Macro- and micro-level behavioral analysis with three-dimensional (3D) spatial reconstruction of swim path.

The reconstructed swim path presented here as an example was obtained from a naïve, wild-type control zebrafish tested in a standard novel tank test for 6 min (see Fig. 6 for more examples). Wild-type fish can be considered “mild anxiety”, compared to both anxiolytic (low anxiety) and anxiogenic (high anxiety) cohorts listed in Fig. 3. Although this fish spent a majority of the trial within the bottom half of the tank, the animal also made large sweeping transitions into the upper half. A detailed spatial dissection of 3D locomotion here revealed that (like temporal 3D reconstructions in Fig. 4) manually scored erratic movement events generally overlap with periods of elevated velocity, rapid movement, high angular velocity, high mobility and sharp turn angles, identified by the computer analysis. For better visuality, the observed endpoints were color-coded, with the legend color scales representing proportional spectrum across min/max ranges of observed experimental values. Overall, this approach strongly supports the utility of 3D-based computer-aided analyses of zebrafish behavior, and for the first time creates 3D reconstructions of zebrafish natural exploratory locomotion, mapping anxiety-related behaviors to these traces. The striking overlap between observer- and computer-generated indices in “real” 3D traces open opportunities for further refinement of video-tracking, eventually leading to fully automated 3D-based neurophenotyping tools to quantify zebrafish anxiety. This method of multidimensional phenotyping of zebrafish locomotion can complement temporal 3D reconstructions (as shown in Fig. 4 and 5).