Integrated metabolomic and behavioral profiling reveals neurotoxicity signatures in zebrafish larvae exposed to pharmaceutical compounds.

Early detection of neurotoxicity remains a challenge, as functional disturbances often precede overt morphological abnormalities. Zebrafish (Danio rerio) larvae provide a suitable in vivo model for integration of behavioral and biochemical endpoints. In this study, we applied a combined metabolomic and behavioral approach to identify biomarkers of neurotoxicity following exposure to pharmaceutical compounds with diverse mechanisms of action. Locomotor activity was evaluated in zebrafish larvae exposed to selected compounds at sublethal concentrations using a light-dark transition assay. In parallel, targeted metabolomic analysis was performed to quantify metabolites associated with neurotransmitter pathways. Behavioral profiling highlighted distinct pharmacological signatures across drug classes: antipsychotics led to pronounced hypolocomotion and coordination impairment, antidepressants produced moderate and phase-specific activity changes, whereas stimulants promoted increased locomotor activity. Metabolomic analysis demonstrated that the most informative changes were associated with serotonergic, catecholaminergic, and γ-aminobutyric acid (GABA)-related pathways. While pathway-specific alterations reflected pharmacological neuroactivity, coordinated perturbations across multiple neurotransmitter-related pathways were observed for selected compounds, including amitriptyline and ethanol. These changes were accompanied by behavioral alterations consistent with a neurotoxicity-like phenotype. Reduced locomotor activity alone was not a sufficient marker of neurotoxicity as pharmacologically distinct compounds provided similar locomotor effects. Instead, the integration of multidimensional behavioral endpoints with metabolomic data allowed discrimination between pathway-specific neuroactivity and broader neurotoxicity-like effects. Overall, combined metabolomic and behavioral profiling in zebrafish larvae provides a sensitive and mechanistically informative approach for early neurotoxicity assessment. The identified alterations in neurotransmitter-related pathways represent candidate biomarkers associated with neurotoxicity-like responses and support the application of integrated omics-based strategies in toxicological screening.