As illustrated above, traditional behaviour-based neuroactive drug discovery is very effective. It is also very low-throughput (). Large-scale behaviour-based chemical screens would likely identify novel drug prototypes. However, it is prohibitively laborious, time consuming and expensive to systematically expose humans, rodents and other mammals to the small amounts of chemicals found in modern chemical libraries. Thus, despite its proven efficacy, behaviour-based drug discovery has largely been limited to serendipitous observations. Due to these drawbacks, phenotype-based drug discovery approaches have largely been replaced over the past 50 years by in vitro
assays that are very high-throughput [18
Ironically, target-based assays are especially problematic for psychiatric disorders, which are poorly understood and difficult to model in vitro
. Unlike phenotype-based chemical screens, target-based in vitro
screens are designed to identify compounds that act on pre-defined molecular targets [19
]. In vitro
assays are incredibly valuable. However, they cannot be applied to poorly understood illnesses for which the appropriate therapeutic targets are unknown [18
]. Biological understanding is a prerequisite, not a result, of in vitro
screens. In vitro
approaches to developing analogues of existing drugs are often profitable and sometimes provide therapeutic benefits. However, truly significant advances in psychiatric pharmacotherapy will require the discovery of novel drug classes and therapeutic reagents.
Unlike other approaches, behavioural assays in larval zebrafish have the potential to combine the advantages of phenotype-based drug discovery with high-throughput chemical screening methodologies (). Zebrafish assays are lower-throughput than in vitro assays; however, they are much higher-throughput than behavioural tests in mammals. Like other chemical screens in zebrafish, behavioural screens will depend on larval phenotypes because only larvae are small enough to be easily arrayed in multi-well plates with chemicals from a chemical library (adult animals are too large). Today, most zebrafish chemical screens evaluate fewer than ten thousand chemicals. In future, the number of chemicals screened will likely increase to tens and even hundreds of thousands as automated robotic screening technologies become more accessible. Thus, zebrafish can make large-scale behaviour-based drug screens possible at a throughput that would be unattainable using mice and other large model organisms.
Figure 1: Zebrafish combine high-throughput screening and physiologically complex phenotyping. Behavioural assays in mammals are a physiologically complex but low-throughput approach to drug discovery. Conversely, in vitro target-based assays are a high-throughput (more ...)
Fish do not suffer from schizophrenia or depression. So how can they be used to identify psychiatric drugs? Because drugs act on highly conserved molecular targets to affect neuronal signaling, behaviours in other species can be used to identify chemicals with potential psychoactive properties in humans (). Behavioural tests in model organisms need not simulate human behaviours or illnesses to be useful for drug discovery. To be useful for drug screening, behaviour only needs to be sensitive and specific to neuroactive drugs [21
]. In theory, almost any behaviour in any animal could be used to discover neuroactive drugs; even simple behaviours in larval zebrafish [22
Psychiatric drugs act though conserved mechanisms to alter behavioural phenotypes. Evolutionarily conserved drug targets mediate different behavioural phenotypes in a variety of organisms.
The behaviour of larval zebrafish is a relatively small but exciting field that is developing rapidly. Zebrafish larvae exhibit a variety of behaviours including the optokinetic response [23
], the optomotor response [24
], prepulse inhibition [25
] and sleep [26
]. In the optokinetic response (OKR) assay, smooth-pursuit and saccadic eye movements are measured in partially immobilized ~5-day-old larvae as they respond to movements in their visual field [28
]. Abnormal smooth-pursuit eye movements are an endophenotype of schizophrenia, suggesting that this behaviour has great potential for identifying potential anti-psychotic drugs. Although the OKR assay requires some labour-intensive manual steps, further refinements to this assay may increase its throughput for large-scale chemical screens. In the optomotor (OMR) assay, changes in swimming direction are measured in response computer-animated patterns of moving images. The OMR assay is high-throughput, capable of evaluating the behaviour of hundreds of larvae in a few minutes, and would likely be a powerful approach for identifying neuroactive chemicals that affect motion perception [24
]. Prepulse inhibition (PPI) is a phenomenon in which a startle reflex to a high intensity stimulus (the pulse) is inhibited by a lower intensity prepulse. An automated behavioural assay for PPI in zebrafish larvae has recently been described that can evaluate hundreds of larvae per trial [25
]. Deficits in PPI are an endophenotype of schizophrenia in humans and, haloperidol, an anti-psychotic drug, can suppress prepulse inhibition defects in larval zebrafish [25
]. Thus, PPI is a promising behavioural test on which to base large-scale chemical screens. Sleep-like behaviour has also recently been described in zebrafish, and may be useful for identifying stimulants, hypnotics and other drugs that affect arousal. For example, benzodiazepines modulate sleep in larval zebrafish [27
Zebrafish larvae exhibit additional behaviours that may be amenable to high-throughput chemical screens including the startle response [32
], feeding [33
], learning [35
] and (of course) swimming [36
]. Zebrafish larvae exhibit a robust startle response to high intensity sound stimuli [32
]. Startle magnitude may be a useful model for post-traumatic stress disorder. Feeding assays may be useful for modeling aspects of eating disorders and zebrafish behaviours that undergo habituation, sensitization and other simple forms learning may be useful models for identifying cognitive enhancers. Finally, simple locomotor swimming behaviours may also be useful for identifying neuroactive drugs. For example, anti-psychotics and anti-depressants produce locomotor defects [37
], and anti-convulsants can suppress seizures in zebrafish larvae [39
]. Like behavioural assays in all model organisms, new behavioural assays in larval zebrafish will be difficult to develop. However, the unique ability to perform large-scale chemical screens in larval zebrafish provides strong motivation to pursue these developments.
Considering the strengths of zebrafish as a developmental and genetic model organism one can imagine many additional potential applications for high-throughput behavioural assays beyond forward chemical screens in wild-type animals. For example, high-throughput behavioural assays would be a valuable tool for phenotyping genetic mutants, performing chemical and genetic suppressor/enhancer screens and analysing large-scale morpholino loss-of-function experiments. Because zebrafish larvae are transparent, transgenic animals expressing fluorescent markers could also be used to identify chemicals that affect neuronal morphology in vivo, or to correlate structural changes in the nervous system with behavioural outcomes. Overall, these examples suggest that larval behaviours have great potential to be used for investigating psychopharmacolgy in general.