REVIEW Open Access Adult zebrafish as a model organism for behavioural genetics William Norton*, Laure Bally-Cuif* Abstract Recent research has demonstrated the suitability of adult zebrafish to model some aspects of complex behaviour. Studies of reward behaviour, learning and memory, aggression, anxiety and sleep strongly suggest that conserved regulatory processes underlie behaviour in zebrafish and mammals. The isolation and molecular analysis of zebra- fish behavioural mutants is now starting, allowing the identification of novel behavioural control genes. As a result of this, studies of adult zebrafish are now helping to uncover the genetic pathways and neural circuits that control vertebrate behaviour. Review Henry David Thoreau wrote “Many men go fishing all of their lives, without knowing it is not fish they are after”. Thus, one of the intrinsic difficulties of studying behaviour is to understand the underlying motivation of complex behaviours. Most behavioural traits are multi- genic and display environmental interactions, further compounding the difficulty of analysing them. However, recent studies using rats, mice, zebrafish, nematodes and fruit flies have begun to identify the genetic toolbox that controls behaviour. The general suitability of zebrafish as a model organ- ism, as well as its use in the genetic and neuroanatomi- cal analysis of larval behaviour has been comprehensively described elsewhere [1,2]. Although more difficult to manipulate than larvae, adult zebrafish display a full repertoire of mature behaviours making their characterisation particularly enticing. Zebrafish (Danio rerio) are a typical cyprinid (carp family) school- ing fish. In contrast to other laboratory behavioural models, zebrafish are naturally social animals that show preference for the presence of conspecifics [3,4]. Zebra- fish are therefore an excellent model to probe the genet- ics of social behaviour. In addition, zebrafish are diurnal allowing behaviour to be measured during their natural day time. Finally, it is crucial to investigate whether complex behaviours such as reward, learning and social behaviour are conserved throughout the animal king- dom. Thus, comparative studies of many model organ- isms, including zebrafish, are necessary to determine general principles of behavioural control. Several groups have already developed protocols to measure aggression, alarm reaction, antipredatory behaviour, anxiety, loco- motion, learning and memory, sleep, reward and social behaviour (see Table 1 and references therein). In this review we consider the brain areas and neurotransmitter systems that have been linked to the control of beha- vioural in adult zebrafish. We also describe the proto- cols and tools that have been developed for zebrafish behavioural studies. Contributions of zebrafish to behavioural genetics: Reward and Learning Reward behaviour Perhaps the most prominent area in which the adult zebrafish has contributed to behavioural genetics is reward. Reward behaviour provides animals with an instinctive drive to search for resources and to repro- duce. However, the brain’s reward pathway can also be hijacked by drugs of abuse such as cocaine, ampheta- mine or opioids. Reward behaviour may thus constitute the first step towards addiction. Reward can been mea- sured in zebrafish by using the conditioned place prefer- ence (CPP) test, which pairs a primary cue (e.g. a drug) with a secondary stimulus such as a coloured aquarium compartment. Drug dependency can also be evaluated by measuring the persistence of CPP following a period of abstinence. * Correspondence: norton@inaf.cnrs-gif.fr; bally-cuif@inaf.cnrs-gif.fr Zebrafish Neurogenetics group, Laboratory of Neurobiology & Development (NED), CNRS, UPR 3294, Institute of Neurobiology Albert Fessard, Avenue de la Terrasse, 91198 cedex, Gif-sur-Yvette, France Norton and Bally-Cuif BMC Neuroscience 2010, 11:90 http://www.biomedcentral.com/1471-2202/11/90 © 2010 Norton and Bally-Cuif; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:norton@inaf.cnrs-gif.fr mailto:bally-cuif@inaf.cnrs-gif.fr http://creativecommons.org/licenses/by/2.0 In line with studies of other animals (e.g. [5]), stimuli that have been shown to be rewarding for adult fish include ethanol [6,7], cocaine [8], amphetamine [9], opi- ates [10], nicotine [7], food [10] and the presence of conspecifics [11]. The major neurotransmitter associated with reward behaviour is Dopamine (DA). Increases of DAergic signalling from the ventral tegmental area to the nucleus accumbens (nAC) motivates mammals to repeat stimulus application. In zebrafish, this key DAer- gic pathway is most likely comprised of projections from the diencephalic posterior tuberculum to the ven- tral telencephalon (subpallium, (Vv and Vd), see [12]). Several other neurotransmitters have also been impli- cated in reward behaviour. Heterozygous mutant zebra- fish lacking one copy of the acetylcholinesterase (ache) gene have enhanced acetylcholine levels in the brain due to decreased breakdown of the neurotransmitter. The increase of acetylcholine in the brain of ache mutants causes a decrease in amphetamine-induced CPP [13]. Mammalian reward pathways also include raphe 5-HTergic neurons [14] as well as a number of inhibi- tory influences including projections from the habenula. The zebrafish ventral habenula appears to be homolo- gous to the mammalian lateral habenula in both gene Table 1 Protocols to measure behaviour in adult zebrafish Stage Behaviour Paradigm Reference Adult Aggression Live observation of two fish [29,37,39,40,45] Adult Aggression Mirror image test [6] Adult Aggression Pigment response [6] Adult Aggression Startle reaction [85] Adult Alarm reaction Response to alarm substance [26,52,86] Adult Antipredation Predator stimulation [87] Adult Anxiety Exit latency test [51] Adult Anxiety Group preference [6] Adult Anxiety Light/Dark preference [48,88] Adult Anxiety Locomotory activity [6] Adult Anxiety Place preference / Thigmotaxis [13,34,49,51] Adult Anxiety Tank diving test [50,53,55] Adult Anxiety Time in enriched T-maze chamber [89] Adult Audition Response to startling noise [64] Adult Courtship Observation of courtship postures [82] Adult Lateralisation Interaction with object [41,77] Adult Locomotion Mean velocity [90] Adult Locomotion Number of lines crossed [36,81,87] Adult Locomotion Total distance moved / Videotracking [80,81,90] Adult Locomotion Turning angle [90] Adult Mate choice Video-stimulus technique [91] Adult Learning / memory Active avoidance conditioning [27,28,92,93] Adult Learning / memory Delayed spatial alternation [35] Adult Learning / memory Learned alarm reactions [86] Adult Learning / memory Spatial alternation, learning and memory [31,32,54,94] Adult Learning / memory T-maze [8,13,33,89] Adult Learning / memory Visual discrimination learning [29] Adult Olfaction Response to amino acids [75] Adult Reward Conditioned place preference [6-9,13] Adult Reward Presence of Conspecific [11] Adult Sleep Locomotor inhibition [61] Adult Sleep Monitoring sleep postures [57,58,63] Adult Sleep Pigment response [61] Adult Social preference Area occupied [85] Adult Social preference Group preference [6] Adult Social preference Nearest neighbour distance [85] Adult Social preference Shoaling [3,78,87] Adult Vision Optokinetic response [76] Norton and Bally-Cuif BMC Neuroscience 2010, 11:90 http://www.biomedcentral.com/1471-2202/11/90 Page 2 of 11 expression and innervation of the raphe [15]. The recent identification of selective molecular markers for both structures [16,17] will make genetic manipulation of the reward pathway possible. Such a targeted approach will allow functional interrogation of the reward circuitry in zebrafish and may highlight both similarities and differ- ences in the mechanisms controlling monoaminergic behaviours in vertebrates. There have been several screens for zebrafish mutant families with altered reward behaviour. Darland and Dowling isolated three families of mutants which were not responsive to cocaine application, although the affected genes were not reported [8]. Other groups have used microarrays to identify addiction related genes. Brennan and colleagues demonstrated a robust change in place preference (PP) following nicotine or ethanol treatment [7]. This PP was also maintained fol- lowing a period of abstinence or when paired with an adverse stimulus (3 seconds removal from the tank water) suggesting that drug dependency had occurred. Microarray analysis comparing the brains of both trea- ted and untreated fish identified 1362 genes that were significantly changed