Behavioral Biomarkers of Neurotoxicity: A High-Throughput Zebrafish Thigmotaxis Assay

Introduction:

Traditional neurotoxicity (NT) testing relies mainly on rodent-based guidelines (OECD TG 424 and 426). Despite their relevance to many neurotoxic mechanisms, these tests are costly and ethically challenging, and they overlook behavioral endpoints related to anxiety. To address the urgent need for animal replacement in NT testing, the European Partnership for the Assessment of Risks from Chemicals (PARC), subtask 5.2.1e, focuses on the development of human-relevant New Approach Methodologies (NAMs). In this context, the zebrafish embryo (ZFe), up to 120 hours post fertilization (hpf), is an in vivo model that has high human homology, but is not included in animal welfare regulations and is not considered an NAM.

Methods:

We developed a high-throughput thigmotaxis assay based on 120 hpf ZFEs to evaluate anxiety-like behavior as a biomarker of NT. Thigmotaxis—the tendency to remain close to the edge of a container—is a robust indicator of altered neural function. We optimized the assay to include visual and acoustic stimuli and evaluated two plate formats (24-well round and 96-well square). A range of known anxiogenics, anxiolytics, negative controls and known neurotoxicants (e.g., nicotine, chlorpyriphos) were tested to establish baseline responses and assay reliability. Thigmotaxis response was compared to general locomotion to test for assay sensitivity.

Results:

The 96-well square plate format offered optimal throughput and reproducibility. Exposure to positive control compounds resulted in significantly elevated/decreased thigmotactic behavior, consistent with anxiogenic/anxiolytic responses. Negative controls showed no behavioral alteration, supporting assay specificity. Comparison with general locomotion showed that thigmotaxis evaluation increased assay sensitivity.

Conclusions:

This thigmotaxis assay is a high-throughput and ethically viable approach for screening chemicals for neurotoxic potential. It effectively captures anxiety-related behaviors and serves as a promising NAM for inclusion in alternative developmental and adult neurotoxicity test batteries. Further validation under PARC is underway to support regulatory adoption.