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DaviesDL1, Khoja S1, Huynh N1, Asatryan L1, Brodie MS2, Jakowec MW1

1University of Southern California, Los Angeles, CA USA
2 University of Illinois at Chicago, Chicago, IL USA
A growing body of literature indicates a role for P2X4 receptors (P2X4Rs) in the regulation of ethanol intake. For example, we recently reported that male P2X4 knockout (KO) mice consume greater levels of ethanol compared to wildtype (WT) controls across several drinking paradigms. Using lentiviral-mediated shRNA knockdown strategies, we and others reported that reduced expression of P2X4Rs in the nucleus accumbens (NAc) or ventral tegmental region (VTA) of rodents resulted in a significant increase in ethanol intake. To gain molecular insights into the potential mechanism linked to changes in ethanol intake, we used western blot to analyze important dopaminergic markers in the ventral striatum (NAc) of P2X4R KO and WT mice. We found that P2X4R KO mice had increased expression of D1 dopamine receptor (DA1), D2 dopamine receptor (DA2), DA transporter (DAT), and phosphorylated cyclic-AMP response element-binding protein (CREB), all of which suggests that elimination of p2rx4 leads to an increase in dopaminergic tone. To further investigate the potential role of P2X4Rs in modulating dopamine neurotransmission, we used a 6-hydroxydopamine (6-OHDA) unilateral lesion mouse model of dopamine depletion. We found that the number of L-DOPA induced contralateral rotations was significantly less in male P2X4R KO compared to WT controls. Taken together, these results suggest that genetic deletion of P2X4R induces aberration in DA homeostasis in regions important for the reinforcing effects of ethanol. In support of this hypothesis, using electrophysiological studies in acute brain slices, we found a clear interaction between P2X4Rs and ethanol in dopaminergic neurons of the VTA (DA-VTA neurons). In this work, administration of ATP acutely resulted in inhibition of spontaneous activity of DA-VTA neurons. Ethanol potently attenuated the inhibition. Further, ethanol-induced reduction of tonic inhibition produced by ambient ATP release suggesting that ATP may contribute to the magnitude of ethanol-induced excitation. Together, the findings indicate that reduction or elimination of p2xr4 significantly increases ethanol intake and that P2X4Rs plays a role in activation of DA-VTA neurons by ethanol. Overall, the findings support investigations focusing on P2X4Rs as a novel target for development for alcohol use disorder pharmacotherapies.