following drug application, including 153 that were responsive to both nicotine and ethanol. Many of these genes are also involved in reward behaviour in other species, by either altering dopaminergic or glutamatergic signalling or modulat- ing synaptic plasticity [7]. In addition, this study also revealed a number of novel genes that were changed upon drug administration, including those coding for Calcineurin B and the Hypocretin receptor. Bally-Cuif and colleagues conducted a screen for mutants that were insensitive to amphetamine application [13]. One of these mutants, no addiction (nad), was used to iden- tify genes that were transcriptionally modified by amphetamine in wild-type fish and were differentially over- or under-regulated in nad. Importantly, gene expression was unmodified in nad mutants in the absence of the drug [9]. This strategy permitted the unbiased recovery of 139 genes linked to amphetamine triggered CPP. A large proportion these genes were developmentally active transcription factors. These include Dlx1a, Emx1a, Lhx8, Sox9 and Tbr1, proteins that are implicated in the control of neurogenesis in the vertebrate embryo and show persistent expression in the adult-neurogenic regions of the mammalian and fish brain [9]. A recent study of rats has also demon- strated altered cocaine (but not sucrose) mediated reward behaviour following a reduction of hippocam- pal neurogenesis [18]. This constitutes an exciting new development in the field of reward behaviour; neuro- genesis-induced plasticity may account for some of the learning aspects of reward and the long-lasting changes in the brain associated with addiction. A comparable approach has led to the identification of too few (tof) a mutant that fails to change place prefer- ence following morphine treatment but not food appli- cation [10]. tof encodes a forebrain specific zinc finger protein, Fezl [19], which establishes neurog1-expressing DA progenitor domains in the basal forebrain [20]. Loss of fezl leads to a reduction of dopaminergic and seroto- nergic neurons in specific nuclei of the forebrain (dien- cephalon and hypothalamus; [21]), defects that are maintained into adulthood. Dissociation between the preference for a natural reward (food) and a drug (mor- phine) has previously been observed in dopamine D2 receptor knock-out mice [22] but is not understood at the molecular or neurological level. Since both mor- phine and food rewards are dependent on opioid recep- tor activity in zebrafish [10], the separable reward behaviour seen in tof suggests that distinct neural sys- tems act downstream of opioid signalling to mediate the response to morphine and food. Alternatively, the rewarding aspects of food and drug treatment may be mediated by different subsets of dopaminergic nuclei in the forebrain. Thus, together with the study of hippo- campal irradiated rats [18], tof presents an excellent opportunity to dissect the neural basis of discrimination between rewarding substances in the brain. Learning and memory Studies of mammals have shown that learning and memory can be controlled by several brain circuits, each of which is neuroanatomically distinct. These include spatial learning (hippocampus), implicit learning (such as simple motor reflexes; cerebellum) and avoidance learning (amygdala). Although the neural basis of learn- ing is not well understood in zebrafish, studies of the closely related goldfish (Cassius auratus) hint at brain areas which could be involved. Focal ablations of the goldfish brain have identified the lateral pallium (Dl, equivalent to the hippocampus), medial pallium (Dm, equivalent to the amygdala) and cerebellum [23-25] as playing key roles in learning. Several paradigms have already been developed to measure learning and memory in zebrafish. Associative learning can be measured by pairing two previously unrelated stimuli such as colour, reward or aversion. For example, Suboski and colleagues paired a neutral stimu- lus (morpholine) with the aversive effects of alarm sub- stance [26]. Avoidance learning can be assessed by using a shuttle box; fish are quickly able to associate a condi- tioned stimulus (e.g light [27] or colour [28]) with an unconditioned stimulus (such as a mild electric shock). Spatial learning can be measured using either a T-maze [8,11,29,30] or