Multi-behavioral fingerprints can identify potential modes of action for neuroactive environmental chemicals

There is a lack of confidence in the relevance of zebrafish-based behavior data for chemical risk assessment. We extended an automated Visual and Acoustic Motor Response (VAMR) new approach method (NAM) in 5-day post-fertilization (dpf) zebrafish to include 26, behavior-based endpoints that measure visual-motor responses, visual and acoustic startle responses, habituation learning, and memory retention. A correlation analysis from 5159 control larvae revealed that more complex endpoints for learning- and memory-related behavior yielded unique behavior patterns. To build confidence in the VAMR NAM, we established neuroactivity fingerprints using concentration-response profiles derived from 63 reference chemicals targeting neurotransmission, neurodevelopmental signaling, or toxicologically-relevant pathways. Hierarchical clustering revealed diverse toxicity fingerprints. Compounds that targeted the N-Methyl-D-aspartic acid (NMDA) or gamma-aminobutyric acid type A (GABA_A) receptors reduced habituation learning. Pathway modulators targeting peroxisome proliferator-activated receptor delta (PPARδ) or gamma (PPARγ), GABA_A, dopamine, ryanodine, aryl hydrocarbon (AhR), or G-protein-coupled receptors or the tyrosine kinase SRC inappropriately accelerated habituation learning. Reference chemicals targeting GABA_A, NMDA, dopamine, PPARα, PPARδ, epidermal growth factor, bone morphogenetic protein, AhR, retinoid X, or α2-adreno receptors triggered inappropriate hyperactivity. Exposure to GABA_A receptor antagonists elicited paradoxical excitation characterized by dark-phase sedation and increased startle responses while exposure to GABA_A/B receptor agonists altered the same endpoints with opposite directionality. Relative to reference chemicals, environmental chemicals known to be GABA receptor antagonists (Lindane, Dieldrine) or agonists (Tetrabromobisphenol A (TBBPA)) elicited predicted behavior fingerprints. When paired with the phenotypically rich VAMR NAM, behavior fingerprints are a powerful approach to identify neuroactive chemicals.