Melatonin Modulates Behavioral and Physiological Responses in a Concentration-dependent Manner in a Pilocarpine-induced Seizure-like Model Using Zebrafish Larvae

Melatonin has been extensively studied for its antioxidant activity, yet its safety profile and concentration-dependent effects remain only partially understood, particularly in aquatic experimental systems. Here, we investigated the effects of melatonin on behavioral and physiological responses in a pilocarpine (PILO)-induced seizure-like model using zebrafish (Danio rerio) larvae. A multibiomarker framework was employed, combining embryo-larval toxicity assessments up to 144 h post-fertilization with behavioral and biochemical endpoints related to seizure-like activity. Across the tested concentration range (0.14-18 µg/mL), melatonin did not affect mortality or hatching rates. However, exposure to higher concentrations (9.0 and 18 µg/mL) resulted in altered spontaneous contractions and increased embryonic heart rate, indicating measurable physiological changes during early development. At lower concentrations (2.25 and 4.50 µg/mL), melatonin was associated with reduced PILO-induced hyperlocomotion, increased latency to seizure-like onset, and reduced progression to more severe behavioral stages. These responses were accompanied by a reduction in reactive oxygen species (ROS) levels at both 72 and 144 hpf. Collectively, the data reveal a concentration-dependent response profile, with distinct behavioral and physiological outcomes emerging across exposure levels. This study contributes to the characterization of melatonin effects in zebrafish larvae and underscores the importance of dose selection in experimental seizure models.