The measurement of multiple behavior endpoints in zebrafish can provide informative clues within neurobehavioral field. However, multiple behavior evaluations usually require complicated and costly instrumental settings. Here, we reported a versatile setting that applied ten acrylic tanks arranging into five vertical layers and two horizontal columns to perform multiple behavior assays simultaneously, such as the novel tank diving test, mirror-biting test, social interaction, shoaling, and predator escape assay. In total, ten behavioral performance were collected in a single video, and the XY coordination of fish locomotion can be tracked by using open source software of idTracker and ImageJ. We validated our setting by examining zebrafish behavioral changes after exposure to low dose ethanol (EtOH) for 96 h. Fish were observed staying longer time at bottom of the tank, less mirror biting interest, higher freezing time, less fear in predator test, and tight shoaling behaviors which indicated the anxiogenic effect was induced by low dosage exposure of EtOH in zebrafish. In conclusion, the setting in this study provided a simple, versatile and cost-effective way to assess multiple behavioral endpoints in zebrafish with high reliability and reproducibility for the first time.
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