Stress ensemble in the BNST mediates comorbid anxiety and sleep disruption

Anxiety and insomnia are highly comorbid; however, the underlying brain-circuit mechanisms remain inadequately explored. Here, we identify a dual-functional ensemble within the bed nucleus of the stria terminalis (BNST) that modulates both anxiety-like behaviors and insomnia through distinct neural circuits. Specifically, a subset of BNST neurons exhibits enhanced activity in response to anxiety-related cues and during wakefulness. Activation of these ensemble neurons exacerbates anxiety-like behaviors and reduces non-rapid eye movement (NREM) sleep in chronic restraint stress (CRS) mice. Conversely, inactivation of these neurons alleviates anxiety-like behaviors and promotes NREM sleep. In addition, anxiolytic-hypnotic medication attenuates BNST stress ensemble hyperactivity, mitigating both phenotypes. Furthermore, BNST stress ensembles project divergently to the dorsal raphe nucleus (DRN) and the preoptic area (POA): DRN-projecting neurons selectively modulated anxiety, whereas POA-projecting neurons specifically regulated sleep-wake states. These findings suggest that hyperactive BNST stress ensembles are responsible for inducing anxiety-like behaviors and disrupting sleep in CRS mice and achieve the modality specificity through their divergent downstream targets.