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

## TL;DR

This study explores how withanolides from ashwagandha interact with ethanol to affect zebrafish larvae behavior and GABA receptor genes.

## Contribution

The study reveals compound-specific effects of withanolides on ethanol-induced behavior and GABAA receptor subunit gene expression in zebrafish larvae.

## Key findings

- EtOH alone caused biphasic locomotor responses in zebrafish larvae.
- Withanolides WITA and WIN preserved or enhanced EtOH-induced hyperactivity, while WTFA potentiated locomotor suppression.
- Withanolides caused subunit-specific changes in GABAA receptor gene expression.

## Abstract

Concurrent consumption of ethanol (EtOH) and herbal preparations containing Withania somnifera (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 (Danio rerio) larvae. Locomotor behavior was quantified under EtOH concentrations ranging from 0 to 4.0%, and the expression of four GABAA receptor subunit genes (gabra1, gabra2, gabrd, gabrg2) 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 gabra1 and gabra2, compound-dependent regulation of gabrd, and complex gabrg2 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.