolated length dependent axonal sensorimotor polyneuropathy of variable clinical severity (axonal autosomal recessive charcot-marie-tooth disease type , cmt ) by whole exome sequencing. although peripheral neuropathy has been described as a secondary feature in a few cases of fatal infantile cardioencephalomyopathy, the disease onset, clinical phenotype and survival in our patients differ significantly from the previously described cases. our patients developed predominantly motor neuropathy; moreover, they are still alive and they have not developed cardiomyopathy, which is the main phenotype and cause of death at early infancy in reported patients. both of our patients harbor mutations adjacent or near the conserved copper-binding motif (cxxxc), including the common reported pathogenic variant e k and the novel change d g. in addition, each patient carries a second mutation located in the same loop region of the protein, p t and r q. western blots from fibroblasts from these cmt patients showed reduced levels of cox , a subunit of complex iv, indicating cox deficiency. our findings demonstrate that cmt can be the predominant phenotype associated with sco mutations, pointing to a broader phenotypic heterogeneity. the mechanism linking mitochondrial respiratory chain dysfunction to isolated axonal loss of variable severity remains to be elucidated. the muscarinic acetylcholine (ach) type receptor (m r) is a metabotropic g protein-coupled receptor expressed by adult sensory neurons. cholinergic signaling through muscarinic receptors can modulate axonal plasticity in invertebrates and lower vertebrates. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) m r antagonists elevate neurite outgrowth and protect from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt, et al., ) . furthermore, we demonstrated that excessive cholinergic signaling due to m r overexpression caused a significant reduction in neurite outgrowth (calcutt, et al., ) . the mechanism of m r-antagonist driven neurite outgrowth remains poorly understood, however, we have proposed that ach constrains axonal outgrowth via m r activation. cholinergic signaling is mediated via recruitment of trimeric g proteins, of which g alpha- and g alpha- regulate cytoskeleton dynamics by control of tubulin polymerization. activated gtp-bound g-alpha proteins destabilize microtubules by increasing the intrinsic gtpase activity of tubulin. we have therefore tested the hypothesis that cholinergic signaling regulates neurite outgrowth via modulation of g protein mobilization and the dynamics of the tubulin cytoskeleton. we found that over-expression of m r in adult sensory neurons induced dissolution of the tubulin cytoskeleton in distal neurites. g alpha- expression in adult sensory neurons was significantly higher (p< . , -fold) than g alpha- . subsequent knockdown the peripheral neuropathy research registry (pnrr) is a multicenter collaborative research project sponsored by the foundation for peripheral neuropathy to advance the science in distal symmetrical polyneuropathies (dsp). the registry was designed to prospectively characterize clinical phenotype and natural history of patients with dsp and obtain biofluids to identify new causes and genetic modifiers of dsp with careful genotype/phenotype correlations, and to develop biomarkers. the enrollment in the registry is still ongoing but an interim analysis was carried at the end of december . eligible study participants were years or older with a diagnosis of idiopathic, diabetic, chemotherapy-or hiv-induced peripheral neuropathy. they were examined by a physician at one of the six consortium members (johns hopkins university school of medicine, icahn school of medicine at mount sinai medical center, beth israel deaconess medical center, northwestern university medical center, university of utah medical center and kansas university medical center). the collected data set included ( ) a detailed questionnaire, discussing their symptoms, medical history and family history, ( ) a standardized neurological examination form, ( ) electrodiagnostic evaluation and ( ) diagnostic laboratory testing. blood samples (whole blood, plasma and serum) were collected for future biomarker and genotyping evaluations. at the end of , complete data sets and blood samples were collected from patients. % had diabetic pn, % had hiv-associated pn, % had chemotherapy induced pn and % were diagnosed with idiopathic pn. detailed analysis of clinical presentation, examination findings and diagnostic investigations will be discussed at the presentation. standardized phenotyping with linked bio-specimen banking will help establish the minimum data set required for neuropathy diagnosis and support genotype-phenotype correlations with next generation sequencing technologies and development of novel biomarkers. pnrr data will improve our understanding of disease mechanisms paving the way for new therapeutic discoveries in painful and non-painful neuropathies. chemotherapy-induced peripheral neuropathy (cipn) is a major side effect of treatment, typically presenting as a sensory neuropathy. symptoms include pain, paraesthesia, and numbness in the extremities, resulting in functional impairment. increasingly, patient reported outcomes (pros) are utilized to accurately examine the impact of cipn symptoms on patient function. however, the links between objective neurological assessment and pro measures remain ill defined. this study aimed to identify links between neurophysiological measures and pros in patients treated with neurotoxic chemotherapy. assessments were conducted in patients (f= , mean age . ± . years) who had completed neurotoxic chemotherapy on average . ± months previously (platinum-based n= , taxane-based n= or taxane/platinum combination therapy n= ). patients reported the presence and severity of neuropathic symptoms via the fact-gog ntx , a validated patient questionnaire. clinical the most important neurological side effect of a large number of anti-cancer drugs is a painful peripheral neuropathy. mainly chemotherapeutics that interfere with microtubules, including the plant derived vinca-alkaloids such as vincristine, are well known to cause chemotherapy-induced peripheral neuropathies (cipn). to date, few treatments are available and focus on symptom alleviation and pain reduction rather than on preventing the neuropathy all together. for the first time, we highlight the potential of specific histone deacetylase (hdac ) inhibitors as a preventive therapy for cipn, using novel rodent models for vincristine-induced peripheral neuropathies (vipn), characterized by a sensory axonopathy. one reason so few therapies are available, is because the exact pathophysiological mechanisms are poorly understood. mounting evidence proposes axonal transport, a pathway frequently disturbed in neurological disorders, as a major player in the pathophysiology of vipn. proper axonal transport requires dynamic microtubules which are highly modulated by post-translational modifications. since vincristine interferes with the polymerization of microtubules, we reason disturbances in microtubule dynamics, and by extension axonal transport, could contribute to vipn. we illustrate that increasing acetylation of -tubulin after hdac -inhibition, can restore vincristine-induced defects in axonal transport in cultured dorsal root ganglion neurons. also in vivo, -tubulin acetylation was restored in the saphenous nerve and dorsal root ganglia, two sensory tissues that are affected by vincristine. ultimately, this correlates to a reduced severity of the neurological symptoms, both on the electrophysiological and on the behavioral level. moreover, we discovered that hdac -inhibition was not only protective against neurotoxicity, but also reduced tumor progression in a mouse model for acute lymphoblastic leukemia. taken together, our results show that hdac -inhibition is an ideal strategy to prevent vipn with beneficial effects both on the neurotoxicity as well as on tumor growth.approximately two-thirds of cidp subjects need long-term corticosteroids or intravenous immunoglobulins (ivig), with ivig being slightly preferred based on safety profiles. subcutaneous ig (scig) is an alternative option for ig delivery but has not previously been investigated in a large-scale clinical trial in cidp.we performed a randomized, double-blind trial in cidp investigating . and . g/kg weekly doses of scig igpro (hizentra ® , csl behring) versus placebo in subjects for maintenance treatment. ivig-dependent adults with definite or probable cidp according to efns/pns criteria were eligible. the primary outcome was the percentage of subjects with a cidp relapse ( -point deterioration on adjusted incat disability score) or who were withdrawn for any other reason during the -week scig-treatment period. multiple secondary endpoints were assessed. superiority of at least one igpro dose over placebo was tested one-sided using the cochran-armitage trend test for the primary outcome and the jonckheere-terpstra tests for secondary outcomes.the primary outcome occurred in % of high-dose scig, % of low-dose scig, and % of placebo subjects (p < . ); cidp relapse occurred in % of high-dose scig, % of low-dose scig and % of placebo subjects (p < . ), respectively. both scig doses were superior to placebo (low-dose vs placebo p = . ; high dose vs placebo p < . ). median incat score, mrc sum score, and grip strength remained stable in scig subjects. high-dose scig prevented the r-ods decline seen with low-dose scig and placebo (p < . ). all placebo subjects deteriorated on measures of strength and disability.causally related adverse events occurred in ( %) subjects ( % placebo, % low dose, and % high dose).scig igpro was efficacious and safe as maintenance treatment. mutations in metalloendopeptidase (mme) gene have been associated with autosomal-recessive late-onset charcot-marie-tooth type- (cmt ). to date, all patients have had at least one truncating mutation, either in homozygosity or in trans with a missense mutation. more recently, loss-of-function and missense heterozygous mutations were also identified in autosomal-dominant cmt. we report the case of a previously healthy caucasian woman, born to healthy unrelated parents, who presented at the age of thirty-nine with numbness and cold sensation in the lower limbs. subsequently she developed progressive gait disturbance and impaired hand dexterity. her homozygous twin presented at the same age with similar symptoms. the family history was otherwise uneventful, in particular neither neuropathy nor dementia were described. neurological examination at the age of fifty-three revealed a steppage gait, distal upper and lower limb atrophy and weakness, distal sensory loss and bilateral pes cavus. deep tendon reflexes were normal in the upper limbs and absent in the lower limbs. nerve conduction studies revealed an axonal sensory and motor neuropathy. a sural nerve biopsy revealed a reduction in myelinated nerve fibers and active axonal degeneration. targeted sanger sequencing of mpz , gjb , gdap , nefl, fkrp, bscl , hspb and mfn were negative. sureselect focused exome sequencing was therefore performed and identified two missense heterozygous mutations [c. g>a,p.c y;c. t>c,p.y h] or lack of efficacy (n= ). most frequently reported adverse events were cognitive ( %), gastrointestinal ( %) and genitourinary ( %). discontinuation due to perception of lack of efficacy was more frequently reported by men ( % vs % p= . ). women presented higher punctuations on pigc scale compared with men ( . ± . vs . ± . , p= . ). pigc scores were significantly higher in patients receiving taxane ( . ± . ) than platinum ( . ± . ) agents (p= . ). no significant differences according severity of neuropathy neither type of chemotherapy were observed in drop-out and retention rates. patients with long-lasting cipn (> months) reported lower pigc scores ( . ± . vs . ± . , p= . ) and higher frequency of suspension due to adverse events ( % vs %, p= . ) and less rate of continuation of duloxetine ( % vs %, p= . ). more than one-third of patients with disturbing cipn discontinued duloxetine prematurely due to intolerable side-effects. low tolerability, male gender and long-lasting cipn may limit duloxetine usefulness in the treatment of symptomatic cipn. verboon c , jacobs bc , and the igos consortium. department of neurology, erasmus medical centre, rotterdam, the netherlands; department of immunology, erasmus medical centre, rotterdam, the netherlands.the efficacy of intravenous immunoglobulin (ivig) in guillain-barré syndrome (gbs) has only been demonstrated in severely affected patients who are unable to walk independently. although there is no proof that ivig is effective in milder forms of gbs, some neurologists are treating these patients with ivig considering that even milder forms of gbs may result in poor recovery, residual deficits, fatigue or pain.we determined the effectiveness of a single course of ivig ( g/kg in - days) in relatively mild forms of gbs in the ongoing observational international gbs outcome study (igos). the gbs disability score, mrc sum score and patient reported outcome measures (prom) were compared at and weeks. ordinal logistic regression analysis was used to determine the effect of ivig on the gbs disability score, adjusted for previously identified prognostic factors.data were analyzed from the first patients enrolled in igos by december , including patients with mild gbs at entry, of which patients ( %) were treated with supportive care, while patients ( %) received ivig (start ivig after onset of symptoms in days, median , iqr - ). at baseline, patients in the ivig treated group compared to the untreated group less frequently had pure motor gbs ( % versus %, p< . ) and axonal damage or unresponsive nerves ( % versus %, p= . ), but a worse gbs disability scores at nadir (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ). at weeks, the median mrc sum scores and prom were not significantly different between treated and untreated patients. however, more patients in the ivig group showed complete recovery of muscle strength at weeks than patients in the control group ( % versus %) (p= . ) and more frequently showed full neurological recovery on the gbs disability scale ( % versus %, p= . ). additional results will be presented at the conference.based on the results of this interim analysis in observational data, we conclude that patients with a relatively mild form of gbs may benefit from a single course of ivig. despite treatment with intravenous immunoglobulin (ivig), many patients with guillain-barré syndrome (gbs) recover insufficiently. we primarily aimed to determine whether a second ivig course ( g/kg in - days) in patients with a poor prognosis improves outcome on the gbs disability scale after weeks. we included patients from the prospective, observational international gbs outcome study (igos) treated with ivig and who had a poor prognosis on the modified erasmus gbs outcome score (megos). of patients enrolled in igos, patients were eligible; patients ( %) were treated with one ivig course (control group); patients ( %) received an 'early' second ivig course ( - weeks after start first course) and patients ( %) a 'late' second ivig course (within - weeks). one week after study entry, patients receiving an 'early' or 'late' second ivig course had significantly worse gbs disability scores and mrc sum scores than controls, implying the need for adjustment of baseline characteristics. the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' group, and . ( % ci . - . ) for the 'late' group, suggesting worse outcomes with a second course of ivig compared to controls. at months, patients ( %) in the 'early' second ivig group, patients ( %) in the control group and only ( %) in the 'late' second ivig group were able to walk unaided (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' second ivig group and only . ( % ci . - . ) for the 'late' second ivig group. in gbs patients with a poor prognosis, we did not find a beneficial effect of a second course of ivig after weeks follow-up. our results suggest that an 'early' administered second ivig course might improve outcome at weeks. given the limitations of this observational study, a randomized controlled trial with a larger number of gbs patients with a poor prognosis being treated early in the disease course is needed to confirm or refute these results. vidal c , bhatt n , agudelo c , mahapatra a , saporta ma . department of neurology, university of miami miller school of medicine, miami, usa.multifocal motor neuropathy (mmn) is an inflammatory demyelinating chronic neuropathy characterized by progressive asymmetric weakness in the distribution of two or more peripheral nerves, without objective sensory loss or upper motor neuron signs. the cardinal neurophysiological finding is conduction block outside the usual sites of nerve compression. supportive clinical criteria include high titers of anti-gm antibodies, good response to ivig, increased cerebrospinal fluid (csf) protein (< g/dl) and magnetic resonance imaging (mri) with diffuse swelling of the brachial plexus. we report a case of a year old woman with a two month history of progressive muscular weakness which began in the right upper extremity, described as difficulty in gripping objects, writing and typing on the computer, followed by progressive lower extremity weakness with difficulty rising from a chair and left foot drop. she also complained of pain in her right trapezius and scapular area. she denied any tingling or numbness. on neurological examination there was right scapular winging and asymmetric weakness predominantly involving the right upper and left lower extremities. reflexes were absent throughout. lumbar puncture revealed albuminocytological dissociation (wbc and protein . g/dl). ncs revealed slow conduction velocities for both ulnar and median nerves and a median nerve conduction block at the forearm segment. sensory nerve action potentials were normal for all tested nerves. needle emg revealed acute denervation of right periscapular muscles. brachial plexus mri showed symmetric bilateral thickening from trunks to peripheral nerves. anti-gm igg/igm were negative. two days after lumbar puncture and the beginning of ivig, patient presented binocular diplopia on extreme left lateral gaze. brain mri with and without contrast showed no intracranial pathologies. after five days of ivig, patient was discharged with significant clinical improvement and recovery of the right scapular winging. diplopia improved a week after discharge. we report an atypical multifocal motor neuropathy case. although this patient fulfills all clinical criteria for mmn, we report some features usually not found in mmn such as scapular winging and a mild and transient left vi nerve palsy. mmn should be included in the differential diagnosis of scapular winging. vlckova e , , raputova j , , srotova i , , sommer c , Üçeyler n , birklein f , rebhorn c , rittner hl , kovalova e , , nekvapilova e , , belobradkova j , olsovsky j , weber p , dusek l , jarkovsky j , bednarik j , . despite many studies addressing biomarkers for pain in diabetic polyneuropathy (dpn), little is known about why it affects only a certain proportion of dpn patients. the somatosensory system plays a key role in the pathophysiology of neuropathic pain (neup) and subgroups with different sensory profiles might respond differently to pain treatment. we aimed to characterize sensory phenotypes of patients with painful and painless diabetic neuropathy and to assess demographic, clinical, metabolic, electrophysiological and psychological parameters related to the presence of neup in a large cohort of well-defined dpn subjects.this observational cross-sectional multi-centre cohort study (performed as part of the ncrnapain eu consortium) of subjects with dpn (non-painful, ndpn, n= ; painful, pdpn n= ) associated with diabetes mellitus of type and (median age years, range - years; women) comprised detailed history taking, neurological examination, laboratory tests, quantitative sensory testing, nerve conduction studies, neuropathy severity scores, and neuropsychological questionnaires. all parameters were analysed with regard to the presence and severity of neup. the presence and severity of neup were positively correlated with severity of neuropathy and thermal hyposensitivity (p< . ). a minority of pdpn patients ( . %) had a sensory profile indicating thermal hypersensitivity; this was associated with less severe neuropathy and better response to pain therapy. the presence of neup was also associated with female gender (p< . ) and with higher cognitive appraisal of pain as assessed by the pain catastrophizing scale (p< . ), while parameters related to diabetes (duration, hba c, microangiopathy) showed no influence on neup presence and severity. this study confirms the necessity of comprehensive dpn phenotyping and underlines the importance of the severity of neuropathy that should be taken into account in the stratification of patients with pdpn for analgesic treatment and drug trials.people with charcot marie tooth disease (cmt) experience slowly progressive muscle weakness, sensory loss and musculoskeletal changes over time. this leads to disability and risk of comorbidities due to inactivity. exercise is important to maintain general health but may also help to improve symptoms of cmt. we conducted a randomised controlled crossover trial of aerobic exercise to ascertain the effect of training on fitness levels, muscle strength, function and general well-being. in addition, we monitored the safety of training and feasibility of participation in this type of exercise in local community gyms. motivation, confidence and barriers to exercise were explored using qualitative interviews. the recruitment target was people. in total people with cmt a were approached to participate and were unable to commit to the trial or did not meet the study criteria on initial screening. thirty-one people underwent more detailed screening but three failed to meet the study criteria, five people withdrew before starting and three withdrew part way through the study. the data for the people who started the study were analysed using a random effects model. there was a % participation level in the training and it was well tolerated with no increases in pain or blood serum creatine kinase. an increase in vo peak (ml/min/kg) was observed in the cmt group with (pre training: n= , . ± . , % ci . to . ; post training: n= , . ± . , % ci . to . ; pre control: n= , . ± . % ci . to . ; post control: n= , . ± . , % ci . to . ) . there was wide between subject variation leading to a small overall effect size with cohen d of . ( % ci:- . to . ). a tentative regression model showed no effect of group or time point. there were no major changes in other measures of impairment, function or patient reported outcome measures. this pilot study showed that a community based model of training had a small effect on cardiopulmonary fitness, and was well tolerated with good participation. ankle-foot orthoses (afo) are commonly prescribed for children with charcot-marie-tooth disease (cmt) to manage foot drop, however the type and severity of functional impairment results in gait deviations that might require alternate orthotic designs. the aim of this study was to identify d gait patterns of children with cmt based on severity of functional weakness (based on heel walk, toe walk and foot drop items during gait using the cmt pediatric scale) to inform a design pipeline for d printed orthoses. d gait data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model in children with cmt ( male; ± . yrs, ± . cm, ± . kg), of various cmt types: cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx and compared to typically developing children ( male; . ± . yrs, ± . cm, ± . kg). data were subdivided into three groups denoting increasing severity of dorsiflexion and plantarflexion weakness: no difficulty heel or toe walking (cmt nd ), difficulty heel walking (cmt dh ), difficulty toe and heel walking (cmt dth ). the cmt nd group showed a near-normal gait pattern. the only significant differences noted at the ankle were reduced peak dorsiflexion in stance (p< . ), indicating that an orthotic intervention may not be required. in addition to reduced peak dorsiflexion, the cmt dh group demonstrated significantly reduced dorsiflexion in swing (foot-drop) and a reduced dorsiflexor moment in loading response (p< . ). this suggest, the cmt dh group would require a flexible afo to allow activation of the plantarflexors during push-off, prevent foot-drop and restore a heel rocker in loading response. in contrast, the cmt dth group presented with significantly delayed and increased peak dorsiflexion in stance and reduced plantarflexion and power at push-off (p< . ). they also had significantly increased mean knee extensor moment (p< . ) revealing early signs of 'crouch gait'. therefore, the cmt dth group would require a rigid afo to limit the amount of dorsiflexion and assist movement of the ground reaction force anterior to the knee during stance. three distinct gait patterns at the ankle were identified in children with cmt, indicating patient-specific orthotic design pathways to target specific functional impairment. wojciechowski e , , chang a , cheng t , , little d , , menezes mp , , hogan s , burns j , . university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia.children with cmt are often prescribed ankle-foot orthoses (afo) to manage lower limb impairment such as foot drop and foot deformities. they are handmade by plaster cast followed by thermoplastic moulding. this traditional approach provides limited design options, can be costly, with long outpatient wait times. d printing, also known as additive manufacturing, has the potential to transform the way orthoses are prescribed, designed and manufactured. the aim of this review was to evaluate the evidence of d printing afos compared to traditional manufacturing methods, for children with cmt. there are currently no studies evaluating the application of d printing afos for children with cmt. however, a small, but emerging evidence base exists for d printed afo's in adults from studies including healthy participants and populations with rheumatoid arthritis, post-polio syndrome, foot drop and ankle weakness secondary to injury. samples sizes ranged from - participants for studies related to in-shoe orthoses and from - for studies related to afos. the methods of d printing included sterolithography, selective laser sintering and fused deposition modelling using materials such as nylon , nylon , polylactide, polycarbonate and abs. d printed afos were comparable to traditional manufactured orthoses in terms of patient-perceived comfort, temporal-spatial parameters, plantar pressure measurements and d gait analysis. however, the effects of long-term usage and durability of d printed afos has not been investigated. d printing orthoses have potential advantages including increased design possibilities, improved productivity, higher compliance, and reduced labour needs. disadvantages include redesigning clinical pathways, limited evidence base for clinical effectiveness, limited biocompatible materials, occupational safety considerations and a high level of expertise required for software operation and fabrication of devices. further research is required to determine the feasibility of d printed afos for children with cmt, and the most appropriate and effective printing pathway, materials to improve health outcomes of affected patients. wong shj , koh sj , lee bjh , chng ysk , pawa c , subramaniam t , cheng ksa , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore; yong loo lin school of medicine, national university singapore, singapore; khoo teck puat hospital, singapore.treatment-induced neuropathy of diabetes mellitus (dm) (tind) is a complication of rapid glycaemic control. individuals present with neuropathic pain and autonomic dysfunction within weeks of improvement in glucose control. the use of both insulin and oral hypoglycaemic agents has been associated with tind. its severity is determined by the rate and quantum of hba c decline. other predisposing factors include anorexia and weight loss. we studied the incidence of tind in dm patients who have undergone massive weight loss and hba c decline after bariatric surgery. we screened electronic records of patients ( dm, non-dm) who underwent bariatric surgery between and . dm patients fulfilled the tind hba c criteria of a decrease of ≥ % over months or ≥ % over key: cord- -rz r sj authors: nan title: abstracts for the th annual meeting of the japan neuroscience society (neuroscience ) date: - - journal: neuroscience research doi: . /j.neures. . . sha: doc_id: cord_uid: rz r sj nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns . sl - - frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl - - neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif a/kif bbeta), nmda type(kif ) and ampa type(kif s) glutamate receptors and mrnas(kif s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least rna related proteins such as hn rnp-u, staufen, and fmrps. kif a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award - what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in - months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v ). after complete ablation of v , the monkeys recover performance of visually guided saccades toward the blind field in months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi , kakenhi sy - - - distinct roles of two -pass transmembrane cadherins in neurite growth control tadashi uemura , , yasuyuki shima , , keisuke sehara , manabu nakayama , shinya kawaguchi , mikio hoshino , yoichi nabeshima , tomoo hirano , graduate school of biostudies, kyoto university, kyoto, japan; school of science, japan; kazusa dna research center at chiba, japan; graduate school of science, japan; graduate school of medicine, japan; crest, jst, japan drosophila flamingo (fmi) and mammalian celsr - , which are pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian -pass transmembrane cadherins celsr and celsr are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr enhanced neurite growth whereas celsr suppressed it. our result suggested that celsr had a stronger activity as g-protein coupled receptor than celsr did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of -pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin- is preferentially localized in axons; and nectin- , in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin- and dendritic nectin- is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi sy - - - lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa , fumikazu suto nagoya university, nagoya, japan; institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema a is expressed in ca pyramidal cells and principally suppresses invasion of mossy fibers to ca . second, the repulsive signal of sema a is mediated by plexin-a expressing in mossy fibers. third, the repulsive activities of sema a are attenuated by plexin-a in the proximal dendritic segments of ca pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on ( ) working memory maintenance and manipulation, ( ) rule representation and task-switching, and ( ) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy - - - towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy - - - neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of − genes on chromosome q . , has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi ( ) sy - - - a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and k songbird cdna array. we used the arrays to reveal a set of genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. - - - stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi sy - - - role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using -photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., ) . furthermore, in response to single spine stimulation using -photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration ( ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy - - - basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼ hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy - - - coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f- fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad- found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca + sensing protein was activated. synchronies of sv fusion are maintained by a ca + sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy - - - sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim . thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka , takeshi baba , sumiko mochida , yoshimi takai department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca + -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab system, munc- , munc- , doc- , and tomosyn. we have previously reported that tomosyn, a syntaxin- -binding protein, works as a molecular clamp that controls free syntaxin- availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin- , resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin- , which increases the affinity of syntaxin- for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca + sensor for neurotransmitter release. however, the physiological contributions of its ca + -binding domains (c a and c b) are still unclear. we have studied the roles of aspartate (asp) residues in the c b ca + -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell , p. ) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca + binding to the c b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca + binding to the c b because mutation in the second asp inhibits ca + binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin- (nt- ) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt- and its low-affinity receptor p genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c t in the noncoding region, bdnf-linked polymorphic region, and val met), three polymorphisms of the nt- gene (g- a, microsatellite in intron , and gly- glu) and a missene polymorphism of the p gene (ser leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val met) in the bdnf gene resulting in a prodomain substitution at position from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns sy - - - processing of bdnf and brain disorders masami kojima , aist, osaka, japan; sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p neurotrophin receptor (p ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); (t/g) and (t/g) of the human bdnf gene, causing amino acid substitution (r m and r l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy - - - pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣ leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣ , the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv- vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il ss+fgf- , and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi ( ) sy - - - hgf gene therapy for the treatment of spinal cord injury masaya nakamura , akio iwanami , kazuya kitamura , , yoshiaki toyama , hideyuki okano department of ophthalmology surgery, keio university, tokyo, japan; department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th level in adult rat days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. or weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap +fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., ) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., ) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., . nature neuroscience. nakamura et al., . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first - months after stroke, suggesting the presence of a critical period. in the cat, illert et al. ( ) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c -c segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. ( ) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c -c propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. ( ) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca + responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l cam(l ) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l . therefore, these syndromes have been reclassified as l syndrome. we performed a nation-wide l gene analysis and identified li gene mutations in families with l syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l -mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l syndrome. research funds: kakenhi ( ), ( ) sy - - - rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein gene (mecp ) located at xq is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like gene (cdkl ) manifest as atypical rtt. the mutations of mecp are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in -ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase- are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp , suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca , and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣ or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣ and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context - days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr , nr a, b, c), glycine transporter , and eaac , by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase ␣ -subunit (pde c), a component of cone-type pde. since genetic mutations of pde c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde in visual functions. shinichi nakagawa , masatoshi takeichi , , fumi kubo , nakagawa initiative research unit, riken, wako, japan; riken cdb, kobe, japan; department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi ( ) sy - - - identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea- to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea- during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea- double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea- -positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea- have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx and crx play critical roles in retinal photoreceptor development. while otx is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi ( , , ) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf found in the mesoderm. we find that although fgf can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf and is actually inhibited by fgf . for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy - - - how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel , encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein e was identified as the gene responsible for this behavioral difference among the species. obp e forms a gene cluster with obp d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp d/e region obtained from species phylogenetically located between d. melanogaster and d. pseudoobscula. sy - - - an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi , , akinori nishi , , toshihiko shiroishi , , tsuyoshi koide , sokendai, kanagawa, japan; national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c bl/ (b ) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b and msm, and those strains have the same genetic background as b except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b - msm, which have msm chromosome , showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi ( . ) sy - - - cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome (capsq ) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv ) was impaired in kjr neurons in contrast to that of c bl/ j mice. by searching the candidate genes, eh domain-containing four (ehd ), a trp-binding scaffold protein encoding gene was found. ehd binds to c-terminal of trpv . three mutations were found in ehd of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv . this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy - - - comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than different strains of mutant mice with the battery. surprisingly, more than % of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi ( , , , ) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least genes and loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt f caused by prx, and scan caused by tdp . sy - - - gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm , who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm /gd synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, ) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa ) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi ( ) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo- is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo- mediates the inhibition of axonal growth as a component of the ngr /p /lingo- and ngr /troy/lingo- signaling complex. inhibition of endogenous lingo- by soluble lingo- or dominant negative lingo- can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo- treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo- treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo- mediates the inhibition of differentiation and myelination. loss of lingo- function using dominant negative lingo- , lingo- rnai, or soluble lingo- or lingo- knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo- led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo- in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc - a sy - - - neuronal avalanches reveal neuronal wirings of layer / cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi ( ) sy - - - spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi , jean-pascal pfister , kazuyuki aihara , , wulfram gerstner department of complexity science and engineering, university of tokyo, japan; school of computer and communication science & bmi, epfl, japan; aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows j and sci. res. on priority areas from mext of japan, and swiss natl. sci. found. - / sy - - - timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as - ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi , ikue otaki , saya suzuki , yasunobu yasoshima , kazuto kobayashi laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin- receptor ␣ subunit (hil- r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil- r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. - - - integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy - - - molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj- is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are ( ) leukemia in patients exposed with atomic bomb during the iind world war, and ( ) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, ( ) many genetic/familial diseases show clinical symptoms at the middle or old age. ( ) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. ( ) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp , which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼ drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc /+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi k/akt signaling, which results in decreased gsk activity, has also been shown to ameliorate ami. both pka and gsk phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk at young ages, declining igf levels during aging may increase gsk activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk causes neuronal dysfunction during normal aging. research funds: kakenhi sy - - - molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, - - tsukiji, chuo-ku, tokyo - , japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy - - - identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue , department of geriatric medicine, university of tokyo hospital, tokyo, japan; research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of - - sigma, a negative cell cycle regulator that causes g arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka , hossein esteky , kiani roozbeh , tadashi sugihara , gang wang riken brain science institute, wako, saitama, japan; institute for studies in theoretical physics and mathematics, tehran, iran; kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of inferotemporal cells to object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to • rotation. research funds: kakenhi alexander thiele , gene stoner , louise s. delicato , mark roberts university of newcastle upon tyne, uk; the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy - - - context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy - - - effects of task demands on color processing in area te of the monkey hidehiko komatsu , , kowa koida national institute for physiological sciences, okazaki, japan; sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development . monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception - . here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy - - - dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui , hirotaka sakamoto , katsunori sasahara , hanako shikimi , kazuyoshi ukena , mitsuhiro kawata laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi ( and to kt) sy - - - roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi ( , ) sy - - - sex steroid receptor function in sexual behavior shigeaki kato , , takashi sato , takahiro matsumoto , imcb, university of tokyo, tokyo, japan; erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., ) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., ) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., ) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., . pnas usa , . sato, t., et al., . pnas, usa , . shiina, h., et al., research funds: probrain sy - - - annexin : a mediator of cell-cell communication in the neuroendocrine system julia buckingham , helen christian , john morris imperial college london, uk; department of human anatomy and genetics, university of oxford, uk annexin (anxa ) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca . the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori , yuki takayanagi , masahide yoshida , yoshiyuki kasahara , masaki kawamata graduate school of agricultural science, tohoku university, sendai, japan; department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) ( ) sy - - - cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f ); f activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m ). local field potential activity in f and m is also tuned to grasp, and there is strong beta coherence between f and m , indicating reciprocal transmission of information. this is also seen in synaptic responses of m neurones to stimulation of f (and vice-versa). single pulse stimulation in f strongly modulates corticospinal outputs from m through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy - - - where tactile signals are ordered in time shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., . s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy - - - decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo , biology/cns, california institute of technology, pasadena, ca, usa; jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. -channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha ( ) ( ) ( ) ( ) ( ) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy - - - why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust ( ) sy - - - recognizing self actions through externalized eyes atsushi iriki , symbolic cognitive development, riken brain science institute, saitama, japan; cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut , existing mostly in cortical pyramidal cells, and vglut , found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut -expressing terminals (n = ), but were found in- % of spines innervated also by vglut -expressing terminals (n = ). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy - - - gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼ -fold decrease in the potassium-chloride co-transporter (kcc ) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer / pyramidal cells in visual cortex share common excitatory inputs from layer and from within layer / , when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer / cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi ( , ) sy - - - local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer of rat visual cortex and layer / of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer , axondescending cells receive excitatory input predominantly from layer / while neurogliaform cells receive stronger input from deeper layers. layer / neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer / , while the cr− martinotti cells also receive strong excitation from layer . irregular-spiking basket cells also receive strong excitatory input from layers / and , but they often have a gap at the top of layer , with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers / and , and only weak input from deeper layers. research funds: nih sy - - - physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers ( - ) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r ey , mcknight neuroscience of brain disorders award sy - - - axon guidance mediated by localized ca + signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca + signals. its turning direction depends on the occurrence of ca + -induced ca + release (cicr) through the ryanodine receptor type (ryr ). the activity of ryr is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr , that serves as a key regulator of axon guidance. attractive ca + signals facilitate intracellular membrane transport to the leading front and subsequent vamp -mediated exocytosis on the side with elevated ca + . in contrast, repulsive ca + signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp -mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy - - - molecular mechanisms for signaling through plexin-a hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema a acts as a guidance cue for axons through a receptor complex comprising neuropilin- as the ligand-binding subunit and plexin-a as the signal-transducing subunit. the ferm domain-containing gef, farp , associates directly with plexin-a . sema a induces the dissociation of farp from plexin-a , resulting in activation of farp 's rac gef activity, rnd recruitment to plexin-a and down regulation of r-ras. simultaneously, the ferm domain of farp sequesters pipki␥ from talin, thereby inhibiting its kinase activity. these activities are necessary for sema a-mediated repulsion of outgrowing axons. plexin-a also functions as a ligand binding receptor of a transmembrane semaphorin, sema d and contributes to cardiac morphogenesis. sema d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a forms a receptor complex with off-track in the ventricle or with vegf receptor type in the conotruncal segment, which are responsible for the effects of sema d on the respective regions. research funds: crest sy - - - axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins a (sema a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema a stimulated the transport of neuropilin- ::egfp, plexin-a ::egfp, and fyn::egfp, which are the components of sema a signaling, but not neuropilin- ::egfp. interestingly, sema a accelerated the anterograde transport of trka::egfp. these facts suggest that sema a selectively facilitates the transport of its own signaling components and that sema a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein- (crmp- ) is enriched in growing axon, and play a crucial role in axon specification. crmp- interacts with tubulin dimers to promote microtubule-assembly, and binds to sra- , an effector of rac to regulate wave-dependent reorganization of actin filaments. crmp- links kinesin- to tubulin dimmers and sra- , and participates in the kinesin- -dependent transport of tubulin dimmers and the sra- /wave complex to growing axons. we also found that the par- /par- complex and the ras/pi -kinase/akt/gsk- b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk- b and inactivates its kinase activity downstream of ras/pi -kinase, thereby increasing non-phosphorylated active crmp- which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy - - - regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki , , takashi abe , , , shigeyuki kan , , satoru miyauchi , national institute of information and communications technology, kobe, japan; japan society for the promotion of science, tokyo, japan; graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; kyushu institute of technology, kitakyushu, japan; crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o -and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p x receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p x receptors produce a reduction of the neuropathic pain behaviour ( . nature , - ) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p x causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury ( ( . nature , ( - . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi ( ) sy - - - pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk , which labeled with carbon- can be used for positron emission tomography (pet). using c-(r)-pk pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy - - - application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy - - - brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality ( . neuropsychobiology , ), aging ( . neuroimage , , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, ) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia ( . biol. psychiatry , ; . neuroimage , ) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every . s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy - - - clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy - - - measurement of language related brain activities during recovery from aphasia eiju watanabe , yumiko muroi , chizuru nakajima department of neurosurgery, jichi medical university, tochigi, japan; department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy - - - neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy - - - neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy - - - neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy - - - bridging neuroscience and public: neuroethics in cultural contexts osamu sakura , interfaculty initiative in information studies, university of tokyo, tokyo, japan; research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura , , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira , argye hillis showa university school of medicine, japan; crest, jst, saitama, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi ( ), crest sy - - - impairment of surface perception in patients with ventral occipital damages shinichi koyama , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , masato taira , argye hillis showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient was a -year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory ) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient was a -year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy - - - how do pictorial cues influence d information processing in the parietal association cortex? masato taira , arish, nihon university, tokyo, japan; department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for d perception. basically, it is thought that the parietal association cortex processes d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence d information processing in the parietal association cortex. research funds: kakenhi sy - - - case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy - - - neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v + for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi ( ) sy - - - neural correlates of visual attention argye hillis , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira johns hopkins university, usa; showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp gene, encoding methyl-cpg binding protein , cause rtt. mecp acts as a transcriptional silencer. abnormal expression of some genes due to mecp dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp -downstream genes. using mecp -null mice (mecp −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp directly regulates expression of insulin-like growth factor binding protein (igfbp ) gene in human and mouse brains. pathological features of mecp −/y have the similarity of igfbp transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp due to lack of mecp may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements- or l (line- ) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute % of the mammalian genome with l elements alone representing %. the fact that l can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy - - - notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima , jun kohyma , tetsuya taga , masakazu namihira grad. sch. biol. sci., naist, ikoma, japan; inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat . a cpg dinucleotide in the stat recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat binding element in the gfap gene promoter. sy - - - neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. ( ) which type of brain infarction will be a future target of this therapeutic approach? ( ) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? ( ) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy - - - gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that ( ) motor function may recover after minor injury to the primary motor cortex, ( ) this recovery is, at least in part, associated with reorganization of cortical motor representation, ( ) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and ( ) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy - - - neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji , toshiya sato , mitsunori yamada department of neurology, the university of tokyo, tokyo, japan; center for bioresource-based researches, japan; department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with cag repeats. the q mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy - - - control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi , sy - - - mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima , dept. of anat., keio univ. sch. of med., tokyo, japan; inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day . mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca + signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca + elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca + and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp , rho gtpase, mapk and pi k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy - - - membranes, water and diffusion denis j. le bihan shfj/cea, france among einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy - - - diffusion tensor fiber tractography using a tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded -t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on -t tractography compared to . t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using -t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy - - - anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to % of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor ( - mg/kg) into male wistar rats (from days to weeks of age) significantly caused hyperactivity at - weeks old. immunohistochemical analyses of the brain tissues at weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy - - - non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about - meters from a base station. another remarkable observation in our studies is the fact that a significant (p < . ) neuronal damage is seen in rat brains days after a hour exposure to gsm at sar values , and mw/kg. we have followed up this observation in a study where animals were sacrificed and days respectively after an exposure for hours to gsm mobile phone electromagnetic fields at sar values , , , . and (controls) mw/kg. significant neuronal damage is seen after days and albumin leakage after . our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after and days. sy - - - the mitochondrial toxin -nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs , france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin -nitropropionic acid ( np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of np toxicity and show that np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by np. in summary, the np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of -ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at days of age showed behavioral hyperactivity at - weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of -ohda-induced neurotoxicity, using pc cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of -ohda. we also characterized the oxidation of cellular proteins by -ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by -ohda, ros and p-quinone. sy - - - environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥ subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥ subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥ subunit-containing receptors in gh and hek cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥ subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi ( ) sy - - - involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca + rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k a at nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k a application was started soon after hfs or when k a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi ( ) sy - - - autocrine mglur activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur -gq/ transduction pathway as well as being initiated by direct mglur activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy - - - regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca + -activated protease calpain or by disturbance of association of gaba a receptor ␥ subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy - - - contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy - - - detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai , , susumu takahashi department of psychology, kyoto university, kyoto, japan; crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl- s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi ( ) sy - - - impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto - , japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy - - - tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad the amount of the tonic inhibition is reduced in ca hippocampal interneurons, while unchanged in pyramidal cells. sy - - - modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca region yoshihiko yamazaki , yasukazu hozumi , kenya kaneko , satoshi fujii , hiroshi kato dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca region. research funds: kakenhi ( ) sy - - - carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of m carbachol induces beta oscillations which occur intermittently with the interval of - s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is . ± . hz (mean ± s.e.m.). the oscillations are induced via muscarinic m , , receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations ( - hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that ( ) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, ( ) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca and ca neurons discharged during both up and down states, ( ) gamma and fast (ripple) oscillations were observed in hippocampal ca area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: ( ) most of the mprf neurons were active during rem sleep; ( ) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; ( ) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi ( ) sy - - - clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) : h and ld : h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per and per rhythms suggest that light-on resets per rhythms in both light conditions, while per rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld : . the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per and per showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy - - - genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified genes that fluctuated between day and night in the livers, lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose- -phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy - - - neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy - - - a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e) . to e . , the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy - - - live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi ( ) sy - - - visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol , , -trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy - - - rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦ (glur␦ ) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦ into glur␦ -null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦ lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi sy - - - gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi ( ) sy - - - physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi ( ) sy - - - deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid- th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy - - - firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no. ) sy - - - characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama , department of neurological surgery, nihon university school of medicine, tokyo, japan; division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate ( . ± . hz, n = ) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca + -atpase isoform that is located on the th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that ( ) d -type dopamine receptors are downregulated presynaptically in the striatum, and ( ) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy - - - metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr ( . nature , ) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat ( . endocrinology , ) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca + imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi sy - - - metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy - - - molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi , sy - - - thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp c is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t and reverse t , but the transport activity of t is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp a , the transport activities of t and t are similar, is also expressed. the uptake of t and t by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t and t . research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy - - - alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi , masaki takahashi , yasuhiro yoshikawa , tomoko tashiro department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to -hydroxy- , , , , pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to -hydroxy- , , , , , , -heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites ( . dev. brain res. , ) . koibuchi et al. ( ) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. ( ) detected some pcb and oh-pcb congeners in human csf, of which oh-cb is the highest concentration. to determine its effects on developing neurons, we examined oh-cb in rodent cerebellar culture cells. interestingly, oh-cb promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or -htp to examine the relevance of serotoninergic function. the treatment of -htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., ) . reference kawano et al., . j. neurosci. res. , - . research funds: kakenhi os a- - neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama , kazumi takahashi , yukihiko kayama cluster of science and technology, fukushima university, fukushima, japan; department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic ( ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣ receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os a- - correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi , , kazuyoshi hirano , , yukimasa muraishi , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan; secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l ) and grey matter volume among students aged - , who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l . os a- - grammar center activation in honorification judgment of japanese sentences kanako momo , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f op/f t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os a- - top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui , , , kimitaka kaga , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, tokyo, japan; dept. of otolaryngology, univ. of tokyo school of medicine, japan; crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m ) (neurosci. res. , s ). in a subsequent study, we confirmed that m was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m elicited by the original songs and that by the modified ones. however, while m without the deviation showed no significant effect of lateralization, m with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os a- - cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os a- - hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima , , naoki fukui , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, komaba, japan; crest, jst; dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os a- - individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext ( ), jsps research fellowship ( ) os a- - brain activity of happy facial recognition in mother-daughter relationship jun shinozaki , nobukatsu sawamoto , toshiya murai , takashi hanakawa , hidenao fukuyama hbrc, kyoto univ. grad. sch. of med. kyoto, japan; neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os a- - anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto , , atsushi maki , , kuniyoshi l. sakai , advanced research laboratory, hitachi, ltd., tokyo, japan; crest, japan science and technology agency, saitama, japan; dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f t/f o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f t and f o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (− , , ) , (− , , ) , and (− , , − ) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os a- - effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada , sunao iwaki , mitsuo tonoike aist, osaka, japan; chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = ), and with those from an inner semantic category (middle, d = ), while others were not changed (normal, d = ). peak amplitudes of waveforms of the root mean square values on the components of ms (f ( / ) = . , p = . ) and ms (f ( / ) = . , p = . ) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the and ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os p- - mechanisms of p y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono , xiaoming zhu , takashi tominaga , fumihito saitow , shiro konishi , waseda-olympus bioscience research institute, singapore, singapore; department of neurophysiology, tokushima bunri university, kagawa, japan; department of pharmacology, nippon medical school, tokyo, japan postsynaptic p y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and - min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were ± . % and ± . % of the controls, respectively. pretreatment with the protein kinase a inhibitor h- , but not the calmodulin kinase ii inhibitor kn- , completely abolished the atp-induced ipsc enhancement. os p- - activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca + permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca + influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein (arip ). activin-induced nmdar activation persists more than h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os p- - roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr phosphorylation following ltp induction in rat hippocampal ca region. like camkii activation following ltp (fukunaga et al., ) , the increased camki-thr phosphorylation remained elevated at least for min after ltp induction. the increased camki-thr phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto- significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing ( ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing ( ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi ( ) os p- - mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p ) rats that had received a single brdu injection at p were substituted with the corresponding region of entorhino-hippocampal slices from p rats. after five days in vitro, newborn gcs, detected by brdu and prox , migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os p- - cdk is required for neuroblast migration in the adult mouse brain yuki hirota , , , toshio ohshima , takuji iwasato , ashok b. kulkarni , hideyuki okano , , kazunobu sawamoto , , bridgestone lab. keio univ., tokyo, japan; dept. physiol., keio univ., tokyo, japan; dev. neurobiol. riken, tokyo, japan; behavioral gen. riken, tokyo, japan; nih, bethesda, usa; sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase (cdk ) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk mice to emx cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk has a cell autonomous function in neuroblast migration in the adult brain. os p- - colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi , wu sheng-xi , yuchio yanagawa , , kunihiko obata , nobuaki tamamaki kumamoto univ., kumamoto, japan; fourth military medical univ., xi'an, people's republic of china; gunma univ., maebashi, japan; sokendai, hayama, japan; riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng , plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad -gfp knock-in mouse neocortex at e -p by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s -␤ expression in the gabaergic neuron progenitors. os p- - role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi , , , masanori sakaguchi , toru yamashita , , , yuko fujita , yukiko gotoh , arturo alvarez-buylla , takeshi kawase , hideyuki okano , kazunobu sawamoto , neurosurgery, keio univ. sch. med., tokyo, japan; bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; physiol., keio univ. sch. med., tokyo, japan; inst. mol. cell biosciences, univ. tokyo, tokyo, japan; neurosurgery, ucsf, san francisco, usa; neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt b. to examine the molecular interactions between c-tsk and wnt b, we co-electroporated them into the optic vesicle at stage - chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) in developing cortical neurons. an expression vector of gfp-tagged hdac was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac was primarily localized in nuclear until week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac contributes to neuronal differentiation in cortical development. research funds: kakenhi ( ) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., ) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum . ns) was measured. in high-voltage electron microscopy (hitachi m, nips, , gap junctions (average size . m long) were present in contacts. in confocal laser-scanning imaging, connexin localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be /m and a single channel conductance is ps, the conductance of each junction would be ns. the presence of seven junctions between a pair will lead to estimate a total junction of ns. the measured conductance could allow to estimate a fraction of open channels as . %. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi ( ), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os p- - multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer stimuli and propagated horizontally in the layer / . in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os p- - presence of two phases in the sensitive period of orientation plasticity shigeru tanaka , toshiki tani , kazunori o'hashi , , jerome ribot brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after weeks of goggle rearing starting around the age of weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os p- - residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida , , , tadashi isa , , dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; sch. life sci., grad. univ. adv. stud., hayama, japan; crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield ( - % correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v lesion is unlike normal, near-threshold vision. research funds: kakenhi , kakenhi and crest, jst os p- - comparison of the angle representation in macaque visual areas v and v minami ito , , hidehiko komatsu , national institute for physiological sciences, okazaki, japan; the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v neurons has angle selectivity. here, we studied the angle selectivity of area v , which is the major source of inputs to area v . we conducted single-unit recordings from the superficial layer of area v , while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v neurons responded mainly to sharp angles ( • ), straight lines ( • ) or right corners ( • ), but not to intermediate angles ( • or • angle width). this contrasted with area v , where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v . os p- - firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os p- - integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v , we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os p- - probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner , , pei sun , keiji tanaka , david j. heeger , kang cheng department of psychology and center for neural science, new york university, usa; laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri ( . mm × . mm × mm voxels) in human v . we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t *-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih ( f ey - ) os p- - relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations ( - ms; isi = s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase ( - ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase ( - ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼ % of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os p- - the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe , kazuto terai , osamu hieda , shigeru kinoshita dept. of ophthal., kyoto second red cross hospital, kyoto, japan; dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from . to . . the logmar average was . . eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was . , improved from . . the residual five eyes showed less or no improvement. the change of logmar average was from . to . . conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os p- - the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada , hideomi tanaka , , satomi nakayama , miki iwasaki , , hitoshi okamoto , laboratory for developmental gene regulation, bsi, riken, japan; crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) and migrate tangentially to r near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr and frizzled a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os p- - disrupted-in-schizophrenia (disc ) regulates the transport of the nudel/lis complex to axons via direct interaction with kinesin- shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc . disc is reported to interact with nudel, which forms a complex with lis . although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc remain largely unknown. here we report that disc interacted with kinesin- , a motor protein of anterograde axonal transport. kinesin- interacted with the nudel/lis complex through disc , and these molecules accumulated at the distal part of axons. the knockdown of disc by rnai of disc induced the delocalization of nudel and lis from the axons and reduced axonal growth. the knockdown of kinesin- induced the delocalization of disc from the axons. taken together, these results indicate that disc links kinesin- to the nudel/lis complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os p- - role of a novel collapsin response mediator protein- interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein (slp ) can mediate the vesicle transport. slp is known to associate with rab . we found that slp associates with collapsin response mediator protein- (crmp- ), which is a key regulator of axon formation. slp could form the trimeric complex with rab b and crmp- , and also associate with kinesin- through crmp- . slp is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp functions as a mediator of recruitment of certain receptor depending on crmp- and kinesin- . os p- - absolute quantification of mdr a, mrp , mrp and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie , , yuki katsukura , , sumio ohtsuki , , xiao-kun cai , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr a, mrp , mrp , and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between and fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr a, mrp , bcrp were . , . and . fmol/g, respectively, while it of mrp was under detection limit of standard curve. this quantitative profile suggests that mrp and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr a. os p- - dominant expression of claudin- in highly purified brain capillary endothelial cells sumio ohtsuki , , hirofumi yamaguchi , saori sato , tmoko asashima , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam- ) and magnetic cell sorting. mrna expression of caludin- - was measured by real-time rt-pcr. claudin- showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin- and - were . % and . % of that of claudin- . claudin- mrna was concentrated in the purified bcecs, while claudin- and - mrna in the purified bcecs were lower than that in the whole brain. rat claudin- mrna was also concentrated in rat brain capillary fraction, but claudin- mrna did not. these results suggest that claudin- is a dominant tjs protein in bcecs, and expression of claudin- and - , which was reported as tj protein in bcecs, are not restricted in bcecs. os p- - effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad , a. pauzi m. yusof , p.d. mourad , m. bainbridge , s. ab ghani universiti sains malaysia; university of washington, seattle, usa; brody school of medicine, east carolina university, nc, usa h o is the main peroxides produced in mammalian cells that consume o . the main source of h o in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h o towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h o microsensor, we investigated the level of h o in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h o level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h o was injected into the brain. fatality occurs when the injected h o exceeds m. these finding showed that the altered paracellular permeability in the presence of h o is caused by a series of events that happen one after another. research funds: short term grants pkimia and pfar-masi os p- - somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t - ) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t - spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os p- - responses of renal sympathetic nerve activity and sodium excretion to days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions ( , , , meq./l nacl) over days. the sodium loading with meq./l nacl suppressed rsna significantly and then it gradually recovered while either meq./l nacl or meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os p- - cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku , haruko masumiya , yasumasa okada dept. physiol., hyogo col. med., nishinomiya, japan; dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c ventral root (c vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: ( ) a continuous column extending from the para-facial region to the pre-bötzinger complex, and ( ) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os p- - slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca + -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi , os p- - the effect of music to sex hormones of elderly person hajime fukui , kumiko toyoshima , kiyoto kuda , katsuhiko iguchi nara univ. of edu., nara, japan; grad. school of human sciences, osaka univ. japan; nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a -kda protein band in csf of % (n = ) of confirmed and % (n = ) of suspected tbm patients out of tbm patients. the -kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv c (ag a) and rv c (ag b) and one host derived protein as the components of the tbm specific -kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q , vitamin d and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os p- - involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh , boon chock , dennis l. murphy , chuang c. chiueh dept. applied pharmacol., univ. toayama, toyama, japan; lab. bioch., nhlbi, nih, md, usa; lab. clin. sci., nimh, nih, md, usa; cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk / and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy y cells. consistently, trx increased the expression of mnsod and bcl- supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl- proteins for protecting against mpp + -induced neurotoxicity. os p- - pgd induces neuronal apoptosis via d- , -pgj tatsurou yagami , noboru okamura , toshiyuki sakaeda facul. health care sci., himeji dokkyo univ., himeji, japan; kobe univ. grad. sch. med., japan prostaglandin d (pgd ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd induced neuronal apoptosis in rat cortical cultures. however, a pgd receptor blocker did not suppress neurotoxicity of pgd . little pgd receptor was detected, suggesting an involvement of pgd metabolites in the apoptosis. among pgd metabolites, -deoxy- , -prostaglandin j ( d- , -pgj ) caused neuronal apoptosis most potently and rapidly. although d- , -pgj is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that d- , -pgj induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ h] d- , -pgj (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd induced apoptosis via d- , -pgj in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the d- , -pgj -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c to t ). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi os p- - compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi ( ) os p- - timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae , xiaofeng lu , , yusuke uchida , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of targets following a gosignal given at a random timing between and ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: ( ) those that showed buildup activity during the delay period until the go-signal, and ( ) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, - h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (> ms) than larvae (> hpf, ms). m-cell activity monitored with confocal ca + imaging during the tail flip (> hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (< %) after hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca + release through ryanodine receptors, triggered by glutamate receptor-mediated ca + influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os p- - distribution of voltage-gated calcium channel ␣ ␦- mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka - , japan the ␣ ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ ␦- to ␣ ␦- . the distributions of ␣ ␦- , - , - mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ ␦- remains to be determined. we cloned ␣ ␦- cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ ␦- mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ ␦- mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ ␦- subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os p- - hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb ) depressed the excitation, while bicuculline depressed the inhibition. h or h histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os a- - activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c bl/ mice. by recording field epsp (fepsp) along m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at - div, then the amplitudes of fepsps in the ventral side began to decrease at - div, and dominated in the dorsal area at div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at - div but the axons terminals in the ventral area were eliminated until div. this synapse elimination from the ventral side was blocked by apv application from div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until div. the number of those ventral axons is reduced before div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during - div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os a- - transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os a- - segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa in r is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa function resulted in selective loss of eph expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa dependent cues are required to determine the territory for whisker representation in r and the assembly of a somatosensory circuit. os a- - the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p and those in the venrtolateral (vl) area were eliminated from p to p and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p , reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os a- - proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi ( ), kakenhi ( ) os a- - the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m , s , sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms ( , , hz, stimulations) was compared in dp patients. the pain relief was evaluated with visual analogue scale. high frequency ( , hz) rtms of m was the only effective stimulation for treating intractable pain in of patients ( %). the pain relief continued for h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m . os a- - involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p x r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p x , , / rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p x , , , , / , / r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p x rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p x r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os a- - the cantor-like patterns in rat hippocampal ca pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after days of age, before which they had been reared with song tutors from birth. when the noise stopped at - days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization ( days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, ) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, ) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada ( ) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca , may be encoded in ca in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca , using -compartment model of neuron, and we found the cantor coding of information of time series in the model ca . it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in patients with chronic ischemia due to diverse vascular occlusive diseases (n = ) and moyamoya disease (n = ). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os a- - hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata , , heng zhao , gary steinberg department of neurology, brain research institute, niigata university, niigata, japan; department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia ( • c) reduced infarct size by %. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < . ). p-␦pkc (t ) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s ) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, -vessle occlusion was induced. fluo- /am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu- , yokogawa, japan) equipped with × objective lens] was inserted into the brain. -vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo- increased up to %, and persistently increased up to % during -min reperfusion, indicating the long-lasting ca + increase in the ca region. in contrast, in the frontal cortex, -min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca + in situ. research funds: kakenhi # , # os a- - reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto , h. watanabe , y. kumon , t. ohnishi , chi ii , y. imai , j. tanaka dept. neurosurgery, ehime university, japan; dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b and cd b recognized with ox antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba + mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba + amoeboid-shaped mg started to accumulate days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os a- - insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki , , chihiro tamaki , sumio ohtsuki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein (lrp- ) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp- expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤( - ), which reached . -fold greater uptake than non-treated control after min treatment. increase of the hepatic uptake of a␤( - ) by insulin was concentration dependent (ec = pm), and was completely suppressed by rap ( m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp- to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os a- - mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used n astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of n cells was inhibited by an inhibitor of ip receptors, -aminoethoxydiphenyl borate ( -apb) or an endoplasmic reticulum ca + -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca + was inhibited by -apb and cpa. moreover, removal of external ca + accelerated the reversal of thrombin effects. these results suggest that refilling of ca + store by ca + entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age and a -year-clinical course. the brain showed severe atrophy ( g). microscopic examination disclosed that all telencephalic neocortices had nft of more than and sp of more than . all limbic cortices and nuclei had nft of more than and sp of more than . although there was no sp, various numbers of nft were observed in the following structures: claustrum , caudate , globus pallidus , hypothalamus , meynert's nucleus , thalamus , substantia nigra , central gray , locus ceruleus , purkinje cells , posterior root ganglion , adrenal medulla . this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os a- - transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv was expressed in soma, dendrites and synapses in the neurons. since trpv was firstly cloned as an osmotically activated channel, we hypothesized trpv might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv -deficient neurons at • c, and found rmp in wild type was more depolarized by approximately mv than rmp in trpv -deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv -deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv is involved in regulation of both neural activity and spine motility in hippocampus. os p- - a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at locations per ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by -photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os p- - d -like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a receptors (adora ) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora in the ng decreased by > % of that in the non-treated ng, being not accompanied by a change in mrna level for a a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase- activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf blockers such as brefeldin a, exo , and arf n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf and subsequent pld may regulate the k + -current response induced by da. os p- - p gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa , toshihiko kuriu , ayako m. watabe , toshiya manabe , shigeo okabe , tadashi yamamoto div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; dept. of cell biol., tokyo medical and dental univ., tokyo, japan; div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p gap, which interacts with the nr b subunit of nmdar. within neurons, p gap was highly concentrated in the postsynaptic density and co-localized with nr b and an actin-binding protein, cortactin. p gap promoted gtp hydrolysis of cdc and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p gap. when over-expressed in dissociated neuron, p gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os p- - non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os p- - spatial frequency tuning of disparity-selective neurons in macaque v hironori kumano , seiji tanabe , ichiro fujita grad. sch. of engineering science, osaka univ., osaka, japan; grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional ( -d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using -d filtered noise stereograms. the disparity tuning of most v neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os p- - predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v neurons. the results suggest that v neurons are involved in mediating stereoacuity. os p- - the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field -d ternary dynamic random noise, generally refreshed every - ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v /v neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v , and especially areas beyond. research funds: mext( ), jsps( ), coe os p- - analysis of center-surround organization of v neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area (v ) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of cells analyzed, % showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ± degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os p- - spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki , izumi ohzawa , grad. school of eng. sci., osaka univ., japan; grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with × dots was presented over an area to times larger than the rf of a complex cell. for each dot location, a nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of nd-order kernels at all locations. nd-order kernels had roughly comparable extent as the rf, and contained . subregions on average (n = ). among complex cells, whose rf envelopes were elongated, cells exhibited the horizontal elongation. research funds: mext( ), jsps( ), coe os p- - orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito , osamu sadakane , masahiro okamoto , hironobu osaki , hiromichi sato grad. sch. med., osaka univ., osaka, japan; grad. sch. front. biosci., osaka univ., osaka, japan; med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about % lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that % of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os p- - temporal dynamics of suppressive receptive field surround in cat v satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v ), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes ( ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (< ms) but not in middle ( - ms) and late responses ( - ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from . deg (early response) to deg (middle response) and . deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi ( ) os p- - spatial-frequency dependent surround suppression in cat v ayako ishikawa , satoshi shimegi , hiroyuki kida , hiromichi sato grad. sch. front. biosci., osaka univ., osaka, japan; grad. sch. med., osaka univ., japan; grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v . we used a stationary flash (duration, ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus ( ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = ) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os p- - contrast-dependency of spatial summation property in cat v and lgn masahiro okamoto , tomoyuki naito , osamu sadakane , hiromichi sato grad. sch. front. biosci., osaka univ., japan; grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v ) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by . and . times in v and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v , the strength of surround suppression did not affected by stimulus contrast on average. we conclude that ) rog model describes the surround suppression better than dog model both in v and lgn, ) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida , satoshi shimegi , ayako ishikawa , hiroshi sakamoto , hiromichi sato grad. sch. eng. sci., osaka univ., japan; grad. sch. med. sci., osaka univ., japan; grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v ), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into flanks ( • step), and stationary stimulated with an annulus, oppositely-faced flanks ( -fk) or a flank ( -fk) stimulus. the durations of stimulus presentation were ms for crf and ms for srf stimulation. localized srf stimulation with either -fk or -fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for -fk stimulation throughout the crf response. however, -fk stimulation of end position to crf had strong and long-lasting suppression on responses during - ms after onset. os p- - temporal-frequency dependency of receptive field size and surround suppression in lgn and v osamu sadakane , tomoyuki naito , hironobu osaki , masahiro okamoto , hiromichi sato grad. sch. med., osaka univ., japan; med. sch., osaka univ., japan; grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v ) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v neurons, suggesting that the subcortical tf tuning property contributes to that in v . ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. ; yang and maunsell ) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using . t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around %) was smaller than that of bold (> %), the temporal profile showed a constant precedence of the diffusion signal by . s. os p- - new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os p- - spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international - system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad ( . ), approaching unity (∼ . ) for oh, and in between for control ( . ). statistical analysis was performed and significant differences were observed for these groups (p < . ). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: ( ) resting, ( ) staring at a standing human object, ( ) thinking of standing, ( ) standing with eyes open, ( ) standing with eyes closed. regional cbf data analyzed by spm were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os p- - potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono , , eiji shigetomi , kiyoharu inoue , fusao kato dept. neurol., jikei univ., sch. med., tokyo, japan; lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os p- - effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan , -methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin ( -ht) release and cause degeneration of -htergic nerve terminals via -ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the -htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on -ht release and -htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant -htergic neurons around the raphe nuclei. treatment with a -htergic neurotoxin , -dihydroxytryptamine dramatically reduced the tissue contents of -ht and its metabolite, which was blocked by a selective -ht reuptake inhibitor. mdma and meth ( . - m) increased -ht release, and reduced the tissue contents of -ht and its metabolite at higher doses. the mesencephalic slice culture including the -htergic raphe nuclei may be useful to examine the mechanisms underlying -htergic neurotoxic effect of mdma in vitro. os p- - studies on drug dependence (rept. ): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os p- - prostaglandin d is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd by hpgds and that astrocytes expressed both dp and dp receptors and were activated by pgd . we found that gliosis and demyelination were well suppressed in hpgds-or dp -null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd is a key molecule of neuroinflammation involved in the demyelination. research funds: , os p- - on a sodium channel distribution enabling high frequency signal processing go ashida , , kousuke abe , kazuo funabiki grad. sch. medicine, kyoto univ., kyoto, japan; grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below khz, while that in the node does up to khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated nl cells from owls and achieved intracellular recordings in of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = ± mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, vision impaired ( blind and low vision) and sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os p- - autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada , runa araya , naoto kitamura , yuji mishinsa yamada unit, riken bsi, saitama, japan; nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba -positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os p- - fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita , , hideomi tanaka , , sachiko tsuruoka , hironori wada , , hitoshi okamoto , riken bsi, wako, japan; jst crest, kawaguchi, japan; riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose , dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi ( ) os p- - initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd -homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin (fas ) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas genetically expressed on a muscle, we found fas accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas is crucial for the postsynaptic localization of fas . in addition, postsynaptic localization of a scaffolding protein dlg, psd -homolog, and glutamate receptors was impaired in fas mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor a (mef a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef a that is sumoylated at lys promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef a at ser and thereby promoted a switch from sumoylation to acetylation at lys , leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns , ag ps a-a characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., ) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., . mol. brain res., , - . research funds: kakenhi ( ) ps a-a the distribution of snap- protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap- derived from exon splicing are expressed in brain. we generated two specific antibodies for snap- a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap- a and b during early postnatal period. snap- b was low at the birth and increased remarkably thereafter. by the contrast, snap- a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap- b but not snap- a was recovered in raft-enriched fractions of triton x- -treated lp membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap- b was widely distributed throughout brain, whereas, snap- a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap- protein are regulated differently in isoformspecific manners, and snap- a and snap- b play different functional roles in brain. ps a-a erc(elks/rab ip /cast) regulates syaptic short-term plasticity by recruiting bmunc - to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain ( . mol. brain res. , - ) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt- , are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt- , the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt- (-/-) mice, and a novel antagonist of glial glutamate transporters, ( s, s)- -[ -( -methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately %) of functional transporters, glast and/or glt- , is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat- gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat- mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat- vglut. a mutation in avr- glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr- functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps a-a biochemical characterisation of the vesicular glutamate transporter stephan schenck, shigeo takamori department of neurology and neurological science, st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut and vglut are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut and vglut has been found. to further characterise the specific properties of the transporters we make use of the vglut -ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut by yeast two-hybrid screening and found endophilin a . as expected for the interacting molecule to vglut , endophilin a was typically localized to vglut -positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in ∼ min, followed by spontaneously recovered after min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) was demonstrated in the lactate medium. results suggested that increased mct expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu , kazuhito tomizawa , shuang liang , iori ohmori , teiichi nishiki , kohji takei , hideki matsui dept. of physiol., okayama univ., okayama, japan; dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca + -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos- cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately ( th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm - and fm - , and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca + channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca + channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca + influx and epscs, suggesting that exocytotic machinery downstream of ca + influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca + or g-proteins. ps a-b spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca + channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap- subunit can directly bind with synprint site. this interaction was ca + -dependent, being weaker at concentrations higher than nm. in contrast, the interaction of synaptotagmin with synprint was optimal at m ca + , being weaker at lower or higher concentrations. the binding domain of synprint for ap- and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca + channel synprint site makes ca + -dependent interactions with ap- and synaptotagmin thereby contributing to vesicular endocytosis. ps a-b acl- , an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl- ˜ , and generated their deletion mutants. the mutants of acl- gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat- mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl- expression, suggesting that certain phospholipid species produced by acl- are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku , hussam jourdi , akiyoshi kakita , tadasato nagano , hitoshi takahashi , nobuyuki takei , hiroyuki nawa dept. mol. neurobiol., brain res. inst., niigata univ., japan; brain resource center, brain res. inst., niigata univ., japan; dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb ) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip and sap . moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima , eriko miura , keiko matsuda , tetsuro kondo , , masahiko watanabe , michisuke yuzaki dept. physiol., sch. med., keio univ., tokyo, japan; dept. anatomy, hokkaido univ., sch. med., sapporo, japan; mol. neurophysiol., aist, tsukuba, japan cbln is a member of the c q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln - , are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln , cbln and cbln were secreted as glycoproteins, whereas cbln was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd- and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd- . more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd- at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf /ra (ra ), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca +independent homophilic trans-cell adhesion activity. ra participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra -/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra interacted with par- being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par- was colocalized in the cell adherent region of p embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra +/+ testis, whereas it was undetectable in the spermatid of the ra −/− testis. ra and jam-c were localized around the head portion of spermatid and ra deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra and par- . research funds: izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd- clusters in cultured hippocampal interneurons. higher density of dendritic psd- clusters was observed in interneurons at div. at div, this difference was less prominent, mainly due to > -fold increase of psds in excitatory neurons. psd- -gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd- -gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps a-c two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in - div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, ; shigetomi & kato, ) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites ( s, -micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi ( ) ps a-c ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of d-and d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps a-c integrative spike dynamics of rat ca neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur connects with syntenin at the dendrites and mglur with pick at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur that was known to be ectopicly expressed in the dendrites of mglur -deficient on cone bipolar cells. we observed no pick but only syntenin at the mglur -deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur immunopositivity. these findings suggest that mglur connects with syntenin in place of mglur that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca + -permeable channels. trpv , expressed in sensory neurons, is activated by noxious heat above • c, whereas trpv , expressed in keratinocytes, is sensitive to moderate temperatures (> • c). here we examined the role of trpv and in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to • c did not induce the change in bt, desensitization of trpv with capsaicin resulted in the increase in bt. on the other hand, in trpv -knockout mouse, moderate thermal radiation (> • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv and respectively. research funds: kakenhi ( ) ps a-c generation and biochemical analysis of a glur␣ knockout mouse hirotsugu azechi , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan glur␣ (glur ) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣ , we generated a conditional glur␣ knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra f using c bl/ (b ) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra f was used as a target of glur␣ line. by crossing gra f and tlcn-cre that expressed cre in germ line cells, glur␣ null ko mice were produced, but most of them died within days after birth. to overcome the lethality, the glur␣ mutation was transferred onto b / or b /cd- genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣ in the distribution of functional ampa receptors in vivo. ps a-c gisp: a novel brain specific protein that binds to the gaba b subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b and gaba b from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b protein and also promotes gaba b surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b /gaba b complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b -binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut - ) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut and vglut seem to participate in this glutamate accumulation. in the present study, we found that vglut mrna is also expressed in the adult rat pinealocytes. vglut immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut -ir or vglut -ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito , , , kenji nakamura , toshikazu kakizaki , , satoe ebihara , masakazu uematsu , shigeo takamori , minesuke yokoyama , shiro konishi , masayoshi mishina , , jun-ichi miyazaki , kunihiko obata , yuchio yanagawa , gunma univ., maebashi, japan; sokendai, hayama, japan; sorst, kawaguchi, japan; mitsubishi kagaku inst. life sci., machida, japan; kumamoto univ., kumamoto, japan; toyohashi univ. tech., toyohashi, japan; tokyo med. den. univ., tokyo, japan; univ. tokyo, tokyo, japan; osaka univ., suita, japan; riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi ( ) ps a-c postnatal changes in the colocalization of vglut and vglut immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura , , akiya watakabe , hiroyuki hioki , fumino fujiyama , yasuyo tanaka , tetsuo yamamori , takeshi kaneko , dept. morphol brain sci., grad. sch. med., kyoto univ., japan; crest, jst, japan; div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut) and vglut accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut and vglut in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p . the cc was higher in area s than areas m and area v throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r and r . the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin ( -hydroxytryptamine, -ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several -ht receptor subtypes, -ht transporter ( -htt) and vesicular monoamine transporter (vmat ) in primary cultured mouse osteoblasts and mc t -e osteoblastic cells. sustained exposure to fluoxetine, a selective -ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc t -e . these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦ glutamate receptor (glur␦ ). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦ were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho j, was caused by a new type of intragenic deletion of the grid gene, which was indeed translated as glur␦ lacking -amino acids in the n-terminus. mutant glur␦ proteins were retained in the soma of purkinje cells and degraded. as a result, ho j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦ signaling and cerebellar functions. research funds: kakenhi ( ) ps a-d role of the dry motif in melanin-concentrating hormone receptor in signaling yumiko saito , yoshimi aizaki , mituse nakano , kei maruyama dept. pharamacol., saitama med. sch., saitama, japan; international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp , arg and tyr in the dry of rat melanin-concentrating hormone receptor (mch r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a r and p y r electrophysiologically using xenopus oocyte expression system. when a r and p y r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/ pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps a-d dual signaling via metabotropic glutamate receptor ␣ is regulated by a cytoskeletal protein . g michihiro tateyama , , yoshihiro kubo , department of biophysics and neurology, nips, aichi, japan; sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor ␣ (mglur ) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur are differentially regulated by different types of ligands, glutamate and gd + . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur . these proteins, such as homer and . g, are also known to change the membrane expression of and modulate the function of mglur . here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur . interestingly, the functional couplings of mglur to gq and gs pathways were altered by co-expression of . g, but not by homer. deletion of the c-terminal tail abolished the effect of . g, indicating that the interaction of . g with the c-terminal tail of mglur regulates the multi path signaling. ps a-d modulation of the eaac -mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto , , saori akiduki , age dimension research center, aist, ibaraki, japan; graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein - (gtrap - ), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier (eaac ). it contains two potential pkc phosphorylation motifs at positions - and - . however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac -mediated glutamate transport activity in c bu- cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine into alanine (s a) or at serine into alanine (s a). as compared with wt, s a had no inhibitory effect on glutamate transport activity under exposure to nm pma, and had increased glutamate transport activity under normal condition. by contrast, s a had the same glutamate transport activity as that of wt. thus, the eaac -mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine in addicsin. kaori akashi , manabu abe , toshikazu kakizaki , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, japan; sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤ - (glur - ) and glur␥ - (ka - ) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤ ko mice from c bl/ es cell line. the glur␤ ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥ and glur␥ proteins were significantly decreased in the crude fraction of ca region of glur␤ ko. furthermore, subcellular localizations of both subunits were also changed in glur␤ ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤ subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps a-d sema d/plexin-b activates gsk- ␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin d (sema d) receptor plexin-b induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b -mediated r-ras gap activity, leading to growth cone collapse. sema d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk- ␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk- ␤ antagonist suppressed the sema d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b -induced dephosphorylation of akt and gsk- ␤. plexin-a also induced dephosphorylation of akt and gsk- ␤ through its r-ras gap activity. thus, we conclude that plexin-b dephosphorylates akt and gsk- ␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur . hops was co-immunoprecipitated with grip (glutamate receptor interacting protein ) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur via grip binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc- . a neural amino acid transporter snat /ata also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc- immunoreactivity (asc- -ir) was detected in dendrites and somata of pyramidal neurons. the snat -ir was widely detected in neurons, whose intracellular localization was similar to that of asc- -ir. deferent from asc- -ir, snat -ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc- and snat to the neuronal uptake of l-serine. snat might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps a-d neuronal glutamate transporter eaat controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake , si-young song , shiro konishi , keiji imoto natl. inst. physiol. sci. (nips), okazaki, japan; mitsubishi kagaku inst life sci, tokyo, japan; tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat /glt- blocker threo- -methylglutamate, but not the glt- blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat was abundant. the results suggest that eaat plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat expression in postsynaptic pcs. chitoshi takayama , yoshiro inoue department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k- cl co-transporter (nkcc ) and k-cl co-transporter (kcc ). in this study, we focused on kcc , which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc with special reference to the neuronal development in the cerebellum. kcc was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc . the kcc -protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc -expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima , hisashi mori molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d -like and d -like according to their pharmacological properties. the d -like receptors consist of d and d receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop ( i loop) domain of d r fused with egfp effectively reduce camp production mediated by d and d receptors. interestingly, we also identified that the i loop domain of d r interfere with gs coupled beta adrenergic receptor signaling. these results suggest that the third intracellular loop of the d receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d -like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama , yoichi ogawa , hiroki yoshino , masayuki yamashita , toshifumi kishimto dept. psychiatr.; dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca pyramidal cells. da ( m) profoundly decreased gaba a receptor-mediated postsynaptic currents to % in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to % in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d -like receptor, since skf , a selective agonist for pi-linked d -like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d -like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina , jinko konno , sachine yoshida , hideki ohmomo , hitoshi kawano , fumihiro shutoh , haruo nogami , setsuji hisano graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut or - mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day (e ), vglut immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e , vglut -ir was seen as puncta close to l -ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l or tag -ir in the imz, whereas vglut -ir was first observed in the sp and upper imz where l -ir existed. these results show that vglut ir corticofugal fibers appose to elongating vglut -ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps a-d developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca regions michie kon , yoichi avil , hiroshi tsubokawa , dept. of information engineering, tohoku univ., sendai, japan; grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine ( . - mg/kg) produced a dose-dependent cpp. using a -day schedule of conditioning, it was found that intra-ca administration of nmda receptor antagonist, mk- ( and g/rat) significantly attenuated the morphine ( . mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda ( . , . and g/rat) significantly potentiated the morphine ( . mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk- receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura , kouichi nakamura , , hiroyuki hioki , masanori uemura , takeshi kaneko , dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d receptor are expressed in striatonigral neurons and d receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d , d , d , d , d ). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p - ) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since ( ) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, ( ) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, ( ) depolarizing agent released tpa from isolated neurosecretosomes, ( ) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u st, galnac - st, d st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda , maki ishii , isao nagata , yumiko shimazaki dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo- epitopes, glca( s)␤ - galnac( s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa , mineko kengaku , nobuaki maeda dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka , tohru marunouchi division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp or sulffp appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of -o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of -hydroxytryptoamine ( -ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day or rat cerebral cortex was cultured in the presence of -ht, na or da. after days, we analyzed dendrite formation using anti-map antibody. after - days, we analyzed synaptogenesis with anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( - nm), na ( - nm) or da ( - nm) increased dendritic length of pyramidal neurons. -ht ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by -ht a receptor, ␣ a receptor and d receptor, while inhibited by -ht a and ␤receptors. in addition, synaptogenesis was promoted by -ht a and -ht c receptors, whereas inhibited by -ht a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in t cell, singar associated with p and p , the subunits of pi kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps a-e lrfn , a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd- naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd- , a postsynaptic scaffold protein. we examined the physical interaction between lrfn and psd- by immunocrecipitation and pull-down assay, since lrfn contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn associated with psd- /nmda receptor complex in the brain extracts and lrfn directly bound to psd- via its pdz domain-binding motif. in this study, we suggest that lrfn may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt - ). among the prmt proteins, the prmt has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt in the rat brain. the prmt was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt in the brain, we tried to identify novel interacting proteins with the prmt in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps a-e proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi , , michihiro igarashi , div mol cell biol, grad. sch. med dent sci; trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi ; project-promoting grant from niigata univ ps a-e netrin- is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda , keisuke watanabe , kazuhiro ikenaka , katsuhiko ono , hiroyuki yaginuma dept. anat., fukushima med univ. sch. of med., fukushima, japan; div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin- is known to be a chemorepellent for a subset of axons, the role of netrin- for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin- in this repulsion. in the mouse embryo at e , netrin- is expressed in the floor plate and the dermamyotome, and the netrin- receptor unc c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin- repelled chick e drg axons. moreover, using function-blocking antibody against netrin- , we revealed the fact that netrin- plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin- to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p neonates, but the ventral neurons became unresponsive after p . the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe , hirohide takebayashi , , kazuhiro ikenaka , katsuhiko ono div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; dev stem cell biol. program, ucsf, usa netrin- is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin- also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin- −/− background, using olig -lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig +/lacz ;netrin- −/− mice, ventral migration of olig cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig -positive cells. these results suggest that netrin- exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g and netrin-g are vertebrate-specific membrane-anchored members of the unc- /netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g and netrin-g proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g and netrin-g differentially bind to lrrcontaining proteins, ngl- and a related molecule nag , in vitro. ngl- and nag in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g and netrin-g , respectively. furthermore, in netrin-g and netrin-g deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang , ryuichi sakai , teiichi furuichi lab. molecular neurogenesis, brain science institute of riken, saitama, japan; division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p -p , stimulation of parallel fibers produces excitation of purkinje cells lasting for - ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p -p . since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p and that they then locate in purkinje cells, in cell body on p and in primary dendrites on p -p . the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya- and mmahya- ) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya- protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya- is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya- for the early cerebellum development. toshitaka morishima , erina fukushi , kazuto kobayashi , naohiro hozumi , sachiko yoshida toyohashi university of technology, toyohashi, japan; honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto , hideyuki dekimoto , tomiyoshi setsu , masahiko watanabe , mikio hoshino , yo-ichi nabeshima , toshio terashima dept. of anat., kobe univ. grad. sch. of med., kobe, japan; dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida , tomoko nishiyama , hitoshi tatsumi , masahiro sokabe , department of physiology, nagoya university graduate school of medicine, nagoya, japan; icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately h and the elongation started at approximately h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca . once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca , which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) or e rat. dendritic outgrowth of pyramidal neurons was analyzed after days using anti-map antibody. synapse formation was analyzed after - weeks, using anti-psd- and anti-synaptophysin antibodies. in the presence of cgrp ( - nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of hydroxytryptoamine ( -ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day rat hippocampus was cultured in the presence of -ht or na. after days, we analyzed formation of dendrites using anti-map antibody. after days, we analyzed formation of synapses using anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( nm) or na ( nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites -ht ( - nm) and na ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, -ht a receptor and ␣ a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di- anepeq) and the imaging system (micam ). in dscam-ko mice, the optical signal which precedes c inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps a-f hippocampal cells cultured on d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after weeks in vitro, neurons formed a dense layer, > . mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, ( ) collagenasespecific inhibitor did not affect cell depth, ( ) limiting gliosis by antimitotics reduced the thickness of the tgl by %, ( ), neither glial nor neuronal cell body were found in the tgl by gfap/map detection, ( ) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca + by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama , kazuto kujira , tetsuya kawabe , takao ito , yoshihiro tsuruo department of cell biology and anatomy, wakayama medical university, wakayama, japan; department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) ( ) ps a-f metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama , hitoo nishino , takehiro yahata nagoya city university graduate school of medical sciences, nagoya, japan; nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit , anand goswami , nobuyuki nukina , nihar ranjan jana national brain research centre, india; laboratory for structural neuropathology, riken brain science institute, - hirosawa, wakoshi, saitama - , japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin- for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc . finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of , ests ( , non-redundant sequences). of them matched known genes of rodents in the genbank databases, but remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of , , or weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps a-f transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb- , an inhibitor of the type tgf-␤ receptor (t␤r ). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps ( h restraint; min forced-swimming; ether anesthesia) then they were put in untouchable situation for days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c bl/ j mice. pups were separated from mothers daily ( h) on postnatal days through . starting at weeks of age they were tested for ( ) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; ( ) responses to social stimuli in social investigation (sit) and social preference tests; and ( ) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of db (so), or were forced to swim (sw). these manipulations were performed for min once a day at weeks after birth (from postnatal days to ) or weeks after birth (from postnatal day to ). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of weeks. it was found that the spatial learning activity of both so and sw mice manipulated weeks after birth was impaired as compared to no mice. so mice manipulated weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo , yukiko yamada , mitsuo iinuma , yasuo tamura , fumihiko iwaku , kazuko watanabe , minoru onozuka dept. oral anat., asahi univ. sch. dent., japan; dept. ped. dent., asahi univ. sch. dent.; dept. physiol., gifu univ. sch. med., japan; dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara , yayoi kitamura , junichi tanaka , yukio hattori , yasushi hayashi department of human nutrition, notre dame seishin university, okayama, japan; department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c bl/ j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a -min interval, to measure the basal temperature (t ) and stress-enhanced temperature (t ), respectively. the difference ( t = t − t ) gives the sih. in control mice, t was approximately • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t . furthermore, spermidine and spermine also lowered t and t at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox- and cox- . it has been known that, under some stressful conditions, cox- is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for h and subjected them to the elevated plus maze task h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox- by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps a-g imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang , takesi sakurai , yasuitirou fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. pharmacol., univ. tsukuba, japan; dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of - weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between and weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between and weeks of age, and in the hippocampus at the age of weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi # ps a-g a systematic analysis of genetic factors associated with behavioral diversity between msm and c bl/ koide tsuyoshi , , aki takahashi , , toshihiko shiroishi , , akinori nishi mgrl, national institute of genetics, mishima, japan; sokendai, hayama, japan; mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c bl/ . in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b - cmsm, which carries chromosome of msm, showed extreme behavioral differences from c bl/ . the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga , yu kajiyama , shigenobu shibata , hiroshi kunugi department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e ) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e ) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps a-g synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi ( ) ps a-g involvement of nr b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary , takanobu nakazawa , yuji kiyama , toshiya manabe , tadashi yamamoto div. of oncology, ims, univ. of tokyo, tokyo, japan; div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr b is tyrosine-phosphorylated, with tyr- being its major phosphorylation site. to investigate the role of tyr- phosphorylation, we generated mice with a tyr phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr- and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr b is involved in the control of emotional responses by regulating crf content. ps a-g increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca + signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for weeks, locomotor activity and longinterval ( , and ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui , , yosky kataoka , , yasuhisa tamura , yasuyoshi watanabe , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at - times/min), inhibition periods (less than times/min) were often observed h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns- , a selective cox- inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps a-g impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima , naoya aoki , andras csillag biology, hokkaido univ., sapporo, japan; agriculture, nagoya univ., nagoya, japan; anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task , chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to s. in task , chicks chose between small reward located at cm and large reward at − cm, where cues signaled the distance of invisible food. task was similar to the task , except that cues signaled the food quantity. in task , total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi ( , ) ps a-g analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe , which had a missense mutation in the casy- gene. several casy- deletion mutants also showed learning defects. casy- has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x l/mint . however, after dissociation of x l, alcadein is susceptible to cleavage by protease(s). we found that casy- was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy- can also be proteolytically cleaved. ps a-g insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama , atsushi kuhara , akiko mohri , , kotaro kimura , , masatoshi okumura , masahiro tomioka , yuichi iino , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; mol. genet. res. lab., univ. of tokyo, japan; inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins- encoding insulin homologue caused defective associative learning, mutations in daf- and age- encoding the homologues of insulin receptor and pi -kinase, respectively, suppressed the defect of ins- , and the mutation in daf- encoding forkhead transcriptional factor caused the learning defect. this suggests that ins- antagonizes daf- insulin-like signaling for associative learning. interestingly, age- animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age- in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins- . we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps a-g analysis of ttx- : novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx- (nj ) and ttx- (nj ) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx- encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx- is similar to ric- , identified in c. elegans at first and conserved among several species (halevi et al., (halevi et al., , . ric- is thought to be required for the maturation of acetylcoline receptor (halevi et al., ) , so ttx- may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps a-g analysis of aho- mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio , akiko mohri , , eiji kodama , atsushi kuhara , mizuho koike , kotaro kimura , , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho- (nj ) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., ) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho- gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho- is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho- gene further. we have demonstrated aversive conditioning in lymnaea using mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b , located in the buccal ganglion. an associative memory, lasting h, was produced with pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura , dai hatakeyama , tetsuro horikoshi , etsuro ito , manabu sakakibara lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno , minoru saitoe , , tim tully tokyo metropolitan institute for neuroscience, tokyo, japan; department of biology, tokyo metropolitan university, japan; cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi ke zhang , jian z. guo , ai k. guo , institute of neuroscience, chinese academy of sciences, china; institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal /uas-shi ts system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (< min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari , , kazuo okanoya , , lab. for biolinguistics, riken-bsi, wako, japan; grad. sch. of sci. and tech., chiba university, chiba, japan; presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day , developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps a-h facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi ps a-h the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment , rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of . m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment , rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi ( ) ps a-h transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps a-h gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c bl/ mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over %, while those in the mscand ptx-injected mice decreased to %. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦ that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps a-h post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao , , takehito okamoto , fumihiro shutoh , lab for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan; dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of h daily training for week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi ( ) ps a-h modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri , , takehito okamoto , shin-ichi nishimura , fumihiro shutoh , , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; univ of the air, chiba, japan; dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., ) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps a-h influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho , , t kobayashi , , e hori , , k umeno , , t ono , h nishijo , system emotional science, univ. of toyama, toyama, japan; crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of place cells recorded, activity of was significantly modulated by the car speed and/or distance between the car and rat; , and cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a . - . mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas and , contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca + but is blocked by mg + in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg + binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l is a component of the ubiquitin system. uch-l is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl . gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca . in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps a-i learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage ). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band ( - hz) was strong at stage and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps a-i longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about . mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal ubiquitously expresses in brain. bmal functions by forming a heterodimer with either clock or npas , which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal function in a forebrain. using a dominant negative mutant of bmal (bmal r a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., ), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal r a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn ( , , or db; intermittent white noise). db bgn induced significant decreases in number of error and time to goal in maze compared with and db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn , , anucha pongpanparadorn , wasana pratchayasakul , anchalee pongchaidacha , nipon chattipakorn fac. dent. cmu, chiang mai, thailand; cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were ± , ± and ± in the groups administered , and mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was . ± . , . ± . and . ± . for , and mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a -h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a -h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses ( , and min). percentage inhibition of cache activity following a single tde injection at doses and mg/kg significantly decreased at and min after tde injection, but not at min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣ and ␣ -nachr and kinases involved in pi k-akt pathway, and increase the levels of phosphorylated akt and bcl- . these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi k-akt pathway via ␣ and ␣ -nachrs. ps a-i arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae , yoshiyuki ishikura , shigeaki fujikawa , yoshinobu kiso , manabu sakakibara lab. neurobiol. engr., tokai univ., numazu, japan; inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in -month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps a-i thimet oligopeptidase co-exists in gfap-and cd b-positive glia in rat pc/rsc treated with mk- takeshi kato , mohammad arif , michiyuki yamada , toshiyuki chikuma , md. mahiuddin ahmed lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; grad. sch. integr. sci., yokohama city univ., yokohama, japan; dept. hygien. chem., showa pharmaceut. univ., machida, japan; dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep . ) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk- activated gfapand cd b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) day after the treatment. mk- also increased ep . and prolyl oligopeptidase. immunohistochem data showed that ep . co-localized with gfap and cd b positive glial cells. since mk- causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep . in the pc/rsc. these data suggest that ep . in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps a-i effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) and in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) and , code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps and ps with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps a-i effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of -week-old adult mice. in this study, a␤ peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤ peptide. we found that treatment of nsc with oligomeric form of a␤ peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤ peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein- (hmgb ) is a non-histone chromosomal protein. here, hmgb was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb immunoreactivity was observed around dying neurons in the kainic acid-and a␤ - (a␤ )-injected rat hippocampi. hmgb was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb delayed the clearance of a␤ and accelerated neurodegeneration in a␤ -injected rats. these results suggest that hmgb released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps a-i x -like protein regulates metabolism of app in the mouse brain yoshitake sano , , tadashi nakaya , shigeyoshi itohara , toshiharu suzuki riken bsi, saitama, japan; hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x -like protein (x l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells ( . j. biol. chem. , . j. biol. chem. , ) . there is no evidence, however, that endogenous x l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x l in app metabolism in the brain, we generated x l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin- protein by d-electrophoresis and tof-mass delineate that mutant at and htt proteins similarly reduce transcriptional factor x and x . immunoprecipitation and pull-down assays support interaction between polyq and factor x and x . immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x and x into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x and x ameliorates poly-q pathology in htt-/at -expressing neurons and transgenic drosophila. these results suggest that factor x and x are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps a-j ba - was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, rats/group (g). group received e at . g/kg per day; groups and received ycj at ml, and ml/kg day, respectively, once everyday. group received ycj ml/kg plus e at . g/kg day twice a week, all for weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e in group was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid - antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer and layer of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group was lesser than ovx and control groups. amyloid deposition was correlated with e serum at r = − . . ps a-j correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki , shigeo kinomura , kazunori sato , shinya uchida , ryoi goto , kentaro inoue , ichiro tsuji , hiroyuki arai , ryuta kawashima , hiroshi fukuda department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; tohoku univ. grad. school of med., sendai, japan; niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h -kenko- ), (h -choju- , h -choju- ) ps a-j effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook , d.g. goodenowe , y. yamazaki , j. flax phenomenome discoveries inc., saskatoon, canada; precisionmed inc., san diego, ca, usa dementia affects about % of the population over the age of and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an -metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the -metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin- (chr ). in this study, we quantitatively investigated the rapidness of the light-gated current of chr expressed in pc cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about -fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori , shigeki ohta , kenji hamada , naofumi yamada-okabe , yonehiro kanemura , hideyuki okano , yutaka kawakami , masahiro toda , neuroimmnology research group, keio univ., tokyo, japan; chugai co. ltd., kanagawa, japan; inst. cli. res., onh, japan; physiology, keio univ., tokyo, japan; cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day were dissociated. after the cells were treated with nm estradiol- beta (e ) or ethanol for days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis ( d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than spots were detected from d dige. compared with ethanol treatment, e increased the expression of spots in the hypothalamic cells and spots in the cortical cells (p < . , difference > . ). proteomics analysis showed different effects of e for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp client protein family. -allylamino- -demethoxygeldanamycin ( -aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. -aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of -aag in a mouse model of sbma. administration of -aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of -aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp function by -aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately - % of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps a-k selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura , yoshiyuki ishii , kazuto kobayashi , yasufumi sato , keiichi itoi lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; dept. mol. genet., fukushima med. univ., japan; inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a , a and a ) and dopaminergic (a , a and a ) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg b has been shown to play an important role in the processing of lc , a mammalian homologue of yeast atg , but the tissue distribution of atg b remains unknown. to understand the role of atg b in rat tissue cells, we prepared an antibody to atg b and pc cells in which atg b expression was knocked down by rnai. in rnaitreated pc cells where atg b expression was % of that in the wild-type pc cells, the expression of cytosolic lc -i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc to lc -i. moreover, the expression of atg b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg b was intense in neurons, especially in the cerebellum. these results suggest that atg b plays a major role in the processing of lc , while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps a-k spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev n strain into the central nervous system was examined after its subcutaneous inoculation ( pfu) in the rat hind footpad. on day post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki , hiroshi kameda , hisashi nakamura , taro okunomiya , koji ohira , kouichi nakamura , , takahiro furuta , takeshi kaneko , dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn , rta , mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣ and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr increased the surface expression of glur in cos cells compared with grip/glur or ␦-catenin/glur transfection. this study points to ␦-catenin as a regulator of glur receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca area in wild type (wt) and gad knockout (ko) mice. this parameter was twice lower in gad ko mice (wt: . ± . pa/pf, n = ; gad ko: . ± . pa/pf, n = ; p = . ). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba ( m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad ko animals (wt: . ± . ms, n = ; gad ko: . ± . ms, n = ; p = . ). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p to p ) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur but not by mglur because it was antagonized by the mglur antagonist ly but not by the mglur antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps p-a involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita , michiko ohtomi cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d antagonist sch into pfc by using a microdialysis method impaired md-pfc ltp but the d antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by - %). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer / pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya - , japan we showed in previous study that somatic inhibitory synapses of neocortical layer pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate ( nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr in the regulatory mechanisms for ltp in the amygdala, we utilized ntr -knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr -ko mice than in wild-type mice. the bla-ltp in ntr -ko mice was attenuated by sulpiride, a d receptor antagonist. these results suggest that d -ntr interaction regulates the extent of ltp in the mouse la-bla synapses. ps p-a facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over -day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca + sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca + at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin- , and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps p-a inhibitory modulation of the hippocampal ca transmission and plasticity by glucagon-like peptide- jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode- (glp- ) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp- (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp- effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp- on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices ( m) were prepared from to days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca in the whole-cell mode using glass microelectrodes (resistance: - m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp- ( nm- m) inhibited spontaneous excitatory postsynaptic current. glp- ( nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp- may play an inhibitory role in the dg-ca transmission. ps p-b quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp- and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a , a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, hz for s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, hz for s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of m apv, m bicuculline, but not m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at − mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine ( - m) or capsaicin ( . - m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin ( m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv ), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji , akira komatu , akinao nose dept. phys., univ. tokyo, tokyo, japan; dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca + -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr * flies did not show mg + blockade, and dnr * flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf- , and notch. we examined how mg + blockade of dnr links to these transcription factors. research funds: kakenhi ps p-b response properties of wind-sensitive giant interneurons in the th-instar nymphs of the cricket tetsuya matsuura , masamichi kanou dept. of welfare eng., iwate univ., morioka, japan; dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) - , - , - and - in the th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the response magnitudes of gi - reached a plateau at a stimulus velocity of mm/s in most stimulus directions. the response magnitudes of gis - and - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi - , the ipsilateralfront and contralateral-rear in gi - , the ipsilateral-rear in gi - and the ipsilateral-front in gi - , designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca . serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca + , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca region. we found that ptp induced by the -hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca + channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca + channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma , kunihiko kaneko , , makoto asashima , grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca + concentration ([ca + ] i ) may change because of nonsynaptic communication. we loaded pc cells with a ca + indicator, indo- am, and the [ca + ] i of more than , cells was measured using flowcytometry. the [ca + ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca + ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca + -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss from human and mouse cns. in mouse cns, four isoforms (types - ) were expressed. subcellular localization analysis revealed that type (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc and nb a derived from neuronal cell express only type , whereas os and kt- derived from astrocyte express both type and type . interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os and kt- . we analyzed promoter region of m-spinesin gene, and identified that -flanking region from − to − bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme -phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca + signals, such as intra-and intercellular ca + waves; however, their physiological roles remain elusive. the overexpression of an ip -hydrolyzing enzyme, ip -phosphatase, suppressed the spontaneous ca + signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca + -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca + -signal-deficient astrocytes. these results indicate that spontaneous ca + signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai , noriko osumi , tohoku univ. sch. med., japan; crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax in postnatal mouse brain and found that pax was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax heterozygote mutant (sey/+) mice at weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, and h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., ) . we previously reported a unique expression of dim ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., ) . we show here that mab dim also recognizes adult neural stem cells and glial cells at postnatal period. dim , brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad / / (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk . lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in s and s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in -ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after h ( and %) and h ( and %) exposure to − and − m -ohda respectively. it was accompanied by morphological changes and induction of gfap, s- and ox . -ohda ( − m, h) was found to cause a significant impairment in h glutamic acid uptake ( %) and gsh levels ( %). further neurons (in coculture with -ohda pre exposed glial cells) on exposure to -ohda ( − m), showed loss of th expression and significant neuronal cell death ( %). the results of the present study suggest that -ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at day after injection, then started to repair at days and returned to the normal levels at weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at ∼ days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng ) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba + cells accumulated in stab wounds and ischemic lesions were found to express ng , of which molecular weight of its core protein was higher by kda than that of ng expressed in contralateral brain region. this was due to the lack of shedding of ng in the brain lesions. we found that iba + cells accumulated in stab wounds and ischemic core lesion, most of which were ng +/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd and von willebrand factor. ng + mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng + mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from < to m . most of the fibers were myelinated and counts produced a mean of , ( , - , ). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from . to . m (from fetal age to years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p - . costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca , ca pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps p-d impact of s b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani , akiko seto-ohshima , shigeyoshi itohara , hajime hirase neuronal circuit mechanisms research group, japan; lab. for behavioral genetics, bsi, riken, wako, japan s b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s b, we have recorded hippocampal spontaneous activities from urethane anesthetized s b ko and wt mice. typical eeg patterns including theta ( - hz) and sharp wave associated fast ripple ( - hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca radiatum, where ictal event was characterized by hyper-synchronous gamma band ( - hz) activity. while both populations developed ictal event within min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s b related pathways could potentially be activated. yoshiko takagishi , erina okabe , xiaoyang sun , sen-ichi oda , yoshiharu murata riem, nagoya univ, nagoya, japan; grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps p-d regulation of hippocampal neurocircuit activity by glutamate transporter glt- noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt- , the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap ) and the glt- blocker (dhk) with the signal subtraction, we could visualize the activity of glt- as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt- by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt- function in the ca region was elicited by much lower concentration of dhk than that in the ca region. ps p-d monoclonal antibody rip specifically recognizes , -cyclic nucleotide -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg- cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was , -cyclic nucleotide phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg- cells. moreover, the same localization was also observed in rat cnp transfected hek t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca + signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca + signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca + signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki , , hisatsugu koshimizu , megumi kashihara , tomoko hara , masami kojima , research institute for cell engineering; sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that -day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p ) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p expressed in the neural progenitor cells at embryonic days (e ) mice brain. to ascertain the function of p on these cells, we treated the cultured e cells with the selective chemical inhibitor for p (sb ) for days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit , takashi yamazaki , , takeshi oya , yoko ishii , masakiyo sasahara pathology ii, university of toyama, toyama, japan; oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on to days after injury, and activation of pdgfrs, mapks, akt and p were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from to days after injury. p-tyr was highly detected from to days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to days after injury and then gradually decreased. activation of akt and p continuously increased from to day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln is a cerebellum-specific protein structurally related to the c q and tumor necrosis factor families. recently, we have shown that cbln is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to -untranslated region (utr) of bdnf mrna to investigate the effect of the utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps p-e bmp expression in the adult rat brain bone morphogenetic protein- (bmp ) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp expression in the adult rat cns using immunohistochemistry. bmp is intensely expressed in most neurons and their dendrites. in addition, intense bmp expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp protein. these data indicate that bmp is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp plays pivotal roles also in the adult brain. ps p-e hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl- protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl- expression, decreasing the level of bax expression in c bl/ mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase . here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of -methyl- -phenyl- , , , tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda , yoko tohyama , shinichi kohsaka , tadashi kurihara , kazuyuki nakajima , soka university, hachioji, tokyo, japan; dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt- , and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤ among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba , chiaki nakagawa , masako tsuzuki , motoko matsuda , satoshi ichisaka , yoshio hata inst. dev. res., aichi human ser. ctr., kasugai, japan; div. neurobiol., tottori univ., sch. med., yonago, japan; div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats h after beginning an -h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin- (nt- ) in selected brain regions from -day-old male rats and compared to those from adults. the changes in levels of bdnf and nt- were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci ( kdyne, ih injury device) at t / and were assigned to three groups; a (placebo), b (mp treated; mg/kg i.v. immediately after injury), c (g-csf treated; g/kg i.v. for days after injury). animals were assessed with the bbb locomotor rating scale for w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi ( ) ps p-e glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons ( div) exposed to glutamate for min at m. glutamate exposure led to a decrease in mrna of nd and nd , which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least - h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk- . these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj- is enhanced by oxidative stresses. although exact functional significance of dj- has still unknown, it is thus proposed that dj- is protective against neural damage under oxidative stresses. in this study, we tested expression of dj- in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj- was markedly increased in the molecular layer of the dentate gyrus on days and post tmt injection. on day post tmt injection, enhanced expression of dj- was observed in the stratum lucidum of the ca . in glutathione-depleted mice, tmt was more effective in enhancing expression of dj- , compared with that in untreated mice. furthermore, double staining of dj- and gfap demonstrated that most of cells highly immunoreactive to dj- were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj- is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt ( . mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus days later, with visually apparent recovery of the granule cell layer days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone ( . - mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of . mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb ( g/ml) on the pc cells was elicited through apoptotic cell death. activation of caspase- / was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin ( nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase- / activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc cells. ps p-e kinesin superfamily protein (kif ) regulates activity-dependent neuronal survival by suppressing parp- enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein (kif ), a microtubule-based molecular motor. the c-terminal domain of kif is a module that suppresses the activity of poly (adp-ribose) polymerase- (parp- ), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif from parp- , resulting in upregulation of parp- activity, which supports neuron survival. after dissociation from parp- , kif enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. research funds: ps p-e expression of hsp , apg- , and apg- in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt ( . mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp , apg- , and apg- in the granule cell layer of the dentate gyrus, but not in ca -ca pyramidal cell layer, h to days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp highly expressed in survival neurons in the culture, apg- and apg- highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day . thirty gray of x-irradiations were performed on the cultured cells at or days in vitro (div). then the cells were fixed at h after the irradiation with dapi. at -div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at -div. this indicates that the radiosensitivity of -div immature neurons is higher than that of -div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦ glutamate receptor (glur␦ ), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦ is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca + , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin- and found a novel gene down-regulated in response to mutant ataxin- in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca molecular pathology. ps p-f permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca + -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca + -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways. interestingly the up-regulation of bcl- , a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat probe. scat is an indicator protein for caspase- activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase- cleavage sequence. using this probe, we could monitor the activation of caspase- at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt ( . mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex days later. in cortical neurons, tmt exposure for h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases and , but not caspase , were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride ( . mg/kg, i.p.) in -( w) and -week-old ( w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in w mice. by contrast, slight, diffuse neuronal damage was found in the ca and ca subfields and dentate gyrus of w mice. the hippocampal putrescine level was elevated markedly in w mice on tmt administration, whereas a minor putrescine increase was detected in w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps p-f establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai , toshinori hongo , kimisato naito , shigeto sasaki department of physiology, kyorin university, school of medicine, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for . - . s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of . s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in volunteers. experiment was performed by three tasks, control task is a forearm rotation, task is simulation of tools-manipulation, task is execution of tools-manipulation. we found different brain regions by this experiment. task to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at khz, to record simultaneously and independently the • horizontal saccades of each eye for subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi ( ) ps p-f dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼ hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from healthy subjects, high and low scorers (n = each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band ( - hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a -t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = . s) with online monitoring of meps. the intensity of tms pulses was varied from % to % at a % step (frequency at ∼ . hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above % stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = . , p = . ). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps p-f shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda , , yukihiro gomi , itsuki imai , nobuaki shimoda , , hiroyuki kato , international university of health and welfare, ohtawara, japan; crest, jst, kawaguchi, japan; nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels ( channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of s task and s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky , , rieko osu , hiroshi imamizu , mitsuo kawato , computational brain project, icorp, jst, japan; atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed hz isometric force pulses to seven targets separated by • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a d strain gauge. twenty repetitions of each condition were performed in s blocks (tr = s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only . % (chance: . %), errors were distributed normally about the target such that . % (chance: . %) of the data was classified correctly to within • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from • c, fish showed repetition of short time swimming in the range . - • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in - days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps p-f to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu , ken-ich morishige , jun nakanishi , , hiroyuki miyamoto , mitsuo kawato atr computational neuroscience labs, kyoto, japan; kyushu institute of technology, japan; jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p pups of the surviving cases increased greatly between p ( % of the control value) and p ( %), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p , dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p and p pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p to p . we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully ( • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative , , and days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps p-g role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa , tatsumi nagahama dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps p-g neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto , sayaka hihara , kazuo okanoya , atsushi iriki lab for symbolic cognitive development, bsi, riken, japan; lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about . • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps p-g prostaglandin e -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, - - toyama, shinjuku, - , japan unilateral microinjection of pge into the region around the rostroventral wall of the third ventricle (av v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of - pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide ( . mm, nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge injection site was examined. this treatment alone had no effect on the o consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge -induced responses were blocked min after the pretreatment with bicuculline, and recovered at ∼ min. pretreatment with vehicle saline had no effect on the pge -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge -induced thermogenesis. tetsufumi ito , hiroyuki hioki , kouichi nakamura , takeshi kaneko , yoshiaki nojyo dept. anat., univ. of fukui, fukui, japan; dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad -ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad -positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad . we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad -ir. after cutting t -t vrs and drs, almost all gad -ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama , ning ma , reiji semba , satoru naruse dept. of anat. ii, mie univ. school of med., tsu, japan; inst. for developmental research, kasugai, japan; dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin (aqp ) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp -immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp -ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp -ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp -ir fibers were found both in the myenteric and submucosal plexi. many aqp -ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp -ir fibers penetrated into the muscle layers. these results suggest that aqp -ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi ( ) ps p-g abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days - . the increase in the volume and frequency setting of the artificial ventilator (fio = %, fico = %) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad -gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = ) in medullary transverse slices. about % of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about % of gabaergic neurons recorded in the superficial rvm were co inhibitory, and all of the remaining neurons were co insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with and mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with mv forehead spla, in comparison to subgroups mv forehead spla. such law was common for both for sg and for cg. in two subgroups of and mv nasal zone spla in sg the greater rt gain after il was in mv nasal spla subgroup, and for cg in mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin ( ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via ht receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c bl/ n) were pretreated with pcpa ( . g/ ml/kg body weight) or saline intraperitoneally for consecutive days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o inhalation was changed to stepwise , and % co in o inhalation every min. hypercapniainduced increases in tidal volume and minute ventilation during % co inhalation were reduced by pcpa pretreatment; these results suggest that ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a . % gosha-jinki-gan diet group. after weeks, continuous cystometry with physiological saline or . % acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with . % acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps p-g differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to db white-noise for min. db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase (gad ), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were . on the small neurons, . on the medium-sized neurons, . on the large neurons, and . on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about % of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large ( . m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps p-h neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c nerve and facial nerve activities. addition of mm k + to the standard superfusate decreased burst rate of c activity. addition of or mm k + caused initial inhibition of c burst and subsequent high frequency c burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi ( ) ps p-h regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp ( m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at − mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin receptor (nk r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps p-h effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min- cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm- , shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda , usa) at the right middle finger from volunteers ( . ± . years). mild thermal stimuli ( ± • or ± • ) were administered every min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm- . we examined the effects of color environments on cognitive function in healthy subjects and patients after traumatic head injury using p components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p latency in the red environment was significantly shorter in controls than in patients. the p amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps p-h neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi , , mikinobu takeuchi , , takahiro omote , naoyuki yosimura , etsuro hori , , kazuo sasaki , taketoshi ono , , hisao nishijo , system emotional science, univ. toyama, toyama, japan; crest, japan science and technology agency, japan; bio-information engineering, univ. toyama, toyama, japan; molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems ( channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate ( ) human facial stimuli with different gaze directions and ( ) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi , sachiko koyama , ayumu matani , andrzej cichocki riken, wako, japan; hokkaido university, sapporo, japan; university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: ( ) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. ( ) the functions were averaged across subjects. ( ) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. ( ) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi ( ) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps p-h the reflection of category perception of sound in the auditory evoked n m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for different fundamental frequencies (f ) and different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n m source strength for nonlinear sounds was significantly larger than that for linear sounds. n m peak latency only for vocal sounds was not affected by f . these results suggest that perceptual categorization was reflected in n m source strength and location (linear or nonlinear), and in n m latency (vocal or nonvocal). sunao iwaki , hiroko kou , kouichi sutani , mitsuo tonoike national institute of advanced industrial science and technology, osaka, japan; chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p , related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p components, suggested the involvement of frontal and anterior cingulate cortex in the early p component (p a) and the contribution of the parietal and temporal regions to the later component (p b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l and l , respectively). we compared the brain activity of chinese learners of japanese as l with that of japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm . while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l and l . neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a . t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya , mie matsui , , tsuneyuki kobayashi , , hisao nishijo , , michio suzuki , , yasuhiro kawasaki , , masayoshi kurachi , , jun nakazawa , kyo noguchi , hikaru seto neuropsychiatry, univ. toyama, toyama, japan; crest, japan science and technology corporation, japan; psychology, univ. toyama, toyama, japan; system emotional science, univ. toyama, toyama, japan; neuropsychiatry, univ. toyama, toyama, japan; developmental psychology, univ. chiba, chiba, japan; radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura , atsushi sekiguchi , keisuke wakusawa , , yuko sassa , , hyeonjeong jeong , , kaoru horie , , shigeru sato , , ryuta kawashima , miyagi university of education, sendai, japan; niche, tohoku univ., japan; dep. pediatrics, tohoku univ. school of medicine, japan; ristex, jst, japan; gsics, tohoku univ., japan; lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain , , points or lose , , points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps p-h brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara , masato taira , , toshio iijima , ken-ichiro tsutsui div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; arish, nihon university, tokyo, japan; appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either yen or a gain or loss of , , or yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps p-i neural activation during experience-based reasoning chisato suzuki , , takashi tsukiura , hiroko mochizuki-kawai , yayoi shigemune , , toshio iijima neurosci. res. inst., aist, japan; div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps p-i the difference of brain activity elicited by different styles of art hiromi yamamura , yasuyuki kowatari , , shigeru yamane , miyuki yamamoto , comprehensive human sciences, university of tsukuba, tsukuba, japan; system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm . while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba , ba ). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j- ) ps p-i effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were - aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi , ps p-i the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo , michio nomura , jun kawaguchi ntt commun. sci. labs., ntt corp., atsugi, japan; dept. psychol., tokai women's univ., kakamigahara, japan; dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo , , , yulri nonaka , katsumi mizuno , kazuo okanoya , riken, bsi, biolinguistics, saitama, japan; chiba university, chiba, japan; jsps, japan; department of pediatrics, showa university, tokyo, japan; presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for neonates within days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three ( ) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around - ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism ( : - : ) and agematched typically developing children ( : - : ) were instructed to name pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = . ± . , loudness = . ± . ) than in the children with autism (duration = . ± . , loudness = . ± . ) (p < . ). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at . /s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser ( nm, mw) to the muscle surface ( . mm ) significantly delayed its attenuation (p < . ). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < . ). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps p-i nedl , a novel e ubiquitin ligase for dishevelled- , targets mutant superoxide dismutase- and interacts with p yuanyuan li , , , kou miyazaki , toshinori ozaki , akira nakagawara division of biochemistry, chiba cancer center research institute, chiba, japan; production technology development center, the furukawa electric co., ltd., ichihara, japan; hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e ubiquitin ligase gene termed nedl . previous study has shown that nedl is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled- , a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl for uniquitination and proteasome-mediated degradation. on the other hand, nedl bound and ubiquitinated mutant (but not wild-type) sod in a mutant sod type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl physically bound p , and induced apoptosis in a p -dpendent manner. taken together, our results suggest that nedl may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod and p . spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc interacting protein), u-box type e ubiquitin ligase, has been shown to interact with hsp or hsp and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca + current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox- inhibitor, rofecoxib in pentylenetetrazol (ptz, mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the th day of ptz treatment. chronic treatment with rofecoxib ( . and . mg/kg, p.o.) for days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced ( mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin ( and mg/kg, p.o.), naproxen ( and mg/kg, p.o.), nimesulide ( - mg/kg, p.o.) or rofecoxib ( - mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib ( mg/kg) or nimesulide ( mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox- inhibitors. cox- inhibitors also antagonized the effect of flumazenil ( mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps p-j cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps p-j a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus ( mg/ min). after min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that -oxoguanine ( -oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that -oxog accumulated greatly in mtdna of the ca microglia. wild-type and mth -null mice, the latter lacking an ability to hydrolyze -oxo-dgtp and -oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca neuron loss after kainate administration, however, levels of -oxog accumulated in mtdna and cellular rna in the ca microglia were significantly increased in mth null mice in comparison to wild-type mice. we thus demonstrated that mth efficiently suppresses the accumulation of -oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps p-j transcription factor nrf regulates brain response to kainate-induced excitotoxicity yukihiko dan , kosuke kajitani , noriko yutsudo , ken itoh , masayuki yamamoto , yusaku nakabeppu kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e related factor (nrf ) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf has been reported to increase neuronal death under increased oxidative stress, nrf seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca subregion of hippocampus. to unveil the functional significance of nrf in the brain, we compared seizure responses between wild-type and nrf -null mice after systemic kainate administration. we found that nrf -null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps p-j synaptic plasticity and -aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine ( -ap) at the concentration below . mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the -ap induced discharges in ca region was studied. ten m -ap induced the epileptic discharges with the frequency of . ± . hz (mean ± s.e.m.; n = ) and the amplitude of . ± . mv. when ap- , an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap- was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of -ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps a-a influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno , hugh p.c. robinson engineering science, osaka university, osaka, japan; university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣ a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the -day-old rat pup spinal subarachnoid space at the rate of l/ . min. the rats were sacrificed h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣ a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps a-a on the membrane potential profile of ca pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga , , yoko tominaga dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; lab. for dynamics of emergent intelligence, riken bsi, hirosawa - , wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps a-a overexpression of inwardly rectifying k + channel . in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) . have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca pyramidal neurons. then, egfp and kir . expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer / neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types and of ryanodine receptors (ryr and ryr ) in the cerebellum of a ca + channel ␣ a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr but not ryr in the rmn cerebellum: a less ryr mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr -mediated ca + release, contributing altered ca + homeostasis in the rmn purkinje cells. ps a-a dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than s. d receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi , , hiroyoshi miyakawa , masashi inoue , masato okada , tokyo institute of technology, japan; brain science institute, riken, japan; tokyo university of pharmacy and life science, japan; the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai , maya yamazaki , , mika tsujita , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan we previously reported that stargazin family (␥ , ␥ , ␥ , and ␥ ) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., ) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥ subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥ subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day , while the reduction was not so significant in adult brain. these results suggested that ␥ might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph . ) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop ). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop -transfected cells. mvsop induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn + sensitivity. neutralization of a positive charge in the s -like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh ). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca region. in the pyramidal layer, the amplitudes of the responses to the -hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the - -hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels (asic ) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic -expressing cells in rat central nervous system (e -p ) using immunohistochemical techniques. asic positive fiber first appeared in brain stem and spinal cord at e - stage. asic -expresseing cells appeared in white matter of brain stem and spinal cord at e stage. in early postnatal stages asic expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval ( st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long st isi by whole-cell recordings in rat slices. application of m apamin, an sk-type ca + -activated k + (kca) channel blocker, shortened the st isi and decreased the amplitude of the slow ahp. the shortening of the st isi was observed when membrane potentials were depolarized. moreover, application of m mibefradil, a t-type ca + channel blocker, shortened the st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca + influx through t-type ca + channels. research funds: kakenhi (c) ( ) jafar vatanparast , , mahyar janahmadi , houri sepehri , ali haeri-rohani , ali reza asgari neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px ( . m, within min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca + spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca + dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm ). repeated menthol application induced trpm desensitization. this desensitization was depended on extracellular ca + , indicating that involvement with ca + -dependent kinase. pkc activator (pma) desensitized trpm and go (pkc inhibitor) abolished pma-induced trpm desensitization. pma similarly desensitized wild type trpm and mutant trpm , in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm . as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm causes pkc to desensitize trpm itself. yosuke sawada , hiroshi hosokawa , kiyoshi matsumura , shigeo kobayashi dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa is activated by pungent compounds stimuli. if trpa responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa is activated by cooling or not is still controversial. here, we investigated that trpa -expressing hek cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was . ± . • c. in inside-out singlechannel recording, cooling activated trpa directly. single channel conductance was . ± . ps. single channel currents showed inword rectification. in conclusion, trpa is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn - ) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn - demonstrated that samples from the nerve and the heart showed an hcn band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn , hcn and hcn could not be found in those samples. when an hcn channel blocker, zd was applied on the stimulus portion of sciatic nerve and the nerve was elicited at . /s by a duration of ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < . ). it is suggested that hcn channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir ) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir . and kir . channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps a-b proliferation of rat c glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c ), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control ( mm) to or mm. an increase in [na + ] i was revealed by an image processor in c cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr- , which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr- level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma ( - m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma ( m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi ( ) il-sung jang , in-sun choi , eun-ju park , jin-wha cho , man-gee lee , byung-ju choi kyungpook national university, school of dentistry, south korea; kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (< m), but decreased those induced by gaba at higher concentrations (> m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav . -deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav . -deficient mice as negative controls to examine nav . distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav . expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav . distribution, in the present study, higher magnification analysis revealed that nav . is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav . antibodies used in these previous studies, none of which utilized nav . -deficient mice. based on our findings, we propose that nav . might be involved in propagating action potential to presynapses. keiji miura , , masato okada , , , shun-ichi amari department of physics, kyoto university, kyoto, japan; "intelligent cooperation and control", presto, jst, japan; department of complexity science and engineering, university of tokyo, chiba, japan; brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. and ) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [ - c] glucose intravenously ( g/kg) or h after the intraventricular injection of colchicine ( g/ l) and the amino acid fractions were extracted from the brains at , or min after the glucose injection. the amount of [ - c] glucose in the cerebra was increased, however, the c incorporation into glutamine, glutamate, gaba and aspartate from [ - c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes or hes gene, phes -and phes -egfp transgenic (tg) mice were generated (ohtsuka et al., ) . in each transgenic mouse, a promoter of hes or hes gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes or hes regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day (e ) to postnatal day (p ). in the phes -egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e to p . in the phes -egfp tg mouse inner ear, gfp-ir cells were observed from e . to p . gfp-ir cells in phes -gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., . mol. cell neurosci. ps a-c expression of zfh- in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine , kenji nakamura , motoya katsuki , tetsuo yamamori national institute for basic biology, okazaki, japan; mitsubishi kagaku institute of life science, machida, japan zfh- is a transcription factor containing three homeodomains and zn fingers and expressed in differentiating neurons. we have reported that the level of zfh- mrna is negatively regulated by antisense transcripts of the zfh- gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh- antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh- mrna starts to be expressed. recently, we have raised an antibody against mouse zfh- and examined the expression profile of the zfh- protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh- protein was not detected even when the zfh- mrna was already expressed. this observation together with other data suggested that the zfh- protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue , maya ota , katsuhiko mikoshiba , jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan; laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic - ) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic /zic compound mutant mice. mice carrying homozygous zic mutant allele together with zic null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic -or zic -single mutant. these observations indicate that zic , in cooperation with zic , have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp , a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp transcripts are first detected at embryonic day in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day - embryos, rp transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e , rp transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c hc-type zinc finger motifs. using p cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf- and nzf- respectively. nzf- null mice are born alive, but die within min after birth with cyanosis. on the other hand, nzf- null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf- and nzf- by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , ) . in the present study, to examine the role of runx in the development of drg in more detail, we examined the development of drg neurons in runx -deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx −/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days . and . . shin hisahara , , susumu chiba , hiroyuki matsumoto , yoshiyuki horio department of pharmacology, sapporo medical university, sapporo, japan; department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt is still unclear. recent studies indicated that sirt interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes promoter. we performed overexpression of sirt and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt and n-cor in the sirt with n-cor. sirt and n-cor suppressed hes transcription by notch-icd in the luciferase assay. hes transcription was suppressed in overexpression of sirt and n-cor, suggesting that interaction between sirt and n-cor represses hes transcription. consistent with this, chip assays revealed that not only n-cor but also sirt bind to the promoter of hes gene. taken together, these results indicate that sirt and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes promoter site and repressing hes transcription. yasushi maruyama , mitsuhiko kurusu , masataka okabe , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, japan; natl. inst. genetics, mishima, japan; inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., ) . furthermore, we announced previously that runx mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi , hirohide takebayashi , takayuki manabe , kazuhiro ikenaka , akio wanaka dept. anatomy, nara med. univ., kashihara, nara, japan; division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng proteoglycan and olig at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig lineage cells in the forebrain is largely unknown. here we examined fates of olig expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig -creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) . or . , when most of olig + cells are observed in the basal forebrain. the olig + cells at e . gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig + cells at e . generated more glial cells than neurons. these results indicate that olig lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi , , masato yano , , satoru hayashi , , hirotaka j. okano , , hideyuki okano , dept. physiol., keio univ. sch. med, tokyo, japan; sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p , p and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf and nf , binding partner of nf , respectively. in mouse embryonic brains, we found that nf / expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf / formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er and brn ) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi ( ) ps a-d protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for - weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng + cells in the neocortex are the opcs. we investigated about subtypes of ng + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp and wnt a, a significant proportion of these neural cells become math + . the induced math + cells mitotically active and express markers characteristic of granule cells precursors (pax , zic , and zipro ). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l + /calbindin-d k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf rather than by wnt a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p cip , p kip and p kip mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p kip mrna was shown. transfection of p kip into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e . rat midbrain and hb .f cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p kip elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g /s phase did not activate mapk and pi -k signaling. yuichi tanaka , yusuke tozuka , dai muramatsu , kin-ichi nakashima , tatsuhiro hisatsune departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng + and olig + cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj- + or map- + neuronal cells by adding retinoic acid or bdnf. more than % of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi , michiko imanishi , teiko kuroda , masao tachibana , masahiko takada department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv . b and kv . were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv . in the frog retina and by kv . b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv . b. thus, the differential expression of kv channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou , , kazunori yamamoto , masayuki hara , akinori homma , akimichi kaneko , masahiro yamada tokyo metropolitan univ., hino, tokyo, japan; astellas pharma. inc., osaka, japan; sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko ( ) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by -hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as . ± . ph unit by mm k + . acidification of pho was suppressed by . m bafilomycin a , a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps a-e analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar -channel field potential recording system in slice preparations obtained from to days old mice. stimulation at ssc induced negative field potential with short latency and short duration ( - ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing m bicuculline, the same stimulus induced a large negative field response with long duration ( - ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya , ken tsutsui , kimiko tsutsui dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap- binding, and the c-terminal sh domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe- , also expresses amph ir. arpe- thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe- cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura , satoshi ichisaka , chihiro hayashi , hirotoshi maki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v ) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi ( ) ps a-e rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut) and vglut , respectively. a complementary distribution of vglut and vglut was established by postnatal day (p) and both signal intensities increased further by p - (peak cp). the immunoreactivity for vglut decreased around layer iv after brief monocular deprivation ( dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area , with a small projection to layer vi of areas and a. cells in layer vi of area projected to dlgn. in addition, cells in layer v of area projected to dlgn, which is not observed in normal rats. in area a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of a. cells in layers v and vi of area a projected to lp. smaller number of cells in layer v of area also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps a-e activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida , takemasa satoh , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v ) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v to the lgn. we focused on the density of type metabotropic glutamate receptor ␣ (mglur ␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur ␣ in the lgn decreased after elimination of v , showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur ␣ signal in the lgn of young rats. yu morishima , hiroshi sakamoto , takahumi akasaki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., japan; div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, ( ) how rapidly the reverse od shift proceeds and ( ) whether the shift is induced only in young animals. we infused the cortex with muscimol in -week-old kittens and in adults. the reverse od shift was observed after days md, but not significant after days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps a-e experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai , nafiseh atapour , john renger , john roder , peter seeburg , takao hensch neuronal circuit dev., riken, bsi, wako, japan; mount sinai hospital, toronto, canada; max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur , ) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation ( d md) rapidly alters both the phosphorylation state and surface expression of glur in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as d md eventually led to full od shifts. in glur ko mice, even d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx promotor and dlx / enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca and revealed a nonpyramidal neuron projecting to ca and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita , takao oishi , motoharu hayashi div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than % of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area ( . %) than in other areas, such as the prefrontal ( . %), primary motor ( . %), somatosensory ( . %) and secondary visual areas ( . %), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than % of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe , guy n. elston , , tomofumi oga , hiroshi tamura , , ichiro fujita , osaka univ., japan; univ. queensland, australia; crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v ). pyramidal cells in prefrontal cortex, such as area , have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v (n = ), area te (n = ), and area (n = ) of a -week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area , followed by area te, and v . thus, considerable heterogeneity in pyramidal cell structure already exists -weeks after birth. hiroaki matsushita , mahito ohkuma , masami watanabe , ei-ichi miyachi department of physiology, fujita health university, aichi, japan; department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura- based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the % of axotomized rgcs. bath application of m carbachol, an ach receptor agonist, rose [ca + ] i in % of normal rgcs. although the % of rgcs responded to carbachol at days after axotomy, no responsive rgcs appeared during - days. ach responsiveness recovered in axonal regenerated rgcs ( %). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi ( to em, to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., ) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor ( hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames ( ms) or only one frame ( ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps a-e motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima , , tohru kiryu , kazuhiko ukai , takeshiko bando div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura , kenichiro miura , masakatsu taki , hiromitsu tabata , naoko inaba , kenji kawano , frederick a. miles grad. schl. med., kyoto univ., kyoto, japan; lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn ) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency f and f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps ( hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the f forwards and the f backwards. the contrast of the f was fixed at , , or %, while the contrast of the f was varied from one-fourth to four times the contrast of the f. when the contrast of the f ( f) was less than about half that of the f ( f), the f ( f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < . ). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito , yaoxing chen , yukiko tanada dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; china agricul. univ., beijing, china twenty white-leghorn chicks (p - ) were perfused with % paraformaldehyde through the heart under deep anesthesia of nembutal ( mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, ). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to in tec-rgcs, in tha-rgcs, and in hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers - ). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie , kenji yuda , eiichi okawa , katsuyoshi maruyama , hiroshi uozato , hiroko horie , satomi nakajima , kenkichi tanioka , yuji ohkawa , tomoki matsubara , wolfram tetzlaff advanced res. centr. biological sci., waseda univ., tokyo, japan; technomaster co. ltd., yokohama, japan; kikuna yuda eye clinic, yokohama, japan; healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; nhk sci. technical res. labo., tokyo, japan; icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between and cm toward and away in a high speed (top speed: mm/s) from the subjects. the device significantly improved visual performances in over % of the school-aged children with myopia and % of adults with presbyopia. hiroyuki miyamoto , toral s. surti , takao k hensch laboratory for neuronal circuit development, riken brain science institute, wako, japan; san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v ) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v . here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa , miquel bosch , shuzo sakata , yoshio sakurai , tetsuo yamamori division of brain biology, national institute for basic biology, okazaki, japan; mit, ma, usa; rutgers university, nj, usa; department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., ) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki , youichi miyawaki , tsunehiro takeda department of complexity science and engineering, university of tokyo, chiba, japan; mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly ( ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = - ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa ( and ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p , n and area under curve of a late positive potential expanding from to ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse , ayumu matani , , tomoe hayakawa , , norio fujimaki university of tokyo, kashiwa, japan; nict, kobe, japan; teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p amplitude becomes. this suggested that a factor of the variability of the p amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. ) hitoshi sasaki , takuya ishida , masayoshi todorokihara , junichiro miyachi , tahei kitamura , ryozo aoki dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama , nobuo usui , izuru nose , , masato taira , department of applied system neuroscience, nihon university school of medical science, tokyo; faculty of human science, bunkyo university, saitama, japan; arish, nihon university, tokyo, japan when an object is moving, perception of its d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps a-f local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a ) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., ) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., ) . moreover, aaf has dense synaptic connections within the area (budinger et al., ) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were -ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima , yasuo takeda , yasushi shimoda , kazutada watanabe department of bioengineering, nagaoka university of technology, nagaoka, japan; department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb- that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb- expression is prominent in the auditory system. nb- knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb- is indispensable for the function of auditory system. here we report the detailed analysis of the nb- localization using anti-nb- monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb- was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb- immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb- regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation ( khz octave band, db, h), and then subjected to electrophoretic mobility shift assay to determine activator protein- (ap- ) dna binding. ap- binding was increased - h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea h later. taken together, ap- may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c bl/ mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli ( - khz for ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by khz were different from those elicited by or khz. however, the areas activated by khz were also activated by the mixture of plus khz but not by that of plus khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps a-f functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by - %, whereas the inhibition of dc resulted in reduction only by - %. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh- . a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato , , kazuo okanoya laboratory for biolinguistics, riken bsi, wako, saitama; graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, ) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented - times at a fixed interval, through a speaker at - db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (< hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (> hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki , , , , , hokkaido university, sapporo, japan; hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan; kobe shoin women's university, kobe, japan; riken, wako, japan; sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children ( - years old, n = ) and adults ( - years old, n = ). there were six gap conditions with durations of , , , , and ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n and p complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps a-f an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua , tamaki miyamoto , atsushi terao , sachiko koyama , takashi omori , harumitsu murohashi , shinya kuriki jst, saitama, japan; hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba ), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation ( subjects, p < . corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi , yuko toyosawa , fumiya takeuchi , michinao matsui , shinya kuriki research institute for elecronic science, hokkaido university, sappro, japan; jst, saitama, japan; hokkaido university, school of medicine, japan; kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = ). four synthesized vowels were used as stimuli (stimulus duration ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used ( / ms). for the native vowels, an early sf ( - ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf ( - ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi ( ) ps a-f spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto , , junsei horikawa department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh ) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli ( - khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe , go ashida , , kazuo funabiki graduate school of informatics, kyoto university, kyoto, japan; hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi , shigenori takebayashi , masahiko bundo , masazumi fujii , toshihiko wakabayashi , jun yoshida , hiroyuki fujisawa , kazunori ando , kazumasa hayasaka department of radiology, national hospital for geriatric medicine, obu, japan; department of neurosurgery, national hospital for geriatric medicine, obu, japan; department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using . tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in out of patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = ) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, ) . surprisingly, subjects tended to misidentify the second toe as the third ( . %), and the third toe as the fourth ( . %), while the reverse misidentification rarely occurred (third as second, . %; fourth as third, . %). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps a-g effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi , , shunjiro moizumi , ayami okuzumi , humine saito , shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, jst, saitama, japan morrone et al. ( ) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade ( • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps a-g function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda , , miki taoka , department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas b and of four conscious macaque monkeys was studied by recording single-neuron activities. a total of electrode penetrations were made in areas b and . in penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining penetrations, however, % of adjacent pairs ( / ) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas b and might be responsible for forming composite rfs of area neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi ( , ) miki taoka , michio tanaka , hisayuki ojima , atsushi iriki secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them ( % contra-, % bi-and % ipsilateral hand movements), we could determine receptive fields for only neurons ( %). most of activities ( neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta , , kouichi nakamura , takeshi kaneko department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata , , shanlin chen , hiroki kitaura , masaharu kudoh , minoru shibata , katsuei shibuki department of neurophysiology, brain research institute, niigata university; department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane ( . g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation ( hz for s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi , nanae fukushima , tetsuhiro fukuyama , akira kakegawa , tetsuji moriizumi department of anatomy, shinshu university school of medicine, matsumoto, japan; department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima , takeshi kawano , hidekuni takao , , , kazuaki sawada , , , hidekazu kaneko , , makoto ishida , , department electrical & electronic engineering, toyohashi university of technology, aichi, japan; issrc, toyohashi university of technology, aichi, japan; aist, hsbe, ibaraki, japan; jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, m in diameter, m in length; array: -m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = . ). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi ( ), the st century coe program "intelligent human sensing" ps a-g a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko , hiroshi tamura , shinya s. suzuki , takahiro kawashima aist, hsbe, ibaraki, japan; laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over h. seiji matsuda , takehiro terashita , tetsuya shimokawa , kyoujy miyawaki , yuji miguchi , takuya doihara , jie chen , shuang-yan gao , chun-yu li , min wang , zhong wang , bing xue , naoto kobayashi , , kazuhiro shigemoto department anatomy, ehime university of medicine, ehime, japan; medical education c, ehime university of medicine, ehime, japan; department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than . m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat , whereas other genes known to be regulated by stat were not detected. stat is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat and stat phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto , susumu hyodo , makoto kawasaki , minori shibata , takeshi saito , hiroaki fujihara , takashi higuchi , yoshio takei , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; department of integrative physiology, university of fukui, japan adrenomedullin (am ) (identical to intermedin) belongs to the super family of am. centrally administered am and am activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am was stronger than those of am and the effects of am on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am would have unknown receptor except cgrp and am receptor. arata oh-nishi , makoto saji , taku uchida , sen-ichi furudate , nobuyuki suzuki division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, ) . here we report that the density and morphology of spines of pyramidal neurons in ca region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca , both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day - , the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day . the staining of somatostain mrna in individual neurons was diminished when examined on day or , albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps a-g short chain sugar acid, -buten- -olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki , tatsushi onaka , hirofumi hashimoto , hiroaki fujihara , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; department of physiology, jichi medical school, tochigi, japan -buten- -olide ( -b o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of -b o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at - min after intraperitoneal (i.p.) administration of -b o ( mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei min after i.p. administration of -b o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of -b o. these results suggest that peripherally administered high dose of -b o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps a-g estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto , , shinpei higo , , yasushi hojo , , kouhei nakajima , , hironori nakanishi , , hirotaka ishii , , suguru kawato , department of biophysics & life science, university of tokyo, tokyo, japan; crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps a-g gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from − to - mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than - mv and most of them were ∼− mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr , galr and galr , in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr selective agonist m induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr . hiroaki fujihara , tomoki fujio , david murphy , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking % saline for days in , , and months old tg rats. in and months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats ( and months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps a-h prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera , hiroaki fujihara , hirofumi hashimoto , makoto kawasaki , tatsushi onaka , takakazu oka , sadatoshi tsuji , yoichi ueta department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; department of physiology, school of medicine, university of occupational and environmental health, japan; department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin , afsaneh eliassi , nasser naghdi , ali ghanbari ghsemi; pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above ( . mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from min up to the end of experiments ( min). at the peak acid response, the vmh microinjection skf ( . , ) significantly reduced the amount of gas (p < . ). there was no any effect by microinjected sch ( . ) into the vmh. injecting skf into the vmh, min after sch , had no effect on gas in compare with control. but, the acid suppressant effect of skf was completely removed by peripheral injection of sch (p < . ). our results show that the vmh d dopamine receptors have regulatory mechanisms of gas by interaction with h receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p y purinoceptors (p y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p y r agonists increased [ca ++ ] i in son, with p y r agonist being the most effective. the possibility that p y r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p y r antagonist, mrs . atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs ( m) in mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs . furthermore, combined treatment with mrs and ppads ( m, a p x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p y r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps a-h analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii , , yasuhiro sonoki , , aizo furukawa , , yasushi hojo , tetsuya kimoto , , suguru kawato , department of biophysics and life science, university of tokyo, tokyo, japan; crest, jst, japan; kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day (pd ) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd to pd with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai , , gen murakami , shirou kominami , john h morrison , william g.m janssen , tetsuya kimoto , , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo - , japan; crest project, jst, japan; faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima , japan; kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc- , mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca , ca , and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud , song t. yao , jing qiu , julian fr paton , david murphy university of bristol, hw-line, uk; department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic ( d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase , has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt - . delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt - cells cultured in dmem supplemented with % fbs for days. bk current was increased by addition of ␤-estradiol (e ) in culture medium in a physiological concentration range. this action of e was blocked by ici- , , a potent estrogen receptor (er) antagonist. we further examined whether e acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e but ppt had no effect. e had no effect on the i k . these results indicate that e increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi , ps a-h myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi , haruko kumanogoh , shun nakamura , seiji miyata , shohei maekawa department of biosystems science, kobe-university, kobe, japan; division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, kda protein, was identified as myelin protein zero (p ) from mass analysis and this result was confirmed by western blotting that a specific antibody to kda band reacted to an authentic p prepared from rat sciatic nerve myelin. p is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan ␤-estradiol (e ) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e (hojo et al., ) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca , ca and dg with a custom made multi-electrode measuring system (med ). ltd was enhanced by e dose-dependently in ca , ca and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito , takushi x. watanabe , tomoko urabe , hirofumi hashimoto , hiroaki fujihara , yukio hirata , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; peptide institute, inc., osaka, japan; department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto , hao-gang xue , yuji ishikawa , yoshitaka oka , hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan; national institute of radiological sciences, chiba, japan; department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki , eiji munetsuna , asuka kamogawa , suguru kawato , shiro kominami graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p arom in the hippocampus. method: the cultured hippocampus slices from days male rat were treated with - nm of tbt for h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p arom was determined by quantification of [ h]estradiol from [ h]testosterone. result: forty-eight hours treatment of hippocampal slices with nm tbt induced increases in mrna contents of p arom, and with nm tbt induced that of ␤-hsd. estradiol content was increased by the treatment with nm tbt, but not affected by nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of , , and ng in nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent h. the ng dose of bicuculline methoiodide had minimum effect whereas and ng produced arousal. maximal wakefulness was observed at dose of ng with no further increase in wakefulness at higher dose of ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe , hiroyuki tsuda department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee ). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, ( antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via -opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d is a potent somnogenic substance, and isomerized from pgh through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl decreased rapid eye movement (rem) and non-rem sleep for h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl administrations. these findings indicate that pgd plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd involved in sleep-wake regulation. under baseline conditions, h r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (< s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., ) . ps a-h dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = nm). the scn neurons responded to the light in intensities above photons/cm /s and were activated maximally at around photons/cm /s, they were about . log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca + in scn neurons. since cytosolic ca + regulates diverse cellular systems, we have hypothesized that the cytosolic ca + rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca + rhythms, mouse bmal and its dominant negative sequence (mbmf r ) are transfected into the organotypic culture of scn with a yellow cameleon ca + sensor by the gene gun. the results demonstrated that over-expression of bmal or mbmf r significantly inhibited the circadian ca + rhythms and thus we concluded that the native bmal rhythm is essential for cellular output processes of the murine clock system. ps a-h the activation of ␣ adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca region of rat hippocampal slices. carbachol ( m) induced ␤ oscillation with the frequency and the amplitude of . ± . hz, and . ± . mv, respectively (mean ± s.e.m.; n = ). epinephrine ( m) significantly increased the frequency of . ± . hz (**p < . ), not change the amplitude. clonidine ( m), an ␣ receptor agonist, alone significantly increased the frequency at the concentration of m (*p < . ). yohimbine, an ␣ receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣ receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart . mm from each other in the hippocampal ca region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency - ms), oscillatory ( - hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties ( / slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca region. ryozo aoki , hiroshi wake , hitoshi sasaki , kiyokazu agata dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as /f, ranging from − to + hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida , hiroki sato , , naoki tanaka , atsushi maki , japan science and technology agency, crest, saitama, japan; advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from to hz) decreases and the sleep spindle power (from to hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from to s in (i), and from to s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim , kazuki nakajima , yutaka oomura , kazuo sasaki div. of bio-information eng., univ. of toyama, toyama, japan; dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca + exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng , wei zhang , akira nakamura , masashi yanagisawa , yasuichiro fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. autonom. physiol., chiba univ., japan; dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb- (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb- . icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co -sensitivity at least during waking periods. research funds: kakenhi , junko hara , taizo matsuki , katsutoshi goto , masashi yanagisawa , takeshi sakurai , department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; yanagisawa orphan receptor project, erato, jst, tokyo, japan; howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps a-i prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto , kazuhiko kubo , shuji aou dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. ) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. , s.a.) shuji aou , tetsuya fujimoto , yumi ichihara , kimiya narikiyo , toru ishidao , hajime hori , yukiko fueta dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan -bromopropane ( -bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to -bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to ppm of -bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. -bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that -bp is the potential candidate of endocrine disruptors which affect brain development. ( ) ps a-i changes in hippocampal excitability of rats prenatally exposed to -bromopropane yukiko fueta , toru ishidao , susumu ueno , yasuhiro yoshida , hajime hori department of environmental management, school of health sciences; department of pharmacology; department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to -bromopropane ( -bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to -bp ( ppm) during gestational day - ( h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca at the pnd - . conversely, s/r curves of fepsp as well as ps in the ca were inhibited at the age of - weeks. our results suggest that -bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca of the offspring during development. research funds: grant-in-aid for exploratory research ( ) ps a-i effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura , , kyoko itoh , takeshi yaoi , tohru sugimoto , shinji fushiki dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously g/kg of bpa daily from embryonic day (e ). