Ethanol–Withanolides Interactions: Compound-Specific Effects on Zebrafish Larvae Locomotor Behavior and GABAA Receptor Subunit Expression

Concurrent consumption of ethanol (EtOH) and herbal preparations containing <i>Withania somnifera</i> (WS, ashwagandha) is increasingly common, but the neurobehavioral and molecular consequences of such interactions remain poorly characterized. This study investigated how three purified withanolides-withanolide A (WITA), withanone (WIN), and withaferin A (WTFA)-modulate the effects of acute EtOH exposure in zebrafish (<i>Danio rerio</i>) larvae. Locomotor behavior was quantified under EtOH concentrations ranging from 0 to 4.0%, and the expression of four GABAA receptor subunit genes (<i>gabra1, gabra2, gabrd, gabrg2</i>) was analyzed by qPCR. EtOH alone induced a biphasic locomotor response, with stimulation at low-to-moderate doses and suppression at higher doses. WITA and WIN modulated this pattern in a dose-dependent manner, preserving or enhancing hyperactivity, while WTFA consistently potentiated locomotor suppression. mRNA profile analysis revealed subunit-specific changes, including downregulation of <i>gabra1</i> and <i>gabra2</i>, compound-dependent regulation of <i>gabrd</i>, and complex <i>gabrg2</i> responses. These results demonstrate that individual withanolides distinctly shape behavioral and molecular outcomes of EtOH exposure, suggesting specific interactions at the level of inhibitory neurotransmission. The findings provide mechanistic insight into the combined effects of WS-derived compounds and EtOH and highlight the importance of considering such interactions in both experimental and applied contexts.