A Unified Approach to Investigating 4 dpf Zebrafish Larval Behaviour through a Standardised Light/Dark Assay

Abstract

Zebrafish have emerged as a dynamic research model in the domains of psychiatry, neuroscience and behaviour. Working with larvae ≤ 4 days post-fertilisation (dpf) offers an avenue for high-throughput investigation whilst aligning with the 3Rs principles of animal research. The light/dark assay, which is the most used behavioural assay for larval research, lacks experimental reliability and standardisation. This study aimed to formulate a robust, reproducible and standardised light/dark behavioural assay using 4 dpf zebrafish larvae. Considerable between-batch and inter-individual variability was found which we rectified with a normalisation approach to ensure a reliable foundation for analysis. We then identified that 5-minute light/dark transition periods are optimal for locomotor activity. No discernible impact of acclimation (length or lighting conditions) on larval performance was observed. Overall locomotion was found to be significantly greater in the early afternoon compared to morning and evening, but time of day of testing had no impact on light/dark transitions. Next, we confirmed the pharmacological predictivity of the standardised assay using ethanol which, as predicted, caused hyperlocomotion at low concentrations and hypolocomotion at high concentrations. Finally, the assay was validated by assessing the behavioural phenotype of hyperactive transgenic (adgrl3.1-/-) larvae, which was rescued with psychostimulant medications. Our standardised assay not only provides a clear experimental and analytical framework to work with 4 dpf larvae, but also facilitates between-laboratory collaboration using our normalisation approach.