Pairing extinction training with vagus nerve stimulation reduces drug-seeking by altering activity in afferents to the medial prefrontal cortex.

Introduction: Relapse triggered by drug-associated cues remains a major challenge in treating substance use disorders, as extinction learning is often weak and context dependent. Vagus nerve stimulation (VNS) enhances learning-related plasticity and, when paired with extinction training, reduces cue-induced reinstatement of cocaine seeking; however, the underlying circuit mechanisms remain unclear. Methods: We examined how VNS paired with extinction reshapes medial prefrontal cortex (mPFC) networks that regulate drug seeking, focusing on afferent inputs from the anterior (aPVT) and posterior (pPVT) paraventricular thalamus (PVT), posterior basolateral amygdala (BLA), and ventral hippocampus (vHPC). Using retrograde viral tracing combined with cFos immunolabeling, we identified pathway-specific effects of VNS on neurons projecting to the infralimbic (IL) and prelimbic (PL) cortex. Results: In the aPVT, VNS selectively increased activation of IL-projecting neurons, whereas in the pPVT it increased activity in PL-projecting neurons. VNS reduced overall BLA cFos expression, decreasing activation of BLA→IL projections while increasing BLA→PL activity. In the vHPC, VNS selectively reduced activation of IL-projecting neurons. Within the mPFC, VNS decreased overall neuronal activity but bidirectionally regulated parvalbumin interneurons, increasing their activity in PL and decreasing it in IL. Discussion: These findings show that pairing extinction with VNS remodels extinction circuits through projection- and cell-type-specific mechanisms, providing a framework for how VNS strengthens extinction learning and reduces relapse-like behavior.