Spicer MM1,2, Weber MA3, Yang J1, Stewart AM6, Luo Z5, Yang J1, Narayanan NS3,4, and Fisher RA1,2,4. 1Department of Neuroscience & Pharmacology, 2Interdisciplinary Graduate Program in Molecular Medicine, 3Department of Neurology, 4Iowa Neuroscience Institute, 5Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242 6Department of Biomedical Sciences, Florida Atlantic University Brain Institute, Florida Atlantic University, Boca Raton, FL 33431 Alcohol (EtOH) is the most commonly abused drug worldwide and chronic EtOH consumption often leads to development of dependence and abuse. EtOH dependence is characterized by progressive, neuroadaptive changes to the brain's reward system resulting from chronic EtOH exposure. The neurobiological mechanisms underlying EtOH-seeking behavior and dependence are not fully understood, resulting in limited therapeutics. Currently, abstinence remains the only effective way to prevent alcohol use disorders (AUDs). We previously discovered that loss of RGS6 in mice leads to reduced EtOH seeking and reward behaviors. GABAB and D2 autoreceptor (D2R) antagonism partially reduced EtOH seeking in RGS6-/- mice, whereas singular inhibition of the dopamine (DA) transporter (DAT) completely reversed this effect. Moreover, RGS6-/- mice exhibited reduced striatal DA content. These findings led us to hypothesize that RGS6 promotes mesolimbic DA neurotransmission by suppressing GPCR-Gαi/o-mediated upregulation of DAT. To investigate this possibility, we examined the effects of RGS6 loss on DAT expression in VTA cell bodies and synaptic terminals in the nucleus accumbens (NAcc). In addition, we examined the effects of RGS6 loss on mesolimbic neuronal activity and DA transmission using c-Fos staining and EtOH-induced release of DA from VTA neurons, respectively. We found robust RGS6 expression in VTA cell bodies and terminals, where its loss led to upregulated expression of DAT. Additionally, RGS6 loss reduced VTA neuronal activity as measured by c-Fos immunostaining. These findings suggest that RGS6 functions to promote EtOH seeking and reward behavior by promoting DA transmission in the mesolimbic circuit by suppressing GPCR-Gαi/o signaling, thereby promoting VTA neuronal activity and suppressing DAT upregulation.