a shuttle box [31,32]. Fish must learn to collect a reward by either navigating a maze correctly or alternating the side of the tank visited. These beha- vioural tests are high-throughput, making them suitable Norton and Bally-Cuif BMC Neuroscience 2010, 11:90 http://www.biomedcentral.com/1471-2202/11/90 Page 3 of 11 for screens for novel genes controlling learning and memory. Pharmacological studies have validated adult zebrafish as a model for learning and memory, making it a very promising area for future research. Several evolutionarily conserved neurotransmitter systems have been impli- cated in learning and memory. ache mutants, with increased acetylcholine levels in the brain, learn to find a food reward faster in a T-maze [33] thereby linking cholinergic signalling to learning. Fish exposed to mod- erate levels of nicotine perform better in a delayed spa- tial alteration task, a type of avoidance learning test. Nicotine acts via nicotinic acetylcholine receptors (nACHRs), thus further underscoring the importance of cholinergic signalling [34,35]. Treatment of fish with a Histidine decarboxylase inhibitor, alphafluoromethylhis- tidine (a-FMH), reduces both levels of histamine and the number of histaminergic fibres in the brain. a-FMH treated animals display defects in long-term memory formation but not initial learning [30]. Finally, NMDA antagonists have also been shown to impair memory formation in zebrafish [27,28]. NMDA receptors are found abundantly in the telencephalon, which contains the teleostean (bony fish) equivalent of the hippocampus and amygdala [36]. The neurotransmitters discussed above have also been connected to learning in other species, suggesting that work in zebrafish may give insight into conserved learning mechanisms. Therefore, adult zebrafish constitute a particularly promising model for research into learning and memory. Emerging fields for genetic analysis: Aggression and Anxiety Aggression Aggression is a complex suite of behaviours serving a number of adaptive purposes. Fish use aggression to protect offspring, monopolise resources such as food, territory and mates and establish dominance hierarchies. Aggression can be measured in the laboratory by recording the interaction of two free-swimming fish or by using mirror induced stimulation (MIS)[6,37]. Fish are unable to recognise their own image and so attack as if an intruder is present [38]. Furthermore, MIS pro- vides immediate feedback to the fish’s activity and avoids damaging the subjects [37]. Zebrafish display characteristic agonistic postures including erection of the dorsal, caudal, pectoral and anal fins coupled to bit- ing, thrashing of the tail and short bouts of fast swim- ming directed against the mirror [6]. A positive correlation between aggression and boldness has also been reported [39]. Aggression is a very plastic beha- viour. Both habitat complexity and rearing conditions can influence the number of interactions [37,40]. Furthermore, different wild-type strains show varying aggression levels suggesting a genetic component to its control. Finally, aggressive behaviour also shows laterali- sation, with adult fish predominantly using the right eye to view predators [41]. Studies in other species have identified 5-HT as the major neurotransmitter controlling aggression. Animals with high levels of 5-HT tend to be timid, whereas those with lower levels are more impulsive and aggres- sive (e.g. [42]). Other neurotransmitters, including GABA, glutamate and nitric oxide as well as the hor- mones vasopressin and testosterone have also been implicated in agonistic behaviour [43]. However, the role of these neurotransmitters has not been explicitly tested in zebrafish. In zebrafish, agonistic behaviour can be modified by exposure to pharmacological com- pounds including ethanol [6] and 17alpha-ethinylestra- diol (a synthetic oestrogen; [44]). The brain areas mediating aggression in fish are not well characterised. Arginine vasotocin-expressing cells of the magnocellu- lar preoptic area change size depending on the domi- nance status of fish. This suggests involvement of the preoptic area in control of social hierarchy and the agonistic behaviour used to establish it [45]. Studies of other fish species have identified additional brain terri- tories that