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e . and e . , whereas those cells increased in the cortical plate at e . , as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant keiko ikemoto , teruko uwano , hisao nishijo , taketoshi ono , masayuki ito , ikuko nagatsu , katsuji nishi , shin-ichi niwa dept. neuropsychiat., fukushima med. univ. sch. med.; toyama med. pharm. univ.; faculty med., mie univ., mie, japan; fujita health univ. sch. med., toyoake, japan; dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day and after fertilization. pups were perfused at postnatal day . in the frontal cortex, the number of largesized (more than m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c /a catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps a-i developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi , , , kaori morohoshi , , rie yanagisawa , erina saita , gen watanabe , , masatoshi morita , kazuyoshi taya , , hirohisa takano , , toshiyuki himi , , hideki imai , dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; res. inst. pharmaceut. sci., musashino univ., tokyo, japan; nation. inst. for environ. stud., ibaraki, japan; grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; wildlife rescue veterinarian associ., tokyo, japan; facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; the united grad. sch. veterinary sci., gifu univ., gifu, japan; div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps a-i the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic ( -ht) systems in stressinduced anxiety, we measured brain da and -ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of -ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and -ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although -ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl - ) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl and nl in autistic disorders. nls have a relative identical structure (∼ %); nl and nl localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl seems express in the olfactory glia, but nl distribution is unknown. here we have generated antibody against human nl , and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl is important for neuroendocrine function. nl cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl were rough and thicker than those of nl or nl transformants. it suggested the unique activity of nl for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting - % of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below . here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura , daisuke horiuchi , tomoyuki miyashita , minoru saitoe , hitoshi okazawa department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein- (pqbp- ) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp- disease that pqbp- mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp- in xlmr pathology, we produced two kinds of flies, human pqbp- overexpression flies (hpqbp flies) and drosophila pqbp- knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp- in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun , sentaro takahashi , yoshihisa kubota , rui zhang , chun cui , yoshihiro fukui natl. inst. radiol. sci., chiba, japan; sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr subunits paired with two nr subunits. nr and nr (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr /nr a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr /nr b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca pyramidal neurons from mice (at weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca region after ischemia. in blood, corticosterone levels increased biphasically at min and h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca region. by days after ischemia, mr and gr were not significantly altered in the ca region. from days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca region, and at days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase- and midkine in the mca area. stroke was induced by a -min mca occlusion using an intraluminal filament. rats were exercised for min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase- expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps a-j reversed astrocytic glutamate transporter glt- crucial to the ca + paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca + paradox" is the phenomenon whereby the intracellular concentration of ca + paradoxically increases during reperfusion with normal ca + -containing media after brief exposure to a low ca + solution. the present study aims to characterize the ca + paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca + solution for min significantly injured only neurons after reperfusion with a normal ca + medium for h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt- . the present findings suggested that ca + paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt- during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c t, which results in pro ser. the second patient carried the missense mutation c g, which results in arg pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps a-j mci - , a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol kinase madinyet niyaz , tadahiro numakawa , yoshinori matsuki , emi kumamaru , yuki yagazaki , harumi kitazawa , hiroshi kunugi , motoshige kudo pathology department of tokyo medical university, tokyo, japan; department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci - , a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci - prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci - exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways and that the mci - -dependent survival effect was blocked by the inhibitors, u (an mapk pathway inhibitor) or ly (a pi k pathway inhibitor). these results suggested that mci - exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru , noriko yoshimura , tetsuro sakamoto , takaaki kitano , naoko nisimaru , isao yokoi dept. physiol., sch. med., oita univ., japan; med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by p-nuclear magnetic resonance ( • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with mm ep (ep- ), acsf replaced by acsf with mm ep after ischemia (ep- ), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep- than in control (p < . ). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) # from mext to t.k. hideaki tamai , kuniko shimazaki , norimasa seo department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc ), yintang (ex-hn ) and sanyinjiao (sp ), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through -bromo- ,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc , ex-hn and sp . the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago , keiko takahashi , shigeo nakamura , akemichi baba , toshio matsuda laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara , tatsuya mishima , takefumi kofuji , kimio akagawa department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc- /syntaxin a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc- /syntaxin a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc- /syntaxin a knock-out mouse will be discussed. research funds: kakenhi ( ) ps a-k effects of local administration of the gaba agonists into the hippocampus ca area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka , hiroko miyakubo , junichi tanaka , yasushi hayashi , yukio hattori , masahiko nomura department of curriculum, teaching and memory, naruto university of education, tokushima, japan; department of human nutrition, notre dame seishin university, okayama, japan; department of physiology, saitama medical school, saitama, japan in fischer male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite -hydroxytryptamine ( -hiaa) concentration in the ca area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad- . fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka , sho koyasu , akinori nishi , tomoyuki furuyashiki , toshiyuki matsuoka , shuh narumiya department of pharmacology, university of kyoto, kyoto, japan; department of physiology, university of kurume, kurume, japan prostaglandins e (pge ) exert their actions in various organs through specific receptor, ep to . the previous study suggests that ep modulates da system. to investigate the roles of ep in da system, we examined ep ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼ % in both wild-type and ep ko mice. however, increase of locomotor activity in ep ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d receptor (d r) signaling, we tested the density of d r and d r signaling with phosphorylation of darpp- . there were no differences in d r binding. d r signaling was significantly attenuated in the striatal slices from ep ko mice. the effect of d r agonist on locomotor activity was also attenuated in ep ko mice. these results indicate that pge has enhancing effects on locomotor activity via ep by potentiating the d r signaling. central serotonin ( -ht) function has been implicated in impulsivity. the present study examined rats with -ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of -ht and its metabolites ( -hiaa) in the brain. for both tasks, significant negative correlations between learning performance and -ht and -hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and -ht and -hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of -ht roles on selective indices of impulsivity. yuki sato , , , tatsushi onaka , norimasa seo , eiji kobayashi dept. anesthesiol., jichi med. univ., tochigi, japan; dept. physiol., jichi med. univ., tochigi, japan; div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p - ; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p ; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p . within the vestibular nucleus of p - rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p in the vestibular nucleus as fuzziness about neuronal cell bodies. from p onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p . during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p . with ul, the pn network on the lesioned side remained compact until days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca region of rat hippocampal slices, hz/ pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at hz. ikuko yao , hiroshi takagi , hiroshi ageta , tomoaki kahyo , ken hatanaka , kaoru inokuchi , mitsutoshi setou , mitsubishi kagaku institute of life sciences, tokyo, japan; university of tokyo, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps p-a presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., mv, s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca + currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca + channel knockout mice. we conclude that the p/q-type specific ca + channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi , koji ikegami , ken hatanaka , , , yoko fujiwara-tsukamoto , mineo matsumoto , ikuko yao , mitsutoshi setou , , mitsubishi kagaku institute of life sciences, japan; presto, japan; school of pharmaceutical sciences, the university of tokyo, japan; okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa (−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki , shingo kimura , reiko fujita , shuji watanabe , kazuhiko sasaki dept. of physiol., sch. of med., iwate medical univ., morioka, japan; dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c , a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦ glutamate receptor (␦ r) plays a crucial role in cerebellar functions. although ␦ r has a putative channel pore domain, and ␦ r displayed ca + -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦ r functions as a channel. here we introduced a ␦ r transgene, which had a mutation (gln arg) in the putative channel pore conserved in ca + -permeable glutamate receptors, into ␦ −/− mice. surprisingly, a mutant ␦ r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦ −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca + influx through ␦ r is not required for its function in the cerebellum in vivo, and that wild-type ␦ r may not function as a ca + -permeable ion channel. research funds: kakenhi ( ) and takeda science foundation ps p-a distribution of tarp - on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya , mika tsujita , maya yamazaki , etsuko kushiya , manabu abe , kaori akashi , masanobu kano , haruyuki kamiya , kenji sakimura , masahiko watanabe department of anatomy, hokkaido university school of medicine, sapporo, japan; department of cellular neurobiology, brain research institute, niigata, japan; department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the - is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized - on excitatory synaptic and extrasynaptic membrane. in - -ko mice, ampars were reduced in hippocampal homogenates ( % of control) and psd fraction ( %). immunogold labeling also exhibited reduction of extrasynpatic ( %) and synaptic ( %) ampars in ca pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites ( %) and spines ( %). ampar-mediated responses were reduced at ca synapses ( %). therefore, - is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa , yugo fukazawa , elek molnar , masahiko watanabe , ryuichi shigemoto , div. cerebral structure, nips, okazaki, japan; mrc, univ. of bristol, bristol, uk; hokkaido univ., sapporo, japan; sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed . to times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed . to times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda , , , hitoshi kita dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; japan society for promotion of science, tokyo, japan; dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp abolished the sipsp. an mglur antagonist, but not an mglur antagonist, partially blocked the sdepo. in addition, cgp enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur -mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps p-a essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto , yoshikazu isomura , , michiko imanishi , tomoki fukai , masahiko takada system neurosci., tokyo met. inst. neurosci., tokyo, japan; neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for > s after the induction. gaba amediated cl-influx lasted for . s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura , yuko sekino , , toshiya manabe , division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < ), using electrophysiological methods and optical imaging. in immature animals (p ), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p ) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p - ). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p - compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣ , ␣ , and ␤ / subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣ , ␣ , and ␤ / subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ -or ␣ -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ -adrenoceptor agonist phenylephrine, while reduced by the ␣ -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi ( ) ps p-b involvement of basal pkc and erk / activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa , takahiko chimura , azumi matsumoto , kazuhiko yamaguchi lab. for motor learning control, riken, bsi, japan; lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk / activities at pf-pc synapses. taisuke miyazaki , kohichi tanaka , masahiko watanabe department of anatomy, school of medicine, hokkaido university, sapporo, japan; department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki , , kouichi hashimoto , taisuke miyazaki , masahiko watanabe , masanobu kano dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto , , takashi kodama , , ryuichi shigemoto , , , yugo fukazawa , , yasuo mori division of neural signaling, nips, okazaki, japan; school of life science, graduate university for advanced studies (sokendai), okazaki, japan; division of cerebral structure, nips, okazaki, japan; sorst, japan science and technology agency, kawaguchi, japan; department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v . (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v . mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v . exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n ) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n ) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps p-b target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from . ± . to . ± . (n = ; mean ± s.d.) by reducing [ca + ] o from to . mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps p-b response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at / s, and were depressed completely by cnqx. when stimulus rate was increased to /s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at /s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at / s, but not at /s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at /sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono , keisuke shiba , ken nakazawa , ichiro shimoyama department of otolaryngology, chiba university, chiba, japan; department of otolaryngology, kimitsu central hospital, chiba, japan; department of integrative neurophysiology, chiba university, chiba, japan; section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps p-b in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada , , eiji shigetomi , , fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan; dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi ( , ) ps p-b laminar sources of synaptic input to layer neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer / . neurogliaform cells receive more excitatory inputs from layers and . the dominant inhibitory synaptic inputs onto both types of cells originate from layer / . these neurons also get inhibitory inputs from layers and - . the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer are highly integrative. takako nishi , tsukasa gotow , shiro nakagawa ins. nat. sci., senshu univ., kawasaki, japan; dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p- , es- , ip- and ip- which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p- /es- of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip- /ip- . the present study was conducted to examine the functional significance of the above ip- /ip- , having a peak sensitivity at nm light. the body wall of the animal transmitted enough nm to hyperpolarize ip- /ip- in the ganglion covered by the body wall. ip- /ip- were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip- /ip- were not coupled to a-p- /es- one another. finally, ip- /ip- , the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps p-c cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the -day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e . it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e -e ), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada , nyberg tobias , nahoko kasai , yuriko furukawa , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, ntt corporation, kanagawa, japan; sorst, jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase -green fluorescence protein (gad -gfp) knock-in mice at postnatal day - were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps p-c pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda , toshiyuki ishii , , yasuhide shigematsu , toshihiko hosoya , yukio shimoda dept. physiol., keio univ. sch. med., tokyo, japan; bsi, riken, wako, japan; mri, tokyo womens' medical univ., tokyo, japan; dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p x -purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p x -purinoceptors. furthermore, a p x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca + . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca + oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of mesatp was observed. the inhibition of mesatp response by p y receptor specific antagonist mrs indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca + oscillations. data will be presented on the p receptor mediated regulation of synchronized ca + oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d -like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf was shown to selectively stimulate plc-linked d -like dopamine receptors. darpp- is a phosphoprotein that regulates the efficacy of d receptor signaling. activation of d receptors that couple to golf/pka signaling leads to darpp- phosphorylation at thr . we investigated the effect of skf on darpp- phosphorylation at thr by using mouse neostriatal slices. treatment of neostriatal slices with skf increased thr phosphorylation. the effect of skf was enhanced by a plc inhibitor, u . in contrast, the effect of skf was partially blocked by a d receptor antagonist, sch , and the sch -insensitive increase in thr phosphorylation was completely abolished by an adenosine a a receptor antagonist, zm . thus, analysis of darpp- phosphorylation revealed that skf activates at least three signaling cascades in the neostriatum: ( ) plc, ( ) d receptor/golf/pka, ( ) adenosine a a receptors. ps p-c thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c bl mouse were dyed with a voltage-sensitive dye rh . in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal ( mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx ( mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no. ps p-c brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur at psd within min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca + transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca + rise, that is, ca + release from ip r and ca + influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca + signaling regulation. research funds: health sciences research grant of nano- hiroyuki konishi , kazuhiko namikawa , , keiji shikata , yuji kobatake , taro tachibana , hiroshi kiyama dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; dept. anat., asahikawa med. col., hokkaido, japan; dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t , is involved in the control of cell morphology via the regulation of rho proteins via rho gdi . however, it is unclear whether t regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c rat glioma cells, which express t endogenously. rho gdi was dissociated from t in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t -specific sequences reduced the effects of bdnf. these results suggest that t regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d , bcl . the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf- in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind in response to igf- both at protein & rna levels but no change was observed in c-myc, bcl levels. interestingly, mapk-erk, which is also known to activate cd in other systems, had no effect on the cd level in neuronal cells. on the other hand, pi kinase not only activated jak/stat pathway but also affected on cyclind level. thus it appears that jak/stat is activated by pi k, which in turn upregulates cyclind level. pi k also activated erk without changing cd level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi k. cd is a one of the major downstream targets of jak/stat. hiroko inoue , haruhisa kawasaki , ritsuko fujii , tohru yoshioka , kazunori sango , toshihiko kadoya , hidenori horie sch. of sci. and eng., waseda univ., tokyo, japan; advanced res. center for biol. sci., waseda univ., tokyo, japan; tokyo metropolitan inst. for neurosci., tokyo, japan; pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin- (gal- /ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal- /ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal- /ox enhanced mrna expression of nerve regeneration-related factors such as il- , il- ␤. however, those of gdnf, ngf, bdnf, nt- , nt- , igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal- /ox. since il - , il- , and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal- /ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca + concentration, ␣camkii is activated by the interaction with ca + /cam. prolonged increase in ca + level leads to further activation of ␣camkii through autophosphorylation at t . to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca + level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy y cells, the increase in ca + level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca + /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦ isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦ - antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦ isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps p-d the role of ca + /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata , , hiroyuki sakagami , keiji imoto , , kunihiko obata , yuchio yanagawa okazaki, japan; sokendai, okazaki, japan; tohoku univ., sendai, japan; riken, wako, japan; gunma univ., maebashi, japan ca + /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k with r to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , # ) ps p-d characterization of ca + -dependent membrane binding proteins of rat brain shohei maekawa , katsutoshi taguchi , haruko kumanogoh , shun nakamura division of biosystems science, grad. sch. of sci/tech, kobe-u kobe - , japan; division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo - , japan ca + -dependent membrane binding proteins were purified through a ca + -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca + -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps p-d generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa ∼ ) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa ∼ ). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal -dbd with mutants of crest; crest c ( ∼ ) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps p-d characterization of iq-arfgef, a novel exchange factor for arf , in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor (arf ) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf . in this study, we determined the entire sequence of a partially identified cdna (mkiaa ) encoding a novel sec domain. the deduced protein contained coiled coil, iq, sec , plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf more selectively than arf . thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf and interaction with various pdz proteins. eriko miura , masahiro fukaya , tokiharu sato , kazushi sugihara , masahide asano , katsuji yoshioka , masahiko watanabe dept. anat., hokkaido univ. sch. med., sapporo, japan; dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap (or jip ) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk ∼ mrnas. in embryos, jsap immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori , shigeo uchino , tomoko isosaka , , shinichi kohsaka , takeshi yagi , shigeki yuasa dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr b phosphorylation and the nmda-induced calcium responses increased after dopamine d -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp : dystrobrevin binding protein ) at p . . however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi , naoko oda , ryuzo shingai , st coe program, iwate university, morioka, japan; department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa , masaki nomura , , toshio aoyagi , tomoki fukai riken brain science institute, saitama, japan; graduate school of informatics, kyoto university, kyoto, japan; crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki , , tatsumi hirata division of brain function, national institute of genetics, sok-endai, mishima, japan; prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps p-d an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc- cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama , masahiro matsuura , nobuhiko yamamoto grad. sch. of frontier biosci., osaka univ., suita japan; riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer . to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a , sema a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a , sema a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi , kakenhi ps p-e expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami , katsuhiko ono dept. anat., juntendo univ. sch. of med., tokyo, japan; div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin- expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin- receptor neogenin. slit- was expressed in the forebrain, but its receptor robo- was not expressed in gnrh neurons. semaphorin (sema) a expression was detected in the olfactory-forebrain region. within the forebrain, sema a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema a receptor neuropilin- , suggesting that sema a may act as a repellent during the migration of gnrh neurons. kazuto fujishima , , qing-hua jin , junko kurisu , tomoo hirano , mineko kengaku riken, bsi, lab. for neural cell polarity, saitama, japan; department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., ) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer /vldlr induces dab phosphorylation, dab is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab . reelin treatment of the neurons derived from dab deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab signal pathways. hidenobu mizuno , , tomoo hirano , , yoshiaki tagawa , dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area / border of the other hemisphere, where they terminate in layers / and . we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the / border and started to invade the cortical plate around p . they generated exuberant axonal arborizations by p then underwent remodeling to form the adult pattern by p . neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir . in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers / . area-specific targeting to the / border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps p-e identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp like protein (nplp) that has a distant homology to pmp /claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata , shin-ichi sakakibara , hirotaka j okano , hideyuki okano , dept. physiol., keio univ., sch. med., japan; dept. histol. neurobiol., dokkyo med. univ., japan ; crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi and msi , which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi and msi deficient mice. msi −/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi −/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi −/− msi −/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu , satoshi yamagoe , kazuo suzuki , michiko ohtomi department of biomolecular science, graduate school of science, toho university, chiba, japan; department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect (leukocyte cell-derived chemotaxin ) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect and tau or map in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect in neurons. at the early stages in cultured neurons, lect was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda , , takahiro moriya , hideyuki terazono , suguru kudo , masahiro yamaguchi , kenji yasuda , izumi nagata , kazuyuki shinohara div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e . nestin-promoter gfp transgenic mice were placed into pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng cells were co-cultured with vascular smooth muscle cells (sm- ). sm- cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin- mrna was specifically up regulated in ng cells. but the western blotting analysis did not show the significant increase of neuropilin- . sema a, a candidate of ligands for neuropilin- , was found in ng cells rather than in sm- cells. the immunocytochemical analysis revealed that neuropilin- was increased in the surface of ng cells co-cultured with sm- cells or cultured with the conditioned medium of sm- cells. these results suggest that neuropilin- may be transferred from cytoplasmic pool to surface of ng cells by some soluble factors released by sm- cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa , j. nakanishi , h. hamada , y. yokomachi , k. yamaguchi , n. takeda inst. biomed. eng., waseda univ., tokyo, japan; riken, japan; kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca- was shown to be required for cdc to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca- . toca- is strongly expressed in developing brain. knockdown of toca- in pc cells significantly enhances neurite elongation. consistently, overexpression of toca- suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca- in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca- regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi ( ) ps p-e pragmin, a novel effector of rnd gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd is specifically expressed in brain. although rnd and rnd display an antagonistic action for rhoa, signaling pathways of rnd are not well known. here we have performed a yeast two-hybrid screen using rnd as bait and identified a novel rnd -effector protein, pragmin (pragma of rnd ). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd among the rho family gtpases in a gtp-dependent manner. rnd -bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd , and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd controls neurite outgrowth by regulating rhoa activity through pragmin. ps p-e regulation of growth cone motility by collapsin response mediator protein- (crmp- ) masumi iketani , yanai shigeki , fumio nakamura , yoshio goshima , , kohtaro takei , dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein- (crmp- ) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp- within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp- in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp- in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp- activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps p-e eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani , , masaru ihara , , hiraki sakuta , , hiroo takahashi , , ikuko watakabe , masaharu noda , division of molecular neurobiology, national institute for basic biology, okazaki, japan; crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps p-e neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa and lpa . here we examine how lpa , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma (pc ) cells. pc cells expressed low levels of endogenous lpa mrna. when pc cells were infected with retrovirus expressing lpa and treated with nerve growth factor under serum-free conditions for days, neurite formation was observed in % of total infected cells, which was similar to that in control virus-infected pc cells. however, the mean length of neurites was increased in pc cells expressing lpa , compared with that in control pc cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa was involved in neuritogenesis and branch formation. masao sakurai , tomoko aoki , kyoko ishikawa , shingo yoshikawa , john b. thomas , chihiro hama cdb, riken, hyogo, japan; salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt as a regulatory factor for glomerular patterning. in the wnt mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt expression in orns. previous studies in embryos have shown that wnt repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt mutant. this and other genetic data suggest that drl is an antagonist for wnt signaling. we propose that wnt signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps p-e a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps p-e differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino , masatomo kobayasi , taiki nakajima , lydia michaut , indrayani ghangrekar , kuniaki takahashi , ryu ueda , kaoru saigo , walter j. gehring , katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan; biozentrum, univ. of basel, switzerland; genet. strains res. ctr., natl. inst. genet.; dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified , genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki , yoshie suzuki , hiroyuki aburatani , akinao nose dept. phys., univ. tokyo, tokyo, japan; rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, and , which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼ genes with more than two-fold difference in expression level. for candidate genes out of ( %), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles or , implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet- . to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet- signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet- . loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps p-e analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi , takaaki hirotsu , eriko kage , hideaki takeuchi , hirofumi kunitomo , yuichi iino , takeo kubo dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; dept. biol., fac. sci., kyushu univ. grad. sch., japan; mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr- is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr- mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr- functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura , ken-ichi ogura , yoshio goshima , dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc- is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc- is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc- is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc- during the axonal guidance of the hsn neurons. silencing of netrin/unc- only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc- only at the vulval precursor cells in unc- null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc- expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn a-hsp :gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal /uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere . these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) masahiko taniguchi , yoshinori mikami , tomoyuki masuda , tomoyuki yoshida , naoto matsuda , masayoshi mishina , takao shimizu department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; department of anatomy, fukushima medical university, japan; dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin d (sema d). the orf of sema d is bp ( aa). zebrafish sema d protein showed a . % identity with mouse sema d protein. to clarify the expression pattern of sema d, in situ hybridization was performed. in the embryos, sema d is expressed in the lens and hindbrain. we also found that sema d has the repulsive activity. these results indicate that sema d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext ( ) mayumi yamada , shohei maekawa , seiji miyata department of applied biology, kyoto institute of technology, kyoto, japan; division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps p-f semaphorin d inhibits ␤ integrin activity through the r-rasgap activity of plexin-b izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin d (sema d) receptor, plexin-b functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema d/plexin-b in cell migration. activation of plexin-b by sema d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi -k activation, and ␤ integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤ integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤ integrin activation and cell migration, and that inactivation of r-ras by sema d/plexin-b -mediated r-ras gap activity control cell migration by modulating the activity of ␤ integrins. shun hamada , emi fukuda , , shota katori , , takeshi yagi , dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; grad. sch. frontier biosci., osaka univ., osaka japan, crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise > cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga is expressed by layer / neurons throughout the neocortex at p mice, but is absent in adults. between e . and late p , stages that span the generation and radial migration of layer / neurons, mdga is expressed in patterns consistent with its expression by migrating layer / neurons, suggesting a role for mdga in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e . to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day , but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps p-f analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono , yoshiyuki kuroda , fumiko matsui , yoshihito tokita , keiko nakanishi , michiru ida , masahito ikawa , masaru okabe , katsuhiko ono , atsuhiko oohira institute for developmental research, aichi human service ctr., kasugai, japan; research institute for microbial diseases, osaka university, suita, japan; national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi ( ) ps p-f phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata , tatsuhisa watanabe , ikuko suzuki , junichi kitagawa , akiko ogawa , kenro kanda , kazunao kuramoto dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats ( - months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura , koichi obata , masafumi sakagami , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk , mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days and after cfa injection. furthermore, time course of phosphorylated-erk level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk in drg neurons may contribute to the development of inflammatory pain. ps p-f activation of erk in drg neurons contributes to acute pain toshiyuki mizushima , , koichi obata , takashi mashimo , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p in dorsal root ganglion (drg) neurons. however, there has been no study examining erk (also known as big mapk or bmk ) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk in small-to-medium diameter sensory neurons with a peak at min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and spinal cord and participate in pain-related behaviors in the l spinal nerve ligation (snl) model. l snl induced an increase in the phosphorylation of erk not only in the injured l drg, but also in the spared l drg at day after surgery. furthermore, l snl induced a striking increase in erk phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai , minoru asada , naoki seno , hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan; pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣- , which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps p-f interleukin- ␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda , jun kadoi , msanori nasu , masayuki takahashi , shigeji matsumoto dep. physiol and res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il- ␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il- ␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il- ␤ induced depolarization in inflamed rats was larger than that in control rats. il- ␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il- ␤ paracrine mechanism following peripheral inflammation. junichi kitagawa , mamoru takeda , jun kadoi , yoshiyuki tsuboi , shigeji matsumoto , koichi iwata dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day after ion treatment and the threshold decrement was lasting more than day . single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day - after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for min. at , , , and h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at h, peaked at h and then decreased. intense gfap immunoreactivities were also detected at and h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for min at m/min. after the running, rats were applied the weight on the gastrocnemius muscles for min, and then spinal cord and soleus muscles were removed from the rats. productions of il- beta, il- and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at h post-running, number of fos-positive neurons was increased, peaked at h and then decreased to control level at h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo , yusuke sakai , aya masawaki , takashi shimoda , masayuki moritani , motohide takemura dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep receptor (ep r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca + ] i ) in a ptx-sensitive manner in cho cells expressing canine ep r and mouse bk b receptor (b r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk ( nm) was applied twice with a -min interval to the cells expressing b r, the second [ca + ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h- ( m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk ( pm) treatment for h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca + ] i was at least in part mediated by pka. ps p-f contribution of peripheral ht a or ht receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral ht a or ht /r in male rats with tmj inflammation. here we tested if these two ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c . fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for days compared to naive rats. we tested if local ht a or ht /r antagonist affects fos expression in both groups. the number of fos-li in the vc/c but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral ht a and ht /rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of -ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼ % of sg neurons examined, -ht induced an outward current. the outward current was mimicked and suppressed by a -ht a agonist and -ht a antagonist, respectively. in ∼ % of sg neurons, -ht evoked an inward current which was mimicked by a -ht agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that -ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of -ht a and -ht receptors, respectively. the reciprocal postsynaptic actions of -ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by -ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps p-g nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi ( ) and km ( ) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about % of neurons examined, melittin ( m) superfused for min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at mv in the presence of bicuculline. this action was visible about min after the beginning of its superfusion and subsided within min after washout. these melittin actions were reduced in extent by a pla inhibitor -bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi ( ) ps p-g presynaptic p y receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied -methylthio adp ( mesadp, m), a p y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about % of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by mesadp with no change in the amplitude. the mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs ( m), a selective p y receptor antagonist. these results indicate that the activation of presynaptic p y receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p y receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi ( ), the japanese health sciences foundation (kh ) ps p-g presynaptic enhancement by proteinaseactivated receptor- agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)- but not par- agonist (each m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par- mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par- agonist (sfllrn, m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at - mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par- antagonist (yfllrnp, m). these results indicate that the activation of par- s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par- activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi ( ) ps p-g effect of tramadol metabolite m on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m , which is one of its metabolites, at mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at − mv. this action was not seen in the presence of a -opioid receptor antagonist ctap ( m). m also reduced the peak amplitudes of epscs which were monosynaptically evoked at − mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk- or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk- injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk- . these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps p-g studies on pain control system (rept. ): changes in phosphorylation of nr b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for weeks. under these conditions, the immunoreactivities of phosphorylated-ser- nr b (p-ser- nr b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr- nr b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps p-g prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, hz, a, s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by -ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps p-g the nav . sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav . , a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav . . four weeks post-sci, nav . protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav . caused a significant reduction in nav . expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda , hiroyuki ichikawa , ryuji terayama , tomosada sugimoto department of oral maxillofacial rehabilitation, okayama university, okayama, japan; department of oral function and anatomy, okayama university, okayama, japan ib -sap is a neurotoxin designed for targeting primary nociceptors with ib binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib -sap ( . l of . % solution for each ganglion) directly into the th and th lumbar (l and ) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib -binding. after counterstain, the cell body size of neurons were measured. ib -sap reduced the total number of drg neurons in l and ganglia combined by % ( ± on untreated side versus ± on treated side). small neurons (< m ) were reduced by % whereas large ones (≥ m ) were not affected. ib -binding neurons were mostly small (≥ %) and were reduced by %. the number of small neurons, that were not stained for ib -binding, increased by % ( ± versus ± ). schuichi koizumi , kaoru nasu-tada , makoto tsuda , emiko kunifusa , , kazuhide inoue div. pharmacol., natl. inst. hlth. sci., tokyo, japan; dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p x receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p x receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p x receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p x receptor in microglia, leading to neuropathic pain. research funds: mf , kakenhi ( ) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi ps p-g lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at mm decreased the protein levels of myelin basic protein (mbp) at h. this action was significantly inhibited by botulinum neurotoxin/c (bont/c ). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp , mag, p in cultured fibers. the maximal decrease was observed all at as early as h after the addition of lpa. bont/c and y abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at and , , , , days after operation. pre-emptive co-administration of mk- and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk- decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama , , naomi akiyama , mikie hirata , takayuki nakagawa , shuji kaneko , masabumi minami dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for h per day between postnatal day and . visceral sensitivities were assessed in stomach and colon at weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research ps p-g change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered to days after exercise in rats ( w old). c-fos expression in the superficial dorsal horn increased in l spinal segment when the edl muscle was compressed days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats , ( - ) and ( - ) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in w old rats, but not significant. after lec, the threshold started to decrease day after lec in all three age groups. it returned to the pre-lec level days after lec in and w old rats only. recovery of w old rats delayed up to days after lec. increased c-fos expression in the superficial dorsal horn was observed in l as well as in l in w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama , masaharu akita kanagawa life-science res., japan; kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day to of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days - of age, they did not show the first estrus before days of age with mean age of . ± . day. in contrast, most of normal mouse igg-treated controls showed the first estrus by days of age with mean age of . ± . day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss- gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days of age (d ), and first estrus on around d . brain tissues were obtained on d , , , and . kiss- mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d to and was kept at a high level thereafter. gpr mrna expression in the medial preoptic area increased significantly from d to . metastin-immunoreactive cells were not found on d but were apparent in the arc-me on d onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito , sei-etsu fujiwara , kenjiro konno , takashi yamaguchi , tetsu nemoto , etsuko kasuya , ryosuke sakumoto anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; grad. sch. sci. & eng., yamagata univ., yonezawa, japan; sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps p-g hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi , teruko uwano , , anh tran , , eturou hori , , taketoshi ono , , hisao nishijo system emotional science, univ. of toyama, japan; integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of hf neurons, responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue , atsu aiba , kaoru inokuchi mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; grad. sch. med., kobe univ., kobe, japan vesl- s/homer- a and vesl- l/homer- c are splicing isoforms encoded by the vesl- gene. vesl proteins bind and regulate mglur / , ip receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl- s is upregulated by tetanic stimulation that elicits l-ltp. vesl- s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl- s gene-targeting mice (ko) and examined whether vesl- s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl- s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta , r. migishima , s. kida , k. inokuchi , mitsubishi kagaku inst. life sci (mitils), japan, tokyo univ. agricul., japan; crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps p-h sex differences in the effects of chronic estrogen treatment on fear conditioning in c bl/ j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c bl/ j mice were implanted with a silastic capsule containing , , or g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps p-h analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps p-h analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps p-h analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor (cb ) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb and lvgccs, we also found that cb is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda , takahiro furuta , kouichi nakamura , , takeshi kaneko , ps p-h difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama , tomo unzai , kouichi nakamura , , sakashi nomura , takeshi kaneko , department of morphological brain science, kyoto university, kyoto, japan; department of physical therapy, kyoto university, kyoto, japan; crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut) , thalamostriatal inputs, were less dense in patch region, whereas vglut -positive corticostriatal inputs were evenly distributed. quantitative analysis revealed % of vglut positive synapses in patch region were formed with spines, whereas % in matrix region were made with dendritic shafts. in contrast, the targets of vglut -positive inputs were mainly spines in both regions. moreover, vglut -positive axospinous synapses in patch region were larger than vglut -positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi ( , , , ( ), ) ps p-h single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai , fumino fujiyama , takeshi kaneko , department of morphological brain science, university of kyoto, kyoto, japan; crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi ( , , , ( ) , ) ps p-h lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui , kayo nishizawa , nobuyuki kai , kazuto kobayashi , dept. of psychology, fukushima univ., japan; dept. mol. genet., fukushima medi. univ., japan; crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of -ohda injections into the nucleus accumbens (acb) in c bl/ j mice. all mice were trained to press the lever for a food pellet using a fixed ratio (fr ) schedule. the mice were injected with ascorbate vehicle or -ohda into the acb, and then tested post-surgically using the fr schedule again. the -ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area , it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area , suggesting different functional roles played by each subdivision of area in spatial memory and behavior. eiichi jodo , yoshiaki suzuki , tadahiro katayama , ken-yo hoshino , yukihiko kayama dep. of physiol., fukushima med. univ., fukushima, japan; dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, %) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about . s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in % out of vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only % out of it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., ) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., ) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., ) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of sessions in phase , although they learned easily the four problems in phases and . our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto , noriyuki higo , munetaka shidara neuroscience research inst., aist, tsukuba, japan; grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d receptor study using antisense cdna showed that d receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d receptor antipeptide antiserum. d receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area of the perirhinal cortex than in area teav. the differential localization between area and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano , natsuko yoshida , kiyohisa natsume kyushu kyoritsu university, fukuoka, japan; kyushu institute of technology, fukuoka, japan; kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f and f was the highest and suggests that the theta rhythm at f and f may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after -month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi ( ) ps p-h neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe , sho yagishita , hideyuki kikyo , department of physiology, the university of tokyo school of medicine, tokyo, japan; department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a . t scanner system was used (te = ms; tr = s; acquisition time = . s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong , motoaki sugiura , yuko sassa , keisuke wakusawa , , kaoru horie , , shigeru sato , , ryuta kawashima , gsics, tohoku university, sendai, japan; niche, tohoku university, japan; miyagi university of education, sendai, japan; department of pediatrics, school of medicine, tohoku university, japan; the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz , christie l. sahley , kenneth j. muller physio. & biophys., univ. of miami, miami, fl, usa; biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps p-h sensory/motor modules regulating the development of peer social relationship mamiko koshiba , , shun nakamura jst, crest, japan; ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata , minoru hattori department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) -s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi -s procedure over days. the -s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the -s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi -s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps p-i the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka , , ritz oshio , , norihiro sadato , , manabu honda , nips, okazaki, japan; jsps, tokyo, japan; nagoya univ., nagoya, japan; ristex, jst, tokyo, japan; sorst, jst, tokyo, japan; ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps p-i sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi ( ) ps p-i selective manipulation of working memory through d and d receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d and d receptors. the computer simulation with this model shows that d receptor activation mainly affect working memory activity itself, while d receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d and d receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or mg/kg mam on the th day of gestation. the activity of male and female offspring was measured for each h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every min interval for h under min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca pyramidal layer and ectopic neuron mass). ps p-i long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur . hamid gholamipour , shirin babri , khameneh saied department of physiology, university of tabriz, tabriz, iran; university of tabriz, iran; university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca region of hippocampus on memory formation. fifty male rats were divided into five groups. ( ) control ( ) sham operation ( ) test ( ) diabetic/saline ( ) diabetic/insulin. groups and were made diabetic by treatment with stz ( mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < . ). compared to sham group diabetic/insulin group showed increased latency (p < . ) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca + /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of per , in india. we studied the park polymorphism in north indian population and parkin expression in early parkinson's disease (n = ) and sporadic parkinson's disease (n = ). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon- , and in early pd and exon- and in sporadic pd, heterozygous mutations in exon and in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul , kittisak sripanichkulchai , david finkelstein faculty of medicine, srinakharinwirot university, thailand; faculty of medicine, khon kaen university, thailand; university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c bl/ mice were pretreated with beta-estradiol (e ), injected with mptp on the day and brains were collected on day . e pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps p-i effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki , fumihiko yoshino , masaichi chang-il lee dept. of food sci. and nutrition, showa women's univ., tokyo, japan; clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi ( ) ps p-i recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi , kaori sawada , haruo okado , atsushi nambu , masahiko takada tokyo met. inst. neurosci., fuchu, tokyo, japan; natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype- raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type- ) of raav (raav ) carrying αs cdna (raav -␣s), and a cocktail of raav -␣s and raav carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav -␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged - s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of . - (i), . - (ii), . - (iii), and . - (iv). the ␤waves were rated with total periods (%) of - hz wavelets in -s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated - % in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about % in group iv. the pvp ␤-waves were - %, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana , , hirokazu iwamuro , , masahiko takada , atsushi nambu , div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; sokendai; dept. neurosurg., univ. tokyo, tokyo, japan; dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd expression on microglia and also enhanced the mt -mmp expression to shed off cd at the cell surface and degrade surrounding ecm to open the migratory way. a t mutant ␣-synuclein showed greater level of cd shedding and cell migration. extracellular treated ␣-synuclein also increased cd and mt -mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant ( grant ( - ps p-j serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai , hiromasa yamada , yoshinari aimi , ikuko nagatsu department of anatomy, shiga university of medical science, otsu, japan; fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba -and tb distribution in microglia were immunohistochemically investigated at h and days after twice mptp-treatments (one treatment comprised of intraperitoneal injections of mg/kg mptp at h interval) to c bl/ and balb/c at months (mo) interval. the recognizable change of iba -and tb -distibution in microglia of both mice strains was observed even mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps p-j activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada , ryohei hishida , yoko hirata , kenji ono , hiromi suzuki , shin-ichi muramatsu , imaharu nakano , kunihiro tsuchida , toshiharu nagatsu , , makoto sawada school of medicine, fujita health university, aichi, japan; division of neurology, jichi medical university, japan; department of biomolecular science, gifu university, japan; research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps p-j phosphorylation of erp in adult rat brain with neonatal -ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of -ohda at (p ) and days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p , tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the -ohda lesioned rats at p , and it was identified as disulfide-isomerase a precursor (erp ) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi ( or nm, h) was applied to cultures at days in vitro and succeeding changes of da neurons were investigated up to days. more than % of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps p-j specific expression of proapoptotic factor pag on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p -activated gene (pag ), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag in the internal capsule of l-dopatreated and constitutive expression in the smi- -immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps p-j an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang , , yuzuru imai , haruhisa inoue , , ayane kataoka , sachiko iita , nobuyuki nukina , ryosuke takahashi , neurology, university of kyoto, kyoto, japan; bsi, riken, saitama, japan since loss of parkin e activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., ) have found that application of an antisense oligodeoxynucleotide specific for nr have successfully knockdown the expression of nr gene expression in the striatum of -hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr was re-addressed using a small interfering rna (sirna) specific for nr . in pc cells, reductions of nr proteins after a single application of nr sirna were found by western blot experiments. and after one single application of nr sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr immunofluorescence were found in the striatum after the sirna treatments. lai et al., . neurochem. int. , - . research funds: faculty research grant, frg/ - /ii- , hong kong baptist university ps p-j homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy ( . - mol in l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on th day. animals with mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps p-j ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca takahiro seki , takayuki shimahara , naoko adachi , naoaki saito , norio sakai dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type (sca ), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca . however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca + homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki , yo sasaki department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat ) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at min, but next increased at h and returned to basal at h after cocaine challenge. actin showed a similar pattern but was maintained at high level at h. both psd- and glur were increased between h and h like actin. by contrast, some proteins such as drebrin were decreased after the peak at h. interestingly, the s proteasome subunit demonstrated a dramatic upregulation at h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps p-k effects of mdma and -meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, , methylenedioxymethamphetamine (mdma) and -methoxy-n,ndiisopropyltryptamine ( -meo-dipt), on sert and dat were examined. as previously reported, mdma ( . - m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, -meo-dipt ( . - nm) evoked an outward current response in sertexpressing cells similarly to that of selective -ht reuptake inhibitors. no current response was observed when -meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that -meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka , nobue kitanaka , tomohiro tatsuta , , yoshio morita , motohiko takemura department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine ( mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of . mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of and mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth ( . mg/kg, i.p.), pretreatment with subchronic clorgyline ( . - mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura , nobue kitanaka , tomohiro tatsuta , , yoshio morita , junichi kitanaka department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline ( . - mg/kg, i.p.) min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum min after drug challenge. these results suggested that the inhibitory effect of lobeline ( . - mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with mg/kg meth and locomotor activity measured daily for days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, nmol ufp- (a nop receptor antagonist) and mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp- strongly suppressed locomotor activity. thus, it was unclear if ufp- suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp- co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine ( - mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine ( . - mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine ( , . and mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine ( mg/kg, i.p.) caused more than % mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine ( mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps p-d regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: ( ) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis (ca ) were increased after fs. the rule of this kind of changing was of region-and time-specific. ( ) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi , , tomoaki shirao dept of neurobiol & behav.; ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only . ± . % of drebrin in the spine is stable, with a turnover time of . ± . min. blockade of ampar by m cnqx reduces the population of stable drebrin ( . ± . %), and has no effect on a turnover time. on the other hand, blockade of nmdar by m ap has no effect on the population of stable drebrin, whereas shortens a turnover time ( . ± . min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu , yoshito saitoh , kaoru inokuchi , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki , , daisuke okada , kaoru inokuchi , , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; yokohama natl. univ. environment information sci., kanagawa, japan; crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto , mitsutoshi setou , , kaoru inokuchi laboratory for molecular gerontology, mitils, japan; laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps a-a an activity that traps vesl- s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl- s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at m, but not nm. because nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps a-a acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosening, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca region in rat hippocampal slices. an application of . dheas for min to the slice augmented instantly the epsp, which was terminated within min. however, even h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosensing, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan to know whether ␤-estradiol (e ) can protect ca neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion ( vo) for min, and the effect of e against this ischemic injury was examined. the electrophysiological properties of ca -ca synapses were examined by a real-time optical recording method days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e ( mg/kg) h before vo was able to protect ca neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt ( mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn ( mg/kg) did not. above results suggest that e can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps a-a non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima , mari ogiue-ikeda , yuki oishi , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca , ca , and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps a-a morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki , gen murakami , , tetsuya kimoto , , suguru kawato , college of humanities and sciences, nihon university, tokyo, japan; department of biophysics and life sciences, university of tokyo, tokyo, japan; crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of nm dex induced rapid modulation of the density and morphology of dendritic spines in ca pyramidal neurons within h. the total spine density increased from . spines/m to . spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura , yuji ikegaya , sadao shiosaka division of structural cell biology, naist, nara, japan; laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: st century coe research ps a-a actin organizations within single dendritic spines in ca pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of . min and min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds ( . - . um/min) and lengths ( . - . um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori , katsushige sato , kohichi tanaka , hiroko hamazaki mol. neurosci., tmdu, tokyo, japan; physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at or days post-hatching (p or p ), and at p . this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p , but they lose this ability at p . in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps a-b effect of estrogen on hippocampus in male and female mice takanori sugawara , shinji hayashi , victoria luine graduated school of integrated sience, yokohama city university, yokohama, japan; department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received ␤-estradiol (e ) or oil-vehicle injections at adult before golgi impregnation. spine densities m of apical dendrites of the pyramidal neurons in the hippocampus ca region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto , , takafumi inoue , , akinori kuruma , katsuhiko mikoshiba , , lab. developmental neurobiology, brain science inst., riken, saitama, japan; div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca + reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p mapk and c-jun nh -terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u , a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb and sp , antagonists for p mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps a-b regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey , chou-chi li , yumiko motoyama , junko yamaoka , masao ito riken brain sci. inst., japan; national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur . glur phospho-ser promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur -vcp interaction. yuichi kitagawa , , shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp- and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps a-b long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from to days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over days old, when song learning is nearly complete, but ltd remained possible in birds over days old who had been isolated from song tutors, and thus retained the capacity for learning. ps a-b involvement of ca + -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that exposures to glu of cultured rat hippocampal slices at h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur and glur , showed that the number of glur -positive puncta increased transiently after the repeated glu exposures, whereas the number of glur -positive puncta increased gradually and persistently. jstx (a ca + -permeable ampar blocker) suppressed fepsp amplitude recorded at ca -ca synapses by - % in the period corresponding to the transient increase of glur -positive puncta. this transient increase should represent the delivery of ca + -permeable (glur -lacking/glur -including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca + -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka - , japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd times ( h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of -ht ( m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the -ht b receptor agonist cp mimicked the presynaptic inhibitory effect of -ht. -ht b receptor antagonist nas- reversed the -ht inhibitory effect, indicating that the -ht induced inhibitory effect occurs by mediating -ht b receptors. the presynaptic inhibitory effect of -ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, -ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this -ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that -ht, upon activating -ht b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku , masato ano , yuki hashimotodani , tadasato nagano , masanobu kano dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, -ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a , a a, a b and a ). adenosine a and a receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a and a receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a agonist (cpa) and a agonist ( -cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and -cl-ib-meca as reported previously. although ca currents were decreased by -cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of -cl-ib-meca. at administration of cpa and -cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a receptor may inhibit adenosine a receptor throughout a intracellular pathway in neurons. ps a-c influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa , j. yamada , k. inoue , y. yanagawa , a. fukuda , dept. physiol., hamamatsu univ. sch. med., japan; dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad -gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa , hiroyuki hioki , kouichi nakamura , , hisashi nakamura , takeshi kaneko , dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter (vglut )-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut -positive cells showed immunoreactivity for -hydroxytryptamine ( ht), a significant proportion ( . %) of vglut -postive cells was ht-negative. in this study, triple immunofluorescence staining was performed for vglut , ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad ) and tyrosine hydroxylase (th). our results showed that all of the vglut -positive/ ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut positive/ ht-negative neurons didn't show any immunoreactivities for gad nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut -positive neurons in these nuclei. research funds: kakenhi , , ps a-c cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig , mika ushimaru , yoshiyuki kubota , akiya watakabe , tetsuo yamamori , yuchio yanagawa , yasuo kawaguchi div. cerebral circuitry, nips, okazaki, japan; div. brain biology, nibb, okazaki, japan; dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin ( -ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, % of the cs were inhibited by -ht through -ht a r and % were excited through -ht a r. however, % of the fs cells were inhibited and % excited. these results show that cs cells are more inhibited by -ht than fs. the expression of -htr was confirmed by in situ hybridization. research funds: jsps pe and ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v , teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer but also in layer and/or . in each of the cases, about - gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad , somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine ( ) their other features, ( ) possible species variability, ( ) their functional significance. supported by riken bsi. withdrawn ps a-c regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge , , susanna b. park , greg j. stuart , yasuo kawaguchi national institute for physiological sciences, japan; div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure ( m) inhibited layer pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers / were generally less responsive to ach, but ach inhibited layer cells in visual cortex. prefrontal layer pyramidal neurons were more responsive to ach than were layer cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations ( mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in % of non-fs interneurons ( of cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki , , kenzi saito , , yuchio yanagawa , department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; sorst, jst, kawaguchi, japan; sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad -deficient mouse fetus. ps a-c gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal to day in egl. its release, especially from premigratory zone, was inhibited by ni + , but cd + couldn't. we suggest that gaba excitation induces granule cell migration. ps a-c gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi , hiroaki wake , junichi nabekura , osamu takahashi department of histology, kanagawa dental college, yokosuka, japan; national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata , kunihiko obata , jonathan davies , mark bellingham , peter g. noakes lab. for memory & learning, riken-bsi, wako, japan; obata res. unit, riken-bsi, wako, japan; sch. biomed. sci., univ. queensland, queensland, , australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., ) . we investigated the involvement of gaba alone in the mn death using gad -deficient mice, in which cerebral gaba is reduced to less than % of the wild-type. mn numbers at embryonic day (e) were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e embryos and subjected to electrical recording from the c and c ventral roots and also gaba measurement. in gad -deficient embryos, increase in number of brachial mns ( %) and decrease in both spontaneous discharges in the c , c roots and gaba content (less than %) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣ ␤ ␥ and ␣ ␤ ␥ combinations ( % reduction) but had no effect at ␣ ␤ ␥ or ␣ ␤ ␥ . in slice recordings dmcm blocked ipscs by % in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥ subunits whereas in the central amygdala some inputs contain ␥ while others contain ␥ subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps a-d hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane , takashi iwai , yasuo nagai , junichiro oka , hideo kimura dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h s. we examined the effect of h s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na s), a donor of h s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na s. these observations suggest that h s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by ( l) lidocaein ( %) min before training. animals were allowed to self administer morphine ( mg/kg per inf.) ten consecutive daily -h session. during all morphine self administration session lever pressing was measured. our results show that: ( ) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. ( ) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca + wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of ␤ estradiol from glucocorticoid activity yuki oishi , suguru kawato department of physics, graduate school of science, university of tokyo, tokyo, japan; graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, ␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. ␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that ␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps a-d the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex ( . mg/ g b.w., sc) or sesame oil (vehicle control) everyday for - days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf- mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf- , estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf- and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps a-d mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of -hydroxydopamine. ps a-d the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba , kimi watanabe , munekazu komada institute for developmental research, aichi human service center, aichi, japan; graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida , , asif k mustafa , kenji hashimoto , kumiko fujii , , kazuhisa maeda , , hiroshi ujike , richard l. huganir , solomon h. snyder , akira sawa dept. of systems biology, obi, suita, japan; depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; chiba univ. forensic mental health, chiba, japan; dept. of psychiat, shiga univ. med. sci., shiga, japan; div. of neuropsychiat, tottori univ., yonago, japan; dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick knockout mice were significantly lower than those of their wildtype littermates at age of p , but not in adults, suggesting regulation of pick on sr at developing stage. in case-control association study, we observed an association of the pick gene with sz, which is more prominent in disorganized sz. our findings suggest that pick contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps a-d epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe , , werner kilb , ileana l. hanganu , taizhe qian , daiichiro nakahara , atsuo fukuda , heiko j. luhmann dept. of physiol., hamamatsu, japan; inst. of physiol., mainz, germany; dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca region of an intact preparation. mm taurine blocked epileptiform activity induced by mg + free acsf and m -ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps a-d responses of pge in undifferentiated and differentiated ng - cells kayoko matsushima , takashi imanishi , akinori kawaguchi , tetsuyuki wada , shigeru yoshida , seiji ichida school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of sci. & eng., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan our previous findings showed that -ht-and bk-induced [ca + ] i increases were enlarged in differentiated ng - cells. for the next stage, we investigated the effect of pge , an inflammatory mediator for -ht and bk, on the cells. ng - cells were loaded with fura- /am, and the change in [ca + ] i was monitored by an image processor. the results showed: ( ) pge -induced response was decreased when ng - cells were differentiated by bt camp, ( ) − m ah and sc irreversibly inhibited pge -induced response by about % and %, respectively, while − m ah and sulprostone had no effect, and ( ) pge -induced response was abolished under ca +free conditions in about % of both ng - cells. these results indicate that the response to pge , via ep and ep receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from -to -day-old rats. bath application of serotonin ( -ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during -ht application. under the current-clamp recording, -ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that -ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps a-d searching for endogenous ligands of trace amine receptors in mammals ( ) akira komatsu , airi yamaguchi , noriko makikusa , osamu koizumi dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by % dms, post-fixed by % formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps a-d effects of mg + on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa , , nahoko kasai , akiyoshi shimada , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, kanagawa, japan; sorst/jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg + plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca + concentration are largely affected by mg + removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg + removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg + concentration and their actions on neural activity using a mg + indicator, kmg- -am together with fluo -am. we observed different effects of the mg + removal on gabaergic and non-gabaergic neurons by using gad -gfp knock-in mice. research funds: jst/sorst ps a-e transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p -p . zn + cortical neurons were also labeled, intrinsic and extrinsic, from p . unlike in the adult, these were in layer , instead of layers , , and . at p , neurons occurred in layers , , and and, in some areas, layer . at p , zn + neurons first appeared in layer ; and at p , there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. ps a-e systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano , , ryohei kanzaki , kei ito , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi , , taizo nakazato , shigeru kitazawa neurol, juntendo univ., tokyo, japan; physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, ) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns ). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse ( to mv for da; to mv for ht) was applied at hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps a-e time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui , , yuko sekino , , tomoaki sirao dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; div. of neural network, inst. med. sci. univ. of tokyo, tokyo; crest, jst, kawaguchi, japan; jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at days in vitro (div), and analyzed the dynamic localization of gfp-da at div. glutamate stimulation started gfp-da translocating within s and completed in min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps a-e distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa , jun shiota , hiroyuki tsutsumishita , hiroyuki sakagami , masaaki tsuda , akiko tabuchi dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr- , c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi ( ) to a.t. shoko shimizu , shinsuke matsuzaki , tsuyoshi hattori , ko miyoshi , masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia (disc ) was identified as a novel gene disrupted by a ( ; ) (q . ;q . ) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc at centrosome and residues - of disc (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc to target to the centrosome. furthermore, we have shown that inhibition of the disc -kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc is important to the disc -kendrin interaction and that a truncated form of disc lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca + or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca + and cl − imaging. we found that the relatively low density a glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca + oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps a-e involvement of ca + influx in the unpolarized non-vesicular release of fgf- hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf- lacking a conventional signal peptide sequence. we found that fgf- is co-released with s a , a ca + binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf- is co-released with s a from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf- and s a required ca + . the overexpression of s a - mutant lacking an ability of interaction with fgf- inhibited the their release, suggesting that s a is a cargo molecule. the release of fgf- upon either stimulation was abolished by voltage-dependent n-type ca + channel blocker. these findings suggest that ca + influx may be involved in the unpolarized non-vesicular release of fgf- . in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that -ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca + ] i ) in pc cells. however, it is unclear how [ca + ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca + ] i on actin dynamics in pc cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a . treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to -ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp is endogenously expressed in pc cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp in actin dynamics in growth cones and found that knockdown of clp expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp deletion mutants that both of pdz and lim domains are necessary for the proper function of clp . ps a-e screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura , tsuyoshi kobayashi , yuchio yanagawa , kunihiko obata , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; grad. sch. of medicine, gunma univ., maebashi, japan; bsi, riken, wako, japan; sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the clones tested, two genes, neuronatin and seizure related gene (sez- ) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu , , tomoko matsumoto , , sakae mikami , takashi nagasawa , fujio murakami , grad. sch. of frontier biosci., osaka univ., japan; sorst, jst, japan; inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf- and its receptor cxcr in the precerebellar migration. we show that cxcr is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr and sdf- knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps a-e involvement of an immunoglobulin superfamily molecule, neph /mkirre in the migration of precerebellar neurons kazuhiko nishida , , kazuhide nakayama , saori yoshimura , , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph , neph /mkirre, and neph , only neph /mkirre was strongly expressed in pcns. expression of neph /mkirre was detected from e . when pcns migrate tangentially. the expression level became weaker at p , when pcns stop the radial migration, raising the possibility that neph /mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph /mkirre in the migration of pcns. hiroki umeshima , , toshio ohshima , tomoo hirano , mineko kengaku lab. for neural cell polarity, riken bsi, wako, japan; department of biophysics, kyoto university, kyoto, japan; lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk (cdk -dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin -gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk -dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito , , takahiko kawasaki , , tatsumi hirata , division of brain function, national institute of genetics, mishima, shizuoka, japan; department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc functions with unc- to regulate axonal transport in drosophila hiroaki mochizuki , hirofumi toda , , emiko suzuki , joseph gindhart , toshifumi tomoda , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; gene net. lab., natl. inst. genet. mishima; beckman res. inst., city of hope, ca, usa; dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc , which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc is essential for neural development in drosophila. loss of function of drosophila unc results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc genetically interacts with unc- , an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc mutants, unc- was separated from synaptotagmin vesicles. these results suggest that unc coordinates kinesin-cargo interaction via unc- to regulate dynamic axonal transport. ps a-e change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi , daisuke takahashi life science inst. sophia university, tokyo, japan; waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi ( ) and kakenhi on priority areas ( ) ps a-e generation and analysis of region-specific rac -deficient mice hidetoshi kassai , masahiro fukaya , eriko miura , mizuho sakahara , masahiko watanabe , , atsu aiba div. cell biol., kobe univ. grad. sch. med., kobe, japan; div. physiol. sci., hokkaido univ. grad. sch. med., japan rac is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac in the cns is poorly understood because of the embryonic lethality of rac knockout mice. in this study, we generated and analyzed region-specific rac -deficient mice (emx -rac ko mice) by the cre-loxp system, in which a promoter for emx homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx -rac ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx -rac ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map b containing nterminal amino acids promoted neuronal death. to reveal the mechanism of map b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map b by two-hybrid system. alpha-tubulin was found to interact with map b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map b n-terminal has not yet been reported. beta-tubulin was also found to interact with map b n-terminal. when ␤ tubulin was divided in fragment at between amino acid and , there was no interaction between ␤ tubulin fragments and map b n-terminal. interaction needs the continuous region over aa and . there were much proportion of round formed cos cells in n-terminal containing map b transfected cells than in n-terminal lacking map b transfected cells. there might be some interference in interaction between map b and tubulin in cells express map b containing n-terminal. otone endo , , masaaki mizuno , yasukazu kajita , jun yoshida department of neurosurgery, ja kainan hospital, aichi, japan; department of neurosurgery, nagoya university, nagoya, japan; department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta , masae yaguchi , yumi matsuzaki , yoshiaki toyama , yutaka kawakami , hideyuki okano , masahiro toda , neuroimmunology research group, keio univ., tokyo, japan; physiology, keio univ., tokyo, japan; orthopaedic surgery, keio univ., tokyo, japan; institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd c + dcs, cd ␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd ␣ + dcs compared to cd c + dcs. these results suggest that cd ␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps a-e von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo , hiroshi kanno , takaakira yokoyama , shuichi nakano , naoki sugimoto , nahoko kobayashi , tetsuhiko yoshida , isao yamamoto dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; fiber and dept. of chemistry, konan university, kobe, japan; toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly %, and peptide with the elongin c binding site (residues - ) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps a-e the effect of pueraria mirifica on erk / and s- following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e ) on the expression of erk / and s- following sciatic nerve crush and transection in rats. protein levels of perk / and s- in distal segments of nerve at day were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e , caused a significant decrease in the expression of perk / levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk / phosphorylation. following treatments, levels of s- were significantly decreased both in crushed and transected nerves with respect to control group at p < . . this estrogenic effect was blocked by ici , . because of their structural similarity to e , pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day . this study suggested that pm as well as g could enhance nerve regeneration like e by interfering with the injury-induced erk signaling pathway. yasuhiro kato , takafumi suzuki , kunihiko mabuchi department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf- and stat , which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p - wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y , a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin ( ht) positive cells increased in number progressively from day to weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and ht cells surrounded the axons and glia. thus, ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda , yukio someya , ryo kadota , chikato mannoji , tomohiro miyashita , atsushi murata , masashi yamazaki department of orthopaedic surgery, togane hospital, department of ortopaedic surgery, graduate school of medicine, chiba university, japan; division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt ( . mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), -bromo- -deoxyuridine- -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps a-f early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski , hanna kozlowska , marcin jurga , aleksandra habich , elzbieta wanacka , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed h, h, h and days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue , , and days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext , one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican- and syndecan- were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe , hiroe sagawa , masahiro ichikawa , yoshihito tokita dept. perinatol., int. dev. res., kasugai, japan; dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y , can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for d. after fixation, the neurites were stained with anti-tuj antibody to obtain number and length of tuj neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day , wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of -days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of -days-old embryonic mice by culturing in dmem/f medium for days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of - div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for - div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for days. dha increased the number of tuj (+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi ( ) ps a-f interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps a-f effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the d structure of surrounding stimuli defined by binocular disparity. ps a-f rotational motion aftereffect in positive direction for -dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for -dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known dimensional mae. this finding suggests that the visual system has a mechanism that detect -dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j takanori uka, ryo sasaki department of physiology , juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch ( deg) of a center/surround display presented degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always percent. for each of subjects, we calculated direction discrimination thresholds (at % correct) at each surround size. consistent with crowding, thresholds increased when surround size was . and degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was and degrees, relative to degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps a-f generation of receptive fields in higher visual areas based on v columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v and v , respond to stimuli with complex shapes. how do these neurons integrate signals from v ? in particular, does the well-known columnar organization of v play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about % of model neurons exhibit responses similar to those of v and v neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a -d surface slant is perceived (blakemore, ) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa ) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext( ), jsps( ), coe ps a-f consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi , kota sasaki , izumi ohzawa , grad. school of frontier biosci., osaka univ., japan; grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and -d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by -d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext( ), jsps( ), coe ps a-f firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate , , hiroshi tamura , , ichiro fujita , graduate school of frontier biosciences, osaka university, osaka, japan; jsps, japan; crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi - ps a-f spatial-frequency dependency of receptive field size and surround suppression in lgn and v hironobu osaki , tomoyuki naito , osamu sadakane , masahiro okamoto , hiromichi sato , med. sch., osaka univ.; grad. sch. med., osaka univ.; grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v ), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v (n = ) and lateral geniculate nucleus (lgn) (n = ) in anesthetized cats. first, we found that v neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura , akichika mikami , kazuo itoh department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v a, we injected a bi-directional tracer biotinylated dextran amine into the v a. our results indicated that the v a has connections with the occipital, parietal and temporal cortices. the v a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v a has commissural connections with the v , the v a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v . these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps a-g activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera ( fps), and were also monitored with a d or d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity - ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps a-g timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki , , arman abrahamyan , catherine stevens , andreas a. ioannides , brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately ms (n ) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto , shoutaro akaho , kenji fujikumi , yasuko sugase-miyamoto , masato okada aist, ibaraki, japan; ism, tokyo, japan; university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of neurons for visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps a-g relationship between color and shape selectivity in area teo of the monkey masaharu yasuda , , hidehiko komatsu , national institute for physiological science, okazaki, japan; sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps a-g neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a -choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was . , a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across - simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant ( ) ps a-g behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki , atsuko komori , tomonori manabe , toshihiko hosoya , hiroshi sagara , emiko suzuki , hitoshi okamoto , ichiro masai masai initiative research unit, riken, wako, japan; riken bsi, wako, japan; ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde c activity is important for the sensitivity of vision. ps a-g localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: - years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from to years of age, and showed a decline and lack of ir in older retinas ( - years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from to years of age, and absent at years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps a-g temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa , norio fujimaki , toshihide imaruoka nict, kobe, japan; kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak ( , ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity ( , ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano , , derek arnold , alan johnston , tsunehiro takeda univ. tokyo, chiba, japan; ntt cs lab., kanagawa, japan; univ. sydney, sydney, australia; ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, ) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around hz and matched with the alpha frequency of meg. as hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps a-g the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya , , kei ito , , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps a-g dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi , , toshiki tani , shigeru tanaka , graduate school of life science & systems engineering, kyushu institute of technology, japan; laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of weeks. we also found that when the goggles were removed after or weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, ) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than . hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps a-g retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., . neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada , yasuhiro enami , hiroshi jouhou , takehiko saito , kaj djupsund tokyo metropol. univ., hino, tokyo; astellas pharma. inc., osaka, japan; suny upstate med. univ., center for vision and ophthal., ny, usa; univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph only . units from the baseline of . , we found a remarkable reduction of the conduction velocity by - %, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps a-g analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi ( ) ps a-g stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of % ( of ) of neuron pairs (inter-neuronal distance > m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in % ( / ) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii , , toshihiko hosoya bsi, riken, japan; dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v ). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v . this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v neurons. masayoshi tsuruoka , masako maeda , bunsho hayashi , ikuko nagasawa , tomio inoue dept. physiol. showa univ. sch. dent. tokyo, japan; dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t -l dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention ( mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil ( %, ml). the increased emg activity significantly reduced following bilateral l -s ventral rhizotomies. a baseline emg did not significantly alter when the l -s ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps a-g hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba ) was significantly greater (p < . , uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < . , uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm agonist (l-menthol: . - mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = ) or boo rat (n = ). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm on the urothelium cell of the urinary bladder. ps a-g caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito , a.d. craig dept. physiol, shimane univ. sch. med., izumo, japan; atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol ) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns ps a-g ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps a-h fasting induced long-chain fatty acid receptor gpr expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor (gpr ) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of h fasting on gpr expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. h fasting induced gpr mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr -like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr at least fasting period. ps a-h ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that hydroxybutylate ( hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls ( v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of v respond to the change in hb. change in the intracellular calcium concentration ([ca + ] i ) in vitro was measured in dispersed ependymocytes taken from the v in rats. the present results showed that the [ca + ] i increased in response to hb, but the increase was blocked by ␣-cyano- -hydroxycinnamic acid, which is a monocarboxylate transporter (mct ) inhibitor. immunohistochemistry showed that mct -immunoreactivities were located on the v ependymocytes. these results indicate that the ependymocytes may sense hb through a mct -dependent mechanism. research funds: kakenhi ps a-h comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin ( . mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with -hydroxydopamine ( -ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at weeks after -ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at mm; anteroposterior, + mm; lateral and − and − mm; dorsoventral from bregma at weeks after -ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better -ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: ( ) retrieving the food with a rake (a motortool), ( ) retrieving the hidden food with a mirror-attached rake, ( ) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, ( ) moving a mirror hung along the rail by hand to find the food, ( ) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada , , shigehiro miyachi , michiko imanishi , masato taira , masahiko takada div. applied system neurosci., nihon univ. sch. med., tokyo, japan; dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area ( m), dorsal area ( d), and ventral area ( v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from m, d, and v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that m, v, and d receive multisynaptic projections from the entorhinal cortex, area , and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps a-h new neural activities of reward anticipation and task errors h. ogawa , h. ifuku , t. nakamura , s. hirata kumamoto kinoh hosp, kumamoto, japan; fac educ, kumamoto univ., kumamoto, japan; nat kikuchi hosp, kumamoto, japan; dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, & - ) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca s after led offset at correct trials. relations between cues and responses were reversed. of reward-related neurons found, . % showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = ) only at correct trial, i-type (n = ) at around possible reward onset only at incorrect trials, and c-i type (n = ) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai , takayuki hosokawa , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps a-h neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue ), and centrally allocated one of the objects presented in cue (cue ). the frequency of which object was presented as cue , i.e., task context, was manipulated across blocks. we focused on cue period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps a-h anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki , barry j. richmond , munetaka shidara , grad. sch. of tsukuba univ., ibaraki, japan; neurosci. ri., aist, tsukuba, japan; lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in conditions: ( ) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], ( ) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition / neurons responded in all trials. in the certain condition / neurons responded in the rewarded trials only. of these showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas ), aist masamichi sakagami , , kosuke sawa , xiaochuan pan , bsrc, tamagawa university, tokyo, japan; senshu university, kanagawa, japan; presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a -b -c and a -b -c . after monkeys could acquire the sequences, new pairs of stimuli (i.e., d and d , e and e , etc) were introduced to associated with b or b (d -b , d -b , etc). the asymmetric reward rule was instructed by pairing c (c or c ) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a and a , which were associated with c and c in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi ( ), hsfp, presto, jst ps a-h reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan , , kosuke sawa , , masamichi sakagami , bsrc, research institute, tamagawa university, japan; presto, jst, japan; department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a -b -c and a -b -c . the asymmetric reward rule was instructed by pairing c (c or c ) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of neurons from the lateral pfc, % showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about % of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps a-h neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima , hajime mushiake , jun tanji department of physiology, tohoku university school of medicine, sendai, japan; brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the -sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps a-h neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. ) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = ) preferentially when the instruction was associated with small reward. the instructions activated groups of putamen neurons preferring small reward-(n = ), large reward-action (n = ) and both types of action (n = ). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by - and - ms, respectively. putamen neuron activation lead that of cm neurons by - ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi ( ) ps a-h encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t ), second (t ) and third (t ) choices with reward probabilities of about , and %, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r , %). most da neurons ( / ) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t to t and to t trials, then decreased in r trial. in another task with two repetition trials (r and r , %), magnitude of start-cue responses decreased gradually from t to r and to r trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi ( ) ps a-h striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada , , hitoshi inokawa , minoru kimura dept. of physiol. kyoto prefect univ. med., kyoto, japan; jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded striatal projection neurons from a monkey. after depressing a start button, the monkey chose of target buttons with correct rates at st, nd, rd and repetition trials of , , and %, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice ( / ) rather than to upcoming choice of target ( / ). during the target choice, another subset of neurons showed firings selective to choosing particular target ( / ) rather than to expected reward probability ( / ). after the target choices, another group of neurons fired related to expected reward ( / ) rather than to chosen action ( / ). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi ( ), jsps fellows ps a-h encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose of target buttons at average correct rates of (first), (second), (third) and % (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans ( / ) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi ( ) ps a-i representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda , k. samejima , k. doya , m. kimura dept. physiol., kyoto pref. univ. med.; brain sci. res. center, tamagawa univ.; irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of , or %. a linear regression of neuronal discharge rates showed: neurons encoded reward values of either action during delay period before go signal, with most ( %) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = ), while other neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi ( ) ps a-i delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c ), first delay (d ), second cue (c ), second delay (d ), and response periods. activity during the d and d periods was analyzed in this study. firing rates during the d period linearly depended on c durations. on the other hand, d period activity depended on trial types (ls and sl), but not on the variety of c durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi ( ) ps a-i neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku , ryoi tamura , teruko uwano , taketoshi ono dept. integrative neurosci., univ. toyama, toyama, japan; dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi ( ) ps a-i reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori , mikio inagaki , wu lisa , taijiro doi , eishi hirasaki , hiroo kumakura , ichiro fujita osaka univ., japan; massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from and takeda science foundation ps a-i response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, ( ) performance of visually guided sex discrimination task and ( ) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) ( ) (sa) and coe program in kit from the mext ps a-i activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa , satoshi katai , masanori saruwatari , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro ) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto , tetsuya iidaka department of psychology, nagoya university, nagoya, japan; department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: - hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the - ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the - ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included images ( objects × views). novel objects were generated by deforming a prototype in four directions. for deg-interval object sets, views were obtained by rotating each object with the interval of deg, deg-interval set and deg-interval set were with deg and deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across deg separated views was significantly higher in the trials with deg-interval sets than those with deg-interval sets. similarly, the difference was also found in the performances across deg separated views between those with deg-interval sets and deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps a-i brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps a-i obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps a-i sirna-induced nr knockdown causes hypofunction of nmda-r and cognitive deficit m. saji , , t. utida , a. ohnishi , k. noda , m. ogata , h. akita , n. suzuki , physiol, health sci. sch. kitasato univ., sagamihara, japan; brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr using hvj-envelope vectors. we confirmed that marked down-regulation ( %) of nr expression occurred only in the hippocampus among various brain regions - days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr knockdown, the nr down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr knockdown, the nr down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka , , hiroshi ageta , ikuko yao , kaoru inokuchi , yutaka kirino , mitsutoshi setou , graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; mitsubishi kagaku institute for life sciences, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps a-i decreased interneurons in the pax mutant mouse limbic system hasumi haba , tadashi nomura , yoshinobu hara , , noriko osumi , div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at weeks, but not at weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp ) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine ( . mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: , ps a-i gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba , , satoko hattori , hiroaki hori , tetsuo nakabayashi , hiroshi kunugi , ryota hashimoto department of mental disorder research, national institute of neuroscience; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of patients with schizophrenia and age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: , ps a-i genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto , hiroko noguchi , hiroaki hori , tetsuo nakabayashi , satoko hattori , sachie chiba , seiichi harada , osamu saito , hiroshi kunugi department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; musashi hospital, ncnp, kodaira, japan; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in healthy volunteers and patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: , ps a-i detection of f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura , nicola bright , toshio yanagida , masatoshi takeda , paul grasby department of physiology, osaka university, japan; department of psychiatry, osaka university, japan; cyclotron unit, imperial college, hammersmith hospital, uk we used f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and f-dopa scans were obtained from a database of scans published in mcgowan et al. , archives general psychiatry. the schizophrenic patients all met dsm-iv criteria on medication and healthy volunteers were compared. we attempted to improve the quality of the f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > . ). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p component of the visual eventrelated potential in patients with schizophrenia and healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p amplitude between the faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area between the faces in controls, while in patients, there was no significant activity difference between the faces. stimulation with crying face induced higher activities in the and right areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps a-j inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura , shoji tanaka , koki yamashita , motoichiro kato , haruo kashima department of neuropsychiatry, school of medicine, keio university; department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps a-j pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc , a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as -ht are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in of the sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps a-j alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta , ryo yamamoto , shigeki sugiura , kaoru inokuchi , nobuo kato dept. integrat. brain sci., grad. sch. med., kyoto univ.; nara med. univ.; mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer a/vesl- s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer a protein by diffusion from patch pipettes. homer a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer a may be a biological mediator of ecs effects. masanori kasai , nozomi miyagi , norio kawashiro , daisuke torizuka dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant ( . mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the nd, th, th, st, th and th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc ( . ± . g/dl, n = ) on the the day was significantly lower than that in intact rats ( . ± . g/dl, n = ). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps a-j molecular cloning of a novel candidate for ethanol-responsive genes, yy ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy ap (yy -associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy -binding and transactivation domains previously demonstrated in yy ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy genes was demonstrated in the same neurons. analogous to yy ap as a co-activator of transcription factor yy , it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps a-j excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr b-selective nmda antagonists ifenprodil ( and m) and ro - ( . and m) markedly inhibited degeneration of orexin neurons induced by h application of nmda ( m) or qa ( . mm). we also show that stimulation of gaba a receptors by muscimol ( and m) or isoguvacine ( and m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba ( m) plus a gaba uptake blocker nipecotic acid ( mm) was abolished by a gaba a antagonist picrotoxin ( m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh ). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps a-j a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi , osamu fukayama , takashi sato , takafumi suzuki , kunihiko mabuchi , dept. biomed. eng., univ. tokyo, tokyo, japan; dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps a-j on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki , yasuhiko jimbo , kenji yasuda department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are khz in ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita , tetsu okumura , kazuo okanoya , jun tani lab. for behavior & dynamic cognition, riken-bsi, japan; lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps a-k local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura , yuichi yamashita , kazuo okanoya , jun tani behav & dynamic cognition, riken-bsi, saitama, japan; biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata , shinya ishii , munetaka shidara dept. of e&e engineering, oita univ., oita, japan; grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [ ] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki , shinya yamamoto , sunao uchida , shigeru kitazawa , faculty of hum sci., waseda univ., tokorozawa, japan; neurosci. res. inst, aist, tsukuba, japan; faculty of sport sci., waseda univ., tokorozawa, japan; dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ± ms, s.d. = ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi ( ) ps a-k single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama , fumiko sato , ken nakazawa , kenichi ono chiba university, japan; field of home economics, faculty of education, chiba univ., japan; department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm- , shi-madzu) over the frontal area ( channels) for volunteers ( - y). thirty tasks were presented visually every s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was − . +/− . between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi , , kosuke hamaguchi , masato okada , , department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; jst, japan; riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi ( ) and ( ) ps a-k auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa , , sachiko koyama , , takashi omori , takashi morotomi research institue for electronic science, hokkaido university, sapporo, japan; japan science and technology agency, saitama, japan; graduate school of information science and technology, hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = ). the erps were elicited by tones ( hz) with different durations ( , , , , , ms) and gaps ( , , , , , ms) embedded in a continuous pure tone ( hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for and ms were high both for the tones ( ms, %; ms, %) and the gaps ( ms, %; ms, %). ps a-k determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps a-k theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to % till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, , -diphenyl- , , -hexatriene, and -[ (trimethylammonio)phenyl]- -phenyl- , , -hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp expression correlates with wnts/bmps in the cortical hem, sp with fgfs in the cop, and sp with shh in the ventral midline and mge. by using in utero electroporation, we show that sp regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp and fgf exhibit reciprocal induction, indicating that sp is a positive feedback regulator of fgf . surprisingly, though, ectopic expression of both sp and its dominant active form shift cortical areas in the opposite manner to fgf , suggesting that sp activates additional targets that overcome fgf function. our results indicate that fgf is an additional target of sp , showing effect on patterning similar to sp . these findings indicate that sp balance the proper cortical arealization through fgf and fgf . research funds: nihr ns ps p-c fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira , kotaro hattori , takeshi yagi , shigeki yuasa dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi , kazuko keino-masu , tatsuyuki ohto , satoshi kunita , satoru takahashi , masayuki masu dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp -deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp /sulfatase- and sulffp /sulfatase- , which have hs -o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp and sulffp . here we report the isolation and the characterization of the third homologue, sulffp . sulffp has about % and % overall amino acid homology with sulffp and sulffp , respectively. at h postfertilization, sulffp is expressed in the ventral region of spinal cord, whereas sulffp is expressed only in the floor plate and sulffp is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp was shown to mediate the repulsive effect of sema a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp , , , and are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps p-c developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa , n. yamamoto , m. yoshimoto , t. yasuda , k. maruyama , t. kage , h. takeda , h. ito nat. inst. rad. sci., chiba, japan; nippon med. sch., japan; tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax -or dlx -positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er homologue (zfer ), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is % identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta , , hiroo takahashi , , takafumi shintani , , kazuma etani , masaharu noda , div. of mol. neurobiol., nibb, okazaki, japan; crest, jst, japan in the developing chick retina, the expression of bmp is relieved by that of bmp at around e with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina , are expressed in a double-gradient fashion from e on like ventroptin and bmp . when bmp expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp knockdown and ephrina -misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp to bmp thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en , wnt b and pcp /l gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and di- asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and di- asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or di- asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru , , takahisa taguchi aist, ikeda, japan; presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a -dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps p-d laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps p-d synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki , toshio aoyagi department of physics, kyoto university, kyoto, japan; graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf- mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into weekold wistar strain rats in several doses followed by successive injections of brdu for the next days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps p-d the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato , james e. goldman division of pharmacology, national institute of health sciences, tokyo, japan; department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p rat svz, sagital sections of forebrain were made at p and cultured for days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣ integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps p-d localization of neurotrophin receptors trka in pc cells: d reconstruction analysis of membrane proteins tomoki nishida , hiroshi jinnai , tatuo arii , akio takaoka , ryoichi yoshimura , yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan; department of polymer science and engineering, kyoto institute of technology, kyoto, japan; national institute for physiological sciences, myodaiji, okazaki, japan; osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin- in pc cells, analyzed by high-voltage electron microscopy, and reconstructed d structure of their subcellular distribution by imod. our results indicated that localization of caveolin- , known as an integral membrane protein of caveolae, was never found in the invagination structure in pc cells, but trka and caveolin- immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd , a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd in the wnt-mediated neuronal development. there are three subtypes of mouse ccd gene products, ccd a, ccd b and ccd c, which are generated by different promotor usage. mouse ccd a as well as zebrafish ccd a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi ( , ) ps p-d analysis of a role of r-spondin on proliferation of the cortical neuroepithelium yumiko hatanaka , masahiro yamaguchi , fujio murakami , masayuki masu grad. school of comprehensive human sci., univ. of tsukuba, japan; grad. school of med., univ. of tokyo; grad. school of frontier biosci., osaka univ r-spondin (rspo ) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. ) . rspo is expressed in the developing medial cerebral wall and transgenic mice expressing rspo in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. ) . since wnt a has a role for expansion of caudomedial cortical progenitor cells (lee et al. ) , these findings lead us to the idea that rspo may synergistically promote proliferation of cortical neuroepithlial cells together with wnt a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day . mouse. an application of wnt a on these cells increased level of the reporter expression, and an addition of rspo further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt a and rspo directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il- ␤, il- , lif, gdnf) or lowered o ( . %), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells ( . -fold) as compared to normal o . cytokine cocktail significantly increased th (+) cells ( . -fold) compared to nontreated control. treatment of lif and il- ␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps p-d structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui , terukazu nogi , mitsuharu hattori , kenji iwasaki , , junichi takagi research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer and vldlr), resulting in tyrosine phosphorylation of dab . in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic d reconstruction of four-domain reelin fragment (i.e. r - ) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r - ) was capable of binding to the receptor (apoer ), and was also able to induce tyrosine phospholylation of dab in primary neuronal culture. ps p-d effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax and pax compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps p-d neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res : , ) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi- k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps p-d fabp is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa , miho matsumata , , yuji owada , shigeki yuasa , noriko osumi , natl. inst. of neurosci., ncnp, tokyo, japan; tohoku univ. sch. of med., sendai, japan; crest, jst pax transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp , a downstream of pax , regulating maintenance of embryonic neural stem/progenitor cells (arai et al., ) . fabp was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). % of fabp -expressing cells co-expressed gfap (a marker for early progenitors), and % of them co-expressed psa-ncam (a marker for late progenitors). fabp expression was also overlapped with pax , and expression of fabp was down-regulated in the dg of pax deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp knockout mice. taking all together, it is concluded that fabp is required for maintenance of neural stem/progenitor cells in dg. ps p-d involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day - . sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani , , shiki okamoto , masato imada , kouichi itoh , atsushi irie , hitoshi sakuraba , hideo kubo department of molecular biologu, ohu univ., koriyama, japan; dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; dept. anatomy, nihon univ. shl. med., tokyo, japan; dept. mol. pharma., univ. tokushima bunri, sanuki, japan; dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam- shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam- is possible. in the present, we show that the isolated randam- high+ cells enrich nscs. the randam- high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam- low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam- is a usefule marker for the isolation of nscs by facs. yasuharu takamori , yasuhisa tamura , , yosky kataoka , , yilong cui , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b and b are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b (++), b (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b (+++), b (+), and mature neurons possess lamin a/c (++), b (+), b (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka , yuichi tanaka , tatsuhiro hisatsune department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps p-e new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba , , hideo namiki , tatsunori seki dept. of anat, juntendo univ. sch. of med., tokyo, japan; integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p . three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., ) . the dividing pattern is currently under investigation. akiya watakabe , noritaka ichinohe , sonoko ohsawa , tsutomu hashikawa , kathleen s. rockland , tetsuo yamamori div. of brain biol, nibb, okazaki, japan; lab. for cortical organization and systematics, bsi, riken, wako, japan, lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer neurons in several areas of monkey neocortex. we previously reported that nurr , ctgf and sema e mrnas are specifically expressed in subsets of layer neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer . by double ish, layer neurons in monkeys are roughly divisible into cck(+) and sema e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi ps p-e rbp-j regulates the cortical laminar formation kenji tanigaki , kazue muraki , norio yamamoto , tasuku honjo shiga medical center, research institute, shiga, japan; department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps p-e search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi , tomoko ikawa , yuya kasukawa , hironori ueda , , kazuki kurimoto , michinori saitou , fumio matsuzaki , lab. for asymmetric cell division, cdb, riken, kobe, japan; functional genomics subunit, cdb, riken, kobe, japan; lab. for systems biology, cdb, riken, kobe, japan; lab. for mammalian germ cell biology, cdb, riken, kobe, japan; crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi ( ) ryosuke tatsuno , tomoaki sai , , masahiro otsu , kuniko akama , takashi nakayama , tosifusa toda grad. sch. of sci. and tech., chiba univ., chiba, japan; lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; dept. orthop. surg., jikei univ. sch. med., tokyo. japan; dept. biochem., yokohama city univ. sch. med., yokohama, japan; proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using -d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps p-e identification of se , a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se in developing and matured cns. teiichi furuichi , akira sto , , yukiko sekine , noriyuki morita , tetsushi sadakata , satoshi shoji , jin-hong huang , toshio kojima laboratory for molecular neurogenesis, riken brain science institute, japan; comparative systems biology team, riken genome sciences center, yokohama - , japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki , makoto mizuno , kazuhisa sakai , yoshimoto kiyohara , kazuhiko yamaguchi , tsutomu hashikawa , hiroyuki nawa institute of biomedical engineering, waseda university, tokyo, japan; department of molecular neurobiology, brain research institute, niigata university, niigata, japan; laboratory for memory and learning, bsi, riken, saitama, japan; laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg knock out mice (nrg type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps p-e psd- protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama , , satoshi wakisaka , shiho honma , akira ito , , kei azuma , , mikihiko kogo department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd- implicates in formation and maturation of excitatory synapses. psd- regulates the localization of the nmda receptor by means of binding with nr . rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd- protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd- expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf . in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps p-e ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps p-e comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi , kenji mizutani , hiroto ogawa , kohji hotta , kotaro oka ctr. for biosci. and info, keio univ., yokohama, japan; neuro, karolinska inst, stockholm, sweden; bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion ( % or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps p-e three dimensional ( d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in monkeys. of a total of p-cells that were examined during both frontal and vergence-pursuit, % discharged for both, % only for vergence, and % only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had d pursuit signals. majority ( %) of these p-cells discharged before onset of vergence eye movements with the typical lead time of ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi ( ) ps p-e information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey , rr aya takemura , yumi murata , , kenji kawano , neurosci. res. insti, aist, tsukuba, japan; dept. integ brain sci., grad. sch. med., kyoto univ., japan; grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid ( mg/ml, - l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps p-e the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara , kenji kunita , kaoru maeda , takeo kiyota department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for ms with -ms intervals. the oscillation trial ( . -hz frequency and . -cm amplitude) ( - s) was repeated times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the th trial. a significant correlation was shown between th- st trial differences in mean speed and vep amplitude (r = . ). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps p-e primary motor cortex contributes to generating manual following response toshitaka kimura , naoki saijo , hiroaki gomi , ntt cs labs, kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m ), that ( ) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and ( ) intracortical inhibition within m assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m . such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in radial directions applied during reaching. the data show that the direction of the initial phase ( - ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase ( - ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps p-e spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps p-f influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka , takashi obu , kazuyuki kanosue asmew, waseda university, saitama, japan; graduate school of human sciences, waseda university, saitama, japan; faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = ) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at . , . , and . hz. over % success rate was observed in all tasks except oppo ( - %). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps p-f simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu , tetsuro muraoka , kazuyuki kanosue , , faculty of human sciences, waseda university, saitama, japan; faculty of sport sciences, waseda university, saitama, japan; asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at , . , and . hz. they were asked to perform tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps p-f activities of erector spinae muscles during jaw clenching in man kayoko yasunaga , , tadachika yabushita , kazuo toda , kunimichi soma orthodontic science, tokyo med. & dent. univ., tokyo, japan; div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada , sachiko motoyama , futoshi mori , shigemi mori department of veterinary physiology, yamaguchi university, yamaguchi, japan; national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for - weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps p-f neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima , futoshi mori , akira murata , masahiko inase dept. of physiol., kinki univ. schl. of med., osakasayama, japan; dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi ( ) ps p-f activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara , , nobuhiko hatanaka , , masahiko takada , atsushi nambu , school of life science, the graduate university for advanced studies, japan; division of system neurophysiology, national institute for physiological sciences, japan; tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu , sei-ichi sasaki , yuichiro cho , kenji sato anatomy and physiological science, tokyo medical and dental university, tokyo, japan; department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s and s spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl -s spinal segments which contain afferent fibers of eas muscles. after weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for consecutive days. we compared adaptation in day and that in day . the gain decrease for the first saccades in day ( . ± . ) was larger than that in day ( . ± . ) (p = . , n = , paired-t test). the rate of adaptation in day ( . ± . × − /sac) was higher than that in day ( . ± . × − /sac) (p = . ). the overall gain change ( saccades) in day ( . ± . ) was larger than that in day ( . ± . ) (p = . ). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi ( ) ps p-f asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v . the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = ) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in % of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida , xiaofeng lu , , shogo ohmae , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi ps p-f frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps p-f projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki , , kei ito , , dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; center for bioinform., imcb, univ. of tokyo, japan; bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening , strains, we identified about candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps p-f immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura , yoshitaka ohtubo , takashi kumazawa , kiyonori yoshii graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi ( ) and the st coe program (center # ) granted by mext of japan ps p-f inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa , atsushi kuhara , hitoshi inada , eiji kodama , takafumi mizuno , ikue mori , lab. of mol. neurobiol., nagoya univ., japan; institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx- gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx- mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx- mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps p-f postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut and - ) during rat ob development from postnatal day (p ) to p by in situ hybridization and immunohistochemistry. at p vglut immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut mrna signals were detectable in the mitral cell layer from p to p . in contrast, vglut ir was prominent in the glomerulus at all days examined, and only at p and p in mitral cells. despite mitral vglut ir disappeared at p , the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps p-f v r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito , , masumi ichikawa , yuji mori , kimiko hagino-yamagishi tokyo metrop. inst. med. sci., tokyo, japan; the univ. of tokyo, tokyo, japan, tokyo metrop. inst. neurosci., tokyo, japan in rodent, v r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v r gene, which is expressed in the olfactory epithelium (oe). to examine when the v rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified v r sequences. these v rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps p-f analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t r /t r heterodimer plays a role as a sweet taste receptor. but, mice lacking t r showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t r -independent sweetener binding site also exist in mice. in this study, to predict binding sites on t r /t r and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [( ) sucrose, glcose, fructose, ( ) saccharin, aspartame, acesulfame-k, glycine, ( ) d-phenylalanine, ( ) d-tryptophan, ( ) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t r and t r , respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu , sachiko saito , yuko murata , ding ming , tatsu kobayakawa , robert f. margolskee , yuzo ninomiya sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; saito sachiko taste and smell research institution, ibaraki, japan; national res. institute of fisheries sci., kanagawa, japan; dept. of physiol. & biophys., mount sinai sch. med., new york, usa; national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t r receptors which coupled with ggustducin. ps p-f newborn infant body odor attenuates their mother's postpartum moods shota nishitani , mayumi kokuryo , tsunetake miyamura , kazuyuki shinohara div. neurobiol. & behav., nagasaki university, japan; obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps p-f human prefrontal activity in taste encoding: an fnirs study masako okamoto , mari matsunami , haruka dan , tomoko kohata , kaoru kohyama , ippeita dan national food research institute, tsukuba, japan; nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = ) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes ( subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes ( subfamilies) were expressed in the dorso-medial zone (zone ) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone . these results indicate that there is a clear boundary between zone and non-zone areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi ( ) ps p-g taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to ( ) best-taste profiles and ( ) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi ( ) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼ members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi ( ) ps p-g identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka , kazuhisa sakai , tsutomu hashikawa , kensaku mori , masahiro yamaguchi dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps p-g nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi ( ), kakenhi ( ) ps p-g integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than % of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi ( ) ps p-g odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi ( gs ) ps p-g odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa , takaaki sato , akira shimizu , ken-ichiro tsutsui , toshio iijima div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range ( hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps p-g chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when m lysine was spotted on ammonium chloride background, . - . m and . m background did not influence lysine chemotaxis, while . m background augmented and . - . m background suppressed the chemotaxis. in contrast, when . m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps p-g glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu , , , takeshi ishihara , eisuke nishida , yuichi iino dept. biol., fac. sci., kyushu univ., japan; mol. genet. res. lab., univ. of tokyo, japan; grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr- , a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr- appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr- . in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr- . these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr- localization in aiy neurons. kohei ueno , yoshiaki kidokoro dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼ %), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal /uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps p-g spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki , graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps p-g medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka , yoshinori sahara , shinichiro horie , masumi ichikawa , shun nakamura st. marianna univ. schl. med., kawasaki, japan; ntl. inst. neurosci., ncnp, tokyo, japan; tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps p-g synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi ( ) ps p-g rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii , , ken yoshikawa , yuki takatori , koichiro kawashima dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca + signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca + signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m , m , m and m machr subtypes, and ␣ , ␣ , ␣ , ␣ , ␣ , ␣ and ␤ nachr subunits. transfection of anti-m , anti-m and anti-␣ small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca + signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m or anti-m sirna. ca + signals evoked by nicotine were reduced by anti-␣ sirna. these findings indicate that m , m machr and ␣ nachr subtypes play major roles in ca + signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi ( ) ps p-g is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto , sachiho kubo , seiji matsuda , naoki maruyama dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, ) research funds: kakenhi ( ) ps p-g dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day . electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day . analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c from kansai medical university ps p-h unusually folded sod species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno , yumiko simazaki , fuminori saitoh , ryosuke takahashi , toshiyuki araki national institute of neuroscience (ncnp), tokyo, japan; dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod protein is thought to be responsible for the selective loss of motoneurons in sod -related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod species are not yet clarified. since we have detected accumulation of unusual sod species in motoneuronal axons from g a sod -tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod species. immunoprecipitation analyses revealed specific interaction of unusual sod species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod -tg compared with wt sod -tg mice. these data strongly suggest that the toxic property of unusual sod proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c ( ) ps p-h relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately % of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day . the mice started to show ncl symptoms on p , and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from % to % of that in the wild-type mice. research funds: kakenhi ( ) ps p-h neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura , katsumi higaki , junichiro matsuda , yoshiyuki suzuki , eiji nanba division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; national institute of biomedical innovation, osaka, japan; clinical research center, international university of health and welfare, tochigi, japan g m -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm accumulation of lysosome and pm and the membrane fluidity was also reduced. gm -bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m -gangliosidosis. katsuya inoue , , katsuaki endo , takamitsu fujikawa , seijyun fukuda , tatsuo nakamura department of physical therapy, university of aino, osaka, japan; institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after min occulusion. ps p-h development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando , yuya kawanaka , minoru saito , hiroaki mochizuki , ken honjo , hirofumi toda , , toshifumi tomoda , akira sawa , katsuo furukubo-tokunaga grad. school of life & envir. sci., univ. tsukuba, japan; molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; beckman res. inst., city of hope. california, usa; dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia- (disc ) gene, originally identified at the breakpoint of a chromosome ( ; ) (q . ; q . ) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc functions in vivo, we expressed disc in drosophila and examined developmental and behavioral phenotypes. overexpression of disc resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps p-h effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake , ayumi kamo , kenji minato , hitoshi haruna , hiritsugu fukuda , yuji murata , takayoshi hosono department of bomedical engineering, osaka electro-communication university, japan; graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of of -day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing % oxygen at • c for min. after the insult, of the rats were put in a box at • c for h (hypothermia, h-group). the other rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps p-h tau hyperphosphorylation in ts cje, a partial trisomy mouse model for down syndrome ebrahim abdul , a. shimohata , w. yu , m. yamaguchi , m. murayama , d. chui , t. akagi , t. takeuchi , k. amano , h.s. karthik , t. hashikawa , h. sago , c.j. epstein , a. takashima , k. yamakawa research scientist; lab. for neural arch.; lab. for alzheimers disease; div. of fetal med. ncchd; ucsf, usa although down syndrome (ds) or trisomy is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts cje, a ds mouse model partially trisomic for chromosome , shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome q , has about genes. importantly, sod and app, which may contribute to the ds phenotype, are excluded from the ts cje trisomic segment. here we report that ts cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk ␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts cje segment other than app and sod can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: ps p-h increased oxidative stress and mitochondrial dysfunction in ts cje, a down syndrome mouse model atsushi shimohata , ebrahim a. s. , m. yamaguchi , w. yu , h. sago , c.j. epstein , k. yamakawa lab. for neurogenetics, riken-bsi, japan; div. of fetal med. ncchd, japan; dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome (hsa ) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome extending from sod to znf , containing genes syntenic with hsa . since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts cje brains. collectively, our results implicate dosage imbalanced genes other than sod and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts cje ds mental retardation-like phenotype. ps p-h polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan , , kouko tatsumi , takayuki manabe , takahira yamauchi , , eri makinodan , juro shimoda , toshifumi kishimoto , akio wanaka department of psychiatry, nara medical university, kashihara, japan; department of nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded ( q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. genes, encompassing around % of the drosophila genome, were screened, resulting in the identification of candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps p-h analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. ( ) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the , ,-cyclic nucleotide -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps p-h dysregulation of sodium channel ␤ subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤ (␤ ) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤ as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤ started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤ expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤ gene in pc cells. forskolin, an activator of the camp/pka pathway, did not affect b promoter activity, indicating that ␤ is not camp-responsive gene. these findings strongly suggest that sodium channel ␤ subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps p-h repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki , yuji takahashi , jun goto , mutsuo oyake , toshiya sato , shoji tsuji department of neurology, the university of tokyo, tokyo, japan; department of neurology, brain research institute, niigata university, niigata, japan; center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of (q ), (q ), (q ), or (q ). in the accelerating rotarod ( w), the latencies of q , q , q and q were %, %, % and %, respectively. in the open field, moving distances of q , q , and q were decreased to %, %, and %, respectively, while that of q was increased to %. home cage activity was decreased depending on the repeat length. the q mice, however, showed increased ratios of the activity during the light time to that during the total day at weeks ( %) and weeks ( %), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps p-h the mice lacking schnurri- show multiple behavioral abnormalities related to psychiatric disorders keizo takao , nobuyuki yamasaki , keiko toyama , tsuyoshi takagi , shunsuke ishii , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; riken, tsukuba, japan schnurri- (shn- ) is a zinc finger transcription factor, a mouse homologue of human hiv-ep , that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn- is known to play important roles in the mammalian immune systems. however, the role of shn- in the central nervous system (cns) is still unknown. to investigate the functional significance of shn- in mammalian brain, we analyzed the shn- knockout (ko) mice using a comprehensive behavioral test battery. shn- ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn- mice. our results demonstrate a critical role of shn- in cns and suggest that shn- ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca + /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi ( , , , ) , jst bird ps p-h effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c bl/ j, c bl/ n, c bl/ c, svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than mice (, including wild type and mutant mice from strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behaivoral analysis of ryanodine receptor type knockout mouse suzuko ohsako , koichi tanda , , nobuyuki yamasaki , keiko toyama , hiroshi takeshima , tsuyoshi miyakawa kyoto university graduate school of medicine, kyoto, japan; dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca + signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr deficiency, we subjected ryr knockoout mice to a battery of behavioral tests. ryr knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr in regulating general locomotor activity and social behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama , koichi tanda , , nobuyuki yamasaki , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi ( , , , ) , jst bird ps p-h primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi , keiko shimizu grad. sch. human and environmental std, kyoto univ., kyoto, japan; primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected -ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. -ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in -ohda injected monkeys. these results suggest that monkeys injected -ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi ( ) ps p-i training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata , , , noriyuki higo , , takao oishi , , , akiko yamashita , keiji matsuda , motoharu hayashi neurosci. res. inst, aist, tsukuba, japan; grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; crest, jst, kawaguchi, japan; dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext ( ) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about , g animals (dba/ j × enu-treated c bl/ j) for home-cage activity, open-field activity, and passive avoidance response, and obtained lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in of mutant lines. hyperactivity was predominant phenotype, and of mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps p-i ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., ) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg -knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., ) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc , and found that in our ncl model mice, positive signals for ubiquitin and lc were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)( ) and creative scientific research ( gs ) ps p-i activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama , eiichi jodo , yoshiaki suzuki , ken-yo hoshino , yukihiko kayama dept. of physiology, fukushima medical university, fukushima, japan; dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch , mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps p-i increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara , hiroyuki sakagami , hisatake kondoh , kohji fukunaga department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps p-i severity of audiogenic seizures is influenced by multiple factors in vlgr -mutated mice hideshi yagi , , makoto sato , division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr -mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c bl/ backcrossed vlgr -mutated mice and /svs backcrossed vlgr -mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr -mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps p-i reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota , ken kadota , department of bioenvironmental medicine, university of chiba, chiba, japan; chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of mg/kg of map i.p. for days (d d ) and then withdrawn from the drug for days (wd wd ). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg ) and mfc. the decrease in the density of pac was first observed in cg and then in mfc. the reduction began on d and advanced to higher levels on d and subsequently wd . these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg and mfc, particularly in layers ii and iii. ps p-i up-regulation of ␤ -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ -adrenergic receptor (␤ -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ -ar-immunoreactive cells were labeled with ki- , a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ -ar-immunoreactive cells also exhibited fgf- -ir. these findings suggest that ␤ -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf- . ps p-i effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita , norihiro muraoka , hiroshi hasuo , takashi akasu , minoru shigemori dept. of physiology, kurume univ. sch. of med., kurume, japan; dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh ). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity ( - v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was - v. the amplitude of slow peak in fpi group was about % larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps p-i comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca + imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca + imaging. the slices ( m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of ml/min. green autofluorescence (> nm) of the slices illuminated by blue light ( - nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca + imaging was also performed in the slices. slices were incubated in acsf medium containing m of fluo / am for h at • c. the ca + image was recorded with an excitation wavelength of - nmand an emission wave length of > nm. the autofluorescence and ca + responses wereobserved in slices perfused with l-glutamate ( mm). takuya hayashi , hiroshi sato , shinichi abe , takashi hanakawa , hiroshi watabe , hidenao fukuyama , babak aldekani , hidehiro iida department of investigative radiology, national cardiovascular center research institute, osaka, japan; human brain research center, kyoto university, kyoto, japan; nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area corresponded to that revealed by the tracer (mncl ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h -kokoro- ps p-i optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata , , yasutake mori , , tsuya taneda , , hiroaki okuda , , masaya tohyama , department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps p-i a model selection of glm applied to fmri data using aic jobu watanabe , fumikazu miwakeichi , andreas galka , , ryuta kawashima , tohru ozaki , sunao uchida , institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; department of medical system engineering, faculty of engineering, chiba university, chiba, japan; institute for statistical mathematics, tokyo, japan; institute of experimental and applied physics, university of kiel, keil, germany; new industry creation hatchery center, tohoku university, sendai, japan; faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for - min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps p-j flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata - , japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps p-j effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune , , takashi tsukiura , hiroko mochizuki-kawai , chisato suzuki , , toshio iijima neurosci. res. inst., aist, tsukuba, japan; div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps p-j different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno , kenji kansaku , yu aramaki , mitsuo kawato atr cns, kyoto, japan; institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = ) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, or yen is provided with a probability of . , but only with . if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < . ) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps p-j brain networks for communicative speech production: feeling inference and speech content production yuko sassa , , motoaki sugiura , hyeonjeong jeong , , keisuke wakusawa , , kaoru horie , shigeru sato , ryuta kawashima niche, tohoku university, sendai, japan; ristex, jst, tokyo, japan; miyagi university of education, sendai, japan; gsics, tohoku university, sendai, japan; department of pediatrics, tohoku university, sendai, japan; the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the st coe program ps p-j the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi , yasuhisa fujibayashi , yoshiharu yonekura , tatsuya asai department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps p-j imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai , kuniyoshi shinya , tetsuo araki , masahiro kusakabe , yasuhisa fujibayashi , yoshiharu yonekura department of nuclear power and energy safety engineering, university of fukui, fukui, japan; department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura , , winfried denk , michael hausser department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; university college london, london, uk; max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps p-k analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka , yasuhiro tanaka , takeshi kaneko , dept. of morphological brain science, kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad -gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after h incubation in nmdg-based solution than in nacl-based solution. cutting at • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at • c. the viability after h incubation was better kept at • c than at or • c. we thus recommend that slices be cut at • c in na-free solution, and incubated at • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi ( , , ) ps p-k contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh , yoshichika yoshioka faculty of engineering, iwate university, morioka, japan; iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal ( . ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals ( . , . , and . ppm). a phantom solution mimicking the normal condition (gsh:gssg = : ) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = : : : ), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was . mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area ) and the right superior and middle frontal gyri (brodmann area ) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps p-k cortical commissural connection in macaque and human callosum using diffusion mri rishu piao , takuya hayashi , hiroshi sato , shinichi abe , , takashi hanakawa , hidenao fukuyama , hidehiro iida national cardiovascular center, osaka, japan; human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps p-k optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong , niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the -d neural cortical activity using the -d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps a-c sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp ) nobuyuki karasawa , kazuko watanabe , keiki yamada seijoh universitry, tokai, japan, dept. physio., sch. med., gifu univ., gifu, japan, dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, , -diamino- -hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp ) mice. time course of immunoreactive intensity for serotonergic ( -ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that -ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and -ht neurons. katsuya yamada , , , yoshihiro matsumura , takashi miki , makoto wakui ␣-smooth muscle actin + arterioles were fewer in kir . (−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir . (−/−) barrel cortex than in wild-type one for short ( s, hz) but not long ( s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps a-h learning to use sensory-tools by japanese monkeys yumiko yamazaki , hiromi namba support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps p-h mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h receptor (h r) in cognition in physiological and pathological conditions by using h r mutant (h −/−) mice. in normal condition, several behavioral studies indicated h −/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h −/− mice. these results indicate h receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h −/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h −/− mice. mutation of h receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h r impairs cognition in normal conditions, whereas h r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi ( ; ) ps a-h -ht a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura , , hirohito-m. kondo , makio kashino , department of psychology, tokai women's university, kakamigahara, japan; ntt communication science laboratories, ntt corporation, atsugi, japan; shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of -ht neurotransmission. we examined whether this -ht a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the -ht a receptor gene polymorphism in impulsive behavior (nomura et al., ) , together with the present findings, this observation indicates the involvement of -ht a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps a-h role of cortical thin arterioles in neurovascular coupling; analyses of kir . -containing atpsensitive potassium channel-deficient mice ps p-g rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal , chinmay k. mukhopadhyay , birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india; special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv- and the direct infection of hsv- are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv- jrfl-infected macrophages or with hsv- . the generation of primary neurospheres did not suppressed by hiv- -infection or by hsv- infection at no more that moi . in the secondary neurospheres, cd + neural stem cells were intact in these infections, although beta- -tubulin + cells were decreased in hiv- infection and intact in hsv- -infection. in the differentiation assay, neun + nfp + neurons in hiv- -infection and gfap + s + astrocytes in hsv- infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv- infection and in hsv- infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv- and hsv- infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi grant-in-aid for young scientists(b) ps p-g effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga , , taeko yamauchi , toshihiro konagaya , hideki ohira department of psychology, nagoya university, japan; department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka , shen jin-song , saburo saito , shigeki yuasa dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori , tomoko yamamoto , kiyoshi matsumura , hiroshi hosokawa , shigeo kobayashi dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e (pge ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge but also induce an enzyme cyclooxygenase- (cox- ) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox- , and whether cox- is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox- was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox- specific inhibitor, ns- . these results indicate that aa itself or its metabolites induces cox- that accelerates the formation of pge from aa, and, hence, enhances fever. mitsunari abe , tatsuya mima , shinichi urayama , toshihiko aso , nobukatsu sawamoto , hidenao fukuyama human brain research center, kyoto university graduate school of medicine, japan; nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at . hz ( min; % of the rest motor threshold) applied over the left primary motor area (m ). we scanned sets of dwi (before, and min, min and min after rtms) using -t mr scanner, and calculated apparent diffusion coefficient (adc). in out of subjects, the adc decreased (mean . × − /mm s − ) in the left m just after the rtms, which recovered at min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v and v a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v a located more dorsal has a larger receptive field size then v . both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v and v a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v and v a.ps p-i measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro -channel magnetometer consisting of a × matrix of superconducting quantum interference device (squid) with its integrated base of . × . mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with - ms peak by the white led flashing stimulation and the aef wave with - ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of bits was used, along with a "blue dye" (rh ) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were . - . % of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto , yoko hoshi , chie sato , susumu terakawa tokyo institute of psychiatry, tokyo, japan; photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals ( and nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around -ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps p-j multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh , a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a -s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba ), superior occipital gyrus around the parieto-occipital sulcus (ba ) and superior parietal lobule (ba ). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps p-j influence of task difficulty during meter inspection: an fmri study naoki miura , , makoto takahashi , jobu watanabe , , shinya uchida , , shigeru sato , kaoru horie , masaharu kitamura , toshio wakabayashi , katsuki nakamura , , ryuta kawashima crest, jst, kawaguchi, japan; niche, tohoku univ. sendai, japan; graduate school of engineering, tohoku univ. sendai, japan; bme institute, waseda univ., tokyo, japan; idac, tohoku univ., sendai, japan; lbc research center, tohoku univ., sendai, japan; department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps p-j neural connectivity among brain areas related to language function shinya uchida , , , naoki miura , , jobu watanabe , shigeo kinomura , kazunori sato , yasuyuki taki , kentaro inoue , ryoi goto , ai fukushima , kaoru horie , shigeru sato , katsuki nakamura , hiroshi fukuda , ryuta kawashima department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; niche, tohoku university, sendai, japan; national institute of neuroscience, ncnp, kodaira, japan; japan science and technology agency, kawaguchi, japan; bme institute, asmew, waseda university, tokyo, japan; graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than % of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa , , motoaki sugiura , yuko sassa , , hyeonjeong jeong , , kaoru horie , shigeru sato , hiroyuki yokoyama , kazuie inuma , ryuta kawashima niche, tohoku univ., sendai, japan; department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; miyagi univ. of education, sendai, japan; ristex, jst, tohoku univ., sendai, japan; gsics, tohoku univ., sendai, japan; lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda , yasumasa okamoto , kazuhiro shishida , akiko kinoshita , shigeru toki , kazutaka ueda , hidehisa yamashita , shigeto yamawaki department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada , , , kenji yoshimi , , noriyuki higo , yong-ri ren , hideki mochizuki , yoshikuni mizuno , shigeru kitazawa , , dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; crest, jst, tokyo, japan; neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba- positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma − . mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections. key: cord- -uj fe y authors: nan title: scientific abstracts date: - - journal: reprod sci doi: . / sha: doc_id: cord_uid: uj fe y nan by reduced placental oxygenation, hypoxia-induced oxidative stress is a predominant mechanism. we investigated the effect of hypoxic pregnancy, with and without antioxidant treatment, on placental and maternal circulatory indices of oxidative stress in rats. methods: on pregnancy day , wistar rats were randomised into: normoxia ( % o litters), hypoxia ( % o litters) and hypoxia + vit c ( % o + mg. ml - vit c in water, litters). on day , dams were anaesthetised, maternal blood taken, pups measured and weighed, placentae weighed and frozen. only placentae from two male pups from any one litter were investigated. blood was processed for ascorbate, urate, l-cysteine and glutathione (gsh) measurement. placental protein was analysed for heat shock protein (hsp ; western). results: hypoxia + vit c did not affect maternal food or water intake. vit c elevated maternal ascorbate by % of baseline; a similar increment to human trials (poston et al. ) . hypoxia elevated placental hsp and maternal plasma urate and l-cysteine, but decreased gsh. vit c in hypoxic pregnancies prevented all stimulated effects, but the reduction in gsh persisted. hypoxic pups had a reduced ponderal index and elevated head diameter: body weight ratio; effects also prevented by vit c. fetal oxygen uptake in normal and gdm pregnancies. emanuela taricco, tatjana radaelli, veronica cozzi, gabriele rossi, danila puglia, giorgio pardi, irene cetin. department of obstetrics gynecology "l. mangiagalli", irccs policlinico, mangiagalli e regina elena, milan, italy. background. diabetes in pregnancy has been associated with alterations of fetal growth probably due to increased nutrient availability and placental transport. fetal hypoxia and acidemia have been reported in pregestational diabetic pregnancies with poor glicemic control but this is still uncertain in well controlled patients. the role of placental function and the relationship between maternal and fetal circulation is crucial for efficient exchanges of oxygen and nutrients. since umbilical blood flow can be obtained by us in utero, we studied normal and gdm pregnancies in order to evaluate fetal oxygen uptake. methods. normal (n) and gdm pregnancies were studied at term, at the time of elective caesarean section. umbilical vein volume flow (qumb) was measured by us before caesarean section and blood samples from umbilical vein (uv) and artery (ua) were obtained. blood gases and acid-base balance were evaluated. results. average fetal weights were similar in both groups (n= ± ; gdm= ± g) while placental weights were significantly different (n= ± ; gdm= ± g). n and gdm pregnancies showed similar values of qumb and qumb/kg of fetal weight. (qumb: . ± . in n and . ± . ml/min in gdm; qumb/kg: . ± . in n and . ± . ml/min/kg in gdm). in fetuses from gdm pregnancies a significant reduction in o sat, o cont and po and a significant increased lactate conc was found in both uv and ua compared to n (table ) . o umb uptake (n= . ± . ; gdm= . ± . mmo/l/min) and o umb uptake/kg (n= . ± . ; gdm= . ± . mmo/l/ min/kg) were significantly lower in gdm compared to n fetuses. conclusions. our data indicate that fetuses from gdm pregnancies show a significant reduction in oxygen supply despite a normal blood flow/kg of fetal weight. these data may suggest that a good maternal metabolic control is not sufficient to ensure normal placental oxygen supply and/or utilization by the gdm fetus. background: synthetic glucocorticoids (sgc) are given to mothers at risk of preterm delivery to promote fetal lung maturation. evidence is emerging indicating long-term effects of such treatment on endocrine function and behavior in offspring. however, virtually nothing is known concerning potential transgenerational influences on growth, endocrine function and behaviour. we hypothesize that repeated treatment of grandmothers (f ) with sgc will alter hypothalamic-pituitary-adrenal (hpa) function in f offspring with no manipulation of the f pregnancy. methods: pregnant guinea pigs (f ) were subcutaneously injected with betamethasone (beta; mg/kg) or vehicle (veh) on gestational days / , / / . adult f female offspring from each group were mated with control males. hpa function was assessed in adult f offspring by non-invasive measurement of salivary cortisol concentrations: ) under basal conditions, ) during and following exposure to psychological stress (high frequency strobe light) or psychological/physical stress (forced swim) and, ) following dexamethasone suppression. results: there was no effect of beta (f ) on bodyweight from birth to adulthood in f offspring. basal salivary cortisol in the betaf females was lower than in the vehf group in the morning but not the afternoon; there were no differences in male f offspring. in contrast, both male and female betaf failed to mount adrenocortical responses to psychological stress compared to vehf offspring that mounted robust responses (p< . ). swim stress induced robust adrenocortical responses in all groups (p< . ), however, the response was consistently lower in betaf offspring. beta exposure also led to a significant difference (p< . ) in the cortisol response to dexamethasone suppression in female (f ) offspring, with a similar trend in males. conclusion: prenatal exposure to sgc (f ) causes transgenerational programming of adrenocortical function in adult f offspring. grand maternal exposure to beta results reduced basal adrenocortical activity in betaf female offspring, and caused stress hypo-responsiveness in both males and females. dexamethasone suppression tests indicate altered central glucocorticoid feedback. these findings have important ramifications for the management of human preterm labor. support: canadian institutes of health research. in the uterine and umbilical vasculatures, we hypothesized that their remodeling would also be blunted in pregnant enos -/-mice, leading to an elevated vascular resistance and decreased blood flow to the placenta contributing to fetal growth restriction. methods: utero-and umbilical-placental blood velocity waveforms and umbilical arterial diameters were measured using mhz ultrasound biomicroscopy in control (c bl/ j) and enos -/-mice at . days of pregnancy (n= mothers). spiral artery and fetal capillary morphologies, and uterine arterial diameters were evaluated from vascular corrosion casts. tissues were collected for hydroxyprobe- and actin immunohistochemistry to identify hypoxic and smooth muscle regions. we calculated resistance index ((s-d)/s) from systolic (s) and diastolic (d) velocities, and blood flow from mean velocity and vessel area. results: calculated uterine blood flow normalized to the weight of the uterus and its contents was % lower (p< . ) in pregnant enos -/-mothers due to large reductions in uterine artery diameter (- %, p< . ) and mean velocity (- %, p< . ). uterine arterial resistance index was % higher (p< . ), and the spiral arteries were less coiled and contained more smooth muscle actin in enos -/-mice than controls. more intense hypoxic immunoreactivity was detected in the spongiotrophoblast and trophoblast giant cell layers of the junctional zone of enos -/-placentas, whereas fainter staining was only detected in the spongiotrophoblast cell layer in controls. in the umbilical circulation, flow normalized to fetal weight was not significantly changed although the resistance index was slightly elevated ( %, p< . ) and capillary lobule length was reduced by (- %, p< . ). fetal organs showed increased hypoxic immunoreactivity suggesting reduced organ oxygen delivery in enos -/-fetuses. conclusions: results suggest that enos plays an important role in uterine and spiral artery remodeling and in augmenting utero-placental blood flow during pregnancy. it also appears to enhance oxygen delivery to the placenta and fetus. enos may contribute to normal fetal growth by these mechanisms. integrin methods: hpmcs isolated from term placentas were assessed for their phenotype markers, mutilineage capacity, and the expression of integrin molecules. the hpmcs were induced to endothelial cell differentiation in the presence of endothelial cell growth medium with % of fcs and ng/ml vegf for to days. the angiogenesis ability of these cells was demonstrated by using an in vitro angiogenesis kit and in vivo chick chorioallantoic membrane assay from ten-day-old embryos. blocking antibodies specific to integrin , associated with gdm. this study was adequately powered to detect association in the caucasian and asian group. the absence of association suggests that gdm and t dm may have more divergent molecular pathophysiology than previously suspected. background: uterine endometrium has a unique cycle of physiological angiogenesis. in mice, endothelial progenitor cells (epc) contribute to endometrial angiogenesis being incorporated after oestradiol administration. objective: to determine whether circulating © epc number and function vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: ten healthy, nulliparous, pre-menopausal, non-smoking women (mean age years) with regular menses ( - days) were studied. venous blood was collected during menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - , - ) . cepcs, serum oestradiol (e) and progesterone (p) were measured at each phase. cepcs were quantified by phenotype using flow cytometry (leukocytes co-expressing cd , kdr and cd ) and function by the epc-colony forming unit (cfu) assay. epc-cfus were stained for endothelial markers including uptake of acetylated low-density lipoprotein, binding of lectin (ulex europaeus) and endoglin. results: luteal p was > nmol/l in all women. cepc numbers increased in the menstrual and follicular phases being -fold higher in the follicular compared to periovulatory phase (p< . ). there was no significant variation in cepc function over the menstrual cycle. there was no correlation between serum e or p levels and cepc number or function. conclusion: cepcs number but not function (epc-cfu assay) vary during the menstrual cycle with numbers increasing during the menstrual and follicular phase and falling in the periovulatory and luteal phase of the menstrual cycle. this may represent mobilisation of cepcs from the bone marrow and subsequent incorporation into the endometrium. neither function nor cepc number correlate with serum p or e. our previous studies demonstrated that tissue factor (tf), which binds factor vii to act as a potent pro-coagulant and angiogenic factor, is over-expressed in endometriotic lesions. thus, we determined whether tf could serve as a target for the elimination of pre-established ectopic human endometrial growth in a mouse model. icon is composed of a mutated factor vii (fvii) domain targeting tf and an igg fc (fvii/igg fc) effector domain that activates antibody-dependent immune responses against tf bearing endothelial cells. methods: athymic, ovariectomized and estrogen-treated mice received intraperitoneal (i.p.) injections of . mg of proliferative phase human endometrial tissue derived from normal (disease-free) women. twelve days after inoculation to establish lesions, icon protein ( ug) was delivered i.p. once a week for weeks. after sacrifice, animals were subject to gross inspection. residual endometriotic tissue was formalin fixed, paraffin embedded and immunostained for von willebrand's factor (vwf) and tf. results: compared to control mice, treatment with ug of icon abolished all lesions in of mice and reduced both size ( . to . mm) and number of lesions ( . to per diseased mouse) in the remaining mice. moreover, residual lesions from icon treated mice were atrophic and displayed significant reductions in vessel areas of % +/- % (p= . , mean +/-sem, n= ) as determined by vwf immunostaining. no hemorrhagic or thrombotic sequelae were observed in icon treated mice. conclusions: unlike other treatments that target developing angiogenesis, icon can target both developing and established human endometriotic lesions in athymic mice. the gross and microscopic vessel analysis suggests that icon directly or indirectly destroys endometriotic vessels. thus, icon presents a novel, non-toxic therapy for endometriosis. compared to the miscarriage and top groups. ihc for crisp demonstrated increased secretion of crisp in the glandular epithelium and expression in the leucocytes of the tubal uterine decidua. conclusions: there are differences in decidual gene expression in tubal compared to iu pregnancies. we believe that potential biomarkers of tubal pregnancy can be discovered by focusing on secreted proteins associated with uterine decidualization. one of these proteins, crisp , is significantly increased in decidua of tubal ectopic pregnancies and we are currently investigating its expression pattern in sera from women with tubal compared to iu pregnancies. progesterone and hoxa regulate gaba-a pi receptor expression, membrane translocation and activation. homayoun sadeghi, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale school of medicine, new haven, ct, usa. objective the expression of the gaba-a pi receptor has been previously described in the human endometrium in both luminal epithelium and stroma. it is upregulated during stromal decidualization in the rat and in the implantation window of human endometrium. the gaba receptor is modulated by progesterone metabolites, with the resultant opening of the receptor ion channels which allow the water flux necessary for trophoblast attachment. here we identified regulators of pi subunit receptor gene expression and activity. the well-differentiated human endometrial adenocarcinoma cell line (ishikawa) and human endometrial stromal cells (hesc) were transfected with hoxa , treated with progesterone, or treated with vehicle or empty plasmid controls. gaba-a pi receptor mrna upregulation was evaluated by real time rt-pcr. protein expression was evaluated using immunohistochemistry. results gaba-a pi receptor mrna expression was increased with either progesterone treatment ( %, p= . ) or hoxa transfection ( %, p= . ). coadministration of progesterone along with increased hoxa transfection had no additive effect on the expression of gaba-a pi receptor mrna (p= . ). gaba-a pi receptor protein expression was similarly increased by each treatment. either hoxa or progesterone independently caused translocation of the gaba receptor from the cytoplasm to the cell membrane in ishikawa cells. conclusion gaba-a pi receptor expression is increased in the human luminal epithelium and stroma in the window of implantation. activation of the pi subunit leads to opening of ion channels, likely allowing flux of water into the epithelial cells and out of the uterine lumen. progesterone and hoxa each increase both pi subunit receptor expression and membrane translocation. the lack of additive effect suggests progesterone induced pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of hoxa expression. finally, after receptor expression and translocation, progesterone mediated gaba receptor ion channel activation mediates water resorption necessary for implantation. cancer. suzy davies, donghai dai, kimberly k leslie. obstetrics and gynecology, university of new mexico, albuquerque, nm, usa. objectives: endometrial cancer is the most frequent gynecologic cancer in women. it affects an estimated , women in the us every year, and long term outcomes for patients with advanced stage or recurrent disease are poor. targeted molecular therapy against the vascular endothelial growth factor (vegf) and its receptors constitute a new therapeutic option for patients that is now under study by the gynecologic oncology group in a phase trial, gog e (now in second stage accrual). the goal of our work was to assess the potential effectiveness of vegf/vegfr blockade in preclinical endometrial cancer models. methods: these studies employed two agents, bevacizumab (avastin, a vegfa blocking antibody) and vandetanib (zactima, a tyrosine kinase inhibitor of egfr and vegfr ). ic experiments were performed on endometrial cancer cells in culture using four established cell line models. xenografted athymic mice were also employed to test the ability of compounds to inhibit tumor growth in vivo. tumors were isolated from controls and treated animals, mrna was extracted, and affymetrix gene expression arrays were performed to determine the genes consistently modulated by treatment. results: compared to vandetanib with an ic of . m, bevacizumab showed little activity on cell proliferation in vitro, and cell numbers were not reduced by % using concentrations up to . m. however, bevacizumab demonstrated robust activity in the athymic mouse model, resulting in a significant decrease in tumor formation and growth compared to vehicle treated animals when dosed bi-weekly in a concentration of . mg/mouse ip. tumors from this model demonstrated that eighteen genes were consistently up or down regulated in the presence of bevacizumab. among the regulated transcripts was microrna , which was significantly down-regulated. microrna is an anti-apoptotic factor, and its inhibition by bevacizumab predicts for increased expression of the tumor suppressor pten, decreased cell proliferation, and a reduced capacity for metastasis. conclusions: these studies confirm that the vegf pathway is a good target for new therapies against endometrial cancer. blocking this pathway not only inhibits angiogenesis, but also results in changes in gene expression that enhance apoptosis and reduce cellular proliferation and tumor invasion. we collected fibroid and adjacent normal tissues following hysterectomy with patient consent and institutional irbs. to date, data points for each gene from arrays have been collected from patients. the fluorescence readings were log-transformed and normalized with the robust quartile normalization method. quality control of the normalized data was performed to remove arrays that deviated from twice the inter-quartile range calculated from the array signals. results: the custom microarray profiling identified genes that were differentially expressed between normal and fibroid tissues (p < . ; fdr< . ). among them, genes encode receptor tks, genes encode tk ligands, and genes encode cell cycle and apoptosis proteins. clustering analysis of the mean log ratios of these genes has led to the division of the patients into two major groups. thirty four ( %) belong to a group characterized by the significant downregulation of the cyr (ccn ) gene in fibroid tissues. the cyr -down group can be further divided into two sub-groups based on the expression of another tk ligand, efn a. other receptor differentially expressed tk did not segregate with the three defined sub-groups. finally, there was no sub-group segregation based on age of the patient, menstrual phase, or weight of the fibroid. conclusion: our results have demonstrated that tyrosine kinases and their ligands are uniquely differentially expressed between normal and fibroid tissues. unique tyrosine kinase ligands in our population were cyr and efn a, and these markers created three molecular classification groups based on their differential expression. these results support the hypothesis that tyrosine kinase ligands are involved in fibroid growth, and may offer targets for a strategy to avoid hysterectomy. microvascular perfusion sonographic imaging to detect early stage ovarian cancer. joanie mayer hope, arthur c fleischer, brian day, stephanie v blank, bhavana pothuri, robert wallach, john p curtin, david a fishman. gynecologic oncology, new york university school of medicine, new york, ny, usa; radiology, vanderbilt university medical center, nashville, tn, usa. objective: epithelial ovarian cancer (eoc) is the th leading cause of death in us women due to the inability to detect early stage disease. recent sonographic developments involving harmonics, pulse inversion, and the use of contrast agents justify the hope that depiction of aberrant tumor microvascularity associated with early disease can occur. this study utilizes these new techniques to assess the unique microvascularity associated with early stage eoc. methods: we used pulse inversion harmonic microvascular imaging (mvi) technology to depict differences between benign and malignant ovarian lesions. contrast enhanced harmonic transvaginal (tv) sonography was performed using the philips iu scanner after intravenous injection of g of definity (bristol-myer-squibb). split screen real-time images were acquired displaying conventional sonographic views adjacent to harmonic, low mechanical index images. morphologic features (thickened wall, papillary excrescence, calcifications) and aberrant vascularity were noted. q-lab quantification of wash-in, peak enhancement, and wash-out times as well as area-under-thecurve (corresponding to microvessel perfusion) were compared using student's t-tests. results: to date, contrast enhancement patterns of ovaries have been analyzed, benign and malignant. of the malignancies ( fallopian tube, ovarian: stage i, stage iii), / women were correctly identified using conventional tv imaging and others were identified using mvi / . all benign lesions were correctly identified by mvi / while tv detected normals ( false negatives). the lesions detected as malignant by mvi were -stage i fallopian tube and -stage i eoc. contrast enhancement kinetics of malignant lesions demonstrated similar wash-in ( . ± . vs . ± . , p = . ), greater peak enhancement ( . ± . vs . ± . , p < . ), longer wash-out ( . ± . vs . ± . (p < . ), and greater perfusion ( . ± . vs . ± , p < . ) when compared to benign lesions. conclusion: contrast enhancement patterns are significantly different in benign vs. malignant ovarian masses. this technique has clear potential in differentiating benign from malignant lesions and for detecting occult stage i disease. identification and characterization of mir- a as regulator of ikk expression and its function in ovarian cancer cells. rui chen, ayesha b alvero, thomas rutherford, gil mor. department of obstetrics and gynecology, yale university school of medicine, new haven, ct, usa. introduction: the proinflammatory environment associated with tumor growth and chemoresistance is produced by both immune cells and cancer cells. the nf-b pathway plays a critical role mediating the capacity of cancer cells to produce pro-inflammatory cytokines. recently, we described a group of epithelial ovarian cancer (eoc) cells characterized by ikk expression as the main factor promoting nf-b activation and cytokine production. in this study we evaluated the regulation of ikk in eoc cells. we describe the identification and characterization of mir- a as a regulator of ikk expression, thus indirectly of nf-b activity and function. materials and methods: human eoc cell lines were established from malignant ovarian cancer ascites. protein expression were determined by western blotting. ikk mrna was measured by rt-pcr. cytokines were profiled by the luminex system. ikk transfection was done with roche fugene transfection reagent. mirna microarray was done with invitrogen ncode multi-species mirna microarray kit. mir- a qrt-pcr was performed with invitrogen ncode sybr greener mirna qrt-pcr analysis kit. mirna transfection was done with ambion siport neofx agent. the ikk '-utr luciferase reporter plasmid was established based on ambion pmir-report mirna expression reporter vector. results: ikk expression was associated with nf-b cyclic activity and the ability of type i eoc cells (but not type ii) to produce inflammatory cytokines. transfection of ikk into type ii eoc cells reversed their phenotype. mirna microarray identified mirnas differentially expressed in type i versus type ii cells, one of which, mir- a, had putative binding sites in the '-utr of ikk mrna. mir- a introduction into type i cells inhibited ikk expression, and direct inhibition through ikk 's '-utr was confirmed by luciferase assay. conclusion: we describe for the first time the identification of ikk as a potential key switch between chemo-resistant and chemo-sensitive phenotypes, by regulating nf-b activity in eoc cells. furthermore, we identified mir- a as a direct regulator of ikk expression. ikk expression may represent an adaptational stage in tumor progression allowing cancer cells to create their own inflammatory environment. these findings may provide novel molecular targets and potential markers for individual therapy selection. regulation of cd in the rat uterus by nitric oxide and the involvement of p kinase pathway. uma yallampalli, rebakah elkins, pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cd is expressed in many cell types including uterine cells and has been shown to play an important role in protecting against compliment attack. we have previously shown in endometrial cell lines that cd levels were down regulated by nitric oxide (no) . in this study we extend our previous observations to determine if no down regulates cd in rat uterine tissues and assess its mechanisms of action. methods. non pregnant rats ( g; b wt) were bilaterally ovariectomized under ketamine anesthesia. groups of ovariectomized rats were implanted with alzet mini pumps to deliver nitro-l-arginine melthylester (l-name) at or /mg/rat/day in saline or saline alone. uteri were obtained from these rats at or hours after infusion. in another set of experiments uteri were obtained from ovariectomized rats and cut in to small pieces and incubated in vitro with either l-name ( mm), diethylenetriamine-no mm) or worthmanin ( . m) in mem without phenol red for hours. uterine tissues were homogenized in trizol and mrna levels for cd were measured using rt-pcr and expressed as a ratio to s. results. results show that in vivo treatment with l-name to ovariectomized rats caused elevations in uterine cd mrna levels in a time and dose dependent manner with maximal responses seen with mg l-name and at hours. in vitro studies show that deta-no suppressed cd mrna levels in the rat uterus. both l-name and pi kinase inhibitor, worthmanin caused increases in cd levels in these tissues and the effects of worthmanin are reversed by deta-no. these results suggest that cd levels in the rat uterus are down regulated by no and are upregulated when no synthesis is inhibited by l-name. further, pi kinase appears to be involved in cd regulation in the rat uterus and no donor appears to modulate this response. lung. lakeitha r foster, daniel b hardy, carole r mendelson. dept of pediatrics; depts of biochemistry and ob/gyn, ut southwestern medical center, dallas, tx, usa. during % of human pregnancy, the maternal uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone (p ). we previously observed that p acting through the progesterone receptor (pr) serves an anti-inflammatory role and inhibits uterine contractility by antagonizing nuclear factor b activation and cyclooxygenase- (cox- ) expression (hardy et al., ) . our previous findings also suggest that the fetus provides an important signal for the initiation of labor near term through augmented secretion of the major lung surfactant protein, sp-a, into amniotic fluid (condon et al., ) . secreted sp-a, in turn, activates fetal macrophages which migrate to the maternal uterus where they release cytokines and promote an inflammatory response, leading to labor. in the present study, we tested the hypothesis that maternal p also maintains uterine quiescence by inhibiting sp-a production by the fetal lung. age matched icr mice were injected s.c. once daily either with sesame oil (control) or p ( mg/ml) from to days post-coitum (dpc). as expected, treatment with p delayed parturition by - h. this also was associated with a decrease in uterine cox- mrna expression, as compared to the vehicle-injected controls. interestingly, maternal p treatment caused a marked decrease in the levels of immunoreactive sp-a protein secreted by the fetal lungs into in amniotic fluid at dpc. furthermore, sp-a protein and mrna levels were reduced in the fetal lungs of p -injected mothers, as compared to controls. this was associated with an inhibitory effect of p treatment on cox- protein and mrna levels in the fetal lungs. these findings were of interest, since cox- expression is markedly upregulated during differentiation of human fetal lung (hfl) explants in culture (hardy et al., ) and endogenous and exogenous prostaglandins increase sp-a expression in hfl (acarregui et al., ) . collectively, these findings suggest that maternal p treatment prevents increased uterine contractility, in part, by inhibiting inflammatory response pathways within the fetal lung. this, in turn, blocks the developmental induction of sp-a expression and its secretion into amniotic fluid. in this manner, maternal p inhibits an important fetal signal leading to labor. supported by nih p hd ; nih r hl . recent evidence suggests that leukocytes infiltrate uterine tissues at the time of parturition implicating inflammation as a key mechanism of human labor. ccl- is a pro-inflammatory cytokine that may contribute to the development of inflammatory reaction in the myometrium. previously we showed upregulation of rat ccl- gene expression in myometrium during term and ru -induced preterm labor. also ccl- was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium. the objective of this study was to investigate the role of mechanical stretch as a possible regulator of myometrial leukocyte infiltration and ccl- as a mediator of this stretch response. we also studied the effect of progesterone (p ) on the myometrial secretion of ccl- . methods. we used primary culture of rat myometrium smooth muscle cells (smcs) to study in vitro ccl- gene and protein induction by static mechanical stretch. ccl- gene expression analysis was performed by real-time rt-pcr and immunoreactive (ir) protein content was measured by elisa assay. we used primary rat monocytes to access whether stretch-induced ccl- production by myometrial smcs resulted in enhanced monocyte chemotactic activity. results. myometrial cells were stretched for - hours and the supernatants collected. analysis of media conditioned by primary myometrial smcs revealed that static mechanical stretch ( % elongation for hours) caused a significant accumulation in ir ccl- which was repressed by pretreatment with p ( um). the rise in ccl- protein levels was preceded by a transient increase on ccl- mrna. the migration of primary rat monocytes in response to conditioned medium from stretched myometrial smcs was much greater than that of conditioned medium from control non-stretched cells. co-incubation with a neutralizing antibody to ccl- significantly reduced the chemotaxis of monocytes in response to the stretch-conditioned medium. conclusion: uterine smcs play an active role in uterine inflammation by producing chemokines and promoting the chemotaxis of immune cells into the myometrium. the blockade of this effect by p offers a potential explanation for the therapeutic actions of this hormone in the prevention of preterm birth. membranes during human labor. nardhy gomez-lopez, , guadalupe estrada-gutierrez, lourdes vadillo-perez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico; escuela nacional de ciencias biologicas, ipn, mexico city, df, mexico. introduction. leukocytes arriving to the choriodecidua (chd) during labor are capable to secrete cytokines and matrix metalloproteinases that may play a role in the fetal membranes (fm) extracellular matrix degradation. objective. the aim of this work was to identify changes in the leukocyte subpopulations in the chd and fm during human labor. methods: fm were obtained from two groups of women: ) term without labor (n= ) and ) term with spontaneous labor (n= ). chd cells were isolated and analyzed by flow cytometry. explants of fm were embedded in paraffin and analyzed by confocal microscopy. in both techniques, cd , cd , cd , cd and cd subpopulations of leukocytes were identified. intracellular mmp- was also identified in these cells. results: major changes in leukocytes subpopulations during labor involved a higher amount of cd +, cd+ and cd + cells, both in the chd and inside the fm. mmp- was associated to cd + cells. cd + exhibited a more widespread localization in the fm and cd + cells were localized in the contact with the trophoblast layer during labor. conclusions: leukocyte populations changes both in the chd and fm during labor and are characterized by arrival and infiltration of specific subpopulation of lymphocytes and monocytes. nk cells are enriched in mmp- , which may be related to a role in extracellular matrix degradation leading to the rupture of fetal membranes. women with a history of a pregnancy complicated by preeclampsia or intrauterine growth restriction (iugr) have an increased risk of future cardiovascular disease. excessive weight, particularly abdominal fat mass, is associated with cardiovascular morbidity and mortality. objectives: the aim of this study was to investigate differences in body composition and fat distribution between women with a history of preeclampsia or iugr and uncomplicated pregnancies. methods: from a genetically isolated population in the southwest of the netherlands, non-pregnant women with a history of preeclampsia (n= ), iugr (n= ) and uncomplicated pregnancies (n= ) were recruited at a mean follow up time of . years after pregnancy. body composition and fat distribution were assessed by dual energy-x-ray absorptiometry (dxa) and anthropometric measurements. results: women with a history of preeclampsia compared to controls had higher mean total-, fat-and lean mass (p < . ) as well as higher mean indices of body mass, fat mass and lean mass (p< . ). no significant differences were found for these variables between women with a history of iugr and controls. women with a history of preeclampsia had higher waist circumferences and waistto-hip ratios (p< . ) as well as excess of android fat mass and increased android-to-fat ratios (p< . ). women after pregnancies complicated by iugr had higher waist-to-hip ratios (p < . ). after controlling for body mass index, both women with a history of preeclampsia or iugr had higher waist circumferences (p < . ) and waist-to-hip ratios (p < . ) as well as smaller hip circumferences (p < . ). conclusion: despite differences in body mass index, both women with a history of preeclampsia and women after pregnancies complicated by iugr have a metabolically adverse fat distribution, marked by an excess of fat deposition in the abdominal region relatively to the hip region. these findings may explain, at least partly, their increased cardiovascular risk. women with a history of preeclampsia. marc ea spaanderman, timo h ekhart, robert aardenburg, louis lh peeters. obstetrics and gynecology, radboud university medical center, nijmegen, netherlands; obstetrics and gynecology, university medical center maastricht, maastricht, netherlands. background: a history of preeclampsia is associated with persistent short-term alterations in circulatory function and remote cardiovascular disease. in this study we tested the hypothesis at least years after preeclamptic pregnancy renal and central hemodynamic function is impaired as compared to women with uncomplicated pregnancy. methods: in formerly preeclamptic women (pe) and healthy parous controls (control) who were normotensive at weeks post-partum follow up, we assessed at least years after delivery blood pressure (mmhg), cardiac output (co, doppler ultrasonography, l/min) and effective renal plasma flow (erpf, pah clearance, ml/min/ . m ) after which we calculated total peripheral vascular resistance (. dyne.s/cm ) and renal vascular resistance (. dyne.s/cm ). data were analyzed parametrically (p< . ). results: age and bmi were comparable between groups. blood pressure was higher in formerly pe. moreover, % of formerly pe women and % of control were hypertensive (p< . ). although cardiac out was comparable between groups, total peripheral and renal vascular resistance were about % higher and erpf % lower in formerly pe women as compared to control. conclusion: at least years after gestational hypertensive disease, women who were normotensive at direct follow up have impaired renal and central hemodynamic function and developed more often chronic hypertension. long-term follow up may also be warranted in apparently healthy formerly pe women who are normotensive at post-partum follow up. circulatory function in formerly preeclamptic women and healthy parous controls co erpf tpvr rvr formerly pe . ± . ± * ± * ± * control . ± . ± ± ± * = p< . pregnancy increases blood-brain barrier permeability coefficient (l p ) to lucifer yellow: role of estrogen. marchien j wiegman, , marilyn j cipolla. neurology, ob/gyn and pharmacology, university of vermont, burlington, vt, usa; ob/gyn, university medical center groningen, groningen, netherlands. background: eclampsia is similar to posterior reversible encephalopathy syndrome in which an acute rise in blood pressure causes breakthrough of autoregulation, blood-brain barrier (bbb) disruption, and cerebral edema formation. we previously showed that late-pregnant (lp) animals developed cerebral edema during breakthrough, a response that was absent in nonpregnant (np) animals. in the current study we hypothesized that pregnancy predisposes the brain to edema during acute hypertension by enhanced bbb permeability. we further hypothesized that the underlying effect of pregnancy on the bbb permeability is due to elevated estrogen levels. methods: permeability coefficients (l p ) to lucifer yellow (ly), a polar compound that does not pass through tight junctions, were compared in posterior cerebral arteries (pca) from groups of sprague dawley rats: np (n= ), lp (d ; n= ), ovariectomized and implanted with -estradiol ( . mg, -day release) and estriol ( . mg, -day release) pellets for days (ovx+e; n= ), and ovariectomized and implanted with placebo pellets for days (ovx; n= ). pcas were isolated, pressurized in an arteriograph, and perfused with . mg/ml ly in saline. concentration changes of ly outside the vessel wall were determined at pressures from - mmhg. the slope of the pressure vs. permeability curve is the rate of flux, or l p for ly. results: l p for ly was significantly increased in pcas from lp and ovx animals vs. np (p< . ; figure ). estrogen was protective of the bbb only in ovariectomized animals, decreasing l p % in ovx+e vs. ovx. however, pregnancy did not afford protection and had a l p that was % greater than np. conclusions: pregnancy significantly increases bbb permeability to ly, an effect that may predispose the brain to edema formation during acute hypertension. these data also show that estrogen modulates l p in ovariectomized animals differentially than pregnancy, suggesting that the increased bbb permeability in pregnancy is caused by a mechanism other than elevated estrogen levels. in both pre-and early pregnancy, the sympathoinhibitory response to volume expansion is blunted in formerly preeclamptic women with low plasma volume. ineke krabbendam, marc ea spaanderman, dorette a courtar, robert aardenburg, ben j janssen, fred k lotgering, louis lh peeters. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; obstetrics and gynecology, university hospital maastricht, maastricht, netherlands; pharmacology and toxicology, university of maastricht, maastricht, netherlands. background: the circulation of formerly preeclamptic women with a low plasma volume (lpv) is characterized by sympathetic dominance. these women respond to a new pregnancy with an aberrant rise in atrial natriuretic peptide (anp) and a times higher chance to develop recurrent gestational hypertensive disease compared to their counterparts with normal plasma volume (npv). anp has sympathicomimetic capacity. we postulate that the sympathetic overdrive in lpv-women is associated with a reduced venous capacitance. to this end, we compared the response to volume expansion (ve) in women with lpv and npv, both before and in pregnancy. method: in non-pregnant normotensive formerly preeclamptic women, we measured pv (hsa i indicator dilution method) at least months post partum. we intravenously infused ml of iso-oncotic fluid over minutes. during the infusion, we recorded changes in heart rate (hr, bpm), blood pressure (bp, mmhg), cardiac output (co, l/min), sympathetic activity (lfsys, mmhg , low frequency component of spontaneous fluctuations in systolic bp, portapress) and anp (nmol/l). eight women became pregnant within year and were evaluated at weeks gestation. changes in circulatory and autonomic function between and within groups were analyzed non-parametrically (p< . ). results: before pregnancy, ve leads to comparable changes in hr, bp and co in women with lpv ( / ) and npv ( / ). in npv, lfsys decreased %, but only % in lpv (p< . ). anp remained unaltered in npv, but increased in lpv. in the pregnant group, women had lpv and had npv. in both groups, pregnancy did not alter the response to ve. conclusion: irrespective of pregnancy, the sympathoinhibitory response to ve is diminished in lpv. these data suggest that in these women ve leads to venous overfill, giving rise to anp-release and consequently sympathetic activation, flattening the normal baroreceptor-mediated sympathoinhibitory response. we speculate that this mechanism contribute to circulatory maladaptation to pregnancy, sympathetic dominance and subsequent gestational hypertensive disease. in both pe and ht, serum sflt- was increased, and plgf reduced at all gestations (p< . ). seng levels were also increased in pe. after weeks (but not before) antihypertensive treatment was associated with a significant fall in serum sflt- and seng, in pe only. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). only sflt- was significantly reduced in the placenta in women who received antihypertensive therapy. conclusion in pe, antihypertensive therapy after weeks' gestation is associated with a significant fall in serum sflt- and seng, and in placental sflt- . these findings raise the possibility that these drugs may have an effect on the pathophysiology of pe other than their known antihypertensive action. the synergistic effect of soluble vegf receptor pre-treatment and small doses of tnf-on endothelial cells. tereza cindrova-davies, debbie a sanders, olivera spasic-boskovic, graham j burton, d stephen charnock-jones. dept of pdn, university of cambridge, united kingdom; dept of obstetrics and gynaecology, university of cambridge, united kingdom. introduction: preeclampsia is marked by an enhanced endothelial inflammatory response manifested by maternal endothelial activation. soluble fms-like tyrosine kinase- (sflt- , svegf-r ), a naturally occurring circulating antagonist of vegf-a and plgf, is one of the secreted factors implicated in the pathogenesis of preeclampsia. in women who develop preeclampsia, sflt rises sharply, preceding the onset of the clinical disease. the aim of this study was to examine the effect of a combined treatment with recombinant sflt- and tnf-on the activation of human umbilical cord endothelial cells (huvec) by examining leukocyte adhesion, and the expression of icam, vcam, endothelin and vwf. methods: huvec were seeded, grown overnight and pre-treated with recombinant sflt ( - ng/ml) in a basic % fbs-dmem medium for hr. on day , low doses of tnf-( . ng/ml) were applied to pre-treated cells for hr. antagonism of the vegf-a action was mimicked at the protein level by pre-incubating huvec with anti-flt antibody ( - g/ml), anti-kdr antibody ( g/ml), anti-vegf antibody ( g/ml), or vegf receptor inhibitor su ( m). at the rna level, the effect of sflt was mimicked by sirna transfection of huvec with siflt or sikdr. at the end of each experiment cells were either harvested for western blotting, fixed in % pfa for immunofluorescence or incubated with labelled hl leukocyte cells, followed by fluorescent detection of adhesion. results: pre-incubation of huvec with sflt and subsequent treatment with low doses of tnf-increased the adhesion of hl leukocytes and increased icam- , vcam- , endothelin and vwf, compared to tnf-treatment alone. similar results were obtained when cells were pre-treated with su , anti-flt, anti-kdr or anti-vegf. transfection knock-down of flt or kdr gene also significantly increased leukocyte adhesion when small doses of tnfwere added. conclusions: pre-incubation with recombinant sflt, anti-flt, anti-kdr, anti-vegf, su or knocking-down flt or kdr transcripts all antagonised the autocrine actions of vegf and/or plgf. this predisposes huvecs to be more sensitive to the effect of tnf-. our study shows that sflt and tnfcombine to induce an enhanced synergistic effect, activating endothelial cells. maternal obesity is associated with increased production of inflammatory cytokines and risk of poor perinatal outcome. inflammatory cytokines can stimulate production of reactive oxygen (ros) and nitrogen (rns) species, which can covalently modify protein function. placental oxidative and nitrative stress are increased in pathological pregnancies and associated with altered placental function. objectives: determine the effect of increasing body mass index (bmi) on placental nitrative stress, measured by the expression and localization of nitrated (nitrotyrosine) and oxidized proteins. methods: placental tissue was collected at term ( - wks) from lean kg/m ), overweight ( - . kg/m ) and obese ( - kg/m ) patients (n= or /group). tissue was sectioned for immunostaining with nitrotyrosine antibody. protein samples were either dot blotted onto nitrocellulose membrane, probed with nitrotyrosine ab, and nitrated proteins detected using ecl, or derivatized using , -dinitrophenylhydrazine (dnph) (oxyblot® kit), separated on sds-page, probed with anti-dnph ab ( : ) and oxidized proteins detected using ecl. protein band intensity was measured by densitometry. oxidized proteins were selected for maldi mass spectrometry analysis. results: nitrotyrosine residues were immunolocalized primarily in the fetal capillary endothelial cells and the villous stroma, but were almost absent in the syncytiotrophoblast. by dot blot, nitrotyrosine expression differed across the three groups (p< . , anova) with expression in tissue from obese women being significantly increased compared to lean (p< . ) and overweight (p< . , tukey test). several oxidized proteins were detected with significantly greater expression seen in the lean versus overweight groups (p< . , mann-whitney u). one oxidized protein was identified by maldi-ms with four peptide matches and . % coverage as -beta hydroxysteroid dehydrogenase ( bhsd). discussion: with increasing bmi, an increase in nitrative stress appears to occur in parallel with a decrease in oxidative stress. oxidative stress is apparently reduced as ros are consumed by the interaction with rns to give nitrative stress. bhsd is involved in the biosynthesis of steroid hormones and glucocorticoids. its activity and hence steroid metabolism in the placenta may be regulated by oxidation with implications for fetal development. background teenagers are susceptible to delivering small-for-gestationalage (sga) infants. previous studies implicate continued maternal growth as a causal factor . growing adolescent sheep have reduced fetal birthweight due to impaired placental development and nutrient transfer . we hypothesized that placental function is impaired in human teenage pregnancy if there is maternal growth. methods placentas were collected from teenagers ( - years) and adults. activity of the amino acid transporter, system a, was quantified by the sodium-dependent uptake of c-methylaminoisobutyric acid into placental fragments. teenagers were defined as growing (> mm increase in kneeheight per days) or non-growing. system a activity was analysed in relation to individualised birthweight centile and maternal growth. results placental system a activity was significantly lower in teenage compared to adult pregnancies (p< . ). this was unrelated to birthweight; teenagers who delivered infants appropriate-for-gestational age (aga) had significantly lower placental system a activity compared to aga infants delivered to adults (p< . ). growing teenagers did not deliver lower birthweight infants than non-growing teenagers (median birthweight g and g respectively). furthermore, system a activity in placentas from growing teenagers was significantly elevated compared to that in non-growing teenagers (p< . ), and was similar to placental activity in adults. conclusions system a activity was reduced in placentas from teenagers compared to adults. this suggests that inherently lower placental function predisposes teenagers to sga, but that other factors (e.g. adequate nutrition) can compensate in those teenagers delivering aga infants. in contrast to our hypothesis, placental system a was elevated in growing teenagers and mimicked that of adults. this may be related to a hormonal-milieu in growing teenagers that is conducive to fetal growth, in part through stimulating placental transport. homocysteine inhibition of system a amino acid transport activity in human placenta. eleni tsitsiou, susan l greenwood, colin p sibley, stephen w d'souza, jocelyn d glazier. maternal and fetal health research group, st. mary's hospital, university of manchester, manchester, united kingdom. background: elevated plasma levels of the amino acid homocysteine (hcy) during pregnancy are associated with vascular-related complications and adverse neonatal outcomes including a reduced birthweight. fetal and maternal plasma hcy concentrations are positively correlated suggesting placental transport of hcy may be an important determinant of fetal plasma hcy. the mechanisms involved in placental hcy transport are uncharacterised. evidence that the system a amino acid transporter, which transports neutral amino acids in a na + -dependent manner, is important in promoting fetal growth and that a reduced system a activity is associated with intrauterine growth restriction (iugr), led to our hypothesis that system a provides one mechanism for placental hcy transport. this hypothesis was tested by measuring the ability of hcy to inhibit system a activity in isolated microvillous plasma membrane (mvm) vesicles and placental fragments. materials and methods: mvm vesicles and placental fragments were isolated from placentas of normal pregnancies at term. system a activity was measured at initial rate ( s and min respectively) as na + -dependent cmethylaminoisobutyric acid (meaib) uptake into mvm vesicles ( . mm) or fragments ( . mm) in the absence (control) or presence of l-hcy and dl-hcy or model substrates. results: mm l-hcy (custom-synthesised) and dl-hcy (commercial source) significantly (p< . ) inhibited na + -dependent c-meaib uptake into mvm vesicles compared to control; comparable in magnitude to other model substrates (meaib, l-ala, l-ser, l-met; n= , kruskal-wallis with dunn's multiple comparison test). l-hcy, l-met and meaib ( . - mm) caused a dose-dependent inhibition of na + -dependent c-meaib uptake into mvm with ec values (in mm; mean ± se) of . ± . , . ± . , and . ± . respectively, n= ). na + -dependent c-meaib uptake into fragments was reduced substantially in the presence of mm l-hcy or dl-hcy causing a ± and ± % reduction (mean ± sd, n= - ) of control respectively. conclusion: these observations suggest that hcy is a relatively high affinity substrate for system a in the human placenta. we speculate that inhibition of placental amino acid uptake by hcy could impact on fetal growth and development. supported by the mrc and action research endowment fund. glucose regulates placental mtor activity and glucose deprivation down-regulates placental system l activity in an mtor-dependent manner. sara roos, theresa l powell, thomas jansson. department of physiology, institute of neuroscience and physiology, gothenburg, sweden; department of obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. placental amino acid transporters are down-regulated in intrauterine growth restriction (iugr). we have previously shown that mammalian target of rapamycin (mtor) regulates placental system l transporter activity and that placental mtor activity is decreased in iugr. however, the upstream regulators of placental mtor are unknown. in iugr, fetal hypoglycemia and reduced maternal glucose levels are common and the placenta may therefore be exposed to low glucose levels. hypothesis: we hypothesized that glucose availability regulates placental amino acid transporter activity mediated by changes in mtor signaling. methods: cytotrophoblast cells were isolated and cultured until syncytialization at hours. cells were cultured for an additional hours in culture media containing . mm, . mm, or mm glucose (control), which corresponds to standard culture media. at hrs, the activity of the mtor signaling pathway was assessed by measuring the protein expression of s k phosphorylated at thr- , the primary site of mtor phosphorylation. in another set of cells, system l activity was assessed by measuring the bchinhibitable uptake of h-leucine. results: as compared to control, phospho-thr- -s k expression was reduced by % in cells incubated in . mm results: fgr pregnancies were delivered earlier ( ± . v ± . w; p< . ), weighed less ( . ± . v . ± . kg; p< . ) and had higher s/d ratios ( . ± . v . ± . ; p< . ). eaa concentrations were similar between groups, but there were differences (non-parametric testing; p< . ) in transport rates between groups with his crossing considerably faster and ile crossing slower in fgr . the table shows fetal vein/maternal (fv/m) ratios standardized to the leu fv/m ratio for all eaa for both groups. amino acids fell into groups for fgr pregnancies: ) his (ratio . ), ) leu, phe, met (ratios ), and ) val, thr, ile, trp, lys with intermediate ratios ( . ns conclusion: this is the st study to compare the relative rates of in vivo placental transport for all eaa between normal and fgr human pregnancies showing striking differences in the transport rates of two eaa. in the absence of eaa concentration differences between groups, the higher his transport rate in fgr suggests higher utilization by the placenta. vasculature of women who received exogenous estrogen compared to those who received placebo. the purpose of this investigation was to identify the gene expression in response to the estrogen, equilin, as the major component of conjugated equine estrogen and genistein, a phytoestrogen in human coronary artery endothelial cells. human coronary artery endothelial cells from a -year old female were used. cells were treated with estradiol [e ], equilin [eq] ( . nm) or genistein [gen] ( micromolar) for hours. focused oligomicroarrays for cardiovascular disease and endothelial cell function were used to study gene expression. rt-pcr was used to confirm the transcription of genes analyzed in oligoarrays. vascular adhesion molecules and genes involved in the inflammatory response were most affected with the three estrogen sources. significant reduction of integrin alphae was seen with all three . , . and . for e , eq and gen respectively. similarly nfkb was also reduced . , . and . fold compared to controls. significant reduction of ccl was demonstrated with eq ( . fold) and gen ( . fold). tnfreceptor a and b were only significantly decreased by gen. vcam- which is regulated by proatherogenic factors and upregulated mainly at atherosclerosis -prone sites, was significantly reduced ( . -fold) with genistein, and a slight reduction was seen with e and no change was observed with eq. our data support the clinical findings that estrogen has favorable effects on the genes involved in atherosclerotic disease. while e has been shown to be slightly more effective than equilin in the modulation of gene expression, genistein was significantly more effective in the favorable expression of genes involved in particularly the inflammatory response. olga n lekontseva, sandra t davidge. physiology and obstetrics/gynecology, university of alberta, edmonton, ab, canada. introduction: the prevalence of cardiovascular disease in women dramatically rises in the postmenopausal period. although deficiency of estrogen has been implicated in the pathophysiology of systemic vascular dysfunction, the effects of estrogen on vasculature are complex and not completely understood. we have previously shown that estrogen exerts a beneficial effect on the aging vascular system by reducing circulating levels of the inflammatory cytokine tnf . tnf is a known regulator of matrix metalloproteinases (mmps), proteolytic enzymes that may modulate vascular tone through cleavage of vasoactive peptides such as big endothelin- (et- ). the role of estrogen in this pathway is unknown. we tested the hypothesis that in aging/estrogen deficiency, tnf -induced mmp activity mediates greater vasoconstriction, in part, through the et- pathway. we further hypothesized that estrogen replacement reduces vascular sensitivity to the constriction by preventing mmp activation. methods: aged ( month old) female sprague dawley rats were ovariectomized and treated with either placebo [ovx] , -estradiol [ovx+e ], or the tnf inhibitor etanercept [ovx+etan] . after four weeks, resistance mesenteric arteries were isolated and studied on the pressure arteriograph. concentrationresponse to exogenous big in the absence or presence of mmp inhibitor (gm , μm) was assessed in the vessels. results: treatment of "menopausal" [ovx] rats with either estrogen or the tnf inhibitor reduced sensitivity of arteries to big et- (ec = . ± . μm in ovx versus . ± . μm in ovx+e or . ± . μm in ovx+etan groups; p< . ). although mmp inhibition attenuated maximal constriction in all of the arteries, there was a significantly greater (p< . ) role of mmps in big et- -induced vasoconstriction in the ovx+e group (reduction in max constriction= . ± . %) compared to ovx ( . ± . %) and ovx+etan groups ( . ± . %). conclusions: both estrogen and tnf inhibition reduced big et- vasoconstriction. however, contrary to our hypothesis, tnf is not contributing to mmp modulation of et- vasoconstriction. interestingly, our study demonstrates a novel role for estrogen to increase mmp contribution to big et- vasoactivity with the net effect being less vasoconstrictive. understanding this unique pathway of regulation by estrogen in the aged vasculature will allow for development of new therapeutic options for women. mo, usa. introduction: the etiology of pelvic organ prolapse is multifactorial, with both inherited and acquired components. the molecular mechanisms of prolapse have not been established yet. we have previously shown that lysyl oxidase (lox) expression is suppressed in uterosacral ligaments of women with pelvic organ prolapse. it has also been shown that lox is a tumor suppressor gene inactivated by methylation in human gastric cancers. hypothesis: the aim of this study was to analyze the dna sequence of the promoter region of the lysyl oxidase gene in tissues from women with pelvic organ prolapse and identify whether methylation is present. our hypothesis is that the promoters of the lox gene in women with pelvic organ prolapse have significantly more methylation sites than women without prolapse. materials and methods: genomic dna was isolated from the uterosacral ligaments of eight women with pelvic organ prolapse and women without prolapse (controls). genomic dna samples were treated with ez dna methylation kit (zemo research, orange, ca). the lox gene promoter region of - to + was amplified by pcr and then cloned into pcr . -topo (invitrogen, carlsbad, ca) and transformed into an e. coli dh a strain. amplified plasmid dna samples containing the lox gene promoter region from each woman were sequenced and methylated cpg islands were identified by sequence comparison. results: a total of methylated cpg sites were found in the patient group with pelvic organ prolapse while only methylated cpg site was found in the non-prolapse control group. conclusion: these findings suggest that methylation in the promoter region suppresses lox gene expression in women with pelvic organ prolapse. background: reports from case-control and cohort studies have suggested an inverse association between lactation and breast cancer risk, but findings have been inconsistent. methods: we conducted a prospective observational cohort study of , parous women participating in the nurses' health study ii from to . our primary outcome was incident premenopausal breast cancer. results: during the study period, cases of premenopausal breast cancer were diagnosed during , person-years of follow-up. women who had ever breastfed had a % lower incidence of premenopausal breast cancer ( % confidence interval [ci] - %) compared with women who never breastfed, adjusting for parity, age at first birth, year of first birth, height, body mass index (bmi), bmi at age , family history, personal history of benign breast disease, participant birth weight, preterm birth, age at menarche, oral contraceptive use, physical activity, and alcohol consumption. no trend was observed with duration of lactation (p= . ). the association between ever-breastfeeding and premenopausal breast cancer was modified by use of medication to suppress lactation (p= . ); in analyses restricted to women who had never used suppressive medication, ever-breastfeeding was associated with a % ( %ci - %) reduction in incident disease. the association between lactation and premenopausal breast cancer was further modified by family history of breast cancer (p= . ). among women who had never used suppressive medication and reported a family history, those who had breastfed had a % lower covariate-adjusted risk of premenopausal breast cancer ( % ci - %) than women who had never breastfed. among women without a family history, ever-breastfeeding was not associated with breast cancer incidence (hazard ratio . , % ci . - . ). among women who had ever breastfed, we observed no association between breast cancer risk and duration of lactation amenorrhea or exclusive breastfeeding. conclusion: in a large, prospective cohort study, ever-breastfeeding was inversely associated with risk for premenopausal breast cancer. at the durations observed in our cohort, we observed no trend with duration of breastfeeding or lactation amenorrhea. the inverse association with ever-breastfeeding was stronger among women with a family history of breast cancer. regulatory and nkt cells at the maternal-fetal interface. thomas f mcelrath, rachael a clark. brigham women's hospital, boston, ma, usa; harvard skin disease research center, brigham women's hospital, boston, ma, usa. introduction: pregnancy represents an immunologically challenging event requiring maternal tolerance of the fetal semi-allograft. an increase in decidual cd + cd + t cells has been documented but it is unclear if these represent foxp + t cells (tregs) . the possibility also exists that other cd + lineages with potential regulatory function exist within the decidua parientalis. we examined if cd + cd + cells are true foxp + tregs and evaluated the frequency of other t cell subsets with possible regulatory potential. methods: we extracted t cells from term deciduas of planned cesarean deliveries. cells were stained with directly conjugated monoclonal antibodies and were analyzed on a six color flow cytometer. results: we found that only a subset (median %) of cd + t cells were true foxp + regulatory t cells. from donors, foxp + tregs accounted for a median of . % of all cd + cells. these cells were memory cd ro + t cells lacking ccr , and l-selectin but expressing cd , ctla- , gitr, and hla-dr. additionally we found that a median % of cd + t cells expressed the nkt marker cd . these cells were a mixture of cd and cd -cd -t cells, with variable numbers of cd t cells, suggesting they do not represent merely recently activated t cells. there was enrichment for t cells with nkt markers after culture on hela cells expressing cd d, suggesting that these cell represent true nkt cells. nkt cells were of the non-classical type, with a diverse t cell repertoire ( . % iv jq) and also expressed cd , hla class ii, cd ro but were ccr and l-selectin low. comments: we find that only a minority of cd -expressing t cell in the decidua are true foxp + tregs. because much of the work on treg in pregnancy has used cd as a treg marker, this suggests additional studies are needed to confirm the role of these cells in pregnancy. we find that a novel population of non-classical nkt cells exists in the human decidua. nkt cells can either promote or antagonize tolerance, depending on the immunologic context. the large number of these cells in the decidua suggests they may play a role equal or exceeding that of regulatory t cells. prolapse. marsha k guess, kathleen a connell, richard bercik, lloyd g cantley. