INTRODUCTION: Brain function relies on tightly regulated excitatory and inhibitory signaling. NMDA and AMPA receptors mediate excitation, while GABA_A receptors provide synaptic inhibition. Disruption of this balance contributes to neuropsychiatric disorders. Neurosteroids fine-tune these receptors and can rapidly strengthen or weaken their activity, yet their behavioural consequences remain poorly understood. The zebrafish has emerged as a model for brain disorders, as its neurotransmitter systems are highly conserved with mammals.

AIM(S): Here, we investigated the neurosteroid 5β-pregnan-20-deoxo-3α-yl-sulfate (PAdO-S) across molecular, synaptic, and behavioural scales to link receptor modulation with system-level outcomes.

METHOD(S): Using whole-cell patch-clamp electrophysiology, we examined PAdO-S actions on recombinant rat and zebrafish NMDA receptors expressed in HEK293 cells and on synaptic transmission in rat hippocampal autaptic cultures. Behavioural effects were assessed by measuring spontaneous locomotor activity in zebrafish larvae.

RESULTS: PAdO-S inhibited recombinant rat and zebrafish NMDA receptors with comparable potency in a dose-dependent manner (0,1 - 10 μM), supporting the validity of our model. At synapses, PAdO-S (10 μM) reduced GABA_A receptor–mediated IPSCs. AMPA receptor–mediated EPSCs were potentiated by 41,4%, while paired-pulse ratio decreased by 43,4% (10 μM), indicating enhanced presynaptic glutamate release. Behavioural benchmarks were established using selective receptor blockers: the GABA_A antagonist picrotoxin (10–30 μM) triggered seizure-like activity in larvae, whereas the NMDA receptor blocker memantine (10–100 μM) reduced locomotion. By contrast, PAdO-S increased locomotor activity without inducing aberrant movement (1–10 μM).

CONCLUSIONS: PAdO-S exerts pre- and postsynaptic actions, and the balance between these effects shapes circuit-level and behavioural outcomes. Exploring these mechanisms may clarify the therapeutic actions of neurosteroids in clinical use and guide future work.

FINANCIAL SUPPORT: This work was supported by the Czech Science Foundation (GAČR), grant no. 23-04922S.