influence aggression. For example, the neural activity maker cfos is expressed in the dience- phalon, thalamus and hypothalamus and a few nuclei in the pons and medulla oblongata of the mudskipper (Periophthalmus cantonensis) following an aggressive episode [46]. Finally, electrical stimulation of the blue- gill (Lepomis macrochirus) implicates the inferior hypothalamus in aggression control [47]. The MIS protocol is simple to establish and perform. Coupled to computer-aided automation, it can be adapted for high-throughput screening studies, thus pro- viding a golden opportunity to uncover novel genes implicated in aggression control. Anxiety Anxiety is a state of constant fear or restlessness caused by anticipation of a real or imagined future event. Multi- ple anxiety tests have been established in fish, although it is not always clear whether fear or anxiety is being measured, or indeed whether the different states even exist [48]. Protocols to measure anxiety tend to assess one of two variables. The first set of protocols record the reaction of adult fish to novel environments, such as the amount of time spent at the edge of a tank [30,49], at the bottom of a novel tank [34,50] or on the dark side of a light/dark tank [28,51]. The second approach analyses locomotory patterns: freezing, long-lasting increases in basal locomotory activity [6,48,49] and tigh- tening of a fish’s shoal [52] have all been reported to be reliable measures of anxiety. The expression and level of anxiety are wild-type strain dependent [49,50]. Norton and Bally-Cuif BMC Neuroscience 2010, 11:90 http://www.biomedcentral.com/1471-2202/11/90 Page 4 of 11 For example, AB wild-types manifest anxiety as a hyper- active swimming response [49]. Similar to other behaviours, anxiety protocols have been validated using pharmacological compounds devel- oped for human patients. Application of caffeine [50,53], pentylenetetrazole [53], alarm substance [50,52], the benzodiazepine partial inverse agonist FG-7142 [49] and withdrawal of cocaine [49] have all been shown to be anxiogenic. Conversely, many anxiolytic substances have been characterised including nicotine [54], diazepam [49,55], the Htr1a (5-HT receptor) partial agonist bus- pirone [55], fluoxetine hydrochloride and ethanol [50]. Finally, a link between anxiety levels and the major zeb- rafish stress hormone cortisol has also been demon- strated [50]. The ease of applying drugs and robust behavioural assays (see [49,50]) make zebrafish an ideal model to study anxiety and related behaviours. Sleep Although sleep is a widespread phenomenon, its beha- vioural and physiological function is not well under- stood. Sleep is characterised by periods of behavioural quietness, species-specific body postures, an increased arousal threshold and a quick return to wakefulness [56]. Furthermore, sleep-deprived animals also show homeostatic rebound, increasing the amount of time needed to sleep following deprivation. The timing of sleep also shows circadian rhythmicity. Several studies have identified sleep-like behaviour in zebrafish. During the night, adult fish have periods of two to four minutes of inactivity in which the fish floats horizontally and makes small pectoral fin movements. There is also a simultaneous reduction of mouth and operculum move- ments suggesting lower respiratory levels [57]. Sleep rebound has been demonstrated in zebrafish indicating homeostatic regulation; disrupting the normal night time routine (using light, vibration, electric shock or forced movement) deprives fish of rest and causes a subsequent increase in sleep duration [57,58]. Finally, zebrafish also show circadian rhythmicity, with higher activity levels in the day [57]. Studies in other species have identified several signifi- cant sleep-related neurotransmitters: Increases of dopa- mine levels in the brain reduces the amount of time asleep [59], whereas GABA signalling promotes sleep and GABAA receptor agonists are used to treat insom- nia [60]. Although these pathways have not been directly examined in zebrafish, treatment with diazepam, pento- barbital [57], alpha2 adrenoceptor agonists [61], and his- tamine H1 antagonists [62] have all been shown to increase sleep, thus implicating GABA, acetylcholine and histamine in its control. Several studies have also