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa; internal medicine/neprhology, yale university school of medicine, new haven, ct, usa. objectives: thy- is a cell surface glycoprotien expressed in human fibroblasts, neurons, hematopoetic stems cells and endothelial cells. thy- expression affects fibroblast proliferation and migration, cell-cell, as well as, cell-matrix interactions. moreover, thy- expression has been shown to play a critical role in fibroblast dedifferentiation into myofibroblasts, as well as in extracellular matrix (ecm) production and fibrosis. women with pelvic organ prolapse (pop) have alterations in vaginal ecm protein expression and metabolism, as well as decreases in smooth muscle fractional content. in the current study, we evaluated thy- as well as the smooth muscle markers alpha-smooth muscle actin (asma) and desmin expression in women with pelvic organ prolapse compared to women with normal pelvic support (controls). methods: anterior apical vaginal wall specimens from women with pop and controls were collected at the time of hysterectomy. messenger rna and protein expression of thy- , asma and desmin were evaluated using semiquantitative rt-pcr, real-time pcr and western blot analysis. gapdh and beta actin were used as internal controls. results: rt-pcr demonstrated the presence of thy- , asma and desmin in vaginal tissue from women with pop and controls. further, thy- mrna expression was downregulated % in women with pop compared to controls (p = . ). a parallel decrease in thy- protein was seen in women with pop compared to controls (p= . ). although a % and a % decease were seen in asma and desmin mrna, these differences were not statistically significant. similarly, no differences were seen in asma and desmin protein expression. conclusion: we demonstrate that there is significantly less thy- expression in vaginal tissue from women with pop compared to controls. differential expression of thy- in prolapsed vaginal tissues suggests that thy- may have a functional role in mediating ecm metabolism in the female genitourinary tract. hur expression is altered in ectopic endometrium. s karipcin, t altun, ua kayisli, e seli. ob gyn, yale u., new haven, ct, usa. introduction: cytokines and growth factors contribute to cyclic turnover of the normal endomterium and to the pathogenesis of endometriosis. cytokine and growth factor messenger rnas (mrnas) undergo rapid turnover that is primarily mediated by au-rich elements (are) that consist of multiple stretches of adenylate and uridylate residues located in the ' untranslated region ( '-utr) of their mrnas. hur is a ubiquitously expressed rna-binding protein that stabilizes are containing mrnas and prolongs their expression. we hypothesized that hur may play a role in the regulation of cytokine expression during normal menstrual cycle and in endometriosis. methods: tissue sections obtained from normal (n= ) and ectopic (n= ) endometrium were immunostained for hur. staining intensity was evaluated by hscore and grouped according to menstrual cycle phase. statistical analysis was done with one-way anova. cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (e ; - m), or progesterone (p, - m) for and h, and hur expression was determined using western analysis and normalized to ß-actin. results: hur immunostaining was nuclear in endometrial cells. hur immunoreactivity was significantly lower in the early proliferative and late secretory phases ( . ± . and . ± . , respectively), compared to the mid-late proliferative ( . ± . ) and early-mid secretory phases ( . ± . )(p< . ). moreover, hur expression was significantly lower in ectopic endometrial cells when compared to normal endometrium in midlate proliferative and early and mid-secretory phases (p< . ). progesterone suppressed hur levels significantly in cultured endometrial stromal cells at both and h compared to control (p< . ) while estrogen did not cause a significant change. discussion: decreased hur levels in the late secretory and early proliferative phases are likely to contribute to degradation of cytokines and result in lower cytokine levels observed mid-cycle. late secretory decrease in hur levels may be mediated by progesterone as suggested by in vitro findings. in ectopic endometrium, persistent low expression of hur compared to normal endometrium most probably results from elevated cytokine levels associated with endometriosis. the effect of lower hur expression in ectopic endometrium on other are-containing transcripts, and on the pathogenesis of endometriosis remains to be elucidated. tissue. hong zhao, joy innes, scott reierstad, mehmet bertan yilmaz, serdar e bulun. department of obstetrics and gynecology, northwestern university, chicago, il, usa. background: aromatase is the key enzyme for estrogen biosythesis, and is encoded by the cyp a gene. thus far, only three unique untranslated first exons associated with distinct promoters in the mouse cyp a gene were described (brain-, ovary and testis-specific exon i). however, it remains unknown whether aromatase is expressed in other mouse tissues via previously unknown tissue-specific promoters activating new exon is. methods: real-time pcr was used to examine the aromatase expression levels in various c bl mouse tissues. '-rapid amplification of cdna ends ( '-race) was used to determine the transcriptional start sites of cyp a transcripts. promoter activity was measured using serial deletion mutants of dna fused to the luciferase reporter gene. results: real-time pcr results showed that aromatase was expressed in male gonadal fat and the expression level is lower than that in testis. the adipose tissue-specific untranslated exon i of cyp a transcript was isolated using '-race and this novel gonadal fat-specific exon i of cyp a mrna did not show sequence similarities to previously reported ones. this new adiposespecific exon i was mapped to kb upstream of the translation start site in the coding exon ii. the genomic region upstream of the adipose-specific exon i was cloned into luciferase plasmids. transfection of murine t -l cells with these plasmids showed that promoter activity was conferred by the sequence located at - to - bp upstream of the transcriptional start site. dexamethasone significantly induced activity of the adipose-specific promoter region. conclusion: taken together, our results suggest that a novel cyp a transcript is regulated by a tissue-specific promoter in male murine gonadal fat. these sacrificed; reproductive and selected other organs were removed, weighed and evaluated histologically by an observer blinded to treatment. results: the table below shows that tcc augmented effects of tp on weights of accessory sex organs. histological assessment revealed that tcc induced greater glandular distention with more secretions compared to the effects of tp alone; furthermore, mitotic figures were seen only in prostates from rats exposed to tp+tcc. conclusions: tcc is a newly identified endocrine disruptor with unique and novel actions resulting in potentiation of androgen effects on sex organs. these observations underscore possible environmental risks related to exposure to tcc. background: blastocyst implantation is dependent on the differentiation of human endometrial stromal cells (hesc) into decidual cells. transforming growth factor family members have well defined roles in cell differentiation and proliferation. activin a, a tgf superfamily member, enhances hesc decidualization and localizes to decidualized cells in human endometrium. other tgf superfamily members, including bmp , bmp , bmp , gdf , gdf (myostatin), gdf and nodal, may also be present in decidual cells and therefore may also play a role during this important process. this study aimed to determine whether activin is the major family member driving decidualization or whether other family members contribute to the process. methods: broad ranging activin inhibitors (activin-m a and sb ) that effect receptor-ligand interactions of other tgf superfamily members were used in hesc decidualization. protein localization was examined in secretory phase endometrium and first trimester decidua by immunohistochemistry and mrna expression was examined in an ex vivo model. the secretion of candidate proteins was measured during hesc decidualization and certain recombinant proteins added during decidualization to examine their effect. results: m a ( nm) and sb ( m) significantly reduced decidualization ( % and % respectively) demonstrating that activin and possibly other tgf family members are involved in decidualization in vitro. bmp , gdf and tgf protein were detected in decidual cells of mid-late secretory endometrium and first trimester decidua whilst all ligands except nodal, were expressed by hesc. both bmp and tgf secretion increased during hesc decidualization and administration of both these proteins significantly enhanced decidualization in vitro. conclusions: these data support a role for activin a, bmp and tgf in hesc decidualization. this is important as the elucidation of factors involved during decidualization will aid in better understanding implantation and fertility. abnormal chromatin remodeling in diabetic murine oocytes. laura lawrence, ann ratchford, cybill esguerra, qiang wang, kelle moley. ob/ gyn, washington university, st. louis, mo, usa. background: diabetic women experience increased miscarriages and adverse pregnancy outcomes. previous studies suggest adverse diabetic outcomes may occur earlier than the preimplantation period, particularly during oogenesis. we hypothesize that diabetes affects chromatin remodeling and chromosomal condensation in murine oocytes. methods: mii oocytes from diabetic and control mice were fixed with % pfa, permeabilized with . % triton x- for min, and immunostained against a-tubulin and the nucleus for hr at rt. images were obtained with laser confocal scanning microscope. protein expression levels of chromatin with dimethyl h k modifications were measured in nondiabetic and diabetic denuded murine oocytes at hours post pmsg ( hour) and at six hours post hcg ( hour) via western immunoblots. mature nondiabetic denuded oocytes were fixed in %pfa and permeabilized with . % triton x- . they were stained via immunohistochemistry against histone protein h at lysine (h k me ) and heterochromatin. results: immunohistochemistry reveals that diabetic mii oocytes have aberrant spindle formations and metaphase chromosome alignment. approximately / ( %) diabetic oocytes examined had abnormal spindles and metaphase alignments compared with only about / normal oocytes ( . %). western blot demonstrated times higher expression of dimethylated chromatin in diabetic oocytes at time compared with nondiabetic oocytes. at time hours, diabetic oocytes had significantly fewer h k modifications than the controls. when staining mature murine nondiabetic oocytes for dimethylation of h k me by immunohistochemistry, we demonstrated h k me expression in a condensed heterochromatin ring surrounding the nucleolus, consistent with transcriptional silencing. conclusions: diabetic mii oocytes have a significant increase in abnormal spindle formation and metaphase chromosome alignment. they also have increased dimethylation compared with normal oocytes at a time point when they should be transcriptionally active, storing maternal mrna in preparation for the silencing period. in addition, after hcg injection to trigger maturation and gene silencing, the diabetic oocytes had decreased dimethylated chromatin changes. our findings suggest that diabetic oocytes may be exiting the transcriptionally active period prematurely and may ultimately experience decreased, partial, and incomplete gene silencing. and xenopus epab genes and proteins were performed. expression of human epab and pabpc mrna was tested in ten different somatic tissues, testes, and ovaries by rt-pcr. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. epab and pabpc expression in human prophase i (pi) and metaphase ii (mii) oocytes, -cell embryos and blastocysts was evaluated using quantitative real time pcr. results: human epab is a aa protein with % identity and % similarity to mouse epab and contains rna recognition motifs and a pabp domain. human epab mrna is detected in ovaries and to a lesser extent in testes and several somatic tissues including kidney, liver, and muscle. similar to its mouse orthologue, human epab mrna is expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. pabpc mrna is ubiquitously present in all tissues as well as -cell, and blastocyst stage embryos. however, its levels are significantly lower than that of epab in oocytes. conclusions: in this study we report the identification of human epab. similar to that observed in xenopus and mouse, human epab is the predominant poly(a) binding protein in oocytes and it is replaced by pabpc following zga, which occurs at -to -cell stage in human. our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans. objective: c-jun nh -terminal kinase (jnk), a member of mitogen-activated protein (map) kinase family, is involved in cell proliferation, differentiation, and survival. fsh is required for granulosa cell proliferation and antral follicle growth but its mechanism of action in preantral stages is not well defined. we previously showed that pharmacological inhibition of jnk pathway halts invitro growth of murine preantral follicles in serum free media supplemented with fsh ( miu/ml). sigcs (spontaneously immortalized rat granulosa cell line) have characteristics similar to preantral granulosa cells and hence they were used in this study to determine whether the jnk pathway plays a key role in preantral granulosa cell proliferation. our specific aims were to determine whether: a) fsh activates jnk pathway in granulosa cells; b) the inhibition of jnk pathway blocks cell cycle progression. material and methods: sigcs were treated with miu/ml recombinant fsh in serum free media two days after serum starvation. activation of jnk pathway was analyzed with if and wb using phospho-jnk and phospho-cjun expression, respectively. fsh receptor expression (fshr) was analyzed with if. the inhibition of jnk pathway on cell cycle progression was analyzed by facs using a jnk inhibitor sp . results: fshr protein was expressed in sigc indicating that they can respond to fsh (fig a) . likewise by if, phospho-jnk expression was significantly increased in sigc hour post fsh exposure (fig- a) . similarly on wb, phospho-cjun expression increased as early as min after fsh exposure and peaked at hrs. cjun phosphorylation was abolished hr after treatment with sp ( mm) (fig b) . facs analysis showed that the inhibition of jnk by sp resulted in cell cycle arrest at g /m transition in a dose dependent fashion (fig c) . conclusion: these results strongly suggest that the proliferative effect of fsh on immature granulosa cells is mediated through the activation of jnk pathway. this is the first experimental observation implicating jnk signaling in granulosa cell cycle control. (nichd - ). the induction of {alpha}-hydroxylase (cyp ) expression in granulosa cells. satin s patel, victor e beshay, william e rainey, bruce r carr. reproductive endocrinology and infertility, university of texas at southwestern medical center at dallas, dallas, tx, usa; physiology, medical college of georgia, augusta, ga, usa. according to the traditional two-cell two-gonadotropin hypothesis of the ovary, androgen production arises exclusively from theca cells. the granulosa cells, in turn, utilize androstenedione, which is aromatized eventually to estradiol. studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein- (ap- ) transcription factor, cfos compared to theca cells, where cfos expression is virtually absent. we hypothesize that cfos functions to inhibit the expression of cyp in granulosa cells, thereby suppressing androgen production. hence, the inhibition of cfos activity might result in cyp expression in the granulosa cell. our objective was to define the role of cfos, in the regulation of cyp expression in granulosa cells. transformed human luteinized granulosa (hgl ) cells were utilized for all experiments. hgl cells were cultured in monolayer for h. cells were treated for h with and without pd (pd), a mapkk inhibitor, which also blocks cfos expression. rna was isolated and real time rt-pcr was performed for cyp . cfos rna interference experiments were carried out using rnaifect, cfos smartpool sirna and scrambled sirna for h. rna was isolated and rt-pcr was also performed for cyp . immunochistochemical studies were performed on normal ovaries, staining for cfos and cyp . treatment of hgl cells with the mapkk inhibitor pd for h, resulted in a -fold increase in cyp mrna expression compared to basal conditions. in cfos gene silenced cells, cyp mrna expression also increased by -fold compared to control sirna conditions. immunohistochemical staining for cfos and cyp showed significant staining of cfos in the granulosa cell layer, but absent staining for cyp . conversely, the theca cell layer did not stain for cfos, but staining was evident for cyp . these results suggest that the ap- transcription factor, cfos, may play a role in the inhibition of cyp expression in granulosa cells. this may provide an explanation for the lack of cyp expression in granulosa cells. the g /m stages of the granulosa cell cycle. as clip- has been identified as an mtor substrate, we hypothesized that its function at the mitotic spindle would be positively regulated by mtor during the late g and m phases of the cell cycle in granulosa cells. during periods of stress (e.g., mtor inhibition), mtor would fail to phosphorylate clip- , leading to spindle checkpoint failure and follicle undergrowth. objectives: the expression of clip- and mtor were evaluated. computational analysis of potential clip- phosphorylation sites and comparison with residues on known mtor targets were performed. clip- threonine and serine were chosen and evaluated as bona fide mtor phosphorylation sites. a preliminary assessment of the effects of mtor inhibition upon clip- function was performed. methods: for protein expression analyses, western blots, immunostaining of tissues and primary granulosa cells in culture were performed. computational analysis of potential clip- phosphorylation sites was followed by in vitro assessment of mtor kinase activity upon clip- and a peptide substrate. results: clip- was expressed in the ovarian stroma, blood vessels (including the endothelial cells of both arteries and veins), granulosa cells, and in the oocytes of primordial and growing follicles. overlapping expression was found between clip- , mtor, and the mtor cofactors raptor and rictor in granulosa cells. this expression was conserved between the mouse and the human. evaluation of clip- phosphorylation supported thr as a bona fide mtor target. conclusions: clip- was supported as an mtor substrate protein during granulosa cell mitosis. the mechanism of mtor action during granulosa cell growth and survival is likely to include the phosphorylation of clip- and subsequent positive regulation of mitotic spindle function. the effect of a selective oxytocin antagonist (barusiban) in threatened preterm labour: a randomised, double-blind, placebo-controlled trial. steven thornton, thomas m goodwin, gorm greisen, morten hedegaard, joan-carles arce. warwick medical school, university of warwick, coventry, united kingdom; maternal-fetal medicine, university of southern california, los angeles, usa; dept of neonatology, rigshospitalet, copenhagen, denmark; dept of obstetrics, rigshospitalet, copenhagen, denmark; clinical research development, ferring pharmaceuticals, copenhagen, denmark. objective: a mixed oxytocin/vasopressin v a antagonist, atosiban, has been shown to reduce uterine contractions in placebo-controlled clinical trials and is useful in the management of preterm labour. the objective of this study was to determine the effect of a selective oxytocin antagonist, barusiban, in delaying delivery and reducing uterine contractions in women with threatened preterm labour at a late gestational age and relatively high risk of delivery. methods: this was a randomised, double-blind, placebo-controlled multicentre study in countries. a total of women between + and + weeks gestation, and with uterine contractions of sec duration during min, cervical length mm, and cervical dilatation > and < cm were randomised to receive a single intravenous bolus dose of either barusiban . mg, mg, mg, mg or placebo. rescue tocolytics were prohibited. the primary end-point was percentage of women who did not deliver within h. uterine contractions were monitored by cardiotocography. obstetrical and neonatal outcomes were determined. results: there were no significant differences between the placebo and any barusiban group in percentage of women who did not deliver within h ( % in the placebo group and % to % in the barusiban groups). there was no dose-effect relationship nor an effect at or h. none of the barusiban groups were associated with a significant reduction in number of uterine contractions compared to placebo at any time point up to h post-dosing. postpartum blood loss and time to established lactation were not significantly increased with barusiban. barusiban was well tolerated and was not associated with safety concerns for the women, fetus or neonates. conclusion: a single intravenous bolus of a selective oxytocin antagonist, barusiban (dose range . - mg), did not delay delivery or reduce uterine contractions compared to placebo in women with preterm labour at late gestational age and with short cervical length. the results contrast those of the mixed oxytocin/vasopressin v a antagonist, atosiban. prolonged delivery intervals in triplet gestations. tracy a manuck, heather l mertz, leah passmore, david c merrill. obstetrics and gynecology, wake forest university health sciences, winston-salem, nc, usa. objective: delayed interval delivery is one management strategy for previable preterm labor affecting multiple gestations. prior reports of asynchronous deliveries have examined twins and higher-order multiples as a group. this study was conducted to analyze the unique situation of asynchronous triplet deliveries. study design: cases of asynchronous triplet deliveries resulting in an ongoing twin gestation were ascertained through medline. data were abstracted and combined with two similar previously unpublished cases. patients were grouped by management with and without rescue cerclage. variables compared included use of tocolytics, antibiotic administration, gestational age at delivery of each fetus, interdelivery interval, delivery mode, birthweights, and short and long term outcomes. chi-square or t-test analyses were used where appropriate. results: fifty-one cases of asynchronous triplet deliveries met inclusion criteria and were analyzed. twenty-three patients ( . %) underwent placement of a rescue cerclage following delivery of the first infant. these patients delivered the first fetus at a significantly earlier gestational age as compared to those patients without a cerclage ( . +/- . weeks vs. . +/- . weeks, p= . ). patients with a rescue cerclage had a significantly longer prolongation of the remaining twin gestation ( . +/- . days vs. . +/- . days, p= . ). no significant differences in use of tocolytics or antibiotics, gestational age at delivery of triplets "b" and "c," mode of delivery, short term outcome (alive at hours), or long term outcome (alive at discharge) were noted, despite delivery of triplet "a" at a significantly younger gestational age. conclusion: rescue cerclage, particularly when placed following previable delivery of a first triplet, may significantly prolong the delivery interval for the remaining twin gestation. mean pregnancy prolongation (days). objective the aim of our study was to evaluate the role of amnioinfusion in pregnancies complicated by pprom. we studied singleton pregnancies with pprom at < weeks gestation. all patients were managed conservatively with bed-rest, prophilactic antibiotics, tocolytics and steroids. only patients without vaginal bleeding and/or contractions were included: patients showed an amniotic fluid pocket (afp) persistently cm and did not undergo amnioinfusion (group b) whereas had a maximum afp < cm and were offered amnioinfusion to restore an adequate amount of amniotic fluid (group a). in patients of group a amnioinfusion was successful (afp cm for hours following the procedure: group a ) whilst in it was unsuccessful (afp< for hours: group a ) and repeated. results were analized with the student t test for unpaired samples and with the when appropriate. p values < . were considered significant. results the group where amnioinfusion was not successful (group a ) showed the worst outcome (see table) . there were intrauterine deaths, all in this group. pulmonary hypoplasia was present in / ( . %) newborns (both survived and deceased) newborns, / in group a . no maternal complications were recorded. conclusions our data confirm that a conservative-active management with amnioinfusion can be considered a reasonable option in women with pprom. in our series it was effective in preventing both neonatal death and pulmonary hypoplasia. group a (infusion successful) background. synthetic progestogens are effective in reducing the risk of spontaneous preterm birth in high risk singleton, but not multiple, pregnancy. we hypothesized that myometrial stretch may inhibit the response of human myometrium to progestogens. methods. myometrial strips obtained with written consent at the time of term planned cesarean section were studied using a modification of the method of young and zhang (jsgi ; : - ) . strips were maintained in individual tubes in tissue culture media in an incubator for a period of three days. the effect of prolonged stretch was assessed by comparing strips connected to a . g weight with those connected to a . g weight. the effect of prolonged exposure to progestogen was studied by adding medroxyprogesterone acetate (mpa, nm or nm). following the day incubation, myometrial strips were transferred to an organ bath containing kreb's solution. all were placed under g tension and responses obtained to mm potassium then oxytocin. contractility was expressed as the ratio of the maximum response to potassium or oxytocin to the wet weight of the tissue (units = g.tension per gram), summarized as the mean (sem) and compared using student's paired t test. prolonged stretch increased the maximum response to both potassium ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ) and oxytocin ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days reduced the maximum response to potassium (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ) and there was a trend towards a reduced maximum response to oxytocin (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days had no effect on either the maximum response to potassium . prolonged stretch increases human myometrial contractility in vitro. . prolonged exposure to a progestogen inhibits the contractility of human myometrium but this effect is blocked by prolonged stretch. these properties of human myometrium may explain the failure of oh progesterone caproate to reduce the incidence of spontaneous preterm birth in multiple gestations. sangeeta jain, william l maner, janet l brandon, gary dv hankins, robert e garfield. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine if transabdominal uterine electromyography (emg) correlates with parturition factors such as measurement-to-delivery time (mtdt), cervical dilation (cd), cervical effacement (ce), and station (s) for preterm labor patients with and without tocolysis. materials and methods: pregnant preterm labor women were included. uterine electromyography (emg) was measured for minutes. cd, ce, and s were assessed at or near the time of uterine emg measurement. the power density spectrum peak frequency (pdspf) was calculated on emg. patients were grouped (g : tocolysis, n= ; g : no tocolysis, n= ). pearson-product-moment test was used for correlation. significant differences were sought between groups using student-t test. p< . significant. results: there was a significantly higher uterine emg activity (pdspf: . ± . vs. . ± . ), but no difference in cd ( . ± . vs. . ± . ), for patients delivering within days of emg recording compared to those who delivered later, regardless of tocolysis. there was no apparent difference in uterine emg in tocolytic vs. non-tocolytic patients, regardless of mtdt (table ) . conclusions: uterine emg activity is significantly greater in patients in preterm labor who delivered within days of measurement, making it a viable alternative diagnostic parameter for assessing the state of parturition. tocolytics may not affect uterine emg, but this should be further verified with larger studies. supported by grant nih r -hd . objective: calcium sensitizers are a novel class of drugs with unique molecular and phsiological actions. levosimendan, the best characterized of these compounds and is used in the treatment of acute and chronic heart failure. levosimendan can exert an inotropic effect via sensitization of myofilaments to calcium. it also exerts a relaxant effect on vascular smooth muscle through the opening of atp-dependent potassium channels and has been shown to be a potent inhibitor of human uterine contractions in vitro. for these reasons we investigated the effects of levosimendan on uterine contractions, both spontaneous and agonist induced, in the presence of glibenclamide, a k-atp channel blocker. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to glibenclmide ( mmol) followed by cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. control experiments were performed simultaneously. results: levosimendan exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human m yometrium in vitro, in comparison to control experiments. the effect of levosimendan was significantly antagonized by glibenclamide with the mean maximal inhibition seen due to levosimendan greatly reduced (n= , p< . ). conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on human uterine contractility in vitro. this action was antagonized by glibanclamide and this study demonstrates that the effect of levosimendan on uterine smooth muscle is mediated at least in part through the k-atp channel. introduction: the determination of the beginning and ending points for "bursts" of electrical activity occurring during uterine contractions is sometimes difficult. if bursts cannot be discerned, the preferred burst-by-burst analysis cannot be performed. one solution to is to analyze any given electrical recording in its entirety. but this approach has often lead to meaningless results when traditional analytic methods are applied. chaos analysis, using lyapunov exponents, may provide an answer. materials and methods: term patients were included in the analysis: were in labor (group ), while were non-labor (group ). minute recordings were analyzed using "lyapunov exponent." for each pair of subsequent trajectories in phase space, only the most positive lyapunov exponent was calculated. the mean largest exponent was found by averaging over all of the trajectories in the recording. the lyapunov exponent is given in units of bits per data sample. thus a value of + means that the separation of nearby orbits doubles on the average in the time interval between data samples. the mean largest exponent was found for each patient recording. these values were compared using t-test (p < . considered significant). results: the mean and sd of the lyapunov exponent for all the patients was . ± . . moreover, the lyapunov exponent calculated for each patient was positive. comparing lyapunov exponents of the two groups showed a statistically low value (low chaos) for the laboring group, compared to the non-laboring group (figure) . conclusions: there is a chaotic component associated with uterine emg traces, since small but non-negative lyapunov exponents were found in all the traces observed. the lyapunov exponent indicated significantly lower chaotic behavior in the whole emg traces of patients who were in labor than found in those who were not in labor, implying that this measure could be a good diagnostic parameter for labor, possibly eliminating the need for tedious burst-by-burst analysis. supported by grant nih r -hd . comparing uterine emg to tocodynamometer for monitoring contractions. robert e garfield, lynette b mackay, sangeeta jain, william l maner. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine whether uterine electromyography (emg) plots contractions similarly to tocodynamometer (toco). study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. uterine emg signals were sampled at hz and band-pass filtered in the . to . hz range. root-mean-square (rms) function was calculated from the uterine emg signals in order to produce a "toco-like" trace from the original emg trace. emg-generated rms contraction plots were then compared to toco contraction plots using the following criteria: contractions were assigned a marker value of " ." in-between contraction periods were assigned a " ." from these marker values, contraction rates were established. correlation was found between the contraction rates of rms and toco. temporal overlap of contractions plotted by the two methods was used to find overall percent agreement (opa), positive percent agreement (ppa), and negative percent agreement (npa). these parameters were corrected for within-patient variation using a bootstrap method. results: uterine rms contraction plots were seen to correspond with toco contraction plots (fig. ) . corrected opa, ppa, and npa were high at . %, . %, and . %, respectively. there was a large, statistically significant correlation between uterine emg and toco contraction frequency (fig. ) . conclusions: the similarity between toco and uterine emg contraction plots (specifically, using rms to convert) will allow emg to be used interchangeably with toco in the clinic. supported by grant nih r -hd . introduction -this is a secondary analysis of women participating in a center randomized placebo controlled trial (rct) evaluating the impact of -ohpc in preterm birth (ptb) prevention among women with twins. objective -to evaluate the relationship between plasma -ohpc concentrations and gestational age (ga) at delivery in women with twins receiving weekly injections of -ohpc. methods -women with twins were randomized between - weeks to receive weekly im injections of either -ohpc ( mg) or placebo until weeks. after a minimum of five consecutive injections had been administered to assure steady state concentrations a plasma sample was collected between - weeks. the sample drawn just prior to the next scheduled injection was analyzed for -ohpc by hplc-ms in a blinded manner. the lower limit of quantification of -ohpc was ng/ml. we conducted univariate analyses to assess the association of -ohpc concentration and ga at delivery. we also conducted a proportional hazards model to evaluate the time from sample draw to spontaneous delivery (censoring indicated preterm deliveries), and a logistic regression to evaluate ptb< weeks; in both analyses we adjusted for bmi, race and ga at sample draw. results - women assigned to -ohpc were included; all received all of their scheduled injections. the concentration of -ohpc was significantly higher in women delivering < weeks compared with those women delivering > weeks (p= . , table) . concentration of -ohpc was significantly correlated with ga at delivery (r = - . , p= . ). each unit increase of -ohpc was associated with a % increased odds of delivering < weeks (odds ratio . , % ci, . - . , p= . ) and a % increase in hazard of spontaneous delivery (hazard ratio . , % ci, . - . , p= . ) after adjusting for confounders. gestational age at delivery mean (sd) -ng/ml < weeks (n= ) . ( . ) > weeks (n= ) . ( . ) conclusion plasma -ohpc concentrations after weekly injections were inversely related to ga at delivery in women with twins. since -ohpc induces its own metabolism it is possible that higher concentrations during initial treatment are associated with lower plasma concentrations and reduced efficacy in later pregnancy . clearly more studies are needed. objective: in many non-human species, maternal circulating progesterone levels fall prior to delivery, leading to the theory that in humans progesterone withdrawal occurs on a local and/or functional level. our objective was to characterize maternal and fetal progesterone in human preterm and term labor. methods: women between . and . weeks' gestation (cases) or term controls ( - weeks) with either labor with intact membranes or premature rupture of the membranes prior to labor (prom) were enrolled in a prospective case-control study. progesterone was measured by immulite assay in maternal serum collected upon enrollment and again within minutes after delivery and in umbilical cord serum obtained at delivery. maternal progesterone treatment was not used in any subjects. results: cases and controls were studied (see table for comparisons). controls p value ga at enrollment, weeks . ± . . ± . < . interval to delivery, days (median, range) ( - ) ( - . ) < . maternal progesterone at enrollment, ng/ml ± ± < . maternal progesterone after delivery, ng/ml ± ± . cord progesterone, ng/ml ± ± . among cases, fetal but not maternal progesterone was significantly lower in preterm labor with intact membranes ( ± ng/ml, n = ), as compared to prom ( ± , n = ), p< . . this difference increased further when cases of clinical chorioamnionitis were excluded. conclusions: serum progesterone in laboring patients prior to delivery is higher at term than in the preterm period, which may be attributable to increased placental mass in late pregnancy. this disparity disappears shortly after delivery of the fetus and placenta. fetal progesterone levels are several-fold higher than peripartum maternal levels. preterm labor with intact membranes is associated with diminished fetal progesterone, a phenomenon unrelated to clinical infection. these findings suggest the possibility of fetally regulated progesterone withdrawal as a mechanism underlying preterm labor with intact membranes. [snps] and ptb. however, many of these studies are inconclusive and non reproducible. the challenge of identifying robust associations between genetic variation and either susceptibility or protection from ptb is enormous. a systematic review of literature followed by metaanalysis was performed to understand true associations between snps and ptb. methods: for systematic review, articles were chosen based on medline and embase searches ( ( -april and relevant articles were chosen based on stringent inclusion criteria. primarily, studies reporting genetic associations between snps in maternal dna in singleton pregnancies and spontaneous ptb were included. other criteria included, but not limited to, provided genetic data in a complete enough format so that it could be evaluated in meta-analysis and defined the clinical outcome clearly. meta-analysis was performed wherever > replication data sets were available results: a total of abstracts were reviewed and were selected for full text review. data were extracted from articles. over associations were reported between snps on various candidate genes and ptb; however only had replication dataset. meta-analysis documented significant association between pon a g (odds ratio [or]= . ( %ci . - . ), pon (rs# )(or= . ; ci- . - . ), tnfrsf - a/g (fas) (or= . ; ci- . - . ) and ptb. two snps pon s c (or= . ; ci- . - . ) and ifn gamma (rs ; or- . ; ci- . - . ) documented protective effect. conclusions: systematic review concludes significant heterogeneities leading to lack of reproducible data in genetic association studies of ptb. heterogeneities are contributed predominantly by lack of adequate power, poor phenotype selection, and population admixture. the functional relevance of the risk and protective alleles needs to be verified. jignesh parvadia, mounira habli, jeff livingstone, william polzin, foong lim, timothy crombleholme. pediatric and thoracic surgery, cincinnati children's hospital medical center, cincinnati, oh, usa; obstetric and gynecology, university of cincinnati, cincinnati, oh, usa; obstetric and gynecology, good samaritan hospital, cincinnati, oh, usa. objective little is known about the response of ttts to treatment either by amnioreduction or selective fetospopic laser in triplet pregnancy, particularly the survival of the bystander fetuses. in order to define the response of triplet pregnancies to treatment for ttts we reviewed our experience with higher order multifetal gestations complicated by ttts. study design retrospective chart review of patients diagnosed with in high order gestation from - was performed. results among cases of ttts / ( . %) patients with high order gestations were identified (n= ) with a mean ga at diagnosis of . ± . weeks. pregnancies ( . %) were dichorionic triamniotic and ( . %) were monochorionic triamniotic. cincinnati modification of quinterro staging was utilized to characterize recipient cardiomyopathy as mild (stage iiia, n= ), moderate (stage iiib, n= ) and severe (stage iiic or iv, n= ) categories. / ( . %) were treated with amnioreduction alone (ar), / ( . %) with selective fetoscopic laser photocoagulation (sflp) alone, / ( . %) with ar followed by sflp and / ( . %) with ar followed by intrafetal radio frequency ablation (rfa). / ( . %) patient had a cervical cerclage. / ( . %) patients were treated but remain undelivered. mean ga at delivery was . ± . weeks. overall survival was / ( . %) with bystander survival was / ( %), donor survival / ( . %), recipient survival was / ( . %). conclusion triplet pregnancies treated for ttts have % survival rate for bystander fetuses and have . % survival rates for donor and recipients comparable to twins treated for ttts. ga at diagnosis . ± . weeks cincinnati modification of quinterro (i), (ii), (iii), (iiia), (iiib), (iv) ga at delivery . ± . weeks live birth -donor / ( . %)* # -recipient / ( . %)*# -bystander / ( %) # birth weight -donor to assess the effect of breast feeding (bf) on perinatal outcome in relation to maternal antenatal methadone dose. study design a retrospective chart review study of methadone dependent mother and infant pairs. patients were categorized into groups based on maternal dose at time of delivery: group : dose mg, group : dose - mg, group : dose > mg. the finnegan's scoring system was used to monitor neonatal abstinence syndrome(nas). treatment for nas was initiated if there were scores of . neonatal outcome data included:% nicu admission, % of babies discharged(d/c) at time of maternal d/c, % nas, % treated for nas and total hospital stay. data were analyzed by t-test and fisher's exact test. maternal characteristics between the groups were similar. regardless of maternal methadone dose, bf infants have shorter hospital stays and higher rates of d/c at time of maternal d/c, lower incidence of nas and fewer nicu admission (table) . in all three groups, breast feeding did not impact the severity of nas as reflected in finnegan's score(fs). (table) conclusion regardless of maternal methadone dose, breast feeding improves perinatal objective: to evaluate the effects of preventive collagen plugging of the fetoscopic access port at the time of balloon removal on pregnancy outcome in fetoscopic endoluminal tracheal occlusion pregnancies. study design: fifty-one pregnancies involving fetuses with severe congenital diaphragmatic hernia (cdh) were studied. all patients underwent feto between - weeks gestational age (ga) and fetoscopic balloon removal around weeks ga. at the time of balloon removal, a purified dried collagen plug was inserted through the fetoscopic access port in consecutive pregnancies but not in the first pregnancies considered as controls. all patients underwent post-plugging ultrasound and magnetic resonance imaging studies to evaluate for membrane dehiscence, amniotic fluid volume and fetal well being. ga at delivery, incidence of premature rupture of the membranes (pprom), bleeding at port retrieval and adverse fetal effects were compared in both groups. results: mean (sd) ga at feto [ . ( . ) vs. . ( . ) weeks; p= ns] and balloon removal [ . ( . ) vs. . ( . ) weeks; p= ns] was similar in the treatment and in the control group. incidence of pprom following the second fetoscopy was / in the study group compared to / in the control group (p< . ). mean (sd) ga at delivery was . ( . ) weeks in the study group, compared to . ( . ) in the control group (p= . ). bleeding from the trocar insertion site occurred in cases in both groups, but clinically significant bleeding occurred only in one of the controls. membrane dehiscence was noted in patient in the treatment group compared to in the control group (p=ns). conclusion: preventive collagen plugging of the fetal membrane defect created by the fetoscopic access resulted in a significant reduction in pprom rates and a trend towards increased ga at birth without adverse fetal effects in feto pregnancies. wider application of this technique should be considered, but needs evaluation in larger, randomized trials. hydrops fetalis is an uncommon fetal condition characterised by the abnormal accumulation of fluid in two or more body cavities, traditionally associated with a poor prognosis. the relative rarity of this presentation has meant that published case series have consisted of small numbers. a retrospective review of case notes of all cases managed at kemh between and was performed. in western australia, kemh is the only tertiary maternity hospital incorporating a maternal-fetal medicine unit. cases were obtained from the mfm database. in the period to there was a total of pregnancies affected by hydrops (incidence . per births). the average maternal age was years. in cases a fetal abnormality had occurred in a previous pregnancy. the median gestational age at diagnosis was weeks (range - weeks). in just over half ( %) of cases, the diagnosis was confirmed prior to delivery. a post-mortem was performed on all but of the babies not born alive. edema was present in at least cavities in over half of cases (n= ). chromosomal anomalies included trisomy , trisomy and turners syndrome. in all cases of infection, parvovirus b was implicated. cardiac arrythmias included svt and atrial flutter. cases classified as other included alpha thalassemia and syndromic disorders. in cases an interruption of pregnancy was performed at a mean gestational age of weeks. of those who did not elect to terminate the pregnancy, there were fetal deaths in utero, live borns with neonatal death. for the live borns, the median gestational at delivery was . weeks (range to . weeks). the causes of hydrops in live birth cases included cardiac arrythmia (n= ), infection (n= ), chromosomal abnormality (n= ), unknown (n= ) and other (n= (dmv) have been noted to play a role in the development of hemorrhagic and periventricular leukomalacic lesions in premature babies. since deep vein drainage system is relatively more prominent in the developmental brain than adult brain, we investigated if dmv anomalies could be associated with clastic lesions in-utero. methods two senior neuroradiologists reviewed fetal brain exam performed between and , seeking for unequivocal anomalies in dmv, such as periventricular venular engorgement. all mr scanning is performed at . tesla, using surface abdominal coils and single-shot fast spin-echo t -weighted - mm thick sections, with . - . mm in planar spatial resolution. we found cases with dmv anomalies (tab. ). most of the dmv engorgement is located at frontal lobes level. from this limited preliminary series it appears that dmv involvement plays a role in the development of periventricular leukomalacia and periventricular hemorrhagic necrosis. the observation that these lesions are mostly located at frontal level may suggest that some of the term neonates carrying sequelae of atypically located leukomalacia (i.e. deep frontal lobe) might have developed these lesions in-utero. it is of interest to notice that most of our cases were related to heart failure. therefore, central venous hypertension affecting immature deep cerebral venous system has to be taken into account. in our center, patients with an estimated risk for chromosomal abnormalities at term greater that in after st trimester combined screening test (ft) are offered non-directive genetic counseling. the aim of this study was to evaluate the responses of women younger than attending this genetic counseling session. material and methods: data from patients referred for a positive ft from september , to july , was retrieved from our database. information concerning women younger than years of age at the estimated date of delivery was extracted and tabulated. results: during the study period women had genetic counseling for positive ft. thirteen patients were excluded from further analysis ( had incomplete clinical documentation and had spontaneous miscarriages prior to weeks gestation). four hundred and twenty-five patients were older than and were younger than at the estimated date of delivery. table depicts summary statistics for studied variables in this younger group of women. conclusions: overall this data suggests that approximately % of this younger group of women opted for chorionic villous sampling (cvs), % for amniocentesis and more than % declined prenatal genetic testing. moreover, this data also suggests that: ) these women opted for cvs when the ft risk (mean = in ) almost doubled the cvs procedure related risk quoted at % and, ) when the ft risk is between in and in almost half ( out of ) declined not only the st trimester cvs but also the nd trimester amniocentesis. we believe that understanding our patient population is important to optimize both the efficiency and efficacy of the alternative prenatal screening programs. acceptance for prenatal genetic testing after a positive first trimester combined screening test in women of less than to determine the optimal diagnostic test using prenatal ultrasonography and mri for predicting pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. methods: relevant papers were identified by searching medline ( medline ( - , embase ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the cochrane library ( issue ). in addition, the specialist literature on the topic and reference lists were hand searched for relevant articles. studies were selected if they examined diagnostic tests for the prenatal prediction of pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. the primary outcome measure was perinatal survival. study selection, quality assessment and data abstraction were performed independently and in duplicate by separate observers. results: of a total number of articles (published studies), there were eighteen studies that fulfilled the entry criteria. six examined entirely unique heterogeneous parameters. of the remainder, all examined the ultrasound measurement of lung to head ratios (lhr) at a 'cut-off of greater than or less than the thresholds of . , . , . , . . in addition, the presence of liver in the fetal thorax was included (if present) as a co-variable (liver "up"). a lhr > . compared to < . provided the strongest association with perinatal survival (peto or . , % ci . - . ). the finding of "liver up"in the fetal thorax had a negative association with survival (peto or . , % ci . - . ). only three studies provided data for lhr in conjunction with the presence of liver in the fetal thorax (peto or survival for lhr > . compared to < . was or . , % ci . - . ). data was also available for liver up and lhr > . which had a peto or of . ( % ci . - . ). discussion: the data supports the view that lhr may be a useful prognostic indicator of perinatal survival in fetuses with congenital diaphragmatic hernia. however, heterogeneity still exists regarding the timing of ultrasound measurement and the use of mri. the majority of studies have small sample sizes. objective: to estimate, in fetal anaemia, the diagnostic value of fetal ultrasonography and doppler blood flow in the evaluation of fetal anaemia methods: literature from to was identified using medline and embase, the cochrane library and relevant specialist register of the cochrane collaboration, and by checking reference lists of known primary studies and review articles. studies were selected if the accuracy of the fetal ultrasound parameters or doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard. diagnostic tests evaluated were ultrasound measurement of the fetal spleen and liver length and doppler studies from the umbilical vein and middle cerebral artery. data from the selected studies were abstracted as x tables comparing the diagnostic test result with the reference standard. results were pooled where appropriate. diagnostic accuracy was expressed as sensitivity and specificity. twenty-nine primary studies were identified containing suitable data. twentyone of these gave data on middle cerebral artery doppler peak systolic velocity (mca-psv) and could be pooled in the meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases in detecting severe anaemia. four studies gave data on spleen perimeter and it was possible to pool three of these giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases. three studies had data for liver length measurements and two were pooled. the sensitivity was . ( . - . ) and the specificity was . ( . - . ) but only cases were used in the analysis. there were three studies on umbilical vein maximum velocity doppler studies and all were suitable for meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) with cases analysed. two studies gave data on middle cerebral artery time-averaged mean velocity score giving cases. the sensitivity was . ( . - . ) and specificity was . ( . - . ). discussion: middle cerebral artery peak systolic velocity dopplers remain the gold standard for non-invasive screening of fetal anaemia. middle cerebral artery time-averaged mean velocity scores require further investigation. other tests perform poorly when diagnostic accuracy is assessed. cochrane review of treatment in twin to twin transfusion syndrome. twin-twin transfusion syndrome is a condition affecting monochorionic twin pregnancies associated with a high risk of perinatal mortality and morbidity. the objective of this review was to evaluate the impact (maternal,fetal and pediatric)of treatment modalities in twin-twin transfusion syndrome. register and cochrane controlled trials register. we also searched conference proceedings and made personal contact with experts active in the area of the review. randomised and quasi-randomised studies of amnioreduction versus laser coagulation, septostomy versus laser coagulation or septostomy versus amnioreduction. eligibility was assessed by one reviewer. study authors were contacted for additional information. two studies were included. this review shows that laser coagulation of anastomotic vessels results in less fetal (rr . ; % ci . , . ) and neonatal deaths (rr . ; % ci . , . ) than amnioreduction ( figure ). there is no difference in perinatal outcome between amnioreduction and septostomy. more babies in the laser arm are alive without neurological abnormality at six months of age (developmental delay at < years rr . , % ci . , . )( figure ). conclusions: endoscopic laser coagulation of anastomotic vessels should be considered in the treatment of all stages of twin twin transfusion syndrome to improve perinatal outcome. further research on the effect of treatment on milder forms of twin twin transfusion syndrome (quintero stage and ) are required. (produced in collaration with the cochrane collaboration, uk). (cvs) concluded that although the risks of pregnancy loss are relatively low, lack of adequate controls tends to underestimate the true added risk of prenatal invasive procedures (obstet gynecol. ; ( ): - ). the west midlands is a large region within the uk containing approximately % of the total uk population. the congenital anomaly register for this region is able to monitor pregnancy outcomes with accurate deminator data. results: there were first trimester cvs performed, by ten operators, in the west midlands (uk) in . this equates to . procedures per births. significantly higher rates were noted in areas of high socioeconomic status. the median number of procedures performed per operator was (range - ). all operators were performing other invasive tests such as amniocentesis or cordocentesis,etc. the most common indication for cvs was: i) fetal anomaly on dating scan ( . %) ii) abnormal (> in ) risk on combined first trimester screening ( . %) iii) molecular genetic diagnosis ( . %) and iv) maternal request ( . %). using a combination of first trimester scanning and cvs, % had abnormal karyotype/structural anomaly.the corrected loss rate (background and procedure-related) following cvs in normally formed, singleton pregnancies was . % ( thci . - . %) up to days postnatal (perinatal loss) and . % ( thci . - . %) with days of procedure. the proportion of cvs in which an adequate sample was not obtained was . % ( th ci . - . %). conclusions: this epidemiological study using accurate demoninator data provide interesting statistics relating to the uptake and prenatal risks of first trimester cvs. with the increasing prevalence of obesity in the last two decades, we have seen a tremendous increase in bariatric procedures in reproductive aged women. malnourishment and vitamin deficiency are common complications after gastric bypass which may impact on fetal development. we present the case of a yo who underwent a duodenal switch procedure in . she was discovered to be pregnant during evaluation for persistent malnutrition in . multiple prenatal ultrasounds were performed; the first at weeks gestation was unremarkable. the week sono revealed a male fetus with shortened femurs and humeri bilaterally, nasal bridge hypoplasia, macroglossia, poorly defined hands, and possible clubbed feet. amniocentesis revealed a normal karyotype. d ultrasound redemonstrated the abnormal facial findings. a fetal echocardiogram was normal. the lagging long bone measurements continued to worsen, ultimately with femurs weeks behind. the fetal thoracic circumference was two standard deviations below the mean, giving rise to concern for pulmonary sequelae. growth restriction was noted at the wk sonogram. delivery by cesarean section was at / weeks secondary to nonreasuring fetal status. the birthweight was gm; apgars were , , and . postnatal radiographs confirmed antenatal ultrasonographic findings and demonstrated evidence of epiphyseal stippling. the infant remained intubated until weeks of life, after which it died secondary to respiratory complications associated with pulmonary hypoplasia. gene mapping studies have not found any point mutations on the recessive gene as an etiology of this disorder. rhizomelic chondrodysplasia punctata refers to a heterogeneous group of bone dysplasias with a familiar radiographic phenotype involving punctate calcifications and epiphyseal stippling. the etiology of this may be secondary to chromosomal abnormalities, mendelian gene disorders, or teratogens, notably warfarin. this case may be explained by vitamin k deficiency of the embryo due to maternal malabsorption after bariatric surgery. the maternal vit k level was <. ng/dl at time of delivery(normal > . ). the teratogenic effects of vitamin k deficiency in this instance highlight the need for strict counseling and screening for vitamin deficiency in those women undergoing bariatric surgery since previous obesity-related anovulation is reversed as patients lose weight, resulting in unexpected pregnancies and potentially preventable fetal abnormalities. term preeclampsia: any risk for the neonate? sindhu k srinivas, jamie bastek, christina m andrela, emmanuelle pare, michal a elovitz. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: preeclampsia continues to be a major contributor to maternal morbidity and mortality worldwide. preeclampsia at term is not associated with the same risk of neonatal morbidty and mortality as preterm preeclampsia . however, neonatal outcomes in term women with preeclampsia have not been adequately studied. we sought to compare short term neonatal outcomes in term infants born to women with and without preeclampsia. methods: this study was part of a large case control study. women with preeclampsia (n= ) and term controls (n= ) were prospectively identified. infants with congenital anomalies were excluded. hospital length of stay (los), admission or transfer to the nicu, and use of mechanical ventilation or cpap within first week of life were assessed. associations between neonatal outcomes and preeclampsia were evaluated using chi-square analysis and wilcoxon rank sum test. significant confounders were controlled for using multivariable logistic regression. results: discharge day of life was significantly different between neonates born to women with preeclampsia (median = ; mean = . ) versus those born to women with uncomplicated term deliveries (median = ; mean = . , p< . ). this difference persisted even when neonates with iugr and those born to diabetic mothers were excluded (p< . ). term infants born to women with preeclampsia have a higher odds of being admitted or transferred to the nicu (aor= . [ . - . ], p= . ) after controlling for iugr, delivery mode, race, and gestational age at delivery. these infants also have a higher odds of mechanical ventilation (aor= [ . - . ], p= . ) and cpap use (aor= . [ . - . ], p= . ) after controlling for the same confounders. there was no difference in ivh or nec between the two groups. conclusion: neonates born to women with preeclampsia have differences in short-term morbidity when compared to neonates born to women without preeclampsia, despite being born at term. whether this increase in neonatal morbidity is attributable to medications used in preeclampsia, such as magnesium sulfate, is unclear. these findings may have implications for patient counseling as well as hospital resource allocation. further investigation correlating these findings with long-term morbidity is warranted. could confined placental mosaicism account for adverse perinatal outcomes in ivf pregnancies? benoit c jacod, gh schuring-blom, kd lichtenbelt, jse laven, d van opstal, mjc eijkemans, nick s macklon. reproductive medicine gynaecology, university medical centre, utrecht, netherlands; clinical genetics, university medical centre, utrecht, netherlands; obstetrics gynaecology, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background: ivf singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. this may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. aim: to compare the incidence of confined placental mosaicism (cpm) in ivf/icsi pregnancies and spontaneous conceptions. methods: multicentre retrospective analysis of karyotype results obtained by chorionic villus sampling (cvs) performed because of advanced maternal age ( years at weeks of gestation) in the netherlands between and . results: from a total of pregnancies, cvs results were analysed: in the ivf/icsi group and in the control group. the mean age of women in both groups was . years (mean difference - . , % ci - . - . ). foetal karyotype was missing in cases of possible cpm, all in the control group. when taking into account missing data, the incidence of cpm was lower in the ivf-icsi group than in the control group, . % vs. . % (odds ratio . , % ci . - . ) whereas the incidence of foetal chromosomal anomalies was increased . % vs. . % (odds ratio . , % ci . - . ) although both differences are not significant. conclusions: the incidence of confined placental mosaicism is not increased in ivf/icsi pregnancies compared to spontaneous conceptions. cpm probably does not account for the adverse perinatal outcomes following ivf/icsi. fetal rh d, cc and ee genotyping using fetal dna from maternal blood is not impaired by the presence of maternal alloimmunization. chad a grotegut, stacey l jeronis, john p gaughan, enrique hernandez, ossie geifman-holtzman. obstetrics and gynecology, duke university, durham, nc, usa; obstetrics, gynecology and reproductive sciences, temple university, philadelphia, pa, usa; biostatistics, temple university, philadelphia, pa, usa. this study was conducted to assess the impact of maternal alloimmunization on the accuracy of fetal rh d, cc and ee genotyping from maternal blood. we performed a literature search of english-written articles describing fetal rh d, cc and ee determination from maternal blood (am j obstet gynecol ; : - ) . using this database, we determined the accuracy of rh d, cc and ee genotyping in the presence of maternal alloimmunization. for each subgroup, % confidence intervals for a proportion were calculated and compared between groups. we found english-written publications reporting non-invasive rhd genotyping and reporting non-invasive rh ce genotyping from maternal blood. fourteen ( . %) of the rh d articles and three ( %) of the rh ce articles provided accuracy results in the setting of alloimmunization. the accuracy results are reported as follows: . % of the samples were determined correctly in the presence of alloimmunization.* this accuracy was significantly lower than the accuracy reported for all rh cc samples. when only studies utilizing free fetal dna for rh cc genotyping were used (vs fetal cells), fetal cc genotype was determined correctly in / ( %, % ci . , ), which was similar to the overall success rate for rh cc determination. overall, there were no differences in the success of rh d, cc or ee determination in the setting of alloimmunization compared to the overall accuracy seen when free fetal dna was used. the presence of maternal alloimmunization does not reduce the accuracy of fetal rh d, cc or ee non-invasive genotyping from maternal blood utilizing free fetal dna. further research into structure and rearrangements of the rh d, cc and ee genes may further improve diagnostic accuracy of free fetal dna from maternal blood. fetal dna, most likely of trophoblast origin, is present in both the plasma of pregnant women and provides a potential basis for non-invasive fetal diagnostic tests. however, fetal dna in maternal plasma is highly contaminated with maternal dna, and this contamination is the main technical challenge in trying to accomplish non-invasive detection of fetal chromosome abnormalities. existing methods for the selective amplification of fetal dna have generally relied on specific sequence differences between the mother and fetus. as an alternative, we have developed a method for selective amplification of fetal dna that makes use of observation that trophoblast dna is globally hypomethylated in comparison with dna from other sources. in this method, a dna mixture is first digested with a methylation sensitive restriction enzyme and then amplified by linker-mediated pcr. after an initial amplification, a second isothermal rolling-circle amplification is performed. this procedure results in the differential amplification of short, relatively hypomethylated fragments. after amplification, the resulting "representations" are comparatively hybridized to a microarray consisting of oligonucleotides that correspond to restriction fragments generated by the initial digest. copy number differences are then detected through statistical analysis of array addresses that show significant amplification. to test the feasibility of this method for detecting aneuploidy, we have prepared mixtures of peripheral blood dna and first trimester trophoblast dna from either normal or from samples with trisomy and trisomy . we present data showing that aneuploidy can be detected even when % of the starting dna sample was derived from a euploid source and only % was from an aneuploid trophoblast sample. two different approaches to data analysis are presented. one relies on prior analyses of trophoblast methylation and the second is independent of any prior knowledge or analysis. both methods provide similar ability to detect aneuploidy. future work will focus on testing whether this approach can be used for non-invasive prenatal diagnosis. chromatin immunoprecipitation and emsa analysis of nf-b and c/ ebp synergism on the otr promoter. shirin khanjani, yun s lee, vasso terzidou, mark r johnson, phillip r bennett. institute of reproductive developmental biology, imperial college, london, united kingdom. we have shown, the transient transfections of the transcription factors nf-bp and c/ebp leads to a synergistic increase in otr promoter activity in human myocytes. this effect is mediated through a bp region of the promoter between - to - from tss. we now report that this sequence binds both nf-bp and c/ebp in vitro and chip studies show binding of both transcription factors to be increased by il- in vivo. materials and methods: emsa studies were performed using a bp oligonocleotide sequence (- to - ) , containing the bp region responsible for the synergistic activation of the otr promoter and nuclear extracts from primary human myocytes treated with il- ng/ml for hours. for chip analysis, dna protein complexes were crosslinked and antibodies recognizing p , c/ebp and h (positive control) and igg (negative control) were used for immunoprecipitation. primers were designed to amplify the region - to - , which includes the bp response sequence. to further confirm the interaction between nf-bp with c/ebp a xnf-b consensus/luc reporter construct was cotransfected with an expression vector for either nf-bp or c/ebp . results: specific nf-bp and c/ebp binding was seen in the emsa study. preincubation with antibodies to nf-bp and c/ebp led, not to supershift, but to elimination of dna binding for both nf-kb p and c/ebp . chip analysis confirmed increased in vivo binding of nf-b p and c/ebp to this region of the otr promoter following stimulation with il- . transfection of the nf-b/luc reporter construct with an expression vector for c/ebp caused a significant reduction in the basal promoter activity suggesting that c/ebp is binding to nf-kb. this interaction was further confirmed using a tf-tf interaction array. conclusion: these data support the role of nf-b and c/ebp in regulation of otr. the bp region contains a c/ebp but not a nf-b dna binding site suggesting that c/ebp primarily binds to this part of the otr promoter but also interacts with nf-b. the emsa data shows that the bp region binds both nf-b and c/ebp . the loss of the supershift observed in previous emsa studies suggests that the antibodies inhibit the interaction between c/ebp and nf-b, therefore inhibiting dna binding. chip analysis supports the concept that il- leads to binding of nf-b and c/ebp to the bp region. regulation of pro-labour genes by c/ebp, nf-b and ap- in human uterine myocytes. suren r sooranna, shirin khanjani, yun s lee, phillip r bennett, mark r johnson. imperial college parturition research group, imperial college, london, united kingdom; irdb, imperial college, london. introduction: the transcription factors c/ebp (lap), nf-b (p ) and ap- (c-fos and c-jun) are implicated in inflammatory processes such as parturition. the promoter regions of the pro-labour genes il- , pghs- and otr contain putative transcription factor-binding sites for these transcription factors. our aim was to determine the effect of transfecting these transcription factors either alone or in combination into uterine myocytes and to determine their effects on the expression of pro-labour genes including il- , pghs- , otr, connexin- and fp. methods: primary cultures of human uterine myocytes (n= ) were grown from myometrial samples obtained at the time of elective lscs. cells were cultured in well plates to % confluence at which point expression constructs for c/ ebp (lap), nf-b(p ), ap- (c-fos and c-jun) were transfected either alone or in different combinations. the empty expression vector psg was used as a filler construct. cells were cultured for and h after which culture medium was collected for elisa and cells frozen at - °c prior to rna extraction. copy numbers of il- , pghs- , otr, fp, connexin- and gapdh were measured by qpcr using a rotor-genetm (corbett research). results: h post transfection with c/ebp (lap), nf-b (p ), c-fos and c-jun alone or in combination showed no significant changes in pghs - , otr and connexin- expression. over expression of p alone or together with either c-fos or c-jun increased fp expression by , and % respectively (p= . ). nf-bp consistently increased il- expression either alone (by %; p= . ) or in combination with lap, c-fos or c-jun (by , and % respectively; n= ; p= , ). rel a, lap, c-fos and c-jun together also increased il- expression (by %; p= . ) and a small but significant increase was seen with a combination c-fos and c-jun (by %; p= . ). the changes observed in il- expression were reflected in the medium il- concentration at h post transfection. in the presence of rela and c-fos il- increased from . ± . to . ± . pg/ml of culture medium (mean ± sem; n= ). conclusions: nf-b (p ) consistently increased fp and il- expression in human myometrium. the data suggest that pghs- activation has greater dependence upon other transcription factor(s) in addition to p . true identity of myometrial pr-c: fact or fiction? yun s lee, suren r soorana, mark r johnson, jan brosens, phillip r bennett. imperial college parturition research group, hammersmith campus, london, united kingdom. progesterone is thought to be central to maintenance of pregnancy. multiple progesterone receptor (pr) isoforms underlie complex and diverse biological action of progestins. previously two human pr isoforms have been identified: pr-b and pr-a. pr-a is n-terminally truncated form of pr-b (initiation site methionine ). in some cells pr-a inhibits pr-b. it has been proposed that increased expression of pr-a in myometrium underlies a 'functional progesterone withdrawal'. the breast cancer cell t d contains a kda progestin-specific binding protein that is not found in pr negative cells. it was proposed that there is a downstream methionine (met ) which serves as a translation initiation site for the generation of a pr isoform of kda. based on such findings condon et al (mol endo ) have focused on the possible role of kda pr-c in human parturition. they found that expression of "pr-c" using pr antibody (sc- santa cruz, sc) is increased in upper segment myometrium with labour and that overexpression of this protein inhibits pr-b function. we cloned the same human pr cdna into psg expression vector. in vivo translation produced a protein of only kda. furthermore overexpression of pr-c did not significantly decrease pr-b activity in human myocytes. we examined other downstream initiation sites, which may produce a kda protein. we constructed potential pr isoforms in psg vector with initiation sites at met and . in vivo translation produced proteins of approximately and kda respectively. to determine the effect of pr isoforms on pr-b function, myocytes were co-transfected with pr-a, pr-c , pr-c and pr-c with constant amounts of pr-b. the progesterone dependent pre/luc was used as reporter. unlike pr-c both pr-c and pr-c significantly inhibited ligand dependent pr-b mediated transcriptional activity. we found in western analysis that the antibodies pgr- (nc) and the sc- (sc) detected both endogenous and overexpressed pr-a and pr-b but none of the pr-c isoforms. the sc- antibody detected only pr-a and pr-b very poorly. our data suggests that the sc- antibody would not detect any pr-c protein and that none of the commercially available antibodies in the uk do so. great care needs to be taken when over-expressing pr isoforms to ensure that proteins are of the expected size. if pr-c does exist in vivo then the kda but not the kda isoform might inhibit pr-b function. tnf receptor antibody (tnf ri ab), nf-b inhibitor (nf-b activation inhibitor) and erk inhibitor (u ) (p< . ), but not by tnf receptor antibody (tnf rii ab), p mapk inhibitor (sb ) and jnk inhibitor (sp ). by western blot analysis, we found that the protein level of tnf receptor associate factor (traf ) was higher than that of tnf receptor associate factor (traf ) (traf >traf ) in untreated ct cells. however, after tnf treatment for h to h, traf protein level was increased, but traf protein level was reduced (traf >traf ). the increase of traf and decrease of traf were blocked by tnf ri ab, but not by tnf rii ab. we also found that tnf rapidly (within - min) and significantly increased phosphorylation of ikk , erk / and jnk / / and the phosphorylation of these protein kinases by tnf was reduced significantly by tnf ri ab, but not by tnf rii ab. moreover, we found that the changes of increased traf and decreased traf in ct cells (traf >traf ) resulted in a dramatic deficiency in phosphorylation of the above protein kinases induced by tnf compared with the normal ct cells (traf >traf ). nuclear localization of nf-b p in tnf treated cells was increased compared to untreated controls. conclusion: we have demonstrated for the first time that tnf stimulates mmp- production in ct cells through tnf ri-trafs-ikk /erk-nf-b signaling pathways, but not through the jnk/p -ap- pathway. these studies reveal steps within this pathway as possible therapeutic targets to inhibit mmp- expression potentially attenuating tnf -induced degradation of extracellular matrix and pre-term rupture of the fetal membranes. objective: women with preterm birth are at elevated risk of cardiovascular disease, but mechanisms that might relate these conditions are not understood. we hypothesized that women with spontaneous preterm vs. term births may have early gestation evidence of activation of the fibrinolytic cascade, as measured by the thrombin-antithrombin iii (tat) complex. we also tested if this relation may be associated with inflammation. methods: tat was measured in plasma collected < weeks gestation (mean . weeks, sd . ) among women without chronic medical conditions, preeclampsia or growth restriction who delivered singleton liveborn infants. inflammation was assessed by c-reactive protein (crp) measured in serum from the same samples. women with spontaneous preterm birth (sptb) < weeks (n= ) and -< weeks (n= ) were compared to women with term births >= weeks (n= ). high tat was defined as > . ng/ml (highest quartile among women with term births) and high crp was defined as >= ug/ml. multinomial logistic regression was utilized to relate elevated tat and inflammation to risk of sptb subtypes. results: women with sptb were more likely to have tat concentrations in the highest quartile compared to women with term births (< weeks, . %; -< weeks, . %; >= weeks . %, p< . ). women with high tat concentrations had a . -fold ( % ci: . - . ) increased risk for sptb < weeks, after adjustment for body mass index, race, age and gestational age at sampling. there was no relation between high tat and sptb -< weeks (or . , % ci . - . ). additional adjustment for elevated crp (>= ug/ ml) did not effect the estimates associated with tat, and elevated crp was independently related to risk for both sptb subtypes (< weeks, or . [ . - . ]; -< weeks, or . [ . - . ]). thus, women with high tat and elevated crp appeared to be at particularly elevated risk of sptb < weeks (or . , % ci . - . ). conclusions: the thrombin-antithrombin iii complex was elevated early in gestation among women with sptb < weeks, perhaps secondary to microvascular injury. this effect was independent of inflammation, suggesting that the elevated fibrinolytic cascade may function independently from inflammation among women with sptb. plasma cortico-releasing hormone and cortisol concentrations and psychological stress among pregnant women. katherine p himes, hyagriv n simhan. obstetrics and gynecology, university of pittsburgh medical center, magee womens hospital, pittsburgh, pa, usa. objective: many studies have found an association between psychological stress and preterm birth. we sought to determine if women with greater psychological stress during pregnancy had higher concentrations of plasma cortico-releasing hormone (crh) or cortisol. study design: this is a secondary analysis of a multicenter case-control study, nested within an observational cohort. of , participants, plasma crh and cortisol concentrations at and weeks gestation were available in controls who delivered after weeks and cases who delivered before weeks. the abbreviated scale for the assessment of psychosocial status in pregnancy (asaps) was available for all women. concentrations of crh and cortisol were compared between women above and below the lowest quartile score on the asaps among cases and controls. the same analysis was done for the portion of the scale related to psychological stress. concentrations of crh and cortisol and psychological stress were also compared between black and non-black cases and controls. univariate analysis was performed with kruskal wallis or chi-square. results: there was no difference in crh or cortisol concentrations at or weeks among women above or below the lowest quartile on the asaps (controls:p= . - . cases:p= . - . ). greater psychological stress was not associated with higher concentrations of crh or cortisol at or weeks(controls:p= . - . cases:p= . - . ). crh concentrations were not different between blacks and non-blacks. among both cases and controls, cortisol concentrations at and weeks were lower in black women than non-black women (controls:p< . cases:p< . ). the median cortisol concentration among control black women was . g/ml at weeks compared to . g/ml among non-black women and . g/ml compared to . g/ml at weeks. black women reported less psychological stress than non-black women (p= . ) conclusion: we found no relationship between psychological stress and plasma crh or cortisol. furthermore, while stress is hypothesized to play a role in the racial disparity of preterm birth, black women reported less psychological stress and had lower cortisol concentrations than non-black women. improved assessments of psychological stress and additional biomarkers involved in the stress response may broaden our understanding of how stress contributes to preterm birth. expression, tissular traffic and activation of mmp- in human fetal membranes during labor. rodrigo vega-sanchez, arturo flores, marisol castillo, nardhy gomez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico. introduction. rupture of the fetal membranes (fm) during human labor occurs as a consequence of extracellular matrix degradation. this process is controlled by increased secretion and activity of matrix metalloproteinases, particularly mmp- .several evidences suggest that mmp- is mainly produced by infiltrating choriodecidual leukocytes that could arrive from placental circulation. characterization of the synthesis, transport and activation of mmp- within the fm is critical to understand the process of membrane rupture during human labor. objectives. expression and secretion of mmp- in placental leukocytes, trafficking of the enzyme through the fm and one possible mechanism for its activation were analyzed. methods. leukocytes were isolated from placental blood of women after active labor. maternal leukocytes were used as controls. cells were cultured for h. relative expression of mmp- by rt-pcr and enzyme secretion by elisa and zymography were followed at different times. to analyze the traffic of mmp- through the fm, fluorescein-conjugated prommp- was added to the choriodecidual side of the fm in an in vitro system that allows the separation of amnion and chorion. labeled mmp- was localized at distinct times by confocal microscopy. the protease responsible for the activation of mmp- was identified using neutralizing antibodies and specific inhibitors. results. no difference in the relative expression of mmp- in leukocytes throughout the culture was found. however, secretion of the enzyme significantly increased since h (p< . ). experiments using labeled mmp- , repeatedly showed that after h in culture, the enzyme was mainly localized within the amniotic epithelium. specific inhibition of mmp- significantly decreased the activation of pro-mmp- . conclusions. our results demonstrate that the increased secretion of mmp- by placental leukocytes is not associated to increased gene expression, suggesting that homing of a specific leukocyte subpopulation to the choriodecidua is occurring during labor. mmp- can be trafficked from the choriodecidua to the amnion, suggesting a transmembranal pathway that may regulate the tissular localization of the enzyme to the area of the fm with high content of connective tissue. once secreted by the placental leukocytes, activation of mmp- depends mainly on mmp- , which seems to be derived from the same leukocytes. objective: preterm labour is a major problem in terms of perinatal morbidity and mortality. the histone-deacetylase inhibitor (hdaci), trichostatin a (tsa) has been shown to have an inhibitory effect on myometrial contractility. the aim of this study was to evaluate the effect of the hdaci's suberic bishydroxymate (sbha) and valproic acid (vpa) on human uterine contractions and hence their potential role as tocolytic agents. methods: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of sbha in the concentration range of nmol/l to mmol/l and vpa ( nmol/l- mmol/l). control experiments were run simultaneously. results: sbha and vpa exerted a potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions, compared to control strips. the mean maximal inhibition (mmi) values for sbha were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). the mmi values for vpa were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). conclusion: these results raise the possibility that hdaci's may have tocolytic potential, in addition to their current clinical indications. the inhibitory effect observed may be linked to the ability of hdac inhibitors to induce the expression of genes involved in the maintenance of myometrial quiescence via epigenetic mechanisms but may potentially also involve non-epigenetic pathways. progestin suppresses thrombin-enhanced interleukin- expression in term decidual cells: implications for abruption-induced preterm delivery. edward kuczynski, lynn f buchwalder, frederick schatz, charles j lockwood. obstetrics/gynecology reprod. sciences, yale university school of medicine, new haven, ct, usa. background and objective: decidual hemorrhage (abruption) promotes binding of factor vii to decidual cell (dc)-expressed tissue factor to generate thrombin. thrombin in turn induces several biological effects leading to preterm delivery via activation of cell surface protease-activated receptors (pars). interleukin- (il- ) is a pleiotropic proinflammatory cytokine induced by pars. this study assessed the separate and interactive effects of thrombin and medroxyprogesterone acetate (mpa) on il- expression in term dcs. methods: term decidua from stripped fetal membranes were isolated and the dcs were purified on a percoll gradient, grown to confluence and passaged until leukocyte-free. confluent dcs were primed in - m estradiol (e ) of e + - m mpa for days, then incubated in a defined medium (dm) with corresponding steroids ± thrombin. after hours, il- levels in conditioned dm were measured by elisa and western blotting. in parallel -hour incubations, il- mrna levels were assessed by quantitative rt-pcr and normalized to -actin mrna. results: secreted il- levels were similar in cultures maintained in e alone ( . ± . ) and e + mpa ( . ± . pg/ml/ug protein; mean ± sem; n = ). the addition of thrombin ( . u/ml) enhanced secreted il- levels by . ± . fold (p< . ) in incubations with e and by . ± . -fold (p< . ) in incubations with e + mpa. the inhibitory effect of mpa was statistically significant (p< . ). in confluent dcs incubated with e + mpa, exogenous thrombin ( . - . u/ml) elicited a concentration-dependent increase in secreted il- levels. hirudin acted as a pure thrombin antagonist, exerting no agonist effects alone, but counteracting thrombin-enhanced il- secretion. western blotting confirmed the elisa results. quantitative rt-pcr confirmed that il- m-rna levels corresponded to protein changes. conclusion: thrombin enhances il- mrna and protein expression in term dcs and progestin blunts these effects. since thrombin-generating abruption is closely associated with preterm delivery, anti-inflammatory effects induced by progestin inhibition of dc-derived il- may contribute to the protection against ptd, and may explain the reported protective effects of administration of -oh-progesterone in recent clinical trials. introduction: catechol-o-methyltransferase (comt) catalyzes the methylation of the phenolic hydroxyl groups in a variety of catechols. during estrogen metabolism, this enzyme converts the catechol estrogen, -hydroxyestrogen ( ohe ), to -ethoxyestrogen ( meohe ). the comt substrate, -ohe , can exhibit an anti-estrogenic effect in multiple biologic assays while the methoxyestrogen ( -meoe ) can exhibit an estrogenic effect. the biologic activities of these estrogen metabolites ( ohe meoe) depend upon their concentrations and tissue type. since comt activity ultimately controls levels of these metabolites, it appears to be a key factor in regulating the cellular estrogenic milieu. we have recently reported that amnion layers of human fetal membranes from laboring women exhibited folds higher comt mrna expression when compared to non-laboring women (wentz et. al. sgi ) . objective: to investigate the impact of comt inhibition on prostaglandin e (pge ) production by human amniotic membrane explants. study design: explants consisting of -cm circular sections of the amnion layer (obtained from term pregnant women who underwent elective repeat cesarean section) were prepared and placed in tissue culture explants media at ºc. after a -hour incubation, explants were treated with the selective comt inhibitor ro - , at and m concentrations. the incubation media was harvested after and -hour intervals. the levels of prostaglandin e (pge ) in the media were measured by sensitive elisa and were normalized against total protein concentration. results: ro - comt inhibitor induced major reductions in pge production in media collected from amnion explants of human fetal membranes. in the group treated with m of ro - for hours there was %± % reduction of pge after hours compared to untreated control (p< . ). in the amnion explants treated with m of ro - , there was %± % after hours and %± % after hours of treatment compared to untreated control (p< . ). conclusions: this finding indicates that comt activity in the amnion layer of human fetal membranes affects pge production. by facilitating a pro-estrogenic milieu in human fetal membranes in late gestation, increased comt activity may indirectly increases production of factors associated with labor such as pge . hypothesis: an extensive remodeling of the human cervical connective tissue takes place throughout pregnancy with a decrease in the total concentration of collagen and proteoglycans due to an altered higher metabolic turnover. we hypothesize that the profound changes in proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. we also hypothesize that proteoglycan production in cervical fibroblasts from preterm partal women are simmilar to the production in fibroblasts from partal women. method: cervical biopsies were obtained from non-pregnant women, women during elective cesarean section, woman after spontaneous parturition and after a preterm vaginal delivery. by explant technique fibroblasts were cultured from the biopsies. produced proteoglycans were metabolically labeled with s during hours and then purified by ion-exchange chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. results: the total proteoglycan production decreases with approximately % in partal and preterm partal cell cultures. the reduction of proteoglycans in preterm partal and partal cell cultures is significant compared to non pregnant fibroblast cultures. the distribution of biglycan and perlecan are similar in partal and preterm partal cells. biglycan are significantly reduced by around % and perlecan are significantly increased by around % compared to non pregnant cultures. preterm partal cervical fibroblasts secreates significantly more heparan sulfate proteoglycans compared to non pregnant cultures. the changes in total proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. both partal and preterm partal cell cultures differ in their proteoglycan production compared to their non pregnant counterpart, suggesting a role for proteoglycans in cervical ripening. the role of the oxytocin/oxytocin receptor system is not well defined in human amnion. previous studies in rabbit amnion have demonstrated an up-regulation of oxytocin receptors in the end of pregnancy and have shown that there is a large increase in the ability of ot to stimulate pge production. we and others have previously shown a role for nf-b in otr regulation. in whole genome array analysis of human amnion we found that otr was the gene with the second highest increase associated with activation of nf-b (the highest being cox- ). the present work was directed towards further understanding of otr expression and function in human amnion at term. we have shown that pge release by pre-labour primary human amnion cells is significantly increased after oxytocin treatment for hrs (ot: - m; n= ; triplicate samples; fold increase p< . ). the expression of otr in labour (+) and labour (-) primary amnion cells was measured with real time rt-pcr. we found a significantly higher level of expression in the labour (+) cells (n= ; duplicate samples; p< . ). western blot analysis confirmed the upregulation of otr in labouring amnion. treatment with il- b resulted in a significant upregulation of otr which peaked with a fold induction after hours (p< . ). il- caused a fold increase in otr mrna levels in labour (-) cells, bringing the expression level up to that found in labour (+) cells. our findings provide further evidence for a role of otr in human amnion. expression of otr in human amnion is significantly increased after the onset of labour. term non laboured amnion can be stimulated with il- to increase otr expression to levels of laboured amnion. one of the functions of otr in amnion cells is stimulation of prostaglandin synthesis. expression of antioxidant defence proteins in human myometrium before and after the onset of labour. vasso terzidou, mandeep s kandola, shirin khanjani, jan j brosens, phillip r bennett. parturition reserach group, irdb, imperial college, london, united kingdom. background oxidative stress is a result of an imbalance between the production of reactive oxygen species (ros) and the antioxidant defence mechanisms present in biological systems. parturition and infection-induced preterm labour resemble inflammatory processes that are linked to the production of ros including super-oxide (o -), hydrogen peroxide (h o ) and peroxynitrite. low concentrations of ros can act as second messengers in the regulation of several cellular functions. in an attempt to maintain redox homeostasis cells are equipped with machineries to both produce and scavenge ros. enzymatic scavengers include superoxide dismutase (sod), glutathione peroxidise and catalase. sod is the first enzymatic step in the defence system against oxidative stress. nuclear factor-kappa b (nf-b), a transcription factor family classically associated with inflammation, is activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. labour is associated with an increase in nf-b activity in human myometrium and in fetal membranes. nf-b is known to regulate a range of genes associated with the onset of labour. in a study using affymetrix whole genome arrays analysis nf-b overexpression was associated with increased expression of sod- ( . fold). to determine changes in ros scavenging potential upon onset of labour, lower segment myometrial biopsies were taken at term before and after the onset of labour (n= ; each group). cdna was extracted from the tissues and real time rt-pcr was performed for sod- , serum/glucocorticoidinduced protein kinase- (sgk- ) and the dna repair enzyme gadd a. we found that labour onset is associated with a two fold increase in the levels of expression of each. oxidative damage at the fetomaternal interphase has been extensively studied in the placenta as part of investigations for first trimester pregnancy losses, iugr and pre-eclampsia. the role of ros is less well defined in human decidua and the underlying myometrium. our results suggest a role for oxidative stress and redox homeostasis in the maintenance of myometrial quiescene during pregnancy and onset of labour. plasma anandamide levels increase during labour induction and appear to delay labour progression. vijianitha nallendran, anthony h taylor, patricia mw lam, stephen c bell, david j taylor, justin c konje. cancer studies and molecular medicine, university of leicester, leicester, united kingdom. background: the evidence for a role of the endocannabinoid, anandamide (aea) in labour is conflicting. we previously showed elevated aea plasma levels in labouring women whilst another group showed that activation of functional receptors for anandamide actually inhibited uterotonin-induced contractions through an adenylate cyclase pathway . the aim of this study was to explore further the relationship between labour and plasma aea levels. methods: plasma aea levels in women undergoing induction of labour for various indications at term, were measured by a sensitive isotope dilution method using hplc-ms/ms. each volunteer had an assessment of her cervix prior to the start of the induction when the first sample for aea was collected. once active labour was established (cervix cm dilated; contractions every - minutes), a second sample was collected. results: seventeen ( %) of the subjects were multigravida. the inductions were for postdates(n= ); decreased fetal movements(n= ), fetal growth restriction(n= ), symphysis pubis dysfunction(n= ), diabetes mellitus(n= ), pre-eclampsia(n= ), fetal cardiac complication(n= ), spontaneous rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes(n= ). plasma aea levels increased significantly once labour was established (figure) and demonstrated in ( %) of the cases. the median (interquartile range) plasma aea level increased from . nm ( . - . ) at induction to . nm ( . - . ); *p= . ; mann-whitney u-test) at active labour. there was a positive correlation between plasma anandamide levels taken before induction and the time taken for the women to enter into active labour (r = . , p= . pearson correlation). plasma aea levels were higher in labouring women compared to non-labouring women after the induction of labour confirming our previous observations and suggesting a direct role for this endocannabinoid in labour. further studies are required to elucidate this role. nuclear factor-kappa b (nf-b) is a transcription factor family classically associated with inflammation, activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. as a cytokineinducible transcription factor it plays a key role in the expression of a variety of genes involved in inflammatory responses and cell survival. nf-b dna binding and transcriptional activity plays an important role in labour associated gene expression in myometrium. in this study we examined the range of genes regulated by nf-b in myometrium using transient transfections and wholegenome array analysis. myometrial cells were extracted from myometrial biopsies taken at the time of elective caesarean sections at term. transient transfections of primary myocytes were performed using expression vectors for nf-b p .the amount of dna was constant and psg was used as a control construct. cdna was made from myocytes transfected with either nf-b or psg . affymetrix genechip u microarray was performed (n= , each group). we found that genes were significantly differentially expressed between control and overexpressed nf-b samples. twenty eight of these genes were upregulated with nf-b and were down regulated. several chemokines and cytokines were identified in the upregulated group. interleukin demonstrated the highest, fold, induction in the upregulated group, followed by tumor necrosis factor, a-induced protein (tnfaip ), chemokine ligand (ccl ), chemokine ligand (ccl ), pentaxin related gene (ptx ), interleukin (il ) and superoxide dismutase (sod- ). nine genes were present in the nf-b group that were absent in the control group. these included chemokine ligand (ccl ), chemokine ligand (ccl ), chemokine ligand (cxcl ) and il . ingenuity pathway analysis demonstrated that immune response, inflammatory, cell growth and proliferation and cell death were the main pathways involved. standardisation experiments have been performed, and the microarray results were confirmed with real time rt-pcr in several candidate genes. our results provide further support in the role of nf-b in human labour and suggest its direct link in upregulation of inflammatory genes, cytokines and chemokines, consistent with the imflammatory nature of the biochemistry of labour. inflammation is widely accepted to be a key feature of human labor. secretory leukocyte protease inhibitor (slpi), an innate immune molecule, has been shown to be an antimicrobial and anti-inflammatory protein. the aims of this study were to verify its expression and localization in human myometrium methods: specimens were obtained at time of cesarean delivery with or without labor. expression and localization of slpi was detected using immunohistochemistry. slpi expression pattern relative to nf-b p subunit was compared between not in labor and in labor subjects, between different tissue sections as well as in in vitro model systems including myometrial explants, uterine smooth muscle cells (usmc) and ishikawa endometrial adenocarcinoma cells. slpi was predominantly localized to the nuclei of myocytes. the observed nuclear immunoreactivity of myocytes was increased during the labor relative to not in labor, paralleled with p nuclear translocation. the nuclear pattern of slpi is specific to myometrium since slpi immunostaining was present exclusively in the cytoplasm of all other tissues examined, including amnion, chorion, decidua and endometrium. slpi staining was also positive in macrophages, indicated by co-localization of slpi with cd positive cells. treatment with il- or tnf-induced nuclear translocation of p in myometrium explants and usmc, but not in ishikawa cells. in human myometrium, slpi is predominantly localized in the nuclei of myocytes and in macrophages. the nuclear expression pattern of slpi is myometrium-specific and increased following the onset of labor and correlated with nf-b activation. further understanding of its physiological significance may suggest new strategies aimed at preventing preterm birth. application of a new proteomic technology on amniotic fluid in prom. sara consonni, niccolo bosso, marianna andreani, agnese pizzardi, fulvio magni, anna locatelli. obstetrics and gynaecology, university of milano-bicocca, monza, italy; experimental medicine, university of milano-bicocca, monza, italy. objective: mass spectrometry (ms) is the obligatory tool for proteomics studies. biological samples must be purified before ms analysis due to the matrix complexity. a recent approach combines active surface prepurification with maldi-tof (matrix assisted laser desorption) analysis. clinprot technology provides the prepurification of the sample through the use of magnetic beads (mb) with activated surface. this technique can be carried out by robot in an automated way on a large number of samples. a unique example of the use of mb before maldi-tof analysis to determine proteomic profiles of amniotic fluid (af) is reported for rapid detection of fetal aneuploidies (wang ) . the objective of our study was to verify the applicability of clinprot prepurification before maldi-tof analysis on amniotic fluid collected noninvasively in premature rupture of membranes (prom). methods: we sampled af from vaginal posterior fornix of women with preterm prom (group , n= ) and term prom (group , n= ). samples were prepared with mb and analyzed with ms maldi-tof in order to generate proteomic profiles. results: it was possible to generate average proteomic profiles in the two study groups and the observed profiles were different. samples of af non invasively collected in prom can be analyzed by ms maldi-tof after preparation with mb. this technique allows to retain part of the eluted sample for characterization of protein peaks of interest. due to the less laborious characteristics of this method in comparison with techniques based on bidimensional electrophoresis its application can be useful in clinical proteomics. progestins accentuate the maternal but not fetal inflammatory response of women with intra-amniotic inflammation. sonya abdel-razeq, irina a buhimschi, michael cackovic, guoyang luo, antonette dulay, victor rosenberg, mert bahtiyar, errol norwitz, edmund funai, catalin buhimschi. ob./gyn. reprod.sci., yale university, new haven, ct, usa. introduction: data from animal research suggests that progestins have a marked pro-inflammatory capacity. recent studies support the administration of -hydroxyprogesterone caproate in women at risk for preterm birth. we sought to determine the impact of progestins during gestation on the extent of maternal and fetal inflammatory responses in pregnant women with intraamniotic inflammation. methods: amniotic fluid, placenta and cord blood were obtained from women who delivered preterm (median[range], ga: [ - ] wks). an amniocentesis was done to rule out infection. women exposed to progestins (n= ) within one week prior to amniocentesis were matched to controls (crl) by age, parity, history of preterm birth ga, membrane status and interval to delivery. proteomic profiling of amniotic fluid [mass restricted (mr) score] identified the presence or absence of intra-amniotic inflammation. an mr score of or confirmed intra-amniotic inflammation. amniotic fluid and umbilical cord interleukin- (il- ) levels were measured by elisa. histological chorioamnionitis was graded based on recognized criteria. results: overall, women and their fetuses exposed to progestin did not exhibit an increased inflammatory response (table) . however, sub-analysis restricted to women with mr or (n= ) showed that in the context of intraamniotic inflammation, progestins were associated with significantly elevated amniotic fluid il- levels compared to unexposed women (progestins (n= ): vs. crl (n= ): [ . - ] ng/ml, p= . ). these relationships were maintained after correction for steroid and antibiotic exposure. such significance was not found for amniotic fluid glucose, ldh, wbc count or cord blood il- . conclusion: our results suggest that progestins may amplify the maternal, but not the fetal inflammatory response of women with intraamniotic inflammation. objective: recently progestins have been shown to reduce the incidence of recurrent preterm labor in women. progestins have long been known to inhibit preterm labor in some species including rats and are also known to delay term labor. nitric oxide (no) donors, including ng, inhibit uterine contractility and have been used as tocolytics. the aim of this study was to examine the inhibitory effects of r on preterm labor in rats induced with a progesterone antagonist (onapristone, zk) with and without ng. materials and methods: charles river s-d timed pregnant rats (n= /group) were treated with zk ( mg/rat, s.c.) alone or vehicle (controls) on day of gestation. other groups of rats were treated with zk in combination with various doses of r ( . , or mg/rat s.c) with and without ng ( mg s.c. pellet) from days to of gestation. all rats were sacrificed on day of gestation and the number of fetuses and implantation sites were counted to determine the preterm delivery rate. one way anova was used for statistical analysis. p< . was considered significantly different. results: rats treated with zk alone delivered all their fetuses prematurely compared with controls (p< . ) treated with vehicle only (ca. % fetuses delivered). ng treatment alone did not affect the delivery rate (p> . ) compared to controls. similarly zk + ng did not reduce the preterm delivery rate compared to zk alone (p> . ). however r dose dependently reduced (p< . at all doses) the number of fetuses delivered prematurely in response to zk and the premature delivery rate was further reduced when treatment included the combination of r plus ng (p< . ). conclusions: zk effectively induces premature delivery. premature delivery produced by zk can be effectively reduced with a r , a progestin known to bind with high affinity to nuclear progesterone receptors. ng by itself, at the dosage used, does not reduce the prematurity rate caused by zk. however, r and ng act synergistically to reduce the preterm delivery rate. this study indicates that combinations of a progestin with an no donor may be an effective treatment for preterm labor and delivery. monkeys. pl grigsby, jp rasanen, , dw sadowsky, m bertolino, m carbonatto, e gillio tos, s canali, j lacy, a chollet, mj novy. reprod sci, oregon primate res ctr, beaverton, or; ob/gyn, oregon health sci univ, or, usa; rbm, merck serono, italy; merck serono, switzerland. objective: to investigate the pharmacokinetics (pk) and pharmacodynamics (pd) of as , a novel orally active non-peptide oxytocin (ot) antagonist in rhesus monkeys. study design: dose finding and pk/pd studies were done in chronically instrumented non-pregnant (n= ) and pregnant ( - dga, term d; n= ) monkeys. maternal and amniotic fluid compartments were serially sampled during dosing and washout. treatment phases included: ot infusion control, ot infusion + as , and ot rescue therapy. ot infusions ( - mu/kg/hr, iv) were given to determine the lowest dose required to produce stable, sub-maximal uterine contractions in each animal. as was administered p.o. at , , mg/kg and the effects on inhibition of uterine activity (hca, mmhg.sec/hr) were compared. to verify antagonist reversibility, objectives: infection such as chorioamnionitis is thought to be the cause of premature rupture of the membrane and induce uterine contractions leading to preterm delivery. however, the mechanism for enhancement of uterine contractility is not well understood. we have reported that non-selective cation channels (nsccs) regulate pacemaker potentials to generate rhythmical contractions and should be targets of magnesium ions used for tocolysis. the purpose of this study is to investigate the changes of the expression of nsccs during normal pregnancy and the effect of inflammation in preterm. methods; atp receptors (p x) and transient receptor potential canonical (trpc) channels were examined as uterine nsccs. the mrna was extracted from the rat myometrium of non-pregnant and pregnant rats at days , , and . the expression of each subtype of p x and trpc channels was measured by real time rt-pcr (abi ) with taqman probes (abi). as an inflammatory model lipopolysaccharide (lps; . mg/kg) was injected into intraperitoneal cavity at day and the tissue was sampled after six hours. results; p x and p x were determined to be dominant subtypes of p x channel. the expression of p x was increased by % at day , compared with day and that of p x was enhanced by %. on the other hands, trpc and trpc were detected dominantly. the expression of trpc was increased three times in the late stages of gestation. however, trpc was suppressed by %. in the lps treated rat myometrium cox- mrna expression was measured to be fold higher than that of the control rat, showing inflammatory effects in the myometrium. in this model the expressions of p x , p x and trpc were enhanced by . , . and . times, but trpc was not changed. the mrna expression of p x , p x and trpc channels in rat myometrium was increased in the late stages of pregnancy. these channels are suggested to be concerned with onset of labor. in the inflammatory model the expression of these channels was accelerated dramatically and these values were much higher than those in normal pregnancy. this finding supposed inflammation may enhance some types of nsccs to accelerate uterine contractility and induce preterm delivery. anandamide ( to determine the mechanism of rho protein activation during oxytocin and carbachol induced contraction, freshly prepared myometrial strips in krebs henseleit buffer were treated with oxytocin ( nm) and carbachol ( μm) under isometric and tension free conditions. control strips were exposed to buffer only. treated myometrial strips were solubilised and separated into membrane and cytosolic extracts and equal aliquots were immunoblotted with rhoa and rhob antibodies. rhoa translocated to the membrane after oxytocin and carbachol stimulation under both isometric and tension free conditions (p< . ). there were no significant changes in rhob membrane to cytosol ratios relative to control. agonist induced contraction in human myometrium is associated with rhoa but not rhob membrane translocation. during pregnancy components of the intracellular camp signalling pathway show increased gene expression resulting in the maintenance of myometrial quiescence until term where a substantial decrease in expression of these genes is observed. protein kinase a regulatory subunit riia (rii ) is upregulated at both the mrna and protein levels in the human myometrium during pregnancy. this particular subunit is membrane-bound and by directing phosphorylation to myometrial cytoskeletal proteins may affect contractile machinery thus playing a role in maintaining uterine relaxation. acetylation of histones promotes a favourable chromatin environment for transcriptional activity of many genes. this process is largely inhibited by histone deacetylases (hdacs), whereby its activity leads to transcriptional repression. hdacs can be recruited to the promoter region of a gene by other transcription factors such as sp proteins. since the rii promoter contains three sp - consensus binding sequences in its proximal part, we investigated whether this gene is a target for transcriptional regulation by hdacs. using dna precipitation assays we found that sp , and as well as hdac and form complexes with biotin-labelled fragments relating to sp - elements in the promoter region of rii . additionally, treatment of myometrial primary cell cultures with hdac inhibitor trichostatin a (tsa), or with the methyltransferase inhibitor -azac resulted in increased mrna and proteins levels. further studies with full and truncated luciferase constructs of the promoter region of the rii gene in transiently transfected myometrial cells confirmed that all three sp - elements are involved in the transcriptional regulation of the gene. this process involves hdacs, as h treatment with tsa significantly increased the luciferase signal. changes in the binding of sp proteins and hdacs to the promoter after tsa and azac treatment were investigated employing chromatin immunoprecipitation assays. alterations in the methylation status of the promoter after treatment were examined by bisulfite modification and dna sequencing. together, this study highlights the importance of chromatin modifications in the maintenance of uterine quiescence during pregnancy as well as identifying a potential mechanistic target for drugs that may reduce the incidence of preterm labour. objective: progesterone maintains pregnancy by promoting myometrial quiescence. typically progesterone effects are thought to be mediated through the classic genomic pathway. there is evidence, however, that progesterone also acts via a non-genomic pathway by interacting with specific membrane progesterone receptors (mprs) and in particular progesterone receptor membrane component - (pgrmc- ). the role of non-genomic progesterone actions in human pregnancy and parturition is not clearly understood. the goal of this study was to measure the extent of mpr expression in biopsy specimens of human myometrium obtained at cesarean delivery, and to determine whether expression changes with advancing gestation or the onset of labor. study design: lower uterine segment myometrial biopsies were obtained at the time of delivery from consenting women who were at term and not in labor (n= ), preterm and not in labor (n= ) and term and in labor (n= ). protein extracts were prepared and subjected to polyacrylamide gel electrophoresis and immunoblot analyses for pgrmc- and gapdh. abundance of pgrmc- protein relative to gapdh was determined by digital densitometry. we also performed immunohistochemistry (ihc) to determine the cellular localization of pgrmc- in the human pregnancy myometrium. results: pgrmc- protein was identified in each biopsy specimen. there was a -fold increase in pgrmc- protein in term compared with preterm biopsies (relative to gapdh p< . ). the relative level of pgrmc- protein was not different between biopsy specimens from laboring and non-laboring women at term (compared to gapdh, p= . ). pgrmc- immunoreactivity was localized to granular cytoplasmic staining. conclusion: this is the first description of the presence of pgrmc- protein in the human myometrium during pregnancy. its presence suggests that progesterone may influence contractility non-genomically via these receptors. the functional significance of the gestational age associated two fold increase in pgrmc- is unclear. changes in expression of this receptor during pregnancy may be important for the hormonal control of parturition and can be the focus of future studies. (ruddock et. al., abstract smfm, ) . the aim was to examine the mechanism of p inhibition. methods: uterine tissues from women (n= ) at term with cesarean section, were suspended in organ chambers and exposed to various agents or solvents. contractility was registered, stored, analyzed and compared before and after addition of agents or kcl. tissues were treated with p alone ( - to - m) or p bound to bovine serum albumin (bsa/p , - to - m p ), a progestin with low affinity to mpr (r , - - - m), or a non-sex steroid (cholesterol, - to - m). other tissues were pretreated with selective inhibitors of adenylate cyclase (sq , - m), guanylate cylase (odq, - m), phosphodiesterase (rolipram, - m), nitric oxide (no) synthases (l-name, - m) or a nuclear p receptor antagonist (mifepristone, mif, - m), followed by p . data were analyzed by anova for statistical differences (p< . ). results: p rapidly (< hour), effectively ( %) and dose-dependently inhibits spontaneous and kclinduced contractility (ed of < - m incubation of strips with zd- , at concentrations up to m for one hour prior to the experiment, led to no significant reduction in the total work done. however, incubation of strips with l- with a concentration of nm for one hour prior to the experiment, led to a statistically significant reduction in the total work done after one and a half hours. furthermore, when increasing concentrations of pge ( - to - ) were added to l- ( nm) pre-treated strips, total work done per contraction was comparable to that of non-treated controls. similar effects were not observed in zd- ( nm) pre-treated strips. since the reduction in contractility caused by l- was greater than that caused by zd- , it is likely that the stimulating effect of pge acts predominantly via ep receptors. taken together with our previous data showing an increase in ep at labour, this data shows that targetting an ep receptor may be a useful strategy in managaing pre-term labour. recently a truncated kda isoform of the er-has been described in human endothelial and testicular cells. we describe the presence of this kda erisoform in pregnant and immortalized non-pregnant human myometrial cells. methods: myometrial tissue obtained from non-laboring pregnant women undergoing cesarean section at term (n= ) was dissected prior to being finely minced. a portion of the tissue was placed in pbs and sonicated, while the remainder of the tissue was dissociated with collagenase prior to filtration and placement on culture plates. cells were then cultured in mem w/ % fetal bovine serum (fbs) until confluence. cultured cells were then dissociated with . % typsin/edta, subsequently re-plated and grown to confluence. immunohistochemistry directed toward smooth muscle protein was used to verify myometrial phenotype.) protein was extracted and quantified. western blot was performed with mouse monoclonal anti-er-receptor antibody (santa cruz biotechnology) to the f- domain of the human er-receptor. an immortalized non-pregnant human myometrial cell line provided by ann word, htert, was cultured and isolated as described above. results: htert cells expressed both the truncated ( kda) and the full length ( kda) er-isoforms. fresh and cultured myometrial cells from non-labored term pregnant patients also expressed both isoforms of er-. subsequent subcultures of myometrial tissue continued to express the kda erisoform, yet the expression of the kda isoform was lost. a representative blot is below. conclusions: we demonstrate, for the first time, the presence of a kda erisoform in cultured and non-cultured pregnant and cultured nonpregnant human myometrial tissue. discovery of this isoform of er-in myometrial tissue could provide insight into the molecular mechanisms involved in parturition. the action of prostaglandin f (pgf ), a potent uterotonic stimulant that is associated with labour at term and preterm, is mediated by its receptor, ptgfr. myometrial ptgfr mrna levels fall during pregnancy and this likely plays a role in uterine quiescence. however, the mechanisms by which this occurs are poorly understood. we previously reported that pgf downregulates fp mrna expression in cultured human myometrial ultr cells in a protein kinase c (pkc) dependent manner. in addition to the downregulation of mrna levels, receptor desensitization may also represent another mechanism of decreasing ptgfr activity because ligand binding to g protein coupled receptors often results in receptor internalization. we therefore hypothesized that pgf treatment of ultr cells also results in pkc dependent ptgfr internalization. methods: near confluent cultured human myometrial ultr cells were treated +/- - m or - m pgf for , , or hr. cells were fixed with formaldehyde and visualized for localization of ptgfr by immunofluorescence. to examine the potential involvement of pkc in the process, ultr cells were treated +/- - m pgf and +/- m myristoylated pkc inhibitor ( - ) and examined for ptgfr cellular localization as described above. results: pgf treatment resulted in a dose dependent decrease in ptgfr membrane signal at , , and h. this decrease was dependent on pkc as cotreatment with the myristoylated pkc inhibitor ( - ) prevented the pgf induced decrease in membrane ptgfr at h treatment. there was no visible effect of the pkc inhibitor on ptgfr membrane signal on its own. conclusion: we conclude that pgf decreases membrane levels of ptgfr protein in human myometrial ultr cells in a pkc dependent manner. these results suggest that pkc may be required for both the pgf induced internalization and desensitization of ptgfr protein and downregulation of ptgfr mrna in human myometrial ultr cells. therefore pkc may play a crucial role in downregulating ptgfr expression and activity and maintaining uterine quiescence during pregnancy. rhoa is a small gtpase that acts as a molecular switch to control a variety of signalling pathways in smooth muscle, including contractility. it is thought that increases in rhoa-gtp levels facilitates phosphorylation of target proteins such as cpi- , promoting contractility at pre-term and term labour in humans. however, in situations of acute or chronic hypoxia in the uterus, it is important that myometrial contractility and subsequent labour is not facilitated prematurely. given the importance of rhoa to a cell's response to hypoxia in other cell types, it was hypothesised that rhoa plays a central role in the mechanism controlling smooth muscle contraction in the uterus too. following acute hypoxia ( . % o ) for one to six hours, rhoa mrna, total protein and activation (rhoa-gtp) levels were analysed, using semi-quantitative pcrs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. next, we investigated whether reduced oxygen conditions affected oxytocin induced activation of rhoa, following a two hour treatment of nm oxytocin. firstly, our results demonstrate that the rhoa itself is significantly activated under low oxygen conditions, resulting in phosphorylation of myosin phosphatase, myosin light chain and cofilin, three proteins known to be central in contraction and actin filament organisation. secondly, hypoxia significantly reduced the coupling of oxytocin to rhoa activation under the conditions examined. we observed a significantly reduced level of rhoa expression and activation which correlated with an increase in the level of another rhogtpase protein, rhoe. we propose that rhoa inactivation occurs through a rhoe-mediated mechanism, suggesting a balance in the activity of these two antagonistic rhogtpases in oxytocin-induced hypoxic human uterine smooth muscle cells. these results provide a possible explanation for the reduced coupling of oxytocin as a stimulant of myometrial contractions during slowly progressing labours. oxytocin-induced release of calcium ions (ca + ) from sarcoplasmic reticulum (sr) and sensitisation of contractile proteins to ca + have been suggested to mediate the oxytocin-induced potentiation of myometrial contractions. objective: we investigated the effects of oxytocin in the presence of nifedipine, a known inhibitor of the l-type calcium channel (ltcc). method: samples of myometrium were obtained from women undergoing term caesarean section with the approval of the local ethics committee. a standard organ bath system (ad instruments, uk) was employed to analyse contractile activity. stable spontaneous contractions were recorded for - minutes before addition of nifedipine. results: in agreement with our previous findings, application of oxytocin to spontaneously active strips produced a two-component effect: a transient tetanus-like contraction, followed by prolonged augmentation of phasic contractions. nifedipine ( um) rapidly abolishes spontaneous contractions, subsequent addition of nm oxytocin produced an initial, transient rise in force, approxiamately % compared to oxytocin alone, followed by high frequency oscillations in > % of strips. calcium-free solutions were used to confirm that oscillations were due to ca + entry. disabling the sr store using thapsigargin ( um) had no effect on oscillations, confirming the sr not to be involved. the t-type calcium channel blocker, mibefradil ( um ) showed no inhibition of oscillations. an ip receptor and store-operated calcium channel inhibitor, -aminoethyldiphenylborate ( -apb) um also had no effect on oxytocin-induced oscillations. the store-operated calcium channel inhibitor skf- ( um) showed partial inhibition of oscillations. conclusions: based on these results, we propose that the most likely mechanism of ca + entry producing oxytocin-induced oscillations in the presence of nifedipine is the transient receptor channel-c (trpc) channel, known to be present in the human myometrium. further work needs to be completed to clarify this further. identification of stim and orai in human myometrium. a new paradigm in store-operated calcium signaling. evonne c chin-smith, mark r johnson, rachel m tribe. division of reproduction and endocrinology, king's college london, london, united kingdom; department of maternal fetal medicine, imperial college school of medicine, chelsea and wesminster hospital, london, united kingdom. background: recent reports have suggested that two novel proteins, stim and orai, are involved in the regulation of store-operated calcium entry. we have previously reported that members of the trpc family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labour associated stimuli il- beta and mechanical stretch. although stim and orai isoforms have been reported in other smooth muscle cell types, there are no published reports of stim and orai expression in human myometrium. the aim of this study was to identify mrna expression of stim , stim , orai and orai in human myometrium. methods: human myometrial biopsies were obtained from women undergoing elective caesarean section at term (prior to labour) with informed written consent and institutional ethics committee approval. whole myometrial tissue was either snap frozen and stored at - o c or used for cell culture. rna was extracted from whole tissue (n= ), primary cultured myometrial cells (n= ) and passaged (p ) myometrial cells (n= ) and stim , stim , orai and orai mrna expression was assesed by quantitative real-time pcr. results: all four genes were expressed in whole myometrial tissue and cells. stim and stim mrna expression in cultured myometrial smooth muscle cells (primary and passaged) was significantly reduced compared to myometrial tissue expression (p< . ). however, there was no significant difference in either orai or orai expression in whole tissue versus cultured myometrial smooth muscle cells. conclusion: to our knowledge this is the first report of stim / and orai / mrna expression in human myometrium. these genes may contribute to the regulation of calcium signalling in human myometrium, but the functional significance of their expression remains to be determined. funded by the bbsrc. obstetrics and gynecology, national university of ireland, galway, galway, ireland. objective: the ability of uterine smooth muscle cells to stimulate collagen contraction has been well established as an in vitro model of myometrial contractility. devost and zingg ( ) reported that the contractility of human myometrial cell lines layered onto collagen matrices was increased by oxytocin while another group described the stimulation of human uterine smooth muscle cell contractility, cultured within collagen lattices, with endothelin- (dallot et al., ) . our study investigated the response of human primary uterine smooth muscle cells cultured within collagen lattices, to various compounds, including the non-specific depolarizing agent potassium chloride (kcl), the inflammatory cytokine tnf , the rock- inhibitor y- , oxytocin, and oxytocin plus its clinically used antagonist, atosiban. methods: human primary uterine smooth muscle cells (utsmc) (lonza) were maintained in dmem high glucose media. cells ( , per well) passage - , were embedded in . mg/ml collagen, in . ml aliquots, in dmem-f media on well plates (invitrogen) (dallot et al., ) . effects on contraction were studied by monitoring changes in gel area (alpha innotech imager, image j software (nih)). statistical significance was determined by the student t test. results: the utsmcs displayed basal contraction of the collagen gels while the non-contractile cell line hek cells, did not. kcl ( nm) stimulated an % increase in contractility (n= , p= . ) while nm tnf resulted in an . % increase (n= , p= . ), in comparison to unstimulated cells embedded in gel. the rock inhibitor y- ( m) inhibited contractility, with a . % decrease in collagen gel contractility (n= , p= . ). a % increase (n= , p= . ) in utsmc embedded collagen contractility was observed with nm oxytocin, which was antagonized by atosiban ( m) (n= ). conclusion: this study highlights the importance of the development and optimisation of a reproducible human in vitro myometrial contractility model, to evaluate the effect of various known labor-associated, and also unknown compounds. this should aid in our understanding of the many complex biochemical pathways involved in myometrial contractility at labor, and ultimately contribute to the prevention of preterm labor. changes in intra-mural myometrial blood flow during spontaneous labour using d power doppler angiography (pda). nw jones, , nj raine-fenning, h mousa, mj taggart, k jayaprakasan, gj bugg. nottingham university hospitals nhs trust, united kingdom; nottingham university, united kingdom; university of newcastle upon tyne, united kingdom. methods d pda was used to measure the percentage (%) change in the vascularization index (vi), the flow index (fi) and the vascularization flow index (vfi) in a volume of myometrium at the uterine fundus of nulliparous women at term, in the first stage of uncomplicated spontaneous labour. d data sets were obtained during a single cycle of uterine relaxation (r ), contraction and subsequent relaxation (r ). measurements were made independantly by two authors (nwj and gjb) using vocal® (ge kretz) and the mean value was used for analysis. the results from each woman are presented as a % change of r . data is presented as medians [interquartile range (iqr)] and analysed using non-parametric tests. the median volume (cm ) of interest for r was . cm ( . - . cm ), for the contraction was . (fig. ) . the % change for all three indices between r and r was not significantly different. however, there was a significant difference between the contraction and r (p< . ). the mean intra-class correlation coefficient and % confidence interval (ci) of vi, fi and vfi for the authors (nwj and gjb) were . ( . - . ), . ( . - . ) and . ( . - . ), indicative of good inter-observer reliability. conclusion d pda is a useful and reliable tool in the assessment of changes in intramural myometrial blood flow, which was found to reduce significantly during a contraction but increase again during the following uterine relaxation. . raine-fenning nj, et al. the inter-observer reliability of d pda acquisition within the female pelvis. ultrasound obstet gynecol. ; : - . the role of pgf on myometrial contractility; studied with a selective fp antagonist. shankari arulkumaran, andre chollet, phillip r bennet. imperial college parturition research group, institute of reproductive and developmental biology, hammersmith hospital campus, london, united kingdom; merck serono, geneva, switzerland. objectives: human myometrial strips established in culture will usually begin contracting after one hour. we have previously shown that stretch upregulates prostaglandin synthesis and have therefore hypothesized that spontaneous contractions occur because of stretch-related prostaglandin synthesis. methods: experiments were performed using x mm human pre-labour, lower uterine segment myometrial strips in a dmt myograph ms in oxygenated kreb's solution, with adi powerlab software. spontaneous contractions required stretch force and initial experiments determined that the maximum number of strips attaining spontaneous contractions was greatest at a force of - g. a novel antagonist to pgf (fpa) was studied in this model. the fpa has a ki of nm for fp and is - fold selective for fp compared with other prostanoid receptors. results: addition of pge and pgf after the commencement of spontaneous contractions caused a statistically significant increase in the total work done by the strips. the effect of pge was being greater than that of pgf . pre-incubation of the baths with a novel and selective fp antagonist (fpa) with concentrations up to m ( - ) did not affect the total work done by spontaneous contractions compared to non-treated controls. however, increasing concentrations of the fpa ( - to - ) decreased the total work done by -fold on strips treated with pgf beforehand in comparison the pgf -treated strips alone. conclusion: these data suggest that stretch and synthesis of prostaglandins is essential for spontaneous contractility in human myometrial strips. since fpa is able to block pgf induced but not spontaneous contractions, it is likely that pgf does not play a role in spontaneous myometrial contractility in vitro, although it may do so in vivo. because other factors may combine to increase contractility, the combination of an fp antagonist with other inhibitors may therefore, be a more effective strategy in reducing pre-term deliveries. objectives: brain natriuretic peptide (bnp) is synthesized in fetal membranes and inhibits oxytocin-induced contraction of preterm human myometrium. we hypothesized that bnp may be a paracrine mediator of human myometrial quiescence. we showed bnp content is higher in membranes from preterm pregnancies absent labor and significantly decreased with idiopathic preterm labor. while bnp activates natriuretic peptides receptors a (npr-a), b (npr-b), and its clearance receptor (npr-c), we have shown bnp does not inhibit myometrial contraction via npr-a or npr-b. herein, we test in part the hypothesis that bnp inhibits myometrial contractions by activating npr-c by quantitating npr-c in human myometrium at different gestations and labor status. methods: myometrial samples were obtained at the time of cesarean section after informed consent from groups of patients: preterm not in labor (pt-nl), preterm in labor (pt-l), term not in labor (t-nl) and term in labor (t-l). myometrial samples were obtained from women - weeks' gestation, and term between and weeks. mrna for npr-a, b and c were semiquantitated by realtime pcr (normalized by s mrna), and npr-c protein by western blot. results: natriuretic peptide receptors a, b and c mrnas were identified in all groups. while there were no differences in mrna levels among groups, npr-c protein was increased in samples from term laboring women. conclusion: since bnp inhibits the contraction of human preterm but not term myometrium independent of npr-a and npr-b, we have previously speculated that bnp activates another "unknown" receptor. herein we find that myometrial npr-c protein but not mrna increases during human labor at term. the increase in npr-c at term could compete with the unknown quiescent receptor to functionally reduce the availability of bnp and thus permit or promote myometrial activation/contraction. (supported by a grant from chilean government fondecyt ). objective: bnp is synthesized within human chorion and amnion and inhibits oxytocin-induced contraction of human myometrium. bnp activates guanylate cyclase (gc) natriuretic peptides receptors a (npr-a) and b (npr-b). bnp also stimulates the clearance receptor (npr-c) whose action is not mediated by gc. the intracellular pathway of bnp/npr-c inhibition is not known. we determined that bnp does not inhibit myometrial contraction via npr-a or -b. we hypothesized that bnp inhibits myometrium by npr-c activation. we test aspects of our hypothesis by determining whether bnp/npr-c pathway inhibits myometrial contractions via myometrial nos pathway. methods: bnp and canp (specific npr-c agonist) were added to primary human myometrial cell cultures and enos/inos activity/expression determined. cell cultures (n= ) were prepared from myometrial samples of nonlaboring, term pregnant women. after confluence ( d), the cells were incubated ( h) with nm bnp and/or canp. nos activity was measured ( l-citruline assay) and transcription/translation semi quantitated (realtime pcr and western blotting). results: bnp had no effect on either nos expression or activity. canp significantly reduced inos but not enos mrna level. canp did not alter the protein level of nos but significantly reduced nos activity. co-incubation with bnp and canp reduced both mrna levels (p< . ) and significantly decreased enos, but not inos, protein without change in overall nos activity. within the female pelvis. ultrasound obstet gynecol. ; : - . conclusion: the activation of npr-c by canp reduces nos activity and expression. the same effect was not observed by bnp. the difference may be explained because bnp (but not canp) activates all natriuretic peptide receptors, and they may have opposite actions on nos pathway. we conclude unlikely bnp inhibits human myometrial contraction by npr-c activation via the nos pathway. ( introduction: ultr is a retroviral immortalized human uterine smooth muscle cell line which we use as a model for uterine hypertrophy studies. however, this cell line has limited usage due to a reduced rate of cell division and eventually replicative senescence in culture. one of the mechanisms of human somatic cellular senescence is un-compensated shortening of telomeres, the specialized dna structures located at the ends of eukaryotic chromosomes. introduction of human telomere reverse transcriptase (htert) has been shown to induce telomerase activity and telomere elongation, and extend life-span of normal human cells. objective: to recover ultr cell division without altering the phenotypic characteristics of smooth muscle cells by introducing htert, therefore improving ultr cell line. methods: ultr cells were transfected with a modified htert expression vector at a relatively early stage (passage ) in the presence of selective antibiotic. ultr cell growth rate, with (ultr-ht) or without transfection, was determined by plating cells in multiple plates at a fixed density and counting cell numbers after days. single cell clones of stably transfected cells were further isolated by plating in well plates. expression of htert, smooth muscle specific genes (smc-sgs), and target genes was identified by rt-pcr of total rna extracted from ultr and ultr-ht cells. results: rt-pcr demonstrated successful introduction of htert into ultr cells. the growth rate of ultr-ht cells was increased and cell morphology was improved (free of the typical aneupoid appearance). at passage , ultr-ht cells grew . fold (p< . ) and . fold (p< . ) faster than ultr cells at passages and , respectively. currently single cell clones have been selected. ultr-ht cells express a set of smc-sgs, including -actin, caldesmon, calponin, myosin heavy chain, sm , and smoothelin, confirming that the smooth muscle phenotype is preserved. a panel of genes involved in angiotensin ii signalling, including angiotensin ii receptors (at / ), nox family (nox , , and duox ), nox-associated genes (p phox, p phox, p phox, and rac / ), was also preserved. conclusion: this is the first report of rescuing uterine smooth muscle cell replicative senescence via activation of telomerase. we have established a human uterine smooth muscle cell line which will provide an improved in vitro model for studying human myometrium. at term, myometrium is characterized by spontaneous contractions which vary in the amplitude, frequency and duration depending on specie and hormonal status. repetitive depolarization of plasma membrane followed by transient elevation in [ca + ] i is thought to underlie the contractions. however, the detailed pathways which regulate the spontaneous contractility remain unclear. objective: this study was designed to elucidate the effect of protein kinase c (pkc) on the spontaneous contractions and [ca + ] i transients in the myometria from term pregnant mice and women. methods: the human samples were obtained from women undergoing cesarean section with the approval from the institutional review board committee while mice myometria were dissected from the days pregnant mice sacrificed according to the animal study protocol. the isometric force and [ca + ] i were measured simultaneously in fura- loaded myometrial strips using spectrofluorometer equipped with force transducer. results: the pkc activator phorbol , -dibutyrate (pdbu) applied at - m first stimulated the amplitude of spontaneous contractions in mice and human myometria followed by their inhibition. under the pdbu treatment the frequency of the contractions was first increased and then decreased in both species. at the same time, pdbu didn't initially increase the amplitude of [ca + ] i transients but attenuated it over time. however, the frequency of the transients was first increased and later decreased upon the pdbu exposure. in addition, pkc activation with pdbu resulted in the elevation of the uterine basal tone without corresponding changes in the basal level of [ca + ] i in human myometrium. in mice myometrium, on the contrary, pkc activation didn't result in an increase of the muscle tone and the basal level of [ca + ] i . conclusions: we propose that pkc causes bi-phasic effect on uterine spontaneous contraction in mice and human first to potentiate the amplitudes and the frequencies and later decreases them. the amplitude of [ca + ] i was not initially potentiated by pdbu suggestive of the dissociation of the contractile and [ca + ] i response. the stimulatory effect of pdbu on the basal muscle tone in human myometrium and the absence of the effect in mouse suggest different mode of pkc action on uterine contraction in human and mice. introduction: mechanical stretch of uterine myocytes is detected through integrins on the cell surface which form part of the focal adhesion complex, this signals through mapk, ultimately leading to the up-regulation of various pro-labour genes including pghs- and il- . on integrin activation fak is recruited to the focal adhesion complex and activated by autophosphorylation at tyr- , creating a src binding site. this promotes further fak phosphorylation at tyr- , and , enhancing fak catalytic activity. however fak can also act as a scaffold protein and its exact role in the expression of pro-labour genes is uncertain. in this study we used inhibitors for the kinase activity of fak and mek / in order to test the affect of fak kinase activity on expression on pghs- mrna. methods: primary human myometrial cell cultures were grown from myometrial biopsies taken from women undergoing elective caesarean section. cells were plated onto -well flexible bottom plates coated in type i collagen. cells were subjected to % static stretch for up to minutes. cells were also incubated with either the rho kinase inhibitor y or the mek / inhibitor u prior to being stretched. western blots were performed using antibodies to fak phospho- , fak phospho- and erk / phospho- / , -actin was used as a loading control. rna was also extracted and levels of pghs- measured using q-pcr. results: stretch increased levels of phosphorylation at both tyr- and tyr- with the greatest increases occurring at and minutes. however the increase at tyr- appeared to be greater than at tyr- . incubation with the rho kinase inhibitor reduced phosphorylation of fak- , however this did not affect phosphorylation of erk / or stretch induced up regulation of pghs- mrna. in contrast incubation with the mek / inhibitor reduced erk / phosphorylation and expression of pghs- mrna, whilst also reducing fak phosphorylation (n= ; p= . ). conclusions: fak has previously been shown to be important in activation of the stretch induced mapk cascade and pghs- expression. however these data suggest that while stretch causes fak phosphorylation fak kinase activity is not essential to pghs- expression. this suggests fak may be acting as a protein scaffold. . our aim was to examine the effects of both natural progesterone and hp on spontaneous myometrial contractions. myometrial biopsies were taken with informed consent and ethics approval from non-labouring women at elective caesarean section weeks gestation. strips of myometrium mm long , mm wide were cut and suspended under a resting tension of mn in organ baths of krebs gassed with % o / % co within hours of collection. progesterone or hp were added in a cumulative manner at -minute intervals. changes in amplitude were recorded. results were compared using anova. following equilibration for hours, myometrial strips contracted in a rhythmic manner (amplitude . ± . mn, n= pairs). progesterone ( nm- m) produced a concentration dependent inhibitory effect on myometrial contractions (fig ), which was greater than that of vehicle (p< . ). maximum inhibition measured . ± . % and . ± . % for progesterone and vehicle, respectively. hp exerted an inhibitory effect, this was not significantly different from the vehicle (p> . ). progesterone exerts an inhibitory effect on myometrial contractility in vitro. this is apparent within minutes suggesting a nongenomic action. our data are in agreement with some reports in the literature but conflict with others[ ]. this acute inhibition of myometrial contractility may contribute to the mechanism by which progesterone prevents preterm birth. in contrast, we were unable to demonstrate an inhibitory effect of hp on contractility despite its demonstrated ability to reduce the incidence of preterm delivery [ ] . our data suggest that natural progesterone may be a more effective tocolytic agent in the acute setting than hp. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: chemokines has been shown to play an important role in regulating uterine function. recent evidence demonstrates that monocyte chemotactic protein (mcp) level in amniotic fluid increases during spontaneous labor. the aim of this study was to examine the expression of mcp in human myometrium. methods: myometrial biopsies were taken from nonpregnant women undergoing hysterectomy and term pregnant women undergoing cesarean section followed written consent and local ethics committee approval. elective cesarean section was performed before the onset of labor while emegency section was done after the onset of labor. immunolocalization (n = each) was performed on paraffin sections by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . reverse transcriptionpolymerase chain reaction (n = each) using gene specific primer against mcp and mcp receptor was performed to identify mcp messenger(m) rna in human myometrium . results: immunohistochemical findings demonstrated mcp in human myometrial cells from nonpregnant, term pregnant women before and after the onset of labor. mcp was labelled on plasma membrane and cytoplasm of myocytes from these three groups of women. similarly, mcp and mcp receptor mrna were found in nonpregnant and term pregnant women before and after the onset of labor. in this prospective study a group of fetuses was consecutively enrolled. one ultrasound examination was performed to each patient within days from delivery. we considered fetal macrosomia a birthweight g and big babies a birthweight g. cut-off points for identifying the best value of fetal abdominal circumference for fetal macrosomia prediction were chosen by receiving operator characteristics' curve (roc) analysis. using the best cut-off indicated by roc analysis, specificity and sensitivity were calculated. mean gestational age at delivery was + weeks ( + sd). neonates weighted less than g, had a weight range between and g and weighted more than g. the fetal ac measurement was the selected criterium to evaluate the risk of fetal macrosomia. to identify macrosomic fetuses the roc curve analysis identified a cut off of ac > mm which allowed to select fetuses. analysing this population we found true negative cases, false negatives, false positives and true positives, with a sensitivity of . % and a specificity of %. to detect big babies the roc curve analysis identified a cut off of ac > mm (n. fetuses), reaching a sensitivity of % and a specificity of %, without false negative cases and with false positives ( true positives, true negatives). the cases that weighted between and g, were included in the cases considered false positives by this cut-off. conclusions. these results clearly indicated that ultrasounds alone can not be used to manage a pregnancy suspected for fetal macrosomia or big babies. in fact, to avoid shoulder dystocia and all other complications strictly related to macrosomic fetuses, clinicians should perform a large number of useless elective cesarean sections. . pathway analysis of differentially expressed genes (pa; z-score . ) showed up-regulation of axon guidance, folate biosynthesis, nitrogen metabolism and down-regulation of steroid biosynthesis, insulin signaling, oxidative phosphorylation, tgf-beta signaling, and ubiquinone biosynthesis pathways in cm vs cf. comparison of mnr vs c in f showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation steroid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, phosphatidylinositol signaling, ketone body metabolism, and ubiquinone biosynthesis pathways and down-regulation of bile acid biosynthesis, cell adhesion molecules, dna polymerase, notch signaling and ti diabetes mellitus pathways in mnr vs c. comparison of mnr vs c in m showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation of jak-stat signaling, autophagy, renin-angiotensin system (ras), and ubiquinone biosynthesis pathways and down-regulation of apoptosis, basal transcription, folate biosynthesis, nitrogen metabolism, protein export, and snare interaction pathways in mnr vs c. only the ubiquinone biosynthesis pathway up-regulation of mnr vs. c was common to both m and f. conclusions: ta and pa demonstrate sex-specific transcriptome expression in fetal kidneys at . g. in addition, these results show sexspecificity in response to mnr. we have seen similar effects of mnr on gene expression for autophagy, apoptosis, ras, ubiquinone and cell adhesion pathways at . g, suggesting these pathways may contribute to persistent affects of mnr. finally, we postulate that stress in utero may contribute to sex differences in risk of hypertension in adult life. leptin and neuropeptied y protein expression paradoxally increased in gestationall food restricted dams. louiza belkacemi, chun-hung chen, andrea jelks, michael g ross, mina desai. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: placental insufficiency is associated with marked increase in placental leptin production. this results in a rise in maternal leptin levels that serves as an early index of placental dysfunction. further increased placental leptin and suppressed neuropeptide y (npy) are associated with preeclampsia. leptin, an anorexigenic hormone and npy, an orexigenic peptide regulate food intake. importantly, leptin also serves as a placental and fetal growth factor. we have shown that maternal food restriction (mfr) results in intrauterine growth ... sf ()to:!)) df(,..)l) ...._ bf(jtolo) ... -m bf(,..lj) =- ( . ) <(u.)) ( . ) )( . )"" ' ~. ) !( . )' d.'cbom< ( ) f( ))""" s( l) background teenagers are more likely to deliver small-for-gestational age (sga) infants than adults, even after adjustment for socioeconomic factors , . previous studies of mostly black and hispanic subjects in the usa have suggested that maternal growth may contribute to reduced infant birthweight, due to preferential nutrient partitioning to the mother . the impact of maternal growth on birthweight and nutrient partitioning in pregnant teenagers in the uk has not been examined. methods skeletal growth (change in knee-height from st to rd trimester), weight gain and skinfold thicknesses were measured in pregnant teenagers (n= , % non-white) in london and manchester. key mediators of nutrient partitioning and metabolism: insulin-like growth factor(igf)- , igf binding protein(bp)- and leptin, were measured in maternal plasma ( weeks gestation). results maternal growth (defined as increase in knee-height > mm/ days) was detected in % of pregnant teenagers. this growth was not associated with sga birth; in fact these mothers were more likely to deliver large-forgestational age (lga) infants (p< . ). maternal weight gain and fat accrual at peripheral and central sites were greater in growers (p< . ). these parameters correlated positively with maternal igf- and leptin but negatively with the igf inhibitor, igfbp- (p< . for all). subjects delivering sga infants gained significantly less weight (p< . ) and had lower igf- levels (p< . ) than those delivering non-sga infants. conclusion maternal growth in teenage pregnancy was not associated with reduced birthweight. indeed the increased weight gain and fat accrual observed in growing teenagers may protect against sga birth and promote fetal growth. igf- and leptin promote fetal growth, primarily through effects on maternal metabolism and nutrient partitioning to the fetoplacental unit. these data suggest that higher maternal igf- and leptin in growing teenagers may provide an anabolic drive for both maternal and fetal growth. background. umbilical oxygen uptake (o umb uptake) has been estimated in human pregnancies only in acute experiments at the time of caesarean section. the recently developed possibility to measure umbilical blood flow by ultrasound in utero, prompted us to study normal and iugr pregnancies in order to evaluate fetal oxygen uptake utilizing the fick principle, i.e. uptake equals umbilical blood flow times (a-v) differences. methods. thirty-six iugr pregnancies were studied at the time of elective caesarean section and compared to twenty-one controls (c) (gestational age: c= . ± . and iugr= . ± . wks). an ultrasound examination was performed within hours from the caesarean section in all the recruited patients. umbilical vein absolute volume flow (qumb) was measured as the result between umbilical vein area and the time-averaged peak velocity * . . blood samples from umbilical vein (uv) and artery (ua) were obtained after the delivery and blood gases and acid-base balance were evaluated. umbilical oxygen uptake was calculated as o umb uptake = qumb*(uv-ua) o content. results. as expected, average fetal and placental weights were significantly different in the studied groups ( ± and ± g in iugr vs ± and ± g in n) . iugr pregnancies showed a significant reduction in qumb ( . ± . vs . ± . ml/min; p< . ) but no differences in the qumb/kg of fetal weight. iugr fetuses showed a significant reduction in o sat, o cont and po in both uv and ua compared to n. (uv-ua)o content ( . ± . vs . ± . mmol/l; p< . ) and o umb uptake/kg ( . ± . vs . ± . mmol/min/kg; p< . ) were significantly reduced in iugr. conclusions. we here report an evaluation of fetal oxygen uptake that proved surprisingly similar to the values reported in chronically catheterized animals. however, iugr fetuses showed a significant reduction in both blood and oxygen supply: this latter was reduced more that % on a per kg basis. iugr fetuses therefore utilize less oxygen than normally grown fetuses. circulating levels of vitamin d and il- in pregnancies with iugr. calvin j hobel, chander p arora, adegoke adeniji, priya arora, susan e jackman, olga miadel, baldjyan lilit. ob-gyn, cedars-sinai medical center, burns ans allen research institute, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: any condition resulting in under exposure to sunlight, including the use of sun block or poor nutrition may result in insufficiency ( . - nmol/l) or even deficiency of vitamin d (< . nmol/l).vitamin d regulates placental development and function. vitamin d deficiency has been linked to increased risk of serious chronic and inflammatory diseases. objective: to determine if the circulating levels of vitamin d and interleukin - (il- ) in maternal plasma correlates to pregnancies resulting in fetus with intrauterine growth restriction (iugr). hypothesis: the metabolism of vitamin d initiates the biochemical cascade of events leading to the expression of il- and the inflammatory response in iugr births. study design: in a behavior in pregnancy study, plasma samples at all three time points were analyzed in a cohort of women for (oh)d using elisa. the samples were also analyzed for il- at three stages of pregnancy: t ( - weeks), t ( - weeks) and t ( - weeks). iugr was defined as birth weight below the tenth percentile for gestational age. none of the iugr cases had spontaneous preterm birth. results: iugr was diagnosed in of women with available samples from a behavior in pregnancy study (bips) . out of these subjects, were selected as matched case controls. circulating levels of vitamin d ( (oh)d) were significantly lower in iugr cases at each visit (p<. ). the levels indicated deficient ( . ± . nmol/l) vitamin d in iugr group at t but sufficient vitamin d levels ( . ± . nmol/l) in controls. subsequent visits also showed lower levels in the iugr cases compared to the control group (t : ± . nmol/l vs ± . nmol /l; t : ± . nmol/l vs ± . nmol /l). at all three time intervals, significantly (p<. ) higher levels of il- were associated with the iugr cases ( pg/ml, pg/ml and pg/ml respectively) as compared to the controls ( pg/ml, pg/ml and pg/ml respectively). conclusions: vitamin d deficiency or even insufficiency may be an unrecognized cause of iugr. it is possible that a primary non-infectious inflammatory process is activated by vitamin d deficiency. combined assessment of vitamin d deficiency and il- expression during different stages of pregnancy may facilitate the resognition of the risk of developing iugr. response to global % maternal nutrient restriction (mnr). nathan drever, thomas j mcdonald, peter w nathanielsz, cun li. obstetrics and gynecology, university of texas health science center at san antonio, san antonio, tx, usa. background: igf-ii is a major growth factor in the developing pancreas and studies in the human fetus demonstrate that the peptide localizes to b cells of the islets (j endocrinology : ). rodent studies show decreased fetal pancreatic growth and igf-ii and ins abundance and increased apoptosis with mnr (j endocrinology : ). we previously demonstrated a fall in most components of the placental and fetal baboon liver igf systems with mnr. here we have evaluated fetal pancreatic igf-ii and ins changes in response to mnr. methods: pregnant baboons were fed ad lib (ctr, n= ) or % of wt adjusted ctr diet (mnr, n= ) from . gestation (g) and fetuses were recovered at c-section under general anesthesia at . (n= ; ctr and mnr) and . g (n= ; ctr and mnr). igf-ii and ins expression were determined by immunohistochemistry (ihc) and quantified by image analysis for fraction (area immunostained/area of the field x %) and density. data are expressed as mean + sem; ctr data are expressed] first; comparison made with two tailed t-test. results: at . g there was no difference in igf-ii or insulin fraction or density between groups. at . g, igf-ii fraction ( . ± . vs . ± . ,p< . ) and density ( . x ± . x vs . x ± . x , p< . ) and insulin fraction ( . ± . vs . ± . , p= . ) were reduced. conclusion: moderate mnr decreases abundance of fetal pancreatic igf-ii and ins at . g. in the fetal baboon and supports the extant evidence for impaired pancreatic development with mnr seen in rodents. maintenance of liver growth in the hypoxic growth restricted fetal sheep: a role for intrahepatic glut ? sheridan gentili, janna l morrison, i caroline mcmillen. sansom institute, unisa, adelaide, south australia, australia. objective: we have previously demonstrated that there is a differential tissue response to chronic placental and fetal growth restriction. growth of fetal tissues such as the brain and the adrenal are consistently spared in the face of chronic substrate restriction, whilst we have demonstrated that the growth of the fetal liver may be either maintained or reduced. it is unclear whether the growth response of the fetal liver to hypoxia and hypoglycemia are determined by intrahepatic metabolic adaptations. hypothesis: we hypothesize that there will be a differential profile of hepatic expression of glut , hsd , the gluconeogenic and glycolytic enzymes pepck and g pdh and the transcription factors pgc and ppar in animals in which liver growth is maintained or reduced. methods: carunclectomy was performed in non-pregnant ewes to induce placental restriction (pr). vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. mean gestation po < mmhg was defined as hypoxic (h; normoxia, n). post mortem was performed at - d. hepatic mrna expression of glut , hsd , pepck, g pdh, pgc and ppar was determined using qrt-pcr. results: four experimental groups were defined by fetal po and liver growth (c-n, pr-n, pr-h and pr-h-reduced liver growth). fetal weight correlated with mean gestational po (r = . , y= . x+ . , p< . ). liver weight was significantly lower in a cohort of pr-h fetuses (c, . ± . ; pr-n . ± . ; pr-h . ± . ; pr-h-rlg . ± . g:kg; p< . ). glut expression was highest in those pr-h fetuses in which liver growth was maintained, whilst the expression of hsd , pgc , ppar and pepck was highest in the pr-h fetuses in which liver growth was reduced (p< . ). conclusions: in the pr-h fetuses in which liver growth was reduced, the increase in hsd expression may be associated with an increase in hepatic exposure to cortisol and an associated increase in pepck, pgc and ppar . interestingly the compensatory increase in hepatic glut expression did not occur in fetuses in which liver growth was reduced. predicting the trajectory of fetal growth. racine n edwards-silva, jeffrey gornbein, calvin j hobel. obstetrics gynecology, los angeles, ca, usa; biomathematics, david geffen school of medicine at university of california, los angeles, ca, usa; obstetrics gynecology, david geffen school of medicine at university of california, los angeles, ca, usa. objective: to evaluate twelve potential predictors of fetal growth trajectory defined as the rate of fetal weight change over time. study design: a longitudinal prospective study of singleton fetal growth trajectory. the twelve potential predictors considered were: fetal gender, gestational age, parity, race, bmi, age, weight at st clinical exam, cumulative weight gain at each exam, smoking, and alcohol use. estimated fetal weight was computed using the hadlock formula and sonographic fetal biometric parameters at - weeks, - weeks, and - weeks. the rate of change in fetal weight was defined as x (fetal wt at exam j -fetal wt at exam i )(j > i)/ (gestational age at exam j -gestational age at exam i ). bivariate statistical analysis included the non-parametric spearman rank correlation and wilcoxon rank sum test. all factors were assessed multivariately using multiple linear regression. results: there were multi-ethnic women included in the study, after were excluded. they underwent a total of , exams. fetal gender (p= . ), maternal weight at st exam (p= . ), and cumulative maternal weight gain at exam (p < . ) were significant predictors of fetal growth trajectory. in this model, male fetuses had an average rate of fetal weight change of . grams per days higher than females. the rate of fetal weight change increased by an average of . grams per days for each lb increase in maternal weight at the st exam. the fetal growth rate increased . grams per days for each lb of cumulative weight gain at the rd exam. conclusions: in this study, maternal weight at the st exam and cumulative maternal weight gain were the significant determinants of fetal growth trajectory. adequate initial maternal weight and cumulative gestational weight gains probably ensure sufficient nourishment for normal placental growth, uteroplacental blood flow, and fetal nutrient uptake. this supports the emphasis on periconceptual nutritional counseling for all pregnant women. the implication of this study is that similar to fetal programming of adult diseases, there is nutritional programming of fetal growth trajectory. high risk patients. other predictors include the known risk factors of age < and > , single, tobacco/alcohol use, and african american race. interestingly, hispanic and native american patients have a lower lbw rate compared to other groups. introduction: catecholamines released by the sympathetic nervous system and adrenal medulla act via b-ars to regulate glucose and insulin function in liver, pancreas, adipose and muscle tissue. b-ar knock out mice show increased fat mass and glucose intolerance (asenslo et al.,diabetes, ) . mnr animal models have increased sympathetic activity. we, therefore, evaluated effects of mnr on baboon fetal liver b -ar. methods: baboons were fed as ad lib controls (ctr) or % of wt adjusted ctr diet (mnr) from . gestation(g) with fetuses retrieved at c-section under general anesthesia at . or . g. protein expression determined by immunohistochemistry for b -ar in the central liver lobule was quantified by image anaysis and expressed as fraction = area immuno-stained/area of the field x %. all data are expressed as mean + sem with ctr data presented first. liver glycogen expression was determined by the periodic accid schiiff (pas) method. comparisions were made with student's t-test with alpha level set at . . results: fetal body and liver wts were not changed by mnr at either age. pas stained liver glycogen at . g = . ± . vs. . ± . %, p< . . b -ar fraction was lower following mnr at . g and at . g (p< . ; fig ) conclusions: mnr decreased b -ar over % at . g and % at . g. decreased fetal liver b -ar in mnr alters glucose metabolisam and may result in reduced lipolysis predisposing to fatty liver. infection with noncytopathic bovine viral diarrhea virus (ncpbvdv) during early bovine pregnancy (< d gestation) results in fetal immunotolerance, persistent infection (pi) and intrauterine growth restriction (iugr). in contrast, infection after the development of adaptive immune competence (> d gestation or postnatal) results in a transient infection (ti). we have previously reported an iugr in pi fetuses presenting as decreased body weight and ponderal index. a growth defect can be the result of many factors, including placental insufficiency and nutrient restriction, however it was hypothesized that the iugr seen in bvdv pi fetuses may be an immunopathological effect caused by the persisting virus. our two part experimental design examined the relationship between bvdv and its pi host. in experiment (exp) , blood cell mrna was collected from pi steers (n= ; confirmed by virus isolation), or uninfected control steers (n= ) and used to identify differentially expressed genes using microarray (affymetrix) and qtrt-pcr approaches. in exp , bvdv naïve pregnant heifers (n= per group) were not infected (control) or infected with ncpbvdv on d. or d. of pregnancy creating pi and ti fetuses, respectively. fetuses were collected by c-section on d. ; infection was confirmed by elisa and qtrt-pcr. histology of placental tissue revealed no placentitis or pathology, and glucose and lactate levels in fetal serum were normal. microarray analysis revealed genes that were differentially regulated in pi vs. controls (p< . , > . fold). qtrt-pcr of steer and fetal blood revealed a significant upregulation of activators and products of the antiviral type-i interferon (ifn-i) pathway. as ifn-i can act as a growthsuppressive cytokine, a long-term upregulation may contribute to the iugr seen in persistent bvdv infection and in other viral infections observed during pregnancy. nricg - from the csrees. patients with a short cervix are at an increased risk for spontaneous preterm delivery. therefore, it is possible that women with a short cervix during pregnancy are also at risk to deliver an sga neonate. this study was conducted to address this question. study design: patients > weeks of gestation were prospectively enrolled into an observational study ( / to / ). transvaginal sonographic examinations were performed every weeks until delivery. the shortest cervical length between - weeks was used for analysis. sga was defined as less than the tenth percentile of birth weight. results: asymptomatic patients were studied. . % of patients delivered an sga neonate ( / ). the median cervical length was mm ( to mm); patients had a cervical length < mm. of these, % ( / ) delivered an sga neonate. similarly, % ( / ) patients with a cervical length < mm had an sga neonate. no relationship was found between a short cervix and sga (short cervix was defined as either < mm and < mm). the frequency of sga was not significantly different between women with a short cervix and those with a long cervix [ % ( / ) vs. % ( / ); p= . ]. newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced background folate is an essential micronutrient for cellular growth. recommendations on periconceptional folic acid use are mainly focussed on prevention of neural tube defects, despite growing evidence that folic acid use may have positive effects on birth weight. objective to examine associations between folic acid use, intrauterine fetal growth and birth weight. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods information on folic acid use was obtained by questionnaires and categorized into three groups: ) preconception start of folic acid use; ) start of folic acid use in first ten weeks of gestation; ) no folic acid use at all. fetal growth measurements included head circumference, abdominal circumference and femur length measured in mid-and late pregnancy, i.e., gestational age - and > weeks, respectively, and birth weight. fetal weight in mid-and late pregnancy was estimated using the haddock method. results data from , pregnant women were available. overall, folic acid use was positively associated with fetal growth. preconceptional folic acid use resulted in an increased growth of grams ( %ci . - . , p< . ) per week from late pregnancy to birth, compared to no folic acid use. similarly, start of folic acid use in the first ten weeks of gestation resulted in an increased growth of grams ( %ci . - . , p= . ) per week from late pregnancy to birth. both preconceptional folic acid use and folic acid use started in the first ten weeks of gestation resulted in higher birth weights of grams ( %ci - ) and grams ( %ci - ), respectively, compared to no folic acid use. a tendency was found for an increased risk of birth weight less than grams when folic acid was not started preconceptionally (or . , %ci . - . ). conclusion periconceptional folic acid use is significantly associated with increased fetal growth resulting in a higher birth weight. ductus venosus isovolumetric relaxation in severely premature growth-restricted fetuses. jason l picconi, katherine drennan, farhan hanif, michael kruger, giancarlo mari. obstetrics and gynecology, wayne state university/dmc, detroit, mi, usa. objective: ductus venosus (dv) doppler waveforms are characterized by two periods in which blood velocity decreases. the first represents the isovolumetric relaxation (ir) at the end of ventricular systole and the second represents atrial contraction at the end of ventricular diastole (a). ductus venosus reversed flow (dvrf) occurring at the time of the a-wave is considered a risk factor for intrauterine fetal demise (iufd). we have previously reported that absent or reversed a-wave flow can be present for weeks before iufd occurs or delivery is performed for non-reassuring fetal testing. the guiding hypothesis for this study is that decreased flow at the time of ir in combination with absent or reversed a-wave flow allows a more accurate prediction of fetal outcome than a-wave absent or reversed flow alone. material and methods: ductus venosus doppler was serially studied in severely premature iugr fetuses (estimated fetal weight < th percentile and umbilical artery pulsatility index > th percentile) from diagnosis until demise or delivery. ductus venosus waveforms were assessed quantitatively for peak systolic velocity (psv), isovolumetric relaxation velocity (irv), and end diastolic velocity (edv). the psv/irv + edv were compared to fetal and neonatal outcome. a kruskal-wallis one way anova, mann whitney u post-hoc test, and a roc, were used for statistical analysis. a p < . was considered statistically significant. results: all fetuses were delivered at < weeks. six cases resulted in iufd, five cases resulted in neonatal demise (nd), and six cases resulted in neonatal survival (ns) at the time of discharge from the hospital. the psv/irv+edv correlated better than a-wave reversal of flow with perinatal outcome. a psv/irv+edv score less than - . resulted in iufd, whereas a score greater than - . resulted in live birth. live births segregated based on estimated gestational age, where those fetuses at less than weeks resulted in nd and those fetuses at or greater than weeks resulted in ns. all results were statistically significant. conclusions: the isovolumetric relaxation velocity is a novel doppler parameter in the assessment of severely premature iugr fetuses. these data indicate that assessment of irv should be considered part of the evaluation of severely iugr fetuses. background: fetal growth restriction has been linked to an increased incidence of chronic hypertension, which may be the result of extracellular matrix changes (ecm) within the vascular tree. in previous studies, ecm changes were observed in the umbilical arteries of preterm growth restricted infants. objective: to determine if there are alterations in collagen subtypes within the umbilical cords from growth restricted fetal rat pups after a period of maternal nutrient restriction. methods: timed pregnant sprague-dawley rats were fed either a % food restricted diet (mfr; n= ) or were fed ad libidum (control; n= ) from d until d of gestation. litter size, fetal weights and placental weights were then noted and umbilical cords from randomly selected pups in each litter were snap frozen. gene expression for collagens i, iii, xiv and decorin was evaluated by real-time rt-pcr with normalization to the gadph housekeeping gene. data were analyzed from fitting general linear regression models with estimation based on the quasi-likelihood estimation (generalized estimating equations) to account for clustering of responses within litters. data are shown as cycles to amplification (ct), which is inversely proportional to mrna levels. results: no difference in median litter size was detected. however, fetal and placental weights in the mfr group were significantly less than those from control dams. no significant differences in umbilical cord gene expression for collagens i, iii, xiv or decorin were detected between mfr and control pups. conclusions: maternal food restriction does not result in any detectable alterations in collagen or decorin expression within the intact umbilical cord, despite causing significant reductions in fetal growth. control (n= ) p-value litter size (median, range) ( , ) ( , ) . fetal weight (mean ± sd) g . ± . . ± . < . placental weight (mean ± sd) g . ± . . ± . . collagen i (mean ± sem) ct . ± . . we have previously demonstrated that the restriction of placental and fetal growth results in fetal hypoxia and fetal brain sparing suggesting a redistribution of cardiac output. furthermore, placentally restricted (pr) hypoxic fetuses are more dependent on their sympathetic nervous system for the maintenance of blood pressure during late gestation. nerve growth factor (ngf) plays a significant role in sympathetic innervation. hypothesis: we hypothesize that the expression of ngf will be higher in the aorta and femoral artery of the pr hypoxic compared to the control fetus. method: carunclectomy was performed in non-pregnant ewes to induce pr. vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. all pr fetuses introduction elevated sflt- levels have been shown to be a feature of pre-eclampsia and is considered to play a significant role in the pathogenesis of the condition. the role of angiogenic factors in fetal growth restriction has not been as well established. this study evaluated the levels of circulating vascular endothelial growth factor (vegf) and its soluble receptor sflt- , in normal pregnancies and isolated placental vascular disease without evidence of pre-eclampsia. method maternal peripheral venous samples were collected antenatally from two groups of pregnant women between - weeks of gestation. group a: uncomplicated normal pregnancies (n = ) and group b: pregnancies complicated by isolated placental vascular disease( n = ) as defined by birth weight less than th centile for gestation and umbilical artery doppler s:d ratio above the th centile for gestation, with no evidence of maternal preeclampsia or pre-existing hypertension. plasma vegf and sflt- levels were measured using standard eliza techniques. comparison between groups were performed by using one way analysis of variance. the maternal plasma sflt- levels (figure ) in group a: normal pregnancies increased with gestation (p < . ). the sflt- levels in group b: isolated placental vascular disease were significantly higher throughout all gestations (p = . ) and did not show a significant variation with gestation ( p = . ). the plasma vegf levels were below the detectable levels of the assay in all samples except normal pregnancies. the significantly increased maternal plasma sflt- levels in established isolated placental vascular disease without evidence of pre-eclampsia suggest a disease of placental origin. the dysregulation of angiogenic factors may be part of a repair and regeneration process in the placenta. objectives: production of -reduced neurosteroids is critical for reducing fetal vulnerability to stressors in pregnancy and inhibition of -reductases ( r) increase acute hypoxia-induced apoptosis in the fetal brain (yawno et al neurosci : . we have developed a model of placental insufficiency that results in fetal growth restriction (gr) in the guinea pig (palliser et al repro sci # ). the aim of the present study was to determine the effects of suppression of -reduced steroid synthesis on apoptosis in vulnerable regions of the fetal brain and on neurosteroid synthetic enzymes in pregnancies compromised by chronic placental insufficiency. methods: placental insufficiency was induced in guinea pig dams by surgical ablation of uterine artery branches at mid gestation (term d). sham operated or gr dams received finasteride (a r inhibitor; mg/kg/day) during late gestation ( d until term). activated caspase- , a marker of apoptotic cell death, was measured by immunohistochemistry and steroidogenic enzymes, r and cytochrome p side chain cleavage (p scc) were measured by real time pcr and western blotting in fetuses ( d) and neonates h after birth. results: placental insufficiency significantly reduced fetal body and organ weight by % whilst sparing brain weight. the number of activated caspase- positive cells was significantly increased in the fetal hippocampus of gr fetuses and further increased in the cortex of gr fetuses receiving finasteride. the neonatal brain also exhibited changes in caspase- activation following gr and finasteride treatment. the fetal adrenal and the placenta responded to the compromise with increased expression of p scc mrna and r protein, respectively. conclusion: the combination of placental insufficiency and suppressed neurosteroid system leads to markedly increased apoptotic cell death in the fetal brain which continues to affect the neonatal brain. the fetus responds to these conditions by increasing steroid synthetic enzyme expression in the placenta and adrenal glands suggestive of a possible neuroprotective feedback process. to study the effect of smoking by mothers on the fetal and neonatal brain using non-invasive magnetoencephalography technique (meg). materials and methods: using fetal magnetoencephalography, cortical auditory evoked responses (aer) were measured from fetuses ranging from to weeks gestational age for a total of recordings. measurements were taken from mothers with a history of smoking (sm) and from mothers with no smoking history (ns). after delivery, five sm and five ns newborns had meg aer measurements twice for a total of recordings. aer was quantified by cross-correlation analysis and its significance was assessed by boot-strap technique. results: aers were detectable in out of fetal recordings in the ns group and of in the sm group. the neonatal response rate was % for each group. the latencies were divided into three components: c ( - ms), c ( - ms) and c ( - ms). in both fetuses and neonates as well, there was a statistically significant difference (p< . ) between the two groups in the c -component and the sm group showed faster aer compared to the lr group. conclusion: meg technique provides a non-invasive approach to study the effects of smoking on developing fetal and neonatal brain. the observed decrease in the latency of fetuses and neonates of the smoking mothers could indicate a hypersensitive cortical response to auditory tone. adenosine (ado) modulates metabolism in adult mammals through multiple mechanisms that involve ado a and a a receptors. objective: this study was designed to test the hypothesis that ado a and a a receptors participate in fetal metabolic homeostasis. methods: experiments were performed in chronically catheterized fetal sheep (> . term). intravascular infusion for h of dpcpx (a receptor antagonist) or zm (zm, a a receptor antagonist) was performed alone or in concert with ado administration. the highly selective ado receptor antagonists were also infused in fetuses in which hypoxia was induced for h by having the ewe breathe a hypoxic gas mixture (fio = . ). data were analyzed by two-way repeated measures of anova. results: blockade of ado a receptors (n= ) increased significantly (p < . ) fetal concentrations of glucose [control (c): . ± . (se)]; experiment (e): . ± . mg/dl] and lactate (c: . ± . ; e: . ± mg/dl) without significantly altering insulin levels and arterial blood gases or ph. antagonism of ado a a receptors (n= ) did not affect plasma levels of glucose, lactate, or insulin. intravenous infusion of ado (n= ), which did not alter pao or paco , increased concentrations of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . ). zm (n= ), but not dpcpx (n= ), abolished ado-induced rise in glucose and lactate concentrations. isocapnic hypoxia (pao torr), which increases ( - fold) fetal plasma ado levels to those similar to ado infusion, decreased arterial ph (c: . ± . ; e: . ± . ), and increased fetal levels of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . mg/dl) without altering changing insulin concentrations. these effects of hypoxia were not altered by dpcpx or zm. conclusions: ) a receptors modulate plasma levels of glucose and lactate in normoxic fetuses; ) a a receptors mediate the adoinduced rise in plasma glucose and lactate; and ) a and a a receptors are not significant modulators of hypoxia-induced changes in plasma levels of glucose and lactate. supported by usphs hd- . objective: to investigate the dynamic changes of the relationship between leptin, adiponectin and resistin in maternal and fetal circulation during pregnancy and in the early post-natal period. materials and methods: thirty pregnant women with uncomplicated singleton pregnancy delivered at term. maternal and fetal/neonatal venous blood samples were obtained at delivery and at hours from birth. leptin, adiponectin and resistin were measured by specific elisa assays. neonatal anthropometric measurements, glucose metabolism and lipid profile, blood pressure information were obtained. statistical analysis was performed by anova followed by student t test or duncan's test whenever appropriate. correlations were calculated by using the pearson coefficient. results: adipokines concentration at birth and at h from birth was showed in table. multivariate regression analysis showed that fetal leptin levels were positively associated with female gender and adiponectin levels, but not with anthropometric characteristics. fetal leptin and adiponectin levels were not correlated with maternal concentration, whereas fetal and maternal resistin levels were. fetal and maternal resistin concentration was positively associated with gestational age and birth weight and fetal resistin levels correlated negatively with lipid profile. after birth leptin concentration in maternal and neonatal circulation decreased dramatically and the correlation between leptin and adiponectin levels was lost. a positive correlation between resistin and leptin concentration in neonatal circulation was found at h from birth. conclusions: in contrast to adult life a positive correlation between leptin and adiponectin is present in fetal life. placental secretion of leptin, but not of adiponectin and resistin, contributes significantly to maternal and fetal circulating levels. significant changes in the relationship among adipokines occurred immediately after birth and may affect growth and development in early post-natal period. adipokines concentration in maternal and fetal circulation ) antagonism has been shown to normalize placental perfusion and fetal growth in several rat models of fetal growth restriction. however, direct administration of et a antagonists to newborn rats within hours of delivery has been consistently associated with neonatal demise due to failure of the ductus arteriosus to close, raising concerns about the safety of their use late in pregnancy. perinatal exposure to et a antagonists (maternal administration in late gestation) and its impact on rat pup survival and oxygen saturation has not been investigated. objective: to determine the impact of a maternally administered et a antagonist on oxygen saturation in newborn and -day-old rat pups. methods: timed pregnant sprague-dawley rats were treated with fr ( mg/kg/day; et a antagonist) or . % nahco vehicle, by subcutaneous osmotic pump connected to an intravenous catheter, from gestational day (term= days) through parturition. all five pregnant rats in each group delivered spontaneously and nursed their pups through postpartum day . oxygen saturation of each rat pup was measured by pulse oximeter on postpartum days and . results are presented as means ± se. newborn -day neonate vehicle . ± . . ± . eta antagonist . ± . . ± . there were no statistically significant differences between the treatment groups. maternal administration of an et a antagonist from gestational day through parturition, at a dose sufficient to ameliorate fetal growth restriction, has no adverse impact on oxygen saturation in neonatal rat pups. helen l torrance, jan b derks, martijn a oudijk, avnesh s thakor, tereza cindrova-davies, frank van bel, gerard ha visser, graham j burton, dino a giussani. perinatal center, university medical center utrecht, netherlands; department of physiology, development neuroscience, university of cambridge, united kingdom. introduction: the management of perinatal asphyxia remains a major concerns in obstetrics. umbilical cord compressions (ucc) induce fetal asphyxia and ischaemia-reperfusion (i/r). i/r increases reactive oxygen species, for instance via activation of the xanthine oxidase (xo) pathway, which may promote oxidative stress in the fetal circulation. while treatment with allopurinol of asphyxic human neonates reduced free radicals and improved cardiovascular status, treatment started postnatally was deemed too late to prevent oxidative damage (benders et al. arch dis child : , ) . consequently, in complicated pregnancy, recommendations to treat the fetus via the mother, rather than the neonate, with allopurinol are being entertained today. we investigated the effects of maternal allopurinol treatment on indices of oxidative stress in the fetal heart following repeated ucc in sheep. methods: at . of gestation, surgically instrumented sheep fetuses were submitted to i/r ( x min repeated ucc) under maternal allopurinol (n= ) or saline vehicle (n= ) infusion. fetal hearts were collected h after i/r and snap frozen for measurement of (anti)oxidant proteins by western blot. hearts from uninstrumented fetal sheep at . gestation served as controls. statistical comparisons were made using one-way anova. results: i/r episodes led to increased expression of cox- , enos, hsp and decreased expression of sod and gluthatione peroxidase (gpx) in the fetal heart, findings consistent with cardiac oxidative stress. maternal treatment with allopurinol ameliorated these effects (fig. objectives: hypoxic-ischemic fetal brain injury (hie) is a major cause of neonatal death and morbidity. evidence suggests that the brain cell injury associated with chronic hpx (in contrast to an acute ischemic reperfusion injury) is a complex process reflecting a series of adaptive intracellular events that are duration and gestational age dependent. we applied advanced proteomic tools as a next step toward ascertaining a more complete understanding of the impact of hpx on the fetal brain proteome. methods: time-mated guinea pigs were housed in a chamber beginning on day for d, breathing either room air (nmx), or . % or % o ( % o hpx or . % o hpx). on day (term), the fetal brains were removed and preserved for study. total protein was extracted, and the proteome first characterized by d gel electrophoresis. the density of the resulting protein spots were acquired and analyzed using the gs densitometer and pdquest software. identified spots of interest were trypsin digested and subject to maldi mass spectrometry using the proteomics analyzer mass spectrometer for peptide mapping or sequencing. the hpx-induced protein spots were identified based on a minimum of a x change from nmx. results: hpx had a clear effect on the fetal brain proteome. superoxide dismutase (sod), heat shock protein (hsp ), actin (actg ), interleukin- (il- ), and glutamine synthetase (gs) were each up regulated, while cofilin- , brain-type creatine kinase (bb-ck), and - gtp binding protein were each down regulated. all protein changes were proportional to the hpx ( % o hpx vs nmx, % o hpx vs . % o hpx, p< . ). conclusions: this initial application of proteomic techniques confirms that fetal brain damage secondary to chronic hpx (in contrast to acute hpx) is a complex processes characterized by fetal adaptations mediated by multiple protein activations and inactivations. while sod, il- , and gs have each been previously investigated, the potential roles of actg , bb-ck, beta- gtp binding protein, and cofilin- in the fetal response to hpx and the associated brain damage are unclear and represent strong candidates for future investigation. acknowlegement: this study was supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). intermittent umbilical cord occlusion occurs in % of human pregnancies.given that elastogenesis within the vascular wall is in part mediated by hemodynamic conditions during development, the blood pressure response to acute hypoxic insults such as cord occlusion may alter arterial composition. elastin content of a central and peripheral artery and blood pressure responses in fetal sheep exposed to varying degrees of cord occlusion were determined. methods: over a day period, near term fetal sheep received total umbilical cord occlusion (uco) lasting min/ hour (mild group; n= ), min/hour (moderate group; n= ), min/hour (severe group; n = ) or no occlusion (control group; n= ). fetal arterial blood samples were drawn min prior to and at the end of cord occlusions. mean arterial pressure (map) was monitored continuously. the carotid and superior mesenteric arteries were excised and a colorimetric assay (biocolor) performed for determination of elastin content. results are presented as mean ± sem. results:umbilical cord occlusions produced decreases in fetal arterial oxygen pressure and oxygen saturation that were progressively more pronounced across mild, moderate and severe uco groups (p < . ). lactate concentration rose during occlusions in the moderate and severe groups, but not the mild group (p < . ).elastin content of the superior mesenteric artery did not differ between the experimental groups and the control group. elastin content of the carotid artery and map response elastin content (μg/mg tissue) max ∆ in map duration of rise in map (min) control . ± . . ± . -mild . ± . . ± . ** . ± . moderate . ± . . ± . ** † . ± . † severe . ± . * . ± . ** † † . ± . † † * p < . ; ** p < . : experimental groups compared to control † p < . ; † † p < . : compared to mild group the max in map was positively correlated with elastin content (r = . , p < . ). conclusions:the transient rise in blood pressure and preferential blood flow to the brain that occur in response to acute hypoxemia during severe intermittent umbilical cord occlusion induce an increase in elastin synthesis in the carotid artery.this may give rise to adaptive programming of postnatal central arterial compliance. electrocortical activity during repetitive umbilical cord occlusions (uco) with worsening acidemia in the ovine fetus near term. martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. obgyn, chri, univ of western ontario, london, canada; obgyn, harbor-ucla med ctr, torrance, ca. objective: uterine contractions during labour can restrict umbilical blood flow compromising fetal oxygenation and leading to adverse neonatal outcome including newborn encephalopathy/subsequent cerebral palsy. while electronic fetal heart rate (fhr) monitoring is widely used for assessment during labour, abnormal fhr patterns as used clinically have a poor positive predictive value for concerning/significant acidosis at birth. there is limited study of fetal electrocortical activity (ecog) in animal models with induced hypoxia/ acidemia as might be seen during labour. thus, we aimed to induce repetitive ucos in fetal sheep leading to worsening acidemia to determine the predictive value of ecog activity for fetal compromise. methods: near-term fetal sheep (n= ) underwent chronic preparation with artery catheters, ecg/ecog electrodes and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) ucos with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood samples for blood gases and ph were taken at selected time points during the baseline, ucos, and recovery periods. arterial blood pressure (abp) and fhr were continuously monitored and spectral edge frequency (sef) was calculated from ecog. correlation of sef with fhr change was calculated for time intervals using sef maxima and fhr minima during uco series. data are presented as means±sem. results: repetitive ucos led to development of a marked acidosis (ph . ± . to . ± . , p< . ). ± min prior to fetal ph drop < . , the sef of ecog began to increase abruptly during each fhr deceleration from ± hz up to ± hz (p< . ) and was correlated to both fhr change and a decrease in fetal abp during each fhr deceleration at this time (p< . ). conclusion: our findings suggest that in the animal model studied ( ) fetal ecog activity is impaired with progressive acidemia accompanied by fhr decelerations and pathological abp decreases; ( ) there is a consistent temporal relationship between the occurrence of ecog alterations and the subsequent critical drop of ph < . . these findings could contribute to improvement in the clinical ability to predict fetal compromise during labour using ecog/fhr monitoring. background: the ductus arteriosus (da) plays a pivotal role in fetal development and circulation. during gestation, patency of the fetal da is maintained by nitric oxide (no) and prostaglandins (pg). no and pgs are downstream effectors of estrogen (e ) and progesterone (p ). however, the roles of e and p in da regulation have not been studied. objective: we hypothesized that: e and p have opposite effects in the da; that rising e and falling p levels help trigger da closure via specific hormone receptors; and that no and/or pgs mediate da responses to e and p . methods: expression of er , er , pra, prb, and the putative membrane receptors mpr-, -, -, pgrmc- , - , and serbp- was examined by rt-pcr and qpcr on days , , (term), and p . the effects of e and p ( - - - m) on the d fetal da were examined in a cannulated microvessel myography system. e and p effects were also studied in the presence of receptor antagonists (ici , ru ) and no and pg inhibitors (l-name, indomethacin). results: er and pr receptors were expressed at low levels; pgrmc- expression was stronger than other membrane prs, and increased with advancing gestation. under fetal o conditions, e induced rapid, concentrationdependent dilation at - - - m (n= ); p induced progressive vasodilation at all doses (n= ). e and p -induced dilation occurred within - seconds of exposure. while e -dilated das did not respond to ici, ici-pretreated das failed to dilate to the same dose of e (n= ) suggesting antagonism of e effects. the vasodilatory effects of e were partially inhibited by pretreatment with l-name. p -dilated das did not respond to ru , whereas ru pretreated das showed a small, significant response to p (n= ), suggesting partial antagonism or signaling via alternative, ru -independent pathways. pre-treatment with indocin did not block the vasodilatory effects of p . conclusions: contrary to our expectation, both e and p have dilating effects on the fetal da, via no, pgs and non-no, non-pg pathways. expression studies and the rapid response of ex vivo das are consistent with non-genomic actions via membrane receptors. hormone shifts in parturition may have longterm effects on da preparation for postnatal closure, but strategies to maintain fetal da patency or treat newborns with pda will require better understanding of this process. background. for the fetus, arterial blood gases are critical in the regulation of cerebral blood flow (cbf) and cerebral oxygenation. however, the relation of cbf, cortical tissue po (tpo ), and electrocorticographic (ecog) activity to arterial o tension (pao ) are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that in the near-term fetus, acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with ecog state. methods. by use of a laser doppler flowmeter with a fluorescent tissue o probe, and with fluorescent-labeled microspheres, and with ecog electrodes, in near-term fetal sheep (n = ) we measured laser doppler cbf (ld-cbf), tpo , ecog (root mean square (rms) voltage with high voltage low frequency, hvlf versus low voltage high frequency, lvhf) and spectral edge frequency- % (sef ) in response to min moderate isocapnic hypoxia. results. ld-cbf, cerebral o delivery, tpo , and several other variables correlated highly with ecog state. in the normoxic control fetus, in association with a shift from hvlf to lvhf ecog activity, tpo decreased briefly to ± from a control value of ± torr; however, as ld-cbf increased ± %, and sef increased to ± from ± %, tpo returned to near normal value. with acute hypoxia (pao = ± torr) when in the lvhf state ld-cbf increased only ± %, as opposed to a ± % increase when in hvlf ecog state. with this degree of hypoxia, tpo decreased to ± torr, sef remained at ± %, and cerebral metabolic rate for o (cmro ) decreased ± % (p< . ). conclusions. for the near-term fetus, normoxia with changes in ecog state was associated with brief periods of decrease in tpo , which were restored quickly by increased ld-cbf. in contrast, acute hypoxia was associated with a significant depression of cortical tpo , cmro , and ecog state, with increased ld-cbf failing to restore cortical tpo . thus, we reject the null hypothesis that in such fetuses, hypoxia demonstrates no compromise in cerebral oxygenation. (supported by usphs hd- ). releasing hormone and arginine vasopressin in the ovine fetus. charles a ducsay, kanchan m kaushal, malgorzata mlynarczyk, kimberly hyatt, dean a myers. ctr. for perinatal biol., loma linda univ., loma linda, ca; ob/gyn, univ. oklahoma hlth. sci. ctr., oklahoma city, ok. background: long term hypoxia (lth) profoundly affects the hypothalamicpituitary-adrenal axis of the fetal sheep. we previously showed that lth causes augmented corticotrope function. the present study was designed to test the hypothesis that lth enhances sensitivity to the acth secretagogues; cortiocotrophic releasing hormone (crh) and arginine vasopressin (avp) resulting in increased anterior pituitary corticotrope secretion of acth. methods: pregnant ewes were maintained at high altitude ( , m) from day to - of gestation (dg), when they were returned to the lab and a maternal tracheal catheter was implanted. maternal po was maintained at a level comparable to that observed at altitude ( mmhg) by nitrogen infusion. on dg, lth (n = ) and age-matched, normoxic control (n = ) fetuses were implantated with vascular catheters. each fetus received a min infusion of either saline vehicle, ng/kg of ovine crh or ng/kg of avp (estimated body weight/min) in a randomized order over consecutive days ( - dg). blood samples were collected at min (baseline prior to infusion), , , and min following the start of the infusion and analyzed for acth, as well as the acth precursors pro-opiomelanocortin and the major processing intermediate kda proacth. results: vehicle had no effect on any of the measured parameters. with crh infusion, acth (pg/ml) increased in both groups over the course of the study. however, peak concentrations (at min) were significantly higher in the lth group compared to control ( ± vs. ± , respectively; p< . ). acth precursor secretion (pm) was greater in lth fetuses compared to controls during the experiment (p< . ). in response to avp, peak acth concentrations were also higher in the lth fetuses compared to control ( ± vs. ± respectively; p< . ), however peak levels were reached at between and min after start of infusion with levels in both groups returning to pre-infusion values. a similar pattern was observed with precursor levels ( . ± . vs. . ± . , p< . , lth vs. control). conclusions: lth significantly increases pituitary sensitivity to both crh and avp. this enhanced sensitivity may be mechanism of our previously observed enhanced corticotrope function. (supported my nih grants hd and hd ). martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. ob/gyn, chri, university of western ontario, london, canada; ob/gyn, harbor-ucla med. ctr., torrance, ca. objective: repetitive uco leading to worsening fetal acidosis with fetal heart rate (fhr) decelerations (fhr dec ) are accompanied by pathological decreases of fetal arterial blood pressure (bp). we hypothesized this bp change may be caused by the bjr, a vagally mediated reflex with bradycardia and hypotension to reduce cardiac workload, via stimulation of cardiac chemoreceptors during systemic acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. after a control period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco each lasting h or until ph decreased to < . . fetal arterial blood samples were taken at selected time points during the control and uco periods. bp and fhr were continuously monitored. individual fhr nadir during each fhr dec and accompanying bp change ( bp = [bp at the time of a fhr nadir] -[bp at baseline preceding a uco series]) were determined during all uco. data are presented as means±sem. results: control period ph ( . ± . ), fhr ( ± bpm) and bp ( ± mmhg) were within the physiological range. average depth of fhr dec for all uco was ± bpm and increased with higher lactate concentrations (r = . , p < . ) and lower ph (r = . , p = . ). bp during all uco demonstrated an initial hypertensive response to fhr dec , which decreased with lower ph (r = . , p < . ). bp increased ± mmhg (p < . ) during mild uco (ph to . ± . ), ± mmhg during moderate uco (ph to . ± . , p < . ) and ± mmhg during severe uco (ph to . ± . , p < . ). conclusion: these results suggest that bjr, a short-acting, ph dependent depressor reflex, blunts the physiologic hypertensive response to cord occlusion insults leading to worsening acidemia as might be seen in the human fetus during labour with repetitive variable fhr dec . the failure to increase bp may prevent optimal blood flow distribution responses necessary for preservation of vital organs. role of nos and pde in placental dysfunction following fetal bypass. mitali basu, r scott baker, christopher t lam, kenneth e clark, pirooz eghtesady. cardiothoracic surgery, cincinnati children's hospital, cincinnati, oh, usa; ob/gyn, university of cincinnati, cincinnati, oh, usa. introduction rising placental vascular resistance following fetal cardiopulmonary bypass (bypass) remains the achilles' heel of fetal cardiac surgery. we have previously shown in real-time that nitric oxide (no) production rises during bypass and falls post-bypass, while cgmp levels rise throughout. using immunohistochemical and western analysis of placenta, we examined the involvement no pathway components in this placental vascular pathophysiology. ovine fetuses at - days gestation were placed on bypass for minutes and followed post-bypass for hours. placental samples were collected immediately prior to bypass and at and min post-bypass (n= ) and compared to a group of similarly instrumented controls, (n= ). placental enos, inos and pde protein expression was measured using standard methods and relative expression normalized to beta-actin. statistical analysis utilized students t-test, and anova for trend and group-wise analysis, (significance at p= . ). pre-bypass protein expression did not differ between groups. pde protein levels and phosphorylated pde- expression were both elevated min post bypass, and reduced min post bypass compared to sham, (p= . ). enos levels in the bypass group increased linearly from pre-bypass to min postbypass (p< . ), and were also elevated compared with shams (p< . ), while shams had declined significantly by min post bypass, (p< . ). similarly, phosphorylated enos expression in the bypass group increased linearly from pre-bypass to min post-bypass, (p= . ), while shams trended towards decline by min post-bypass. simultaneously, placental inos expression remained stable within groups, but was lower in the bypass group at and min post-bypass, (p< . ). the preceding data correlated with observed immunohistological changes in the same placental cotyledons. fetal bypass leads to significant increases in placental protein levels of pde , phosphorylated pde- , enos and ostensibly phosphorylated enos. increased pde expression may be a response to increased no and the generated cgmp. this data suggests a compensatory upregulation of pde and enos that eventually fails, leading to increasing placental vascular resistance and subsequent lethal placental dysfunction. angiotensin receptors (rat-ii) in neuronal nuclei of the brainstem have been implicated in integration of baroreceptor responses. in near term fetal sheep, we have previously shown that days of mild chronic hypoxemia increases heart rate (hr) and blood pressure (bp). the aim of the present study was to investigate the effects of chronic mild hypoxemia on the expression of rat-ii in the medulla oblongata and on baroreflex control of the circulation. methods: at days of gestational age, pregnant sheep were submitted to days of hypoxemia ( % of fetal arterial po ; at day fetus ± vs ± . ; mother ± vs ± mmhg). hr and arterial bp were continuously recorded from mother and fetus in control (n= ) and hypoxemic (n= ) animals. baroreflex sensitivity (brs) as well as hr and diastolic bp variability were analyzed (nevrokard software). brainstem was collected and rat-ii expression in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) was measured by autoradiography using i-sarthran labeling. data are shown as mean±sem and were analyzed by t-test. results: hypoxemia induced a greater total binding of i-sarthran in nts and dmnx resulting from an increased expression of at receptors. in the time-domain analysis of brs hypoxemia induces lower baroreflex sensitivity in the fetus (brs in ms/mmhg; . ± . vs. . ± . , p< . ). in the frequency domain, hypoxemia changes the relative power of low frequencies (lf: . - . hz) and high frequencies (hf: . - . hz) of rri and dbp with an increased value of the lf/hf ratio (rri lf/hf . ± . vs ± . , p< . ; dbp lf/hf . ± . vs . ± . , p< . ). also the alpha index for baroreflex sensitivity is decreased in hypoxemic animals (alpha lf . ± vs . ± . , p< . ; alpha hf . ± . vs . ± . , p< . ). no differences were observed in maternal variables. conclusions: our results suggest a link between a prenatal insult, alterations in cns receptors and functional alterations of baroreflex responses. a higher sympathetic outflow, suggested by a greater lf/hf ratio, and impaired reflex gain, both possibly mediated by increases in nts rat , may have potential long-term consequences for the development of hypertension. hd ;hd ;hl . objective : we evaluated velocity profiles, relative wall distension rate (rwdr), and wall shear rate (wsr) of fetal descending aorta (fda) in uncomplicated singleton gestations. study design: ninety seven uncomplicated singleton fetuses were studied throughout gestation using multigate spectral doppler analysis (msda) working with gasp software. this consists of a personal computer (pc) add-on board including a single high-speed digital signal processor. the analysis of echo-signals backscattered from range cells located along the axis of the interrogating ultrasound (us) beam. post-processing was accomplished using gasp software. statistical analysis consisted of spearman correlation and chi-square test. results: velocity profiles, wall distension, wall shear rate were obtained from fetal descending aorta throughout gestation establishing gestational age specific norms. wdr[%] is highly correlated with gestational age in appropriate growth fetuses ( . ± . , rs= . p< . ) with linear regression with standardized coefficient of . (p< . ). in contrast, wsr ( ± ) is unchanged during the first , second and third trimesters (p= . ).conclusion: we speculate that the relative wdr changes observed during gestation, in normally grown fetuses, may be secondary to adaptive vascular and autonomic responses and the evolving composition of the vessel wall, particularly with respect to elastin. conversely, the mean wsr for the study group was independent and constant throughout the gestation. these findings suggest that there is an increase in the diameter of the fetal aorta, which provides adaptation to the progressive flow demands, while preserving other key hemodynamic parameters. in this study, we have established normative values of rwdr and wsr, which are new rheological parameters that may be useful in distinguishing normal and pathologic hemodynamic states. objective: placental dysfunction is a key barrier to successful fetal cardiopulmonary bypass (cpb) for repair of congenital heart defects in utero. endothelial cells regulate vascular tone during fetal cpb through interactions of vasodilation by nitric oxide (no) and endothelin- (et- )-mediated vasoconstriction. the objective was to determine the time during fetal cpb when endothelial cell-mediated changes occur. methods: human umbilical vein endothelial cells (huvec) were cultured in media containing % serum collected from ovine fetuses (n= ) that underwent min of cpb, then were maintained for min. serum was collected before cpb, from pump prime before initiation of cpb, min on cpb, or and min after fetal cpb. control cells were cultured in normal fetal serum. cells were harvested and hr after addition of fetal serum. no production was measured in real time with an electrochemical detection system (inno-t, harvard apparatus). et- was measured in the culture media by elisa. results: no production by huvec after and hr was stimulated above control levels by fetal serum collected during and up to min after fetal cpb (p< . ). serum collected from fetuses that were surgically instrumented, but not yet subjected to cpb, decreased no levels below controls (p<. ). stimulation of et- after and hr of huvec culture peaked with serum collected at min after fetal cpb (p<. compared with control), but was elevated above control levels at each collection time point (p<. , table ). conclusions: fetal cpb releases serum proteins that elevate endothelial cell no and et- production during and for at least min after cpb. although the specific regulatory proteins remain to be identified, the no and et- pathways share circulating mediators and participate in a feedback loop to modulate vascular tone. to test the hypothesis that hypoxia-induced upregulation of no is linked to cardiac inos expression, a selective inos inhibitor, l-nil (l-n -( -iminoethyl)-lysine), was administered in vivo to fetal guinea pigs and no levels measured in fetal hearts. methods: pregnant guinea pigs were exposed to either normal room air (normoxia; %o ) or . %o (hypoxia) in a hypoxic chamber for days prior to term (term= d). l-nil was administered to pregnant normoxic and hypoxic guinea pigs via their drinking water at a dose of - mg/kg/d for days. at d gestation, pregnant sows were anesthetized and near-term fetuses removed via hysterotomy. the fetal hearts were excised, weighed, and normalized to their respective fetal body weights. left cardiac ventricles were obtained and frozen in liquid n and stored at - o c until ready for analysis. the effect of total no product (no and no -, nox) of left ventricles of fetuses exposed to normoxia (n= ), hypoxia (n= ) and hypoxia plus l-nil (n= ) was quantified by a commercial fluorometric no assay kit. results: intrauterine hypoxia significantly reduced fetal body weight by % and increased placenta/fetal body wt by % as expected for hypoxic stress. hypoxia induced a slight increase in heart/fetal body wt by %. fetal cardiac nox levels (pmoles/mg) were increased by hypoxia ( . + . ) by . fold compared to normoxic controls ( . + . ). l-nil significantly decreased (p< . ) nox levels in hypoxic hearts by % ( . + . vs . + . ; hypoxic vs hypoxic+l-nil, respectively). conclusion: l-nil inhibits inos-derived no generation in the hypoxic fetal guinea pig heart. since previous study in our lab showed a significant increase in inos expression but a decrease in enos and no change in nnos expression, we hypothesize that hypoxia upregulates cardiac no generation via the inos pathway. further study is needed to identify the important role of cardiac inos in the adaptive response of fetal hearts to chronic intrauterine hypoxia. objective: fetal asphyxia-mediated metabolic acidosis results in a ph decrease and an increase of lactate and base deficit in blood (bd blood ) and extracellular fluid (bd ecf ). it is not clear whether bd blood and bd ecf are similarly altered with worsening fetal acidosis. in the present study we sought to study the dynamic relations of bd blood and bd ecf to lactate in the ovine fetus subjected to repetitive uco with worsening acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood was sampled at baseline, immediately before and during the first mild, moderate and severe uco and at min intervals during the moderate and severe uco. bd gap (bd blood -bd ecf ) was studied in relation to lactate. presented as means±sem. results: lactate correlated to bd blood (r= . , p< . ). bd ecf correlated strongly with bd blood (r= , p< . ). bd ecf increased by . ± . mmol/l more than bd blood from baseline to ph nadir, with bd gap therefore decreasing with increasing lactic acidemia ( fig. ) . lactate, bd blood , bd ecf and bd gap correlated to ph (r= . , r= . , r= . and r= , respectively, all p< . ) and ph could therefore be predicted with any of the four acid-base parameters. conclusion: the increases of bd blood and bd ecf and the decrease of bd gap with increasing lactic acidemia suggest a relatively more rapid accumulation of [h+] in ecf during uco of increasing severity. this may be because the metabolic build-up of acidosis occurs primarily in the tissues and the endothelial permeability for [h+] is impeded during increasing acidosis with atp depletion thus decreasing [h+] movement from ecf to plasma. previous studies have shown that intraperitoneal transplantation of human umbilical cord blood (hucb)-derived mononuclear cells led to the specific 'homing' of these cells to a hypoxic-ischemic brain lesion in perinatal rats. motor deficits resulting from the lesion were alleviated upon transplantation. thus, the presence of hucb cells at the lesion site seems to be a major prerequisite for their potential beneficial effect. however, the mechanisms of cell 'homing' are still unclear. in this study, we focused on elucidating mechanisms underlying the specific migration of hucb-derived mononuclear cells to the brain lesion. one possibility to induce cell 'homing' are chemotactic signals present at the lesion site. the cxc chemokine stromal derived factor- (sdf- ), which was previously shown to be a potent chemoattractant for directed migration of other stem and progenitor cells, is a putative candidate in our lesion paradigm. therefore we investigated the spatial and temporal expression of sdf- in brain hemispheres with or without hypoxic-ischemic lesion. sdf- expression was substantially increased at the lesion site during the investigated period of fourteen days after the insult. furthermore, hla-positive hucb cells were mainly detected in sdf- expressing brain regions and we were able to show that these cells express the sdf- receptor cxcr on their surface. the functional implication of sdf- in directing hucb cell migration was determined by application of neutralizing sdf- antibodies in vivo, resulting in a reduced number of hucb-derived mononuclear cells at the lesion site. with these functional effects, together with the observed timing and location of its expression, the involvement of the chemokine sdf- in hucb cell 'homing' seems conceivable. novel pathways in inflammation-induced fetal brain injury. michal a elovitz, jinghua chai. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. intro: while survival of extremely preterm infants continues to improve, the number of children with cognitive impairment and/or cerebral palsy is increasing. if preterm birth (ptb) cannot be prevented, then strategies to identify and treat fetal brain injury in the setting of a ptb must be investigated. these studies were performed to explore novel pathways involved in fetal brain injury in the setting of inflammation-induced ptb. methods: cd- mice on e receive intrauterine injection of lipopolysaccharide (lps) or saline. hours after injection, fetal brains from the left upper horn were harvested. fetal brains were removed from each dam with dams per treatment group. separate rna samples were prepared and used for microarray (ma) analysis. all protocols were conducted as described in the affymetrix genechip expression analysis technical manual in the ma core facility using the moe av chip. data analysis was performed using significance analysis for microarray (sam). the brain samples from the same dam were considered as dependent samples. pathway analysis and functional annotation clustering tools were used with david. validations studies using qpcr with fetal brain samples (n= - per group) were performed. results: while there was significant differences in gene expression between lps and saline exposed fetal brains, variability existed even between pups from the same dam. genes were significantly differentially expressed between lps and saline brains (p< . ). with a p value of < . , genes were differentially expressed. pathway analysis revealed significant involvement of ) the cadeherin, cadherin-like, cell fraction and calcium binding (enrichment score . ) and ) ribosomal processing, rna metabolism, pyrimidine metabolism (enrichment score . ). specifically, genes involved in neurogenesis, synaptic function, and neuronal and glial metabolism were most differentially regulated. qpcr confirmed observed fold changes in / genes analyzed. inflammatory pathways were not differentially regulated. conclusions: current theories regarding fetal brain injury in ptb focus on activation of inflammatory processes as essential events. this data suggests that long-term neurological injury in a ptb may be secondary to altered neuronal function, metabolism and/or communication. disruptions in these pathways should be explored as key mechanisms to adverse neurological outcomes in preterm neonates and should be targets for future investigations. regulation of microglial activation in the developing murine brain. mariya hristova, virginia zbarsky, daniel cuthill, adam wallace, donald peebles, gennadij raivich. institute for women's health, university college london, london, united kingdom. introduction: the aim of this study was to assess the process of microglial differentiation in developing white matter, an area of the brain that is particularly vulnerable to damage pre-myelination (approx weeks gestation). microglia form a distinctive non-neuronal component. although related to peripheral macrophages they undergo highly specific processes of regional maturation and differentiation inside the brain with a slimming of the cell body, development of very elaborate crenulated arborised branches and downregulation of most macrophage activation markers. this process is relatively rapid in most grey matter brain regions, but is retarded in and around the subcortical white matter (swm) giving rise to the phagocytic fountains of microglia (fom). methods:we examined the process of deactivation and morphological differentiation in the cortex and swm of mice - days after birth (p -p ) using confocal microscopy for monoclonal antibodies against alpha and beta integrin subunits and the costimulatory factor b . , colocalised with standard microglial marker iba . results: strikingly, only the fom macrophages, but not cortical microglia, strongly expressed typical activation markers alpha- , alpha- , alpha-m, alpha-x, beta- and b . . the data for alpha-x are shown as an example in the figure; cortical microglia are shown in the grey bars and swm in black. fom activation was maximal at p , decreased linearly over p and p and disappeared at p . this process followed the presence of ingested phagocytic material but correlated only moderately with ramification, demonstrated by non-ramified but inactive p cortical microglia and formation of stubby processes in p fom. conclusion: these data describe strong and selective biochemical activation of fom phagocytes in p -p swm, roughly equivalent to early rd trimester human foetal development. this presence of highly active phagocytes in the neighbourhood of vulnerable wm could play an important role in the genesis of axonal damage in the foetus and premature neonate. , pp. - ) . mbp is expressed in mature oligodendrocytes (jakovcevski and zecevic, glia ) . although myelination in the fetal sheep brain, a model extensively used to evaluate fetal development, has been described using conventional staining techniques (barlow, j comp neurol ) the use of specific mbp staining allows a more precise determination of the onset of myelination (antonow-schlorke et al., reprod sci . , , a) . aim: to use mbp expression to determine the trajectory of development of different white matter tracts of fetal sheep brain. methods: the ontogenetic profile of mbp expression was estimated in healthy fetuses at . (n= ), . (n= ), . (n= ), . (n= ), . (n= ), . (n= ) and . (n= ) of gestation (term days). after brain fixation and embedding in wax, sections at the level of the optic chiasm were stained with a monoclonal anti-mbp antibody using the abc-technique. immunohistochemical distribution of mbp was morphometrically assessed in the dorsal internal capsule and cortical white matter tracts, i.e. the lateral centrum semiovale, superficial white matter and median corpus callosum using an image analysis system (scion image . , nih, usa). results: mbp expression of various fetal white matter structures started at different time points; initially in the internal capsule at . of gestation followed by the cortical white matter structures at . of gestation. cortical myelination advanced from the cortical deep white matter via superficial white matter to the corpus callosum reflecting different rates of progression. conclusions: the onset of mbp expression estimated here explicitly antedates previous observations in sheep (barlow, ) the way the embryonic heart functions before cardiac morphogenesis is completed is still subject of many studies. to gain more insight into the early cardiac structure-function relationship, doppler blood flow velocity waveforms at four different locations in the embryonic chicken heart during cardiovascular development were assessed. we collected waveforms using high frequency ultrasound biomicroscopy with a -mhz transducer at hh stages , and which are comparable to humans at to weeks of gestation. waveforms were obtained at the inflow tract, the primitive left ventricle, the primitive right ventricle, and at the outflow tract in ten different embryos per stage. by exploring the time relation between the waveforms, cardiac cycle events were outlined. our results demonstrate that stage and location dependent, intracardiac blood flow velocity waveforms can be obtained in the chicken embryo which reflect stage dependent pumping mechanisms. the blood flow profiles assessed at the four locations in the embryonic heart demonstrated a developmental related increase in velocity. in the primitive ventricle the passive filling wave decreased, whereas the active filling wave increased resulting in a decreased p to a ratio in the course of time. high frequency derived cardiac blood flow velocity characteristics support previous findings that the embryonic heart functions like a suction pump at early development and transforms towards another pumping mechanism at later developmental stages. these findings are of importance for the interpretation of human first trimester cardiac velocimetry studies. figure a . changes in the perimeter of the thymus with gestational age by fetal gender are depicted in figure b . background. for the fetus, the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (cbf) and cerebral metabolic rate for oxygen (cmro ), cortical tissue po (tpo ), and electrocorticographic (ecog) activity. in addition, metabolites such as adenosine (ado) are important in this regard. nonetheless, the relation of adenosine and its metabolites to these indices of cerebral oxygenation are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with adenosine. the most common cause of perinatal brain injury is chronic hypoxia/ischemia. the mechanisms are complex and poorly understood. applying proteomics tools, we identified a set of hypoxia-related proteins in the fetal guinea pig (gp) brain (companion abstract). one protein, cofilin- , which regulates the rapid cycling of actin assembly and disassembly, was dramatically altered by chronic brain hpx. herein, we test the hypothesis that confilin- modifications during hpx contributes to brain damage via apoptosis and is an example of an adaptive response that becomes maladaptive. methods: time-mated gps were housed in a chamber from d to d (term) with either room air (nmx) or . % or % o . fetal brains were removed and sections prepared for double staining specific to bax and dephosporylated cofilin- . total protein was isolated and equal amounts loaded on a sds gel, separated by electrophoresis, and transfered to pvdf membranes probed with primary antibodies to dephosphorylated and phosphorylated cofilin- , bax and bcl-xl, and incubated with second antibody. protein signals were detected, quantified by densitometry, and normalized to -actin. total rna was isolated, reverse-transcribed, and quantified by real-time pcr using sybr green i labeling. expression was calculated by the ct method using s for control. melt analysis was performed to confirm specificity of the amplification, and efficiency determined by the slope of the standard curve. the mitogen activated protein kinase (mapk) signalling pathway is upregulated in perinatal hypoxic-ischaemic brain. the role of the extracellular signal-regulated kinase (erk) cascade, a pivotal component of mapk signalling, remains unclear with reported actions ranging from mitogenic and trophic effects to a neuronal death-promoting role. the aim of this study was to assess the role of neuronal erk activity using selective neuronal inhibition in a perinatal model of hypoxic-ischemic brain injury. methods: we explored the effects of selective neuronal inhibition of erk activation using transgenic mice expressing dominant-negative (dn) mek , the upstream activator of erk / , which was under the control of the pan-neuronal talpha alpha-tubulin promoter. p-erk immunoreactivity was quantified following a hypoxic inschemic (hi) insult in p c bl/ mice, caused by unilateral occlusion of the carotid artery, followed by hypoxia with % oxygen for mins at °c. the volume of surviving brain on the affected hemisphere was expressed as a % relative to the control side. results: expression of the mek dn was associated with a reduction in erk / activation following hypoxia ischaemia, as assessed by p-erk immunoreativity. compared with the wild type, littermate controls, mek dn animals exhibited significantly decreased volume of forebrain damage brain following unilateral, min hi insult (*p< . , **p< . , anova). similar protective effects of mek dn were observed in cerebral cortex, hippocampus, thalamus and striatum when compared to wild type littermate controls and correlated with a significant reduction in microglial activation in all brain areas. conclusion: overall, these results suggest that neuronal mek and its downstream signals have an important death-inducing role in this model of perinatal brain injury and could serve as potential targets for therapeutic intervention. oup, ) . however, whether melatonin protects placento-fetal development during hypoxic or undernourished pregnancy is unknown. we investigated in rats the effects on placental and fetal growth of maternal treatment with melatonin in hypoxic and undernourished pregnancy. methods: on pregnancy day , wistar rats were divided: control ( % o ) + melatonin ( g.ml - drinking water), hypoxia ( % o ) + melatonin, and undernutrition ( % reduction in food intake) + melatonin (n= per group). on day , dams were anaesthetised, the pups, placentae and fetal brain were weighed. results: relative to controls (fetal weight: . ± . g; brain/body weight ratio: . ± . mg/g; n= ), both hypoxic ( . ± . g; . ± . mg/g, n= ) and undernourished pregnancy ( . ± . g; . ± . mg/g, n= ) promoted asymmetric iugr (p< . ), without affecting placental weight. melatonin treatment had no effect on iugr, but it decreased placental weight in normoxic ( . + . , n= ), hypoxic ( . ± . , n= ) and undernourished ( . ± . , n= ) pregnancies relative to untreated controls ( . ± . , n= ; p< . ). when fetal body weight was expressed relative to placental weight, a measure of placental efficiency, melatonin prevented the fall in the ratio in undernourished, but not hypoxic pregnancies (fig. objective: midkine (mk) is a -kda heparin-binding growth factor with various functions including cell proliferation, migration, differentiation and angiogenesis. mk expression is strictly regulated in temporal sequence; it is highly expressed during midgestation. we recently studied mk expression in the midgestation human fetus, and showed that the highest mk expression were observed in the adrenal gland, brain, lung and kidney. in the present study, we investigated the profile of mk in human amniotic fluid (af) and amnion (am). methods: amniotic fluid and amniotic membranes were collected at diagnostic amniocentesis, preterm no labor, and term no labor. mk protein levels were analysed by western blot. expression of transcripts encoding mk and putative mk receptors were examined by rt-pcr and real-time quantitative rt-pcr (qpcr). results: ) western blot analysis demonstrated abundant mk protein in the human am; mk levels were higher at midgestation than at term ( -fold: wks vs. wks). ) tissue transglutaminase known to polymerize mk was abundant in af. ) qpcr revealed that mk mrna was not expressed in am whereas it was highly expressed in the fetal adrenal, kidney and lung (positive controls). ) among the receptors implicated in mk signaling, lowdensity lipoprotein receptor-related protein and syndecan- were expressed in am while protein-tyrosine phosphatase, anaplastic lymphoma kinase, and syndecan- were not. conclusions: abundant mk protein in the midgestation af is likely to be derived from the fetus. mk in af may play a role in feto-amniotic communications and/or development of fetal organs exposed to af. short term hfix expression. we hypothesized that long term hfix expression could be achieved in fetal sheep using an aav vector, without stimulating an immune response to the transgenic hfix protein. we injected aav hfix vector ( - x p/kg) into the peritoneal cavity of fetal sheep under ultrasound guidance in early (n = ) or late (n = ) gestation. fetal blood was retrieved by ultrasound guided sampling from the intra-hepatic umbilical vein. fetal and lamb blood was tested for hfix expression using elisa, antibody responses using functional assays, and for liver damage up to a year after birth. vector spread was detected in maternal and fetal tissues by quantitative pcr analysis. results: the highest level hfix was detected days after late ip injection ( % and % normal human levels). early gestation ip injection gave . % and . % at and days after injection. hfix levels dropped rapidly correlating with the increase in size of the fetal liver and lamb. however, hfix was detectable at a low levels ( . %) year after birth in early and months after birth in late gestation injected lambs. up to year after birth, liver function tests and bile acid levels were normal, showing no evidence of liver pathology. no functional antibodies to the hfix protein or aav vector were detectable. high vector levels were detected in the fetal liver, and other peritoneal organs; no vector was present in fetal gonadal tissue. conclusion: hfix expression is detectable up to year after delivery of aav vector to the fetal sheep using a clinically applicable method. this is the first study to show long term hfix expression after fetal gene therapy in a large animal. further work will include testing for immune tolerance to exogenous hfix protein in these animals. objective: placental estrogens play a pivotal role in the endocrine control of pregnancy and may be involved in the key changes occuring during parturition. it has been established that crh interacting with crh receptor has a positive effect on estrogen production throughout pregnancy. urocortin (ucn ), a novel peptide of the crh family binding exclusively crh receptor , is expressed by human placenta and the aim of the present study was to evaluate the influence of ucn on estrogen biosynthesis in cultured trophoblast cells. methods: cultured term placental cells were treated with various concentrations of ucn in the presence of the estrogens precursors dehydroepiandrosterone sulphate (dhea-s), androstenedione or testosterone. estradiol secretion was measured in the culture medium using a specific elisa, p arom mrna and protein expression were evaluated by real time pcr and western blot analysis respectively. results: the addition of ucn , in presence of dheas significantly increased e levels at , and hours treatment, while in presence of androstenedione and testosterone an increase in e secretion was detected only at , and hours (at different ucn doses). both p arom mrna and protein were up-regulated in presence of each estrogen precursor and the addition of ucn caused a synergistic increase. anti-sauvagine (a crh type receptor antagonist) resulted in a significantly attenuated ucn effects on e secretion and on p arom mrna and protein expression. conclusions: the present study supports a possible role of ucn on placental e biosynthesis: e secretion and p arom transcript and protein expression were significantly increased after ucn treatment. in conclusion, the crf family may play a major role on placental steroidogenesis, stimulating dheas secretion in fetal adrenals by crh and controlling placental estrogen biosynthesis through ucn . a possible influence on the mechanisms of parturition may be hypothesized. increased expression of kiss- gene in preterm placenta. letizia galleri, michela torricelli, chiara voltolini, fernando m reis, felice petraglia. department of pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. introduction placenta expresses a large number of peptides involved in delivery. neuropeptides, cytokines, are expressed and secreted by human placenta at the time of preterm delivery. human placenta is a major source of kiss- , a -amino-acid peptide encoded by a putative metastasis suppressor gene. kiss- acts on its placental g protein-coupled receptors (kiss- r); this peptide stimulates release of oxytocin in rats, the most potent known uterine stimulant, suggesting a possible role of kiss- in the mechanisms of labor. aim of study the aim of this study was to evaluate placental expression of kiss- at preterm labor. material and methods placental tissue and plasma samples (both maternal and fetal) were collected at term in the absence of labor (tnl), at term spontaneous vaginal delivery (tl), and at preterm labor (ptl). changes in placental mrna expression were determined by real-time quantitative rt-pcr analysis. kiss- protein levels were measured by specific immunoenzymatic assay (elisa). results placental kiss- mrna expression was significantly higher (p< . ) in ptl than in tnl and in tl. however, maternal and fetal plasma kiss- levels did not differ among tnl, tl, and ptl samples. conclusion the present study showed that placental kiss- mrna expression is increased in preterm delivery. further studies are needed to better understand the role of kiss- on cascade leading term and preterm labor. placental angiogenesis is essential for placental development, which occurs under a hypoxic environment ( - % o ) during normal pregnancy. it has been reported that in transformed human dermal microvascular endothelial cells, hypoxia activates erk / , which stabilizes hypoxia-inducible transcription factor- (hif- ). however, signaling mechanisms governing placental angiogenesis under hypoxia is largely unknown. herein, we tested whether hypoxia affected fgf -and vegf-stimulated cell proliferation partly via activating erk / and stabilizing hif- protein levels in human placental artery endothelial (hpae) and transformed human placental microvascular endothelial (hpme) cells. methods: cells cultured under normoxia ( % o ) or hypoxia ( % o ) for days were treated with fgf and vegf. after days, the number of cells was determined. activation of erk / and levels of hif- protein were determined by western analysis. results: under normoxia, fgf and vegf stimulated (p < . ) cell proliferation and induced ( min, p < . ) erk / phosphorylation in hpae and hpme cells. hypoxia promoted (p < . ) fgf -and vegf-stimulated cell proliferation in hpae cells, whereas hypoxia blocked (p < . ) such actions in hpme cells. hypoxia enhanced fgf -, but not vegf-induced erk / phosphorylation in hpae cells. in contrast, hypoxia promoted (p < . ) vegf-, but not fgf -induced erk / phosphorylation in hpme cells. hypoxia increased (p < . ) hif- levels in the hpae cells: first detected at . hr and maintained up to hr. hpme cells had high basal levels of hif- ( folds; p < . ) compared with hpae cells. hypoxia did not alter hif- levels up to hr and decreased (p < . ) hif- levels at hr in hpme cells, conclusions: in hpae cells, hypoxia enhances fgf -and vegf-stimulated cell proliferation possibly partially via promoting activation of different protein kinases. this stimulatory effect is associated with increased hif- protein levels. however, inhibition by hypoxia on fgf -and vegf-stimulated hpme cell proliferation is associated with relatively high hif- levels in the first hr and decreased hif- levels at hr. thus, these data suggest that different signaling mechanisms are involved in hypoxia-modulated growth factor-induced placental angiogenesis in which an increase in hif- levels plays a critical role. objective: corticotrophin releasing factor (crf) is a well established neurohormone involved in the initiation of the stress response. we recently documented that rat placenta is analogous to human placenta in the expression of crf-mrna and protein, and that placental crf release may contribute to in utero meconium passage. time-of-day variation in crf expression is a highly recognized phenomenon at the brain cellular sites of crf synthesis. we sought to determine whether crf expression in rat placenta is subject to time-of-day variation. method: time-dated pregnant rats were obtained on day of gestation (term= days), housed in under h- h light-dark conditions (lights on ). lab chow and water were continuously available. on day , pregnant rats were quickly anesthetized by exposure to isoflurane, abdomen opened and fetuses and placenta exteriorized either at - hr (zeitgeiber time, zt ) or - hr (zt ). individual placentas were processed either for rna extraction or immunohistochemical investigation. the levels of crf-mrna were assessed by pcr using rat specific pcr primers. pcr bands were subsequently cloned and sequenced. bouin's solution fixed paraffin sections of placenta were subjected to crf immunohistochemistry with antibodies specific to rat species and intensity of immunostaining was analyzed using image pro-plus software and expressed in arbitrary units (au). results: one specific pcr band of bp size was consistently identifiable in placenta harvested at zt but not at zt . nucleotide sequence of the pcr band confirmed its identity as crf-mrna. intensity of crf-immunostaining was significantly greater at zt in giant trophoblast (gt) cells (gt-crf: zt = . ± . au, zt = . ± . au, p= . ) but not in spongiotrophoblast cells (stc) (stc-crf: zt = . ± . au, zt = . ± . au, p=ns) or labyrinth cells (lbc) (lbc-crf: zt = . ± . , zt = . ± . au, p=ns) . conclusion: similar to adult brain, rat placenta expresses crf mrna and protein. time-of-day variation of crf expression originally seen in central nervous system neurons is also identifiable in giant trophoblasts cells at zt . these findings suggest that stress-mediated placental crf release, and potentially fetal meconium passage, may be dependent upon time-of-day. objectve: transplacental water flow is essential for the provision and maintenance of fetal body water and amniotic fluid. water flow across membranes is regulated by aquaporin (aqp) water channels in many tissues. thus aqp , expressed in the placenta, is a candidate to regulate maternal to fetal fluid exchange. maternal beta-mimetics have been hypothesized to augment fetal growth by increasing the availability of nutrients and perhaps water. as camp acts as a second messenger to increase expression of selected aqps in other tissues, we sought to determine if betamimetics acting through camp could modulate placental aqp , and potentially influence placental water transfer. methods: trophoblastic cell cultures were established in first trimester-derived extravillous htr- /svneo cells and term placenta-like trophoblast carcinoma cell line jeg- . cultures were treated with sp-camp, a membrane-permeable and phosphodiesterase resistant camp, and forskolin, an adenylate cyclase stimulator, in doses of . , and m for hrs (aqp mrna expression) and hrs (aqp protein expression). for time course experiments, cells were incubated with μm of either sp-camp or forskolin for , and hrs at °c, % co . after cell harvest, mrna and protein expression were assayed using real time pcr and western blotting. results: sp-camp and forskolin increased aqp mrna expression in both cell lines after hrs (p< . ) in a dose-dependent manner. protein expression paralleled the increase seen in the mrna. m of sp-camp and forskolin stimulated aqp mrna expression after hrs in htr- /svneo cells and after hrs in jeg- cells (p< . ). m of either sp-camp or forskolin stimulated aqp protein expression in both cell lines after hrs (p< . ) and the expression remained high at hrs. conclusion: aqp gene expression in trophoblast cells is up-regulated by camp agonists. these results suggest that maternal beta-adrenergic agonists or antagonists may modulate maternal-fetal water flux via modulation of aqp water channels. rat placenta expresses corticotrophin releasing factor-binding protein and mrna. jayaraman lakshmanan, thomas r magee, bindu cherian, sharon k sugano, hanalise huff, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor-binding protein (crf-bp), a kda secreted glycoprotein, was originally isolated from human plasma. it binds crf and urocortin i with an equal or greater affinity than does the crf receptor. crf-bp expression has been reported in target tissues such as brain and placenta. based on its ability to inhibit crf actions in vitro, it is speculated to function as a "gate keeper" for crf-initiated stress responses. we recently documented expression of crf and urocortin- in rat placenta. in the present study we sought to establish the expression of crf-bp in rat placenta by immunohistochemical and pcr-analyses. methods: placental tissues (n= ) collected from sprague-dawley pregnant rats on day of gestation (term= ) were either fixed in % paraformaldehyde solution (ph . ) and paraffin embedded or processed for total rna extraction. paraffin sections of five micron thickness were subjected to immunohistochemical analysis with goat polyclonal antibodies to human crf-bp precursor (sc- santa cruz biotechnology, ca) by avidinbiotin-peroxidase complex technique. the structure of cloned human crf-bp precursor exhibit significant amino acid sequence homology among all species studied. immunoreactivities on the sections were quantified using image pro . software and staining intensity (od/area) expressed as arbitrary units (au). all values are expressed as mean±sem. for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, clone into plasmid vector and dna sequenced. results: the crf-bp antibody elicited strong positive staining in decidua, giant trophoblasts, spongiotrophoblasts and labyrinth cells. the results of image analyses revealed (au): decidua: . ± . , giant trophoblast cells: . ± . , spongiotrophoblasts: . ± . , fetal membranes: . ± . . pcr analyses identified a single bp band, consistent with crf-bp. conclusion: our study establishes for the first time that rat placenta is analogous to humans in that both express crf-bp mrna and protein. immunohistochemical findings reveal that crf-bp protein expression occurs at multiple sites within the placenta. expression of per clock protein in rat placenta: an internal timer for placental functions. jayaraman lakshmanan, reuben lakshmanan, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor (crf) expression time-of-day variation occurs in giant trophoblast cells on the maternal side of the rat placenta. this temporal change in crf expression pattern is very similar to that of central nervous system neurons, suggesting that placental cells may use a similar mechanism to read time of the day. the self-sustaining rhythm generating capacity of the suprachiasmatic nuclei is believed to be derived from cell-autonomous, transcriptional feed-back loops dependent on a number of canonical clock genes. in the present study we sought to determine whether rat placenta expresses period gene (per ), one of the clock/cycle related genes. methods: time-dated pregnant sprague-dawley rats were received on day of gestation and housed in a controlled environment ( - h lights on) with free assess to food and water. placentas collected on days and of gestation (term= days) between and . am were fixed in % paraformaldehyde and paraffin embedded. for each gestational ages a total of placentas from different pregnant rats were used. paraffin sections ( sections per placenta) were subjected to immunohistochemical analysis with antibody to per ( : , santa cruz biotechnology, ca) by abc technique and , 'diaminobenzidine as a chromagen. sections were examined under microscope, imunostaining quantified by image pro . software and expressed in arbitrary units (au). data are presented as mean ± sem. statistical significance was analyzed by anova with a p value < . as significant. conclusion: the present results indicate that rat placental cells, devoid of any neural innervations, express per clock protein, similar to central nervous system neurons. based on the differences in the relative intensities between trophoblasts and labyrinth cells on day of gestation, we conclude that fetalmaternal interactions in per regulation disappear at term (or at the time of birth.). our findings imply that per may function as internal physiological modulator in placenta. to determine the mechanism(s) underlying placental time-of-day crf variation, we examined the effect of maternal administration of betamethasone (a synthetic glucocorticoid) on nuclear gc receptor expression in placental cells. method: time-dated pregnant rats (n= ) on day of gestation were given a single subcutaneous injection (at : am) of betamethasone ( μg/kg body weight) while control pregnant rats (n= ) received saline. dams were exposed to isoflurane anesthesia at : am, and placentas harvested, fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analysis with gc-receptor antibody (sc- , santa cruz biotechnology), with immunoreactive material identified by abc technique using , ' diaminobenzidine as a chromagen. percentages of gc-nuclear receptor (gc-nr) positive cells and their intensities (od/area) were quantified using image pro . plus software. all values are expressed as mean ± sem. statistical analysis was by anova with p< . considered significant. results: in placentas of saline exposed pregnant rats, isolated labyrinth (lb) cells (< %) were positive to gc-nr. no positive staining for gc-nrs was seen either in giant trophoblasts (gt) or in spongiotrophoblasts (st). betamethasone administration was associated with a significant and marked increase in gc-nr staining in all three placental cell types (p< . ). among the cells, gt ( ± %) demonstrated a greater percentage of cells expressing gc-nr than did st ( ± %) or (lb= ± %) (p< . ), though there was similar immunoreactive intensities (gt: . ± . , st: . ± . , lb: ± . au; p=ns) conclusion: our findings indicate that all placental cells respond to gc with upregulation of gc-nr with an enhanced response among gt cells. these results suggest that endogenous or exogenous maternal stress-induced gc exposure may influence signaling responses within both maternal and fetal placental compartments. ovine placenta at very-preterm gestation expresses corticotrophin releasing factor (crf), crf-binding protein (crf-bp) and glucocorticoid receptors (gr). jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: in humans, the placenta is the major source of maternal and fetal plasma crf. based on its critical functions, crf is considered as a "placental clock" of parturition. we recently reported that ovine fetal as well as maternal plasma contain measurable amounts of crf at near-term but not at very preterm gestation. we interpret the absence of crf in plasma at very preterm gestation is either due to lack of placental crf expression and/or placental crf release. here, we examined the expression status of crf, crf-bp (a known specific binding protein for crf and a known regulator of crf functions in human placenta) and gr (a known positive regulator of crf expression in human placenta) in ovine placenta collected at very-preterm gestation. method: placenta harvested from time-dated pregnant ewes on ± days gestation were fixed in bouins's solution and processed for paraffin embedding. paraffin sections were subjected to immunohistochemical analysis with polyclonal antibodies specific to ovine crf( : - : , phoenix pharmaceuticals, ca,) crf binding protein ( : - : , sc ) and gr ( : - : , sc: , santacruz biotechnology, ca). immunoreactive material on the sections were identified as brown staining by abc technique using diaminobenzidine as chormagen. immunoreactive material was quantified by image analysis using image pro . plus software and the immuoreactive intensity (od/area) expressed as arbitrary units (au). results: strong positive staining for crf ( . ± . au), crf-bp ( . ± . au) and nuclear-gr ( . ± . au) were noticed in syncytiotrophoblast cells in all placental sections obtained from pregnant ewes at days gestation. control sections exhibited no positive staining. conclusion: our findings indicated that ovine placenta at very-preterm gestation expresses crf, crf-bp and gr. these results suggest that absence of measurable crf in maternal and fetal plasma at very-preterm gestation is not due to lack of placental crf expression but rather due to the absence of regulated crf secretory mechanisms. rat placenta expresses urocortin i protein and mrna. thomas r magee, michael g ross, john d richard, sharon k sugano, jayaraman lakshmanan. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: urocortin i (ucn- ), a amino acid neuropeptide belongs to corticotrophin releasing factor (crf) stress hormone family. in humans, the placenta and several gestational tissues have been reported to express ucn- protein and ucn- mrna. a number of published studies indicate that ucn- is similar to crf in expression pattern and biological functions. we recently reported that rat placenta is a site of crf protein and crf mrna expression. in the present study we sought to determine whether rat placenta expresses ucn- protein and ucn- mrna. methods: placenta (n= ) collected from pregnant rats at day gestation were either fixed in bouin's solution and processed for paraffin embedding or placed in rna later preservative and frozen for rna extraction. for immunohistochemical localization, five micron thickness paraffin sections were cut and immunostained with rabbit polyclonal antibodies to ucn- ( : to : , sigma) by standard abc technique. control sections were incubated with omission of ucn- antibody. immunoreactive materials on the sections were identified using , ' daminobenzidine as chromagen. immunostaining intensity (od/area) was quantified by image-pro plus software and expressed in arbitrary units (au). for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, cloned into a plasmid vector and dna sequenced. all values are given as mean ± sem. results: ucn- polyclonal antibody elicited strong immunostaining in placental trophoblast cells with variable intensity. the pattern of immunostaining is as follows: giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au. the relative intensity in labyrinth cells was significantly lower than the other two cell types (p< . ). pcr analyses revealed the presence of a single band of bp, consistent with ucn- mrna. conclusion: similar to human placenta, rat placenta expresses ucn- protein and ucn- mrna, with most expression localized to the maternal side trophoblast cells. these results support our hypothesis that rat placenta can be used as a model to understand the role of this peptide in feto-maternal stress. the role of the nuclear hormone receptors fxr, pxr and car in placental bile acid homeostasis in normal and pathological pregnancy. victoria geenes, peter dixon, selina raguz, jenny chambers, kishore bhakoo, catherine williamson. imperial college, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder characterised by raised maternal serum bile acid levels and associated with adverse fetal outcome. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from an accumulation of bile acids in the fetal circulation. bile acids are the toxic end products of hepatic cholesterol metabolism and are synthesised from weeks gestation. in common with other waste products, accumulation in the fetal compartment is prevented by excretion across the placenta into the maternal compartment. however, studies of maternal and cord serum from normal and oc pregnancies have suggested that bidirectional transfer of bile acids is possible. hepatic bile acid transport and metabolism is regulated by members of the nuclear hormone receptor family, namely fxr, pxr and car, but the mechanisms for regulating placental transfer are unknown. objectives: this study used placenta from normal and cholestatic pregnancies to investigate the expression of genes involved in hepatic bile acid homeostasis. methods: villous trophoblast samples from oc and normal pregnancies (np), and human livers were collected and preserved in rnalater. explant cultures were prepared from a further np placentas and cultured for days at % oxygen. on day they were treated with chenodeoxycholic acid, lithocholic acid or vehicle for hours prior to fixing in rnalater. total rna was extracted using trizol, and reverse transcribed to cdna. quantitative real-time pcr was performed using sybr green. target gene mrna abundance was calculated from a standard curve and normalised to l . results: the expression of fxr, pxr and car, the nuclear hormone receptors responsible for hepatic bile acid homeostasis and several fxr target genes (shp, mdr and bsep) was found to be very low in np, and unaffected by the presence of maternal cholestasis. furthermore, the expression of these genes could not be induced by bile acid treatment in an in vitro model. here we have shown that the nuclear hormone receptors (fxr, pxr and car) involved in hepatic bile acid homeostasis are expressed at very low levels in normal and pathological pregnancy and are thus likely to be of limited importance in placental bile acid transfer. a placental phenotype for obstetric cholestasis. victoria geenes, jenny chambers, jo wyatt-ashmead, kishore bhakoo, catherine williamson. imperial college, london, united kingdom; hammersmith hospital, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder that affects . % of pregnant women in the uk. biochemically, oc is characterised by liver dysfunction with elevated maternal serum bile acids (sba), and clinically by an increased risk of fetal complications including fetal distress, meconium staining of the amniotic fluid, preterm labour and sudden intrauterine death. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from the toxic effects of bile acids, which can cause vasoconstriction in the placenta and fetal cardiac dysrrhythmias. furthermore, in a rodent model of oc, bile acids cause oxidative stress in the placenta and a reduction in litter size. these changes are absent in animals treated with ursodeoxycholic acid (udca), a drug used to treat oc. however human data are lacking. objectives: this study used whole placenta and explant cultures to investigate the effects of bile acids on human placental architecture and to establish whether udca protects the placenta against bile acid induced damage. methods: villous trophoblast samples were collected from oc and normal pregnancies (np) and fixed in formalin. explant cultures were prepared from a further np placentas, and cultured for days at % oxygen. on day a subset were treated with udca overnight, and on day the explants were treated with taurocholic acid, taurochenodeoxycholic acid or vehicle for hours prior to fixing. m sections were stained with haematoxyllin and eosin. slides were reviewed by a perinatal pathologist and syncytial knots (sk) counted. results: oc tissues showed marked alterations in morphology, including congestion of the terminal villi, and loosening of the stroma and fibrotic change of the membranes of the stem villi. there were significantly more sk the oc samples (mann-whitney u test p= . ). furthermore, sk were increased in explants treated with bile acids, but not those treated with udca prior to the addition of bile acids. conclusions: here we describe several morphological abnormalities of the placenta associated with maternal cholestasis. these changes were confirmed using a placental explant model, which also showed that udca protects the placenta against bile acid induced damage. in summary, this study indicates that udca is likely to protect the fetus in oc. the placenta has complex metabolic and endocrine activities and is essential for the growth and survival of the fetus in utero. ultrasound is the most sensitive and less invasive method to evaluate placental size, morphology and function. the three-dimensional approach allows to calculate placental volume in the i and ii trimester of pregnancy. pregnancies from assisted reproduction technologies (art) show an increased rate of pathologies potentially related to placental insufficiency such as intrauterine growth restriction and preterm delivery. aim. to determine placental volume in art pregnancies in a cross sectional study in the first trimester of gestation. design. using three-dimensional ultrasound machine (ge ) placental volume measurement from art pregnancies (n= ; - wks) were compared with data from normal controls (n= ) matched for gestational age. data were analysed with software stata . results. mean placental volume was . ml (sd± . ) in art pregnancies and . ml (sd± . ) in normal controls. mean difference resulted in . ml (- - . ) and was statistically different (p= . ). multiple linear regression analysis showed a statistically significant interaction (p= . ) between gestational age and case status, i.e. differences in placental volume increase significantly with advancing gestational age between cases and normal controls. mean gestational age at birth was not essentially different between the two subsets ( . weeks ± . ) however a statistically significantly lower mean fetal birthweight was found in art pregnancies: g (sd± ) vs .(sd± . ) (p= . ). conclusions. placental volume is slightly decreased in art pregnancies. measurements of placenta volume by d ultrasound may play a role in identifying the degree of placental growth early in gestation in a risk population. in vitro effects of adenovirus-mediated gene transfer of insulin like growth factor- (ad-igf- ) in trophoblast cells. mounira habli, datis alae, foong yen lim, jignesh parvadia, timothy crombleholme. obstetrics and gynecology; pediatric surgery, university of cincinnati/children's hospital, usa. objective:recently, ad-igf- corrected both fetal and placental weights in rat model of fetal growth restriction (fgr) . to elucidate the mechanism of action of ad-igf- ;we examined the effect of ad-igf- on trophoblast proliferation, differentiation and invasion. methods: rcho- trophoblast cell line derived from rat choricarcinoma was used. ad-igf- and galactosidase transgene (ad-lacz) were given at moi of : and : in all the experiments.transduction efficiency was assessed hr after infection with ad-lacz by galactosidase enzyme activity. the invasiveness of rcho- was measured by using bdmatrigel invasion chamber kit. rcho- proliferation cell density was assessed by crystal violet assay. morphologic analysis of rcho- differentiation in response to treatment (differentiated cells are multinucleated giant cells) was assessed byimunocytochemistry staining using placental lactogen- antibodies(specific hormone produced by giant cells). results: % efficiency of gene transfer of ad-lacz to rcho- cells was observed at both moi's.there was no significant difference in proliferation between treated control group regardless of the dose at and hrs. ad-igf- induced morphologic differentiation of trophoblast cells compared to control (fig ) at hrs.ad-igf- induced higher rate invasion in a dose dependent fashion as compared to control(ad-lacz) group( % at moi vs . % in control p< . )(fig ). conclusion: ad-igf- gene transfer induces differentiation and invasiveness of trophoblast cells. these may be the mechansim of correction of fgr in rat model of placental insufficiency. placental gene transfer may be an effective treatment strategy for placental insufficiency. types of human placenta. martina dieber-rotheneder, ursula hiden, gernot desoye, mila cervar-zivkovic. department of obstetrics and gynaecology, medical university graz, graz, austria. background and hypothesis: endothelin- is a polypeptide with a wide range of functions. in the placenta it acts as a potent regulator of vasotonus on endothelial and smooth muscle cells, whereas on the trophoblast it regulates cell proliferation, invasion and apoptosis. endothelin- action is differently signaled through two endothelin receptor (etr) subtypes, etr-a and etr-b. several alternative splice variants of etr were identified. here we hypothesize that the etr-a splicing varies with gestational age and is different in trophoblast vs endothelial cells of the human term placenta. methods: mrna of placental tissue from first trimester (pregnancy terminations, missed abortions) and term of gestation, first trimester and term trophoblasts, arterial and venous placental endothelial cells as well as cell lines representing first trimester trophoblast (ach p) and term placental endothelial cells (hpec) were analyzed for full length etr-a and known as well as unknown splice variants by sqrt-pcr using primers spanning the exons - . the predominant dna bands in agarose gel were excised and sequenced. results: all tissues and cells expressed full length etr-a. in all tissues an additional -deletion variant was identified which was not found in the isolated cells. trophoblasts expressed another yet unidentified splice variant. the endothelial cells expressed a -deletion variant and a novel splice variant, which was identified as partial -partial deletion. this novel splice variant was found regardless of the arterial or venous origin of the cells as well as in the cell line (sv -transformed). in tissues und trophoblast no difference in splicing was found between first trimester and term. conclusion: gestational age does not alter splicing of endothelin receptor-a. splicing is different between trophoblasts and endothelial cells. the endothelial cells contain a novel splice variant. the differential splicing may allow maternal and fetal endothelin- to induce different effects on the two major cell types of the placenta. (supported by grant , jubilee fund, austrian national bank, vienna). adrenomedullin production and secretion by human trophoblast cells are regulated by glucocorticoids and hypoxia. francesca ciardo, katia pacioni, emanuela marinoni, giovanna corona, massimo moscarini, alfredo patella, romolo di iorio. department of gynecology, perinatology and child health, university "la sapienza", rome, italy; department of obstetetrics and gynecology, university of ferrara, ferrara, italy. objectives: adrenomedullin (am) is produced by intrauterine tissues and is involved in the regulation of implantation, placental hemodynamics and endocrine function. circulating am is increased in pregnancy complications such as preeclampsia and intrauterine growth retardation. we investigated whether am output by human trophoblast cells is regulated by hypoxia and/or glucocorticoids. study design: trophoblast cells obtained by human placentas at term (n= ) were cultured in presence or absence of hypoxia ( % o ) and treated with or without betamethasone at the dose of - , - , - m. media and cells were collected at h and at , and h from syncytiotrophoblast cultures. am was measured in cultured media by specific ria kit. protein expression in trophoblast cells was evaluated with immunohistochemistry and western blot. results: hypoxia stimulated am output and protein expression by cytotrophoblast and syncytiotrophoblast cells. betamethasone induced an increase in am production and secretion in a time-and dose-dependent manner (figure). effects of hypoxia were partially reversed by betamethasone in a dose and time-dependent fashion. conclusions: am production in trophoblast cells is up-regulated by hypoxia and glucocorticoids, independently. increased am levels in pregnancy complications characterized by placental insufficiency might derived by an increase in am placental secretion stimulated directly by hypoxia or indirectly by an increase in fetal cortisol levels. preeclampsia complicates - % of pregnancies, and while associated with significant maternal and fetal morbidity and mortality, the mechanisms responsible for preeclampsia are not completely understood. recent advances suggest there are imbalances of pro-and antiangiogenic factors, present from the time of implantation affecting vascular responsiveness of the fetal placental unit and maternal vasculature. in this study, we investigate vasomotor responsiveness of placental arteries from normal and preeclamptic (pet) pregnancies using an in vitro muscle bath contractility assay. transverse rings of placental arteries were cut and equilibrated in the muscle bath containing a bicarbonate buffer aerated with % o / % co at °c. viability was demonstrated by contraction to mm kcl prior to all experiments. cumulative logarithmic dose dependent responses to four different contractile agents: pgf , serotonin, carbochol, and norepinephrine were compared. placental arteries precontracted with pgf at half maximal concentration were relaxed with three vasorelaxants: sodium nitroprusside (snp), forskolin (fsk) and lactate (lct). agonist studies revealed contraction to both pgf and serotonin but not to carbochol or norepinephrine. there were no statistically significant differences between the responses of normal and pet arteries. both normal and pet arteries demonstrated heightened responsiveness to sodium nitroprusside compared with forskolin and lactate. this study reveals that the placental vessels are more sensitive to sodium nitroprusside, that mediates relaxation through nitric oxide -cyclic gmp signal transduction pathway, than forskolin that induces relaxation through the cyclic amp pathway. cancer complicates approximately one in , pregnancies. depending on the type of cancer and trimester, patients can terminate the pregnancy or choose treatment such as chemotherapy. since there is limited knowledge on the safety of chemotherapy on fetal tissues in utero, clinicians cannot efficiently analyze the risks of particular anticancer agents when deciding on a course of therapy. since carboplatin is among the most common anticancer agents used for treatment of cancer during pregnancy the primary objective of this study was to evaluate if carboplatin will readily cross to placental barrier and determine total potential platinum fetal exposure. this project utilizes an ex vivo placenta perfusion model to determine the concentration of carboplatin that crosses the human placental barrier. placentas are obtained within minutes of spontaneous delivery and re-perfused. two carboplatin concentrations were selected of ng/ml and ng/ml to represent clinically relevant maternal plasma concentrations. antipyrine was used as the internal control. serial samples were collected every minutes for total minutes in open-open model then experiment repeated with closed circuit model. antipyrine concentrations were determined by hplc methods. platinum concentrations in media samples were determined with a validated atomic absorption assay. a cell culture-based approach will be used to determine whether cell cycle or apoptotic proteins are altered (p , bax, bcl- , aif, and pro-caspase- ) using western blotting as a detection method. a total of two placentas have been completed at the low carboplatin concentration and one at the high concentration. the mean carboplatin clearance was . +/- . ml/min and . +/- . ml/min at the low and high carboplatin concentrations, respectively. an estimated % increase in the transport fraction was observed in the high concentration experiments compared to the low concentrations model. fetal carboplatin exposure ranged from to ng/ml. the placental perfusion experiments conducted at carboplatin and ng/ml indicate that carboplatin crosses the placenta through simple diffusion. the toxicology assays are ongoing to determine potential effect on fetal tissues. preterm delivery represents one of the predominant causes of perinatal mortality and morbidity, and is one of the most unpredictable gestational disturbances. urocortin is a peptide expressed by trophoblast and gestational tissues (amnion and chorion), whose maternal levels correlate with gestational length, since they are increased at preterm delivery and decreased in post-term pregnancy, when compared to term pregnancy. the addiction of urocortin enhances contractility of human myometrial strips, suggesting a possible role on uterine contractility. high maternal urocortin levels in threatened preterm delivery correlates with the timing of delivery, suggesting a possible role in the predictivity pf preterm delivery. in the present study urocortin concentrations in amniotic fluid collected at mid gestation for amniocentesis were correlated with gestational age at delivery. a case-control study with amniotic fluid obtained from healthy women undergoing amniocentesis for genetic indications was performed; urocortin concentrations were measured by a specific elisa. amniotic fluid urocortin concentrations resulted significantly lower (p= . ) in women delivering preterm (n= ; ucn= . ± . pg/ml) (m ± se) than in those delivering at term (n= ; ucn= . ± . pg/ml). in conclusion, the present preliminary data showed that amniotic fluid urocortin concentrations at mid gestation may represent predictive marker of preterm delivery. human placentas throughout pregnancy. annunziata mastrogiacomo, elisabetta federico, francesca caprio, maria teresa schettino, gabriele coppola, antonio de luca, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; department of medicine and public health, section of anatomy, second university of naples, nales, italy. hypothesis apoptosis is intimately involved in placental homeostasis, growth and remodeling and the apoptotic rates increase progressively during normal pregnancy as part of normal placental development. moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction, diabetes. in the present study, we describe differences in the expression of pro-apoptotic protein bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. material and methods human placental samples were obtained with informed consent from patients undergoing surgery such as first trimester voluntary termination of pregnancy (n= ), first trimester abortion (miscarriage) (n= ), delivery section (n= ), spontaneous birth (n= ), preeclampsia (n= ) and diabetes (n= ). the gestation period ranged from to weeks. the specimens were immediately fixed in formalin for immunohistochemistry. we first observed a strong increase of bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. secondly, we showed a more intense immunopositivity for bax in the third trimester spontaneous birth respect to the third trimester caesarean birth. thirdly, we observed an increase of bax expression in preeclamptic placentas compared to the normal full-term placentas. on the contrary, we observed a moderate bax expression in diabetic placentas only slightly decreased compared to the normal full-term placentas. our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies. objectives: maternal endothelial activation in preeclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. the application of proteomic technologies such as mass spectrometry promises the reward of identifying and characterising these factors. using a placental explantconditioned culture media model system we have developed a proteomics workflow to obtain relative quantification of proteins released into placental explant culture media. methods: term villous explants were cultured in serum-free conditions and exposed to differing oxygen concentrations ( % %) to mimic physiological and non-physiological intervillous o tensions. the d media was concentrated, immunodepleted to remove fibrinogen (using beckman igy- columns) and proteins labelled using an itraq (applied biosystems) kit following the manufacturer's protocol. labelled peptides were fractionated by strong cation exchange and then analysed using lc-maldi on a maldi-tof/tof (applied biosystems) mass spectrometer. data was analysed using protein pilot . (applied biosystems). results: when matched pooled (n= ) media samples were subjected to our proteomics workflow over proteins were identified with a calculated false positive identification rate of < %. of these proteins a total of display a statistically significant difference in protein levels between the % % o samples (p=< . ). from a list of proteins of interest we selected proteins for validation using elisa/western blotting to measure their relative abundance in both pooled and individual explant media samples. interleukin is an example of a low-abundance differentially expressed protein identified in our proteomic workflow and subsequently validated by elisa; il- (pg/ml) in % o : . , range . to . ; % o : . , range . to . ; values are median with interquartile range. *** p< . , mann-whitney test (n= ) conclusions: we demonstrate a successful reduction to practice of a relative quantitative proteomics strategy applied to placental explant-conditioned culture media. having optimised and validated this proteomic workflow we will apply the same methods to compare proteins released by normal and preeclamptic placentas. a proteomics screen of human placental microvillous syncytiotrophoblast (stb) revealed the expression of dysferlin (dysf), a membrane repair protein associated with certain muscular dystrophies (vandré et al., ) . a second ferlin protein, myoferlin (myof), was also discovered which has not yet been characterized in the placenta. in human c c myoblasts, dysf and myof are reciprocally expressed as a function of differentiation into multinucleate syncytial myotubes, with dysf predominating in differentiated cells. we hypothesized that dysf and myof would show a similar reciprocal expression pattern in human trophoblasts as a function of syncytialization. methods term placentas from uncomplicated pregnancies were obtained with informed consent (n= ) and either fixed for immunofluorescence (if) labeling or flash frozen for subsequent immunoblotting (ib). human trophoblastic (bewo, jar, and jeg- ) and other cell lines (mrc- , hl- , huvec) were cultured in the absence or presence of μm forskolin or solvent control for - days. dysf and myof expression was examined by rt-pcr, ib, and if. results ib validated proteomics data showing myof expression in term placenta. by if, myof labeling was predominant in apical and basal stb plasma membranes. myof was expressed in trophoblastic cell lines (bewo, jar, and jeg- ), cultured endothelium (huvec), and a fibroblast cell line (mrc- ), but was not detected in a leukemic cell line (hl- ). dysf was constitutively expressed in jar, but minimally expressed in unstimulated bewo. following forskolin-induced syncytialization, we observed a time-dependent increase in dysf expression in bewo concomitant with increasing syncytin- levels. by if, dysf expression was restricted to bewo cells in syncytial structures. in contrast, myof expression was robust in mononuclear bewo cells and not down-regulated over the course of days of differentiation. conclusions two ferlin-family genes are expressed in trophoblasts. fusion-competent bewo cells behave similarly to cultured myoblasts and ctbs with regard to dysf expression, which is restricted to syncytializing cells. myof, in contrast, is expressed constitutively in bewo and other models. while both proteins are likely to function in stb plasma membrane repair (e.g., following syncytial sprouting), the relative contributions of each to this process awaits clarification. features of chronic placental insufficiency are significantly more common in small for gestational age placentas from infants with intrauterine growth retardation. emily king, violetta kolesnikova, carri tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: there is a strong association between intrauterine growth restriction (iugr) and small for gestational age (sga) placentas (heinonen et al. placenta ( ), : - ) . the cause is likely multifactorial, but we hypothesize that chronic uteroplacental insufficiency may play a role. our objective was to test whether sga placentas from human neonates with iugr show significantly more pathologic features of placental insufficiency compared to controls. design: we performed a retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ), excluding elective terminations and spontaneous abortions before weeks gestation. clinical records were reviewed and pregnancy outcomes recorded, including: ) neonatal sex; ) weight (iugr calculated by routine methods), ) trimmed weight of the placenta (sga calculated by routine methods), ) gross placental infarctions, ) gestational age at delivery, and ) maternal features (e.g. race, gravida). controls were defined as cases within the series without sga or iugr (e.g. submitted for meconium, infection, etc). histologic sections were scored by two pathologists while blinded to clinical diagnoses as positive or negative for features of placental insufficiency, including accelerated villous maturation (avm), chorangiosis, and microscopic infarctions. significant associations were tested by analysis and logistic regression for multiple variables. results: similar to prior reports, we observed a strong association between iugr and sga placentas ( . ; p < . ). this relationship was independent of maternal race, fetal sex, and parity, although it was more common in primigravidas. avm and placental infarctions were significantly more frequent in sga placentas with superimposed iugr. controls ( introduction: messenger rna expression peripheral blood cells (pbc) has been recently used as biomarkers of environmental exposures (ionizing radiation or tobacco), physiological conditions (stress) and diseases (hypertension, neurological disorders). pbc evaluation is a useful diagnostic tool in an era of individualized medicine. since there is an urgent need for non-invasive methods for determination of fetal (f) and placental (p) function, this study was designed to evaluate the genes differently and commonly expressed in p tissue and leukocytes in maternal (m) and f circulation.material and methods. p (n= ), f (n= ) and m blood (n= ) were obtained during cesarean section in pregnant baboons at term. total rna from a buffy coat pellet was isolated using a modified procedure of the qiagen rneasy mini kit (qiagen). anti-sense rna (arna) was synthesized and purified using the illumina rna amplification kit (ambion, usa). hybridization of arna to illumina sentrix human whole genome (wg- )beadchips and subsequent washing, blocking and detection was performed using illumina's beadchip ´ protocol. samples were scanned on the illumina beadarrayer gx reader using illumina beadscan image data acquisition software (ver. . . . ). differential gene expression data analysis was performed using illumina beadstudio software (ver. . . . ). results. the detection level of gene transcripts using illumina methodology with control human rna was - at p< . . gene transcripts were detected in f blood and in maternal blood. transcripts were uniquely expressed in fetal blood. transcripts were found in m, but not in f leukocytes. the number of gene transcripts expressed in p tissue was and of these genes were not expressed in m leukocytes. conclusion. despite white blood cells trafficking through the p barrier there is a set of unique genes expressed only in p or in the m or f circulation. the application of these genes as the biomarkers of p barrier function still need to be evaluated. objective: to evaluate if a relationship exists between duration of placental exposure to meconium in vivo and histologic evidence of severity and extent of meconium uptake by macrophages. study design: from a cohort of / ( %) consecutive singleton liveborn infants delivered at term with thick meconium-stained fluid, ( %) had placental histologic examination performed, and in / the timing of meconium appearance after membrane rupture was documented. placental histologic examination quantitatively evaluated the intensity of meconium uptake by resident macrophages based on the number of macrophages/field, and the extent of uptake based on histologic location, graded in a score to . results: mean interval between meconium appearance and delivery was . ± . min (range - ). after exclusion of cases in which severe placental inflammation interfered with analysis, meconium uptake by macrophages was documented in / cases at the level of amniochorionic membranes, in / cases at the placenta, and in / cases at the umbilical cord. there was no correlation between the interval meconium appearance-to-delivery in relation to presence of meconium in the membranes (p= . ), in the placenta (p= . ), in the cord (p= . ), or score of severity of meconium uptake (p= . ). the results did not change after correcting for gestational age, oligohydramnios, presence of placental acute inflammatory or vascular lesions. conclusion: there is no relationship between duration of placental exposure to meconium and the extent and intensity of its uptake by macrophages in cases with exposure up to . hours. transfer of bisphenol a across the human placenta. biju balakrishnan, , kimiora henare, , eric thorstensen, , murray d mitchell. , the liggins institute, university of auckland; national research centre for growth and development, auckland, new zealand. introduction: there are growing concerns over the effects of developmental exposure to the xenoestrogen bisphenol a (bpa). animal studies have shown that bpa is transferred through the placenta and can cause deleterious effects to the fetus. the presence of bpa in feto-placental tissues in humans has also been reported. however, a detailed study of the time-course of bpa transfer across the human placenta has not been performed. the aim of this research was to study the transfer of bpa in ex-vivo perfused human placental tissues. methods: a dual recirculating single cotyledon perfusion was used to monitor the placental transfer of bpa. bpa ( ng/ml), antipyrine (ap) ( μg/ml), and fitc dextran (fitc-dx) ( . μg/ml) were added to the maternal perfusate, and perfusion was continued for hours. perfusate samples were collected from both reservoirs at timed intervals and analysed. bpa, ap, and fitc-dx were determined by hplc with fluorescent detection, hplc with uv detection, and spectrofluorometry respectively. the viability and metabolic activity of the placentae were assessed by measuring -hcg (elisa), glucose utilization, and lactate production (autoanalyzer). fetal pressure, ph, flow rates and fluid shifts were monitored continuously. results: the biochemical validation parameters (glucose consumption, lactate production and -hcg secretion) indicated that the placental tissue was metabolically active and viable throughout the -hour perfusion period. the physical parameters observed (fetal pressure < mmhg, ph range . - . ) were in concordance with other published works for placental perfusion. membrane integrity was confirmed by fluid shifts from either circuit of < ml/hr, and by < % materno-fetal transfer of fitc-dx. an observed ap transfer of - % further validated our model. bpa first appeared in the fetal compartment within minutes of perfusion and reached a peak of about - % of maternal concentration within hours of perfusion. this figure is likely to be an underestimate since it does not include conjugated bpas. conclusion: this first study of bpa transfer in ex-vivo perfused human placental tissue shows that our model can serve as a useful tool to study the transfer kinetics and metabolism of bpa in human term placentae. we found that bpa rapidly crosses the human placenta at environmentally-relevant doses, with potentially harmful effects on the human fetus. angiogenic growth factor secretion by uterine natural killer cells in co-culture with extravillous trophoblast. gendie e lash, katsu naruse, , barbara a innes, stephen c robson, judith n bulmer. institue of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom; obstetrics and gynaecology, nara medical university, nara, japan. objectives. uterine natural killer (unk) and extravillous trophoblast (evt) cells have been proposed to play roles in remodeling of uterine spiral arteries through secretion of various angiogenic growth factors. we have previously demonstrated that unk cells are a significant source of angiogenic growth factors within the decidua, many of which alter with gestational age. however whether the secretion of these proteins is altered by interactions of unk cells with evt is unclear. hypothesis. unk cell angiogenic growth factor secretion is regulated by evt in early pregnant decidua. methods. placental and decidual samples were collected from women undergoing termination of pregnancy with written informed consent ( - and - weeks gestation, n= each group). . × cd + unk cells were positively selected from decidua and co-cultured with evt ( . × ) or cytotrophoblast (ctb; . × ) purified from the same placenta for h in direct or indirect contact (n= each group). angiogenin, ang , pdgf-bb, fgf-basic, timp , icam and vegf-a were measured by fast quant ® angiogenesis multiplex assay system, and vegf-c, ang and plgf by elisa. results of unk co-culture with evt or ctb (negative control) at each gestational age were analyzed with wilcoxon rank test. in addition, the effect of direct and indirect co-culture of unk cells with evt at each gestational age was compared with mann whitney u test. results. at - weeks gestation unk secretion of ang (p= . ), icam (p= . ) and plgf (p= . ) was increased in the presence of evt compared with ctb. no differences were observed at - weeks gestation. in addition, at - weeks gestation unk secretion of ang (p= . ), vegf-c (p= . ) and ang (p= . ) was increased in direct co-culture with evt compared with indirect co-culture. at - weeks gestation unk secretion of timp- (p= . ) was reduced in direct co-culture with evt compared with indirect co-culture. conclusions. unk cell secretion of several key angiogenic growth factors was altered by direct culture with evt. these data suggest that a membrane bound molecule (such as hla-g) mediates this modulation of unk cell activity. abnormalities in placentation and impaired placental circulation can lead to fetal growth restriction. -d ultrasound can be used to evaluate this through the quantification of the power doppler signal that may be expressed as a percentage of colour voxels within a user-defined volume (vi: vascularisation index). we aimed to test the hypothesis that increased fetoplacental blood flow correlates with an increased vi, using the in vitro dual perfusion model of the human placental lobule. three term lobules were dually perfused through both circulations with earles bicarbonate buffer (ebb), and supplemented on the fetal-side only with adult erythrocytes, prepared to a % haematocrit. following initial equilibration perfusion at normal flow values, fetal-side flow was varied between and ml/min, whilst maternal-side flow was held at ml/min. images were obtained with a 'voluson i' ultrasound machine and a neonatal transducer (pulse repetition frequency = . hz, wall motion filter = low , and gain = . ). three -d datasets were acquired at each flow rate from each placental lobule and these were measured in triplicate using vocal. a sphere was centred on a visibly perfused cotyledon along the chorionic-decidual axis, with a diameter corresponding to placental thickness. linear regression analysis was used to assess the relationship between the total fetal-side flow and mean vi. the mean vi showed a high degree of correlation with total fetal-side flow for each lobule ( figure ) suggesting increased vascular perfusion and the inclusion of perfused vessels that cross the detection threshold with increased flow. this data provides qualifying information for translation to a clinical application, where early gestational fetoplacental blood flow will be assessed to predict the onset of fetal growth restriction. anthony n imudia, brian a kilburn, anelia petkova, samuel s edwin, roberto romero, d randall armant. objective survival of first trimester cytotrophoblast cells depends on their upregulation of heparin-binding egf-like growth factor (hbegf), which is downregulated in placental tissues diagnosed with preeclampsia. we have examined the expression and cytoprotective activity of hbegf in term villous explants subjected to hypoxic stress in vitro. non-pathological placentas were collected by cesarean section at term (n= ). chorionic villous explants were prepared and cultured at either % or % o and treated with the hbegf antagonist crm or recombinant hbegf. paraffin sections were assayed for trophoblast cell death by the tunel assay, proliferation by immunohistochemical labeling of nuclear ki and hbegf expression by semi-quantitative immunohistochemistry. data were compared using anova and the student-newman-keuls posthoc test. results crm ( mg/ml) increased trophoblast cell death after culturing villous explants h at % o (p< . ), but only slightly affected proliferative capacity. culture at % o increased trophoblast cell death % above explants incubated at % o (p< . ). trophoblast cell proliferation decreased after h in explants cultured at either % or % o (p< . ). exogenous hbegf ( nm) prevented the elevation of cell death during hypoxia (p< . ) and maintained nuclear ki expression at %, but not %, o . contrary to first trimester trophoblast, hbegf was not upregulated by hypoxia in term trophoblast. the failure of term trophoblast to elevate hbegf expression in response to hypoxia could contribute to their decreased survival at low o compared to early gestation. endogenous hbegf signaling appears to facilitate survival of term trophoblast during villous explants culture. exogenous hbegf supplementation prevented cell death due to hypoxia and maintained trophoblast proliferation rates under in vitro conditions. therefore, hbegf, which is downregulated in preeclampsia, could have significant impact on trophoblast survival during late gestation. supported by the intramural research program of nichd. conspicuous meconium-laden macrophages in the chorionic plate are an independent predictor of clinically significant fetal distress. violetta kolesnikova, emily king, carrie tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: meconium is a common indication for placental examination, present in approximately % of placentas routinely submitted to pathologists (beebe et al. obstet gynecol ; : - ) . prolonged meconium exposure leads to accumulation of meconium-laden macrophages in the chorionic plate (estimated to require at least hours). our objective was to test whether prolonged meconium exposure is associated with clinically significant fetal distress. design: retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ) was performed, excluding abortions before weeks gestation, and placentas without representative sections of the chorionic plate. clinical records were reviewed and pregnancy outcomes recorded, including: ) evidence of fetal distress prompting c-section (non-reassuring fetal heart rate), ) gestational age, ) maternal diagnosis, ) apgar scores, and ) neonatal length of hospital stay. routine histologic sections were independently scored by two pathologists while blinded to clinical diagnoses and outcomes. cases were scored as positive or negative for: ) diffusely conspicuous meconium-laden macrophages in the chorionic plate (at least / hpf), ) chorioamnionitis, and ) features of placental insufficiency. significant associations were tested by the mann-whitney u test for paired comparisons, x analysis, and logistic regression for multiple variables. results: conspicuous meconium staining of the chorionic plate was common ( / , %) and was significantly more frequent in c-sections performed for fetal distress ( / cases, %) (x . ; p-value < . ). logistic regression modeling showed that this association was independent of chorioamnionitis and features of placental insufficiency. there was no association between meconium and neonatal outcome, including no difference in apgar scores or length of hospital stay. conclusions: our data support the hypothesis that meconium-laden macrophages in the chorionic plate are associated with fetal distress prompting c-section. whether meconium is the cause or consequence of this distress is uncertain. however, given the acute time frame between clinical diagnosis and c-section, we suspect prolonged meconium exposure may be a significant cause of non-reassuring fetal heart rate changes. apoptotic index in normal and intrauterine growth-restricted rat placentas. elissa scotland, tri nguyen, s chiang, radmila runic. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: intra-uterine stress caused by maternal food-restriction may have adverse fetal and placental effects. we sought to assess the effect of maternal food restriction during pregnancy on placental apoptosis. methods: rat placentas were analyzed from control dams fed ad libitum and dams % food-restricted from day of gestation. placentas were harvested at embryonic days and (n= ). placentas were fixed in % pfa and two methods of analysis were utilized to determine the proportion of apoptotic to non-apoptotic cells within maternal food restricted and control rat placentas: immunohistochemistry (ih) using fas-ligand and in situ tunel (terminal deoxynucleotidyl transferase biotin-dutp nick end labeling). tunel: after rehydration, slides were subjected to tdt enzyme. dab was used to visualize brown apoptotic nuclei. dna-se treated slide was used as a positive control. ih: primary rabbit polyclonal fas-ligand antibody was used at : dilution, after which secondary antibody linked to peroxidase and dab staining was performed. results: food-restricted placentas demonstrated . % relative apoptotic index when compared to . % in the control group. the iugr iod (integrated optical density) per unit area in the placental membrane was higher than in the control group. fasl demonstrates an iod of . + . in food restricted placentas as compared to . + . per μm surface area in controls. apoptosis was seen in the amnion as well as trophoblast (syncytialtrophoblasts and some cytotrophoblasts). conclusion: the increased apoptotic index in maternal food restricted placentas suggests that the accompanying iugr may be a result of both maternal/fetal nutrient restriction and increased fetal stress. this study found that maternal food restriction during pregnancy affects placental apoptosis. there is more apoptosis in the iugr placental bed at e than at e , with the reverse being true for the placental membrane. more research is required to statistically validate the data. the suggested next step is to evaluate fas and fas-l and to address possible mechanisms of placental apoptosis. cord. jayaraman lakshmanan, avish arora, lilit baldjyan, sharon k sugano, olga miadel, adegoke adeniji, michael g ross, calvin j hobel. ob-gyn, harbor-ucla medical center, torrance, ca, usa; ob-gyn, cedars-sinai medical center, los angeles, ca, usa. background: crf-bp is a kda protein, that specifically binds corticotrophin releasing factor (crf). the structure of cloned crf cdnas in all species examined predicts that the precursor is larger than the kda in size and contains one n-glycosylation site. marked reductions in plasma crf-bp levels seen in pregnant women prior to both preterm and term delivery led to the notion that crf-bp is a "gatekeeper" of crf responses. recently we identified crf-bp expression in term human umbilical cord (uc) by immunohistochemical analyses. objective: to characterized the expression of crf-bp by immunohistochemical and biochemical analyses in human uc. methods: freshly obtained human preterm ( to < weeks, n= ) umbilical cord ( pieces of mm thickness taken at - cm intervals close to placenta) were fixed in bouin's solution and paraffin embedded. also, pieces of ucs were dissected, arteries and vein separated, weighed and frozen at - c. for immunohistochemical localization, uc sections were subjected to immunostaining with polyclonal antibodies to human crf-bp precursors. for western blot analyses, whole uc as well as isolated arteries and vein were homogenized in a buffer containing detergents and protease inhibitors. the homogenate supernatant proteins were subjected to western analyses by standard protocol. immunoreactive protein bands were identified by chemiluminescent reagent. results: both goat and rabbit crf-bp polyclonal antibodies elicited weak to moderate positive staining in uc epithelial layers, vascular musculature and barely endothelial cells. they identified a strong kda band in whole uc as well as in isolated artery and venous preparations. in addition minor immunoreactive protein bands of , , kda in size were noticed in all three preparations. conclusion: we conclude that preterm uc expresses crf-bp. we postulate that the kda major band is either glycosylated crf-bp precursor or glycosylated kda mature protein. the low molecular weight immunoreactive proteins likely represent proteolytically processed, glycosylated crf-bp precursor or a proetolytically processed mature da crf-bp. uc obtained at delivery could be useful as a tool to understand the critical functions of crf-bp in feto-placental unit. objective: adenosine, known to be released from inflammatory sites and tisse ischemia, has many important biologic roles. four specific adenosine receptors have been cloned to date, termed a , a a, a b, and a . recently our study has shown that increased a receptor in the trophoblast of preeclamptic pregnancy was noted and non-vascular and trophoblast-mediated a receptor may play an important role in the pathogenesis of preeclampsia. there are evidences of impaired trophoblast invasion related to matrix metalloproteinase (mmp) in preeclampsia and the relationship between adenosine receptor and mmp in other fields. the objective of this study is to evaluate the effect of mmp expression by adenosine a receptor in preeclamptic villous explants at different oxygen conditions. methods: placental villous explants from normal (n= ) and preeclamptic (n= ) pregnancies were cultured at high ( %) and low ( %) oxygen levels for days. explants were analyzed for mmp- /- and timp- /- by rt-pcr and western blot. preeclamptic villous explants in hypoxic culture condition were treated with a receptor agonist, cl-ib-meca and a receptor antagonist, mre. mmp- / - expression was determined in a time-and dose-dependent manner by rt-pcr, western blot. also mmp- /- activity was evaluated by zymogram assay. results: there were significantly increased a receptor intensity and reduced mmp- /- and timp- /- expression at low oxygen level in normal and preeclamptic villous explants. interestingly, in preeclamptic villous explants, after high oxygen culture mmp- /- and timp- /- expression were recovered to almost same level compared to those in normal villous explants. treatment of preeclamptic villous explants with cl-ib-meca in low oxygen level resulted in a time-and dose-dependent enhanced expression of mmp- /- . this cl-ib-meca-induced expression of mmp- /- was inhibited by pretreatment with mre. conclusion: to our knowledge, this study is the first to evaluate modulation of mmp secretion by adenosine a receptor in preeclamptic villous explant. our results provide evidence for the existence of functional adenosine a receptors in the trophoblast and suggest that adenosine a receptor will be investigated as a therapeutic target in preeclampsia. murray d mitchell, timothy a sato, anderson wang, jeffrey a keelan, anna p ponnampalam, michelle glass. liggins institute; department of pharmacology and clinical pharmacology, university of auckland, auckland, new zealand. introduction: it is well established that prostaglandins play critical roles in multiple aspects of pregnancy and that the fetal membranes are an important site of intrauterine prostaglandin production. endocannabinoids have been implicated in the maintenance of pregnancy and parturition in women and are a source of arachidonic acid which is a substrate for the production of prostaglandins. the aim of the present study was to determine the effects of endocannabinoids on the production of prostaglandins in extraplacental membranes -amnion, chorion and decidua. methods: explants of term amnion and choriodecidua were established and treated with endogenous endocannabinoids -arachidonoyl glycerol ( ag) and anandamide (aea) and with the synthetic cannabinoid cp , , to determine the ability of these substances to modulate prostaglandin e (pge ) production. pge was measured by radioimmunoassay. the explants were also treated with cp , in the presence of either sr a (a selective antagonist of the cannabinoid receptor cb ) or ns (a cox- inhibitor), to determine whether any observed stimulation of pge production was mediated through cox- activity and/or the cb receptor. cox- , cox- , cpla and pgdh protein levels were measured by western blotting. results: all three cannabinoids caused a significant increase in pge production in amnion but not in choriodecidua. however, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. the enhanced pge production caused by cp , was abrogated by co-treatment with either sr a or ns , illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by cb agonism and cox- . preliminary data from western blotting show that cannabinoid treatment results in the up-regulation of cox- expression. however, there was no change in cox- expression and no evidence either for up-regulation of cpla or for down-regulation of pgdh expression. conclusion: this study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labour and membrane rupture. inactivation of vegf receptor- , but not vegfr- or vegfr- , during the peri-implantation period prevents normal pregnancy development in the rodent through disruption of uterine angiogenesis. nataki c douglas, hongyan tang, raul gomez, bronislaw pytowski, daniel j hicklin, jan kitajewski, mark v sauer, ralf c zimmermann. division of reproductive endocrinology and infertility, columbia university, new york, ny, usa; imclone systems, inc., new york. objective: vegf is involved in the regulation of uterine angiogenesis and implantation in both rodents and non-human primates. vegfr- , r- , and r- are expressed in the uterine decidua and are involved in the regulation of vessel formation in many systems. to determine if these receptors have a functional role in the regulation of post-implantation angiogenesis and pregnancy development, we examined the effects of blocking vegfr- , r- , and r- function. design: prospective animal laboratory material and methods: to avoid effects of vegf receptor neutralization on ovarian function, we utilized a progesterone replaced, ovariectomized mouse model. vegf receptor blocking antibodies were administered on ed . , prior to embryonic expression of these receptors. embryonic development was evaluated on ed . , blood vessel density and apoptosis on ed . , and cellular proliferation on ed . (n= per time point in each group). anova with bonferroni correction was used to compare sample means. results: see tables. conclusions: neutralization of vegfr- and vegfr- , but not vegfr- resulted in a significant reduction in cellular proliferation and decidual angiogenesis. vegfr- mediates decidual angiogenesis, but is not required for normal pregnancy development. in contrast, an intact vegf/vegfr- pathway is required for the decidual angiogenesis that mediates early pregnancy development. effect of vegfr neutralization on ed . control anti r- anti r- anti r- no. of implantation sites . +/- . +/- . . +/- . . +/- . hoxa encodes a transcription factor required for endometrial receptivity and embryo implantation. our objective was to identify and to characterize those molecular markers regulated by hoxa in multiple cellular model-systems. using microarray technologies, we identified putative hoxa target genes involved in early implantation. liposome-mediated transfection delivering either empty vector or the same plasmid constitutively expressing hoxa was introduced into newly impregnated mice during laparotomy or layered onto cultured human endometrial stromal-cells (hescs). rna products from these in vivo and in vitro transfections were used to identify targets and to validate the microarray screen employing semi-quantitative real-time pcr (qrt-pcr). we identified statistically-significant genes regulated by hoxa overexpression of which genes were down-regulated greater than -fold when compared to controls. cellular ontogenies of differentially-expressed genes include: cell adhesion molecules, signal transduction factors as well as metabolic regulators. furthermore, we identified the -phosphoglycerate dehydrogenase gene, (pgdh) whose products are regulated by hoxa during implantation in both murine model systems in and cell culture. this genes codes for an enzyme critical to de novo l-serine biosynthesis via a phosphorylation-dependent pathway. microarray analysis demonstrated a fold expression decrease when hoxa is overexpressed. this diminution in pgdh expression was noted in the validation experiments using qrt-pcr and corroborated in hesc cells where the mrna levels decreased to % when compared to controls. the repression of pgdh during the implantation window may represent a conservation of activity as secretory-phase protein synthesis may be suppressed in order to promote cellular differentiation and resultant implantation. these regulatory relationships identified in mouse implantation likely function to enhance uterine receptivity and may have a role in human implantation. objective: hoxa is expressed in endometrium, where it is regulated by sex steroids and is necessary for endometrial receptivity and implantation. hoxa is also expressed in leukocytes. here we hypothesized that hoxa would be regulated by sex steroids in both cell types. we further hypothesized a correlation between expression of hoxa in peripheral blood cell (pbcs) and endometrium in both mice and in humans. methods: real-time pcr was used to determine differential expression of hoxa mrna in u cells, a monomyelocytic cell line, in response to increasing concentrations of estradiol. to determine if hoxa is expressed in leukocytes in vivo, peripheral leukocyte hoxa mrna expression was measured over sequential estrus cycles in mature cd nulliparous mice and correlated to vaginal smear-cytology. additionally, peripheral leukocytes were isolated either from pregnant or normally-cycling women to assess hoxa mrna expression. results: there was a direct, dose-responsive correlation between exposure to increasing estradiol and hoxa mrna expression levels in u cells. in a murine model, we demonstrated that hoxa mrna expression-levels varies throughout the estrus cycle with a marked increase in expression following vaginal plug detection. the nadir of hoxa expression is prior to proestrus and increased up to -fold during the receptive phase. this increased expression continues throughout gestation. the heightened expression in murine leukocytederived hoxa mrna also is demonstrated across species. our preliminary data suggests that the greatest fold-increase of expression occurs during the window of implantation of the secretory phase in normal, cycling women. this level was sustained in pregnancy. there appears to be a trend with the highest levels of expression associated with viable gestations. women with attenuated expression profiles had non-viable gestations. conclusions: hoxa expression is regulated by sex steroids in both leukocytes and endometrium. the temporal pattern of peripheral hoxa transcript expression demonstrated in mice and humans mimics the differential rna expression documented within the uterus. leukocyte hoxa expression during the reproductive cycle in mice and humans is a marker of endometrial receptivity. peripheral leukocyte expression of hoxa mrna may correlate with implantation success. carolien m boomsma, annemieke kavelaars, marinus jc eijkemans, gijs teklenburg, bart cjm fauser, cobi heijnen, nick s macklon. reproductive medicine and gynaecology and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands. the purpose of this study is to assess the association between the intra-uterine cytokine expression profile at the time of embryo transfer and successful embryo implantation following ivf/ icsi treatment. materials and methods women undergoing ivf/ icsi underwent endometrial secretion aspiration prior to embryo transfer. known soluble mediators of implantation were measured using a multiplex immunoassay, namely il ß, il , il , il , il , il , il , il , tnf , vegf, ifn , eotaxin, mcp- , ip- , dkk- and hbegf. mif was determined using an elisa. the total protein concentration was measured for normalization purposes. data were log transformed to obtain normal distribution. multivariable logistic regression analysis with a backward elimination procedure (p< . ) was used, potential confounders (age, blood contamination, embryo quality) were included in a forward stepwise model. ten mediators of the analysed were detectable in - % of the samples. il was detectable in % of samples, dkk- %, il- %, il- %, il- % and hbegf was detectable in % of samples. ifn-was not detectable in any of the samples. multivariable logistic regression showed only logmif concentrations to have a significant correlation with achieving clinical pregnancy (p = . ). higher mif concentrations were correlated with a higher chance of conceiving. endometrial secretion analysis represents a novel means of assessing the intra-uterine milieu encountered by the embryo and offers new perspectives in the study of endometrial receptivity. in this large prospective study assessing an array of cytokines, mif was found to be significantly correlated with pregnancy. mif, macrophage migration inhibitory factor, is a cytokine with numerous proinflammatory, immunomodulatory, angiogenic and tissue remodelling properties. mif induces the synthesis and secretion of matrix metalloproteinases by endometrial cells, which may contribute to embryo invasion. its expression is particularly increased during the secretory phase, suggesting a role in reproductive processes. analysis of aspirated endometrial secretions offers a direct clinical test of endometrial receptivity which can be applied during treatment cycles without disrupting implantation. endometrial receptivity and secretory differentiation require progesterone (p). it has been hypothesized that low p levels result in delayed endometrial differentiation and infertility due to reduced receptivity. objective: test the effects of low luteal p on histologic and molecular markers of differentiation and function. methods: normal cycling women (n= ) were treated with daily leuprolide ( . mg/d) beginning in the midluteal phase and continuing through the protocol. after menses, subjects received transdermal estradiol (e, . mg/d) for days. after day of e, subjects also received daily i.m. injections of p, randomized to mg/d (sub-physiological) or mg/d (physiological). endometrial biopsy was performed after days of combined e and p treatment. additional untreated women had biopsies performed days after spontaneous lh surge. endometrial histologic dating was performed by two individuals according to the criteria of noyes et al. mrna levels were assessed using real-time rt-pcr. results: mean(s.d.) of peak and trough p serum concentrations in the mg/d p group were . ( . ) and . ( . ) ng/dl, respectively, while those in the mg/d group were . ( . ) and . ( . ) ng/dl. there were no differences between treatment groups for histologic dating; the mean(s.d.) histologic date was . ( . ), . ( . ), and . ( . ) for the mg, mg, and spontaneous cycle groups, respectively. there also were no differences among the three groups in mrna levels of ten functional markers (er , pr, integrin subunit, osteopontin, cyr , egr- , fkb , c-fos, and cd ), although variability of gene expression was greater in those who received mg/d p than in those who received mg/d p. there was also no correlation between serum progesterone level and gene expression or histologic date. conclusions: sub-physiological levels of progesterone, in the range seen in ovulatory women, do not induce detectable changes in expression of marker genes or histological dating, although low p levels were associated with greater variability of gene expression. these data suggest that abnormalities in endometrial histologic development and function likely result from intrinsic abnormalities rather than from low levels of p secretion. (supported by unc nova carta fund and nih u hd- ). endometrial ip attracts trophectoderm through cxcr interaction. simcha yagel, caryn greenfield, hen sela, jacob hanna, irit manaster, orna singer, ronit haimov-kochman, shira natanson-yaron, diana prus, benjamin reubinoff, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical centers, jerusalem, israel; lautenberg center for immunology, jerusalem, israel; human embryonic stem cell research center, jerusalem, israel. introduction: implantation is initiated in part by attraction of the blastocyst to the endometrium lining the uterus. we hypothesized that this process is partly accomplished by chemokines expressed by the endometrium interacting with chemokine receptors on the blastocyst, suggesting that they play an important role in implantation. materials and methods: chemokine receptors were characterized in jeg cells, placental villi, primary trophoblast cell culture, trophectoderm cells derived from human es cells, blastocyst trophectoderm, and st trimester placental tissue sections. expression of chemokines was tested in decidua, endometrium, and ishikawa and hec- cell lines. immunohistochemistry, intracellular staining, elisa, facs analysis, and rt-pcr were employed to characterize chemokine receptor and ligand expression. functional testing was performed using transwell migration assays and in a nude mouse model using a matrigel gel plug cell attraction assay followed by facs analysis. results: trophoblasts demonstrated expression of various chemokine receptors, most prominently cxcr and cxcr . immunohistochemistry of trophoblast from placental villi plated on matrigel expressed cxcr and cxcr as well as hla-g. noteworthy is that trophectoderm cells derived from hes cells treated with bmp- and jeg cells, and blastocyst trophectoderm expressed principally cxcr . elisa and immunohistochemistry showed that decidua and endometrium expressed chemokines ip and il . migration assays demonstrated that ip significantly attracted various trophoblast and trophectoderm cells in vitro, and in the mouse model in vivo. taken together these results demonstrate the interaction between trophoblasts and endometrial cells is mediated by cxcr and cxcr , and il and ip . these interactions are important in the attraction of trophoblasts at the feto-maternal interface. however, ip -cxcr is the most relevant to early implantation as only cxcr is expressed consistently by trophectoderm. the endometrial proteome: changes from proliferative to secretory phase. lois a salamonsen, jenny i-c chen, xian mak, peter j stanton, david m robertson, andrew n stephens. prince henry's institute of medical research, melbourne, vic, australia. global gene analyses have demonstrated major changes across the menstrual cycle, but which of these are reflected in the proteome is not known. this study aimed to globally assess proteins differentially expressed in the endometrium between the proliferative and. secretory phases. d page analysis with dige minimal dye labelling was conducted across the pi range - on endometrial tissue from either the mid-proliferative or midsecretory phases (n= /group). profiles were assessed using samespots software. differentially expressed proteins were identified using maldi-tof ms and the interrelationship of proteins examined using ingenuity software. a total of spots were detected: were differentially expressed (p < . ) with spots having an overall false discovery rate < % (q < . ). hierarchical clustering analysis revealed that these proteins lay within three main branches in the protein dendrogram. one cluster had proteins upregulated in the proliferative phase and two contained proteins up-regulated in the secretory phase. the unique protein profiles were also revealed using principle component analysis (pca): proteins clustered into two main groups, according to cycle phase. pca thus indicated similar unique protein signatures as suggested by hierarchical clustering. thirty one of the differentially expressed proteins were identified using maldi-tof ms. these proteins could be grouped into seven categories, which included structural ( ), transport ( ), regulatory ( ), membrane ( ), enzyme ( ), motor ( ) and others ( ). proteins involved in matrix assembly and those needed for subsequent establishment of secretory endometrium were up-regulated in proliferative endometrium. proteins important for cellular organisation and communication as well as products responding to environmental stress and the immune system were highly up-regulated during the secretory phase. biological pathways were constructed based on the proteins identified. the top network for secretory endometrium clustered around tgf-b. others related to inhibition of cell death / cell viability and leukocyte extravasation. these studies provide a global approach to the cyclic changes of the endometrium and highlight the complex dynamics of protein expression in human endometrium. hormonal regulation of prokineticins in the human fallopian tube: potential regulators of embryo transport. aw horne, hn jabbour, p lourenco, s wright, s battersby, arw williams, hod critchley. reproductive and developmental sciences, university of edinburgh, edinburgh, united kingdom; mrc human reproductive sciences unit, queen's medical research institute, edinburgh, united kingdom. background: understanding the factors regulating embryo transport in the fallopian tube (ft) has important clinical implications. embryo retention in the tube due to ft dysfunction is thought to lead to tubal pregnancy, a considerable cause of morbidity and occasional mortality. transport of the embryo through the ft is, for the greater part, accomplished by smooth muscle contraction. a group of multi-functional proteins and their receptors, called prokineticins, have been shown to affect smooth muscle function in other tissues, such as the intestine. the expression pattern of prokineticins, and their receptors, was examined in normal human ft obtained throughout the menstrual cycle, and the effect of the sex steroids on prokineticin expression was examined in an in-vitro model of the ft. methods: ft biopsies (n= ) and sera (for measurement of oestradiol and progesterone for endocrine staging) were collected from women undergoing gynaecological procedures for benign conditions. using a combination of quantitative taqman rt-pcr and immunohistochemistry, the mrna and protein expression pattern of prokineticins, and their receptors, were examined in the ft throughout the menstrual cycle. tubal explant culture was established using surgical tissue from the biopsies and exposed to varying concentrations, and time courses, of oestrogen and progesterone. results: prokineticin (pk ) and prokineticin receptor (pkr ) mrna are up-regulated in the progesterone-dominant mid-secretory phase. pk and pkr protein are expressed in the epithelium, smooth muscle and around the blood vessels of the ft. stimulation of tubal explant cultures with a physiological concentration of progesterone showed an up-regulation of pk and pkr . conclusions: prokineticins show temporal variation in expression in human ft and appear to be regulated by progesterone. their role in embryo transport needs to be investigated to further understanding of pregnancy complications, such as tubal pregnancy. translating mouse to human: a dynamic model of xenografted human endometrium. alex j polotsky, liyin zhu, nanette santoro, jeffrey w pollard. albert einstein college of medicine, bronx, ny, usa. in the mouse endometrium, the hormonal environment controls cellular proliferation and cell cycle activity. estradiol (e ) inhibits glycogen synthase kinase beta (gsk- ), resulting in nuclear accumulation of cyclin d and progression of the cell cycle, as well as dna replication licensing. in utero administration of the gsk- inhibitor, licl, results in epithelial cell proliferation in the absence of e (zhu, pollard. pnas. ; : ) . in this study, we derived a functional model of xenografted human endometrium to perform mechanistic studies of human endometrial proliferation. methods: human endometrial samples were obtained from volunteers aged - . immuno-compromised mice were transplanted with disaggregated/ recombined human epithelial glands and stroma under the kidney capsule. after weeks of out-growth, mice were ovariectomized, and replaced with e or licl. xenografts were harvested and processed for immunohistochemistry (ihc) and glandular labeling index (li). t test was used to compare group means. results: - % of engraftments were successful, resulting in a vascularized endometrium with characteristic architecture (a, b). hoechst staining confirmed that xenografts were made up of human cells ©. e (e) induced significantly greater proliferation compared to control (d) as assessed by ihc for ki , with li of . ± . and . ± . , respectively, p = . ). minichromosome maintenance- , a protein involved in dna replication licensing, was more sensitive for e -treated cells synthesizing dna than was ki staining (i). estrogen (g) and progesterone receptors (h) were expressed in xenotransplant tissue, the latter being up-regulated by e . licl (f) induced proliferation similar to e and greater than control (li = . ± . , p= . for e vs. licl). conclusion: xenografted human endometrium provides a dynamic model of endometrial proliferation that is well suited for translational studies. administration of licl in the absence of e induced glandular proliferation, supporting the notion that similar mechanisms are operative in human proliferation as in the mouse. gnrh analogs have been extensively used in assisted reproduction. although the main effects of gnrh analogs are via gnrh receptors on the pituitary gonadotrope, gnrh and gnrh receptors have been identified in many reproductive tissues including human endometrium, suggesting their potential action at endometrial level. in the present study, we examined the potential regulatory action of gnrha, la on sex steroid mediated gene expression in human endometrium. human endometrial surface epithelial (hes) cells and isolated endometrial stromal (esc) cells were used as in vitro models. all experiments lasted for h. the cells were treated with estradiol (e , nm, h), progesterone (p , nm, h), gnrha (la μm, h), e ( h) followed by p (last h) and gnrha plus e plus p where, gnrha added either first, second or last in order at h intervals. total rna was extracted, reversed-transcribed and subjected to real-time pcr simultaneously, measuring the expressions of il- , il- , il- , il- , il- , il- , il- , il- p , il- p , il- , ifn-and tnf . both hes and esc expressed majority of these cytokines with the exception of low to undetectable levels of il- , il- , il- and ifn-. treatment with e and p , either alone, or in combination significantly upregulated the expression of many of these cytokines at varying extend as compared to controls. however, gnrha either alone or in combination with e and p significantly diminished e , p or e plus p induced mrna expression of cytokines. the suppressive effects of gnrha on some of the cytokines varied significantly by the order which gnrha was introduced into the culture medium. we conclude that gnrha by acting directly on the endometrial cells effectively suppresses the mrna expression of several key proinflammatory cytokines upregulated by ovarian steroids. our results imply that gnrha therapy during assisted reproduction may modify endometrial receptivity via downregulation of proinflammatory cytokines induced by estradiol and progesterone. supported by nih grant hd . introduction: endometrial angiogenesis is characterized by a rapid increase during the early proliferative phase that peaks midcycle, followed by a gradual decrease in the secretory phase, while menses involves generalized endometrial inflammation, necrosis, and vascular thrombosis. vascular endothelial growth factor (vegf), whose expression is regulated by sex steroids, is a mediator of endometrial angiogenesis. recently, myoferlin, a kd transmembrane protein, was identified in endothelial cells where it mediates vegf-dependent endothelial cell proliferation, migration, and nitric oxide synthesis by affecting vegf receptor- function and stability. myoferlin also appears to play a role in vesicle trafficking and membrane repair. objective: to characterize myoferlin protein expression in endometrium. methods: western analysis of cultured human endometrial stromal cells (hesc) and human endometrial endothelial cells (heec) treated with physiologic concentrations of estradiol and progesterone was performed using a polyclonal rabbit antibody against myoferlin. subsequently, immunohistochemistry (ihc) was performed on human endometrium samples obtained from various stages of the menstrual cycle. results: myoferlin protein was expressed in hescs and heecs, but expression was not affected by sex steroids. ihc staining for myoferlin was specific, intense, and localized to the apical membrane of glandular epithelial cells and endothelial cells, with less intense staining in the stroma. h-score quantification showed that in endometrial endothelial cells, myoferlin protein expression was highest during the early proliferative and early secretory phases, while glandular and stromal myoferlin expression peaked during the late proliferative/early secretory phase. conclusion: myoferlin expression in human endometrium correlates with periods of greatest endometrial angiogenesis, and expression is not limited to endothelial cells, but also includes glandular and stromal cells. given the involvement of myoferlin in vegf signaling as well as membrane repair, understanding its role in human endometrium may further elucidate an understanding of endometrial development both under physiologic and pathologic conditions. the human embryo is the primary regulator of embryo-endometrial molecular cross talk during early implantation. gijs teklenburg, , cobi heijnen, esther baart, karima amarouchi, carolien boomsma, janet carver, helen mardon, annemieke kavelaars, nick macklon. reproductive medicine and gynaecology, and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands; nuffield department of obstetrics and gynaecology, university of oxford, women's centre, john radcliffe hospital, oxford, united kingdom. introduction: uterine receptivity and implantation are controlled by locally acting trophic factors and cytokines. in humans, the regulation of the embryo-endometrial dialogue beyond the early blastocyst stage is poorly understood. we hypothesized that the interaction between a healthy conceptus and the receptive endometrium is associated with a distinct local regulation of cytokine production favouring implantation. methods: human embryos, cryopreserved at day after fertilization and donated for research, were thawed and cultured under standard conditions until day five. forty-two embryos from donors developed to the blastocyst stage. following removal of the zona pellucida, they were placed in individual coculture on a confluent monolayer of endometrial stromal cells. on day , the developmental potential of each embryo was assessed as early arrested, late arrested or developing. culture supernatants were analysed for concentrations of il- , il- , il- , il- , il- , il- , il- , il- , tnf-, mcp- , ip- , eotaxin and hb-egf using a multiplex immunoassay. day supernatants from culture systems in which no embryo had been placed were also analysed as controls. results: out of ( %) embryos continued to develop and were able to attach and invade into the stromal cell compartment of the co-culture environment. twelve late arrested embryos showed signs of degradation on day and the totally disintegrated embryos were assigned to the early arrested group. supernatants from both early and late arrested embryo cultures contained significantly lower levels of a number of cytokines and growth factors in comparison to developing embryo cultures. moreover, the levels of these mediators in co-cultures were significantly lower than those in non-embryo control stromal cell culture supernatants. conclusion: these data suggest a pivotal role of the embryo in embryoendometrial cross talk. whether reduced mediator expression in co-cultures reflects a selective down regulation of stromal cell cytokine and growth factor production is now being investigated. epithelial cell dynamics during human implantation. hiroshi uchida, tetsuo maruyama, toru arase, masanori ono, takashi kajitani, maki kagami, hideyuku oda, sayaka nishikawa, yasunori yoshimura. department of obstetrics and gynecology, keio university school of medicine, shinjuku, tokyo, japan. epithelio-mesenchymal transition (emt) is thought to play a role in functional differentiation of endometrial epithelial cells during human implantation. molecular mechanism of epithelial sheet remodeling caused by embryo invasion remains elusive. to address this, we investigated cellular dynamics of n-cadherin and vimentin, the two representative major markers of emt, during implantation. in in vitro implantation assay using a human endometrial epithelial cell line, ishikawa, and a human choriocarcinoma cell line, jar (uchida et al., hum reprod ), we pre-treated human ishikawa cells with or without ovarian steroid hormones ( -estradiol + progesterone; ep), fa- (n-cadherin blocking ab), or suberoylanilide hydroxamic acid (saha), one of histone deacetylase inhibitors, which has a potential to improve in vitro implantation (ibid). implantation or treatment with or without ep or saha enhanced the expression of n-cadherin and vimentin but down-regulated ecadherin. furthermore, treatment with ep or saha accelerated ishikawa cell motility and increased the number and spreading area of jar spheroids. in vitro implantation assay, the most prominent staining intensity of n-cadherin was observed just around the adhered spheroid from which its intensity decreased away. functional blockade of n-cadherin by fa- resulted in the complete suppression of ishikawa cell motility, the unique distribution of n-cadherin around jar spheroids, and the spreading area of jar spheroids, while it did not affect the number of the adhered spheroids. human implantation consists of the multiple steps, including apposition, adhesion and penetration. thus, these results collectively indicate that emt may take place after the apposition and that n-cadherin may be required for the remodeling and emt of the epithelial sheet during embryo invasion. n-cadherin may enhance the recruitment of spheroid-neighboring cells, suggesting its role in the covering-up of the invading embryo through acceleration of epithelial cell motility. endocannabinoid regulation in human endometrium. jessica g scotchie, marc a fritz, steven l young. obstetrics and gynecology, university of north carolina, chapel hill, nc, usa. background: research in mouse models demonstrates two cyclically regulated endocannabinoids produced in murine endometrium, anandamide and -arachidonoyl glycerol ( -ag); both play critical roles in murine embryonic implantation. no studies of endocannabinoids in human endometrium have been performed. objectives: determine menstrual cycle expression and localization of synthetic and degradative enzymes for anandamide and ag in human endometrium. methods: human endometrium was collected from volunteers across the menstrual cycle (n= ). quantitative rt-pcr was performed analyzing the expression of: n-acylphosphatidylethanolamine (nape) and fatty acid amide hydrolase (faah), the synthetic and degradative enzymes for anandamide, respectively; sn- -diacylglycerol lipase-a (dagla), and b (daglb), the synthesis enzymes for ag; and monoacylglycerol lipase (magl) and cyclooxygenase- (cox ), the degradative enzymes for ag. the constitutive gene ppia was used for comparison. immunohistochemical localization of nape protein was performed using nape-pld polyclonal antibody (cayman chemical, # ). anova and student's t-test analysis performed on samples grouped by proliferative (pro), early, mid-, and late secretory (es, ms, ls) phases. results: mrna expression of all enzymes responsible for synthesis and degradation of anandamide and ag was detected throughout the cycle. no significant cyclic change in nape, faah, or daglb gene expression was seen. a decrease in dagla gene expression in the ms and ls phases compared to the pro and es phases (p= . ) was seen. magl gene expression was higher in the secretory phase than the pro phase (p= . ). cox gene expression was detected at low levels in the pro, es and ms phases, with marked increase in the ls phase (p< . for all comparisons). protein localization of nape showed a cytoplasmic epithelial location, with increased staining on pro and es samples compared to ms and ls samples. immunolocalization of remaining proteins is ongoing. conclusion: this is the first report documenting the presence of endocannabinoid synthetic and degradative enzymes in human endometrium. genes controlling anandamide expression do not fluctuate significantly across the cycle. however, ag's degradative enzymes increase in the secretory phase, suggesting that lower ag levels may be advantageous for embryo implantation. our findings suggest that human endometrial endocannabinoid regulation differs from murine regulation. interleukin- beta (il- ) regulates il- signaling in decidua-implication in the pathophysiology of preeclampsia (pe). sj huang, cf yen, cp chen, f schatz, cj lockwood. obstetrics, gynecology and reproductive sciences, yale university, new haven, ct, usa; ob/gyn, chang gung memorial hospital, tao-yuan, taiwan; ob/gyn, mackay memorial hospital, taipei, taiwan. objective: previously, we found much higher cytoplasmic immunoreactive il- levels in the preeclamptic decidual cells than in adjacent interstitial trophoblasts. such decidual cell-derived il- contributes to the systemic endothelial cell dysfunction that elicits the proteinuria and hypertension of the maternal syndrome. il- promotes the transition from innate to adaptive immunity. moreover, by skewing monocyte differentiation from a dendritic to a macrophage phenotype, decidual il- may promote the macrophage excess observed in the preeclamptic decidua. macrophages impair trophoblast decidual invasion to foster incomplete spiral artery remodeling that elicits placental ischemia and hypoxia. the current study: ) localized il- mrna levels in preeclamptic versus normal decidual sections; ) evaluated mechanisms regulating il- synthesis by targeting intracellular signaling pathways with specific inhibitors; ) identified potential il- targets by immunolocalizing the il- receptor (il- r) to specific cell types in placental bed biopsies. methods: in situ hybridization localized il- mrna in normal versus preeclamptic decidua. il- r was immunolocalized in placental bed biopsies. leukocyte-free first trimester decidual cells were incubated with e and mpa ± il- ( ng/ml) ± an inhibitor of p mapk (sb ) or protein kinase c (calphostin c) or nf b (activation inhibitor iii) for hrs. an elisa measured secreted il- levels. results: il- mrna was present primarily in decidual cells with increased il- mrna levels observed in pe. preferential expression of the il- r was observed on decidual cells in placental bed biopsies. compared with basal il- levels ( . ± . pg/ml/ g cell protein) by decidual cells, il- enhanced il- output by decidual cells ( . ± . pg/ml/ g cell protein). only the p mapk inhibitor significantly reduced this output to . ± . pg/ml/ g cell protein (n= , p< . ). our results indicate that inflammatory cytokine enhances il- synthesis in decidual cells of the preeclamptic decidua by a mechanism involving p mapk. such il- is likely to act as an autocrine/paracrine effector via decidual cell-expressed il- r to contribute to the macrophage excess observed in the preeclamptic decidua. heparanase is up-regulated by estrogen and during the secretory phase of the human endometrium. ronit haimov-kochman, shira natanson-yaron, caryn greenfield, achinoam lev-sagie, lichtenstein michal, haya lorberboum-galsky, israel vlodavsky, simcha yagel, arye hurwitz. ob/ gyn, jerusalem, israel; cellular biochemistry human genetics, hadassah hebrew university medical centers, jerusalem, israel; cancer and vascular biology research center, technion school of medicine, haifa, israel. introduction: heparanase is an endoglycosidase that cleaves heparan sulfate (hs) proteoglycan of the extracellular matrix. the full-length proheparanase is activated by cleavage into an active isoenzyme, resulting in the release of hsbound cell-differentiation factors, such as hb-egf. the cycling endometrium involves remarkable steroid hormone-induced tissue remodeling. in vivo, increasing exposure to unopposed estrogen may lead to endometrial malignant transformation. aim: to investigate heparanase expression and regulation in the cycling endometrium. materials and methods: heparanase mrna levels were measured by quatitative rt-pcr in naturally menstruating women and in hec a, estrogen receptor (er)-negative and ishikawa, er-positive endometrial carcinoma cell lines exposed to increasing doses of estradiol. heparanase isoenzymes were localized by immunohistochemistry using specific anitbodies in murine endometrium and human normal, hyperplastic and malignant endometrium. results: heparanase mrna level increased fold in secretory phase (d ) compared to proliferative phase (d ) endometrium. heparanase transcript levels increased fold during hr culture in er positive adenocarcinoma cell line exposed to increasing doses of estradiol, but not in hec a, er negative cell line. both heparanase isoforms were localized to murine glandular endometrium. human glandular endometrium at both proliferative and secretory phases was immunoreactive with the active isoform of heparanase. proheparanase was detected in basal membrane of endometrial glands and endometrial stroma during secretory phase. along with malignant transformation of the endometrium the presence of proheparanase increased dramatically from none in stroma of normal and hyperplastic endometrium to abundance in malignant tumors. conclusions: heparanase gene expression is higher during the window of implantation and up-regulated with estrogen in endometrial cells via er in vitro and vivo. heparanase is differentially localized in the secretory phase of the endometrium compared to the proliferative phase, suggesting a role for this molecule during the window of implantation in man. interleukin- (il- ) system mrna and protein expression in the human fallopian tube with ectopic implantation. hong-yuan huang, , tien-hung huang, chin-jung li, chyi-long lee, , hsin-shih wang, , yung-kuei soong. , obstetrics and gynecology, chang gung memorial hospital, kwei-shan, tao-yuan, taiwan; obstetrics and gynecology, chang gung university and school of medicine, kwei-shan, tao-yuan, taiwan. objective: ectopic pregnancy, an abnormal implantation of a fertilized ovum outside the uterine cavity, has been increasing in number at a staggering pace of all pregnancies. il- system is one of the major cytokines involved in human endometrium during embryo implantation and might perform a defensive role against maternal immune response. very little information is available regarding the expression and synthesis of cytokines in the pathogenesis of fallopian tube with ectopic gestation. the purpose of this study is to investigate il- system expression in human fallopian tubes with ectopic pregnancy. methods: paired segments of human fallopian tubes with ectopic implantation site and side portion close to ectopic gestation (n= ) were collected from women undergoing laparoscopic salpingectomy after informed consent and irb approval. segments of fallopian tubes from women undergoing tubal ligation (n= ) were used as control groups. total extracted rna was reverse transcribed and amplified by pcr using specific primers for gapdh ( bp), il- ( bp), il- bp ( bp) and il- r ( bp). quantitative il- and il- bp mrna expression in human fallopian tube was determined by real-time pcr. to determine the presence of il- system proteins, tissues were fixed and processed for immunohistochemical study. data analysis was done with anova and pearson's correlation. results: il- and il- bp as well as il- r mrna were all expressed in tubal ectopic implantation and normal tubes. according to real-time pcr with c t value quantification and -ct method, a significantly higher il- expression in tubal ectopic implantation and lower ratio of il- antagonist to agonist in portion close to ectopic implantation is demonstrated in comparison to normal tubes (p< . ). immunoreactive il- system at the protein levels was also present in human fallopian tubes with ectopic implantation and normal tubes. conclusions: these results suggest that fallopian tube il- system expression may play a crucial role during the process of early embryonic implantation. the expression and ratio of antagonist to agonist in fallopian tubes may indicate an earlier "dialogue " in human fallopian tubal gestation prior to uterine implantation. replacement. marcia c ferreira, ines kd cavallo, fernando m reis. gynecology, ufmg, belo horizonte, mg, brazil. activin a is a growth factor expressed in the endometrium, where it modulates tissue remodelling and enhances decidualization. the effects of activin a are counteracted by two binding proteins, namely follistatin and follistatin-like (fstl ). while the endometrial expression of activin a increases during the secretory phase of menstrual cycle, the effects of ovarian steroids on these proteins and their mrnas has not been assessed yet in postmenopausal women or in ovariectomized animals. we have evaluated the effects of estrogen alone or estrogen plus progestin on the endometrial expression of activin beta-a subunit, follistatin and fstl in ovariectomized rats. adult female wistar rats (n= ) were ovariectomized and received one week later a single dose of estradiol benzoate ( . mg/kg body weight, i.m. injection), either alone (n= ) or associated with depot medroxyprogesterone acetate ( . mg/kg body weight, i.m. injection, n= ), or oil vehicle (control group, n= ). one week after the hormone or placebo treatment, the animals were sacrificed and their uteri were removed and processed by immunohistochemistry and real-time pcr. data were normalized to the expression of ribosomal phosphoprotein p (rpp ) and analyzed with the delta-delta ct method, anova and newman-keuls test. activin beta-a subunit mrna levels increased significantly in the uteri of rats treated with estradiol alone ( . fold increase over controls, p< . ) and to the same extent in rats receiving estradiol plus medroxyprogesterone ( . fold increase over controls, p< . ). this was accompanied by increase of beta-a subunit immunostaining in estradiol and estroprogestin-treated rats, which was noted only in the surface endometrial epithelium. follistatin mrna expression, conversely, showed a significant decrease in the groups treated with estrogen alone ( . fold compared to controls, p< . ) and estrogen plus progestin ( . fold compared to controls, p< . ), while follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprotestin-treated rats compared to controls. fstl expression was similar in the groups. in conclusion, the expression of activin beta-a subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. endometrial nk cells are a unique inert nk subset until pregnancy. simcha yagel, irit manaster, jacob hanna, ronit haimov-kochman, miri godin, yuval bdolach, caryn greenfield, shira natanson-yaron, arye hurwitz, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical center, jerusalem, israel; lautenberg center for tumor immunology, hadassah-hebrew university medical center, jerusalem, israel. introduction: we recently demonstrated that nk (natural killer) cells play a critical role in trophoblast migration and angiogenesis at the fetal maternal interface. nk cells populate the endometrium at the secretory phase of the menstrual cycle, the time of anticipated blastocyst implantation. peripheral blood (pb) nk cells and decidual nk (dnk) cells express a variety of activating receptors, including nkp , nkp and nkp , collectively known as natural cytotoxicity receptors (ncrs), and nkg d which regulate nk cell killing and growth factor production. to compare endometrial nk cell (enk) activating receptor expression and function to pbnk and dnk cells and endometrial ligand expression, with a focus on their roles in blastocyst implantation. patients and methods: subjects were ivf patients undergoing natural menstrual cycles. endometrium samples were collected on treatment days and . a lymphocyte profile of the endometrial cells and pb was performed. facs analysis was performed on isolated endometrial nk cells, pbnk cells and dnk cells for cd , cd , nkp , nkp , nkp and nkg d. ncr ligand expression was characterized on adherent endometrial cells using ncr-ig fusion proteins and nkgd -ig and nkg d specific ligands as well as control ccmi-ig. redirected killing assays and cytokine secretion assays of ifn , vegf, plgf, and il- with and without il- were performed. results: endometrial lymphocytes of day and in these women are mostly cd bright cd -nk cells, with a significant amount of t cells, similar to pbnk cells and in marked contrast to dnk cells. unlike pbnk and dnk cells, enk receptors do not express nkp , nkp . nkp and nkg d are the only activating enk receptor expressed. like decidual cells, adherent stromal endometrial cells expressed the ligands for nkp , nkp and nkg d, suggesting that these nk cells have potential for activation. finally enk cells could not kill or secrete cytokines. conclusions: these findings of a unique activating receptor profile on endometrial nk cells, unlike that of dnk and pbnk, suggest that enk cells are a special local population of nk cells that change dramatically and are activated at the onset of pregnancy. variation in platelet activation throughout the menstrual cycle. fiona c denison, amy o robb, imogen b smith, nicholas l mills, hilary od critchley, david e newby. centre for reproductive biology; centre for cardiovascular sciences, the university of edinburgh, united kingdom. background: platelet-monocyte aggregation (pma) is a sensitive and novel measure of platelet activation with important proinflammatory consequences including release of cytokines and chemokines. previous studies using less sensitive techniques suggest that platelet activation alters during the menstrual cycle in response to circulating concentrations of sex steroids. the effect of sex steroids on circulating (c) pmas during a single menstrual cycle is not known. objective: to determine whether cpmas, platelet surface (ps) p-selectin and plasma (p) p-selectin vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: healthy, nulliparous, pre-menopausal, non-smoking women (mean age years), with regular menses ( - days) were studied. subjects gave written informed consent and the study had ethical approval. serial venous blood samples were taken at menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - and - ). cpmas (monocytes positive for the platelet marker cd a) were measured by flow-cytometry. psp-selectin expression was calculated on cd a positive cells. isotype-matched controls were used. serum oestradiol (e) and progesterone (p), plasma and pp-selectin were measured by elisas. data were analysed by one-way anova with repeated measures and bonferroni's post-tests for multiple comparisons. results: luteal phase p was > nmol/l in all women. numbers of cpmas and expression of psp-selectin were both significantly higher during menstrual compared with periovulatory phase of the menstrual cycle ( . ± . vs. . ± . %, p= . and . ± . vs . ± . %, p< . , respectively). there was no significant difference in pp-selectin concentration during the menstrual cycle (p= . ). there was no correlation between levels of serum e or p and numbers of cpmas, expression of psp-selectin or pp-selectin concentration. conclusions: numbers of cpmas and expression of pspselectin are maximal at menstruation with neither numbers of cpmas nor expression of psp-selectin correlating with serum e or p levels. this study suggests that activated platelets may potentially contribute to the inflammatory response at menstruation by releasing inflammatory mediators. by angiogenic factors and peri-cellular proteases in decidual secretory endometrium (dse), decidua parietalis (dp), and basalis (db) of miscarriage patients and matched controls. comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages. methods: decidua was obtained during st trimester termination of pregnancy (control group) and vacuum aspiration of missed abortions (case group). vascularization was studied by cd -immunohistochemistry. the expression of vascular endothelial growth factor-a, placental growth factor, flt- , kdr, angiopoietin- , angiopoietin- , tie- and the membrane-type matrix metalloproteinases mt -, mt -, mt -and mt -mmp were determined at mrna and antigen level and cd -positive unk cells, cd -positive macrophages, proliferation (ki ) and apoptosis (activated caspase- ) were evaluated by immunohistochemistry in consecutive serial sections. results: the decidual vascularization pattern showed differences between cases and controls: i.e. fewer vessels with larger circumference in cases, and this correlated with the differential expression of various angiogenic factors and proteases at mrna and antigen level. moreover, the endothelial protein expression of flt , kdr, mt -and mt -mmp was increased at the implantation site of cases. ki and active caspase- showed similar levels in the two groups and also the immune cells, both unk cells and macrophages, showed no differences at the implantation site between both groups. conclusion: differences between cases and controls appeared not to be based on altered proliferation, apoptosis, and/or inflammation. the differences in vascularization pattern and in the expression of angiogenic factors and proteases between both study groups suggest a correlation between decidual vascularization and the occurrence of miscarriages. respond to adrenomedullin. yaun-lin dong, hong y wen, janice endsley, alison hogg, hui-qun wang, manubai nagamani, chandra yallampalli. background: natural killer (nk) cells are the predominant lymphocytes present in human implantation site. decidual nk cells express perforin, an essential molecule required for lysis. formation of the placenta involves cooperation between maternal nk cells and fetal trophoblast cells that remodels the blood supply; however, the interaction between trophoblasts and decidual nk cells is largely unknown. adrenomedullin (adm) has been implicated in regulating early placental function and fetal growth. objective: to determine the role of multifunctional peptide adm in the decidual nk cells and fetal trophoblast cells interactions. methods: decidual and placental tissues were obtained from normal firsttrimester pregnancies terminated for social reasons. ethical approval to use these tissues was obtained from the irb of university of texas medical branch. cell preparations containing all decidual mononuclear cells were isolated by collagenase enzymatic disaggregation. cd decidual nk cells were purified by magnetic bead isolation. results: ) immunohistochemical analysis showed that adm is expressed primarily in decidual cells and trophoblast cells at the human implantation site; ) confocal imaging analysis demonstrated that decidual nk cells, which were identified by anti-cd staining, express adm receptor components crlr/ramp /ramp and their mrna expressions were futher confirmed by rt-pcr; ) k target cell killing assay indicates that adm inhibits cytokine il- /il- -induced decidual nk cell cytotoxicity; and ) immunofluorescent labeling and flow cytometric analysis revealed that adm suppresses perforin expression by decidual nk cells. conclusion: trophoblast-derived adm inhibits decidual nk cell cytotoxicity via suppressing perforin expression, thus, our results provide evidence for a new paradigm of embryonic-maternal communication involving a adm mediated interaction between decidual nk cells and fetal trophoblasts. leandro g oliveira, gendie e lash, judith n bulmer, barbara a innes, roger f searle, stephen c robson. institute of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom. background: we have previously demonstrated that co-culture with extravillous trophoblast cells (evt) (expressing hla-g) alters cytokine secretion by uterine natural killer (unk) cells, particularly at - weeks gestation. we have also reported that unk cells can stimulate evt invasion, but only at - weeks gestation (not at - weeks gestation). in addition, unk cell cytokine profiles alter with increasing gestational age. other reports have suggested that evt or hla-g expressing cells may alter the expression of cytokines and angiogenic growth factors by unk cells. hypothesis: hla-g expressing cells alters unk secretion of cytokines. methods: cd + unk cells were isolated from early pregnancy decidua ( - and - weeks gestation, n= each group) using enzyme digestion and positive immunomagnetic bead separation. the human b lymphoblastoid . transfected with either hla-g ( g) or a mock cdna ( cdna) were obtained as a kind gift from mr r apps (university of cambridge, uk). isolated unk cells were cultured in the presence or absence of the two cell lines in either direct or indirect contact (n= each group and each gestational age) for hours. cell supernatants were analysed for cytokines using a fastquant® th /th multiplex protein assay (il- , il- , il- , il- , il- , tnf-, il- , il- , ifn-) or by standard elisa (tgf- ). the effect of direct co-culture of unk cells with g compared with co-culture with cdna at each gestational age was tested using mann whitney u test. the effect of co-culture of unk cells with g in both direct and indirect contact was also tested using mann whitney u test. results: there was no difference in the level of cytokines secreted by the g or cdna cells. cytokine secretion by unk cells was not altered after direct co-culture with either g or cdna cells at either gestational age. in addition, direct or indirect co-culture of g or cdna with unk did not alter cytokine secretion at either gestional age. conclusions: hla-g does not alter the secretion of cytokines by unk cells from either - or - weeks gestation. other evt or decidua derived factors (including hla-e) may be responsible for the alteration in secretion of cytokines by unks with increasing gestational age. introduction: natural killer (nk) lymphocytes are central to innate immunity and contribute to tissue homeostasis by eliminating altered cells. their nkg d receptor pathway plays a fundamental role in target elimination through binding nkg d ligands on the cell surface. reduction in the nkg d ligand, ulbp , expression is associated with immune resistance in neoplastic processes. we have previously shown that fibroblasts from adhesion tissue (at) are characterized by increased extracellular matrix molecules and inflammatory cytokines compared with normal peritoneal (np) fibroblasts. objective: to determine if there is a difference in nk lymphocyte-mediated elimination between np and at fibroblasts and to investigate potential role of nkg d pathway in this process. material and methods: expression of nkg d ligands; ulbp , ulbp , mica, and micb was evaluated by flow cytometry and western blot in primary cell cultures of fibroblasts from np and at, established from two patients. peripheral blood nk lymphocytes (cd +cd -) from three healthy volunteers were isolated using macs system with purity greater than % and kept in interleukin overnight. fibroblast elimination with and without ulbp blocking was investigated following -hour co-incubation with allogeneic nk lymphocytes using our established flow cytometric cell mediated cytotoxicity assay. paired t test was used in statistical analysis. results: the flow cytometry studies showed that nkg d ligands (ulbp , ulbp , mica and micb) were lower in at compared to np fibroblasts, reaching a statistical significance in ulbp expression (p = . ). western blot analysis also revealed a lower ulbp protein level in at than np fibroblasts. furthermore, nk lymphocyte-mediated elimination was % lower in at in comparison with np fibroblasts. blocking ulbp expression resulted in decreased nk lymphocyte-mediated np fibroblast elimination by %, supporting the role of nkg d receptor pathway in the process. conclusions: our results demonstrate that nkg d pathway is operational in at fibroblast resistance to immune elimination, and extends our prior observations of the potential role of immunological mechanisms in the pathogenesis of adhesion development. objective: galectin- is an anti-inflammatory lectin that has pleiotropic regulatory functions at the crossroad of innate and adaptive immunity. human galectin- is expressed in the placenta and immune privileged sites and it has been implicated in establishing immune tolerance. the aim of this study was to examine the evolution and placental expression of the lgals gene in primates. methods: seven primate nucleotide sequences were generated, aligned to vertebrate orthologs from all classes and subjected to phylogenetic analysis. deduced amino acid sequences were analyzed for functionally important substitutions. placental galectin- expression was studied by immunohistochemistry and western blot. results: ) the lgals gene had high sequence identity among all investigated species. ) phylogenetic analysis revealed that intense purifying selection had been acting on the lgals gene in placental mammals (dn/ds= . ); ) residues responsible for sugar binding or molecule stabilizing were highly conserved in primates. ) immunostaining showed a uniformly abundant and ubiquitous galectin- expression pattern in human, old and new world monkey and prosimian placentas, regardless the type of placentation. the lgals gene has conserved sequence and placental expression pattern in primates that may suggest its important function in maternal-fetal immune interactions. these results support the view that immune interactions at the maternal-fetal interface evolved in concert with invasive placentation and that these interactions have been maintained regardless of the degree of placental invasion in primates and other mammals. expression of interleukin- in human endometrium throughout the menstrual cycle and early pregnancy. yesim h uz, , william murk, umit a kayisli, aydin arici. department of obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa; department of histology and embryology, trakya university school of medicine, edirne, turkey. background: interleukin- (il- ) is a recently discovered heterodimeric cytokine, comprised by a novel p subunit and a p subunit shared by il- . it has biological activities that are similar to but distinct from il- , and is known to be involved in th /th cell class switching and the regulation of cytokines such as ifn-gamma, il- , tnf-alpha, and il- . early pregnancy is associated with alterations in the maternal immune response, such as changes in cytokine expression, and leukocyte recruitment and subtype switching. we hypothesized that expression of il- in the human endometrium is menstrual cycle-and pregnancy-dependent. materials and methods: endometrial samples from women (n= ) undergoing surgery for benign gynecologic conditions, and decidual tissues from women (n= ) with clinically normal pregnancies terminated voluntarily in the first trimester, were obtained after receiving informed consent. endometrial samples were grouped according to menstrual phase. paraffin sections were stained with il- p antibodies and evaluated semi-quantitatively with hscore. statistical analysis of the data was done using anova, with p< . considered significant. results: il- immunoreactivity was predominantly located in the cytoplasm of both endometrial stromal (esc) and glandular (egc) cells. escs showed mild il- immunoreactivity without significant changes in intensity throughout the menstrual cycle. on the other hand, first trimester decidual cells showed significantly stronger il- staining compared to escs from non-pregnant endometrium (p< . ). il- immunoreactivity in egcs was high in the late proliferative phase, as compared to other cycle phases and first trimester tissues (p< . ). moreover, egcs from the early secretory phase (p< . ) and first trimester tissues (p< . ) showed higher il- immunoreactivity compared to the early proliferative and late secretory phases. conclusions: this is the first study describing il- expression in the human endometrium and decidua. these results suggest that il- has a cycledependent expression in endometrial cells and may be involved in regulating cytokine expression and immune cell modulation during the menstrual cycle and early pregnancy. gercel-taylor, douglas d taylor. obstetrics, gynecology, and women's health, university of louisville, louisville, ky, usa. objective: estrogen appears appear to be a critical regulator of the immune system. since hypoestrogenism is present in the postmenopausal woman, our objective was to determine whether t cell activation and function, defined as il- production and signaling molecule expressions at the transcriptional and translational levels, were affected by a low estrogen environment. design: prospective study in a university research laboratory. materials and methods: jurkat . t cells, initially grown in estrogen free media, were incubated in pm (representing postmenopausal levels) or pm (premenopausal levels) of estradiol (e ) for hours. cells were either resting or activated with a phorbol ester, -phorbol -myristate -acetate (pma), and ionomycin. enzyme-linked immunosorbent spot assay (elispot) was performed to analyze production of il- . expression of signaling protein components, cd and jak, were determined by western immunoblotting. real time-polymerase chain reaction was performed to quantify cd , jak , and jak gene expression. a p value of < . was considered significant. results: jurkat cells exposed to pm e and activated exhibited significantly diminished numbers of il- producing colonies compared to t cells exposed to pm ( . ± . vs. . ± . colonies, p< . ). analysis of cellular cd and jak protein demonstrated that jurkat cells incubated in pm e expressed a . -fold decrease in cd and . -fold decrease in jak compared with cells incubated in pm e (p< . ). these diminished protein levels appeared to be the consequence of suppressed transcription, as the mrna levels of cd , jak and jak were significantly decreased in jurkat cells incubated in low levels of estrogen ( . , . , and . fold, respectively, compared to pm). conclusions: jurkat cells exposed to low postmenopausal estrogen levels produce significantly less il- following activation, which was associated with a significant decrease in signaling proteins. the diminished levels of signaling proteins appear to result from decreased cd , jak and jak gene expression in the presence of low estrogen. these findings support the observation of decreased cellular immune response in postmenopausal women and may provide a basis for the increased risk of infections and cancer proliferation associated with aging. support: dept. of ob/gyn research seed fund. the expression pattern of novel cytokines (il- and ) in human fetal membranes. judith eckardt, , stephen j fortunato, holger maul, ramkumar menon. the perinatal research center, nashville, tn, usa; womens' hospital, university of heidelberg, heidelberg, baden-wuerttemberg, germany. objective: interleukin (il) and are novel cytokines produced by various immunological cells in response to microbial antigens. the functions of these cytokines in reproductive system is unknown. this study examines the expression pattern of il- and il- in human fetal membranes from preterm and term births and in in vitro in normal term membranes in response to bacterial endotoxin (lipopolysaccharide-lps). methods: fetal membranes collected (n= ) from cesareans at term (normal, not in labor) were placed in an organ explant system for hours and were stimulated with lps for an additional hrs. fetal membranes were also collected (n= ) either at preterm or term after vaginal deliveries. in a case -control study (preterm birth vs. normal term deliveries) amniotic fluids (af) (n= ) were collected to document the role of il- and il- in ptb. tissue expressions of il- and il- were studied by rt-pcr using specific primers. elisa documented culture media and af cytokine concentrations. statistical analysis was performed using non-parametric mann-whitney u test. results: both il- and il - expressions were seen in fetal membranes in culture (in vitro) regardless of stimulation. in vivo in membranes from preterm and term deliveries and membranes at term not in labor also documented the expression of these cytokines. culture media analysis documented higher concentration of il- after lps stimulation (lps- . vs. . pg/ml; p= . ) whereas no difference was noticed in il- concentration (lps- . control- . pg/ml; p= . ) between the two groups. af analysis, regardless of the status, did not document detectable concentrations of either of the cytokines (lower limit . pg/ml for both). conclusion: this is the first study to document the expression of two novel cytokines in laboring and non laboring human fetal membranes and also in membranes from preterm deliveries. il- production was stimulated by lps whereas il- was not affected. these cytokines are not physiological components of af and their role in fetal membranes is unclear. higher il- concentration in response to lps but lack of its presence in term or preterm af is suggestive of an autocrine immune response during pregnancy in response to a microbial antigen. evidence for a selective migration of fetus specific cd + cd bright regulatory t cells from the peripheral blood to the decidua in human pregnancy. tamara tilburg, , dave l roelen, barbara j van der mast, godelieve m de groot, carin kleijburg, sicco a scherjon, frans hj claas. department of obstetrics, leiden university medical centre, leiden, netherlands; department of immunohematologie and bloodbank, leiden university medical centre, leiden, netherlands. during pregnancy the maternal immune system has to tolerate the persistence of fetal alloantigens. many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. murine studies suggest that cd + cd + t cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. previous studies by our group demonstrate that a significantly higher percentage of activated t cells and cd + cd bright t cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy ( ) . in this study we examined the phenotypic and functional properties of cd + cd bright t cells derived from maternal peripheral blood and decidual tissue. depletion of cd + cd bright t cells from maternal peripheral blood demonstrates regulation to a rd party umbilical cord blood cells comparable to non-pregnant controls, whereas the suppression capacity to umbilical cord blood cells of her own child is absent. furthermore, maternal peripheral blood shows a reduced percentage of cd + cd bright foxp + and cd + cd bright hla-dr + cells compared to peripheral blood of non-pregnant controls. in contrast, decidual lymphocyte isolates contain high percentages of cd + cd bright t cells with a regulatory phenotype that are able to down regulate fetus-specific and non-specific immune responses. these data suggest a preferential recruitment of fetus-specific regulatory t cells from maternal peripheral blood to the fetal-maternal interface where they may contribute to the local regulation of fetus specific responses. ( ) tilburgs t, roelen dl, van der mast bj, van schip jj, kleijburg c, de groot-swings gm, kanhai hh, claas fh, scherjon sa. differential distribution of cd (+) cd (bright) and cd (+) cd (-) t-cells in decidua and maternal blood during human pregnancy. placenta. apr; suppl a:s - . alicia del toro-arreola, lourdes nunez-atahualpa, juan velazquez-rodriguez, laura gonzalez-lopez, jorge i gamez-nava, adrian daneri-navarro. there is evidence that the maternal immune system is influence by changes in the hormonal levels during the menstrual cycle (mc). so far, the information related to the levels of t, treg, nk cells and receptors of activation and inhibitors is scarce. aim: to analyze the populations of t, treg, nk cells and their receptors of peripheral blood of healthy women and their correlation with hormones during mc. material and methods: we studied to women not using hormonal contraceptives in the day th of the follicular phase and st of the luteal phase of the mc. pbmc subsets and their receptors were determined by flow cytometry and hormone levels by chemiluminescence method. we found that the progesterone and prolactin were positive correlated (rho= . , p< . and rho= . , p< . , respectively) with cd /nkg in t cells and negative correlated (rho= - . , p< . and rho= - . , p< . , respectively) with nk cells. meanwhile cortisol was positive correlated (rho= . , p< . ) with the receptor nkg d expressed in nk cells. the results observed in this study in the luteal phase of mc on the expression of cd /nkg inhibitor receptor and nkg d activator receptor were related to a particular hormone (progesterone, prolactin and cortisol) might contribute to understanding the physiological role of the neuroendocrine axis on the expression of some receptors of the immune system in order to keep the homeostasis milieu of the mc. objective: sp-d, a key component of the innate immune system, is detected in amniotic fluid (af) and believed to originate in the fetal lung. however, sp-d is produced by other cells and therefore extra-pulmonary sources must be considered. the objective of this study was to determine the maternal and fetal plasma and af concentrations of sp-d to gain insight into the behavior of this natural antimicrobial peptide in pregnancy. moreover, we studied sp-d expression in maternal and fetal peripheral leukocytes. methods: maternal and fetal plasma and af samples were obtained from patients in the following groups: ) term not in labor (tnl; n= ); ) term in labor (til; n= ); ) preterm labor without histologic chorioamnionitis (ptl; n= ) and ) preterm labor with histologic chorioamnionitis (ptl-hc; n= ). sp-d concentration was measured by elisa. sp-d mrna expression in maternal and fetal leukocytes was evaluated by real-time qrt-pcr. flow cytometry and confocal microscopy were used to study the localization of sp-d in leukocytes. results: ) the af sp-d concentration increased as a function of gestational age (mean, til: , . ng/ml vs. ptl: , . ng/ml; p< . ); ) in contrast, the maternal and fetal plasma sp-d concentrations decreased with advancing gestational age (mean, til: . ng/ml vs. ptl: . ng/ml; p< . , and til: . ng/ml vs. ptl: . ng/ml; p< . , respectively); ) the maternal plasma sp-d concentration was lower than that of fetal plasma (mean: . ng/ml vs. . ng/ml; p< . ); ) however, sp-d mrna expression in maternal leukocytes was . fold higher than that of fetal leukocytes (p< . ); ) neutrophils (both maternal and fetal) expressed sp-d as demonstrated by flow cytometry and confocal microscopy. conclusion: ) the concentrations of sp-d in the maternal and fetal circulation decreased with gestational age while the af concentration increased; ) the expression of sp-d mrna is higher in maternal leukocytes than in fetal leukocytes; ) we report for the first time that maternal and fetal neutrophils are a source of sp-d and propose that this molecule plays a role in host defense against infection and in the modulation of the maternal and fetal immune response. introduction intrauterine insemination (iui) is a fertility technique that allows for couples to have intercourse after the procedure is performed. it has been postulated that intercourse after iui may increase the pregnancy rate by either endometrial stimulation or because it may represent a second spermatic flow in the periovulatory period. in the present study we evaluate the effect of intercourse on the pregnancy rate of patients undergoing iui. material and methods: from to patients were enrolled in the study. every couple undergoing iui was instructed at the moment of insemination to decide whether to have or not intercourse on the same day of the procedure. all couples were abstinent three to seven days before iui. the information regarding intercourse was recorded the day after treatment. ovulatory, insulin resistant, cervical, male, tubal and endometrial factors as well as parity and time of infertility were compared between the two groups. all these factors were analyzed based on number of follicles that ovulated in each group. our principal outcome was to determine the pregnancy rate. intercourse was practiced by . % of the couples. the global pregnancy rate was . %. the pregnancy rate for the couples who had intercourse was . % and . % for those who did not have intercourse (p< . ). even though age, parity, time of infertility and stimulation protocols were similarly distributed in both groups, the proportion of tubal and endometrial factors were higher among those who had intercourse (p< . ). when subjects with tubal and endometrial factors were excluded, the pregnancy rate between both groups (n= ) was similar ( . % vs . % for positive and negative intercourse, respectively). the average number of ovulatory follicles was . + . for the group that had intercourse and . + . for those who did not. according to our results, intercourse after iui does not improve pregnancy rate after this procedure is performed. furthermore our study indicates that iui does not interfere with sexual intimacy since almost % of the couples decided to have intercourse on the same day of the procedure. . we sought to investigate the effects of gravidity on mmc and fmc in healthy, parous women. methods: mc was assayed in dna extracted from peripheral blood mononuclear cells (pbmc). hla-genotyping was first conducted and mc quantified employing a q-pcr assay targeting a non-shared maternal-or fetalspecific hla polymorphism. gravidity was dichotomized as a history of one pregnancy compared with two or more, and the prevalence of mc was analyzed using logistic regression. possible confounders were included as appropriate, including subject age and time since last pregnancy. adjustment for possible correlation between values was also made when there were repeated measures for the same subject. results: for the mmc analysis, there were subjects with observations. for the fmc analysis, there were subjects with observations. table provides a summary of mmc and fmc by gravidity. mmc was significantly decreased with higher gravidity. fmc was not affected by gravidity. gravida gravida or more adjusted* or ( %ci) mmc / ( %) / ( %) . ( . - . ) fmc / ( %) / ( %) . ( . - . ) *adjusted for possible correlation between values within a subject, subject age, and time from last pregnancy, as appropriate. increasing gravidity is significantly associated with a decreased prevalence of mmc. despite additional sources of fmc, there does not appear to be an increase in fmc prevalence with increasing gravidity. the biology of mc is incompletely understood, and the nature of mmc and fmc are likely to be different given that acquisition of the former, but not the latter, occurs within a nascent immune system. these data raise interesting questions when considered as interactions of acquired grafts within a host, including whether emergence and persistence of one dominant source of mc may be most advantageous for an individual. anti-igd antibody treatment as a novel immunosuppressive agent for autoimmune diseases and its effects on th /th gene expression. tommie g nguyen, eileen d gallery, , jonathan m morris. elevated t-helper cell type- (th ) and type- (th ) cytokine expression have a role in autoimmune diseases, allograft rejection and pregnancy-related complications. thus, molecules that can shift the immunity away from th /th responses toward a th response represent a novel therapeutic treatment for these conditions. we have previously demonstrated that pregnancy is associated with a suppression of t-bet in peripheral t cells. in this study, we examined a novel effect of anti-igd antibody on t-bet expression, th /th gene expression in human peripheral blood mononuclear cells (pbmc) and its therapeutic effects in an animal model of collagen-induced arthritis. methods: human pbmc were isolated and then cultured in the presence of anti-igd antibody at various time points followed by stimulation with pma/ionomycin (p/i) for hrs. gene expression was examined by rt-pcr, western blotting and elisa. for in vivo study, arthritis-prone dba/j mice were induced to undergo joint inflammation by intradermal injections with bovine type-ii collagen. these mice were then given daily doses of mg/kg of intravenous injection with anti-igd antibodies as preventive or therapeutic treatments (n = per group). results: treatment with anti-igd antibodies significantly suppressed p/iinduced expression of t-bet (a master regulator of th immunity), tnf-(a classical pro-inflammatory th cytokine), and il- (a critical proinflammatory th cytokine) in human pbmc. this suppression is highly specific to these genes because anti-igd antibodies have no effects on the expression of ifn-g and il- (two classical th cytokines). in vivo experiment showed that anti-igd antibody treatment markedly reduced clinical severity of joint inflammation when comparing the clinical score of control mice group ( . ± . , mean ± s.e.m), preventive group ( . ± . ) and therapeutic group ( . ± . ) . conclusions: our study has demonstrated that suppression of t-bet by anti-igd antibodies, similar to the changes seen in human pregnancy is a novel in vivo anti-inflammatory effect. given the essential role of t-bet, tnf-and il- in the pathogenesis of human autoimmune diseases, anti-igd antibodies may represent a novel immunosuppressive treatment that needs further studies and evaluation. objective: women with circulating anti-phospholipid antibodies (apl) are at risk for recurrent miscarriage, preeclampsia and preterm labor. apl antibodies directly target the placenta by binding to phospholipids or phospholipid-binding proteins expressed on the surface of viable trophoblasts. the objective of this study was to determine the effects of apl antibodies on first trimester trophoblast cells. methods: two mouse igg anti-human beta -glycoprotein i monoclonal antibodies (mabs), designated id and iic , were used in these studies. the first trimester trophoblast cell line, htr , was incubated with either medium, a mouse igg control, id or iic ( - g/ml), in the presence or absence of unfractionated heparin ( ng/ml). trophoblast cell death and apoptosis was determined using a viability assay, hoechst staining and a caspase activity assay. cytokine production was evaluated by multiplex analysis. results: following a hour incubation, significant trophoblast cell death was induced by iic ( . ± . %) and id ( . ± . %) at the high dose of g/ml, when compared to the medium and mouse igg controls (p< . ). hoechst staining showed that id -and iic -induced trophoblast cell death was a result of apoptosis. moreover, id and iic induced a significant increase in caspase- , - and - activity (p< . ). treatment of trophoblasts with heparin significantly inhibited the effects of iic and id on cell death by . ± . % and . ± . % %, respectively (p< . ). following a hour incubation at lower concentrations ( g/ml), treatment of trophoblast cells with id or iic resulted in a significant upregulation of il- , mcp- , gro production (p< . ), and a significant reduction in il- secretion (p< . ). conclusion: this study demonstrates that at low levels apl antibodies can modulate trophoblast cytokine production, while at higher levels, the same antibodies induce trophoblast apoptosis in a caspase-dependent manner. these findings shed new light on the mechanisms by which apl antibodies may impact placental survival and function. antigenic targets for the diagnosis of premature ovarian failure. hc bohler, c gercel-taylor, lt ku, st nakajima, dd taylor. obstetrics, gynecology, women's health, university of louisville, louisville, ky, usa. objective: premature ovarian failure (pof) is a premature depletion of ovarian follicles before the age of , affecting approximately % of women < years. the involvement of autoimmune mechanisms in pof has been suggested and similar mechanisms have been postulated for other ovarian pathologies, including idiopathic infertility, polycystic ovary syndrome (pcos), or endometriosis. while the association of autoantibodies has been demonstrated for these ovarian pathologies, variation in specificity and frequency of false positivity have limited the diagnostic use of autoantibodies. the objective of this study was to develop an antigen array to differentiate antibody recognition patterns of pof from other infertility pathologies. design: prospective study in a university research laboratory. materials and methods: patients diagnosed with infertility were included in this pilot study: endometriosis (n= ), pcos (n= ) and pof (n= ). autoantibodies were assayed by dot immunoblotting using an antigen array derived from endometrial and ovarian cells. for the cellular antigen preparations, solubilized total proteins were separated by reverse phase-hplcquid chromatography and the individual proteins were blotted onto nitrocellulose membranes and reactivity visualized by peroxidase-labeled antihuman igg. results: patients with pof, endometriosis, and pcos all exhibited autoantibodies reactive with these cellular proteins. while some antigenic reactivities were shared by all infertility patients, the pattern of antigen recognition was distinct for patients with pof. patients with pof all recognized a common antigenic proteins (row , antigens a,b,d-g). conclusions: alterations in autoreactivity are observed in patients with the diagnosis of infertility; however, distinct patterns of autoantibody recognition can be demonstrated for patients with different pathologies. while this study needs to be expanded to reliably establish the specificity, sensitivity and positive and negative predictive values, patients with pof clearly exhibit a shared recognition pattern that may be useful a diagnostic marker. cicek gercel-taylor. ob/gyn, university of louisville, louisville, ky, usa. objective: americans' consumption of nutraceuticals is one of the most rapidly expanding health markets, growing at a rate of % annually. multiple nutraceuticals containing phytoestrogens have been marketed as "immune boosters" despite suboptimal evidence-based medicine to support such statements. as immunomodulatory therapies should affect downstream cytokine expression, the relative effects of estradiol and genistein in regulating expression of cd -and jak were tested. these markers were chosen since they are central to t cell signaling. cd -is a critical transducer of tcr activation and regulates t cell proliferation and cytokine production. jak upregulation is a specific marker for hematopoietic cell stimulation. methods: to test the immunomodulatory effects of phytoestrogens and estrogen, jurkat . (t cell leukemia) cells were grown in estrogen-free, phenol red-free media for hours. these cells were then exposed for hours to pm, pm (postmenopausal), or pm (premenopausual) of estradiol in the presence of increasing concentrations of genistein ( , . , . , . , , and m). cells were then solubilized and cellular protein quantitated. protein concentrations were standardized and western blots for each set of culturing conditions were run in triplicate. cd -and jak expression were quantitated following visualization with chemiluminescence by digital pixel quantification. results: our findings show that in the absence of estradiol and at postmenopausal levels of estradiol, genistein induced a dose dependent increase in cd -reaching a maximum of fold. although cultivation of t cells in pm of estradiol significantly increased the levels of cd -and jak relative to hypoestrogenic conditions, the genistein mediated dose response was not observed. conclusions: these in vitro results indicate that genistein can at least partially reverse suppression of signaling molecules observed in postmenopausal estrogen environments. clinically, this suggests that phytoestrogens may have greater immunomodulatory properties for postmenopausal females than those that are premenopausal. maternal serum il- as a biomarker of acute immunologic rejection of pregnancy. joaquin santolaya-forgas, juan deleon-luis, isabel galan. obstetrics and gynecology, brigham and women's hospital, boston, ma, usa; amarillo women's health research institute, texas tech university health science center, amarillo, tx, usa. objective: markers of acute rejection of pregnancy are very scarce. in this study we aimed at determining if rapid changes in maternal serum concetration of a variety of biomarkers could be used for this purpose. we used an established baboon model for in utero stem cell therapy to introduce at - days from conception and via ultrasound-guided celocentesis, human hematopoietic stem cells with different proportions of natural killer t-cells (nk). maternal blood was collected before and hours after celocentesis for quantification of hormones and il- using solid phase, enzyme labeled, chemiluminescent sequential immunometric assays. pearson correlation analysis was used for determination of significant changes from baseline (p< . ). results: all animals survived their pregnancies. seven animals receiving < % concentration of nk delivered at term ( days gestation) while animal receiving more than % concentration of nk had dead fetuses on ultrasound evaluation hours after celocentesis. table depicts mean maternal serum concentration of the biomarkers investigated (all n.s.). figure shows mean il- changes from baseline in continuing (n.s.) and rejected pregnancies (p< . ). conclusions: we have described a model in which in utero graft vs host disease can be studied. these preliminary results suggest that of all the biomarkers investigated, il- might be the most sensitive for detection of an acute rejection of pregnancy. biomarkers of acute immunologic rejection of pregnancy biomarker unit pre-celocentesis ( ) the activity of cytotoxic cd + t cells during pregnancy protects the mother and fetus from infection. however, pregnancy's effect on the proliferation and apoptosis of cd + t cells has not been clearly defined. objective: to determine if normal pregnancy changes the number of proliferating or apoptotic splenic cd +t cells. methods: female c bl/ mice were used unmated (um) or underwent timed mating. one day prior to harvest, mice were i.p. injected with bromodeoxyuridine (brdu), which is incorporated into replicating dna. harvested spleens were homogenized, enumerated, and stained for cell surface expression of cd and t cell receptor beta chain (tcr ). apoptotic cells were detected by treatment with terminal transferase and fitc-dutp (tunel). the numbers of cd +tcr + cells that were brdu+ or tunel+ were calculated from the percentage of positive cells obtained by flow cytometry and the absolute number of cells counted. for each experiment, the ratio of the number of positive cells in pregnant to um mice was compared by anova with dunnett's post-test. results: at day of pregnancy (n= ), the number of brdu positive cd + t cells was two fold higher than that found in um (n= , p< . ). the number of proliferating cd + t cells continued to be non-significantly elevated at day ( . x, n= ), day ( . x, n= ), and day of pregnancy ( . x, n= ) compared to um. by day of pregnancy (n= ) the number of proliferating cd + t cells returned to the um level, however by this time the total number of splenic cd + t cells was . fold higher than um (n= p< . ). on gestational day , the number of proliferating cd + t cells declined further ( . x, n= ), and the number of splenic cd +t cells returned to the um level (n= , p> . ). compared to um mice, there was no significant difference in the number of cd + t cells undergoing apoptosis at any gestational day examined ( . - . x, p> . ). in normal murine pregnancy, the number of cd + t cells is increased in late gestation, and then returns to baseline at the end of pregnancy. this is due to an early increase then gradual decline in cd + t cell proliferation, accompanied by a steady rate of apoptosis. this argues that the maternal immune system undergoes dynamic homeostatic changes, and is not globally suppressed. supported by nihro hd - niht ai . arturo cerbulo-vazquez, cun li, , gene hubbard, natalia e schlabritz-loutsevitch, , peter w nathanielsz. objectives: early thymocyte (t) maturation occurs in the cortex while later stages occur in the medulla. thymic epithelial cells (tec) synthesize gc and t express gr. tec may influence t cell maturation by regulating apoptosisinduced gc-gr interactions. igf- (also synthesized by tec) may support thymocyte prolifetarion. human fetuses of mothers in premature labor are exposed to gc. gc administered to pregnant baboons at . , . , and . gestation (g) alters fetal lymphocyte populations at . (g) (j repro immunol, , : ) . we determined if fetal gc exposure alters thymic ) structure; ) gr and igf-i protein. methods: pregnant baboons received saline (control ctr; n= ) or betamethasone i.m. ( μg/kg daily for two days at . , . and . g; gc group; n= ), c-sectioned at at . g under general anesthesia and thymic gr and igf-i evaluated by immunohistochemistry. results: gr localized to medulla and a few cortical cells. igf-i localized to cortex with little medullary expression. medullary necrosis was greater in ctr than gc fetuses. t gr was located in cytoplasm. no gross differences were observed between ctr or gc fetuses for either igf-i or gr. conclusions: a) early thymocyte maturation may be supported by igf- , b) later differentiation involves gr, and c) after exposure to gc doses equivalent to human therapy, no gross effects were detected on gr or igf, but d) natural thymic necrosis was inhibited. lindsay s christensen, peyman bizargity, elizabeth a bonney. ob/gyn, university of vermont, burlington, vt, usa. background: the exact mechanism by which bacterial products trigger preterm delivery and the immune cellular circuits involved remain unclear. our recent data in normal c bl/ (b ) or recombinase deficient c bl/ mice (rag-ko) indicates that t and b cells are not critical for lps-induced preterm delivery and stresses the importance of related innate mechanisms. rag-ko mice are more susceptible to lps, suggesting that t or b cells may control the innate response. macrophages are vital to innate immunity and produce proinflammatory cytokines that can activate prostaglandin synthesis and myometrial contraction. we questioned whether differences in susceptibility between the strains are due to differences in the uterine macrophage response to lps and thus examined macrophages at the maternal-fetal interface early after injection. objective: to compare uterine macrophages levels at and hours after lps injection in pregnant b and rag-ko mice. methods: b and rag-ko mice were mated and on gestation day , females were injected intraperitoneally with μg lps in l saline (pbs) or l pbs alone. euthanasia and uterine harvest occurred or hours after injection. frozen uterine sections were stained with the macrophage marker f / or an isotype-matched control followed by an alexafluor -conjugated secondary and a nuclear stain (dapi). sections were visualized by fluorescence microscopy. for each mouse, f / + dapi+ and total dapi + cells were counted in areas of representative section and the percentage of f / + dapi+ cells was calculated. the mean percentage for at least representative areas per experimental group was analyzed by anova. results: two hours post-injection, macrophages levels were similar in b and rag-ko mice injected with pbs (b , n= , . ± . ; rag-ko, n= , . ± . % + per area). lps injection increased macrophages (p< . ) in both strains (b , n= , . ± . ; rag-ko, n= , . ± . ,); no difference was evident between strains. the percentage of f / + cells was similar hours post-injection (b , n= , . ± . v. rag-ko, n= , . ± . ), and not elevated relative to the hour time point. objective. decay-accelerating factor (cd ), is expressed in the plasma membranes and protects mammalian cells against the lytic action of serum complement. phosphoinositide -kinases (pi ks) are involved in the regulation of cell functions by synthesizing a second messenger molecule ptdins ( , , ) p . akt, a serine-threonine kinase acts downstream of pi k and regulates cell survival, growth and proliferation. the pi k-akt activity is controlled by tumor suppressor gene pten. in this study we assessed whether the pi k-akt activity affects the expression of cd in human endometrial and cervical cells. methods. endometrial and cervical cell lines which differ in the constitutive pi k activity were used in this study. ishikawa and rl - endometrial cell lines harbor pten mutation and have high levels of phosphorylated akt (p-akt). hec- -a and kle endometrial cell lines and hela cells express wild-type pten and have minimal or no demonstrable levels of p-akt. the expression of cd was evaluated by rt-pcr, immunoblotting and flow cytometry. the pi k activity was assessed by immunoblotting with anti-p-akt antibodies. the effect of inhibition of pi k-akt pathway on cd expression was evaluated in cells treated with wortmannin ( nm), ly ( mm), or with akt inhibitor sh ( mm). results. immunoblotting densitometry and measurements of mean fluorescence intensities showed that the level of cd expression correlates with the status of pi k-akt pathway. the cd expression was lowest in hec- -a, ishikawa and rl - cells which constitutively express p-akt. higher cd expression was found in hela cells and kle cells which express wild-type pten product and has no detectable phospho-akt. mean fluorescence intensities were . -fold higher for kle cells and -fold higher for hela cells compared to hec- -a cells. treatment of cells with akt inhibitor led to . - . -fold increase in cd expression. the . - . -fold increase following treatment with pi k inhibitor wortmannin was found in ishikawa cells, rl - and kle cells. conclusions. human endometrial cell lines with elevated pi k-akt activity express lower level of cd compared to cell lines with intact pten gene function. these findings may indicate that structural alteration at the dna level and resultant overexpression of pi k-akt pathway are involved in the downregulation of cd . endometrial and cervical cells. pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cell shape is determined by the cytoskeleton, which provides the mechanical support and is involved in cellular signaling. apoptotic cells undergo major morphological changes such as rounding and contraction, a process regulated by caspases, the cysteine proteases responsible for events controlling the cell disassembly. the motifs in certain cytokeratins make them substrates for caspase degradation. anti-apoptotic serine/threonine kinase akt provides a survival signal by phosphorylating downstream effector molecules including caspase- . while studying the akt distribution in human endometrial cell line we found that akt shows filamentous pattern of staining resembling cytoskeleton organization. in this study we evaluated whether akt staining correlates with the microfilaments (mf), microtubules (mt) or intermediate filaments. endometrial ishikawa, rl - , hec- -a, kle and cervical hela cell lines were used in the study. incubation with cytochalasin d, ( ug/ml) or nocodazole ( mg/ml) and labeling with bodipy fl phallacidin, anti -tubulin or anti-akt antibody were used to assess whether cytoskeleton disruptors affect akt distribution and mf and mt organization. for colocalization, cells were stained with anti-cytokeratin mouse antibody followed by anti-mouse alexa conjugate, and then stained with anti-akt rabbit antibody and anti-rabbit alexa conjugate. the scans collected with laser scanning confocal microscope from channels filtered for alexa and alexa were combined digitally and evaluated with imaris colocalization analysis software. filamentous pattern of akt staining was most pronounced in ishikawa and less obvious in hec- -a cells. treatment with cytochalasin d or nocodazole resulted in disruption of mf and mt but had no effect on cytokeratin organization and akt distribution. double staining with anti-cytokeratin- and anti-akt antibody showed overlapping staining for cytokeratin and akt. analysis of digitally acquired images showed highest correlation for colocalized channels in rl - cells ( . ) followed by ishikawa ( . ) and hela cells ( . ). lowest correlation was found for kle ( . ) and hec- -a cells ( . ) conclusions. this study indicates a strong colocalization pattern of serine/ threonine kinase akt with cytokeratins, and suggests a mechanism by which cytokeratins might be protected against cleavage by caspase- and caspase- in the early apoptotic stages. background: cd + cd + t regulatory cells (t-reg), express foxp ,suppress antigen-specific immune responses and are important for allograft tolerance. during pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus), requiring substantial changes in immune regulation over a programmed period of time. the presence of t-reg cells (cd + cd highfoxp +) was assessed in the peripheral venous blood of non-pregnant, pregnant and seven postnatal healthy women by flow cytometry. human decidua was obtained by surgical termination of pregnancy in the first (n= ), second (n= ) and third (n= ) trimester of human pregnancy. paraffin sections were immunostained for foxp and cd . foxp +cells were quantified in x fields and results compared between first, second and third trimester samples and according to the presence of extravillous trophoblast. results: fluorometric studies of blood samples indicate an increase % of circulating cd + cd highfoxp t-cells in pregnant ( . % [range . - . %]) vs. non-pregnant controls ( . % [range . - . %]; p< . ). a progression from st, nd and rd trimesters indicated the % of cd + cd highfoxp t-cells was . %, . % and . %, respectively. low numbers of foxp + cells were detected in all decidua samples and their distribution mirrored that of cd + cells. in st trimester samples, foxp + cells were often detected in lymphoid aggregates adjacent to endometrial glands. increased numbers of foxp + cells were detected in st ( . ± . ) compared with nd trimester decidua ( . ± . ; p< . ) but there was no difference between st and rd trimester ( . ± . ), nor between nd and rd trimester decidua. in st trimester decidua, numbers of foxp + cells were increased in areas without extravillous trophoblast. conclusion: normal human pregnancy is associated with an increase in the number of circulating cd + cd highfoxp t-cells. the presence of foxp + cells in early gestation human decidua may be important in the initiation of materno-fetal tolerance at an autocrine level. (supported by mrc). aims: -defensins are small cationic peptides with antibiotic and antimicotic activity. hyaluronan and its degradation products have been described as endogenous ligands for tlr and tlr , whose involvement in -defensin expression has been reported in different epithelial tissues and cell lines. we aim to investigate weather low molecular weight hyaluronic acid induces -defensin release by keratinocytes, via tlr and tlr . methods: the induction of -defensin production following in vitro treatment of human keratinocytes with a low molecular weight hyaluronic acid solution was evaluated by pcr-analysis and elisa techniques. studies on the involvement of tlr and tlr in -defensin production have been performed using specific blocking antibodies. results: pcr and elisa revealed an intense -defensin production following hyaluronic acid treatment in human keratinocytes. the -defensin production induced by hyaluronan was abolished following block of tlr and tlr by specific antibodies, demonstrating the involvement of these receptors. the same hyaluronic acid treatment did not induce activation of inflammatory genes, such as il- , tnf-, il- and il- . conclusion: our data show that hyaluronic acid is an efficient inducer ofdefensin production in keratinocytes, via tlr and tlr . this observation might be important to open new perspectives in the development of possible topical products containing hyaluronic acid, to improve the release ofdefensins by keratinocytes, ameliorating the self-defence of the skin in case of skin infections. therefore, one of the possible applications for this kind of topical products might be the treatment of infective vulvitis, one of the most distressing gynaecological diseases for adult women. pregnancy outcome? kiera von besser, serena wu, mary d stephenson. , obstetrics and gynecology, university of chicago, chicago, il, usa; obstetrics and gynaecology, university of british columbia, vancouver, bc, canada. objective: to investigate whether gender of an ongoing pregnancy impacts the probability of a successful outcome, and, to ascertain whether the gender of prior live birth(s) impacts subsequent pregnancy outcome, among women with rm/aps. materials and method: cohort-control study. rm subjects were evaluated by mds between - (stephenson, . couples who met aps criteria (wilson et al, ) , restricted to rm only, were followed prospectively. cohort data was compared to live birth data from the vital statistics agency of british columbia from - . secondary sex ratios (ssrs) among successful pregnancy outcomes were calculated by dividing the number of male live births by female. sex ratios were calculated for all pregnancies weeks, regardless if they ended in success or demise. pearsons test with yates continuity correction was applied. results: subjects were identified. subjects had prior live births of known gender ( male/ female), giving a ssr of . . there were also prior fetal demises wks of known gender ( / ) giving a sex ratio for all prior pregnancies at wks of . . subjects delivered subsequent live births of known gender ( / ), giving a ssr of . . there were also subsequent fetal demises ( / ) giving a sex ratio for all subsequent pregnancies of . . subjects delivered both prior and subsequent live births. the ssr was . ( / ) among their prior and . ( / ) among their subsequent live births. including fetal demises, subjects had ongoing prior and subsequent pregnancies. the sex ratio was . among their prior pregnancies and . among their subsequent. as the control, a ssr of . ( , / , ) was calculated from vital statistics data. when the prior and subsequent ssrs of the cohort were compared to each other, as well as to the control, there were no statistically significant differences. conclusions: our findings, from the largest study of its kind to date, suggest that, in patients with rm/aps, the gender of an ongoing pregnancy does not significantly affect the probability of a successful outcome, to any greater degree than it does in the general population. also, the gender of a prior ongoing pregnancy does not significantly impact the likelihood of developing rm/aps. oocyte maturation. jk friend, fb bezirci, e seli. ob gyn, yale u., new haven, ct, usa. introduction: oocyte maturation is associated with repression of transcription. during oocyte maturation, fertilization, and early embryo development until the onset of zygotic gene expression, proteins are synthesized from maternallyderived mrnas. the regulation of protein expression from these maternal mrnas is post-transcriptional, and occurs mainly via poly(a)-tail elongation. the embryonic poly(a)-binding protein (epab) is the predominant poly(a)binding protein before the activation of the zygotic genome, and plays a critical role in the activation of certain maternal mrnas, those bound by cpeb and probably pumilio. we are characterizing additional functions of epab during the process of oocyte maturation. methods and results: our model system is the xenopus laevis oocyte where oocyte maturation is induced by the addition of progesterone. our preliminary findings indicate that epab is phosphorylated, and that levels of phosphorylated epab increase upon progesterone-induced oocyte maturation. moreover, glycerol gradient centrifugation revealed that nonphosphorylated and phosphorylated epab are contained in distinct complexes that change mobility upon oocyte maturation. furthermore, oligo-dt selection for poly(a)-containing mrnas strongly suggests that these mrnas are bound exclusively by phosphorylated epab. using affinity purification, we have determined that nonphosphorylated epab exists primarily in a large protein complex prior to oocyte maturation that is later disassembled after the addition of progesterone. conclusions: based on these preliminary findings, we conclude that prior to oocyte maturation, the bulk of epab is nonphosphorylated and is found in a protein-rich complex separate from poly(a)-containing mrnas. upon oocyte maturation (when certain maternally-derived mrnas are activated for translation), the majority of epab becomes phosphorylated, and this phosphorylated form of epab is likely bound to translationally-active mrnas. we are currently investigating what kinase phosphorylates epab and whether this phosphorylation plays a role in translational up-regulation of epab-bound mrnas. introduction: reactive oxygen species (ros) play important roles in all aspects of cellular fate. nadph oxidase isoforms, a family of genetically preserved enzyme complexes, have been shown to be the main sources of ros in various cell types. however, the role of nadph oxidase isoforms in human myometrium proliferation and differentiation has not been defined. in the myometrium, different smooth muscle phenotypes maybe associated with specific physiologic functions. we have shown that angiotensin ii (angii) stimulates hypertrophy but not cell proliferation in ultr cells, an in vitro model of human myometrium. ultr cells at greater than passages display a replicative senescent phenotype. by introducing human telomerase reverse transcriptase (htert) gene, we have obtained a stable cell line (ultr-ht) which has a significantly increased division rate and distinct cellular morphology than the original ultr cells. objective: to determine the relationship of expression of nadph oxidase to ultr cellular fate. methods: early and late passages (p - ) of ultr and ultr-ht (p - ) cells were grown on either plastic or collagen iv (cn )-coated surfaces. ultr-ht cells were further stimulated with angii ( . um) for hrs. expression of nadph oxidase core (nox - and duox / ) and associated subunits (p phox, p phox, noxo , p phox, noxa , p phox, and rac / ), and angii receptors at / was identified by rt-pcr from cellular total rna. fluorescent immunohistochemistry (ihc) was employed to determine protein expression and localization. results: the mrna level of house keeping gene -actin was unchanged by any cellular manipulation. the senescent phenotype of ultr cells was accompanied by an apparent down-regulation of nox , p phox, and noxa genes, and an up-regulation of at / . overexpression of htert did not reverse nox , p phox and noxa expression while cell division rate was increased. however, there was a down-regulation of nox , at / and rac . plating ultr-ht cells on cn induced nox / down-regulation and up-regulation of duox / , with no apparent change of at / . however, exposure to cn re-directed cellular response to angii such that only nox was induced by angii stimulation. conclusion: expression of nadph oxidase isoforms nox , , , and duox / are correlated with ultr cell differentiation and cell fate control. data also suggests that ang ii-induced myometrial hypertrophy involves nox mediated ros generation. fluids from reproductive women -the influence of aging. eriko y fujii, masahiro nakayama. women's health, national center for child health and development, tokyo, japan; aska reproductive clinic, nara, japan. [introduction] receptor for advanced glycation end products (rage) is a multiligand type glycoprotein, and is characterized based on its ability to bind ages, adducts formed by non-enzymatic glycation and oxidation of protein and lipids. this process occurs during normal course of aging. ages/rage interaction regulates various physiological function, such as inflammation, angiogenesis through vegf inducement. a soluble form of rage (srage) works as a decoy in the body and inhibits intracellular signaling. [objectives] the balance of these factors may contribute to reproductive dysfunction by aging. we aimed to measure the ages, srage and vegf concentrations in plasma and follicular fluids from reproductive women, and examined the differences of those factors between young group and old group. [material and methods] patients' plasma and follicular fluid were collected with consent based on regulations of the ethical committee, and we measured ages (pentosidine, cml), srage and vegf in duplicate using commercially available elisa kits (fushimi co, r d and cyelex). concentrations were calculated from each standard curve, and compared between the young group under years old, and the old group over y.o. data were evaluated for the difference in two groups by student's t test , and the significance was determined by p< . . [results] ) srage in plasma, ± pg/ml (mean±sd), n= in the young group was significantly higher than ± pg/ml, n= in the old group. there was no significant difference in plasma vegf. ) vegf in follicular fluid was ± pg /mg protein, n= in the young, and ± pg /mg protein, n= in the old was increased significantly. ) we could not see statistical difference of pentosidine nor cml concentrations between two groups in plasma and follicular fluid samples. [conclusions] it has been reported that higher concentration of vegf in follicular fluid may relate to worse pregnancy rate in art. there was a significant decrease of plasma srage in older group in our result, and because of this decrease of 'decoy', focal ages-rage-vegf signaling might be activated in older women. our results showed the possibility that ages/rage and vegf regulation may contribute to the reproductive dysfunction by aging. and leiomyosarcoma cells. qun pan, xiaoping luo, nasser chegini. ob/ gyn, university of florida, gainesville, fl, usa. as a part of a novel endogenous rna silencing machinery, a noncoding short rna strand referred to as "microrna" (mirna) has been identified to regulate the stability of the target gene expression through mrna degradation and repression. we have identified the expression of many of these mirnas in leiomyoma, myometrium, their isolated smooth muscle cells (lsmc and msmc), transformed lsmc (t-lsmc) and sklm (leiomyosarcoma cell line), including mir- which is predicted to target the expression of many genes, including tgf-b and tgf-b type ii receptor (tgf-brii). however, the biological significance of these mirnas in various cellular processes remains to be established. as such in the present study we examined the expression, regulation and function of mir- in lsmc, msmc, t-lsmc and sklm. we found that mir- is expressed and regulated by b-estridiol and medroxyprogesterone acetate ( - m) in these cells (p< . ). we further assessed the regulatory function of gain of and loss of function of mir- on the expression of tgf-brii. transfection of lsmc, msmc, t-lsmc and sklm with pre-mir-and anti-mir- oligonueclotides resulted in a significant increase and/or inhibition of mir- expression in these cells, respectively as determined by realtime pcr (p< . ). over-expression of mir- resulted in a significant reduction, while transfection with antimir- increased the expression of tgfbrii mrna and protein in these cells as compared to controls (p< . ). we concluded that mir- is expressed in leiomyoma and myometrial cells, its expression is regulated by the ovarian steroids and it functions by targeting the expression of tgfbrii and possibly other genes with key regulatory action on cell growth, angiogenesis, transcription regulation, ecm turnover and apoptosis that results in leiomyomas growth and regression. (supported by nih grant hd ). objective: placenta and a number of gestational tissues are well recognized to express corticotrophin releasing factor (crf), urocortin (ucn ), ucn , ucn and crf -r and crf-r receptor subtypes together with crfbinding proteins locally. ucn and ucn are implicated in the reversal of stress responses initiated by crf. in the present investigation, we evaluated functions of crf and ucns by quantifying their contents in venous smooth muscle layers using image pro . in human umbilical cords collected at preterm and term gestation methods: umbilical cord specimens ( - mm thickness, pieces per umbilical cord) collected at preterm and term (n= each) at delivery were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analyses with polyclonal antibodies to crf ( : ), ucn ( : ), ucn ( : ) and ucn ( : ) (peninsula laboratory, pa and sigma aldridge, ms) by standard abc technique. immunoreactive materials on the sections were identified using , '-diaminobenzidine as a chromagen. immunostaining intensities (od/area) on uc-sections were quantified by image pro . software and expressed as arbitrary units (au). all values were expressed as mean ± sem. differences between groups were evaluated by anova, followed by the post-hoc tukey test for multiple comparison. results: antibodies to crf and ucns elicited positive immunostaining of variable intensity in venous smooth muscle layers in uc-sections of preterm and term gestations. immunostaining intensity (au) of venous smooth muscle layers at preterm (pt) and term (t) are as follows: crf-pt= . ± . vs crf-t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p=ns); ucn -pt= . ± . vs . ± . (p< . ). conclusion: crf content in venous smooth muscle layer markedly increased at term, while ucn and ucn contents significantly decreased and no significant change occurred in ucn content. based on the opposing changes in crf vs ucn and ucn immunostaining, we speculate that crf, but not ucn or ucn , is the major key player of vasodilation and function locally at the level of venous smooth muscle cells at term. ucn and ucn may perform cytoprotective functions at preterm. physiol. ; : - , am j physiol. ; : - ) . these studies showed that when ad libitum (al) feeding was resumed in cr females, fertility was sustained well beyond the age at which al-fed females became infertile. however, much of what is known on the effects of cr on fertility derives from models in which cr was initiated at weaning. further, there is large variation in how the experiments were conducted. objective: herein we tested if adult-onset cr could delay age-related infertility in females. methods: cr ( %, nia protocol as described in j geront. a:b -b ) was initiated in c bl/ female mice at months and continued until . months of age, at which time al feeding was resumed. matings were initiated at months of age. for mating during cr, a male mouse was housed overnight in a cage with a female and removed the next morning, so that the female mice could be fed their dietary food ration. al-fed and cr females followed the same mating regimen. the number of offspring born and that survived to weaning (day ) were recorded. results: fertility was lost in of al-fed females by months of age and continued to decline through . months of age. age-matched females maintained on cr during the same period exhibited poor fertility, with a total of pregnancies achieved out of females. although cr females showed poor fertility while on cr, their fertility improved dramatically after the reinitiation of al feeding at . months of age. while only out of , al-fed females achieved a pregnancy between . - . months of age, out of age-matched cr-then-al-fed females achieved a total of pregnancies in this -month time. notably, % of the pups born to cr-then-al-fed females between . - . months of age survived and developed to weaning without complications. conclusions: adult-onset cr allows maintenance of female fertility into advanced maternal age after the reinitiation of al feeding. how long fertility can be maintained and the minimum time needed for the beneficial effects of cr to be realized remain to be investigated. nonetheless, these observations suggest that there may be ways to safely extend fertility in females at ages when reproductive function is suboptimal (nih r -ag ). bile acids in human ovarian follicular fluid. laura p smith, , kaila deiorio-haggar, jason reindollar, alan s penzias, , anny usheva-simidjiyska. ob/gyn reproductive endocrinology infertility, bidmc, boston, ma, usa; boston ivf, boston, ma, usa; endocrinology, bidmc, boston, ma, usa. introduction: bile acids are known to serve important functions in the hepatobiliary and gastrointestinal systems. the presence of bile acids in the human ovary and relation with fertility potential have never been previously evaluated. methods: human follicular fluid (ff) from large follicles and small follicles was obtained at vaginal oocyte retrieval. human serum was obtained before and hours after human chorionic gonadotropin (h-cg). follicular fluid and serum samples were analyzed for total bile acids by spectrophotometry. bile acid concentrations were correlated with age, number of retrieved and mature oocytes, number of fertilized oocytes, and pregnancy. results: bile acid concentrations were analyzed and compared to the normal human serum bile acid concentration which ranges from to micromoles/l. bile acids are present in follicular fluid with a mean concentration of . micromoles/l in large follicles and . micromoles/l in small follicles (p= . ). pre and post h-cg serum bile acid concentrations differed significantly ( . micromoles/l vs. . micromoles/l, p= . ). there was a trend toward higher bile acid concentration in large follicles of young patients < years old compared to older patients years old ( . ± . vs. . ± . , p= . ). there was also a trend toward higher pre h-cg serum bile acid concentrations in older patients ( . ± . vs. . ± . , p= . ). there was no correlation between serum and follicular fluid bile acid concentrations and number of oocytes retrieved or number of mature oocytes, but there did appear to be a positive correlation between pre h-cg serum bile acid concentration and number of fertilized oocytes (spearman's correlation coefficient . , p= . ). conclusions: this is the first demonstration of the presence of bile acids within human ovarian follicular fluid. there may be a relationship between bile acid concentration and fertility potential. the precise function of bile acids in human ovarian follicular fluid is under investigation. objective to investigate the impact of ovarian hyperstimulation treatment on the biomarkers of the homocysteine pathway in blood and follicular fluid, and their association with the follicle diameter as a measure of follicular maturation. methods in women undergoing ivf/icsi treatment blood samples were collected on cycle day and the day of hcg administration. during oocyte retrieval in each woman the diameter of two follicles was measured and the corresponding follicular fluids were collected. in blood and follicular fluid total homocysteine (thcy), folate, cobalamin and pyridoxal' 'phosphate (plp) concentrations were determined. women with a serum folate . nmol/l were classified as folic acid supplemented. results ovarian hyperstimulation significantly decreased thcy and cobalamin blood levels (both p . ). the blood and follicular fluid concentrations of thcy, folate, cobalamin and plp were significantly correlated (all p . ). in the total group, a two-fold increase of thcy in follicular fluid resulted in a . mm decrease of the follicular diameter (p . ). in non-supplemented women this decrease was . mm (p . ). in supplemented women a twofold increase of follicular fluid folate resulted in a . mm decrease of the follicular diameter (p . ). conclusions ovarian hyperstimulation reduces thcy blood levels independent of folic acid supplementation. however, high follicular fluid thcy and folic acid supplementation may have detrimental effects on the maturation of the follicle. ), post-fixed for rain in % w/v osmium tetroxide in the same buffer, quickly dehydrated in a series of ethanol solutions, and embedded in epon. thin sections were stained with uranyl acetate followed by lead citrate and were observed at electron microscope. results: ten blastocysts from each group were collected and analyzed. compared to the in vivo embryos, blastocysts generated in vitro exhibited significant differences. the surface of their trophectoderm (te) layer had a reduced number of microvilli, the number of the apoptotic cells in the inner cell mass (icm) was higher and the presence of non functional mitochondria was elevated. in this study we have, for the first time, compared the ultrastructure of the in vivo and in vitro conceived blastocysts. taken together, these results suggest that both, the higher rate of apoptosis and the morphological alterations in the mitochondrial structure in ivf embryos, are associated with stress during in vitro embryo culture. therefore, these parameters can be used, in the future, as markers for the assessment of the embryo wellbeing in the ivf settings. deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age.here, we explore a newly discovered role of nitric oxide (no) in the sustenance of oocyte quality. methods: sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (icsi) with cauda-epididymal spermatozoa following exposure to either the no donor, s-nitroso n-acetyl penicillamine (snap, . m/min); a soluble guanylyl cyclase inhibitor, h-[ , , ] oxadiazolo [ , -a] quinoxalin- -one (odq, m) or an no synthase inhibitor, n w -nitro-l-arginine methyl ester (l-name, mm). their sibling oocytes were subjected to icsi either before (young) or after culture for the corresponding period (old). outcomes of fertilization, cleavage and development to the morula and blastocyst stages were compared. some embryos from each subgroup were also subjected to tunel assay for apoptosis. results: oocytes deteriorated in their ability to undergo normal fertilization and development to morulae/blastocysts after aging in culture, as compared to their sibling cohorts subjected to icsi immediately after ovulation (p< . ). this deterioration was prevented after oocyte exposure to snap. while, exposure to l-name or odq resulted in a significant compromise in fertilization and development to the morulae/blastocysts (p< . ) with detection of apoptosis, which was also noted in embryos derived from aged oocytes but not in those from young or snap exposed oocytes. conclusions: no is essential to sustain oocyte fertilizability and developmental ability, and to prevent blastomere apoptosis. objective to investigate the effects of the levels of the biomarkers of the homocysteine pathway on ivf outcome. methods from women undergoing an ivf or icsi procedures, two blood samples and two mono follicular fluid samples were collected for determination of folate, cobalamin, and total homocysteine (thcy). total protein was determined in follicular fluid to adjust the biomarker concentrations for follicular maturation. primary endpoint of the study was oocyte quality measured by fertilization and embryo quality (range - ; with being best quality). secondary endpoint was the occurrence of pregnancy. results % of the included women used a folic acid supplement (serum folate . nmol/l). in non-supplemented women higher cobalamin levels in follicular fluid correlated with a better embryo quality (estimate - . ; p . ) and higher thcy levels (median . mol/l, range . - . ) correlated with a worse embryo quality (estimate . ; p . ). in supplemented women higher follicular fluid thcy (median . mol/l, range . - . ) correlated with better embryo quality (estimate - . ; p . ). the follicular fluid folate level of oocytes that did not fertilize was . -fold higher than in the fluids of a fertilized oocyte ( % ci . - . ; p . ). a two-fold increase of follicular folate corresponded with a . higher chance to achieve pregnancy ( % ci . - . ). conclusions cobalamin levels in follicular fluid are correlated with embryo quality. folic acid supplementation modifies the thcy and folate levels in follicular fluid and thereby affects oocyte quality. the level of folate in follicular fluid is important in the fertilization process. we recently reported that increasing relative oocyte immaturity is associated with worsening outcome, and that cycles with many immature oocytes are more common in younger women. (moore et al, asrm annual meeting, ) to further investigate this trend, we conducted a case/control analysis of patients with repeated cycles of high-level oocyte immaturity (hloi). methods: oocyte maturity data was collected on all icsi cycles starting in . we defined a cycle with hloi as having > % immature oocytes (> sd's above the median). a case was defined as a patient with hloi on more than one cycle. control subjects were age-matched and defined as having </= % ( sd above median) immature oocytes on all cycles. results: from subjects, we identified cases of recurrent hloi (comprising icsi cycles) and control subjects. at baseline, cases had more oocytes retrieved per cycle than controls ( . vs. . , p<. ). all other baseline characteristics were similar. all case subjects were younger than . outcomes are shown in table . discussion: clinical pregnancy and live births were reduced in the case group, but more than expected based on % immaturity. during cycles, there were no live births among the case group. patients with recurrent hloi represent a previously undescribed group of young patients with a very poor prognosis during icsi treatment. this study may suggest that the problem underlying the immaturity may relate to overproduction of oocytes during stimulation, a factor which may be modifiable. however, the additional trend (though not significant) that even the mature oocytes from the case patients tend to have poor outcomes suggests the possibility of a maturation defect and warrants further consideration. data on response to gonadotropins, fert rates, and implantation rates will be presented. amongst those, were performed for an initial diagnosis of premature ovarian aging (poa), for the diagnosis of premature ovarian failure, and for repeated pregnancy loss. in women under age , b-fsh levels over miu/ml and up to < miu/ml denoted a diagnosis of poa, while levels of miu/ml were considered diagnostic for pof. all patients signed consent permitting review of medical records for clinical research purposes. results: day fsh levels ranged from . to . miu/ml (mean . ± . miu/ml). amh levels ranged from < . to . ng/ml (mean . ± . ) and allele repeats ranged from to , (mean for allele- , . ± . ; allele- , . ± . ). mixed model analysis of variance for all patients, adjusted for age, race, and allele- revealed a statistically significant correlation between b-fsh levels and number of repeats in allele- ( figure ; p < . ). we did not observe a significant linear relationship between serum amh levels and cgg repeat counts, however cgg repeat counts above were significantly associated with serum amh levels less than ng/ml (chi square = . ). conclusions: triple repeats above , a level currently considered well within normal, are associated with evidence of premature decline in ovarian function. the evaluation of triple repeats in frm gene offers a first objective assessment of ovarian function and should be predictive of autologous reproductive life span in most women. objective: the levels of mis in the serum and in the ovary have been demonstrated to decrease in response to increasing doses of cisplatin. we hypothesize that ovarian stimulation also will reveal dose related decreases in ovarian and serum mis levels after cisplatin exposure, further demonstrating the follicular damage from chemotherapy. methods: adult female sprague-dawley rats were treated with saline, . mg/kg cisplatin or . mg/kg cisplatin as two weekly injections. five days following the last cisplatin injection, the rats were injected with pregnant mare serum gonadotropin (pmsg) and euthanized hours following the pmsg injection. serum was collected and both ovaries were obtained for protein analysis. serum and ovarian levels of mis were determined using an elisa kit. western blot analysis, immunohistochemical analysis, and tunel analysis were also performed on the ovarian proteins. results: stimulated mis levels declined in a linear fashion in response to increasing cisplatin doses (p< . ). ovarian protein levels measured by elisa and western blot both indicated that stimulated ovarian mis levels also decrease in a linear fashion (p= . and p< . respectively.) immunohistochemical analysis revealed that while the total number of follicles remains the same, both the total number of mis positive follicles, and the percentage of mis positive follicles declines in a linear fashion (p= . for both). tunel analysis reveals that there is no change in the incidence of apoptosis in the stimulated ovaries from any treatment group. conclusions: the administration of pmsg after treatment with cisplatin causes a dose dependent decrease in the levels of mis in both the serum and in the ovary. serum mis levels following ovarian stimulation may be useful as a serum biomarker in this animal model to assess ovarian damage following the administration of cisplatin. correlations of micro-rnas a, b, - pp, and - p with inflammatory and steroidogenic factors in human granulosa/cumulus cells. tannaz toloubeydokhti, orhan bukulmez, kenneth c drury, r stan williams, nasser chegini. obstetrics and gynecology, university of florida college of medicine, gaineville, fl, usa. as part of the novel endogenous rna silencing machinery, noncoding short rna strands, referred to as micrornas (mirnas), have been identified to regulate the stability of the target gene expression through degradation and repression. the expression of many of these mirnas has been identified in human tissues however, their biological significance in various cellular processes remains to be elucidated. in this cross-sectional study of human granulosa cells, we aimed to study the potential correlations between five selected mirnas, mir- a, mir- b, mir- - p, mir- and mir- - p, and mrna expressions of some of their predicted target genes namely, cyclooxygenase (cox)- , il- , steroidogenic acute regulatory protein (star), aromatase and estrogen receptor (er) . granulosa/cumulus cells were obtained from women (age range - ; mean age . ± . ) undergoing oocyte retrieval for assisted reproduction. total rna was extracted from these cells and reversed-transcribed and real-time pcr was performed to measure steady-state levels of mirnas and mrna transcripts. we observed that the expression of mir- a, mir- - p, mir- and mir- - p displayed a significant and positive correlation with the expression of cox- . moreover, mir- b significantly correlated with star mrna expression, but not with other genes tested (p< . ). with advancing age, the expression of mir- - p and cox- was significantly decreased, with cox- expression displaying a positive correlation with aromatase, star and er mrna expressions (p< . ). none of the factors tested showed any significant correlations with peak estradiol levels and number of oocytes retrieved. receiver operating characteristic curve analysis revealed that female age cut-off value of . y best predicted whether mir- - p was below its mean arbitrary value. in conclusion, given the importance of mirnas' regulatory function in gene expression, our results suggest that mir- b may play an important role in gene expression related to granulosa cell luteinization, while variation in mir- - p expression with female age may suggest its potential role as a predictor of oocyte quality and granulosa cell function. given the importance of mirnas in gene regulation, the role of mirnas in ovarian physiology and aging requires further investigation. support: nih grant . introduction: a symbiotic relationship between ovarian granulosa cells (gc) and the enclosed developing oocyte is critical to reproductive efficiency. genetic modulations in gc's can lead to reproductive insufficiency, highlighting the critical role of gc's in reproductive competence. defects in the oocyte-gc repertoire in women with dor include lower e levels, luteal deficiency, poor fertilization rates, higher incidence of aneuploidy, lower pregnancy and higher miscarriage rates. utilizing global gene expression analyses in cumulus gc's, we attempt to enhance our understanding of biological mechanisms that may contribute to poor reproductive capacity in young women with dor. methods: infertile women (< years old) undergoing ivf were prospectively enrolled into two groups based on ovarian reserve (normal reserve, fsh< dor, fsh > miu/ml). at egg retrieval, cumulus gc's were isolated, rna extracted transcribed into cdna. microarray targets were generated cdna hybridized to affymetrix human genome u plus . genechips. microarray data were analyzed (array assist) and normalized (robust multichip analysis). a difference in gene expression of > fold was considered biologically relevant. results: of the , genes identified to be differentially expressed in young women with dor compared to normal reserve, genes demonstrated consistency of expression across five different normalization schema; were down regulated and up-regulated. expression of gremlin, a member of the dan family of genes known for its highly regulated expression pattern during folliculogenesis, was noted to be down-regulated -fold over two probe sets (- . ) in women with dor versus normal reserve; this down-regulation was confirmed by real-time pcr (- . ) . conclusions: this is the first demonstration linking differential expression of gremlin with etiology of infertility in women. gremlin is a downstream effector of oocyte-derived gdf- which facilitates cumulus cell expansion, a critical event in reproductive physiology. our finding of a significant downregulation of gremlin expression in cumulus gc's associated with dor may partly explain the physiology of poor reproductive performance. nih k cd . nih k rr . ferring pharmaceuticals. the objective of the present study was to investigate the effects of the gnrh antagonist ca on expression of mis and aromatase (cyp ) using luteinized human granulosa cells obtained during in-vitro fertilization cycles and an immortalized human granulosa cell line (hgl ). the granulosa cells were cultured +/-dibutyryl camp ( mm) and incubated +/-the gnrh antagonist, ca, for - h. rna was isolated, reversed-transcribed and real-time pcr was performed to measure mrna transcripts for ovary-specific cypiia and mis. we observed that camp markedly induced aromatase mrna in both the primary and immortalized human granulosa cells. interestingly, camp treatment of these cells also caused an upregulation of mis mrna. objectives: bisphenol a (bpa), a known endocrine disruptor, is a chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins and is present in multitude products, including the interior coating of food cans, milk containers, and baby formula bottles. bpa can leach into foods during heat processing and is known to exert a variety of endocrine-like effects on different cell types acting as an estrogen because it contains two hydroxyl groups in its diphenyl structure. in this study we focused on the effects of bpa on aromatase expression and estradiol production in the human granulosa kgn cell line. we also evaluated its effects on several transcription factors crucial in cyp expression. materials and methods: kgn cells were cultured in f- dmem and were starved for h before experiments. subsequently they were treated for h with vehicle (control), fsh ( ng/ml), and/or bpa ( , , , , um) . messenger rna expression was quantified by real time pcr and estradiol secrection was measured in supernatants by elisa. results: fsh induced a -fold increase in aromatase expression. bpa induced a dose-dependent decrease in cyp production, with the greatest effect at um (p< . ), resulting in +/- % (mean+/-sem) inhibition, compared to aromatase expression induced by fsh alone. bpa also reduced levels of estradiol secretion in a dose-dependent manner, with the greatest inhibition at um (p< . ) resulting in +/- % decrease. we also evaluated expression of transcription factors known to be involved in regulating the activity of the ovary-specific aromatase proximal pii promoter. interestingly factors known for induction of aromatase such us steroidogenic factor- and gata- , mimic the pattern of cyp expression after bpa treatment, whereas, other receptors previously reported to act as aromatase inhibitor, such as ppar gamma were up-regulated by the addition of bpa. moreover, expression of creb remained virtually intact, suggesting that most likely mechanisms governing endocrine disruption by bpa are highly selective. conclusions: bpa inhibited fsh stimulated aromatase expression and downstream estradiol secretion. we propose that constant exposure to this chemical may result in endocrine disruption which may contribute to reduced fertility and early ovarian senescence. oocyte maturation occurs during folliculogenesis as a result of complex cell-tocell communications between the granulosa cells and the oocyte. maintaining the granulosa cells' spherical structure and network of gap junctions surrounding the oocyte is critical. this project tests the ability of a novel substrate-free threedimensional culture system to form viable granulosa cell spheroids. methods: after irb approval, freshly obtained follicular fluid from in vitro fertilization was obtained and granulosa cells were purified by percol gradient. nonadhesive agarose hydrogels, containing cylindrical round bottom recesses m in diameter, were cast from micro-molds designed using computer-assisted rapid prototyping. granulosa cells seeded at a density of , cells per gel were incubated for up to days. cellular viability was assessed with live:dead assay. results: after three days in culture, granulosa cells formed spheroids of densely packed cells that were difficult to disperse with multiple pipettings. the cells remained viable for at least days. conclusions: granulosa cells can be cultured in a novel substrate-free threedimensional culture system. the cells form tightly adherent spheroids that remain viable for extended culture. the cohesiveness of the cells suggests the formation of gap junctions. this is under investigation with immunohistochemistry and electron microscopy. these experiments suggest that a substrate-free threedimensional hydrogel culture system may be ideal to reassemble follicular structure important for future in vitro evidence testing and oocyte maturation. known to function as responders to pathogens, inflammation, and tissue injury. previous studies in our laboratory demonstrated that saa was produced in mouse granulosa and production was regulated by cytokines. ovulation has long been considered an inflammatory reaction and patients with chronic inflammatory conditions often experience infertility. the present study was undertaken to explore the role of saa in human ovarian function. methods: ovarian granulosa and luteal cells were obtained from surgically removed specimens and mural and cumulus granulosa-luteal cells were obtained from ivf aspirates. rna was extracted from fresh or cultured cells. some cells were treated in vitro with tnf or other cytokines for h. expression of saa was assessed by quantitative rt-pcr. in addition, serum levels of saa were determined using a commercial elisa in women undergoing ovulation induction (oi) and art. saa levels were measured at baseline, during oi, on the day of hcg administration and at the time of the pregnancy test. results: saa mrna was expressed in theca, granulosa, and granulosa-luteal cells. in granulosa-luteal cells both saa and saa mrnas were expressed at higher levels in cumulus compared to mural granulosa. expression of saa and saa in theca was increased following treatment with tnf in vitro. serum levels indicated that patients with ovulatory dysfunction had increased levels of saa at the time of hcg injection while patients without ovulatory dysfunction had lower saa levels as compared to the baseline level. in addition, patients undergoing oi who achieved pregnancy exhibited increased levels of saa at the time of the pregnancy test compared to baseline levels, whereas patients who did not become pregnant had lower post-cycle levels of saa. interestingly, saa levels did not change in art patients that became pregnant without undergoing oi (donor egg or frozen embryo transfer) conclusions: human ovarian cells express saa mrnas which can be altered in vitro. serum levels of saa may correlate with the responsiveness of the ovary to gonadotropins as evidenced by altered levels in women with ovulatory dysfunction, and by an increase in pregnant patients following ovarian stimulation. yeh, beom su kim, felicia hercules, jennifer peresie, armando arroyo. gynecology-obstetrics, university at buffalo, buffalo, ny, usa. objective: cisplatin is a common chemotherapeutic agent given to women for treatment for a wide variety of cancers. we hypothesize that one mechanism by which cisplatin may cause damage to ovarian structures is by decreasing the amount of anti-oxidant activity in the ovary. we examined super-oxide dismutase (sod ), a critical anti-oxidant enzyme that has been shown to be affected by cisplatin in other tissues, in the ovaries of cisplatin treated animals. methods: adult female sprague dawley rats were injected with saline, cisplatin . mg/kg or cisplatin . mg/kg as weekly doses. five days following the last injection, the rats were euthanized and both ovaries were excised. one ovary was processed for immunohistochemistry and the other was processed for protein analysis using western blot techniques for sod . the anti-sod antibody was purchased from santa cruz. the immunohistochemical sections were scored using a semiquantitative h scoring method. results: immunohistochemistry analysis of the expression pattern of sod following cisplatin administration revealed that there was a significant linear decrease in a dose response pattern in the expression of sod in antral follicles and in corpora lutea (p< . for both). no change was found in the h score of sod in other ovarian structures. western blot analysis of sod in the ovaries following increasing doses of cisplatin revealed no changes in the overall protein levels of sod in the ovary. conclusions: this is the first report that administration of cisplatin causes changes in the expression pattern of sod in antral follicles and in copora lutea. cisplatin decreases the amount of sod available in these structures, possibly leading to increased oxidative stress and free radical damage, thereby leading to ovarian damage found after cisplatin administration. is there evidence for aromatase activity in the stroma of postmenopausal ovaries? mf landay, rh fogle, rb allen, s patel, fz stanczyk, rj paulson. ob/gyn, usc, keck school of medicine, los angeles, ca, usa. background: following menopause, the ovaries continue to secrete androgens and estrogens. we have recently confirmed the production of androstenedione, testosterone and estradiol (e ) up to ten years after menopause by measuring gradients from ovarian venous effluent and peripheral blood. anti-müllerian hormone (amh) and inhibin b have been shown to be markers of follicular activity. peripheral levels of these hormones have previously been found to be undetectable in menopause, suggesting the absence of follicular activity in the postmenopausal ovary. objective: to investigate if the postmenopausal ovary continues to demonstrate evidence of follicular activity as the source of steroid production. design: observational study materials and methods: eight subjects aged ± . yr (range - ) were enrolled. postmenopausal status was confirmed by preoperative fsh levels of more than u/l and/or amenorrhea greater than months. serum was collected from the ovarian veins during total abdominal hysterectomy and bilateral oophorectomy. peripheral blood was also collected pre-operatively, intraoperatively and postoperatively. all samples were analyzed for amh and inhibin b using elisas with sensitivities of . ng/ml and pg/ml, respectively. androgen and estrogen levels in these samples have previously been reported, and documented a gradient between ovarian venous effluent and peripheral serum in all cases. results: ) six patients had no detectable follicular activity by amh and inhibin b levels. ) one patient demonstrated detectable inhibin b levels with an -fold gradient between ovarian venous effluent ( pg/ml) and peripheral blood ( pg/ml), however no amh was detected. ) in one patient, aged and months postmenopause, both amh and inhibin b were detected. peripheral inhibin b levels were high at pg/ml. amh was detectable at levels of . ng/ml. conclusions: ) in the majority of patients, continued e and androgen production in the ovary occurs in the absence of follicular activity as detected by amh and inhibin b production. ) some patients have evidence of follicular function up to ten years after menopause. ) since e is produced in post-menopausal ovaries in the absence of follicular activity, these data provide evidence for aromatase activity in the stroma of post-menopausal ovaries. to elucidate the process by which prostaglandin f (pgf ) mediates luteal regression, this study examined the role that the transcription factor yin yang (yy ) and histone deacetylase (hdac ) play in altering luteal cholesterol uptake by the scavenger receptor class b type i (sr-bi), intracellular cholesterol transport by steroidogenic acute regulatory protein (star), and cholesterol processing by p side chain cleavage enzyme (p scc) expression. rat luteal cells ( days post-ovulation) were treated with pgf ( hr) and trichostatin a (tsa; hr), a potent hdac inhibitor. protein expression was measured post-treatment by western blot and cholesterol was localized via filipin staining. star and sr-bi promoter activation was also assessed in hek t cells treated with yy , myy , a deletion mutant lacking an essential region required for transcriptional repression, and tsa. pgf caused a significant -fold decline in star (p< . ), and smaller declines in sr-bi and p scc which occurred concomitantly with an increase in yy ( -fold, p< . ) and intracellular lipid staining ( % increase). in contrast, nm tsa treatment caused a dose dependent increase in sr-bi, star, and p scc protein levels, . -fold (p< . ), . -fold (p< . ), . -fold (p< . ), respectively, and a . -fold decline (p< . ) in intracellular lipid levels. tsa prevented the pgf -induced decline in sr-bi, star, and p scc expression. promoter analysis demonstrated that wildtype yy , but not myy , repressed sr-bi and star activation while the addition of tsa overcame the repressive effects. this study shows the critical role that yy plays in pgf induced luteal regression by recruiting a histone deacetylase to block three essential processes in steroid production. in luteal cells yy -mediated global repressive action prevents cholesterol metabolism by inhibiting cholesterol uptake, intracellular transport, and processing thus leading to functional and structural luteal demise. supported by nih hd and nih hl . regulation of intermedin expression in cycling rat ovary. madhu chauhan, rebekah elkins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd) is a ct/cgrp family peptide involved in a variety of physiological functions, including vasodilatation and fetoplacental growth. this peptide is expressed in a variety of tissues such as stomach, placenta, uterus, pituitary and ovary suggesting its different functions including in reproduction. imd gene has an estrogen response element and we have shown that the plasma concentration of immuno-reactive imd is elevated in rats with pregnancy. however, expression of imd in the ovary and its regulation during estrous cycle is not known. we hypothesize that imd is expressed in the ovary and its expression is hormonally regulated throughout the estrous cycle in rat. objective: ) to assess expression of imd mrna and its receptors components calcitonin receptor like receptor (crlr), and receptor activity modifying proteins, ramp and ramp mrna in rat ovary; on diestrus, proestrus and estrus stages of rat estrus cycle and ; ) to demonstrate immunohistolocalization of imd, crlr, ramp , ramp and ramp in rat ovary. methods: groups of sprague-dawley non-pregnant and pregnant rats on day of gestation were used in this study. non-pregnant rats were sacrificed on diestrus, proestrus and estrus stage. ovaries were collected and total rna was extracted using trizol method. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). immunohistolocalization of imd, ramp , ramp and ramp proteins were assessed in tissue sections of ovaries from pregnant rats sacrificed on day . results: ) imd mrna is regulated in rat estrus cycle and its expression is significantly downregulated in estrus stage compared to the diestrus and proestrus; )expression of imd receptor crlr is highest in the diestrus stage, followed by a decline in proestrus which further declined during estrus; ) expression ramp mrna is higher in proestrus compared to diestrus and estrus (p< . ) but there is no significant change in the ramp expression during the estrus cycle and ; ) imd, ramp , ramp and ramp are immunolocalized in rat ovary in granulose cells of all follicles and granulosa cells of the corpus luteum. conclusion: imd mrna and protein are expressed in rat ovary suggesting a possible role for imd in ovarian function. defining and defying deterioration in oocyte quality with advancing chronological age: role of nitric oxide. pt goud, ap goud, mp diamond, b gonik, hm abu-soud. div reprod endocrinology, dept ob/gyn, wayne state university, detroit, mi, usa; div maternal and fetal medicine, sinai grace hospital, wayne state university, detroit, mi, usa. nitric oxide (no) is a ubiquitous free radical essential for oocyte maturation, function and sustenance of oocyte quality post-ovulation. the current study investigates the role of no insufficiency in the causation of oocyte quality deterioration with advancing chronological age. methods: in set , oocytes were retrieved from the following superovulated b d f mice: (a) young breeders (yb, n= ); (b) retired breeders (rb, n= ), and © old animals (oa, n= ), aged - , - , and - weeks respectively; and studied for zona pellucida dissolution time (zpdt), spindle ( -tubulin fluorescence immunocytochemistry, sigma) and chromosome morphology (dapi, vector), ooplasmic microtubule (mt) dynamics (omd) in response to taxol [ ], and cortical granule (cg) status (rhodamine conjugated lectin, vector). in set (n= ), oocytes from retired breeders were studied after exposure in vitro to an no-donor, s-nitroso acetyl penicillamine (snap in m- , . m no/min , n= , h, °c, % co ), while their sibling control oocytes were cultured for corresponding period under identical conditions without snap. [ ]. zpdt, spindle and chromosome morphology, omd and cg status were assessed with a confocal microscope and compared between the subgroups using anova, chi square and/or fisher's exacts test and appropriate post-hoc tests. results: in set , a significantly fewer oocytes were retrieved per animal (mean) in rb ( ) and oa ( ) compared to yb ( . , p< . ). advancing age was also associated with a significant increase in zpdt, omd and cg loss in rb compared to yb (p< . ). furthermore, significantly fewer oocytes from rb than yb had normal spindle and chromosome morphology (p< . ). oocytes from oa had significant spindle and chromosome disarray, a near total cg loss and significantly harder zp (p< . ). these oocytes also exhibited diminished omd in response to taxol although, metaphases were exquisitely sensitive to disruption with taxol. in set , exposure to snap in oocytes from rb resulted in a significantly lowerzpdt, omd and cg loss, and significantly higher incidence of normal spindles ( the gamma-aminobutyric acid (gaba), its biosnthetic enzyme gad and gaba-a receptors have been found in the oviduct and ovary. objective: this study examined the expression of gaba-a receptor subunit genes and gad and whether the gaba-a receptor could alter cytosolic ca + in gc. methods: for qrt-pcr, both human cumulus and mural gc were obtained at the time of oocyte retrieval for ivf and cultured separately. total rna was isolated separately from each gc type and from human brain ( positive control). the total rna from patients was pooled for each gc type and all rna was treated with dnase i. two-step qrt -pcr was performed using gene-specific lux™ primers for all gaba-a receptor subunits, gad and gad plus gapdh. single and specific qrt-pcr products were verified by melting curve analysis, gel electrophoresis, and dna sequencing. for ca + study, gc were cultured on coverslips. gc were loaded with fura- -am and changes in ca + concentration of gc were studied using a dynamic digital ca + imaging system. gaba-a agonist, muscimol, was used to study any dose-dependent effects on gc. gaba-a antagonist, m bicuculline, were perfused min. prior to and during application of muscimol. the responses were represented as changes in the / nm fluorescence ratio over time. m atp was used as positive control. results: the qrt-pcr results indicated that all gaba-a receptor subunits were expressed to various degrees in both types of gc, with the expressed highest in both cell types. gaba-a receptor subunits showing the next highest expression in both cell types were , and . gad isoenzyme was more abundantly expressed in cumulus and mural gc than gad . ca + imaging showed that muscimol, had the ability to increase ca ++ in gc, about % gc (n= ) cells responded to muscimol. muscimol increased intracellular ca + in a dose-dependent manner. the muscimol responsive cells was reduced by bicuculline, from % to % (n= , p< . ). conclusion: qrt-pcr indicates that gaba-a receptor subunit gene and gad expression occurs in both cumulus and mural gc. the ability of bicuculline to inhibit the ca + response to muscimol suggests the activation of gaba-a receptor. the current study confirms the presence of functional gaba-a in gc for the first time, and suggests that gaba may exert trophic effects in the ovary via gaba-a receptor. the present study test the hypothesis that administration of l-nitro-l-arginine methyl ester (l-name), a nos inhibitor, prior to hypoxia prevents the hypoxiainduced activation of p mapk, erk and jnk in the cerebral cortex of the guinea pig fetus during gestation. to test this hypothesis guinea pig fetuses at and days gestation were assigned to normoxic (nx, n= ), hypoxic (hx, n= ) and hypoxic pretreated with nos inhibitor (hx+l-name, mg/kg i.p., n= ) groups. hypoxia in the fetuses was induced by exposing the pregnant guinea pigs at both gestational ages to an fio of . for min. cerebral tissue hypoxia was documented biochemically by determining the tissue levels of atp and phosphocreatine (pcr). neuronal nuclei were isolated, purified and proteins separated by sds-page, and then probed with specific phosporylated erk, jnk and p antibodies. in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx) . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx) . ± . (hx+l-name). the data show that administration of l-name prior to hypoxia decreased the expression of phosporylated p , erk and jnk at both gestation ages however expression of phosporylation was higher at term as compared to preterm. since a nos inhibitor prevented the hypoxia-induced phosphorylation of p , erk and jnk in both gestational ages, we conclude that the hypoxia-induced activation of p , erk and jnk in the cerebral cortical nuclei of preterm and term guinea pig fetus is no-mediated. we speculate that no-mediated modification of cysteine residue leading to inhibition of map kinase phosphatases results in increased activation of p , erk and jnk in the guinea pig fetus. (nih-hd , nih-hd and st. christophers foundation for children). the the endocannabinoid, anandamide (aea), is involved in the hormone-cytokine network that regulates implantation and early pregnancy maintenance with levels at the endometrial level considered a major checkpoint . high levels ( nm) in culture are associated with embryo death while plasma levels (> nm) at weeks in women undergoing ivf-et are associated with failed pregnancy . what is uncertain is whether systemic aea levels after spontaneous conception in women presenting with threatened miscarriage are predictive of pregnancy outcome. our aim was therefore to measure plasma aea levels in women presenting with threatened miscarriage and to relate these to outcomes. methods: plasma aea levels were measured using a sensitive and previously validated hplc-ms method at - weeks gestation in women (nonsmokers, bmi < kg/m ) presenting with threatened miscarriage and in whom a viable pregnancy was confirmed by ultrasound scan. results: nine of the women subsequently had a miscarriage. the plasma aea levels in those women who had live births was . -fold lower than that in those who subsequently miscarried ( . ± . nm versus . ± . nm, mean ± sem; respectively; p< . unpaired student's t-test). the roc analysis revealed an area under the curve of . ± . with a sensitivity of % ( %ci of . % to . %) and a specificity of . % ( % ci of . % to . %). using an aea level of . nm as the optimal cut-off point, a single plasma aea measurement provided a sensitivity of % and a specificity of % with a negative predictive value of % and a positive predictive value of % for subsequent miscarriage. conclusion: these findings suggest a possible predictive role for plasma aea in women presenting with threatened miscarriage. the data also indicate that systemic aea levels may reflect local endometrial levels and therefore the local hormonal milieu in the early stages of normal pregnancies and in those complicated by threatened miscarriage. introduction: follicle stimulating hormone (fsh) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation. the ovarian response of different women to fsh is variable, ranging from poor response to ovarian hyperstimulation and has been partly attributed to two common variants of the fsh receptor (fshr). we have previously identified four abnormal fshr splicing products ( exon deletions and intron insertion) in women with low and high response to fsh. two of the splice variants, deletion of exon and deletion of exon , showed a correlation with low and high response to fsh, respectively. in the current study, we evaluated the functional competence of the mutant fshrs in vitro. methods: we established stable hek cell lines expressing wild-type (wt) and splice-variant (del) fshr under the control of the inducible tet on/off system. the cells were transfected with a camp-responsive luciferase reporter plasmid and stimulated with fsh. results: the subcellular distribution of all splicing variants was the same as the controls and the protein localized mainly on the cell surface. all four splicing variants showed markedly decreased camp activation compared to controls when stimulated with fsh. however, all variants were able to form functional heterodimers with the wt receptor when co-expressed. interestingly, the heterodimer containing the form of fshr lacking exon , found in patients with decreased response to fsh, resulted in lower intracellular camp compared with the wild-type homodimer. conclusion: our findings suggest that fshr variants can contribute to abnormal response to stimulation in certain women undergoing ivf treatment. the variants require the presence of wild-type receptor in order to initiate signaling in response to fsh. further analysis of this signaling cascade in granulosa cells is underway to estimate the final production of estrogen from these heterodimeric receptors. objective: to compare the proteome of human endometrium in the prereceptive versus the receptive phases of the menstrual cycle. m m: endometrial biopsies were collected and days after urinary lh surge in the same menstrual cycle from three fertile women (n= ). proteins were extracted using sample grinding kit (ge healthcare) and interfering substances removed using -d clean-up kit (ge healthcare). labelling was performed with cydye dige fluors (ge healthcare) and proteins were separated using difference gel electrophoresis (dige). for the isoelectric focusing, cm ipg-strips in the nonlinear range of ph - were used. the second dimension was carried out using sds-page. differentially expressed proteins were detected by image analysis using decyder v . and the statistical module eda (ge healthcare). the spots of interest were subject to protein identification based on in-gel digestion, maldi-tof/tof mass spectrometry and database searching. western blot analysis were performed in the same biopsies in order to validate some candidate proteins. results: table displays the differentially protein abundance between days lh+ and lh+ (> -fold change). of these proteins, were increased at lh+ in comparison with lh+ , whereas only proteins were decreased. stathmin was found more than -fold decrease in lh+ compared to lh+ in the three patients studied in the validation studies performed by western blot. conclusions: this study shows that the human endometrium has a differential protein repertoire during the window of implantation compared to the prereceptive phase. the role of these proteins in the molecular events directed to embryo implantation is under research. differential protein abundance in human endometrium ( the extracellular signal-regulated kinases and (erk / ) are a mitogen-activated protein kinase (mapk) subfamily that act as key links in eukaryotic intracellular signaling transduction. activated by phosphorylation in response to specific stimuli, erk / is known to play a role in the regulation of cellular proliferation and survival. the human myometrium is a tissue known to undergo cycle-dependent proliferative and apoptotic changes in response to sex steroids. we hypothesized that erk / activity is involved in mediating menstrual cycle-dependent changes in the myometrium. materials and methods: immunostaining for phospho-erk and total-erk was performed on myometrial tissues obtained from normal women (n= ) at different phases of the menstrual cycle. staining intensities were evaluated by hscore. myometrial smooth muscle cells were isolated and cultured from normal women and treated with vehicle, estrogen ( - m), and progesterone ( - m) for and minutes, and then subjected to western blot analysis for p-and t-erk. statistical analysis was performed using one-way anova. results: tissue staining revealed that p-erk was mostly nuclear in all tissues, while t-erk was cytoplasmic and nuclear. p-erk staining was significantly stronger in the secretory phase and strongest at the early secretory phase, compared to other phases (p< . ). t-erk staining intensity was moderatehigh without variation across the menstrual cycle. in cultured myometrial cells, progesterone significantly increased p-erk levels within and minutes (p< . ) when compared to control. our results suggest that erk activity in the human myometrium is regulated in a menstrual cycle-dependent manner. the increased phosphorylation in the secretory phase suggests the involvement of progesterone in erk activation, as supported by our in vitro results. this increased erk activity may play a role in regulating myometrial proliferation. measures of insulin resistance (ir) including: fasting serum insulin, gir and homa. measures of plasma levels of endothelin- (et- ), soluble intercellular adhesion molecule- (sicam- ), soluble vascular cell adhesion molecule- (svcam- ) and high sensitivity c-reactive proteins (hscrp). results: women with pcos exhibited significantly higher levels of et- (p < . ), sicamp- (p < . ), svcam- (p < . ) and hscrp (p < . ) as compared with age-matched controls, respectively. positive correlations were evident between et- and fai (r = . ; p < . ) but et- negatively correlated with shbg (r = - . ; p < . ). svcam- positively correlated with total t (r = . ; . ), hscrp correlated with: bmi (r = . ; p < . ), and homa (r = - . ; p < . ), respectively. conclusions: women with pcos exhibited abnormal levels of endothelial and inflammatory markers, which appear to be inter-related to hyperandrogenaemia. ( ), and a higher expression of fatty acid amide hydrolase (faah) in peripheral lymphocytes post-ovulation ( ), suggest an involvement of the endocannabinoid system in menstrual cycle control. our aims were to investigate changes in plasma aea levels and in endometrial faah expression throughout the menstrual cycle. methods: plasma aea levels were measured using a hplc ms/ms method from women, median age yrs (range - ) and bmi kg/m (range - ), with regular menstrual cycles, with no medical problem and not on any medication for the preceding months. the menstrual cycle was divided to early follicular d - (n= ), late follicular d - (n= ), ovulatory d - (n= ), early luteal d - (n- ) and late luteal phases d - (n= ). uterine biopsies were taken from hysterectomy specimens taken for benign conditions and subjected to immunohistochemistry for faah with polyclonal antibodies. results: aea levels were significantly higher around ovulation in comparison to the pre-ovulatory or post-ovulatory phases as shown in the figure. faah expression in the endometrial stroma was unchanged throughout the follicular phase but increased during the mid to late luteal phase reaching a maximum in the late luteal phase. a high aea level in the early follicular phase and during ovulation suggests a role for aea in ovulation. the lower levels of aea in the luteal phase, essential for successful implantation, may be regulated by increased faah expression at the uterine level. the modulation of plasma aea levels during the menstrual cycle strongly suggests that it is regulated by gonadal steroid hormones. ( , , , , , , and , as well as at pregnancy. binding activities of igfbp and their protein levels (igfbp , , , , , ) were assessed by western ligand blot (wlb) and western blot (wb), respectively. the glyscosylation and phosphorylation status of igfbps were examined by deglycosylation treatment. our results showed that both glycosylated and unglycosylated igfbp elevate in fetal and newborn rat and graduately decline to a lower level at day and kept lower constant level since then. interestingly, biding activity of glycosylated igfbp was not detected by wlb assay. the igfbp- cleaved products were observed after rat day age day , which associated with a decrease in full length of igfbp- , suggesting endoproteolytic processing may involved in decreasing igfbp content. igfbp- , with heterogeneous glycosylation, were appeared after age day and disappeared during pregnancy, recurrence again postpartum. glycosylation of igfbp has no effect on its binding activity. unglycosylated and glycosylated igfbp were constant in life time. physiologically constant igfbp were detected by wb in protein, but not by wlb in binging activity. conclusion: highly elevated circulation igfbp suggest physiological role in new born and early postnatal development. its binding activity are well regulated by its posttranslation modification, such as glycosylation of igfbp in inactivating binding activity and possible involvement of active endoproteolytic processing in maintaining binding active igfbp- at low background: cigarette smoking affects hormone biosynthesis, storage, release, binding, transport and clearance, resulting in changes in circulating hormone levels. we used metabolomics to analyze the effects of cigarette smoking and second hand smoke (shs) on changes in hormone levels in women of childbearing age. methods: this is a three arm study; women aged - years who are active smokers, exposed to shs (passive smokers), or non-exposed were recruited from the washington d.c. area. all women completed a detailed staffadministered questionnaire probing their medical history, occupational, lifestyle factors and diet. blood and urine samples were collected at the follicular phase. hormone profiles were determined using metabolomics for serum thyroxine and triiodothyronine and steroid hormones, as well as for cotinine using isotopedilution tandem mass spectrometry (lc/ms/ms-api ). in addition, all samples were analyzed for serum lh, fsh, tsh and creatinine. results: the relationships of cigarette smoking, age, relative weight, and dietary intake to serum thyroxine, triiodothyronine, estradiol (e ), estrone (e ), progesterone (p), -hydroxyprogesterone ( ohp),dehydroepiandrosterone (dhea), dehydroepiandrosterone sulfate (dheas), androstenedione, cortisol, -deoxycortisol, corticosterone, testosterone, aldosterone and vitamin d were analyzed using lc/ms/ms. mean tsh in non-exposed, shs-exposed and smokers: . , . , and . miu/ml respectively. similarly, mean t . , . ( % increase) (p= . ), . μg/dl (- %) in active smokers; (active vs. exposed p= . ). shs increased dhea levels ( % higher, p = . ), dheas ( % higher, p = . ), cortisol ( % lower, p = . ), aldosterone ( % lower, p = . ) and androstenedione ( % higher, p = . ). these data suggest that active smoking and shs can have a profound effect on serum t , adrenal steroid and sex hormone concentrations in women of childbearing age. objective: to determine the prevalence and predictors of the metabolic syndrome (mbs) among in saudi women with polycystic ovary syndrome (pcos) in comparison to women without pcos and to assess the role of androgens and insulin resistance (ir) in mbs development. design: a prospective case control study. setting: tertiary referral university hospital. subjects: six hundreds saudi women living in the jeddah area were classified as follows: with pcos and age-matched women without pcos. interventions: blood samples were collected from all women with or without pcos between : - : , after an overnight fast. main outcome measures: measures of abdominal obesity, blood pressure and that of serum levels of lh, fsh, tsh, ft , -ohp, -a, dheas, total t, free t, shbg, insulin, hdl-c, triglycerides and plasma levels of glucose. measures ir including: fasting serum insulin, gir and homa. results: age-adjusted prevalence of mbs was higher in women with pcos ( . %, % ci: . - . %) as compared with women without pcos ( . %, % ci: . - . %) (p< . ). in the same age group, the risk of mbs in women with pcos was greater than that for women without pcos (p< . ). markers of ir were significantly abnormal in women with both pcos and mbs in comparison to those without mbs (p< . ). the most common abnormal components of mbs in women with both pcos and mbs (after adjustment for age) were: decreased hdl-c ( . ± . %); increased triglycerides ( . ± . %); and increased bmi ( . ± . %), respectively. the prevalence of mbs from lowest to highest tertile of free t level was . , . and . %, respectively; in women with both pcos and mbs. in women with pcos, % exhibited all components of mbs; . % had components, and . % had components. conclusions: women with pcos exhibited significantly higher prevalence of mbs ( . -fold) as compared with age-matched control without pcos. ir is a possible common pathogenetic factor for both mbs and the pcos. it is suggested that more intensive screening and/or therapy monitoring of mbs among women with pcos should be part of the therapeutic modalities of the condition. case of sisters with complete androgen insensitivity syndrome and discordant mullerian remnants. jennifer l nichols, jennifer j gell, eric j bieber. reproductive endocrinology and infertility, geisinger medical center, danville, pa, usa. complete androgen insensitivity is an x-linked recessive disorder resulting in the abnormal expression of the androgen receptor. affected individuals are most commonly phenotypically female but genotypically male. the prevalence of this disorder is in , live male births. we present a case of complete androgen insensitivity in members of the same family with differing residual mullerian tissue. sister a presented at age for evaluation of primary amenorrhea. a karyotype revealed ,xy. an mri of the pelvis showed a hypoplastic uterus but no ovaries. this patient underwent laparoscopic bilateral gonadectomy and hemihysterectomy. on examination under anesthesia, she was noted to have a normal vagina with no cervix noted. at laparoscopic evaluation, she was noted to have bilateral elongated gonads and what appeared to be a remnant of uterine tissue. pathology revealed portions of immature testicles and fragments of smooth muscle in what grossly appeared to be the uterine remnant. the patient's total testosterone following surgery was noted to be elevated at . ng/ml. other laboratory evaluation showed fsh . miu/ml, lh . miu/ml, free testosterone . pg/ml, and estradiol . pg/ml. approximately two years later sister b presented at age for evaluation of primary amenorrhea. no uterus or ovaries were visualized on pelvic ultrasound. again a karyotype revealed ,xy. laboratory evaluation demonstrated fsh . miu/ml, lh . miu/ml, estradiol . pg/ml, total testosterone . ng/ml, and free testosterone . pg/ml. she underwent a laparoscopic bilateral gonadectomy. no uterus, cervix or pelvic masses were identified on exam under anesthesia. at laparoscopy, both gonads were visualized and removed without difficulty but no uterus was visualized. pathology reported two testicular and epididymal-like structures. this case demonstrates the presentation and laparoscopic results of complete androgen insensitivity syndrome discovered in two siblings. although both girls are genotypically male, they differ in the presence of vestigial mullerian tissue. the case shows with complete androgen insensitivity, as an x-linked defect, one should consider apparent sisters of affected individuals, as well as offspring of unaffected individuals with a family member diagnosed. background: anandamide (n-arachidonoylethanolamine, aea) is an endocannabinoid that binds to and activates the cannabinoid receptors, cb and cb and may have important roles in the regulation of human reproduction. the traditional lipid extraction methods used for aea are cumbersome, slow and of low efficiency. the aim of this study was to determine whether the use of a solid phase (spe) method of aea extraction from human plasma would offer any advantages over the traditional liquid phase (lpe) method. methods: pooled human plasma was obtained from the local blood transfusion unit and aliquots stored at - °c prior to extraction. an internal standard of . pmol of deuterated aea (aea-d ) was added to each plasma sample and aea extracted from aliquots on each occasion over three days using the lpe and spe methods. spe was performed with waters oasis hlb cc/ mg cartridges on a waters vacuum manifold. cartridges were activated with methanol and water, the samples applied and washed with % methanol at ml/min. aea was eluted with ml acetonitrile and the eluents dried under a stream of nitrogen before reconstitution in ml acetonitrile. aea levels were measured using uplc-ms/ms against authentic standards. results: these are shown in the table. conclusion: the spe method was -fold more efficient at extracting aea compared to the traditional lpe method. the intra-day and inter-day assay variability were similar for both techniques, although the spe method was quicker, cheaper and required less plasma to generate data similar to that from the traditional lpe method, suggesting that the spe method may be more efficient than the lpe method, and thus making it more suitable for routine analysis of multiple plasma aea samples. background: the precise role of the endocannabinoid, anandamide (narachidonoylethanolamine, aea) in reproduction has been hampered by difficulties in its accurate measurement. aea levels have previously been measured by tlc, gc-ms and hplc-ms but these are laborious. our aim was to improve the analysis of aea using uplc and tandem ms/ms with a standard isotope-dilution method , . methods: authentic non-labelled and deuterium-labelled aea (aea-d ) diluted in acetonitrile were maintained at °c before analysis and separation by uplc on a c ( . x mm) column maintained at °c using a gradient of % a, . min: % a, . min: % a, . min: % a, . min: % a with a flow rate of . ml/min. the mobile phases were a ( mm ammonium acetate, . % formic acid) and b (acetonitrile, . % formic acid). the analytes were quantified using multiple-reaction monitoring in positive ion mode with a quattro premier mass spectrometer. entry, collision and exit energies were - , and - ev, respectively. calibration curves were performed in triplicate with . pmol aea-d as the internal standard. transitions employed were . > . and . > . for aea and aea-d , respectively. results: calibration curves ( . to fmol aea on column; n= ) were linear, producing a mean (±sd) gradient of y = . ± . x, crossing the y-axis very close to the origin ( . ± . units). variability was limited, with an r = . . measurements were precise, fmol aea produced a cv of only . %, and the retention time was consistent at . ± . min (n= ). the limit of quantification (signal/noise> ) was . fmol on column and the limit of detection (lod) was . fmol on column (signal/noise= ). accuracy for . fmol, . fmol and fmol aea was . ± . %, . ± . % and . ± . %, respectively. conclusions: the method described is an improvement over other lc-ms/ms methods , with a lower lod [ . fmol v. fmol or fmol ] and shorter run time [ min v. min or . min ]. thus, an improvement in terms of speed, increased sensitivity and better reproducibility will allow for a more accurate assessment of aea concentrations in a number of biological samples. objectives: the gata family of transcription factors consists of six zinc-finger proteins with a critical role in tissue-specific and developmentally-regulated gene expression. gata factors exert their effects alone and through interactions with cofactors, such as friend of gata (fog), as well as with nuclear hormone receptors, including steroidogenic factor- (sf- ), a well-described activator of gonadotropin gene expression. the objective of these studies was to define the role of gata family members in the gonadotrope. methods: ) total rna was extracted from the gonadotrope cell line, l t , and analyzed by standard rt-pcr analysis. ) the cv- fibroblast cell line was transiently transfected by the calcium phosphate precipitation method with a rat - /+ lh promoterreporter vector as well as cmv-driven expression vectors for gata- , gata- , dngata- , fog- and/or sf- . results: gonadotrope l t cells were found to express transcripts encoding gata- , gata- , and gata- as well as fog- and fog- . gata- and gata- stimulated lh gene promoter activity by approximately -fold (p< . ) and synergistically increased the sf- response ( -fold versus -fold for sf- alone; p< . ). the gata-mediated increase in lh gene expression was nearly eliminated with co-transfection of fog- . similarly, co-transfection with a gata- dominant negative construct blunted wild-type gata- effects in a dose-dependent fashion. conclusions: these data demonstrate expression of both gata and the functionally related fog proteins in a well-characterized gonadotrope cell line. furthermore, they demonstrate a functional role for these factors in regulation of gonadotrope function, specifically expression of the lh gene. low-dose dexamethasone (dex) therapy early in pregnancy is used in fetuses with suspected risk of congenital adrenal hyperplasia. several adverse neurological events in prenatally treated children have been reported and the fetal hypothalamic-pituitary-adrenal (hpa) axis may be involved. aim: to investigate the immediate and long-term effects of early maternal dex administration on fetal growth and pituitary-adrenal activity in sheep. method: pregnant ewes carrying singleton fetuses (total n= ) were randomized to control ( ml saline/ewe) or dex treatment ( . mg/kg ewe weight) consisting of four intramuscular injections at -hourly intervals over hours on - days of gestation (dg). at , , , and dg fetal weights were recorded. i -ria, qrt-pcr and in-situ hybridisation were used to measure fetal plasma cortisol and acth levels and to analyse adrenal and pituitary mrna expression. results: dex-exposed fetuses were lighter than control animals at dg*, but not at other times; in general fetal organ weights were similar between treatment groups. fetal plasma acth was unaffected by dex and did not differ between genders. similarly, pomc and pc- mrna in pars distalis were unaltered after dex. however, fetal plasma cortisol was reduced after dex in both male and females at dg*, was similar at and dg, then elevated at dg*. plasma cortisol in female fetuses in control and after dex was significantly higher than in males. the increases in cortisol after dex at dg* were associated with increased fetal adrenal expression of p c and bhsd mrna in females, reduced expression of mc r in males, but no difference in star mrna. conclusion: we conclude that in sheep, early dex programs the developmental trajectory of the fetal hpa with increased activation directly of the adrenal, but not pars distalis function. in females this effect may be attributed to increased fetal adrenal steroidogenic activity. the effect of dex in increasing cortisol in males, albeit at a significantly lower level than in females, appears to be independent of the enzymes that we have measured.