The Establishment of a Three-Chamber Social Preference Assay in Larval Zebrafish for Neurotoxicity Assessment and Neurodevelopmental Gene Phenotyping

Zebrafish serve as a vertebrate model for assessing environmental neurotoxicity and exploring neurobehavioural phenotypes. However, rapid detection systems for larval zebrafish social preference are still lacking, which restricts efficient behavioural phenotyping and toxicological screening. Here, we developed and validated a 3D-printed three-chamber social preference assay for larval zebrafish that is fully compatible with the Noldus behavioural analysis platform. We confirmed that 14 days post fertilisation (dpf) zebrafish larvae display social preference toward conspecifics, whereas 7 dpf larvae have no significant social bias, defining the key developmental time window for larval social preference. The utility of this method was validated using multiple chemical and genetic models. Acute ethanol and developmental bisphenol A (BPA) exposure significantly disrupted larval social preference with altered locomotor activity, while equal-dose bisphenol AF (BPAF) exposure only impaired locomotion without affecting sociality, indicating distinct neurotoxic effects of BPA analogues. Additionally, two models with neurodevelopmental gene knockdown showed distinct alterations in larval social preference when assessed using this assay. Collectively, this study establishes an efficient larval social preference assay in zebrafish. It provides a feasible methodological tool for environmental neurotoxicity evaluation and phenotypic analysis of neurobehavioural phenotypes.