PROJECT SUMMARY/ABSTRACT Cannabis is the most frequently used illicit drug in the United States. Onset of cannabis use usually occurs during adolescence, which represents a vulnerable period in neurobiological development. Due to uncertainty surrounding the long-term consequences of adolescent cannabis exposure, there is mounting concern that relaxing legal restrictions will result in increased adolescent use. Approximately 30% of users develop cannabis use disorder (CUD), and nearly half of all regular cannabis users develop cannabis withdrawal syndrome which is marked by negative affective symptoms and craving that may drive relapse. During abstinence craving may increase over time or “incubate” resulting in a period of heightened vulnerability to relapse in response to drug-related cues. Withdrawal symptoms during abstinence are clinically significant because they may impact efforts to reduce cannabis use, promote use of other substances of abuse, and adversely influence treatment outcomes. Currently there are no approved pharmacotherapies for CUD. The primary objective of this proposal is to determine how chronic cannabinoid use in adolescence impacts glutamatergic transmission and plasticity in the nucleus accumbens (NAc), and the contribution of these adaptations to later relapse-like behavior and craving. Our central hypothesis is that cannabinoid self- administration and withdrawal induces changes in glutamate transmission and synaptic connectivity in the NAc that promote negative affective processes and ultimately trigger relapse. To test this hypothesis we will use behavior, whole mount immunohistochemistry and light sheet microscopy, optogenetic aided neurophysiology, and chemogenetic manipulation techniques to assess the involvement of pathway specific glutamatergic neuroplasticity in cannabis seeking. We will use a model of adolescent intravenous D9-tetrahydrocannabinol (THC) self-administration and withdrawal to confirm the presence of an incubation of drug seeking effect at an intermediate or protracted withdrawal time point. We will assess somatic and non-somatic symptoms of spontaneous withdrawal based on alignment with DSM-5 criteria and the Research Domain Criteria framework. Unbiased mapping of whole brain c-Fos immunoreactivity will be used to identify regional neural activation associated with cannabinoid withdrawal and cue-reactivity. We will examine input-specific cannabinoid withdrawal-induced plasticity at glutamatergic synapses in identified D1 and D2 medium spiny neurons in NAc core and shell coupling in vitro slice electrophysiology with optogenetically evoked EPSCs. Finally, we will utilize chemogenetic neuromodulation to test the importance of one of these circuits for regulating relapse-like behavior and endophenotypes of withdrawal. These studies will inform our understanding of the mechanisms underlying cannabis withdrawal syndrome, an important and understudied facet of CUD, and how glutamate transmission and sy...