*p< . . synaptophysin and gonadotropin-releasing hormone (gnrh) are colocalized in rat hypothalamus. armando arroyo, beom su kim, amanda biehl, blenna cl bett, , john yeh. gynecology-obstetrics, university of buffalo, buffalo, ny, usa; physiology and biophysics, university at buffalo, buffalo, ny, usa. the cellular and molecular mechanisms that control gonadotropin-releasing hormone (gnrh) release are not completely understood. gnrh is stored in synaptic vesicles and released by exocytosis at gnrh nerve terminals. there are currently nine families of synaptic vesicle proteins that are involved in neurotransmitter release by exocytosis. synaptophysin is one of the most common synaptic vesicle proteins present in synaptic vesicles in neurons. the hypothesis of this study is that synaptophysin is expressed in gnrh neurons. we obtained sections from the hypothalamus of female sprague dawley rats. double-label fluorescence immunohistochemistry was performed on free-floating sections. sections were incubated with a mixture of mouse monoclonal antibody against gnrh ( : -chemicon international) and with a rabbit polyclonal antibody against synaptophysin ( : -santa cruz biotechnology) for h. after incubation the sections were washed and incubated with a mixture of alexa conjugated goat antimouse and alexa conjugated goat antirabbit ( : ; molecular probes) for h. slices were visualized with confocal microscopy (zeiss lsm- ). fifteen out of a total of fifteen gnrh cell bodies in the medial preoptic area and most gnrh neuron terminals in the median eminence showed intense immunostaining for synaptophysin. this is the first study to demonstrate that synaptophysin is expressed in rat gnrh neuron terminals. this suggests that synaptophysin is present in gnrh neuron vesicles. thus, gnrh release by exocytosis may be mediated by synaptic vesicle proteins. objective: our aim was to identify the effects of early gestation gc exposure on fetal and postnatal hpa axis development and function in postnatal life. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dex groups ( . mg/kg), consisting of four at h intervals on days - of pregnancy. at months postnatal age, catheters were implanted; a bolus injection of crh ( . mg/body weight) and avp ( . mg/body weight) were administered and arterial blood samples were taken at - , - , , , , , , , and min. levels of hepatic cbg mrna were determined by qpcr, expressed relative to s rrna. plasma cortisol and cbg levels were measured by radioimmunoassay. results: both total and free cortisol levels in the dex females (n= ) were lower than in dex males (n= ) from to min (p . ) and lower than in control females (n= ) at minutes (p . ), also in the dex-m group were higher than in control males (n= ) at min (p . ). plasma cbg levels and cbg mrna expression were not altered by dexamethasone exposure or sex. conclusions: these findings suggest that prenatal glucocorticoid exposure alters the development of the hpa axis differentially according to the sex of the exposed fetus. to determine maternal injections of synthetic glucocorticoids in early gestation can alter expression of hippocampal corticosteroid receptors at months postnatal age. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dexamethasone treatment ( . mg/kg) consisting of four injections at h intervals on days - . hippocampal was collected from the offspring at months postnatal age. levels of mrna of gr and mr were determined using qpcr and levels relatived to s rrna. results: dexamethasone-treated male animals (n= ) had significantly higher levels of mr gene expression than both control males (n= ; p= . ) and females (n= ; p= . ). gr gene expression levels were higher in treated vs. control males (p= . ), but in females levels in treated and control animals were similar. total body, brain and hippocampus weights were similar. conclusions: maternal dexamethasone administration in early pregnancy resulted in gender-dependent changes in hippocampal gene expression when measured in the offspring seven months after birth. objective: diminished ovarian reserve (dor) affects many younger women. we analyzed superovulation with intrauterine insemination (so) cycles in vitro fertilization (ivf) cycles of women with dor to determine if women < with dor differ from their older counterparts. method: irb approved retrospective review of so ivf cycles from / - / with follicle stimulating hormone (fsh) levels based on age < or . results: so cycles were performed in women. no differences were noted in clinical pregnancy. women < were more likely to have inseminates with a lower mean tms/ins, median tms/ins was . among pregnancy cycles compared to for unsuccessful cycles. for ivf, mean total gonadotropin dosage was significantly lower in women < , mean number of follicles mm peak e were significantly higher in women < . so ivf measures are in tables , respectively. conclusions: similar clinical pregnancy outcomes were seen despite age. in ivf, women < required less gonadotropins and generated more follicles but with no significant difference in number of mature oocytes or clinical pregnancies. of note, pregnancy rates for so in women < with dor are substantially lower than expected in our clinical practice. as ivf yielded a substantially higher chance of pregnancy, consideration should be made to expedite progression to ivf. to assess the effects of intraovarian injection of ad-fshr on the reproductive system of fshr (-/-) mice. methods: about l containing x pfu of ad-hfshr were injected directly into each ovary of treated group and same amount of ad-lacz were injected into the ovaries of control animals. vaginal smears were collected and body weight was measured daily. four weeks after the injection animals were sacrificed and all organs were weighted and evaluated by h e. fsh, e and p measured before and after treatment. results: ad-hfshrtreated mice showed obvious estrogenic changes in vaginal smear while in control animals vaginal smear remained at diestrus stage. significant increase in total body weight and estrogen dependent organs weight (uterus, ovary, vagina) was observed in treated animals compare to control group (p< . ). no significant weight changes were observed in other organs. h e evaluation of the ovaries showed significant increases in both the total number of follicles and the collective diameter of the follicles in treated animals compare to controls. on average follicles/ovary were observed in ad-hfshr-treated group of which follicles were at the antral stage while only follicles observed in ad-lacz control group, with zero follicles at antral stage. a . to folds increase in e and about % decrease of fsh observed in treated animals compared to control mice. there was no significant change in serum progesterone level between treated and control groups. conclusion: intraovarian injection of an adenovirus expressing human fshr gene is able to restore folliculogenesis and resume estrogen hormone production in female forko mice. objective: the sentinel issue surrounding multiple gestations following ivf is the inability to precisely estimate the reproductive potential of individual embryos with the currently used embryo grading systems based on embryo cleavage rate and morphology. recently, metabolomic profiling of spent culture media using raman and near-infrared spectroscopy have been reported to predict reproductive potential of embryos. in this study we applied proton nuclear magnetic resonance (¹h nmr) spectroscopy to analyze metabolomic profile of embryo culture media and to identify components of the media that correlate with reproductive potential. methods: eighteen spent media samples from embryos that failed to implant, and samples from embryos that resulted in pregnancy and delivery were individually collected after embryo transfer on day , and evaluated using ¹h nmr. the spectra obtained were analyzed using a selective genetic algorithm (ga) to determine regions predictive of pregnancy outcome as determined by logistic regression. to avoid random correlations, a leave-one out crossvalidation was used. sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. results: using the ga, two areas in the ¹h nmr spectral region were identified as most discriminatory between the two groups. viability indices calculated by ¹h nmr using these regions were significantly higher in culture media of embryos with proven reproductive potential ( . ± . ) compared to those that failed to implant ( . ± . ) (p< . ). compiled outcomes from the leave-one-out cross-validation of the logistic regression using the ¹h nmr measurements resulted in a sensitivity of % and a specificity of . %. quantification by integration showed significantly decreased glutamate levels (p= . ) and a trend toward an increase in pyruvate levels (p= . ) in culture media of embryos that did not cause pregnancy. conclusion: metabolomic profile of spent embryo culture media using ¹h nmr correlates with the reproductive potential of embryos. the lower glutamate levels detected in culture media of embryos that failed to implant could potentially be due to the toxicity associated with increased embryonic glutamate uptake. additional studies using complementary approaches are needed to further delineate molecular components associated with reproductive potential. objective: beta-carotene and other carotenoids are known antioxidants previously identified in human follicular fluid (ff). in addition to their inherent antioxidant properties, carotenoids have been identified as precursors of the antioxidant retinol in bovine ff. high retinol levels in bovine ff are associated with non-atretic follicles. this would suggest a possible role for retinol and its carotenoid precursors in follicular heath and the general oxidative state of the follicle. we sought to measure carotenoids and retinol in the ff of women undergoing ivf and correlate these levels with normal fertilization as a marker of follicular/oocyte health. design: prospective cohort study materials and methods: ff from a single - mm follicle was obtained from women (age - ) undergoing ivf and intracytoplasmic sperm injection (icsi). serum was also obtained at the time of oocyte retrieval. retinol, vitamin e ( , , and tocopherol) and carotenoids ( -carotene,cryptoxanthin, lycopene and lutein/zeaxanthin) were measured using hplc. we correlated ff carotenoid and retinol levels with oocyte fertilization status following icsi. results: as previously reported, retinol, vitamin e and carotenoids were all identified in the ff. each fat-soluble vitamin level was significantly lower in ff compared to serum (p< . for all analytes) and the levels were strongly correlated (r > . , p< . for all analytes). mean levels of ff -carotene were significantly higher in those follicles that resulted in a fertilized oocyte ( . +/- . g/ml vs. . +/- . g/ml, p= . ). other carotenoids, retinol, and vitamin e levels did not correlate with fertilization outcomes. conclusions: our finding of a strong association between ff -carotene concentration and subsequent normal fertilization of the oocytes suggests an important antioxidant role for -carotene in the health of the human ovarian follicle/oocyte. the lack of correlation with other carotenoids, retinol and vitamin e suggests that the antioxidant properties of -carotene act by preventing singlet oxygen and scavenging the peroxyl radical and may directly influence oocyte competence. mature oocytes obtained during egg retrieval have been shown to undergo spindle depolymerization as a result of cooling. for this reason, ivf programs strive to maintain constant temperature during follicle aspiration. we sought to elucidate if there was a difference in temperature of fluid aspirated into a hand held (hh) or heating block (hb) encased collection tube. the experiment was performed in an ambient temperature of °c. thermistors, pinhead sized sensors with an accuracy of %, were used to monitor temperature differences between control fluid (cf) (sterile water ºc) and aspirated fluid. data was recorded using an -channel data acquisition system from dataq instruments. one thermistor was placed into the cf, the other was placed into an empty collection tube set in the heating block or held in a gloved hand. a cm, gauge, single lumen needle connected to cm of tubing was used to aspirate into the empty collection tube. temperature was recorded x/second, each experiment was repeated times. baseline empty collection tube temperature was significantly cooler in the hh vs the hb group ( . ± . °c vs . ±. °c, p=. ). two seconds after aspiration, the lowest aspirate temperature was observed, (hh . ±. °c, hb . ±. °c, p>. ) no difference between groups. in both groups, temperature quickly increased as aspiration progressed (hh . ±. °c, hb . ±. °c, p>. ). the hb group took an average of . min to return to baseline ( °c), the hh group never returned to baseline. substantial cooling of aspirated fluid occurs during oocyte retrieval, with a mean temperature decrease of . ±. °c corresponding to . °c. considering this dramatic decrease, the difference between temperatures in the hh vs. the hb group is negligible. current aspiration systems poorly regulate temperature, thus the choice of aspirating into a test tube warmed by hand or by heating block is inconsequential. clinical management of twin gestations with recurrent preterm labor symptoms. lesley de la torre, luis roca, niki b istwan, debbie j rhea, gary j stanziano, victor hugo gonzalez-quintero. obstetrics and gynecology, university of miami medical center, miami, fl, usa; clinical research, matria healthcare, marietta, ga, usa. objective to examine pregnancy outcomes in women with twin pregnancies receiving nifedipine tocolysis (nt) who experienced recurrent preterm labor symptoms (rptlsx). study design twin pregnancies enrolled for outpatient preterm labor (ptl) surveillance services prescribed nt following an initial episode of ptl were identified from a database (n= ). eligible for inclusion were patients later hospitalized with acute rptlsx (n= ). included were those < weeks' gestational age (ga), with intact membranes, remaining undelivered for > hours after rptlsx . pregnancy outcomes of women resuming nt (rnt group, n= ) following hospitalization were compared to those having an alteration in treatment (alttx group, n= ) to continuous subcutaneous terbutaline. per normality assumptions, either independent student´s t or mann-whitney u test statistics were used for continuous variables; pearson´s chi-square for categoric. all p-values presented as two-sided, significant at < . . results overall, ( . %) of twin pregnancies prescribed nt experienced rptlsx; ( . %) were not eligible for continued tocolysis. pregnancy outcomes are presented in table. conclusion rptlsx are common. in twin pregnancies receiving nt, alteration of tocolytic treatment following rptlsx had a positive impact on pregnancy prolongation and neonatal outcomes. routine cervical dilatation during elective cesarean section -is it really necessary? arie koifman, avi harlev, eyal sheiner, fernanda press, arnon wiznitzer. obstetrics and gynecology, soroka university medical center, ben-gurion university of the negev, beer-sheva, israel. objective: the purpose of this study was to examine the necessity of routine cervical dilatation during elective cesarean section. material and methods a retrospective cohort study was performed, including all cases of elective cesarean sections performed at a tertiary medical center during . stratified analysis, using the mantel-haenszel technique, was done to control for confounders. results out of a total of elective cesarean deliveries (cd), underwent routine cervical dilatation. the overall rate of febrile morbidity was . %. no significant differences in postpartum febrile morbidity were noted between the groups ( . and . %; p= . ). in addition, hospitalization duration did not differ between the groups ( . ± . and . ± . days p= . ). about % of all elective operations were repeated cd. there was no difference in febrile morbidity between the groups even in that subgroup of the elective cd. likewise, there was no difference in anemia rate between the two groups (hemoglobin . ± . mg and . ± . mg p= . ). controlling for a previous vaginal delivery, using the mantel-haenszel technique, no significant association was noted between cervical dilatation and fever (weighted or= . ; % ci . - . ; p= . ). nevertheless, in a subgroup of patients following a previous vaginal delivery, cervical dilatation was significantly associated with post-operative fever (or= . the majority of women with an unfavorable cervix undergoing iol at term deliver vaginally, even with a prolonged first stage of labor. this is important information to discuss with women prior to iol when establishing labor expectations. providers should consider the ongoing success of labor induction when contemplating a diagnosis of "failed induction". objective: this study was performed to investigate and compare changes of the lipid peroxide levels and the protein carbonyls formation in the maternal venous plasma of preterm premature rupture of membrane (pprom) during antibiotics administration. materials and methods: thirty-six pregnant women with pprom between and weeks of gestation were chosen for this study. eighteen patients (group ) were treated with amoxicillin and erythromycin for day period while the other patients (group ) were treated with rd generation cephalosporin and erythromycin for the same period. samples of maternal blood were obtained from the two groups at before the antibiotics administration, day , and day after the antibiotics administration. lipid peroxide levels were measured by thiobarbituric acid reaction and protein carbonyl contents were determined by the , -dinitrophenylhydrazine method. results: . the lipid peroxide levels and protein carbonyls formation in the maternal venous plasma of pprom was significantly higher than that of normal pregnancy ( . ± . vs . ± . nmol/mg protein, p< . ), ( . ± . vs . ± . nmol/mg protein, p< . ). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the maternal venous plasma with pprom mixed and incubated by amoxicillin, cefodizime, and erythromycin (in vitro). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the venous plasma of group among before the antibiotics administration, day , and day after the antibiotics administration. . the protein carbonyls formation in the venous plasma of group was significantly decreased at day and day after the antibiotics administration than that of before the antibiotics administration ( . ± . , . ± . , . ± . nmol/mg protein, p< . ). conclusion: in the maternal venous plasma of pprom, the lipid peroxide levels and protein carbonyls formation were increased. the results suggest that reactive oxygen species formation by inflammatory reaction is suppressed by combined treatment of rd generation cephalosporin and erythromycin. objective: the aim of the present prospective cohort study was to evaluate the correlation between blood flow pulsatility index in fetal middle cerebral artery (mca) and the onset of spontaneous labor. study design: doppler evaluation of fetal mca were performed between and weeks gestation in consecutive pregnant women with a singleton pregnancy and known gestational age. the study population was divided according to the mca pi (< . or >/= . mom) and, using survival analysis and cox regression, the two subgroups obtained were compared. in these analyses, the event was delivery (following spontaneous labor) and the time variable was time elapsed since doppler exam. results: the median time elapsed between doppler evaluation and spontaneous labor was significantly shorter in the women with mca pi lower than . mom ( . days, interquartile range - ) in comparison with the women with mca pi higher or equal than . mom ( . days, interquartile - . days (p< . , mann-whitney test). after correction for birth weight and umbilical artery pi, survival analysis and cox regression confirmed that mca pi was independently associated with the number of days elapsed from doppler to spontaneous labor and delivery (p< . , exp(b) . , ci % . - . ). conclusions: the present data show that, at term of pregnancy, fetal cerebral resistance reduction anticipates the onset of spontaneous labor. novel calcium sensitizers and human uterine contractility. mark p hehir, audrey t moynihan, terry j smith, john j morrison. department of obstetrics and gynaecology, national university of ireland, galway, ireland. objective: the factors regulating contractility of uterine smooth muscle are central to the occurrence of preterm labour and delivery. calcium sensitizers are a novel class of drugs with unique molecular actions. levosimendan, the best characterized of these compounds, is used in the treatment of acute and chronic heart failure and is a compound which exerts a number of effects on smooth muscle. it can exert an inotropic effect via sensitization of myofilaments to calcium and also exerts a relaxant effect by opening atp-dependent potassium channels. for these reasons we hypothesized that levosimendan may have an effect on myometrial contractility and investigated its action on both spontaneous and agonist induced contractions. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. two sets of control experiments were performed simultaneously as follows: . strips exposed to either physiological salt solution (pss) only, for spontaneous contractions, or . nmol/l oxytocin; . strips exposed to pss/oxytocin and vehicle for levosimendan. results: levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, compared to control strips. the mean maximal inhibition values were as follows: . ± . % for spontaneous contractions (n= ; p< . ) and . ± . % for oxytocin-induced contractions (n= ; p< . ). no significant difference was found between control and control for both spontaneous and oxytocin induced contractions. conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on uterine contractility in vitro. this action was seen in both spontaneous and agonist induced contractions. the results from this study raise the possibility of calcium sensitizers holding potential tocolytic properties in vivo and further studies are required to investigate the potential benefits of this novel class of drugs. to determine to what degree extensive patient movement affects uterine emg signals and toco signals. study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. baseline recordings were obtained when patients were still, and these were used as control records. test recordings for both devices were obtained from all patients by asking the patients to perform movements. area under the rectified-voltage amplitude curve of the uterine emg signals and area under the curve for the toco signals were found. mean %-increase was calculated for the uterine emg and toco devices (each device %-increase values were averaged over all patients). mann-whitney rank-sum test was used to look for any statistical differences in %-increase in area for uterine emg vs. toco methods (p < . considered significant). results: there was a large increase in activity (artifact) on both devices' signals during patient movement (figure ) . both devices' traces eventually returned to baseline after the patient movement stopped. toco movement artifact was significantly higher than emg movement artifact ( table ) . conclusions: both uterine emg and toco signals experience artifact when patients move the uterine emg electrodes and toco pressure transducer, respectively. toco seems to be more adversely affected by such movements. uterine emg may be a preferred method for monitoring contractions of laboring patients in the clinic. supported by grant nih r -hd . results: % of obstetricians completed the questionnaire. none would counsel patients against labour unless there were contraindications. the majority would recommend labour for all indications for previous caesarean section investigated although in all instances, personal preferences were lower (p< . ); after a failed instrumental delivery, % obstetricians would recommend labour but only % would choose that option for themselves (p< . ). overall, female obstetricians would contemplate and recommend labour more readily than male obstetricians. labour augmentation and induction were recommended to patients more frequently ( % and % respectively) than chosen for personal care ( % and %). reluctance for labour augmentation and induction was greatest among younger consultants. conclusion: consultants have responded to consumerism and aim to meet the requests of their patients. they more readily recommend labour than they would choose for personal care, and a majority would recommend labour induction when necessary. informed patient choice is paramount rather than attaining a target figure for women attempting to labour, and the views of those requesting delivery by caesarean section should be respected. (table) . cd increased in nullipara singleton, cephalic, term pregnancy in spontaneous labor, mostly due to dystocia; nullipara singleton, cephalic term pregnancy with induced labor, mostly due to non-reassuring fetal status and dystocia; singleton, cephalic term pregnancy with previous cd, mostly due to elective cd; singleton cephalic preterm. no changes in neonatal outcome were observed. conclusion: clinical audit was useful to keep under control cd rate in our institution in comparison with italian reality, but it was not sufficient to maintain stable cd rate suggesting the need of other, multifaceted strategies. elective delivery at weeks' gestation is a common obstetric intervention. the purpose of this study is to estimate the maternal mortality rates (mmr) associated with this acog -approved intervention. there are no reliable us data describing mmr by mode of delivery. therefore, we base our estimates on british data indicating a procedure related mmr of . / , , . / , and . / , for vaginal, elective cesarean section (c/s) and emergency-unplanned c/s deliveries, respectively. a decision tree model was constructed assuming that all eligible patients (approx. , , /annually in the u.s.) would be delivered at weeks by either an elective c/s or an induction of labor. we further assumed that % of inductions would result in a vaginal delivery. our estimates show that the annual, delivery-related maternal mortality associated with an elective delivery of all patients at weeks would be and for elective c/s and induction of labor, respectively. because vaginal delivery results in the lowest mmr, we performed a oneway sensitivity analysis to identify the impact of changing the success rate of induction on the estimated mmr. the results indicate that once the success rate exceeds %, this intervention would be associated with a lower mmr as compared to elective c/s. our estimates indicate that although the overall mmr associated with elective delivery at weeks is relatively low (approximately . / , deliveries), it is certainly not negligible. in addition, the mmr is highly dependent on the likelihood of a successful vaginal delivery following induction of labor. when the success rate for induction of labor falls below %, an elective c/s appears to be the safer delivery method. background: the first obstetric visit is an opportunity to provide the pregnant patient information regarding substances that can cause potential harm to the pregnancy. little is known about how obstetric care providers handle these topics. objective: to examine patient-provider communication about substance use during the first prenatal visit. methods: we audiotaped first prenatal visits and qualitatively analyzed those tapes in which patients disclosed substance use. we invited patient participants to return for a semi structured interview during which they reviewed their audiotaped conversations and described their reactions to the providers' communication styles. results: providers ( residents, midwives, nurse practitioners) and patients participated. providers asked about smoking, alcohol and drug use in all ( %) visits. patients reported being smokers, reported alcohol use, and reported drug use. provider responses to smoking disclosures included brief discussions of smoking effects on pregnancy, encouragement to quit/cut down, and referral to smoking cessation programs. provider responses to alcohol or drug disclosures included only general statements regarding effects on pregnancy (e.g., "we find that this is bad for babies.") and referral to ultrasound/genetics for reassurance. few alcohol or drug discussions assessed whether the patient had intentions or concerns regarding continued use during the pregnancy. few discussions addressed strategies for behavioral change. none included assessment for motivations, readiness, or barriers to change. in follow up interviews, patient participants said they expected to be asked about substance use but advised providers to ask non-judgmentally. those who used alcohol/drugs wanted more information on potential effects of these substances on the pregnancy/fetus and appreciated the reassurance from referrals to ultrasound/genetics. discussion: counseling for risky behaviors in the first obstetric visits contained only limited discussion of the effects of the risky behaviors and primarily focused on referral-which may be a proxy for avoiding a difficult and time consuming conversations. background. there are conflicting results regarding the association of maternal psychiatric disorders or psychological distress due to stressful life events with pre-term birth and low birth weight. aims. to investigate the association between maternal psychiatric disorders and/or stressful life events and intrauterine growth abnormality, low birth weight or preterm birth. method. three mutually exclusive and homogeneous groups of pregnant women ( with actual psychiatric disorder, with stressful life events, and healthy comparisons) underwent serial fetal ultrasound examinations and uterine and umbilical artery doppler velocimetry at (+ ), (+ ) and (+ ) weeks of gestational age. subjects were recruited from all consecutive women attending two antenatal clinics. the presence of any maternal medical illness, drug treatments, fetal chromosomal and/or structural malformations, were exclusion criteria. all women recruited underwent a structured interview at - weeks for the psychiatric diagnosis (mini international neuropsychiatric interview -mini) (sheehan et al, ) ; moreover, the -item hamilton rating scale for depression and the hamilton rating scale for anxiety were included in the assessment. the person who obtained obstetrical clinical data was blind to the results of psychological evaluations. results. the three groups were comparable for: age, parity, socioeconomic status, smoking, alcohol consumption, body mass index. gestational age at birth was not different in the three groups. infants of women with psychiatric disorders had significantly lower birthweight ( + g) and higher percentage of birth weight below the th centile for gestational age ( %) than infants of healthy mothers ( + g and %, respectively). there was also a trend towards lower mean birth weight ( + g) and higher incidence of birth weight below the th centile ( %) in the stressful life event group. there was no significant difference among groups in the percentage of abnormal uterine or umbilical doppler results. conclusions. maternal psychiatric disorders are associated with a lower birth-weight, but the effect is unlikely to be due to abnormal utero-placental or feto-placental vascularisation. objective: the purpose of this study was to evaluate antenatal ultrasound as a tool for the detection of intrauterine growth restriction (iugr) and small for gestational age (sga) infants among subjects with elevated human chorionic gonadotropin (hcg) levels on second trimester serum screening. although iugr has been linked to elevated hcg levels, the optimal screening regimen for antenatal sonographic surveillance has not been previously established. methods: a retrospective cohort study was performed at saddleback memorial medical center where serial ultrasounds from weeks-delivery are generally recommended for patients with hcg levels > . mom. all pregnancies were dated by second trimester ultrasound ± last menstrual period. subjects with an hcg > . mom, who had at least one antenatal ultrasound evaluation for iugr, were identified from an electronic ultrasound database used for clinical report generation. ultrasound data were then linked to an obstetrical birth outcomes database for relevant demographic/delivery information using unique identifiers. iugr was defined as a sonographic estimated fetal weight (efw) < %ile for the estimated gestational age (ega). sga was defined as an actual birthweight < %ile for the ega at the time of delivery. results: from - , there were subjects with elevated hcg levels who underwent antenatal ultrasound surveillance for iugr and who had known delivery information. a total of ultrasound examinations were performed. the median number of examinations per subject was with a range from - examinations per subject. the incidence of iugr and of sga were . % (n= / ) and . % (n= / ), respectively. no fetus with iugr demonstrated absent or reverse end diastolic umbilical artery doppler flow. antenatal ultrasound examinations only identified . % (n= / ) of sga infants. however, the sensitivity for the detection of sga was % when an efw cut-off of % was used. conclusions: although the majority of sga infants did not demonstrate growth restriction on antenatal ultrasound, a sonographic efw > %ile appears to be a safe cut-off to rule out fetuses at risk for sga. neonatal dry lung syndrome after pprom: reason to change intrauterine referral practice in the netherlands? ellen s hoogakker, christiaan v hulzebos, gerda g zeeman. obstetrics and gynecology, university medical center groningen, groningen, netherlands; pediatrics, division of neonatology, university medical center groningen, groningen, netherlands. background: neonatal dry lung syndrome (dls) is a distinct clinical entity following pprom, mimicking pulmonary hypoplasia but with dramatic respiratory improvement during the first - h . its pathogenesis implies complete collapse of small airways to a degree that capillary forces impede distension by ordinary ventilatory pressures. in the netherlands, women with pprom remain admitted at a level iii nicu perinatal center until they reach wks. when they are referred back to their community hospital, unless they live in the neighborhood of the tertiary center. we question this referral pattern because we still see severe respiratory problems occur > wks. we sought to determine the prevalence of such morbidity, in particular dls, in women with pprom who deliver > wks. methods: retrospective descriptive study of neonatal outcome data of singleton pregnancies complicated by pprom between - wks, who deliver > wks (latency > h) , during the -yr period of - at a single academic center. data were extracted from medical records and electronic department databases. results: pprom pregnancies were identified. all but received at least full course of steroids. ( %) delivered > wks. newborns born at the community hospital needed emergency transportation to a level iii nicu for respiratory morbidity. neonatal outcome data (means ± sd) are listed in the table. there were no cases of late onset sepsis, nec or perinatal mortality. conclusions: respiratory morbidity still occurs after pprom with delivery > wks. further investigation of pregnancy-related characteristics, such as the presence of anhydramnios and the latency period, with regards to dls is needed. modification of current referral practice depends upon complete data derived from all dutch level iii perinatal care centers (n = ). tertiary care center (n = ) to determine if an association exists between macrosomia (birthweight > g) and perinatal outcomes in women with and without gestational diabetes mellitus (gdm). study design: this is a retrospective cohort study of , singleton pregnancies. the study cohort was stratified by the diagnosis of gdm, with the presence or absence of macrosomia as the dependent variable. perinatal outcomes examined included neonatal hyperbilirubinemia, hypoglycemia, respiratory distress syndrome (rds), shoulder dystocia and erb's palsy. chisquare tests were performed as well as multivariable analyses controlling for confounders, using p< . to indicate statistical significance. results: in women diagnosed with gdm, macrosomia is associated with a higher frequency of hypoglycemia, respiratory distress syndrome, shoulder dystocia and erb's palsy. though the prevalence of these outcomes is relatively decreased in patients without gdm, they are still more prevalent in macrosomic patients. macrosomia is associated with a higher prevalence of adverse perinatal outcomes in women with and without gdm. therefore, it is important to evaluate neonates with birthweights greater than grams for hypoglycemia and unrecognized erb's palsy. conclusion a significant proportion of our obstetrical patients are obese and many do not perceive themselves to be obese. while our finding of an inverse correlation between level of education and bmi may be confounded by socioeconomic status, our results suggest that in order to address the problems of obesity in our population an important first step will be improving the education of our reproductive age women regarding normal weight gain and nutrition in pregnancy. this may have a significant impact on improving pregnancy outcomes of today's obstetrical population. objective: the aim of the study was to evaluate the impact of a "flat" oral gct upon the outcome of pregnancy. the gct was considered flat when the difference between the basal value and the after load value was %. methods: we prospectively analyzed the outcomes of pregnancies who delivered at our department. inclusion criteria were singleton pregnancy; bmi < kg/m , absence of major risk factors for diabetes and of pregestational diabetes. g -hour oral gct was performed at - weeks of gestation. women were subdivided into groups according to the result of the gct: group = negative (load glucose > % and < mg/dl) women ( . %); group =flat (load glucose % than basal and < mg/dl) women ( . %); group =positive gct/negative ogtt, women ( . %). data are mean ± sd. differences were calculated with the student t test for unpaired samples and test. regression analysis were performed by the least squared method. p-values were considered significant at p< . . results: the characteristics and obstetric outcomes in the three groups are presented in the table . in all patients there was a significant linear relationship between the load and basal glucose values (load value= . + . basal value; r= . ; p< . ) and between birthweight and load values (birthweight= . + . load value; r= . ; p< . ). the relationships were significant also in group and separately but not in group . in this preliminary study we found no major outcome differences in women with flat gct compared to women with normal and gct positive/ ogtt negative results. it seems important, however, to futher investigate the meaning of a flat curve in a bigger population and/or by means of metabolic studies with the use of stable isotopes. results characteristic of the study population and obstetric outcomes are presented in table . the probability to be primiparous and to deliver babies < ° centile decreased significantly with bmi whereas the risk of cesarean section, of post partum hemorrhage at vaginal delivery and to deliver babies > ° centile increased significantly. also the risk to develop preeclampsia and gestational diabetes was increased, although not significantly, with bmi. compared to lean and normal, obese were more likely to be hypertensive and diabetic before pregnancy (p< . ) and to start pregnancy without any medical and obstetrical risk but obesity ( . % vs . and . %; p< . ). african women exhibited the highest bmi and the highest rate of obesity (table ). conclusions we confirm that obese women have an increased risk of prepregnancy and pregnancy complications: less than % have an uncomplicated pregnancy. at delivery there is an increased risk of cesarean section and post partum haemorrhage. objective: overweight and obese women often give birth to larger babies, which is associated with traumatic birth injuries and an increased risk to develop obesity, diabetes and hypertension in childhood and later in life. the mechanisms underlying fetal overgrowth in obese pregnant women are largely unknown. the aim of this study was to establish a mouse model of obesity/high fat diet in pregnancy. we hypothesized that a moderately high fat diet prior to and during pregnancy would result in increased maternal adiposity, fetal overgrowth and a metabolic profile similar to that of obese newborn offspring with persistent pulmonary hypertension, despite enhanced pregnant women. method: c bl/ j female mice were fed control (c, % of energy from fat) or isocaloric high fat (hf, % of energy from fat) diets ad libitum for weeks prior to mating and during gestation. at gestational day . maternal blood samples were obtained to measure adiponectin, leptin and cytokine levels and maternal fat pads were isolated and weighed. in a sub set of animals measurements of transplacental transport of neutral amino acids and glucose were performed in vivo under ketamine anesthesia using h-meaib, and c-glucose. results: no significant differences were observed in maternal pre-pregnancy bodyweight, total caloric intake, weight of the dam at e . or litter size between treatment groups. fetal weight was increased in the hf group by % (p< . ). maternal adiponectin levels were significantly (p< . , n= ) decreased (hf ± , c ± g/ml) and leptin levels increased by % in animals fed a high fat diet, but this difference did not reach statistical significance (n= ). adiposity (fat pad weight) was increased by % (p< . , n= in the dams fed high fat diet, however no difference was observed in maternal il- levels and neither group had measurable levels of tnf-. maternal red blood cell lipid profiles were altered in high fat animals with an increase in stearic and linoleic acids but decreased oleic acid levels. preliminary data showed that placental uptake and transfer to the fetus of glucose and meaib were increased by at least % in dams fed high fat diet. conclusion: this murine high fat diet model has several features consistent with human obesity in pregnancy and the maternal metabolic environment is similar to that seen in the human. the increase in placental uptake and transfer of nutrients constitute a key mechanism underlying fetal overgrowth in overweight/obesity in pregnancy. objective: epidemiological studies have demonstrated a positive relationship between maternal overnutrition and the development of the metabolic syndrome in the offspring. we previously reported that lambs of well fed ewes have increased plasma glucose levels in early life, this may be a consequence of altered hepatic glucose production. increased hsd expression is associated with an increase in intracellular cortisol, promotion of hepatic insulin resistance and a consequential increase in gluconeogenic activity. hypothesis: we hypothesised that maternal overnutrition in late gestation in the sheep results in increased hepatic expression of hsd in the lamb before and after birth. methods: ewes were provided with either % (control,c) or % (well fed, wf) of maintenance energy requirements from d gestation until delivery. post-mortem was performed on either ± d gestation (c= ,wf= ) or and postnatal day (c= ,wf= ). plasma glucose and leptin concentrations in the fetuses and lambs were determined. the relative hepatic mrna expression of hsd and reference gene rpp were determined by qrt-pcr. results: relative liver weight was significantly higher in lambs of wf ewes compared to c at d (p< . ). the expression of hsd mrna was significantly higher in the postnatal compared to the fetal lamb (p< . ) independent of maternal nutritional treatment. however, there was no effect of maternal overnutrition on the hepatic expression of hsd mrna before or after birth. there was no effect of prenatal nutrition on fetal or postnatal plasma cortisol concentrations. the expression of hsd in the liver of lambs of wf, but not c ewes, was inversely related to plasma glucose concentrations in the first hrs after birth (r =- . , p= . ). we have therefore demonstrated that exposure to prenatal overnutrition results in an inverse relationship between hsd mrna expression in the liver at d and plasma glucose concentrations on the first day of life. this suggests that exposure to high glucose levels before and immediately after birth results in a reduced expression of hepatic hsd . we would expect a reduction, rather than promotion of intra-hepatic cortisol production, and therefore it is unlikely to explain the hyperglycemia present in lambs of wf ewes in early life. ninety-seven ( %) of mothers were classified as obese (bmi> ) based on their first pregnancy weight. glycemic control at weeks was superior in the non-obese group (table ) . there were no differences in glycemic control during the last week of pregnancy. obesity was significantly associated with increased maternal weight gain during pregnancy. mean birth weights, ponderal indices and rates of macrosomia were significantly higher in infants born to obese women when compared to non-obese (table ) . primary cesarean deliveries rates were comparable. the rate of neonatal hypoglycemia, hyperbilirubinemia, phototherapy and neonatal icu admissions did not differ between obese and non-obese diabetic women (table ) . although not statistically significant, there was a trend towards an increased rate of birth injuries in the obese group. a similar comparison between obese and non-obese women treated with medication (glyburide or insulin) demonstrated a higher mean birth weight ( g+ vs. g+ , p=. ) and higher rate of macrosomia ( vs. %, p= . ). there were no differences in glycemic control, cesarean delivery rates and other neonatal outcomes between the obese and non-obese treated with medication. conclusion: obese women with type ii and gdm give birth to larger infants than their non-obese counterparts and have a higher incidence of fetal macrosomia. there was a trend towards increased rate of birth injuries in the obese group. despite these differences other maternal and neonatal outcomes were similar which may be a reflection of glycemic control. introduction: tlrs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. the placenta, and more specifically the trophoblast, may play an important role in the response to infection. previously, we described the expression of tlr- by human trophoblast and their ability to respond to polyinosinic-polycytidylic acid (polyi:c), a synthetic double strand rna which mimics viral rna. in the present study we evaluate the effect of polyi:c in mouse pregnancy and characterize the local and systemic response. material and methods: human first trimester trophoblast cell line, htr , was treated with polyi:c. c b/ wild type and tlr- knock out mice were injected intraperitoneally with polyi:c at . gestation day. cytokine and chemokine level were determined in supernatant and lysates using the bio-rad multiplex assay and analysis was done using the bio-plex is. results: polyi:c induced cytokine (il , il and il ) and chemokine (il , rantes, gro , mcp and mip ) secretion and production by human cultured trophoblast in a time ( - h) and a dose ( - g/ml) dependent manner. injection of polyi:c to c b/ wild type mice induced preterm delivery within h at a dose of mg/kg body weight. no effect was observed in tlr- knock out mice. a robust systemic (spleen and serum) and local (placenta and amniotic fluid) inflammatory response was observed and h following polyi:c treatment. trophoblast cell cultures from tlr- ko mice confirmed that the response to polyi:c is tlr- dependent. conclusion: we demonstrate that viral infection may trigger an immune response leading to preterm labor. furthermore we show that the trophoblast is able to recognize and respond to viruses through the expression of tlr- . our findings provide a novel mechanism of pathogenesis of preterm labor associated with tlr- mediated inflammatory response. introduction: adverse neurological outcome is a major cause of neonatal morbidity after a preterm birth (ptb). a growing body of evidence demonstrates the involvement of inflammatory pathways in ptb and implicates these pathways as a causative factor in fetal brain injury. however, activations of cytokine pathways in normal labor (whether iatrogenic preterm or at term) has been observed. what remains understudied is the effect of labor on the preterm fetal brain and whether an inflammatory stimulus is essential for fetal brain injury. methods: mouse models were utilized: ( ) a model of intrauterine inflammation (lps into uterine horn) (n= ); controls received intrauterine saline (n= ) and ( ) autism is a neurodevelopmental disorder with a strong genetic component and several known environmental risk factors, such as infection. in addition, its onset of etiology is likely to occur during prenatal development. we propose that subjecting fetal sheep via amniocentesis to the bacterial endotoxin lipopolysaccharide (lps) injected to the amniotic fluid at gestational day (gd) will result in morphological alterations in the offsprings' cerebellum resembling alterations found in the cerebellum of patients with autism. using high precision design-based stereology, we investigated mean total-and layer-specific volume and mean total granule and purkinje cell (pc) number in the cerebellum of lps infected animals and controls. the results of the present study showed preserved volumes of the total cerebellum as well as of the molecular layer, outer and inner granular cell layers and white matter. interestingly, compared to controls, the lps infected brains showed a statistically significant increase (+ . %) in the mean total number of granule cells, whereas the pcs did not show any difference between the groups. these seemingly paradoxical results might be explained by ( ) the so-called time of origin of these neurons, i.e. the pcs develop prenatally whereas the granule cells develop postnatally or ( ) the direct correlation between pcs and granule cell number in the cerebellum. these results might contribute, as an animal model, to our understanding of the biological basis for interindividual differences in morphological alterations found in the brains of patients with autism. the previous studies have shown that there is a higher incidence of spontaneous preterm birth and poorer neonatal outcome in pregnancies with a male fetus. in vitro studies also report a sex dependent pattern in different placental enzymatic systems. we have shown previously that lactobacillus rhamnosus gr- supernatant is able to antagonize the actions of lps on cytokines and ptgs in placental trophoblasts. we hypothesize that fetal sex will influence the production of cytokines and prostaglandin regulating enzymes in lps and lactobacilli treated placental trophoblast cells. methods: term placentae were collected from women undergoing elective caesarean section. placental trophoblasts were isolated using established primary culture protocols. cells were pretreated with lactobacilli supernatant and subsequently treated with lps. pgdh and ptgs expression levels were measured by western blot analysis and tnf-, and il- concentrations measured by elisa. results: lps stimulation caused a marked increase in production of tnf-( . pg/ml to . pg/ml, n= , p< . ), an effect that was greater in placentae of the male fetuses ( . pg/ml, n= ) compared to female fetuses ( . pg/ml, n= ). lactobacilli supernatant abolished this response in both sexes. lps-activated trophoblasts from placentae of the male fetuses showed an increase in il- production (n= , p< . ) and ptgs expression (n= , p< . ). however, there was no response to lps in placentae of the female fetuses. lactobacilli supernatant up-regulated pgdh (n= ) by %, and this effect was greater in placentae of the female fetuses (n= ). conclusion: we conclude that human placentae from pregnancies carrying male fetuses are more responsive to lps by producing more pro-and anti-inflammatory cytokines, as well as ptgs . conversely, placentae of the female fetuses upregulate pgdh with lactobacilli treatment. these findings may explain the underlying mechanism for the higher incidence of preterm birth and adverse pregnancy outcomes seen with male fetuses in the clinical setting. , ) . this study investigates whether iai is associated also with altered expression of neuropilin- . methods: (i) immunohistochemistry (ihc) was performed on tissue sections of term decidua with or without clinical / histologic evidence of iai (n= for each). neuropilin- expression was scored by an investigator blinded to the identity of the samples. (ii) cultured term dscs were retrieved from elective cesarean (n= ), purified, and depleted of leukocytes. after treatment with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days, dscs were stimulated with il- b ( - ng/ml), tnfa ( ng/ ml), or thrombin ( . iu/ml) for h. since no elisa exists for neuropilin- , protein expression was determined by immunocytochemistry (icc). (iii) total rna was extracted and the effect of il- b on neuropilin- mrna expression measured by real-time rt-qpcr and corrected for b-actin mrna. results: neuropilin- expression in term decidua was increased in tissues with iai vs controls (p< . ), and localized primarily to dscs. using icc, an increase in neuropilin- was noted after stimulation with il- b and tnfa, but not thrombin. il- b increased neuropilin- mrna expression in dscs by . ± . -fold (from . ± . to . ± . neuropilin- mrna/b-actin mrna; p= . ). conclusions: iai is associated with increased expression of the vegf receptor, neuropilin- , in term decidual tissues. il- b and tnfa (but not thrombin) stimulated neuropilin- expression in term dscs, and this effect appears to be mediated at the level of gene transcription. since aberrant vegf function alters vascular permeability, these data provide a mechanism by which iai can promote 'decidual activation' and preterm labor. inflammation, university of glasgow, glasgow, united kingdom. objective: asthma is associated with inappropriate activation of airway smooth muscle, chemokine expression and accumulation of mast cells which drive smooth muscle reactivity. labour is similarly associated with smooth muscle activation and expression of cxcr and cxcr ligands. the role of mast cells in human parturition is unknown; however, mast cell products can stimulate myometrial contractions and preterm labour in animal models. we have quantified mast cells in association with human labour and determined whether they express cxcr and cxcr . methods: lower segment myometrial and cervical biopsies were taken at term caesarean section from women not in labour (nil) (myometrium n= ; cervix n= ) and in labour (il) (myometrium n= ; cervix n= ). mast cells were localised in myometrial and cervical sections by icc with a primary antibody against c-kit. the number of cell transects in randomly selected high-powered fields ( x) was quantified blindly by two observers for each specimen, with median density and interquartile range (iqr) calculated. backto-back icc was performed to determine whether c-kit co-localised with the chemokine receptors cxcr and cxcr . results: mast cells were in close association with myometrial smooth muscle in non-labouring lower segment myometrium. labour was associated with a significant influx and increase in mast cells numbers (nil median . , iqr . - . ; il median . , iqr . - . , p= . ). in contrast no significant increase in mast cells was observed in cervical tissue in association with labour (nil median . , iqr . - . ; il median . iqr . - . , p= . ). analysis of chemokine receptor expression demonstrated co-localisation of cxcr to c-kit positive cells present within the myometrium. conclusions: human labour at term is associated with an increase in mast cells within the myometrium, with close approximation to smooth muscle bundles. these mast cells express the chemokine receptor cxcr , the ligands of which we have previously shown to be up-regulated in labouring myometrium. mast cells are not accumulated in cervix in association with labour suggesting a less critical role in cervical ripening. further analysis of the role of mast cells in modulating myometrial smooth muscle physiology is warranted. progestins and the glucocorticoid receptor in human myometrial and amnion-derived wish cells. alison j tyson-capper, stephen c robson. reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. background and objectives: progesterone (p) can reduce the risk of preterm birth in some high risk women. in this context there is accumulating evidence that this, at least in part, may be due to anti-inflammatory and immunoregulatory properties of p. target tissue responsiveness to p is considered to be determined by the progesterone receptors (pr) and nuclear co-factors that directly interact with pr. pr and glucocorticoid receptors (gr) share several structural and functional characteristics, including similarities in dna sequence recognition by binding to the same hormone response elements. pr and gr interact with similar chaperones in the absence of ligand and with a similar group of co-activators in the presence of hormones; both can display comparable anti-inflammatory activities under specific physiological conditions. in this study we aimed to investigate whether the anti-inflammatory properties of progestins may be mediated by pr and gr signalling. methods: primary cultures of non-pregnant and term pregnant human myometrial (passage - ) and wish cells were serum starved for hrs and treated with -hydroxyprogesterone ( -hp), progesterone (p), dexamethasone (dex) and immunofluorescent staining and immunoblotting analyses performed. in some experiments cells were pre-treated with ru (a pr/gr antagonist) or org (a pure pr antagonist). results: in the absence of hormone gr appeared to be predominantly cytoplasmic, whereas, upon treatment with -hp and p ( and m) and dex ( nm) gr was abundant within the nuclei of myometrial cells. immunoblotting analyses demonstrated that levels of gr progressively increased within the nuclear fractions of both pregnant myometrial and wish cells in response to increasing concentrations of p ( nm to m), and decreased sequentially within cytoplasmic fractions. in the presence of org gr protein levels remained constant within the cytoplasm. there also appeared to be a slight increase in gr expression, though not statistically significant (p> . ) within both cells types in response to p. conclusion: in this study we show that gr is activated by -hp and p and translocates to the nuclei of human myometrial and amnion-derived cells. in addition, levels of gr increase in response to p. whether p and -hp act as agonists or antagonists for gr in the regulation of hormone response genes associated with the onset of term and preterm labour remains to be elucidated. objective: using a mouse model of inflammation-induced preterm birth (ptb), we have demonstrated dramatic cytokine elevations in the uterus and placenta with concomitant, though less dramatic, cytokine elevations in the fetal liver and brain, associated with neuronal injury. because precise mechanisms of fetal injury in ptb remain unclear, we sought to examine inflammatory cell trafficking, and target organ damage by histopathologic assessment of the placenta, fetus, and fetal brain. study design: hours after intrauterine infusion of saline or lps into the right lower uterine horn of cd- mice, the left upper horn, with the gestational sacs(gs) in situ, was removed en bloc(n= per group) each with - gs with fetuses/treatment group. specimens were fixed, bisected and processed for histology and ihc. inflammatory and hematopoietic cells were quantified using pas, gata- (erythroid precursors), cd , and bm (macrophage-mp) within the placenta, liver, extremity mesenchyme, brain and leptomeninges. the presence of hemorrhage, necrosis, and apoptosis (h ax stain) was assessed. erythopoietin (epo) levels were measured in brain and liver by elisa. results: more neutrophils were present in maternal decidual vessels in lps compared to saline (p= . ). in lps-exposed, fetal mp were increased in the placenta (p= . ), fetal extremity mesenchyme (p= . ), fetal liver (p= . ) and leptomeninges (p= . ) but not in the brain or spinal cord compared to saline. no necrosis, hemorrhage or increased apoptosis was noted in the fetal brains. % of lps-exposed fetuses and % of saline-exposed had liver hemorrhages (p< . ). increases in nucleated erythrocytes and erythroid precursors were found in fetal vasculature of the placenta in lps-exposed (p= . ). epo levels were not elevated in either group. conclusion: intrauterine lps infusion induces acute inflammation predominantly in the maternal circulation of the placenta. in the fetus, there is widespread mp activation, liver hemorrhage and increased erythroid precursors seen in the fetal circulation of the placenta. although histologic evidence of cns damage was not evident, the increased mps present in the leptomeninges may play an important role in inflammatory-mediated cns damage. non-toll-like innate immune proteins: do they change during pregnancy? juan m gonzalez, hua xu, ella ofori, michal a elovitz. obgyn; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: trem- (trigger receptors expressed on myeloid cells) is an important regulator of innate immunity. the natural ligand for trem has not been identified. activation of trem- in the presence of toll-like receptors results in substantial amplification of the host inflammatory response (klesney-tait et al nature immunology ). since inflammatory pathways are implicated in adverse pregnancy outcomes, this novel mediator of inflammation may play a critical role in preterm birth (ptb). therefore, we sought to determine trem- expression in the uterus, cervix, and placenta across gestation and to determine if trem- levels are altered by intrauterine inflammation. methods: in cd- mice, trem- was investigated in non-pregnant (np) and throughout gestation e , e (n= - per group). uterine, cervical, and placental tissues were harvested. using an established mouse model of inflammation-induced ptb, uterine tissue was collected hours after intrauterine infusion of saline (n= ) or lipopolysaccharide (lps) (n= ). for a non-pregnant model, using cd- mice, lps (n= ) or saline (n= ) was injected into the uterine horn following same procedures as with pregnant mice. uteri were harvested hrs later. quantikine ® mouse trem- immunoassay was utilized for these studies. statistical analysis was performed using oneway anova followed by pair-wise comparison if statistical significance was reached (p< . ) results: trem levels are significantly different between np and pregnant uterine tissues (p= . ). e and e trem expression is significantly increased . and . -fold compared to np (p= . and . respectively). trem- levels in the placenta and cervix were not significantly different between e and e . trem levels increased about -fold in the uterus after intrauterine infusion of saline or lps compared to e controls. in nonpregnant, trem levels were significantly different (p= . ) with a -fold increase in trem expression in uteri exposed to lps or saline compared to controls. conclusions: non-toll-like innate immune proteins are differentially regulated during pregnancy compared to the non-pregnant state. the role of trem- in inflammation-induced ptb requires further study. research is warranted to determine if uterine up-regulation of trem in gestation is associated with an increased likelihood of responding to pathogens or severe as a protective mechanism. reduced plasma levels of vitamin d in caucasian women at term are associated with increased rate of infection. chander p arora, adegoke adeniji, susan e jackman, babak forooghi, isaac mostadim, phillip yadegari, calvin j hobel. og-gyn, cedars-siani medical center, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: vitamin d plays an important role in human pregnancy by acting as a regulator of immunity at the fetal-maternal interface. inflammatory changes associated with pro-inflammatory cytokines were reduced by vitamin d while anti-inflammatory cytokines were increased in t lymphocytes. vitamin d status has been defined as deficiency (< . nmol/l), insufficiency ( . to nmol/l) and sufficiency (> nmol/l) . objective: to assess the involvement of vitamin d in the occurrence of maternal infection during pregnacy in women with term deliveries. hypothesis: vitamin d metabolism could affect the rate of infection during pregnancy. study design plasma levels of (oh)d were determined by elisa and the rate of infection was recorded in a behavior in pregnancy study. in this study, ethnically diverse women were evaluated at - weeks (t ), - weeks (t ) and - weeks (t ). maternal infections were documented at each stage as well as at baseline visit with history of infection in current pregnancy. of these subjects who delivered at term two groups ( with no infection during pregnancy, with infection or history of infection) were matched further for non-smoking status, non-diabetics, ethnicity and maternal age. plasma from these were assayed for (oh)d and analyzed for the rate of maternal infection using fisher´s exact test or chi-square test. results although the women delivered at term, the levels of (oh)d in caucasians were significantly lower in the subjects with infection than the ones without (p<. ). women with vitamin d insufficiecy in the first trimester were more likely to develop infection during pregnancy ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ) but not subjects with sufficient vitamin d at t ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ). conclusion the results reveal a positive association between (oh) d concentrations and greater risk of infection. vitamin d deficiency or even insufficiency may, therefore be involved in the pathogenesis of maternal infection during pregnancy. it is probable that vitamin d deficiency or even insufficiency could modulate the maternal susceptibility to infection during pregnancy by a proinflammatory mechanism. in vitro and in vivo observational data suggest that infection leads to caspase activation and apoptosis in the placenta and membranes, however currently there are no data on the role of apoptosis in the pathogenesis of infection associated preterm delivery. here we used group b streptococcus (gbs) as a model pathogen and examined the role of caspase dependent and independent apoptosis in preterm delivery. methods: we injected ( . x ) heat killed group b streptococcus organisms (hk-gbs) intraperitoneally (i.p.) in . day pregnant c b/l mice. the mice were euthanized at hr (n= ) and hr (n= ), the placentas and membranes were removed and assessed for apoptosis by tunel staining. caspase activation and expression were determined by immuno-histochemistry (ih) and western blotting. the effect of apoptosis on preterm delivery was assessed by i.p. treating the pregnant mice with pbs (n= ), dmso (n= ) or pancaspase inhibitor z-vad-fmk (n= ) prior to hk-gbs and observing the animal for delivery for hrs. results: there was a time dependent, gbs-induced increase in apoptosis by tunel assay and caspase activation in the placenta and membranes. in addition hk-gbs-induced the expression of caspase and caspase independent m-calpain in the placenta. z-vad-fmk ( mg/kg), at the maximum concentration that did not induce maternal illness, did not prevent hk-gbsinduced preterm delivery. conclusions: caspase dependent and independent mechanisms are activated in the placenta upon exposure to gbs. systemic adminstration of a pan-caspase inhibitor did not impact upon the occurance or timing of bacterially induced preterm delivery. further studies are needed to assess the role of apoptosis in the pathogenesis of infection associated preterm delivery. early and ( . %) had a late sptb. the mean + sd gestational age at blood draw was + weeks. the median level of bb was higher in women with early as compared with late sptb or term births (p= . for trend). women with bb in the top quartile were . times more likely to have an early sptb as compared with women who had lower levels of bb ( % ci . to , p = . ). there was no association between bb and late sptb (rr= . , % ci = . to ). the adjusted or of an elevated bb for early sptb was . ( % ci = . to , p= . ). when the analysis was restricted to the women with sptb the rr of an elevated bb for early sptb was . ( % ci . to . , p = . ). conclusions: a significant relationship was found between an elevated bb in early pregnancy and early sptb suggesting inflammatory events in early pregnancy, perhaps infection-related, are part of the pathogenic mechanisms. objective: genital tract infection and/or inflammation appears to contribute to the majority of ptds preceding weeks of gestation. ptd in humans has been associated with colonization and/or infection with a variety of different organisms including gram positive and negative bacteria, mycoplasma, ureaplasma, trichomonads, malaria parasites and viruses. the innate immune response to these pathogens is produced by a family of pattern-recognition cell membrane receptors known as the toll-like receptors (tlrs). these studies sought to characterize the tlr isoforms expressed in the preterm mouse uterus, and their modulation during lipopolysaccharide (lps)-induced ptd. methods: using sterile surgical technique, day- pregnant cd- mice underwent intrauterine injection of g lps. subsequently, the mice were euthanized at , , , , and hours after lps injection. uterine tissue was harvested and placed in rnalater; subsequently total rna was isolated using the trizol reagent and genomic dna was removed using turbo dnafree. cdna was made using iscript cdna synthesis kit. pcr primers were designed using published mouse tlr gene sequences. real-time quantitative rt-pcr was performed using the power sybr green master mix and an abi prism multicycler. to confirm tlr amplicon sizes, the rt-pcr products were visualized on a % agarose/tbe gel stained with gelred. results: these studies have confirmed mrna expression of all of the reported mouse tlr isoforms. these tlr amplicons range in size from to bp as expected; amplicon sequences are pending. quantitative rt-pcr studies performed using uterine tissues from five mice at each time point demonstrated that at hours after lps injection, tlr increased -fold and tlr increased -fold (both p< . ). in contrast, the expression of tlr (the ligand for lps) remained stable during the hours after lps; the expression of tlr , , , , , and also remained stable. tlr expression trended upward and tlr trended downward, although neither was statistically significant. conclusions: these studies have confirmed expression of all tlrs within the preterm pregnant mouse uterus, along with characterization of their modulation during lps-induced ptd. these observations are important because they contribute to our understanding of the immunologic signaling events leading to ptd. (funded by nih hd ). udp-glucose and its receptor p y as a new innate immune system in the female reproductive tract. toru arase, tetsuo maruyama, hiroshi uchida, takashi kajitani, masanori ono, maki kagami, hironori asada, yasunori yoshimura. obstetrics and gynecology, keio university, shinjukuku, tokyo, japan. objective: innate immune system involving toll-like receptors has recently emerged in the female reproductive tract (frt). we hypothesize that there may exist new other mucosal immunity in frt. recently, it has been reported that p y , a g protein-coupled p y receptor for udp-glucose (udp-g), is involved in the lung epithelial immune system. the aim of this study is to investigate whether udp-g and p y have a potential as the defense immune system in frt, in particular endometrium. materials and methods: we obtained human endometrial tissues from consenting reproductive-aged patients. the spatiotemporal expression of p y in human and mouse endometrial tissues was analyzed using rt-pcr and ihc. isolated human endometrial cells and a human endometrial epithelial cell line, ishikawa, were cultured, treated with udp-g, and subjected to rt-pcr analysis for il- mrna expression. we also measured the il- secretion using elisa. small interfering rna was used to knock down p y expression. the chemotactic activity of udp-g on neutrophils was tested using transwell assay with ishikawa cells. lastly, mouse uterine tissues were incubated with udp-g and subjected to rt-pcr analysis for mrna expressions of kc and mip- , the il- homologues in mice. results: p y was exclusively expressed in the glandular and luminal epithelium both in human and mouse uteri. treatment with udp-g induced the secretion of il- in ishikawa and human endometrial glandular cells, but not stromal cells, in a dose-and a time-dependent manner. p y knockdown abrogated udp-g-induced il- production. treatment with udp-g also significantly increased the chemotaxis of neutrophils, which was attenuated by co-addition of anti-human il- neutralizing antibody. in the mouse uterus stimulation of udp-g significantly up-regulated the expressions of kc and mip- mrna. conclusions: udp-g is an endogenous molecule and released into the extracellular environment in a lytic manner after cell damage. taken together, our results collectively substantiate a model in which udp-g released from endometrial cells damaged by infection stimulates il- production via p y in endometrial glandular epithelium, which, in turn, recruits neutrophils thereby preventing the progression of infection. thus, udp-g and its receptor p y may be involved in the trans-species mucosal immune system in frt. (jsgi ; , (suppl) , abst # ) but in utero effects on fetal lung remain to be established. we have examined the relationship between duration of iai and subsequent azi treatment on the severity of fetal lung histopathology. we hypothesized that early treatment would prevent the development of advanced lesions, while late treatment may reduce the severity of lung damage. study design. thirteen chronically instrumented rhesus monkeys received intraamniotic inoculation of u. parvum (serovar ; - x cfu) at ± . days gest. age (dga, mean ± sem, term= d). six of these animals received maternal i.v. azi ( . mg/kg q h or q h for d) either alone (n= ) or in combination (n= ) with dexamethasone (dex; mg/kg/d i.v. for d) and indomethacin (indo; mg/d p.o for d). tissues were obtained at cesarean section for histopathologic assessment. leukocytic infiltration of aveolar spaces and septal walls, type ii pneumocyte hyperplasia and peribronchiolar lymphocytic aggregates were scored as absent ( ), minimal ( ), moderate ( ) and severe ( ). results. inoculation-to-delivery interval was - d for combined treatment groups and was similar to long duration infection without treatment ( - d). treatment effects were tabulated as mean scores and compared as follows: control (n= ), score ; short duration infection ( - d; n= ), score ; long duration infection ( - d; n= ), score ; short duration infection + treatment (n= ), score ; long duration infection + treatment (n= ), score . conclusions. histopathologic findings of fetal pneumonia progressively worsen with duration of u. parvum iai. early maternal azi treatment prevents development of advanced lung lesions, while later treatment may reduce the severity of fetal lung damage. our results suggest that in utero treatment of iai may lower the risk of neonatal bronchopulmonary dysplasia. support: nih hd , rr . brain associated with adverse neurodevelopmental outcome. lps triggers proinflammatory responses through toll-like receptor- (tlr ). mitogen activated protein kinases including c-jun-n-terminal kinase (jnk) have been reported to be implicated in tlr signalling pathways and play important role in both onset of labor and brain injury. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of specific inhibitor of jnk signaling, d-jnk inhibitory peptide (d-jnki) can (i) inhibit jnk activity in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr- specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. in vitro kinase assay demonstrated the activation of jnk in response to lps in the maternal uterus and fetal brain. furthermore, the brain specific jnk was found to be the major jnk isoform activated by lps in the fetal brain. co-administration of d-jnki with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and reduced pup mortality up to %. this was associated with inhibition of jnk activity in both maternal uterus and fetal brain. in addition, we have found that treatment with lps significantly up-regulated cox- , cxcl (il- equivalent) and ccl in myometrium and this is significantly suppressed after co-administration of d-jnki. we conclude that specific inhibition of jnk signaling may have a potential of controlling preterm labor and preventing fetal brain damage as a result of infection/inflammation. the prostaglandin -deoxy- , -prostaglandin j delays lps-induced preterm delivery and reduces mortality in the mouse. intrauterine infection is a common trigger for preterm birth, and is also a risk factor for the development of neurodevelopmental abnormalities in the neonate. bacterial lipopolysaccharide (lps) binds to toll-like receptor- (tlr ) to activate pro-inflammatory signaling pathways. the transcription factor nuclear factor kappa b (nf-kb) is a key player in the orchestration of the inflammatory response and has a central role in parturition. we have previously shown that exposure to the anti-inflammatory cyclopentenone prostaglandin -deoxy- , -prostaglandin j ( d-pgj ) inhibits il- b-induced nf-kb activity and cox- expression in human myometrial and amnion epithelial cells in vitro. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of d-pgj can (i) inhibit nf-kb in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. co-administration of d-pgj with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and conferred protection from lps-induced fetal mortality up to %. we have looked at the expression profile of several labor associated genes in myometrium hours after lps administration. (otr, connexin and , cox- , cox- , cxcl (il- equivalent) and ccl ). we have found that treatment with lps significantly up-regulated cox- , cxcl and ccl and this is significantly suppressed after with co-administration of d-pgj . western analysis for ser -phosphorylated p and ikkb in-vitro kinase assay has demonstrated that lps induced activation of nf-kb at both h and h. co-administration of d-pgj was associated with inhibition of nf-kb activation in both the maternal uterus and the fetal brain. conclusion d-pgj may have potential as a therapeutic agent in the management of preterm labor and, by targeting the player nf-kb, may have the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation. synergistic macrophage response to co-activation of tlr- and tlr- : mechanisms and implications for bacterial/viral co-infection. vladimir ilievski, emmet hirsch. , department of ob/gyn, evanston northwestern healthcare, evanston, il; department of ob/gyn, feinberg school of medicine, northwestern university, chicago, il. background: toll-like receptors (tlrs) recognize structural components of pathogens and initiate host defenses. tlr- responds to gram-positive organisms and peptidoglycan (pgn), a gram-positive cell wall constituent. tlr- is activated by viral infection in response to double-stranded rna. polyinosinic-cytidylic acid (poly(i:c)) is a tlr- ligand. we have shown that pgn and poly(i:c) have a synergistic effect on the expression of downstream genes for both tlr- and tlr- . here we identify mechanisms underlying this synergy. methods: mouse peritoneal macrophages or a mouse macrophage cell line (raw . ) were treated in tissue culture with either pgn ( g/ml), poly(i: c) ( g/ml) or both pgn and poly(i:c) either simultaneously or sequentially for - hours. total rna was extracted and duplex rt-pcr was performed for inducible nitric oxide synthase (inos), interleukin (il- ), tumor necrosis factor (tnf), the chemokine rantes and tlr- , normalized to the housekeeping gene gapdh. results: compared to stimulation with either pgn or poly(i:c) alone, costimulation of raw . cells with both pgn and poly(i:c) resulted in synergistic expression of inos, il- , tnf and rantes (p< . for all) at and hours . sequential stimulation with either pgn or poly(i:c) for h followed by incubation for an additional h with the alternate ligand also induced synergistic expression of the same rnas, albeit at lower levels than were elicited by simultaneous stimulation. in contrast, incubation with either pgn or poly(i:c) for h followed by medium for h induced minimal to no gene expression. both pgn and poly(i:c) induced tlr- mrna after h but not h. tlr- mrna was not detectable by rt-pcr. in primary peritoneal macrophages, similar synergy due to pgn and poly(i:c) was seen. conclusions: simultaneous or sequential exposure to pgn and poly(i:c) exerts a synergistic effect on the expression of inflammatory mediators in macrophages. interestingly, either one of these two tlr pathways can prime cells for activation of the other pathway. a possible mechanism for this effect may be induction of tlr- by either tlr- or tlr- activation. these observations have implications for bacterial/viral co-infection. this is a secondary analysis of a prospective cohort study. after irb approval, daily blood samples were obtained from pprom subjects and analyzed for il- by elisa. paired maternal serum il- levels from subjects were divided into groups: il- levels obtained - hours prior to completion of antibiotics and those obtained - hours after completion of antibiotics. the wilcoxon signed rank test was used to perform the data comparison on the analyze-it statistical software program. statistical significance was defined as p< . . of the pprom subjects, the maternal age was . yrs; gestational age at admission was . weeks; latency was . days; gestational age at delivery was weeks; and infant birth weight was grams. median maternal serum il- levels obtained off antibiotics were significantly higher when compared to those on antibiotics ( . vs. . pg/ml, p< . ). the results of this investigation suggest that maternal serum il- levels rise after discontinuation of antibiotics. the optimal duration of antibiotics administration in the setting of pprom is unknown. this data suggests a role for continuation of antibiotics in women with pprom in order to prolong the latency period and potentially decrease neonatal morbidity. to identify clinical and pathologic factors associated with fetal inflammatory responses in the placenta from term parturients. methods: retrospective cohort study of consecutive term parturients with submitted placentas in . placentas with histologic chorioamnionitis were divided into two cohorts: group -maternal inflammatory responses only, and group -maternal and fetal inflammatory responses. maternal and fetal inflammatory responses in the placenta were classified according to guidelines established by the amniotic fluid infection nosology committee of the perinatal section of the society of pediatric pathologists. selected demographic, intrapartum, newborn and placental characteristics were analyzed with t-tests and chi-square tests as appropriate. multiple logistic regression was used to identify independent predictors of fetal inflammatory responses in the placenta. results: of consecutively submitted placentas, had histologic chorioamnionitis: with maternal inflammatory responses only (group ) and with both maternal and fetal inflammatory responses (group ). fetal inflammatory responses observed in group were associated with higher stages of maternal inflammatory responses (p<. ). % of fetal inflammatory responses were stage i (acute chorionic vasculitis or phlebitis).group patients were more likely to have had amnioinfusion ( % v %, p=. ) and less likely induction of labor ( % v %, p=. ). group was more likely to have had intrapartum fever ( % v %, p=. ) and maternal tachycardia ( % v %, p=. ). newborns from group were more likely to have been observed for sepsis ( % v %, p <. ) and have an apgar score at minutes ( % v %, p=. ). a logistic regression model showed that stage ii or greater maternal inflammatory responses (or . ) and amnioinfusion (or . ) were independent predictors of fetal inflammatory responses. conclusion: higher stages of maternal inflammatory responses in the placenta and amnioinfusion were independent predictors of fetal inflammatory responses in term parturients with histologic chorioamnionitis. objective: previous studies have shown that sulfasalazaine (sasp) inhibits lipopolysaccharide (lps)-induced nf-b activation and decreases lpsstimulated interleukin- (il- ) production by cultured explants of placenta, amnion and choriodecidua with no effect on cell viability. bacteria-induced il- production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. it is unclear, however, whether sasp can inhibit il- production by endocervical cells. therefore, we performed a series of in vitro studies to determine if sasp inhibits il- production by endocervical epithelial cells stimulated with bacterial pathogens associated with preterm birth. methods: human endocervical epithelial cells were incubated with - . mm sasp overnight and then stimulated with ccu/ml ureaplasma parvum, cfu/ml escherichia coli, or x cfu/ml gardnerella vaginalis for another overnight incubation in -well cultures. conditioned medium was then harvested and production of il- was quantified by elisa. viability of the cells was ascertained at the end of the experiment with the mtt-assay. each treatment was applied in quadruplicate wells and experiments were repeated times using different batches of cells for each pathogen. results were evaluated using the general linear models procedure of sas for a randomized block design. results: sasp had no detectible effect on il- production by endocervical cells not treated with bacteria. at the highest concentration tested ( . mm), sasp significantly inhibited il- production by cultures stimulated with e. coli (p< . ), u. parvum (p< . ), and g. vaginalis (p< . ). viability of the cells, however, was significantly reduced by sasp at . and . mm in the both the presence and absence of bacteria for all experiments. conclusion: although high concentrations of sasp inhibit il- production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be due to toxicity of the antibiotic on the cells. the effect of -hydroxyprogesterone caproate on lipopolysaccharide stimulated peripheral blood mononuclear cells from pregnant women. richard h lee, aimin li, frank z stanczyk, jinwen i lin, t murphy goodwin. obstetrics and gynecology, university of southern california, los angeles, ca, usa. introduction: -hydroxyprogesterone caproate ( -ohpc) reduces the rate of recurrent preterm birth in high-risk women. however, there are lines of evidence to suggest that -ohpc alters inflammatory response in the setting of gram-negative infection. in a mouse model, -ohpc decreased the rate of preterm birth but was associated with significantly increased maternal morbidity when mice were exposed to lipopolysaccharide (lps). in non-pregnant women, -ohpc pre-treatment of whole blood exposed to lps significantly increased the production of tnf-. objective: to determine if -ohpc has an effect on the production of proinflammatory cytokines from peripheral blood mononuclear cells (pbmc) in pregnant women. methods: pbmc were isolated from fresh whole blood samples of pregnant women between and weeks. pregnant women had no prior history of preterm birth. cells were treated with -ohpc ( m) and escherichia coli lipopolysaccharide (lps, g/ml) alone or in combination. after and hours of culture, supernatants were collected and tested for tnf-and il- levels by enzyme-linked immunosorbent assay (elisa). statistical analysis was performed using non-parametric tests. p < . was considered significant. results: pbmc exposed to lps significantly increased tnf-and il- secretion compared to untreated controls (p= . and p= . ). tnf-concentrations were not significantly different between lps and lps+ -ohpc treated cells at both and hours (p= . and p= . ). il- production was significantly increased after -hour treatment with lps+ -ohpc compared to lps alone ( , [ , - , ] background. recent clinical trials indicate that progestins reduce the incidence of preterm birth in some high risk pregnancies. it has been proposed that progesterone promotes uterine quiescence, in part, via its antiinflammatory properties with inhibition of pro-inflammatory gene expression. it is intriguing that progestins are clinically effective given the considerably increased background circulating levels of the hormone during pregnancy. we hypothesised that non-classical progesterone signalling pathways contribute towards mediation of the anti inflammatory effects of progestins. to the endotoxin lipopolysaccharide (lps) by activation of inflammatory pathways. myometrial cell cultures were treated with lps ( g/ml)+/progestin ( nm). two progestin compounds were investigated. natural progesterone (p) is known to have a strong affinity with progesterone receptor (pr) analogues in contrast to -hydroxyprogesterone ( -hp) which, in the absence of esterification with caproate or acetate, has been reported to have no progestogenic activity at pr. the effect of p and -hp on the activation of two inflammatory genes known to be associated with labour (cycloxygenase [cox- ], toll-like receptor [tlr- ]) was evaluated. cox- and tlr- were detected at the protein and mrna levels using sds-page and rt-pcr. results. lps-induced expression of cox- and tlr- proteins were significantly inhibited by both p (p< . and p< . , respectively) and -hp (p< . and p< . , respectively). furthermore, preliminary results indicate that co-incubation with the anti-progesterone mifepristone, fail to abrogate the anti inflammatory effect associated with progestin treatment. conclusion. non-classical progesterone signalling pathways have a role in mediating the anti-inflammatory properties of progestins. further elucidation of the molecular actions of progestins may allow novel approaches to ameliorate the inflammatory response associated with preterm labour. detection and transcriptional expression of tlr- , tlr- and tlr- at the maternal-fetal interface. jacqueline p tilak, karen zakharian, alexandra tungol, gabriel o schubiner, david m svinarich. patient care research, providence hospital, southfield, mi, usa. preterm labor and delivery remains a leading cause of neonatal morbidity and mortality and bacterial infection is considered to be the most common etiology. toll-like receptors (tlr's) and the associated components of the innate immune system may represent a first line of defense against these pathogens. tlr's belong to a family of pattern-recognition receptors that bind to highly conserved pathogen-associated molecular patterns (pamps) including lipopolysaccharide (lps), lipotechoic acid (lta) and cpg dna, and are a key component of the innate immune system. this study was undertaken to characterize the transcriptional expression and regulation of tlr- , tlr- and tlr- within gynecologic and gestational tissues. human first trimester trophoblasts, endometrial mesoderm, ectocervix, choriocarcinoma and neonatal epithelium, were cultured in media alone or in the presence of either lps ( mg/ml) or lta ( mg/ml) for , , , , and hours. total rna was isolated and semi-quantitative rt-pcr was performed using intron-spanning amplimers to tlr- , tlr- , tlr- and gapdh. following gel electrophoresis, the integrated optical densities were determined for the corresponding pcr products and normalized with respect to gapdh levels. inducible transcription of tlr- with lta was observed in choriocarcinoma cells ( -fold, h), and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in ectocervical cells ( -fold, h), choriocarcinoma cells ( -fold, h) and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in choriocarcinoma cells ( fold, h) and neonatal epithelial cells ( -fold, h). all cell lines showed at least constitutive levels of transcription for tlr- , tlr- and tlr- . these data demonstrate that tlr- , tlr- and tlr- are transcriptionally expressed either constitutively or inducibly in both gynecologic (endometrial mesoderm, ectocervix) and gestational (chorion, trophoblast), tissues. translation of these receptors suggests that the innate immune system may function at the maternal-fetal interface to help protect the fetus against infection and prevent or diminish the likelihood of prematurity. objective: further to our development of a sheep model of intrauterine ureaplasma spp infection, we aimed to examine the capability of ureaplasma colonization in the amniotic fluid to infect the fetus and alter lung and brain development. methods: at days of gestation (d, term= d) ewes bearing single fetuses were given a single ultrasound-guided intra-amniotic injection of (a) x colony forming units (cfu) of u parvum (serovar , n= ; serovar , n= ), (b) x cfu of u parvum (serovar , n= ; serovar , n= ) or (c) media control (n= ). at d, fetuses were delivered by cesarean section. amniotic fluid and umbilical arterial blood samples were collected. fetal body weight was recorded, fetal cerebrospinal fluid (csf) collected and a descending pressurevolume curve constructed after inflation of the lungs to cmh o. fetal membranes were immersion fixed and the fetal brain was perfusion fixed with % paraformaldehyde. the fetal brain and membranes were blocked, paraffin embedded, stained and viewed under the light microscope. results: chronic intra-amniotic colonisation with u parvum serovar or (ureaplasmas) did not result in fetal abortion or death. amniotic fluid ureaplasma titers at delivery were not dose-dependent. chronic ureaplasma exposure did not affect fetal body or brain weights, or result in a fetal systemic inflammatory response. umbilical arterial blood gases at delivery were similar between ureaplasma-and media-exposed fetuses. chronic intra-amniotic exposure to ureaplasmas resulted in higher inflammatory cell scores in the fetal membranes compared to media controls (p< . ). lung compliance was increased in ureaplasma-exposed fetuses compared to controls (p< . ). there were no gross anatomical changes observed in the white or grey matter in the cerebral hemispheres of fetuses that had been exposed to ureaplasmas; even in animals (n= ) that had csf ureaplasma colonisation. conclusion: chronic ureaplasma colonisation enhances fetal lung compliance without gross anatomical changes in the fetal brain. background: an important source of vitamin d is its synthesis by skin from uv solar radiations. the skin pigment melanin absorbs uv photons thus reducing the vitamin d synthesis by more than % making african americans at high risk for vitamin d deficiency. low maternal vitamin d status during pregnancy has been linked to molecular pathways for adverse outcomes. objective: the nf b transcription factor regulates genes involved in inflammation and immune processes, and is proposed to play a major role in the successful outcome of pregnancy and labor. prior immunohistochemical (ihc) and biochemical studies have been conflicting regarding nf b activation in human intrauterine tissues. the aim of this study was to quantify nuclear localization of p , the major tranactivating nf b subunit, in full-thickness fetal membranes (fm) and myometrium using ihc. methods: a retrospective analysis of paired fm, decidua, and myometrial samples was conducted using tissues collected from women in cohorts: preterm no labor (pnl, n= ), preterm labor (ptl, n= ), spontaneous term labor (stl, n= ), and term no labor (tnl, n= ). subjects were delivered between gestational ages - weeks (preterm) and - weeks (term) by cesarean. paraffin sections were stained with polyclonal (rabbit) anti-human p and microscopically examined. myometrial samples were categorized in a blinded fashion to groups of staining: no nuclear p (-), rare nuclear p (+), and > % nuclear p (++). a p nuclear labeling index (nli; % nuclear p /total cells) was calculated for each histological layer in full-thickness fm specimens using a blinded targeted randomization scheme consisting of non-overlapping low-magnification fields. results: nuclear p labeling was rare in amnion and inconsistently present in chorion. in decidua, p nuclear labeling was observed in all cases; however, decidual nli did not vary significantly across cohorts [pnl- % ( - %); ptl- % ( - %); tnl- % ( - %); stl- % ( - %); all values are median (iqr)]. in myometrium, ++ p nuclear labeling was significantly associated with labor, but present only in a portion of cases (ptl- %; stl- %). despite a trend, decidual nli was not significantly correlated with myometrial nuclear p labeling: myometrial specimens classified as -, +, and ++ corresponded with decidual nli of % ( - %) [median (iqr)], % ( - %), and % ( - %), respectively. conclusions: in a comprehensive ihc analysis, significant nuclear p immunolabeling was observed in myometrial cells following labor. nuclear p labeling in decidua was present in all cases, and did not vary significantly among the cohorts. the inflammatory cytokines interleukin- and tnf-increase g-csf expression in term decidual cells. objectives: chorioamnionitis (cam) elicits premature rupture of the membranes and pre-term delivery. previously, we found that the decidua from cam patients contains much higher neutrophil numbers than control decidua, but macrophage numbers are similar in both groups. granulocyte colony-stimulating factor (g-csf) enhances granulocyte colony formation chemoattraction and activation. the amniotic fluid during cam contains elevated tnf-and il- levels. to account for the marked influx of neutrophils infiltration in cam-complicated decidua, tnf-and il-effects were assessed on g-csf expression in term decidual cell (dc) monolayers. methods: confluent leukocyte-free term dcs from normal term deliveries were primed with - m estradiol (e ) + - m medroxyprogesterone acetate (mpa) for days, then switched to serum-free defined medium (dm) with steroids ± tnf-or il- . after h, aliquots of conditioned dm supernatants, cell lysates and extracted rna from h parallel incubations were frozen. secreted g-csf was measured by elisa in conditioned dm and normalized to cell protein and mrna was assessed by quantitative real time rt-pcr and normalized to -actin mrna. results: in cultured first trimester dcs, basal g-csf levels in conditioned dm were . ± . pg/ml/ug cell protein [mean ± sem, n= ] and did not differ from e +mpa basal levels. the addition of ng/ml of tnf-or il- significantly elevated g-csf output to . ± . and ± respectively (p< . ), representing more than a -fold and , -fold change; respectively. western blotting confirmed the elisa results. quantitative rt-pcr demonstrated corresponding changes in g-csf mrna levels as found for the elisa measurements. concentration-dependent effects for both tnfand il- from . to . ng/ml were observed. conclusions: when extrapolated to the placental milieu of cam, the marked increase in g-csf elicited in term dcs by tnf-and il- may provide a mechanism to account for the selective increase in decidual neutrophils versus macrophages. background. the immunological mechanisms of the female reproductive tract are unclear. toll-like receptors (tlrs), innate immune receptors that combat microbial infections and establish adaptive immunity, may play a role. infection in pregnancy has been associated with preterm birth and tlrs may modulate the host response to such infections. we hypothesised that the distribution of tlr and tlr in cervical epithelial cells may differ with pregnancy, and that oestrogen may contribute to the modulation of these receptors. aims and objectives. . to compare tlr and tlr protein expression, using immunocytochemistry, in the cervical epithelium of pre-menopausal non-pregnant women with pregnant women at term. methods. fresh non-pregnant (n= ) and pregnant (n= ) human cervical cells were obtained by smear and tlr and tlr expression investigated by immunocytochemistry. cervical epithelial cells from nonpregnant women obtained by smears were then coincubated with varying concentrations of estradiol, and tlr and tlr protein expression quantified by immunocytochemistry. results. using pixel-based image analysis software, we demonstrated a reduction in tlr expression in late pregnant compared with non-pregnant cervical epithelium (p= . ), whilst tlr did not appear to differ. there was an apparent up-regulation of tlr protein expression in response to beta-oestradiol (n= ) objective: to evaluate in vitro antimicrobial activity of cranberry-exposed urine against common urinary pathogens. subjects were pregnant women enrolled in a clinical trial evaluating the effect of daily cranberry juice cocktail or matching placebo on asymptomatic bacteriuria and other urinary tract infections. methods: -hour urine samples from pregnant women who were randomized to cranberry or placebo in three treatment arms: a. cranberry two times daily with meals (c, c; n= ), b: cranberry in the am, then placebo at dinner (c, p; n= ), c: placebo two times daily with meals (p, p; n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment groups and collected -hour urine specimens from them. the ph of all urine specimens was adjusted to ph= and filtered. aliquots of each were independently inoculated with overnight culture of - cell/ml each of single strains of e. coli with fimbriae type i and type ii, k. pneumoniae, and c. albicans. after hours of incubation for e. coli and k. pneumoniae, and hours for c. albicans cfu/ml of each specimen were enumerated by subculture with quantitative plate counts in duplicate. results: there were no differences for any of the antimicrobial activities between pregnant and non-pregnant groups, or based on treatment allocation. we were able to perform antimicrobial assays on the urine of women exposed to cranberry juice or placebo, but unable to demonstrate differences based on treatment allocation or pregnancy. this may be due to beta-error. further studies are planned. supported by r dk - ; clinical trials registration nct . ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • objective: to measure compliance, we sought to evaluate the use of a bioassay for cranberry in the urine of women enrolled in a clinical trial designed to determine the effect of daily dosing of cranberry juice cocktail or matching placebo on the incidence of asymptomatic bacteriuria (asb) and other urinary tract infections (uti) during pregnancy. methods: we collected -hour urine specimens from pregnant women who were randomized to ingest cranberry or placebo in three treatment arms: a: cranberry two times daily with meals (n= ), b: cranberry in the am, then placebo at dinner (n= ), c. placebo two times daily with meals (n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment regimens (group a: n= , group b: n= , group c: n= ), and collected -hour urine specimens from them. bacterial anti-adhesion effects of the cranberry-exposed urine were evaluated utilizing a human red blood cell hemagglutination assay specific for uropathogenic p-fimbriated e. coli. activity was quantified as , , and %. results: when combining all subjects and dosing regimens, the sensitivity of the assay was %, range % in the pregnant group assigned single daily dose of cranberry to % in the non-pregnant group assigned to multiple daily doses. the specificity ranged from % to %. conclusions: these data indicate that the bioassay can be applied to the pregnant patient population, although the sensitivity of the assay is variable. higher daily dosing appears to confer greater sensitivity. further studies are needed to evaluate the utility of this bioassay for compliance. supported by r dk - . clinical trials registration nct . objective: increasing evidence suggests that inflammation plays a crucial role in initiation of labour both at term and preterm. we have previously shown upregulation of pro-inflammatory cytokines in myometrium, cervix and membranes at term labour. we have also shown that myometrium and cervix is invaded by leukocytes at the time of labour, and these leukocytes express pro-inflammatory cytokines. in this study, we aimed to test the hypothesis that pro-inflammatory cytokines and toll-like receptor and (tlr and ) mrna expression are up-regulated in circulating leukocytes during term and preterm labour. methods: peripheral blood samples were taken from pregnant women: - weeks gestation (w) preterm not in labour (ptnl) n= ; - w preterm in labour (ptl), n= ; - w term not in labour (tnl) n= ; and - w term in labour (tl) n= . leukocytes were isolated using dextran sedimentation. total rna was isolated and reverse transcribed using high capacity cdna archive kit, and mrna expression determined by real-time pcr (abi, taqman). student's t-test was used to compare outcomes between groups. results: messenger rna expression of il- , icam- , mcp- , tlr and tlr was significantly increased in tl compared to gestation matched tnl. the expression levels of il- b, il- and tlr- were significantly greater in ptl compared with gestation matched ptnl. (table i) . conclusions: we show up-regulation of pro-inflammatory cytokine production in circulating leukocytes in both term and preterm labour. thus, the pathophysiology of labour seems to involve the upregulation of proinflammatory cytokine production in peripheral blood leukocytes, followed by their invasion into the myometrium and cervix. this work further contributes to our understanding of the pathophysiology of parturition, and suggests that peripheral blood leukocytes may be potential targets for therapeutic agents aimed at modifying the time course of parturition. introduction. the mechanisms of innate immunity and tolerance in the female reproductive tract (frt) are ill-understood but involve the pattern recognition toll-like receptors (tlrs). we have previously demonstrated high expression levels of tlr gene and protein in fresh human cervical epithelium. aims. in order to gain insight into the immunological role of cervical epithelial cells (cecs), we sought to determine cec responses to the following tlr- agonists: macrophage-activating lipopeptide (malp- ), pam csk , and peptidoglycan. methods. cecs were isolated by smears and compared between pregnant ( rd trimester) and nonpregnant women. following cell preparation, flow cytometry was performed using a direct staining procedure with mouse anti-human tlr primary antibody and its igg , isotype control, to determine tlr- protein expression. a further nonpregnant smear samples were each prepared, and seeded at a concentration of , cervical epithelial cells/ml into -well cell plates. cells were incubated at °c in % co overnight with the tlr agonists malp- and pam csk (at concentrations of and ng/ml), peptidoglycan( ng/ml), il- ( ng/ml, positive control) and rpmi + -glutamine media only (vehicle). following centrifugation, all resulting supernatants were stored at - °c until the concentration of il- was determined by elisa and an array of cytokines by bead assays. results. both pregnant and nonpregnant cecs expressed tlr (specific mean fluorescence . vs . respectively) but did not differ (p = . ). unstimulated cells incubated with buffer alone demonstrated high il- levels ( pg/ml), which did not differ following stimulation with malp- ( pg/ml), pam csk ( pg/ml) or peptidoglycan ( pg/ml). results of an array of pro-and anti-inflammatory cytokines following incubation of cells stimulated with tlr agonists are pending. conclusion. high basal il- levels suggest constitutive expression by cecs but the physiological relevance of this intriguing observation is unclear. that cec stimulation with tlr- agonists did not lead to further release of il- may epitomise the resistance of these cells to antigenic challenge by the vaginal commensal flora. cecs may play a pivotal role in modulating the immunological tolerance necessary to minimise inappropriate inflammation in the cervix. there are several epidemiological and clinical studies that omega- fatty acids and related fish oils can decrease the premature labor and birth. however, the scientific mechanisms are not well elucidated. this study was carried out to investigate the effects of omega- fatty acids on producing pge and il- , in human umbilical vascular endothelial cell(huvec) media with artificial inflammation as an infection-induced premature labor tissue model. also, if there are some significant effects with omega- fatty acids, we want to investigate the possible mechanisms. materials and methods; human umbilical vascular vascular cell primary culture was done. in the adequate cell confluence in each cell dish, pretreatment with various concentrations of epa, dha and troglitazone (another ppar-ligand) and incubation were done for hours in °c, co incubator. after that, g/ml conc. of lipopolysaccharide(lps) was treated to the each cell dishes and incubated for hours. the cell media were collected, and pge and il- concentration were checked by elisa. the each cell lysates were collected and western blot anaysis for cyclooxygenase(cox)- were done. on the other hand, nuclear factor kappa b (nf b) luciferase vector was transfected and then did the same pretreatment with epa, dha and troglitazone and lps treatment to each cell dishes. after that, nf b luciferase activity was checked with luminometer. statistical analysis was done by student t-test. results: epa, dha and troglitazone decreased the pge (p< . ) and il- (p< . ) significantly in elisa. cox- expression revealed the significant reduction with pretreatment of epa, dha and troglitazone in higher concentration ( , m) in the western blotting (p< . ). nf b luciferase activity were also significantly decresed with pretreatment of epa, dha and troglitazone in higher concentration (p< . ). conclusion; this study offers some scientific mechanisms, by which omega- fatty acids (epa, dha) and troglitazone may be one kind of the preventive medicine for infection-induced preterm labor and delivery. also, these effects may come from the common mechanism of epa, dha and troglitazone, ppar-ligands. the next study would be how the cox- , nf b and pparare related. ( mg/ml). cytokine expression was measured in medium using the bio-plex suspension array system (bio-rad). mean expression was compared between the two groups using t test. results: -aminopurine significantly inhibited lps stimulated cytokine production by human myometrium. in contrast, there were no significant differences in expression after mpa treatment, although a trend towards inhibition of proinflammatory cytokine and an increase in il- production was noted. introduction: intrauterine infection is now recognised as a major antecedent of white matter injury (wmi) in the preterm infant brain, which can manifest later as cerebral palsy or as varying degrees of cognitive dysfunction. wmi in these infants is characterized by damage to immature oligodendrocytes, and has been linked both to microglial activation and to elevated levels of tnfa, il- b and il- . we have developed a fetal sheep model for diffuse and focal wmi, based on repeated administration of e. coli lipopolysaccharide (lps) as the stimulus for an inflammatory response. methods: surgery to implant fetal vascular catheters was performed on pregnant ewes carrying single fetuses at d - of gestation. fetuses received repeated iv injections of lps ( ng/kg estimated fetal weight/day) between d and d . plasma levels of pro-inflammatory cytokines were determined in fetal arterial blood samples taken between d and d . at d ewes and fetuses were euthanized and fetal brain tissue samples collected for histological analysis. results: five days after final administration of lps to fetuses we observed a pattern of wmi similar to that seen clinically, ranging from focal to diffuse injury within the periventricular region, impairment of white matter (cnpase; marker for immature/mature oligodendrocytes) tracts, and thinning of the corpus callosum, characterised by oligodendrocyte disruption and microglial proliferation in the surrounding and sub-cortical white matter. we also found a progressive rise in fetal plasma tnf levels between days and (day two of treatment to third day following final dose of lps). background: plasma fatty acid (fa) levels are associated with altered host inflammatory responses, blood pressure regulation, and insulin resistance, characteristic features of pregnancy-induced hypertension (pih). most studies compare the n- and n- polyunsaturated fatty acids (pufas). in addition, recent data demonstrate that saturated and trans-fas promote inflammation. based on the immunomodulatory activity of various fas, we explored their effects on placenta inflammatory responses ex vivo. methods: placenta cells were isolated from fresh human (anonymous), term placentas and treated with/without lipopolysaccharide (lps) with various fas, including saturated fas, trans-fas, and n- and n- pufas (at physiological concentrations). after an overnight treatment, tnf-alpha (tnf) production was determined. data were analyzed by analysis of variance (anova) followed by the dunnett's test. results: oleic acid ( : n- ), a cis-monosaturated fa common in the mediterranean diet, did not induce significant placenta tnf production (fig. a) . by contrast, elaidic acid ( : n- ), a trans-monosaturated fa, induced tnf production by -fold compared to vehicle (fig. a) . likewise, palmitic acid ( : ), a saturated fa, induced placenta tnf production by -fold (fig. a) . to mimic inflammation, placenta cells were treated with lps ex vivo in the presence/absence of fas. docosahexanoic acid ( : n- , dha) reduced placenta tnf production by up to % following stimulation (fig. b) . similarly, treatment of placenta cells with linoleic acid ( : n- , la) or arachidonic acid (n : n- , aa) suppressed tnf production by up to % and %, respectively (fig. b) . conclusions: both saturated fas and trans-fas, which positively correlate with hypertension, induce placental tnf production. the n- fa precursors to prostaglandins, aa and linoleic acid, reduce placental tnf production following stimulation. likewise, dha, a product of n- fa metabolism commonly consumed in fish oil and associated with improved blood pressure, suppresses tnf production by stimulated placenta cells. vegf and flk mediated glomerulogenesis in offspring exposed to maternal hypernatremia. roy z mansano, mina desai, ahmed abdel-hakeem, thomas r magee, tazmia q henry, cynthia nast, john s torday, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: growth restricted human and animal offspring, resulting from maternal nutrient restriction or uteroplacental insufficiency, consistently demonstrate reduced glomerular number and a predisposition to adult systemic hypertension and renal compromise. in contrast, maternal water restriction (wr) produces newborns with a unique programmed phenotype of increased glomerular number. glomerulogenesis is dependent, in part, on appropriately timed and quantified vascular development. as vegf and its receptor flk have crucial stimulatory effects on renal vasculogenesis, we hypothesized that maternal wr-induced morphologic renal changes are secondary to vegfmediated vasculogenic effects. methods: from day to term gestation, pregnant rats received either ad libitum food and water (control, n= ), or wr to produce an increment of meq/l in plasma sodium (n= ). following delivery, all dams received ad libitum food and water. at d after birth, offspring kidneys were extracted for mrna. vegf and its receptor, flk , mrna levels were determined using real-time rt-pcr (presented as fold difference normalized to s). at d after birth, offspring glomerular number were determined in formalin fixed m sections using histomorphometric analysis. values are means±se. results: wr offspring (day ) had higher glomerular number than control (wr ± , control ± per mm , p< . ). flk mrna expression was increased in wr offspring kidneys (wr . ± . , control . ± . , p< . ) as compared to controls. in contrast, vegf mrna expression in wr offspring kidney was comparable to control (wr . ± . , control . ± . , p= . ). conclusion:prenatally wr offspring demonstrate significantly increased glomerular number. as vegf recruits angioblasts to the developing glomerulus via its receptor flk , increased receptors (flk ) with the same level of the ligand (vegf) suggest that enhanced vasculogenesis may represent a putative mechanism for increased nephrogenesis in wr offspring. modulation of newborn vasculogenesis via vegf and flk expression may represent a therapeutic option for growth restricted offspring with decreased glomerular number. objective: maternal food restriction (mfr) during gestation (embryonic day to birth) reduces rat kidney glomerular number by % at weeks of age. undernutrition during human gestation leads to similar impaired nephrogenesis and increased hypertension in adults. renal development may be delineated into stages including ureteric bud branching, mesenchymal to epithelial transformation and glomerulogenesis. previously, we detected dysregulation of genes controlling nephrogenesis at gestational ages, e -e , suggesting that mfr has significant effects on ureteric bud branching. in the present study we evaluated the effect of this dysregulation on early nephron development in e fetal kidney explants from dams with mfr. methods: e fetal kidneys were collected from % mfr pregnant rats and ad libitum control rats (n= per group and time point), incubated in dmem/f k medium for , , , , and days, and fixed with % paraformaldehyde. kidneys were stained with fluorescein-labeled dolichos biflorus agglutinin (dba), images captured and terminal ureteric buds quantitated. all values are presented as mean ± sem. differences were considered significant at p< . . results: mfr ex-vivo kidneys at day demonstrated an initial moderate reduction in terminal branches versus control (c) samples (mfr: ± vs c: ± terminal branch ends per kidney). both mfr and control (c) kidneys demonstrated significant (p< . ) increases in branching in explant culture to maximum values at days (mfr: ± vs c: ± ). with increasing culture days, the percent reduction in mfr branching increased with terminal branch point decreases of % at day , % at day , % at day , % at day , and % at day (p< . , mfr vs c at day ). conclusion: kidney explant cultures from mfr treated fetuses display basal and culture-based decreased branching compared to controls. this decrease in terminal ureteric buds, in combination with our previous findings of dysregulation of branching-associated kidney transcription and growth factors, suggests mfr induces early (e ) dysregulation of branching as a major mechanism of the associated nephropenia. these results indicate that early programming events in kidney development induce nephropenia and renal disease in adults. intrauterine growth restriction (iugr) has important implications for the neonate not only at the time of birth, but also as an adult. in humans and animals with iugr, the elevated risk of developing hypertension is thought to involve an upregulation of the renin-angiotensin system (ras). however, the link between the intrauterine insult and enhanced ras is not known. a novel mechanism leading to hypertension has emerged recently involving two orphan g-protein coupled receptors (gcr and gcr ) and their endogenous ligands, succinate and -ketoglutarate. infusion of succinate into adult mice induces hypertension, an effect which was eliminated in gcr knockout mice or by pretreatment with ace inhibitors. interestingly, succinate levels have been shown to increase in the circulation under conditions of oxidative stress, one of the hypothesized mediators of developmental programming of hypertension. thus, we hypothesized that gcr and gcr are upregulated in a rat model of iugr and may contribute to in utero programming of hypertension. timedpregnant rats were fed either control (c, % casein) or low protein (lp, % casein) diet throughout gestation. kidneys were collected on embryonic day (e ), post-natal day (d ) or (d ), n . real-time pcr was used to compare gcr , gcr and renin expression. data were standardized to a housekeeping gene (s ) and are expressed as fold increase/ decrease compared to control. a student's t-test was used to determine significance between groups (p< . ). offspring from lp dams were significantly smaller at birth and did not display catch-up growth. in kidneys from lp fetuses, both gcr and gcr were significantly elevated compared with controls ( . fold and . fold respectively; p= . ), whereas renin was . fold lower. on post-natal d , there was a trend towards increased expression of both gcr ( . fold; p= . ) and renin ( . fold; p= . ) in offspring from lp dams. at d , there were no significant differences between groups. in conclusion, these preliminary data suggest that in iugr offspring from lp rats, the gcr / pathway may be enhanced, indicating a novel mechanism for the programming of hypertension. objective: in addition to excess adipose tissue, obesity is accompanied by increased fat storage in organs such as the liver. peroxisome proliferatoractivated receptors (ppars) are ligand-activated transcription factors involved in the regulation of lipid metabolism, and lipid-associated inflammatory response. obesity represents a state of chronic low-level inflammation, with ppar and ppar implicated in this process. we have previously shown that nutrient restriction in pregnancy results in intrauterine growth restricted (iugr) newborns which develop adult obesity with elevated c-reactive protein (crp) levels. as crp is derived from the liver, we hypothesized that iugr-induced obesity inhibition of hepatic ppar and ppar is associated with an increased inflammatory response. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to to produce iugr newborns. all pups were nursed by control dams and weaned at weeks to ad libitum feed. at day and months of age, male offspring were analyzed for hepatic ppar , ppar and crp mrna (real time rt-pcr) and protein (western blot) expression. data was normalized to -actin and presented as fold change for protein levels. at months, hepatic triglyceride content was determined enzymatically. results: at d of age, iugr pups showed significant downregulation of ppar ( . -fold) and ppar ( . -fold) expression, though crp expression was significantly upregulated ( -fold). findings persisted at months of age, with continued downregulation of ppar and ppar ( . -fold) and upregulation of crp expression ( -fold). furthermore, iugr adults had significantly increased hepatic triglyceride content ( ± vs. ± mg/g liver, p< . ). conclusions: reduced expression of hepatic ppar and ppar in iugr offspring may contribute to elevated hepatic crp levels and triglyceride content. thus, developmental hepatic dysregulation leads to programmed obesity-induced inflammation in iugr offspring. offspring. mina desai, ederlen casillas, guang han, darran n tosh, , michael g ross. dept. of ob/gyn, labiomed at harbor-ucla med. ctr., torrance, ca, usa; dept. of physiology, univ. of adelaide, australia. objective: leptin and insulin mediate central anorexigenic signaling responses via different receptor molecules: leptin binds to the obrb receptor activating jak-stat and pi k pathways, whereas insulin activates pi k pathway by binding to its receptor (ir) and substrate (irs ). maternal food restriction in pregnancy results in iugr newborns that develop hyperphagia and adult obesity. the iugr newborns have significantly decreased plasma leptin levels with increased hypothalamic expression of basal obrb, stat and decreased expression of ir and irs , suggesting altered anorexigenic pathways. we studied the response of hypothalamic leptin/insulin signal molecules to peripheral leptin in iugr newborns. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to . day old male offspring were given saline or leptin ( g/g, i.p). hypothalamus was dissected at , and minutes and protein expression of total stat , phosphorylated stat (p-stat ), inhibitor of leptin signal (socs ), ir, irs , total akt and phosphorylated akt was determined by western blot (normalized to -actin). data is compared between leptin and saline treatments in iugr and controls. results: in response to peripheral leptin, iugr newborns showed marked dysfunction in stimulated hypothalamic leptin and insulin signaling responses. ( ) jak-stat : leptin-treated controls show progressively increased pstat ( -fold) with initial suppression of socs ( . -fold) as compared to salinetreated controls. conversely in leptin-treated iugr, the pattern is reversed such that there is sustained decline in pstat expression ( . -fold) with failure to downregulate socs . ( ) pi k pathway: leptin-treated controls showed a significant reduction in irs ( . -fold) and pakt ( . -fold) as compared to saline-treated controls. however, leptin-treated iugr newborns exhibited a paradoxical increase expression of irs ( -fold) and pakt ( -fold). conclusion:the iugr offspring demonstrate persistent upregulation of leptin receptor, a reduced phosphorylated stat (p-stat ) response in conjunction with an enhanced socs- response. the persistent increase in insulin responses indicates a dysfunction in dynamic signaling, leading to altered anorexigenic response and development of programmed obesity. we have previously demonstrated that maternal food restriction (mfr) in rats induces a marked increase in the expression of vegf protein in the aorta and mesenteric arterioles, accompanied by an increase in tgf-and collagen in both vessel types in adult rat offspring (am j physiol, ) . the aim of this study was to determine if this in vivo finding could be reproduced in an in vitro preparation. methods: two types of preparations were used in this study. we isolated endothelial cells from week old male control rat aortas. these cells were used after the third passage. staining with von willerbrand factor demonstrated that these cells were pure endothelial cells. the second type of preparation was aortic explants from week old male control rats. endothelial cells and aortic explants were transfected with a vegf adenovirus ( - viral infective particles) or a -galactosidase-adenovirus as a control. after hours of culture protein was isolated from cells and explants for western blot analysis using rat specific antibodies. culture media was assayed for vegf by elisa. results: transfection of vegf adenovirus induced a dose-dependant increase in the expression of vegf protein in primary endothelial cells and aortic explants. the transfection of vegf into the endothelial cells showed a bell shaped curve, and was accompanied by an increase in media levels of vegf protein. maximal secretion of vegf was found with viral infective particles. vegf adenovirus transfection induced a dose-dependant increase in c-reactive protein (crp) (inflammatory marker), and tgf-protein in both aortic explants and primary endothelial cells. these results indicate that upregulation of vegf in blood vessels induces inflammation and tgf-expression which in turn can induce collagen synthesis. thus the increased collagen expression and reduced compliance previously reported by us in vessels of mfr offspring can be explained by the over-expression of vegf which we reported. therapeutic intervention aimed at prevention of the increase in vascular vegf expression in mfr offspring could potentially prevent programmed hypertension. maternal regulation of high fat nourishment during lactation period reduce a hypertension of male offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med.univ., fukushima, fukushima, japan. objective: exposure to undernutrition or high fat nourishment during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of maternal feeding regulation during lactation period on blood pressure of offspring is unclear. our objective was to investigate the effects of either high-fat diet (hfd) during gestation to lactation period or restrictive fed a hfd during lactation period on blood pressure in male rat offspring. we use types pregnant wistar rats as fed with normal nutrition (group a), with a high fat diet (hfd) during gestation to lactation period (group b) and with hfd nutritionally restricted by feeding with % of the normal lactationmatched dietary intake from the day of delivery to the end of lactation period (group c). the male offspring was measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using oneway annova. results: body weight was significantly reduced in c offspring compared to a and b in male offspring at day after delivery (p< . ). at weeks old, the body weight of c offspring was no difference to catch up compared to a and b offspring. systolic and diastolic blood pressures were significantly elevated at all , and weeks in offspring of b > c >a. (p< . , vs. a) conclusions: under high-fat nutrition during gestation to lactation period induced hypertension in male rat offspring. maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. background: uterine artery (uta) doppler velocimetry has been validated in populations of heterogeneous parity in the second trimester for prediction of obstetric outcomes requiring preterm delivery to include: fetal growth restriction, fetal demise, hypertensive disorders of pregnancy, abruption, and indicated preterm delivery. understanding that parity may affect uta doppler indices in subsequent pregnancies, we sought to validate these predictive values in the first trimester in a homogeneous population of multiparous women. study design: multiparous women undergoing first trimester screening of singleton pregnancies were enrolled and followed prospectively until delivery (n= ). these women were divided into controls, ri < . (n= ) and cases, ri . (n= ), based on prior studies. demographic, clinical, and sonographic data (including uta indices and assessment of notching) were obtained. statistical analysis included student's t test and chi square. results: cases were not significantly different from controls in terms of maternal age, ethnicity, bmi, or medical history. uta doppler indices were significantly different between the two cohorts in terms of the presence of unilateral or bilateral notching ( % vs, % p< . and % vs. %, p< . , respectively). in contrast to that observed in patients of heterogenous parity previously, ri . was not associated with adverse pregnancy outcomes despite an average ri of . , significantly above this threshold. conclusions: in this multiparous cohort ri was not predictive of adverse obstetrical outcome, in contrast to that observed in cohorts including nulliparous patient. parity may affect uta vasculature and obscure the ability of doppler velocimetry to predict adverse obstetric outcome in multiparous women. presence of uta doppler notching in the first trimester remained a robust predictor of adverse obstetric outcomes in multiparous patients. objective: epidemiological studies have shown that offspring exposed to preeclampsia during fetal development are more susceptible to airway disease later in life. we have shown previously that gender, but not sflt- over-expression during pregnancy determines higher reactivity in the offspring airways at months of age. the objective of this study was to examine the effect of preeclampsia on the trachea from female and male offspring in our model of sflt- induced preeclampsia at year of age and compare responses between the two age groups. methods: cd- mice at day of gestation were injected via the tail vein with adenovirus carrying sflt (adsflt , pfu/ l) or mfc (admfc, pfu/ l). mice were allowed to deliver. tracheas were isolated from female and male offspring at months and year of age, and rings were mounted in organ chambers for isometric tension recording. responses to potassium chloride (kcl, mm), the mast cell degranulating agent compound / ( / , g/ml), and concentration-responses curve to acetylcholine ( - - - m) were obtained. results: there was no significant difference in responses to acetylcholine, kcl, or compound / between year old offspring born to the sflt and mfc groups. when comparing offspring within the same pregnancy exposure groups, responses to acetylcholine in adsflt -treated group were significantly higher in year old females than males. comparison between age groups by pregnancy exposure revealed that in the mfc group, year old male offspring had higher responses to compound / and acetylcholine than months old males. responses to kcl were significantly higher in months old males than year olds independent of maternal treatment during pregnancy. in females, the only difference between age groups was observed in the mfc group, where months old offspring demonstrated significantly higher responses to acetylcholine compared to year old offspring. conclusions: our findings did not show that airways of year old offspring born to mice with a preeclampsia-like syndrome induced by sflt- over-expression have airway hyperreactivity. however, sex and age differences in airway responses dependent on maternal exposure during pregnancy were observed, and needs to be explored further to elucidate underlying mechanisms. objective: maternal food restriction (fr) results in iugr newborns that when normally nursed exhibit rapid catch-up growth and adult obesity. continued fr during nursing delays catch-up growth and prevents adult obesity. igf- , which modulates growth and is secreted by the liver, may contribute to these morbidities. igf- is epigenetically regulated involving two promoters, alternative exon splicing and multiple transcription termination sites. we determined if hepatic igf- mrna levels correlate with obesity, and whether these changes are due to programmed epigenetic modification. methods: control pregnant rats received ad libitum food from gestation day to and lactation, whereas study dams were % fr. fr pups were nursed by either control (fr/adlib) or fr dams (fr/fr) and weaned to ad libitum feed. at day and months, male livers were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k trimethyl, and associated levels of each igf- species were measured by pcr. results: at months, obese fr/adlib males showed increased mrna levels of igf- a, igf- b, igf- exon , and igf- exon as compared to controls ( ± , ± , ± , and ± %). comparing fr/adlib month to newborn offspring, h /k was increased at igf -promoter , promoter , exon , utr# and utr# ( ± , ± , ± , ± , and ± %), though there was no differences between control month and newborns. in contrast, month fr/fr males had comparable mrna levels to the controls except for igf- b (% of control: ± ). further, fr/fr month h /k was only different from newborns at utr# (% of newborn: ± ). conclusion: iugr newborns with rapid catch-up growth and adult obesity have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. conversely, iugr with delayed catch-up growth and absence of adult obesity have levels similar to that of controls. thus, modulation of the rate of iugr newborn catch-up growth may protect against igf- epigenetic modifications. introduction: igf-ii is synthesized as a pro-hormone (proigf-ii; -amino acid peptide) which is then processed into its active forms: "big" igf-ii ( - ) and mature igf-ii ( - ). these active forms are essential for placental and fetal development and have also been shown to persist into postnatal life. since maternal smoking is known to adversely affect feto-placental growth and postnatal development, we postulated that these effects might be mediated through nicotine-induced alterations in igf-ii processing. methods: in the present study, nulliparous female wistar rats ( - g) were given nicotine ( mg/kg/day) or saline for days prior to mating, during pregnancy, and throughout lactation. at gestational day , and , dams were euthanized and we collected serum (fetal and maternal), amniotic fluid and recorded fetal body weight. a subset of dams were allowed to deliver at term. following parturition, serum samples from the offspring were collected at birth (pnd ) and weaning (pnd ). body weight was recorded weekly from birth to weaning. pro, "big" and mature igf-ii levels were determined by western blot analysis. results: maternal nicotine exposure during pregnancy resulted in a significant reduction in fetal body weight by gestational day . however, there was no effect of nicotine on fetal serum or amniotic fluid igf-ii levels at any gestational age examined. in maternal serum, mature igf-ii in control animals decreased with advancing gestational age such that igf-ii levels were lowest at gestational day . nicotine administration prevented this decline, which resulted in significantly higher mature igf-ii levels in nicotine-exposed mothers at gestational day . in postnatal life, nicotine exposed offspring had significantly lower levels of "big" igf-ii expression at weaning (pnd ). conclusions: these data demonstrate that nicotine can alter the amount of the active forms of igf-ii in the mother and the newborn. dysregulation of maternal igf-ii occurs concomitantly with suboptimal fetal growth. results from this study suggest a mechanism by which maternal smoking causes impaired fetal growth and adverse postnatal health outcomes. objective: maternal food restriction in pregnancy results in iugr newborns which develop adult metabolic syndrome. programming of both increased appetite-mediated hyperphagia and enhanced adipogenesis contribute to the development of obesity. transcription factors, peroxisome-proliferatoractivated-receptor (ppar ), ccaat/enhancer binding-protein (c/ebp ), and sterol regulatory element binding-protein (srebp c) regulate adipogenesis and lipogenesis. although iugr offspring exhibit acute upregulation of the adipogenesis signaling cascade prior to the development of obesity, we determined if this increased adipogenic potential was an intrinsic cellular response, and thus maintained in cell culture. we further examined the responses to adipocyte stimulators (ppar activator-ligand rosiglitazone) and inhibitors (ppar repressor-ligand badge). methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to term. adipocytes from day old iugr and controls were isolated and cell proliferation rate was determined (mtt). primary adipocyte cell cultures were established and following % confluence, iugr and control adipocytes were treated to two doses ( and m) of either rosiglitazone or badge for h. mrna and protein was extracted for expression of ppar , c/ebp , srebp c. data was normalized to -actin and compared to the respective untreated cells. results: iugr adipocytes had significantly increased protein expression of ppar ( . -fold) and c/ebp ( -fold) as compared to control adipocytes, though srebp c levels were unchanged. mrna levels showed similar changes in iugr newborns. importantly, iugr adipocytes exhibited increased cell proliferation ( % of control, p< . ) and showed greater response to rosiglitazone ( . -fold), though similar response to badge, as the control adipocytes conclusion: iugr primary adipocytes cell culture exhibit basal phenotypic characteristic of programmed upregulation of adipogenic transcription factors which promote adipose cell proliferation. the enhanced response to the adipogenic stimulant is further evidence of the predisposition to obesity. in contrast, the normal suppressive response to the inhibitor suggests that iugr adipocytes may respond to pharmacologic approaches to prevent obesity during this period. objective: maternal nutrient restriction results in intrauterine growth restricted (iugr) newborns which develop programmed obesity despite a normal post-weaning diet. the epidemic of obesity has been attributed in part to programmed "thrifty phenotype" and exposure to "western" diets. hepatic igf- is epigenetically regulated involving two promoters, alternative exon splicing, and multiple transcription termination sites. iugr offspring with normal post-weaning diet have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. we hypothesized that iugr newborns that develop programmed obesity would demonstrate discernable hepatic igf- changes which are distinct from diet-induced obesity. we determined igf- hepatic mrna levels and epigenetic characteristics in programmed (iugr) and dietinduced (dio) offspring. methods:: control pregnant rats received ad libitum food whereas study dams were % maternal food restricted from day to . all pups were nursed on ad libitum fed dams. controls were weaned to high-fat (fat, %) diet whereas iugr were weaned to normal ad libitum diet (fat, %) to produce diet-induced (dio) and programmed obese groups, respectively. at months, male hepatic igf- were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k dimethyl and h k trimethyl, and associated levels of each igf- species were measured by pcr. result: relative to dio control males, iugr had increased mrna of igf- a, exon and exon ( ± , ± , ± %). chip with h k dimethyl showed increased igf- exon ( ± %) and with h k trimethyl, increased igf- promoter and promoter ( ± , ± %) as compared to dio controls. conclusion: adult obese iugr males exposed to normal postweaning diet have increased hepatic igf- a mrna and h k dimethylation and trimethylation of igf- than dio controls. changes in igf- in adulthood from a prenatal insult thus suggest that igf- is programmed during the fetal period and may be associated with programmed adult obesity. rebekah elkins, pandu gangula, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa; internal medicine, university of texas medical branch, galveston, tx, usa. objectives: we previously reported that the offspring of rats fed % protein during gestation develop hypertension at two to four months and that the hypertension is exacerbated in males. this study is to evaluate: ) changes in estrogen receptor (er) angiotensin ii subtype receptor (at -r) and endothelial nitric oxide synthase (enos) in the mesenteric artery and aorta of offspring and assess if these changes, if any, are gender specific. methods: pregnant sprague dawley rats were fed either % protein (ctrl) or % protein (lpd) from day of gestation. the offspring were evaluated for hypertension by means of systolic blood pressure measurements. at four months for the males and nine months for the females, mesenteric artery and aorta were collected in rnalater. expression of estrogen receptor a (er-a) and b (er-b), at -r, and enos were analyzed by western immunoblotting and rt-pcr and expressed relative to b-actin or s. results: mesenteric artery shows no differences between ctrl and lpd female offspring in at -r, enos, er-a or er-b. similarly mesenteric artery shows no diet exposure related changes in at -r, er-a or er-b in male offspring. however, enos expression was lower in mesenteric artery of lpd male offspring. on the other hand, in the aorta both er-a and er-b levels are lower in lpd female offspring while there were no changes in at -r or enos. no changes in at -r, er-a or er-b were observed in male offspring aorta of ctrl and lpd rats. conclusion: the in utero exposure to lpd results in adult hypertension in both male and female offspring. some mechanisms for hypertension include the decrease in er-a and er-b but not at -r or enos in females, and the decrease in enos but not at -r or er in males indicating gender related differences. offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. our objective was to investigate the effects of either severe undernutrition during late gestation or lactation period on blood pressure and the development of vascular function in male rat offspring. we use normal pregnant wistar rats (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b) and % restricted after delivery to the end of lactation period (group c). the offspring was measured blood pressure at and weeks by using indirect tail-cuff method. rings of thoracic aorta with intact endothelium from the male offspring of a and b at weeks, were equilibrated at g passive tension in organ chambers filled with krebs-henseleit solution continuously bubbled with %co in air ( °, ph . ) for isometric tension recording. concentration-response relationships to norepinephrine (ne) and angiotensinii(atii) were obtained in the absence or presence of n(omega)-nitro-l-arginine methyl ester (l-name) or a selective atii type- receptor blocker (valsartan). responses to cumulative concentrations of sodium nitroprusside (snp) and to - m oxyhemoglobin (hb, nitric oxide scavenger) were also determined. contractions were expressed as a percent of the reference contraction induced by potassium chloride ( mm). statically analysis was performed using one-way annova. results: body weight was significantly reduced in b offspring compared to a and c in male offspring at day (p< . ). at weeks the body weight of offspring of b was no difference to catch up compared to a and c offspring. systolic and diastolic blood pressures were significantly elevated at both and weeks in offspring of b > c >a. ne concentration-dependently stimulated tension of aortic rings from in a and b offspring, which was not significantly (n= ). maximal contractions to ne were significantly stimulated by l-name in a (p< . ), but not b offspring. valsartan significantly inhibited aortic contractions by ne in r (p< . ), but not a offspring. there was no significant difference on responses of aortic rings by atii, snp and hb in a and b offspring. conclusions: severe under nutrition during not only late gestation but also lactation period induced hypertension in male rat offspring in adulthood. fetal origin of adult hypertension might be vascular endothelial dysfunction. fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. objective: exposure to undernutrition during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of either high fat nourishment or undernutrition during lactation period on blood pressure is unclear. our objective was to investigate the most effective maternal nourishment and feeding period for offspring induced adulthood hypertension in using high-fat diet (hfd). study design: we use types pregnant wistar rats as fed with normal nutrition (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b), % restricted after delivery to the end of lactation period (group c), with a high fat diet (hfd) during gestation to lactation period (group d) and with hfd nutritionally % restricted from the day of delivery to the end of lactation period (group e). the offspring was measured body weight (bw) and measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using one-way annova. results: bw was significantly reduced in b offspring compared to another (a, c, d, e) male offspring at day (p< . ). at day after delivery, bw was significantly reduced in c, e offspring compared to a, d in male offspring (p< . ). at weeks old, bw of all type offspring was no difference. systolic and diastolic blood pressures were significantly elevated at weeks in offspring of d> b > e > c >a. (p< . , vs. a). at weeks, hypertensive offspring as b>d>e>c>a (p< . , vs. a). at weeks, d> e > b > c >a (p< . , vs. a). conclusions: maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. in case with normal food, restrictive feeding during late gestation is more effective than lactation period for inducing hypertensive male offspring. regulation of maternal feeding not only during late gestation but also lactation period may control adulthood hypertension. the strongest epigenetical factor of maternal nutrition is high fat feeding during pregnancy to lactation period for f blood pressure, respectively. or residence at high altitude, impacts fetal growth and development. in a preliminary study, we observed a significant decrease in birth weight, subsequent compensatory postnatal growth, and an increase in relative right ventricular (rv) weight at postnatal (pn) day in female offspring of rats exposed to hypoxia ( , ft; . % po ) from days thru of gestation (dga). thus, our objective was to further elucidate the impact of prenatal hypoxia on fetal growth and postnatal development. methods: pregnant dams (hx, n= ) were hypoxic from - dga with additional control dams either fed ad libitum (al, n= ), pair-fed with the hx dams throughout gestation (pg, n= ), or only pair-fed during the window of hypoxia (ph, n= ). female offspring from hx, pg, and ph dams were cross-fostered onto additional al dams (n= /litter) by h after birth. results: at birth, there was no difference in litter size; however, body weight (bw) of the hx, pg, and ph pups was lower (p< . ) than that of al pups, and hx pups were lighter (p< . ) than ph pups. weight of hx offspring remained lower (p< . ) than al pups until the termination of the study at pn , while the pg and ph pups reached weights comparable to the al offspring by pn . relative to bw, heart weight and left ventricular/septal (lvs) weight was not different among groups; however, right ventricular weight (rv/bw) was greater (p< . ) in the hx offspring at pn as was rv/lvs (p< . ). cardiac function was evaluated by echocardiography at pn . rv wall thickness was % greater (p< . ) in hx pups as compared to al pups, confirming the significantly higher relative rv weight observed at necropsy. pep, pep/at, and pep/et were %, %, and % higher respectively in the hx offspring relative to the al offspring. lv end diastolic and end systolic diameters were smaller (p< . ) in hx and ph offspring relative to the al group. myosin heavy chain (mhc) and mrna concentrations in the rv were evaluated by qrt-pcr, and the mhc / mrna ratio was greater (p< . ) in the hx pups. conclusion: prenatal hypoxia from - dga impacted both fetal and postnatal growth, altered postnatal heart development and function, with the primary impact being on the rv. supported by nih hd . reproductive sciences; pharmacology experimental therapeutics, university of maryland, baltimore, md, usa. background: exposure to nicotine (nic) is a significant risk to normal fetal development. fetal nic, which readily crosses the placenta, can be acquired from pregnant mothers by smoking or nicotine replacement therapy. the impact of nic on fetal organs may be mediated directly and/or via intrauterine hypoxia (hpx) via constriction of the uterine circulation. in adult hearts, both nic and hypoxia stimulate gene expression of matrix metalloproteinases (mmp), although the study of nic and hypoxia on gene expression in fetal organs remains incomplete. because mmps are involved in the regulation of extracellular matrix turnover and cardiac remodeling, we tested the hypothesis that prenatal nic and intrauterine hpx upregulate protein expression and activity of mmp in the fetal guinea pig heart. methods: pregnant guinea pigs were placed in either normoxia (nmx) or hpx ( . %o in chamber) for d prior to term ( d). in two separate groups, nic was also added to the drinking water ( mg/kg/d) for d at a dose that generates fetal nic levels ( ng/ml cotinine) equivalent to a moderate smoker. anesthetized near-term fetuses ( d) were excised and weighed. left ventricles of hearts were obtained and frozen at - c for storage. mmp protein levels and enzymatic activity were measured by western analysis and gel zymography, respectively. results: nic alone (nmx+nic) decreased (p< . ) fetal body wt by %, increased (p< . ) the relative fetal brain wt (brain wt/fetal body wt ratio) by . % and had no effect on relative placental or fetal heart wts. hpx alone decreased (p< . ) fetal body wt by . %, increased the relative fetal brain wt by % but, in contrast to nic alone, increased relative placental wt by . %. both mmp protein levels (mmp /a-actin density values) and activity (clear band density) were increased (p< . ) by nic alone (by . and . fold, respectively) and hpx alone (by . and . fold, respectively). in addition, both protein and activity levels of hpx hearts were further increased by nic (by . and . fold) compared to hpx alone. conclusion: prenatal nic upregulates mmp expression in nmx fetal hearts and is potentiated by hpx. this suggests that under conditions of intrauterine stress cardiac remodeling by mmp activation may be an important mechanism by which nic and hpx affect fetal heart function. objective: in addition to peripheral hypoglycemic effects, insulin induces central anorexigenic responses via stimulation of the phosphoinositide- kinase (pi k) pathway and cellular growth by mitogen activated protein kinase (mapk) pathway. the pi k signaling cascade is activated by insulin binding to its receptor (ir), recruiting ir substrate (irs- ), and phosphorylating pi k. activated pi k in turn causes phosphorylation of protein kinase b/akt which subsequently modulates hypothalamic anorexigenic responses. in contrast, the mapk (erk /erk ) signaling pathway likely involves irs- . further, insulin signaling is inhibited by the lipid phosphatase pten. we have previously shown that maternal food restriction (mfr) during pregnancy results in iugr newborns that develop hyperphagia, obesity and insulin resistance as adults. we sought to determine if altered hypothalamic basal insulin signaling expression of pi k and mapk pathways contribute to reduced satiety responses and thus enhanced growth in iugr newborns methods: pregnant control dams received ad libitum (n= ) food, whereas study dams were % mfr (n= ) from pregnancy day to to produce iugr newborns. at day , hypothalamic region was dissected and analyzed for mrna levels (real time rt-pcr) of insulin signaling components via pi k (ir, irs , pi k and akt) and mapk (irs , erk , erk ) pathways, and pten. data is presented as fold difference normalized to beta- -microglobulin. results: at d of age, iugr pups exhibited downregulation of the entire pi k pathway with significantly decreased (p< . ) mrna levels of ir ( . -fold), irs- ( . -fold), pi k ( . -fold) and akt ( . -fold). further, iugr pups showed similar decreased mrna expression of erk ( . -fold) and erk ( . -fold). however pten expression was similar to the controls. conclusion: reduced insulin-mediated pi k signaling likely contributes to the suppressed anorexigenic responses and development of obesity in iugr offspring. reduction of central mapk signaling suggests a potential maldevelopment of additional neuronal pathways in iugr offspring. objectives: in the rat, uteroplacental insufficiency restricts fetal growth and impairs mammary development further compromising postnatal growth. both male and female growth-restricted offspring have a reduced nephron endowment but only males develop hypertension with glomerular hypertrophy, which can be reversed by improving the lactational environment. this study used cross-fostering to assess the influence of the prenatal and postnatal environments on renal development and nephrogenesis. methods: bilateral uterine vessel ligation (restricted, r) or sham surgery (control, c) was performed on day of gestation in wky rats. control and restricted pups were cross-fostered onto c or r mothers on postnatal day (pn ). post mortem was carried out on pn (c and r) and pn (c-on-c, c-on-r, r-on-c, r-on-r). results: body and kidney weights were decreased in r and r-on-r pups on pn and pn (p< . ). there was some evidence of accelerated pup growth for r-on-c relative to r-on-r on pn . male, but not female, relative bmp mrna expression on pn was higher in r than c (p< . ) while gdnf, tgf and at receptor were not different. on pn , wnt (but not at r, vegf-a) mrna expression (males only) was relatively higher in r-on-r (p< . ) when compared to c-on-c (p< . ). this and the histological analyses suggests an up-regulation of nephrogenic activity with more immature nephrons (males and females) in r-on-r (p< . ) when compared to c-on-c, while r-on-c remained intermediate. intrauterine growth-restricted pups were born lighter and with smaller kidneys. this was partially rescued by improving lactational nutrition (r-on-c) at pn . higher bmp mrna expression indicates impaired branching morphogenesis in pn r male, but not female kidneys, suggesting the timing and/or molecular mechanisms underlying the nephron deficit may be sex specific. at pn there was evidence of extended and increased nephrogenic activity in r-on-r, however, this was unable to restore the later nephron deficit. improved lactation for r-on-c, which prevented the adult nephron deficit and hypertension, increased and extended nephrogenesis to a lesser degree than r-on-r suggesting that the restoration of nephron endowment was likely to have occurred prior to pn . amy m tetrault, sarah b lieber, marya shanabrough, tamas l horvath, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa. objective: classically recognized for its role in energy balance, body weight and appetite, ghrelin has also been implicated in reproduction. ghrelin (-/-) mice are infertile while administration of ghrelin to wt mice results in decreased litter size and constrained embryonic growth. here we investigate the effect of maternal ghrelin deficiency on in utero developmental programming of the female reproductive tract. hox genes determine developmental identity of the paramesonephric duct. we determined that hoxa is regulated by ghrelin in vitro and that in utero ghrelin deficency alters f hoxa gene expression and reproductive success. methods: wild-type females mice parented by ghrelin +/-b d f (ghrellin deficient) mice were analyzed for litter size, oocyte, and corpus luteum number. rna was extracted from the uterus of mice exposed to ghrelin deficiency in utero. ishikawa cells were treated with ghrelin with/without receptor (ghsr) antagonist, or saline and rna extracted. in both hoxa expression was analysed by real time rt-pcr normalized to -actin and also determined by ihc. experiments were repeated in triplicate and mrna expression compared by student's t-test. results: wild-type female offspring of ghrelin deficient dams had smaller litter sizes than controls (n= , . ± . pups; n= , . ± . pups, respectively; p< . ). no differences were seen in oocyte or corpus luteum number suggesting a uterine defect. hoxa mrna and protein expression were decreased in the uterus of the f females. ghsr was expressed in uterine endometrium. treatment of ishikawa cells with nm to nm ghrelin resulted in a to % increase (p< . ) in hoxa expression. treatment with ghrelin and ghsr antagonist resulted in similar increases in hoxa expression indicating a non-receptor mediated mechanism. conclusion: ghrelin contributes to reproductive tract developmental programming; in utero ghrelin deficiency compromises reproduction in female offspring. the developmental effects of ghrelin were mediated by alteration in hox gene expression and not through the classic ghsr receptor. obesity and decreased ghrelin may lead to defects in developmental programming of the reproductive tract. these findings demonstrate the importance of nutrition, energy utilization and appropriate ghrelin levels on normal uterine development. we have previously studied the deleterious effects of lack of the endothelial nitric oxid synthase (nos ) in mouse dams and their offspring. our laboratory demonstrated that adaptive responses in subsequent pregnancies may offset the harmful effects of the genetic deficiency of nos . in this study we aimed to determine hepatic and renal histopathologic damage in nos deficient pregnant mice comparing animals carrying their first versus their second pregnancy. study design: gravid nos +/+wt and nos -/-ko mice during their first (p ) or second (p ) pregnancy were sacrificed at day of gestation. livers and kidneys were stained and analyzed for the presence and extent of histopathologic lesions. results: nos +/+wt dams displayed a low incidence of significant renal or hepatic lesions in either the first or the second pregnancy. in nos -/-ko mice the incidence of liver necrosis and inflammation during the first pregnancy was % and %, respectively. in nos -/-ko dams sacrificed during the second gestation the incidence rates for the same lesions were % and %, respectively (p< . ). this correlation persisted when we analyzed the relative severity of hepatic lesions between p and p animals. although a similar trend was observed, the difference between p and p animals with regards to kidney lesions did not reach the level of statistical significance in our study. conclusions: a second pregnancy in this animal model of hypertension was associated with a significantly improved hepatic histopathology compared with the first pregnancy. this observation is consistent with our previous studies showing a decrease in systemic vascular resistance in p versus p nos -/-ko mice. the beneficial effects of a prior pregnancy may partially underlie the phenomenon of a decreased risk of preeclampsia in multiparous versus nulliparous women. further studies are required to delineate the counterregulatory mechanisms leading to improved cardiovascular function in subsequent pregnancies in these genetically modified animals. maternal hypomethylation is associated with congenital heart defects in down syndrome. lmjw van driel, , r de jonge, wa helbing, bd van zelst, j lindemans, eap steegers, rpm steegers-theunissen. , , , obstetrics gynecology; pediatrics; clinical chemistry; epidemiolog y biostatistics; clinical genetics; erasmus university mc, rotterdam, netherlands. background: maternal age and hyperhomocysteinemia are risk factors for having a child with down syndrome (ds) and congenital heart defects (chds), respectively. evidence is rising that ageing is associated with a state of hypomethylation. objectives: to investigate whether the risk of a child with ds and chd is associated with maternal hypomethylation. methods: we conducted a case-control triad study at months after the index-pregnancy. case-children (n= ) were included if they had ds and chd. children (n= ) without a major congenital malformation served as controls. the concentrations of s-adenosyl methionine (sam), s-adenosyl homocysteine (sah), sam/sah ratio, and homocysteine in maternal blood were measured as biomarkers for methylation. the data were analyzed using the mann-whitney u test and a logistic regression model. results: maternal age was included in the model as potential confounder. the levels and the crude and adjusted or( %ci) of the biomarkers are shown in table . an increase of the sam/sah ratio with unit decreases the risk of a child with ds and chd with percent. moreover, every increase of mol/l of homocysteine . fold increases this risk. conclusions: maternal hypomethylation is significantly associated with an increased risk of having a child with ds and chd. since, the effects are confounded by maternal age, hypomethylation can be considered as feature of ageing. the developmental origins hypothesis postulates that during critical ontogenetic periods, transient environmental stimuli perturb developmental pathways and induce permanent changes in gene expression, metabolism, and chronic disease susceptibility. one likely mechanism is via early nutritional influences on epigenetic gene modification consisting of the presence of a methyl group on the carbon of a cytosine residue. this modification is responsible for an important form of gene regulation in eukaryotes. in the present study, we have tested the hypothesis that maternal low-protein diet altered epigenetic regulation of specific gene of the offspring. c bl/ female mice were mated and on the day the plug was detected, these females were then randomly allocated to be fed isocaloric diets consisting % protein or % protein. at delivery, offspring were killed and the livers were removed immediately, frozen in liquid nitrogen and stored at - c. genomic sequencing after bisulfite modification is used to study site-specific dna methylation. dna methylation status of oct- and sphk- gene upstream regions in the mouse liver was analyzed. hepatic oct- or sphk- promoter methylation was not significantly different between both groups. however, dna methylation pattern of the genomic dna is specific in low-protein diet group. aberrant oct- and sphk- gene expression may cause perturbations in cell differentiation. we suggest that the epigenetic mechanism consisting of dna methylation underlies the fetal programming theory. primary human cytomegalovirus (hcmv) infection during pregnancy can have devastating consequences for both the mother and fetus. hcmv infection has been implicated in the development of pre-eclampsia and intrauterine growth retardation (iugr), as well as congenital cmv syndrome in newborns exposed in utero. previously, we have shown that hcmv infection of placental cytotrophoblasts inhibits their normal invasion, proliferation, and migration. however, the mechanisms occurring during early establishment of placental infection are largely unknown. we assessed the impact of hcmv infection on cytotrophoblasts by performing immuno-based assays for various cytokines and cellular growth factors. we detected significant cytokine dysregulation at both and hours after in vitro hcmv infection of cytotrophoblast cells. soluble cytokines involved in recruitment of monocytes and macrophages (gro-a, mcp- ) were downregulated at both and hours after infection. sdf- , which is chemotactic for lymphocytes during early inflammation, was also decreased. these results suggest that recruitment of cells involved in the anti-viral immune response is being interrupted early in the course of infection by hcmv. additionally, a large decrease in the amount of soluble hgf was seen. hgf normally induces migration of cytotrophoblasts along the invasive pathway, and downregulation of this factor could severely affect these processes. finally, we saw increased amounts of soluble icam- , contrasted by decreased amounts of vcam- , indicating dysregulation of adhesion molecules that are necessary for successful placental invasion to occur. all together, these data indicate significant alterations in cytokine profiles as early as hours after hcmv infection, which could provide important clues to the pathogenesis of hcmv in placental invasion and inflammation. additional studies will further elucidate this dysregulation, and determine whether these effects are due to alterations in pre-existing cellular factors or if transcriptional alterations are involved. method: studies were performed in pregnant ewes (n= in each group) with twin fetuses at - days (very preterm) and - days (near-term). neither mother nor fetuses were instrumented prior to the time of blood collection. maternal blood was collected from the jugular vein before sedation. anesthesia was then induced with isofluorane, the abdomen was opened, fetuses exteriorized and blood collected from umbilical cords. blood samples were transferred to plastic tubes containing ethylene-diamino tetraacetic acid and reduced glutathione, plasma separated and stored at - c. commercial radioimmunoassay kits for ovine-crf (phoenix pharmaceuticals, b : . ± . pg/ml) and cortisol (diagnostic laboratory, b : . ± . ng/ml) were used according to the manufacturer's instructions. results: in very-preterm gestations, maternal and fetal plasma crf levels were undetectable. maternal, but not fetal, plasma cortisol levels were measurable ( ± ng/ml). in near-term gestations, both cortisol and crf were measurable in maternal (crf: ± pg/ml; cortisol: ± ng/m) and fetal plasma (crf: ± pg/ml; cortisol: ± ng/ml). plasma crf levels were higher in nearterm fetuses than in their maternal ewes (p < . ). conclusion: ovine plasma crf levels are measurable in maternal and fetal plasma in near-term but not very-preterm gestations. the absence of crf in preterm plasma, perhaps due to reduced placental expression and/or placental crf release, may contribute to the rarity of in utero meconium passage in preterm gestations. interleukin - objectives: interleukin- (il- ) is a pro-inflammatory cytokine produced in adipose cells. recent studies suggest il- may be a marker of maternal obesity and in utero fetal programming. our hypotheses were ) il- correlates with maternal obesity and ) il- mediates the effect of maternal obesity on infant birth weight. methods: the parity, inflammation, and diabetes (pid) study is a longitudinal study of adipokine levels in a diverse sample of pregnant women. we present a cross-sectional analysis of first trimester il- levels from non-diabetic women who underwent a live birth. the independent variable was il- (pg/ml), measured with monoclonal antibody elisa assays. the dependent variable was infant birth weight (gms). maternal bmi categories were: normal/underweight (< kg/m ); overweight ( - kg/m ); and obese (> kg/m ). data on demographic and clinical factors, nutrition and physical activity were collected at baseline. average il- levels were compared across bmi categories using anova. the association of il- levels with infant birth weight was estimated using multiple linear regression, adjusting for covariates. results: average il- levels were significantly higher in obese women ( . ± . ) compared to overweight ( . ± . ) and normal/underweight women ( . ± . ) [p< . ]. after adjustment, il- levels was positively correlated with pre-pregnancy bmi [regression coefficient (rc) . ; % ci: ( . , . )]. as shown in the table below, elevated levels of il- were statistically significantly associated with a . gm higher infant birth weight after full adjustment. each unit increase in pre-pregnancy bmi was associated with a gm higher infant birth weight. table . association of il- with infant birth weight characteristic regression coefficient % confidence interval interleukin- . . , . pre-pregnancy bmi . , gestational weight gain - - , bmi = body mass index; coefficients adjusted for demographics, clinical factors, pre-pregnancy bmi, gestational weight gain, non-fasting first trimester glucose levels, gestational age, nutritional intake and physical activity conclusion: il- and pre-pregnancy bmi were associated with infant birth weight after adjustment for covariates. our findings suggest that the effect of maternal obesity on infant birth weight may be mediated through il- or an alternative independent pathway. we have demonstrated that -tetrahydrocannabinol (thc), in physiologically relevant concentrations, inhibits the growth and tight transcriptional control of the bewo trophoblast cell line . the mechanism involved the decreased expression of the transcriptional regulator histone deacetylase (hdac ). in these experiments we sought to answer the question, 'does anandamide (aea) work in the same manner as thc?' methods: the first trimester human trophoblast cell, bewo were plated at or x cells /well to -well and -well plates for growth and rna experiments, respectively. after growing to - % confluence, cultures were treated with varying concentrations of aea up to a maximum of m for hr. cell numbers were determined using the xtt apoptosis/proliferation assay. total cellular rna was prepared and the relative levels of hdac and gapdh determined by end-point rt-pcr. aea exhibited an inhibitory effect on the bewo cell cultures, but only at concentrations in excess of m where confluency was significantly reduced from % at m to - % at m and m (*p< . ; one-way anova with tukey's hsd test; n= ). cultures treated with m and m aea did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase in the shedding of cells into the spent medium. bewo cells treated with aea showed a dose-dependent decrease in hdac mrna expression with a significant effect at . m (*p< . ; oneway anova with tukey's hsd test; n= ). at this dose, the effect of aea had reached an effective maximum decrease in hdac mrna levels ( %) because hdac mrna levels were not decreased further by either m aea ( %) or m aea ( %). the alteration of hdac gene expression by aea in bewo cells with its associated decrease in cell number suggest that the trophoblast cell may be an important target for circulating endocannabinoids during the st trimester of pregnancy. the data indicates that although exocannabinoids and endocannabinoids both inhibit bewo cell growth, they do so using different transcriptional mechanisms. further understanding of the mechanism(s) by which aea alters placental physiology may lead to new strategies for the prevention of pregnancy complications such as st trimester miscarriages. ( ) taylor background: anandamide (aea) exerts its effects by acting on two cannabinoid receptors, cb and cb with the main regulator for aea levels being the metabolising enzyme, fatty acid amide hydrolase (faah). aea, cb , cb and faah constitute the endocannabinoid system and previous studies have shown that faah and cb are expressed in term human placenta( ) suggesting that the endocannabinoid system might be present earlier in gestation. this study aimed to document changes in faah, cb and cb expression in the placenta during the first trimester of pregnancy. methods: first trimester samples ( to weeks gestation) were fixed in % neutral-buffered formalin for days before embedding into paraffin wax or frozen in liquid nitrogen for rna analysis. for immunohistochemistry, faah polyclonal antibodies were used at an optimal dilution of : in pbs, cb at : and cb at : . transcripts were measured using q-pcr with gene-specific primers. results: immunoreactive faah, cb and cb were detected in all samples. faah immunoreactivity in the syncytiotrophoblast increased between the th and th gestational week and by week faah was barely detectable within large parts of the placenta. simultaneously, faah immunoreactivity increased in the mesenchymal core of the developing villi. immunoreactive cb and cb localised to the syncytiotrophoblast, cytotrophoblast and mesenchymal core with cb immunoreactivity showing diminished intensity after week , although this did not reach significance at the transcript level. cb immunoreactivity was absent from fetal blood cells and infiltrating maternal plasma cells, whereas cb and cb immunoreactivity was detected in endothelial cells but not in the vascular smooth muscle cells of blood vessels. the intensity of cb immunoreactivity in the syncytiotrophoblast differed from that of cb and faah in that it remained constant throughout. conclusion: the data suggest that placental faah and cb levels do not alter significantly during the first trimester, but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core. the significant loss of cb expression from the syncytiotrophoblast after the th week of gestation, a point of critical alteration in the developing placenta, suggests that its retention may be detrimental to normal placental development. reference: park, b. et al., ( ) hankins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: numerous angiogenic proteins synthesized in the placenta are thought to be involved in placental vascularization and development; however, the molecular mechanism modeling the angiogenic process in early pregnancy remains elusive. calatonin gene-related peptide (cgrp) is a multifunctional peptide expressed at the human implantation site; but its influence on in vitro angiogenesis by human micro vascular endothelial cells is not known. objective: the present study was designed to determine the influence of cgrp on angiogenesis by human dermal microvascular endothelial cell (hdmvec) in vitro. methods: hdmvecs (vec technologies) were cultured in mcdb- complete solution containing cgrp ( - m), cgrp plus its antagonist cgrp - ( - m), in well plates with x cells per well. the existence of cgrp receptor components calcitonin receptor-like receptor (crlr) and receptor activity modifying protein (ramp ) was determined using immunofluorescent staining. cell proliferation was examined using methylthiazoltetrazolium (mtt) assay. the pro-angiogenic bioactivity of cgrp was evaluated using cell migration and capillary like tube formation on the matrigel. results: ) immunofluorescent staining showed that cgrp receptor components crlr and ramp are abundantly expressed by hdmvecs. replacement of the primary antibodies with preimmune serum resulted in a negative staining; ) cgrp dose-dependently ( - to - m) stimulated hdmvec proliferation, and this effect was totally blocked by cgrp antagonist, cgrp - ; ) quantitative analysis for cell migration revealed that cgrp increases hdmvec migration in a dose and time-dependant manner; and ) cgrp promotes hdmvec capillary like tube formation, and the length of capillary tube induced by cgrp ( - m) was significantly increased over that of the untreated controls. this increase was observed at hours of treatment and further increase was noted at hours of culture. conclusion: cgrp induces in vitro angiogenesis by promoting microvascular endothelial cell proliferation, migration and capillary like tube formation. therefore, trophoblast derived cgrp at the implantation site may play a role in placental angiogenesis and fetal growth. over-expression of socs- gene promotes il- production by jeg- trophoblast cells. qin dong, , ruping fan, yang gu, david f lewis, yuping wang. biochemistry, harbin medical university, harbin, heilongjiang, china; obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: suppressor of cytokine signaling- (socs- ) plays an important role in negative regulation of inflammatory response in biological cells. evidence has shown anti-inflammatory cytokine il- expression was significantly reduced in trophoblasts of preeclamptic (pe) placentas. we sought to determine if over-expression of socs- in placental trophoblasts could promote il- production. methods: full-length socs- open reading frame (socs- cdna) was generated by rt-pcr from total rna samples isolated from human leukocytes and cloned into a pzsgreen -n vector, which encodes a green fluorescent protein zsgreen . successful socs- cloning was confirmed by sequencing. for transfection, jeg- cells were placed into well/cluster plates at a concentration of . x /well. the tranfection was carried out with . g of socs- /zsgreen plasmid (psocs- /zsgreen ) for hours when cell reached - % confluence. siport lipid were used. jeg- cells transfected with zsgreen plasmid (pzsgreen ) only was used as control. after approximately hours of transfection, cells were treated with il- at and ng/ml. medium was then collected and measured for il- by elisa. il- production was calculated as the percentage of increase by psocs- /zsgreen transfected cells compared to the cells transfected with pzsgreen only. result: il- production was increased by psocs- /zsgreen transfected jeg- cells compared to the cells transfected with pzsgreen only when stimulated by il- , control: . % increase; ng/ml il- : . % increase and ng/ml il- : % increase, p< . , respectively. data are means from three independent experiments. conclusion: over-expression of socs- gene could promote il- production by placental trophoblast cells. reduced sil- r release and increased ratio of sgp /sil- r production by placental tissues from women with preeclampsia. shuang zhao, ruping fan, jingxia sun, yang gu, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa; obstetrics and gynecology, first hospital, harbin medical university, harbin, heilongjiang, china. objective: placental tissue/trophoblasts release more inflammatory cytokines (il- , il- and tnf ) in preeclampsia (pe) than in normal pregnancies. however, the reason for increased inflammatory cytokines released by pe placentas is not clear. soluble il- receptor (sil- r) and membrane receptor il- /gp complex play an important role in the negative regulation of cytokine signaling in suppressor of cytokine signaling (socs) pathway. in contrast, soluble gp (sgp ) is an antagonist for il- /il- r trans-signaling. this study was undertaken to determine sil- r and sgp production by villous tissues from normal and pe placentas. methods: placentas delivered by normal and pe pregnant women were used in this study. placental explants were incubated with dmem for h. the culture medium was collected. placental villous tissue productions of sil- r and sgp were measured by elisa. all samples were assayed in duplicate. data are expressed as mean ± se and analyzed by mann whitney test. a p level < . was considered statistically different. results: placental tissues from pe produced significantly less sil- r than tissues from normal pregnancies, . ± . vs. . ± . pg/mg of wet tissue, p< . . soluble gp production was relatively compatible between pe and normal placental tissues: . ± . vs. . ± . pg/mg of wet tissue. the ratio of sgp /sil- r release was significantly higher in pe than in normal placentas, . ± . vs. . ± . , p< . . conclusion: reduced sil- r production and/or increased ratio of sgp /sil- r production by pe placentas suggest less cytokine inhibitory activity in pe placentas, which may contribute to the increased toxic cytokine production in placentas from pe. using chemiluminescence immunoassays. peter s uzelac, jing dai, frank z stanczyk, daniel r mishell, jr. obstetrics and gynecology, university of louisville, louisville, ky, usa; obstetrics and gynecology, university of southern california, los angeles, ca, usa. objective: characterizing human chorionic gonadotropin (hcg) levels throughout normal pregnancy is critical to its use as a bio-marker for abnormal gestations. there is a paucity of data describing hcg trends during pregnancy and most of the relevant studies use older, less specific assays. our first objective was to characterize hcg levels throughout normal gestation using two different contemporary chemiluminescence immunoassays. our second objective was to compare hcg patterns in healthy gestations with pregnancies affected by diabetes, a common obstetrical complication. methods: a single blood sample was collected from healthy pregnant women and diabetic pregnancies. gestational age was confirmed by ultrasound. serum hcg levels were quantified by chemiluminescence immunoassays using the acs- and immulite systems. data was grouped in -week intervals until weeks of gestation and -week intervals thereafter, with to samples in each interval for healthy women and to samples in each interval for diabetic women. paired t-test and wilcoxon rank sum test were used for statistical analysis. results: using the acs- system, mean hcg levels (miu/ml) for healthy pregnant women were , at - weeks, , at - weeks, , at - weeks, , at - weeks, , at - weeks, and , at - weeks. mean hcg levels obtained from the immulite system were similar. for diabetic pregnancies, mean hcg levels (miu/ml) were , , , , , , , , , and , , respectively. between - weeks of gestation, the hcg levels were significantly lower in diabetic pregnancies (p= . ) compared to healthy controls. ) compared to healthy pregnancies, diabetic gestations have significantly lower peak hcg levels. the cause of this difference is an area deserving further investigation. background: mouse and human placentae share several cellular and molecular features, including a haemochorial interface, allowing the murine labyrinth to be compared with the human fetal placenta. there is an autonomous embryonic ras from at least the time of implantation. ace converts angiotensin i to the active angiotensin ii (angii). angii's actions as a pro-inflammatory agent, in promoting cell migration, angiogenesis and cellular growth and apoptosis, strongly suggest a rôle in placentation. hypothesis: there would be counterregulation of the placental ras if the effect of ace were removed. this study investigated for the first time whether the knockout of somatic ace affected the expression and localisation of various components of the ras in the placentae of wild-type (wt/wt), heterozygous (wt/ ) and ace knockout ( / ) mice. methods: immunohistochemistry (dako envision plus) was used to localise and semiquantify ang type receptor (at r), ang type receptor (at r), ace, and ace- in the three genotypes (n= /group). placental sections were blinded to genotype; a score range of - was used. the test was used (spss version . ) to analyse the difference in staining score by genotype. results: immunoreactivity of all antigens increased in the placental labyrinth of / mice compared to wt/wt and wt/ mice (at r p< . ; at r p<< . ; ace p<< . ; ace- p<< . ). at r and ace- displayed increased staining in the fetal vascular endothelium of / mice (at r p<< . ; ace- p= . ), and in the cells lining the maternal central artery (at r p<< . ; ace- p<< . ). ace- expression was very high in cytotrophoblast lining the maternal blood space in all genotypes. no gross structural differences were seen. comment: the antibody used did not differentiate between ace and the membrane-bound "testicular"-ace (tace). immunohistochemically-identified ace expression was upregulated in / placentae despite the loss of somatic ace, suggesting that t-ace can be expressed placentally. we believe this is the first demonstration of such expression. ace and t-ace are catalyticallysimilar in converting angi to angii. furthermore, angii acting via the at rs is vasodilatory and ace- catalyses production of the vasodilatory ang ( - ); placental blood flow was presumably well-maintained and pregnancy outcome is normal in / mice. it is generally accepted that prostaglandin production plays a crucial role in both term and preterm parturition, and recently the administration of alpha hydroxyprogesterone acetate has been shown useful in preventing preterm labor. what is not clear is the biochemical pathway of prostaglandin production during labor and what, if any, effect alpha hydroxyprogesterone acetate has on this pathway. in order to address this question, we used immunohistochemical staining techniques to evaluate the effect of treating human placental amnion and chorion decidua with alpha hydroxyprogesterone acetate. membranes from unlabored patients were obtained at cesarean section and immediately seperated into control and drug treated specimens. controls were from the same placenta and were subjected to all experimental procedures except for the addition of alpha hydroxyprogesterone acetate to the culture media. specimens were compared at zero, six, and twenty four hour intervals. at the appropriate time, each specimen was formalin fixed and then paraffin blocked. tissue sections were then mounted on slides which were immunohistochemically stained using appropriate primary and secondary antibodies and standard techniques. the slides were then analyzed via light microscopy for changes in staining of three enzymes involved in prostaglandin production--cyclooxygenase (cox- ), cyclooxygenase (cox- ), and -hydroxy prostaglandin dehydrogenase (pgdh). compared to control, the slides treated with alpha hydroxyprogesterone acetate had differing amounts of enzyme expression. cox- was relatively unchanged and pgdh was only slightly increased, but cox- was noticeably decreased in the treated slides. these results were time dependent. this data suggests that alpha hydroxyprogesterone acetate decreases prostaglandin production in fetal membranes primarily by downregulation of the cyclooxygenase enzyme. objectives: in the human placenta, proliferation, differentiation and fusion of cytotrophoblasts (ct) are essential events in the formation of the multinucleated syncytiotrophoblast, however the regulation of these processes is poorly understood. using an explant model of human first trimester placenta we have established that both igf-i and -ii enhance ct proliferation, differentiation and survival mediated via igf r signalling. we have also shown that non-specific inhibition of protein tyrosine phosphatases inhibits igf-mediated signalling in trophoblast; therefore, we have now used sirna-mediated knockdown to investigate the role of the tyrosine phosphatase shp- in this pathway. methods: amaxa nucleofector technology was used to deliver shp- or scrambled sirna ( nm) to bewo cells or first trimester villous tissue fragments. knockdown was confirmed by q-pcr and western blotting. transfected cells and tissue were maintained in culture for hours, then treated with igf-i or igf-ii ( nm) for a further hours before immunohistochemical (ihc) analysis for cell proliferation (ki , brdu) or apoptosis (m ). results: sirna-mediated knockdown of shp- in bewo cells ( % reduction on western blot) demonstrated that igf-induced proliferation was reduced from . ± . % to . ± . % (p< . , n= ). ihc analysis of tissue demonstrated that shp- is localised to ct. following knockdown ( % decrease by q-pcr), igf-i-and igf-ii-induced ct proliferation was decreased by . ± . % and . ± . % respectively (p< . , n= ). furthermore, the ability of igf-i-and igf-ii to prevent ct apoptosis (m staining) was reduced by . ± . % and . ± . % respectively (p< . , n= ) after shp- knockdown. conclusions: igf stimulation of cytotrophoblast proliferation is mediated by shp- . exogenous igf rescues cytotrophoblast from apoptosis, and this pathway is also shp- -dependent. villous endothelial cells. emily j su, zhi-hong lin, ping yin, scott reierstad, joy innes, serdar e bulun. obstetrics and gynecology, northwestern university feinberg school of medicine, chicago, il, usa. background: within the human vascular system, estrogens have been shown to enhance vasodilatation in both normal and abnormal endothelium. estrogenic function occurs by activation of one or both of two estrogen receptors, estrogen receptor-alpha (esr ) and estrogen receptor-beta (esr ). these estrogen receptors are expressed in a wide variety of tissues. within the vasculature, estrogen receptors regulate the expression of multiple vasodilator and vasoconstrictor proteins. specifically, esr has been shown to be critical in maintaining normal vascular physiology in a murine model, where esr knock-out mice demonstrate significant systolic and diastolic hypertension. we hypothesize that within placental endothelium, estrogen plays an important role in maintaining normal vascular function that is critical for normal fetal growth and development. methods: term placentas from uncomplicated pregnancies were obtained, and the decidua was removed. an iv cannula was inserted into the umbilical vein, which was perfused with a collagenase/dispase solution. the perfusate was collected and subjected to further purification. these cells were cultured in complete medium, and after the initial passage of these cells, purity was confirmed via immunofluorescence and flow cytometric studies. estrogen receptor expression was determined in these cells via western blotting. additionally, these endothelial cells underwent treatment with varying doses of estradiol ( - m to - m), and quantitative real-time pcr was performed thereafter for mrna levels of various genes important in prostanoid production. results: western blotting demonstrated that esr is the only estrogen receptor expressed within villous placental endothelial cells. estradiol induced cyclooxygenase- (cox- ) mrna levels -to -fold, as quantified by real-time pcr (p< . ). conversely, there was no effect of estradiol on cyclooxgenase- (cox- ). conclusion: these results suggest that estradiol and esr are important in mediating the balance of prostanoid production that is essential in maintaining placental vascular health. future studies will further delineate estrogenic effects on prostanoid production within placental endothelial cells in health and disease. supported by the smfm/aaogf scholarship award and the nih grant u -hd . previously we established that proteins secreted by the decidua promote the differentiation of extravillous trophoblasts (evt) from a proliferative phenotype (characterized by cx , her- and alpha integrin protein expression) to an invasive phenotype (characterized by her- and alpha protein expression). the ability of decidua-conditioned media (dcm) to induce trophoblast differentiation was inhibited in the presence of the her- receptor antagonist, ag . furthermore, dcm-induced jar cell migration was also attenuated in the presence of ag . thus, the purpose of this study is to define the role of her signaling in evt differentiation and invasion. methods: evt differentiation was assessed in placental villous explant outgrowths and jar cells using antibodies against markers of the proliferative and invasive phenotypes. trophoblast migration was assessed using jar cells in transwell migration assays. results: treatment of placental villous explants with egf, a her- ligand, resulted in the downregulation of her- and an upregulation of her- expression, as well as an induction of alpha integrin expression. pre-treatment of placental villous explants with ag blocked this effect. in the jar cell line, egf treatment mimicked the differentiation-promoting effects of dcm by downregulating her- and upregulating her- expression, effects that were both blocked when jar cells were pre-incubated with ag . in contrast to dcm however, egf stimulation did not induce jar migration. stimulation of jar cells with hb-egf, a her- /her- heterodimer ligand, induced jar migration in a dose-dependent manner. analysis of dcm using antibody arrays confirmed the presence of many members of the egf family including hb-egf. immunohistochemical assessments of placental villous explants verified the expression of her- in evt outgrowths and in jar cells; her- expression was not affected by stimulation with either egf or hb-egf. conclusions: her signaling is an important and necessary component of the invasive evt differentiation cascade. our data supports a role for her- signaling in the induction of the invasive evt phenotype. chandrasekhar thota, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: parathyroid hormone related peptide (pthrp) is expressed in trophoblast cells and may play a role in placental growth and function. studies conducted in pregnant rats using pthrp antagonist showed decreases in fetoplacental growth during mid gestation. objective: to assess the effects of pthrp silencing on the expression of growth factors in immortalized first trimester trophoblast cells (htr- /svneo cells). methods: htr- /svneo cells cultured at º c and %co in rpmi- medium supplemented with % fbs were transiently transfected with nm of three different sirna sequences of pthrp, si , auaccuaacucaggaaacuuu; s i , g a g c u g u g u c u g a a c a u c a u u ; a n d s i , caagauuuacggcgacgauuu. for control, a scrambled sirna sequence was used. at % confluency, cells from each well were split and transfected again in triplicates with respective sirna sequences. total rna was isolated using trizol reagent h after transfection, and protein was extracted using lysis buffer h after transfection. the isolated rna and protein were subjected to reverse transcription and polymerase chain reaction (rt-pcr) and western analysis, respectively, using primers and antibodies specific for pthrp, plgf, vegf and lif. the results are expressed relative to either s for changes in mrna expression or -actin for changes in protein expression. results: rt-pcr of total rna obtained from htr cells subjected to double transfection with sirnas for pthrp showed a significant decrease in pthrp expression. expression of growth factors plgf, vegf and lif showed decreases with all the three sirna sequences used compared to the scrambled sequence. western analysis of cell lysates obtained from htr cells subjected to transfection with sirnas for pthrp showed a significant decrease in protein expression of pthrp and vegf. however the protein expression for plgf, and lif decreased in cells transfected with only si sequence of pthrp. conclusions: our studies showed that transient transfection of htr cells with sirna for pthrp caused decreases in both mrna and protein expression of pthrp. our results further suggests that decrease in pthrp peptide in transfected cells resulted in a decrease in vegf, plgf and lif suggesting that pthrp may play role in regulating trophoblast cell functions. objective. maternal obesity poses an increased risk to the fetus during pregnancy, and has long term consequences for the progeny. we tested the hypothesis that maternal caloric excess effects growth-related gene expression changes in the murine placenta. methods. female c bl/ mice were fed a hypercaloric diet ( % fat, % sugar) or standard chow for six weeks prior to mating and throughout pregnancy. near-term (day gestation) the dams ( controls, overfed) were sacrificed. following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on regulated genes. results. maternal overfeeding was associated with a two-fold increase in body fat mass. probe sets, corresponding to genes showed differential expression (p < . ); twenty-seven of which were up-regulated, and ten down-regulated, as compared to the placenta of control fed dams. of note, several genes related to obesity, diabetes, dna methylation, and the tgf beta pathway were differentially expressed. conclusions. diet-induced obesity in mice was associated with altered placental gene expression, including genes involved in tgf beta signaling and dna methylation pathways. these findings may have important implications for placental growth and epigenetic regulation. (supported by usphs hd- , earnest eu framework , tommy's the baby charity, uk). objective. maternal dietary protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. to comprehend more completely stress responses to maternal protein restriction, we measured gene expression changes in the mouse placenta. methods. pregnant fvb/nj mice were fed an isocaloric diet containing % less protein than normal chow ( % vs. % protein content) from embryonic day . (e . ) to e . . following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on the regulated genes, and used both qrt-pcr and immunohistochemistry to verify the results. results. the weights of the e . pups were decreased % (p< . ). probe sets, corresponding to genes, were regulated by protein restriction (p< . ); ninety-one being up-regulated and down-regulated. of particular note, several genes related to the p pathway were up-regulated. along with p itself, positive regulators of p (zmiz , jmy, hipk ) and genes activated by p (inpp d, cebpa) were induced. for selected genes we confirmed these results using qrt-pcr and immunohistochemistry. conclusions. by microarray analysis, we have described the genetic response to maternal protein deprivation in the mouse placenta. we observed that pups were growth restricted, and genes related to the p pathway were regulated. we propose a model through which intrauterine growth restriction is triggered, in part, by activation of the p pathway. (supported by usphs hd- and the sgi medical student grant to cpg). purpose: human placental villous tissue cultures have been underused in the study of placental drug disposition. thus we assessed the utility of this model by studying the effect of time in culture on the viability and integrity of the tissue and the, expression and function of proteins involved in the formation and efflux of -chloro- , -dinitrobenzene (cdnb) conjugate , -dinitrophenyl-s-glutathione (dnp-sg) as a model system for phase ii metabolism and cellular efflux. methods: placental tissue samples were obtained within minutes of cesarean deliveries following normal pregnancies in three patients. villous tissue was cultured in m medium to hr. at , , , , , and hr post culture, villous tissue was preincubated without or with atpase inhibitor sodium orthovanadate, exposed to μm cdnb, rinsed and incubated in buffer at °c to determine formation and efflux of dnp-sg, which was assayed by hplc. changes in expression of gstp - , abc transporter isoforms b , c and g (abcb , abcc , and abcg , resp.) were assessed by immunoblotting. lactate dehydrogenase (ldh) release, methyl tetrazolium thiazolyl blue (mtt) incorporation, and total tissue glutathione content were monitored up to hr. villous tissue morphology was assessed by immunohistochemistry. results: villous tissue structure and protein expression of glutathione-stransferase isoform p - (gstp - ) and abcg remained unchanged over hr in culture. expression of abcb and abcc , and total tissue glutathione decreased with culture time. ldh release was unchanged up to hr and increased at hr, while mtt incorporation remained constant to hr and decreased at and hr suggesting a decline in tissue integrity and viability at hr. however, dnp-sg formation, dnp-sg buffer/tissue ratio, and the extent of inhibition of dnp-sg efflux by sodium orthovanadate remained unchanged through hr. sodium orthovanadate decreased the dnp-sg buffer/tissue ratio by . ± . % (p< . ), consistent with inhibition of apical abc transporters. conclusions: these results support the use of this model to study the coordinated function of metabolizing enzyme gstp - and apical abc transporters in the formation and efflux of the model substrate dnp-sg. the model may be useful to study metabolism and transport of other compounds. syncytiotrophoblast. shauna f williams, ewa fik-rymarkiewicz, stacy zamudio, nicholas p illsley. obstetrics, gynecology and women's health, umd-new jersey medical school, newark, nj, usa. introduction: multiple inputs influence placental protein synthesis. nutritional, endocrine and metabolic factors have been implicated but its regulation has not been investigated. one of the factors shown to be associated with the inhibition of protein synthesis is hypoxia. the goal of this study was to determine the effects of hypoxia on a marker of placental protein synthesis. eukaryotic initiation factor (eif ) is a subunit of eif which is required for initiation of translation however when phosphorylated, eif is unable to participate in the assembly of the initiation complex. hypoxia has been shown previously to cause increased phosphorylation of eif . we hypothesized that hypoxia would increase the levels of phosphorylated eif in term syncytiotrophoblast, thus inhibiting protein synthesis. methods: primary syncytiotrophoblast cultured from term cytotrophoblast were incubated for hr in atmospheres of , , or % o or in the presence of the hypoxia-mimetic, dimethyloxalylglycine (dmog, . - . mm) in % o . cell extracts were analyzed by western blotting to determine the degree of eif phosphorylation. results: incubation in , , or % o did not increase eif phosphorylation relative to the % o control (n= , separate placental preparations). incubation in dmog concentrations up to . mm did not affect eif phosphorylation however incubation in . mm dmog increased eif phosphorylation by ± % (p < . , n= ). conclusions: contrary to our expectations, inhibition of protein synthesis via the eif regulatory pathway was not apparent except when induced by the highest concentration of dmog, consistent with severe hypoxia. thus while eif phosphorylation does occur, we did not observe changes at dissolved oxygen levels of %. these data suggest that a reduction in syncytial protein synthesis via the eif pathway takes place only under severe hypoxic stress. (supported by nih hd ). the increasing prevalence of overweight and obese women of childbearing age is a growing public health concern. the impact of maternal obesity on placental aa transport, which is essential for normal fetal development, remains poorly defined. there are three sub-types of the placental na-dependent system a transporter, snat , and which mediate neutral aa transport. snat is ubiquitously expressed in mammalian tissues and is likely responsible for the majority of placental system a activity. objective: to examine the impact of maternal obesity and over-nutrition on the fetal: maternal (f:m) aa ratio and placental protein abundance for snat . methods: nonpregnant ewes were randomly assigned to a control (c, % of nrc recommendations) or obesogenic (ob, % of nrc) diet from - to days of gestation (dg). under isofluorane anesthesia, maternal and fetal blood samples were collected for aa analysis by hplc from five twin bearing ewes in each dietary group. after euthanasia, placental cotyledonary (cot) tissue was separated from caruncular tissue, frozen in liquid nitrogen and stored at - c for western blot analysis. results: fetuses from ob ewes were % heavier (p< . ) than those from c ewes at dg ( ± vs. ± g). blood concentrations of asn, thr, cit, arg, tau, tyr, trp, val, phe, leu and orn were higher (p< . ), or tended to be higher (met and lys, p< . ) in ob than c ewes. in contrast, f:m ratios, for asn, ser, gln, his, gly, thr, cit, arg, b-ala, tau, ala, tyr, trp, met, val, phe, leu, orn and lys were reduced (p< . ) in ob compared to c ewes. snat content in cot tissue was reduced in ob when compared to c ewes ( . ± . vs. . ± . arbitrary units; p< . ). conclusions: maternal obesity in pregnancy reduced expression of placental snat protein and efficiency of placental aa transport in ewes, providing a mechanism whereby fetuses may mitigate excessive delivery of aa under conditions of maternal obesity and over-nutrition. decreased aa transport to the fetus may play a role in altered cellular structure and function. nih inbre p rr . early gestation utero-placental hemodynamics in an ovine model of fetal growth restriction. lucia dohnal, james s barry, henry l galan, randall b wilkening, russell v anthony. perinatal research center, university of colorado health sciences center, aurora, co. objective: fetal growth restricted (fgr) pregnancies, during late gestation, exhibit altered placental hemodynamics, and reduced capacity for o and nutrient transfer. it was our objective to examine utero-placental hemodynamics and o uptake during early gestation in an ovine model of fgr. methods: singleton-bearing ewes were instrumented with uterine artery flow probes, uterine venous and femoral artery catheters before being placed into a highambient temperature (fgr; n= ) or normothermic (con; n= ) environment at days of gestation (dga). maternal arterial and venous blood, uterine artery flow, heart rate, arterial pressure and respiration rate was collected until dga, at which time umbilical venous blood, fetal weight, placental weight and tissue were harvested. data reported here were analyzed by students t-test. results: maternal respiration rate ( . ± . vs . ± . breaths/min) and arterial po ( . ± . vs . ± . mmhg) were increased (p . ), whereas maternal heart rate ( . ± . vs . ± . beats/min), blood pressure ( . ± . vs . ± . mmhg) and arterial pco ( . ± . vs . ± . mmhg) were reduced (p . ) in fgr pregnancies. at dga, fetal weight was not different (p . ), but placental (total placentome) weight ( . ± . vs . ± . g) was reduced (p . ) in fgr pregnancies. while uterine artery (pregnant horn) flow ( . ± . vs . ± . ml/min) tended (p= . ) to be reduced in fgr pregnancies, relative uterine artery flow ( . ± . vs . ± . ml/min/g fetus; . ± . vs . ± . ml/min/ g placenta) was not different (p . ). uterine o uptake (mmol/min), relative uterine o uptake (ml/min/g fetus or ml/min/ g placenta) and uterine o extraction (%) were not different (p . ) between fgr and con pregnancies. at dga, umbilical vein po (mmhg), o content (mm) and o capacity (mm) were also not different between fgr and con pregnancies. conclusions: reduction in absolute uterine artery flow (ml/min) did not impact utero-placental o uptake or transfer to the umbilical vein, and may have resulted from reductions in maternal cardiac output. relative uterine artery flow was not reduced, suggesting that uterine blood flow and delivery of o to the conceptus does not mediate the ongoing placental growth restriction initiated during early gestation. supported by nih hd . barton c staat, anna maria marconi, cinzia paolini, alex cheung, henry l galan, frederick c battaglia. obstetrics gynecology and pediatrics, univ of colorado at denver health sciences center, aurora, co, usa; dept of obstetrics and gynecology, san paolo institute of biomedical sciences, university of milano, milano, italy. objective: to determine relative contributions of transplacental flux vs fetal production for myo-inositol and mannose in normal term pregnancies using stable isotopic methodolgy. background: myo-inositol and mannose are important in biologic functions. an external supply of mannose may be required for glycoprotein synthesis. low maternal myo-inositol is associated with spina bifida. mannose concentrations are known to be higher in the mother than the fetus. in contrast, myo-inositol concentrations are higher in the fetus than the mother. what remains unknown is whether fetal levels of these polyols are a result of direct maternal transport or from conversion of glucose. design: four term uncomplicated pregnancies undergoing an elective ceasaran section were infused with c labeled isotopes of glucose, myo-inositol and mannose over hours prior to delivery. maternal samples were obtained prior to infusate being administered, and at hour (h), . h and h. fetal concentrations were measured from umbilical artery and vein plasma. the concentrations of labeled and unlabeled glucose, mannose and myo-inositol were measured using high pressure anion exchange chromatogrpahy permitting detection of polyols and sugars at concentrations in the μm range. the feto-maternal molar percent enrichment (mpe) ratio was calculated for each glucose, mannose, and myo-inositol as the ratio between fetal plasma enrichment and the maternal plasma enrichment at steady state. steady state was calculated as the mean of the three maternal samples taken during infusion. results: the feto-maternal mpe ratios of mannose ( . ± . , p= . ) and glucose ( . ± . , p= . ) were not significantly different from . , consistent with transplacental supply. the feto-maternal ratio for myo-inositol ( . ± . , p= . ) indicates little transplacental flux ( % of fetal inositol derived from maternal plasma). conclusion: in normal term pregnancies, fetal mannose and glucose concentrations are dependent upon maternal transplacental supply. in contrast, fetal myo-inositol concentration is not dependent upon transplacental supply, but fetal demands are met by placental conversion, likely from glucose. christian wadsack, manuela augsten, christian guelly, ursula hiden, ingrid lang, manfred moertl, uwe lang, gernot desoye. clinic ob/gyn; center of med res; inst cell biol, histol embryol, med univ graz, austria. background placenta and fetus need lipids for growth and synthesis functions. part of the lipids is supplied from maternal sources by transplacental transfer. recently, we identified in human placenta the high density lipoprotein (hdl) receptor scavenger receptor class b type i (sr-bi). among other functions it mediates hdl-induced ser -phosphorylation of endothelial nitric oxide synthase (enos) resulting in enos activation. this mechanism allows hdl to contribute to regulation of vasotonus in arteries. we hypothesized that term placental endothelial cells (ec) express sr-bi at levels different between arteries and veins. methods sr-bi was localized by ihc and quantified by qrt-pcr in rna isolated from arterial and venous vessels. arterial (eca) and venous (ecv) placental ec were rigorously characterized. sr-bi levels were measured by qrt-pcr and immunoblotting. hdl binding and uptake was measured in eca and ecv with i-labelled hdl. hdl from human donors was used to stimulate ser enos phosphorylation. epigenetic regulation was studied by methylationspecific pcrs for cpg-rich promoter regions of sr-bi. pdzk a key adaptor for sr-bi mediated enos activation was measured by sqrt-pcr. in situ analyses (ihc, qrt-pcr) showed more sr-bi in arteries than in the vein. the differential expression persisted in vitro in isolated eca and ecv even after passages and culture under same conditions suggesting epigenetic mechanisms regulating sr-bi. however, no methylation was found in eca or ecv. sr-bi was functional since hdl cell association was -fold higher in eca than in ecv ( ± . vs ± . ng hdl/mg prot). hdl did not induce ser enos phosporylation in eca or ecv, which was stimulated by ionomycin about -fold in both cell types. pdzk was undetectable in eca and ecv, whereas it was expressed in placental tissue. conclusion more sr-bi is expressed in ec from arteries than from veins in situ and in vitro. this is not the result of different methylation of sr-bi promoter and, hence, unlikely an epigenetic phenomenon. mechanism of differential expression and its functional consequences for vasotonus regulation is yet unknown. the lack of pdzk may account for the failure of hdl to activate enos. (grants , , oenb). cells. juan a arroyo, brad ziebell, mi-hye park, henry l galan. obstetrics and gynecology, university of colorado andgealth sciences center, aurora, co, usa. introduction: mtor is a protein that regulates cell growth in response to nutrients and growth factors. downstream effectors of the mtor pathway include the p and the ebp proteins. activation by phosphorylation of these proteins increases protein synthesis. given that various signaling pathways are regulated by hypoxia in human trophoblast and that mtor is expressed in human trophoblast, our objective was to determine the effects of hypoxia in the activation of mtor, p and ebp in cultured human trophoblast. study designs: trophoblast cell were isolated from term uncomplicated placentas using a trypsin, dnase and disapase solution. cytokeratin immunocytochemistry confirmed trophoblast cells culture purity. trophoblast cells were treated with hypoxia ( % o ) or normoxia ( % o ) for and hours. western blot for p-mtor, mtor, p-p , p , p- ebp , and ebp were done for each time studied. results: trophoblast cells demonstrated: ) positive staining for cytokeratin, ) non significant differences for mtor at either ( . -fold; p= . ) or hours ( . -fold, p= . ), ) no differences in p protein at ( . fold; p= . ) or hours ( . -fold, p= . . ), ) no differences for ebp at either or hour. conclusion: we conclude that the mtor pathway is not regulated under hypoxic conditions in cultured trophoblast, which suggests that hypoxia does not affect protein synthesis in cultured human trophoblast. however, this may not reflect what happens in vivo in iugr. (supported by nih grant r hl - a ). increased expression of phospho-mtor, phospho-p , phospho-akt and phospho-erk in an ovine model of fetal growth restriction. juan a arroyo, brad ziebell, henry l galan. obstetrics and gynecology, university of colorado and health sciences center, aurora, co, usa. objective: both phosphorylated (p) mtor and p are known to be involved in protein synthesis and are regulated by physiological conditions such as fetal growth restriction (fgr). in a hyperthermic (ht) ovine model of fgr we hypothesize that mtor, p , ebp , erk and akt will be phosphorylated (activated) in the placentae of age (dga) animals. study design: ewes were exposed to ht conditions for days to induce iugr and were placed in ambient conditions. at necropsy ( dga), placentomes were separated into the maternal (caruncle) and fetal (cotyledon) components and frozen for western blot analysis with antibodies against (p) mtor, mtor, (p) p , p , (p) ebp , ebp , (p) erk, erk, (p)akt and akt. results: compared to control animals, fgr animals had smaller fetuses ( ± g v. ± g; p= . ) and smaller placentae ( ± g v. ± g; p= . ) at dga. fgr cotyledon showed an increase in p-mtor ( . -fold; p= . ), p-p ( . -fold; p< . ), p-erk ( . -fold; p< . ) and p-akt ( . -fold; p< . ). in contrast, caruncle (maternal) did not show any changes for the mtor pathway. conclusion: in fgr ovine pregnancies, the fetal placental tissues (cotyledons) showed upregulation of the mtor pathway for protein synthesis via phosphorylation of the p but not ebp while this was not seen in the maternal (caruncle) tissues. in addition neither the cotyledon or caruncle tissues at mid-gestation ( dga) showed changes in these endpoints, which is prior to the exponential fetal growth that starts at mid-gestation. (supported by nih grant r hl - a ). hyperuricemia has long been recognized as a common clinical finding in preeclamptic (pe) women. to date, elevated uric acid concentrations in these women have been considered a marker of disease severity. however preeclamptic pregnancies with hyperuricemia, are associated with an increased frequency of preterm birth and fetal growth restriction. over the past decade several pathogenic roles for uric acid have become evident, raising the possibility of a role(s) for uric acid in the altered vascular and placental functions associated with pe. objective: examine the effects of syncytial uric acid uptake on system a amino acid transport across the human placenta using a primary placental villous explant model. methods: placental villous explants from placentae of healthy, term pregnancies were incubated for hours with uric acid ( . mg/dl), corresponding to concentrations of uric acid observed in pe women. these experiments were conducted in the presence or absence of probenecid ( m), a uric acid cellular uptake inhibitor. system a amino acid transport was subsequently assayed using a radiochemical assay in which na+-dependant uptake of radio-labeled system a substrate, [ c] methyl-amino-isobutyric acid, was measured over minutes. data were analyzed using a paired student's t-test and presented as mean ± sem. results: uric acid attenuated system a amino acid placental transport by % (± . %, p< . ). this inhibitory effect of uric acid on system a activity was prevented by probenecid. conclusions: uric acid reduces placental amino acid transport at concentrations observed in pe women. this inhibitory effect of uric acid is dependant upon syncytial uptake of uric acid, being inhibited by the uric acid transporter inhibitor probenecid. these results may be relevant to the increased frequency of fetal growth restriction observed in hyperuricemic pe. additionally the results of this study, indicating a detrimental effect of hyperuricemia on placental function, also suggest a role for uric acid in the pathophysiology of pe. hyperuricemia, a well-documented clinical finding in preeclamptic women, is associated with pre-term birth and intrauterine growth restriction. uric acid is higher in women destined to develop preeclampsia as early as weeks of gestation at a time when cytotrophoblast are invading decidua and myometrium and remodeling uterine spiral arterioles. we propose that elevated concentrations of uric acid may have detrimental effects on placental development in part through inhibition of trophoblast invasion through the decidua. objective: examine the effects of increasing concentrations of uric acid on trophoblast invasion through a reconstituted extracellular matrix. methods: using the in-vitro matrigel invasion assay, the effects of increasing concentrations of dissolved uric acid ( . mg/dl, . mg/dl and . mg/dl) on the ability of immortalized first trimester extravillous trophoblast cells (htr -svneo) to invade through a reconstituted extracellular membrane were assessed. the concentrations of uric acid used were comparable to those measured in healthy pregnant women and preeclamptic women with an increase in uric acid of two or four standard deviations above normal. cells that successfully invaded through the matrigel membrane within hours were fixed with methanol, stained with hematoxylin and counted. data were analyzed using a one-way analysis of variance with fisher's post-hoc analysis. results: uric acid attenuated trophoblast invasion in a dose-dependent fashion (p< . ), with decreases of % (± . %), % (± . %) and % (± . %) respectively compared to untreated controls. conclusions: exogenous uric acid, at physiological and pathological concentrations, is capable of attenuating trophoblast invasion through a reconstituted extracellular membrane in a dose dependent fashion. these results suggest uric acid is a potential contributor to the pathophysiology of altered placental perfusion in preeclamptic pregnancies. previous studies have shown that transforming growth factor (tgf)-is a key inhibitory factors in the invasion of early trophoblast cells, suggesting therefore that overcoming tgf-beta signaling may be necessary for successful implantation. smad ubiquitin regulatory factor (smurf ), a hect type e ubiquitin ligase, is a key regulator of tgf-signaling pathway, targeting tgf-receptors and various smads for proteasome-mediated degradation. in this context, smurf has been shown to play important roles in embryonic development, cell senescence and tumor formation. as a key regulator of tgfbeta signaling, we wished to determine whether smurf has a physiological role during embryo implantation, especially in trophoblast invasion. we have examined the spatio-temporal expression of smurf in human placental villi during pregnancy. we have also investigated the possible function of smurf in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, htr /svneo. our results showed that expression of smurf in placental villi was the highest during the first trimester and the expression decreased in the nd trimester. expression of smurf was lowest in placental villi at parturition. overexpression of smurf in htr /svneo cells reduced tgf beta type i receptor levels and attenuated the inhibitory effect of tgf-on cell migration and invasion. conversely, rnai-mediated down-regulation of smurf resulted in significant increase of tgf-type i receptor protein levels. in contrast, the levels of smad , another potential target of smurf , was unchanged. in conclusion, the present study suggests that smurf participates in trophoblast cell migration and invasion by down-regulating the expression of tgf-type i receptor. our previous data demonstrated the extensive expression of mmp- in various kinds of trophoblast cells in human placenta at the early pregnancy. however, the modulation of the enzyme in trophoblasts is largely unclear. in the present study, the effects of the two types of gonadotropin releasing hormone (gnrh) on mmp- expression were examined in an immortalized human cytotrophoblast cell line, b tert- that has been established in this lab. real-time quantitative pcr and western blot analysis revealed that both types of gnrh (gnrh i and gnrh ii) could increase mmp- mrna and protein levels in b tert- cells in time-dependent manners. in particular, regulatory effect of gnrh i on mmp- expression was concentration-independent, whereas that of gnrh ii was dose-dependent. moreover, both gnrh i and gnrh ii could evidently activate jnk kinase, and sp , an inhibitor of a jnk kinase, reversed the up-regulation of mmp- induced by either gnrh i or gnrh ii. on the other hand, it is not likely that erk / pathway participates in the signaling of gnrh i or gnrh ii. collectively, our observations suggest that gnrh i and gnrh ii elicit their modulation effects in human trophoblastic cells through jnk pathway leading to up-regulation of mmp- . during the first trimester of pregnancy, the oxygen tension of the developing trophoblast cells is less than %. however, the majority of studies on primary trophoblast cell development have been performed at % oxygen. primary third-trimester trophoblast cells are believed to be nonproliferative syncytiotrophoblast cells. we have previously demonstrated that low oxygen tension dramatically affects the differentiation pathway of these cells. we now hypothesize that cell culture in low oxygen tension will improve cell growth and restore proliferation. methods: primary trophoblast cells were purified from third-trimester placenta by enzymatic dispersion and cd- negative selection and cultured at %, % or . % oxygen tension for up to days. the number of cells in culture was assessed by cell counting and by measuring genomic dna. live:dead and mtt assays were used to determine viability. proliferation was assayed with brdu and immunohistochemistry for proliferating cell nuclear antigen. to assess cellular activity, radioactivity of protein precipitated from cells cultured in the presence of tritiated leucine was measured. results: there were no obvious morphologic changes in the cells cultured in different oxygen tensions. the amount of cell loss was directly proportional to oxygen tension: at % oxygen % of the cells remain in culture; at . % oxygen tension % of the cells remained. the cells at . % oxygen tension were proliferating and had a five-fold increased metabolic activity. conclusions: it was previously believed that third-trimester trophoblast cells are non-proliferative. we have demonstrated that low oxygen tension increases the survival of primary third-trimester trophoblast cells. this may reflect the change in the differentiation pathway of these cells. however, the cells also begin to proliferate and increase their metabolic activity. trophoblast cells in vivo form a three dimensional structure which promotes critical complex cell-to-cell interactions that cannot be studied with traditional monolayer cell culture. we developed a substrate-free three-dimensional trophoblast culture system capable of studying cellular interactions without a confounding artificial matrix. methods: nonadhesive agarose hydrogels containing cylindrical recesses m in diameter were cast from molds designed using computer-assisted prototyping. tcl trophoblast cells were seeded into the gels ( , cells per) for up to days. viability and cellular stress were assessed and the threedimensional structures of the spheroids were analyzed. results: tcl trophoblast cells formed uniform spheroids within three days of seeding. the spheroids remained intact after being removed from the mold. when placed in traditional cell culture dishes the cells adhered to the plate within one hour and rapidly proliferated into a monolayer. repetitive reseeding allowed easy transition between monolayer and spheroid without affecting cellular morphology. serial sectioning on days , and revealed central vacuolization forming a trophoblast vesicle with an outer rim . m (+/- m) thick. this rim size remained constant for at least days. live:dead assay demonstrated that the outer cells remained viable and staining against proliferating cell nuclear antigen demonstrated that the cells were proliferating. the inner cells undergo apoptosis as demonstrated by caspase- staining. there is an abundance of vegf staining in the cells remaining in the on the inside of the sphere suggesting a gradient of nutrient or oxidative stress. the formation of a vesicle has been confirmed with confocal imaging. em imaging revealed the structure of the rim. conclusions: trophoblast cells cultured in a novel substrate-free three dimensional system form trophoblast vesicles within days of seeding. these vesicles remain viable after long-term culture and can be repeatedly reformed with repetitive seeding. this new cell culture technique allows us to better study placental cell-cell interactions with the potential of forming microtissues. the transcription factor glial cell missing- (gcm ) mediates cell cycle arrest and differentiation of human trophoblast progenitors into villous syncytiotrophoblast and invasive extravillous cytotrophoblast (evt). micro-array analysis of total rna extracted from cultured bewo cells, in which gcm mrna and protein were repressed using sirna, identified tissue inhibitor of metalloproteinase- (timp- ) in the highest ( -fold) upregulated group of genes. confirmatory rtpcr demonstrated a -fold mrna induction. in placental villi, gcm acts as a transcription factor promoting expression of the fusogenic protein syncytin that mediates syncytial fusion into the overlying syncytiotrophoblast. by contrast, syncytial fusion is uncommon in evt. rather these cells invade several millimeters into the distal myometrium where they transform spiral arterioles. to investigate the role of timp and gcm in the trophoblast we assessed its mrna by qrt-pcr and protein by western blot in cellular extracts from both bewo cells grown under standard cultivation conditions (synchronized by prior thymidine exposure) and floating cultured first trimester villous explants cultured in % oxygen with prior exposure to either gcm sirna or anti-sense oligo-nucleotides to gcm . gcm inhibition in the bewo system was associated with a - % increase in timp- protein expression and alteration of cell proliferation and differentiation in both models. we are presently utilizing the explant model of evt invasion (explant tips cultured on matrigel in % oxygen) to test the hypothesis that gcm mediates metallo-proteinase expression and evt invasion via timp- . presently we conclude that gcm -mediated evt differentiation involves more than an arrest of mitosis and may include promotion of invasion via repression of timp- . funding: cihr. scott h purcell, jeremy d cantlon, virginia d winn, russell v anthony. , colorado state university, fort collins, co; university of colorado health sciences center, aurora, co. background: periattachment factor (pf) is a nuclear protein first described in the bovine conceptus. our research in sheep has shown pf mrna concentration peaks when the conceptus is undergoing elongation and initial apposition to the endometrium, and that pf is a nuclear protein localized to the trophoblast. in silico analysis identified a human homolog, hprr . objective: the objective of this experiment was to determine if pf was expressed in the human placenta, and to develop short-hairpin (sh) rnas for hpf to begin investigating its function. materials and methods: immunohistochemistry was performed on paraffin embedded first and second trimester human placental samples. placental sections were immuno-stained using rabbit polyclonal antiovine pf or anti-human cytokeratin- . cytotrophoblasts from first trimester pregnancies (n= ) were subjected to an in vitro invasion assay and rna was harvested following , , and h. quantitative rt-pcr was performed on these samples with intron-spanning primers for hpf, and normalized on hs mrna concentrations. based on the human pf sequence, four putative shrna constructs were generated and cloned into a lentiviral expression vector. bewo human choriocarcinoma cells were treated with one of four shrna contructs or an empty vector for h and then rna was harvested from cells for analysis by quantitative rt-pcr. results: periattachment factor was present in the nuclei of both first and second trimester cytotrophoblasts. hpf mrna concentration increased as invasion occurred from , , to h in all samples; while hypoxia decreased expression at h of invasion compared to h under normoxic conditions. the four lentiviral vectors expressing shrna against hpf resulted in hpf mrna concentrations at , , and % of hpf mrna concentration with the control vector. conclusion: the presence of pf in the human placenta and the increase in pf mrna during cytotrophoblast invasion may indicate this gene plays a role during implantation. we have developed shrnas against pf that result in greater than % mrna knockdown and will be using these to begin to elucidate the function of pf in the human placenta, specifically during the invasion process. recently we demonstrated that infusion of imd antagonist (imd - ) in rat caused distorted labyrinth indicative of a deficient vasculature in placenta. we hypothesize that imd has a role in migration of first trimester trophoblast cell (htr- /svneo) via regulating human leukocyte antigen (hla-g) and stimulating mek / / erk / phosphorylation. objectives: ) to asses the effect of imd on migrating capacity of htr- sv/neo cells using scratch assay in presence or absence of mek and ras/raf inhibitor, u and manumycin a, respectively ; ) to assess the effect of imd peptide on phosphorylation of mek / and erk / in first trimester htr cells ) to analyze the effects of imd on the expression of human leukocyte antigen, hla-g, a critical factor involved in the invasion and vascular remodeling of spiral uterine arteries and subsequent pregnancy in human. methods: htr- sv/neo cells were used to assess the effect of imd ( - m) on the expression of hla-g mrna and phosphorylation of erk / and mek / protein by reverse transcriptase polymeration chain reaction (rt-pcr) and western blot analysis respectively. scratch wound assay was used to determine the migration capacity of htr cells. total rna was isolated from cells using trizol reagent and processed for rt-pcr and results are expressed relative to s mrna. trichloroacetic acid was used for the extraction of total protein for western blot analyses. results: our data demonstrates that, ) imd enhances the migrating capacity of htr cells (compared to the untreated cells) and these effects are inhibited by mek and ras/raf inhibitors, u and manumycin a, respectively; ) imd ( - m ) stimulates phosphorylation of erk / and mek / proteins in htr cells, ) imd increases the expression of hla-g mrna in htr cells. conclusion: imd promotes migration of first trimester htr cells through mek/ erk signaling pathway and modulates the expression of immunoregulatory molecule, hla-g in these cells. chymotrypsin-like protease promotes the placenta tissue release of sflt- . yang gu, shuang zhao, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: the placenta is a major source of soluble vegf receptor- (sflt- ) in the maternal circulation during pregnancy. increased placental release of sflt- is believed to play an important role in the pathophysiology and pathogenesis in pe. however, the mechanism of increased placental sflt- release in pe is unknown. we recently reported increased chymotrypsin-like protease (clp) activity and expression in placental trophoblasts from pe. in this study, we tested if proteolytic effects of chymotrypsin may play a role in promoting sflt- release by placental trophoblasts. methods: placentas delivered by normal pregnant women (n = ) were used. we tested if chymotrypsin could promote sflt- release by placental tissue, in which villous explants were cultured with dmem containing chymotrypsin at . , . , and . g/ml for hours. the culture medium was then collected for measuring sflt- . we then determined the specificity of chymotrypsin induced sflt- release. villous tissues were cultured with or without chymotrypsin inhibitor (ci) in culture and then the medium was collected and measured for sflt- . soluble flt- was measured by elisa. all samples were assayed in duplicate. data are presented as mean ± se and analyzed by anova. a p level < . is considered as statistically different. results: ) sflt- concentrations in the medium were increased when chymotrypsin was present in culture and the increased sflt- release induced by chymotrypsin was in a concentration-dependent manner: control: . ± . ; . g/ml: . ± . ; . g/ml: . ± . ; and . g/ml: . ± . (p< . ) pg/mg tissue/hour. ) ci could attenuate sflt- release. this inhibitory effect was also revealed in a concentration-dependent manner: control: . ± . ; ci . g/ml: . ± . ; and ci . g/ml: . ± . (p< . ) pg/mg tissue/hour. conclusions: increased placental sflt- release stimulated by chymotrypsin and decreased placental sflt- release inhibited by chymotrypsin inhibitor suggest that the proteolytic effect of clp may play a role in sflt- generation. therefore, increased clp activity in placental trophoblasts may contribute to the increased placental sflt- production in pe. (supported nih grants hl and hd ). the change of autophagy-related proteins, lc and beclin- , by tnfa stimulation in cultured primary trophoblasts. soo-young oh, kyung hee kim, suk-joo choi, jong-hwa kim, cheong-rae roh. department of obstetrics and gynecology, samsung medical center, sungkyunkwan university school of medicine, seoul, korea. objective: our previous work have demonstrated that the expression of lc , but not beclin- , was increased in placentas from pregnancies complicated by severe preeclampsia (sgi abstract # ) . to understand the regulatory mechanism of these autophagy-related proteins in trophoblast cells, we investigated the changes in these proteins in response to cytokine or hypoxic stimulation in cultured primary trophoblast. material and methods: primary human cytotrophoblasts obtained from normal term placenta were cultured with stimulation of tnf-a or cocl for a given time and the changes of beclin- and lc were assessed using immunoblot analysis. paired t test was used for statistic analysis. results: tnf-a stimulation induced a significant increase of the expression of lc -ii in cultured primary trophoblasts while decreasing the expression of beclin- (p< . for each). however, cocl stimulation did not induce a significant change of both lc -ii and beclin- . conclusions: our data suggests that tnf-a stimulation in cultured primary trophoblasts is associated with increased autophagic activity. background: thyroid hormones play vital roles in the development of the fetal brain. mutations in mct , recently recognised as a specific thyroid hormone transporter, define a novel syndrome of severe x-linked psychomotor retardation accompanied by elevated serum t . we previously reported that mct expression in n-tera- (nt ) cells (a human embryonal cell line with characteristics of cns precursors), as well as mct -null jeg- choriocarcinoma cells, resulted in markedly reduced cell proliferation. further, the s x mct mutation, as reported in males affected by severe psychomotor impairment, resulted in a similar repression of proliferation to wild type, whereas the l p mutant failed to influence cell turnover compared with control. methods: we now examine the effect of "knocking down" mct via sirna and evaluate the effects of cell proliferation (mtt and [ h]-thymidine assays) and tri-iodothyronine (t ) uptake. results: repression of endogenous mct expression in nt cells by % caused a significant increase in proliferation compared to matched-dose nonspecific sirna treatment, independent of t concentration ( . %, . % and . % induction at , and nm t , n= , p< . ). we also sought to examine the role of mct in t uptake. in jeg- cells, wild type mct induced a . -fold increase in the uptake of i-labelled t . by contrast, mutants s x and l p failed to significantly augment t uptake, though r h caused a mild but significant . fold induction in uptake, hence retaining approximately % of wt activity. in parallel experiments, co-transfection of mu-crystallin, a t binding protein, resulted in a similar increase in t uptake compared with control ( . -fold; n= ; p< . ), implying that mct plays only a minor role in thyroid hormone efflux in jeg- cells. mutants s x, l p and r h showed analogous responses to those in the absence of mu-crystallin. conclusion: these results further extend the evidence of a potential role for mct in the modulation of cell proliferation, independent of t transport. to determine predictors of failure for labor induction in women with preeclampsia. we conducted a retrospective cohort study to examine cesarean delivery rates in all the preeclamptic women at a single institution undergoing labor induction between - with a singleton pregnancy >= weeks gestational age (ga). bivariate analyses informed the creation of multivariable logistic models to predict the risk of cesarean delivery using multiple predictors (maternal age, race/ethnicity, unfavorable cervix, gestational diabetes, diabetes, and gestational age). analyses were stratified by parity. our study population included , preeclamptic women undergoing labor induction. in the bivariate analyses, the risk of cesarean delivery ranged from as low as . % (p= . ) among multiparous women - weeks ga to as high as . % (p< . ) among nulliparous women with diabetes. a total of , women had adequate data to be included in the multivariable analyses. odds ratios of the predictors are presented in the objective: preeclampsia and cardiovascular disease share many risk factors, and women with preeclampsia are at increased risk of cardiovascular mortality later in life. we investigated whether risk factors associated with cardiovascular disease and preeclampsia remain elevated months postpartum. methods: we measured plasma sflt , endoglin, plgf, cellular fibronectin (cfn), uric acid, homocysteine, and asymmetric dimethylarginine (adma) in women with uncomplicated normotensive pregnancies compared to women with preeclampsia in samples collected at pre-delivery and again several months postpartum (average . ± . months). data are mean±sd or median (interquartile range). statistical analysis was by wilcoxin rank-sum or students unpaired t-tests with statistical significance accepted at p< . . results: the mean concentration of sflt , endoglin, plgf, homocysteine, adma, cfn, and uric acid were all significantly different in samples collected pre-delivery in subjects with preeclampsia compared to controls (table). adma, cfn and uric acid remained significantly higher postpartum in subjects with previous preeclampsia compared to postpartum controls (table) . conclusions: biological markers associated with altered vascular function or cardiovascular risk are elevated in women with preeclampsia, and some remain significantly higher in postpartum preeclamptic women. these data suggest that vascular dysfunction persists in women with previous preeclampsia, and may contribute to the increased risk of future cardiovascular disease. funded in part by national institutes of health nih- mo -rr and nih- po -hd . (ajp, ) . as leptin is a known potent angiogenic factor we hypothesized that leptin deficiency and/or resistance to leptin-induced vegf expression might be a mechanism for reduced angiogenesis in mfr offspring. