Comparison between two- and three-dimensional scoring of zebrafish response to psychoactive drugs: identifying when three-dimensional analysis is needed.

Zebrafish () have recently emerged as a valuable laboratory species in the field of behavioral pharmacology, where they afford rapid and precise high-throughput drug screening. Although the behavioral repertoire of this species manifests along three-dimensional (3D), most of the efforts in behavioral pharmacology rely on two-dimensional (2D) projections acquired from a single overhead or front camera. We recently showed that, compared to a 3D scoring approach, 2D analyses could lead to inaccurate claims regarding individual and social behavior of drug-free experimental subjects. Here, we examined whether this conclusion extended to the field of behavioral pharmacology by phenotyping adult zebrafish, acutely exposed to citalopram (30, 50, and 100 mg/L) or ethanol (0.25%, 0.50%, and 1.00%), in the novel tank diving test over a 6-min experimental session. We observed that both compounds modulated the time course of general locomotion and anxiety-related profiles, the latter being represented by specific behaviors (erratic movements and freezing) and avoidance of anxiety-eliciting areas of the test tank (top half and distance from the side walls). We observed that 2D projections of 3D trajectories (ground truth data) may introduce a source of unwanted variation in zebrafish behavioral phenotyping. Predictably, both 2D views underestimate absolute levels of general locomotion. Additionally, while data obtained from a camera positioned on top of the experimental tank are similar to those obtained from a 3D reconstruction, 2D front view data yield false negative findings.