Larval zebrafish, with a simple and transparent vertebrate brain composed of ~100 K neurons, is well suited for deciphering entire neural circuit activity underlying behavior. Moreover, their small body size (~4–5 mm in length) is compatible with 96-well plates, making larval zebrafish amenable to high content screening. Despite these attractive features, there is a scarcity of behavioral characterizations in larval zebrafish compared to other model organisms as well as adult zebrafish. In this study, we have characterized the physiological and behavioral responses of larval zebrafish to several easily amenable stimuli, including heat, cold, UV, mechanical disturbance (MD), and social isolation (SI). These stimuli are selected based on their perceived aversive nature to larval zebrafish. Using a light/dark choice paradigm, in which larval zebrafish display an innate dark avoidance behavior (i.e. scotophobia), we find that heat, cold and UV stimuli significantly enhance their dark avoidance with heat having the most striking effect, whereas MD and SI have little influence on the behavior. Surprisingly, using the cortisol assay, a physiological measure of stress, we uncover that all stimuli but heat and SI significantly increase the whole body cortisol levels. These results identify a series of stressors that can be easily administered to larval zebrafish. Those stimuli that elicit differential responses at behavioral and physiological levels warrant further studies at circuit levels to understand the underlying mechanisms. The findings that various stressors enhance while anxiolytics attenuate dark avoidance further reinforce that the light/dark preference behavior in larval zebrafish is fear/anxiety-associated.

Complex behaviors are often observed at a spectrum in the population, and psychiatric disorders represent extremes of such behavioral spectra. While grasping the underlying cellular and molecular basis of these disorders represents a major challenge, it is believed that studies of complex behaviors in model organisms, where genotyping and phenotyping can be more conveniently carried out and cause–effect relationships can be further discerned, will help address this challenge. Here we report the characterization of a natural dark aversion behavior in larval zebrafish, which is previously shown to be fear or anxiety-associated. Phenotyping ∼200 individuals using a light/dark choice assay uncovered that, while a majority of individuals displayed medium level of dark aversion (mda), a small number of individuals exhibited strong dark aversion (sda), and a third small cohort showed variable dark aversion (vda). Through selective breeding and phenotyping of the next generation, we demonstrated that both the sda and vda traits are heritable, with sda being invariable while vda being highly variable across multiple trials. Additionally, sda appears to be recessive and vda appears to be dominant over the common allele(s) in the population. Moreover, compared to vda, sda showed increased thigmotaxis (preference for the walls in an open field), another measure of anxiety. Together, these findings reveal a naturally heritable variation of anxiety-like behavior in a tractable model organism, thereby laying foundation for future dissection of the underlying molecular and cellular mechanisms.

Salient sensory stimuli are perceived by the brain, which guides both the timing and outcome of behaviors in a context-dependent manner. Light is such a stimulus, which is used in treating mood disorders often associated with a dysregulated hypothalamic-pituitary-adrenal stress axis. Relationships between the emotional valence of light and the hypothalamus, and how they interact to exert brain-wide impacts remain unclear. Employing larval zebrafish with analogous hypothalamic systems to mammals, we show in free-swimming animals that hypothalamic corticotropin releasing factor (CRF^Hy) neurons promote dark avoidance, and such role is not shared by other hypothalamic peptidergic neurons. Single-neuron projection analyses uncover processes extended by individual CRF^Hy neurons to multiple targets including sensorimotor and decision-making areas. In vivo calcium imaging uncovers a complex and heterogeneous response of individual CRF^Hy neurons to the light or dark stimulus, with a reduced overall sum of CRF neuronal activity in the presence of light. Brain-wide calcium imaging under alternating light/dark stimuli further identifies distinct and distributed photic response neuronal types. CRF^Hy neuronal ablation increases an overall representation of light in the brain and broadly enhances the functional connectivity associated with an exploratory brain state. These findings delineate brain-wide photic perception, uncover a previously unknown role of CRF^Hy neurons in regulating the perception and emotional valence of light, and suggest that light therapy may alleviate mood disorders through reducing an overall sum of CRF neuronal activity.

Anxiety is a fear-like response to stimuli perceived to be threatening. Excessive or uncontrollable anxiety is a debilitating psychiatric disorder which affects many people throughout their lifetime. In unravelling the complex genetic and environmental regulations of anxiety-like phenotypes, models measuring the natural dark avoidance of larval zebrafish have shed light on the individual variation and heritability of this anxiety-related trait. Using the light/dark choice paradigm and selective breeding, this study aims to validate previous findings of the variable (VDA) and strong dark aversion (SDA) heritability in AB-WT larval zebrafish using the outbred zebrafish strain EK, which offers more genetic diversity to aid in future molecular mapping efforts. 190 larvae (6 days post fertilization [dpf] and 7 dpf) were tested across four trials and divided into variable (VDA), medium (MDA) and strong (SDA) dark aversion for further in-crosses. VDA and MDA larvae became more explorative with time, whereas SDA larvae rarely left the preferred light zone. The SDA and VDA in-crosses significantly increased the respective phenotypes in the second generation of larvae, whereas VDA × MDA inter-crosses did not. For the second-generation SDA cohort, dark aversion correlated with increased thigmotaxis, which reinforces SDA as an anxiety-like phenotype. Our finding that the dark aversion trait and SDA and VDA phenotypes are heritable in an outbred zebrafish population lays an important foundation for future studies of genetic underpinnings using whole-genome mapping methods. This conserved fear/anxiety-like response in a highly accessible model organism also allows for further pharmacological and behavioral studies to elucidate the etiology of anxiety and the search for novel therapeutics for anxiety disorders.

Animals survive environmental challenges by adapting their physiology and behavior through homeostatic regulatory processes, mediated in part by specific neuropeptide release from the hypothalamus. Animals can also avoid environmental stressors within seconds, a fast behavioral adaptation for which hypothalamic involvement is not established. Using brain-wide neural activity imaging in behaving zebrafish, here we find that hypothalamic neurons are rapidly engaged during common avoidance responses elicited by various environmental stressors. By developing methods to register cellular-resolution neural dynamics to multiplexed in situ gene expression, we find that each category of stressor recruits similar combinations of multiple peptidergic cell types in the hypothalamus. Anatomical analysis and functional manipulations demonstrate that these diverse cell types play shared roles in behavior, are glutamatergic, and converge upon spinal-projecting brainstem neurons required for avoidance. These data demonstrate that hypothalamic neural populations, classically associated with slow and specific homeostatic adaptations, also together give rise to fast and generalized avoidance behavior.