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 multi-behavioral phenotyping approach in larval zebrafish to identify and mechanistically elucidate neuroactive chemicals, with particular focus on chemical compounds that affect non-associative habituation learning.

Methods We devised a battery of automated behavior assays in larval zebrafish. The battery captures stereotypical visual and acoustic behaviors including habituation, a form of non-associative 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 multi-neurotransmitter (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 non-mammalian vertebrate test system to screen chemicals for neurotoxicity. Together with in silico target predictions and mouse- and human-based models, our findings establish multi-behavioral phenotyping in zebrafish as a powerful toolkit for neurotoxicity testing and mechanism identification, with relevance for humans.