<p>Three-dimensional reconstructions of zebrafish swimming paths (Cachat et al. 2011) are a new technique to discover novel, meaningful behavioral patterns evoked by different experimental manipulations. Compared to traditional 2D traces, 3D swim path reconstructions enable both macro-level (general) and micro-level (specific/repeated) analyses of potentially meaningful behavioral patterns, offering a complete picture of fish behavior that can be easily combined or re-analyzed. ‘Temporal’ 3D reconstructions, which plot horizontal (x) and vertical (y) spatial data across time (t), reflect zebrafish activity over testing time. ‘Spatial’ 3D reconstructions require two cameras to plot horizontal (x, front view), vertical (y, front view) and side-side (x or y, top view) data, depicting zebrafish 3D activity within the actual testing arena. These reconstructions are highly sensitive to anxiolytic, anxiogenic and hallucinogenic effects in adult zebrafish. For example, we have recently characterized unique movement profiles of ibogaine (a hallucinogen with psychedelic/dissociative properties) which reversed natural zebrafish behaviors, and whose comprehensive characterization would not have been possible without using 3D reconstructions (Cachat et al., 2012). We also applied Track3D, a supplement for EthoVision XT (Noldus IT, Netherlands) developed originally for insects, to adult zebrafish neurophenotyping. Our data represents the first successful application of Track3D in adult zebrafish, showing strong (R>0.07) significant correlation of automated behavioral endpoints with manual data. Track3D provided a precise calculation of movement parameters (i.e., distance traveled, velocity, angular velocity, path tortuosity) within 3D space, also offering accurate spatiotemporal integration of two-camera recordings. We are actively using the 3D-based reconstructions as a window into qualitative and quantitative descriptions of behavioral profiles of various psychotropic drugs. The spatiotemporal data generated from 3D approaches also permits the application of advanced moving object statistical techniques (e.g., movement pattern analysis) to identify and compare movement profiles of zebrafish under different experimental conditions, including testing anxiogenic/anxiolytic and hallucinogenic drugs, as well as identifying unique movement profiles for screening novel psychoactive compounds.</p
