Association of stress-sensitive mid-insula activity with alcohol drinking and negative affect-like behavior during abstinence in mice.

Stress is central to many neuropsychiatric conditions, including alcohol use disorder (AUD). Stress influences alcohol initiation, escalation, progression to AUD, and relapse. Identifying stress-activated neurocircuits and individual variability in these responses is critical for developing new AUD treatment targets. This study investigates the relationship between adult stress response and AUD hyperkatifeia-a prolonged negative emotional state in protracted abstinence. In C57BL/6J mice, repeated restraint stress did not alter ethanol consumption but heightened aversive behavior during abstinence. We examined the mid-insula, a key network hub for emotional regulation and stress response, as a potential mechanism driving this effect. Mid-insula GCaMP activity was higher during active stress-coping behavior, and negatively correlated with ethanol consumption, and positively correlated with GCaMP activity during the novelty-suppressed feeding test in abstinence. Next, we assessed whether stress-induced activity in the stress-sensitive insula-BNST circuit is sufficient to alter ethanol drinking behavior and abstinence-induced avoidance behavior. Chemogenetically inhibiting mid-insula-BNST neurons during stress had sex-specific effects-reducing ethanol consumption in males and abstinence-induced aversive behavior in females. Clustering analysis revealed two distinct phenotypes-one characterized by high active coping during stress, low ethanol consumption, and low avoidance behavior in abstinence, and a second cluster with the opposite pattern. Insula-BNST inhibition during stress shifted female mice toward the former cluster, but had no impact on male cluster identity. Collectively, this study implicates the insula-BNST circuit as a key mediator of stress response, stress-induced drinking, and abstinence-related affective vulnerability, positioning this circuit as a potential biomarker and therapeutic target for hyperkatifeia.