demonstrated a conserved role for hypocretin/orexin (HCRT) in sleep-wake regulation. Zebrafish contain a single HCRT receptor gene (hcrtr), which is expressed in a small number of glutamatergic neurons of the adult hypothalamus [58,63]. Loss of hcrtr function causes sleep fragmentation but not cataplexy or decreased wake bout length, suggesting that HCRT may function to consolidate sleep in fish [58]. HCRT acts by stimulating the endogenous melatonin sleep-promoting system found in the pineal gland [63]. Taken together, studies of zebrafish have confirmed that the control of sleep appears to be evolutionarily conserved. Although zebra- fish sleep research is still in its infancy, the high throughput nature of the set-ups used to measure sleep demonstrates that zebrafish are an ideal model in which to conduct screens for novel hypnotic mutants. Practical considerations: strain differences, screen design and duplicated genes Strain differences in wild-type fish The examples discussed in this review highlight the suit- ability of adult zebrafish for studies of complex verte- brate behaviours. However, there are several considerations that need to be taken into account before initiating behavioural work. For example, care must be taken to dissect the influence of neurotransmitter signal- ling pathways and the specificity of drugs used to modu- late them. Finally, another important consideration when designing behavioural studies is the background strain of the fish used. Strain differences in adult beha- viour have already been reported [13,49,50,64]. Thus, in order to avoid some of the known difficulties in repro- ducing behavioural work, all behavioural studies should be carried out on well defined laboratory strains. Although no inbred strains exist, the AB line, available from the ZIRC stock centre is an excellent choice for a reference strain. The line has been maintained in the laboratory for more than 70 generations and is freely available to the zebrafish community. Screen design Genes do not directly control behaviour. Rather, genes influence behavioural output by either modulating neural circuit formation (neural specification, differentiation and connectivity) or function (e.g. neurotransmitter release or reuptake). High throughput forward genetic screening has long been one of the goals of zebrafish research, and in this regard the nascent behavioural field is no different. However, behavioural phenotyping is subject to large variability between animals. This can make it difficult to phenotype mutants with certainty, and so complicates positional cloning of the mutations. Furthermore, careful consideration needs to be given to the choice of mutagen. The most commonly used mutagen N-ethyl-N-nitro- sourea (ENU; Fig. 1) efficiently induces intragenic point mutations in the germline [65], but the subsequent clon- ing steps needed to recover the mutagenised gene are Norton and Bally-Cuif BMC Neuroscience 2010, 11:90 http://www.biomedcentral.com/1471-2202/11/90 Page 5 of 11 laborious. As an alternative to ENU treatment, insertional mutagenesis looks particularly promising (Fig 2). Although insertional mutations occur at a lower fre- quency, isolation of the genetic lesion is much simpler [66,67]. The mutagenic cassette may also be coupled to a fluorescent reporter line thus highlighting the expression profile of the mutated gene. This technique will allow fas- ter and more reliable identification of animals carrying the same insertion and so will facilitate mapping. This has recently been powerfully demonstrated in juvenile fish by using a reporter-tagged insertional mutagenesis strategy to clone two nicotine-response mutants [68]. Finally, the usefulness of zebrafish is not limited to screening paradigms. The advent of TILLING [69] and zinc-finger nuclease technology [70] has opened the door to targeted modification of the zebrafish genome, thus allowing the behavioural function of known genes to be probed. Gene duplication and redundancy in zebrafish In common with all ray-finned fishes (actinopterygii), zebrafish underwent a third whole genome duplication Figure 1 Three-generation breeding scheme for chemically-induced mutant fish. Male fish are mutagenised and then crossed to wild-type females to produce an F1 generation. An F2 generation is made by in-crossing