Visual–acoustic thigmotaxis in zebrafish larvae: a high throughput NAM for neurotoxicity assessment

Introduction Current regulatory neurotoxicity guidelines do not include behavioral endpoints that capture stress-related responses. Zebrafish larvae prior to independent feeding offer a promising vertebrate model for developing new approach methodologies (NAMs) because they combine neurobiological relevance with high-throughput potential. In this study, we developed and evaluated a larval thigmotaxis assay to detect behavioral alterations induced by neuroactive substances. Methods Zebrafish larvae at 120 hpf were exposed for 1 h to model compounds and then challenged with visual (light/dark) and acoustic (tapping/silence) stimuli. Thigmotaxis, defined as edge-preference behavior, and locomotor activity were assessed. To increase throughput, we compared the conventional 24-round-well format with a 96-square-well format. Assay performance was evaluated using caffeine and diazepam as reference compounds, followed by additional neuroactive substances (chlorpyrifos, nicotine, dexamethasone, ethylenethiourea) and low-neuroactivity comparators (saccharin, amoxicillin). Benchmark dose modeling was used to compare the sensitivity of thigmotaxis and locomotor endpoints. Results The 24-well and 96-well formats produced equivalent results, supporting use of the higher-throughput system. Reference compounds confirmed assay performance, with caffeine increasing thigmotaxis and diazepam decreasing it under specific stimulus conditions. Additional neuroactive substances produced stimulus-dependent behavioral responses, whereas saccharin and amoxicillin caused little or no effect. Across compounds, benchmark dose modeling showed that thigmotaxis was generally more sensitive than traditional locomotor activity endpoints. Discussion This multiplexed visual–acoustic thigmotaxis assay is reproducible, scalable, and sensitive for detecting neuroactive effects in zebrafish larvae. It can be used either as a stand-alone behavioral NAM or integrated into a broader test battery for neurotoxicity assessment. The method provides a practical and ethical tool to support chemical safety assessment in both ecotoxicology and human toxicology.