A High-Throughput Behavioral Assay for Screening Novel Anxiolytics in Larval Zebrafish

<b>Background:</b> Anxiety disorders affect millions of people worldwide, but current treatments often have limited effectiveness and produce unpredictable responses in patients. This underscores the need for novel anxiolytics. While behavior-based screens are valuable for discovering new small molecules, high-throughput anxiety-related assays in vertebrates are lacking. The larval zebrafish dark avoidance, which corresponds to aversive responses to a predator's shadow, can be reduced by known anxiolytics and involves neural pathways known to regulate human anxiety. <b>Methods:</b> Larval zebrafish exhibiting strong dark avoidance (SDA) have been characterized, reflecting pathological anxiety. We developed a high-throughput behavioral assay using a 96-well plate and showed that SDA larvae displayed significant dark avoidance in this setup. We tested known anxiolytics, Chlordiazepoxide and Buspirone, and found that they significantly reduced dark avoidance in this 96-well assay. We then tested a new candidate compound, Analgesic Screen 1 (AS1), previously shown to reverse avoidance of noxious stimuli such as high temperature in larval zebrafish. <b>Results:</b> The optimized 96-well plate assay reliably detected the anxiolytic activity of Chlordiazepoxide and Buspirone and revealed the effect of AS1 in reducing dark avoidance, thereby establishing the platform's sensitivity and validity. <b>Conclusions:</b> This study demonstrates that the dark avoidance assay is scalable to a 96-well plate format in a small arena. This finding provides an effective platform for discovering novel anxiolytic compounds.