F1 siblings. Dominant behavioural mutants can be identified in this F2 generation (black fish). For recessive mutant carriers, a second in-cross is performed and the progeny screened for behavioural alterations (red fish) - if the inheritance in Mendelian then one quarter of the progeny should show the behavioural defect. Norton and Bally-Cuif BMC Neuroscience 2010, 11:90 http://www.biomedcentral.com/1471-2202/11/90 Page 6 of 11 around 350 million years ago and often have two copies of genes found in other vertebrates [71]. The most likely fate of a duplicate gene is loss of function. However, in some cases both copies can be retained and subfunctio- nalisation (splitting of the ancestral function between the two new genes) or neofunctionalisation (acquisition of a new function through mutation) can occur [72]. Redundancy can make analysis of a gene’s function more difficult by masking mutant phenotypes. However, redundancy can also be useful, exposing late functions of genes that cause embryonic defects in other animals. For example, zebrafish lacking activity of one copy of fibroblast growth factor 1 (fgfr1a) have a surprising lack of developmental phenotype compared to mice and medaka deficient in the gene [73,74]. Rather, adult fgfr1a mutant fish exhibit several behavioural alterations, including increases in aggression, boldness and explora- tion (W Norton, personal observation). Conclusion Although anecdotally fish are thought to have poor memories and display few complex behaviours, numer- ous studies have disproved such beliefs. In this review we have demonstrated ways in which studies of adult zebrafish have contributed to our understanding of the genetic basis of behaviour. We have described set-ups to measure behaviour (e.g. Table 1) and some of the phar- macological treatments that have already been employed Figure 2 Breeding scheme for the production of insertional mutants in zebrafish. Single-cell to blastula-stage embryos are injected with a mutagen and grown to adulthood. The mature fish are then inbred twice to produce first an F1 and then an F2 generation. Mutants with behavioural phenotypes (black and blue spotted fish) can be identified by in-crossing the F3 fish. The number- and position of insertions can be monitored by western blot and PCR analysis. Norton and Bally-Cuif BMC Neuroscience 2010, 11:90 http://www.biomedcentral.com/1471-2202/11/90 Page 7 of 11 in zebrafish (Table 2). However, fish also manifest other behaviours, the discussion of which is unfortunately beyond the scope of this review. These behaviours include olfaction [75], vision [76], behavioural lateralisa- tion [77], shoaling [3,78,79], locomotion [80,81] and reproductive behaviour [82]. Finally, studies of adult fish are also beginning to give clues about the initiation of locomotion, an assay that might be modified to probe the motivation to move. In the adult spinal cord, appli- cation of 5-HT modifies the cyclical pattern of locomo- tory activity by increasing mid-cycle inhibition and reducing the onset of the next cycle, so reducing the initiation of locomotion [83]. Larval zebrafish are also useful for studying simple behaviours, and protocols have been established to measure locomotion and visuomotor behaviours such as prey capture [1]. Coupled to the transgenic lines avail- able and the emergence of optogenetic technology (e.g. [84]), larvae may allow the dissection of behavioural cir- cuits at the cellular level in intact living fish. Moreover, in an elegant recent study by Engert and colleagues, neural circuit activity has been analysed at the single- cell level by recording bioluminescence in free-swim- ming larvae [85]. In summary, zebrafish have many attributes that make them an ideal model organism for the study of beha- vioural genetics. Although to date there have been rela- tively few studies of adult zebrafish behaviour, the ease of carrying out pharmacological studies coupled to the ever increasing number of available genetic tools suggest Table 2 Pharmacological treatments with known behavioural effects on adult zebrafish Behaviour Modulating agent Function / Activity Effect Reference Aggression Ethanol GABA-A receptor modulator Increases aggression [85] Aggression 17a-ethinylestradiol Synthetic oestrogen Reduces aggression [44] Antipredation Ethanol