Drug use causes the formation of strong cue/reward associations which persist long after cessation of drug-taking and contribute to the long-term risk of relapse. Breaking these associations is an important goal in the treatment of substance use disorders. Extinction is a form of learning that inhibits behavioral responses to a learned stimulus. Modulating extinction processes and consolidating the newly-formed memories has clinical potential to reshape maladaptive behavior and to prevent relapse. We have recently shown that vagus nerve stimulation (VNS) facilitates extinction learning and reduces cue-induced reinstatement of drug-seeking in cocaine self-administering rats. These changes correlate with altered activity in a network that centers on the infralimbic (IL) cortex. Our preliminary data also show that drug-taking and reinstatement reduce AMPA-receptor currents at layer 5 IL pyramidal neurons, and that VNS reverses these changes. Systemic blockade of TrkB receptors for the brain-derived neurotrophic factor (BDNF) during extinction abolishes VNS’ effects on cue-induced reinstatement and on glutamatergic transmission in the IL. We hypothesize that extinction training reverses drug-induced changes in synaptic AMPARs in the IL. Pairing extinction with VNS leads to (additional) BDNF release which consolidates these changes and reduces reinstatement. In this application we will use a combination of behavioral, electrophysiological, and celltype-specific morphological analyses to 1) further investigate the time- and circuit-specific dysfunctional neuroadaptations in the IL that contribute to drug-seeking and relapse, and 2) to determine the mechanisms through which VNS consolidates extinction memory to reduce relapse. Experiments in Aim 1 will use patch-clamp electrophysiology and optogenetic stimulation of afferents from the basolateral amygdala (BLA) to the IL to determine how these inputs are altered by drug-seeking, extinction, and VNS. We will perform voltage-clamp recordings from 2 types of IL projection neurons (to the BLA and Nucleus accumbens shell) and determine VNS-induced changes in postsynaptic glutamate receptor function and presynaptic release during extinction and reinstatement. These experiments will be supported with morphological analyses in the same type of IL projection neurons to determine VNS-induced changes specifically in cells that were activated by reinstatement (labeled by the activity marker pCREB) to test our hypothesis that VNS preferentially modulates networks relevant in behaviors paired with VNS. In Aim 2 we will determine whether the VNS-induced consolidation of extinction depends on endogenous BDNF levels in the IL or its inputs. To this end we will use CRISPR/Cas9 to knock down BDNF produced either from cells within the IL or from cells in the hippocampus that project to the IL, respectively, in order to determine how VNS-evoked BDNF modulates extinction and reinstatement. Experiments in Aim 3 will determine...