Flowchart illustrating the experimental strategy of this study.

The rationale (A) includes examining traditional, manually recorded novel tank test behavioral endpoints across several treatments and trials (Step 1). Video-tracking analysis was then performed to generate additional automated behavioral endpoints and raw spatiotemporal data for three-dimensional (3D) swim path reconstructions (Steps 2–3), followed by hierarchical clustering (Step 4) across all behavioral endpoints and experimental treatments in order to discover potential overlaps between manual and automated endpoints. These overlaps were reconfirmed using the 3D swim path reconstructions (Step 5). Finally, our interpretation of the observed affective states was verified with measured endocrine responses (Step 6). The experimental process (B) was standardized for all novel tank trials. Naïve, wild-type zebrafish were placed in an unfamiliar, novel tank for 6 min. Animal behavior was manually observed and two cameras recorded videos for automated analysis in EthoVision XT7 (during which manual, event-based scoring was also performed). Track data for each subject was exported, processed and visualized in a 3D scatter plot with RapidMiner 5.0.