Three-dimensional (3D) temporal and spatial reconstructions of adult zebrafish swim paths rapidly expose overall affective phenotype.

After indicated experimental manipulations (Fig. 3), zebrafish novel tank behavior was manually observed and video-tracked using EthoVision XT7 program. Raw track and behavioral endpoints were processed, formatted, and visualized in a 3D scatter plot using RapidMiner 5.0 software; traditional computer-generated two-dimensional (2D) swim path traces were placed at t = 0.0 s (top left part of each panel) for reference. Representative 3D reconstructions were selected by comparing swim paths of all subjects within a cohort, ranking them from 1 to n based on similarity to each other (low/no to high activity) and choosing the middle for the illustrations. For better visuality and consistency, fish used for spatial 3D imaging were the same as those used for the respective temporal 3D reconstructions. For a more detailed analysis of 3D reconstructions, the average velocity (m/s) of each fish was reflected by changes in color from blue to green, yellow and red, as the velocity increases. Note that any other computer-generated behavioral indices (Table S1 of Supporting Information) may be expressed in color on 3D reconstructions of zebrafish locomotion paths. Overall, these 3D traces reveal striking differences between zebrafish high- and low-anxiety behaviors, thereby enabling a rapid visualization and interpretation of the observed phenotypes.

在完成指定实验操作（图3）后，采用EthoVision XT7软件对斑马鱼的新缸环境行为开展人工观测与视频追踪。原始追踪数据与行为终点指标均经RapidMiner 5.0软件处理、格式化，并以三维散点图形式可视化；同时将计算机生成的传统二维（2D）游泳路径轨迹置于t=0.0s时刻（每个子图左上角）作为参照。代表性三维重构结果的筛选流程为：比对同队列内所有受试个体的游泳路径，按彼此相似性（从活动度极低/无到极高）排序为1至n，选取位次居中的结果作为图示内容。为保障可视化效果与结果一致性，用于空间三维成像的斑马鱼个体，与对应时序三维重构所用受试鱼完全一致。为实现三维重构的精细化分析，每条斑马鱼的平均运动速度（单位：米/秒，m/s）通过颜色渐变表征：随速度升高，颜色由蓝色依次过渡为绿色、黄色与红色。需注意，其余任意计算机生成的行为指标（详见支持信息表S1），均可通过颜色编码呈现在斑马鱼运动路径的三维重构结果中。综上，上述三维轨迹清晰展现了斑马鱼高焦虑与低焦虑行为间的显著差异，可实现观测表型的快速可视化与解读。