Multi-behavioral phenotyping in zebrafish identifies a novel disruptor of non-associative learning with environmental and human relevance

The vertebrate nervous system is vulnerable to chemical toxicity and the widespread release of chemicals into the environment outstrips the capacity to assess their safety. We devised a battery of automated behavior assays in larval zebrafish (Danio rerio), a 3R-compliant model amenable to higher-throughput chemical screens. The battery captures stereotypical visual and acoustic behaviors including habituation, a form of non-associative learning. Known pharmacological modulators of habituation in zebrafish evoked distinct behavioral patterns. By screening chemicals positive for ex vivo N-methyl-D-aspartate receptor (NMDAR) modulation, we identified chlorophene, a biocide that caused sedation, paradoxical excitation, and reduced habituation in zebrafish. Using in silico target predictions and pharmacological interventions, we discovered that chlorophene acts via gamma-aminobutyric acid A receptors (GABAARs), a previously unknown target site. Orthogonal validation in cultured mouse cortical neurons and human stem cell-derived BrainSpheres confirmed chlorophene's interaction with GABAARs. Together, multi-behavioral phenotyping in zebrafish can accelerate the identification of neurotoxicants and their underlying mode of action.