Ethanol–Withanolide Interactions: Compound-Specific Effects on Photokinetic Behavior and NMDA Receptor Subunit Expression in Zebrafish Larvae

Concurrent use of Withania somnifera (WS, ashwagandha) preparations and ethanol (EtOH) is increasingly common, yet the consequences for glutamatergic signaling remain uncharacterized. Because N-methyl-D-aspartate (NMDA) receptors are a principal target of acute EtOH, we examined whether three purified withanolides modify this response. Zebrafish (Danio rerio) were exposed to withanolide A (WITA), withanone (WIN) or withaferin A (WTFA) at 3–5 days post-fertilization (dpf) and to EtOH (0–4.0% v/v) for 1 h at 5 dpf. Photokinetic behavior was quantified with a normalized dark–light contrast index (DLCI; withanolides at 0–500 µg/L) and grin2aa, grin2bb, grin3a and grin3ba transcripts were measured by RT-qPCR (withanolides at 100 µg/L). EtOH caused a concentration-dependent reduction in DLCI, and photokinetic differentiation was markedly impaired at 2.0% and 4.0%. EtOH increased grin2aa and grin2bb expression and decreased grin3a and grin3ba expression, with grin2bb responding at lower concentrations than grin2aa. The three withanolides produced distinct modulatory profiles. WITA partially preserved photokinetic differentiation at high EtOH concentration and attenuated selected transcriptional responses, whereas WIN showed limited effects. WTFA produced the strongest EtOH-dependent changes in NMDA receptor subunit expression but did not prevent behavioral impairment. These findings indicate compound-specific modulation of acute EtOH-induced behavioral responses and NMDA receptor subunit transcription.