Identification of environmental stressors and validation of light preference as a measure of anxiety in larval zebrafish

Abstract

BackgroundLarval 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.ResultsIn 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.ConclusionThese 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.

Heritable natural variation of an anxiety-like behavior in larval zebrafish

“…Since the dark avoidance behavior in larval zebra fish is considered to be anxiety-associated(Bai et al, 2016; F. Chen et al, 2015; Steenbergen et al, 2011), we asked whether the sda and vda traits are correlated with the extent of thigmotaxis, another measure of anxiety-like behavior (Schnörr, Steenbergen, Richardson, & Champagne, 2012; Treit & Fundytus, 1988): the increased tendency to prefer edges of a chamber over the center area is indicative of a heightened anxiety-like state.…”

“…In both zebrafish and mammals, the behavior has been suggested to be aversion/fear/anxiety-related (Bai et al, 2016; Crawley, 1985; Lau et al, 2011; Maximino et al, 2010; Steenbergen et al, 2011). In larval zebrafish, anxiolytic drugs decrease dark aversion whereas environmental stressors enhance dark aversion (Bai et al, 2016). In this study, through high throughput behavioral phenotyping of individual larvae across multiple trials, followed by selective breeding and statistical analyses, we uncovered an individual variation in dark aversion that is heritable.…”

“…Light dark choice behavioral recording was carried out as previously described(Bai et al, 2016). One hour before the behavioral test, larvae were moved to the behavior room and kept on a blue background.…”

“…Light/dark preference is an evolutionarily conserved trait (Gong et al, 2010)–(Bourin & Hascoët, 2003). In both zebrafish and mammals, the behavior has been suggested to be fear/anxiety-related (Bai, Liu, Huang, Wagle, & Guo, 2016; F. Chen, Chen, Liu, Zhang, & Peng, 2015; Crawley, 1985; Lau, Mathur, Gould, & Guo, 2011; Maximino, Marques de Brito, Dias, Gouveia, & Morato, 2010; Steenbergen, Richardson, & Champagne, 2011).…”

“…Rather than computing the average choice indices (% time in dark −% time in light) as we have done previously(Bai et al, 2016; Lau et al, 2011), we calculated them on an individual basis across four trials in each larva. This analysis uncovered a remarkable dark aversion spectrum, with individuals that displayed strong dark aversion ( sda ), medium dark aversion ( mda ), and variable dark aversion ( vda ).…”