Zebra_K+ : High-throughput analysis of acoustic startle response plasticity in zebrafish embryos and larvae in neurotoxicity testing

The acoustic startle response (ASR) is a conserved sensorimotor reflex widely used to investigate neural plasticity, sensorimotor gating, and neurotoxicity. While zebrafish is an established vertebrate model for ASR analysis, most existing platforms were originally optimized for 6 dpf larvae, which constrains applications requiring reliable assessment of earlier developmental stages. Here, we introduce Zebra_K⁺, a modular extension of the previously developed Zebra_K platform, designed for high-throughput kinematic analysis of ASR in zebrafish embryos (5 days post-fertilization, dpf) and early larvae (6-7 dpf). The system enables simultaneous quantification of ASR kinematics, sensitivity, short-term habituation, and prepulse inhibition (PPI) in up to 25 individuals. Using the NMDA receptor antagonist ketamine, the dopamine receptor agonist apomorphine, and the D₂ receptor antagonist haloperidol, we validated the platform's ability to detect pharmacologically induced and developmentally specific alterations in startle plasticity. Ketamine reduced habituation and PPI at all developmental stages, whereas apomorphine selectively impaired PPI, an effect that was reversed by haloperidol only at 7 dpf. These results demonstrate the neurodevelopmental progression of glutamatergic and dopaminergic modulation of sensorimotor gating and establish Zebra_K⁺ as a modular technological platform that supports the development of New Approach Methods (NAMs) for neurotoxicological screening and developmental neuropharmacology.