A dorsolateral BNST-parabrachial nucleus pathway regulates adolescent alcohol-induced negative affect in females

Adolescent alcohol use is a major predictor of alcohol use disorder in adulthood and coincides with a critical developmental window for the emergence of affective disorders. These comorbidities may heighten relapse vulnerability, particularly in females. Negative reinforcement mechanisms, in which alcohol alleviates negative emotional states, are thought to drive continued drinking. We previously demonstrated that chemogenetic stimulation of the dorsolateral bed nucleus of the stria terminalis (dlBNST) induces negative affect-like behavior in adult female mice with a history of adolescent intermittent ethanol (AIE) vapor exposure, accompanied by enhanced activation of the parabrachial nucleus (PBN), a key brainstem area involved in stress response and homeostatic regulation. Here, we show that selective chemogenetic stimulation of the dlBNST-PBN pathway is associated with increased negative affect-like behavior, with effects observed in AIE females and not detected in air controls or males. Using retrograde tracing combined with RNAscope in situ hybridization, we identified direct dlBNST projections to the PBN originating from neurons expressing both somatostatin and prodynorphin. Notably, intra-PBN kappa opioid receptor (KOR) antagonism did not block this behavioral response, suggesting that local KOR signaling is not the primary mechanism. Together, these findings identify a sex-dependent dlBNST-PBN circuit altered by adolescent alcohol exposure that contributes to persistent negative affect in adulthood.