Dissection of zebrafish behavior using three-dimensional (3D) temporal reconstruction of swim path.

The reconstructed swim path was obtained from a zebrafish tested in a standard novel tank test (Fig. 1) for 6 min following repeated morphine withdrawal, an anxiogenic manipulation (Fig. 3). Manual, event-based behaviors were scored by a human observer during automated video acquisition in EthoVision XT7 program. Subsequently, spatiotemporal (X,Y,time) coordinates, computer-generated movement parameters and manually-scored behaviors were integrated into a single track file for each subject using RapidMiner 5.0 software. After X,Y-coordinates were plotted over experimental time, behavioral endpoints were actively cycled across the swim path as the color attribute and examined for overlaps and patterns. The experimental manipulation used here caused long, prominent freezing bouts separated by short bursts of bottom swimming – a profile typically observed in zebrafish high-anxiety states (Fig. 3). A detailed dissection reveals that manually scored erratic movements on a 3D reconstruction map within episodes of elevated velocity, rapid movement, high angular velocity, high mobility and sharp turn angles (generated by the computer). Conversely, manually scored periods of freezing correlate with lower velocity, slow movement and immobility bouts. 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. This experiment shows that 3D temporal reconstructions permit rapid and objective macro- and micro-level behavioral analysis, thereby improving high-throughput phenotyping of zebrafish behavior. This method of multidimensional phenotyping of zebrafish locomotion can complement spatial 3D reconstructions (as shown in Fig. 5 and 6).