GABA-A receptor modulator Impaired by high doses [6] Anxiety Diazepam Benzodiazepine Reduces anxiety [49,86] Anxiety FG-7142 Benzodiazepine inv. agonist Increases anxiety [49] Anxiety Pentylenetetrazole GABA antagonist Increases anxiety [53] Anxiety Ethanol GABA-A receptor modulator Reduces anxiety [50,53] Anxiety Buspirone Htr1A partial agonist Reduces anxiety [55] Anxiety Alarm substance Hypoxanthine-3N-oxide Increases anxiety [50,52] Anxiety Nicotine NachR agonist Reduces anxiety [34] Anxiety Methyllycaconitine Nicotinic antagonist Anxiolytic [55] Anxiety Dihydro-b-erythroidine Nicotinic antagonist Anxiolytic [55] Anxiety Mecamylamine Nicotinic antagonist Anxiolytic [34] Anxiety Morphine Opiate Reduces anxiety [53] Anxiety Cocaine (withdrawal) Psychostimulant Increases anxiety [49] Anxiety Fluoxetine 5-HT reuptake inhibitor Reduces anxiety [50,53] Anxiety Caffeine Xanthine alkaloid Increases anxiety [50,53] Group preference Ethanol GABA-A receptor modulator Reduced at high conc. [6] Learning a FMH HDAC inhibitor Impairs long term memory [30] Learning Nicotine NachR agonist Improves learning [34,35] Learning MK-801 NMDA antagonist Impairs memory [27,28] Learning L-NAME NO synthase inhibitor Impairs memory retention [27] Light/Dark pref Ethanol GABA-A receptor modulator Decreased at high conc. [6] Locomotion Ethanol GABA-A receptor modulator Reduced at high conc. [6] Reward Acetylcholine Cholinergic agonist Non-rewarding [13] Reward Ethanol GABA-A receptor modulator Rewarding [7] Reward Nicotine NachR agonist Rewarding [7] Reward Food Nourishment Rewarding [10] Reward Morphine Opiate Rewarding [10] Reward Morphine Opiate Rewarding [10] Reward Cocaine Psychostimulant Rewarding [8] Reward Amphetamine Psychostimulant Rewarding [9] Sleep Dexmedetomidine alpha2 adrenoceptor agonist Sedative [61] Sleep Pentobarbital Barbiturate Hypnotic [57] Sleep Diazepam Benzodiazepine Hypnotic [57] Norton and Bally-Cuif BMC Neuroscience 2010, 11:90 http://www.biomedcentral.com/1471-2202/11/90 Page 8 of 11 that zebrafish are about to enter the limelight. Finally, their small size and cheap maintenance costs suggest that zebrafish are ideally suited for large-scale beha- vioural screens. We look forwards to the next steps in the establishment of this fascinating field. Acknowledgements We thank Christina Lillesaar, Ina Rothenaigner, Marion Coolen and Philippe Vernier for critically reading an early version of this manuscript, and all members of the Bally-Cuif laboratory for their enthusiastic discussions about zebrafish development and behaviour. Authors’ contributions WN conceived the review and wrote the manuscript. LB-C suggested subject areas to include in the review and worked on the manuscript. All authors read and approved the final manuscript. Received: 27 July 2010 Accepted: 2 August 2010 Published: 2 August 2010 References 1. Fero K, Yokogawa T, Burgess HA: The behavioural repertoire of larval zebrafish. Zebrafish Models in Neurobehavioral Research Cambridge University PressKalueff AV, Cachat JM 2010. 2. Westerfield M: The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). Univ of Oregon Press, Eugene, 4 2000. 3. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Norton and Bally-Cuif BMC Neuroscience 2010, 11:90 http://www.biomedcentral.com/1471-2202/11/90 Page 11 of 11 http://www.ncbi.nlm.nih.gov/pubmed/14690532?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/14690532?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/14583542?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/14583542?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/10235674?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/10235674?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/10235674?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/10861564?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/10861564?dopt=Abstract Abstract Review Contributions of zebrafish to behavioural genetics: Reward and Learning Reward behaviour Learning and memory Emerging fields for genetic analysis: Aggression and Anxiety Aggression Anxiety Sleep Practical considerations: strain differences, screen design and duplicated genes Strain differences in wild-type fish Screen design Gene duplication and redundancy in zebrafish Conclusion Acknowledgements Authors' contributions References