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. mfr and control offspring were sacrificed on day of life (p ). some tissues were used to determine the expression of leptin by western blot analysis. for culture experiments, thoracic aortas were dissected, cut into - mm explants and incubated with leptin ( - ng/ml) in dmem ( % fbs). after hours of culture, rna was extracted from the tissues and subjected to real time rt-pcr using specific rat primers for vegf, vegfr and r , and ob-ra, stat and socs ( s mrna as control). culture media was analyzed for vegf protein by elisa. results: expression of leptin mrna and protein in p mfr aortas was significantly reduced. in culture, leptin significantly increased expression of vegf, vegfr and vegfr mrna in explants of aortas obtained from the control but not mfr tissues. as expected, control but not mfr aortic explants secreted significantly more vegf in vitro. to determine the mechanism for resistance to leptin-induced vegf in mfr offspring, we assessed expression of leptin receptor (ob-ra) in explants treated with leptin. leptin was found to induce the expression of ob-ra in aortas from both dietary groups. this upregulation of leptin receptor was accompanied by significant upregulation of stat and socs mrna in the control tissues. in contrast, in mfr explants only the ng/ml concentration of leptin induced an increase in stat mrna, and the magnitude of socs mrna increase by both concentrations of leptin was significantly less in the mfr explants. conclusion: these results indicate that reduced angiogenesis in mfr vessels is in part due to reduced leptin expression and ability of leptin to stimulate vegf expression. although in vitro leptin induced the expression of its receptor in both groups, it was only in the mfr group in which leptin up-regulated vegf and its receptors. our results suggest that this defect in leptin receptor function in mfr vessels is due, in part, to defects in jak/stat signaling. objective: women with a history of early-onset preeclampsia are at increased risk of developing major cardiovascular disease (cvd) related events, that have a detrimental effect on their long-term health and life expectancy. in this follow-up study, we measured established risk factors predictive of first cvd events after early-onset preeclampsia. study design: over a -year interval, primiparous women with a history of early-onset preeclampsia (delivery < weeks gestation) were included and tested for major cardiovascular risk factors at least six months after delivery, in addition to a population-based control group of healthy non-pregnant women. women with chronic hypertension were excluded. results: mean age was . years for cases compared to . years for controls (p<. ). after adjustment for age, we observed significantly increased mean values for weight (p=. ), body-mass index (p<. ), systolic blood pressure (p<. ), diastolic blood pressure (p<. ), total cholesterol (p=. ), ldl cholesterol (p<. ), triglycerides (p=. ), fasting blood glucose (p<. ), and lower hdl cholesterol (p<. ) in women with previous early-onset preeclampsia. no difference was found for height, smoking, diabetes, and ethnicity. estimated -year risk of first cvd events by framingham risk scores remained < % for all women (low-risk). nonetheless, at mean (sd) . ( . ) years after early-onset preeclampsia, % of women met the criteria for metabolic syndrome, % of women exhibited >= , % of women >= and % of women >= major cvd risk factors. conclusion: the majority of women with a history of early-onset preeclampsia exhibit at least one modifiable risk factor for future cvd. although most of these women are classified as low-risk according to the current aha guidelines, this is mainly due to their young age masking other, mostly modifiable, major risk factors. our data thus support life-style intervention programs aimed at primary prevention of cvd in women with a history of early-onset preeclampsia. it has recently been shown that antihypertensive drugs can stimulate cytokine release in normal and hypertensive pregnancy. there is evidence that these cytokines alter the secretion of inhibin a. inhibin a and activin a levels are increased in pre-eclampsia (pe), but it is not known if antihypertensive therapy can affect their secretion. patients and methods we recruited women with hypertensive disorders in pregnancy ( pe and non-proteinuric hypertension [ht]) and matched normotensive controls. inhibin a and activin a levels, before and - hours after initiating antihypertensives, were measured in serum and urine, using an elisa. the same markers were measured using validated assays in placentas delivered at cesarean section at similar gestational age ( pe, ht and controls). analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. marker levels before and after antihypertensive therapy were compared using paired t-test. we compared placental concentrations between the group which received antihypertensive therapy and the group which did not, using an independent t-test. data were analysed using spss®. in pe, both serum and urine levels of inibin a and activin a were increased at all gestations (p< . ). activin a (but not inhibin a) level was also increased at all gestations in ht (p< . ). after weeks' gestation (but not before), antihypertensive treatment was associated with a significant fall in both inhibin a and activin a serum levels, and urinary inhibin a, in both pe and ht. the placental concentration of inhibin a, but not activin a, was significantly higher in women with pe compared with controls (p< . ). there was no significant difference in either marker between controls and women with ht. the fall in serum levels of inhibin a and activin a following antihypertensive treatment after weeks' gestation may indicate that these drugs have an effect on the pathophysiology of pe other than their known antihypertensive action. pre-eclampsia (pe) is a placental disease of unknown etiology. anti-angiogenic factors, such as soluble fms-like tyrosine kinase (sflt- ) and soluble endoglin (seng), and pro-angiogenic factors, such as vascular endothelial growth factor (vegf) and placental growth factor (plgf), are believed to play an important role in its pathophysiology. maternal plasma concentrations of these markers are altered in pe, even weeks before the clinical manifestations. the aim of this study was to compare the concentration of these markers in placental extracts of normotensive pregnant women, and women with pe and non-proteinuric hypertension (ht). patients and methods placental samples were collected at cesarean section from women with pe (n = ), ht (n = ) and normotensive pregnancies of similar gestational age (n = ). these samples were stored at - ºc. the frozen tissue samples were homogenised and these four markers measured by specific, validated enzymelinked immunosorbent assays. analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. data were analysed using spss®. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). there was no significant difference in plgf concentration between controls and women with pe or ht. placental vegf concentrations in both pe and ht were higher than in controls (p< . ); there was no significant difference between the levels in pe and ht (p= . ). the fact that placental concentrations of sflt- and seng mirrored the known rise in serum levels in pe suggests that the placenta is the main source of these circulating factors. although sflt- was significantly raised in pe, plgf was not reduced. this suggests that the lower levels of free plgf found in the serum of women with pe are not the result of impaired placental production or secretion, but are due to increased binding by (the increased levels of) sflt- in the serum. objective. the purpose of this study was to evaluate whether systematic screening with uterine artery doppler (utad) and serum biochemical markers of oxidative stress, endothelial dysfunction and vasculogenesis performed during the first trimester predict efficiently pre-eclampsia (pet), specifically early-onset pet, in an unselected chilean population. methods. this nested case-control study involved asymptomatic pregnancies scanned at + - + week of gestation. the subjects for biochemical testing were women who were delivered due to pet (n= ) and normotensive controls (n= ) that were enrolled during the first trimester scan. mean pulsatility index (pi) of the utad was calculated. blood samples were obtained and stored at - o c until biochemical analysis of oxidative stress, endothelial dysfunction and vasculogenesis were performed. normally distributed data were analysed by the unpaired t test, and non-normally distributed data by the mann-whitney rank sum test. chi-square tests were used for the comparison of categorical variables. a probability level of p< . was considered significant. multiple logistic regressions were used to develop a combined predictive index. results. there was % and % significantly increased of the mean pi utad in women who later developed pet or early-onset pet compared to control pregnancies during the first trimester scan. although oxidative stress and endothelial dysfunction biochemical markers were not different between all pet pregnancies and control groups, plasma levels of sflt ( . ± . vs. . ± . pg/ml, p< . ) and placenta growth factor ( . ± . vs. . ± . pg/ml, p< . ) were significantly higher in women who subsequently developed early-onset pet compared to controls. multivariate logistic regression showed that a combination between abnormal utad and both biochemical markers of abnormal vasculogenesis were the best predictor test for early-onset pet, being its detection rate % with % false positive rate. conclusion. this study has shown early and selective changes in markers of impaired placentation and angiogenic state in women who later developed earlyonset pet, without alteration in oxidative stress and endothelial dysfunction. supported by fondecyt . currently it is unknown whether maternal inflammatory changes are specific to pregnancy or reflect an innate susceptibility to inflammation. c-reactive protein (crp) and interleukin- (il- ) are markers of the acute-phase inflammatory response and predictive of future cardiovascular events. we compared crp and il- levels after influenza vaccination, as an in vivo model for lowgrade inflammation, in non-pregnant women with a history of early-onset preeclampsia and controls with only uneventful pregnancies. methods: forty-four women with a history of early-onset preeclampsia (delivery < weeks' gestation) and twenty-nine controls with at least one uneventful pregnancy received an influenza vaccination. we then compared plasma levels of crp and il- at baseline, . days and . days after vaccination. results: median baseline crp and il- levels of women with a history of early-onset preeclampsia were comparable to controls ( . versus . mg/l; p= . and . versus . pg/l; p= . , respectively). however, high crp and il- responses to vaccination were more common in cases (ors for response > th, > th, > th, > th and > th percentile based on the distribution of control values of . , . , . , . and for crp [p for trend . ] and of . , . , . , . and . for il- [p for trend . ], respectively). the relationship between high il- responses and early-onset preeclampsia persisted after adjustment for body-mass index (p for trend . ). conclusion: women with a history of early-onset preeclampsia more frequently exhibit an innate pro-inflammatory phenotype not specific to pregnancy. background altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (hellp) syndrome. we examined whether allelic variants of the innate immune receptors toll-like receptor (tlr ) and nucleotide-binding oligomerization domain (nod ), that impair the inflammatory response to endotoxin, are related to preeclampsia and hellp syndrome. we determined five common mutations in tlr (d g and t i) and nod (r w, g r and l fs) in primiparous women with a history of early-onset preeclampsia, of whom women developed hellp syndrome and in women with a history of only uneventful pregnancies as controls. in addition, we assessed plasma levels of pro-inflammatory biomarkers c-rp, il- , sicam- , fibrinogen and von willebrand factor in a subset of women included at least six months after delivery. after adjustment for maternal age and chronic hypertension, attenuating allelic variants of tlr were more common in women with a history of early-onset preeclampsia than in controls (or . [ % ci . - . ]). highest frequencies for tlr variants were observed in women who developed hellp syndrome (adjusted or . [ % ci . - . ]). in addition, high levels of il- and fibrinogen were associated with a history of early-onset preeclampsia. combined positivity for any of the tlr and nod allelic variants and high levels of il- was . -fold more common in women with a history of early-onset preeclampsia ( % ci . - . ) compared to controls. we observed an association of common tlr and nod gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and hellp syndrome, that suggests involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy. thus, we sought changes in pbef in the serum of patients with mild and severe pe, compared with matched controls. immunodistribution of pbef in fetal membranes and placentas from similar patients was also studied. methods: serum samples ( ) were collected with clinical data including; gestational age, medications, ethnicity and recognized complications. patients in labor or infection were excluded. the standard bp and proteinuria criteria was utilized to classify cases for pe grouping; no pe (n= ), mild pe (n= bp; / - / , proteinuria; trace to + or - mg/ hr urine) and severe pe (n= bp; > / , proteinuria; > + or > g/ hr, or other associated symptoms). pbef concentration was determined by eia (phoenix pharmaceuticals) in accordance with the manufacturers instructions. fetal membranes and placentas of additional patients, no pe (n= ) and with pe (n= ) were fixed and embedded in paraffin. sections ( um) were immunostained with pbef antibody / (pheonix) and treated with abc reagent (vector labs) followed by dab ( . % mg/ml), washed, counterstained, mounted and viewed under brightfield microscopy. results: the concentrations of pbef in serum were between - ng/ml and were significantly higher in those patients with mild pe (p= . ) and further significantly elevated in those with severe pe (p= . ) compared with the matched controls. pbef was detected by immunocytochemistry in the placental syncytiotrophoblast and in the amnion and choriodecidua of the fetal membranes. conclusions: pbef was elevated in the serum of patients according to the degree of pe severity and may be derived from the placenta and/or fetal membranes. (supported by nih #u rr - to the pacific research center for early human development, university of hawaii). background: platelet-monocyte aggregation (pma) is a novel sensitive measure of platelet activation and indicates a proinflammatory state (cytokine release). less sensitive techniques demonstrate platelet activation during pregnancy and pre-eclampsia (pe) but platelet activation has not been assessed by pma.objective: longitudinal study of pma in normal pregnancy and pe. methods: healthy, non-smoking primigravida with an uncomplicated pregnancy and primigravida women with pe were studied. pe was defined by standard definitions. informed consent was obtained and the study had ethical approval. serial venous blood collected at , , , wks in controls, at time of diagnosis in pe cases and wks post-natal (pn) in all. pma, platelet surface p-selectin (psp-sel) and monocyte cd expression (mcd ) were analysed by flow-cytometry and plasma (p) p-sel by elisa. results: groups were matched for mean age and bmi. in controls, pmas, psp-sel and mcd expression and pp-sel increased with gestation and decreased post-natally (table ) . for pe analysis, data was divided into pre-term (sampled at mean wks), and term (mean wks). pp-sel was lower in pre-term pe than control (normal pregnancy wks; p= . ) there was no significant difference in other measures between pe and control ( objective: much effort has been put into the evaluation of novel markers to identify pregnant women at risk for the development of pre-eclampsia. soluble endoglin (seng) and soluble fms-like tyrosine kinase (sflt ), two antiangiogenic agents, appear to be involved in the pathogenesis of pre-eclampsia. despite several studies describing higher midtrimester serum concentrations of these markers in women with subsequent pre-eclampsia, information on first trimester serum levels is scarce. the aim of this study was to assess seng and sflt as first trimester serum markers for the prediction of pre-eclampsia. methods: sera were obtained between + and + weeks of gestation from women who later developed late-onset pre-eclampsia and from controls matched for gestational age, maternal age, maternal pre-pregnancy weight, and storage. using commercially available microplate enzyme immunoanalytical methods, seng and sflt were determined and the results analyzed using non-parametric statistical tests. results: the serum concentration of seng was found to be increased in women with subsequent pre-eclampsia when compared to controls (mean ± sd, . ± . ng/ml versus . ± . ng/ml, p = . , unpaired mann-whitney test). similarly, the serum levels of sflt were higher in women later developing late-onset pre-eclampsia ( ± ng/ml) when compared to controls ( ± ng/ml, p = . ). sensitivities and specificities for predicting pre-eclampsia were % and % for seng and % and % for sflt- , respectively. the combination of the two markers by multiplication yielded a sensitivity of % and a specificity of %. conclusion: seng and sflt , showing increased first trimester serum levels in women with subsequent pre-eclampsia, might both fulfill the characteristics of first trimester markers to predict pre-eclampsia. the combination of the two, however, did not improve the sensitivity nor the specificity compared to their individual determinations. moderate sensitivities and specifities, however, limit the clinical use of these molecules as single markers. pregnancy is associated with increased monocyte/platelet aggregate formation in whole blood. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is associated with increased rates of platelet clearance, changes in platelet function and platelet activation. the goal of the current study was to examine basal levels of platelet activation through pregnancy beginning prior to conception, and to examine the association of platelet activation with the development of hypertensive complications during pregnancy. methods two indices of platelet activation, platelet cd expression (%cd ) and monocyte/platelet aggregate (%mp) formation, were measured in whole blood by flow cytometry using specific, fluorescentlylabeled monoclonal antibodies in healthy, nonsmoking women during the follicular phase of their menstrual cycle (pp, cycle day . + . ). all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - wks) and late pregnancy (lp, - wks). five of these women were diagnosed with hypertensive complications ( hypertension, preeclampsia) at term although hypertension was not observed at any study time point. data are expressed as mean±sem. p< . was accepted for significance. results subjects were . + . years old with a bmi of . + . kg/m at the time of prepregnancy studies. a significant increase in the %mp formed over time of pregnancy was observed (p= . ). there was little change in the %mp formed between pp and ep (pp, . ± . % and ep, . ± . %, p= . ). however, the %mp increased significantly in lp ( . ± . %) as compared to pp (p= . ) and ep (p= . ). this increase occurred independent of the development of hypertensive complications (p= . ) and independent of pp platelet activation status. although statistically significant increases in cd expression were not observed, the change in cd expression over pregnancy correlated with the change in %mp over pregnancy (r= . , p= . ) and cd expression correlated with %mp in lp (r= . , p= . ). conclusion these combined observations suggest that pregnancy is associated with increases in levels of unstimulated platelet activation and that these increases occur in the presence or absence of subsequent hypertensive complications. furthermore, we observed a correlation between the changes in two distinct platelet activation events, %mp and cd expression, during pregnancy. supported by nih hl . backgound sildenafil citrate (sc) has been proposed as a therapy to improve uterine perfusion in pregnancies complicated by iugr or preeclampsia. we sought to determine the effects of sc on uterine vascular resistance, uterine blood flow and cardiac output in young healthy nulliparous women. methods eleven young healthy nulligravid women were studied during the luteal phase (cycle day + ) of the menstrual cycle. women were randomized in a double-blind fashion to receive placebo (pl), or sc at a dose of or mg. uterine artery vascular resistance, uterine artery volumetric flow, brachial artery volumetric flow and cardiac output were measured at baseline and at and hours post dosing employing color doppler ultrasound. comparisons were made by anova between those randomized to pl (n= ) versus sc (n= ). p< . was accepted for significance. data are expressed as mean + s.e.m. results there were no significant differences in subject age, cycle day, body mass index, uterine blood flow, brachial blood flow or cardiac output at baseline comparing the two groups. there was a tendency towards increased uterine blood flow in subjects randomized to receive sc ( % increase) compared to pl (no change), changes in uterine blood flow, brachial blood flow and cardiac output are outlined in the objective reduced maternal plasma volume in the third trimester has been associated with both fetal growth restriction and preeclampisa. we sought to determine the degree to which third trimester plasma volume is dependent on plasma volume prior to pregnancy. methods sixteen young ( . + . years) healthy nulligravid women had their plasma volume measured during the follicular phase (cycle day . + . ) of the menstrual cycle and subsequently conceived. subjects were predominantly caucasian ( / ) with a mean prepregnancy bmi of . + . kg/m . plasma volume was re-estimated at - weeks gestation. all patients were placed on sodium and total calorie balanced diets for days prior to each plasma volume determination. plasma volume was determined employing evans blue dilution with multiple post injection sampling time points. data are expressed as mean + s.d. results baseline prepregnant plasma volume was , + ml or + ml/unit bmi. third trimester plasma volume was , + ml representing a % increase (p< . ). the range of plasma volume expansion was - % dependent upon prepregnant plasma volume. plasma volume in the third trimester of pregnancy was strongly correlated to prepregnant plasma volume r= . (p= . ). plasma volume expansion was consistent across the range of prepregnancy plasma volume. conclusions pre-pregnancy plasma volume contributes approximately % of the variance in third trimester plasma volume. the observed increase is plasma volume is independent of prepregnancy volume resulting in a greater percentage increase for those starting at the lower end of the plasma volume range. as third trimester plasma volume is strongly associated with pregnancy outcome the correlation of prepregnancy plasma volume to third trimester plasma volume suggests that prepregnancy status contributes to these adverse reproductive events.this work was supported by nih hl . background: hydatidiform mole is a rare disorder of pregnancy and may predispose the mother to severe morbidity. molar pregnancies are known to be associated with high risk for the development of early onset preeclampsia. in recent years, the expression of sflt- (soluble vegfr- ) was found to be increased in preeclampsia, and contributes to the pathogenesis of the maternal systemic disease. the objective of the present study was to examine the expression of sflt- in placentae from molar pregnancies. methods: placental samples from unique cases of twin pregnancies with complete molar pregnancy in one sac and developing fetus in the other sac were prospectively collected (n= ). the first set delivered at weeks due to excessive bleeding. the second set delivered at weeks due to severe iugr and elevated blood pressure. mrna level of sflt- was measured by quantitative real-time pcr using specific taqman primers and probe. protein expression of sflt- in placental tissue lysates were measured by western blot analysis using a polyclonal antibody against flt- . immunohistochemistry of paraffin embedded samples was performed using specific antibody for sftl- . results: mrna level of sflt- was increased by . fold in the molar placentae compared to matched controls. the placentae of the developing fetuses which were growth restricted exhibited . fold increase compared to controls. sflt- protein expression in the molar placentae was increased by . fold compared to controls, while the co-twin placentae exhibited a . fold increase compared to controls. immunohistochemistry revealed strong positive immunoreactivity for sflt- in the trophoblast layer of both molar pregnancies and iugr co-twin relative to controls. conclusion: our data suggest that sflt- expression is increased in placentae from molar pregnancies and thus may explain the increased risk for developing early onset preeclampsia. the expression of sflt- in the growth restricted twin placenta is also increased compared to controls and support our previous observation (supported by cihr and owh/igh). (n= ); ) neonates of patients with pe (n= ); and ) sga neonates (n= ). cord blood was collected immediately after delivery and pz plasma concentrations were measured by elisa. pz deficiency was defined as a cord plasma concentration < th percentile of the normal pregnancy group. non-parametric statistics were used for analysis. results: ) cord plasma pz concentration differed significantly among the study groups (kruskal wallis, p< . ); ) neonates of patients with pe and sga neonates had a significantly lower median cord plasma pz concentration than those delivered after normal pregnancy (pe: median . g/ml, range . - . , p< . ; sga: median . g/ml, range . - . , p= . ; normal pregnancy: median . g/ml, range . - . ); ) there were no differences in the rate of pz deficiency among the groups; and ) there was no relationship between placental histologic findings and median cord plasma pz concentrations between and among the sga and pe groups. conclusions: ) at the time of delivery, the median cord plasma pz concentration was lower in sga neonates and those born to women with pe than in neonates born to normal pregnancies; ) there was no difference in the rate of pz deficiency among the study groups, suggesting that the lower median pz cord blood concentrations in pe and sga groups may result from activation of the coagulation cascade rather than an inherited pz deficiency. objective: periconceptional multivitamin (mv) use may be related preeclampsia risk. we examined the relation between timing and frequency of periconceptional multivitamin use and the risk of preeclampsia. methods: women in the danish national birth cohort who delivered singleton liveborn infants (n= , ) reported upon enrollment at . weeks (sd . ) the number of weeks of regular multivitamin use during a week periconceptional period (lmp- to lmp+ ). preeclampsia cases were identified using icd- codes (n= , . %). logistic regression was used to estimate the effect of frequency (number of weeks of use) and timing of use to lmp+ ] and post-conception [lmp+ to lmp+ ]). results were stratified a priori by overweight status. results: overall, , women ( %) reported mv use in the periconceptional period. after adjustment for bmi, smoking, parity and chronic hypertension, infrequent mv use (< weeks of use) had no relation to risk (or . ; % ci . , . ) but regular use (>= weeks) was associated with modestly reduced risk ( . ( . , . ). similarly, when mv use was modeled as a continuous variable, each additional week of use was related to reduced risk for preeclampsia (or . , % ci . - . ). this potential dose effect of periconceptional mv use appeared to be limited to normal weight women (bmi < kg/m², or . ; % ci . - . ), with no apparent effect among overweight women (bmi kg/m², or . ; % ci . - . ). a total of , women reported regular mv use in both the preconception and post-conception periods, and , women reported regular use only in the post-conception period. among normal weight women, regular use in the preconception period had no effect on preeclampsia risk (or . , % ci . - . ). in contrast, use in the post conception period was associated with reduced risk for preeclampsia (or . , % ci . - . ). conclusions: regular periconceptional mv use was associated with a modestly reduced risk for preeclampsia among normal weight women. if causal, mv use immediately after conception appeared to be the critical exposure window. background: pre-eclampsia (pe), a disorder of pregnancy characterized by maternal inflammation, results in immune, cardiovascular and metabolic dysfunction. in non-pregnant persons, inflammatory disorders are treated with and prevented by pharmaceuticals and lifestyle methods such as physical activity (pa). while most pharmaceuticals are contraindicated for pregnant women, pa during pregnancy has been found safe, healthy and beneficial for both mother and baby. clinical evidence has found pa can beneficially affect pregnancy outcome, decrease excessive inflammation and decrease the risk of pe. epidemiological studies indicate that pa may be useful in preventing pe. unfortunately, previous studies have quantified pa based on recall of postpartum women and have not controlled for differences in women's interpretations of amount, type or intensity of pa. however, investigating pa utilizing a laboratory-based exercise intervention to control these variables inflicts difficulties translating the intervention into a community-based program that attracts and retains pregnant women in order to enhance public health. method: a retrospective study was performed to determine the rates of pe among the , women who gave birth at yale-new haven hospital (ynhh) during and , and a person subset of this group who performed prenatal pa in a community-based program that is evidence-based and standardized, thereby controlling for type and intensity of pa. additionally, the program is established in the community, has been offered to the public for years and is internationally known. results: during during - , the pe rate for the general population at ynhh was . %. for the pa group, the rate was . %. two women in the pa group were diagnosed with pe in the last month of pregnancy and delivered normal infants at term. no pe was observed in this group (pa group) during the second or early third trimesters nor was there any prematurity in this group. significance: these findings support the hypothesis that adequate physical activity provided in a standardized community-based group setting may provide a non-pharmacological approach for preventing pe. growth restriction. margreet plaisier, esther streefland, pieter koolwijk, frans m helmerhorst, jan jaap hm erwich. department of gynecology and reproductive medicine, leiden university medical center, leiden, netherlands; department of obstetrics and gynecology, university medical center groningen, groningen, netherlands; department of physiology, vu univerity medical center, amsterdam, netherlands. objective: disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications, like pre-eclampsia (pe) or fetal growth restriction (fgr). whether the regulation of decidual vascular adaptation to implantation is altered in these illnesses, is not elucidated yet. the present study focused on the role of first-trimester angiogenic factors in the pathogenesis of pe and or fgr. methods: first-trimester decidua samples were obtained during routine chorionic villous sampling. the expression of vascular endothelial growth factor (vegf-a), placental growth factor (plgf), flt- , kdr, angiopoietin- (ang- ), angiopoietin- (ang- ) and tie- mrna was determined by rt-pcr. the expression of the angiogenic factors was related to the pregnancy outcome, i.e. uncomplicated, pe or fgr. results: the first-trimester decidual tissues expressed all angiogenic factors. mrna levels of vegf-a, plgf, kdr, ang- , ang- and tie- appeared increased in fgr cases compared to matched controls. in addition, plgf, ang- and tie- mrna appeared increased in pe cases compared to matched controls. the differential expression of angiogenic factors was more pronounced in cases with fgr than pe. the large inter-individual variation disallowed a significant outcome. conclusions: various angiogenic factors showed differential mrna expression in st trimester decidua of patients developing pe or fgr in later pregnancy compared to their matched controls. the first-trimester decidual samples provided a unique opportunity to obtain information regarding the onset of pe and fgr. early st trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. in turn, this may set the stage for increased non-branching angiogenesis and altered decidual and placental vascular adaptation, which may be part of the pathogenesis of pe and/or fgr. complications. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is characterized by endothelial dysfunction. the goal of the current study was to prospectively measure plasma levels of the soluble endothelial cell adhesion molecules, sicam- and svcam- , beginning prior to pregnancy and determine if subjects destined to develop hypertension complicating pregnancy had differences in the concentrations of these molecules. methods serum levels of sicam- and svcam- were measured in healthy, nonsmoking women (cycle day . + . , prepregnancy) by elisa. all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - weeks) and late pregnancy (lp, - weeks). five of these women developed hypertensive complications ( gestational hypertension, pre-eclampsia) near term. all subjects were normotensive at all study time points. data are expressed as mean ± sem. p< . was accepted as significant. results subjects were . + . years old with a mean bmi of . + . kg/m at the time of prepregnancy studies. significant differences in sicam- levels as a function of pregnancy were observed (p= . ) and are outlined in table p= . differences were dependent upon the stage of pregnancy in those women who were not diagnosed with hypertensive complications with a decrease in sicam- levels in ep (p= . ) followed by an increase in sicam- levels in lp (p= . ). in women with hypertension in pregnancy, these differences in sicam- levels were not evident (p= . ). there were no differences in sicam- levels comparing women with or women without hypertensive complications prior to pregnancy (p= . ). in contrast to sicam- , we observed no significant differences in svcam- levels over pregnancy or between those with and without hypertension. conclusions these combined observations suggest that levels of the soluble adhesion molecule sicam- change significantly over time in normal pregnancies. subjects destined to develop hypertension did not demonstrate the early pregnancy reduction in sicam- . supported by nih hl . yuval bdolah, uriel elchalal, shira natanson-yaron, hadas caspi, tali bdolah-abram, angelika bord, caryn greenfield, debra goldman-wohl, ariel milwidsky, franklin h epstein, s ananth karumanchi, simcha yagel, drorith hochner-celnikier. ob/ gyn, jerusalem, israel; ob/gyn medicine, beth israel deaconess medical center, harvard medical school, boston, ma, usa. objectives: nulliparity is a risk factor for preeclampsia (pe) with a reported incidence of up to - times higher than multiparous pregnancies. soluble fms-like tyrosine kinase- (sflt ), a circulating anti-angiogenic molecule of placental origin plays a pivotal role in pe by antagonizing placental growth factor (plgf). increased sflt and sflt /plgf have been shown to antedate clinical signs in pe. we therefore hypothesized, that the higher risk of pe in nulliparous pregnancies is associated with high sflt (or sflt /plgf). methods: maternal serum samples from nulliparous (n= ) and multiparous (n= ) term singleton pregnancies without pe, at the time of admission to delivery room, were used. serum samples were analyzed for levels of sflt and plgf by elisa. statistical analysis was performed applying t-test and the kruskall-wallis test and using spss software. results: for nulliparous and multiparous pregnancies, the mean serum sflt levels were , ± and , ± , (p= . ), the mean serum plgf levels were ± and ± (p= . ), and the mean ratios of sflt- /plgf were ± and ± (p= . ), accordingly. in a subgroup of multigravidous nulliparous pregnancies, sflt levels were , ± . correcting for maternal age did not alter the results. moreover, results did not differ between multiparous pregnancies with a - years interpregnancy interval compared with a - years interval. conclusions: in nulliparous pregnancies, circulating sflt levels and sflt / plgf ratios are significantly higher than in multiparous pregnancies. these findings suggest that the increased risk of pe in nulliparous pregnancies may involve anti-angiogenic imbalance. nulliparity may be more substantial than primigravity, as a risk factor for pe, suggesting that first semester abortions in primigravidas may not protect from pe in a subsequent term pregnancy. nevertheless, even - years intervals from the previous gestation do not increase the risk for pe. different normograms of angiogenesis should be used, when assessing the risk for pe in multiparous versus nulliparous pregnancies. objective: we determined whether maternal serum levels of angiogenic proteins namely soluble fms like tyrosine kinase (sflt- ), soluble endoglin (seng), and placental growth factor (plgf) -measured during the first trimester are associated with the subsequent development of placental abruption. methods: we performed a prospective, nested case-control study of women enrolled in the massachusetts general hospital obstetric maternal study (moms). first trimester serum samples from placental abruption cases and normal pregnancies were measured for angiogenic factors. cases and controls were matched by body mass index and age. placenta abruption was diagnosed by standard clinical findings and pathological examination of the placenta. women with confirmed preeclampsia or chronic hypertension were excluded. results: compared to controls, cases had more pregnancies, delivered infants at an earlier gestational age and with lower birth weight. first trimester levels of seng were significantly increased in cases compared to controls: . ± . ng/ml vs. . ± . ng/ml, p < . . there were no significant difference in serum levels of plgf, . ± . ng/ml versus . ± . ng/ml, p=ns, although sflt levels were lower in cases: . ± . ng/ml vs. . ± . ng/ml, p=ns. in logistic regression analysis adjusted for age, race, smoking, number of pregnancies, gestational age at delivery, gestational age of blood sampling, and blood pressure at first prenatal, seng levels remained independently associated with subsequent risk (odds ratio . , % ci . - . ) of placental abruption. examining this relationship by tertiles of seng, in the unadjusted model, women in the second (or . , % ci . - . ) and third (or . , % ci . - . ) tertiles were at increased risk of developing placental abruption compared with women in the lowest tertile. after adjusting for known risk factors of placental abruption, women in the second (or . , % ci . - . ) and third (or . , % ci . . ) tertiles remained at increased risk for placental abruption. conclusion: increased first trimester maternal serum levels of seng are associated with increased risk of subsequent placental abruption. ob/gyn, univ of vermont. shear stress is the most potent physiologic stimulus for elevating endothelial no production for flow-mediated vasodilatation. we measured in vivo shear stress during the proliferative and secretory phases and at and weeks of gestation hypothesizing that uterine blood flow (ubf) elevations in turn increase shear stress in early and late gestation. methods: during proliferative, secretory phases, and at and - weeks of pregnancy ua blood velocity and internal radius were measured bilaterally using color doppler ultrasound. blood viscosity was measured at shear rates in excess of /sec. results: compared to the proliferative phase viscosity was decreased at weeks and more so at weeks gestation (p< . ). . ± . . ± . nsd . ± . *** . ± . *** ua velocity (cm/sec) . ± . . ± . *** . ± . *** . ± . *** ubf (ml/min) . ± . ± *** . ± . *** . ± . *** shear stress (dynes/cm ) . ± . . ± . *** . ± . *** . ± . *** internal ua radius was not altered by the menstrual cycle, and was greater at weeks (+ %) and weeks (+ %). compared to proliferative, secretory phase showed significant rises in unilateral ubf and velocity that rose progressively during gestation. in contrast, shear stress increased in secretory ( %) and did not rise further in early pregnancy but by weeks shear stresses was further elevated %. conclusions: equivalent rises in shear stress during the secretory phase and weeks gestation demonstrate increases in radius and profound remodeling of uas that reflect the physiologic process of "normalization of shear". by late gestation, continued but modest rises in radius illustrate that further increases in shear stress occur almost solely due to rises in ubf via falls in down stream impedance. continued rises in shear stress into late gestation provide progressive stimuli for no production by ua endothelium. nih hl , hd , hl background: hypertension during pregnancy is associated with altered uterine vascular reactivity and blood flow, although its effects on arterial myogenic behavior have not been explored. the purpose of this study was to evaluate the effects of hypertension and no inhibition on myogenic tone in pregnancy, as the ability of a vessel to constrict and dilate in response to pressure plays a key role in regulating blood flow to the uterus. methods: three groups of sprague dawley late pregnant (day ) rats were used: control (n= ), hypertensive ( . g/l l-name in the drinking water, n= ), and treated with l-name and hydralazine (also in the drinking water, . g/l, n= ) to prevent the blood pressure increase, yet maintain no inhibition. resistance-sized radial arteries (< m) were mounted in a pressure arteriograph and equilibrated at mmhg (in pss containing l-nna and indomethacin) to induce a myogenic response. vessels were then subjected to pressure steps from to mmhg. tone (%) was calculated by comparing the vessel diameter at each pressure with the passive diameter at the same pressure (determined by incubation with . mm papaverine and m diltiazem). results: myogenic tone developed in controls ( ± % maximal), and was maintained over a broad range of transmural pressures . arteries from the l-name group did not develop tone at any pressure. co-treatment with hydralazine reinstated tone ( ± % maximal) over the same range of pressures as in the control group. the reduction in average placental weights in the l-name group ( vs. mg, p< . ) was restored by hydralazine ( mg, p< . vs. l-name). average fetal weights were also reduced in the l-name group ( . vs. . g, p< . ), but only partially restored by hydralazine co-treatment ( . g, p< . vs. control and l-name groups). conclusions: surprisingly, radial uterine arteries from the l-name group did not develop tone over any range of pressures, despite the fact that matched arteries from late-pregnant controls developed significant myogenic tone. this abolishment of tone was reversed by hydralazine, which also had beneficial effects on fetal and placental growth. these results implicate hypertension rather than no inhibition as the key factor in the suppression of myogenicitiy and dysregulation of uterine blood flow. charles r rosenfeld, timothy roy. division of neonatal-perinatal medicine, ut southwestern medical center at dallas, dallas, tx, usa. background: upbf b rises -fold in ovine pregnancy; but the mechanisms responsible for the rise and maintenance are unclear. we (jsgi ) reported that uterine vascular smooth muscle bk ca k + channels contribute to uterine vasodilation and upbf b maintenance; but up-stream activators are unclear. uterine vascular prostacyclin synthesis increases in pregnancy, but cyclooxygenase inhibition does not alter upbf b (ajp ) . vascular nitric oxide synthase (nos) also increases; but acute inhibition with l-name decreases upbf b only % (jci ) . it is unclear if l-name doses were insufficient or if prolonged nos inhibition has a greater effect. objective: to determine if local nos inhibition with l-name dose-dependently decreases upbf b and if prolonged inhibition exerts a greater effect on upbf b . methods: pregnant ewes were studied at - d gestation age (ga). had doseresponse studies with uterine artery l-name infusions to achieve . - . mg/ml over min. had h arterial l-name infusions to achieve and maintain levels of . mg/ml at (n= ), (n= ) and d (n= ) ga, while measuring arterial pressure (map), heart rate (hr) and upbf b before, during ( , , , , , , and h) and after ( , h) infusions. uterine arterial and venous cgmp were measured. data were analyzed by anova. results: acute nos inhibition decreased upbf b - %, but was not doserelated. h arterial l-name infusions decreased upbf b by - h at each ga (p . , anova) and values returned to baseline by h postinfusion. sensitivity did not differ between ga (p= . , anova), upbf b falling - % at each ga. contralateral upbf b was unaffected at all ga. map rose and hr fell during infusions at and d ga, p . ; but were unaffected at d ga. venous-arterial cgmp concentration differences were seen at d and absent at h of l-name infusion, p= . . conclusions: uterine vascular nos increases in ovine pregnancy, but its inhibition decreases upbf b %, suggesting study doses were insufficient to fully inhibit vascular nos activity. alternatively, nos contributes to the maintenance of upbf b , but other mediators, not yet identified, are more important in activating bk ca and regulating upbf b . notably, l-name reached the systemic circulation, and although further diluted, map rose - %, suggesting the systemic vasculature may be more sensitive to nos inhibition than upbf b . charles r rosenfeld, xiao-tie liu. division of neonatal-perinatal medicine, university of texas southwestern medical school, dallas, tx, usa. background: uteroplacental blood flow (upbf) rises -fold in ovine pregnancy, reflecting increases during pre-implantation, placentation and finally, in the last third of gestation. we reported that bk ca density in uterine vascular smooth muscle (uvsm) increases in ovine pregnancy (sgi ) and inhibition with tetraethylammonium ( . mm) dose-dependently decreases basal upbf % in the last third of pregnancy (jsgi ) . it is unclear how bk ca subunit expression changes and activity is regulated in pregnancy; since uterine vascular nitric oxide is increased, signaling via cgmp-dependent protein kinase g (pkg) is one potential pathway. objective: to determine simultaneous changes in uvsm bk ca density and regulatory -subunit expression and the cgmp signaling pathway for activation in ovine pregnancy. methods: endothelium-denuded segments of nd generation uterine arteries obtained from nonpregnant (n= ), pregnant (n= , - d gestation; term d) and postpartum (n= , - d) sheep were used to measure expression of bk ca -and regulatory -subunits and signaling proteins in uvsm by immunoblot analysis and immunohistochemistry. results: uvsm bk ca density, reflected as a change in the kda -subunit, rose % with placentation (p< . ) and was unchanged thereafter. expression of the kda regulatory -subunit paralleled the rise in bk ca density during placentation, increasing % (p< . ), but increased another -fold and exponentially in the last third of pregnancy (p< . , r = . , n= ). changes in subunit immunostaining in uvsm paralleled increases in protein. although uvsm soluble guanylyl cyclase was unchanged in pregnancy (p= . , r= . , n= ), pkg expression rose -fold (p= . , r= . , n= ) and gradually returned to nonpregnant levels by d postpartum (p= . , r= . , n= ). uvsm cgmp is being measured. conclusions: these are the st data suggesting that increases in ovine upbf during placentation involve vascular growth and bk camediated vasodilation. the rise in upbf in the last third of pregnancy reflects bk ca -mediated vasodilation due to enhanced channel activation via increases in uvsm pkg, bk ca phosphorylation and changes in subunit stoichiometry due to an exponential rise in the regulatory -subunit. it is unclear what initiates and directs these changes in bk ca expression and sensitivity. relaxin (rlx) is a hormone traditionally associated with changes in the female reproductive tract during pregnacy. recent evidence suggests that rlx may play a pivotal role in regulating cardiovascular function during gestation. analogous to pregnancy, administration of recombinant human relaxin (rhrlx) to nonpregnant female rats reduces systemic vascular resistance, as well as increases global arterial compliance. additionally, we demonstrated that, concurrent with rlx´s influence on overall cardiovascular function, small renal arteries (sra) from rhrlx-treated mice and rats are characterized by reduced passive stiffness and increased arterial wall area whereas external iliac arteries (eia) are not. we hypothesized that rlx regulates arterial passive mechanical properties by altering the cellular and biochemical composition of the arterial wall. nonpregnant female mice were administered rhrlx for days after which sra and eia were isolated. we measured arterial collagen, elastin, and total protein using the sircol collagen assay, the fastin elastin assay, and the pierce bca protein assay, respectively. additionally, we quantified arterial smooth muscle cell (smc) density using immunofluorescent techniques. sra isolated from rhrlx-treated mice were characterized by a significant reduction in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein; mean±sem; p< . ) as well as a significant increase in smc density ( . ± . vs . ± . cells/ μm ; p< . ) compared to control mice with no significant change in elastin content ( ± vs ± μg elastin/mg dry weight). in contrast, there were no significant changes in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein), smc density ( . ± . vs . ± . cells/ μm ) or elastin content ( ± vs ± μg elastin/mg dry weight) in eia from the rhrlx-treated mice compared to control mice. of note, comparable results were observed for rlx knock-out (rlx -/-) and wild-type mice with rlx -/mice exhibiting increased arterial collagen and decreased smc density. we conclude that the rlx-induced decrease in passive stiffness of sra that we previously reported is, at least in part, due to rlx-induced alterations in arterial wall cellular and biochemical composition. further, our findings suggest that these vessel wall remodeling effects of rlx are artery-type specific. relaxin is a peptide hormone that emanates from the corpus luteum of the ovary and circulates during pregnancy. this hormone plays an important role in renal vasodilation and hyperfiltration, two fundamental maternal adaptations in pregnancy. chronic administration of recombinant human relaxin (rhrlx) to both virgin rats and mice inhibits myogenic reactivity and increases compliance of small renal arteries, thus further mimicking pregnancy. we hypothesize that these arterial responses to rhrlx are mediated by the lgr , and not the lgr receptor. both lgr and lgr receptor-deficient, and wild-type virgin mice were investigated. the mice were chronically infused with rhrlx or vehicle (veh) for days. small renal arteries were isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a mmhg step increase in intraluminal pressure). small renal arteries from rhrlx-infused lgr wild-type mice showed inhibited myogenic reactivity with a . ± . % increase in diameter whereas the arteries from rhrlx-infused lgr knock-out mice exhibited robust myogenic reactivity with only a . ± . % change in diameter (p= . ). in contrast, myogenic reactivity of small renal arteries was inhibited in both the rhrlx-infused lgr knock-out and wild-type mice. the veh-treated lgr and lgr mice, regardless of genotype, exhibited robust myogenic reactivity. arterial compliance was also assessed for each genotype/treatment group. rhrlx infusion increased arterial compliance of small renal arteries from lgr wild-type, but not from lgr knock-out mice (p= . ). in contrast, the arteries from rhrlx-infused lgr wild-type and knock-out mice showed increased compliance relative to veh-infused animals. conclusion: relaxin-induced inhibition of myogenic reacitivity and increase in compliance of small renal arteries is mediated by the lgr , and not the lgr receptor. smoking is associated with adverse pregnancy outcomes including fetal growth restriction. pathologic effects of smoking on maternal vasculature is a potential mechanism leading to fetal growth restriction. the objective of this study was to determine whether cigarette smoke exposure during pregnancy affects the functional properties of uterine and peripheral arteries using a gravid murine model. study design: pregnant and virgin c bl/cj mice were exposed to whole body side stream smoke using an inhalational chamber for hours/day. smoke exposure was increased from day of gestation until late pregnancy (day - ) with mean total suspended particle levels of mg/m , representative of moderate to heavy smoking in humans. control animals were exposed to ambient room air. late pregnant and virgin mice were sacrificed and uterine, mesenteric, and renal arteries were isolated and studied in a pressure arteriograph system (n= - in each group). plasma cotinine was measured by elisa. means were compared using t-test or analysis of variance. results: fetal weights were significantly reduced in mice exposed to smoke compared to control fetuses ( . ± . g vs . ± . g, p= . ), while litter sizes were not different. cotinine levels in smoking mice were significantly elevated compared to control mice ( . ± . vs . ± . ng/ml for virgin mice and . ± vs . ± . ng/ml for pregnant mice). there was no significant difference in phenylephrine responses between groups. endothelial mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. no difference in endothelial-mediated relaxation was seen in isolated renal arteries in pregnant mice exposed to cigarette smoke, however relaxation was significantly reduced in renal arteries from smokeexposed virgin mice. conclusions: passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in a gravid murine model. functional vascular perturbations during pregnancy, specifically reduced uterine blood flow and impaired peripheral vasodilation, may be a mechanism by which smoking results in fetal growth restriction. human chorionic gonadotropin (hcg) is essential during early human gestation for "rescue" of the corpus luteum. however, its potential contribution to the widespread maternal vasodilation of pregnancy that occurs at this stage of pregnancy remains uncertain. our objective was to use the renal circulation of conscious rats as an experimental model in which to test the vasodilatory potential of hcg. in addition, we investigated both myogenic reactivity and relaxation responses in small renal and mesenteric arteries isolated from rats, as well as in small human subcutaneous arteries using a pressure arteriograph. we chronically instrumented rats for measurement of renal function. five were ovariectomized, and sham-ovariectomized. after days of surgical recovery, baseline glomerular filtration (gfr) and effective renal plasma flow (erpf) were measured on two separate days and the values averaged. then, an osmotic minipump containing hcg ( iu/min) was implanted s.c. and renal function was again assessed and days thereafter. gfr and erpf significantly increased and calculated effective renal vascular resistance decreased from baseline in the intact (p< . vs. baseline), but not ovariectomized (p=ns) rats on both days and of hcg administration. in the intact, but not ovariectomized rats, plasma osmolality declined and progesterone increased (both p< . vs baseline). plasma hcg concentrations were , miu/ml and comparable in both groups of rats. incubation of small renal arteries from rats with recombinant human relaxin (rhrlx, - ng/ml), but not hcg ( , - , miu/ml) in vitro inhibited myogenic reactivity and relaxed phenylephrine (pe)-constricted arteries. in contrast, both rhrlx and hcg inhibited myogenic reactivity and relaxed pe-constricted small mesenteric arteries from rats and small human subcutaneously arteries. in conclusion, consistent with our earlier work showing a virtually exclusive role for relaxin in mediating the renal circulatory changes of pregnancy, hcg is likely to play little or no role. in contrast, hcg and relaxin are both likely to contribute to the vasodilation of other organ circulations during pregnancy. pierre-andre scott, , michele brochu, jean st-louis. , research centre, chu ste-justine, montréal, qc, canada; pharmacology, université de montréal, montréal, qc, canada. uterine vasculature undergoes major structural and functional changes during pregnancy. estrogens have been shown to induce increased uterine blood flow in this circulation. we have reported that estradiol ( -e ) induced direct vasorelaxant response on smooth muscle of the uterine arteries that, for it major part, is not mediated through tissue nitric oxide. to investigate the cellular effectors mediating this vasorelaxant effect of -e , we set-up uterine arteries of non-pregnant rats in wire myograph systems for microvessels. -e and -e were equipotent in relaxing phe ( μmol/l)-preconstricted uterine arteries, although the later produced significant smaller relaxations. genistein, a phytoestrogen presumed to inhibit phosphatases, also produced uterine artery relaxation with significant lower potency. to try interfere with the vasorelaxant effect of -e , tissues were preincubated with different substances. cycloheximide (protein biosysthesis inhibitor), ici , (estrogen receptor modulator), and kt (pka inhibitor) did not significantly influenced the response to -e . rp- -pcpt-cgmps (pkg inhibitor) slightly displaced the concentration-relaxation curve to -e to the right. inhibitors of potassium channels, penitrem a (bkca) and glybenclamide (katp), showed opposite effects for -e concentration-response curve; the former producing a right shift and the latter a small not significant left shift. these data indicated that the direct acute effect of -e in uterine artery is the result of complex interactions within smooth muscle cells, involving potassium channels, and protein kinases and phosphatases. the renin-angiotensin-aldosterone system is paradoxically activated during pregnancy, since blood pressure decreased. earlier data showed that the high levels of aldosterone present during pregnancy may be involved in cardiovascular adaptation to pregnancy. in order to delineate this effect of aldosterone on vascular tone, potassium canrenoate, an antagonist of mineralocorticoid receptors (mr), was administered ( mg/kg/day) to pregnant rats from the day to of gestation. rats were sacrificed at day , and of gestation together with untreated and day pregnant rats. the thoracic aorta was quickly removed and set up in tissue baths as ring ( - mm) preparation. as observed previously, canrenoate enhanced responsiveness to phe for all time of treatment tested, but only at day ( days treatment) for kcl responsiveness. aortic contractile responses to tea (tetraethylammonium) gradually decreased during pregnancy to almost disappear at the end of gestation. in the present results, canrenoate treatment made the response statistically different by day of pregnancy. finally, the activity of na/k-atpase was measured by the relaxant effect of kcl added to physiological solution without potassium. the activity of the pump was decreased when approaching parturition compare to day of pregnancy. canrenoate treatment abolished this effect. the present data show that vascular changes that occurred during pregnancy are markedly modified by treatment of rats with an antagonist of mineralocorticoid receptors, suggesting that aldosterone may be involved in vascular adaptation to pregnancy. background impaired endothelium-dependent vasodilatation has previously been demonstrated in small myometrial arteries from women with gestational diabetes. this impairment may play a role in mediating the complications observed in diabetic pregnancies. it is not known which mechanisms of endothelium-dependent vasodilatation are affected in myometrial arteries by gestational diabetes. aim to investigate mechanisms of endothelium-dependent vasodilatation in uterine arteries using a mouse model of pregnancy complicated by diabetes. methods diabetes was induced in female c bl /j mice (streptozotocin; mg/kg) prior to mating. mice were culled at day of pregnancy (term) and uterine arteries dissected, mounted on a wire myograph, normalised to mmhg and equilibrated ( °c; %co /air). arteries were constricted with phenylephrine ( m) and a concentration-response curve to the endotheliumdependent vasodilator acetylcholine (ach; . nm- m) constructed in the presence and absence of a nitric oxide synthase inhibitor (l-nna; m), a cyclooxygenase inhibitor (indomethacin; m) or a combination of the two to determine the contribution of nitric oxide (no), prostacyclin and endothelium derived hyperpolarising factor (edhf) to vasodilatation. results sensitivity to ach was comparable between diabetic and vehicle treated mice (ec . ± . nm vs . ± . nm). the contribution of individual endothelium-dependent vasodilators was significantly altered in arteries from diabetic mice. at m ach, edhf-mediated relaxation was significantly reduced (p= . , one-way anova) compared with controls ( . ± . vs . ± ). in comparison, no-mediated relaxation was significantly increased (p= . , one-way anova) compared with controls ( . ± . vs . ± . %). conclusions endothelium-dependent relaxation was not reduced in uterine arteries of diabetic mice compared with controls. however, there was a profound change in the contribution of endothelium-dependent vasodilators in arteries of diabetic mice. this may alter compensatory capacity as disease progresses. supported by mfn training grant (cihr). raf- serine/threonine protein kinase has been extensively studied as the upstream kinase linking ras activation to the mek/erk module. mek/erk has been shown to play a role in the modulation of vascular contraction. however, the role of raf kinase in vascular contraction and its possible involvement in alteration of maternal vascular function during pregnancy is not known. objectives: to determine ( ) if raf kinase contributes to phenylephrine (pe)induced contractile response, ( ) the role of raf kinase inhibitor (gw ) in regulating vascular tone during pregnancy, and ( ) mechanism by which gw produces vasodilatation in rat mesenteric arteries. methods and results: conscious non pregnant (np) and pregnant (p) sprague dawley rats received increasing doses of pe in the absence or presence of gw . pe induced a dose-dependent increase in map in both np and p rats but responses were substantially depressed in pregnancy. gw shifted the pe-induced dose response to the right in both np and p rats. gw itself did not affect basal blood pressure. isometric tension studies in mesenteric arteries showed that gw did not change the kcl-evoked contraction but significantly inhibited the contraction to pe in both np and p arteries. interestingly, at a given concentration, gw produced greater inhibition of pe-induced contractile response in p than in np arteries. also, in p arteries the inhibitory effects of gw were greater as the pregnancy progressed from day to day . raf kinase expression and activity was significantly decreased in arteries of p compared to np rats. in mesenteric vascular smooth muscle cells (vsmcs), pe stimulated activation of raf kinase as indicated by phosphorylation of its immediate target, mek / in a time-and dose-dependent manner. measurement of [ca + ] i with fura- showed that gw -mediated inhibition of pe-induced contraction was not associated with decrease in [ca + ] i . vsmcs treated with pe exhibited higher levels of the contractile proteins, p-mypt and p-mlc which was inhibited by gw . conclusion: to our knowledge, for the first time we show that raf kinase plays an important role in the regulation of vascular contractility. decreased expression and/or activity of raf kinase during pregnancy may explain in part, for decreased systemic vascular reactivity during late gestation. the mechanism(s) that contribute to reduced vascular sensitivity to phenyleperine (pe) during pregnancy is not well understood. pe, in addition to activating the classical contractile pathways, also stimulates growth factor pathways that results in activation of ras/mitogen-activated protein kinases. it is not clear whether these pathways play a role in the modulation of vascular contraction. hence, the present study was designed to determine ) if ras is involved in mediating the pressor response to pe in non pregnant (np) and pregnant (p) rats, ) if so, the mechanism by which ras protein contributes to pe-induced vascular contraction, and ) any differential expression and/or activity of ras during pregnancy that might contribute to altered vascular response. methods: ) mean arterial pressure (map) was measured in conscious np and p rats. ) isometric tension was measured in mesenteric arteries of np and p rats. ) expression of contractile proteins, p-mlc and p-mypt were studied in cultured vascular smooth muscle cells (vsmcs). ) expression and activity of ras in mesenteric arteries of np and p rats were measured by western blot analysis. results: ( ) intravenous administration of pe resulted in a dose-dependent increase in map but responses were substantially depressed in pregnancy. inhibiting ras activation with manumycin, decreased pe-induced increase in map in both np and p rats. manumycin by itself had no effects on basal map. ( ) isometric contraction studies in myograph with mesenteric arteries showed that manumycin shifted pe-induced dose response curve to the right with a decrease in e max in np and p rats. higher concentration of manumycin was required to inhibit pe-induced contraction in np ( μm) compared to p ( - μm) rats. ( ) pretreatment with manumycin inhibited pe-induced increase in the extent of phosphorylation of both mlc and mypt in mvsmcs. ( ) compared to p rats, mesenteric arteries of np rats revealed increased basal and pe-induced expression and activity of ras. reduced expression of this contractile ras pathway might contribute to decreased vascular reactivity during pregnancy. conclusion: ras protein plays a role in regulation of vascular contraction. the decreased amount and activity of vascular ras may be a novel mechanism that explains the reduced vascular resistance during pregnancy. overweight affects the relaxin-induced response of mesenteric arteries. joris van drongelen, arianne van koppen, marc ea spaanderman, paul abm smits, frederik k lotgering. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; pharmacology and toxicology, radboud university nijmegen medical centre, nijmegen, netherlands. objective: overweight affects pregnancy-induced vascular adaptation and predisposes to gestational hypertensive disease. relaxin plays a key role in normal gestational vascular adaptation by increasing endotheliumdependent vasodilation and compliance, and reducing myogenic reactivity. we hypothesized that overweight blunts the vascular response to relaxin. the vascular responses to flow and pressure of mesenteric arteries after pre-treatment to human recombinant relaxin (rlx) or placebo (plac) were examined in overweight (ow) and normal weight (nw) rats in a pressure-perfusion myograph. overweight was established by a high-fat diet. the endothelium-dependent vasodilatation was measured in response to flow: e (flow inducing % dilatation) and e max (maximal dilative effect). active contractile (myogenic reactivity) and passive dilative (compliance) vascular responses to pressure were determined. all vascular responses were calculated as the proportional change in diameter to % precontraction with u . results: in nw rats rlx decreased e and increased e max to flow and attenuated myogenic reactivity without affecting vascular compliance. in ow plac-treated rats, compared to nw rats, an upregulated vasodilative state was present (decreased e and increased e max , and lower myogenic reactivity). in ow rats rlx increased e to flow and unaffected e max . rlx increased myogenic reactivity mildly in absence of changes in vascular compliance. values are presented as mean±sem; * p< . between nw/ow, # p< . within nw/ow. whereas rlx stimulates endothelium-dependent vasodilation to flow and myogenic reactivity in nw rats, ow overturns the flow-induced response and decreases myogenic reactivity to a lesser extent than present in nw rats. we speculate that these overweight-induced adverse effects of rlx prelude to vascular maladaptation and gestational hypertensive disease. compared to the luteal (lut) phase, uterine blood flow is increased in vivo during the follicular (fol) phase and more so during pregnancy (preg). both of these are physiologic states of high estrogen and shear stress. endothelial cells express enos and produce greater amounts of nitric oxide (no) in response to elevations of shear stress. phosphorylation of enos is a signaling marker of activation. we have recently validated lut, fol and preg uaec culture models for evaluating enos phosphorylation responses to shear stress and vascular mediators. we hypothesized that uaecs derived from fol and preg sheep will show greater enos phosphorylation than lut phase uaecs, and with more robust responses in the presence of estrogen (e ). methods: uaecs were cultured until % confluence, and then subjected to (static control), or dynes/cm for hrs in the absence or presence of e ( nm). western analysis was used to compare optical densities of ser -penos (penos) normalized to total-enos (mean + sem). results: in lut uaec penos was equally increased two fold by and dynes/cm (from . + . to . + . and . + . , respectively). compared to lut uaecs, the static control fol uaecs appeared to have higher penos levels ( . + . ) and this was further increased . - . fold with dynes/cm ( . + . ) and dynes/cm ( . + . ). as seen with fol uaecs, preg uaec static penos ( . + . ) appeared higher than lut uaec, but unexpectedly, neither nor dynes/cm significantly raised these levels of penos ( . + . and . + . ). regardless of shear stress level, e replacement in the culture media only increased the penos levels in the nonpregnant, but not the pregnant derived uaecs. conclusions: increasing amounts of shear stress have a corresponding increase in the ratio of enos that is phosphorylated at serine in lut and fol uaecs. pregnant uaecs do not appear to increase the constitutive ratio of serine phosphorylation of enos at either or dynes/cm . in contrast to our hypothesis, chronic treatment with e for hrs did not augment the ratio of enos phosphorylation in pregnancy. nih hl , hd , hl . cells. honghai zhang, wu xiang liao, dong-bao chen. reproductive medicine, university of california san diego, la jolla, ca, usa. s-nitrosylation (sno) is a rapid, reversible, and nitric oxide (no) dependent post-translational protein modification critical for signal transduction. estrogen stimulates endothelial cell (ec) no production but yet to be determined is if this leads to increased formation of sno-proteins in ec. hypothesis: we hypothesize that estrogen stimulates the formation of sno via a receptor and endogenous no dependent pathway in huvec or ovine uterine artery ec. methods: cells on glass coverslips were treated with mm of no donor gsno or dea nonoate, estradiol- ( nm) in the presence of l-name ( mm) for min. the cells were fixed with methanol. in situ sno-proteins were detected by a rapid biotin derivatization method by blocking thiols by methylmethanethiosulfonate (mmts), followed by ascorbate reduction and labeling with texas red-labeled ethylmethanethiosulfonate (mtsea) and examined by fluorescence microscopy. cells ( x /group) were lysed in hen buffer, and then similarly prepared but labeled with biotin-mtsea. a portion of the samples were separated on sds-page and blotted with anti-biotin antibody for total sno-protein patterns. the biotin-labeled sno-proteins were captured by avidin, followed by immunoblotting with specific antibodies. results: basal sno-protein labeling was apparent in both ec types. exogenous no donated by gsno or dea nonoate significantly increased red fluorescence labeling of sno-proteins in the cytosol of both ec types. treatment with estradiol- also increased total sno-protein labeling in both ec types. pretreatment with l-name significantly reduced sno-protein labeling. sds-page and immunoblotting with anti-biotin antibody analyses identified multiple bands of sno-proteins. in avidin captured biotinylated samples, multiple proteins were indentified. conclusion: exogenous no donated by gsno or dea nonoate and endogenous no upon estrogen stimulation increased s-nitrosylated proteins in ec (supported by nih ro hl and ). background and objective: the renin-angiotensin system (ras) functions both systemically and locally as a primary regulator of blood pressure and fluid volume and is therefore involved in the etiopathogenesis of essential hypertension as well as preeclampsia, a pregnancy-induced hypertensive disorder in pregnant women. while much progress has been made in the development of strategies to block the systemic ras and thus treat essential hypertension, relatively less advance has been achieved in the development of effective local ras inhibitors to treat preeclampsia, primarily due to the fact the conventional systemic ras inhibitors generate potential teratogenic risks to pregnant women during critical stages of their pregnancies. ginger has been widely and effectively used in pregnant women to treat pregnancyinduced nausea and vomiting with minimal adverse effects. the present study was designed to investigate whether ginger could down-regulate renin secretion by human decidual cells (the major source of renin in the tissue-based uteroplacental ras), as an initial step toward a long-term goal to develop potential safe and effective ras inhibitors for use in obstetric patients. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were treated with ginger juice at various doses in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. the ginger juice used was freshly prepared from the ginger roots purchased from a local grocery store and different batches of juice preparations were standardized based on their optical density values at a wavelength of μm on a spectrophotometer. results: a dominant band of renin at approximately kd was detected in all samples. when compared with the control (i.e. %), ginger juice decreased the renin protein contents in the culture supernatants in a dose-dependent manner. no significant difference in the cell morphology was observed between the control and treatment groups. conclusion: ginger root juice down-regulates renin secretion in human decidua, showing a great potential of a novel ras inhibitor, particularly, in obstetric patients. noninvasive quantification of the autonomic nervous system throughout pregnancy. dietmar schlembach, karoline pickel, daniela ulrich, philipp klaritsch, isa alkan, uwe lang, manfred g moertl. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. introduction: the analysis of heart rate variability (hrv), blood pressure variability (bpv), and baroreflex sensitivity (brs) has become a powerful tool for the assessment of autonomic control. although hrv analysis was initially developed for risk stratification in cardiology, the field of clinical application has broadened in recent years. the aim of our study was to investigate the adaptation of autonomic control during pregnancy based on analysis of heart rate variability and baroreceptor sensitivity. methods: patients with uncomplicated pregnancy were measured with the taskforce® monitor i made by cnsystems. all measurements were performed in supine position under standardized resting conditions. autonomic parameters were recorded at rest and within the "deep breathing method" as a "cardiovascular challenge test". results: throughout pregnancy a shift to higher sympathetic activity respectively a lower parasympathetic activity was observed. , and . ± . [> wks]) (figure ). during the deep breathing maneuver we could show an increase of the sympathetic / parasympathetic ratio (figure ). the noninvasive determination of autonomic parameters throughout pregnancy is possible. these results can be used as basic parameters for classifying and assessing autonomic changes in pathological conditions in pregnancy such as hypertensive disorders. how far the characterization and challenge of these autonomic functions can be utilized for diagnosis and prediction of preeclampsia has to be shown in future studies. hemodynamic parameters measured noninvasively throughout pregnancy. daniela ulrich, manfred g moertl, karoline pickel, philipp klaritsch, isa alkan, uwe lang, dietmar schlembach. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. background: hemodynamic changes throughout pregnancy have been measured predominantly by invasive techniques. the discussion on the valence und the low acceptance of these invasive procedures by pregnant women demands a noninvasive method for evaluating and distinguishing cardiovascular adaptative mechanisms throughout normal and especially complicated pregnancy. method: healthy patients with uncomplicated pregnancy were measured with the taskforce® monitor i (cnsystems, austria) at different time points throughout pregnancy. cardiovascular parameters were recorded in supine and left lateral position under standardized conditions. results: throughout pregnancy an increase of global parameters such as heart rate (hr), blood pressure (bp), total peripheral resistance (tpr) and total peripheral resistance index (tpri) were observed. stroke volume (sv), stroke index (si), cardiac output (co), contractility index (ci), acceleration index (aci), and left ventricular ejection time (lvet) decreased throughout pregnancy (table). discussion: the noninvasive determination of cardiovascular parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular changes in pathological conditions in pregnancy such as hypertensive disorders. assigning pregnant hypertensive women into hyper-and hypodynamic groups may aid in planning individual therapeutic strategies. objective: both gnrh i and gnrh ii are expressed in humans. gnrh i is produced by the hypothalamus under the regulation of gonadal steroids and stimulates pituitary gonadotropins. during pregnancy gnrh i is also produced by the placenta and affects hcg. gnrh ii, the ancient isoform, is produced by numerous human tissues including immune and reproductive tissues and circulates in blood in quantifiable levels. we have demonstrated that gnrh ii analogs directly affect fertilization and uterine function, and propose that it acts via immune regulation. during pregnancy it is known to regulate numerous hormone productions, yet the levels of gnrh ii has not been reported. in these studies we have determined the circulating levels of gnrh ii throughout pregnancy and in early pregnancy loss. study design: thirty-three women having normal pregnancies were followed prospectively. plasma samples were drawn at , , , , , , weeks gestation and during labor. plasma was also collected from patients have spontaneous abortions (n= ). circulating gnrh ii and crh and gnrh i were measured by specific radioimmunoassays. results: circulating gnrh ii increased from non-pregnant levels ( +/- pg/ ml, mean+/-sem) to +/- pg/ml by -week lmp. gnrh ii concentrations continued to increase through weeks gestation over the -week levels, although a significant increase was only attained by weeks. gnrh ii continued to increase in late term, attaining levels of +/- pg/ml which was significantly higher than that of early gestation. during labor and delivery gnrh ii in maternal plasma was further increased to +/- pg/ml, i.e., . times that of -week gestation ( +/- pg/ml). circulating gnrh ii did not parallel gnrh i in early pregnancy but did in late gestation. gnrh ii did correlate with crh in early pregnancy but not in late gestation. patients having spontaneous abortion had increased circulating gnrh ii at -weeks lmp ( +/- pg/ml) as compared to normal pregnancies ( +/- pg/ml). conclusion: gnrh ii increased throughout pregnancy attaining highest concentrations during labor and delivery. patient with early pregnancy loss had increased gnrh ii expression. the function of gnrh ii or factors affecting this increased gnrh ii in normal or abnormal human pregnancy should be investigated. introduction: plasma levels of the endocannabinoid, anandamide (aea) decrease during the luteal phase of the menstrual cycle and early pregnancy and increase during parturition. high plasma aea levels at weeks in women undergoing ivf-et was associated with a failure to achieve an ongoing pregnancy . what is uncertain is what happens to aea levels when the pregnancy has already failed. we aimed to quantify aea levels in women presenting in early pregnancy with a diagnosed non-viable pregnancy and to compare them to those of a viable pregnancy. methods: plasma aea was measured by a sensitive isotope dilution hplc-ms/ms method from women in early pregnancy ( - weeks) of whom had a viable pregnancy and had a non-viable pregnancy. serum hcg and progesterone were measured in blood samples by standard elisa methods. results: the ages and bmi of both groups were similar ( ± . versus ± . years; mean ± sd; p= . ; student's unpaired t-test and ± . versus ± . kg/m ; p= . ) respectively. plasma aea levels in women with non-viable pregnancies were significantly higher than those in women with viable pregnancies ( . ± . versus . ± . nm; p= . ). there was no correlation found between aea levels and hcg or progesterone levels p= . and r= . ; p= . , respectively) , despite progesterone being significantly lower in the non-viable group ( . ± . versus . ± . ng/ml; p= . ). conclusion: higher plasma aea levels were associated with early pregnancy failure, and this association appeared to be independent of serum hcg or progesterone concentrations. these data suggest that plasma aea levels are linked with pregnancy failure through a mechanism that does not involve hcg or progesterone production. the precise involvement of anandamide in early pregnancy needs to be investigated further. n- ) and arachidonic (aa, : n- ) acids, the major brain long-chain polyunsaturated fatty acids (lc-pufa) are generated by elongation-desaturation of dietary essential fatty acids (efa). the maternal liver is principally responsible for efa elongation-desaturation. objetive. we determined effects of protein restriction in pregnancy on expression of d, d and elongases and (elov and ) in the maternal liver rat. methods. pregnant rats were fed control ( % casein; c) or restricted ( % casein; r) isocaloric diets. at day gestation maternal blood and livers were collected. serum triglycerides, cholesterol and glucose were determinate with the synchron cx autoanalyser and leptin and insulin by ria. liver fat determination was performed by the soxhlet method. liver ara and dha were calculated by gas chromatography based upon retention times from methyl ester standards. liver d, d, elov and mrna were measured by rt-pcr and northern blot. data are m ± sem; analysis by t-test. results. liver weights did not differ in c and r. total liver fat ( ± vs ± mg) serum leptin ( ± . vs ± . ng/ml) and insulin ( . ± . vs . ± . ng/ml) differed in c and r respectively (p< . ). serum triglycerides, cholesterol, and glucose did not differ. desaturase and elongase mrna and % of maternal liver aa and dha were lower in r (fig ) . conclusion. low liver fat content and desaturase and elongase mrna in r indicate impaired lc-pufa synthesis which may adversely impact fetal development, especially the brain. objective: to test the hypothesis that obstetrical dic results from an excessive leak of fetal material into the maternal circulation. a secondary objective was to assess maternal morbidity. methods: all cesarean hysterectomy cases for hemorrhage at our hospital from to were included. intravascular presence of fetal material was determined by two pathologists, blinded to each other and any clinical information. the percentage of those with any fetal material in the maternal circulation was calculated for each diagnosis for hemorrhage. for a given diagnosis, the percentage of intravascular fetal material in those with the given diagnosis was compared to those without that diagnosis using fisher's exact test. most patients had multiple hemorrhage diagnoses. a two sample t-test was used to evaluate the difference in mean blood loss between those with and without intravascular fetal material. results: primary outcome* % of patients received blood products and % received clotting agents. the average blood loss was cc. there were no maternal deaths and injuries to adjacent organs, all to the bladder. conclusions: there was no association between the presence of intravascular fetal material and any specific hemorrhage diagnosis or amount of blood loss. although the power to detect a relationship was low, the excessive leakage of fetal material as a specific and exclusive mechanism of obstetrical dic, as postulated, seems unlikely. while % of the population was transfused, there were few intraoperative complications and no maternal deaths. hypertension, tel-aviv university, sheba medical center, ramat-gan, israel. objective: the joint national committee on high blood pressure (jnc ) determined blood pressure of - / - in adults to be prehypertension that requires health promoting life style modifications to prevent cardiovascular disease. similarly, we hypothesize that gestational prehypertension in pregnancy is associated with increased risk of pregnancy complications and its management would improve pregnancy outcome. methods: prospective and retrospective recruitment resulted in a study group consisting of patients who were diagnosed in the first and early second trimester (< weeks) with blood pressure of - / , and a control group consisting of patients with blood pressure < / at similar gestational age. comparisons between the two groups were accomplished for demographic characteristics and outcome measures using the chi-square test statistic and two-sample t-tests for categorical and continuous variables, respectively. results: all outcome measures analyzed resulted in poorer results being associated with the study group compared with control as follows: % versus % of the study and control group pregnancies, respectively, experienced preeclampsia (p= . ); and % versus % of the study and control group, respectively, were associated with gestational hypertension (p= . ). % of the control group deliveries were associated with admission to the nicu, compared to only % in the control group (p= . ); % versus % of the study and control group deliveries, respectively, were preterm (p= . ); twenty-nine percent of the study group were cesarean deliveries, compared to % in the control group (p= . ). on admission mean sbp and dbp was and mmhg, respectively, in the study group, compared to and mmhg in the control group (p= . for sbp and p= . for dbp). conclusions: "gestational pre-hypertension" may be a real pregnancy condition that when is recognized, physician attention, patient close monitoring during prenatal care and delivery and early intervention in patients at risk, may all promote improved pregnancy outcome. larger scale study is in progress to evaluate and confirm these findings in the larger pregnant population. are contractions at - weeks gestation less painful than those at full term? kristy a ruis, karin blakemore, abimbola aina-mumuney, valerie jones. gynecology and obstetrics, johns hopkins hospital, baltimore, md, usa. objective to determine if gestational age plays a role in severity of pain associated with uterine contractions. study design self-reported pain from women in labor, on a scale of - , is assessed and recorded in an obstetrical database by nurses at regular intervals at two hospitals within our medical system and was retrospectively reviewed from - . pain at various dilatations ( - cm) of women who were laboring and then delivered at - weeks' gestation was compared to women in labor at term. maternal demographics of age, race, education level, bmi, presence of support person, request for analgesia at any point in labor, and epidural placement were abstracted from maternal records. categorical data were analyzed using chi square or fisher exact test; continuous variables using student t-test. results laboring patients between - weeks gestation and at term were identified. term and preterm patients did not differ with respect to maternal demographics. pain reported by preterm patients was significantly less at - cm dilatation ( . vs. . ‚ p= . ) and significantly greater at - cm ( . vs. . ‚ p= . ). pain reported at - cm and - cm was not statistically different between the groups. of note, fewer of the preterm patients received an epidural despite the request for analgesia. conclusion preterm laboring patients between - weeks gestation may perceive less pain at - cm dilatation compared to term laboring patients; however, their pain perception at advanced dilatation was comparable to those at term despite the fact that they were less likely to have an epidural in place at the time of pain evaluation. in light of these findings, the widely accepted etiology of labor pain as the result of cervical dilatation may need to be re-examined. also, factoring in the patient's discomfort level into the evaluation for onset of early active labor may not be valid in the preterm patient. background and objective: asthma is a risk factor for adverse pregnancy outcome. this has been attributed to the effect of allergy in pregnancy. mast cell mediators can induce uterine contractility. the objective of the study was to test the hypothesis that pregnant women with asthma have different uterine electrical signals from those generated by normal pregnant women. materials and methods: uterine electromyography (emg) recordings from gestational age matched pregnant women at term were analyzed. the cases consisted of patients with asthma and the controls, those women without asthma. emg was recorded for approximately minutes from surface electrodes placed upon the maternal abdomen, with the electrical signals filtered in the uterine-specific range of . to . hz to remove noise components. the recordings were analyzed in their entirety first by power spectrum analysis and then by lyapunov analysis. the power-spectrum-largest-peak frequency and the largest lyapunov exponent were calculated for each patient, and then the mean and standard deviation of these parameters was compared using student's t-test. results: patients with asthma had significant lower power spectrum frequency and higher lyapunov exponent than women without asthma. see ) the power spectrum frequency and the lyapunov exponent used to analyze uterine emg signals were different in women with and without asthma; ) the results suggest that uterine electrical activity is altered in women affected by asthma; ) these findings may explain the observed increased frequency of preterm birth in women with asthma. further studies are required to dissect the mechanisms. fetal microchimeric cells trafficked into the maternal circulation persist in blood and tissues for years after pregnancy. increasing data suggest that microchimerism occurs after every pregnancy, but the biological role of fetal microchimerism or the cell types involved is unclear. while persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest these fetal cells play a broader role in response to tissue injury. methods: appendix specimens were acquired from women undergoing appendicectomy during pregnancy. detailed reproductive histories were obtained. fluorescence in situ hybridisation (fish) with two different probes allowed investigation of the presence of male presumed-fetal cells and nested pcr amplification of sry gene confirmed male dna in the appendix. immunostaining was used to determine the fetal cell phenotype. results: male cells were identified in appendix tissues from women with known male pregnancies (n= ) and also from a woman with no sons and a previous miscarriage of undetermined gender (n= ). no male cells were observed in the control (n= ), a woman with daughters. male cells of presumed fetal origin were evenly distributed in the muscle, mucosa and submucosa layers of the appendix. morphology and co-localisation analysis suggested the identified male cells had differentiated primarily into muscle cells or lymphocytes. combined immunostaining and y-fish demonstrated male desmin+ muscle cells and cd + and cd + lymphocytes. finally, the presence of male dna in the appendix specimens was confirmed by nested pcr. male cells of presumed fetal origin were identified in the appendix of pregnant women. microchimerism frequency varied according to the reproductive history and the degree of inflammation. microchimerism rates were higher in the appendix from women with current male pregnancies than in those with previous male pregnancies and were higher in those with histological acute inflammation, when compared to milder cases. male microchimeric cells identified were of haematopoietic and mesenchymal origin. this study suggests that fetal cells are present in sites of tissue injury and may participate in tissue repair during pregnancy. collagen i and collagen-binding integrins mrna expression in rat cervix during gestation. huiling ji, tanya l dailey, vit long, edward ks chien. obstetrics and gynecology, maternal fetal medicine, women and infants' hospital of rhode island, providence, ri, usa. objective cervical remodeling is associated with cell proliferation and extracellular matrix (ecm) remodeling. integrins are a family of multifunctional cell adhesion receptors which mediate ecm-cell interactions including binding collagen. of the integrin (i) heterodimers, , , and are primary receptors for collagen. the predominant cervical collagen is collagen type . the purpose of this study is to investigate collagen and integrin expression in the pregnant rat cervix. the cervix was harvested from timed pregnant sprague-dawley rats. non pregnant (np)and timed pregnant day , , , , and animals were euthanized using a protocol approved by the iacuc. four animals were sacrificed on each day of gestation. quantitative rt-pcr was used to evaluate mrna expression and normalized to -actin. a standard curve was generated from a single sample. data was analyzed using anova and multiple comparisons testing. collagen i mrna expression increased through mid-gestation but decreased to np levels on day . a similar pattern was seen with the integrin beta subunit. the pattern of integrin alpha subunit expression was different for each subunit during pregnancy. the changes in collagen, integrin alpha , , and integrin beta were found to be statistically significant. (figure) . the pattern of collagen binding integrin alpha subunit expression during the rat gestation appears to vary independently of each other. the expression of the subunit paralleled col expression. collagen binding integrins may play independent roles in signaling and biomechanics during gestation. funding: women infants' hospital research fund; phs nih-ncrr p rr p rr . background. the effects of chemotherapy on human fetal development are poorly studied, mainly due to the lack of adequate animal models in which placentation and embryological development resembles humans and pharmacokinetic studies, prenatal sonography as well as histological studies can be performed. aim of the study. to test the baboon as model for studying pharmacokinetics and fetal effects of chemotherapy administered during pregnancy. methods. experiments were performed in pregnant baboons at a mean gestational age of (+/- ) days. detailed ultrasound examination (biometry, doppler, screening) was performed days before, at and day after the drug administration. the administration of different schemes of chemotherapy and the fetal samplings occurred under endotracheal anaesthesia. maternal blood samplings, as well as percutaneous ultrasound guided fetal blood ( ml) and amniotic fluid ( ml) samplings were performed at least once immediately after drug administration. in case of fetal demise, the fetus underwent detailed macro-and microscopic examination. in the other animals mother and fetus were euthanized h after the experiments, with collection of blood, amniotic fluid and tissues for further analysis. results. all mothers survived the experiments, one mother developed paralitic ileus. none of the fetuses died during the acute phase of the experiment but / ( %) fetuses died within h following the experiment. none of the animals showed significant clinical or histological signs of infection or anaemia. a total of ultrasound examinations were performed in fetuses, allowing the creation of sonographic growth charts in this model. at the time of drug administration the mean maternal weight was . kg (range . - . ) and the estimated fetal weight was gr (range - ). sixteen out of cordocenteses ( %) and all amniocenteses ( %) were successful in obtaining the required samples for analysis. conclusion. the pregnant baboon can be used as a model for studies on pharmacokinetics and fetal effects of chemotherapy administered during gestation. prenatal ultrasound is comparable to the human situation, and amniocentesis and cordocentesis can be performed with a high success rate and short term survival. therapy to prevent preterm birth is limited". moreover, -hydroxyprogesterone ( ohp) caproate is a category d drug according to the fda (evidence of fetal harm). p is produced in the adrenal glands, gonads and brain. after weeks gestation p is secreted from the placenta independently to the mother and the fetus. p is the precursor of aldosterone and after conversion to ohp, of cortisol and androstenedione. baboons have similar reproductive system and development to humans and are used to study mechanisms of labor and prevention of preterm labor. currently, there are no normative data on the concentration of p, ohp, cortisol ©, estradiol (e ) or inhibin (i) throughout their pregnancy. therefore, a doseresponse or the teratogenic effects of p or ohp caproate cannot be studied in a controlled manner in this animal model. the aim of this study was to generate reference values for these hormones during baboon gestation and early postpartum life. material and methods: hormonal quantitative measurements were performed (immulite analyzer) results: the mean concentrations of these hormones are depicted in figure (maternal serum, from conception to term days) and figure (newborn serum) conclusion: this study provides reference values for hormones quantified during the baboon gestation and early postpartum life. this may be useful for future research concerning the effects of p and ohp administration during pregnancy. ( )non-pregnant women eligible for ivf treatment and ( )pregnant women receiving prenatal care. the pre- / samples were drawn in the year prior to / / ; the post- / samples were drawn within months after. the pre- / and post- / normal pregnant samples were drawn at + weeks' gestation. the non-pregnant samples were from ivf candidates with serum e levels< newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced pg/ml and fsh serum levels< . miu/ml. the samples were assayed for acth and cortisol by a commercially available immulite™ system. hsp and hsp were measured by commercially available elisa. these results were compared in the patient populations before and after / . t-tests were used for analysis. statistical significance was set at p< . . results: in the pregnant subjects, there were no statistical differences in the mean levels of acth, cortisol, and hsp pre-and post- / (table ) . only serum hsp levels were significantly increased in the pregnant women post- / .* mean serum acth, cortisol, hsp , and hsp levels were all significantly different in non-pregnant subjects pre-and post- / . conclusions: except for hsp , serum markers for stress were unchanged in pregnant subjects after the stress of / / in comparison to non-pregnant women whose serum hsp , hsp , cortisol, and acth levels were significantly different. the lack of change in serum markers of stress in pregnant women suggests that the hormonal milieu of pregnancy may buffer against acute environmental stressors. objective: in human pregnancy, maternal body composition provides an indicator of maternal nutritional status and metabolic capacity. previously, we reported that ß hsd- activity was significantly reduced in term placentas from thin women and women with a smaller mid-upper arm circumference before conception. this suggests that these fetuses may have been exposed to inappropriate levels of maternal cortisol, which is a mediator of gestation length. in this study, we hypothesize that the duration of gestation will be shorter in women who tend to be thin and have a lower mid-upper arm circumference prior to conception. methods: within the longitudinal, population-based southampton women's survey (sws), analyses were performed on women whose estimated date of conception was set using an algorithm that combined menstrual and early ultrasound scan data and who had a spontaneous onset of labour and delivered after weeks of gestation. linear regression was used to examine the relationships between maternal body composition and the duration of gestation. results: within the women surveyed, lower maternal body mass index, mid-upper arm circumference and arm muscle area before conception were all associated with shorter duration of gestation at delivery (r= . , p= . ; r= . , p= . ; and r= . , p= . , respectively). a lower subscapular skinfold thickness, sum of skinfolds and height were also associated with shorter duration of gestation (r= . , p= . ; r= . , p= . and r= . , p= . , respectively). mother's age, own birthweight and ratio of subscapular/triceps skinfold thickness were not related to the duration of gestation. conclusions: in this study, we found that thinner women with a lower midupper arm circumference and arm muscle area tended to have a shorter duration of gestation. our findings of shorter gestation length in thinner women are in keeping with our previous observation of lower ß hsd- activity in term placentas from thinner women. we conclude that metabolic capacity prior to conception could influence duration of gestation through mechanisms that include alteration in placental metabolism and fetal cortisol exposure. fecal incontinence (fi) is a debilitating condition affecting - % of the us. our prior studies found that % of women report new onset fi after childbirth. the goal of our study was to examine the impact of fi on postpartum quality of life (qol). women reporting fi on a statewide survey who agreed to participate in a -year study of qol were included in the analysis. women were considered to have fi based upon the nih definition of fi. the quality of life survey was based upon the uebersax incontinence impact questionnaire and was administered every months for years. qol in women with fi was examined using chi square, and impact of severe fi (stool incontinence) was determined by multivariate logistic regression. results women with fi were surveyed and of those, ( %) returned at least survey during the study period, completed all survey questions, and were included in the final analysis. among women with fi, % felt frustrated due to fi, % reported fi impacted their emotional health, . % reported fi impacted child-caring abilities, and . % reported a negative impact on social activities. qol was similar across survey periods. one out of three women with fi reported severe symptoms (incontinence of stool). women with severe symptoms were - times more likely to report negative impacts on qol compared to milder (e.g. flatus) fi after adjusting for age, parity and urinary incontinence. . % felt their stool was stored elsewhere before bowel movements, . % reported using digital defecation and more than half ( %) reported symptoms of urinary leakage. despite the substantial impact on postpartum quality of life, few women sought medical help with only % of women at months, . % at year, and . % at years ever reporting their symptoms to a medical provider. postpartum women report that fecal incontinence has a substantial negative impact on their qol after delivery including their emotional health and ability to care for their newborn. despite this profound impact, few women will discuss fi with medical providers. these data suggests there may be a benefit for providers to inquire about fi at postpartum visits. objective: the objective of this study was to determine what characteristics contribute to racial/ethnic differences in breastfeeding rates. study design: a retrospective cohort study of all women who delivered a viable infant (n= , ) was conducted. the primary outcome was breastfeeding upon discharge of the hospital. we first examined the association between race/ ethnicity and breastfeeding. next, we conducted stratified analyses examining a variety of predictors of breastfeeding within the racial/ethnic groups including maternal demographics, obstetric interventions, and perinatal complications. results: we found that both asians (or . , % ci . - . ) and blacks ( . , % ci . - . ) had statistically significant lower rates of breastfeeding, while latinas (or . , % ci . - . ) showed a trend towards higher rates of breastfeeding. in latinas, we found that rd and th degree lacerations were significantly associated with lower rates of breastfeeding, but were not predictive of breastfeeding in the other racial/ethnic groups. epidural use was predictive of lower rates of breastfeeding in caucasians and blacks, but was not predictive in latinos and asians. some of the other predictors which differed between the racial/ethnic groups were obesity and induction of labor (table ) . conclusion: race/ethnicity is significantly associated with breastfeeding, with blacks and asians having the lowest rates of breastfeeding at discharge. a variety of other factors are associated with breastfeeding and interestingly, their effect appears to differ between the racial/ethnic groups. future studies might elucidate the sociocultural and biomedical reasons that explain the differences. these results help focus our efforts during the peripartum period to advocate for mothers who have factors that might impede breast feeding. epidemiology, erasmus medical centre, rotterdam, netherlands; pediatrics, erasmus medical centre, rotterdam, netherlands; public health, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background recommendations on folic acid use to prevent neural tube defects are launched in several countries. however, the adequate use of folic acid supplements during the periconception period seems to be low. objective to assess the prevalence of adequate folic acid use, defined as the preconception start of supplements, and to identify its determinants in a multi-ethnic population. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods from all women in the cohort who delivered between april and january information on folic acid use and potential determinants was obtained by questionnaires and physical examination. logistic regression models were used to identify determinants of periconception folic acid use. results. data from , pregnant women were available. of all women % adequately used folic acid supplements. the most important risk factors for inadequate use were unplanned pregnancy (or . , p< . ), nonwestern ethnicity (or . , ci . - . , p < . ) and a low educational level (or . , ci . - . , p< . ). other risk factors were single marital status, smoking, multiparity (all p< . ) and alcohol use (p< . ). prior spontaneous abortion was associated with increased adequate folic acid use (p< . ). conclusion adequate periconceptional folic acid use is low. improved preconception care and public health education programs are necessary to improve the uptake of folic acid. although methamphetamine (ma) use has been front and center of the united states drug control policy since , little attention has been given to ma use in pregnancy, despite the fact that pregnant admissions to drug treatment for ma have been rising sharply. to determine trends in the prevalence of ma treatment admissions during pregnancy, we undertook a secondary analysis of the treatment episode data set (teds), an administrative data set that captures at least % of all known treatment admissions in the us. in particular, we investigated risk factors for ma use and how these characteristics have changed over time. demographic, geographic and substance use data were collected for the , pregnant admissions captured between and . logistic regression models were constructed by year. confounding was assessed via backwards elimination with a change-in-estimate criteria of . considered substantial. trend results were reported as adjusted proportions and represented graphically. overall ma prevalence, reported as the primary drug of use upon admission, rose from . % in to . % in . although white women had times the odds of using ma in (adjusted or ( %ci) . [ . , . ]), this had dropped to . [ . , . ] by , mostly due to an increase in latina ma use in pregnancy. pregnant women admitted for ma had fewer prior treatment admissions, were more likely to be unemployed, and less likely to use alcohol or marijuana. we found great geographic variability in use. ma was more common in the pacific region and least common in new england. there was little change in regional variation over the study time period. less is known about the perinatal effects of ma compared with other substances, yet it is the primary drug of choice for pregnant women admitted into treatment. as pregnant women occupy a unique place in drug treatment, analysis of national trend data is essential to guide both policy and research. background: epidemiologists have grouped the multiple disorders that lead to extremely preterm delivery in a variety of ways. we sought to identify characteristics that would support the combining or dividing of the disorders that lead to preterm delivery. methods: we enrolled , women who delivered a live born singleton infant between and completed weeks gestation at tertiary centers in the united states. each delivery was classified according to the complication that prompted presentation: preterm labor, preterm premature rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes (pprom), preeclampsia, placental abruption, cervical incompetence, and fetal indication/intrauterine growth restriction (iugr). we compared these entities on the frequency of characteristics identified by standardized interview, chart review, histological examination of the placenta, and culture of placenta parenchyma. results: the percents of women who presented with each antenatal complication were: preterm labor ( ), pprom ( ), preeclampsia ( ), placental abruption ( ), cervical insufficiency ( ), and fetal indication/iugr ( ). after considering antecedents and correlates of the processes leading to preterm delivery, we observed two overarching epidemiologic patterns. the first pattern, characterized by recovery of organisms from the placenta and by histologic chorioamnionitis, tended to be associated with preterm labor, pprom, placental abruption, and cervical insufficiency. the second pattern, characterized by a paucity of organisms and inflammation and the presence of histologic features of dysfunctional placentation, tended to be associated with preeclampsia and fetal indications/iugr. conclusions: disorders leading to preterm delivery can be categorized broadly into two groups: those associated with intrauterine inflammation and those with aberrations of placentation. predictors of compliance with tuberculosis screening in pregnancy. nadav schwartz, sarah wagner, sean keeler, may tam, julian mierlak, , aaron caughey. obstetrics and gynecology, nyu school of medicine, new york, ny; obstetrics and gynecology, ucsf, san francisco, ca; obstetrics and gynecology, gouverneur health care services, new york, ny. objective: poor compliance with tuberculosis screening and treatment is a major obstacle to the containment of this disease. we sought to identify predictors of ppd and cxr compliance during pregnancy. methods: a retrospective cohort study at a single institution which serves a largely immigrant population in nyc, from november , through june , . data on maternal age, ethnicity, country of origin, level of education, ppd and cxr status were collected. results: of the , pregnancies, asian and hispanic race/ethnicities accounted for . % and . % of the population, respectively, with . % being us-born. the mean age was . years and the overall ppd+ rate was . %. there was % non-compliance with ppd testing, and % of ppd+ patients were non-compliant with their chest x-rays. asian women were more likely to be ppd-compliant than hispanic or caucasian women. ppd+ asian women were also more likely to be compliant with cxr. us-born women were significantly less likely to be compliant with their ppd or with their cxr. women > years old were less likely to be compliant with their cxr, while women with an elementary school education or less were more likely to be compliant. (see table for odds ratios and %ci) conclusions: age, education, immigrant status and other cultural and ethnic factors appear to play a role in compliance with tuberculosis screening. further elucidation of these effects may help clinicians target at-risk subpopulations and improve overall compliance, working towards better control of this disease. background: in the us, differential outcomes in cervical cancer among medically underserved women are linked to multiple barriers impacting loss to follow-up and failure or delay in diagnostic resolution. prior studies have found risk factors for acquisition of hpv and for inability to obtain a pap smear. no study has explored health literacy and physician communication as a key factor. methods: this research represents the formative phase of a randomized controlled trial of african american (aa) and hispanic women aimed to improve communication and abnormal pap smear follow-up in chicago. semi-structured interviews and focus groups were conducted face to face in spanish or english. each interview lasted minutes, and each focus group lasted - minutes. we recruited patients ( hispanic, aa) and providers from a purposive sample representative of two large clinics that serve low-income women. results: all interviews were transcribed by two investigators. each interview was coded by two investigators separately and then a third investigator reviewed the transcripts and coding to achieve triangulation. codes for these themes were developed and the responses were tabulated using the coding scheme. atlas ti was utilized to analyze all qualitative data. from these data, we uncovered provider-patient challenges in cancer communication including: the providers' trade off between medical accuracy and/or literacy when communicating with their patients. for the patients, the word cancer was important to hear since they wanted the truth and needed to hear this word in order to encourage them to respond more quickly. however, many providers believed that the word cancer was too "scary" and to "extreme" of a word that may communicate too much exaggerated information. both the hispanic and aa patients did not seem to differ in their responses. conclusion: results exemplify not only the importance of health literacy and patient provider communication, but demonstrate the wealth of information gained from qualitative research-a method of data collection seldom utilized in ob/gyn investigations. funding: women's reproductive health research award: hd - ; r ca (spring). population. kristine beckers, isabelle guelinckx, greet vansant, roland devlieger. obstetrics and gynaecology, university hospital gasthuisberg, leuven, belgium; clinical nutrition, katholic university leuven, leuven, belgium. objective: to generate reference charts for weight gain during pregnancy for the different bmi-categories (underweight, normal weight, overweight, obesity), based on recent data in a homogeneous caucasian population. methods: in a retrospective study at the department of obstetrics of the leuven university hospital (belgium), weight gain and pre-pregnancy bmi were determined in belgian pregnant women with accurately dateable, uncomplicated singleton pregnancies. centile curves for the different bmicategories were constructed using of the linear mixed model, one set of charts based on the absolute weight gain, another set based on the relative (expressed as percentage) weight gain. the effect of parity on weight gain was examined. results: overall mean weight gain was . ± . kg ( . ± . lbs). mean weight gain was . ± . kg ( . ± . lbs) in the underweight population, . ± . kg ( . ± . lbs) in the population with normal weight, . ± . kg ( . ± . lbs) in the overweight population and . ± . kg ( . ± . lbs) in the obese population. weight gain (pattern and amount) of the underweight and normal weight patients differed significantly of the overweight and obese patients. parity had a statistical, but no clinical significant influence on amount and evolution of weight gain. conclusion: by using strict inclusion criteria, bmi-category-specific reference charts were generated representing the optimal gestational weight gain, rather than the mean weight gain. this enables the weight charts to be used as a clinical tool during the counselling of pregnant women. further studies are required to assess the effectiveness of this clinical tool. female fshr(-/-) mice are sterile, because of a block in folliculogenesis at the primary follicle stage, show decrease in e and elevated fsh. this animal model is an appropriate model for studying hypergonadotropic ovarian dysgenesis and infertility, caused by c t mutation in fshr gene. objective: to investigate the effects of bmt on serum hormonal levels, follicular maturation and fertility of fshr(-/-) mice. methods: fshr (-/-) mice at - weeks of age were randomized into treated versus control groups.bm from syngenic female donor was injected into the tails of recipients. control group received vehicle alone. vaginal smears were collected, body weight was measured daily. sample animals were sacrificed at , , , , and weeks post bmt. all organs were weighted and examined by h e. fsh, e , and p were measured before and after treatment. for donor cell tracking, dna was extracted from various organs. specific primer sets were designed for normal and mutant hfshr gene. pcr amplification was done and pcr products were analyzed in % agarose gel. results: total body weight significantly increased in treated animals(p< . ). significant increase in both the total number of follicles, and the collective diameter of the follicles in treated animals observed(p< . and p< . ). six out of treated animals showed estrogenic changes in daily vaginal smear. e level increased . - . times and fsh level dropped to % in treated animals. normal (donor allele) fshr gene was amplifiable in out of recipient mice, and was detected only in the ovaries and uterus but not in any other tested organs.control group did not show any changes in vaginal smear, hormonal level, and normal fshr allele. conclusion: intravenously injected syngeneic bone marrow cells were able to home to the ovaries of female fshr (-/-) mice and restore folliculogenesis and resume steroid hormone production. potential mechanisms for these observations will be discussed. conclusion: neural stem cells (nscs) give rise to progenitors, which expand by rapid proliferation until cell cycle arrest followed by differentiation along one of cns cell lineages: neurons, astrocytes and oligodendrocytes. increased differentiation of neuronal cells with ins and even more with leptin suggests that iugr-associated reduction of these growth factors may contribute to impaired hypothalamic pathway development. objective: to develop a technology of modulating hucb in order to achieve neuronal cells for future potential replacement of damaged neuronal tissue. design: we developed a two-dimensional ( d) tissue culture technology for isolation and differentiation of collagen-adherent hucb neural progenitors (hucbnps). we further used the extracellular matrix protein collagen, organized in a three-dimensional ( d) gel, supplemented with neuronal conditioning medium and nerve growth factor (ngf), to facilitate ex-vivo long term neuronal differentiation of hucbnps. we developed a stable green fluorescence protein (gfp)-pc cell model, to be used as a positive control for monitoring neuronal outgrowth for proper evaluation of the hucbnps growth in the d scaffold, mimicking a neural tissue organization. results: our experimental data indicate that d collagen environment is neuronal biocompatible, supporting attachment, long-term survival, proliferation and differentiation of both hucbnps and gfp-pc cells. conclusions: improvement of the d technology with cultures of hucbnps that is in progress in our laboratory might be the first step in validating the concept for the feasibility of generating a neuronal d tissue construct for future potential treatment of neurodegenerative disorders. piane, justin tsai, gnanaratnam giritharan, emin maltepe, paolo rinaudo. reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: ivf embryos are often used to generate stem cells. however, it is unknown if stem cell lines originated from in vitro generated embryos are different from stem cell originated from in vivo embryos. trophoblastic cells, due to their external position within the embryo, are most susceptible to environmental factors encountered in vitro. furthermore, our previous data showed that trophoblast transport functions may be impaired after culture in vitro and that the number of trophoblastic cells is reduced in the embryo after ivf. in this study, we assess the differentiation characteristics of ts cell lines obtained from in vivo and in vitro fertilized embryos. methods: oocytes were isolated from superovulated c bl/ j female mice and in vitro fertilized with sperm from male c bl/ j mice. the resulting late-cavitating blastocysts were harvested. in vivo controls were obtained by flushing the blastocysts from the uteri of superovulated pregnant mice days post hcg. ts cells from the two groups were allowed to develop and maintained in vitro in the presence of fgf and heparin, using a feeder layer of human placental fibroblasts. ts cells were then allowed to differentiate without fgf and heparin, fixed on day , stained with -tubulin and zo- antibodies and then observed under fluorescence microscopy. three ts cell lines per group were analyzed and their differentiative capacity was evaluated using morphological criteria. results: in vivo and ivf derived ts exhibit a similar differentiation pattern. in particular, the number and timing of trophoblast giant cells and spongiotrophoblasts derivation is similar in the two groups. there are no obvious abnormalities in the immunologic staining morphology of the different cell lines at different time points. conclusion: there are no apparent morphological alterations in ts cells lines derived from ivf embryos as compared to in vivo embryos. this finding in an animal model increases our confidence in the reliability of human stem cells derived from ivf. as a further investigation, markers of trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast and chorionic trophoblast cells will be examined and compared in the two different groups by northern blot hybridization. genomewide high density snp-cgh reveals several new deletion copy number variants on the x chromosome in pof patients. erik ah knauff, cisca wijmenga, ruben van 't slot, lude franke, bart cjm fauser. reproduction gynecology, university medical centre, utrecht, netherlands; complex genetics group, university medical centre, utrecht, netherlands. introduction: around % of women have a post-menopausal hormonal profile before age , referred to as premature ovarian failure (pof). pof could act as a genetic model for accelerated follicle loss and may render useful information about the polygenetic background of major individual differences in menopausal age. macrodeletions on the x chromosome are associated with pof but karyotyping has a maximal resolution of mb. when using genotyping whole genome arrays it is now possible to detect submicroscopic deletions and duplications (copy number variants (cnv)) up to kb effective resolution. we have screened for microdeletions on the x chromosome in a well-phenotyped cohort of pof patients. methods: our study included caucasian, ,xx patients with spontaneous secondary amenorrhea before age , fsh > iu/l and absence of (low) x/ xx mosaicism and fmr premutations. dna analysis was performed using illumina k cnv arrays containing , probes. , probes were located on the x chromosome ( probe for every kb), of which , probes were specifically designed to detect known cnvs. ten samples with call rates < % were removed from the analysis and one sample gave inconsistent x probe intensities. we screened for deletions ( kb) using an algorithm detecting at least five consecutive probes with intensities (logr > - . ) below the mean probe intensity. we identified a total of x chromosomal microdeletions, divided in loci and varying between - kb in size. of the samples showed at least one deletion on the x chromosome. % of the identified deletions had already been recorded in the database of genomic variants. in the newly identified newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced loci, we found coding regions containing genes. eight of these are established or potential pof candidate genes, including five that are clustered on the terminal xq critical pof region. conclusions: we observed abundant variation in the cnv regions, a proof-of-principle for this specially designed cnv-chip. snp comparative genomic hybridization revealed possible new deletions specific to pof on the x chromosome. data on duplications, validation and whole genome cnv analysis, compared to a control cohort, will become available soon and will be presented at the sgi annual scientific meeting. unexplained intrauterine fetal death (iufd) is associated with long qt syndrome. irene cetin, patrizio antonazzo, sabrina cozzolino, stefania calabrese, lia crotti, francesca ferrari, roberto insolia, fabio facchinetti, peter j schwartz. dept. of obst/gynecol, univ. of milano, milano, italy; molec cardiol lab, policlinico san matteo, pavia, italy; mother-infant dept., univ. of modena/reggio emilia, modena, italy. introduction: in developed countries, nearly in every pregnancies ends in late fetal loss. many iufds can be attributed to maternal disorders, fetal pathology, placental pathology and fewer to complications of labor and delivery. however, - % of cases remain unexplained. we recently demonstrated that % of sudden infant death syndrome (sids) cases carry functionally relevant genetic variants in long qt syndrome (lqts) genes. aim of the study was to analyze whether lqts genes are associated with iufd. materials and methods: patients with iufd were enrolled in two years, as part of an italian multicentre study. iufd was defined as fetal death at weeks or more of gestation according to the definition of late fetal death of the who. out of cases were classified as "unexplained stillbirths" according to the wigglesworth and aberdeen classifications. at birth placental and/cord samples were collected and dna extracted. the main lqts genes kcnq , kcnh , scn a, kcne and kcne were screened through dhplc and sequence analysis. any amino-acid substitution identified in the samples was checked for in a control population of caucasian women with uneventful pregnancies. preliminary data on the first cases (gestational age of death: - weeks) are reported. results: a total of missense mutations were identified in of stillbirths ( %), two on scn a and one on kcnh . the two mutations on scn a (v l; p a) were observed in two iufd occurred at term; they had been previously associated to sids and shown to increase the late sodium current. the mutation on kcnh is a novel genetic variant absent in reference alleles, never described in any control populations; this mutations was present in a case of iufd diagnosed at weeks of gestation. we are currently performing the electrophysiological cellular studies to define its functional effect. conclusions: these preliminary data indicate that a potentially significant number of currently unexplained iufd might be caused by ion channel diseases such as lqts. the potential prevention of sids or iufd recurrence and the identification of other affected family members could have important implications for the affected families. alternative splicing of epab is regulated by exonic splicing enhancers. e seli, a yaba, o guzeloglu-kayisli, md lalioti. ob gyn, yale u., new haven, ct, usa. introduction: alternative splicing is an important mechanism by which the genome gives rise to the observed diversity of proteins. embryonic poly(a) binding protein (epab), expressed exclusively in oocytes and early embryos, mediates translation of maternal mrnas. we identified an alternatively spliced form of epab lacking exon (cex del), and investigated its regulation as a model for alternative splicing in early development. specifically, we evaluated: imprinting (expression from maternal or paternal allele only); rna editing (post-transcriptional single nucleotide substitution); and exonic splicing enhancers (eses, exonic sites that bind splicing proteins). methods: a single nucleotide polymorphism (snp) detected in exon (c a/g) served as a marker for the parental origin of the spliced form. snp genotyping was performed by pcr amplification of exon followed by restriction enzyme digestion. to evaluate imprinting, we characterized heterozygous mice (a/g) that inherited the snp from either the mother or the father. to test for rna-editing and exonic enhancer contribution we tested mice homozygous for the exon snp (a/a or g/g). efficiency of alternative splicing in different genetic backgrounds was tested using real-time pcr normalized to actin expression. results: in mice heterozygous (a/g) for the exon snp, cex del was only expressed from the (a) allele. however, this was independent of the parental origin of the allele, ruling out imprinting. in mice homozygous (g/g) for the exon snp, the cex del variant also contained (g). therefore, rna editing did not occur. further sequence analysis led to the identification of an additional snp in exon (c g/c) that co-segregated with the exon snp. presence of c g led to the formation of an ese that binds splicing regulatory protein srp , leading to efficient exclusion of exon . real time pcr revealed a five-fold increase in the expression of the cex del alternative splicing variant in animals carrying the enhancer (homozygous g/g) for the exon snp compared to those that did not (homozygous c/c) at the same locus (p < . ). conclusions: in this study, we found that eses mediate the alternative splicing of oocyte-specific transcripts. our findings suggest that single nucleotide polymorphisms may alter the ratio between alternative splicing variants of oocyte-specific proteins. the role that these subtle differences play in determining individual reproductive outcome remains to be identified. epigenetics and chromosomal abnormalities in human oocytes. ilse van den berg, , joop se laven, robert jan galjaard, j hikke van doorninck. , obstetrics gynaecology; clinical genetics, erasmus medical center, rotterdam, netherlands. humans have a low fertility rate compared to other mammalian species. moreover their fertility declines with increasing age. both phenomena are largely due to an increasing number of chromosomal abnormalities in human oocytes during life. identifying factors that cause aneuploidy in oocytes may offer possibilities to diminish these abnormalities in vitro or in vivo. chromosomal segregation errors can result from aberrant recombination but little is known about epigenetic factors that may cause aneuploidy. epigenetic modifications such as histone acetylation and subsequent de-acetylation in oocytes are necessary for a correct progress through meiosis. if disturbed it may lead to aberrant segregation of chromosomes or chromatids due to decreased kinetochore function and imperfect spindle figures resulting in aneuploidy. mice oocyte data have shown a correlation of abnormal histone de-acetylation and aneuploidy and a correlation between age, remaining histone acetylation and aneuploidy (akiyama et al., ) . our research focused on human oocytes and investigated whether a similar relationship between histone acetylation, age and aneuploidy is present. human oocytes were surplus from standard ivf/icsi treatments (ic+). human oocytes showed immunostaining for histone , lysine acetylation (h k ) at the germinal vesicle stage and complete deacetylation at the mii stage in % of the oocytes, while % keep high levels of h k acetylation. treatment of germinal vesicle oocytes with a histon deacetylase inhibitor (tsa) during in vitro maturation until mii stage resulted in high levels of acetylation. remaining acetylation in tsa treated oocytes was correlated significantly with abnormal spindle figures (tubulin staining), a hallmark of developing aneuploidy. similarly, % of oocytes with naturally remaining h k acetylation showed abnormal spindle figures. in contrast % of the normal de-acetylated oocytes showed normal spindles (p= . ). this suggests that defective de-acetylation of h k in human oocytes leads to abnormal spindle figures and subsequent aneuploidy. advanced maternal age is associated with a reduction in de-acetylation during in vitro maturation and an increase in spindle abnormalities. these results may stimulate the development of assays for histone modifications as biomarkers to follow oocyte quality in in vitro maturation studies or in optimizing general ivf treatments. objective: racial disparity in preterm birth (ptb) is unexplained and genetic risk factors are suspected as a major cause. this large scale candidate gene study examines differences association of single nucleotide polymorphisms [snps] in caucasians (c) and african americans (aa) to help elucidate racial disparity in preterm birth (ptb) methods: in this case (preterm birth < weeks) control study (term birth > weeks) maternal and fetal dna from ( cases and controls) c and ( ptb and term) aa were collected. a high-throughput candidate gene association study was performed examining snps in genes of selected from hypothesized ptb pathways. single locus association analyses were performed separately on maternal and fetal samples. results: snps in genes associated with ptb (p< . ) were common between races with both maternal and fetal dna analyses. however, snps in genes in c and in aa in both maternal and fetal dna differed in its association with ptb. in c maternal dna, the single strongest association between ptb and snp was in plasminogen activator tissue (plat) gene (c- t; rs ) at both allelic (p = . x - ) and genotypic (p= . x - ) level with an odds ratio (or) of . ]. the single strongest effect in c fetal dna was observed in a snp in the interleukin- receptor antagonist gene (a g; rs ) for both allele (p= . ) and genotype (p= . x - ), or . ). in aa, the strongest associations were in interluekin- (c t-rs , allele p= . x - , genotype p= . x - ) in maternal dna with an or= . [ci . - . ] and in fetal dna interleukin- receptor b (a g; rs , allele p= . x - , genotype p= . x - ) with an or . ]. conclusion: large scale high throughput analyses of snps in candidate genes of ptb pathway reveals significant differences between races at both maternal and fetal levels. we found that the strongest single locus associations differed in the two races in both maternal and fetal dna samples. these findings support the hypothesis that underlying genetic predispositions may differ between these populations, perhaps partly explaining racial disparity in preterm birth. candidate gene association study indicates differential etiologies in gdm and in t dm. johann urschitz, tarik sultan, kenneth ward. obgyn, jabsom, university of hawaii, honolulu, hi, usa. objective: recently several genome-wide association studies have associated multiple single nucleotide polymorphisms (snps) in multiple genes with increased risk of type diabetes. as risk factors are similar between t dm and gestational diabetes (gdm), we investigated a possible association of those snps with gdm. study design: blood was collected from caucasian ( cases, controls) women who met coustan-carpenter criteria for gdm and non diabetic controls. dna was extracted and a candidate gene association study was performed (taqman, abi). results: chi contingency tests were used to analyze genotype and allele frequencies in controls and gdm affected pregnancies (see table below). none of the snps showed a significant association after bonferroni correction. conclusion: several polymorphisms which displayed highly significant associations with type diabetes are not associated with gestational diabetes. these results suggest that gdm is not a simple unmasking of a t dm predisposition by the metabolic demands of pregnancy; rather it appears that different biological mechanisms are responsible for the respective diseases. introduction: histone acetylation/deacetylation plays an important role in the regulation of gene expression. histone deacetylase inhibitors (hdaci), in general, maintain gene expression, although in some cases they cause repression of specific genes. in this context we have previously shown that the hdaci tsa suppresses activation of the pro-contractile gene cox- in human myometrial and amnion-derived cells [reproductive sciences, vol , no (supplement, # )]. we have also shown that expression profiles for hdacs ( - , ) differ significantly within upper and lower regions of the myometrium during pregnancy and in labour. objectives: since changes in both acetylation and phosphoryation status can influence the activity of individual hdacs we aimed to define whether there are any changes in the levels of acetylation and phosphoryation of myometrial hdac , and during pregnancy and labour. methods: protein homogenates prepared from non-pregnant, term and labouring myometrium were treated +/-shrimp alkaline phosphatase and subjected to sds-page and western immunoblotting (wb) using antibodies to hdac , and . the acetylation status of the hdacs was assessed by a co-immunoprecipitation assay using anti-hdac and anti-acetylated lysine antibodies. results: distinct patterns of acetylation were observed for the individual hdacs; hdac and appeared to be more acetylated in non-pregnant and labouring lower tissues when compared to pregnant myometrium. in contrast, hdac appeared to be slightly more acetylated in lower uterine samples from pregnant and labouring myometrium. in experiments to evaluate changes in phosphorylation status we observed that ) myometrial hdac appeared to less phosphorylated in both upper and lower labouring samples when compared to non-pregnant and pregnant samples; ) hdac appeared to be more phosphorylated in labouring samples than non-pregnant and pregnant myometrium; ) no changes in phosphorylation levels were observed for hdac using this test system. conclusions: the difference in hdac acetylation and phosphorylation levels in the human myometrium may indicate differential regulation of the activity of the hdacs within the distinct myometrial regions, perhaps leading to the alteration of their epigenetic effect on genes related to myometrial quiescence and contraction and the subsequent onset of both term and preterm labour. objective: native hawaiians and pacific islanders experience a higher perinatal morbidity secondary to the increased incidence and earlier onset of heart failure. this increased incidence is likely multi-factorial and includes co-morbidities and genetic factors. mutations in the human adrenergic receptor gene may play a role in the determination of heart function. mutations in the b -adrenergic receptor (adrb ) located on chromosome have been identified as a cause progressive cardiomyocyte loss leading to a dilated cardiomyopathy. the minor allele frequencies for most of these polymorphisms have been determined for caucasian, japanese, african-american and chinese as part of the hapmap project. the frequencies have not been determined in the pacific islander groups. this study helped determine the hawaiian allele frequency and determine the allele frequency in the presence of cardiac or other vascular co-morbidities such as hypertension, preeclampsia, and gestational diabetes. study design: real time pcr technology (taqman genotyping assays, applied biosystems) was used to screen maternal dna (n= ) from the phenotyping sample core of the pacific center for early human development (prcehd) for the rs single nucleotide polymorphisms (snps). genotype frequencies were also determined in % complete ethnic controls as determined by a four grandparent descent. results: chi square was used to analyze allele and genotype frequencies differences between controls and affected pregnancies. the results are summarized in the following tables. conclusion: the rs snp was not significantly associated with hypertension, preeclampsia or gdm in our overall hawaiian population. the genotype frequency in the % pacific islander group was significantly different from the caucasians (p= . ) and asians (p= . ) in our overall hawaiian population. angiotensin-converting enzyme and -adducin polymorphisms in preeclamptic mothers and fetuses. c mando, p antonazzo, s tabano, f colleoni, a martinelli, s calabrese, c benedetto, l marozio, f facchinetti, m miozzo, i cetin. inst. obstetrics and gynecology, fondaz. irccs policlinico mangiagalli regina elena, univ. milan, milan, italy; dept. biology and genetics, univ. milan, italy; dept.obstetrics and gynecology, univ. torino, italy; univ. modena and reggio emilia, modena, italy. background the genes encoding angiotensin-converting enzyme (ace) and -adducin (add ) share the potential of influencing blood pressure. previous studies demonstrated in humans the association of hypertension with the combined effect of both ace insertion/deletion (i/d) polymorphism, which leads to a different activity of the enzyme, and add g w non-sense single nucleotide polymorphism (snp). ace i-i genotype has been associated with low serum ace activity. controversial studies concerning the association of ace polymorphism with preeclampsia (pe) were reported and the possible combined effect of both ace i/d and add g w polymorphisms yet remains to be investigated. population association study we genotyped polymorphisms (ace i/d and add g w) in women: with pe ( / with severe pe) and controls. moreover, we investigated a subset of their fetuses: from severe pe, from mild pe and controls, in order to identify specific maternal and/or fetal genotypes conferring a higher risk to develop pe. we both evaluated the single and the combined effects of ace and add genotypes on mother-fetus couples and singularly on mothers and fetuses. ace i/d genotype was analyzed using a pcr method; an allelic discrimination approach was performed to detect the add snp. in mother-fetus genotype couples, neither ace nor add polymorphisms are associated with pe, nor are the combination of their genotypes separately in mothers and in fetuses. nevertheless in mothers with mild pe ace i-i genotype is significantly less frequent ( % vs %; p< . ) and ace i-d is significantly more frequent ( % vs %; p< . ) compared to controls. ace i allele frequency is not significantly different in mild pe compared to controls ( % vs %). in women with mild pe the i allele seems to move from i-i to i-d genotype. it leads to the increase in mild pe women of those genotypes with a higher ace activity conferring a higher susceptibility to develop pe. this association with mild pe and not with severe pe could be due to the contribution in the latter of many other genetic and environmental factors. introduction: maternal mrnas stored in the oocyte are critical for early development as transcription ceases upon oocyte maturation, and gene expression until zygotic genome activation (zga) is mediated by translation of maternal transcripts. cytoplasmic polyadenylation element binding protein (cpeb) plays a central role in this process by stabilizing maternal mrnas and regulating their timely activation. this function has been well characterized in xenpous, and a similar role in mammals is suspected as the cpeb knockout mouse displays infertility as a result of arrested oogenesis. in order to investigate if translational regulation of maternal transcripts by cpeb is maintained in mammals, we characterized the spatial and temporal expression of cpeb- in the mouse and human. methods: ten different somatic tissues, testes, and ovaries were tested by rt-pcr for the expression of cpeb mrna in mouse and human. cpeb mrna expression in mouse was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. in human, pi and mii oocytes, -cell embryos and blastocysts were evaluated. sequencing of the pcr products was performed to confirm specific amplification of cpeb. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results and discussion: highest level of cpeb mrna expression was detected in ovaries and testis of both mouse and human. in addition, differential expression of cpeb in somatic tissues was also observed. among these, prominent expression was present in brain, where a role for cpeb in facilitating gene expression through cytoplasmic polyadenylation has been proposed. these data would suggest that translational control of mrna by cpeb may be a mechanism utilized by multiple somatic tissues to regulate gene expression. in the mouse, cpeb was expressed in pi and mii oocytes and -cell and -cell embryos, and became undetectable in -cell or more advanced embryos. human cpeb was expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. zga occurs at late -cell stage and -to- -cell stage, in mouse and human, respectively. the tightly controlled temporal expression of cpeb prior to zga in both mouse and human is consistent with a conserved role for cpeb in the regulation of maternal mrna expression during early development in mammals. background embryonic stem cells (esc) express specific transcription factors that are indicative of their ability to maintain pluripotency. these factors are activated during preimplantation embryo development. the interplay between the transcription factors pou f (oct / ) and caudal-homeobox domain (cdx ) is thought to regulate inner cell mass (icm) and trophectoderm (te) differentiation; however, the mechanism of cell fate determination in mammalian embryos is poorly understood. genetic ablation of oct / in mice prevents icm development, while cdx knockout embryos fail to implant. esc deficient in nanog, a second key regulator of pluripotency, fail to maintain pluripotency and undergo differentiation. expression of nanog in primate embryos has not been investigated, therefore the localization of oct / , nanog and cdx was determined in rhesus macaque blastocysts. methods oocytes were collected from rhesus macaque females, fertilized and cultured in vitro to the blastocyst stage. embryos were collected hours post insemination, then fixed prior to incubation with primary antibodies directed against oct / , nanog and cdx . following incubation with cy conjugated secondary antibodies, nuclei were counterstained with dapi and embryos visualized using an olympus bx fluorescence microscope. results nanog protein was restricted to the icm of monkey blastocysts. unlike the mouse embryo, oct / protein was detected in both the icm and te, based on two different antibodies. the expression of cdx was localized specifically to the te. conclusions the ubiquitous pattern of oct / expression is consistent with observations in human, cow and pig embryos. significantly, lack of restricted oct / protein, and icm localization of nanog in primate blastocysts, suggests that nanog more specifically determines cell fate in primate embryos. these results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of nanog. importantly, this difference may underlie observations that regulatory mechanisms in esc differ between mice and primates. further investigations will focus on determining the onset of marker expression, and the upstream regulators of nanog activation. supported by nih grants r hd r rr , and the intramural research program of nichd. in vitro-conceived mice tend to be smaller at birth and throughout their life. luisa delle piane, annemarie donjacour, francesca di sebastiano, gnanaratnam giritharan, paolo rinaudo. obstetrics, gynecology and reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: epidemiological evidence indicates that ivf is associated with an increased incidence of low birth weight. this phenomenon has not been studied in a mouse model; in addition, it is unknown if the resulting offspring show different growth pattern later in life. we therefore created an ivf mouse model to follow growth patterns till adulthood. methods: we generated experimental groups of b mice: one cohort of in vivo generated mice (in vivo group), one cohort of in vitro generated animals (ivf group) transferred to cd foster mothers and one cohort of animals fertilized in vivo and transferred to cd foster mothers (flushed blastocysts group, fb). the fb group was generated because our preliminary results showed that embryo transfer to b foster mothers was not successful. all pups were delivered at term, measured and weighed at birth and then weekly up to weeks. parametric tests (anova with bonferroni correction) were used as appropriate. a mixed model was used to compare growth curves. results: average litter size was . (in vivo), . (fb) and . (ivf). birth weight of male mice both ivf ( . mg) and fb ( . mg) was larger than male in vivo mice ( . mg) (p< . ). ivf mice tended to be smaller than fb mice in both sexes (p= . ). the bmi (body mass index) was not different among all the groups. male fb growth curves were different from in vivo mice (p< . ) and more importantly from ivf growth curves (p= . ) (fig. ) . conclusion: the method of conception and the maternal environment play a significant role in determining birth weight in this mouse model, emphasizing that the fb group is the best control for the effects of ivf in this model. these preliminary results confirmed for the first time an ivf effect on growth and interestingly the ivf males did not show a catch-up growth. the finding of smaller ivf mice when compared to fb is both noteworthy, because it confirms human data, and worrisome, because lower birth weight is associated with long term sequelae according to the barker hypothesis. the effect of betamethasone exposure in mid-gestation on renal sodium excretion in male sheep. lijun tang, luke carrey, nancy valego, philip deibel, james perrott, jorge figueroa, mark chappell, james c rose. obestetrics and gynecology, wake forest university, medical school, nc, usa; hypertension center, wake forest university, medical school, nc, usa. objective: whereas prenatal exposure of ovine fetuses to clinically relevant doses of glucocorticoids during the time of peak nephrogenesis results in a reduction in nephron number in adulthood, there is little information about its effect on sodium excretion. in the present study, we evaluated the effect of exposure to betamethasone on renal sodium excretion in adult male sheep. methods: we studied nineteen conscious adult rams at . years of age which were exposed to either vehicle or betamethasone at - days gestation. we implanted vascular and bladder catheters and then allowed the animals a - days recovery period prior to study. inulin and para-aminohippuric acid (pah) clearances were performed for estimating glomerular filtration rate (gfr) and renal plasma flow (rpf) respectively. following determination under basal conditions, an acute hypertonic sodium load was administrated intravenously by a continuous infusion of nacl ( . meq/kg/min at . ml/min) for minutes. urine was continuously collected for determination of na + excretion. results: basal gfr was decreased in steroid exposed adults ( . ± . ml/min/kg) compared with the vehicle animals ( . ± . ml/min/kg) (p= . ). rpf was similar in the vehicle and steroid exposed group ( . ± . ml/min/kg vs . ± . ml/min/kg). at basal conditions, na + excretion (u na v) was similar in vehicle and steroid exposed group ( . ± . μmol/h vs . ± . μmol/h). this similarity was also present after normalization by body weight ( . ± . μmol/h/kg vs . ± . μmol/h/kg). the vehicle group excreted . ± . % of the dose of na during the experiment while the betamethasone exposed group excreted only . ± . % (p< . ). gfr and prpf did not change during the experiments. these results suggest that prenatal exposure to glucocorticoids affects renal function in adult male sheep which results in a decreased basal gfr and an attenuated ability to excrete on acute sodium load. the elevated blood pressure previously observed associated with this prenatal steroid treatment may be related to the alteration in renal function. the study is supported by nih grants hd , hl and hd . characterisation of human fetal cardiac stem cells. marah alfakir, nicholas dawe, annette meeson, stephen c robson. school of surgical reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. objectives: for many patients with severe cardiac disease treatment is limited to surgical intervention and/or heart transplantation. the heart has a limited regenerative capacity but it is insufficient to repair extensive damage. cellular therapies might provide an alternative treatment. we have identified stem cells (sp cells) in the adult mouse and human heart and have shown that the mouse cardiac sp cells can differentiate along a cardiac lineage. sp cells can be identified using facs combined with hoechst dye efflux. this ability to efflux hoechst dye is due to expression of abcg (an abc transporter). we have hypothesized that the fetal heart would contain more cardiac stem cells, relative to the adult, and the aim of this project was to determine the spatial and temporal expression of known stem cells markers in the embryonic/fetal heart. methods: human fetal hearts were collected aged between - wk. we used rt-pcr, using primers for several stem cell (abcg , cd , cd ) and cardiac specific (mlc- v, mhc) markers, and ihc on frozen and paraffin sections, using a panel of antibodies (abcg , cd , cd , islet ). cdna was generated from the following regions of the heart at different developmental stages: right and left atrium, right and left ventricle, and outflow tract. results: abcg mrna was highly expressed in all regions of the fetal heart between - wk. expression was down regulated at - wk especially in the la and lv. a similar pattern of expression was observed for cd . cd showed low/moderate expression in all heart regions examined; however, this expression was absent in the lv at - wk. mrna and protein analysis showed similar results. whilst protein expression of abcg was robust at wk (approximately - %), it declined with increasing gestational age. cd was also strongly expressed at weeks of age. there was no co-localisation between abcg and islet . conclusion: abcg and cd are both expressed between - wk of age. based on this analysis, further studies are underway to isolate and characterise both the abcg and cd expressing fetal cardiac cells which might be a potential source of cardiac stem/progenitor cells. xanthine oxidase plays a significant role in the fetal cardiovascular defence to hypoxia. ja hansell, a kane, e herrera, da giussani. physiology, cambridge university, united kingdom. prenatal hypoxia remains a major concern in obstetrics. the fetal defence to hypoxia includes redistribution of the cardiac output, away from peripheral and towards essential ciculations, such as those perfusing the brain (cohn et al . ajog : , ) . the physiology underlying this response is well characterised and involves chemoreflex and endocrine responses (giussani et al. fet mat med rev : , ) . more recently, local factors such as nitric oxide (no) and reactive oxygen species (ros), and the interaction between them, have been shown to play a role. antioxidants, such as vitamin c, scavenge hypoxia-induced ros, maintain no high and thereby depress peripheral constriction (thakor et al., sgi ) . in this study, we address the source of hypoxia-induced ros production and investigated the effects on the in vivo fetal femoral constrictor response to hypoxia of maternal treatment with the xanthine oxidase inhibitor allopurinol in sheep. methods: under anaesthesia, sheep at . gestation, were instrumented with maternal and fetal catheters and a fetal femoral transonic probe. five days later, all animals were subjected to h normoxia, . h hypoxia (mat fio to reduce fetal pao to ca. mmhg) and h recovery, either following maternal i.v. treatment with vehicle or allopurinol in low ( mg.kg - over min) or high ( mg.kg - over min ) doses. treatments finished min prior to hypoxia. the low allopurinol dose was adopted from human studies (benders et al. arch dis child : , ) . the timing of the hypoxic challenge coincided with peak concentrations in fetal sheep plasma of oxypurinol (active metabolite). we evaluated the effect of bpa exposure on uterine gene expression in a nonhuman primate model. method: a total of nine vervet monkeys (cercopithecus aethiops sabaeus) were ovariectomized. three monkeys were treated with bpa via alzet pumps ( microgram/kg/day), two with estradiol benzoate and three received combined treatment with bpa and estradiol. the remaining monkey was untreated and served as a control. following days of treatment the monkeys were hysterectomized and immunohistochemistry performed to examine endometrial gene expression. we evaluated hoxa , proliferating cell nuclear antigen (pcna), and caspase iii gene expression as markers of differentiation, proliferation, and apoptosis respectively. differential expression was evaluated by h-score. results: exposure to a combination of estradiol and bpa resulted in increased expression of hoxa and pcna compared to control (p< . ). lower, but increased, levels of hoxa and pcna gene expression were seen in the specimens exposed solely to estradiol (p< . ). those specimens exposed to bpa alone demonstrated a small induction in hoxa expression (p< . ) with no change in caspase iii or pcna expression when compared to the control. conclusion: bpa induced the expression of hoxa in the uteri of a non-human primate. exposure in the presence of endogenous estrogen further augmented estrogenic stimulation confirming that bpa is a xenoestrogen. bpa's effect of developmental gene expression may alter uterine differentiation. bpa acts as an endocrine disruptor by altering estrogen regulation on a gene required for fertility. contractility and meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, ahmet karadag, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: endogenous glucocorticoids (gcs) increase with advancing fetal age suggesting that gcs function as a hormonal modulator of organ maturation. although fetal colonic motility and meconium passage primarily occur near term, the role of gc in colonic maturation is poorly understood. we sought to examine gc-nuclear receptor (gc-nr) expression in ovine fetal distal colon smooth muscle from very-preterm to term gestation to define the cascade of gc initiated biochemical changes as a prerequisite for maturation-associated meconium passage. methods: ovine fetal distal colonic segments removed at very-preterm (vpt: - d gestation), preterm (pt: - d), near term (nt: - d) and term (t: - d) (n= fetuses in each group) were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochmical analysis with gc-receptor antibody ( : , sc- , santacruz biotechnology, ca) using standard abc regimen. immunoreactive material identified by , 'diaminobenzidine as chromogen. the percentage of gc-nr staining in smooth muscle-enteric unit was analyzed by counting dark brown immunostained nuclei at different fields at x magnification. all values are expressed as mean ± sem. results: the gc-receptor antibody immunostained nuclei both in smooth muscle and enteric units and the observed pattern (expressed as percentage of total nuclei) are as follows: nuclear gc expression varies with gestational age with maximal expression occurring near-term in smooth muscle and enteric neurons, in a synchronized manner. a near total disappearance of gc-nr expression occurs at term. these results suggest that peak gastrointestinal gc-nr mediated effects may occur prior to term with secondary signaling effects resulting in distal colonic motility maturation and meconium passage. the rna-binding protein hud co-localizes with choline acetyl mechanisms of in utero meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, octavio balbuena, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: we have previously speculated that crf, acting through its receptor crf-r , increases gastrointestinal (gi) motility and potentiates in utero meconium passage. patients with paraneoplastic syndrome with auto-antibodies to hud, a neuronal rna binding protein, develop severe gi dysmotility, indicating a role for hud in gi motility. hud binds mrna for actylcholinesterase implying a role in the cholinergic neurotransmitter system. based on these known functions, we hypothesized that hud may regulate post-transcriptional activity in fetal colonic cholinergic neurons expressing crf-r . method: bouin's solution-fixed paraffin-embedded sections of distal colonic segments were prepared from very preterm (vpt: - days), preterm (pt: - days), near term (nt: - days) and term ( - days) ovine fetuses (n= at each age). sections were immunostained with anti-human neuronal protein huc/hud antibodies ( : to : ) with abc reagents and examined at x. neurons positive for hud staining were quantified. double immunofluorescence and laser confocal analyses evaluated co-expression of hud in neurons expressing peripheral choline acetyl transferase (pchat) (a marker for peripheral cholinergic neurons) and crf-r receptor. results: hud immunostaining was seen in either entire cytoplasm (c) or cytoplasm and nuclear regions (c+n) in both submucosal and myenteric neurons. a greater percentage of submucosal (vpt: ± , pt: ± , nt: ± , and t: ± %) than myenteric neurons (vpt: ± , pt: ± , nt: ± , t: ± %) exhibited positive staining at all ages. the percentages of neurons with c+n staining in submucosal neurons were significantly lower at very-preterm gestation. confocal studies co-localized the hud staining with pchat and crf-r receptor immunoreactivity both in submucosal and myenteric neurons. conclusion: in the fetal enteric nervous system hud may function both as a rna-binding protein and as a nuclear cytoplasmic shuttling protein. colocalizaion studies suggest that both pchat-mrna and crf-r mrna species are target transcripts for hud in myenteric neurons. we speculate upregulation of hud contributes to in utero meconium passage. meconium passage during mild stressors. jayaraman lakshmanan, guo l liu, john d richard, sharon k sugano, raina khan, octavio balbuena, kimberly chap, virender rehan, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: mr exhibit a greater affinity to glucocorticoids (gc) than do glucocorticoid receptors (gr). thus low circulating gc levels may preferentially bind mr during mild-stress periods, while the high gc levels may bind gc-receptors. mr antagonism increases gc-mediated responses, suggesting that mrs have a regulatory impact on gc-mediated stress responses. we recently documented that fetal in utero meconium passage is a neurovisceral stress response. to test our hypothesis that mrs play a primary role in mediating suppressive gc effects, we examined the expression patterns of mr in ovine fetal distal colon. methods: bouin's solution fixed paraffin sections of distal colonic segments collected from ovine fetuses (n= for each gestational ages) at very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - days) and term (t: - days) were subjected to immunohistochemistry with polyclonal antibodies to mr. digital photos ( field per colonic ring, rings each gestational age) taken at x were used to count the number of intensely stained neurons, referred to as "mr-capped neurons". differences over time were determined with anova. results: mr antibody elicited a punctate staining pattern in all layers of ovine fetal distal colon. significant immunoreactive intensity was observed in submucosal and myenteric ganglia at all ages: submucosal ganglia: vpt= . ± . , pt= . ± . , nt= . ± . , t= . ± . ; a subpopulation of enteric neurons exhibited dense staining ("mr-capped"). advancing gestation was associated with a significant decreased in the percentage of mrcapped myenteric (vpt = ± , pt = ± , nt = ± , t = ± %; p< . ), though not submucosal ganglia neurons. results indicate a significant decrease in the percentage of mr capped myentric neurons with advancing gestation. in view of the potential inhibitory effect of mr on gc-mediated stress responses, these results suggest that the decrease in mr expression may contribute to near term and term in utero meconium passage. the ambiguity of myometrial progesterone receptor expression in pregnancy and labour. alison j tyson-capper, elizabeth a shiells, stephen c robson. surgical reproductive sciences, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom. background and aims: in contrast to many other species, human parturition is not due to a reduction in circulating levels of progesterone (p) but appears to be related to changes in expression, ratios or signalling events of p receptors (pr-a and pr-b). the literature on pr expression in human myometrium in pregnancy and labour remains conflicting. we hypothesise this is due, at least in part, to differing specificities of the various pr antibodies employed. we carried out a comprehensive analysis of pr expression using ten 'pr-specific' antibodies that recognise different amino acid epitopes within the pr proteins. two phosphorylation-specific antibodies for pr were also included to define whether phosphorylation status of myometrial pr changes in pregnancy and in labour. methods: western immunoblotting and semi quantitative rt-pcr were undertaken using protein lysates and rna prepared from myometrial tissue from: -first (n= ) and second (n= ) trimesters, paired upper and lower segment myometrium from preterm ( - wks, n = ), term not in labour (n = ), term spontaneous labour (n = ) and non pregnant (n = ). results: using the same set of myometrial lysates for each antibody, we found that the specificity of individual pr antibodies, in particular those raised against internal and c-terminal epitopes of the pr proteins, varied considerably resulting in different patterns of expression. there also appeared to be temporal and spatial differences in levels of myometrial pr proteins ( - kda/ - kda) in pregnancy and in labour with use of the phosphorylation-specific pr antibodies, however, it remains to be resolved which pr isoforms these may be. in contrast, data using four antibodies all of which react with the amino terminal domain of pr consistently indicated that expression of pr, in particular pr-b, decreased significantly at term (p < . ) and in labour (p < . ) when compared to non-pregnant levels. a decrease in levels of pr-b protein was also observed when comparing preterm levels to non-pregnant levels. rt-pcr using primers specific to pr-b consistently indicated that levels of pr-b mrna decreased at term and in labour. conclusion: interpretation of gestation-related changes in myometrial pr expression and phosphorylation status must take into account the pr antibodies used. further studies are now underway to validate the specificity of the pr antibodies. objective to test the hypothesis that expression of fshr in mural gl cells from ivf follicles varies with the infertility diagnosis and correlates with the outcome of ovulation induction (oi). materials and methods women undergoing ivf were classified as: . "no ovarian factor", (tubal or male factor and egg donors, nof;n= ); . endometriosis (endo; n= ); . poor responders (pr; n= ); . polycystic ovary syndrome (pcos; n= ). ovulation induction was carried out using a long or microflare or antagonist protocol based on clinical parameters and gonadotrophin doses were selected based on ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual response. after ultrasound guided egg retrieval, mural gl cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-cd immunobeads to eliminate wbcs, viability was assessed by trypan blue. fshr was measured by rt-pcr as relative expression compared to beta actin. statistical analysis was performed with the spss using pearson´s correlation, one way anova and student´s t-test. results fshr expression in nof ( ± . ) was statistically significantly higher than in pr ( ± . ) and lower than in pcos ( ± . ). both endo ( ± . ) and pr levels of fshr were statistically significantly lower than in pcos. analysis of all cycles showed that fshr expression correlates positively with the number of total (r= , ; p< , ) and mii (r= , ; p< , ) oocytes and negatively with the units of fsh (r=- , ; p< , ) and lh (r=- , ; p< , ) administered for oi. separate analysis of nof showed a positive correlation with the number of total and mii oocytes retrieved and with estradiol levels on day of hcg but not with the total dose of gonadotropins received during oi. fshr expression correlates negatively with day fsh levels in all patients except in pcos (r=- , ; p< , ). conclusions these results suggest that expression of fshr in the hyperstimulated ovarian follicle is "average" in normoresponders, high in high responders (pcos) and low in poor responders (pr and endo). knowledge of the level of expression of fshr might be useful to individualize gonadotrophin doses and oi protocols thus improving pregnancy rates. comparison of intracellular atp levels between non-vitrified and surviving post-vitrification/thawed human oocytes. somjate manipalviratn, zhi-bin tong, eric widra, , alan h decherney. reproductive biology and medicine branch, nichd/nih, bethesda, md, usa; department of obstetrics and gynecology, georgetown university hospital, washington, dc, usa; shady grove reproductive science center, washington, dc, usa. objective: to compare intracellular atp level in non-vitrified and surviving post-vitrification/thawed human oocytes using an atp bioanalysis assay. design: prospective match-controlled laboratory study material and methods: all oocytes were obtained from women undergoing controlled ovarian stimulation for ivf/icsi. oocytes were discarded due to nuclear immaturity at the time of planned icsi. all immature oocytes (gv and mi) were incubated overnight. oocytes from patients with or more discarded eggs which matured to mii stage were used in this study. oocytes from each women were randomly divided into groups. in group , the oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. in group , the oocytes were vitrified using % ethylene glycol, % dimethyl sulphoxide and . m sucrose as cryoprotectant. these oocytes were kept in liquid nitrogen for - days before thawing with a rapid thawing method. oocyte survival was determined by morphological assessment after minutes of incubation then oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. intracellular atp level was determined using luciferin-luciferase bioluminescent assay. result: ninety-one discarded human oocytes were obtained from women. oocytes were vitrified/thawed before measuring for atp content. the other oocytes were measured for their atp content without undergoing vitrification/thawing process. oocyte survival rate after vitrification/thawing is . % ( / ). mean oocyte atp levels in non-vitrified oocytes is significantly higher than surviving post-vitrification/thawed oocytes (table ). coefficient of variation of luciferin-luciferase bioluminescent assay is less than %. conclusion: oocyte atp level in surviving post-vitrification/thawed human oocytes is significantly lower than non-vitrified oocytes. objective to study the expression of genes involved in cell proliferation and steroidogenesis in the human follicle (kl , kl , fshr, papp, p ) and its relationship with ivf outcome. materials and methods patients with different infertility diagnosis underwent ovulation induction with either a long or microflare or antagonist protocol based on clinical parameters; the dose of gonadotrophin used for ovulation induction was selected based on the ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual patient response. ultrasound guided egg retrieval was performed hours after administration of . iu of hcg. mural granulosa-lutein cells (gl cells) were isolated from pooled follicular fluids (ff) from each patient using a percoll gradient and anti-cd immunbeads to eliminate wbcs, viability was assessed by trypan blue. the genes under study were measured by rt-pcr as relative expression compared to actin. statistical analysis was performed with the spss statistical software using pearson´s correlation and mann-whitney u. results kl expression correlates positively with kl levels (r= , ; p< , ) and with genes implicated in granulosa cell function such as fshr (r= , ; p< , ), papp (r= , ; p< , ) and p (r= , ; p< , ). the number of mii oocytes obtained is positively correlated with both fshr (r= , ; p< , ) and papp (r= , ; p< , ) expression, but not with the other genes. kl expression in women who became pregnant during the ivf cycle studied was higher ( , ± ) than in non pregnant women ( , ± , ).(n= , mann-whitney u p< , ). conclusions patients who become pregnant have increased expression of kl , which is associated with optimal granulosa cell proliferation and maturation. this is in accordance with the presence of lower apoptosis in granulosa cells in the same patients. syndrome during assisted reproduction treatment. k jayaprakasan, r jayaprakasan, h al-hasie, js clewes, bk campbell, ir johnson, nj raine-fenning. school of human development, university of nottingham, nottingham, united kingdom. objective: to test the hypothesis that an increased pre-treatment ovarian blood flow is associated with the development of ovarian hyperstimulation syndrome (ohss) and to evaluate ovarian vascularity as a predictor of ohss during invitro fertilization (ivf). methods: subjects undergoing first cycle of ivf had d transvaginal ultrasound in the early follicular phase of the menstrual cycle preceding ivf. of them developed ovarian hyper-response, defined as retrieval of oocytes and ohss. subjects had normal ovarian response with retrieval of to oocytes in the absence of ohss. antral follicle count (afc), ovarian volume (ov), and ovarian vascularity (vascularisation index, vi; flow index, fi and vascularisation flow index, vfi) were measured and an unpaired t-test was used to compare these parameters between the ohss and the control groups. multiple logistic regression analysis was used to assess the predictive value of these variables against age, bmi and basal fsh for the development of ohss. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both the groups. afc and ov were significantly higher (p< . ) in the ohss group ( . ± . and . ± . cm respectively) than in the control group ( . ± . and . ± . cm respectively). afc was the only significant (p= . ) predictor of ohss on multiple regression analysis (table ) . conclusion: women developing ohss during ivf do not demonstrate an increased pre-treatment ovarian blood flow as measured by d ultrasound but do have a significantly higher afc, which is the only significant predictor of ohss. compared to other species, little is known about the steroid biosynthetic capacity and gene expression profiles of human cumulus cells. objective: to examine the ability of human cumulus cells in primary and long-term culture to synthesize steroids and respond to gonadotropin or camp-dependent stimulation. methods: human cumulus cells were isolated from cumulusoocyte complexes during assisted reproductive technology (art) and placed in primary culture or propagated to third passage. at subconfluence, cells were transferred into serum-free conditions in the presence of vehicle, forskolin, fsh, or hcg. at h, media was collected and progesterone (p ) and estradiol (e ) levels were determined by eia. aromatase activity was measured by the tritiated water assay. aromatase (cyp ) and cholesterol side-chain cleavage (cyp a ) mrna abundance was determined by quantitative real-time pcr (qrt-pcr). transcriptional regulation of the cyp and cyp a promoters was investigated by transient transfection of cumulus cells with promoter luciferase constructs. results: substantial amounts of p and e were synthesized by cumulus cells in culture, which were further elevated by forskolin, hcg, or fsh treatment. the presence of cyp and cyp a mrna in cumulus cells was confirmed by qrt-pcr, and the relative mrna abundance of both transcripts was induced by forskolin, hcg, or fsh treatment. changes in cyp and cyp a gene expression in response to camp and gonadotropin stimulation were associated with increased transcriptional regulation of both the cyp and cyp a gene promoters. our studies demonstrate that human cumulus cells are sites of significant p and e biosynthesis and respond to camp-and gonadotropin-stimulation in vitro. the ability to examine human cumulus cells in primary and long-term culture provides a unique model system to investigate cumulus cell function, and the paracrine role of cumulus cells in oocyte development, maturation, and subsequent fertilization. background: there is increasing evidence suggesting that events occurring during fetal development may result in increased predisposition to adult conditions, such as diabetes. in vitro fertilization (ivf) is a new environmental stressor and the long-term effects of these manipulations are currently unknown. there is some evidence that lower number of insulin-secreting cells at birth signals predisposition to diabetes later in life. in this study, we compare areas of pancreatic insulin-secreting cells in newborn mice conceived in vivo versus in vitro. methods: oocytes were collected from super ovulated cf- mice and fertilized in vitro with cauda epididymal sperm from b d f /j mice. fertilized eggs were cultured in whitten medium under % co in humidified air at °c for h. blastocysts were transferred to the uteri of pseudo-pregnant recipients. control mice were allowed to conceive in vivo. the newborn animals were sacrificed within hours of birth. pancreases were sectioned, immunostained with anti-insulin, and total areas and stained areas were compared using t-test with two-tailed distribution. p value of < . was considered significant. results: there were total of newborn animals: females ( ivf, controls) and males ( ivf, controls). percentage of total pancreatic area occupied by insulin-secreting cells was lower in female ivf ( . mm , . %) animals compared to female controls ( . mm , . %) and higher in male ivf ( . mm , . %) animals compared to controls ( . mm , . %). the average of males and females was slightly lower for ivf ( . mm , . %) than controls ( . mm , . %). none of these differences were statistically significant. there was a trend in newborn female mice towards lower amount of insulin-secreting cells in the ivf offspring, suggesting possible predisposition to diabetes later in life. this effect was not observed in newborn males. while our study failed to demonstrate consistent and significant differences in the areas of insulin-secreting cells between newborn mice conceived in vitro and in vivo, the number of animals in the study may have been too small to show significant results. larger studies are needed to further investigate this question. peter s uzelac, phyllis risch, kassi shelton, steven t nakajima. obstetrics and gynecology, university of louisville, louisville, ky, usa. objective: several fertility preservation methods currently available may not be viable options to certain patients due to their high cost and limited number of centers offering these services. for women undergoing bilateral oophorectomy, in vitro maturation (ivm) of oocytes retrieved from unstimulated whole ovary specimens may represent a more practical solution. here we describe out initial experience in offering ivm as a fertility-preserving measure to two patients undergoing total abdominal hysterectomy and bilateral salpingo-oophoectomy (tah-bso). case one was a year old nulligravid with chronic pelvic inflammatory disease unresponsive to medical management. case two was a year old nulligravid with stage ia endometrial carcinoma. surgery was planned for the mid-follicular phase in order to collect prophase i oocytes before the onset of late-follicular phase atresia. upon surgical removal, suction aspiration of all identifiable follicles was performed. ovaries were then serially sectioned and all tissue was examined for the presence of prophase i oocytes. results: total number of prophase i oocytes retrieved was and , maturation rate after hours was % ( / ) and % ( / ), fertilization rate after hours was % ( / ) and % ( / ), maturation rate after hours was % ( / ) and % ( / ) and fertilization rates after hours % ( / ) and % ( / ), for case and case respectively. ivm of oocytes retrieved from unstimulated whole ovary specimens may be a simple and inexpensive approach to fertility preservation in women undergoing bilateral oophorectomy. by requiring minimal adjustments to in vitro fertilization protocols, this treatment could easily be implemented in centers which currently have no fertility preservation program. patient age at time of retrieval may be predictive of developmental potential. continued patient enrollment and follow-up studies on the developmental potential of embryos derived from this technique are necessary to fully evaluate its potential for a role in fertility preservation. the routine use of progesterone as luteal phase support in women with a diagnosis of polycystic ovary syndrome (pcos) undergoing ovulation induction cycles with oral agents has not been fully elucidated. we hypothesize that women with pcos utilizing either clomiphene citrate or letrozole, an aromatase inhibitor, should administer intravaginal progesterone in the luteal phase to increase pregnancy rates. design: retrospective chart review. materials and methods: cycle data from women with pcos undergoing ovulation induction with clomiphene citrate or letrozole at the university of cincinnati medical center from to were evaluated. diagnosis of pcos was based on rotterdam criteria and all other infertility diagnoses were excluded. clinical pregnancy rates (presence of fetal cardiac activity on ultrasound at - weeks gestation) in women who received intravaginal micronized progesterone ( mg bid) following ovulation induction (with and without intrauterine insemination) were compared to those who did not receive progesterone. results: no significant differences were noted in demographic parameters, including patient age or bmi. a total of cycles were evaluated in women treated with clomiphene citrate. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to . % ( / ) of the non-progesterone group (p < . ). forty-three cycles were evaluated in patients treated with letrozole. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to none ( / ) in the non-progesterone group (p < . ). conclusions: patients with pcos who used letrozole for ovulation induction had superior clinical pregnancy rates when using intravaginal micronized progesterone compared to women who did not receive luteal phase support. there was a trend toward increased clinical pregnancy rates in pcos patients utilizing luteal support following clomiphene citrate for ovulation induction. therefore, in women with pcos undergoing ovulation induction with oral agents, luteal supplementation with progesterone should be strongly considered, especially in those using letrozole. were measured and an unpaired t-test was used to compare these parameters between poor and normal responders. multiple logistic regression analysis was used to assess the predictive value of these variables against age and basal fsh for poor ovarian response. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both poor and normal responders. afc and ov were significantly lower (p< . ) in poor responders ( ± . and . ± . cm respectively) than in normal responders ( . ± . and . ± . cm respectively). afc was the best (p< . ) predictor of poor ovarian response on multiple regression analysis ( objective: gay male couples increasingly seek parenthood through in vitro fertilization using an oocyte donor and a gestational carrier, but no studies describe this unique experience. the purpose of this study was to determine medical and psychosocial issues unique to gay men using art. design: qualitative analysis of semi-structured interviews with gay male couples seeking parenthood through art. materials and methods: sixteen gay males (eight couples) entering an art program were assessed through the use of a semi-structured interview. characteristics evaluated included age, relationship status, duration of their relationship, psychological health and stability, how the decision was made concerning who would donate the sperm, the decision to use an anonymous or known oocyte donor, and whether their gestational carrier was someone previously known to them or not. results: the average age of the men in this study was years. all eight couples were in a committed relationship and had been together for an average of . years. five of the couples ( %) had been joined in a civil union which is legal in the state of connecticut. all subjects were psychologically stable and in good health. six couples ( %) were very clear about which partner would inseminate the oocytes. of those couples, two felt that the older partner should donate; two felt that the partner who cared more about a genetic connection to the child should donate; and two felt that the partner with "better genes" should donate. the remaining two couples chose to inseminate equal numbers of oocytes in order to transfer an embryo from each partner. all couples chose an anonymous oocyte donor, two couples chose relatives as their gestational carriers, while the others chose carriers recruited through an agency. conclusions: participants in this study were determined to become parents through assisted reproduction. they had given thoughtful consideration to the medical and psychosocial issues unique to this process including which partner would be the genetic parent, and why. clomiphene superovulation. rb allen, az steiner, rh fogle, mj kalan, rj paulson. ob/gyn, usc keck school of medicine, la, ca, usa; ob/gyn, unc, chapel hill, nc, usa. intro: micro-dose hcg has been demonstrated to increase ovulation and pregnancy rates following clomiphene administration in women resistant to clomiphene. since micro-dose hcg stimulates growth in follicles expressing the lh receptor, we hypothesized that its use following clomiphene in women with unexplained infertility would increase the number of follicles that ovulate and subsequently, pregnancy rates. m m: irb approval was obtained for this prospective pilot study of women with unexplained infertility. on day # a baseline ultrasound was performed and serum fsh and e levels were measured. subjects were given mg of clomiphene daily from days - and then returned for serial ultrasounds on day # . when at least follicles mm were present, iu of hcg im daily was initiated. cycles were monitored by ultrasound every days until a +lh surge occurred. all subjects had previously undergone a cycle using clomiphene alone for superovulation followed by iui and these cycles were used for comparison. results: subjects, aged . ± . yrs (mean±sem) and bmi . ± . kg/m with . ± . yrs of infertility were enrolled. the mean fsh and e levels were . ± . miu/ml and . ± . pg/ml, respectively. there was an average of . ± . days from starting clomiphene to the attainment of follicles mm. out of the subjects had follicles mm by day # . a mean of . ± . days of treatment were required until ovulation occurred. there were twice as many follicles in the micro-dose hcg cycles, although due to the small sample size this did not reach statistical significance ( . ± . follicles, measuring . ± . mm in the micro-dose hcg cycles, compared to . ± . (p= . ), measuring . ± . mm (p= . ) in the control cycles). there was no difference in the endometrial thickness at the time of ovulation between the micro-dose hcg cycles and the control cycles: . ± . mm vs. . ± . mm (p= . ), respectively. all subjects had at least ovulatory sized follicles in the study cycles. % of subjects had an iui performed, however there were no pregnancies resulting from this study. conclusions: ) the addition of micro-dose hcg to clomiphene may allow more follicles to reach an ovulatory size, which should theoretically increase pregnancy rates ) this pilot study demonstrates that adding micro-dose hcg may increase the effectiveness of clomiphene when given in a superovulatory protocol. jessica salas, donald maier, john nulsen, claudio benadiva, lawrence engmann. obstetrics gynecology, university of connecticut, farmington, ct, usa. objective: to evaluate the antepartum, intrapartum, and neonatal complications in patients undergoing ivf using a gnrh-antagonist protocol where a gnrhagonist was used to induce final oocyte maturation. materials and methods: a retrospective review of data from high responders undergoing their st or nd ivf cycle using a gnrh-antagonist protocol who were triggered with leuprolide acetate (study group) or hcg (control) and achieved at least a singleton pregnancy reaching the third trimester. patients younger than years old were included. both groups received luteal phase support with intramuscular progesterone. the study group received estrogen patch supplementation. outcomes measured were antenatal and intrapartum complications, order of gestation, gestational age at delivery, birth weight, neonatal adverse outcomes, and congenital anomalies. pearson chi square, fisher's exact test, or independent t-test were used as appropriate. results: the baseline characteristics were different (table ) . maternal antenatal and intrapartum complications were similar in both groups ( . % vs . %, p= . ). there were more singletons in the study group ( . % vs . %, p< . ). a subgroup analysis of gestational age at delivery, birth weight, neonatal complications and congenital anomalies in singletons showed no difference (table ) . conclusions: this is the first study reporting the maternal and perinatal outcomes after gnrh-agonist trigger. differences in response to ovarian stimulation may dictate use of gnrh-agonist triggering for prevention of ohss, which may explain the differences in baseline characteristics. maternal and neonatal complications remain unaffected. table lupron (n= ) objective: moderate to severe ovarian hyperstimulation syndrome (ohss) occurs in - % of assisted technology cycles and carries the potential for severe complications. traditional management consists of hospitalization with intravenous fluids (ivf), bedrest, and close monitoring. early and aggressive paracentesis is an alternative method of treatment for ohss in an outpatient setting, but the economic consequences of the two treatment regimens have not been compared. design: cost-effectiveness analysis materials and methods: two scenarios, outpatient management with transvaginal paracentesis and conservative therapy with hospitalization, were compared. potential initial outcomes were analyzed for the conservative group to include hospitalization either in a ward hospital bed or the intensive care unit (icu) for an average of seven days. costs included ivf administration, ultrasound, and daily bloodwork. initial outcomes for the outpatient management group included no further therapy beyond the initial transvaginal paracentesis, bloodwork, ivfs, and ultrasound versus admission for an average of three days to a ward bed with similar management. the probability of the negative outcome for the conservative group was set at % (icu admission) and % for the outpatient group (regular hospitalization). results: the cost of conservative therapy including first tier complications ranged from a low of $ , to a high of $ , . the cost of outpatient management with aggressive paracentesis and its first tier complications ranged from a low of $ to a high of $ , . this resulted in an estimated cost burden of $ , to $ , for conservative management with hospitalization. the main improvement factor was that patients in the outpatient management group had a much lower likelihood for prolonged hospitalization than the conservatively managed group. conclusions: aggressive outpatient treatment of moderate to severe ohss with early paracentesis appears to be a cost-effective strategy. induction. zeynep alpay, michael p diamond, michael l kruger, elizabeth e puscheck. obstetrics and gynecology, division of reproductive endocrinology and infertility, hutzel hospital, wayne state university, detroit, mi, usa. introduction: aromatase inhibitors (ai) are a new method of ovulation induction and is proposed to replace clomiphene citrate (cc) due to their reported advantages. their superior effect is thought to be due to up-regulation of the estrogen receptors and increase in the sensitivity of the endometrium, resulting in better proliferation despite low estrogen levels in the circulation. objective: to compare the effect of different ovulation induction methods, including ai, cc and gonadotropins (gt) on the endometrium in infertility patients. methods: we reviewed ovulation induction cycles performed in our institution in the last one-year period retrospectively. there were gt, cc and ai cycles. age, gravida, day (d et) and midcycle (mcet) endometrial thickness, endometrial pattern (ep) on day of hcg injection, endometrial growth during the induction cycle (d-et), which was calculated by the difference between mcet and d et, and the pregnancy rates (pr) were compared. the ep was categorized as type a (homogenous and hyperechogenic), type b (intermediate isoechogenic pattern and a poorly defined central echogenic line), and type c (multilayered triple-line). chi-square, anova, t test and pearson correlation were used for statistical analysis. results: in all cycles, ep was closely related to the d-et (p < . ). this correlation appeared to be more pronounced when observed ep was compared with d-et (r = . ; p < . ) rather than with mcet (r = . ; p = . ). there was a reverse relationship between the age of the patients and ep (r = - . ; p < . ). a negative correlation was also found between the gravida and ep (r = - . ; p < . ). pregnancy rate was significantly correlated with ep (r = . ; p < . ). pregnancy rate was % in type a ep, . % in b, % in c when all cycles were analyzed. aromatase inhibitors and gt treatments each resulted in % type c pattern in contrast to % with cc (p = . for ai vs. cc; p = . for gt vs. cc). the d-et was greater in gt cycles compared with ai or cc (p < . ). conclusion: these results show that ep has a strong correlation with pr in ovulation induction cycles. additionally, ai and gt treatments have similar effects on ep. both ep and the growth of the endometrium during the induction (d-et) are good prognostic variables for the successful pregnancy initiation. ovarian response in patients undergoing ovarian stimulation after myomectomy. hyacinth browne, , desiree mccarthy-keith, , barbara stegmann, , alicia armstrong. , reproductive biology and medicine branch, nichd, nih, bethesda, md, usa; reproductive endocrinology and infertility, walter reed army medical center, washington, dc, usa. objective: the impact of myomectomy on ovarian function has not been wellstudied. other surgical treatments of fibroids, such as hysterectomy and uterine artery embolization, have shown an increase of fsh into the peri-menopausal range. the objective of this study is to examine ovarian response in infertile women undergoing ovarian stimulation after abdominal myomectomy. design: retrospective analysis. materials and methods: a retrospective analysis of all infertile women with known fibroids who had a failed art cycle, from january to , followed by an abdominal myomectomy and a subsequent art cycle was performed. women served as their own controls. ovarian function pre and post-myomectomy was assessed by age, day and fsh levels, days of stimulation, total gonadotropins used, peak estradiol level, number of oocytes retrieved, embryos obtained, and high-grade embryos, and pregnancy outcome. quantitative results are presented as mean + sd. results: four women had a failed art cycle and underwent an abdominal myomectomy prior to a subsequent art cycle. the mean age was and pre-and post-myomectomy, respectively. all subjects had uterine factor infertility. two of these women also had tubal factor infertility, and one had endometriosis and male factor infertility. we found no difference in ovarian response pre and post-myomectomy. pre-myomectomy post-myomectomy age (years) + . + . day fsh (u/l) . + . . + . day fsh (u/l) . + . . + . objective: anti-mullerian hormone ( amh) is produced by developing primordial follicles. amh levels are stable through the menstrual cycle and therefore can be drawn randomly. the objective of this study was to assess the association of anti-mullerian hormone ( amh) and oocytes obtained at ivf retrieval. design: cross-sectional cohort study. materials and methods: the study cohort is comprised of thirty women in cycles of in vitro fertiization (ivf). serum levels antimullerian hormone (amh) were drawn before starting an ovulation induction cycle for ivf. standard ovulation induction was performed with leuprolide flare and miu/ml per day of follicle stimulating hormone. we performed linear regression of log convert d oocyte number against the log converted amh level. serum amh was measured using an enzymatically amplified two-site immunoassay dsl- - active mis/amh elisa. statistical analysis was performed using spss version . . continuous values are presented as mean std error. results: the log number of oocytes retrieved was directly related to the log value of amh (p = . ). conclusions: our data demonstrate an association between serum amh and oocytes produced in response to ovulation induction. using amh levels in addition to fsh levels in evaluating for ovarian reserve and pregnancy outcome may help improve predictions of reproductive performance. support: foundation for reproductive medicine. context: prediction of outcome after in vitro fertilization (ivf) can be difficult due multiple factors. human chorionic gonadotropin (hcg) levels correlate with pregnancy outcome, but data that can be used to easily counsel patients on their possible outcome is lacking. objective: to investigate the use of hcg levels along with other significant factors to predict the likelihood of an ivf pregnancy progressing to the point of detection of cardiac activity by ultrasound design: retrospective data analysis of ivf cycles performed from january to july resulting in pregnancies. multiple logistic regression analysis modeling was performed to determine the factors most predictive of an ongoing early pregnancy and to assess possible confounding variables. setting: an academic fertility center. patients: patients undergoing in vitro fertilization using autologous fresh embryos. intervention: none main outcome measure: pregnancy continuation to the documentation of cardiac activity by ultrasound. results: maternal age, day (post-oocyte aspiration) hcg level, and day hcg levels were significant in predicting pregnancy outcome. day and day hcg levels were highly correlated and can be considered proxies for each other. the most accurate predictive model used only a single day hcg level and maternal age. the type of fertilization method used, the cycle number for that patient, and the number of embryos transferred were not found to be significantly different. ongoing pregnancy rates were directly proportional to day hcg level, and inversely proportional to maternal age. the incidence of multiple pregnancies also increased proportionally to the initial hcg level. % of ongoing pregnancies had hcg level > miu/ml. conclusions: a single day post-oocyte aspiration hcg level and maternal age are the most predictive of an ongoing ivf pregnancy. there was no difference in outcome between fertilization methods or number of embryos transferred. there is no benefit to obtaining serial hcg levels after the initial one. erk activation with u ( m)) reduced fsh stimulated cyclin d mrna expression by %. fsh has also been shown to stimulate mtor, a regulator of growth and proliferation of many cell types. inhibiting mtor activation with nm rapamycin for min significantly reduced fsh-mediated cyclin d mrna expression. dht exposure for hours also inhibited fsh stimulated mtor signaling, as shown by a % reduction in the phosphorylation of its down stream target p s kinase. furthermore, pretreatment with dht resulted in significantly reduced fsh-mediated tsc- phosphorylation, which is an upstream regulator of mtor pathway. further studies revealed that fsh mediated tsc- phosphorylation and mtor signaling is regulated by erk, but independent of akt. based on these results we conclude that elevated reduced metabolites of androgens inhibit fsh-stimulated pka-erk pathway resulting in the inhibition of multiple mitogenic signaling pathways leading to defective follicle maturation culminating in anovulation. thus, rescuing the erk activation might serve as a potential therapeutic target to restore normal granulosa cell proliferation in hyperandrogenic states. (supported by nih grant hd- ). searching pcos-genes: results of a genome screen for pcos in a dutch founder population. olivier valkenburg, annemarie g mulders, aida bertoli-avella, ben a oostra, joop se laven. department of gynecology and obstetrics, erasmusmc, rotterdam, netherlands; department of internal medicine, erasmusmc, rotterdam, netherlands. context: although the etiology of pcos is not yet fully understood, evidence has accumulated for a complex genetic background i.e. a combination of multiple genetic and environmental factors. to reduce genetic heterogeneity, this study was conducted in a genetically isolated population in the netherlands . objective: in order to identify genomic loci that are associated with pcos, a whole genome screen using highly polymorphic microsatellite markers was performed in a founder population. subjects and methods: pcos patients ( rotterdam criteria) were identified in the founder population (rucphen, the netherlands) of ± . inhabitants. patients underwent a standardized screening procedure that included clinical, ultrasound and endocrine evaluation. all patients plus unaffected first-degree family members were genotyped using polymorphic markers on (microsatellites) from the abi prism ® linkage mapping set md- (average spacing cm). association-analysis was performed with the transmission disequilibrium test (tdt, genehunter software). linkage analysis was performed in clusters of or more closely related patients (simwalk ). results: there was an average number of . alleles per polymorphic marker. the tdt identified two non-adjacent markers on chrome (d s and d s ) that showed significant association with pcos (p , ). other markers that showed significant association were positioned on chromosomes (d s ), (d s ), (d s ) , (d s ), (d s ) and seventeen (d s ) (all p values . ). linkage analysis in sub-pedigrees revealed no significant linkage for these, or other, loci with pcos. moreover the results of a prior report of linkage of a marker on chromosome (d s ) could not be confirmed. conclusions: the lack of consistent results suggests the absence of a consistent genetic background in this select group of pcos patients. this supports the hypothesis of a complex genetic background for pcos that allows relatively small contributions of multiple risk-genes to be involved in the pathogenesis of this syndrome. in this founder-population the genetic heterogeneity was not sufficiently reduced to find risk-loci in a genome wide screen using highly polymorphic markers with an average spacing of cm. objectives: to objectively quantify uterine and endometrial blood flow in women with polycystic ovarian syndrome (pcos) and to examine if this was different in women with different phenotypic expressions of the disease. methods: transvaginal d and d ultrasound was performed in women with pcos, as defined by the rotterdam criteria, and sub-group analysis conducted based on the subjects' body mass index (bmi), ovulation status, and hirsutism score. anova was used to compare the mean values between the groups. results: pcos women with clinical hyperandrogenaemia had significantly lower endometrial and subendometrial blood flow than their anovulatory normoandrogenic counterparts (table ). there were no differences between lean and obese women or between anovulatory and ovulatory women with pcos. the pulsed wave doppler parameters were similar in all three phenotypic groups. conclusions: hirsute women with pcos have impaired endometrial perfusion compared to their normoandrogenic counterparts which is only evident with d ultrasound and not conventional pulsed wave doppler. nusayba a bagegni, jill blaine, anuja dokras. obstetrics gynecology, university of iowa hospitals clinics, iowa city, ia, usa; obstetrics gynecology, university of pennsylvania, philadelphia, pa, usa. background: there is conflicting evidence on the association between pcos and early and late obstetric complications. it is unclear if the reported risks are independent of bmi, preexisting hypertension and diabetes. we examined the risk of early and late obstetrical complications in a large group of women with pcos compared to controls. methods: we reviewed pregnancy records of women with pcos (rotterdam criteria, n= ) and controls (tubal infertility, n= ) after in vitro fertilization at university of iowa from - . the wilcoxon rank sum test and fisher's exact test were used to evaluate differences between variables and logistic regression analysis was used to determine the independent risk of pcos. results: subject demographics and medical history are shown in table. the first trimester miscarriage rate was % in women with pcos and % in controls. after logistic regression analysis pcos was not associated with miscarriage (p= . ). the prevalence of gestational dm (gdm) was similar in both groups % pcos vs % controls. pcos was not associated with gdm after adjusting for age and bmi (p= . ). however, bmi was significantly associated with gdm after adjusting for age and pcos (p= . ). risk of both pre-eclampsia and pih was % in pcos and % in controls, but not statistically significant after adjusting for age, bmi and twin gestation. preexisting htn showed a significant association with preeclampsia (p< . ). there was no significant difference in preterm delivery, cesarean section, twin gestation, intrauterine fetal death and intrauterine growth restriction in the groups. conclusion: despite adequate power, our study did not detect an increased risk of miscarriage in women with pcos. obesity was a significant contributor to late obstetric complications, namely gdm. these findings may warrant aggressive counseling of women with pcos on the potential benefits of weight loss prior to pregnancy. objective: to assess the prevalence of polycystic appearing ovaries (pcao) as defined by the rotterdam criteria; and to determine whether metabolic parameters differ between regularly cycling women with pcao and those with normal ovaries. studies have demonstrated a population frequency of pcao of - %, with - % among women with regular cycles. most were performed in a young reproductive age population; none examined the impact of age. all were performed prior to adoption of the rotterdam criteria. recent studies have shown an increased prevalence of metabolic syndrome among women diagnosed with polycystic ovarian syndrome (pcos). however studies focused on women in their s found no increase in bmi or fasting insulin with pcao but regular menses. participants: women aged - with regular cycles (every - days) enrolled in the ova study, a population-based study of ovarian aging. each subject underwent a transvaginal ultrasound for assessment of ovarian volume and antral follicle count (afc). outcomes collected included waist measurements and hdl, ldl, total cholesterol, triglycerides, fasting glucose and insulin. pcao were determined by the rotterdam criteria (afc of on one ovary or ovarian volume > cc). student's t-test and chi-square tests were used to assess differences between those with and without pcao for continuous and categorical variables, respectively. the prevalence of pcao decreased with increasing age (table ) . of the women with pcao, % met the afc criterion only, while % met the volume criterion only, and % met both. women with and without pcao did not differ in waist measurements, or in fasting lipids, insulin, or glucose. the rotterdam criteria, while less subjective than those described by adams in , have led to an increased prevalence of pcao among women with regular menses, over / of women in their s. women with pcao do not differ from those with normal ovaries in metabolic parameters associated with pcos. consideration should be given to adopting an age-adjusted criterion for afc, or a combination of afc and ovarian volume, for diagnosing pcos. background: pcos, especially accompanied by obesity, has been reported to be associated with a characteristic dyslipidemia comprising elevated triglycerides (tgs) and depressed hdl, especially the hdl- fraction. this is an atherogenic profile; in fact, studies suggest low hdl- may correlate most strongly with cardiovascular disease risk. weight loss is a mainstay of treatment and improves all manifestations of the disease, but the optimal diet to recommend remains undetermined. preliminary studies show a high protein diet may improve total hdl levels and insulin responses. however, the effect of weight loss and dietary composition on hdl- levels in pcos has not been investigated. objective: to evaluate the fasting lipid profile in newly diagnosed obese pcos patients and to determine the effects of a high-protein diet with or without metformin on weight loss, hdl- and other lipoproteins, and menstrual cyclicity. methods: in this pilot retrospective observational study, the fasting lipid profile of obese women newly diagnosed with pcos was determined. they were then placed on a high protein ( - g/day), low carbohydrate ( - g/day) diet with or without metformin ( and %, respectively) and followed monthly for an average of months (range - ). results: at diagnosis, % had low hdl and % had low hdl- ; only % had elevated tgs. on the diet, the patients demonstrated an average weight loss of . lbs ( . to . , p< . ) and decreased bmi of . kg/m ( . to . , p< . ). hdl levels increased significantly ( % increase from . to . , p< . ), especially the hdl- fraction ( % increase from . to . , p< . ). triglycerides decreased as well ( . to . , p< . ). ldl decreased but did not reach statistical significance. resumed menstrual cycles, were started on oral contraceptives, and had a hysterectomy. pregnancies occurred. no difference was seen with metformin use. conclusion: a majority demonstrated decreased hdl and hdl- at diagnosis. the high protein diet resulted in significant weight loss and improvement in hdl- levels, as well as improvements in total hdl, tgs and menstrual cyclicity. metformin produced no added benefit. prospective trials based on these data will help determine the optimal diet to reduce the significant short-and long-term morbidity in the large population of women with pcos. resistin is an adipokine that has been associated with obesity and insulin resistance in animal models. studies on the role of resistin on insulin resistance in humans have been controversial. recently resistin has been shown to exert atherosclerotic effects and elevated resistin levels have been observed in women with coronary heart disease (chd). women with polycystic ovary syndrome (pcos) are at high risk for chd. our present study investigates potential association of resistin and markers of inflammation, c-reactive protein (crp) and insulin resistance in women with pcos. methods: thirty two women with pcos participated in the study. all were hirsute and had irregular cycles. nineteen women with normal ovulatory cycles who matched the pcos patients in bmi served as controls. fasting glucose, insulin, resistin and crp levels were measured in all women. after a high carbohydrate diet for days, a standard oral glucose tolerance test (ogtt) was performed. blood samples were collected for glucose and insulin before and , and hrs after g oral glucose. the area under the curve (auc) for insulin and glucose was calculated. women with overt diabetes were excluded from the study. results: fasting insulin levels ( . + . μu [±se] /ml) and insulin response to oral glucose (auc) ( . + . μu/ml) were higher (p < . ) in women with pcos compared to controls. all were insulin resistant with homa-ir value > . mol x μu / l . resistin levels in women with pcos ( . ± . ng/ml) was significantly (p < . ) higher compared to control women ( . ± . ng/ml). crp levels in women with pcos ( . ± ng/ml) was also significantly (p < . ) higher than the controls ( . ± ng/ml). there was a significant positive correlation between resistin and crp levels (r = . , p < . ). there was no correlation between resistin levels and fasting insulin levels or insulin auc. there was also no correlation between resistin levels and fasting glucose or glucose auc. conclusions: our results indicate that ( )women with pcos and insulin resistance have increased resistin levels, ( ) there is a strong association between resistin and crp ( ) elevated resistin and crp levels may predict women who are at increased risk for chd ( ) ) it is unlikely that resistin plays a major role on insulin resistance in women with pcos. vuk p jovanovic, enrico carmina, prati vardhana, michel ferin, rogerio a lobo. department of obstetrics and gynecology, columbia university, new york, ny, usa; department of internal medicine, university of palermo, palermo, italy. current diagnostic criteria for pcos includes both ovulatory (ov) and anovulatory or "classic" (c) phenotypes. in an effort to further characterize differences and /or similarities between these phenotypes, we studied hyperandrogenic women with pcos (age . ± . , bmi . ± . ) and age matched controls (age . ± . , bmi . ± . ). women with pcos were divided into weight matched (ov) n= and (c) n= groups. fat and weight distribution were assessed by dexa (total fat, r fat, trunk fat) as well as fasting levels of lh, e , mis/amh, kisspeptin and testosterone, the adipocytokines (leptin, adiponectin, visfatin and retinol-binding-protein- rbp ) and serum glucose, insulin and crp. the hyperandrogenic pcos groups had characteristically altered hormone profiles compared to matched controls. although total fat mass was comparable, women with c-pcos had a significantly larger waist circumference ( . . vs. cm, p< . ) trunk fat, r fat and %trunk and %r fat compared to ov-pcos (p< . ). leptin, rbp and visfatin did not significantly differ among the pcos subgroups although adiponectin was lower in the c-pcos group (p< . ). quicki was significantly lower in c-pcos ( . ± . vs. . ± . , p< . ) and insulin was higher ( . ± . vs. . ± . , p< . ). serum lh was also higher ( . ± . vs. . ± . , p< . ) but kisspeptin, testosterone and estradiol were similar. mis/amh ( . ± . vs. . ± . ng/ml, not significant) and crp ( . ± . vs. . ± . , p< . ) were higher in c-pcos. significant correlations (p< . ) were noted among kisspeptin/rbp (r . ), mis/testosterone (r . ), insulin/ %trunk fat (r . , p< , ), total fat/leptin (r . , p< . ), %trunk fat/adiponectin (r - . ). in conclusion, women with c-pcos when compared to similarly hyperandrogenic women with ov-pcos with similar bmi, have increased abdominal fat, and appear to have differences in serum lh, crp and increased insulin resistance. the latter, as well as subtle differences in the adipocytokines may explain these differences in anthropometric findings. problems of normal oogenesis and folliculogenesis but also disturbed oogenesis and folliculogenesis in polycystic ovaries are not fully understood. oocyte specific genes play an essential role in oogenesis and folliculogenesis. there are suggestions about possible role of some oocyte specific genes in etiopathophysiology of pcos. zona pellucida gene (zp ) is recently identified gene, which belongs to zona pellucida genes such as zp , zp and zp . the role of zp , contrary to above-mentioned other zp genes is not well described. the aim of this study was to analyze zp coding sequence and expression in patients with polycystic ovary syndrome. material included blood received from patients (mean age , +/- , years; mean bmi , +/- , kg/m ) with polycystic ovary syndrome. all patients with pcos were diagnosed with the use of eshre/asrm criteria from . dna was isolated from blood cells( after separation of blood cells from serum) using a dna isolation kit (qiagen ). genomic dna was used for in vitro amplification by pcr with a specific set of primers complementary to the coding sequence of the zp gene. products from each pcr reaction were examined by sscp method. samples with changes detected by sscp in comparison to control probes were cloned into plasmid vector and then automatically sequenced from a total of patient samples with pcos, we identified nucleotide changes in the zp coding seguence : silent nucleotide changes in exons , , , , and nucleotide change in the exon ( position , t>g). the mutation in exon ( t>g ) results in substitution of cystein for glycin of amino acid in position of zp protein. in summary, our data demonstrate that zp nucleotide changes account for % of patients with pcos. relationship between serum mullerian inhibiting substance levels and insulin in women with polycystic ovary syndrome. rebecca a chilvers, shilla chakrabarthy, summer james, xin ma, manubai nagamani. ob/gyn, university of texas medical branch, galveston, tx, usa. müllerian-inhibiting substance (mis) is a member of the transforming growth factor-superfamily of growth factors. it is expressed exclusively in granulosa cells and is believed to play a role in the regulation of follicle selection and maturation. women with pcos have anovulation and most of them have hyperinsulinemia. the purpose of our study is to investigate possible association between insulin and mis in the dysregulation of folliculogenesis in pcos. methods: twenty one women with pcos who had anovulatory cycles and hyperandrogenism were recruited for the study. sixteen women with ovulatory cycles and matched the pcos patients in age and bmi served as controls. an oral glucose tolerance test (ogtt) was performed in all women. blood samples were obtained at fasting and , and hours after glucose ingestion for measurement of glucose and insulin. to investigate the effect of hyperinsulinemia on mis secretion, mis levels were measured in ten patients during the ogtt. fasting mis, testosterone, dheas, fsh, and lh levels were measured in all patients. results: fasting insulin levels ( . + . μu/ml) vs . + μ u/ml [+ se]) (p < . ) and area under the curve (auc) of insulin ( . + . vs . + . μu/ml) (p < . ) were significantly increased in women with pcos compared to control women, while the glucose levels were normal indicating insulin resistance. mis levels were significantly (p < . ) increased in women with pcos ( . + . ng/ml) compared to controls ( . + . ng/ml). there was no correlation between age and mis levels in pcos patients while there was a highly significant negative correlation between the age and mis levels in the control women ( r = - . , p< . ). there was significant negative correlation between mis and fasting insulin levels (r = - . , p < . ) and insulin auc during the ogtt (r = - . , p < . ). there were no changes in mis levels during ogtt. conclusions: results of our study indicate that in women with pcos, ( ) there is an increase in secretion of mis, ( ) higher insulin levels are associated with lower mis levels, ( ) acute increase in insulin levels has no effect on mis levels, ( ) lower mis levels in women with severe hyperinsulinemia could be due to associated increased follicular atresia and a decrease in the ovarian reserve. further studies are needed to investigate the role of insulin on mis secretion. hiroyuki asakura, noriko tanaka, kyoko nishio. ohgimachi ladies' clinic, osaka, japan. objective: elevation of serum anti-müllerian hormone (amh) levels among polycystic ovary syndrome (pcos) patients has been reported. however, the regulatory factors of amh in pcos remain unknown. we examined correlations between amh values and various indices of insulin resistance in pcos women. methods: infertile women compatible with rotterdam criteria for pcos were recruited under informed consent. the subjects underwent g oral glucose tolerance test ( ggtt, sampling at , , minutes), and -step elisa for amh (sensitivity . pmol/l, inter intra-assay c.v. : . %, . %, respectively). p< . was considered as statistical significance. results: average amh level of the subjects (age: . + . years, bmi: . + . kg/m , mean+s.d.) was . + . pmol/l, which was higher than normo-ovulatory women ( . + . pmol/l, n= ). amh levels had significant positive correlation with total ovarian volume by ultrasound ( . + . cc), but not with bmi, waist-hip ratio ( . + . ), and levels of serum lh ( . + . iu/l), total testosterone ( . + . ng/dl). although fasting glucose levels ( . + . mg/dl) were positively correlated with total ovarian volume (r= . ), amh levels had no significant correlation with fasting insulin ( . + . iu/l), fasting glucose/insulin ration ( . + . ), homa-ir ( . + . ), and the sum of insulin levels ( . + . iu/l)during ggtt. conclusions: increased serum amh level of pcos and its positive correlation with total ovarian volume implies that determination of serum amh level would aid in confirming diagnosis of the ovulatory disorder. absence of relationship between amh and various clinical indices of insulin resistance suggests alternative regulatory factors of amh gene expression in the follicular compartment. reproductive tissues. amisra a nikrodhanond, keeley l mui, helen h kim. ob/gyn, university of chicago, chicago, il, usa. background: although primarily a neuroendocrine hormone, gonadotropinreleasing hormone (gnrh) is also produced in reproductive tissues, where it acts locally. the study of gnrh regulation in these tissues is limited by low levels of expression. objective: to elucidate mechanisms that regulate gnrh expression in male tissues, our objective was to identify regions of the gnrh gene that target expression in vivo. methods: transgenic mice were generated with different fragments of the mouse gnrh gene promoter (- /+ and - /+ bps), fused to the luciferase reporter gene. in these mice, luciferase activity (detected as light) reflects gnrh promoter activity. using bioluminescent imaging, gnrh promoter activity was assayed in live gnrh-luc mice and in their reproductive tissues ex vivo. to confirm imaging results, luciferase activity was also measured as relative light units (rlu) in tissue homogenates. for each dna construct, male mice were examined. with whole-body imaging, bioluminescence was detected in the genital region of the gnrh-luc transgenic mice (fig.a) . in mice that incorporated the - luc transgene, examination of reproductive tissues ex vivo revealed bioluminescence in the prostate and penis, but not in the seminal vesicles, epididymis, or testes (fig.b) . in contrast, in the - luc mice, bioluminescence was detected in the testes, but not in other tissues (fig.c) . examination of luciferase activity in tissue homogenates from - luc mice confirmed gnrh promoter activity in the prostate ( ± rlu) and penis ( ± rlu) and absence in the testes ( ± rlu). in the - luc mice, however, luciferase activity was detected only in the testes ( ± rlu) and not in the prostate ( ± rlu) or penis ( ± rlu). conclusions: our studies demonstrate that gnrh is present in male reproductive tissues, but may be differentially regulated in the testes vs. prostate/penis. promoter elements, contained within the proximal - bp of the mouse gnrh gene, are sufficient to mediate testicular gnrh expression while - bp of the gene promoter are necessary to direct expression to the prostate and penis. characterization of mouse ringo/speedy homologues. z walton, s uckac, o guzeloglu-kayisli, md lalioti, d sakkas, e seli. ob gyn, yale u., new haven, ct, usa. introduction: ringo/speedy (ringo/spy) is a recently discovered cyclindependent kinase (cdk) activator that functions similar to cyclins in controlling the cell cycle. ringo/spy plays a crucial role during meiotic maturation in xenopus by inducing meiotic g /m progression and germinal vesicle breakdown (gvbd). more recently, padmanabhan and richter demonstrated that ringo/spy mrna is repressed in the xenopus oocyte cytoplasm by pumilio . upon meiotic reactivation, pumilio loses its interactions with ringo/spy permitting its translation. ringo/spy is then expressed, leading to activation of cpeb by phosphorylation, which in turn elicits polyadenylation and translation activation of the mrna for a critical oocyte maturation factor, the mos kinase. in this study, we investigated the expression of ringo/spy in mouse. methods: ten different somatic tissues, testes, and ovaries were tested by reverse transcription-polymerase chain reaction (rt-pcr) for the expression of ringo/spy homologues and their alternative splicing variants. ringo/spy mrna expression was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results: we analyzed the two previously identified homologues of ringo/spy (a and b). the two alternative splicing variants of ringo/spy a (a and a ) were separately studied for their expression profile. we also identified an additional alternative splicing variant of ringo/spy b and evaluated similarly. ringo/spy a ( and ) were expressed in testes, ovaries, and certain somatic tissues including brain, and spleen. ringo/spy b (both variants) were only expressed in testis. ringo/spy a or b were not present in mouse oocytes or early embryos. conclusions: our findings indicate that the previopusly described ringo/spy homologues a and b are not expressed in mouse oocytes or early embryos suggesting that either an alternative cdk-activator or a yet to be identified homologue of ringo/spy may be mediating meiotic g /m progression in mouse. testis specific expression of ringo/spy b, and previously described role of ringo/spy in meiosis suggests a role for this protein in male gametogenesis in mouse. treated with glyburide. jennifer aguayo, gladys a ramos, alethea hanley, carri r warshak, thomas r moore. reproductive medicine, university of california, san diego, san diego, ca, usa. objective: to determine the risk factors that may predict inadequate response to first-line glyburide monotherapy in pregnant women with type diabetes mellitus (dm) or gdm and to assess if non-responders are at increased risk of adverse pregnancy and neonatal outcomes. study design: this was a retrospective cohort of women diagnosed with type ii or gdm initially treated with glyburide at a single institution from - . maternal characteristics, adequate glycemic control defined as more than % of fasting glucose < mg/dl and one-hour post-prandials < mg/dl, and neonatal outcomes were assessed. non-responders were defined as failure to achieve adequate glycemic control on maximum daily doses of glyburide or intolerance due to side effects, necessitating switch to insulin therapy. statistical methods included bivariate analyses. results: of the women initially treated with glyburide, ( %) failed to achieve adequate glycemic control or did not tolerate glyburide. reasons for glyburide non-response were maximum dose ( mg/d) reached ( %), maternal hypoglycemia ( %) and other side effects ( %). there were no statistically significant differences between non-responders and responders with respect to family history of diabetes ( % vs. %, p= . ), prior history of gdm ( % vs. %, p= . ) and macrosomia (> g) ( % vs. %, p= . ) or hour glucose challenge test ( + vs. + mg/dl, p= . ). non-responders had a higher rate of obesity (bmi> ) ( % vs. %, p= . ) and earlier gestational age at initiation of therapy ( + . vs. + . wks, p= . ). there were no differences between the groups in the mean -week fasting ( + vs. + mg/dl, p= . ) and post-prandial glucose values ( + vs. + mg/dl, p= . ). no significant differences were observed in incidence of pre-eclampsia, primary cesarean delivery or birth weight. neonatal outcomes including ponderal index, neonatal hypoglycemia, nicu admission, and birth injuries also did not