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 reproduce. However, the brain's reward pathway can also be hijacked by drugs of abuse such as cocaine, amphetamine or opioids. Reward behaviour may thus constitute the first step towards addiction. Reward can been measured in zebrafish by using the conditioned place preference (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.

In line with studies of other animals (e.g. 5), stimuli that have been shown to be rewarding for adult fish include ethanol 67, cocaine 8, amphetamine 9, opiates 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 DAergic pathway is most likely comprised of projections from the diencephalic posterior tuberculum to the ventral telencephalon (subpallium, (Vv and Vd), see 12). Several other neurotransmitters have also been implicated in reward behaviour. Heterozygous mutant zebrafish 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 inhibitory influences including projections from the habenula. The zebrafish ventral habenula appears to be homologous to the mammalian lateral habenula in both gene expression and innervation of the raphe 15. The recent identification of selective molecular markers for both structures 1617 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 differences 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 following 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 treated 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 modulating 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 identify 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 demonstrated altered cocaine (but not sucrose) mediated reward behaviour following a reduction of hippocampal neurogenesis 18. This constitutes an exciting new development in the field of reward behaviour; neurogenesis-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 preference following morphine treatment but not food application 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 serotonergic neurons in specific nuclei of the forebrain (diencephalon and hypothalamus; 21), defects that are maintained into adulthood. Dissociation between the preference for a natural reward (food) and a drug (morphine) has previously been observed in dopamine D2 receptor knock-out mice 22 but is not understood at the molecular or neurological level. Since both morphine and food rewards are dependent on opioid receptor activity in zebrafish 10, the separable reward behaviour seen in tof suggests that distinct neural systems 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 hippocampal irradiated rats 18, tof presents an excellent opportunity to dissect the neural basis of discrimination between rewarding substances in the brain.

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 learning 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 232425 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 stimulus (morpholine) with the aversive effects of alarm substance 26. Avoidance learning can be assessed by using a shuttle box; fish are quickly able to associate a conditioned 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 8112930 or a shuttle box 3132. Fish must learn to collect a reward by either navigating a maze correctly or alternating the side of the tank visited. These behavioural tests are high-throughput, making them suitable 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 implicated 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 moderate levels of nicotine perform better in a delayed spatial alteration task, a type of avoidance learning test. Nicotine acts via nicotinic acetylcholine receptors (nACHRs), thus further underscoring the importance of cholinergic signalling 3435. Treatment of fish with a Histidine decarboxylase inhibitor, alphafluoromethylhistidine (α-FMH), reduces both levels of histamine and the number of histaminergic fibres in the brain. α-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 2728. 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.

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)637. Fish are unable to recognise their own image and so attack as if an intruder is present 38. Furthermore, MIS provides 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 biting, thrashing of the tail and short bouts of fast swimming directed against the mirror 6. A positive correlation between aggression and boldness has also been reported 39. Aggression is a very plastic behaviour. Both habitat complexity and rearing conditions can influence the number of interactions 3740. Furthermore, different wild-type strains show varying aggression levels suggesting a genetic component to its control. Finally, aggressive behaviour also shows lateralisation, 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 aggressive (e.g. 42). Other neurotransmitters, including GABA, glutamate and nitric oxide as well as the hormones 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 compounds including ethanol 6 and 17alpha-ethinylestradiol (a synthetic oestrogen; 44). The brain areas mediating aggression in fish are not well characterised. Arginine vasotocin-expressing cells of the magnocellular preoptic area change size depending on the dominance 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 territories that influence aggression. For example, the neural activity maker cfos is expressed in the diencephalon, 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 bluegill (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 providing a golden opportunity to uncover novel genes implicated in aggression control.

Anxiety is a state of constant fear or restlessness caused by anticipation of a real or imagined future event. Multiple 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 3049, at the bottom of a novel tank 3450 or on the dark side of a light/dark tank 2851. The second approach analyses locomotory patterns: freezing, long-lasting increases in basal locomotory activity 64849 and tightening 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 4950. For example, AB wild-types manifest anxiety as a hyperactive swimming response 49.

Similar to other behaviours, anxiety protocols have been validated using pharmacological compounds developed for human patients. Application of caffeine 5053, pentylenetetrazole 53, alarm substance 5052, 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 4955, the Htr1a (5-HT receptor) partial agonist buspirone 55, fluoxetine hydrochloride and ethanol 50. Finally, a link between anxiety levels and the major zebrafish stress hormone cortisol has also been demonstrated 50. The ease of applying drugs and robust behavioural assays (see 4950) make zebrafish an ideal model to study anxiety and related behaviours.

The examples discussed in this review highlight the suitability of adult zebrafish for studies of complex vertebrate 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 signalling pathways and the specificity of drugs used to modulate them. Finally, another important consideration when designing behavioural studies is the background strain of the fish used. Strain differences in adult behaviour have already been reported 13495064. Thus, in order to avoid some of the known difficulties in reproducing 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.

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-nitrosourea (ENU; Fig. 1) efficiently induces intragenic point mutations in the germline 65, but the subsequent cloning steps needed to recover the mutagenised gene are laborious. As an alternative to ENU treatment, insertional mutagenesis looks particularly promising (Fig 2). Although insertional mutations occur at a lower frequency, isolation of the genetic lesion is much simpler 6667. 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 faster 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.