Project Summary/Abstract Polysubstance use (PSU) is extremely common, with the majority of substance use treatment seekers reporting the use of multiple substances. Not only is the prevalence of PSU high, but the consequences of PSU are also thought to be worse than for individuals who use single substances. One of the most common substances to be used with any other drug of abuse is nicotine. Marijuana and nicotine co-use is particularly common, with higher overall incidence of marijuana use in the general population and up to 80% of marijuana users reporting that they also use nicotine. Recent epidemiological studies indicate that the co-use of nicotine with THC containing products is increasing and those that use both drugs at the same time have worse overall outcomes than those individuals that consume both, but on separate occasions. Thus, there is a need to increase our understanding of the neural mechanisms affected by interactions between nicotine and THC. In ongoing studies, we have developed procedures for understanding how nicotine influences intravenous THC self-administration in rats. We have found that nicotine acutely and reversibly enhances the amount of THC self-administered, and in concurrent choice procedures have found that nicotine availability enhances acquisition of THC self- administration and leads to preference for THC over nicotine. In the present proposal we aim to determine the mechanisms by which nicotine enhances the reinforcing properties of THC and the consequences of concurrent nicotine and THC self-administration on the function of dopamine (DA) neurons in the ventral tegmental area (VTA). In aim 1 we will use dual-color fiber photometry in the VTA and nucleus accumbens shell (NAc shell) to determine the effects of nicotine on THC-induced DA neuron activity and DA release. In aim 2, we will use the concurrent choice procedure to determine if nicotine-enhanced THC self-administration is mediated via actions at α7 and/or β2/4 subunit containing nicotinic acetylcholine receptors (NAchRs) using specific antagonists. We will further determine if either antagonist affects nicotine-THC mediated changes in DA neuron activity or DA release. Finally, in aim 3, we will use a novel method for performing cell-type specific, mass spectrometry-based, proteomic and phosphoproteomic analysis to determine the effects of nicotine-THC self-administration on DA neuron function relative to either substance alone. Proximity labeling of proteins in DA neurons will be accomplished through Cre-dependent viral expression of the APEX construct in TH-Cre rats. At the conclusion of the proposed experiments, we will have determined: 1) if nicotine enhances the reinforcing effects of THC through promotion of increased DA neuron activity and DA release in the NAc shell, 2) which NAchRs are responsible for the enhanced reinforcement, and 3) what are the protein signaling consequences of co-self- administration of the two substances on DA neuron ...