Three-Dimensional Neurophenotyping of Adult Zebrafish Behavior

Abstract

The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior.

Perspectives on Zebrafish Models of Hallucinogenic Drugs and Related Psychotropic Compounds

“…For a more detailed analysis of 3D reconstructions, the average velocity (m/s) of each fish was reflected by the changes in color (from blue, green, yellow, to red) as the velocity increases. 63 In particular, LSD and ketamine evoked a clear top dwelling, with ketamine exposed fish also exhibiting some decrease in velocity relative to controls. Similarly, MDMA also reduced anxiety-like behaviors in zebrafish, increasing time in top and reduced latency to top.…”

“…At the same time, as advanced videoanalysis software and detailed zebrafish ethograms become available, research laboratories are now able to rapidly obtain reliable behavioral data, supplemented with video capture and video tracking, and followed by a multistep assays of physiological biomarkers. 63 Several other potential problems exist. For example, the dissection between hallucinogenic behaviors per se and additional domains (likely to be affected by these drugs, e.g., anxiety, activity, and cognition) requires a thorough investigation using more specific behavioral tests.…”

“…Recently, we have introduced a novel automated neurophenotyping methodology based on analyses of 3D reconstructions of swimming paths, and have applied and validated this method for various experimental manipulations. 63 These studies have developed detailed 3D-based approaches to phenotyping of zebrafish motor and anxiety-related behaviors, offering an innovative high-throughput data-dense methodology for automated visualization and quantification of fish swimming activity in both X, Y, Z (spatial) and X, Y, Time (spatiotemporal) coordinates. The use of 3D reconstruction of movement patterns (see Figure 1 for example) to study hallucinogenic drugs 66,70 enables a more precise deconstruction of zebrafish behavior affected by various agents.…”

Automated image-based tracking and its application in ecology

“…Tracking systems designed for 2D can provide some resolution for behavior in a third spatial dimension [34), but ultimately developers must produce tracking systems that can successfully track large numbers of animals in 3D space (Movie S8 in the supplementary material online). Tracking unconstrained flying or swimming animals can be achieved in several ways, but most often multiple cameras are employed [18,29,[35][36][37][38][39][40][41][42][43][44][45] (Movie S6, Movie 88, and Movie S22 in the supplementary material online). Although only two calibrated cameras taking images of the same point in space are required for triangulation, information from additional cameras can incrementally improve localization, especially if some cameras are limited by occlusion or low contrast [18) .…”

A Simple Setup to Perform 3D Locomotion Tracking in Zebrafish by Using a Single Camera

“…Basically, our method (with one mirror) is similar to Zhu and Weng's method (which requires two mirrors) [32] that also uses only one camera to capture 3D images. Other methods need at least two cameras to capture images simultaneously from different axes [46,[57][58][59], which make the operation more difficult and more expensive. In addition, with the application of idTracker, we are able to perform 3D tracking on multiple subjects.…”