Osmotic Stress Uncovers Correlations and Dissociations Between Larval Zebrafish Anxiety Endophenotypes

Abstract

Larval zebrafish are often used to model anxiety disorders. However, since it is impossible to recapitulate the full complexity and heterogeneity of anxiety in this model, examining component endophenotypes is key to dissecting the mechanisms underlying anxiety. While individual anxiety endophenotypes have been examined in zebrafish, an understanding of the relationships between them is still lacking. Here, we investigate the effects of osmotic stress on a range of anxiety endophenotypes such as thigmotaxis, dark avoidance, light-dark transitions, sleep, night startle, and locomotion. We also report a novel assay for stress-induced anorexia that extends and improves on previously reported food intake quantification methods. We show that acute <30 min osmotic stress decreases feeding but has no effect on dark avoidance. Further, acute osmotic stress dose-dependently increases thigmotaxis and freezing in a light/dark choice condition, but not uniform light environmental context. Prolonged >2 h osmotic stress has similar suppressive effects on feeding while also significantly increasing dark avoidance and sleep, with weaker effects on thigmotaxis and freezing. Notably, the correlations between anxiety endophenotypes were dependent on both salt and dark exposure, with increased dissociations at higher stressor intensities. Our results demonstrate context-dependent effects of osmotic stress on diverse anxiety endophenotypes, and highlight the importance of examining multiple endophenotypes in order to gain a more complete understanding of anxiety mechanisms.

Larval Zebrafish as a Model for Mechanistic Discovery in Mental Health

“…However, it is worth noting that heat stress and mechanical disturbance did not trigger the expected correlated increase in dark avoidance and cortisol levels. We show that prolonged, but not acute osmotic stress enhances dark avoidance in larvae (Cheng et al, 2022). This could suggest that different stressors and stressor durations generate distinct anxiety states that are regulated by diverse pathways.…”

“…However, dark avoidance was unaffected, suggesting that dark avoidance and thigmotaxis behaviors are dissociable traits with potentially different underlying pathways. In contrast, prolonged, high salt stress enhanced dark avoidance but had weaker effects on thigmotaxis (Cheng et al, 2022).…”

“…When anxiolytic aconitine was added, the extent of thigmotaxis behavior observed in the dark periods was reduced significantly (Ellis et al, 2012). In a recent study we observed that acute salt stress increased thigmotaxis in a light/dark choice assay (see Section "Light/Dark Choice") relative to uniform light condition, suggesting a potential interaction of darkness with thigmotactic behavior (Cheng et al, 2022).…”

“…Feeding assay Low Multiple (Jordi et al, 2018;Wee et al, 2019b) Stress-induced anorexia Salt stress feeding assay High HPA (De Marco et al, 2014;Cheng et al, 2022) Avoidance of social eating Social feeding assay Medium Oxytocin Social avoidance Social aggregation/avoidance/ orienting/preference assays Medium Pth, disc1, egr1, monoamines, basal forebrain (Tunbak et al, 2020;Harpaz et al, 2021;Anneser et al, 2022;Tallafuss et al, 2022) Response to addictive substances Preference/selfadministration assays High HPA, monoamines, glutamate, cholinergic system (Darland and Dowling, 2001;Ninkovic et al, 2006;Mathur and Guo, 2010;Bossé and Peterson, 2017;Müller et al, 2020) Arousal, (2) Flight or Freeze, (3) Exploration and Avoidance, and (4) Responses to Appetitive Stimuli (Figure 1).…”

“…There are certainly correlations between measures of sleep and locomotion (see also Cheng et al, 2022) though sleep can be distinguished from general activity using specific criteria (Prober et al, 2006). Similar to locomotion, sleep dysregulation itself is not exclusively an endophenotype for mental disorders, and needs to be considered alongside other more specific behaviors.…”