Analyzing the Effects of Age, Time of Day, and Experiment on the Basal Locomotor Activity and Light-Off Visual Motor Response Assays in Zebrafish Larvae.

The recent availability of commercial platforms for behavioral analyses in zebrafish larvae based on video-tracking technologies has exponentially increased the number of studies analyzing different behaviors in this model organism to assess neurotoxicity. Among the most commonly used assays in zebrafish larvae are basal locomotor activity (BLA) and visual motor responses (VMRs). However, the effect of different intrinsic and extrinsic factors that can significantly alter the outcome of these assays is still not well understood. In this work, we have analyzed the influence of age (5-8 days post-fertilization), time of day (8:00, 10:00, 12:00, 14:00; 16:00, 18:00, and 20:00 h), and experiment (three experiments performed at different days) on BLA and VMR results (4004 analyses for each behavior) in 143 larvae. The results from both behaviors were adjusted to a random-effects linear regression model using generalized least squares (GLSs), including in the model the effect of the three variables, the second-way interactions between them, and the three-way interaction. The results presented in this manuscript show a specific effect of all three intrinsic factors and their interactions on both behaviors, supporting the view that the most stable time period for performing these behavioral assays is from 10:00 am to 04:00 pm, with some differences depending on the age of the larva and the behavioral test.