The anoxia escape test as a novel and sensitive protocol to assess despair-like behavior in zebrafish

Behavioral despair-like states are commonly modeled in rodents using inescapable stressors that suppress active coping, yet comparable paradigms for zebrafish remain limited.

Here, we introduce and validate a simple anoxia escape test (AET) in juvenile zebrafish based on brief air exposure (60 s out-of-water emersion), an ethologically relevant and strongly aversive challenge. This assay quantifies escape motivation by measuring the number of jump attempts. To validate the test, we compared baseline control fish with animals subjected to established manipulations that induce depressive-like phenotypes and with fish treated with the antidepressant fluoxetine (1 μg/mL). Depressive-like states were induced using three approaches: a pharmacological model (reserpine, 40 μg/mL), a behavioral protocol (unpredictable chronic stress; UCS), and early developmental exposure to a drug of abuse (0.5% and 1.0% embryonic alcohol exposure), a translational model within the fetal alcohol spectrum disorder (FASD).

Baseline control and fluoxetine-treated fish showed high escape responding, predominantly at the beginning of the test, indicating robust escape drive. In contrast, UCS and embryonic alcohol exposure significantly reduced escape attempts relative to controls, consistent with impaired active coping under an uncontrollable stressor.

Compared with the Forced Swim Test, the AET offers a zebrafish-adapted, minimally invasive, and quantifiable assessment of despair-like behavior.

Taken together, the AET provides a rapid and scalable method to quantify stress-induced reductions in escape motivation in zebrafish, supporting its use to investigate despair-like states and to screen candidate treatments.