Multibehavioral Phenotyping in Early-Life-Stage Zebrafish for Identifying Disruptors of Nonassociative Learning

BACKGROUND: 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. The zebrafish (Danio rerio) is a powerful vertebrate model that can bridge the gap between in vitro- and mammalian-based in vivo studies. However, the behavior-rich repertoire of larval zebrafish, a 3R-compliant model amenable to higher throughput chemical screens, has yet to be fully deployed to identify and characterize chemical compounds that cause neurotoxicity. OBJECTIVE: We sought to establish a multibehavioral phenotyping approach in larval zebrafish to identify and mechanistically elucidate neuroactive chemicals, with particular focus on chemical compounds that affect nonassociative habituation learning. METHODS: We devised a battery of automated behavioral assays in larval zebrafish. The battery captures stereotypical visual and acoustic behaviors including habituation, a form of nonassociative learning. To elucidate mechanisms underlying exposure-induced behavioral alterations in zebrafish, in silico target predictions, pharmacological interventions, patch-clamp recordings in cultured mouse cortical neurons, and human multineurotransmitter (hMNR) assay in 3D BrainSpheres were used. RESULTS: 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 (GABA_ARs), a previously unknown target site. Orthogonal validation in cultured mouse cortical neurons and human stem cell-derived BrainSpheres confirmed chlorophene's interaction with GABA_ARs. Chlorophene's behavioral profile resembled that of flupirtine, a Kv7 potassium channel (M-current) activator, suggesting that habituation deficits stem from M-current rather than GABA_AR modulation. CONCLUSIONS: These studies combined a series of behavior assays in a phenotypically rich, rapid, and inexpensive nonmammalian vertebrate test system to screen chemicals for neurotoxicity. Together with in silico target predictions and mouse- and human-based models, our findings establish multibehavioral phenotyping in zebrafish as a powerful toolkit for neurotoxicity testing and mechanism identification, with relevance for humans.