The scototaxis (dark/light preference) protocol is a behavioral model for fish that is being validated to assess the antianxiety effects of pharmacological agents and the behavioral effects of toxic substances, and to investigate the (epi)genetic bases of anxiety-related behavior. Briefly, a fish is placed in a central compartment of a half-black, half-white tank; following habituation, the fish is allowed to explore the tank for 15 min; the number and duration of entries in each compartment (white or black) are recorded by the observer for the whole session. Zebrafish, goldfish, guppies and tilapias (all species that are important in behavioral neurosciences and neuroethology) have been shown to demonstrate a marked preference for the dark compartment. An increase in white compartment activity (duration and/or entries) should reflect antianxiety behavior, whereas an increase in dark compartment activity should reflect anxiety-promoting behavior. When individual animals are exposed to the apparatus on only one occasion, results can be obtained in 20 min per fish.

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The scototaxis test has been introduced recently to assess anxiety-like phenotypes in fish, including zebrafish. Parametric analyses suggest that scototaxis represents an approach-avoidance conflict, which hints at anxiety. In this model, white avoidance represents anxiety-like behavior, while the number of shuttling events represents activity. Acute or chronic fluoxetine, buspirone, benzodiazepines, ethanol, caffeine and dizocilpine were assessed using the light-dark box (scototaxis) test in zebrafish. Acute fluoxetine treatment did not alter white avoidance, but altered locomotion in the higher dose; chronic treatment (2 weeks), on the other hand, produced an anxiolytic effect with no locomotor outcomes. The benzodiazepines produced a hormetic (inverted U-shaped) dose-response profile, with intermediate doses producing anxiolysis and no effect at higher doses; clonazepam, a high-potency benzodiazepine agonist, produced a locomotor impairment at the highest dose. Buspirone produced an anxiolytic profile, without locomotor impairments. Moclobemide did not produce behavioral effects. Ethanol also produced a hormetic profile in white avoidance, with locomotor activation in 0.5% concentration. Caffeine produced an anxiogenic profile, without locomotor effects. These results suggest that the light-dark box is sensitive to anxiolytic and anxiogenic drugs in zebrafish.

Modeling of stress and anxiety in adult zebrafish (Danio rerio) is increasingly utilized in neuroscience research and central nervous system (CNS) drug discovery. Representing the most commonly used zebrafish anxiety models, the novel tank test (NTT) focuses on zebrafish diving in response to potentially threatening stimuli, whereas the light-dark test (LDT) is based on fish scototaxis (innate preference for dark vs. bright areas). Here, we systematically evaluate the utility of these two tests, combining meta-analyses of published literature with comparative in vivo behavioral and whole-body endocrine (cortisol) testing. Overall, the NTT and LDT behaviors demonstrate a generally good cross-test correlation in vivo, whereas meta-analyses of published literature show that both tests have similar sensitivity to zebrafish anxiety-like states. Finally, NTT evokes higher levels of cortisol, likely representing a more stressful procedure than LDT. Collectively, our study reappraises NTT and LDT for studying anxiety-like states in zebrafish, and emphasizes their developing utility for neurobehavioral research. These findings can help optimize drug screening procedures by choosing more appropriate models for testing anxiolytic or anxiogenic drugs.

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Due to the fish-specific genome duplication event (~320-350 mya), some genes which code for serotonin proteins were duplicated in teleosts; this duplication event was preceded by a reorganization of the serotonergic system, with the appearance of the raphe nuclei (dependent on the isthmus organizer) and prosencephalic nuclei, including the paraventricular and pretectal complexes. With the appearance of amniotes, duplicated genes were lost, and the serotonergic system was reduced to a more complex raphe system. From a comparative point of view, then, the serotonergic system of zebrafish and that of mammals shows many important differences. However, many different behavioral functions of serotonin, as well as the effects of drugs which affect the serotonergic system, seem to be conserved among species. For example, in both zebrafish and rodents acute serotonin reuptake inhibitors (SSRIs) seem to increase anxiety-like behavior, while chronic SSRIs decrease it; drugs which act at the 5-HT1A receptor seem to decrease anxiety-like behavior in both zebrafish and rodents. In this article, we will expose this paradox, reviewing the chemical neuroanatomy of the zebrafish serotonergic system, followed by an analysis of the role of serotonin in zebrafish fear/anxiety, stress, aggression and the effects of psychedelic drugs.

Alarm reactions to a substance secreted by the damaged skin of conspecifics and closely-related species are increasingly being recognized as fear-like responses in fish. The neurochemical underpinnings of these effects are so far unknown; however, given the role of the serotonergic system on defensive behavior, it is possible that the alarm reaction is mediated by this monoamine. Exposure to conspecific alarm substance (CAS) increased anxiety-like behavior in the light/dark test in zebrafish and decreased nocifensive behavior. These effects were accompanied by increases in blood glucose, hemoglobin, epinephrine and norepinephrine levels, as well as extracellular levels of serotonin in the brain. Pretreatment with fluoxetine blocked the anxiogenic effects of CAS on the light/dark test as well as all physiological parameters and the increase in extracellular brain 5-HT, but not the reduction in nocifensive behavior. Conversely, pretreatment with the 5-HT1AR antagonist WAY 100635 blocked the effects on nocifensive behavior, but not the effects on anxiety-like behavior nor on physiological parameters. These results point to an important and complex role of the serotonergic system in the mediation of fear-potentiated behavior in the light/dark test and in fear-induced analgesia in zebrafish.

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