A novel behavioral paradigm to measure anxiety-like behaviors in zebrafish by the concomitant assessment of geotaxis and scototaxis.

Pathological anxiety is a set of diseases characterized by specific clinical manifestations and the use of alternative models may provide novel insights in translational neurobehavioral research. In zebrafish, the separate performance of novel tank and light dark tests in different order to assess anxiety using a same animal may provide conflicting data due to the battery effect and/or time-drug-response and variability across tests. To improve data reliability, we aimed to characterize a novel behavioral paradigm to measure geotaxis and scototaxis as anxiety-like responses in the same trial. The novel apparatus consisted of four colored-compartments, with specific white- and black sections delimited in both bottom and upper areas of the tank. The main baseline responses of zebrafish in the novel apparatus were measured and animals were further exposed to modulators of anxiety. Zebrafish showed robust habituation to novelty stress during the 6-min trial with preference for the black section while exploring the top area. Fluoxetine (100 μg/L, 15 min) reduced geotaxis and scototaxis and ketamine (20 mg/L, 20 min) decreased geotaxis and increased the distance traveled in the black section while exploring the top, possibly due to the increased circling behavior. As anxiogenic modulators, conspecific alarm substance (3.5 mL/L, 5 min) exacerbated risk assessment, geotaxis, and scototaxis, whereas caffeine (10 mg/L, 15 min) increased geotaxis and exploration in the black section of the top area. Since important correlations were also found for relevant anxiety-like behaviors, our findings support the predictive validity of this novel paradigm to simultaneously assess geotaxis and scototaxis in zebrafish. Moreover, it fully adheres to the 3Rs principle of animal experimentation of reducing the number of subjects tested, execution time, also minimizing a potential battery effect.