Multi-behavioral phenotyping in early-life-stage zebrafish for identifying disruptors of non-associative learning

Abstract

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. https://doi.org/10.1289/EHP16568.