Ontogeny of schooling behavior and stage-specific vulnerability to valproic acid in Xenopus laevis larvae

Perinatal exposure to chemicals such as valproic acid (VPA) increases the risk of neurodevelopmental disorders, including autism spectrum disorder (ASD). Because maternal drug metabolism complicates the assessment of fetal impacts in mammalian models, Xenopus laevis tadpoles have emerged as an effective alternative for neurotoxicology. Tadpoles exhibit "schooling," a polarized social aggregation behavior. Previous studies, which exposed tadpoles to VPA continuously from stage NF42 to NF49, suggested that VPA-induced social behavior abnormalities are triggered during this period. In this study, we re-evaluated the ontogeny of schooling behavior and its vulnerability to VPA by administering short-term exposures at specific developmental stages. Using computational modeling and statistical analyses, we compared the spatial distribution of live tadpoles with computer-simulated random models. Untreated tadpoles exhibited significant, non-random social aggregation as early as stage NF42, indicating that the sensorimotor circuitry for schooling is established earlier than previously assumed. Furthermore, tadpoles exposed to VPA for 4 days starting at stages NF42 and NF48 maintained schooling behavior, whereas VPA exposure starting at stage NF52 significantly disrupted spatial cohesion. These findings indicate that while social behavior is acquired prior to NF42, NF52 represents a critical window of heightened pharmacological vulnerability to teratogens, providing new insights into the etiology of environmentally induced neurodevelopmental disorders.