Fentanyl and alcohol co-exposure induce robust, sustained hyperlocomotion and neural circuit disruption in larval zebrafish.

The increasing prevalence of poly-substance misuse, particularly co-use of synthetic opioids and alcohol, poses significant health risks, yet their combined neurobehavioral effects remain poorly understood. Here we used larval zebrafish to investigate interactions between fentanyl and ethanol, employing behavioral assays and in vivo whole-brain calcium imaging. Co-exposure induced a distinctive biphasic locomotor response characterized by initial suppression followed by sustained hyperlocomotion, which was replicated with other opioids but not with GABAA modulators, indicating an opioid- and ethanol-specific effect. Imaging revealed widespread neuronal hyperactivity and dysregulation during co-exposure compared to individual treatments. These findings reveal complex neurobehavioral mechanisms underlying opioid and alcohol co-use, highlighting ethanol's potentiation of opioid effects and suggesting larval zebrafish as a valuable model for studying poly-substance abuse. This work provides foundational insights that may inform future therapeutic strategies for managing opioid-alcohol co-intoxication.