Protocol for investigating light/dark locomotion in larval stage zebrafish using a standardized behavioral assay

Zebrafish (Danio rerio) larvae offer a unique avenue for high-throughput in vivo investigation. The light/dark locomotor assay is widely used but lacks experimental consistency. Here, we present a protocol for a standardized light/dark assay by describing the steps for plating, acclimatizing larvae, performing the assay, and preparing drug exposure solutions. We also detail procedures for substance exposure and data analysis. For complete details on the use and execution of this protocol, please refer to Hillman et al.1 [Display omitted] •Steps for plating larvae for randomized selection and automated system acclimatization•Guidance on recording pre-exposure locomotion and post-exposure behavior•Instructions for preparing chemical solutions and exposing larvae•Procedures for analyzing behavioral data with the custom R code Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Zebrafish (Danio rerio) larvae offer a unique avenue for high-throughput in vivo investigation. The light/dark locomotor assay is widely used but lacks experimental consistency. Here, we present a protocol for a standardized light/dark assay by describing the steps for plating, acclimatizing larvae, performing the assay, and preparing drug exposure solutions. We also detail procedures for substance exposure and data analysis.