The incidence of post-traumatic stress disorder (PTSD) among combat-experienced Veterans is ~20%, which is substantially larger than in the general population (~3.5%). Moreover, 40-50% of Veterans suffering PTSD are also diagnosed with substance use disorders (SUDs). Patients with comorbid PTSD and SUDs have greater drug use severity and show poorer treatment outcomes than patients diagnosed with either PTSD or SUDs alone. This special need of returning combat Veterans is largely unaddressed by preclinical research. PTSD and SUDs share the DSM-V characteristic that environmental stimuli associated with a stressor or drug use can precipitate symptoms of each disorder. Repeated drug use or acute stress produce enduring changes in synaptic plasticity in a circuit containing the nucleus accumbens and ventral pallidum. Also, stress- and drug- conditioned cues elicit drug seeking and stress behaviors by inducing transient synaptic plasticity at these synapses. The first overarching hypothesis in our Merit renewal is that the neural circuits and cellular mechanisms mediating conditioned stress contribute strongly to comorbid PTSD and SUDs. Cannabis is among the addictive drugs most widely abused by Veterans. During the current Merit Award funding we used a rat model of cannabis (THC+CBD) self-administration in combination with acute restraint stress as a rodent surrogate of comorbid PTSD and CUD (Cannabis Use Disorder). By associating an odor with acute stress we studied the behavioral and neurobiological effects of conditioned stress and how drug use alters those effects. We discovered that cannabis use changes and appears to exacerbate conditioned stress behaviors. Given the high comorbidity between PTSD and CUD in Veterans, this is a clinically relevant discovery that is the focus of the renewal Merit application. We are proposing to explore the neurbiological underpinnings of how cannabis use is changing responses to stress-conditioned cues. We will focus on a brain circuit known to be critical for conditioned responses, the projection from the nucleus accumbens to ventral pallidum. Discoveries during the current Merit Award revealed that quantifying stress-cue induced synaptic changes in the canonical pre- and postsynapse of the accumbens was insufficient to understand the behaviorally relevant transient plasticity produced by stress cues. Accordingly, will also quantify signaling in the protein-rich extracellular matrix that surrounds the synapse, and changes in perisynaptic astroglial processes that regulate synaptic transmission through the patterned expression of proteins adjacent to the synaptic cleft. Together, these four synaptic compartments are referred to as the tetrapartite synapse, and the second overarching hypothesis of the Merit renewal is that cannabis use alters tetrapartite synapses in the accumbens to pallidum pathway in a manner that changes cue-induced stress responses. The proposed Aim 1 characterizes the behavioral and syna...