Overt physiological responses and atypical behavior evoked in adult zebrafish in the novel tank test by two acute ‘survival’ stressors

Zebrafish (Danio rerio) are increasingly utilized in neurobehavioral research. The novel tank ‘diving’ test is presently the most popular and sensitive behavioral assay for adult zebrafish. Characteristic bottom dwelling ('diving') in this test is commonly interpreted as stress-related fear/anxiety state, and top swimming - as anxiolytic-like behavior. However, mounting evidence suggests a more complex nature of zebrafish behavioral responses in this paradigm. Here, we report ‘atypical’ top preference in the novel tank test caused by two distinct severe survival stressors (live predator exposure and hydrodynamic turbulence), accompanied by elevated whole-body cortisol levels and brain expression of early protooncogenes. Collectively, this suggests complex zebrafish behavioral profile in the novel tank test driven by multiple other factors beyond anxiety per se. Calling for further systematic analyses of experimental modifiers in this and other aquatic novelty-based behavioral assays, these findings show that the novel tank data need to be carefully interpreted in various contexts, and jointly with physiological endpoints, to ensure valid conclusions.