differ between the groups. conclusion: women who are obese and who require earlier initiation of glyburide therapy are at increased risk of non-response to glyburide monotherapy. however, despite non-response, these women can be managed with subsequent insulin therapy to achieve similar glycemic control and pregnancy and neonatal outcomes. a different diagnostic strategy using the gram, -hour glucose tolerance test for the diagnosis of gestational diabetes mellitus. yvonne w cheng, ingrid block-kurbisch, jennifer lydell, aaron b caughey. obstetrics, gynecology and reproductive sciences, university of california, san francisco, san francisco, ca, usa. objective: to examine whether a simplified strategy using the gm, -hour glucose tolerance test (gtt) may be useful for the diagnosis of gestational diabetes mellitus (gdm). methods: this is a retrospective cohort study of women with singleton pregnancy who received the -gm, -hour glucose challenging test (gct) for initial screening and the gm, -hour gtt as confirmatory tests for the diagnosis of gdm between and . various combinations of the gm, -hour gtt results were examined and compared to the diagnostic criteria for gdm established by the carpenter and coustan criteria using the receiver-operator characteristic (roc) curves. perinatal outcomes of women who would have had a false-positive or false-negative test results were compared to those who did not have gdm using the carpenter and coustan criteria. potential confounding factors were controlled for using multivariable regression models. results: , women had gm, -hour gtt results available for analysis during the study period. using gdm diagnosed by the carpenter and coustan criteria as reference, various diagnostic strategies was compared using roc curves (figure ) . summation of the -hour and -hour gtt results with a diagnostic threshold of mg/dl yielded the most optimal balance between sensitivity ( . %) and specificity ( . %). when compared to women without gdm, women who were diagnosed with gdm by c c criteria but not by summation of -hour and -hour gtts had higher odds of operative vaginal delivery (aor= . , % confidence interval [ci] . - . ) and neonatal birthweight > gm (aor= . , % ci . - . ). conclusion: using only the summation of only the -hour and -hour gtt results of the gm -hour gtt offers an alternative test strategy which may be more convenient and less costly for the diagnosis of gdm. however, women who would have gdm by the carpenter and coustan criteria but not by the summation method have higher odds of having an operative vaginal delivery and neonatal birthweight > gm. outcome of induction of labor indicated for term prom among women with or without a uterine scar. yifat ochshorn, avital skornick rapaport, adi reches, joseph b lessing, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. objective: we aimed at comparing the outcome of induced term deliveries presenting with prom with or without a previous uterine scar. methods: the computerized files of women delivered following induction of labor due to term prom, were reviewed. perinatal outcome parameters such as the mode of delivery, indication for cesarean section, rate of low ' apgar scores and nicu admissions were compared between women with and without a previous cesarean. results: during the study period women delivered in our institution following prom and induction of labor , of them had a previous uterine scar. parturients of both groups (with and without a scar) were similar with regard to age, gestational age at delivery and parity. cesarean section rate was higher for the previous scar group ( % vs %, p< . ). the most common indication for cesarean was arrest of dilatation and/or descent among women with previous scar accounting for % and % (p< . ) for women with and with no uterine scar, respectively. no differences were noted in neonatal outcome parameters such as rate of low ' apgar scores and nicu admission rate between the two groups. conclusion: induction of labor due to prom culminates in higher cesarean rate in women with one previous scar compared with parturients with no scar. perinatal outcome is similar between the two groups. acid base balance and immediate perinatal outcome of vertex compared with breech presentation in elective cesarean section. avital skornick rapaport, yifat ochshorn, joseph b lessing, yuval yaron, michael kupferminc, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. backround: apgar scores, umbilical blood ph and bicarbonate are generally lower and pco levels are higher in vaginally delivered breech neonates compared to cephalic deliveries. although cesarean delivery improved apgar scores there is a debate wether it improved the acid base status. this retroprospective study compared umbilical cord blood acid-base values and perinatal outcome of elective cesarean breech-delivery with those of elective cephalic cesarean delivery and to determine whether a different metabolic status and perinatal outcome should be expected in neonates in breech presentation. study design: the study group included singleton pregnancies delivered by elective cesarean section at term between january and march . computerized files of singleton breech presentation elective cesarean sections were compared to those of singleton vertex neonates delivered by elective cesarean section. demographic data included: maternal age, pregnancy week at delivery and parity. perinatal outcome measures checked were: birth weight, apgar scores at 'and ', umbilical cord venous and arterial ph and base excess. results: during the period between january and march there were singleton elective cesarean sections, of them were breech and vertex. the mean age, gravida and parity were significantly different between groups ( . vs. . , . vs. . . and . vs. . respectively, p< . ). the birth weight was significantly different - . gram for the vertex and . gram in the breech deliveries (p< . ) there were no differences in either apgar scores or umbilical ph between the breech and vertex neonates delivered by cesarean section at term ( - + w). venous and arterial po and pco levels were significantly different, though the differences were very small and we doubt if these differences have any clinical importance. conclusion: although vaginal breech deliveries are associated with increased risk of asphyxia during delivery, elective cesarean breech deliveries are not at increased risk for lower ph levels or apgar scores. the clinical significance of bleeding during the second trimester of pregnancy. arie koifman, amalia levi, yaron zaulan, avi harlev, eyal sheiner. obstetrics gynecology, soroka university medical center, ben gurion university of the negev, beer-sheva, israel; epidemiology and health services evaluation, faculty of health sciences, ben gurion university of the negev, beer-sheva, israel. objective: this study aimed at investigating clinical importance and pregnancy outcome in women suffering from bleeding during the second half of their pregnancies. methods: a population based study including all deliveries which took place in the soroka university medical center between the years - were examined. comparison was performed between patients with and without second trimester bleeding pregnancies terminated before weeks, multiple gestations and women lacking prenatal care were excluded . stratified analysis, using the mantel-haenszel technique, and a multiple logistic regression model were performed . results: during the study period, , singleton deliveries occurred in our institute. of these, ( . %) were complicated with bleeding upon admission during the second half of pregnancy. the cases were attributed to placental abruption ( . %; n= ) and placenta previa ( . %; n= ). independent risk factors associated with bleeding, were oligohydramnios, polyhydramnions, (odds ratio [or]= . ; % confidence interval [ci] . - . ; p= and . ; . - . ;p< . respectively ), suspected intra uterine growth restriction (iugr, . ; . - . ; p<. ), gestational age, previos abortions and maternal age. these patients subsequently were more likely to deliver by cesarean section (cs, . % vs. . %, or= . ; %ci . - . ; p< . ). perinatal mortality among patients admitted due to second half bleeding was significantly higher as compared to patients without bleeding (p<. ). conclusion: bleeding upon admission during the second half of pregnancy is an independent risk factor for perinatal mortality. careful surveillance, including fetal monitoring, is suggested in these cases in order to reduce the adverse perinatal outcome. crude and adjusted odds ratios for perinatal mortality among patients with vaginal bleeding. characteristics or % ci p crude or for perinatal mortality . . - . < . or adjusted for: < . iugr . . - . < . oligohydramnios . . - . < . premature rupture of membranes . . - . < . the predictors included neonatal pi, bmi, or birthweight while the outcomes included hypoglycemia, shoulder dystocia, acidemia and hyperbilirubenemia. roc curve analyses were utilized to evaluate the relationship between sensitivity and specificity for each of the predictors and outcomes, with an area under the curve (auc) significantly greater than . indicating a screening test that is better than chance. results: neonatal pi, bmi and birthweight are poor predictors of nicu admission, acidemia and hypoglycemia with all auc values not statistically significantly different than chance alone. they are a reasonable predictor of shoulder dystocia, with birthweight functioning the best (p< . ). in general, neonatal anthropometric measurements do not appear to be good predictors of short term neonatal outcomes. although they are a reasonable predictor of shoulder dystocia, they are not useful clinically for this outcome since they cannot be calculated until after birth. in future studies, new models of neonatal anthropometrics should be created to better predict adverse neonatal outcomes. neonatal anthropometric measurements and their relationship to perinatal outcomes expressed as auc and % confidence intervals history of at least one spontaneous preterm delivery were offered protocolbased prenatal care including first-or second trimester bacterial vaginosis screening, repeated ultrasound cervical length assessment, and supportive care by specialized nurses. women with a positive test for bacterial vaginosis were treated with oral metronidazol therapy, and women with a cervical length below . cm (before weeks of gestation) underwent a vaginal cerclage. progesterone administration was not provided. data on obstetrical and medical history, pregnancy, delivery and neonatal outcome were prospectively collected and evaluated. results: in total, pregnant women were prospectively followedup. eighty-seven women had experienced a preterm delivery in their last pregnancy, of whom had delivered before weeks. three women were diagnosed previously with a bicornual or septal uterus. twelve women received metronidazol therapy and women underwent a vaginal cerclage. eighty-four women ( . %) delivered at weeks or beyond. one-hundred and three women ( . %) delivered no earlier than weeks, and only one woman delivered at weeks. two women gave birth to a twin (at and weeks, respectively). two neonatal deaths due to pulmonary hypoplasia were recorded. conclusion: women at high risk of preterm delivery who were offered protocol-based prenatal care not including progesterone therapy had a better pregnancy outcome than would be expected from previous studies. our findings may question the reported beneficial effects of prenatal progesterone administration. premature rupture of membranes. tracy a manuck, alexandra g eller, m sean esplin, robert m silver. obstetrics gynecology, university of utah health sciences, salt lake city, ut, usa. objective: historically, outcomes following expectant management of midtrimester preterm premature rupture of membranes (pprom) have been uniformly poor. thus, many patients elected pregnancy termination. however, outcomes may be improved with recent advances in neonatal medicine. our purpose was to assess outcomes in expectantly managed early pprom in an era of improved maternal and neonatal care. study design: this is a retrospective cohort of patients from tertiary healthcare systems from - experiencing pprom . weeks gestation. patients electing immediate termination of pregnancy, carrying fetus(es) with lethal anomalies, or delivering within hours of pprom were excluded. survival without major morbidity was the primary outcome. data were analyzed using student t-test and chi-square as appropriate. results: a total of women carrying fetuses ( singleton, twin, triplet, quadruplet) met inclusion criteria. only the fetus in the "ruptured sac" was studied. pprom occurred at a mean of . (+/- . , range . - ) weeks. the average latency period was . (+/- . , range . - ) days, with a mean delivery gestational age of . (+/- . , range . - ) weeks. this communication provides the first report of fundal uterine necrosis following placement of multiple uterine compression sutures. only two previous published cases report occurrence of uterine necrosis following application of a uterine compression suture --both identified as the b-lynch technique --and neither of these cases report necrosis confined to the fundus. in our case, uterine atony was refractory to pharmacologic therapy and manual compression of the uterus appeared to decrease the amount of blood loss. therefore, we applied a traditional b-lynch which effectively contracted the majority of the uterus, while the fundus remained atonic. a second horizontal, square suture was then placed between the cornua and carried over the fundus (see figure one: red reflects b-lynch suture; blue represents second fundal, square stitch). the patient subsequently experienced significant, persistent fevers despite antibiotic therapy. a repeat laparotomy was performed, at which time we found uterine necrosis confined to the uterine fundus (see figure : photograph taken at the time of hysterectomy). the placenta showed no histologic evidence of chorioamniotis --hence, the patient's main risk factor for fundal necrosis was the compression sutures. physicians should be aware of the risk of uterine necrosis, especially after placement of multiple compression sutures and, more specifically, after placement of a compression suture confined to the uterine fundus. background: migraines are far more common in women than in men. the incidence of migraines increases in girls after puberty, reaching an incidence of % in women who experience migraine at least once a year around middle age. migraine has been postulated as one of the major risk factors for stroke during pregnancy and the puerperium.triptans are a class of serotonin receptor agonists used in the treatment of migraine headaches. triptans administered in combination with other drugs have been known to precipitate serotonin syndrome, a rare but potentially life-threatening condition clinically manifested by a triad consisting of mental-status changes, autonomic hyperactivity, and neuromuscular abnormalities. objective: to determine whether triptan monotherapy is associated with serotonin syndrome methods: using data mining techniques we analyzed the entire food and drug administration's (fda) adverse event reporting system (aers) database. results: after excluding reports of concomitant serotonergic medication or other potentially confounding medication, eleven reports remained of serotonin syndrome associated with triptan use without concomitant serotonergic medication. the mean age for these eleven cases was . years; nine cases occurred in females and two occurred in males. there were no apparent instances of overdose among these eleven cases. conclusions: serotonin syndrome is a rare but serious occurrence with the use of triptan monotherapy. such cases were seen with eletriptan, rizatriptan, sumatriptan, and zolmitriptan. because of the spontaneous nature of voluntary reporting to aers, the actual number of occurrences of serotonin syndrome in patients using triptans is probably higher and cannot be assessed from aers data. users of triptan in combination with an ssri or snri should be warned of this rare but serious adverse effect. during periods of drug initiation, dose escalation, or the addition of another serotonergic agent, patients should be particularly vigilant for symptoms of concern and seek urgent medical attention if any occur. the offending agents should be withdrawn and the patients closely monitored and treated with supportive measures as required. rising maternal mortality in the midst of modern technology. oormila p kovilam, jane khoury, padmini c sekar, ralph c buncher. obstetrics gynecology, university of cincinnati, cincinnati, oh, usa; center for epidemiology and biostatistics, cincinnati children's hospital medical center, cincinnati, oh, usa; environmental health, university of cincinnatic, cincinnati, oh, usa. introduction: maternal mortality rate (mmr) is an index of overall wellbeing of the community and safe motherhood should be given utmost priority in obstetric care. as per , who estimates % of maternal mortality occurs in developed countries . this rate has established without much decline in recent years. the mmr for ohio for to was reported by cdc to be . per , live births with % confidence interval . to . . objective: our objective was to audit the trend in maternal mortality in the state of ohio for the last years. methods: information from the death certificates completed by attending physicians, medical examiners, coroners, funeral directors filed with ohio state registration offices were analyzed. we only used women who were residents of the state of ohio at the time of death, and cause of death was coded as a "complication of pregnancy, childbirth and the puerperium", icd codes to and icd codes o to o . five year maternal mortality pattern and long term trend was assessed. results: the number of maternal deaths recorded as due to pregnancy complications varied over the years from a high of in to a low of in and . in general the five-year mortality rate was decreasing over time until the last four years to , when we may be seeing an upward trend compared to to (p= . ). the rates and associated % confidence intervals (ci) are shown in the table below. years mmr % ci ci - ci . . - . ci - . . - . - . . - . - . . - . - . . - . the rate of maternal mortality is on the rise after a period of downward trend. we should develop a multidisciplinary approach to analyze and target the root causes of maternal death. introduction. women with thrombophilia are at higher risk of vte during pregnancy due to the acquired hypercoagulable state. it is likely that not only maternal veins but also placental vessels are more prone to the development of thrombosis. our objective was to compare maternal and fetal placental circulation disorders in women with intra-uterine fetal death (iufd) and thrombophilia and women without thrombophilia. methods. in a dutch multi-centre study on iufd, during the period - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. factor v leiden and prothrombin g a were tested at induction of labour. panel classification of cause according to the tulip classification was performed by assessors after individual investigation of structured patient information. we studied the cause of death group "maternal and fetal placental circulation disorders": placenta bed pathology with abruption or infarction as origin of mechanism and placental parenchyma pathology with fetal thrombotic vasculopathy and massive perivillous fibrin deposition as origin of mechanism. results. of the women tested for factor v leiden, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of factor v leiden. of the deaths with another cause of death ( . %) mothers were carriers of factor v leiden (p= . ). of the women tested for prothrombin g a, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of prothrombin g a mutation. of the deaths with another cause of death ( . %) mothers were carrier of prothrombin g a mutation (p= . ). in women with iufd and factor v leiden or prothrombin g a mutation "maternal and fetal placental circulation disorders" did not seem to cause iufd more often than in non-carriers. , university medical center groningen, groningen, netherlands; pathology, university medical center groningen, groningen, netherlands; trial coordinating center, dept of epidemiology, university medical center groningen, groningen, netherlands; hematology, university medical center groningen, groningen, netherlands. introduction. growing evidence suggests that women with thrombophilic defects may be at higher risk of fetal loss. although some paternal components to the predisposition of preeclampsia have been demonstrated it is not known whether paternal components contribute to intra-uterine fetal death (iufd). our objective was to investigate the relation between paternal thrombophilic defects and iufd. methods. in a dutch multi-centre study on iufd, from - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. we tested male partners of women with iufd for antithrombin (at), protein c, protein s type i and iii, factor v leiden, prothrombin g a (factor ii) and factor viii: ag. standard tests were performed in one laboratory. normal ranges were determined in healthy male blood donors. we compared prevalence of thrombophilic defects to reference values from the literature. . of the men tested for factor v leiden, ( . %) were carrier versus ( . %) non-carriers. prevalence of factor v leiden in the normal population is % (p= . ). men were tested for prothrombin g a, ( . %) were carriers and ( . %) non-carriers. all were heterozygous. prevalence of prothrombin g a mutation in the normal population is % (p= . ). decreased levels of antithrombin, protein c, protein s type i and iii and increased levels of factor viii: ag were observed significantly more often in male partners of women with iufd compared to the normal population. conclusion. in our iufd group the prevalence of male factor v leiden carriers was comparable to the normal population, prevalence of prothrombin g a mutation was lower. the difference in other factors imply an as yet unexplained association of male thrombophilia with fetal death in their partner. objective fetal macrosomia, the most important complication of gdm, increases the risk of shoulder dystocia, erb's palsy and intrauterine hypoxia. we compared frequencies of fetal macrosomia and erb's palsy in two gdm cohorts with different severity of glycemic disturbance and in healthy controls. methods we studied consecutive gdm women with singleton childbirth and or abnormal values in -h ogtt with g of glucose. abnormal plasma glucose values of the ogtt were: fasting . , -h . and -h . mmol/l. insulin treatment was started when values were . preprandially or . mmol/l postprandially in the -h glucose profile done within days of diagnosis. if the same woman had more than childbirth during the study period, only the last pregnancy was included. the control group consisted of women from a nearby town with singleton childbirth in the same hospital. women with diabetes were excluded from controls. macrosomia was defined as birth weight (bw) > . sd above the mean of a standard population. erb's palsy was diagnosed by pediatric surgeons. results gdm women were older, more obese, had more childbirths and more often a previous child with bw > g than controls. insulin was started in gdm women ( . %). c/s rate was . % in controls, . % in diettreated and . % in insulin-treated gdm women. macrosomia rate was . % in controls, . % in diet-treated and . % in insulin-treated gdm women (p< . compared with controls or diet-treated gdm women). the frequency of macrosomia did not differ between the diet-treated gdm and control women. erb's palsy occurred in . % of controls, in . % of diet-treated and in . % of insulin-treated gdm women. the frequency of erb's palsy was significantly higher in both gdm groups than in controls (p= . ). by regression analysis, previous child with bw > g (p< . ), previous c/s (p= . ), mother's age (p= . ), bmi (p= . ), insulin treatment (p= . ) and fasting plasma glucose of the -h profile (p= . ) were significant independent predictors of fetal macrosomia. conclusions the -h glucose profile done shortly after the diagnosis of gdm clearly distinguishes between low-risk (diet-treated) and high-risk (insulintreated) gdm pregnancies for fetal macrosomia. unfavourable fetal body composition of diet-treated gdm women is the likely explanation for the high rate of erb's palsy in this group. . '-:·:tpartq,-, '-:·:tpartq,-, '-:·:tpartq,-, % p= . ). first trimester uta doppler indices were similar in the two cohorts in terms of the resistance and pulsatility indices (ri . vs. . , p= . ; pi . vs. . , p= . ) . however, bilateral notching was much more common in the cohort of prior adverse outcomes ( % vs. % p= . ) as well as in patients destined to have a subsequent poor outcome (p= . ). conclusions: not surprisingly, prior poor obstetric outcome was strongly associated with recurrent adverse obstetric outcome. uta notching was robustly associated with prior poor obstetric history as well as a recurrent poor outcome. this clinical history had no discernable influence on ri or pi. reassurance may not be offered based on first trimester uta ri and pi. anti-retroviral therapy is associated with increased blood loss and uterine atony for patients undergoing primary cesarean section. carey eppes, alice cootauco, melissa russo, jessica bienstock. gynecology and obstetrics, johns hopkins university school of medicine, baltimore, md, usa. objective: anti-retroviral therapy has been associated with gastrointestinal smooth muscle dysfunction. it has also been hypothesized that myopathies can occur secondary to anti-retroviral therapy due to mitchondrial alterations. in our experience, we have noticed an increased incidence of uterine atony in our hiv patients on anti-retroviral therapy. we sought to examine the incidence of postpartum hemorrhage and uterine atony in patients currently on anti-retroviral therapy. study design: a retrospective case-controlled study was conducted on all hiv positive pregnant women on anti-retroviral therapy undergoing a primary low segment transverse cesarean section from through . these patients were obtained from an irb approved database containing hiv positive patients within our institution. patients' medical records were abstracted for demographic data, use of uterotonics, preoperative and postoperative hematocrits, and incidence of blood transfusions. controls were matched for age, parity, gestational age and surgical indication. data was analyzed using the t-test, fischer's exact test and chi square. results: there were no differences in demographics, incidence of chorioamnionitis or magnesium sulfate use between groups. patients on anti-retroviral therapy had a statistically greater decrease in hematocrit and estimated blood loss compared to controls. they also had an increased need for uterotonics and blood transfusions. conclusion: anti-retroviral therapy may impact uterine smooth muscle contractility in pregnancy, as evident by the increased incidence of uterine atony and change in hematocrit. additional research is needed to elucidate the mechanism. clinicians should be aware of the potential for uterine atony and excessive blood loss in patients on anti-retroviral therapy. background: adolescent pregnancy is frequently associated with adverse outcomes, especially small-for-gestational age (sga) deliveries. some studies have implicated maternal nutritional status in these poor outcomes. methods: in a prospective longitudinal study, ethnically-diverse, pregnant adolescents were studied from booking to parturition, with collection of anthropometric and nutritional variables. blood samples ( - weeks' gestation; n= subjects) were assayed for a spectrum of nutritional biomarkers. logistic regression was used to determine significant associations between studied variables and pregnancy outcomes. results: median age at recruitment was . years (iqr: . - . ). outcome data was available for subjects. ( . %) had uncomplicated pregnancies. median birthweight was , g (iqr: , - , g) and median birthweight centile was . % (iqr: . - . %). ( . %) infants were born sga and ( . %) were preterm. spontaneous vaginal deliveries occurred in . % of cases. there were ( . %) cases of pre-eclampsia and ( . %) admissions to neonatal care. . % of subjects reported smoking at booking. iron deficiency anaemia was prevalent in . % of subjects by - weeks and was strongly associated with higher infant birthweight centile (p< . ). . % had serum -hydroxy vitamin d concentrations < ng/ml, although this was not associated with any outcomes. low folate status, as indicated by low red cell folate (p= . ), low serum folate (p= . ) and high serum homocysteine (p= . ) concentrations, was associated with higher rates of sga birth. conclusion: adolescent pregnancy in inner-city populations is associated with a high risk of sga and preterm birth, increasing the likelihood of health problems in later life and perpetuating social disparities in health. the association between anaemia and higher birthweight is unexplained. impaired maternal folate status may contribute to impaired fetal growth. we suggest that sga birth in this population could be reduced by the use of antenatal supplements or the mandatory fortification of flour with folic acid. amount of cigarette use pre-pregnancy and during pregnancy were self-reported and subgrouped into nonsmokers, quitting during pregnancy, and continued smoking throughout pregnancy. categorical outcomes were compared using chi-square test. multivariable logistic regression analyses were used to control for potential confounders (continued smoking compared to quitting during pregnancy). results: women who quit smoking during pregnancy had higher rates of pregnancy-associated hypertension and cesarean delivery but lower rates of preterm delivery and neonatal birthweight < gm compared to non-smokers. compared to women who quit smoking during pregnancy, those who continue to smoke have lower odds of pregnancy-associated hypertension and cesarean delivery, but higher odds of preterm delivery, neonatal birthweight < gm, and apgar score < at minutes (see table) conclusion: quitting cigarette smoking during pregnancy appears to reduce undesirable neonatal outcomes, though an increase in pregnancy associated hypertension. these findings should be emphasized to women who are smoking during pregnancy. to achieve effective tamponade the balloon was inflated initially up to - ml with incremental increases of volume by ml until bleeding stops. to measure intrauterine balloon pressures we used a standard pressure transducer (edwards lifesciences) connected to the balloon port and to a pressure monitor (ge dash ). the pressure transducer was mounted on the bedrail at uterine level, primed with sterile saline and then connected to the balloon port of the catheter. the system was zeroed to atmospheric pressure, the stopcock of the intrauterine balloon port was opened and the pressure was recorded (p : the total pressure on the fluid). to verify the accuracy of measurements we used another balloon catheter (reference), inflated with the same amount of saline, connected to the pressure transducer and zeroed to atmospheric pressure. the reference balloon measured p (the intrinsic balloon pressure caused by distension at that volume). the system was then zeroed to the reference balloon. the pressure transducer was then reconnected with the intrauterine balloon and the actual intrauterine pressure was recorded (p ). we calculated the correlation coefficient between p and systolic, diastolic and mean blood pressure using a linear regression (statsdirect statistical software there is a growing body of evidence to suggest that peripartum assessment of fetal or neonatal lactate levels are as good as or better than standard blood gas analysis in the prediction of neonatal outcome. in this study we have evaluated the ability of umbilical cord blood gases and lactate levels in the prediction of neonatal hypoxic-ischaemic encephalopathy (hie). department of neonatal paediatrics, king edward memorial hospital, perth, western australia, australia; women and infants research foundation, king edward memorial hospital, perth, western australia, australia. objective: current evidence suggests that umbilical cord ph at delivery provides the most sensitive reflection of birth asphyxia. paired umbilical artery/vein blood gases have been routinely collected at king edward memorial hospital over the last years. the objective of this study was to determine: local reference ranges; accuracy of sampling; and the rates of metabolic acidosis. of the , births ( ) ( ) ( ) ( ) , accurate paired results were available on % of births. over the study period there was a progressive improvements in accuracy rates of paired sampling (p< . ). the median ( . th , . th centile) values for cord arterial blood gases were: ph . ( . , . ); po . mmhg ( . , . ); pco . mmhg ( . , . ); base excess - . (- . , . ); and lactate . mmol/l ( . , . ). there was a progressive improvement in all blood gas measures over the years of this study (all p< . ). moreover, there were significant reductions in all measures of metabolic acidosis (see table) . the progressive improvement in the measures of metabolic acidosis remained significant after multivariate analysis including obstetric, fetal, and demographic factors associated with metabolic acidosis. the introduction of universal umbilical cord blood gas analysis to all births is associated with significant improvements in all markers of metabolic acidosis. together with other compelling evidence in the literature, these data support the routine use of cord arterial and venous gases at all births; however, improved accuracy rates on paired sampling requires an ongoing education program umbilical artery indicators of acidosis, percentage of total ph < . ph < th centile* objective: intrapartum pcn prophylaxis aims to prevent early-onset gbs sepsis by interrupting vertical transmission from colonized mothers to their newborns. however, despite its wide clinical use, systematic pharmacokinetic evidence in support of the current pcn dosage regimen is lacking. current cdc guidelines recommend intensified surveillance and testing of infants exposed to < h of prophylaxis. our goal was to examine the relationship between maternal time of exposure to pcn and fetal serum pcn levels among maternal-fetal dyads exposed to short durations of pcn prophylaxis (< h) compared to those exposed to longer durations. study design: ninety-eight laboring gbs positive women were administered million units (mu) of intravenous pcn to be followed by . mu every hours until delivery (cdc ) . subjects with renal disease, multiple gestation, and preterm delivery (< wks) were excluded. umbilical cord blood samples were collected at delivery and pcn levels measured by high-performance liquid chromatography. intra and inter-assay coefficients of variation were < %. results: the pcn concentrations (mean±sd) by duration of prophylaxis were: < h, . ± . g/ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ml (n= );> h . ± . g/ml (n= ); and for those without a second dose after h, . ± . g/ml (n= ). fetuses exposed to short duration (< h) had higher levels of pcn than those exposed to > h (p= . ). in multivariable linear regression analysis, fetal pcn levels were determined by total duration of exposure, time since last dose, dosage, and number of doses, but not maternal bmi. pcn levels in cord serum increased linearly until hour; thereafter, they decreased rapidly, but all groups were significantly above the minimum inhibitory concentration (mic) for gbs ( . g/ml)(p< . ). furthermore, every sample individually remained - fold above the mic. conclusion: in this study, even short durations of prophylaxis achieved levels above the mic, suggesting a benefit to prophylaxis even in precipitous labors. the data also suggests that the current cdc designation of infants exposed to < h of pcn prophylaxis as particularly at risk for gbs sepsis may be inaccurate from a pharmacokinetic standpoint. objective: -oh aa is a metabolite of tryptophan with pro-oxidant and proapoptotic properties and has been shown to increase in umbilical cord blood in pregnancies with intra-uterine infection. since labour-related events may also activate inflammatory pathways, we sought to determine the placental release of -oh aa into the umbilical circulation in labouring vs non-labouring patients at term. methods: twenty-six patients were studied (term labour n= , and term elective cesarean section n= ) with blood sampling from a clamped segment of umbilical cord after delivery of the fetus and from the cord at its insertion into the placenta after delivery of the placenta, with subsequent measurement of blood gases/ph and -oh aa (isocratic hplc using fluorometric detection with assay sensitivity at pmol). results: -oh aa measurements from respective umbilical and placental cord vessels were all variably higher in the labouring group vs the elective cesarean group patients (table ) . for labouring group patients, the -oh aa levels from the umbilical vein were significantly higher than those from the umbilical artery, indicating net release from the placenta into the fetal circulation. placental vein levels were also significantly higher than those from the umbilical vein, indicating continued placental release of -oh aa into the cord blood after delivery of the fetus. conclusion: labour at term is associated with changes in the placental metabolism of tryptophan resulting in the increased release of -oh aa into the fetal circulation with the potential for pro-oxidative and apoptotic effects in many tissues, including the brain. obstetrics, gynecology, and reproductive sciences, new brunswick, nj, usa; department of obstetrics and gynecology, mineola, ny, usa. objective: ischemic placental disease (preeclampsia, small for gestational age, sga and placental abruption) is a major contributor to pregnancy-related morbidity. although the placenta is considered a fetal organ, it is accepted that ischemic placental disease (ipd) can present clinically with either fetal or maternal manifestations. we hypothesized that the pattern of diagnosis (maternal versus fetal) varies by gestational age and would provide insights in to origins of indicated and spontaneous preterm birth. methods: this was a retrospective cohort study utilizing the maternallylinked reproductive history data for missouri residents , restricted to singleton live births. women who experienced spontaneous onset of labor and subsequently delivered preterm were classified as spontaneous preterm birth. medically indicated preterm birth included women who delivered preterm through a labor induction or (prelabor) cesarean delivery. ipd was classified as maternal (preeclampsia only), fetal (sga only) or both (preeclampsia with sga or abruption, and all conditions). results: among term births with ipd, . % presented as maternal disease only, . % as fetal disease, and the remainder ( . %) as both. among spontaneous preterm births with ipd, a greater proportion were of fetal presentation ( bethesda, md, usa; dept ob/gyn, wayne state univ, detroit, mi, usa; dept pathology, wayne state univ, detroit, mi, usa; dept ob/gyn, soroka univ medical center, israel. objective: hemoglobin (hg) and its catabolic products have been observed in cases of amniotic fluid (af) discoloration, which is a risk factor for intraamniotic infection/inflammation (iai). the study aimed to determine the association between af fetal hg concentration and gestational age, term and preterm labor and iai. study design: this cross-sectional study included: ) mid-trimester (n= ); ) term not in labor (tnl) (n= ); ) term in labor (tin) (n= ); ) preterm labor (ptl) who delivered at term (n= ); ) ptl without iai (n= ); ) ptl with iai (n= ); ) preterm prelabor rupture of membranes (pprom) with (n= ) and without iai (n= ). af fetal hg concentrations were determined by elisa. results: ) fetal hg was detected in . % of all af and . % of mid-trimester samples; ) women at tnl had a higher median af fetal hg concentration than patients at mid-trimester ( . ng/ml, iqr - vs . ng/ml, iqr . - . , p= . ); ) no differences were found in median af fetal hg concentration among patients with and without labor at term (til: . ng/ml, iqr - . ; p= . ); ) median af fetal hg concentration was not significantly different among the ptl subgroups [ptl with iai: . ng/ml, ptl who delivered preterm: . ng/ml, ptl without iai who delivered at term: . ng/ml, p= . (kruskal wallis) ]; ) in pprom, there were no differences among patients with and without iai ( . ng/ml, respectively; p= . ) ; ) median af fetal hg concentrations were significantly higher in ptl or pprom, with or without iai than in pregnant women at term, with and without labor (p< . for all comparisons). conclusions: ) immunoreactive af fetal hg increases with gestational age; ) among women with ptl or pprom, the median af fetal hg concentration is not associated with iai; ) the median af fetal hg concentration is higher in pregnancies complicated with ptl or pprom than in term pregnancies. the impact of latency time to delivery after preterm premature rupture of membranes (pprom) on neonatal outcome. dan nayot, deborah penava, barbra de vrijer, orlando da silva, bryan s richardson. obstetrics and gynaecology; pediatrics, university of western ontario, london, on, canada. objective: there continues to be controversy as to the management of pprom with conservative management to advance gestational age (ga) versus aggressive management with early induction to avoid chorioamnionitis. we have therefore used the perinatal and neonatal databases of a large regional patient population to determine the association of pregnancy variables with latency time to delivery after pprom and the impact of latency duration on adverse neonatal outcomes. methods: the perinatal/neonatal database of st. joseph's health care, london, ontario was used to obtain demographic and neonatal outcome information for all patients with pprom > and < weeks gestation, singleton and no major anomalies, delivering between january , and december , . patients were grouped according to ga at pprom stratified for latency time < hrs vs > hrs with incidences for those pregnancy related variables and neonatal outcomes available from the database then compared with the use of logistic regression analysis. results: there were patients who met the inclusion criteria of whom %, %, and % had pprom at - wks , at - wks, or at - wks, respectively, and with these pprom groupings showing a stepwise decrease in the percentage of patients with latency to delivery > hrs, at %, %, and %, respectively. pregnancy related variables and neonatal outcomes for these patient groupings are as shown in table . conclusion: despite a to fold increase in the incidence of chorioamnionitis with latency to delivery > hrs, a policy of conservative management to advance ga after pprom will result in decreased severe infant morbidity until weeks, and moderate infant morbidity until weeks. hospital. outcomes studied were prolongation of latency period with intact membranes and prom using magnesium sulfate (mgso ), nifedipine, terbutaline and tocolytic agents. secondary outcomes examined were maternal complications. statistical analysis performed were t-test and . comparisons were expressed as odds ratios ( % ci). results: in a -year period, twin gestations were admitted in ptl and administered tocolytic agent(s) with mean gestational age of . weeks. when tocolytic agents were used, maternal complications were: ( . %) with intact membranes and ( . %) with prom had pulmonary edema (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had chorioamnionitis (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had postpartum hemorrhage (or= . , ci . - . ). conclusion: there is no difference in prolongation of latency period achieved in twin gestations in ptl with intact membranes or prom using tocolytic agents. similarly, there is no difference between the groups with latency period hrs. there is an increased likelihood of pulmonary edema and chorioamnionitis with use of tocolytic agents. however, results are not significant due to type ii error. mean latency period and latency ≥ hrs using single or multiple tocolytic agent ( introduction: high sensitivity crp (hscrp) is a serum marker of inflammation and has proven clinical utlility in predicting cardiovascular disease (cvd). considering the hypothesized association between preeclampsia (pre) and inflammation and cvd, it is plausible that hscrp may have utility in predicting pre. prior to widespread utilization of this marker, the affect of labor on levels of crp needs to be clarified. we assessed the association between labor and hscrp levels in term deliveries and between elevated hscrp and adverse perinatal outcomes. a secondary analysis comparing hscrp in women with preeclampsia (pre) to those without was performed. methods: women presenting for term delivery or pre were prospectively identified as part of a case-control study. clinical data and serum were collected for all subjects. a standard immunoturbidimetric assay was used to measure hscrp levels. women presenting for induction of labor or planned cesarean delivery (non-labor) were compared to women presenting in labor. a secondary analysis comparing non labor women with and without pre was performed. serum was collected prior to labor induction in the non-labor group. nonparametric comparisons were made using wilcoxon rank sum tests. mvlr was used to evaluate dichotomous outcomes and control for confounders. results: women were included (non-labor group (n= ), labor group (n= ), non-labor pre (n= )). the median and mean hscrp levels were and and and in the non-labor and labor groups respectively (p= . ). elevated levels of hscrp in these term deliveries were not associated with chorioamnionitis (p= . ), maternal postpartum complications (endometritis, hemorrhage, transfusion) (p= . ), mode of delivery (p= . ), or admission to the nicu ( . ). levels of hscrp were significantly greater in the non-labor pre group compared to non labor without pre (mean . vs. . , median vs. , p< . ). conclusion: use of hscrp as a biomarker may improve clinical prediction of obstetrical complications such as pre. levels of hscrp are affected by labor and this should be taken into account when studying the utility of this biomarker. further, crp levels are elevated in women with pre even after excluding patients in labor. further investigations to determine if crp elevation in term labor is associated with adverse outcomes may be warranted. the role of hscrp as a valid and discriminating biomarker in pre should be assessed. review of the electronic labor record facilitated collection of maternal demographic, intrapartum, and newborn data. data analyzed with t-tests, chi-square tests, and calculated odds ratios with % cis as appropriate. a forward stepwise logistic regression analysis was used to identify predictors of histologic chorioamnionitis. results: of submitted placentas, had histologic chorioamnionitis (cases) and did not (controls). the groups were similar with respect to age, race, gbs status, and mode of delivery. gestational age, birthweight, duration of labor and ruptured membranes, and number of vaginal exams were greater in the cases (p . ). the cases were more likely to have had epidural anesthesia (or . ), internal monitoring (or . ), fever (or . ), maternal tachycardia (or . ) and fetal tachycardia (or . ) and less likely to have had induction of labor (or . ). newborns in the histologic chorioamnionitis group were more likely to have been observed for sepsis (or . severe sepsis defined as sepsis associated with acute respiratory distress syndrome (ards) or cardiovascular dysfunction(cvd) or with or more other organ dysfunction.all patients were resuscitated with fluids and treated with broad spectrum antibiotics and supportive care as needed. outcome data were: etiology, management, maternal complications, duration of icu stay and perinatal survival. results patients were young (mean age= . ± . years) with a mean gestational age at delivery . ± . weeks. etiologies were pyelonephritis(n= ), septic abortion(n= ), endomyometritis (n= ), chorioamnionitis (n= ), ruptured appendix(n= ), pneumonia( n= ) and one unknown. eighteen ( %) were diagnosed during antepartum and ( %) postpartum period.there were ( %) maternal deaths and high rate of major morbidities (table) . among the antepartum patients,there were, abortions, iufd, neonatal death for a perinatal survival rate of only %. conclusion pregnancies complicated with severe sepsis/septic shock are associated with substantial maternal and perinatal morbidities. the low maternal mortality rate in our study as compared to previous reports is attributed to early diagnosis and aggressive management of maternal complications. fetal loss rate, however continues to be high when septic shock develops antepartum. and tnfa levels are elevated. we developed a dynamic computer (in silico) model of pregnancy (uterine myometrial environment -infection/inflammation and endocrine crosstalk). mathematical differential equations were used to describe the interactions between molecules. infection was represented as increased levels of activated, nuclear transcription factor nf-kb. in the model ru inhibited both the glucocorticoid receptor and progesterone receptors. simulations were run adding different concentrations of ru ( . um= dose used in patients, . um, . um) at different time points during infection (before, at the time of or after nf-kb activation). ru degradation kinetics was also included. the effect of ru on nf-kb induced il- and tnfa levels was assessed. results: infection induced nf-kb activation led to increased il- and tnfa levels. there was a subsequent increase in cortisol that led to dampening of nf-kb activation, il- and tnfa levels. in the presence of ru , il- and tnfa levels continued to rise. the effect of ru on nf-kb induced il- and tnfa was dose dependent and was more prominent in slow metabolizers who had ru in the system for a longer time. addition of ru after the onset of nf-kb activation led to increased il- and tnfa levels above those observed without ru . the addition of misoprostol (prostaglandin e analogue), at the concentrations used together with ru for medical abortion, did not add to the effect of ru on nf-kb induced il- or tnf. conclusions: ru has dose dependent effects on infection induced immune activation and may contribute to the pathogenesis of c sordelii induced sepsis syndrome. the increased susceptibility of neonates to infection remains a major clinical problem. we previously found that after intraperitoneal (ip) listeria monocytogenes infection, neonatal mice have an ld that is orders of magnitude lower than adults. we also found that the inflammatory response of neonatal mouse macrophages, but not neutrophils, in the peritoneal fluid was deficient, and that this correlated with low levels of macrophage chemokines mcp- and rantes. given that the liver and spleen are important organs in listeria infection, we sought to characterize the innate immune response in these organs. a sublethal dose of listeria was injected intraperitoneally into balb/c - week old adult mice and - day old neonatal mice. liver and spleen was collected at , , and hours, sectioned serially for staining with hematoxylin-eosin and primary antibodies (rabbit anti-listeria monocytogenes igg; mhc class ii rat anti-mouse igg, a marker for activated macrophages; f / rat antimouse igg, a marker for macrophages) and secondary antibodies (cy- goat anti-rabbit igg; af goat anti-rat igg) were applied. negative controls used only secondary antibody. real time pcr was used to compare the levels of the chemokines mcp- and rantes in adult and neonatal liver. after ip infection, both adults and neonates showed similar influx of neutrophils to the sites of infection within the liver and spleen. at hours post-infection with listeria, adult liver and spleen showed increased staining for f / and class ii, which increased further and became confluent surrounding microabscesses at and hours. in contrast, the listeria infected neonatal mouse showed some increase in f / around microabscesses but no apparent increase in staining for mhc class ii. the neonates showed greater staining for listeria at each time point. mcp- and rantes levels were higher in infected neonatal liver compared to adults. in the neonatal mouse, the innate immune response in the liver and spleen was characterized by a deficiency of activated macrophages. this deficiency was not correlated with hepatic expression of macrophage chemokines. is there a seasonal pattern in the incidence of post-cesarean endometritis? tamula m patterson, alan tn tita, william w andrews. obstetrics and gynecology, the university of alabama at birmingham, birmingham, al, usa. objective: several theories, including one suggesting a peak in july coincident with resident turnover, postulate seasonality in post-cesarean infections. we assessed whether there is seasonal variation in endometritis. a retrospective cohort study of post-cesarean endometritis at our university-based institution using our obstetric computerized database to compare annual variation in monthly incidence patterns from to . prior to establishing an average aggregate seasonal pattern for all years, years were assessed separately for a recurrent pattern of peaks and nadirs in incidence. peak incidence (or nadir) was defined as any monthly incidence that differed from the mean incidence for the year by over %. results: a total of . % ( , ) of , deliveries from to were by cesarean. annual cesarean rates increased by an absolute rate of over %; while, post-cesarean endometritis rates decreased from % to %. monthly incidence of post-cesarean endometritis did not reveal a consistent recurrent pattern of peaks (figure ) or nadirs. the month of july accounted for only out of a total of peaks for all years. the adjacent months of june and august accounted for only each. the month with the highest number of peaks was april with only . these findings contraindicated the establishment of an average aggregate monthly pattern for all years. the incidence of post-cesarean endometritis did not follow a seasonal pattern. chi-square analyses were used to compare associations between race (black (bl) vs non-black (nbl)) and dichotomous characteristics. student´s t-test was used to compare continuous variables. results , patients were evaluated ( cases and controls). % and % of cases and % and % of the controls were bl and nbl respectively. the baseline prevalence of chtn in bl and nbl controls was . % and . % (p= . ). when comparing bl and nbl cases, bl women had a higher mean systolic blood pressure and screening bmi. bl women also had a trend toward being discharged on post partum blood pressure medicine when compared to nbl women. there was no difference in chtn, diabetes, severity of disease, iugr, or delivery < wks between the two groups (table) . to determine the leading causes of death in a case series of stillborn infants examined in a large hospital autopsy service, and to describe the most common post-mortem observations. study design: one hundred and sixty one stillborn infants were examined. gross pathology observations were recorded at autopsy and during the placental exam, and tissue sections were collected and examined microscopically. immediate and underlying cause of death (cod) were recorded, along with contributory cod, concomitant/significant cod, and incidental findings. statistical analysis was conducted using the software spss v. . . results: the immediate anatomic cod could be determined in . % of all infants examined. in over % of these cases, cod was attributable to placental or umbilical cord findings affecting the maternal-fetal blood supply. the most prevalent among these findings were placental lesions (maternal floor infarction, placental abruption, fetal thrombotic vasculopathy), umbilical cord lesions (entanglement, true knot, compression, excessive length/twisting), and infectious/inflammatory processes (chorioamnionitis, chronic villitis objective: advanced maternal age (ama) is associated with increased risk of intrauterine fetal demise (iufd). antenatal testing (at) is widely used in clinical practice to prevent iufd due to uteroplacental insufficiency and has been suggested to reduce the risk of iufd in ama women. we sought to assess the impact of at on obstetrical interventions and compliance with new practice recommendations. methods: retrospective cohort of ama women ( and older at their due date) who delivered at or after weeks. non-exposed women delivered from july to december (when at for ama was not routinely recommended); exposed women delivered from july to december (after at for ama was introduced at our institution). subjects were identified through the perinatal database; records were abstracted for demographics, medical history, and labor/delivery variables. outcomes included rates of at and induction of labor (iol) and mode of delivery. associations between at for ama and outcomes were tested using t test and chi square. assuming a baseline rate of iol of %, we had % power to detect an increase to % after the introduction of at. results: women met the inclusion criteria: delivered before the introduction of at (non-exposed=before at) and delivered after the introduction of at (exposed=after at). baseline clinical characteristics were similar in both groups. as anticipated, at was more common in the after at group than in the before at group ( % vs %; p< . ). women were not eligible for or declined trial of labor, thus not "at risk" for iol and not included in all analyses. ob intervention rates were increased after at compared to before at (table ) . the corrected iufd rates were similar in both groups ( / vs / ). conclusion: at an academic center, compliance with new practice recommendations was excellent. introducing at testing for a new indication seemed to increase iol and cs rates. these findings should be considered when assessing the risks:benefits ratio of antenatal testing. . we stratified indications for cesarean into the following: failed induction (cervix < cm dilated), arrest of dilation, arrest of descent, failed operative delivery, fetal intolerance of labor (fil), and "other" reasons (e.g., pre-eclampsia, abruption, chorioamnionitis, malpresentation). both fil and "other" reasons were more likely to occur among the medically indicated group (rr . , . physicians in solo practice had higher rates of elective inductions (p< . ), but there was no association between cesarean and practice type. conclusions: inductions accounted for . % of cesareans. these results suggest an increased risk for cs for patients undergoing medically indicated inductions at our institution. there was no association between cesarean and type of practice, whether solo or group, suggesting institutional clinical policies may be more important than practice type in determining delivery outcome after induction. further research is needed to understand how age, race/ethnicity, or other unmeasured patient factors may impact these findings. given rising rates of both cesarean delivery and inductions, this information may be pertinent to women considering elective induction prior to weeks. objective: fetal demise can lead to a consumptive coagulophathy ("fetal death syndrome") traditionally attributed to the release of "tissue thromboplastin", now known as "tissue factor" (tf). tf is the most potent activator of coagulation. despite the appeal and acceptance of this proposed pathophysiology, there is no evidence supporting this view. this study was undertaken to determine if fetal death prior to development of fetal death syndrome is associated with changes in maternal plasma concentration of cd l (a marker of platelet activation), tf and its soluble inhibitor (tfpi). methods: a cross-sectional study included the following groups: ) women with normal pregnancy (n= ) and ) patients with fetal demise without disseminated intravascular coagulation (n= ). plasma concentrations of scd l, tf and tfpi were measured by elisa. standard coagulation tests were performed. non-parametric statistics were used for analysis. results: ) patients with fetal demise had a higher median maternal plasma scd l concentration than women with normal pregnancy (median . pg/ml, range - vs. median . pg/ml, range . - . , p< . ); ) there was no significant difference between the groups in the median maternal plasma tf concentration and ) in contrast, the median maternal plasma tfpi concentration was significantly lower in patients with fetal demise than in women with normal pregnancy (median . ng/ml, range . - . vs. median . ng/ml, range . - . , p< . ). conclusions: ) a change in the plasma concentration of tf was not demonstrated; ) a change in the ratio of tf/tf inhibitor pathway may predispose to thrombin generation and activation of the coagulation cascade; ) however, maternal platelet activation is present in patients with a fetal demise without fetal death syndrome and ) the role of tf in fetal death syndrome remains to be proven. lipoic acid inhibits matrix metalloproteinase production, activity and prostaglandin e secretion by cultured amnion epithelial and mesenchymal cells. r moore, j novak, d kumar, j moore. case western reserve university, cleveland, oh, usa. introduction: cytokines, free radicals, matrix metalloproteinases (mmp) and prostaglandins (pg) have been implicated in processes of fetal membrane rupture and labor. dietary anti-oxidant supplementation has been suggested as a possible therapy for high risk patients, however, clincal evidence supporting the efficacy of agents such as vitamin c or n-acetylcysteine remains controversial. in fact, we have previously shown that vitamin c increases matrix metalloproteinase (mmp) activity in isolated fetal membrane fragments and fails to inhibit tumor necrosis factor (tnf) induced fetal membrane weakening in vitro. in this study, we examine the effect of the naturally occurring anti-oxidant, -lipoic acid, on tnf induced mmp activity/protein and pge secretion in isolated amnion epithelial and mesenchymal cells. methods: amnion epithelial and mesenchymal cells were pre-treated with increasing doses of -lipoic acid ( - mm/ h), then with increasing doses of tnf ( - ng/ml/ h). medium and cells were analyzed by gelatin zymography/western blotting for mmp /mmp activities/protein. pge output was determined by immunoassay. results: tnf induced a dose dependent increase in mmp production, secretion and activity in amnion epithelial cells. tnf ( ng/ml) induced an fold increase in cellular active mmp production and fold increase in secreted mmp enzyme activity by amnion epithelial cells. these increases were reduced - % following h pre-treatment with . - . mm -lipoic acid. mmp protein/activity and pge secretion by amnion epithelial cells were barely detectable and unaffected by tnf and/or -lipoic acid treatment. in striking contrast, mesenchymal cells exhibited little basal or tnf induced mmp protein/activity. mmp protein/activity in mesenchymal cells were unaffected by either tnf and/or -lipoic acid. however, tnf treated mesemchymal cells exhibited a dose dependent increase in pge production ( fold increase/ ng/ml tnf/ h) that was inhibited by %- % following . objective: nearly fifty years after the discovery of microphthalmia-associated transcription factor (mitf), its gene was identified as a specialized transcription factor that dictates cell-specific differentiation. unique mitf isoforms are generated from alternative promoter usage. an isoform of mitf (mitf-cx) is down-regulated in cervical stromal cells of the ripened cervix. further, mitf-cx inhibits il- gene expression and thereby suppresses signaling of the final pathway in cervical ripening. since mitf binds to canonical eboxes (canntg) in promoter regions of target genes, we sought to determine if mitf regulated its own promoter through ebox motifs. methods: the kb genomic dna sequence upstream of the mitf-cx transcription start site was cloned into pgl luciferase reporter vectors which were co-transfected with wild type or mutmitf-cx (impaired dna binding) into cervical stromal cells or hek cells. at h, promoter activity was determined and normalized for transfection efficiency. results: gel-shift assays conducted with oligonucleotides corresponding to eboxes in the mitf-cx promoter revealed two strong binding sites (eboxes - to - and - to - ). specific binding was established using oligonucleotides with or without mutated ebox, antibody supershift experiments, competition with cold probe, and absence of binding to mutmitf-cx. binding specificities were confirmed in nuclear extracts from cells that overexpressed mitf-cx, but not control or mutmitf-cx. reporter gene studies indicated that mitf-cx, but not mitf-m, increased mitf-cx promoter activity -to -fold. whereas mutations in ebox or resulted in significant decreases in mitf-stimulated promoter activity, mitfinduced increases in promoter activity were abolished by mutations in both eboxes. conclusions: collectively these experiments indicate that mitf-cx is a vital regulator of its own promoter activity and acts in a positive feedforward loop through two specific binding sites in its promoter. moreover, isoform-specific amino acids are important to mediate mitf-induced mitf-cx promoter activity. decreasing mitf protein or mutating its promoter would interrupt this loop resulting in rapid reduction of mitf synthesis. since mitf-cx suppresses il- gene expression in cervical stromal cells, we suggest that preservation of mitf-cx-induced mitf gene expression is an important mechanism to maintain the "brake" on cervical ripening and ensure cervical competency during pregnancy. the role of cd in cervical remodeling. denisse sanchez, brenda timmons, mala mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa. objective: prior to the onset of parturition, the uterine cervix undergoes a remodeling process from a closed, rigid structure, to one that is soft and dilatable. many changes occur, including increases in hyaluronan (ha), a glycosaminoglycan that facilitates loosening of the collagen matrix. in the postpartum period, the concentration of ha is reduced to that of the nonpregnant state. cd , a transmembrane glycoprotein expressed in hematopoietic and epithelial cells, is a receptor for ha. cd expression by immune cells is important in extravasation of leukocytes into tissue. cd may also be required for ha catabolism through the action of hyaluronidases and . in the cervix, cd is expressed in the endo-cervical epithelia as well as in immune cells localized in the stromal matrix. to study the importance of cd in cervical remodeling, mice with a null mutation for cd (cd -/-) were evaluated during pregnancy, parturition and postpartum. methods: changes in ha amount and size distribution were assessed using ha molecular weight gels and fluorophore assisted carbohydrate electrophoresis. to identify defects in cervical remodeling in the cd -/mice, differences in expression for genes regulated in the cervix were studied by quantitative real time pcr . to determine whether cd plays a role in the recruitment of immune cells during cervical ripening and postpartum repair, immunohistochemistry with antibodies against leukocytes was done. in the postpartum period, there is a higher ratio of high molecular weight ha relative to low molecular weight ha in the cd -/mice. this suggests that postpartum breakdown of ha in cd -/cervices is delayed. as compared to wt cervix, there was a significant increase in hyaluronidase (hyal- ) mrna in the postpartum period. the distribution and relative numbers of immune cells in the cd -/cervix was similar to wt. conclusion: these studies provide evidence that cd may play a role in remodeling of the postpartum cervix back to the nonpregnant state. our current data suggests that the catabolism of ha after birth is delayed in the mutant mice and upregulation of hyal may compensate to allow ha removal. furthermore, the activity of hyal- may be dependent on cd . little difference in the recruitment of immune cells between cd and wt animals suggest that cd expression is not required for this process. these experiments provide a greater understanding for the role of cd and ha in cervical remodeling. in background: during in vitro experiments we have shown that separation of amnion from choriodecidua occurs as an integral part of the process of fetal membrane (fm) rupture. although spontaneous amnion and choriodecidual separation is seen in fm after both svd and elective c/s deliveries, its etiology is uncertain. biochemical degradation at the amnion-choriodecidua interface may be a key contributing factor. our previous biomechanical studies have demonstrated that separated fm require less physical work to rupture than intact membranes. the purpose of this study was to determine whether fm separation was associated with clinical differences in the birth process. hypothesis: during term, normal labor, spontaneous separation of fm is associated with differences in the clinical parameters of labor and delivery. study design: fm from consecutive term deliveries were cut off the placental disk. separated areas of fm were cut from the intact areas. both were weighed and their weight ratios determined. maternal medical, pregnancy, and delivery data were collected and analyzed. results: term fm had the following characteristics: maternal age ± . yr, gravida . ± . , gestation ± . wks, elec. cs . %, duration of rom ± min, duration of contractions ± min, african american %. % of the fm had < % separation; % had more than % separation. srom fm with > % separation (vs. < %) had significantly shorter duration of rom (p= . ) and admission to birth (p= . ) times. srom fm with > % separation (vs. < %) had even shorter rom (p= . ), duration of contractions (p= . ) and admission to birth (p= . ). the > % group (vs. < %) was further along, gestationally (p= . ). srom fm (vs. arom) had shorter admission to birth (p= . ), but longer rom to birth (p< . ) times. absence of epidural (p= . ), srom mode of rupture (p= . ), svd (vs. elec. c/s) (p= . ), and the presence of meconium (p= . ), were all associated with increased fm separation. conclusion: spontaneous separation of fetal membranes is nearly universal and is associated with increased gestation, spontaneous rupture of membranes, shorter duration of contractions, and svd. speculation: we speculate that programmed biochemical changes initiate fm separation which then facilitates rom and childbirth. whole genome array and si-rna investigation of the function of nfkb in human amnion epithelial cells. sheri e lim, shirin khanjani, yun s lee, tg teoh, , philip r bennett. institute of reproductive and developmental biology, imperial college, london, united kingdom; maternal fetal medicine, st. mary's hospital, london, united kingdom. introduction: labour is associated with activation of nfkappab in the amnion. nfkappab increases prostaglandin synthesis through the upregulation of cyclooxygenase- (cox- ), which is essential to the labour process. cox- mrna expression increases with gestation in the amnion. primary amnion epithelial cells cultivated from tissue collected prior to the onset of labour display a spectrum of nfkappab activation, similar to the spectrum of cox- expression presumably relating to the nearness of labour. our aim was to investigate the full range of genes under nfkappab control in amnion epithelial cells by using whole genome arrays. methods: amnion from women undergoing elective caesarean section was collected and primary cell cultures established. total rna and protein were extracted from each culture. nuclear localization of nfkappab is required for its activation. western analysis of nuclear p was therefore performed to identify the samples displaying the lowest and highest nfkappab activity. the corresponding rna samples displaying the three lowest and three highest nuclear p protein concentrations were used for whole genome analysis using affymetrix u arrays. results and conclusions: we identified significantly regulated genes. the gene with the highest fold change was cox- (x . ) followed by oxytocin receptor (x . ), ch orf (x . ), integrina (x . ), and connective tissue growth factor (x . ). other significant genes included interleukin- (il- ) (x . ). pathway analysis revealed the majority of other nfkappab associated genes were involved in cell signaling, turnover and proliferation. we used real-time pcr (rtq-pcr) to validate cox- and il- expression. to prove that cox- is directly regulated by nfkappab, primary amnion epithelial cells were then transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna pool. western blot analysis confirmed knockdown of nfkappab p associated with inhibition of cox- demonstrating that nfkappab is essential for cox- expression. objective: premature birth is a major public problem accounting for over , deaths and , surviving infants with life-long morbidity yearly. in order to develop a rational basis for treatment and prevention of premature fetal membrane (fm) failure, we first need to understand the sub-failure fm structural and mechanical behavior at near full term. methods: we utilized planar biaxial mechanical testing, which approximates the physiologic loading state, for mechanical evaluation of the fm, and a structural constitutive model approach was used to offer insight into the structure-strength of the fm by integrating information on tissue composition and structure. small angle light scattering (sals) was used to nondestructively quantify the collagen fiber architecture of both intact and separated fm layers. results: in the stress free state, the gross collagen fiber architecture of the fm and separated layers were not homogenously align but exhibited small regions of fiber alignment. the amnion layer displayed the greatest alignment. the model fit the equi-biaxial strain data well (r = . ) and indicated that fm collagen fibers were rapidly recruited and straightened well below failure stress levels. collagen fibers were gradually recruited followed by a drastic increase in fiber recruitment. conclusion: this study provided the first data on the effective collagen fiber stiffness in the intact fm under physiologic biaxial loading, which was related to quantitative collagen fiber architectural measures. modeling results indicated that the collagen fibers became fully loaded and straighten well below physiological loading levels. failure did not occur during physiological loading, indicating that fibers do not begin to fail until all collagen fibers are fully straightened and bearing load. this result suggested modest structural reserve in the fm collagen architecture, and may be an important aspect of its failure properties. previously, we demonstrated that a physically "weak zone" exists overlying the cervix in the fm, evident of collagen remodeling and cellular apoptosis. we are currently extending the present study to include the "weak zone" tissues, allowing us to elucidate the micro-mechanical mechanisms that facilitate failure in this newly identified fm zone. supported by nih . the extracellular matrix of the cervix undergoes extensive remodeling during parturition. hyaluronan (ha) is a major constituent of the extracellular matrix of the term pregnant cervix. the onset of labor is preceded by an increase in ha and after delivery, the concentration of cervical ha gradually decreases to that of the non-pregnant state. these dramatic changes suggest that ha plays an important role during parturition. hyaluronan synthase (has ) is one of three known ha synthases and the most abundant isoform in the pregnant cervix. transcripts for has are regulated by two alternative promoters, one upstream of the first coding exon (proximal promoter) and another upstream on an untranslated exon (distal promoter). the focus of the current study is to further our understanding of the transcriptional regulation of has during cervical ripening. methods: rna blotting was carried out using transcript specific probes corresponding to the distal and proximal promoter of the mouse has gene. the regulation of has was evaluated in a cervical epithelial cancer cell line (caski cells) which we have previously shown to express endogenous has . regulation of has expression by epidermal growth factor was assessed in the caski cells by western blotting and quantitative real time pcr assessment of transcripts. results: has mrnas in the nonpregnant (np) and pregnant cervix are transcribed from the distal promoter upstream of exon . two transcripts of approximately . kb and . kb were detected that arise from use of polyadenylation sequences. as compared to np, the expression of has is increased , , and fold on gestation days , and shortly postpartum respectively. has mrna expression is increased upon treatment of caski cells with epidermal growth factor ( ng/ml) and is suppressed in cells treated with ag ( m), an inhibitor of egf receptor phosphorylation. maximal stimulation was observed at and hours of treatment. conclusion: has is the major ha synthase expressed during cervical ripening and the majority of transcripts are driven by the distal promoter in the has gene. in vitro studies using caski cells suggest has is regulated in part by the egf signaling pathway resulting in a several fold increase in has expression. these results provide an understanding of has gene regulation at the time of cervical ripening which will ultimately enhance our understanding of the molecular mechanisms important to cervical ripening. chorion and decidua cells. chad a grotegut, bernard j canzoneri, liping feng, phil heine, amy p murtha. obstetrics and gynecology, duke university, durham, nc, usa. objective: preterm premature rupture of the fetal membranes accounts for approximately % of all preterm deliveries. cigarette smoking independently carries a fourfold increase risk for pprom. our laboratory has previously demonstrated that the chorion layer undergoes apoptosis in women with pprom. this study was conducted to determine if extract of cigarette smoke causes cell death in specific cells of the fetal membrane. fetal membranes were collected at the time of elective cesarean section from women without labor and at term. the chorion and decidua layers were separated and purified on a gradient spin column and then plated near confluence. cigarette smoke extract (cse) was collected in cell media and used to treat chorion and decidua cells in culture at concentrations ranging from % to % in -well plates. cell viability was determined at , and hours following treatment with a non-radioactive cell viability assay. data were analyzed using paired t test (analyse-it, leeds, uk). chorion and decidua cells underwent cell death when exposed to cse in a dose dependent fashion. increasing concentrations of cse resulted in increased cell death at hours in both cell types (figure ). at hours, chorion exhibited greater percent cell death compared to decidua at concentrations of , and % cse (p= . , . , and . , respectively). for any given concentration of cse, the degree of cell death increased with increasing length of exposure ( , and hours) for each cell type. chorion cells routinely exhibited greater percentage of cell death following treatment with cse at concentrations ranging from - % compared to decidua cells. human chorion and decidua cells in primary cell culture exhibit a dose-response and time dependent cell death in the presence of cse. human chorion cells show greater sensitivity to cell death when compared to decidua cells. further studies are needed to determine the mechanisms through which these cell types undergo cell death and the implications for the differential sensitivity to cigarette smoke. yoon ha kim, tae-bok song, cheol hong kim, jong woon kim, moon kyoung cho, sung yeul yang, bong whan ahn. obstetrics gynecology, chonnam national university medical school, gwangju, korea; biochemistry, chonnam national university medical school, gwangju, korea. objective: to investigate the lipid peroxide levels and protein carbonyls levels in the amniotic fluid of pregnant women with preterm premature rupture of membranes (pprom). the lipid peroxide levels in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced measured by thiobarbituric acid reaction. the protein carbonyl contents in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were determined by the , -dinitrophenylhydrazine method. after amniotic fluid of them were mixed and incubated up to hours with . ml of mm moxalactam, cefodizime, amoxacillin, erythromycin, the lipid peroxide levels and protein carbonyl contents in them were measured. results: . the lipid peroxide levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein, p< . ). . the protein carbonyl levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein p< . ). . the lipid peroxide levels and protein carbonyls formation by moxalactam in the amniotic fluid of pregnant women with pprom was significantly higher than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . the lipid peroxide levels and protein carbonyls formation by cefodizime in the amniotic fluid of pregnant women with pprom was significantly lower than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . there were no significant differences in the levels of lipid peroxide and protein carbonyls by amoxacillin and erythromycin in the amniotic fluid of pregnant women with pprom between antibiotics-induced and basal levels. background: chorioamnionitis (cam) is a major antecedent of preterm delivery (ptd) associated with elevated amniotic fluid tnf and il . we hypothesized that these cytokines enhance the term decidual cell (dc) expression of the matrix metalloproteinases (mmp) and , which can then promote ptd by degrading the extracellular matrix of the decidua, fetal membranes, and cervix. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on cam-complicated (n= ) and gestational age-matched control decidua (n= ), and staining intensities were evaluated by hscore. confluent, leukocyte-free term dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ), and quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in cam-complicated decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in control decidua ( ± , and ± respectively; p< . ). in cultured term dcs incubated with e , tnf and il significantly increased secreted levels of mmp- compared to e alone (pg/ml/ g protein: . ± . and . ± . , respectively, vs. . ± . ; p< . ). in parallel incubations with e +mpa, basal mmp- output was lowered by %, and tnf -and il -elicited mmp levels were blunted by % and %, respectively. rt-pcr confirmed that tnf and il increased mmp mrna levels (p< . ), although mrna levels in e +mpa incubations were not different from those of e alone. mmp levels in all treatments were similar. conclusions: mmp- and mmp- are elevated in cam decidua compared to controls. our in vitro results suggest that mmp- expression is enhanced by the high levels of il and tnf associated with cam, and that mpa may be able to blunt this effect. we have previously found a similar regulatory mechanism of mmp- and mmp- and their over-expression in cam-complicated tissues. synergy among these mmps may represent a potent pathogenic mechanism of cam that can be targeted through the therapeutic use of progestins in preventing cam-induced ptd. domerudee preechapornprasert, patama promsonthi, wasun chantratita, mana rochanawutanon, patcharee karnsombut, chutatip srichunrusami, stephen j lye, boonsri chanrachakul. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: cervical ripening is an inflammatory process involving chemokines, cytokines and various mediators. recent study has shown that the level of monocyte chemotactic protein (mcp) , a chemokine, increases in amniotic fluid during spontaneous labor. the aim of this study was to examine the localization and expression of mcp in human cervix before pregnancy, during pregnancy and after the onset of labor. methods: this study was approved by the local ethics committee and written informed consent was obtained from each participant. cervical biopsies were taken from groups of women; nonpregnant women, first trimester pregnant women, term pregnant women with and without labor. tissue samples were fixed in % formal saline for paraffin section. immunohistochemistry (n = each) was performed by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . the mcp messenger(m) rna was identified by reverse transcription-polymerase chain reaction using gene specific primer against mcp and mcp receptor (n = each). results: immunohistochemistry demonstrated mcp in cervical tissues from all four groups of women. mcp was localized on plasma membrane and cytoplasm of both squamous epithelial and columnar cell lining of endocervical gland. mcp and mcp receptor mrna were identified in nonpregnant, first trimester and term with and without labor human cervix. conclusion: mcp and mcp receptor were located in cervical tissues of nonpregnant and pregnant women at different gestation both before and after the onset of labor.ongoing studies are investigating the role of this chemokine during pregnancy and labor. whether preterm cervical ripening is just an aberrant regulation in timing or whether divergent mechanisms and pathways are involved in preterm versus term cervical ripening remains to be elucidated. methods: cervical tissue was collected from groups of cd- mice. group : mouse model of ptb that utilizes intrauterine infusion of lipopolysaccharide (lps). group : e dams representing preterm controls. group : e . - dams selected from a timed pregnant batch where half of the dams had delivered representing term cervical ripening. n= dams/treatment group. separate rna samples were used for microarray analysis (ma). significance analysis for ma and partek software was used for biostatistical analysis. pathway analysis was performed using david. quantitative pcr was performed to confirm the most differentially regulated genes. results: using a cut-off of -fold change with p value of < . , genes in the cervix were differently regulated between the groups. principal component analysis revealed three distinct groups (see graph). functional annotation clustering demonstrated the following pathways: ) in preterm cervical ripening (e lps vs e ): immune and inflammation response, defense response ) in term cervical ripening compared to preterm controls: negative regulation of cellular process and biological process, ecm, cell-cell communication. qprc confirmed the highly significant differences found in ma (see table) . conclusions: the molecular mechanisms and pathways governing preterm and term cervical ripening are distinctly different. elucidating these unique pathways can lead to improved therapeutics for prevention of ptb as well as for postdate pregnancies. cervical ripening at term involves activation of apoptotic enzymes. maria kb sennstrom, valentina ciani, gunvor e ekman. obstetrics and gynecology, women and child health, karolinska institute, stockholm, sweden; obstetric and gynecology, university of siena, siena, italy. aim: to investigate if the human cervical ripening at term involves programmed cell death. during the final cervical ripening the extracellular matrix dominated cervix undergoes an extensive remodelling of the tissue. inflammatory mediators such as the cytokines increase in ripening cervical tissue at term. programmed cell death, apoptosis, has been suggested as important in this process. apoptosis can be induced by inflammatory mediators such as cytokines. we also looked upon the distribution of inflammatory cells in cervical tissue. materials and methods: cervical biopsies from pregnant women at term and post partum women with fully ripened cervix were studied. biopsies from non-pregnant women served as controls. immunohistochemical analysis of the apoptotic enzyme caspase- and the inflammatory cell marker cd was performed on paraffin embedded sections of cervical tissue. double staining was performed. mann-whitney u-test was used for statistical analysis. results: there was a significantly higher frequency of caspase- staining in the post partal sections from ripened cervical tissue compared to tissue from term pregnant (p= . ) with unripe cervix and from non-pregnant patients (p= . ). the inflammatory cells staining for cd increased in post partal and term pregnant sections compared to non-pregnant (p= . ). there was a higher frequency of caspase- positive cells in the post partal tissue than of cd positive cells (p= . ). the localization of cd- positive staining was highest in the epithelia and basal lamina while caspase- staining was most pronounced in stromal tissue and around vessels. conclusion: our data show apoptotic activity in stromal tissue in fully ripened human cervix at term of pregnancy, suggesting that apoptotic mechanisms are involved in the extracellular matrix remodelling at term. the apoptotic activity is not co-localized with inflammatory cells suggesting non-infectous inflammation with apoptosis as important for cervical ripening at term. objective: cervical biomechanical responses are important for accommodating the increased stress induced by an enlarging uterus. a mechanical testing system was modified to evaluate the stress relaxation response in the pregnant cervix. methods: tissue harvested from the non pregnant and timed pregnant (days , , , , and ) sprague-dawley rats underwent tensile testing using an instron material testing system. the testing regimen consisted of tissue extension to near maximal strain over seconds followed by a period of constant strain for minutes, then return to rest over seconds. this cycle was repeated additional times with a minute rest period between cycles. strain and force measurements were recorded at second intervals. - animals were used for each time point. in addition, stress and strain at the yield point was also determined for each gestational time point. results: the pregnant and non pregnant samples exhibit marked differences in response in both stress and strain. the timed pregnant tissue demonstrated progressively increased compliance and lower nominal stress compared to non pregnant tissue. peak nominal stress declined with each successive cycle. this was demonstrated in the peak nominal stress and strain values at the yield point. conclusion: the cervix becomes more distensible (compliant) but less resistant to force with increasing gestational age. ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • introduction: a growing body of evidence supports that inflammatory processes are implicated in spontaneous preterm birth (ptb). using an inflammatory and non-inflammatory mouse model of ptb, we sought to determine if activation of these inflammatory pathways are essential for ptb and/or cervical ripening to occur. methods: timed pregnant cd- mice were used in these two models of ptb: ) a model of intrauterine inflammation where lipopolysaccharide (lps) is injected into the uterine horn (n= ); controls for this model received intrauterine saline (n= ) and ) a non-infectious model of ptb using ru sq ( ugrams/dam) (n= ); controls for this model received no intervention (n= ) were used for these studies. for both models, hours later uterine and cervical tissues were harvested. the tissues were processed for protein and rna studies. elisas were performed to assess il- and tnf-alpha in the uterine tissue. mrna expression of ifn , il- , il- , il- , tnf-alpha were assessed in cervical tissue from both models by quantitative pcr. results: in uterine tissues, both il- and tnf were significantly elevated in the lps-induced ptb when compared to control, saline, and non-infectiousinduced preterm birth (p< . ) (figure ). in cervical tissue, an increase in il- , il- beta and tnf mrnawas observed in both models, while ifn-gamma and il- were only increased in the lps model. (figure ). conclusions: up-regulation of pro-inflammatory cytokines in the uterus do not appear to be essential for ptb. cytokine expression in the cervix is greater in an inflammatory model of ptb but is also present in a non-infectious model. these studies suggest that targeting a cytokine response in the cervix may hold the most promise in prevention of ptb. the initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (af) and infiltration of the myometrium by neutrophils and macrophages (m ). whereas, in preterm labor, intra-amniotic infection may provide the stimulus for increased af interleukins and inflammatory cell migration, the stimulus for these events at term has remained uncertain. in studies using pregnant mice, we observed that the m that invade the maternal uterus near term arise from the fetus. furthermore, we obtained compelling evidence that surfactant protein-a (sp-a), a developmentally regulated c-type lectin secreted by the fetal lung into af near term, activates af m , which migrate to the uterus where they promote an inflammatory response culminating in labor. we propose that interactions of m surface receptors with sp-a, at term, or bacterial lipopolysaccharide at preterm, initiate changes in m phenotypic properties, resulting in the enhanced expression of genes that promote their migration to the uterus. the objectives of the present study were to analyze the numbers and phenotypic properties of mouse af m during late gestation and to identify their putative tissue source(s) of origin. to assess changes in the number of m in af during late gestation, af cells were isolated and stained for the m marker f / . the density of adherent f / + cells greatly increased in equivalent volumes of af between . and . days postcoitum (dpc) ( . dpc = term). interestingly, the f / + cells at . dpc were highly similar in morphology to those present in . dpc fetal lung, but distinctly different from those in fetal liver, suggesting their pulmonary origin. the af m were foam cell-like, suggesting the presence of lipid inclusions, a property shared by adult alveolar m . to further analyze gestational changes in the m population(s) in mouse af, we used flow cytometric analysis. in our initial studies, cells isolated from af were stained for f / and for cd , a pan-leukocyte marker. we observed that af from . and . dpc mice contained two sub-populations of cd + f / + cells. studies are in progress to analyze these af m populations for expression of cell surface antigens indicative of their maturity, activation state and chemotactic properties in association with the developmental induction of sp-a synthesis and secretion by the fetal lung. april bleich, patrick keller, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. the role of prs, proinflammatory cytokines, cell adhesion molecules, cox- , and toll-like receptors in mediating cervical ripening prior to labor is not clear. the objective of this study was to quantify pr isoforms and determine the relative expression of certain inflammation-related genes in cervical stroma from nonpregnant and pregnant women in early gestation (eg), term before and after cervical ripening, and during labor. methods: standard curves of pr-b, -a, pra+b and qpcr were used to quantify total and pr-b in cervical stroma from nonpregnant (proliferative, n = ; progestin treatment, n = ) and pregnant women undergoing hysterectomy (eg, n = ; term before ripening, n = ; after ripening, n = ; in labor, n = ). cervical status was determined by modified bishop scoring. results: total pr expression was maximal in nonpregnant women in the proliferative phase ( . ± . pg/ug cdna) and decreased % by progestins ( . ± . pg/ug cdna). this level was maintained in stroma from pregnant women before labor ( . ± . , eg.; . ± . before ripening; . ± . after ripening pg/ug cdna). in contrast, total pr was decreased significantly in the dilated cervix ( . ± . pg/ug, p < . ). interestingly, whereas pr-b was ± % that of total pr in the nonpregnant cervix, pr-b mrna levels were ± % to ± % in cervical tissues from all pregnant women and did not vary with labor status. using immunoblot analysis and pr-specific antibodies (pgr ), pr-b immunoreactivity was % that of total pr in all samples from pregnant women, and both pr-a and -b were downregulated significantly in the dilated cervix (from ± to ± units/atub). decreased expression of pr in the dilated cervix was accompanied by significant increases in il- , mcp- , tlr- , cd l, cox- , and s a mrna (all p < . , anova) but not cd b or tlr- . pgdh mrna was decreased significantly in the dilated cervix. with the exception of cox- , expression of these genes was similar before labor regardless of cervical ripening. conclusions: both pr and pr-b are decreased proportionately in the dilated cervix, but not during cervical ripening. further, a number of inflammatory gene products are increased dramatically in the cervix during labor, but not before. taken together, the results suggest that cervical ripening is distinct from cervical dilation and involves upregulation of cox- but not il- , mcp- , or toll-like receptors. ifn-y il- il- , il- tnf- "p value < . lps/saline ru /controls . "" . " . " . * " " . * . . " association between interleukin- (il- ) and il- to examine association of amniotic fluid interleukin (il- ) concentration with il- and its receptor il -r haplotypes in term and preterm caucasians (c) and african americans (aa) samples. methods: in this study case (preterm birth -ptb [< weeks]) and control (term [> weeks]) amniotic fluid (af) il- concentrations were analyzed for association with haplotypes of the il- and il r genes in aa and c separately. in il- , eight, and in il- r, single nucleotide polymorphisms (snps) were examined. aa and c maternal and fetal genotypes were assessed (aa: maternal:cases- controls- fetal: cases- fetal controls-; c: maternal cases- , controls- , fetal:cases- ; controls= ). haplotype associations were performed by using a sliding window with outcome il- concentration. analyses were performed separately on maternal and fetal dna. results: the strongest haplotype associations were observed in il- r rather than in il- . in c fetal dna il- r haplotypes defined by markers - bp from the transcription start site associated most strongly with af il- concentrations (global p= . x - ) and in aa maternal il- r haplotype markers at - - - (global p= . x - associated with il- concentrations. in the c fetal cases the - haplotype with the highest concentration was a-g (log(cytokine) = . pg/ml). in aa maternal samples the highest concentration was observed for haplotype t-t-g-c at - - - .this was seen in both cases and controls at (case log (cytokine) = . ; control log(cytokine) = . pg/ml). significant associations from haplotype analyses converged on three regions of the il- r in both races. no strong differences were observed between the haplotypes of cases with and without microbial invasion of the amniotic cavity (miac), with the exception of aa fetal samples that showed two overlapping haplotypes in il- that associated in cases with miac but not in cases without miac (- and - ; - and - )(both with p < x - ) conclusion: differences in the af il- concentration in ptb do not result from single snp effect on il- but are a result of complex relationships between il- and il- r haplotypes. these associations exhibit racial disparity. the role of tnf-in parturition. helen alexander, amanda tattersall, mark tattersall, suren sooranna, peta grigsby, leslie myatt, mark johnson. obstetrics gynaecology, imperial college, london, united kingdom; obstetrics gynaecology, university of cincinnati college of medcine, cincinnati, oh, usa. introduction: tumour necrosis factor-alpha (tnf-) is thought to play a role in inflammation-induced preterm labour since the decidua produces tnfin response to bacterial products and amniotic fluid tnf-concentrations are increased in the presence of intra-amniotic infection. the aims of this study were to (i) investigate the expression of myometrial tnf-and its receptors in relation to the onset of preterm and term labour; (ii) to identify which intracellular pathways are activated by tnf-; and to investigate the effect of tnf-alone and in combination with il- or il- on gene expression in uterine myocytes. methods: biopsies of human myometrium were taken at caesarean section from women before and after the onset of preterm and term labour and analysed for tnf-and its receptor mrna expression. a further samples were obtained before the onset of labour (n= ) from which myocytes were isolated and cultured in -well plates. when cells were - % confluent either tnf-at a concentration of ng/ml was added to the cells for , , , and min and the cells analysed by western blotting or tnf-at concentrations of , . and ng/ml was added either alone or in combination with similar concentrations of il- or il- to cells for hours and mrna was extracted and converted to cdna to determine il- and gapdh gene expression by qpcr. results: there was no change in tnf-mrna expression in relation to the onset of labour, but the expression of tnfr and tnfr mrna levels were significantly increased with gestation and further increased with the onset of labour. incubation of uterine myocytes with tnf-( ng/ml) activated all three mapk substypes: erk, jnk, and p , activation peaked between - minutes. preliminary data suggest that tnf-induces il- mrna expression but exposure to il- or il- itself did not enhance this response. conclusions: although there is no significant increase in tnf-concentration from baseline during labour we have shown tnf receptor mrna levels do increase with labour at term. exposure of isolated uterine myocytes to tnfcauses activation of all mapk subtypes and an increase in il- mrna expression. this enhanced mapk-dependent il- expression at term may be mediated via increased myometrial sensitivity to tnf-through increased tnf receptor expression. remodeling. brenda c timmons, anna-marie fairhurst, mala s mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa; immunology, ut southwestern medical center, dallas, tx, usa. objective: the molecular mechanisms involved in cervical ripening are not well understood. immunohistochemical studies from our lab report a recruitment of inflammatory cells to the cervical stroma one day before birth in the mouse using a neutrophil/monocyte marker (neutrophil / ). in this study, we sought to identify and quantitate inflammatory cells migrating into the mouse cervix and to determine if this recruitment was affected by changes in progesterone levels. peripheral blood was also evaluated to see if changes in the cervix was paralleled in blood. methods: flow cytometric analysis was performed using cervical cells and peripheral blood obtained before and during cervical ripening along with - h postpartum. dispersion of cervical cells was optimized. these cells were stained with a panel of fluorescent conjugated antibodies directed against leukocyte antigens and analyzed on an lsrii flow cytometer. cells were also sorted and stained to visualize cell morphologies. to determine the effect of progesterone on the migration of leukocytes, gestation d mice were treated for h with a progesterone receptor (pr) antagonist prior to tissue collection. results: neutrophils do not appear to increase in the cervix until after birth. monocyte (mo) numbers do increase during cervical ripening (late day , d . ) and remain high through postpartum (pp). macrophages (mØ) are present prior to cervical ripening and steady state levels are maintained during labor and pp. pr antagonist treatment on d resulted in a premature increase in mo but not neutrophils or mØ. in contrast to the cervix, mo and neutrophil numbers do not significantly increase in the peripheral blood until pp. results from lymphocyte studies suggest a low level of b and t cells in the cervix. in the peripheral blood, b cells remain consistent through parturition and the t cells decrease by d . and continue to decrease pp. conclusion: tissue mo are increased in the cervix during ripening. this recruitment is dependant on loss of pr function. in contrast, neutrophils are increased in the pp cervix while mØ numbers appear constant. timing of changes in mo and neutrophil numbers in the peripheral blood differed from that observed in the cervix suggesting quantitation of these cell types in blood is not reflective of what is occurring in the cervix during ripening, dilation and pp repair. novel interactions between nf-b and other labour-associated transcription factors identified by a tf-tf array. shirin khanjani, yun s lee, suren r sooranna, mark r johnson, phillip r bennett. irdb, imperial college, london, united kingdom. introduction: external stimuli lead to changes in cellular gene expression through activation of inducible transcription factors. nf-b is a ubiquitous transcription factor classically associated with inflammation, which is activated in response to infection and proinflammatory cytokines such as those prevalent during the labour. the tf-tf interaction array uses a novel technology for detecting interactions between transcription factors based on binding of tfs to their own consensus dna binding sequence. materials and methods: primary myometrial cells were grown until - % confluent and stimulated with ng/ml il- prior to nuclear protein extraction. the nuclear extracts were incubated with the provided set of biotin-labeled, double-stranded oligonucleotide probes, which represent a known library of cis-elements. during the incubation step, these tf probes bind to their specific tfs in the nuclear extract. next, immunoprecipitation was performed using an antibody against nf-bp , which pulled out nf-bp and any tfs bound to it, bound to corresponding cis-elements. normal igg was used in a parallel experiment to represent a negative control. free cis-elements and nonspecific binding proteins were washed away and the cis-elements were finally eluted and hybridized to the array membrane, which is spotted with different tf consensus sequences. results: table shows the different tfs interacting with nf-bp based on the degree of binding stimulated by il- compared to no-il- control. conclusion: these data show that il- stimulation causes nf-b to bind to a wide variety of other treansciption factors. of particular interest in the area of parturition is binding to the other pro-inlammatory tfs such as c/ebp and ap- , which are known to regulate labour-associated genes in synergy with nf-b. the lack of association between nf-b and pr without il- stimulation supports the concept that with the onset of labour inflammation leads to functional progesterone withdrawal, rather than pr acting to inhibit inflammation. table high ap- , c/ebp, cbf, creb , c-myb, e f- , ets, ets- /pea ,fast- , gas/isre, hse, mef- , mef- , myc-max, nf- , nfatc, nf-e , nf-e , pax- , objective: pre-partum cervical ripening involves remodeling of collagen structure and inflammatory immune cell activity (jsgi : , ; reprod biol endo : , ) . although parturition is associated with systemic or local progesterone withdrawal (ajog : , ) , effects of a decline in progesterone on cervical ripening is not known. the present study tested the hypothesis that progesterone withdrawal promotes collagen degradation, innervation, and immune cell trafficking in the cervix of nonpregnant mice. methods: adult virgin female c bl mice received capsules (sc) with oil vehicle (v) or estradiol (e) and progesterone (p) to simulate concentrations in pregnancy (hum reprod : , ) . after days, mice in the v and e+p groups were euthanized. the p capsule was removed from some mice on day (e-p) and groups killed on days and . cervix sections were stained for collagen, nerve fibers, macrophages, or neutrophils (n= /group/day; sections/ cervix; biol reprod : , ; jsgi : , ) . stained macrophages and neutrophils were counted (image pro-plus , media cybernetics). results: e+p treatment for days promoted hypertrophy of the cervix compared to v controls, i.e., collagen content and structure diminished, cell nuclei density declined, and nerve fibers increased. removal of p did not affect these endpoints. for immune cells, e+p for , , or days decreased immune cell numbers. by contrast, p removal increased macrophages and neutrophils in the cervix on days and (p< . , e-p vs e+p groups, respectively). the census of resident immune cells in e-p groups at and h after p removal equaled that in the v group. conclusions: mimicking gonadal steroid concentrations in circulation during pregnancy promotes hypertrophy and suppresses immigration of immune cells in the cervix. in this non-pregnant murine model for parturition, progesterone withdrawal recruits immune cells, but fails to promote further remodeling or hyperplasia of nerve fibers in the cervix. the findings raise the possibility that ripening of the cervix requires not only recruitment, but also activation of immune cells. whether proinflammatory activities by specific immune cells affect nerve fiber hypertrophy or neural activity, as part of the mechanism for ripening of the cervix, remains to be determined. we have previously identified and characterized a truncated pr (pr-m), that localizes to the mitochondrion by multiple experimental techniques, including confocal imaging of a recombinant gfp fusion protein, western blot analysis after cellular fractionation of nuclear pr negative t d-y breast cancer cells and western blot analysis of purified human heart mitochondrial proteins. initial studies with nuclear pr negative mcf- a breast epithelial cells shown to express pr-m demonstrated an increase in mitochondrial membrane potential (mmp) with progesterone/progestin treatment. these studies led to the hypothesis that progesterone modulates cellular respiration via the mitochondrial receptor, pr-m. objectives: the present studies sought to further localize pr-m to the outer, inner or matrix portion of the mitochondrion and to correlate the increase in mmp with total cellular atp production. additionally, the potency of progesterone and synthetic progestins on the change in mmp was evaluated. methods: the location of pr-m was determined by western blot analysis after fractionation of human heart mitochondria with digitonin treatment and differential centrifugation. mmp was determined in mcf- a breast epithelial cells and a rhabdomyosarcoma cells by the change in fluorescent emission of jc- . total atp was determined by a bioluminescent assay. results: western blot analysis after mitochondrial fractionation showed pr-m localization exclusively in the outer membrane. a dose-dependent increase in mmp was seen in cell lines with - min treatment with progesterone and r which were inhibited by a specific pr antagonist, rti- - b, and not affected by the translational inhibitor, cycloheximide. similar changes in mmp were seen with the same concentration of progesterone, mpa and r . progesterone/progestin treatment for min led to an increase in total cellular atp without a change in cell number. conclusions: progesterone/progestin treatment results in an increase in cellular respiration in cells expressing an outer mitochondrial membrane pr and known to lack nuclear pr expression. this may represent a mechanism whereby progesterone enhances cellular energy production to meet the demands of pregnancy. introduction pge is a major product of the fetal membranes, decidua and myometrium and plays an important role in cervical ripening and myometrial contractions. there are four pge receptors, ep- and ep- mediate contractions whilst ep- and ep- mediate quiescence. ep- contains multiple consensus sequences for transcription factors known to be of importance in labour, in particular nfkappab. we therefore performed experiments to determine the effect of activation and inhibition of nfkappab upon ep- expression. myocytes plated in well plates were treated with il- ( ng/ml), to activate nfkappab. myometrial cells were also transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna to knock down nfkappab p . rna was extracted for amplifying ep- using quantitative rt-pcr with amplification of l as a control to normalise data. il- caused an increase in expression of ep- . knock down of nfkappab using si-rna resulted in a further increase in ep- . this data suggests that although il- stimulates expression of ep- it does so through an nfkappab independent mechanism. the upregulation of ep- with sirna knock down of p suggests that activation of nfkappab would inhibit ep- expression consistent with the concept that activation of nfkappab at term causes the myometrium to adopt a more contractile phenotype. introduction: a role for the pro-inflammatory cytokine il- is suggested in preterm and term birth, independent of the presence of infection. we previously showed that mice with a null mutation in the il- gene (ko) delivered one day later than wild type (wt) mice due at least in part to altered timing of uterine expression of prostaglandin (pg) f receptor, ptgfr, mrna. we also observed differences in mrna expression of other uterine activation proteins (uaps), pg h synthase (pghs)- , oxytocin receptor (otr) and connexin- (cx- ), suggesting multiple physiological effects for il- in term delivery. objective: to examine the effect of il- deficiency on the peri-partal uterine mrna expression of the pge receptors (ep) , and ; the post-partum changes of all uaps; and the relationship of these to serum progesterone (p ) concentrations. methods: gestational length was observed in pregnant c bl/ wt (n= ) and ko (n= ) mice. uap mrna levels were measured by real time rt-pcr in wt and ko dams sacrificed from d through delivery and up to h postdelivery. serum p was determined by ria. data were analyzed by one-way and two-way anova using the holm-sidak test to differentiate treatment effects at p< . . results: birth was delayed in the il- ko mice ( . ± . d vs. . ± . d), and this affected the timing of peri-partal changes for all uaps similarly. both ep and ep (relaxatory receptors) were elevated at d in ko dams, but returned to low levels before delivery and were elevated at delivery (ep ) or afterwards (ep ). ep levels did not change. otr increased several hours before delivery in all dams. cx- increased at delivery, then fell, while pghs- increased at delivery and remained elevated afterwards. ptgfr mrna increased - -fold at delivery and a further - -fold after delivery, suggesting loss of pgf permitted enhanced ptgfr expression. p serum concentrations fell pre-partum in both groups. conclusions: il- ko alters the expression pattern of several pregnancy and parturition-related genes and may delay the pre-partum p fall, suggesting a potential ovarian effect. a uterine role for il- in regulating the timing of normal term parturition cannot be ruled out. objective: recent studies have highlighted the prevalence of vitamin d deficiency in pregnant women, particularly in those from ethnic groups with darker skin who require higher levels of uv light to make parental vitamin d. as the active form of vitamin d, , -dihydroxyvitamin d ( , (oh) d ) is a potent immunomodulator, we postulated that vitamin d deficiency may lead to dysregulated placental immunity. both trophoblast and decidua express the enzyme -hydroxylase (cyp b ) which catalyzes synthesis of , (oh) d from the inactive pro-hormone -hydroxyvitamin d ( ohd ). in view of the role of trophoblast as a barrier site protecting the fetus against infection, we investigated the impact of cyp b , ohd and , (oh) d on innate immune responses in trophoblastic cells. methods: a trophoblast cell line was used to assess the effect of vitamin d on innate immune responses. the cells were treated for hrs with various concentrations of , (oh) d ( - nm) and antimicrobial cathelicidin expression was assessed by real time pcr. to assess whether expression of cyp b was affected by pathogenic stimuli, a cells were treated with ligands for toll-like receptor (tlr) - , cyp b expression (real time pcr) and ohd utilization were assessed. results: a trophoblast cell line; which shows temperature-sensitive differentiation, revealed expression of cyp b with higher levels of enzyme activity under conditions of syncytiotrophoblast development. cells treated for hrs with , (oh) d showed dose-dependent induction of the antimicrobial defensin cathelicidin expression ( . - fold induction), whilst cells treated pro-hormone ohd ( nm) showed . -fold induction of cathelicidin expression. activation of tlr (poly i:c) and tlr (lipopolysaccharide) enhanced the expression of cyp b ( -and . -fold) and increased the sensitivity to ohd as a consequence. conclusion: these data show that autocrine synthesis of , (oh) d from ohd can stimulate trophoblast immune responses in a similar fashion to macrophages. as ohd is the major circulating form of vitamin d, we hypothesize that trophoblast innate immunity may be significantly compromised under conditions of vitamin d deficiency. introduction: secretory leukocyte protease inhibitor (slpi) is a potent -kda protein inhibitor of neutrophil elastase and it is a mediator of mucosal immunity and an inhibitor of nfkb regulated inflammatory responses. however, its source, function and regulation within the uterus during pregnancy and at parturition are not well defined. it has previously been shown to be present in fetal membranes and cervical mucus and in amniotic fluid, where its levels is increased from second trimester to term and with a further increase at parturition. slpi has also been shown to be responsive to progesterone in human epithelial cells. our aim was to determine the effects of il- on slpi gene expression in human myometrium. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates and when cells were - % confluent they were serum starved overnight and incubated with m methyl- -hydroxy-progesterone acetate for h and with or without ng/ml il- for a further hours. at the end of incubations rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of slpi, gapdh and beta-actin were measured by qpcr. results: h incubation of uterine myocytes with ng/ml il- caused a marked increase in slpi by % (n= ; p< . ). incubation of uterine myocytes with m progesterone for h also increased slpi by % (n= ; p< . ). incubation with il- for h in the presence of h with progesterone increased slpi: gapdh mrna ratio from . ± . to . ± . (mean ± sem; p< . ). slpi expression was similar in the upper and lower segment myometrium in preterm patients. in term myometrium the slpi: beta-actin mrna ratio was increased by -and -fold in term labour versus term non labour samples in the lower and upper segment respectively (mean ± sem; p< . ). these data show slpi is present in human myometrium and that it is increased by il- . progesterone also increases its expression. its expression is increased in term labour where its anti-bacterial, anti-fungal and anti-viral properties could allow it to act as an endogenous block to infections. objective: pre-b cell colony-enhancing factor (pbef) downstream mapk signaling and transcription factor activation. introduction: pbef is expressed in all layers of the human fetal membranes and the myometrium and is upregulated by labor, infection, nf-kb, ap- and stretching. pbef has the ability to protect a variety of cells from apoptosis, however it also appears to act as an insulin mimetic via the insulin receptor (fukuhara et al. science : ; - ) . its levels are elevated in obese patients, those with type diabetes and in gestational diabetes. because pbef has poorly understood biological activities an investigation of mapk signaling and transcription factor activation induced by pbef has been undertaken in order to gain insights into its mechanisms of action. methods: primary aec were isolated from fetal membranes and treated with rhpbef ( ng/ml) for h, lysed and the resultant proteins labeled with cy or cy (amersham). there were used on a protein microarray containing constituents of the mapk signaling pathways (sigma). isolated aec were transfected with luciferase constructs for nf-kb, ap- , cre, hse and gre response elements using the exgen reagent. the cells were treated with rhpbef ( . , . , , ng/ml) hrs, lysed and ul of sample was analyzed for luciferase activity with dual-luciferase reporter assay system (promega). co-transfection with gfp and sv luciferase constructs to control for transfection efficiency and cell number (respectively) was also performed for each experiment. results: pbef significantly upregulated mapk signaling components including functioning as part of the erk pathways and belonging to p signaling. it also activated nf-kb and camp response elements. however, it significantly down regulated signaling molecules belonging to the jnk pathway that resulted in decreased c-jun phosphorylation. conclusions: pbef signaling in aec is consistent with its anti-apoptotic ability and its upregulation of the pro-inflammatory cytokines. although some mapk components responsive to pbef could be associated with insulin receptor signaling, some may not. therefore, it appears likely that pbef interacts with another potentially unique, but currently unidentified receptor. was the first effector to be characterised, but camp is now known to have other effectors, including camp receptor protein, cyclic nucleotide-gated channels and camp-guanine nucleotide exchange factor/exchange protein directly activated by camp (camp-gef/epac). two epac's have been identified and they consist of functional domains: camp-binding domains, a dep domain, a ras exchange motif and a gef domain. our aim was to determine the presence of epac's in human myometrium and to study the effect of camp responses on prolabour genes such as il- , pghs- , otr and fp. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates until - % confluent. cells were serum starved overnight and incubated with μm methyl progesterone, . mm sodium -bromo-camp, . mm forskolin, μm kt , ng/ml brefeldin a and . mm -pmeopt- 'o-me-camp either alone or in combination for h. rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of epac , epac , il- , pghs- , otr, fp, gapdh and -actin were measured by qpcr. results: epac and epac were present in the upper and lower segment of myometrium with epac levels being some -fold higher than those of epac . there was no change with the onset of labour at or before term. treatment with il- for h significantly increased epac (n= ; p< . ) but had no effect on epac . when conditions mimicked pregnancy, (the presence of forskolin and progesterone), brefeldin a, an epac antagonist, increased basal pghs- and fp mrna expression (n= ; p< . ), but had no effect on il- and tended to reduce otr. the il- -induced increase in pghs- and il- , but not fp, was greater with the epac antagonist. conclusions: these data show epac's are present in human myometrium and that camp acts via epacs to reduce pghs- and fp expression during pregnancy. background: inflammatory events have been implicated in the process of labour. glucocorticoids mediate strong anti-inflammatory effects through binding to the glucocorticoid receptor (gr), which on activation translocates to the nucleus and either increases or decreases the expression of responsive genes thereby suppressing inflammation. objective: to characterise the expression profile for gr protein and mrna in human myometrium during fetal maturation and parturition. methods: western immunoblotting (wb) was employed to characterise gr protein expression in first (n= ) and second trimester myometrium (n= ), and in paired upper and lower segment pregnant (non-labouring, p, n= ) and labouring (l, n= ) myometrium; as compared to non-pregnant (np, n= ) control samples. immunofluorescence staining with confocal microscopy and rt-pcr were also undertaken. results: detection of gr protein by wb revealed two bands at - kda, representing the alternatively spliced isoforms, gr-and gr-. densitometric analysis showed that gr levels decreased significantly during pregnancy and remained at very low levels at term and in labour when compared with np samples (p< . ); this decrease was seen for gr-and gr-. no significant temporal variations were observed in gr protein levels at term or during labour. gr protein was localised by immunofluorescence staining to the nuclei and cytoplasm of cells. less intense staining was apparent in p compared to np tissues; consistent with wb data. rt-pcr showed a consistent predominance of gr-to gr-mrna in all the tissues used. the observed decrease in protein expression was also mirrored at the mrna level: gr-mrna levels appeared to gradually decrease throughout gestation (p< . ). differences between the upper and lower myometrial regions were only observed in the labouring samples, where gr-mrna levels were significantly decreased in the lower segment (p< . ). nested pcr was additionally used to amplify gr-, but no consistent pattern of mrna expression was obtained. conclusions: these data are the first to characterise gr expression in human myometrium. spatial and temporal variations have been found with expression evolving through the three trimesters of pregnancy and in labour. further studies are now underway to evaluate whether gr contributes to the sequence of inflammatory events implicated in triggering term and preterm labour. these studies sought to determine the mechanism by which pas modulate the immune response. methods: ) an in vitro co-culture model mixed with human cervical epithelial hela cells and pma-induced human macrophage u cells at epithelial/macrophage cell ratio of : was employed. ) the co-culture was pre-treated with medroxyprogesterone acetate (mpa), progesterone (prog) and dexamethasone (dex) at concentrations of , , and nm for hrs followed by day stimulation of g/ml lipopolysaccharide (lps). these experiments were repeated using hela cells transfected with sirna for glucocorticoid receptor (gr). the production of cytokines il- , il- and il- , il- and tnf were determined using elisas. ) the co-culture was pre-treated with nm of each pas for hours prior to lps stimulation at g/ml for . , . , , , and hours. the phosphorylation of p mapk and gr and the expression of mapk phosphatase (mkp ) were determined by western-blotting. results: il- , il- and il- , il- and tnf were elevated by . fold, . fold, fold, . fold and . fold in the co-culture model in response to lps(p< . for all). pretreatment of mpa and dex, but not prog, inhibited the lps-induced cytokine production. the anti-inflammatory effect of mpa occurs in a dose-dependent manner. in the absence of gr, the inhibitory effect of mpa and dex on il- , but not on il- , was lost. mpa and dex, but not prog, induced the phosphorylation of gr and expression of mkp . p mapk was activated by lps for up to hrs and pretreatment of mpa and dex attenuated this response. the ability of mpa and dex to suppress the inflammatory response in cervical tissues appears to be mediated through a gr-dependent pathway, specifically though p mapk. our studies suggest that prog is not a significant immunomodulator in these tissues. background: progesterone (p ) has been shown to play a critical role in maintaining pregnancy and preventing preterm birth. these effects are likely related to its immunomodulatory properties. we investigated whether camp plays a role in the immunosuppressive action of progesterone on fetal mononuclear cells. methods: umbilical cord blood mononuclear cells were isolated using density gradient centrifugation. to establish a p effect and optimize concentrations, cells were pretreated with p ( - m- - m), dexamethasone ( um) or vehicle for hour prior to overnight lps ( ng/ml) stimulation. supernatants were assayed for tnf-using elisa. ldh assays confirmed the absence of a cytotoxic effect. next, cells were pre-incubated with forskolin (adenylate cyclase activator) or db-camp (camp agonist) prior to lps to determine the effects of camp on tnf production. finally, cells were pretreated with rp-camp (camp antagonist) prior to p incubation and lps stimulation to determine whether the p effect was reversed by a camp antagonist. results: p significantly inhibited lps-induced tnf production in a dose dependent manner, with maximum suppression observed at - m. both forskolin and db-camp suppressed lps-induced tnf production in a doserelated manner (fig ) . finally, a dose-dependent partial reversal of tnf production was observed when cells were pretreated with rp-camp. (fig ) conclusions: progesterone suppresses the inflammatory response in fetal mononuclear cells as measured by tnf expression. this effect is mimicked by adenylate cyclase activators and camp agonists and partially reversed by camp antagonists. this implicates the camp pathway in mediating the immunomodulatory actions of p . objectives estrogen improves endothelial function after vascular injury via largely unknown mechanisms. endothelial progenitor cells (epcs) are known to be implicated in various vascular events requiring endothelialization. we hypothesized that estrogen and progesterone could influence the function and the change of epcs in menstrual cycle. material and methods peripheral blood mononuclear cells (pbmcs) were isolated from peripheral blood of healthy young women in each menstrual period by density gradient centrifugation with ficoll separating solution. pbmcs were seeded in endothelial basal medium with or without estrogen or progesterone. on the th day in culture, nonadherent cells were removed. on the th day in culture, adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. the number of ldl-and lectin-positive cells was measured as epcs using flowcytometry. the expression of estrogen and progesterone receptor mrna in epcs were measured by real time pcr in menstrual and luteal period. the number of epcs was significantly increased in the menstrual and luteal period compared with the follicular phase. estrogen and progesterone significantly increased the number of adherent epcs dose dependently in menstrual period, but not in luteal period. the expression of estrogen-alpha receptor in menstrual period was higher than luteal period. also, estrogen-beta receptor in luteal period was strongly expressed compared with menstrual period. these results suggest the expression of estrogen receptor and progesterone receptor play important roles to regulate epcs' proliferation during menstrual cycle. objective: elevated levels of fetal plasma avp are associated with the development of oligohydramnios, in part a result of avp-mediated reduction in fetal urine production. as avp urinary concentration effects are mediated via upregulation of renal tubular aqp water channels, we propose that avp modulates placental aqp channels, influencing bidirectional maternal-fetal water flow. we sought to study the effect of avp on trophoblast aqp gene expression using the first trimester-derived extravillous htr- /svneo cells and the term placenta-like trophoblast carcinoma cells jeg- . methods: cultures of both cell lines were treated with a physiological concentration of avp ( . nm) to determine aqp mrna and protein expression. negative controls consisted of cells incubated in medium supplemented with % fbs without avp. to determine whether avp regulation of aqp occurs by a camp signaling pathway, jeg- cells were preincubated with μm -(tetrahydro- '-furyl) adenine (sq ), a cell-permeable camp inhibitor, before being treated with avp ( . nm). cells were incubated for hrs for aqp mrna expression and for hrs for protein extraction at °c. after harvest, real time pcr and western blotting analysis were used to detect the aqp mrna and protein expression levels, respectively. results: avp increased aqp mrna expression in both cell lines by . and . fold after hrs. aqp protein expression paralleled the increase seen in the mrna (p< . ). pretreatment of jeg- cells with sq inhibitor completely blocked the stimulatory effect of avp. conclusion: aqp gene expression is up-regulated by avp in first trimester and term trophoblast cells, with a higher induction in the later. avp activation of aqp gene expression occurs via a camp mediated pathway, as the adenyl cyclase inhibitor blocked avp effects on aqp gene expression. these results suggest that increased fetal plasma avp may contribute to oligohydramnios by an increase in aqp-mediated fetal to maternal water flow. objective: changes in expression, ratio and activity of progesterone receptors within human myometrium have been proposed as potential contributory mechanisms for the functional progesterone withdrawal effect which precedes labour. progesterone receptor c (pr-c) is an n terminally truncated isoform of the full length progesterone receptor; pr-c has been shown to be abundant in fetal membranes, placenta and upper segment of laboring myometrium ( , ). the function and regulation of pr-c is at present unclear. in this study we aimed to investigate the regulation of pr-c in cultured human myometrial and amnion-derived wish cells. methods: myometrial primary cell cultures were prepared from non pregnant and term pregnant uteri. both myometrial and wish cell cultures were treated with natural progesterone ( , nm, mm, mm, mm) for , and hrs. western immunoblotting was employed using nuclear and cytoplasmic extracts prepared from treated cells to observe the effect of progesterone on a) expression and b) subcellular localisation of pr-c within both cell types. immunofluorescent staining and confocal microscopy were also employed. experiments were also undertaken whereby nuclear and cytoplasmic extracts were subjected to shrimp alkaline phosphatase treatment to evaluate the phosphorylation status of pr-c in response to hormone. results: data indicates that a kda cytoplasmic protein, representing pr-c is abundant in myometrial and amnion cell cultures. we show that expression of pr-c increases in both cytoplasmic and nuclear fractions within both cell types in response to progesterone. evidence also suggests that pr-c is phosphorylated in a progesterone-independent manner. concurrent treatment with progesterone and the pr-antagonists ru and organon in amnion-derived cells still led to an increased abundance of pr-c but seemed to alter the phosphorylation status of pr-c. conclusion: pr-c expression and subcellular localisation within myometrial and wish cells alters in response to progesterone. speculation is generated about potential role of pr-c in reproductive tissues and its contribution to functional progesterone withdrawal. . taylor smooth muscle responses have been studied, data on regional blood flow (bf)distribution are scarce. we have studied the effect of a single dex course on relative bf distribution within the brain, skeletal muscle, heart, omental fat, placenta and adrenal in fetal sheep at . g, the equivalent of weeks. methods: fetal sheep received amniotic, jugular, carotid and femoral artery and vein catheters at days g (dg). experiments started at dg in dex ( mg) and saline treated controls (ctr), each receiving injections h apart. fetal blood pressure was recorded continuously (windaq pro+). microspheres ( . x total; sterispheres, biopal) were injected at : am into fetal jugular and femoral veins before maternal i.m. injection (t ) and h after the rd injection (t ) in both groups. tissues were collected hours after the th injection for assessment of bf (biopal). results: fetal bp increased above baseline after dex ( . + . mmhg; p= . ) with no change in ctr (- . + . mmhg). regional bf distribution is presented as a percent of the total counts per g tissue (table ) and after normalization within each animal to placentomal flow, shown previously to be unchanged by dex, in table . no significant differences in flow were found. discussion: although dex altered bp, regional distribution of bf among key organs was unaffected. whether gc does not affect regional bf, or the rise in bp is the result of a non-specific, system-wide vasoconstriction, or the impact of gc on bf is similar in all organs, will be subject of further investigation that will include determination of regional vascular resistance and absolute flow. stimulator while crf-r is an inhibitor of gi motility at times of stress. we recently hypothesized that stress-induced in utero meconium passage in fetuses is analogous to stress-induced defecation in adult rats and likely mediated by crf pathway. in support, we demonstrated hypoxia-induced meconium passage in conjunction with marked increases in fetal plasma crf levels. as the mouse is a common experimental model, with known genome, we sought to determine the presence of crf-r and crf-r receptors in mouse fetuses. methods: time-dated cd- pregnant mouse were anaesthetized and fetuses (n= ) were collected at e (term = e ). whole gi tracts were dissected, fixed in bouin's solution, paraffin embedded and sectioned at five micron thickness. sections were immunostained with rabbit polyclonal antibodies to crf-r and crf-r by abc technique with vector abc reagents. immunoreactivity was identified as brown staining using ', diaminobenzamidine as chromagen. slides were counter stained with mayer's hematoxylin and examined under the microscope. immunoreactive signals in the smooth muscle layers of gi tracts were quantified by image analysis using image pro . plus software. results: immunostaining for both crf-r and crf-r was seen in the smooth muscle layers of all lumens examined ( lumens per section). the immunostaining intensity expressed as intensity over density (iod) in arbitrary units for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) and for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) greatly varied between lumens. the mouse fetal gi tract expressed both crf-r and crf-r receptors, suggesting that the mouse is an appropriate model for fetal-stress induced neurovisceral motor responses. the non-uniform distribution of crf receptors in the fetal gi tract suggests the existence of regional differences in the expression patterns of crf-receptors of both types. we speculate that mouse devoid of crf and crf-r or r receptors will be useful to confirm the participation of crf pathway in stress-induced in utero meconium passage. antenatal exposure to glucocorticoids (gc) is associated with a reduction in nephron number and hypertension in adult life. one of the mechanisms for the development of hypertension is thought to be the long term effect of single nephron hyperfiltration. however, in most studies there is only a - % reduction in nephron number with no change in gfr. thus, although a reduction in nephron number may be a contributing factor it is unlikely the only variable. the aim of the present study was to evaluate nephron mass, renal function and blood pressure in a cohort of adult sheep exposed antenatally to betamethasone. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hs apart at days gestational age and allowed to deliver at term. at . yr of age in female (f) and male (m) offspring, glomerular filtration rate (gfr) was measured as inulin clearance and effective renal plasma flow (erpf) as pah clearance. blood pressure was measured though an indwelling catheter in the femoral artery over a -hour period. nephron number was measured by the acid maceration technique. data mean±sem were analyzed by anova and/or two sample t test. results: antenatal bm was associated with an elevation in arterial blood pressure and a reduction in nephron number in both f and m offspring. in contrast, a reduction in gfr and erpf was present only in males. plasma and urinary electrolytes as well as urinary protein excretion were not different from ctr. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on blood pressure regulation. interestingly, while the decrease in nephron number was of similar magnitude in m and f, evidence of alterations in renal function was present only in males. these suggest that the decrease in renal function observed in males is not solely a consequence of the decrease in nephron number. furthermore, it is possible that different mechanisms are responsible for the elevation in arterial blood pressure observed in m and f. hl ; hd p hd . clr l>t-x to evaluate the effects on blood pressure, urine output, sodium excretion and gfr of the intrarenal infusion of angiotensin - in the male sheep with or without prenatal exposure to b. we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation and were born at term. catheters were placed in the femoral artery, femoral vein, left renal artery and the bladder. the right kidney was removed. after days recovery the sheep received an infusion of ang - ( ng/kg/min) with or without its antagonist, [d-ala ]-ang-( - )(d-ala, ng/kg/min), into the renal artery for hours. blood pressure, gfr, urine output and sodium excretion were measured before and after the infusion. two-way analysis of variance (anova) was used to test mean values between the groups. with or without d-ala, map and urine output didn't change significantly during ang - infusion. however, the infusion of ang - resulted in a decrease (change from baseline) (p= . ) in na + excretion of . ± . meq/kg/ h in the vehicle animals and . ± . meq/kg/ h in the b animals. there was no significant change in gfr during ang - infusion. gfr was lower in the b group during the combined ang - and d-ala infusion ( . ± . ml/min vs . ± in vehicle group, p= . ). intrarenal infusion of ang - at ng/kg/min is followed by a significant decrease in sodium excretion but no significant change in urine output and gfr. the decrease tended to be greater in the vehicle animals and was not markedly attenuated by the antagonist d-ala. this suggests the effect of ang - may be mediated by a receptor other than the mas receptor for ang - and may be of lesser magnitude in animals exposed to b before birth. and norepinephrine ( . - . g/kg/min) and to an l-name ( mg/kg) bolus were studied. vascular reactivity was analyzed by curve fitting of either the blood pressure or hr responses to obtain the maximal response. data are expressed as mean±sem of change from baseline. statistical significance was established using t test. results: bm-exposed animals displayed a higher blood pressure response to at-ii (atii max ± vs ± % of baseline, p< . , figure left panel). no differences in blood pressure or heart rate responses to norepinephrine infusion were detected in bm when compared to control animals (figure right panel) . maximal response to l-name was also elevated in bm-exposed animals but did not reach statistical significance with the current sample size. conclusion: we have shown that antenatal gc treatment significantly increases blood pressure in sheep. here we show an enhanced in vivo response to at-ii. this response may contribute to the increased blood pressure observed. the greater response to l-name most likely represent an increased no production as a compensatory mechanism to the higher blood pressure observed. hl . women at risk of delivering preterm are frequently treated with glucocorticoids to facilitate fetal lung maturation. animal studies have revealed that prenatal exposure to glucocorticoids may alter aspects of hypothalamic-pituitary adrenal functioning in later life. in this study we examined the effects of clinically relevant prenatal betamethasone treatment on the responsiveness of pituitary cells (in terms of adrenocorticotropin, acth, secretion) isolated from adult sheep. time-dated pregnant sheep were injected with betamethasone ( . mg/kg) or vehicle on days and of gestation. thereafter pregnancy was allowed to continue unimpeded and offspring were born. pituitaries were isolated from adult sheep aged between and months and cells were dispersed and plated at a density × cells per well in well plates. after h, cells were stimulated with normal medium, nm arginine vasopressin (avp), nm avp, nm corticotropin releasing factor (crf) or nm crf for h. medium was collected and analyzed for acth using a commercially available kit. acth secretion (given as % increase from untreated cells) was similar between the control and betamethasone groups following nm avp ( . ± . vs. . ± . ) and nm crf ( . ± . vs. . ± . ). at higher stimulatory concentrations of both avp and crf, acth secretion remained similar in control cells ( . ± . and . ± . respectively), but was significantly decreased in betamethasone cells ( . ± . and . ± . respectively). these findings suggest that prenatal exposure to clinically relevant doses of betamethasone can alter pituitary responsiveness to both avp and crf in adulthood. this research was supported by nih grants hd and hd . lcc is supported by an rjr-leon golberg fellowship in pharmacology and toxicology. objective: newborn meconium (mec) passage normally occurs within the first - h after birth. in contrast, more than half a million infants born annually in the us pass mec in utero. neither the physiological mechanism(s) nor causes for in utero mec passage are well understood, though both fetal maturation and stress are associated. we recently reported in utero mec passage and marked increases in plasma crf levels in term fetal rats exposed to maternal hypoxic stress. we hypothesize that stress-induced in utero mec passage is mediated by the crf pathway in a manner analogous to stress-induced defecation in adult rats. in the present investigation we examined placental crf content prior to and following maternal hypoxia, to determine the source of increased plasma crf. methods: time-dated pregnant rats (n= ) were exposed to a paradigm of graded, stepwise hypoxia on day (term= ) (pediatr. res. : - , ) . control pregnant rats were exposed to % oxygen for a similar duration as experimental animals. at the end of the study, placentas were harvested, fixed in % paraformaldehyde and paraffin embedded. sections (n= per placenta) were subjected to immunohistochemical analyses with rat/human crf antibody (peninsula laboratory) by abc technique. immunoreactivities on placental sections were quantified using the image pro . software and intensity expressed as arbitrary unit (au). all values are mean± sem. results: in control maternal rats exposed to normoxia, positive staining for crf was seen in decidua ( . ± . au) and in all three major placental cells types (giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au.) in contrast, in animals exposed to hypoxia, there was a total absence of crf staining in the placental cells, with only positive crf staining seen only in the decidua ( . ± . au). conclusion: the total absence of crf staining in both the basal and labyrinth zones in placentas of pregnant rats subjected to hypoxic stress suggests that placental cells rapidly release crf into the maternal and fetal circulation in response to hypoxic-stress. our findings support our hypothesis that the peripheral crf pathway mediates in utero mec passage. offspring. angela g massmann, jie zhang, jorge p figueroa. department of obstetrics and gynecology, wake forest university school of medicine, winston-salem, nc, usa. objective: in rats and sheep, exposure to glucocorticoids (gc) in the perinatal period induces hypertension in adult life. recent evidence suggests that endothelin b (etb) receptor plays an important role in the regulation of sodium balance and blood pressure. renal medulla is an important site of expression and action of etb receptor. furthermore, in kidney it is the medulla (km) where the highest concentrations of immunoreactive et- and etb receptor are found. the aim of this study was to measure eta and etb expression in adult sheep kidney exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (v) hours apart at days of gestational age and allowed to deliver at term. at . yr of age, male and female offspring exposed to either vehicle or bm were euthanized and the kidneys harvested. kidney medulla (km) was obtained by sharp dissection. eta and etb protein and mrna expression were evaluated by western blot and rnaase protection assay. data are expressed as mean±sem and were analyzed by two way anova. results: a significant decrease in etb (f= . , p= . ) but not eta protein was observed in km of bm exposed male and female adult sheep. at the mrna level, bm exposure also affected etb, but not, eta expression. however, bm treated animals had higher mrna levels than control sheep (f= . ; p= . ). conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects. the activation of etb receptor by et- inhibits sodium transport function in the collecting duct and the selective gene deletion of the etb in medulla results in hypertension.therefore, our finding of a significant reduction in etb protein suggests that there may be an impairment in the kidney's ability to regulate sodium reabsorption. the functional relevance of the alterations in etb protein expression as well as the discrepancies in mrna regulation need to be established. hl ; hd p hd . in the placenta, -hydroxysteroid dehydrogenase type ( hsd ) regulates the transfer of cortisol. maternal glucocorticoid (gc) administration has been shown to affect the ovine fetal growth trajectory and regulate hsd expression in mid and late gestation. however, little is know about the effects of gc administration in early gestation. we hypothesized that maternally administered low-dose dexamethasone (dex) in early gestation would affect hsd expression, which will subsequently alter fetal birth weight. pregnant ewes were randomized and received intramuscular injections consisting of either saline ( ml saline/ewe) or dex ( . mg/kg) given hours apart starting on days of gestation (dg). the animals were sacrificed at , , , and dg and fetal weights were recorded and placental tissue was collected. qrt-pcr was used to measure hsd placental mrna expression. statistical analysis was done by anova with student's t-test posthoc. overall, there was no significant effect of dex treatment on placental hsd mrna expression across gestation. however at dg, there was a significant increase in hsd expression after dex (p= . ). there was an increase in placental hsd mrna progressively from dg (mean = . ) to dg (mean = . ), independent of treatment. overall, a positive correlation between hsd and fetal weight (r = . ) was apparent at dg and at dg (p= . ) in both control (p= . ) and dex (p= . ). significant positive correlations between placental hsd and fetal weight (r = . ) were found in females (p= . ) and a similar trend was found in male fetuses (p= . ). we conclude that in the sheep placenta, hsd expression increases throughout gestation and may respond, at least transiently in early gestation, to elevations in maternal gc. the positive correlation between hsd and fetal weight is consistent with the thesis that the fetal growth trajectory may be influenced by maternal cortisol levels and that placental hsd is an important mediator of these effects. antenatal gc exposure induces hypertension in the young adult rat (neuroendocrinol; ; : ) and increases femoral vascular resistance in the lamb (jphysiol; ; : ) . unpublished own examinations in preparation of this study have shown age dependency of the vascular reactivity. vascular contractility of the middle cerebral artery (mca) decreased in response to k + and noradrenaline (na) between mo and , y of age (p< . ). vascular relaxation to acetylcholine (ach) but not to pge decreased during the same time (p< . ). vascular contractility of the renal artery (ra) increased in response to k + (p< . ). aims: to examine if antenatal gc alter vascular reactivity in the aged individual when cardiovascular diseases are predominant. we studied the ra because its vascular tone is involved in modulation of arterial pressure control (amjphysiol; ; :r ) and the mca because depressive disorders that are associated with dysregulation of the hpa axis (jcem; ; : ) increase stroke mortality for unknown reasons (amjpsychiatry; ; : ) . to be clinically relevant, we administered dexamethasone at the dose used to enhance lung maturation in babies threaten premature labor. methods: pregnant dams received saline (n= ) or μg/kg dexamethasone (n= ) i.p. at day e / equivalent to x mg dexamethasone administered to a kg pregnant woman. at . years of age, vascular response of the ra and mca to endothelium-dependent and independent mediators was measured using wire myography. vessels were inspected histologically for intact endothelium. results: basal vasoconstrictory and dilatory responses to all mediators were less pronounced in the mca than in the ra probably reflecting autoregulatory properties of cerebral vessels (p< . ). after prenatal dexamethasone exposure, contractility but not sensitivity to k + and na was enhanced in the mca and even more pronounced in the ra (p< . ). relaxation to ach and pge was similarly diminished in both vessels after precontraction with na (p< . ). conclusions: prenatal dexamethasone exposure at the dose used clinically increases renal and cerebral vascular tone in the aged rat by endotheliumdependent and independent mechanisms. this effect may be a potential mechanism of fetal programming of cardiovascular diseases in later life. betamethasone (bmz) therapy during pregnancy to improve fetal lung maturity may result in long term side effects, including hypertension, during adulthood. the kidney is an important organ which regulates systemic blood pressure via the local renin angiotensin system (ras). the major enzymes of the intrarenal ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the hypothesis of this study is that prenatal bmz exposure will result in alterations of the enzymes regulating the intrarenal ras. specifically, sheep that are treated with bmz in utero will have higher amounts of ace activity in kidney cortex compared to control animals. pregnant sheep of known mating date were either treated with vehicle (n= ) or with two doses of bmz (n= ), . mg/kg, hours apart at days of gestation. renal cortex from the male offspring was harvested at - months of age. cortical membranes were then solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were separated by reversephase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results: bmz treatment resulted in a significant increase in ace activity compared to control animals (p= . ). ace and neprilysin levels were not significantly different between treatment groups. when expressed as a ratio of ace/ace activity, bmz treated animals exhibited a . fold greater proportion of ace activity versus control animals (p= . ). this study demonstrates that bmz exposure during fetal life results in a significant increase in renal ace activity during adult life. this increase in enzyme activity would be expected to be associated with increased levels of ang ii in the kidney and na + retention, possibly underlying the development of hypertension. hd , hd . we recently hypothesized that stress-induced in utero meconium passage in term fetuses utilizes peripheral and/or central crf pathways in a manner analogous to stress-induced defecation in adult rats. as participation of central crf pathway requires the crf circuitry system in brain-gut axis, we exploited immunohistochemical techniques to address whether term fetal colon is wired by crf-nerve fibers. methods: fetal distal colon segments (n= ) were dissected from term ovine fetuses ( - d gestation). in addition, lumbosacral spinal cord with spinal roots and dorsal root ganglia (drg) (n= ) were dissected from ovine fetuses, and vagal nerve trunk with nodose ganglia (n= ) were dissected from mouse fetuses. paraffin sections fixed either in bouin's solution or zamboni's were subjected to immunohistochemistry with rabbit polyclonal antibodies specific to ovine-crf (ocrf). immunoreactivity on the sections was identified by standard abc technique, immunostaining quantified by image-pro plus software and expressed (intensity over area) as arbitrary units (au). results: ocrf antibody elicited strong positive staining on the serosal surface (port of entry for extrinsic nerve fibers) in distal colonic segments objective: we hypothesize that stress-induced in utero meconium passage is mediated by the crf pathway. ucn-i, similar to crf, is a potent contractility inhibitor of preterm ovine fetal distal colon, which has abundant crf-r receptors. both ucn- and crf thus may inhibit colonic motility and prevent preterm in utero meconium passage via crf-r receptors. however, ucn-i is reported to stimulate contractility of adult rat colonic smooth muscle strips more strongly than crf. we sought to study the pattern of ucn-i innervation in distal colon of ovine fetuses to delineate whether ucn- functions as a facilitator or inhibitor of colonic propulsive motility. methods: bouin's solution fixed, paraffin sections of very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - ) and term (t: - days) ovine fetal distal colon rings were subjected to immunohistochemistry with polyclonal antibodies to human ucn- ( : sigma) by abc system. intensity of ucn- immunostaining in smooth muscle layers and myenteric neurons were quantified by image-pro plus software and expressed (intensity/area) in arbitrary units (au). differences over time were assessed with anova. in all groups very preterm was significantly greater than term (p< . ). conclusion: ucn- immunoreactivity in all three muscle layers, as well as myenteric and submucosal neurons, is highest at very preterm as compared to more mature gestations. these results suggest that the reduction of ucn- inhibitory function may facilitate stress-induced meconium passage at term. to evaluate the effect on blood pressure (bp), urinary output (uop), sodium excretion (nae) and gfr of the intrarenal infusion of ang ii in the male sheep after prenatal exposure to b. methods: we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation. catheters were placed in the femoral artery and vein, left renal artery and in the bladder. a right side nephrectomy was performed. after days of recovery, the sheep were infused with ang ii ( ng/kg/min) with or without candesartan ( ng/kg/day) or pd (pd g loading dose and ng/kg/min infusion) into the renal artery over hours. bp, gfr, uop and nae were measured before and after the infusion. two-way analysis of variance was used to test mean values between the groups. results: bp and uop did not change with ang ii infusion with or without candesartan. the infusion of pd did not affect uop but did increase bp in the b sheep (p= . ). ang ii decreased the nae in both groups(p= . ). candesartan increased nae among controls(controls . and b . meq/kg/h, p= . ). pd infusion decreased nae among controls but this was not significant. baseline gfr was higher among vehicle compared to b animals (p= . ). during ang ii infusion there was a decrease in gfr among control compared to b sheep (- . vs. - . ml/min, p= . ). this difference was not seen during candesartan or pd infusion. conclusion: ang ii infusion led to a decrease in nae in both groups and gfr among controls but not b animals. infusion of ang ii with candesartan increased nae while pd decreased nae among controls but not b animals. this suggests that prenatal steroid exposure can alter at receptor mediated response in renal function by decreasing the effect of ang ii on renal sodium excretion. the differences in gfr suggest that this may be due to an effect on tubular sodium reabsorption rather than glomerular perfusion. antalarmin antagonism of acth-induced cortisol secretion by ovine adrenal cells probably not at acth receptor. nancy k valego, james c rose. center of research for ob/gyn, wake forest u. school of medicine, winston-salem, nc, usa. in addition to regulating the hpa axis, crh and its type receptor (crhr- ) occur peripherally and in the female reproductive system. late in human pregnancy, a surge in placental crh probably stimulates adrenal secretion of cortisol and dheas requisite to parturition. we previously reported that, in dispersed fetal or adult ovine adrenal cortical cells, hour incubation with the specific crh-r antagonist, antalarmin (ant), significantly reduced both cyclicamp and cortisol responses to acth, suggesting an interaction with acth-r (like crh-r , a g-protein-coupled membrane receptor). however, forskolin (fsk; direct stimulant of adenylyl cyclase)-stimulated cortisol secretion was also attenuated by hour co-incubation with ant. our objective was to clarify the effect of ant on binding of acth to its receptor after a short ( . hours) treatment. method: acth binding: the adrenals from adult sheep were obtained at necropsy and the cortex cells dispersed and plated @ cells/well. after hours in culture, wells were rinsed x and refilled with serum-free dmem/ f containing vehicle (dmso) with or without ant. after . hours, wells were washed very gently and the binding assay (adapted from rainey et al; j biol chem, : , ) completed. triplicate vehicle or ant wells were treated with i -tyrosine human acth ( - ) with or without - m acth (for non-specific binding). after hour, wells were chilled, washed, and the cells lysed and counted on a gamma counter. fsk treatment: cells were treated as above except that fsk ( - m) was added to the wells. after . hours, medium was removed and stored @ - ºc for camp eia and cortisol ria. results (mean±se) of . hour incubation on acth binding and fskinduced secretion. vehicle antalarmin acth binding (net cpm) n= ± ± cortisol (ng/ml/ . hr) n= ± ± * (p=. ) camp (pmol/ml/ . hr) n= . ± . . ± . * (p=. ) * indicates significant difference from vehicle alone. conclusion: the crh-r antagonist, antalarmin, attenuates acthstimulated cortisol secretion but not by preventing acth receptor binding. however, its effect is early in the secretory process and is associated with a reduced camp response. objective glucocorticoids are often administered to pregnant women to prevent neonatal respiratory distress syndrome. prenatal steroid treatment increases blood pressure in adult sheep. exposure to excess corticosteroids before birth is hypothesized to be a key mechanism underlying the fetal origins of adult disease hypothesis and effects on the renin-angiotension system (ras) may modulate the steroid-induced increases in blood pressure. we therefore sought to determine if renin processing and secretion were altered in adult female sheep exposed antenatally to betamethasone ( ) and to compare them with data from males studied previously. methods pregnant sheep were randomized to receive doses of . mg/kg of or vehicle, at and days of gestation; the offspring were studied at and months of age. in female offspring, active renin concentration (arc) and total renin concentration in plasma were measured by ria of angiotensin i generated by incubation with excess substrate. prorenin concentration (prc) is the difference between total and active renin. nine or control exposed female animals born at term ( - days of gestation) were brought from the farm at - months of age, and had vascular and bladder catheters placed. five days after surgery, a sodium load of hypertonic nacl ( . meq/kg/min at . ml/min) was given for min. blood samples were obtained. data are expressed as mean sem and were analyzed by t test. the prc was significantly higher in the females than in the males ( . ± . vs . ± . p< . ) but there was no effect of prenatal steroid treatment. arc was similar in both genders. however, arc was a significantly greater percent of the total plasma renin concentration in the males ( . ± . vs . ± . p< . ) during the na infusion experiment, the exposed females had lower arc than did the control females ( . ± . vs . ± . p< . ). conclusion the data suggest that prenatal exposure to didn't alter the processing and secretion of renin in adult female sheep. prenatal steroid treatment does not appear to alter the effect of gender on plasma renin levels in adult sheep.it seems unlikely that the elevated blood pressure seen in adult ewes after prenatal exposure is the result of increased secretion or processing of renin. supported by hd and hd . background: mrap is a recently discovered protein with alpha and beta isoforms, a common amino terminal region and divergent c-terminal sequences generated by alternative splicing. in human and mouse mrap plays an essential role in the generation of a functional, g-protein coupled acth receptor (melanocortin- receptor; mc r) but has not been described for ruminants. we previously reported that lth fetal sheep exhibited elevated basal acth - yet decreased expression of key steroidogenic enzymes and the mc r in the adrenal cortex while basal cortisol levels were not different from control. we hypothesized that mrap could play a key role in mediating the effect of lth as well as developmental regulation of fetal adrenal cortisol synthesis in fetal sheep. the goals of the present study were to determine the ontogenic pattern of mrap expression in the sheep fetus and to determine if lth alters mrap expression. methods: we searched the bovine genomic sequence database (www.ncbi.nlm. nih.gov/genome/guide/cow/) and found a sequence with > % homology to the human mrap n-terminal residues, with partial homology to the beta isoform in the carboxyl region ( %). using primers based on the bovine sequence, we confirmed the presence of mrap in the ovine fetal adrenal cortex. adrenal cortical tissue was collected from sheep fetuses from - (n= ) and near term ( - ; n= ) days of gestation (dg) as well as from lth (n= ) fetuses (exposed to high altitude hypoxia from to - dg) and age-matched normoxic controls (n= ). cyclophilin was used as a housekeeping mrna. data are expressed in fg mrna/ ng total rna. results: the expression of mrap, based on quantitative rt-pcr, was low between - dg ( . ± . ) and increased (p< . ) approx. -fold near term ( - dg; . ± . ). levels of mrna for mrap were highly correlated to cyp mrna in individual samples. mrap expression in control ( . ± . ) and lth ( . ± . ) did not differ between groups. gender differences in hypertension are well described and there is growing evidence that the regulation of the renin angiotensin system (ras) is influenced by sex hormones. the major enzymes of the ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the peptide products of these enzymes have opposing actions. angiotensin ii (ang ii), the product of ace, is a potent vasoconstrictor. on the other hand, the peptide products of ace and neprilysin, ang ( - ) and ang ( - ), exhibit vasodilatory properties. thus, modifications in the relative proportions of these enzymes and their peptide products can result in alterations of systemic blood pressure. the purpose of this study was to describe the gender differences in angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin (nep) enzyme activities in adult sheep kidney cortex. renal cortex from male (n= ) and female (n= ) sheep were harvested at - months of age. the tissue membranes were solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were then separated by reverse-phase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was then quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results ace activity was over times greater ( ± . vs. . ± . fmol/mg/min, p< . ), ace activity was . times greater ( ± . vs. . ± . fmol/mg/min, p= . ) and nep activity was nearly times greater ( . ± . vs. . ± . fmol/mg/min, p= . ) in female kidney cortex. in adult sheep, key enzymes of the intrarenal ras have signficantly greater activity in female kidney cortex. these findings suggest that there are fundamental, gender specific, differences in the regulation of the enzymes of the intrarenal ras. the physiologic significance of these findings remain to be elucidated. hd , hd . in rats and sheep exposure to glucocorticoids (gc) in the perinatal period is associated with a reduction in nephron number and hypertension in adult life. furthermore, antenatal exposure to gc alters glucose tolerance in animals and in people. the aim of the present study was to determine ) if insulin resistance is a contributing factor for the development of hypertension in adult sheep exposed antenatally to gc and ) if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hours apart at days gestational age and allowed to deliver at term. at mo of age, female sheep were randomly allocated to be fed at either % of recommended nutritional allowance or ad libitum for three months. sheep were chronically instrumented under general anesthesia to place intravascular catheters. insulin sensitivity was evaluated both by iv glucose tolerance test (ivgtt) and euglycemic clamp (hec)techniques. for the ivgtt a . g/kg glucose bolus was used and for the hec mu/kg human insulin was used. data mean±sem were analyzed by anova and/or two sample t test. results: ad lib fed sheep gain > % of the original weight. antenatal bm was associated with an elevation in basal and ivgtt plasma insulin values. as shown on the figure, diet-induced obesity significantly increased insulin plasma levels during ivgtt in bm-exposed adult female sheep (f= . ;p< . ). insulin sensitivity derived from hec was significantly decreased by obesity in bm-exposed adult female sheep. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on glucose metabolism regulation. bm-exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. these abnormalities are exagertated by diet-induced obesity. hl . syngergistic induction of -hydroxysteroid deyhdrogenase type expression by cortisol and interleukin- in human fetal lung fibroblasts. z yang, p zhu, cm guo, l myatt, k sun. school of life sciences, fudan university, shanghai, china; obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. objectives: glucocorticoids acting via glucocorticoid receptor (gr), serve as crucial hormones in fetal lung maturation. glucocorticoids and proinflammatory cytokines to induce b-hydroxysteroid dehydrogenase type ( b-hsd ) which converts inactive cortisone to active cortisol, but their effect on b-hsd expression has not been addressed in human fetal lung. we examined the interactions and mechanism of cortisol and interleukin- b (il- b) effect on b-hsd in human fetal lung fibroblasts (hfl- cells). methods: the expression of b-hsd in hfl- was examined with immunocytochemistry and pcr. b-hsd , prostaglandin h synthase- (pghs- ) and cytosolic phospholipase a a (cpla ) mrna levels in cultured human fetal lung fibroblasts treated with cortisol and il- b were measured with real time pcr. the roles of gr and c/ebps in the effect of cortisol and il- b were studied using a gr antagonist (ru ) and transfection of plasmid carrying c/ebp-specific dominant-negative gene (cmv -a/cebp). results: hfl- cells expressed a high level of b-hsd . both cortisol ( - - - m) and il- b ( . - ng/ml) induced b-hsd mrna expression in a concentration-dependent manner, an effect blocked by the mrna transcription inhibitor , -dichlorobenzimidazole riboside ( μm). ru ( - m) blocked the induction of b-hsd by cortisol. induction of b-hsd mrna expression by cortisol ( - m) was synergistically increased by co-treatment with il- b ( . - ng/ml) in a concentration-dependent manner. in contrast, the induction of cpla and pghs- expression by il- b was inhibited by cortisol, suggesting a different mechanism of interaction. transfection of the cells with c/ebp-specific dominant-negative plasmid attenuated induction of b-hsd mrna expression by either cortisol or il- b. these data suggest induction of b-hsd expression by cortisol is a gr dependent process involving c/ebps, which also mediate induction of b-hsd expression by il- b. conclusions: cortisol and il- b synergistically induce b-hsd expression in human fetal lung fibroblasts. this would lead to greater local cortisol production, perhaps providing either a self-attenuating mechanism for control of inflammation or a mechanism for enhancing fetal lung maturation when the fetus is exposed to cytokines e.g. with infection-induced preterm labor. do alterations in placental -hydroxysteroid dehydrogenase ( hsd) activities explain differences in fetal hypothalamic pituitary adrenal function following periconceptional undernutrition or twinning in sheep? kl connor, pl van zijl, cw rumball, , al jaquiery, , je harding, , mh oliver, , fh bloomfield, , jrg challis. medicine, university of toronto. periconceptional undernutrition (pcun) leads to activation of fetal hypothalamic pituitary adrenal (hpa) function, whereas twinning results in delayed fetal hpa activation. we hypothesized that these differences in fetal hpa activity were the result of altered patterns of expression of placental of hsd isozymes and hence of the maternal glucocorticoid (gc) effect on the fetus. we developed a mass spectrometric assay for the measurement of hsd- and - activities and validated this method against a widely used thin layer chromatography method. sheep were randomly assigned to ad libitum (n control) concentrates throughout gestation or were undernourished (un) from days before until days after mating to reduce maternal body weight by %, with ad libitum feeding thereafter. placentomes were collected on days , , , and of gestation (term, d) and hsd- and - activities were determined by measuring the rate of interconversion between cortisone to cortisol. with un, there was a trend towards lower hsd- activity at d (p= . ), a significant reduction at d (p< . ), but no difference at d or d . hsd- activity was not different between n and un animals at any time. there was no effect of twinning on hsd- or - at d . however, with twins both hsd- (p= . ) and - (p< . ) activities increased at d . hsd- activity was reduced in twins (p= . ) at d but was higher than any singleton pregnancies at d (p= . ). hsd- was not different between singletons and twins at either d or d . overall, hsd- was lower in placentae of male compared to female fetuses in late gestation; hsd- was higher in male than female fetuses. there was no interaction with un in either sex. we conclude that pcun and twinning result in alterations of placental hsd activities in sheep. modifications in these enzymes during critical periods of fetal development may affect transplacental transfer or placental generation of gcs reaching the fetus potentially influencing the timing of activation of the fetal hpa axis, fetal maturation and later life development. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. control animals had ad libitum food. offspring were sacrificed on day of life (p ) and at months (n= - per group). adrenals were dissected and snap frozen in liquid nitrogen for later extraction of rna. real time rt-pcr using specific rat primers was used to quantify mrna levels ( s as control). we evaluated the expression of -beta hydroxylase (cyp b ), aldosterone synthase (cyp b ), acth receptor (mcr ), p side chain cleavage enzyme (cyp a ), star protein, -hydroxysteroid dehydrogenase type (hsd ) and type (hsd ), glucocorticoid receptor (gr), and mineralocorticoid receptor (nr c ). fold changes in mrna expression in controls and mfr offspring was compared by student's t-test. results: there was a marked downregulation in expression of cyp b (p=. ), cyp b (p=. ), hsd (p=. ), p (p=. ), acth receptor (p=. ), star (p=. ) and nr c (p=. ) mrna in p mfr offspring, with no changes in hsd and gr. gender specific differences were found in the adult mfr offspring. in the male mfr offspring the expression of hsd and gr were significantly upregulated with a trend towards an increase in acth receptor (p=. ) whereas in the female mfr the expression of acth receptor (p=. ) was increased and nr c (p=. ) and cyp b were decreased (p=. ). in combined data for adult male and female offspring the expression of acth receptor was significantly (p=. ) increased. conclusion: these results indicate that mfr has a suppressive effect on steroidogenic enzymes of the newborn offspring regardless of gender. this may be an adaptive mechanism in the fetus/newborn to offset the high circulating maternal glucocorticoids in response to undernutrition. in adult male mfr offspring, increased hsd indicates an increase in gc synthesis whereas in the females there were no changes in gc synthesizing enzymes with a suppression of mc synthesizing pathway. the common finding of an increased acth receptor expression in both genders would suggest an increased sensitivity of adult mfr offspring adrenals to the effects of stress. objective: during gestation the fetal adrenal undergoes phases of active growth ( - d), quiescence ( - d) followed by reactivation (> d) before birth. insulin like growth factors play an important role in stimulating adrenal growth throughout late gestation. interestingly the prepartum activation of the adrenal is delayed in the female compared with the male fetus, but the mechanisms underlying this delay are unknown. hypothesis: we hypothesize that there are gender specific differences in the gestational profile of adrenal igf mrna expression between male and female fetuses. methods: a total of twin fetuses were used in this study. post mortem was performed at either - d, - d or - d gestation. adrenal mrna expression of igf , igf , igf r, igf r and cyp was determined by qrt-pcr. results: fetal weight was not different between males and females, and increased (p< . ) with increasing gestational age. the relative adrenal weight was lower however after d when compared to the earlier gestation age group (p< . ). adrenal igf mrna expression was lower (p< . ) at - d when compared to the earlier gestation age groups. interestingly, igf r expression was highest at - d (p< . ). there was an interaction between the effects of age and gender on adrenal igf and igf r mrna expression such that the expression was higher in males compared to females at - d (p< . ), but was not different to either the earlier or later gestational ages. there was no effect of either gender or gestational age on adrenal cyp mrna expression. conclusions: it has been speculated that a delay in prepartum activation of the adrenal in female fetuses may be due to gender specific differences in the intra-adrenal bioavailability of igf . in this study we have demonstrated there is an increase in both igf and igf r mrna expression in males compared to female fetuses. this may be evidence that adrenal igf expression plays a role in the earlier activation of the adrenal gland in male fetuses. we have previously shown that in response to a secondary stressor, in vivo cortisol secretion is elevated in long term hypoxic (lth) ovine fetus despite lower acth receptor mrna expression, and no differences in plasma adrenocorticotropic hormone (acth) levels when compared to normoxic controls. the present study was designed to determine the potential mechanism(s) of this enhanced cortisol secretion. specifically we tested the hypothesis that post receptor signaling events including camp production and expression of steroidogenic acute regulatory protein (star) are enhanced following lth. methods: for the lth group, pregnant sheep were maintained at high altitude ( , m) from day to near term. on days - (term = days), fetal adrenal glands were collected from lth (n= ) and age-matched, normoxic signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. the roles of extracellular signal-regulated kinase / (erk / ) and p map kinase in the differentiation and invasion of human trophoblasts have been studied. however, the in vivo expression and activation of erk / and p at the placental bed has not been elucidated. the study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n= ) and with preeclampsia (n= ) between and weeks of gestation. we evaluated the expressions and phosphorylations of erk / and p map kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. results: p and phospho-p map kinase were not detected in invasive trophoblasts in cases or controls. erk / and phospho-erk / were positive in invasive trophoblasts albeit with variable staining. phosphorylation of erk / was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. conclusion: these findings suggest that preeclampsia is associated with decreased activation of erk / in invasive trophoblasts in vivo. objective: placentae from preeclamptic pregnancies are characterized by an excess of immature hyperproliferative trophoblast cells, however the molecular mechanisms regulating cell cycle progression in this pathology are unclear. our aim was to examine the expression of g phase cell cycle regulators in normal and preeclamptic placentae and to establish whether the hyperproliferative state of trophoblast cells in preeclampsia may result from a developmental delay. methods: human placental samples were collected from normal pregnancies throughout gestation (n= ) and from severe early onset preeclamptic (n= ), and age-matched control placentae (n= ). protein expression of cyclin e , cyclin d , cyclin d and cell cycle inhibitors, p , p , p , and p was assessed by western blot analysis. spatial and temporal localization of cyclins e , d , d , p and p was determined by fluorescence immunohistochemistry. expression of cyclin e and cyclin d mrna was evaluated by qpcr analysis. results: immunohistochemical analysis showed cyclin e and cyclin d to be localized to cytotrophoblast (ct) cells of the chorionic villi; additionally cyclin e was also expressed in the extravillous trophoblast cells of the anchoring columns. in contrast, cyclin d expression was predominantly restricted to the villous stroma. cell cycle inhibitor p was expressed in both ct and syncytiotrophoblast (st) cells whereas p was restricted to the st. during normal placentation, levels of both cyclin e and cyclin d were high in the first trimester and decreased with advancing gestation. the expression of cyclin d , p and p showed an inverse correlation to cyclins e and d whereby their expression increased towards term. levels of p and p remained constant throughout pregnancy. preeclamptic placentae showed a significant increase in both cyclin e and p and a decrease cyclin d and p expression, as compared to age-matched control tissues. interestingly, preeclampsia was associated with an increased number of cyclin e positive progenitor ct cells in the floating villi and an increased expression of p in the endothelial cells lining the villous vessels. conclusion: preeclampsia displays an altered expression of g phase cell cycle regulatory molecules portraying an expression profile that closely resembles that of first trimester placentae. (supported by cihr, owh/igh). we have also demonstrated elevated, hif- -mediated placental and circulating sflt- at high altitude. we sought to correlate circulating levels of plgf, free vegf and sflt- with maternal and fetal oxygen tensions. we hypothesized that circulating sflt- and free vegf would be increased at m, and that plgf, known to be up-regulated by higher oxygen tension, would be decreased. methods: we collected both serum and plasma samples, the latter treated with inhibitors of platelet activation. maternal and umbilical samples were obtained from and healthy mother-infant pairs living at m and m respectively. plgf, free vegf and sflt- were measured in both serum and plasma using commercially available elisa kits (r d). data were log-transformed and analyzed by unpaired student's t test or anova as appropriate. results: plgf did not differ between altitudes. free vegf ( ± pg/ml vs. ± pg/ml, p<. ) and sflt- ( . ± . vs. . ± . ng/ml) were increased at m. however concentrations of all the angiogenic growth factors were reduced when platelet activation was prevented (- ± % plgf; - ± % free vegf; - ± % sflt- , p<. ). cord blood free vegf was -fold greater than in the mothers, but inhibition of peripheral cell activation abolished detectable levels in % of the babies. similar results were obtained for sflt- . thus, while free and total maternal circulating vegf and sflt- are elevated at high altitude, these increases are due to activation of peripheral blood cells. moreover, free vegf does not exist in detectable amounts in human pregnancy. there was no relationship between variation in the angiogenic growth factors and maternal or fetal oxygen tensions. the objective of this study is to identify candidate genes responsible for the variance between severe preeclampsia (spe) and hellp syndrome (hs). placental biopsies from spe (n= ) and hs (n= ) were collected. diagnosis of spe was confirmed by blood pressure and protein criteria. hs was diagnosed in preeclamptic patients who developed characteristic laboratory abnormalities. placental tissues were embedded in oct for sectioning, h e staining and rna isolation (invitrogen, carlsbad). gene expression data was obtained by hybridizing fluorescently labeled reverse transcription products to spotted cdna microarrays. the microarrays were produced by the laboratory of microarray technology at the van andel institute (grand rapids, mi) using a custom microarrayer. microarrays were scanned using an agilent g b scanner. images were analyzed using genepix . (axon). limmagui was used to generate lists of discriminating genes for these data, while david provided functional annotations. there were differentially expressed genes between spe and hs (p<. ). among these candidate genes, were up-regulated and were downregulated. the most up-regulated genes are ( )-deoxyribonucleotidase (dnt- ), superoxide dismutase , hydroxy-delta- -steroid dehydrogenase, caspase , and general transcription factor iih. further analysis of functional groups revealed the most enriched gene categories are related to cellular energy (mitochondria), cell cycle regulation, and protein metabolism. the underlying role of the placenta in the development of hs is currently unknown. this study shows that there is a relatively mdest number of genes that are differentially expressed between hs versus spe placentals. thes data provide the molecular context for placental changes seen in this variant of preeclampsia and provides an opportunity to study the role of the effected genes in disease variance. ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and control group ( patients) uncomplicated pregnancies. total rna was isolated and reversely transcribed to c-dna. the mrna level of tert was detected with a probe-specific real-time quantitative pcr assay using ß-actin as the reference gene. crossing point (cp) values were obtained during the pcr amplification and the relative expression level of htert equals to (cp htert -cp ß-actin) . statistical analysis was performed using the student's t test. immunohistochemistry (ihc) staining was employed to localize htert protein on placenta tissue sections using abc method, incubation with rabbit anti-htert antibody followed by application of a goat anti-rabbit antibody with results evaluation using microscope. objective tgf s are involved in the regulation of trophoblast differentiation and invasion, and we have previously reported that tgf- is over expressed in pre-eclamptic placentae. tgf s signal via a receptor complex composed of type ii (t r-ii) and type i (t r-i) receptor. to date, seven type i receptors, designated as activin receptor-like kinase (alk - ) have been identified. our aim was to investigate the expression pattern of alk and alk receptors, known to exert tgf signaling, in preeclamptic and iugr placentae. methods human placental tissue throughout gestation was used in order to determine the development profile of the receptors. in addition, placental tissue from preeclamptic and iugr pregnancies and from age matched controls was collected. all iugr pregnancies were characterized by absence of end diastolic velocity in the umbilical artery and had no evidence of preeclampsia. expression of alk and alk mrna was measured by real-time pcr analysis, and protein by western blot analysis using alk and alk antibodies. immunoblot analysis demonstrated a unique developmental profile whereby alk expression increased with advancing gestation. in preeclamptic placentae alk expression was significantly increased compared to preterm and term controls, whereas alk expression was significantly decreased. preeclamptic placentae also exhibited decreased phosphorylation of smad , a tgf signaling molecule, which is activated by alk and increased phosphorylation of smad , which is triggered by alk . the expression of alk and alk in iugr placentae differed from that of preeclampsia as both alk and alk mrna levels were significantly increased in iugr compared to preterm and term controls. however, only alk expression was significantly increased in iugr placentae at the protein level, while no differences in alk protein levels were noted between iugr and controls. conclusions imbalance between alk and alk signaling pathways might play a role in the pathogenesis of preeclampsia and iugr. as tgf signaling via either alk or alk has been found to differentially regulate vasculogenesis, changes in these signaling pathways may contribute to the altered vasculogenesis found in these pregnancy-related disorders. (supported by cihr and owh/igh). ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and patients, the control group who had uncomplicated pregnancies. total protein was isolated from placental tissue. gadd a and its downstream signal proteins--phospo-p , phospho-mkk /mkk were assessed by western blot. immunohistochemistry (ihc) staining was employed to localize the expression of gadd a and sflt- proteins in placenta tissue sections using abc method. huvec cells were cultured to - % confluence and were divided into groups: control, stress induction (sorbitol, . m, h), p inhibition (sb- , ug/ml, ug/ml and ug/ml, hour) + sorbitol ( . m, h). total protein was isolated from cells and the supernatant of huvec was collected. western blot was processed to detect the induction of gadd a and phospho-p . supernatant sflt- was measured with an eia kit and the results were read at nm wavelength. results: gadd a protein was elevated in the preeclamptic placentas with its downstream proteins (mkk and p ) activation compared with control. overexpression of gadd a and sflt- in preeclamptic placentas was observed with ihc staining. in huvec cells, gadd a was induced by sorbitol, triggering the activation of the downstream p- pathway and the accumulation of sflt- in the supernatant. the up-regulation of sflt- secretion by inducing gadd a was depleted when treated with p- inhibitor. conclusions: our study reveals that gadd a and its down stream p- pathway were activated in preeclamptic placentas and this stress inducible signal pathway regulates the secretion of sflt- , which is a key player in preeclampsia. it provides novel evidence that links placental stress to sflt- secretion via the gadd . trophoblast adipose triglyceride lipase (atgl) expression is upregulated in preeclampsia. beth a plunkett, jennifer a doll, emily j su, serdar e bulun, mona cornwell, susan e crawford. obstetrics and gynecology, northwestern university, chicago, il, usa; pathology, northwestern university, chicago, il, usa. objective: preeclampsia is characterized by placental endothelial cell dysfunction and elevated maternal triglyceridemia (tg). tg traverse the placenta in a process of uptake followed by lipolysis with subsequent release of fatty acids to the fetus. although three placental lipases (hormone sensitive lipase, endothelial lipase and lipoprotein lipase) have been identified, they do not account for all lipolytic activity. here, we introduce a new lipase, adipose triglyceride lipase (atgl), which is responsible for the hydrolysis of triglycerides to diglycerides in adipocytes and was recently identified as a receptor for endothelial cell modulator pigment epithelium-derived factor. the purpose of this study is to determine if expression of atgl, a potential modulator of both lipid metabolism and vasculature, is upregulated in preeclampsia. methods: immunohistochemical studies were performed on placental tissues from normal pregnancies (n= ) and those complicated by severe preeclampsia (n= ) with anti-atgl antibodies. the degree of positivity in the trophoblasts and endothelial cell was scored ( =none, =spotty, light, =consistent, dark). to determine if atgl is a product of placental endothelial cells (plec), microvascular cells were isolated from normal placental tissue. purity of the sample was confirmed using flowcytometry (> / positivity for factor antigen). atgl was detected immunohistochemically and via western blot using anti-atgl antibodies. results: mean atgl expression in preeclamptic trophoblast was significantly higher than normal placentas ( . + standard deviation versus . + . , p = . ). endothelial expression was not significantly different in preeclamptic ( . + . ) versus normal placentas ( . + . , p> . ). atgl stained intensely and demonstrated a beaded pattern in the endothelial cells, suggestive of a lipid droplet pattern. immunohistochemistry of plec and western blot analysis of cell lysates revealed strong immunopositivity for atgl, although at a smaller size than anticipated. conclusion: these findings demonstrate that a novel lipase, atgl, is produced by trophoblasts and is upregulated in severe preeclampsia. plec express high levels of atgl, suggesting that atgl could prove to be important in the vascular dysfunction and lipid abnormalities characteristic of preeclampsia. increased endothelial chymotrypsin-like protease (chymase) expression is responsible for endothelial activation in preeclampsia. yuping wang, yang gu, yanping zhang, david f lewis. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: endothelial (ec) activation is an important component of inflammatory phenotypic changes in preeclampsia (pe). our previous study showed enhanced chymotrypsin-like protease (clp)/chymase expression in the maternal vessel endothelium in women with pe. in this study, specific effect of placental-derived clp on ec activation was examined. methods: human uterine microvascular endothelial cells (utmvecs) were used. placental conditioned medium (cm) was prepared by culturing villous explants from normal and pe placentas. confluent utmvecs were treated with placental cm with or without depletion of chymotrypsin. ec adhesion molecule expressions for icam, vcam, p-selectin and e-selectin were determined by a colorimetric assay at od nm. depletion of chymotrypsin from cm was performed by immunoprecipitation. to further determine if activation of endogenous clp/chymase in ecs is responsible for up-regulation of p-selectin and e-selectin expression, chymase sirna was applied to ec culture before the cells were treated with normal or pe cm and then ec adhesion molecule expressions were examined. data was expressed as mean ± se and analyzed by anova. a p level < . was set for statistically different. results: ) expressions of vcam, p-selectin and e-selectin, but not icam, were significantly increased in pe-cm treated utmvecs compared to those of normal-cm treated cells and untreated controls, p< . ; ) there was no difference for adhesion molecule expression in utmvecs between normal-cm treated with untreated controls; ) utmvecs transfected with chymase sirna significantly reduced p-selectin and e-selectin expressions when exposed to pe-cm, p< . . conclusion: placental-derived clp/chymase is responsible for activating ecs and inducing ec adhesion molecule expression. activation of ec chymase may be directly related to the inflammatory phenotypic changes that occur in ecs in pe. (supported nih grants hd and hl ). corin is a transmembrane serine protease that is important in processing natriuretic peptides (nps) and maintaining normal blood pressure. genetically modified mice without corin function develop a syndrome during pregnancy similar to preeclampsia. corin is present in the pregnant uterus and a deficiency in the enzyme may lead to hypertension and preeclampsia. we tested the hypothesis that corin expression was increased in human myometrium from women with preeclampsia. methods: myometrium was obtained from groups of women at the time of primary cesarean section: (i) preterm no labor with preeclampsia (n= , ptspe, . weeks); (ii) preterm labor (n= , ptl, . weeks); (iv) term no labor (n= , tnl, . weeks); (v) term labor (n= , tl, . weeks). microarray gene profiling was performed using affymetrix human genome u plus . arrays. women who were not in active labor at the time of delivery (tnl) served as the control group. conventional and real time pcr was performed to verify corin expression in the arrays was directionally accurate. corin protein expression was examined by western blot. results: compared to tnl control, corin levels decreased nearly -fold in myometrium from women in term labor (tl). there was a small increase in corin gene expression of preeclamptic women (ptspe) and little-to-no change in myometrium from women in preterm labor (ptl). these results were confirmed by pcr analysis. conclusion: blood volume surges during pregnancy, increasing the potential for hypertension and preeclampsia in the mother. the corin-np control system could contribute to this condition. however, there are no reports on corin message or protein levels in women with preeclampsia. our preliminary results suggest that preeclampsia is not a consequence of a corin deficiency (decreased corin preeclampsia). acknowlegement: supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). the expression of gp at implantation site: implication for the pathogenesis of preeclampsia. objective: gp is a common shard signal transducing subunit of the il- cytokine family which is critical for implantation, and viewed as marker of endometrial blastocyst receptivity. preeclampsia (pe) is highly related to the restricted trophoblast invasion, which leads to impaired spiral artery remodeling. our hypothesis was that the poor placentation of pe is associated with the altered expression of gp at the implantation site. methods: human decidua from patients with pe and uncomplicated term deliveries (n = , respectively) were immunostained for gp . the intensity and distribution of immunostaining on decidual cells and extravillous traphoblasts were evaluated with hscore. statistical analysis of the data was performed using student's t-test and kruskal-wallis one way anova on ranks followed by post hoc test. results: immunostaining of gp was significantly higher in decidual cells of patients with pe compared with normal specimens, with hscore (median, [interquartile range]) value [ . - . ] and , respectively (p< . ). in pe specimen, the hscore of decidual cells was also significantly higher than that of trophoblast ( [ - ]; p< . ). there were no difference in the comparison of hscore of trophoblasts between the pe and normal specimens, and in normal specimens between decidal cells and trophoblasts. conclusions: the increase of gp expression in the decidual cells of preeclamptic placenta may be implied to the pathogenesis of poor placentation in preeclampsia. we have previously demonstrated that decidual cells are the major source of renin at the human uteroplacental interface, but little is known regarding the human decidual renin expression in hypoxic conditions. therefore, the present study was undertaken to determine the effect of hypoxia on renin secretion by human decidual cells in vitro. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were exposed to normoxia or hypoxia ( % or % oxygen) in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. a dominant band of renin at approximately kd was detected in all samples. when compared with the cells cultured in the normoxic condition, the cells cultured in both hypoxic conditions (i.e. % and % oxygen) had significantly lower renin protein contents in their culture supernatants. conclusion: our data for the first time show that hypoxia down-regulates renin secretion in human decidua, suggesting a link between the uteroplacental ras and oxygen tension during human gestation. the effect of nucleated fetal red blood cells derived from preeclamptic patients on endothelial progenitor cell proliferation. keiichi matsubara, emiko abe, yuko matsubara, shinji hyodo, masaharu ito. obstetrics and gynecology, ehime university school of medicine, toon, ehime, japan. objectives inadequate uteroplacental circulation results in placental ischemia and the development of preeclampsia (pe). endothelial progenitor cells (epcs) are thought to be a key player in the fetal angiogenesis. vascular endothelial growth factor (vegf), which is up regulated in pe, is involved in epcs proliferation. recently, it was reported that fetal nucleated red blood cells (nrbcs) have the capability to generate vegf. we hypothesized that nrbcs could influence epcs proliferation in the placenta and may be involved in the pathogenesis of pe. material and methods mononuclear cells (mncs) were isolated from the umbilical venous blood of normal pregnant women and preeclamptic patients by density gradient centrifugation. mncs were incubated with anti-cd antibody conjugated with microbeads. nrbcs were collected using a mini macs separator. nrbcs were incubated for hours with or without angiotensin ii (ang ii) and erythropoietin (epo). vegf and placental growth factor (plgf) concentrations in the supernatant were measured using elisa. also, pbmcs without nrbcs were seeded in endothelial basal medium with or without nrbcs using a boyden chamber. these samples were incubated for days with or without ang ii and epo. the adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. di-ldl and lectin positive cells was considered to represent epcs and the number was measured using flowcytometry. the number of nrbcs derived from umbilical venous blood was significantly increased in pe. both ang ii and epo significantly increased vegf concentration in the supernatant of nrbcs derived from normal pregnant women. however, ang ii and epo did not influence the nrbcs' vegf production in pe patients. plgf was not detectable in the supernatant. the number of epcs in the umbilical venous blood was significantly decreased in pe and the number was not changed by nrbcs. on the other hand, the number of epcs was significantly decreased in the culture with nrbcs. epo significantly increased the number of epcs in pe at the lower concentration of epo compared with normal pregnant women. conclusions it appears that fetal nrbcs may inhibit fetal epcs proliferation in pe. since epo reduced the inhibitory reaction of nrbcs without vegf production epo may affect epcs proliferation independently of nrbcs. relationship between the hypoxia-inducible factor- (hif- ) and the receptor svegf-r /sflt- : implication for phatophysiology of preeclampsia. julio e valdivia-silva, , juan c gonzalez-altamirano, keisy lopez- the trophoblast invasion is critical for the establishment of the uteroplacental circulation. at early phases of this process local oxygen pressure in the placenta is lower, that pathologically in preeclampsia remain constant. because of this, is important to understand the response of placental cells against these stimuli. in the present work, we use primary cultures of trophoblast cells, fibroblasts of the villous, and human umbilical endothelial cells, isolated of preterm and term placentas (with and without preeclampsia), to explore the effect of the oxygen pressure in the expression and synthesis of vegf, svegfr- /sflt- , hif- and- . our results show that the low pressure of oxygen resulted in a significant increase of the mrna and the protein of the receptor svegf-r selectively in the cts. the vegf's expression and synthesis was raised in three cellular types, but the free protein (not bounded to svegf-r ) of the cts was diminished. on the other hand, the expression of the arnm of hif- or - in cells was comparable in all the types of placentas, nevertheless, the protein hif- was more increased in the cts of preeclamptic placentas. to evaluate the relation of hif- and the increase of the receptor svegfr- , we used sirna-hif . in response to the inhibition, the expression of the receptor svegf-r diminished dramatically. the blockade of hif- did not alter vegf's expression. our data are the first that propose that the protein of the factor of transcription hif- is one of the molecules involved in the selective expression of the receptor svegf-r in trophoblast cells during hypoxia. figure: reduction of the expression to soluble receptor flt- /svegfr- after transfection with sirna-hif in trophoblast cells. sirna= interference rna, lu=luciferase. luc-sirna was used as control. effects of estradiol on synthesis, secretion, and activation of von willebrand factor in endometrial endothelial cell. shumei zhao, chainarong choksuchat, michael s scholfield, todd d deutch, thomas d kimble, david f archer. obstetrics and gynecology, eastern virginia medical school, norfolk, va, usa. objectives: heavy menstrual bleeding (hmb) is a serious clinical condition affecting % of women. due to inefficient and ineffective medical interventions, women with hmb often elect endometrial ablation or hysterectomy to eliminate hmb symptoms. morbidity and loss of fertility linked to these surgical treatments support the search for more effective medical remedies. von willebrand factor (vwf), a principle initiator of blood clotting produced by endothelial cells. women with von willebrand disease (vwd), have a high incidence of hmb indicating poor clotting. these findings suggest vwf heavily impacts the amount of blood loss during menstruation. estrogen stops/reduces hmb and has been used to treat hmb in women with vwd, although the mechanism(s) is unknown. this proposal addresses the hypothesis that estradiol (e ) increases the synthesis and activation of vwf, promoting clotting. materials and methods: immortalized human endometrial endothelia cells (heecs) were used for the studies. to determine if e increases synthesis of vwf, we treated heecs with e at . μm, . μm and . μm for hours. vwf protein and mrna levels were determined by western blotting and real time pcr, respectively. to establish if e can convert vwf from an inactive to an active conformation, the release of activated vwf by cells into culture medium will be assessed by elisa. decreased adamts (a disintegrin and metalloproteinase with thrombospondin type motif) activity results in increased amounts of active vwf. rrelease of activated adamts will be determined by fret. to ascertain if e regulated vwf secretion is by genomic pathway, heecs will be exposed to the estrogen receptor antagonist ici , . elisa and fret will assess the release of active vwf and adamts , respectively. results: western blotting demonstrated e increases vwf mrna and protein levels in a dose-dependent manner in heecs.conclusion: the project will determine if e acts at the heecs to increase synthesis, secretion and activation of vwf. preliminary results show e increases intracellular vwf protein and mrna levels in heecs, supporting our hypothesis that e increases synthesis of vwf in heecs in vitro. if e increases activated vwf, it could reduce/stop hmb by increasing activated vwf, providing justification for a clinical trial of e to treat hmb. over-expression of vegf-d does not induce lymphangiogenesis in the mouse endometrium. peter a rogers, jacqui f donoghue, marc g achen, steven a stacker, jane e girling. obstetrics gynaecology, monash university, melbourne, victoria, australia; ludwig institute for cancer research, melbourne, victoria, australia. background: the human endometrial functionalis has reduced lymphatics compared to the basalis and myometrium . this study examines the distribution of lymphatics in mouse uterus and investigates if over-expression of the lymphangiogenic growth factor vascular endothelial growth factor-d (vegf-d) stimulates growth of new endometrial lymphatic vessels. methods: the distribution of uterine lymphatics was examined in c bl/ jxcba mice collected during the oestrus cycle, early pregnancy and following oestrogen and progesterone treatment. human ebna cells with/without stable transfection of vegf-d were injected into the uterine horn of nod/scid mice. uteri were collected after weeks. serial sections were immunostained with lyve- and/or vegfr (lymphatic endothelial cell markers), cd (blood endothelial cell marker), mab (human vegf-d), mab (human mitochondria) and pcna (proliferative cell nuclear antigen). results: lymphatic vessel profiles were mostly found in the connective tissue between the longitudinal and circular muscle layers of the myometrium. they were rare in the endometrium and only observed in % of the sections. when present in endometrium, lymphatic vessel profiles were usually situated adjacent to the endometrial/myometrial border. ebna tumours formed inside and outside the uterine horn of both the control (n= of ) and vegf-d group (n= of ). localization of ebna cells within the mouse uterus was confirmed by anti-human mitochondrial expression. vegf-d immunostaining confirmed that transfected ebna cells expressed vegf-d in vivo. over-expression of vegf-d did not stimulate endometrial lymphangiogenesis, although there was an increase in vessel diameter of lymphatics in the myometrium adjacent to tumours. initial analysis shows no significant effect of vegf-d ebna cells on endometrial blood vascular density or endothelial cell proliferation. conclusions: minimal lymphatics are present in the mouse endometrium, as is the case for lymphatic vessels in the human endometrial functionalis. the lack of endometrial lymphangiogenesis in response to vegf-d suggests the presence of an inhibitory factor limiting lymphatic growth in this tissue. in early pregnancy, decidual-derived vegf mediates angiogenesis and is required for implantation and placentation. the role of decidual vegf in later pregnancy is poorly understood. decidual hemorrhage (placental abruption) generates excess thrombin and is a major risk factor for pprom and preterm birth. this study compares immunohistochemical (ihc) localization of vegf in decidual tissue sections from term pregnancies complicated by abruption and gestational age-matched controls, and investigates the effect of thrombin on vegf expression by cultured human term decidual stromal cells (dscs). study design: ihc was performed on serial sections of term placental tissues (no labor) with (n= ) and without (n= ) abruption. purified term dscs were passaged until > % free of cd + cells by facs. confluent dscs were primed with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days. after h incubation in defined medium with corresponding steroids ± thrombin ( . - . iu/ml), conditioned supernatants were analyzed for vegf by elisa. extracted total rna was used to assess vegf mrna levels by quantitative rt-pcr using established primers. results: vegf expression was localized by ihc primarily to dscs in placental tissue sections, and was increased in tissues from placental abruption vs controls. in term dscs, thrombin increased vegf secretion in a dosedependent fashion irrespective of the hormonal milieu (eg, . -fold stimulation by . iu/ml thrombin from . ± . to . ± . pg/ml per mcg protein for e +mpa; p= . ). this effect was abrogated by the thrombin inactivator, hirudin. vegf mrna were similarly increased by thrombin with or without steroid hormones (eg, . -fold for e +mpa; p< . ). conclusions: placental abruption is associated with increased vegf expression in term decidual tissues in vivo with thrombin enhancing vegf mrna and protein expression in term dscs in vitro. excess thrombinmediated vegf expression in term decidua aberrantly increases endothelial cell permeability to further generate thrombin by continuous exposure of tissue factor-expressing decidual cells to circulating factor vii. thrombin-enhanced matrix metalloproteinase expression in term dscs would degrade decidual and fetal membrane extracellular matrix to induce pprom and preterm birth. basal directional release of angiotensin ii by endothelial cells stimulated by chymotrypsin-like protease (clp)/chymase. yuping wang, david f lewis, yang gu. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: chymotrypsin-like protease (clp)/chymase is a serine protease which plays a major role in angiotensin ii (ang ii) generation in the human heart. our previous study showed a higher clp activity in the maternal plasma in women with pe than in normal pregnancies. we also found enhanced chymase expression in the maternal vessel endothelium in women with pe. in this study, we determined if clp could promote endothelial cell (ec) generation of ang ii. methods: we specifically examined basal directional release of ang ii by cultured ecs. ecs were grown on cell culture insert ( well/plate, micron pore size). when ecs reached confluence, chymotrypsin (chy) at concentrations of . , . , . , . , and . g/ml were added to the upper chamber of the cell insert. after hours of culture, medium in the lower chamber was collected. medium concentrations of ang ii were measured by enzymelinked immunoassay (eia). all samples were measured in duplicate. data are expressed as mean ± se and analyzed by anova. a p level < . was considered statistically different. results: chymotrypsin produced a concentration-dependent increase in basal directional release of ang ii by cultured ecs, control: . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml (p< . ); chy . : . ± . g/ml (p< . ), respectively. data are means from independent experiments. conclusion: apical exposure of ecs with chymotrypsin-like protease could promote basal directional release of ang ii. our result implicates that in pe, elevated clp levels in the maternal circulation are very likely to affect ec generation of ang ii. basal directional released ang ii may bind to its receptor on underlying vascular smooth muscle cells and contribute to the increased vasoconstriction in pe. (supported nih grants hd and hl ). vegf stimulates angiogenesis and vasodilation critical for dramatic rises in materno-feto interface blood flows directly linked to fetal growth/survival. extracellular signal-regulated kinase (erk / ) pathway mediates partially vegf-induced angiogenic and vasodilatory responses in placental endothelial cells (ec). it is, however, unknown how this vegf-induced signaling is organized in placental ec. objectives: ovine fetoplacental artery ec (ofpaec) and its transformed counterpart, sv -of to test whether: ) vegf-activated erk / signaling is compartmentalized in the caveolae and disruption of caveolae interferes vegf-induced erk / activation and; ) caveolin- , the structure protein of caveolae, regulates vegf-stimulated erk / phosphorylation. methods: ofpaec or sv -of cells were cultured in mcdb- / % fbs/antibiotics. serum-starved subconfluent ( %) cells were treated with rhvegf ( to ng/ml) for various times. caveolae were disrupted by -cyclodextrin ( -cd, mm, min) or caveolin- scaffolding domain (cav-sd, m, hr). sv -of cells were used for fractionation of caveolae membranes by discontinuous sucrose gradient ( %/ %/ %) ultracentrifugation. activation of erk / signaling pathway were analyzed by western-blotting with specific antibodies. results: in total cell extracts, vegf stimulates erk / phosphorylation in a time-and dose-dependent manner. erk / phosphorylation maximized by vegf ( ng/ml) at - min, which was abrogated by -cd or cav-sd. all the molecules for compromising the erk / signaling module, plc , pkc , src, ras, raf- , mek / and erk / , were detectable in purified caveolae membranes positive for various markers including caveolin- , enos, flotillin- , and -adaptin. in caveolae, vegf dramatically increased phosphorylated erk / without altering total erk / in a time-dependent manner similar to that in total cell extracts, which also maximized at - min. pretreatment with -cd or cav-sd blocked vegf stimulation of erk / phosphorylation in caveolae. conclusion: vegf activates the erk / signaling pathway in caveolae and caveolae integrity is essential for vegf-activated erk / signaling pathway. we conclude that caveolae/caveolin- serves as a platform for compartmentalizing the vegf-induced erk / signaling pathway in placental ec (hl and hl ). hypoxia upregulates gcm in human placenta. david mccaig, fiona lyall. institute of medical genetics, university of glasgow, glasgow, united kingdom. introduction: studies in transgenic mice have shown that a variety of genes regulate the differentiation of trophoblast cells. these genes include gcm . gcm is also expressed in the human placenta. placental gcm- protein has been reported to be reduced in pre-eclampsia, in view of the close link between hypoxia, hypoxia-reoxygneation, pre-eclampsia, placental development and the reported reduction in gcm we hypothesised that gcm expression would be affected by hypoxia. aim: the aim of this study was to determine the effects of hypoxia on gcm expression in the human placenta. two model systems were used; ( ) free floating villous explants and ( ) cultured primary cytotrophoblast and syncytiotrophoblast cells as described previously*. methods: explants or cell cultures were exposed to either hypoxia or hypoxia followed by re-oxygenation. western blot analysis was used to assess gcm protein levels. bands on the gels were quantified using scanning densitometry. statistical differences (n= experiments for both models used) were calulated by anova and turkey's post-hoc test. results: gcm protein was detectable at a low level in villous explants maintained for h in % o . a striking increase in gcm protein was observed when villous explants were incubated for h in % o (p< . ). incubation of villous explants for h in % o followed by re-oxygenation for h in % o resulted in a marked decline in gcm protein (p< . ). expression of gcm was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in % o or reduced oxygen ( % o ) conditions. gcm protein was not detected in any of the experimental conditions used. discussion: the present study has shown that acute hypoxia increases gcm- protein in villous explants. the experiments with purified trophoblast do not support a role for hypoxia increasing gcm- in these cells under the experimental conditions used. the present findings are in keeping with the complex effects of oxygen depending on the conditions used. the observed hypoxic effects on gcm warrant further investigation. the effect of acute alcohol exposure on histone -lys modification in the mid-gestation embryonic lung. xiangyuan wang, debra wolgemuth, , , laxmi baxi. ob/gyn; genetics development; human nutrition, columbia university medical center, new york, ny, usa. objective maternal alcohol abuse during pregnancy produces an array of birth defects comprising fetal alcohol syndrome. lung development depends on a balance between cell proliferation and apoptosis. we have previously shown that the acute alcohol exposure in the mid-gestation embryo can delay lung development and induce apoptosis. acute exposure to ethanol of selected tissues in mouse embryos has been reported by others to initiate apoptosis within hours after exposure and result in histone modifications. specifically, histone acetylation and deacetylation are involved in transcriptional activation and repression, respectively, but can also involve apoptosis. in the present study, we have investigated the effect of alcohol on acetylation of histone at lysine (ach lys ) in the mid-gestation embryonic lung. pregnant c bl/ j mice at day . of gestation (e . ) were injected intraperitoneally with doses of % ethanol ( . g/kg ), hr apart (alcoholexposed: ae) or with ringers solution (controls: c). ae and five c fetuses were retrieved and hr later and the lungs were fixed and processed for morphological evaluation and staining with rabbit polyclonal anti-ach ly antibody. the entire lung tissue field was evaluated for the levels of ach lys staining and scored as (-) to (++++). three areas were selected randomly from each sample and the total number of cells and staining positive cells were counted in the bronchial epithelium and in the mesenchyme. twelve hr after alcohol exposure at e . , the morphology of ae embryonic lungs was normal. however, high levels of ach lys were detected in % of the bronchial epithelial cells and % of the mesenchymal cells. the expression level in both lineages decreased hr after alcohol exposure. in the controls, the expression of ach lys was virtually undetectable in both the bronchial epithelium and the mesenchymal cells. our previous study showed that ae e . lungs significantly increased apoptotic cell in both bronchial epithelium and mesenchyme hours after alcohol treatment. we now observe that elevated expression of ach lys in the embryonic lung preceded the observation of apoptosis, suggesting that alteration in the acetylation of h could be one of the molecular mechanisms involved in the induction of apoptosis following acute alcohol exposure. objective: our purpose was to evaluate the effect of vitamin c and e supplementation on lipid peroxide levels, total antioxidant ability, and antioxidant levels in the umbilical venous plasma. materials and methods: women at risk for preeclampsia (nullipara, previous preeclampsia, chronic hypertension) were recruited at to weeksgestation and randomly assigned to receive either mg of vitamin c and iu of vitamin e (study group, n= ) or placebo (control group, n= ) daily until delivery. umbilical venous blood were collected after full term delivery. lipid peroxide levels, oxygen-radical absorbance capacity (orac) values, antioxidant levels were measured by each method (thiobarbituric acid reaction, cao's method, and high performance liquid chromatography). results: . the lipid peroxide levels in the umbilical venous plasma of study group were significantly lower than that of control group ( . ± . vs. . ± . nmol/mg protein, p< . ). . the orac values in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . u/ml, p< . ). . the -tocopherol levels in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . nmol/ml, p< . ). . there were no significant differences in the ascorbic acid, uric acid, -carotene, retinol, and -tocopherol levels in the umbilical venous plasma between control and study group. conclusion: this suggested that maternal vitamin c and e supplementation may affect the oxidant-antioxidants balance of the utero-placenta unit and fetus. placental tnf-related apoptosis-inducing ligand (trail) in normal pregnancy and pre-eclampsia. xilian bai, jenny e myers, philip n baker, john d aplin, ian p crocker. maternal and fetal health research group, the university of manchester. introduction: enhanced placental trophoblast apoptosis is well known to occur in pre-eclampsia. however, the potential role of membrane associated or soluble trail, an apoptosis-inducing ligand, and its death receptor, dr , is not known. we tested the hypotheses that the trail/trail-receptor system is compromised in pre-eclampsia and that soluble trail is a circulating factor which triggers the vascular complications of pre-eclampsia. method: this study was conducted on placental samples and plasma (edta) from women with uncomplicated pregnancies (n= ) and with pre-eclampsia (n= ) at - weeks gestation. protein expression levels and tissue localisation of trail and dr were defined in villous tissue by western blotting and immunohistochemistry. soluble trail (strail) was measured in maternal plasma from both groups using a commercial elisa (diaclone). results: placental villous trail and dr protein was unaltered in preeclampsia compared to normal pregnancy. whilst there was differential distribution of trail and dr within the component cells, this also was unaffected in pre-eclampsia. within the villi, trail was mainly confined to the cytoplasm and perinuclear regions of cytotrophoblast, syncytiotrophoblast, stromal cells and the fetal capillary endothelium. conversely, dr was restricted to trophoblast only, distributed evenly between cytoplasm, plasma membranes and nucleus. strail was present in plasma from non-pregnant women of childbearing age [ . ( . - . ) , median (interquartile range), pg/ml, n= ]. however, there was no significant increase either in pregnancy [ . ( . - . ) pg/ml, n= ] or pre-eclampsia [ . ( . - . ) pg/ ml, n= ] (kruskal-wallis test, p> . ). conclusions: these results suggest that placental villous trail is not adversely regulated in pre-eclampsia. the absence of dr in stromal and endothelial cells may increase resistance to apoptotic stimuli in cells of the villous core. the co-localisation of trail and dr in trophoblast suggests a role in autocrine regulation of cell turnover in this cell type. introduction: preeclampsia is associated with apoptosis of the syncytial layer of placenta and the release of particulate and soluble factors which are deleterious to maternal endothelial function. the goal of the current study was to determine whether laser capture microdissection (lcmd) and western blotting could be used to assess levels of syncytial fas ligand (fasl), a key protein in the apoptotic cascade. methods: frozen sections of term placenta delivered from uncomplicated pregnancies at term were used for study (n= ). following staining with mayer's hematoxylin (n= ), lcmd (leica instruments) of an intact terminal villus was carried out using a focused laser pulse directed to the area of interest using a microscope. in the first round of microdissection the placental villus core consisting of fibroblast, macrophages, fetal vessels, and connective tissue, were removed. in the second round of lcmd, the syncytial layer of that same villus was removed and collected in lysis buffer containing detergent and protease inhibitors, and the number of nuclei per sample was recorded. this procedure was repeated until syncytial tissue from - villi were collected. electrophoretic separation of lysate proteins was then carried out, and western blotting and immunodetection using an anti-fasl antibody was performed. results: we observed that fasl was detected in microdissected syncytial specimens at a molecular weight of approximately kda ( figure) , consistent with our previous reports. it is of note, that western blotting of samples containing approximately (lane ), (lane ), (lane ), and (lane ) nuclei revealed that fasl could be reliably detected in specimens containing as few as nuclei. conclusions: since fasl is a cytokine of low to moderate abundance in placenta, this suggests that lcmd coupled with western blotting will be a valuable methodology to elucidate pathways of syncytial apoptosis and pathophysiology in pregnancies complicated by preeclampsia. supported in part by nih grant hd . the placenta of preeclamptic pregnancies shows oxidative and nitrative stress. we have shown low protein abundance but paradoxically high activity of inducible nitric oxide synthase (inos) in the placenta with severe preeclampsia compared with normal pregnancies. protein nitration and phosphorylation are post-translational modifications possibly involved in nos activity. objectives: examine inos localization and tyrosine phosphorylation in the preeclamptic placenta and the effect of a peroxynitrite generator (sin- ) on inos expression in primary human placental microvascular endothelial cell (hpmec) cultures. methods: placental lysates from normal (n= ), mild (n= ) and severe (n= ) preeclamptic pregnancies were western blotted for inos and what are the roles played by peroxynitrite in preeclamptic women? yoshikatsu suzuki, tamao yamamoto, yoshimasa watanabe, takeo itoh, hidetaka izumi. obstetrics and gynecology, nagoya city university, nagoya, japan; pharmacology, nagoya city university, nagoya, japan; obstetrics and gynecology, izumi women's hospital, fukuoka, japan. aim preeclampsia is characterized by hypertension plus proteinuria. it is hypothesized that the endothelial cell function might be activated by placental faculty in early pregnancy and the activation might cause the vascular disease in preeclampsia. peroxynitrite, which is produced by combination with superoxide (o -) and nitric oxide (no), is strong oxidant as well as o -.we investigated whether or not the localization of peroxynitrite in both placenta and resistance artery might play important roles of developing preeclampsia. omental arteries or placentas were obtained from severe preeclamptic and term-normotensive pregnant women at cesarean section. they were stained by ant-nitrotyrosine (nt, a marker of peroxynitrite) antibody. furthermore, the concentration of nt was measured in omental arteries. in formed consent was obtained from all patients in written. preeclampsia was diagnosed according to the criteria of japan society of obstetrics and gynecology. the localization of nt was seen in placentas obtained from sever preeclamptic women ( / ), although it was not seen from normotensive pregnant women ( / ). the localization was also seen in placentas from of severe preeclamptic women with intrauterine fetal restriction. in omental arteries from both groups, the localization of nt was seen to same degree, and the concentration of nt was similar ( . ± . for sever preeclampsia and . ± . for normotensive pregnant women). from these results, it was suggested that an increase in o production, a decrease in no as well as peroxynitrite production in the placentas might cause the vascular dysfunction and subsequently damage uteroplacental blood circulation in preeclampsia. however, the role played by peroxynitrite in resistance artery might be different and more complicated. background: preeclampsia (pe) is associated with shallow cytotrophoblast (ct) invasion of the decidua, leading to impaired vascular transformation and poor uteroplacental perfusion. ct invasion requires the selective proteolysis of the peri-decidual cell (dc) extracellular matrix. we hypothesized that il and tnf , cytokines that have been linked to pe, may induce aberrant expression of the matrix metalloproteinases (mmp) and in dcs, thereby preferentially degrading the decidual ecm and interfering with sequential ct invasion. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on decidua from normal (n= ) and preeclamptic (n= ) women, and staining intensities were evaluated by hscore. confluent, leukocyte-free first trimester dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ) and confirmed by western blotting. quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in preeclamptic decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in normal decidua ( ± and ± , respectively; p< . ). in cultured first trimester dcs incubated with e , tnf increased secreted mmp- and mmp- levels by ± and ± -fold, respectively, while il increased them by ± and ± fold, respectively (p< . ). in parallel incubations with e +mpa, basal mmp- and mmp- output were lowered by approximately % and %, respectively, while tnf -and il elicited mmp- and mmp- levels were blunted by - %. western blotting confirmed the elisa results, and mrna levels corresponded to changes in mmp- and mmp- secreted protein levels. conclusions: over-expression of mmp- and mmp- in decidual cells may promote pe by disrupting decidual ecm and impairing normal ct invasion. the high levels of il and tnf associated with pe may be contributing to this over-expression. our in vitro observations that mpa blunts tnf -and il -elicited mmp expression suggests that exogenous progestin may offer a novel therapeutic approach in preventing pe. to produce -methoxyestradiol ( -me), a compound with diverse biological activities including inhibition of hif- , a transcription factor that mediates cellular response to hypoxia. circulating levels of -me and placental comt activity are significantly reduced in preeclampsia, raising the possibility that reduced production of -me contributes to the pathophysiology of preeclampsia by altering placental response to hypoxia. genetic variation in the comt gene is linked to comt activity and has been associated with intrauterine fetal growth restriction. we determined if a snp in exon of the comt gene (rs ), which does not change amino acid sequence ( leu ), but reduces comt mrna translation, was associated with preeclampsia. we analyzed comt genotypes in paired dna samples extracted from maternal and cord blood from normal pregnancies and pregnancies complicated by preeclampsia by allele discrimination. the study population was predominantly (> %) african-american. the frequency of the minor rs "g" allele, which is associated with low comt activity, was similar in maternal cases and controls (cases: %; controls: %, p= . ), but was significantly greater in fetal dna from pregnancies complicated by preeclampsia compared to control pregnancies (g allele frequency cases: %; control: %; p< . ). likewise, fetal carriage of the rs "g" allele conferred a significantly greater risk of preeclampsia (odds ratio: . ; % c.i.: . , . , p< . ). there was also a significant discordance between paired maternal and fetal rs genotypes with significantly greater discordance for the "g" allele in fetuses hosted in preeclamptic pregnancies (odds ratio: . ; % c.i.: . , . ). we conclude that genetic variation in the comt gene is associated with risk of preeclampsia, possibly through a mechanism involving reduced production of -me. supported by nih p md . smoking is associated with elevated adma in preeclampsia. michael p frank, robert w powers. , magee-womens research institute, pittsburgh, pa, usa; obstetrics gynecology, university of pittsburgh, pittsburgh, pa, usa. objective: cigarette smoke exposure paradoxically reduces the risk of preeclampsia. asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nitric oxide synthase (nos), an independent risk factor for cardiovascular mortality, adma is elevated in women who develop preeclampsia, and adma has been reported to be both higher and lower in smokers. the objective of this study was to investigate the concentration of adma in pregnant smokers and nonsmokers with and without preeclampsia. study design: case-control study of women with uncomplicated pregnancy (controls), and women with preeclampsia matched for gestational age at sample collection. adma was measured by hplc. cigarette smoke exposure was determined by questionnaire and confirmed by plasma cotinine. data are mean±sd. analysis was by two factor anova with fishers post-hoc testing, significance accepted at p< . results: as previously reported, maternal plasma adma concentrations were higher in women with preeclampsia compared to controls (p< . ). in addition, the concentration of adma was significantly higher in preeclampsia smokers compared to controls and preeclampsia nonsmokers (p< . ). in contrast, there was no difference in adma concentration between control smokers and nonsmokers. conclusion: these data may suggest a differential effect of cigarette smoke exposure on circulating adma concentrations between women who do and do not develop preeclampsia. previous data has suggested that cigarette smoking is associated with lower adma in low risk elderly patients, and higher adma in high-risk subjects with diabetes. therefore, the data in preeclamptic and non-preeclamptic subjects may be consistent with these studies, however, the underlying biological explanation for this differential effect has yet to be determined. objective: eclampsia is similar to hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, hyperperfusion and edema formation. we previously reported that the pressure of breakthrough was similar between nonpregnant (np) and late-pregnant (lp) rats, but only lp animals developed edema. this study tested the hypothesis that lp animals have decreased in cerebrovascular resistance (cvr) and hyperperfusion in response to breakthrough vs. np. we further hypothesized that acute hypertension would cause greater blood-brain barrier (bbb) permeability in lp rats due to elevated hydrostatic pressure. methods: in vivo models of bbb permeability and cerebral blood flow (cbf) were used in np (n= ) and lp (d - ; n= ) rats that were either normotensive or hypertensive (np-htn, lp-htn) by infusion of phenylephrine to raise mean arterial pressure. permeability was determined in anterior and posterior brain regions by calculating the flux of kd dextran into the brain tissue, measured by a fluorescent spectrophotometer after flushing the vasculature with saline. cbf and cvr were measured by infusion of m fluorescent microspheres and determined based on the flow rate and fluorescence intensity of a reference sample for each animal. animals were ventilated to maintain blood gases within normal ranges (po > mmhg, pco = - mmhg). results: although the pressure change was similar between np and lp ( and mm hg), lp animals responded to acute hypertension with hyperperfusion. cbf increased from ± to ± ml/ g/min in np ( %) and ± to ± ml/ g/min in lp ( %; p< . vs. np). hyperperfusion in lp animals was associate with decreased cvr vs. np ( . ± . vs. . ± . mm hg/(ml/ g/min); p< . ). bbb permeability was significantly increased in lp animals at breakthrough vs. np in both anterior and posterior brain regions. the flux of dextran in anterior and posterior brain regions for np vs. lp animals was: ± vs. ± for anterior (p< . ) and ± vs. ± for posterior (p< . ). conclusions: these data demonstrate that pregnancy decreases cvr and causes hyperperfusion of the brain during acute hypertension. because increases in cvr is a protective function in the brain, impairment of these mechanisms during pregnancy may predispose the brain to edema when blood pressure is elevated, as in eclampsia. introduction: this study used the in vitro dually perfused human placental lobule to test the hypothesese that placental release of vegf and the fetoplacental vasodilatory response to exogenous vegf- are altered by tissue oxygenations that mimic healthy and preeclamptic pregancies. methods: lobules were dually perfused for six hours under one of two oxygenation conditions, representing 'normoxia' and 'hypoxia' (n = each): delivering maternal side inflow perfusate at oxygen concentrations of . % and %, respectively, distributed via cannulae; and fetal side inflow perfusate oxygen concentration of - % in both systems. venous perfusates were sampled and assayed, appropriate to side of release, for erythropoietin (epo), macrophage inflammatory protein- alpha (mip- alpha) (reference oxygen sensitive hormones), free vegf, svegfr- and plgf. in separate perfusions, fetoplacental vasodilation in response to pm vegf was investigated, following preconstriction of the fetoplacental vasculature to steady state fetal-side inflow hydrostatic pressure (fihp) with the thromboxane mimetic, u , (n = each). results: maternal-side mip- alpha release was higher in the 'hypoxic' than the 'normoxic' system ( ± and ± pg/ml, respectively, at hours, mean ± se; -way anova: p< . ). maternal-side epo and fetal-side soluble free vegf and svegfr- release were not different between groups. fetal-side release of plgf was higher in the 'hypoxic' group than the 'normoxic' group ( . ± . and . ± . pm, respectively, at hours, mean ± se; -way anova: p < . ). there was no difference in the vasodilatory response to vegf- in the fetoplacental vasculature between the groups ( . ± . and . ± . % change in fihp, mean ± se). discussion: differences in mip- alpha and plgf release provide evidence for metabolic separation of the adapted systems, caused by a changed oxygen environment. our failure to observe differences in epo, vegf and svegfr- release may be explained by longer lag-times for up regulation of their gene expression. vegf associated endothelial signalling appears to be unaffected by 'hypoxia' in the placenta over the time course studied here. background: there is a placental renin-angiotensin system (ras) from very early pregnancy. ang iv mediates various effects by binding to its specific receptor, the at r, the active site of which is an insulin-regulated aminopeptidase (irap). there is at r expression in both endothelial and smooth muscle cells. ang iv at low concentrations is vasodilatory, increasing blood flow via the at rs; it also stimulates nf-kappa beta and modulates glut- . to date at r expression has not been investigated in the placenta. we propose that at r plays a part in the placental vascular development necessary for successful pregnancy, and that reduced at r expression may be associated with inadequate vascular adaptation contributing to pre-eclampsia (pe). aim: to identify and locate at r expression in both np and pe placentae. methods: the study had hospital ethical approval; written informed consent was obtained from all women. placental samples were obtained from np and pe at delivery (gestational ages: and weeks respectively). samples were taken from areas, near cord, middle and outer edge of the placenta. paraffin embedded sections were immunostained for irap reactivity using a rabbit polyclonal antibody (gift from professor david james, garvan institute, australia). immunoreactivity of trophoblast and uterine cell populations was assessed using a semi-quantitative grading system. grade = no positive labelling, = - %, = - %, = - % and = - % of cells positively labelled. median (max, min) are shown. results: ) at r immunostaining was prominent in the syncytiotrophoblast and hofbauer cells of all placental villi examined, with no differences in expression between sampling sites. ) at r positivity was reduced in near cord pe samples ( . ( . , . )) compared to np ( . ( . , . ) p= . ). conclusion: we have shown for the first time, dense at rs in syncytiotrophoblast and hofbauer cells in np placentae, and their down-regulation in pe. reduced ang iv/at r binding may contribute to increased placental vasoconstriction resulting in increased ischemia/reperfusion. this in turn may stimulate xanthine oxidase, which is itself stimulated by angii, leading to increased superoxide production. further work is needed to clearly define the role of this newly identified component of the renin-angiotensin system in normal pregnancy and pe. pre-eclampsia is associated with lower percentages of regulatory t cells in maternal blood. jr prins, hm boelens, jj hm erwich, j heimweg, s van der heide, ajm van oosterhout, aej dubois, jg aarnoudse. obstetrics, university medical center groningen, groningen, netherlands; laboratory of allergology and pulmonary disease, university medical center groningen; pediatric allergology, beatrix children's clinic, university medical center groningen. pre-eclampsia is a serious disease of human pregnancy and immunological mechanisms play a role in its pathophysiology. normal pregnancy is associated with an increase in regulatory t (treg) cells and with a predominant th immune profile. treg cells are a subpopulation of cd + lymphocytes and are specifically characterized by the lineage specific transcription factor foxp . treg cells seem to induce immunological changes that have a protective role in maintaining normal pregnancy. we hypothesised that percentages of treg cells are decreased in pregnancies complicated by pre-eclampsia. methods in total, women with pregnancies complicated by pre-eclampsia and healthy pregnant controls were enrolled. to obtain control umbilical cord blood as well, control group i consisted of eighteen healthy pregnant women at term. in addition, since women with pre-eclampsia delivered preterm, control group ii (peripheral blood only) consisted of women during normal pregnancy with a gestational age matched for the preterm pre-eclamptic group. treg cells were measured from whole blood using four-color flow-cytometry. women with a pregnancy complicated by pre-eclampsia had a significantly lower percentage of cd + foxp + treg cells ( . vs . %; p< . ). in the pre-term group the pregnancies complicated by pre-eclampsia showed a significantly lower percentage of cd + foxp + cells in the peripheral blood as compared to the healthy pregnant controls. at term this percentage was also lower but not significantly so. between pre-term and term pregnancies both complicated by pre-eclampsia no significant difference was found in the percentage of cd + foxp + treg cells. no difference was found in umbilical cord blood ( . vs . %). conclusions our data suggest that pre-eclampsia is associated with a diminished percentage of treg cells in peripheral blood. we conclude that a deficiency of regulatory t cells may play a role in the pathophysiology of pre-eclampsia. background: preeclampsia and eclampsia are significant causes of maternal and fetal death. however, the pathophysiology of these conditions is unclear. we and others have reported that inhibition of endogenous nitric oxide (no) synthesis produces symptoms similar to preeclampsia in pregnant rats. several studies demonstrate that fetoplacental weights are altered in pregnancies of spontaneous hypertensive (shr) rats. in addition, impaired synthesis of tetrahydrobiopterin (bh ), a major co-factor for endothelial nitric oxide synthase (enos) activity and enhanced expression of enos has been observed in the pathogenesis of hypertension. in the current study, we examined whether supplementation of bh and sepiapterin (sep, a precursor for bh biosynthesis in the salvage pathway) reduces increased blood pressure and improves fetoplacental weights in shr pregnant rats. methods: groups ( - ) of shr pregnant rats were either treated with bh , sep ( mg/k.g body weight/day/rat, oral tablets) or vehicle (normal diet) beginning from day of pregnancy until day of gestation. animals were sacrificed on day of gestation and fetoplacental weights were recorded immediately. western blot analysis was performed to determine vascular enos expression (enos/gapdh). results: significant (p< . ) elevations in blood pressure (bp, mmhg) were observed in shr (shr, ± . vs. wky, ± . mmhg) compared to wistar-kyoto (wky) group. supplementation of either bh or sep ( ± . ), significantly (p< . ) reduced elevated bp beginning from day of pregnancy. fetal but not placental weights were significantly (p< . ) reduced in shr ( . ± . grams) compared to wky ( . ± . ) rats. bh ( . ± . ) treatment partially (p< . ) increased fetal weights compared to the shr group. vascular enos expression is significantly (p< . ) elevated in shr ( . ± . ) compared to wky rats ( . ± . ). further, treatment with bh ( . ± . ) but not sep ( . ± . ) significantly (p< . ) reduced elevated enos protein expression in shr rats. conclusions: bh may be beneficial treatment of preeclampsia to reduce blood pressure and improve fetal perfusion to increase fetal weights. genetic risk factor for severe preeclampsia: significance of endothelial nitric oxide synthase gene t- ->c and missense glu asp variants. toshihiro yoshimura, michihiro yoshimura, masafumi nakayama. obstetrics and gynecology, kumamoto university school of medicine, kumamoto, japan; cardiovascular medicine, jikei university school of medicine, tokyo, japan; cardiovascular medicine, kumamoto university school of medicine, kumamoto, japan. introduction: we recently identified two endothelial nitric oxide synthase (enos) gene polymorphisms, a glu asp missense variant in exon and a t- -->c variant in the '-franking region, which are associated with coronary spasm and myocardial infarction in japanese population. and we also identified a missense glu asp variant is associated with severe preeclampsia and placental abruption. our objective was to analyze the association between the t- -->c and severe preeclampsia. materials and methods: the study participants included patients with histories of severe preeclampsia. this is a preliminary study, therefore, the comparisons were made with the general normal population. results: the analyses revealed that the frequency of the missense glu asp variant (n= / , %) was significantly higher than the general population (n= / , %), as we previously published. however, the frequency of the t- -->c variant (n= / , %) was not different from the general population (n= / , %). interestingly, only one patient had both t- -->c and missense glu asp variants, and she developed placental abruption. conclusion: although our sample size is small, it is very unlikely that the t- -->c variant is associated with severe preeclampsia. the t- -->c variant may not be a genetic susceptibility factor to severe preeclampsia. the t- -->c may have some reproductive significance in combination with missense glu asp variant, however huge number of patients would be needed to analyze such rare ( . %) combination of the variance. the association between the development of preeclampsia and methylenetetrahydrofolate reductase, angiotensinogen, vascular endothelial growth factor single nucleotide polymorphism genotype combinations. hyun soo park, jong kwan jun, chan-wook park, joong shin park, bo hyun yoon, hee chul syn. obstetrics and gynecology, dongguk university international hospital, goyang-si, gyeonggi-do, republic of korea; obstetrics and gynecology, seoul national university college of medicine, seoul, republic of korea. objective this study was conducted to investigate if there exists any genotype combination of multiple single nucleotide polymorphism (snp)s which is frequently found in preeclampsia patients. study design one hundred sixty two preeclampsia patients and normotensive pregnant women were included in this study between jan and jul . diagnosis of preeclampsia and assignment of severity were made according to the criteria by national high blood pressure education working group and american college of obstetricians and gynecologists. the patients were reclassified as early ( weeks or before) and late-onset ( weeks or beyond) disease. genotypes were measured with pcr-rflp for methylenetetrahydrofolate reductase (mthfr) c t, angiotensinogen (agt) m t, vascular endothelial growth factor (vegf) c t with the dna extracted from maternal blood. case-control study for each snp was done and the frequencies of genotype combination were compared. anova, t-test, chi-square test, fisher's exact test and logistic regression analysis were used for statistical analysis. a p value of < . was considered statistically significant. results genotypes of mthfr polymorphism showed significant difference between late onset preeclampsia and control (cc+ct/tt, or: . , p< . ) but agt and vegf polymorphism did not show statistical difference between any case-control combination. only out of possible genotype combinations were found and there was no statistical difference in the frequencies of genotype combination between case and control group. conclusion mthfr polymorphism might be associated with the development of preeclampsia, but there was no combination of mthfr, agt and vegf polymorphisms which is associated with the development of preeclampsia. diffuse staining and vascular smooth muscle (vsm) staining. resistance-sized vessels ( - μm) were evaluated. results: for mpo, the intensity of staining (fig) and the % vessels with neutrophil, diffuse and vsm staining was significantly greater for obese than for overweight or normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . , p< . ); % vsm staining ( . ± . vs. . ± . vs. . ± . , p< . ). for mmp , obese and overweight patients had a greater (p< . ) % vessels with neutrophil, diffuse and vsm staining than normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . ); % vsm staining ( . ± . vs. . ± . vs. . ± . ). conclusions: neutrophils infiltrate the systemic vasculature of obese women and release mpo and mmp . speculation: obesity may put women at risk for pe because their vasculature may already be dysfunctional due to neutrophil infiltration and release of mpo and mmp . hl . collagen is an important protein that maintains the structural integrity of tissues. disruption of vascular smooth muscle collagen could result in vascular dysfunction in women with preeclampsia. recently, neutrophil infiltration of the systemic vasculature was demonstrated in preeclamptic women. neutrophils produce inflammatory mediators, such as reactive oxygen species (ros) and tnf-. we hypothesized that neutrophils, ros and tnf-would alter expression of collagen regulating genes. methods: primary cultures of human vascular smooth muscle cells (vsmc) were seeded into t- flasks ( , cells/flask) and grown for days to % confluence. the cells were treated for hours with medium control, ros (hx, . mm + xo . u/ml), tnf-( ng/ml); and neutrophils ( , ) activated with arachidonic acid, μm, ( : ratio of neutrophils to vsmc). rna was extracted from cell homogenates and analyzed for gene expression with an rt profiler pcr array system for human extracellular matrix genes (superarray). to determine the fold-change of gene expression, the results were first normalized to a housekeeping gene and then ct was calculated across two rt-pcr arrays where group was the control and group was the experimental treatment. results: table . conclusions: neutrophils, ros and tnf increased mmp expression. interestingly, genes involved in collagen synthesis (col a ) or inhibition of mmp- activity (timp ) were either not affected or down-regulated. these data suggest that neutrophil infiltration in preeclamptic women could cause vascular dysfunction by creating an imbalance between collagen synthesis and collagen breakdown favoring breakdown. hl , fogarty d tw , p md . objective: hypoxia increases membrane attack complex (mac) binding to cultured human trophoblasts, and mac enhances apoptosis in trophoblasts exposed to low compared to normal fio . trophoblast microparticles and cellular fragments released into the maternal circulation in vivo may contribute to the systemic pathophysiology of preeclampsia. we tested the hypothesis that hypoxia induced mac deposition on cultured human trophoblasts yields microparticles and fragments coated with mac. study design: primary cytotrophoblasts from term human placentas (n= ) were cultured h in % and % oxygen in dmem with % human serum with active mac or heat inactivated serum (control). media were centrifuged to obtain pellets of microparticles and cell debris which were immunostained for mac or exposed - h to confluent, phorbol myristate acetate differentiated u macrophages. the percentage of macrophages that ingested trophoblast debris was quantified by counting the number of macrophages with immunofluorescence for trophoblast cytokeratin filament staining, as assessed by confocal microscopy. results: cultures exposed to normal human serum, but not heat inactivated control serum, showed mac immunofluorescence on microparticles and fragments in medium, with the highest level of mac in cultures exposed to extreme hypoxia. the maximal percentage of macrophages that ingested the trophoblast debris coated with mac from cultures with % oxygen was . %, not different from the . % from cultures exposed a % fio and control serum. conclusion: trophoblasts exposed to hypoxia and active complement release microparticles and cellular fragments coated with mac into the extracellular environment. mac coating does not influence phagocytic removal of the debris by macrophages suggesting that placental derived, membrane bound mac could circulate to yield systemic affects on maternal endothelium. supported by nih hd and hd . is there a role for fatty acids in the pathogenesis of pre-eclampsia? nicola j robinson, laura j minchell, jenny e myers, philip n baker, carl a hubel, ian p crocker. maternal and fetal health research group, the university of manchester; obstetrics, gynecology and reproductive sciences, university of pittsburgh. objectives: women with pre-eclampsia (pe) display altered lipid metabolism as characterized by elevated circulating triglycerides and non-esterified fatty acids (nefa) and these changes are evident before the disease is clinically apparent. we have tested the hypothesis that the increased circulating levels of nefa contribute to endothelial dysfunction in pe. methods: human umbilical vein endothelial cells were incubated for h with pooled plasma ( %) from normal or pe pregnancies, or with palmitic, oleic and linoleic acid in culture media at the concentrations and molar ratios to albumin identified in normal ( , , m, ratio . ) and pe pregnancies ( , , m, ratio . ) , . lipid droplet accumulation was determined using an oil red o absorbance assay. endothelial metabolism was measured using the mtt test and mitochondrial membrane potential determined by jc- assay as a marker of early apoptosis. results: plasma from pe pregnancies increased endothelial cell lipid droplet accumulation compared to normal plasma (p< . , wilcoxon signed ranks, n= ). this change was replicated following exposure to nefa at the combined concentrations found in pe compared to normal pregnant controls (p< . , n= ). plasma from women with pe caused a significant decrease in mitochondrial dehydrogenase activity (mtt test; p< . , n= ) and a reduction in jc- fluorescence (p< . , n= ), compared to normal plasma, suggestive of mitochondrial membrane depolarization and increased cellular apoptosis. again these effects were replicated using nefa in culture medium at the levels found in pe compared to normal pregnancies (mtt test: p< . , n= ; jc- assay: p< . , n= ). conclusions: in endothelial cell cultures, plasma from women with pe caused increased lipid droplet accumulation, decreased cellular metabolism and increased apoptosis. these changes to cellular function were mirrored using nefa in culture medium at the concentrations and molar ratios to albumin previously reported in pe. these findings provide evidence that the changes in endothelial cell function induced by plasma from women with pe may be due to the increased nefa circulating levels and that increased palmitic, oleic and linoleic acid, in combination, could play a role in pathogenesis of pe. lorentzen ( ) bjog; endresen ( ) ajog. background: skewing of the maternal endothelial phenotype in pre-eclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. we hypothesise that factors secreted from pe placental tissue will impair endothelial cell function. we have tested the effect of soluble factors by menopausal status, there was a significant increase in bmi in pre-but not in postmenopausal women. in postmenopausal women there was insufficient power to note a statistically significant change in bmi. results are summarized with the follow-up for each group represented by "n" in the graph below. premenopausal patients were divided into hysterectomy with oophorectomy (pbso) versus hysterectomy alone (ph). the ph group showed an increase in bmi that plateaus at time . in the pbso group the bmi continued to increase over time. subgroup analysis comparing ph to pbso demonstrates initial weight loss in pbso but a significant increase in bmi from baseline at time compared to ph. conclusions: hysterectomy appears to be associated with an increase in bmi over time. subgroup analysis suggests that, in premenopausal women, oophorectomy is more strongly associated with continuing weight gain than hysterectomy alone. purpose: obesity is implicated as a key risk factor in chronic disease, but no studies have associated central obesity to the presence of chronic abdominal and/or pelvic pain. we set out to identify the prevalence of chronic abdominal/ pelvic pain in an underserved, primarily latina population by a cross-sectional study in the olive view-ucla outpatient gynecology clinic. we sought to identify an association between the presence and severity of abdominal/pelvic pain and central obesity. methods: nonpregnant women presenting to the gynecology clinic were prospectively evaluated and grouped according to the presence of abdominal/ pelvic pain ('none-mild' or 'moderate-severe' pain). body mass index (bmi) and abdominal circumference (ac) were measured. patients with 'moderate-severe' pain completed standardized questionnaires for pelvic pain and global health scores. results: / ( %) of patients has 'none-mild' pain, and ( %) had 'moderate-severe' pain. pain prevalence was not significantly associated with bmi (mean: 'none-mild' . + . kg/m ; 'moderate-severe' . + . kg/m , p= . ), nor was pain severity (p= . ). pain prevalence was significantly associated with ac (mean: 'none-mild' . + . inches; 'moderate-severe' . + . , p= . ). a borderline positive association exists between ac and pain severity (p= . ). conclusions: we demonstrate an association between both the presence and severity of chronic abdominal/pelvic pain and central obesity, independent of bmi. ac appears to be a more relevant factor than other traditional measures of habitus in patients with this chronic malady. to improve preventative care in women's health management, further evaluation of the role of central obesity in the pathogenesis of chronic pain is necessary. aims: vulvitis is one of the most frequently diagnosed gynaecological infections. we aim to assess the efficacy against infective vulvitis of a new topical medical device containing low molecular weight hyaluronic acid (lmw-ha). the ability of this molecule to stimulate -defensin release in keratinocytes has been recently shown. methods: we report preliminary data regarding women suffering from infective vulvitis, as assessed by a gyneacologist: patients were randomly selected to receive sphg ( patients), a cream containing low-molecular weigh hyaluronic acid, or vehicle ( patients). patients were asked to apply the cream to the vulva twice-daily for days. at the end of treatment, evaluation of efficacy, tolerability and acceptability of the cream was assessed by a specific questionnaire. results: preliminary results show that patients receiving sphg report a significative improvement of vulvitis symptoms, in terms of itch, redness of the skin and burning, in comparison to vehicle. sphg also showed a good tolerability, cosmetic acceptability and symptomatic relief perceived by patients. conclusion: sphg seems to be efficacious in ameliorating the symptoms of infective vulvitis. this activity may be probably related to the lmw-ha presents in this formulation: in fact, low molecular weight hyaluronic acid has been recently shown to induce -defensin production by human keratinocytes. since this peptide exerts antimicrobial and antimicotic activity, the improvement of symptoms assessed in patients receiving sphg might be linked to a reduced infective charge, attributable to the activity of -defensin . background: allogeneic hematopoietic stem cell transplant (hsct) is a treatment used for many malignant and nonmalignant diseases of the bone marrow and immune system. hsct may be complicated by chronic graft-versus-hostdisease (cgvhd) in to % from matched unrelated donors. genital cgvhd complicates about % of hsct and may uncommonly result in labial fusion. case: year old woman with a history of ewing's sarcoma and acute myelogenous leukemia, had received chemotherapy and total body irradiation (tbi) followed by a matched unrelated donor hsct. menarche occurred at years of age after normal pubertal development. she menstruated regularly until cancer diagnosis. premature ovarian failure resulted after chemotherapy and tbi and oral contraceptive pills were used for hormone replacement. after transplant, she developed chronic gvhd involving the skin, eyes, mouth and joints, and concomitantly complained of vulvar pruritus. she was presumed to have a yeast infection which was treated with fluconazole without a pelvic exam. she was evaluated by a gynecologist when she was unable to insert a tampon. pelvic exam revealed dense labia minora adhesions from the clitoris to urethral meatus and posteriorly leaving a cm opening at the urethra. pelvic mri revealed a normal uterus and ovaries. after weeks of topical estrogen cream, the adhesions remained dense and were lysed under general anesthesia. vaginal examination revealed pale, minimally rugated, normal mucosa. cervical cytology was normal. post-operatively she used daily topical estrogen and hydrocortisone creams. at months after surgery, her urinary stream was stronger. on pelvic exam, the labial opening was cm, but a small posterior forchette adhesion elicited severe pain. after using dilators coated with topical steroids and estrogen, she was able to insert a tampon. conclusion: genital gvhd should be considered in women with genital tract complaints after hsct. labial fusion secondary to chronic gvhd may be treated successfully with surgery and medical therapy. support: rbmb/nichd/nih. dana r ambler, mazen e abdallah, rahi victory, michael p diamond, elizabeth e puscheck, jay m berman. obstetrics and gynecology, wayne state university/detroit medical center, detroit, mi, usa. objective: to determine which factors are predictive of a ruptured ectopic pregnancy, and whether endometrial stripe thickness can be used as an alternative to such criteria in the diagnosis of an ectopic pregnancy. design: retrospectively collected ectopic pregnancy database. setting: detroit medical center, detroit, michigan. patients or participants: women with a diagnosis of an ectopic pregnancy were studied, with surgically confirmed tubal rupture cases. interventions: abstracted data included hcg(iu), gestational age (days), presenting symptoms of pain and/or bleeding, hemoglobin (hgb), hematocrit (hct), historical risk factors, ultrasound-determined ectopic size, endometrial stripe thickness (mm), amount of cul de sac fluid (cds), tubal rupture at time of surgery, and estimated blood loss (ebl)(ml). covariates included demographics. results were significant when p< . . results: chi square analysis revealed that there is a relationship between endometrial stripe thickness and hcg levels. logistic regression models demonstrated that endometrial stripe thickness was not predictive of ectopic rupture, (or . , p= . ) . however, logistic regression, both forced and forward stepwise analysis, demonstrated that hcg (or= . , p< . ), a large volume of cul-de-sac fluid (or= . , p< . ), and increasing pain (or= . , p= . ) were associated with increased risks of rupture. gestational age, and ectopic related risk factors were also not predictive of rupture. conclusions: endometrial stripe thickness is not a useful predictor for the diagnosis of a ruptured ectopic pregnancy. serum hcg measurement, cds fluid volume and the presence of pain are much stronger diagnostic indicators of ectopic pregnancies. clinical profile of migraineurs in a university hospital gynecology department in japan. [objectives] the changes in hormonal milieu associated with menarche, pregnancy, menopause, and post-menopause are frequently accompanied by changes in the patterns and frequency of migraine. little is known on the relationship of women's issues of migraine though the balance between estrogen and progesterone is critical in the elimination of migraine. our aim was to investigate the relations among the prevalence of migraine, the reproductive stage, and gynecologic diseases. [materials and methods] female patients (average age: . years old) who consulted a physician and agreed to answer about the questionnaires during september, -june, . migraineurs were diagnosed with the migraine screener by the japanese headache medical treatment promotion committee in . and the patients answered questionnaires that screened about menopausal disorder and abnormal menstruation at once. they were conducted a survey in the form of a questionnaire and sometimes were taken blood samples. [results] in patients had migraine ( . % average age: . years old). prevalence were . % in one's twenties, higher than another ages. most patients with migraine was complicated by menstruation disorders (premenstrual syndrome %, dysmenorrhea . %), sterility ( . %), and severe menopausal disorder ( %). when trh and the lh-rh test were examined for the sterility patient, migraineurs had higher prolactin basal level and lh level after lord but lower fsh level before and after lord than nonmigraineurs. on the other hand, the prevalence of migraine for postmenopausal women and women who had gynecology cancer treatment was low, and there was no relation between migraine and pregnancy history. [conclusions] this study provides migraine headache is influenced by reproductive stage and that women with migraine are frequently complicated by menstruation disorder. it is thought that migraine account for abnormality of hormone milieu including abnormality of the hypothalamus-pituitary system. objective: this study evaluated the efficacy of doses of estradiol (e ) gel . % (divigel ® ), a novel formulation of e consisting of mg e per g transdermal gel, to reduce frequency and severity of vasomotor symptoms and signs of vulvar and vaginal atrophy (vva) associated with menopause. design: postmenopausal women were evaluated in a -week study comparing placebo to e gel . % at doses of . g/day, . g/day, and . g/day with estimated nominal daily deliveries of . mg, . mg, and . mg of e respectively. endpoints included mean change from baseline in daily frequency and severity of moderate to severe hot flashes (msvs). vaginal ph and % superficial cells were collected at baseline and end of study. results: e gel . % showed statistically significant improvements from placebo gel as early as week (table) that were maintained throughout treatment. signs of vva (vaginal ph and % of superficial cells) showed statistically significant improvements from baseline with all doses of e gel . % compared to placebo. conclusion: e gel . % significantly decreased the frequency and severity of msvs at all doses evaluated in this trial. e gel . % offers multiple dosing options to individualize patient therapy, including the lowest effective dose that was studied ( . mg e , delivering . mg e /day), to treat vasomotor symptoms associated with menopause. estradiol (e ) gel . % (divigel ® ) is a novel formulation of e consisting of mg e per g transdermal gel for the treatment of vasomotor symptoms associated with menopause. safety and tolerability of e gel . % were evaluated in a large placebo-controlled trial. design: postmenopausal women participated in a -week study comparing placebo to . g/day, . g/day, and . g/day of e gel . %. circulating e and estrone (e ) concentrations were measured. safety analyses included the incidence of adverse events (aes) and clinical laboratory evaluations, including plasma levels of sex hormone binding globulin (shbg). application site tolerability was assessed using the draize scale. results: all doses of e gel . % produced physiologic e :e ratios similar to those seen in premenopausal women. e :e increased from a baseline mean of . to . , . , and . with, respectively, the . , . , and . g/day doses of e gel . %. the most frequently reported aes were breast tenderness and postmenopausal bleeding that appeared to be dose-related and would be expected with increased circulating estrogen concentrations. there were no remarkable changes in hematology, blood chemistry, urinalysis, lipid, coagulation, and carbohydrate values following treatment with e gel . %. shbg levels remained unchanged after weeks of treatment at all doses. the vast majority of patients had no evidence of skin irritation throughout the treatment period. mean draise scale scores after , , and weeks of treatment were . for all treatment groups except for a mean value of . ± . for the . g dose group after weeks of treatment. conclusion: e gel . % is a safe and well-tolerated therapy for the treatment of menopausal symptoms. design: pharmacokinetic parameters, dosing, efficacy, and safety information for divigel ® , elestrin ™ , evamist ™ , estrogel ® , and estrasorb ® were obtained from current prescribing information (obtained from manufacturer websites) and the data were compared. results: together, these new transdermal therapies offer multiple dosing/ delivery options and contain a wide range of e ( . mg to . mg), with the lowest systemic daily delivery of e attained by the divigel ® . g dose. following weeks of treatment, across the dosing options, each treatment significantly reduced both the frequency and severity of moderate to severe vasomotor symptoms (msvms) compared to placebo. change in frequency of msvms ranged from - . (divigel ® . g) to - . (estrasorb ® ); change in severity scores for msvms ranged from - . (divigel ® . g) to - . (divigel ® . g). all treatments are safe and well tolerated. breast tenderness was the only adverse event reported in % of subjects, occurring with all therapies. conclusions: current treatment guidelines recommend using the lowest effective dose of estrogen for the treatment of menopausal symptoms. this side-by-side comparison of the recently available e non-patch transdermal options to the standard transdermal therapies is meant to assist physicians in individualizing treatment for their patients. introduction: cdb- (cdb) is a relatively new progesterone receptor modulator being clinically evaluated for contraception and treatment of fibromas. its use in an intrauterine device/system (ius) has not been reported. in this study we prepared cdb-filled intrauterine devices (cdb-ius) and evaluated their effects on endometrial growth and bleeding patterns in rhesus macaques. methods: short ( . - . cm) lengths of silastic tubing (od . mm), either empty (n= ),or filled with silicone rubber matrix containing % of micronized cdb (n= ), were inserted into the uterine lumens of ovariectomized rhesus macaques. animals were induced to cycle by sequential treatment with systemic estradiol and progesterone (p) as reported (brenner et al, ann ny acad, ) . after . cycles, at the end of the follicular phase, the uterus was removed and processed. results: when systemic p treatment was withdrawn at the end of each cycle, animals with empty ius menstruated normally, while animals with cdb-ius bled little or not at all. over the whole . cycles, animals bearing a blank ius bled for an average of . ± . days while cdb treated animals bled for an average of only ± . days. at the end of treatment, animals exposed to blank ius had mean endometrial wet weights of ± mg while the cdb-treated endometria weighed only ± mg. the proliferation markers ki- and phospho-h were substantially lower in the cdb-ius treated than in the blank treated animals. histologically, the cdb exposed endometria were atrophied with evidence of glandular degeneration while the blank controls were proliferative and normal. summary: in cycling rhesus macaques, a cdb-ius prevented progestational development, blocked menstruation after p withdrawal, and suppressed endometrial proliferation. if these effects are confirmed in women, the cdb-ius could provide estrogen-free and bleed-free contraception, and could help control heavy menstrual bleeding. supported by rr , hd and the population council. introduction: irregular uterine bleeding is a major side effect and cause for discontinuation of ltpoc use. while endometria of ltpoc-exposed women display abnormally enlarged, fragile blood vessels (bv), decreased blood flow and evidence of oxidative stress, the mechanisms by which structural and vasomotor endometrial dysfunction occurs remains unknown, in part by the difficulty of manipulating hormone levels in women. the aims of this study were ) to validate the guinea pig (gp) as a model to study uterine effects of ltpoc and ) to investigate ltpoc-effects on endometrial histology and oxidative stress markers. methods: oophorectomized gps were implanted s.c. with time release pellets containing either placebo (crl,n= ); estradiol (e ,n= ); medroxyprogesterone acetate (mpa,n= ) or e +mpa (n= ). after days, uterine horns were weighed and frozen or paraffin embedded. angiogenesis was assessed by quantitative image analysis of vonwillebrand factor staining and included bv density and size. oxidative stress was detected by isoprostane and -oh-deoxyguanosine ( oxog). apoptosis was investigated by the tunel method. statistical analysis was by -way and -way anova. results: gp uteri were enlarged by both e (p< . ) and mpa (p= . ). effects of mpa on uterine weight differed significantly depending on e levels (p< . ), where mpa opposed the e effect in combined treatments. angiogenesis parameters were similarly impacted upon. thus, mpa alone increased bv density (p= . ) and bv average area (p= . ). the presence of e significantly decreased these parameters (bv density mean± sem: crl: . ± . %, e : . ± . %, mpa: . ± . %, e +mpa: . ± . %, p= . ). these changes were associated with highly elevated -isoprostane content in e +mpa-treated uteri compared to all other groups (p< . ). abnormalities in the e +mpa group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased nuclear oxog staining and a marked increase in tunel labeling. conclusions: ltpoc exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. the gp is an excellent model for the study of ltpoc effects on the uterus. physiologic and psychological symptoms associated with injectable and oral contraceptive use. abbey b berenson, susan odom, carmen r breitkopf, mahbubur rahman. ob/gyn, utmb, galveston, tx, usa. objective: to compare physiologic and psychological symptoms over mo among users of depomedroxyprogesterone acetate (dmpa), an oral contraceptive with micrograms ethinyl estradiol (oc), and non-hormonal (nh) contraception. methods: a total of women reported the presence of symptoms prior to initiating contraception ( dmpa, oc, nh) and every mo thereafter for mo. longitudinal relationships between symptoms and contraceptives (reference: nh), as well as race/ethnicity (non-hispanic black, non-hispanic white, and hispanic) were assessed by gee-analysis after adjusting for age, visits and baseline status of symptoms. persistence, resolution, and new development of symptoms were noted by method in mo increments for mo and compared with that of nh controls. the gee analyses showed that oc was protective against mastalgia (or= . ), cramping (or= . ), hair loss (or= . ), acne (or= . ), nervousness (or= . ) and mood swings (or= . ). when race was considered, oc was protective for all women against acne, for whites against mastalgia and cramping, and for non-whites against nervousness. for whites, it was a risk factor for bleeding between menses. oc use resulted in resolution of acne within mo in nearly % of those with it at baseline, but no significant resolution after mo. also mastalgia ( % at baseline) was less likely to persist at and mo. bleeding between menses was reported for the first time at mo by % of oc users. dmpa users of all races had an increased risk of missed periods (or= . ), bleeding between menses (or= . ), bleeding > d (or= . ) and loss of libido (or= . ) relative to nh users. it reduced cramping (after mo) and bloating (all mo). racial differences were observed with dmpa protective against mastalgia and mood swings in whites, cramping in whites and hispanics, and bloating in non-whites. it was a risk factor for loss of energy in whites only. neither method affected depressive symptoms. conclusion: side effects of these two methods are mostly related to abnormal bleeding. very low dose pills can be protective against symptoms (mastalgia, cramping, hair loss, acne, nervousness and mood swings) commonly associated with pills while dmpa protects against cramping and bloating. knowledge about racial differences will allow physicians to individualize therapy. counseling should include that some symptoms can develop after mo while resolution often occurs within mo. methods: twenty-four women were enrolled in irb approved prospective, randomized, cross-over trial comparing months of oc (ortho cyclen) vs. tc ortho evra). the daily oc administers micrograms of ee; the weekly tc contains . milligrams of ee. each treatment was followed by months of washout and months of the alternative contraceptive. blood was drawn at baseline and final week of treatment for each arm of the study. ee was quantified by ria, with preceding organic solvent extraction and celite column partition chromatography. data were analyzed by t test with boferroni's correction, p < . . results: after two months of treatment the mean (+/-sem) ee levels for tc = . pg/ml (+/- . ) and oc = . pg/ml (+/- . ). the ee level is not significant different for the two medication (p = . ). conclusions: there is no difference in ee levels with the use of these oral and transdermal contraceptives. this suggests the transdermal contraceptive, despite the lack of the hepatic first pass effect, has similar levels of ethinyl estradiol compared to oc. the continuous elevation in ee seen with the tc route of administration, versus the episodic increases seen with the oc route, raises concerns of constant exposure to ee. this may explain the increased risk of estrogen-induced thrombotic events with this tc route of administration. impact of paracervical block, in combination with general anesthesia, on post-abortion pain. gweneth b lazenby, tod aeby. obstetrics and gynecology, university of hawaii, honolulu, hi, usa. objective to evaluate the impact of paracervical block with a long-acting local anesthetic, in conjunction with general anesthesia, on post-operative pain. methods a power analysis determined patients per arm were needed to demonstrate a significant difference of in mean pain scores. seventy-two patients were allocated to one of two arms using urn randomization. all patients received standardized anesthesia; intravenous sedation for gestational age under weeks and general anesthesia for over weeks. thirty-nine patients were randomized to receive a paracervical block with . % bupivacaine and thirtythree were randomized to no local anesthesia prior to surgical abortion. patients completed visual analog scales for pain and anxiety prior to the procedure, upon awaking, and minutes post-operatively, and prior to discharge. data were analyzed using an anova and students t-test the experimental and control groups were equivelent in age, ethnicity, gravidity, parity, prior abortions, prior vaginal deliveries, prior c-sections, gestational age, number of laminaria, pre-operative and intra-operative dilation, operative time, estimated blood loss, and reported complications. pain and anxiety were not significantly affected by placement of a paracervical block. these data do not support the hypothesized benefit of local anesthesia, prior to surgical abortion under general anesthesia, to reduce post-operative pain. we do not recommend the routine use of a paracervical block to decrease post-operative pain. age, parity, history of abortion and contraceptive choices affect the risk of repeated abortion. oskari heikinheimo, mika gissler, satu suhonen. ob gyn, university of helsinki, helsinki, finland; national research and development centre for welfare and health, helsinki, finland. objective the rate of repeat abortion varies from to % in northern europe. however, risk factors for repeat abortion are poorly understood. we characterized risk factors (demographic, as well as those related to abortion and postabortal contraception) of repeat abortion. design a prospective cohort study of women undergoing medical abortion between august and december . the subjects were followed by means of finnish registry on induced abortions until december ; the follow-up time (mean ± sd) was . ± . months. results altogether ( . %) of the subjects requested repeat abortion within the follow-up time. in univariate analysis previous abortion, parity, young age, smoking and failure to attend the follow-up visit were associated with increased risk of repeat abortion. immediate -in contrast to postponed -initiation of any contraceptive method was linked to lower risk of repeat abortion. in comparison to combined oral contraceptives, use of intrauterine contraception was most efficacious in reducing the risk of another pregnancy termination. in multivariate analysis the effects of young age, parity, previous abortion and type of contraception on the risk of another abortion persisted. conclusions increased focus on young, parous and those with the history of an abortion may be efficacious in decreasing repeat abortion. contraceptive choices made at the time of abortion have an important effect on the rate of reabortion. postabortal use of intrauterine contraception, specifically that of lng-ius, might decrease the rate repeat abortion. objective. to determine the rate of failure and to analyze factors associated with failure for the essure permanent birth control device at the detroit medical center (dmc). methods. a chart review was conducted on patients who underwent essure placement at the dmc from january through june . patient demographics, past medical and surgical history, anesthesia type, procedure time, intraoperative complications, and procedure failures were noted. data were analyzed for statistical significance using spss. results. there were essure procedures attempted at the dmc from january through june . of the attempted procedures, there were failures ( . %). of the failures were attributed to difficulty visualizing the ostia ( %). other causes of failure included expulsion of the device ( ), tubal spasm ( ), uterine perforation ( ), and tubal ostia too large for the device ( ). there were cases of failed placement for undocumented reasons, one case requiring a laparoscopic tubal ligation secondary to postprocedure tubal patency, and post-procedure pregnancies. age, race, body mass index, gavidity, parity, history of sexually transmitted infections, medical history, history of cesarean section, tobacco or illicit drug use, anesthesia type, and physician experience with the procedure were not significantly associated with placement failure or difficulty visualizing the ostia. a longer procedure time was significantly associated with failure ( . vs . min, p = . ), and history of ectopic pregnancy was significantly associated with difficulty visualizing the ostia ( . % vs . %, p = . ). conclusion. the failure rate for placement of the essure permanent birth control device at the dmc is . % with a pregnancy rate of . %. the majority of failures may be attributed to difficulty visualizing the ostia. a history of ectopic gestation was significantly associated with difficulty visualizing the ostia; thus, it may be reasonable to advise these women that success in essure placement may be reduced. introduction. the essure permanent birth control device is a relatively new form of minimally invasive sterilization for women. under hysteroscopic guidance, a dynamically expanding micro-insert is introduced into the proximal portion of the fallopian tube. the micro-insert induces local fibrosis and ultimately occlusion of the tubal lumen. a hysterosalpingogram (hsg) is performed three months after the procedure to confirm bilateral tubal occlusion. objective. to determine the follow-up rate for the post-essure hsg for a clinic population. methods. a retrospective chart review was conducted on university health center (uhc) patients who underwent placement of the essure permanent birth control device at the detroit medical center from january through june . follow-up for the post-essure hsg as well as the result of the hsg were noted for each patient. results. placement of the essure permanent birth control device was attempted in uhc patients of which were successfully completed. of the patients, ten underwent a post-essure hsg ( . %). the hsg was performed three to six months after placement of the essure permanent birth control device. bilateral tubal occlusion was documented in all ten patients. conclusion. despite counseling patients prior to their procedure that a hsg is needed and providing an information sheet, the follow-up rate for the post-essure hsg for this clinic population is only . %. for those in whom a hsg was performed three to six months after essure placement, bilateral tubal occlusion was confirmed in all. steps and or approaches to improve compliance with post-procedure confirmation of tubal occlusion should be employed to increase follow-up in the future. towards fibroids gene therapy: adenovirus mediated delivery of herpes simplex virus thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the eker rat model. memy hassan, dong zhang, salama salama, cheryl walker, hala el-mazar, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. aim: assessment of the efficacy of gene therapy of uterine leiomyoma in the immune-competent eker rat model using adenovirus mediated delivery of herpes simplex- -thymidine kinase gene followed by ganciclivir treatment (ad-tk/gcv). method: female eker rats with mri-confirmed uterine fibroid lesions were randomized to a single treatment with direct intratumor injection of ad-tk/gcv, ad-lacz/gcv, or medium.the tumor volume was evaluated by serial mri scanning and confirmed with caliper measurement at time of euthanasia. sample rats were selected randomly and killed at the following time points; , and days post treatment. samples were collected from tumors, other body organs and blood to assess the safety and efficacy of the treatment. results: ad-tk/gcv treatment produced dramatic shrinkage of the total uterine fibroid volume by % ± , % ± and %± of pretreatment volume at days , and respectively. the tumor size in negative control animals receiving ad-lacz/gcv continued to grow by + %± , + %± , + % ± while receiving media continue to grow by + % ± , + % ± and + % ± at same time points. ad-tk/gcv induced significant increase in caspase activity, bax expression, decrease in bcl and parp proteins expression and increased tunnel apoptosis index. additionally ad-tk/gcv treatment decreased cyclin d and pcna expressions. ad-tk/gcv did not produce any significant change in liver function tests or relative uterine horns weight to total body weight. the adenovirus transfection did not disseminated significantly to other distal organs except to liver and myometrium in limited number of animals. h e staining of non targeted organs did not revealed any sign of tissue damage. ad-transfection increased local cd and cd expressions as well as serum anti-ad antibodies. conclusion: ad-mediated delivery of hsv tk gene by direct intra-tumor inoculation followed by sc treatment of gcv for ten days effectively shrinks uterine leiomyoma lesions in eker rats. this effect is mediated via induction of apoptosis and decreasing the proliferation. the treatment regimen is well tolerated. these studies provide essential preclinical data for the development of gene therapy as an alternative non-surgical treatment option for women with symptomatic uterine fibroids. inhibitors of catechol o-methyl transferase shrinks uterine fibroids in the eker rat model. memy hassan, dong zhang, hala el-mazar, cheryl walker, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. background :the sex hormone dependent pattern of uterine leiomyomas and their high content of catechole -o-methy transferase (comt) raise the possibility for the development of novel treatment option using comt inhibitors.aim : to assess the potential therapeutic utility of a synthetic comt inhibitor (ro - ) in the eker rat model of uterine leiomyoma. methods female eker rat were evaluated by mri to confirm the prescence uterine fibroid lesion, then randomized for sc treatment with ro - mg /kg ,twice/ day for days versus vehicle injection.fibroid tumor burden was evaluated by serial mri measurement and confirmed by direct caliper measurement at time of euthanasia. sample animals were euthanized at and weeks. at that time tumor tissue, blood and most of animal organs including long bones were collected and subjected for further evaluations. hours urine samples were collected for evaluation of estrogen (e ) metabolites and bone resorption marker. results:animals treated with ro - exhibited significantly lower uterine fibroid tumor burden ( % and %) of pretreatment volume at and weeks post treatment respectively. conversely, the tumor size in control animals continued to grow and reached %, and % of pretreatment size at the same time points. ro - treatment resulted in an increase in urinary / hydroxy e metabolite ratio. in addition ro - increased bax expression and decreased parp , pcna and cyclind experssions . all ro - treated animals tolerated the treatment protocol with no signs of toxicity. h e staining of different body organs did not reveal any signs of tissue damage. furthermore, there was no significant change in both liver function tests (alt, ast, billirubin) and bone resorption marker , deoxypyridinoline croslinks, between treated and control rats. conclusion ro - , a synthetic selective comt inhibitor, caused immediate arrest of the growth of eker rat uterine leiomyoma. this effect might be in part due to modulation of various estrogen dependent genes regulating leiomyoma apoptosis (parp, bax) and proliferation (pcna, cyclin d ). this anti-estrogenic effect is due to the accumulation of the the antiestrogenic metabolite hydroxy estrogen secondary to comt inhibition.comt inhibitors might present an alternative non-surgical option for the treatment of women with symptomatic uterine fibroids. background development of uterine leiomyomas (fibroids) is the most common pathological feature in the female reproductive tract. they negatively impact patients of virtually every gynecologist. despite such morbidity, leiomyoma development is poorly understood. we have recently demonstrated that leiomyomas have a genomic expression pattern that limits retinoic acid (ra) exposure. our group and others have demonstrated that tgf-beta regulation is altered as well. these two pathways likely play central roles in leiomyoma development. the central feature of uterine leiomyomas, the extracellular matrix (ecm), is regulated by both all-trans retinoic acid and tgf- , focusing on versican as a critical ecm component. human uterine leiomyoma and patient-matched myometrium were obtained from surgical specimens under an irb-approved protocol. these tissues were immortalized and treated with either all-trans retinoic acid, tgf- , or anti-tgf- antibody. rna was isolated for qrt-pcr. human immortalized leiomyoma cells demonstrated the same increased template expression of tgf- ( . ± . fold), retinoic acid metabolizing protein (cyp a ; . ± . ), and versican variant v ( . ± . fold) as was found in the progenitor tissue. when treated with all-trans ra, expression of versican variant v decreased to levels found in myometrial cells ( . ± . fold). conversely, when treated with tgf- , expression of versican variant v increased . ± . fold. to confirm that tgf- was central to the overexpression of v , we treated leiomyoma cells with anti-tgf- antibody, and found that baseline over-expression of v template was decreased to expression levels similar to untreated myometrial cells. finally, we elucidated a link between the ra and tgf-pathways by assessing the impact of ra treatment of tgf- expression, demonstrating that tgf- template decreased to levels comparable to myometrial cell expression ( . ± . fold). the disrupted leiomyoma ecm, of which versican is a central component, defines the leiomyoma phenotype. in this study, the leiomyoma fibrotic phenotype regressed when treated with ra and increased when treated with tgf- , providing the basis for novel therapeutic interventions directed at cell differentiation and ecm formation. objectives: uterine fibroids are the leading cause for hysterectomies in the us. the lack of an appropriate in vitro cell model for the initiation of fibroid growth has hindered advancement in understanding the cellular and molecular basis for the development and progression of uterine fibroids. fibrosis is the underlying mechanism of uterine fibroid formation and myofibroblasts cells are the principal fibrogenic cell type in the uterus. we sought to develop a myofibroblast in vitro cell model for analyzing the initiating molecular events of uterine fibrosis. methods: smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and cultured in the presence of % serum until % confluent. for the next h cells were cultured in serum-free media followed by replacement with serum containing media. cells were fixed at , , , , m later. cell fine structure and cytoskeletal organization were evaluated by transmission electron microscopy. smooth muscle specific alpha-actin ( -sma) and progesterone receptors (pr) were detected by western blot. results: we observed smc differentiation into myofibroblasts, marked by the presence of notched nuclei ( figure) and the increased expression of -sma m after serum replacement. pr-a and pr-b were detectable at , and m. conclusions: the development of myofibroblasts is important in wound healing and fibrosis. we show for the first time that uterine myofibroblasts can be derived in culture from myometrial smcs. thus, these cells will be utilized as a model for developing "in vitro fibroids". this model will enable the study of myofibroblast activation, cytokine signaling, intracellular regulation of uterine fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. the presence of prs in our model enables us to evaluate pr mediated events in fibroid pathogenesis and treatment. this model will be more useful in determining the molecular biology of fibroid initiation than cell models derived from established fibroids that are already well past their initial stages of development. novel approach to genome-wide expression profiling analysis. liping feng, morgan walls, insuk sohn, millie behera, sin-ho jung, phyllis leppert. obgyn; biostatistics and bioinformatics, duke university medical center, durham, nc, usa. background: microarray studies have examined the differential gene expression between uterine fibroid and normal myometrium. all previous studies considered the fibroid as a whole and analyzed only fold changes. we have developed a novel statistical approach to genome-wide expression analysis comparing two fibroid tissue sites to myometrium. methods: using affymetrix tm u a genechip, we have compared the gene expression between c and e and matched adjacent m. data has been analyzed by considering the specimens per subject and subjects as individuals. we used a block one-way anova method to test if each gene was differentially expressed among the three sites. the p-value is calculated using a permutation method accounting for possible dependency among three lesions. the multiple testing issue was addressed by controlling the false discovery rate. expression values were calculated using the robust multichip average (rma) method. rma estimates are based upon a robust average of background corrected perfect/mismatch (pm) intensities. normalization was done using quantile normalization. expression values were then transformed by taking logarithm base . confirmatory rt-pcr was performed. results: we applied a hierarchical clustering analysis to all raw data sets and then displayed a dendrogram, where the height of each branch point indicates the similarity level at each generated cluster. identical gene expression among sites clusters together. due to a strong site effect, m tissues clustered separately from e and c combined. genes were differentially expressed when we used a . q-value cut off. expression data revealed concordant changes in genes regulating cholesterol biosynthesis, gene transcription, estrogen and extracellular matrix formation when both e and c were examined. cyp was detected and we report for the first time that scc- (a cell cycle-regulated molecule) folliculin and l-selectin are differentially expressed suggesting that they may be involved in the regulation of cell growth and proliferation of uterine fibroids. conclusions: our novel robust analysis of gene expression provides new clues to the relevant pathways of fibroid development. this new statistical approach that can be used in clinical and/or translational studies to identify differentially expressed genes comparing treatment regimens, cells or tissues. objectives: thrombospondin- (tsp- ) is a large matricellular glycoprotein secreted by many cell types. matricellular proteins modulate interactions between cells and their environment, regulate cell adhesion and are expressed during tissue formative processes. they are especially important in fibrosis. tsp- plays an important role in angiogenesis and is an activator of tgf - . in a previous study, we found that differential expression of tsp- in uterine fibroids may contribute to an altered healing process leading to fibrosis. this alteration in tissue response to injury initiates the development of abundant, nonaligned collagen fibrils and changes in other components of the ecm. methods: we measureed the pattern of mrna and protein expression of tsp- by rt-pcr and western blot in an in vitro serum-deprived differentiated myometrial cell (myofibroblast) model. specifically, smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and grown in primary culture to % confluence. then smc were serum deprived for h and treated back with % serum for , , , and m. cells were collected for rna and protein, and tsp- expression was evaluated. in addition, cells were stained using the combination of anti-cd and anti-smooth muscle -actin or combination of anti-cd d and anti-smooth muscle -actin as well as the appropriate single and double negative controls. stained sections were analyzed using zeiss axio imager widefield fluorescence confocal microscopy. results: tsp- mrna and protein was present in cells in this serumstarvation model after the addition of serum and the expression level remained elevated for m following the addition of serum. fluorescence staining analysis indicated that these cells were positive for human smooth muscle -actin, but negative for leukocyte antigen cd and platelet marker cd d suggesting that the myofibroblasts cells themselves were the source of the tsp- . conclusions: unlike skin wounds, where tsp- is derived from the blood macrophages, monocytes and platelets, differentiated myometrial cells appear to produce tsp- . elucidating the roles of tsp- in myometrium physiology and pathobiology will increase our understanding of the etiology of uterine fibroids and may lead to improved therapies. further studies utilizing this cell model to determine the role of tsp- in the activation of tgf - are indicated. phospholipid s p via gi, rac and rho pathways. yoel smicun, armando wu pimentel, jennifer gilman, david a fishman. obstetrics gynecology, new york university school of medicine, new york, ny, usa. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of ovarian cancer patients. we have demonstrated that low concentration s p enhances while high dose s p inhibits invasion of epithelial ovarian carcinoma (eoc) cells in a dose and attachment mode dependent manner. we sought to further dissect the pathways by which s p affects invasion, using specific inhibitors. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi, p -mapk, rac and rock, thereafter cells were detached and tested for invasion towards m lpa chemoattractant in matrigel-coated chambers. conditioned media from pretreated cells and invading cells were quantified for upa activity using colorimetric assays. the significance of results was calculated by student's t-test. results: inhibition of gi mildly increased invasion of both control ( p= . ) and m s p treated cells ( p= . ). inhibition of both p -mapk and rac did not affect m s p treated cells, in contrast invasion of control cells was mildly increased (p= . , . ). inhibition of rock, a protein effector downstream of rho, highly elevated invasion of both control and m s p treated cells ( fold, p= . , fold, p= . ). both upa and gelatinase activities were higher in conditioned media of invading cells than of attached cells. gelatinase activity was enhanced by both concentrations of s p (p= . , . ). ptx fully inhibited gelatinase activity of control and . m s p treated cells, and partially of the m s p treated cells (p< . ). . m s p significantly increased upa activity of attached (p= . ) but not of invading cells. this increase was sensitive to ptx and rac inhibitor. m s p inhibited upa in both attached and invading cells (p= . ), this inhibition was rock dependent. conclusions: these findings suggest a strong inhibition of invasion and upa by the rho pathway, of both control and m s p treated cells. this inhibition is induced partially by upstream gi protein. increased invasion by . m s p is associated with elevation of gelatinase activity through gi, rac and rock pathways. this suggests that attached cells and invading cells affect upa activity through different pathways. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of epithelial ovarian cancer (eoc) patients. we have demonstrated that invasion of attached eoc cells differentially react to s p as compared to invading cells. we examined the impact of the inhibitors for gi and rac on attached and invading eoc. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi (pertussis toxin (ptx)), and rac (nsc , (rac-i)), and cells were detached and assayed for invasion towards m lpa in matrigel-coated chambers. to distinguish the response of attached from invading cells, inhibition of cells pretreated with inhibitors was either continued or not in the invasion chambers. conditioned media (cm) of invading cells were quantified for upa and gelatinase activity by fluorometric and colorimetric assays. significance of results was calculated by student's t-test. results: the significant (p= . ) increase of invasion by . m s p was inhibited by both ptx and rac-i, either by pretreatment alone or by continuous treatment (p= . - . ). however, the invasion was higher in cells inhibited continuously than cells inhibited only in dishes. both inhibitors did not affect cells treated with m s p. control cells invasion was increased ( . fold, p= . ) by continuous rac-i treatment. . m s p increased gelatinolysis in cm of invading cells. this and control cells activity was inhibited by ptx pretreatment. continuous treatment with ptx or rac-i elevated and fold gelatinase activity of control and m s p treated cells. in contrast upa activity was inhibited by both . m and m s p. activity of control cells was inhibited by both pretreatment and continuous treatment with both ptx and raci. upa activity of cells treated with . m s p was increased only by pretreatment with ptx and raci. in contrast, in cells treated with m s p upa was inhibited only by continuous inhibition with ptx and raci. conclusions: invasion of s p induced eoc cells correlated with the gelatinase and upa activities in their microenvironment. ptx and rac-i affect attached and invading cells in different manner, inhibition of invasion and gelatinolysis of attached and increased invasion of invading cells. this suggests a dual effect of the gi-rac pathway, inhibiting attached and stimulating invasion via gelatinolysis of invading cells. lysophosphatidic acid (lpa) levels are elevated in the ascites and plasma of early-and late-stage ovarian cancer patients, underscoring the unique role this bioactive lipid plays in the pathobiology of epithelial ovarian cancer (eoc). lpa binding to its cognate g-protein-coupled receptor can transactivate the receptor tyrosine kinase, egfr, which is often overexpressed in ovarian tumors. in the current study, we investigated the role that lpa activation of egfr plays in the processing of the metalloproteinase, mmp- , and eoc dissemination. lpa stimulates tyrosine phosphorylation of egfr in ovarian cancer cells, and egfr kinase activity is required for optimal lpa induction of pro-mmp- activation. lpa-dependent pro-mmp- activation does not require the egfr ligands, amphiregulin, b-cellulin, egf, hb-egf, and tgf-a. we previously reported that when cells are cultured at high density, lpa represses rhoa activity to induce loss of stress fibers, and these changes in actin microfilament organization contribute to pro-mmp- activation. in the current study, inhibition of rho-kinase/rock with y- reversed the repression of lpa-stimulated pro-mmp- processing observed with treatment of the egfr kinase inhibitor, ag . correspondingly, lpa induced the loss of stress fibers, while inhibition of egfr kinase restored stress fiber formation in lpa-treated cells. this suggests that lpa acts through egfr to modulate microfilament organization and pro-mmp- processing. finally, lpa-induced cellular haptotactic migration and invasion are abrogated when egfr kinase activity is blocked. taken together, these results suggest that egfr signaling plays a critical role in lpa regulation of metastatic pathways by mediating changes in the cytoskeleton which impact protease activity. objectives: membrane-derived vesicles are active modulators of tumor dissemination; they promote and contribute to extracellular matrix degradation and tumor cell invasion. we have isolated vesicles from ascites of ovarian cancer patients and from ovarian cancer cells in vitro. here we analyzed the functional consequences of exposure of cancer cells to vesicles derived from a different type of malignancy in order to evaluate their proinvasive properties. methods: ovarian cancer (dov ), breast cancer (mda mb ) and mesothelioma (hp- ) cells were grown in media supplemented with % fbs. after serum deprivation, % vesicle-free fbs was added for hr to induce vesicle release. vesicles were isolated from media by differential centrifugation and quantified with a bradford assay. fusion to cells was followed by fluorescence of the lipophilic tracer dii. each cell line was stimulated with and g/ml of each type of vesicles and tested in a matrigel invasion assay. changes in proliferation were evaluated in an mts assay. gelatin zymography was used to assess matrix metalloproteinase activity of vesicles. results: zymographic analysis showed that vesicles contained mmp- and . fusion experiments showed that all vesicles fused to all cell types. all vesicles induced the invasion of their respective cell types in a dose-dependent manner. when vesicles were used at g/ml they induced significantly high levels of invasion in all cell types tested. at g/ml they significantly inhibited invasion in different cell types. both concentrations of vesicles stimulated cell proliferation in all cell types tested. conclusions: membrane derived vesicles are potent mediators of invasion for mesothelioma, breast and ovarian cancer cells in vitro. this effect occurs in a dose-dependent manner when vesicles induce invasion of the same cell type from which they were isolated. when vesicles are used to induce a different cell type, low concentrations induce invasion while high concentrations inhibit invasion, this effect was independent of cellular proliferation. these results suggest that a novel mechanism may be at play where activation of recognition of self and non-self specific pathways may determine the invasive potential of the tumor cell upon fusion to microvesicles. the identification of signaling pathways responsible for this heterotypic signaling is a current effort. vegfr- as a potential target to inhibit lpa-induced epithelial ovarian cancer (eoc) invasion. sonia dutta, fengqiang wang, elaine barfield, david a fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objective: vegf and vegf receptors (vegfrs) play important roles in ovarian cancer metastasis. in this study, we examined the expression profiles of vegf and vegfrs (vegfr , vegfr , co-receptor nrp and nrp ) in established eoc cells lines (dov , r , ovca , skov ) and an immortalized normal ovarian epithelium (iose- ). the effect of lysophosphatidic acid (lpa) on vegf and vegfrs expression and the effect of vegfr- silencing by rnai on lpa-induced invasion were also evaluated. methods: vegf and vegfrs expression in ovarian cancer cell lines and normal ovarian epithelium was quantified by real time pcr. cell invasion differentiate into either a pro-tumor or anti-tumor phenotype depending on the specific tumor microenvironment. previously, we described a subgroup of epithelial ovarian cancer (eoc) cells with a functional tlr- -myd -nf-b pathway (type i eoc cells). these cells have constitutive nf-b activity and constitutively secrete il- , il- , mcp- , and gro . we hypothesize that type i eoc cells, but not type ii, can promote macrophage differentiation into a tumor-supporting immune cell. methods: eoc cell conditioned media (cm) was prepared by incubating eoc cells in log-phase growth in optimem for h. migration assay was done using an in vitro cell culture insert with m-size pore and pkh red fluorescentlabeled thp- . cytokine profile of thp- cells co-cultured with eoc cell cm was determined using xmap technology. modulation of response to apoptotic bodies was determined by "pre-educating" thp- cells with eoc cell cm for h prior to exposure to apoptotic bodies ( : ratio with thp- cells). results: monocytes migrate toward eoc cells. however, migration is significantly higher towards type i eoc cells. type i, but not type ii, eoc cells alter monocytes' cytokine profile by inducing the secretion of high levels of progrowth and angiogenic cytokines il- , il- , mcp- , and gro . furthermore, type i eoc cells modify monocytes response to apoptotic bodies by inducing a significant increase in the secretion of il- , il- , mip- , mip- , and gro ( -fold, . -fold, -fold, -fold, and -fold respectively). conclusion: we demonstrate for the first time a differential interaction between two subtypes of eoc cells and monocytes. we showed that the microenvironment created by type i eoc cells is able to modify the function and differentiation of immune cells towards a tumor supporting phenotype. understanding the molecular mechanisms mediating this tumor-immune interaction will help to design appropriate immune therapies that will take into consideration the tumor microenvironment. objectives: the standard of treatment for patients with ovarian cancer (oc) is intravenous combination chemotherapy (ct) after primary cytoreductive surgery. although initial response is above %, most of these patients experience recurrence. the only approach for these patients is salvage ct which may prolong their lives for months. early detection of patients who are not responding to current therapy or are at risk of experiencing an early relapse of disease might improve response rates and survival if alternative therapy is possible. no single predictive marker has yet been proven sufficiently sensitive or specific to find a place in the daily clinic. in the present study we use a newly described biomarker test for the detection of oc (visintin et al clinical cancer research) and evaluated the ability of the test to monitor ct response methods: patients with oc, figo stage i-iv, were included in this study. all patients recieved postsurgery first line combination ct (paclitaxel/carboplatin). samples were evaluated at ) baseline (mean days after surgery), prior to the first cycle of ct, and ) after cycles of ct. μl serum samples were analyzed by multiplex assay (beadlyte® cancer biomarker panel kit) for six markers. changes during ct and differences in markers between patients with short time to progression (ttp) and patients with long ttp were determined. results: positive test for oc was observed in % of the patients evaluated at baseline. all patients had residual disease after surgery. from patients with long ttp, / patients (specificity %) had a negative test after cycles. from the patients with short ttp, / had a positive test (sensitivity %) p= . ( ²). the risk of experiencing a ttp shorter than months when having a positive test after cycles of ct was %. conclusion: we describe for the first time the use of a panel of biomarkers for oc that might have an application for monitoring ct response and risk of relapse. the test detects the presence of residual disease following debulking surgery, and differentiates between long term and short term progression. a longitudinal study is performed to determine how early during ct the test can identify responder versus non responder. ksp inhibitor (arry- ) as an alternative for paclitaxel in myd -positive epithelial ovarian cancer cells. ki h kim, ayesha b alvero, yanhua xie, david trollinger, gil mor. obstetrics, gynecology, and reproductive sciences, yale university school of medicine, new haven, ct, usa; array biopharma inc., boulder, co, usa. background: we previously described that epithelial ovarian cancer (eoc) cells ubiquitously express tlr- , but not the adaptor protein myd . when treated with paclitaxel, a known tlr- ligand, myd -positive eoc cells exhibited nf-b activation, increased secretion of il- , il- , mcp- , and gro , and increased p-erk levels . since majority of eoc patients are given paclitaxel in combination with a platinum drug, it is not only important to distinguish those patients that should not be given paclitaxel; it is also important to identify alternative chemotherapy agents that would benefit this sub-group of patients. the objective of this study is to determine if the ksp inhibitor, arry- , can be an alternative for paclitaxel in myd -positive ovarian cancer patients. methods: myd -positive and myd -negative eoc cell lines isolated from either ascites or tumor tissue were treated with increasing concentrations of arry- ( to nm) or paclitaxel ( . to m) for , , and hours and cell viability was determined using the celltiter aqueous one solution cell proliferation assay. cytokine profiling was performed from supernatants using xmap technology. nf-b activity was determined using a luciferase reporter system. p-erk levels were measured by western blot analysis. results: arry- and paclitaxel exhibited the same cytotoxic effect on myd -negative and positive eoc cells. the ic at h for myd -negative eoc cells was . m and . um for arry- and paclitaxel, respectively. for myd -positive eoc cells, the ic at h was m and m for arry- and paclitaxel, respectively. however, unlike paclitaxel, arry- did not induce nf-b activation or enhance the secretion of il- , il- , mcp- , and gro , and did not induce erk phosphorylation on myd positive cells. conclusions: administration of paclitaxel to patients with a myd -positive tumor could have detrimental effects due to the paclitaxel-induced enhancement of cytokine production which promotes chemoresistance and tumor growth. arry- has similar anti-tumor activity in eoc cells as that of paclitaxel. however, unlike paclitaxel, it does not induce cytokine production in myd positive eoc cells, and therefore, the ksp inhibitor arry- may represent an alternative to paclitaxel in this subgroup of eoc patients. introduction: nf-b activation has been associated with cell proliferation, angiogenesis, metastasis and suppression of apoptosis in ovarian cancer. in addition, nf-b activity induces the production of pro-inflammatory cytokines which may contribute to chemoresistance. inhibition of nf-b may represent a new approach to prevent tumor growth and reverse chemoresistance. in the present study we described the characterization of a novel nf-b inhibitor, eriocalyxin b (erib) that is able to re-establish the apoptotic cascade in chemoresistant ovarian cancer cells by suppressing pro-inflammatory cytokines and antiapoptotic proteins. materials and methods: eoc cell lines were isolated from malignant ovarian cancer ascites. caspase activity was determined by caspase-glotm assay. cytokine production and secretion were determined using multiplex assay. erib effect on cancer cells was evaluated in a time and dose dependent manner using celltiter cell proliferation assay. protein expression was determined by western blot analysis. combination studies were done with paclitaxel and carboplatin in addition to erib treatment. nf-b activity was determined by monitoring the expression of a nf-b luciferase reporter. results: erib decreased cell viability of ovarian cancer cells with an ic of . - μm in hours and was associated to increasing levels of caspases , and activity. intracellular changes induced by erib included: ) inhibition of nf-b activity; ) decrease in cytokine production; ) down regulation of anti-apoptotic proteins xiap and flip, and reversal of resistance to tnf-and fasl-mediated cell death; and ) chemosensitization to carboplatin and paclitaxel. at present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating, stem-like cells within various tumor types of distinct origins. these cancer stem cells (csc), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. we propose that ovarian cancers arise from csc, and are using microarray-based technology to identify specific genes/cell surface markers associated with ovarian csc that will permit distinction of these rare cells from the remaining tumor bulk. to identify unique gene signatures associated with an ovarian tumor-initiating cell population, we have utilized an in vivo serial transplantation model. this model selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreases with successive serial transplant despite fewer cells injected. our initial studies used cells derived from a human ovarian clear cell carcinoma, serially transplanted for three passages in nod/scid mice. rna derived from these transplanted tumors was analyzed on human genome microarrays. from these analyses, several differentially expressed genes were identified. the differential expression noted for potential genes of interest is currently being validated by rt-pcr. of particular interest, expression of the transmembrane glycoprotein muc was found to increase both at the rna and protein level with successive transplant of this clear cell carcinoma. further studies are ongoing to determine the functional significance of muc and other identified differentially expressed genes in ovarian clear cell cancer. in addition, we are carrying out parallel microarray analyses in other ovarian tumor subtypes. background recent observations suggest a decreased incidence of neoplastic lesions in hiv infected individuals treated with highly active anti-retroviral therapy comprised of protease inhibitors, such as ritonavir. the polycomb group protein ezh is associated with aggressive human malignancies via transcriptional suppression of dna repair proteins. objective objective of the present study was to assess the antineoplastic impact of ritonavir on epithelial ovarian cancer (eoc) cell lines. methods eoc cell lines (mdah and skov- ) were treated with serial dilutions of ritonavir ( - mm) dissolved in dmso. normal diploid human fibroblasts served as controls. growth curves, apoptosis and cell cycle analysis were performed with cell counting kit- , annexin v and flow cytometry. signal transduction was studied with western blotting. dna double strand breaks (dsb) were induced with . mm etoposide treatment for hours. homologous recombination (hr) repair of dna damage was measured by assessment of rad foci formation in the nucleus of the cells as visualized by fluorescence microscopy according to previously published criteria. untreated eoc cells expressed higher levels of ezh and lower levels of rad and xrcc as compared to controls and in turn, had lower prevalence of rad repair foci formation in response to dsb. serial treatments of eoc cells with ritonavir resulted in a decrease in expression of ezh and an increase in expression of rad and xrcc when compared to untreated eoc cells. after induction of dsb, the rad repair foci formation was significantly more prevalent in eoc cells treated with ritonavir as compared to untreated eoc cells. in addition, ritonavir induced apoptosis in ovarian cancer cell lines by down-regulation of akt pathway and caused g cell cycle arrest mediated by down modulating levels of prb phosphorylation and depleting the cyclindependent kinase and . ritonavir effectively induces apoptosis, cell cycle arrest and improves repair of dna damage by hr in ovarian cancer cell lines. as impaired hr is a key event in causation and progression of neoplastic lesions, ritonavir may have a role in chemoprophylaxis and treatment of human malignancies. a rare case of ovarian cystic lymphangioma. tomer singer, tamer seckin, noa feldman, susan jormark, michael divon. department of obstetrics and gynecology, lenox hill hospital, n.y., ny, usa; department of pathology, lenox hill hospital, n.y., ny, usa. cystic lymphangioma (cl) is a rare, benign malformation of the lymphatic system whose exact etiology remains uncertain. cl may arise in different sites: the spleen, the mediastinum, the axillary region, the retroperitoneum, and the mesentery. retroperitoneal cl is extremely rare and its true incidence is unknown. the majority of cases are symptomatic during childhood. clinical presentation of adult cl is variable and may be misleading. typically, this is a slow-growing tumor and it remains asymptomatic for a long period of time. it is most often found incidentally during abdominal or pelvic imaging studies, surgeries or autopsies. total surgical removal of the lesions with microscopically clear margins is the best approach when it is possible. we report, for the first time, a case of cystic lymphangioma arising from the ovary in a post-menopausal woman and present the feasibility and the advantages of laparoscopic surgery, allowing accurate diagnosis, optimal treatment and minimal risk for the patient. lgsc) is a chemoresistant ovarian neoplasm that has been molecularly linked to low malignant potential tumor (lmp), which often behaves in a non-invasive fashion. micropapillary features within lmp (lmp-mp) are associated with increased invasive behavior. the aim of this study was to determine the differential gene expression of lmp, lmp-mp, and lgsc to identify genes involved in malignant transformation and carcinogenesis. methods: laser capture microdissection was used to isolate epithelial cells from snap-frozen lmp (n= ), lmp-mp (n= ) and lgsc (n= ). rna was extracted, amplified, reverse transcribed to cdna and hybridized to affymetrix u plus arrays. data was analyzed by significance analysis methods: medical records were reviewed for patients who underwent hysterectomy for all indications at wsu hospitals from / / - / / . pathology reports were reviewed to identify the incidence of adenomyosis. data obtained from medial records included: age, race, insurance, bmi, social history, medical history, and presenting symptoms. the correlation between adenomyosis and all the above factors was tested using the appropriate statistical methods. results: patients were included. adenomyosis was confirmed by pathology in patients, an incidence of . %. incidence was not significantly different among races after controlling for parity. . % in african americans, . % in caucasians, and . % in others. incidence was not statistically different between uninsured( . %), privately insured ( . %), and medicaid ( . %) patients. p=. . incidence of adenomyosis was not different between smokers ( . %) and nonsmokers ( . %). p= . . table i shows the correlation between adenomyosis and different risk factors. conclusion: adenomyosis was diagnosed in . % of hysterectomy specimens. race, socioeconomic status or social habits didn't affect its incidence. diabetes and endometrial cancer were negative risk factors for adenomyosis, whereas htn, hypothyroid, breast cancer, fibroids, polyps and endometriosis didn't affect its incidence. menorrhagia, dysmenorrhea, dyspareunia, and chronic pelvic pain but not metrorrhagia had a positive correlation with adenomyosis. there is a protective adipocytokine profile. we hypothesize that this finding may precede some ill-defined threshold of fat mass and/or insulin resistance after which adiponectin decreases. the objective: to correlate reproductive hormone production with menstrual bleeding patterns among women in the menopause transition. methods: a sub-cohort of the swan study consisting of women age - was studied. each woman collected daily first void urine samples for one complete menstrual cycle or days (whichever came first) once a year for years. urine was assayed for excreted levels of fsh, lh, estrogen metabolites and progesterone metabolites which were normalized for creatinine concentration. ovulation was detected by a validated algorithm. menstrual bleeding parameters were derived from daily calendars. correlations between bleeding characteristics, hormone concentrations, and other potential clinical predictors were analyzed using multivariate logistic regression models. results: the cohort was ethnically diverse with a median age of and with % in the early perimenopause at the start of the study. % of all cycles were anovulatory. short cycle intervals (< days) were associated with the early perimenopause (or . , ci . , . ) and with anovulation (or . , ci . , . ). long cycle intervals ( + days) were associated with late perimenopause (or . , ci . , . )and with anovulatory cycles (or . , ci . , . ). both short ( - days) and long ( + days) duration of menstrual bleeding were significantly associated with anovulation (or . and . , respectively). women with anovulatory cycles were less likely to report heavy menstrual bleeding than women with ovulatory cycles. menorrhagia was not associated with steroid hormone concentrations but was associated with obesity (or . , ci . , . ) and with the self-reported presence of fibroids (or . , ci . , . ). conclusions: among women in the menopause transition, abnormalities in timing of menstrual bleeding (cycle intervals or bleeding duration) have a hormonal basis and are frequently associated with anovulation. in contrast, abnormally heavy periods do not have a hormonal basis and are less likely following anovulatory cycles. heavy periods are associated with obesity and fibroids. to undetectable levels. the use is vaginal e is contraindicated, although these women have even higher rates of av. topical testosterone (tt) has successfully treated vulvar atrophy; testosterone receptors are also present in the vaginal epithelium. objective: assess the effect of tt on vaginal maturation index (mi) and relief of av symptoms. methods: postmenopausal women on aromatase inhibitor with symptomatic atrophic vaginitis were enrolled in prospective, irb approved study. estradiol (e) and testosterone (t) levels, av questionnaires (score - ; none to most severe symptoms of dryness, irritation, and dyspareunia), gynecologic exam, and vaginal smears (for ph and vaginal maturation index, mi, by meisels criteria) were performed at baseline and after month of daily treatment with mcg of tt. data was assessed by t test and fischer's exact test; significant p < . . results: e levels were undetectable at baseline and following treatment. t levels increased (mean +/-sem) from baseline ( +/- . ) to treatment ( +/- . ); the difference was not significant; p = . , although one patient had an appreciable rise in serum t level. two av symptoms improved significantly with tt use; comparing baseline to treatment scores: vaginal dryness ( . vs. . ; p = . ) and dyspareunia ( . vs. ; p = . saliva analysis is a convenient, non-invasive and rapid method for assessing estradiol (e ) levels. however, particularly in postmenopausal women, the low salivary e levels are often near or below the sensitivity of available assays, compromising both accuracy and precision. we present results using an extraction step prior to e assay, which concentrates the sample to increase sensitivity and removes potentially interfering substances. morning saliva samples were obtained from premenopausal (mid-luteal phase, n= , ) and postmenopausal women (n= , ) not taking hormones, and from postmenopausal women receiving oral conjugated equine estrogens (cenestin, n= ; premarin, n= ), oral micronized e (estrace, n= ; compounded e , n= ), transdermal e patches (climara, n= ; vivelle, n= ); or topical e cream (compounded e , n= ). e levels were determined by an automated enzyme immunoassay (eia) after solid phase extraction. the functional sensitivity of the assay was determined to be . pg/ml, compared with > pg/ml without extraction. . . . . . oral conjugated equine estrogens; oral e ; e patch; topical cream salivary e levels corresponded with the hormone dosage, suggesting a reliable assessment of unbound e levels with each formulation, dosage and type of estrogen therapy. extraction prior to eia in an automated assay dramatically increased precision and accuracy at low concentrations. omitting the extraction step may have contributed to poor serum versus saliva correlations in other studies. this method may therefore allow reliable monitoring of estrogen therapy without the need for expensive and inconvenient blood tests. the role of fibulin- in pelvic organ prolapse. david d rahn, jesus f acevedo, patrick w keller, lihua marmorstein, r ann word. ob-gyn, ut southwestern, dallas, tx, usa; visual sciences, univ arizona, tuscon, az, usa. objectives: fibulin- (fib- ) is crucial for normal elastic fibers in the protein showed a statistically significant reduction in its expression (p= . ). lox, loxl , and proteins were detected by immunohistochemistry in all three layers of vaginal skin biopsies: ( ) stratified squamous epithelium; ( ) the lamina propria and ( ) the muscularis layer from both patients with pop and controls. significantly, in both groups we detected a numerous macrophages scattered throughout the vaginal thickness which displayed a very strong immunostaining to loxl . patients with severe pop showed reduced expression of proteins regulating collagen and elastin biogenesis. our finding raises the possibility that failure of ecm homeostasis could underlie the etiology of pop in women. fibroblast proliferation is regulated by hoxa : molecular implications for pelvic organ prolapse. kathleen a connell, marsha k guess, richard bercik, hugh s taylor. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa. objectives: the integrity of the extracellular matrix (ecm) is maintained by a delicate balance between collagen synthesis and degradation. previously, we have demonstrated that hoxa is essential for the development of the uterosacral ligament in mice and regulates the expression of collagen type iii and mmp . we have also shown that hoxa is deficient in the uterosacral ligament of women with pelvic organ prolapse (pop) compared to women with normal support. the exact mechanisms by which hoxa regulates pelvic organ integrity and repair remains to be elucidated. the aim of this study was to determine the effect of hoxa expression on fibroblast proliferation, and its potential role in pop. methods: nih t cells, a murine fibroblast cell line, were seeded onto a six well plate ( x cells/well) and transfected with either a vector carrying a hoxa cdna or empty vector alone as a control. immunohistochemistry using bromodeoxyuridine (brdu) was performed to evaluate cell proliferation. cell division in the uterosacral ligament (usl) was also compared in women with and without pop. usl specimens were obtained at the time of hysterectomy for benign disease. immunohistochemistry was performed on paraffin embedded sections of the usl to evaluate expression of two mitotic markers, cyclin b and phospho-histone . results: constitutive expression of hoxa in murine fibroblasts resulted in significantly higher proliferation. cells transfected with hoxa had a mean brdu incorporation of . + . cells/ cells compared with . + . cells/ cells in controls (p= . ). in the usl obtained from women with and without pop, cell proliferation as determined by cyclin b and phospho-histone expression was not significantly different. cyclin b and phosphor-histone were expressed in comparable numbers of cells in both groups. conclusion: hoxa is necessary for usl development and promotes proliferation of adult fibroblasts. hoxa may have a similar function in vivo in usl, and may regulate fibroblast proliferation during growth and the acute phase response following trauma when fibroblasts are activated to proliferate and remodel the ecm. it is likely that hoxa mediated proliferation of usl fibroblasts contributes to the tensile strength and resilience of these structures and thereby prevents pop. supported by nichd wrhr: k hd - . connective tissue composition, in term of collagen i, iii and proteoglycans content, in support and nonsupport structures of women with uterine prolapse. elisabetta trabucco, gennaro acone, sara d'avino, marco torella, gennaro trezza, annamaria marenna, nicola colacurci, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; loreto mare hospital, naples, italy. objective. connective tissue consists mainly of collagen and elastic fibers, glycoproteins and proteoglycans (pgs) and is considered an important factor of the support and nonsupport structures of the genitourinary region. it has been already demonstrated altered morphologic features in the pelvic support connective tissue in women with genital prolapse. however, analysis of nonsupport tissue may provide a more accurate reflection of body collagen. the objective of our study was compare the expression of collagen i, iii and four pgs (decorin, fibromodulin, lumican, biglycan), essential for synthesis and regular assembly and diameter of the collagen fibrils, in uterosacral ligaments and in a nonsupport tissue, the uterine cervix, between premenopausal women with uterine prolapse respect to age matched controls. we characterized uterosacral ligaments and uterine cervix of premenopausal women with uterine prolapse and controls. this immunohistochemical study was performed on paraffin-embedded sections. results. uterosacral ligaments of the prolapsed uteri are characterized by a higher expression of collagen iii, decorin, fibromodulin and byglican and a lower quantity of collagen i. no differences in the immunoreactivity of lumican between the two patients groups. the abnormalities of support connective tissue composition are not observed in the uterine cervix of patients with prolapse. conclusions. our results suggest an altered remodeling of connective tissue in the ligaments of premenopausal patients with prolapse, with a significant decrease in collagen i content and an increase in collagen iii and pgs expression . in the prolapse patients this abnormal collagen metabolism and organization, mainly related to the observed change in pgs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the suspensory apparatus to the uterus. the alterd remodeling of support tissues, not detectable in nonsupport tissues, may suggest a predominant role of local biomechanical stresses (childbirth, chronic straining) in the pathogenesis of prolapse respect to systemic connective tissue deficiency. objective: to achieve vaginal delivery with minimal maternal injury, vaginal smooth muscle (sm) contractility likely decreases. the objective of this study was to characterize changes in rat vaginal sm contractility in pregnancy that affords circumferential vaginal distension. methods: a tubular segment of the vagina of virgin, late pregnant , immediate and late post vaginal delivery rats were mounted in an organ bath between a force transducer and an adjustable support block. dose response curves to norepinephrine (ne) and potassium (k+) were used to assess contractility. relaxation capacity was determined in ne preconstricted vagina, using cumulative doses of sodium nitroprusside (snp). differences in active vs. passive length-tension curves were used to measure sm contractility relative to the vaginal wall forces. sm -actin levels were measured using quantitative confocal immunofluorescence. results: cumulative doses of ne induced a maximum constriction force of . ± mn/g in virgin vagina while no measurable force was generated in response to ne in late pregnant or postpartum vaginas. virgin vagina relaxed with cumulative doses of snp; no measurable relaxation response was observed from pregnant or postpartum animals. in the presence of the high dose k+ ( mm), virgin vagina generated the greatest contractile force ( . ± mn/g) vs. late pregnant vagina ( . ± . mn/g, p< . ) or immediate post partum vagina ( . ± mn/g, p= . ). four week postpartum k+ induced forces were similar to virgin levels ( . ± mn/g). sm force generation (difference in active vs. passive length tension curves at mm of displacement) was decreased in late pregnancy ( . ± mn/g) compared with virgin ( ± mn/g, p < . ) and week postpartum vagina ( ± mn/g, p < . ). the expression of sm -actin was lowest in late pregnancy ( ± . , p < . ) and immediate postpartum vagina ( ± . , p < . ) relative to virgin ( ± ) with a return to virgin levels week postpartum ( ± ). conclusions: vaginal sm contractility diminishes in pregnancy. the altered contractility is mirrored by a decrease in sm -actin protein expression. near complete recovery to pre-pregnancy levels occurs by weeks post partum. these functional and biochemical changes likely represent maternal adaptations designed to minimize trauma during passage of the fetus(es). background: diagnosis or exclusion of endometriosis usually requires laparoscopy and peritoneal biopsy. we have described a novel and consistent observation of small nerve fibers in the functional layer of eutopic endometrium in women with endometriosis (tokushige et al, ) . these nerve fibers are not present in women without endometriosis. this finding allows the possibility endometriosis in the nude-mouse and inhibited mmp- expression in human endometrial stroma. the present findings may lead to the development of novel treatments of endometriosis involving statins. supported by nih u hd . k-ras actived immunocompetent retrograde menstruation model of endometriosis. ching-wen cheng, , di licence, , cristin g print, , d stephen charnock-jones. , pathology, university of cambridge, cambridge, united kingdom; obstetric gynaecology, university of cambridge, cambridge; department of molecular medicine pathology, university of auckland, auckland, new zealand. objective: to establish a new murine model of endometriosis that mimics the retrograde menstruation theory using immuno-competent mice. method: ovariectomised donor female k-ras v +/-/cre +/+ /rosa r-lacz +/+ transgenic mice were treated sequentially with steroid hormones. to induce decidualization and activation and k-ras transgene, mg/kg b-nf dissolved in maize oil was injected into the uterine lumen. tissue degeneration mimicking menstruation was induced by hormone withdrawal. menstruating endometrial tissue was collected from donor mice hours after last hormone treatment, re-suspended in matrigel, and implanted subcutaneously in female c bl/ recipients. immunohistochemical and morphometric methods were used to characterize the endometriosis-like lesions. microarray analysis (illumina, n= in each group), was used to study the molecular changes in "menstruating" uteri following k-ras activation. result: simple transplantation of decidualised endometrial tissue into immunocompetent animals does not lead to endometriotic lesion development but using tissue with the genetic modification described here overcomes this. viable endometriosis-like lesions are visible days after implantation. these lesions are histologically similar to those seen in man with intact glands, functional blood vessels, leukocyte infiltration and collagen deposition. statistical analysis revealed that transcripts were differentially regulated in the ras activated and control tissue. gene ontology (go) analysis indicated that transcripts associated with epithelial cell function and differentiation were over represented within this gene set (chloride transport and epidermis development) as were those associated with the acute inflammatory response and neutrophil chemotaxis. we developed a new model of endometriosis using immunocompetent mice to mimic retrograde menstruation induced endometriosis. this permits for the first time, the ready use of transgenic and knock-out tools to investigate the cellular and molecular mechanisms underlying endometriosis. since the animals have an intact immune system it also allows the testing of therapeutic agents that modify the inflammatory response. stra is expressed and induced by progesterone in the endometrium and is absent in endometriosis. mary ellen pavone, serdar e bulun, scott s reierstad, joy innes, magdy p milad, you-hong cheng. obstetrics and gynecology, northwestern univeristy, chicago, il, usa. objective: retinoic acid (ra) plays important roles in development, growth and differentiation by regulating the expression of target genes. in the mouse endometrium, ra deficiency leads to hyperkeratinization, while high concentrations of retinoids promote secretory characteristics. this leads many to believe that ra mediates important actions of progesterone in the endometrium and may account for progesterone resistance in endometriosis. the mechanism for regulation of ra production by progesterone is unknown. moreover, the conversion from retinol to retinoic acid is not different between normal endometrium and endometriotic tissue. we hypothesize that retinol intake into cells rather than conversion of retinol to ra is the critical step that determines ra activity in the endometrium and endometriosis. stra , a widely expressed multitransmembrane domain protein and member of a group of "stimulated by retinoic acid" genes, has been identified as the retinol binding protein receptor. it is strongly expressed in adult mice in several tissues including the female genital tract. we hypothesize that ra activity in the endometrium may be regulated by stra . here we investigate the differential expression of stra in the endometrium and endometriosis. design: we studied primary stromal cells isolated from the eutopic endometrium of disease free women and walls of ovarian endometriomas from women with endometriosis with respect to stra expression and regulation by progesterone. materials and methods primary culture of eutopic endometrial and endometriotic stromal cells were treated with - m estradiol (e ), - m r , a progesterone agonist, or vehicle for hours. real-time pcr was employed to quantify stra mrna levels. we treated cells for hours and quantified protein levels by immunoblotting. results basal mrna of stra levels in endometrial cells (n= ), were > , fold higher than those in vehicle-treated endometriotic cells (n= , p<. ). in endometrial stromal cells (n= ), r stimulated stra mrna levels by . - fold. r induced stra protein levels in endometrial stromal cells (n= ). conclusions stra is highly expressed and regulated by progesterone in endometrial stromal cells, whereas it is practically undetectable in endometriotic cells. stra may mediate important actions of progesterone in endometrium. upregulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity (pc) may influence their survival and persistence via local estrogen synthesis. the inducing factors for the upregulation of aromatase in endometriosis are not well defined, but increased expression of sf- has been suggested to play an important role. given that the aromatase substrate androstenedione (a ) is the predominant steroid in peritoneal fluid, we aimed to determine whether a has a role in the regulation of aromatase expression in human endometrium. we found that culture of primary endometrial stromal cells and explants with a ( - nm) for h significantly upregulated expression of aromatase mrna transcripts containing exon iia at their '-ends. moreover, in human endometrial surface epithelial cells (hes), dose-response studies with a ( - nm) revealed that nm a maximally upregulated expression of both aromatase and sf- . when tissue samples were evaluated from women with endometriosis and control endometrium, expression of aromatase mrna mirrored sf- . treatment of hes cells ( h) with tritiated a demonstrated its metabolism to estradiol (e ), testosterone (t), dihydrotestosterone (dht) and androstanediol (a-diol). although equimolar concentrations of a , t and e upregulated aromatase and sf- mrna expression in hes cells, the nonaromatizable androgens, dht and a-diol, had no effect. a positive feedback role of estrogen in aromatase upregulation was suggested by the finding that the estrogen receptor antagonist, ici , , markedly diminished aromatase and sf- mrna expression induced by a . finally, chromatin immunoprecipitation revealed that a significantly enhanced recruitment of sf- to its response element (- bp) upstream of cyp exon iia. collectively, our findings strongly suggest that exposure of endometrial cells within the pc to c -steroids may cause an acute upregulation of cyp gene expression through their aromatization to estrogens. thus, estrogen may play a critical positive feedback role in the pathogenesis of endometriosis. supported by nih r -dk . the hpa axis and depression/anxiety scores in chronic pelvic pain patients. b stegmann, b frank, j gemmill, g chrousos, m ballweg, p stratton. rbmb, nichd/nih, bethesda, md; som, wake forest university, winston-salem, nc; endometriosis association, milwaukee, wi. stress, pain, anxiety and depression adversely affect the hypothalamic-pituitaryadrenal (hpa) axis. we examined the influence of depression and anxiety on the hpa axis response to corticotropin-releasing hormone (crh) testing in women with and without chronic pelvic pain (cpp). methods: healthy women, aged - , with without cpp, with regular menses and off hormonal contraception were studied. none had pelvic infections, untreated depression, manic depression, fibromyalgia, or chronic fatigue syndrome. after ovine crh injection ( mcg/kg), serial blood samples (- , , + , + , + minutes) were obtained for acth and cortisol measurements. depression and anxiety were scored using the duke quality of life questionnaire. hpa response was abnormal if peak acth levels were > pg/ml without a rise in cortisol levels. subject groups were: no pain and normal acth response (npnr), pain and normal acth response (pnr), no pain with an abnormal acth response (npar) and pain with an abnormal acth response (par). student t-test was used for comparisons. results: women ( cpp, controls) had a mean age of . ± . years (range: - ). women responded normally ( pnr, npnr) and women responded abnormally ( par, npar). peak and absolute rise in acth were significantly higher in abnormal (a) vs normal (n) responders; (peak: . a vs . pg/ml n, p< . ; absolute: . a vs . pg/ml n, p= . ). there was a significant difference within groups: peak acth: . par vs . pg/ml pnr, p= . ; . npar vs . pg/ml npnr, p< . ; absolute acth: . par vs . pg/ml pnr, p= . , . npar vs . pg/ml npnr, p< . ). however, total cortisol level was not different between or within the groups ( . a vs . mcg/dl n, p= . ). mean depression score did not differ among cpp patients ( . par vs . pnr, p= . ), but differed among controls ( npar vs . npnr, p= . ). anxiety scores did not differ between or within groups ( . npar vs . npnr p = . ; par vs . pnr, p= . ). conclusions: chronic pelvic pain is associated with an abnormal hpa response, regardless of anxiety or depression symptoms. abnormal hpa responses in control women, however, appear to be influenced by depression. the mechanism and clinical significance of these findings should be explored. support: rbmb/nichd/nih and endometriosis association. objective: the eutopic endometrium in women with endometriosis demonstrates altered endometrial receptivity and altered gene expression. it is unknow if the endometrium is defective giving rise to a predisposition toward endometriosis or alternativly if the endometriosis causes the altered endometrial receptivity. here we created experimental endometriosis in a mouse model through allotransplantation of the uterus to the peritoneal cavity in immunocompetent mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. materials and methods: the uterus of -week-old cd female mice was transected at cervicovaginal junction and each horn divided and transplantated into the abdomidnal cavity of eight cd mice. seven controls received sham surgery only. after weeks the uterus was removed, snap-frozen in trizol. total rna was extracted and cdna generated. quantitative real time rt-pcr using sybrgreen was performed and normalized to -actin. fold changes in normalized hoxa , hoxa , bteb , emx , igfbp , integrin - , total progesterone receptor (pr-ab) and progesterone receptor-b (pr-b) were assessed. all experiments were conducted in duplicate, repeated at least three times and compared using mann-whitney rank sum test. results: hoxa , hoxa , bteb , emx , and igfbp mrna expression showed . -fold (p= . ), . -fold (p< . ), . -fold (p= . ), . fold (p= . ), and . -fold (p< . ) decrease in the uterus of mice with experimental endometriosis compared with controls. pr-ab and pr-b mrna showed . -fold (p= . ) and . -fold (p= . ) increase in endometriosis group compared to controls, respectively, however the ratio of pr-b to pr-ab (b/ab) showed no significant change in both groups ( . vs. . , endometriosis vs. control, p> . ). there was no significant change in integrin - mrna expression ( . -fold, p> . ). conclusion: we demonstrate significant changes in multiple markers of endometrial receptivity in eutopic endometrium after induction of endometriosis. these findings suggest that origionally normal endometrium can develop defects with the creation of endometriosis; an abnormal endometrium is not a prerequisite for the development of endometriosis or associated abnormalities. this data also suggest the existance of a signal conduction pathway from endometriosis that alteres endometrial gene expression. endometrial expression patterns of relaxin and relaxin receptor mrna suggest involvement of relaxin in endometriosis. sara s morelli, felice petraglia, gerson weiss, stefano luisi, pasquale florio, jeff gardner, andrea wojtczuk, laura t goldsmith. obstetrics, gynecology and women's health, new jersey medical school, newark, nj, usa; pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. objective: in normal endometrium, relaxin is a potent inhibitor of matrix metalloproteinases, which have been implicated in the invasive process of endometriosis. we tested the hypothesis that relaxin plays a role in endometriosis by comparing expression of relaxin and its lgr receptor in normal human endometrium to levels in samples from patients with endometriosis. materials and methods: total rnas, extracted from ectopic (n= ) and eutopic (n= ) endometrium of patients with endometriosis and from endometrium of normal controls (n= ), were reverse transcribed into cdnas. real-time pcr was performed using primers previously shown to identify human lgr relaxin receptor mrna, h relaxin mrna, and beta-actin mrna, with sybr-green detection of double stranded dna products. the comparative c t method ( -ct ) determined relative lgr and relaxin mrna expression (normalized to beta-actin mrna expression). relaxin mrna was detectable in normal endometrium from of ( %) control patients. in contrast, relaxin mrna was detectable in a lower proportion of samples [ of ( . %)] from patients with endometriosis, among whom relaxin mrna was detectable in a lower proportion of ectopic samples [ of ( %)] than in eutopic samples [ of ( . %)]. lgr relaxin receptor mrna was detectable in all samples, with lower expression in endometriosis samples than in endometrium from normal controls. relaxin receptor lgr mrna levels vary with cycle phase, with greater expression in the secretory phase (sp) than in proliferative phase (pp): in normal controls . -fold higher levels in the sp than pp; and in endometriosis patients . -fold higher levels in the sp than pp. in both phases, lgr mrna levels were lower in ectopic samples than in either eutopic samples ( . -fold lower in pp and . -fold lower in sp) or endometrium from normal controls ( . -fold lower in pp and . -fold lower in sp). eutopic endometrium had similar lgr mrna expression to controls throughout the cycle. decreased local expression of relaxin and relaxin receptor mrna in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis. ovarian reserve after laparoscopic cystectomy of endometriotic ovarian cysts. shinichi hayasaka, takashi murakami. obsterics and gynecology, tohoku university, sendai, japan. objective laparoscopic ovarian cystectomy is generally recommended for endometriotic ovarian cysts because it has been associated with a higher pregnancy rate and a lower recurrent rate. however, residual ovarian function after laparoscopic cystectomy of endometriotic ovarian cysts has been questioned. in this study, we retrospectively evaluated ovarian response to hyperstimulation in women selected for ivf and icsi cycles who previously underwent laparoscopic cystectomy of a monolateral endometriotic ovarian cyst. methods a retrospective review of the patients between january and september was performed. the operated ovary and contralateral intact ovary were compared in terms of number of oocytes retrieved, number of follicles with a mean diameter > mm at the time of hcg administration. the patients who had recurrent endometriotic ovarian cysts were excluded. in total, ten patients were identified. the mean(±sd)number of oocytes retrieved was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the mean(±sd)number of follicles with a mean diameter > mm was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the age of patients and diameter of the operated ovary had little influence on the difference between the response of the control ovary and one of the previously operated ovary. laparoscopic cystectomy of endometriotic ovarian cysts is associated with a significant reduction in ovarian reserve. background pre-eclampsia (pe) is associated with systemic maternal endothelial dysfunction, which is thought to result from the presence of circulating factors released following placental damage. it is hypothesised that this occurs as a result of reduced oxygen (o ) delivery. some features of placental pathology seen in pe, such as increased apoptosis, can be reproduced by culture of placental explants in % o . metabolomics operates to study 'all' metabolites within a biological system. this strategy has previously identified differences in maternal plasma between normal pregnancies and those complicated by pe. in these experiments we used metabolomics to investigate differences in the metabolic profile of explants cultured in different o tensions. hypothesis the metabolic profile of placental villous explants is altered by culture in different o tensions. methods placental villous explants were cultured with either % serum (n= ) or in serum-free conditions (n= ) for h in %, % and % o . after h, conditioned culture medium and tissue were collected and snap frozen. tissue was homogenised in % ice cold methanol prior to analysis. samples were analysed using gas chromatography-mass spectroscopy (gc-ms). samples cultured at %, % and % o were compared to identify concentration differences in metabolites in conditioned cultured medium and tissue lysate. kruskal-wallis test was used for statistical analysis. due to the large number of metabolites identified, a p value of . was considered significant. results the mean intra-assay variability was . %. there were no differences in the metabolic profile of conditioned culture medium from % and % o . several metabolites differed in culture medium from % compared to % and % o including: deoxyribose, glycerol and threionic acid. these changes were present in both serum and serum-free conditions. using these metabolites alone, culture in % o could be completely discriminated from % and % o . deoxyribose was also elevated in tissue lysate from % o compared to % o . conclusion this metabolomic strategy can identify differences in metabolic profile in placental tissue cultured in % o . these novel compounds may provide further insight into pathophysiology of pe. objective a strategy of metabolic footprinting (the study of extracellular metabolites, which are related to intracellular metabolism) was used to detect a wide array of low molecular weight metabolites. metabolic profiles were employed to differentiate between placental explants cultured at different oxygen tensions. two separate studies were combined to validate initial observations. methods metabolic footprints of placental villous explants, obtained from uncomplicated pregnancies, (n= ) were cultured for h in %, % and % oxygen. conditioned culture medium was then collected and frozen, prior to analysis by uplc/ltq-orbitrap mass spectrometry in both negative and positive ion mode. samples cultured at %, % and % were compared to identify differences in the metabolic profiles. this procedure was repeated for different explants and the results compared across the studies in order to validate initial findings. approximately unique peaks were detected in both sample sets in negative ion mode and peaks in positive ion mode. a number of metabolites were identified as being significantly different using kruskal-wallis (pval< . ) under different oxygen tensions. some differences were seen between the results obtained from both runs due to separate batch preparation of the medium but good reproducibility was obtained for many metabolites between batches. metabolites of biological interest included amongst others -deoxyribose, valine, tyrosine and aspartic acid. the largest differences were those seen between o % and o %. comprehensive metabolic profiles were detected and employed to identify differences in the metabolic footprints of explants cultured under differing oxygen conditions. a number of biologically interesting metabolites were characterized. objective: brain weight and dna content were reduced in leptin deficient (lep ob /lep ob ) mice postnatally. leptin is detected in the sera and its receptors are expressed in the brain of the mouse fetus. we examined the role of leptin in the proliferation and differentiation of neural lineage cells in the mouse fetus. methods: the number of total cells in the cerebral cortex was compared between (lep ob /lep ob ) and wild type fetuses from embryonic day (e) to . the number of brdu positive cells was also compared on e and e . brdu uptake, the ratio of viable colony number to plated cell number, the proportion of multipotent, neuronal or glial progenitor colonies, and the expression levels of hes mrna were compared between leptin-and non-treated neurosphere cells. moreover, we examined stat phosphorylation by leptin stimulation with elisa to investigate whether or not jak/stat pathway transduces the leptin signal in the prenatal period as in the adult. results: lep ob /lep ob fetuses had reduced total cell numbers in the ventricular zone (vz) on e and e , and in the cortical plate on e . the number of brdu-positive cells was reduced in vz of e and e lep ob /lep ob fetuses. brdu uptake and the number of viable colonies were increased by days-leptin treatment in the neurosphere culture. the proportions of glial-restricted and multipotent precursor colonies were increased by leptin, whereas that of oligodendrocyte precursor colonies were decreased. hes mrna expression was enhanced in neurosphere cells by leptin. neither the amount of phosphorylated stat nor that of stat was increased in neurosphere cells by leptin stimulation. conclusion: our study suggests that leptin maintains the neural stem and glial-restricted precursor cells through upregulation of hes expression and increases the number of cerebrocortical cells in the mouse fetus, however, jak/stat pathway does not mediate the leptin signal in undifferentiated neural lineage cells. a vaginal wall, and > % of fib- knockout (ko) mice develop pelvic organ prolapse by weeks of age. in contrast, fib- and - deficiencies do not result in prolapse, and fib- kos die shortly after birth. herein, we evaluated the role of fib- in pelvic organ support. methods: two observers serially measured the degree of vaginal, perineal, and anal prolapse in fib- ko (fib -/-) and in fib -/-mice severity of prolapse was significantly related to age, but not parity, and the average age at diagnosis was wks. fib -/-animals with advanced prolapse had attenuated uterosacral ligaments and descent of the bladder and uterus caudal to the symphysis. pro-mmp ( -fold, p= . ), active mmp ( -fold, p= . ), and prommp ( -fold, p= . ) were increased in vaginal tissues from mice with gross prolapse compared with age-, strain-, and cycle-matched controls. this increase in protease activity, however, was also accompanied by increased expression of fib- and tropoelastin ( . -fold, p< . ). regardless of prolapse maternal morbidtties tf ards-n(%) ( ) acute ren~m f lure-n (%) ( ) car~tovascular ~ysfunrtion-n ("/o) ( ) hepatc fa..lure-n (%) dt sst'dlm atl:d inlfavascul..-coagul tipalhy-n(%) ( ) durallon of !cu suy (days. mean+sd) :t . hospital my (days.. me +sd) . condon, j. c., hardy, d. b., kovaric, k., and mendelson, c. r. ( ) mol endocrinol ( ), - . objective: innervation of the uterine cervix is important for the process of parturition (physiol beh : , ; j histochem cytochem : , jsgi : , ) . the hypogastric nerve is a major pathway that innervates the uterine cervix, yet its contribution to processes associated with cervical ripening and parturition is not known. the objective of this study was to determine the effect of hypogastric nerve transection on processes associated with cervical remodeling and parturition. methods: time-dated pregnant rats were sham-operated or the hypogastric nerve bilaterally transected just anterior to the inferior mesenteric ganglion on day post-breeding. live pups were born spontaneously by all rats at term on days - post-breeding. on the day of birth, the cervix was excised, postfixed overnight, sectioned, and processed to evaluate collagen content and structure (nih image j). sections were also processed by immunohistochemistry to assess cell nuclei density, the census of resident macrophages, and area of tissue that contained nerve fibers. results: hypogastric neurectomy did not affect cell nuclei density, the number of macrophages, or density of nerve fibers in the cervix. the failure of hypogastric nerve transection to affect indices of cervical remodeling is consistent with the finding that duration of pregnancy and timing of birth were not different in sham-operated and hypogastrectomized rats. conclusions: nerve fibers in the hypogastric nerve that innervate the lower uterus and cervix, including sympathetic and neuropeptidergic projections, are thus not essential for birth. these novel findings provide support for the contention that innervation of the uterine cervix other than through the hypogastric nerve contributes to processes associated with cervical ripening and parturition. receptor system in prostaglandin synthesis in pregnancy. eugenie r lumbers, , della m yates, carolyn m mitchell, jonathan j hirst, , tamas zakar. , school of health sciences, university of newcastle, newcastle, nsw, australia; mothers and babies research centre, hunter medical research institute, newcastle, nsw, australia; obstetrics and gynaecology, john hunter hospital, school of medical practice and public health, university of newcastle, newcastle, nsw, australia. the fetal membranes and decidua contain prorenin, which requires proteolytic activation. ngyuen (j clin invest. : ) described a renin/prorenin receptor that conformationally activates prorenin, so that it can form ang i from aogen. in addition, receptor-bound prorenin can stimulate intracellular pathways directly. prostaglandins (pgs) participate in the control of term and preterm labour, and renin can stimulate pg production by amnion and decidua when angiotensin's action is blocked mitchell placenta : ) . the prorenin receptor may mediate these effects. our aim was to find out if the prorenin receptor gene is expressed and colocalised with prorenin and prostaglandin h synthase- (pghs- ) in human placenta, amnion, chorion and decidua. we have also determined if there is any change in the expression of the prorenin and prorenin receptor genes with labor. placentae with attached membranes were collected after term birth either by elective caesarian section or by spontaneous labor, and prorenin, prorenin receptor and pghs- mrna levels were quantitated relative to beta-actin mrna by real-time rt-pcr in amnion, chorion, decidua and placenta. before labor, mean prorenin mrna levels were . times higher in decidua and . times higher in chorion and placenta than in amnion (p< . ). there were no significant changes with labor. proenin receptor mrna was expressed in all tissues. proenin receptor mrna levels in prelabor amnion, chorion and placenta were similar, while levels in decidua were % of those in amnion (p= . ). this low level of prorenin receptor mrna in decidua persisted after labor (p< . ). pghs- mrna expression was highest in amnion; in decidua and placenta it was only and % of amnion. similar differences were present after labor. we conclude that the decidua is the principal source of prorenin within the pregnant uterus, and all gestational tissues are targets for prorenin. decidual prorenin may affect pghs- expression in amnion through the prorenin receptor forming a maternal-fetal paracrine system that stimulates pg production at labor. the immuno-suppressive effects of progesterone on umbilical cord blood mononuclear cells and the effect of culture media estradiol content. nadav schwartz, xiangying xue, christine n metz. obstetrics and gynecology, nyu school of medicine, new york, ny, usa; feinstein institute for medical research, manhasset, ny, usa. objective: progesterone (p ) is known to supress the maternal immune response and similar effects have been seen with fetal cells. we sought to ascertain whether p action was modulated by the phenol red and/or estrogen content of the culture media. methods: umbilical cord blood mononuclear cells were isolated using a density gradient centrifugation. the cells were incubated with p ( - m) hour prior to overnight stimulation with lps. supernatants were assayed for tnf-using elisa. the following culture media were used: a)'red' media: rpmi+ % fbs, b)'yellow' media: phenol red-free rpmi+ % fbs, and c)'clear' media: phenol-free rpmi+ % dextran/charcoal treated fbs. finally, the 'clear' media experiments were repeated with estradiol add-back. results: p suppressed tnf production in all medias. tnf levels were suppressed to . % (p< . ) of controls using 'red' media and to . % (p< . ) in 'yellow' media. the degree of suppression was not as substantial using 'clear' media where tnf levels were . % (p< . ) of control. (fig ) adding estradiol to the 'clear' media had little or no effect on the degree of tnf suppression. (fig ) conclusions: progesterone exerts an immuno-suppressive effect on lpsstimulated fetal mononuclear cells which is more pronounced when using media containing untreated fetal bovine serum. the phenol-red/estradiol content of the culture media did not modulated the p effect. dextran/charcoal treatment of fbs appears to deplete the media of factors other than estradiol that are necessary for p to exert its full effects. culture conditions should be optimized when studying progesterone's effects on immune response. ovine fetal regional blood flow following prenatal dexamethasone (dex) at . gestation (g). antonine d van heesewijk, jan g nijhuis, susan l jenkins, peter w nathanielsz, mark j nijland. ob/gyn, academisch ziekenhuis maastricht, maastricht, netherlands; ob/gyn, cpnr, uthscsa, san antonio, tx, usa. background: pregnant women at risk for premature labor receive antenatal glucocorticoids (gc) to reduce neonatal morbidity and mortality. while fetal blood pressure (bp), heart rate and vascular endothelial and control fetuses (n= ). dispersed adrenal cortical cells ( . x cells/tube; in duplicate) were challenged with - m acth. samples were collected at time (baseline), , , , and min after acth treatment, and tissue and media samples were frozen for determination of cortisol, camp, and star. results: cortisol output (ng/ . x cells) was higher in the lth group compared to the control, (p< . ) at min ( . ± . vs. . ± . ), min ( . ± . vs. . ± . ), and min ( . ± . vs. . ± . ). peak camp levels (fmol/ . x cells) were observed at min but did not differ between control ( . ± . ) and lth ( . ± . ) adrenal cells. western analysis demonstrated that star protein expression was higher in the lth adrenal cortex compared to control (p< . ) at min ( . ± . vs. . ± . ), and at min ( . ± . vs. . ± . ), relative optical density units. conclusions: results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in lth fetuses is the result of increased adrenal acth sensitivity. this enhanced sensitivity is not due to differences in camp generation. star, which is a key element involved in cholesterol transfer at the level of the mitochondrial membrane appears to play a key role in the enhanced cortisol response to acth in the lth group. (nih grants hd , hd and llu-nih imsd r gm - ). expression. hiroaki itoh, makoto kawamura, shigeo yura, haruta mogami, tsuyoshhi fujii, norimasa sagawa. obstetrics and gynecology, national hospital organization osaka national hospital, osaka, japan; gynecology and obstetrics, kyoto university graduate school of medicine, kyoto, japan; obstetrics and gynecology, mie university school of medicine, tsu, japan. objective: epidemiological evidences suggested that undernutrition in utero develops risk factors for adult cardiovascular disorders, such as blood pressure increase. the present study was designed to prove the hypothesis that maternal iso-caloric high protein diet alleviates blood pressure increase in the adult offspring by affecting placental beta-hydroxysteroid dehydrogenase type ( beta-hsd ) expression. methods: the % calorie restriction was applied to pregnant mice by using either standard protein diet (spd; % casein protein; spd-un) or high protein diet (hpd; % casein protein; spd-un). in some groups, these pregnant mice were sacrificed at . d.p.c.; then maternal and fetal plasma as well as placental tissues were corrected. while in other groups, systolic blood pressure was measured in the offspring at wks. fetal and maternal corticosterone levels were measured by elisa. the placental beta-hsd gene expression was measured by quantitative taqman pcr. results: systolic blood pressure in the spd-un offspring at weeks ( mmhg) was higher than that in spd-nn offspring ( . mmhg). by contrast, systolic blood pressure in the hpd-un offspring ( . mmhg) was similar to that in spd-nn offspring. maternal calorie restriction with spd and hpd caused similar elevation of maternal plasma corticosterone concentrations. by contrast, fetal plasma corticosterone levels in hpd-un offspring ( ng/ml) was lower than those in spd-un offspring ( ng/ml), indicating the amelioration of fetal exposure to high corticosterone by maternal hpd. the placental beta-hsd gene expression in the hpd-un was % higher than that in spd-un, suggesting that maternal hpd augmented placental beta-hsd gene expression in the placenta and probably facilitated the inactivation of maternal cortocsterone in the process of placental transfer to fetal circulation. conclusion: the preset study suggests that maternal high protein ingestion may elevate placental beta-hsd gene expression and ameliorate blood pressure increase in the adult offspring via modification of fetal exposure to maternal corticosterone. elevated cortisol levels at birth exert critical maturational effects through action at glucocorticoid receptors, gr. in contrast, the low cortisol concentrations in the preterm fetus, before the time of increased fetal cortisol secretion, are near to the kd for cortisol at the higher affinity mineralocorticoid receptor, mr. we hypothesized that endogenous cortisol in the fetus exerts physiologic actions at mr in tissues without appreciable cortisol-inactivating enzyme, hsd , including the fetal lung and brain. we therefore tested the effect of infusion of a mr-antagonist, ru (mra, . mg/h over h) into - day fetal sheep. we tested for effects on lung liquid production rate, lung liquid and plasma electrolytes, plasma acth and cortisol, and hematocrit at - h after start of the infusion. although there was no significant difference in the liquid production rate in fetuses infused with ru , the ratio of na + to k + in lung liquid was significantly greater in mra-treated fetuses than in the control fetuses ( . ± . vs . ± . ). plasma acth was significantly greater in the mra-treated fetuses than in control fetuses at h ( ± vs ± pg/ml); in the mra-treated fetuses, acth concentrations at h were greater than in the same fetuses at h ( ± pg/ml), wheras there was no increase in plasma acth in the control fetuses ( h: ± pg/ml). similarly, plasma cortisol concentrations at and h were greater in the mra infused fetuses than in control fetuses ( h: . ± . vs . ± . ng/ml), and cortisol increased significantly over time in the fetuses infused with mra, but not in control fetuses ( h, mra: . ± . , control: . ± . ng/ml). infusion of mra did not alter fetal plasma electrolyte, fetal blood pressure or fetal heart rate. however, fetal packed cell volume was significantly increased at - h of infusion of mra ( h: ± vs ± %) and fetal pco was significantly greater at h of infusion of mra as compared to control fetuses ( . ± . vs . ± . ). these results suggest that endogenous cortisol concentrations in the preterm fetus exert negative feedback control of acth via action at mr in the fetal brain, and play a role in regulation of fetal lung liquid composition and in fetal fluid balance. preparturient increases in fetal acth secretion are cyclooxygenase- (cox- ) dependent. charles e wood. dept. physiology and functional genomics, university of florida college of medicine, gainesville, fl, usa. maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. in sheep, increased preparturient activity of the fetal hpa axis appears to be responsible for triggering parturition itself. this study was designed to test the hypothesis that prostaglandin generation, mediated by cox- in the fetal brain, is critically important for stimulation of the preparturient increase in fetal acth secretion. singleton fetal sheep were chronically catheterized with vascular, amniotic, and lateral cerebral ventricular catheters. nimesulide, a selective cox- inhibitor, was infused ( mg/day, n= ), and vehicle ( % dimethylsulfoxide, n= ) was infused. arterial blood samples were drawn from fetuses at hour intervals for measurement of plasma hormone concentrations and arterial blood gases. in the vehicle-treated fetuses, fetal plasma acth, pomc, and cortisol concentrations increased exponentially (p< . by anova) before spontaneous parturition at ± . days. in the nimesulide-treated fetuses, fetal plasma acth and pomc were constant and did not increase prior to parturition (p=ns by anova). in contrast, fetal plasma cortisol increased independent of acth (p< . by anova) and the fetuses were born spontaneously on ± . days gestation. the slight delay in spontaneous parturition in the nimesulide-treated fetuses was statistically significant (p< . by mantel-cox test). intracerebroventricular nimesulide infusion did not decrease fetal plasma concentrations of prostaglandin e , demonstrating that the action of the nimesulide was restricted to the fetal brain. fetal blood gases were normal in all of the fetuses, and there were no differences in blood gases between groups. we conclude that the spontaneous increase in fetal acth and pomc prior to parturition is cox- dependent. however, we also conclude that the increase in fetal plasma cortisol concentration can occur independent of increases in fetal acth, suggesting that the increase in cortisol can result from increases in adrenal sensitivity to acth. the results of this study demonstrate that the timing of parturition in the sheep is not dependent upon increased acth secretion, and the results suggest that parturition is regulated primarily by changes in adrenal sensitivity. expression of extracellular signal-regulated kinase / and p mitogen-antiphosphotyrosine. immunolocalization of inos was performed in the same tissues. hpmec were used to determine effect of sin- ( mm) on inos protein expression ( min, and h). results: inos protein was detected in microvascular endothelium, smooth muscle and syncytiotrophoblast of the placenta. inducible nos levels in placentas from severe preeclampsia were significantly decreased compared to normal (p< . ) but were not different from mild preeclampsia. aligning western blots of anti-phosphotyrosine and inos revealed a phosphorylated band corresponding to the molecular weight of inos. the proportion of tyrosine phosphorylated inos was reduced by % in severe preeclampsia compared with normotensive. preliminary data with ip for phosphotyrosine and crossblotting with inos confirmed this finding. sin- treatment decreased inos protein abundance at and h in hpmec. conclusions: our results demonstrate decreased tyrosine phosphorylation of inos in preeclamptic placenta. phosphorylation of tyrosine in inos has been reported to negatively regulate its activity. we therefore postulate that decreased phosphorylation of inos may be responsible for the increased catalytic activity of the enzyme that we have previously observed. the decreased levels of inos observed on sin- treatment may be due to nitration, an effect analogous to preeclampsia, where the presence of peroxynitrite has been well established. it remains to be seen if protein nitration has an effect on enzyme stability. objective: -isoprostane ( -iso), a prostaglandin-like compound formed in vivo, is a reliable measure of oxidative stress which occurs in response to many different stimuli, including infection. periodontal disease is a chronic oral infection associated with fetal growth restriction and preeclampsia. our objective was to determine if maternal periodontal disease is associated with oxidative stress as measured by -iso. methods: a secondary analysis was conducted using prospective data from the oral conditions and pregnancy study. a cohort of healthy women enrolled < weeks underwent oral health examination and serum sampling. maternal periodontal disease was categorized as healthy, mild, or moderate-severe by clinical criteria. maternal serum was analyzed for -iso by ultra-sensitive elisa. elevated -iso was defined by a value th %tile. maternal factors associated with elevated -iso were determined using chi-square or t-tests as appropriate. a logistic regression model was created to determine adjusted odds ratios ( th %ci) for elevated -iso. results: women had complete data for analysis. the median -iso level (iqr) was , ( - , pg/dl). using bivariate analysis, moderate-severe periodontal disease, non-white race, use of wic/food stamps, unmarried status, obesity, and lack of insurance were associated with elevated -iso. the logistic regression model for elevated -iso is shown below. adjusted or ( th ci)* mild periodontal disease . maternal periodontal disease and utilization of public assistance are associated with oxidative stress in pregnancy. the relationship between periodontal disease, social hardship, oxidative stress, and adverse pregnancy outcome remains to be determined. antioxidant therapy could represent novel therapy for the prevention of adverse pregnancy outcome. severe transient immunological thrombocytopenia in a preeclampsia patient whose bone marrow production of platelets had been restricted. toshihiro yoshimura. obstetrics and gynecology, ntt-west kyushu general hospital, kumamoto-city, kumamoto, japan. introduction: idiopathic myelofibrosis is a chronic myeloproliferative disorder in which clonal haemopoetic stem cell proliferation is accompanied by reactive fibrosis. case report: a -year-old primiparous woman was referred to our hospital for prenatal care after weeks of gestation. her medical history was significant for idiopathic myelofibrosis, and congenital agenesis of the spleen. the laboratory workup showed the patient's white blood cell count to be , / l; hemoglobin, . gm/dl; and platelet count, , / l. her bleeding time was normal ( min). until the th week, the patient's pregnancy was uneventful. during the th week, however, the patient's platelet count declined to , / l, when proteinuria became evident. her bleeding time was prolonged to min. the coagulation system was normal. by the th week, the patient's blood pressure was elevated to / mmhg, and her urinary protein excretion exceeded g/day. our initial diagnosis was thrombocytopenia due to bone marrow suppression. the patient had no history of platelet transfusion, but we expected it would increase the platelet count, particularly since in the absence of destruction by the spleen, the lifespan of the platelets would be prolonged. this was not the case, however. elective induction of labor was carried out after weeks. at the same time, the patient was transfused with units of platelets, but her platelet count remained unchanged ( , / l). it was then that we realized that immunological thrombocytopenia may be involved, and massive doses ( mg/kg) of gamma globulin were given intravenously for one day. thereafter, platelets ( units) were again transfused, this time raising the platelet count to , / l. cesarean section was promptly carried out under general anesthesia. we later found that the patient's serum platelet associated immunoglobulin (paigg) level was positive, though antiplatelet and antinuclear antibodies were negative. the postoperative course was uneventful. the maternal platelet count declined to the pretransfusion level within days after delivery, but gradually increased to the prepregnancy level by weeks. comment: this idiopathic myelofibrosis patient in whom bone marrow production of platelets had been severely restricted demonstrates that immunological destruction may play a major role in the development of thrombocytopenia in preeclampsia. early l-arginine therapy improves notably the fetal growth in preeclamptic women. a randomized controlled trial. keisy lopez-molina, juan c gonzalez-altamirano, julio e valdivia-silva. , oncoinmunología y biología vascular, facultad de medicina, universidad nacional san agustín, arequipa, peru; cardiología y cirugía de tórax, hospital nacional case essalud, arequipa, peru; inmunología, instituto de investigaciones biomédicas, méxico, distrito federal, mexico. objective: to assess the benefit of early administration to l-arginine, the precursor to nitric oxide, on fetal growth. methods: one hundred women with preeclampsia were randomized to receive either l-arginine or placebo until day postpartum. live singleton infants were born after preeclamptic pregnancies and compared those with other control infants. birth size was expressed as the ratio between observed and expected birth weights, and infants smaller than two standard deviations from expected birth weights were classified as small for gestational age (sga). all the women had neither previous precedents of the preeclampsia nor other factors that they cause sga. results: no significant differences existed between the groups with preeclampsia before randomization. preeclampsia was associated with a % ( % confidence interval [ci] %, %) reduction in birth weight. women with hellp syndrome had to leave the study. the risk of sga was three times higher (relative risk [rr] = . ; % ci . , . ) in infants born after preeclampsia without l-arginine therapy than in control pregnancies, and two times higher (relative risk [rr] = . ; % ci . , . ). in infants born after preeclampsia with l-arginine therapy. conclusion: fetal growth improve markedly with l-arginine therapy in women with preeclampsia. introduction: although the pathogenesis of severe preeclampsia (pe) and intrauterine growth restriction (iugr) is poorly defined, inadequate remodeling of uterine spiral arties may promote reperfusion injury leading to focal infarction and reduced nutrient flow between mother and fetus. our goal was to determine whether an intravillous inflammatory cytokine cascade was associated with pe and iugr. methods: immunohistochemistry (ihc) examined il- and il- expression in preterm placentas with severe pe+iugr, and idiopathic preterm controls (ptc) with no evidence of clinical or histological chorioamnionitis. cultures of fibroblasts (fibs) and syncytiotrophoblasts (scts) from term placentas (n= ) were treated with il- and cytokine levels in conditioned media were determined using elisa or multiplex array. results were analyzed by student's t test or anova. results: the gestational age at delivery was not significantly different in pe+iugr and ptc groups ( . ± . vs . ± . wks). ihc of pe+iugr samples revealed intense villus core staining of il- adjacent to infarcts. villi distal to infarcts stained with a lower intensity. in contrast, staining was virtually undetectable in ptc. the pattern of il- staining was nearly identical in pe+iugr and ptc groups, was localized to the syncytium, and did not differ with respect to distance from infarct. to identify cellular targets of il- action, primary cultures of fibs and scts were incubated for h in serum-free medium ± ng/ml il- . by elisa we observed that il- treatment of fibs increased il- levels from ± to ± pg/ g protein (p< . ). similarly, multiplex array revealed that levels of il- , il- , and il- were induced -, -, and -fold, respectively in fibs. the presence of ng/ml il- receptor antagonist reduced the il- -mediated increase in il- levels ± % (p< . ), indicating that this response was mediated through il- receptor. in marked contrast, il- treatment did not significantly affect levels of il- , il- , il- or il- in scts, indicating that this response was cell type-specific. conclusions: these results indicate that increased expression of il- in the placental villus core in pe and iugr may promote an inflammatory cascade in fibs at this site leading to focal infarction and reduced flow of nutrients to the fetus. introduction: in human pregnancy, decreased responsiveness to angiotensin ii (angii) starts in week , promoting an expanded vascular bed. at the same time levels of the vasodilatory hemopexin increases . during pre-eclampsia the vascular bed is contracted and the responsiveness upon angii persists. this may be due to the increased levels of extra cellular atp, a natural inhibitor of hemopexin . in the present study we tested whether extra cellular atp is toxic in the pregnant condition. methods: pregnant rats were infused with either atp ( g/kg bw; n= ) or saline (n= ) on day of pregnancy (permanent jugular vein cannula/ hr infusion). non-pregnant rats (n= ) were infused with atp identically. one day before and , , days after infusion, hr urine and blood samples (wbc count and hemopexin activity) were collected. seven days after the infusion, rats were sacrificed and kidney tissue processed for immunohistology. results: urinary albumin excretion was increased in pregnant atp infused rats only (fig ) . wbc were also increased only in pregnant rats infused with atp (days and vs pre-infusion value). in pregnant atp infused rats intraglomerular influx of monocytes was increased, which correlated with urinary albumin excretion on the same day (r = . ). staining of glomeruli for the angii receptor (at- r) showed decreased at- r expression in control pregnant rats as compared to non-pregnant rats, while at- r expression in atp infused pregnant rats was increased as compared to control pregnant rats. hemopexin activity was increased on days and in control pregnant rats as compared to all other groups. discussion: these data support the notion that atp is toxic exclusively in the pregnant condition. it may be suggested that atp induced an inflammatory response, although the exact mechanism by which atp induced its effects needs further investigation. background: hepatocyte growth factor (hgf) is a multifunctional cytokine that is known to promote division, motility, invasion and morphogenesis of a wide range of cell types and to inhibit apoptosis. the effects of hgf are mediated through its interaction with the tyrosine kinase receptor c-met. in pregnancy hgf/met system is involved in the physiologic growth and development of the feto-placental unit. hgf/met effects are counteracted by transforming growth factor- (tgf- ), that is known to promote progressive fibrosis in human tissues. moreover tgf- plays a major role in trophoblast growth and differentiation. tgf- inhibits hgf expression as well as hgf inhibits tgf- expression. an understanding of the mechanisms regulating placental balance of these growth factors may provide insights into the processes that occur in complications of pregnancy, such as preeclampsia (pe) and fetal growth restriction (fgr). objective: to verify the hypothesis that hgf, c-met and tgf- are differently expressed in tissues of normal and pe placentas. methods: we studied placentas from pregnancies complicated by pe ( with normal fetal growth and with fgr) and placentas from normal pregnancies. from each placenta random samples were excised and rna was extracted; quantitative real-time rt-pcr analysis was used to investigate mrna expression of hgf, c-met and tgf- in pe (with or without fgr) and in normal placentas. results: gestational age, neonatal weight and placental weight were, as expected, lower in pe groups. the mrna expressions of the three molecules are higher in pe than in normal placentas, but the difference is statistically significant only for c-met. the higher values are mainly due to the increase in the pe group without fgr for c-met and tgf- and the increase in the pe-fgr group for hgf. conclusion: increased levels of expression of hgf/met system in pathological placentas could be explained as an attempt to repair placental damages; nevertheless placental regeneration could be inhibited by the increase of tgf- , that promote fibrosis. placental expression of hgf, c-met and tgf- is increased in all pe placentas, but particularly in placentas of pe with normal fetal growth, where placental tissue is probably less compromised. obesity is a risk factor for preeclampsia (pe), but the reason for this risk is unknown. previously, we found that neutrophils infiltrated into the vasculature of pe women released myeloperoxidase (mpo) and matrix metalloproteinase (mmp ), products that can cause oxidative stress and vascular dysfunction. if neutrophils infiltrate the vasculature of obese women and release mpo and/or mmp , this may help explain why they are at risk of developing pe. hypothesis: systemic vascular tissue of obese women will have a significant presence of mpo and mmp as a result of neutrophil infiltration. methods: subcutaneous fat, which is highly vascularized, was obtained at abdominal surgery from normal weight, overweight and obese women. formalin fixed, paraffin embedded μm sections of fat biopsies were stained using immunohistochemistry with specific antibodies for mpo and mmp . data were evaluated for intensity of vessel staining by visual score ( - ), density of staining using image analysis software, and % vessels with neutrophil staining, liberated from serum-free placental villous explant cultures from normal and pe pregnancies on human endothelial cells, and identified candidate mediators of their differential effects on metabolism and behaviour. methods: term placental villous tissue from normal (n= ) or pe (n= ) pregnancies were explanted for days at % oxygen. conditioned h medium (day - ) was applied to human umbilical vein endothelial cells (huvecs). an angiogenesis assay was conducted in which tubule length and number were measured by morphometric imaging following seeding on % matrigel. the effect of explant conditioned medium on endothelial cell metabolism was determined by mtt and bioluminescent atp assay. the release of vasoactive metabolites (nitrite, endothelin- , prostacyclin) from huvecs exposed to this medium was also measured. finally, a luminex bead array was used to screen the explant media for a panel of chemokines/cytokines. results: in the angiogenesis assay, tubule length (p< . , mann-whitney u test) and number (p< . ) were significantly decreased in pe compared to normal pregnancy medium. there was no change in mtt reduction, but endothelial cellular atp levels were also significantly reduced following exposure to pe explant medium (p< . ). both normal and pe-derived medium stimulated huvecs to produce vasoactive metabolites. the following cytokines were detectable in the explant media: interleukin (il)- , il- , gro-, monocyte chemotactic protein- and macrophage inflammatory protein- (mip- ). higher levels of both il- and gro-were present in the normal medium compared to pe (both p< . ). mip- was present in pe conditioned media but undetectable in media generated from normal placental explants. conclusions: these results suggest that pre-eclampsia stimulates the release of soluble factors from the placenta which have adverse effects on endothelial cell angiogenic potential and metabolism. altered levels of several cytokines were detected in the explant medium, and the effects of these on endothelial cell function are currently being addressed. yang gu, davis f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: placentas from women with preeclampsia (pe) release more chymotrypsin-like protease (clp). the purpose of this study was to determine if placenta-derived clp was responsible for altering endothelial (ec) barrier function in pe. approaches: endothelial junctional protein complex ( -catenin/ve-cadherin/ p ) expression and junctional protein ve-cadherin distribution were examined in ecs treated with pe placental conditioned medium. methods: ) confluent ecs were treated with pe placental conditioned medium (cm). the association of ec junctional protein complex ve-cadherin/catenin/p was examined by a combined immuno-precipitation (ip) and immuno-blotting (ib) assay, in which total cellular protein was immunoprecipitated with monoclonal antibody against -catenin and then immunoblotted with antibodies against ve-cadherin or p- . ) confluent ecs grown on cover slips were exposed to pe placental cm with or without depletion of chymotrypsin. ec junction protein ve-cadherin distribution was examined by fluorescent microscopy. results: ) ve-cadherin and p are expressed in control ecs but not in ecs exposed to pe cm, which indicate that the junctional protein complex vecadherin/ -catenin/ p is lost in ecs exposed to pe cm. ) ecs exposed to pe placental cm showed a discontinuous distribution and reduced expression for ve-cadherin at cell contact areas. the zipper like structure was lost and cleft was formed at cell-cell contacts. these observations indicate that the homotypic cell-cell adhesion and junction protein intracellular partner complex are disrupted. these disruptive phenomena in cells treated with pe conditioned medium were not present in control cells and in cells treated with cm after depletion of chymotrypsin. conclusion: clp released by the placenta could be a candidate agent that is responsible for disrupting ec integrity and inducing endothelial permeability in pe. (supported by nih grants hl and hd ). introduction: chronic pelvic pain (cpp) syndromes such as endometriosis, irritable bowel syndrome, and interstitial cystitis are associated with visceral hyperalgesia, and often coexist in the same patient. one possible explanation for this phenomenon is viscero-visceral cross-sensitization in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input from an unaffected organ to the same dorsal root ganglion (drg). nociception induces upregulation of perk and substance p. the purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of perk-and sp-positive neurons in sensory ganglia innervating both uterus and colon. methods: colonic and uterine drgs were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon and uterus. ganglia were harvested, cryoprotected and cut for fluorescent microscopy. results:approximately % neurons were colon-specific and % were uterus-specific. among these uterus-or colon-specific neurons, up to % of labeled drg neurons in the l -s levels innervated both visceral organs. uterine inflammation increased the number of perk and sp-immunoreactive neurons in drg neurons innervating colon, uterus, and those innervating both organs. furthermore, this effect was specific as non-retrograde labeled drg neurons did not manifest a significant increase in perk or sp immunoreactive cells. conclusions: localized uterine inflammation leads to increased expression of sp and perk in uterine afferents as well as dichotomizing afferents innervating both uterus and colon, suggesting that viscero-visceral convergence is present at the level of drg primary afferent cell bodies. this visceral sensory integration may underlie the co-morbidity of female pelvic pain disorders and may provide basic information regarding the etiology of cpp syndromes. purpose: success rates of medical and surgical modalities for the treatment of severe fecal incontinence due to obstetric trauma are modest. we tested whether the intrasphincteric injection of autologous myoblasts is clinically safe and feasible for postobstetric fecal incontinence. methods: using ultrasound guidance a suspension of autologous myoblasts was injected in the anal sphincter muscle complex in three women with severe postobstetric fecal incontinence. main outcome measures were safety, feasibility and the wexner grading score. secondary outcome measures were incontinence episodes, rockwood fecal incontinenece quality of life scale, external anal sphincter morphology and anal pressure values. results: all procurement procedures and injections were performed without complications; there were no clinically or laboratory signs of infection or inflammation. three months post myoblast injection, wexner continence grading scores and rockwood incontinence scales were markedly improved and incontinence episodes reduced in all three patients. no change could be observed in sonographic anal sphincter morphology. mean anal squeeze pressure improved. conclusion: autologous myoblast injection for fecal incontinence is clinically feasible and safe; further studies will evaluate the efficacy of this approach. objective: to evaluate whether hysterectomy is associated with a change in postoperative bmi. methods: a retrospective cohort study was conducted of hysterectomies performed for benign indications from june to june . institutional review board approval was obtained. the data were collected by a medical student blinded to the hypothesis. basic demographic data were recorded. body mass index (bmi) was determined at time points: time (immediately postoperatively), time ( weeks to months postoperatively), time ( months to year postoperatively) and time ( to . years postoperatively). one-way anova was performed using ncss statistical software. a bonferroni multiple comparison test (p< . ) was used to compare median changes in bmi from baseline. the starting bmi served as the control group. results: there was a statistically significant increase in bmi at time compared to baseline in all women ( . to . ). when sorted was measured by matrigel invasion assay. small interference rna targeting vegfr- were predesigned by ambion inc. and cells were transfected using ambion siport neofx according to the optimized protocol. results: of the four receptors (vegfr- , vegfr- , nrp- and nrp- ), vegfr- was the predominant receptor that expressed in the more invasive cell lines (dov , skov and r ). lpa, at - m, significantly induced vegfr- expression in dov and skov cells (p< . ), without significantly affecting vegfr- expression. lpa ( - m) also significantly induced the expression of vegf and vegf (p< . ) in dov and skov cells. by small interference rna (sirna) transfection, we demonstrated that inhibition of vegfr- expression could significantly decrease dov cells' invasiveness (p< . ) and moderately decrease skov cells' invasiveness. moreover, silencing of vegfr- by sirna significantly suppressed lpa-induced dov and skov invasion. conclusion: these results suggest that knocking down vegfr- expression by rnai may be an effective strategy to inhibit lpa-induced ovarian cancer metastasis. cancer. fengqiang wang, david fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objectives: expression of matrix metalloproteinases, such as mmp- and mmp- , has implicated in epithelial ovarian cancer (eoc) invasion and metastasis. osteopontin (opn) was also expressed in various human cancers and associated with tumor progression, invasion and metastasis. in the present study, we examined the correlation of mmp- , mmp- and osteopontin expression with tumor stage in ovarian tumor tissues and normal ovaries using a commercial tissue scan array. methods: complimentary dna (cdna) from normal ovaries (n = ) and ovarian tumors (n = ) of different stages (stage i, n = ; stage ii, n = ; stage iii, n = ; stage iv, n = ) was amplified by real time pcr using gene specific primer pairs for mmp- , mmp- , and osteopontin. gapdh was also amplified as a reference control. the expression level of mmp- , mmp- and osteopontin was calculated as relative expression normalized to that of gapdh in each tissue sample, and the expression in sample that has the lowest target gene expression was arbitrarily set as . the average expression of mmp- in ovarian tumor tissues ( ± ) is fold of that in normal ovaries ( ± , p = . ). using an arbitrarily set standard, of normal ovaries ( . %) has elevated mmp- expression; in ovarian tumor tissues, the percentage is . % ( of ), significantly higher than in normal tissues (p = . ). in early stage tumors (stage i/ii, n = ), of them have elevated mmp- expression ( . %, p = . vs. normal ovaries), whereh the average expression of mmp- is fold of that in normal ovaries (p = . ) and . fold of that in late stage tumors (stage iii/iv, n = ). the mmp- expression in ovarian tumors (n = , ± ) is fold of that in normal ovaries (n = ), however, the difference is not significant (p > . ) due to the extremely high expression of mmp- in two tumor tissues. osteopontin expression in ovarian tumor tissues is . fold of that in normal ovarian tissues ( ± . , n= vs. . ± . , n = , p< . ). in early stage (i/ii) ovarian tumors, osteopontin expression is . fold of that in normal ovaries, while in late stage (iii/iv) ovarian tumors, its expression is . fold of that in normal ovaries (p< . ), suggesting an increase trend associated with disease stages. conclusions: our results suggest that mmp- , mmp- and osteopontin alone or combined may have clinical value for ovarian cancer detection. objectives: -tubulin acetylation has been proposed to control the dynamic nature of microtubule stability. acetylated -tubulin plays a role in regulating microtubule functions in mitosis and cell migration. here, we sought to identify the relationship between post-translational -tubulin acetylation and the expression of epithelial cell adhesion molecules (ep-cam) after exposure to microtubule interacting agents in ovarian cancer cells. methods: epithelial ovarian cancer cell line, hey was treated with microtubule stabilizing agents (taxol, epothilone b and discodermolide), microtubule-destabilizing agent (vinblastine), hdac inhibitor trichostatin a (tsa), anti-metabolite fluorouracil ( fu), or alkalyting agents (cisplatin and carboplatin). cells were separately treated with either ic or -fold ic of each agent, and incubated at °c for h, h and h. acetylation oftubulin and pan--tubulin were evaluated by western blot analysis followed by protein quantification. cell surface ep-cam expression was examined by flow cytometry. results: increased acetylation of -tubulin was seen with taxol, epothilone b, discodermolide, vinblastine and tsa. -tubulin acetylation was time and dose dependent. the highest level of -tubulin acetylation ( . -fold) was observed with vinblastine at -fold ic after h. exposure to microtubule interacting agents and tsa resulted in increased cell surface expression of ep-cam in a time and dose dependent manner. the highest level of cell surface ep-cam expression ( . fold) was observed with -fold ic of vinblastine at h. the increase in acetylated -tubulin and ep-cam expression was clearly detectable after h treatment. this data reveals a similar dose and time dependent increases between the acetylation of -tubulin and the increase of ep-cam expression. conclusions: these data demonstrate the promotion of -tubulin acetylation and cell surface ep-cam expression by a microtubule destabilizing agent and by microtubule stabilizing agents. interestingly, vinblastine induces the highest -tubulin acetylation and ep-cam expression. acetylation of alpha-tubulin may be associated with redistribution of cell surface antigens in ovarian cancer cells. objective: epothilone b (epob) is similar to taxol in its ability to enhance tubulin polymerization and to stabilize microtubules. epob is currently being evaluated as an antitumor agent and is in phase iii clinical trials. the tumor suppressor gene, p plays an important role in the induction of apoptosis by a variety of anticancer drugs. p mitogen-activated protein kinase is activated by a wide array of stress stimuli including chemotherapeutic agents and promotes apoptosis. since both p and p activation induces apoptosis, we hoped to evaluate the relationship between p , p-p and parp cleavage, an early indicator of apoptosis, in ovarian cancer cells after treatment with epob. methods: hey cells were treated with epob ( to nm) for h. parp cleavage product p as well as p-p , p , phospho-p (ser- ), p and survivin were determined by western blot analysis. a wild type ovarian cancer cell line hey, was treated with or without m sb , a specific inhibitor of p-p , followed by treatment with epob ( or nm) for h. lysates were prepared and western blot analysis was performed with polyclonal phospho-p and anti-phospho-p antibodies. results: epob induces p activation and apoptosis demonstrated by increased parp cleavage product. time course studies indicated that phosphorylation of p precedes phosphorylation of p in hey cells. the expression of p targeted gene, p (survivin) were differentially expressed depending on the dose of epob and the duration of drug exposure. pretreatment with specific inhibitor of p markedly inhibited the p phosphorylation at serine . conclusions: nm epob (a concentration that triggered mitotic arrest) causes a decrease in p expression and an increase in survivin expression. epob induces parp cleavage. p inhibitor sb inhibits epo-b -induced p phosphorylation. these results suggest that phosphorylation of p may lead to p activation and these signaling events may be related to epo-b induced cell death in ovarian cells. conclusions: nf-b has been shown to control the expression and function of anti-apoptotic proteins and pro-inflammatory cytokines. in the present study we demonstrated that specific inhibition of nf-b by erib reversed the anti-apoptotic state of chemoresistant ovarian cancer cells, therefore may provide a new potential venue for the treatment of ovarian cancer patients. expression in cervical cancer. madhu chauhan, chandra yallampalli. gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd)/adrenomedullin is a ct/cgrp family peptide. imd is expressed in a variety of tissues such as pituitary, stomach, placenta, uterus, and ovary .we have shown that imd plays a role in a variety of physiological functions, including vasodilatation and fetoplacental growth regulation. further we have data indicating an angiogenic activity of imd in first trimester trophoblast cells. we hypothesize that imd is expressed in cervix and may have a role in the pathology of cervical cancer objective: ) to analyze expression of imd mrna in human cervix, rat cervix from non-pregnant and pregnant rats; ) to assess the differences in the expression of imd in normal human cervix and cervical carcinoma tissues and ) to analyze the effect of imd on the expression of tnf-alpha and il- beta in human epithelial cervical carcinoma cells (hela). methods: groups of sprague-dawley, non-pregnant and pregnant rats on day of gestation were used in this study. cervical tissues were collected from the non-pregnant and pregnant rats, women undergoing hysterectomy and from women diagnosed with cervical carcinoma. total rna was extracted using trizol method. hela cells were grown to % confluency in rpmi medium supplemented with % fbs. the cells were starved in %fbs for hrs followed by treatment with imd ( - m) in presence or absence of imd antagonist, imda ( - m). cells were further incubated for hrs and total rna was extracted using trizol reagent. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). the rt-pcr data was normalized to that of s. results: ) imd mrna is expressed in rat and human cervix and in hela cells. ) expression of imd is elevated in pregnant rat cervix as compared to the non-pregnant. ) imd has no effect on tnf-alpha expression in hela cells treated with imd but caused a decline in the expression of il- beta mrna and, ) expression of imd mrna is significantly elevated (p< . ) in cervical carcinoma as compared to the normal cervix. conclusion: imd is expressed in both rat and human cervix and is elevated in pregnant rat suggesting that it may have a role in cervical function during pregnancy in rat. in addition we demonstrate that imd is involved in the pathology of cervical carcinoma and thus may have a clinical significance in the pathology of cervical cancer. objective: there is minimal information about the impact of treatment delays or dose reductions on chemotherapeutic treatment responses and overall outcomes in ovarian cancer patients. the primary objective of this study was to quantify the impact of treatment delays and dose reductions in an in vivo xenograft ovarian cancer model and to evaluate if the growth factors could improve overall survival. methods: heya- and skov -ip xenograft mouse models to determine the effect of treatment delays or dose reductions on tumor response. results: the results indicated that full dose of paclitaxel ( mg/kg) and carboplatin ( mg/kg) delivered on timeevery daysachieved better tumor response in both aggressive (heya- ) and metastatic (skov -ip ) human ovarian tumors compared to the non-treatment and vehicle groups. in heya- mice, paclitaxel/carboplatin alone and paclitaxel/carboplatin followed by growth factor support agents including pegfilgrastim ( μg/kg) and darbepoetin ( μg/kg) improved overall survival rate. growth factors also improved the tolerability in heya- model. for skov -ip mice, the treatment delays resulted in a significant reduce in overall survival time compared to full dose, on time treatment (p< . ). for the dose reduction group, there was a significant different survival comparing to full dose, on time treatment (p< . ). conclusion: treatment delays had a negative impact on tumor response and overall survival compare with treatment controls. in addition, use of growth factor agents also improved treatment response and tolerability of chemotherapy and ultimately overall survival. - ) . median age at recurrence was ( - ), and median interval to recurrence . months ( - ). ( %) patients died of recurrent disease and ( %) of other causes. site of recurrence and was local in , groin in and distant in . of the local recurrences were managed with wide local excision (wle) radical surgery ( radical excisions with skin flaps, posterior exenterations, anterior exenteration, total exenteration), wle and radiotherapy (rt)/chemort, chemo/chemort/rt, and palliation. groin recurrences were managed surgically ( adjuvant rt), rt alone, chemotherapy alone, and palliation. patients with distant metastases were managed surgically, with rt/chemo/chemort, and palliation. overall median survival after recurrence was months. median survival (months) by site of recurrence was: local , groin , distant , groin or distant . there was a significant difference in survival in local vs groin node recurrence (p< . ), local vs groin and distant (p< . ), and groin node vs distant (p . ). and years after recurrence survival rates were: local % and %, groin % and % distant , . conclusion patients with vscc remain at risk of recurrent disease therefore long term follow up these patients is essential. patients with local recurrence often have a good prognosis. outcomes for groin and distant recurrences are poor, but a proportion of those with groin recurrences can be salvaged. therefore early identification of local and groin recurrences is essential for improving outcomes in recurrent vscc particularly in cases with more conservative management of primary disease. introduction: endocrine disrupting chemicals (edcs) are environmental agents possessing hormone-like activity. exposure to edcs during differentiation can interfere with hormonal signaling, resulting in predisposition to some cancers. it has been suggested that some of these effects may be epigenetic, mediated by changes in dna and histone methylation. we hypothesized that edcs, diethylstilbestrol (des), and bisphenol-a (bpa), may act by altering the expression of dna and histone methyltransferases (dnmts and hmts). methods: ishikawa (endometrial) and mcf (breast) cells were cultured in steroid-free, phenol-free media with bpa ( m), des ( x - m) or vehicle for hours. pregnant cd- mice were treated with intraperitoneal injections of des ( mg/kg) or vehicle on days - of gestation. weeks after birth, offspring were sacrificed and tissue obtained. mrna was extracted, cdna was generated and quantitative real time rt-pcr was performed and normalized to -actin. all experiments were conducted in triplicate, repeated three times and compared using anova. results: dnmts (dnmt , a, and b) and hmts (mll and ezh ) were screened after in vitro exposure to edcs (table ) . ezh mrna expression was significantly increased in ishikawa cells after treatment with des or bpa. a similar response in ezh expression was seen in mcf cells treated with des or bpa. due to the consistent induction of ezh in all cells with either treatment, we examined the effect of des exposure on ezh expression in vivo. in adult mice exposed to des in utero, ezh expression was persistently increased ( . ± . fold, p< . ). conclusions: breast and uterine cell lines show increased expression of ezh in response to bpa or des exposure. this differential expression persists in the adult offspring of mice exposed to des in utero. ezh has been identified as a risk for neoplastic progression in the breast and for increased proliferation in uterine cancers. des induced ezh expression may be a mechanism for the increased incidence of breast and reproductive tract cancers seen in des exposed women. defects in cellular programs that control apoptosis lead to an imbalance of cell proliferation and cell death in ovarian cancer. recent evidence suggests that the use of some anti-inflammatory drugs decreases risk of ovarian cancer. natural curcumin-based anti-inflammatory therapies were shown to be beneficial in preclinical models of ovarian cancer (lin et al., ) . in this study, we examined the effects of plant derived curcumin on cell proliferation, apoptosis, and vegf expression in cultivated ovarian cancer cells. ovarian cancer igrov cells were cultured under standard conditions to study the effects of curcumin on cell kinetics and on vegf expression. cell proliferation was measured by srb and mtt assays. apoptosis was determined by measuring cytoplasmic histone-associated-dna-fragments (cell death detection elisa, roche, germany). vegf gene promoter-reporter activation and real-time quantitative reverse transcription polymerase chain reaction were used. conditioned media concentrations of vegf were measured with a commercially available enzyme-linked immunosorbent assay (quantikine, r d systems, minneapolis, mn). we observed that curcumin dose-dependently suppressed cell growth in igrov cancer cells (ic = um). treated cells showed a - fold increase in dna fragmentation compared to controls. curcumin also resulted in a significant decrease of vegfmrna expression and vegf protein secretion into the conditioned media in a dose-dependent manner. in this study, we have demonstrated that curcumin induces apoptosis, suppresses growth, and inhibits vegf gene and protein expression in an ovarian cancer cell line. experiments are underway to identify specific mechanism of curcumin action. curcumin may act as a chemosensitizing drug by potentiating the antitumor effects of standard treatments including taxols and platins in ovarian cancer. supported by the caldwell family foundation and the vesa w. and william j. hardman, jr. charitable foundation inc. daniel r christie, faheem m shaikh, john a lucas iii, susan l bellis. obsterics and gynecology, university of alabama at birmingham, birmingham, al, usa; physiology and biophysics, university of alabama at birmingham, birmingham, al, usa. introduction: previous work has shown that an upregulation of the enzyme st gal i, which is responsible for the , sialylation of integrins, confers a more tumorigenic/metastatic phenotype to colon carcinoma cell lines. it is not known, however, if this is unique to colon cancer, or if it is more broadly applicable to other forms of cancers. quantitatively increased expression of st gal i has been reported in ovarian cancers versus controls, but no biochemical or functional assays have been described to date. objective: because integrins are involved in cell adhesion and migration, and because metastasis of epithelial ovarian cancers is largely an intraperitoneal dissemination, we hypothesized that upregulation in the expression of st gal i in an ovarian cancer cell line would enhance binding with the extracellular matrix, increase invasiveness, and alter migration. methods: in the present study we forced a stable transduction of st gal i into the ov ovarian tumor cell line, which we found to be lacking the st gal i enzyme, establishing a parental (par), an empty lentiviral vector (ev), and an st gal i expressing (st ) cell line. a collagen i cell adhesion assay was performed and quantified by staining adherent cells and measuring absorbance. a haptotactic collagen i cell migration assay was performed by seeding cells in boyden chambers lined with collagen i concentration gradient, and quantified with cell staining and absorbance measurement. cell invasion through a reconstituted basement membrane (matrigel) was quantified as previously described for the cell migration assay. results: we were able to demonstrate successful creation of the ov -st gal i cell line by western blot analysis. functional assays demonstrated increased adhesion to collagen i (p < . ), increased haptotactic collagen i cell migration (p < . ), and increased invasiveness (p < . ) in the st cell line as compared to par and ev when analyzed by one-way anova. conclusion: this initial study into st gal i in ovarian cancer may have future therapeutic implications, and, in addition, lend greater insight into the intraperitoneal dissemination of disease.of microarrays (sam), arrayassist and binary tree structured vector quantization. differentially expressed genes were integrated with a database of known predicted protein-protein interactions (ophid) and key genes are being validated with real-time pcr and immunohistochemistry. results: unsupervised hierarchical clustering revealed collective clustering of all tumors, irrespective of their pathological classification. sam analysis has highlighted probe sets as differentially expressed between lmp and lmp-mp, probes between lmp and lgsc, and probes between lmp and lmp-mp+lgsc. no differential gene expression was detected between lmp-mp and lgsc. ophid analysis demonstrated gene members of the egfr and mapk / pathways to be differentially regulated between the non-invasive and the invasive tumors. to date, we have successfully validated members of the mapk / pathway-poly adp ribose polymerase (ppol) and traf family associated nf kappa b activator (tank)-using real-time pcr. conclusion: our data demonstrate that although the tumors have related genetic profiles, lmp-mp and lgsc are similar to each other and different from lmp, in keeping with their clinical behavior. members of the mapk / and egfr pathways appear to play a key role in low grade serous cancer. identification of novel genes associated with malignant transformation, may lead to development of more effective targeted therapy for lgsc. background: approximately % of pap smears with the ambiguous diagnosis of atypical squamous cells, cannot exclude high grade (asc-h), are negative for dysplasia in follow up colposcopic examination and biopsy. although hpv testing provides excellent negative predictive value (npv) ( %), the prevalence of high risk hpv infection is high in young women and the positive predictive value (ppv) in asc-h pap smears is no better than cytologic diagnosis alone ( %). recent studies have shown that immunostaining for p ink a , or proex tm c, supports a diagnosis of dysplasia in surgical biopsies. our objective was to determine whether staining for p or proexc provides sufficient predictive value to reliably distinguish high grade dysplasia in asc-h pap smears. design: we retrospectively collected samples from liquid based pap smears diagnosed as asc-h at either ohsu or portland oregon kaiser permanente ( - ). known hsil and negative pap cases were included as immunostaining controls. asc-h cases with followup cervical biopsies (n= ) were included for subsequent immunostaining according to manufacture's instructions. samples were also sequestered for hpv testing (pending). immunostained slides were scored as positive or negative by two independent cytopathologists (as and tm) while blinded to pap diagnoses and surgical biopsy outcomes. results: we observed excellent agreement between pathologists' scores (pairwise kappa statistic . ). the correlation between p and proexc scores was moderate (kappa . ). chi-square analysis comparing staining to biopsy outcome revealed a significant association between proex c positivity and cervical dysplasia (*** p< . jing lin, zhenmin lei, ch v rao. ob/gyn women health, university of louisville health sciences center, louisville, ky, usa. introduction: matrix metalloproteinases (mmps) are a family of highly homologous zinc-dependent endopeptidases, which degrade all kinds of extracellular matrix proteins. the degradation process is required for tissue growth and remodelling. these enzymes are probably involved in uterine growth and devolopment and its differentiated functions. ovarian steroid hormones, estradiol (e ) and progesterone (p ), are known to regulate some of the uterine mmps. since it is now known that lh/hcg can directly regulate uterine growth and devolopment and its functions, we questioned whether these gonadotropins could also regulate mmps in the uterus. methods: sixty day old wild type (wt) and lh receptor knockout (lhrko) mice and -day e and p treated -day old animals were used. in addition, primary cultures of uterine epithelial cells from wt animals were treated for hrs either with no hormone or with a single or a combination of pg/ml of e or p , ng/ml of hcg. then mmp- , - and - mrna levels were quantified by the semiquantitative rt-pcr. results: while the uterine mmp- mrna levels were unaffected, mmp- mrna levels significantly decreased and mmp- mrna levels significantly increased in lhrko animals as compared with wt siblings. e and p treatment reversed mmp- and mmp- changes in lhrko animals. treatment of wt type primary endometrial epithelial cell cultures with hcg had no effect on mmp- mrna levels, but it did increase mmp- mrna levels. this increase was synergistic with both e or p . conclusion: while lh/hcg do not regulate uterine mmp- and mmp- , they seem to co-regulate mmp- with ovarian steroid hormones. cancer. radhika gogoi, christine ardalan, dorota popiolek, leslie gold, john curtin, stephanie v blank, bhavana pothuri. new york university, new york, ny, usa. objective: megesterol, a synthetic progestin with strong androgenic properties, is used in the medical management of patients (pts) with atypical endometrial hyperplasia (aeh) or endometrial carcinoma (emca). our hypothesis was that androgen receptor (ar) expression in the endometrium of aeh and emca pts would correlate with degree of response to treatment. methods: pre-and post-treatment endometrial biopsy specimens were obtained from pts treated with megesterol for aeh or emca. ar expression was investigated by immunohistochemical (ihc) analysis with appropriate positive and negative controls. ihc staining was scored for intensity ( - ) and percentage of positive cells ( - ) in the glandular and stromal compartments by a pathologist, blinded to clinical response data. a composite score utilizing both intensity and percentage of positive cells was calculated. we evaluated pre-and post-treatment ar expression in responders and nonresponders as well as ar expression in emca, aeh and normal endometrium. the mann whitney u and the wilxocon signed rank tests were utilized for statistical analysis. results: eight pts' pre-and post-treatment samples were obtained; with emca and with aeh. three pts had no response, , a partial response and , complete responses. ar expression in emca samples when compared to the normal endometrium was significantly lower in both the glands (mean . vs. ; p< . ) and the stroma (mean . vs. ; p< . ). although there was no statistically significant difference in glandular ar expression in the pretreatment biopsies of responders compared to nonresponders (mean . vs. . ; p= . ), there was a significantly higher level of glandular ar expression in the post treatment biopsies of responders compared to non-responders (mean . vs. ; p< . ). furthermore, we noted a trend towards higher levels of glandular ar expression in the post-treatment versus the pre-treatment biopsies in the responders (mean . vs. . ; p= . ). we noted a significant decrease in ar expression in emca compared to the normal endometrium. increased ar expression after treatment in responders suggests an important role of the ar in pts treated conservatively with progestational therapy, and needs further prospective validation as a means to predict treatment response in these pts. objectives: study the correlation between adenomyosis and some potential non-surgical risk factors. objective: o -methylguanine-dna methyltransferase (mgmt) acts to repair dna damaged by alkylation of guanine residues. mgmt promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes. the loss of mgmt activity is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas. we sought to determine if mgmt promoter methylation plays a role in endometrial cancer. methods: one hundred and twenty primary endometrial cancers were analyzed for mgmt promoter methylation by combined bisulfite restriction analysis (cobra). the cohort included endometrioid endometrial cancers, endometrial tumors of adverse histologic type, and endometrial cancer cell lines. twenty one endometrioid and mixed endometrioid ovarian cancers were also analyzed. a subset of the primary tumors was analyzed for mgmt expression by immunohistochemistry. results: no mgmt promoter methylation was seen in the endometrial cancers evaluated or the endometrial cancer cell lines. one of the ovarian cancers showed methylation. immunohistochemistry for mgmt expression is ongoing. conclusion: mgmt promoter methylation is an infrequent event in endometrial cancer. mgmt expression in tumor cells and repair of alkylguanine residues could explain in part the limited response of endometrial tumors to alkylating chemotherapy. objective: uterine sarcomas ( % of gynecological malignancies) originate from uterine mesenchyma. patients with high-risk disease (i.e. high grade) or at advanced stages have poor -years overall survival (< - %). pelvic radiation and/or chemotherapy have not demonstrated to improve survival. identification of new therapies for this malignancy is a major goal. few in vitro models have been established to test therapeutic agents for uterine sarcoma. here we sought to establish a new human uterine sarcoma cell line and to test the effects of chemotherapeutic drugs: tnf-related apoptosis inducing ligand (trail) used alone or in combination. methods: tissue sample was obtained from a woman with uterine sarcoma undergoing hysterectomy. sarcoma cell lines were established using a published protocol for endometrial cancer. phenotypic characterization was made through the different passages ( to ) by western blot. levels of estrogen (er), progesterone (pr) and trail receptors were also studied (rt-pcr, w-b). cells were incubated with chemotherapy agents (cisplatin, paclitaxel and doxorubicin and trail) and cytotoxicity (mts assays) and apoptosis (flow cytometry, parp cleavage by w-b, and dna laddering) measured. results: human uterine sarcoma cell line was established from a highgrade uterine sarcoma. through the different passages the cell line remains expressing cytokeratin, vimentin, tissue factor, caveolin- and -actin. this cell line expresses low er and pr levels. trail receptors (r and r ) were also detectable (rt-pcr, w-b). cisplatin, paclitaxel and doxorubicin ( um for h) produced low cell cytotoxicity (< %). trail ( ng/ml for h) induced about % cell cytotoxicity. apoptosis was confirmed by parp cleavage. doxorubicin significantly enhances trail mediated cytotoxicity (up to %), this was demonstrated by a significant increase in the sub g /g region in the dna histogram. conclusions: we establish a human uterine sarcoma cell lines using protocols for endometrial cancer. more importantly, we demonstrated that doxorubicin enhances trail effect on this uterine sarcomas cell line. thus, this combination might be considered as a treatment for high-risk uterine sarcomas. (financial support fondecyt ). defining the tumor initiating capacity of cd + human endometrial cancer cells. anne m friel, petra a sergent, christine l cummings, rosemary foster, current data suggest rare subpopulations of cells with tumor initiating capabilities are a common feature of solid tumors. several investigators have recently identified cd , a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages, as a potential tumor initiating cell (tic) marker in solid tumors of the brain, colon and pancreas. in our efforts to investigate such a population in human endometrial cancers (enca), we developed an in vivo model that is based on serial transplants of tics from endometrial tumor explants. serial transplant experiments were initiated in nod/scid mice that were injected with primary human enca cells. a subset of cells derived from the generated tumor was subsequently transplanted into new recipients. tumors formed following serial transplantation retained the original histological phenotype of the primary enca, and the number of cells required to initiate tumor formation was reduced at each successive transplant stage suggesting an increase in the ratio of tics to non-tics. we exploited this model in our initial efforts to investigate the tumor initiating potential of cd -expressing enca cells. to evaluate the tumorigenic potential of cd + cells, the tumor initiation capacity of xenograft derived cd + and cd cells were compared following subcutaneous injection of each population into nod/scid mice. only the cd + fraction was tumorigenic consistent with the hypothesis that tumors are generated and maintained by a subpopulation of cells with phenotypically distinct profiles. we are further investigating the functional significance and characteristics of this fraction in vitro and in vivo. objectives. central issues in tumor biology are the understanding of factors that control tumor cell proliferation and the identification of extracellular matrix cues controlling the signaling transducing repertoire that make cancer cells proliferate and invade the host tissues. among these factors, small leucine-rich proteoglycans (srlps):decorin, fibromodulin, lumican, biglycan, are emerging as powerful modulators of angiogenesis and cell growth, by affecting several key elements including matrix assembly, growth factor binding, and receptor tyrosine kinase activity. recently has been demonstrated that srlps can act as a pan-erbb ligand and, in doing so, down-regulate the activity of one of the most potent oncogenic proteins, erbb , whose overexpression is linked to poor prognosis and increased cancer mortality in breast, ovary, and prostate. since srlps have not been previously investigated in the endometrial cancer biology, we investigated their role in the benign and malignant endometrial neoplasia. method. the sampling has been obtained in women (n= ; mean age ± . ) with endometrial hyperplasia (n= ), polyps(n= ), and cancer(n= ), during therapeutic and diagnostic procedures (hysterectomy, colpohysterectomy, hysteroscopy). physiological endometrial samples (n= ) were obtained from menopausal women (n= ; mean age ± . ), during procedures for others gynaecologic indications. immunohistochemestry was the biology technique used for the detection of srlps.results. in the physiological endometrial samples immunoreactivity for decorin, fibromodulin and biglycan was intense (+++), while it was weak in polyps and hyperplasia (+) and absent (-) in cancer. no significant difference in the staining of lumican between the physiological and pathological samples. conclusions. these results could provide a mechanism by which naturally occurring proteins normally synthesized by fibroblasts and smooth muscle cells, the two key components of the tumor stroma, may play a protective role in the genesis and progression of endometrial neoplasia counteracting the growth of neoplastic cells and suppressing tumor cell-mediated angiogenesis. although further studies are necessary to understand mechanisms whereby srlps might influence endometrial cell growth and survival, these molecules may represent potential target for pharmacological cancer therapy. myostatin is a member of the tgf-beta superfamily of protein and a wellknown inhibitor of skeletal muscle proliferation. the muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiologic conditions under the influence of steroids. we determined the expression of myostatin mrna in immortalized pregnant human myometrial (phm ) cells and we verified its biological activity. functional assays showed myostatin induced phosphorylation of smad- and reduction of proliferation of phm cells in a time and dose-dependent manner. to investigate the physiological relevance of our in vitro findings, the expression of myostatin in rat uterus was examined at various phases of the estrous cycle. for the first time we report that myostatin is expressed in rat uterus and that levels of myostatin mrna change during distinct phases of the estrus cycle. uterine levels of myostatin peaked during late estrous and were the lowest at pro-estrous. to further examine the role of steroids in myostatin regulation, we examined the effects of gonadal steroid administration in ovariectomized (ovx) rats. ovaryectomy increased myostatin expression compared to normal cycling controls. estrogen treatment strong decreased myostatin levels while progesterone produced less robust effects on myostatin expression. these findings taken together suggest an important role for myostatin in the regulation of myometrial functionality. her neu over-expression and pi kinase/akt pathway activation in paget's disease of the vulva. amy stenson, bita behjatnia, jaime shamonki, jianyu rao, amer karam, jonathan berek, oliver dorigo. ob/gyn and pathology, ucla, los angeles, ca, usa; ob/gyn, stanford university, palo alto, ca, usa. background: paget's disease of the vulva is rare with high recurrence rates. treatment of recurrent disease is challenging due to its extent and location. non-surgical approaches have limited clinical efficacy, obviating the need for novel therapies. in contrast to paget's of the breast with well-described overexpression of her neu, molecular features of vulvar paget's are poorly characterized. our objective was to study therapeutic targets in vulvar paget's, including the her neu protein and the phosphorylated oncoprotein akt (pakt). in addition, detailed clinical characteristics were correlated with molecular expression. methods: specimens with vulvar paget's disease were retrieved from ucla's department of pathology. protein expression was evaluated by immunohistochemistry on slides from paraffin embedded tissue using the hercep test (dako) for her neu expression and a standard protocol to assess expression of activated pakt. slides were scored by two independent pathologists based on a nominal scale of (negative) to (strongly positive). clinical data was retrieved via chart review. results: between and , patients with vulvar paget's were identified. median age was yrs ( - yrs). a family history of cancer was found in / ( %), / ( %) were smokers and / ( %) had a history of hormone use. intraepithelial lesions accounted for the majority ( / , %), while / ( %) demonstrated invasion and / ( %) were associated with underlying gi malignancy. / ( %) had at least one recurrence, with median time to recurrence months. so far, specimens were stained for her neu and pakt. overexpression of her neu was found in / ( %.) positive staining for pakt was evident in / ( %.) statistical analysis suggested a correlation between her neu and pakt expression. conclusions: our study demonstrates that overexpression of her neu and activation of the pi kinase/pakt pathway are important features of vulvar paget's disease. to the best of our knowledge, this is the largest series evaluating these molecular pathways in vulvar paget's. our data suggest that her neu and pakt may be important molecular targets for novel therapies using for example the monoclonal antibody trastuzumab directed against her neu, or a pi kinase pathway inhibitor like rapamycin. introduction: uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. these tumors occur in up to % of adult women, and their prevalence is especially high in africa-american women. currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. epigallocatechin gallate (egcg) constitutes the main solid extract of green tea and is believed to contributes most of the antioxidant and antiinflammation capacity of green tea. egcg has been shown to have beneficial effects on prostate cancer and breast cancer by inducing apoptosis and inhibiting proliferation of cancer cells. in this study, we investigated the ability of egcg to inhibit proliferation of human leiomyoma cells (hlm) in vitro. methods: human immortalized leiomyoma cells were cultured at ºc in a humidified atmosphere of % co - % air in smbm medium supplied with %fbs, . % insulin, . % hfgf-b, . % ga- and . % hegf(lonza). the cells were plated at density of × cells/well in -well plates and grown under the same conditions above. the monolayer cultures at approximately % confluence were treated with various concentrations ( , . , . , , , and μm) of egcg (sigma) for days. a fluorometric assay using hoechst dye (sigma) for dna quantitation was conducted at the following designed time points, day , , , and post egcg treatment. the cells were lysed and dna content was determined using hoechst dye solution ( μg/ml). fluorescence was measured after excitation at nm and emission at nm. results: egcg exhibited marked anti-proliferation effect on the growth of hlm cells. compared with untreated control, the inhibitory effect of egcg on hlm cells was observed at μm and peaked at μm concentration. the difference was statistically significant (p = . ). evaluation of the mechanism of action of egcg is cuurently under investigation in the lab.conclusion: egcg significantly inhibited the proliferation of human leiomyoma cells in a dose-depended pattern. egcg may present a potential effective and safe medicinal treatment for uterine fibroids. objective: choriocarcinoma is an aggressive form of germ cell tumor that exhibits rapid growth with early metastases. choriocarcinomas autonomously secrete hcg which acts as an autocrine/paracrine growth factor in these cancers. we hypothesize that a novel hcg antagonist (hcg-ant) can limit tumor expansion by blocking hcg-induced expression of pro-invasive genes. we investigated if hcg-ant could alter rna expression of matrix metalloproteinases (mmp- and mmp- ), which facilitate basement membrane degradation and hence invasion, and metastin (kisspeptin), a strong suppressant of metastasis, in the choriocarcinoma cell line jeg- . design: in vitro experiments using the jeg- cell line. materials and methods: after plating jeg- cells in a well tray overnight in rpmi media containing % fbs, cells were washed twice with pbs and then cultured in ul of serum-free rpmi media containing one of four treatments: ) saline; ) hcg ( iu); ) hcg ( iu) plus hcg-ant ( iu); or ) hcg-ant ( iu). rna was extracted from each well using trizol and reverse transcribed using sensiscript (qiagen). the relative expression of mmp- , mmp- , and metastin mrna was quantified using sybr-green based real time pcr. the expression of the housekeeping gene hprt was used to normalize expression data. results: treatment of jeg- cells with hcg-ant vs. hcg alone reduced expression of mmp- ( . ± . vs. . ± . ) and . hcg-ant reduced mmp- and mmp- expression by % and %, respectively (p< . ). treatment with hcg-ant vs. hcg increased metastin expression ( . ± . vs. . ± . ). metastin expression was increased by % following hcg-ant treatment. conclusion: treatment of the jeg- choriocarcinoma cell line with hcg-ant reversed the increased expression of mmp- and mmp- following treatment with hcg. metastin expression was increased by hcg-ant. this data suggests that hcg antagonism is capable of altering gene expression thereby inhibiting invasion in a choriocarcinoma cell line. the role of hcg-ant as an adjuvant therapy in hcg sensitive tumors offers promise. retinoids, but not progesterone, directly induce differentiation and apoptosis of endometrial cancer cells. you-hong cheng, serdar e bulun. ob/gyn, northwestern university feinberg school of medicine, chicago, il, usa. objectives: the opposing actions of estrogen and progesterone during the menstrual cycle regulate endometrial proliferation, differentiation and secretion. the unopposed action of estrogen contributes to the development of type i endometrial cancer. however, the mechanisms for progesterone protection of estrogen-induced carcinogenesis in endometrium remain unclear. methods and results: in the current study, we demonstrated that retinoids ( -cis retinoic acid (ra) or all-trans ra (atra)) significantly inhibited basal and hormone-stimulated ishikawa cell proliferation by over % using mtt assay. the same experiment indicated that estrogen had no significant effect, whereas progesterone slightly induced, on cell proliferation. cell cycle analysis indicated that atra significantly increased the g /g cell population by % and decreased s phase cells by %, suggesting that ra induces cell cycle arrest at the s phase. knock-down of rar alone or both rar and rxr significantly increased proliferating cell nuclear antigen (pcna) levels in epithelial ishikawa cells, suggesting that ra signaling via rar/rxr activation is critical for normal endometrial growth and differentiation. to determine whether retinoids are naturally secreted by the endometrial stromal cells, we cultured primary stromal cells and analyzed the culture media using hplc. we found that retinol is the predominant retinoid form secreted by stromal cells. the average concentration of retinol in the cultured media of eutopic endometrial stromal cells was approximately to ng/ml/ cells (n= ). although there was less than . ng/ml/ cells of all-trans retinal or atra in the cultured media, we did find a small peak for all-trans retinal and atra in the media using hplc analysis. furthermore, progesterone significantly increased secreted retinol levels from eutopic endometrial stromal cells, but decreased retinol levels secreted from endometriotic stromal cells. retinol is a precursor for ra that is converted to retinal and then to ra. conclusions: we demonstrated that progesterone signaling via pr induces endometrial stromal cells to secrete paracrine retionids that in turn control the phenotype of adjacent epithelial cells. conversely, this interaction is disrupted in diseased endometrial tissues, such as endometrial cancer and endometriosis. the effects of hormonal contraceptives on antimullerian hormone by obesity status. anne z steiner, frank z stanczyk, stan patel, alison edelman. ob/gyn, university of north carolina, chapel hill, nc, usa; ob/gyn, usc keck school of medicine, los angeles, ca, usa; ob/gyn, oregon health and sciences university, portland, or, usa. background: antimullerian hormone (amh) is emerging as a predictor of reproductive potential. serum levels of fsh, a commonly used measure of ovarian reserve, are suppressed with the use of oral contraceptives (ocs) thereby limiting its use. the impact of ocs and on serum amh levels in normal and obese women is unknown. objective: to examine the impact of ocs on serum amh levels by obesity status in reproductive-age women. materials and methods: ovulatory women, ages - years, of normal (< kg/m ; n = ) and obese (> kg/m ; n = ) body mass index (bmi) received a low dose oc ( mcg ethinyl estradiol/ mcg levonorgestrel) for two cycles. serum was obtained at three time points: after days of active pills (cycle , day ), at the end of the -day hormone-free interval (cycle , day ), and during the first week of active pills in cycle (cycle , day , , or ). amh levels were quantified by elisa; fsh and lh levels were determined by chemiluminescent immunoassay. amh at the three time points was compared using repeated measures anova. models were used to assess the relationship between amh and cycle day by obesity status. amh and gonadotropin levels were compared using spearman's correlation. results: amh levels did not differ by oc cycle day (p anova = . ) or by active versus placebo pill use ( . ng/ml ± . vs. . ng/ml ± . , p= . ) among normal bmi women. among obese women, amh levels differed by oc cycle day (p anova = . ), but not by use of active or placebo pill ( . ng/ml ± . vs. . ng/ml ± . , p = . ). across the cycle, cv(standard deviation/mean) averaged . %± . in the obese and . %± . in the normal bmi women ( p= . ). modeling to determine differences in amh throughout the cycles based on obesity status showed a significant interaction (p = . ) and lower amh levels in the obese group (p< . ). among both bmi groups, serum amh and fsh levels did not correlate during active pills or after days of placebo pills. conclusions: in young, normal bmi women serum amh levels do not appear to fluctuate during oc use. among obese women, amh levels are lower and fluctuate significantly. these fluctuations do not appear to mirror changes in gonadotropins and may complicate clinical interpretation of amh. background: menopausal hormone therapy (ht) is a confusing topic for many clinicians and patients. objective: to assess comprehension of basic clinical trial features among prospective participants for the keeps trial, designed to study the effects of ht initiated within years of menopause on chd markers. methods: screening materials were provided giving an overview of study purpose, duration, medications, likelihood of receiving drug vs. placebo, ht related risks and side effects. at a subsequent interview, a -item questionnaire assessed the participant's level of comprehension. a score of % was adequate to complete the informed consent process. those scoring < % were re-counseled and retested. demographic variables determining the likelihood of an initial score > % were evaluated by univariate and multivariable analyses. results: % ( / ) scored > % on initial testing. all women scored > % after re-counseling and retesting. likelihood of an initial response > % correct was unrelated to age or time since menopause. ability to correctly respond was influenced by highest educational level attained. none of women whose furthest educational level was high school scored > % on initial testing, significantly less than those with a college education (p= . ). a higher proportion of college graduates ( / ) scored > % compared to those attaining further education ( / ) (p= . ). comprehension was greatest for study purpose and duration ( / and / correct responses respectively) and least for questions related to results of the whi hormone trial breast cancer and chd. that e alone was not associated with an increased risk of chd ( %) or breast cancer ( %) was poorly understood. conclusion: comprehension of the risks and benefits of ht by potential research subjects is low despite provision of reading materials prior to the informed consent process. (supported by the montefiore medical center/albert einstein college of medicine site for the kronos longevity research institute and k -hd to ns). variation in menopausal symptoms within a sample of hispanic women: swan, the study of women's health across the nation. robin r green, alex j polotsky, aileen p mcginn, carol a derby, rachel p wildman, lhasa ray, kavitha t ram, gerson weiss, nanette f santoro. albert einstein coll of med, bronx, ny; univ of med and dent of new jersey, newark, nj. background: menopausal symptoms are experienced by over % of women. purpose: to describe symptom frequency in a sample of midlife hispanic women from different countries of origin. methods: the study of women's health across the nation (swan) recruited women at baseline who self-identified as hispanic. their baseline responses to validated questionnaires regarding common menopausal symptomatology were examined. symptoms were reported over the previous two weeks and scored on a frequency scale ranging from (not at all) to (every day). for all analyses, symptoms were dichotomized into "absent" vs. "present" variables. responses were correlated with acculturation ( -item scale: marin, hisp j behav sci : , ) and analyzed by sub-ethnicities: central/south american (c/s am), puerto rican (pr), dominican (dr), and cuban (cu). associations between symptoms and sub-ethnicity were tested by chi-square. logistic regression was used to test for associations with acculturation and being us-born. there was significant variation in several menopausal symptoms. while puerto-rican women had the highest likelihood of reporting trouble sleeping (or= . , %ci: . - . ) and headaches (or= . , %ci: . - . ), dominican women were most likely to report vaginal dryness (or= . , %ci: . - . ) acculturation and being us-born did not explain the variation between subethnicities in any of the models tested conclusion: there appear to be significant differences among hispanic women with respect to menopausal symptomatology. these differences were not readily explained by measures of acculturation. (supported by the study of women's health across the nation (swan) has grant support national institutes of health, dhhs, through the national institute on aging, from the national institute of nursing research and the nih office of research on women's health (grants nr ; ag , ag , ag , ag , ag , ag , ag , ag and cd ). purpose: to compare the relative effects of conjugated equine estrogen, raloxifene, and tamoxifen therapies on cognition among women aged years or older. participants and methods: annual modified mini mental state ( ms) examinations were used to assess global cognitive function among the , women enrolled in the two randomized placebo-controlled clinical trials of the women s health initiative memory study (whims) and women enrolled in co-star, the cognitive substudy of the nsabp's study of tamoxifen and raloxifen (star) trial who had baseline ms testing. associations between baseline cognitive risk factors common to both trials and baseline ms scores were assessed and interactions used to examine whether risk factor relationships differed between cohorts. factors for which relationships were similar were used as covariates in analyses comparing ontrial ms scores. factors for which relationships did not appear to be similar were used to stratify analyses. results: compared to placebo, each of the active therapies was associated with a small mean relative deficit in ms scores of . units or less, which was fairly consistent between women with and without prior hysterectomy. overall, relative deficits appeared to be most marked for tamoxifen (unadjusted p= . ) but were also evident for raloxifene (p= . ) and cee (p= . ). conclusions: while unmeasured differences between trials may have confounded our analysis, these findings suggest that both tamoxifen and raloxifene may adversely affect cognitive function in older women. weight gain and increased abdominal fat have been found in women after menopause and is associated with higher levels of leptin, and decreased levels of the cardioprotective adipocytokine adiponectin. at the same time, bmd characteristically decreases. in an effort to determine the evolution and correlates of these changes, we studied postmenopausal women (pm) within years of menopause (age . ± yrs) of normal weight (bmi . ± ) and compared them to weight matched (bmi . ± ) premenopausal (pre) controls (age . ± yrs.) all subjects had bmd and body composition studies by dexa and measurements of leptin, adiponectin, visfatin and retinol-binding protein (rbp .) while total fat was similar in the groups, pm had more trunk and abdominal fat ( ± ; ± g) compared to pre ( ± g p< . ; ± g p< . ) pm also had greater %trunk fat and %central abdominal fat compared to pre, p< . .serum leptin ( . ± . vs ± pg/ml) and visfatin ( . ± vs. . ± . ng/ml) were similar but adiponectin ( ± vs. . ± g/ml) and rbp ( . ± vs. . ± ng/ml) were higher, p< . in pm. while in pre, abdominal fat correlated negatively with adiponectin (p< . ) in pm only leptin correlated with various parameters of fat mass, p< . , and adiponectin did not correlate but correlated positively with age (r . , p< . .) as expected, pm had reduced bmd at the lumbar spine and hip ( . ± . vs. . ± . g/cm ; . ± . vs. . ± . g/cm , p< . ) but there was a correlation between total and trunk fat in pm and lumbar bmd (r . , . , p< . ) but not with hip bmd; or any correlations in pre. there was a correlation between leptin and lumbar bmd in pm (r . , p< . ) but not in pre. in summary, in normal weight early pm, abdominal fat is increased, but only adiponectin and rbp are altered with an increase in the former correlating with age. lumbar bmd correlated with fat mass in pm and is partially explained by increases in leptin. it is suggested that in spite of increasing abdominal fat in normal weight early pm, (which correlates with a higher lumbar bmd) david d rahn, jesus f acevedo, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. objectives: matrix metalloprotease (mmp) activity is increased in the postpartum vagina of wild type (wt) animals, and this activity is accompanied by a burst in elastic fiber synthesis. the mechanisms that precipitate these changes are unclear. the goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. methods: nonpregnant ( ) and pregnant (d ) wt mice underwent either vaginal distention with a μl balloon x min (to simulate parturition) or sham procedure. tissues were obtained at and h for immunoblot analysis, zymography, and histology. distention was also performed in young fbln -/-, fbln +/-, and wts, and prolapse was quantified for weeks. results: distention resulted in marked increases in mmp activity in nonpregnant animals (prommp , . -fold; active mmp , . -fold; prommp , . -fold; active mmp , . -fold; all p < . compared with sham) which was accompanied by fragmented and disrupted elastic fibers in the vaginal wall. abundant amounts of tropoelastin and fibulin- in the nonpregnant vagina were not increased further by distention. in pregnant animals (which normally have suppressed vaginal wall fibulin- and tropoelastin), however, distention resulted in -fold upregulation of both proteins (p< . ). distention also induced increased mmps in pregnant animals (mmp- , . -fold; prommp- , . -fold; active mmp- , . -fold; all p < . ). thirteen young fbln -/-( - wks prior to age of prolapse development), het siblings, and age-matched wts underwent serial exams after ballooning. distention induced rapid increases in perineal bulge and vaginal diameter (within d) in fbln -/mice which never recovered. conclusions: in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. the finding that distention results in accelerated pelvic organ prolapse in fbln -/animals, but not wt, indicates that distention results in loss of pelvic organ support if elastic fiber synthesis is compromised. further, the data suggest that elastic fiber synthesis is crucial for recovery of the vaginal wall from parturition/distention-induced increases in vaginal protease activity. the molecular etiology of pelvic organ prolapse (pop) is complex and not yet well understood. defects in the connective tissue, such as a decrease in extracellular matrix (ecm) protein content may predispose women to pop. our objective was to study the expression and the enzymatic activity of matrix metalloproteinases (mmps) and their tissue inhibitor (timps) in vaginal tissue of patients with advanced pop and controls. after informed consent, pre-menopausal caucasian patients affected by pop ( grade by pop-q), and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer. western immunoblot analysis was performed (patients: n= , controls: n= ) to study the protein expression of mmp- , - , timp- , - , - . gelatin zymography was used to quantify the activity of mmp- and - . immunohistochemical analysis for timp- and - was performed on pfa-fixed vaginal biopsy tissue (n= ) in each group. both patient and control vaginal biopsy samples expressed latent and active forms of mmp- , and mmp- . the protein expression of the kda active form of mmp- was significantly increased in patients with pop compared to controls (p= . ). zymography detected the enzymatic activity of the proform and active form of both mmp- and mmp- . we found a significant increase in gelatinolytic activity of both kda pro-form and kda active forms of mmp- (p= . and p< . respectively) as well as kda active form of mmp- (p< . ) in patients with pop compared to controls. timp- protein expression in vaginal tissue showed a significant reduction in pop patients compared to controls (p= . ). timp- and - immunostaining were mainly localized in the smooth muscle cells at the muscularis layer of the vaginal biopsies. in vaginal tissue of patients with pop, we have shown a decrease in timp protein expression paralleled by an increase in mmp protein expression and activity. these findings reflect an active ecm remodelling that may compromise the structural integrity of the pelvic floor leading to pop. aberrant elastin and collagen synthesis may play a role in the pathogenesis of pelvic organ prolapse (pop). the lysyl oxidase (lox) family of enzymes direct cross linking of collagen and elastin polymers, however to-date no information is available on the expression and localization of these proteins in human vaginal tissue. our objectives were to study the expression and in situ localization of lox, loxl , loxl and loxl proteins in the vaginal tissue of patients with advanced pop and asymptomatic controls. pre-menopausal caucasian patients affected by pop ( grade by pop-q) and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer and western immunoblot analysis was performed (patients: n= , controls: n= ). pfa-fixed vaginal biopsy tissues (n= for each group) were used for immunohistochemical study. vaginal biopsy samples from both patient and control groups expressed all four members of lox family proteins: lox ( kda pro-form and kda active form), loxl ( kda pro-form and kda active form), loxl ( kda) and loxl ( kda). the expression of all lox family proteins was reduced in patients with pop compared to controls; however only the pro-form of lox of making the diagnosis of endometriosis from an endometrial biopsy. objective: to evaluate the diagnostic value of examining endometrial biopsy specimens for small nerve fibers in women with pelvic pain or infertility in a double-blinded prospective comparison with laparoscopy. methodology: we undertook to compare the detection of endometrial nerve fibers with laparoscopy and peritoneal biopsy for the diagnosis of endometriosis in women (aged . years; range - years) who presented with chronic pelvic pain or infertility. small nerve fibers were detected in the functional layer of endometrium using immuno-histochemical staining with the highly specific pan-neuronal marker pgp . , using a carefully validated technique and blinded assessment of nerve fiber density. laparoscopic assessment of the presence of endometriosis and peritoneal biopsies was undertaken by three experienced gynecologic endoscopic surgeons. data from these assessments were recorded independently of endometrial findings and maintained under blinded coding until the codes were broken. results: small nerve fibers were detected in all of the women in whom endometriosis was surgically diagnosed and in none of the women in whom endometriosis was excluded at laparoscopy, giving the specificity and sensitivity of %. the density of nerve fibers in the endometriosis cases was . per mm²± . (mean ± sd). conclusions: endometrial biopsy provided a reliability of detection or exclusion of endometriosis which was equal to that of diagnostic laparoscopy carried out by experienced gynecologic laparoscopists. background: erk / are mapk intracellular signaling proteins involved in cell survival and differentiation. endometriosis requires angiogenesis for ectopic implant growth. we hypothesized that the endometriotic peritoneal environment, known to be high in estrogen (e ), vegf, and cytokines such as il- and mcp- , stimulates angiogenesis in human endometrial endothelial cells (heec) through erk signaling. methods: serial sections from normal (n= ) and ectopic (n= ) endometrium were immunostained for total-(t-) and phospho-(p-) erk / and cd (an endothelial progenitor cell marker); results were quantified by computer densitometry and grouped by menstrual phase. cultured normal heec were treated with control, e ( - m), il- , mcp- , and vegf (all ng/ml) for min, and western blotted for p-/t-erk (n= ). heec were treated with peritoneal fluid (pf; diluted : in basal media) from normal (n= ) and endometriotic (n= ) women, with or without pd erk / inhibitor ( m) for h, and cell viability was analyzed by mtt assay. statistical analysis was done with one-way anova. results: tissue staining revealed that ectopic cd + endothelial progenitor cells undergoing angiogenesis (vessel sprouting and/or angiogenic cell cord formation) exhibited the strongest levels of p-erk. heec of ectopic foci showed moderate-high p-erk staining, while surrounding mesothelial capillaries were weak for p-erk. in normal endometrium, p-erk was cycledependent, with low levels in the late secretory phase vs. other phases (p< . ). p-erk of ectopic heec showed no cycle dependence, with moderate-high levels in all phases. t-erk in all tissues was high and invariable. in cultured heec, treatment with pf from endometriotic women significantly increased heec viability after h compared to normal pf (p< . ). this effect was abrogated by erk / inhibitor. among factors known to be high in endometriotic pf, vegf increased p-erk in cultured heec in and min (p< . ), while e , il- , and mcp- had no effect. conclusions: high p-erk found specifically in sprouting endothelial progenitor cells and focal ectopic capillaries suggests that erk / is involved in endometriotic angiogenesis. the peritoneal microenvironment of endometriosis may be persistently stimulating erk-mediated endometrial angiogenesis through vegf. further investigation into erk / inhibitors may offer a novel treatment of endometriosis. the events leading to the development of post operative adhesions are unknown, though recent observations emphasize the crucial role played by the superoxide ion generated by hypoxia. exposure of normal peritoneal fibroblasts to hypoxia caused the development of the adhesion phenotype, which is characterized by increased extracellular matrix molecules and inflammatory cytokines as well as high protein nitration and low nitric oxide. superoxide dismutases (sod) are a family of metalloenzymes that eliminates superoxide by converting it to hydrogen peroxide, protecting mammalian organisms against superoxide. objective: to determine whether sod is differentially expressed between normal peritoneal and adhesion fibroblasts and whether its expression is modulated by hypoxia. methods: fibroblasts from normal peritoneal and adhesion tissues were cultured under normal ( % o ) and hypoxia ( % o ) conditions for and hours. real time rt/pcr was utilized to measure the absolute mrna levels for sod in both cell lines before and after hypoxia exposure. results: normal peritoneal fibroblasts exhibited significantly higher basal mrna levels for sod ( . pg/ grna, p< . ) as compared to adhesion fibroblasts ( . pg/ grna, p< . ). short exposure to hypoxia resulted in a significant increase in sod mrna levels to . and pg/ grna in normal and adhesion fibroblasts respectively, p< . . in contrast, long exposure to hypoxia resulted in a significant decrease in sod mrna levels to . and . pg/ grna in normal peritoneal and adhesion fibroblasts respectively, p< . . conclusion: sod mrna levels are lower in adhesion as compared to normal fibroblasts, both basally and following short and longer exposure to hypoxia. reduced sod expression creates an milieu with greater free radical levels, which leads to the development of the adhesion phenotype. enhancement of sod levels and/or function are likely to be of benefit for reduction of postoperative adhesion development. objectives: development of endometriosis requires ectopic attachment and proliferation of endometrial tissue. the invasive process required to establish endometriosis may involve matrix metalloproteinases (mmps), including mmp- . recently, we have demonstrated that statins inhibit proliferation of endometrial stroma. this study evaluated the effects of simvastatin on a nude mouse model of endometriosis and on modulation of mmp- . methods: proliferative phase human endometrial biopsies were established as organ cultures or utilized for stromal cell isolation. to establish endometriosis in the nude mouse, endometrial tissues were maintained in nm estradiol (e) for hrs and subsequently injected intraperitoneally into ovariectomized nude mice. mice were treated with e ( μg, silastic capsule implants) and simvastatin ( - mg/kg/day) by gavage for days beginning day after tissue injection. control mice received e+vehicle. subsequently, animals were sacrificed and endometrial implants were evaluated. studies of endometrial stroma involved plating the cells in the presence of e ( nm) or e+medroxyprogesterone acetate (mpa; pm) with and without simvastatin ( - μm). some cultures additionally received interleukin- (il- , ng/ml). conditioned media was subjected to western analysis for expression of mmp- . results: in vivo studies demonstrated a dose-dependent inhibitory effect of simvastatin on number and volume of endometrial implants in mice. at the highest dose of simvastatin ( mg/kg/day), the number of endometrial implants was decreased by % and the volume by % in comparison to the control group. isolated stromal cells expressed abundant levels of mmp- following treatment with e, but minimal levels in cultures additionally receiving mpa or simvastatin. although il- induced a dramatic increase in mmp- secretion from cells pretreated with e alone, treatment with either mpa or simvastatin prevented this induction. cultures receiving e+mpa+simvastatin were the most resistant to mmp- induction by il- . objective: to increase awareness of potential presentations of adenomyosis and the differential of a uterine mass. material and method: in a tertiary medical center, a patient was being evaluated for a uterine cystic mass and cyclic dysmenorrhea. the patient is a year old nulliparous woman who has been complaining of cyclic dysmenorrhea for several years. the pain starts with the onset of menses and lasts around weeks. the patient did not improve on contraceptives. the patient had prior laparoscopy and imaging studies which misdiagnosed the mass as either a leiomyoma or an adnexal hemorrhagic cyst. the patient underwent exploratory laparotomy and resection of the mass. results: on ultrasound, the mass appeared as an echodense cyst within the uterus. intraoperatively, a cm thick-walled well circumscribed uterine chocolate cyst was identified. the resection of the cyst was performed in similarly to an ovarian cystectomy. tissue examination confirmed the diagnosis of cystic adenomyosis. following surgical intervention, the patient reported significant improvement of symptoms. conclusion: this case highlights an unusual presentation and treatment of adenomyosis and cyclic dysmenorrhea. cystic adenomyosis should be on the differential of uterine cystic mass, particularly in young women with cyclic dysmenorrhea and menorrhagia. the earlier misdiagnosis probably resulted from the unfamiliarity of physicians with this condition. similar clinical presentations may occur with congenital uterine anomalies (with an obstructed uterine segment) and cystic degeneration of a leiomyoma. the incidence and pathogenesis of cystic adenomyosis are unknown. oral contraceptives may be helpful as a first line therapy. however, resection of the adenomyotic cyst appears to be more effective, particularly in women seeking fertility. objective: it is hypothesized that abnormalities within the eutopic endometrium of females with endometriosis can cause the ectopic growth of the endometrial tissue at extrauterine sites. previous studies have shown that gene expression in eutopic endometrium of women with endometriosis is markedly different from disease-free females. inflammatory cytokines and receptor-dependent kinase pathways are widely recognized as therapeutic targets for immune disorders, which is believed to be the underlying pathogenesis of endometriosis. in this study we asked whether responses of primary eutopic endometrial cell cultures are dysregulated between females with or without endometriosis. methods: a total of biopsies of endometriotic eutopic endometrium (eee) and disease-free endometrium (dfe) were obtained from proliferative phase females. the primary cell cultures established from these biopsies were treated with tnfa to induce expression of inflammatory mediators. parallel cultures were also treated with kinase inhibitors of p , mek, pi k and ikk. after a period of or hours, concentrations of il- , mcp- , and gm-csf in cell culture supernatants were analyzed by elisa. results: in eee, basal concentrations of mcp- and gm-csf were - times higher, while il- was times higher compared to dfe. as expected, tnfa treatment stimulated higher levels of cytokine secretion in dfe mimicking disease state, however, the same treatment had almost no effect on eee. kinase inhibitors were very effective in lowering the cytokine levels of dfe, however, their effect on eee were less dramatic. conclusion: eee expresses higher levels of inflammatory cytokines under basal conditions, which is in concert with the current literature. our results validate that high il- levels in endometrium are diagnostic for females with disease. the increase of gm-csf, il- and mcp- following tnfa treatment was expected in dfe however; tnfa's effect was blunted in eee, which implies that eee are already highly activated. the effect of kinase inhibitors on cytokine production from eee was unaltered relative to dfe, which implies that tnfa stimulated kinase pathways are modified even in eee. endometrial cancer is the fourth leading cause of cancer in women, and hyperplasia and adenocarcinoma are more commonly seen in women with polycystic ovary syndrome (pcos). mig- , a negative inhibitor of egf signaling, is expressed in normal secretory phase endometrium and associated with hyperplasia in mig- knockout mice. objective: we examined and compared endometrium from normal and pcos women for mig- expression and characterized its regulation using in vivo and in vitro models. methods: immunohistochemistry (ihc) and real-time pcr were performed in endometrium from normal (n= ) women and pcos (n= ) women. regulation of mig- was studied in ishikawa and ecc- endometrial cell lines, treated with sex steroids or egf. endometrial xenografts from normal and pcos women were implanted in ovxd rag -(c) immunocompromised mice, treated with e or e + p pellets to mimic a natural cycle. results: mig- protein expression was low in the functionalis of the proliferative phase and high in the secretory phase; this pattern was reversed in the basalis layer. pcos secretory phase endometrium had significantly less mig- protein and mrna than normal endometrium (p < . ). xenografts using pcos samples had paradoxically elevated expression of mig- compared to normal, and appeared unresponsive to steroid treatments. mig- expression in endometrial cell lines was regulated by egf but not by ovarian steroids, e or e + p. conclusions: mig- expression is low in proliferating endometrium and regulated by egf. risk of hyperplasia or cancer in pcos women can be explained by altered expression of mig- . reduced expression mig- provides insight into endometrial function and may lead to better treatment options for disorders of endometrial proliferation including endometriosis, adenomyosis, endometrial hyperplasia and cancer. supported in part by nih-u -hd (sly), u hd (lcg) and u hd (fjd). could androgens influence human luteal cells function? anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. in pcos patients reproductive dysfunctions are frequently observed even if ovulation occurs. an impaired luteal function could partially explain this subfertility, since luteal steroidogenesis deficiency and premature luteal degeneration have been described in pcos patients. based on the frequent observation of high plasmatic levels of androgens in pcos, we investigated whether hyperandrogenism could negatively affect luteal function.to this aim, we tested the in vitro effects of androgens on progesterone (p) and on vascular endothelial growth factor (vegf) production by human luteal cells. indeed, vegf is essential for normal luteal development and function being an important regulator of angiogenesis and vascular permeability. since prostaglandins (pgs) play a central role in modulating luteal function, the influence of androgens on pge and pgf a secretion was also investigated. in order to investigate whether testosterone and androstenedione act directly or after local aromatisation, we tested the in vitro effects of estriol, estrone and --estradiol on p, vegf, pge and pgf secretion by human luteal cells. moreover, we tested the effects of testosterone and androstenedione in presence of exemestane -an aromatase inactivator. highly purified human luteal cells were cultured for h with medium alone (control) or in presence of increasing concentrations of testosterone or dihydrotestosterone or androstenedione (from - to - m) or in presence of increasing concentrations of estriol, estrone and --estradiol (from . to ng/ml). moreover, testosterone and androstenedione - m were tested in presence of exemestane (from to nm). in the culture medium vegf, p, pge and pgf secretion was assayed by commercially available elisa kits. in order to evaluate the influence of androgens and estrogens on vegf mrna expression on luteal cells real-time rt-pcr has been performed. our results demonstrated that testosterone, androstenedione and dihydrotestosterone were all able to significantly reduce vegf secretion in human luteal cells, while no effect was seen on vegf mrna expression. androgens were also able to significantly decrease p and pgf secretion, while an increase was observed on pge production. moreover our preliminary results demonstrated that in human isolated luteal cells estriol, estrone and -estradiol at all tested doses are able to mimic androgens effects on p and pge production. on the contrary estrogens were able to increased vegf release. estrogens seemed to have no effect on pgf  released. data regarding exemestane inhibition of testosterone and androstenedione are still in progress. increased levels of androgens were able to decrease luteal vegf, p and pgf release and might be involved in pcos-luteal phase defect. nevertheless, the observed effects could probably be attributed -at least in part -to estrogens locally produced via the aromatase enzyme, rather than be directly mediated by testosterone and androstenedione. the in vitro effect of proghrelin-derived peptides on purified human luteal cells. anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. ghrelin, well known mediator of neuroendocrine effects, has been demonstrated to have a role as signal for energy insufficiency. several evidences suggested that ghrelin might also operate as regulator of reproductive function. indeed, we recently demonstrated that both p and vegf released were significantly decreased by ghrelin in isolated human luteal cells. moreover ghrelin was also able to reduce prostaglandin (pg) e and increase pgf luteal cells release. in the present work we investigated whether two ghrelin-related peptides derived by the same ghrelin precursor (unacylated ghrelin and obestatin) might affect human isolated luteal cells function. conventionally regarded as an inert form of ghrelin, unacylated ghrelin has been recently proven as biologically active in specific cellular contexts. obestatin has been suggested to directly control porcine ovarian cell functions. in these assumptions, we investigated whether unacylated ghrelin and obestatin could directly affect luteal progesterone (p) release. moreover, since vascular endothelial growth factor (vegf) is known to play a critical role in luteal development and function, the influence of unacylated ghrelin and obestatin on luteal vegf, pgf and pge production was also analysed. highly purified human luteal cells were incubated for h with medium alone (control) or with hcg ( ng/ml) or with cocl ( μm) or in presence of increasing concentrations of unacylated ghrelin ( - nm) and obestatin ( - nm). in the culture medium vegf, pgf , pge and p secretion was assayed by commercially available elisa kits. moreover real-time rt-pcr has been performed in order to evaluate whether unacylated ghrelin and obestatin could affect vegf mrna in human luteal cells. our preliminary results demonstrated that either unacylated ghrelin or obestatin are able to negatively affect luteal steroidogenesis. moreover, both peptides seemed to increase the release of two luteotropic factors -vegf and pge and to reduce pgf release -a luteolytic prostanoid -from isolated human luteal cells. finally data regarding the expression of vegf mrna are still in progress. our results suggest that unacylated ghrelin and obestatin -two ghrelin-related peptides -could play a role in regulating luteal function affecting both luteal steroidogenesis and luteotropic/luteolytic imbalance. the mechanisms responsible for labor progression have yet to be fully elucidated. in a previous study, over-expression of small conductance calcium-activated k + channel isoform (sk ) in transgenic mice compromised parturition, suggesting a role for sk channels in this process. based on these findings, we hypothesized that sk channel expression is reduced late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. we investigated the effects of sk channel expression on gestation and parturition by comparing transgenic mice over-expressing mice sk (sk t/t ) mice to wild-type (wt). in wt mice, sk transcript and protein are significantly reduced during pregnancy. the force produced by uterine strips from sk t/t mice on the day of delivery was significantly less than wt or sk knockout control (sk dox ), and the contractile force reached wt levels by application of the sk channel inhibitor, apamin. moreover, lipopolysaccharide and ru , which induce pre-term labor in wt mice, failed to result in completion of delivery in sk t/t mice. thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when sk channel activity is in excess. the mechanism(s) down-regulating this channel in the uterus during pregnancy is unknown. the sk gene promoter region contains two specificity protein (sp) binding sites; ) sp , is a transcription factor known to enhance the transcription of genes in response to estrogen, and ) sp , which competes for the same binding motif as sp , reduces gene expression. sp protein expression in mice uteri dramatically decreases in late pregnancy, while sp protein level remains consistent. our data indicate that sk channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice. the changes in sk channel expression may be transcriptionally regulated by sp and sp .