Larval zebrafish as a translational model for neurotoxicity screening of emerging psychoactive substances.

The rapid emergence of novel psychoactive substances poses a growing neurotoxicological concern, characterised by poorly defined mechanisms, high potency, and escalating overdose fatalities. Effective treatments remain limited, highlighting the need for scalable in vivo systems capable of identifying hazardous pharmacological profiles before widespread harm occurs. Here, we evaluated larval zebrafish (Danio rerio) as a medium/high-throughput model for early hazard assessment of γ-aminobutyric acid (GABA)A positive allosteric modulators (PAMs) and N-methyl-D-aspartate (NMDA) receptor antagonists. Behavioural analysis of 4 days post-fertilisation (dpf) larvae revealed concentration-dependent locomotor effects consistent with mammalian pharmacodynamics, while whole-body bioanalysis confirmed compound uptake, revealing substance-specific differences in internal exposure. Notably, diazepam and tiletamine deviated from expected class profiles, highlighting the model's sensitivity to compounds with distinctive neuropharmacological signatures. These findings demonstrate the translational value of larval zebrafish for rapid neurotoxicity screening and pharmacodynamic profiling, offering an ethically advantageous, 3Rs-aligned platform to inform overdose treatment development and prioritisation of emerging psychoactive threats.