PROJECT SUMMARY The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), has previously been shown through genetic knockout to suppress ethanol intake, but the brain regions and protein isoforms through which this occurs remain to be identified. While the more highly expressed of the two PACAP isoforms, PACAP38, is found to affect a range of behaviors, including anxiety and depression, our recent studies focus attention on the more selectively-expressed PACAP27, which appears to have few such associations. We have found PACAP27 to be significantly more dense than PACAP38 in neurons of a key node of the limbic system, the paraventricular nucleus of the thalamus (PVT), which has a major role in pharmacologically-relevant ethanol drinking. Moreover, these PACAP27-containing neurons are particularly dense in the posterior (p) subregion of the PVT, and we have previously shown that activation of the pPVT can decrease ethanol drinking. Thus, building on published and preliminary results, we hypothesize that PACAP27 in neurons of the PVT, specifically in the pPVT, suppresses ethanol intake (Aim 1); and these effects are exerted through PACAP27 projections to the nucleus accumbens shell (NAcSh) (Aim 2). To test this, Aim 1 investigates the specific hypothesis that expression of PACAP in cells of the pPVT functions to inhibit ethanol drinking, with minimal effects on anxiety- and depressive-like behavior. To accomplish this, the proposed experiments will use an overexpression AAV or an shRNA silencing AAV approach to (1) determine the effects of increasing PACAP expression in the pPVT on ethanol drinking, (2) assess the effects of decreasing endogenous PACAP expression in the pPVT on ethanol drinking, and (3) investigate the effects of increasing PACAP expression in the pPVT on emotional behavior. Next, Aim 2 investigates the hypothesis that PACAP27 from the pPVT suppresses ethanol drinking through projections to the NAcSh. Thus, the proposed experiments will use anatomical, immunohistochemical, pharmacological, and chemogenetic techniques, to (1) identify the primary projections of PACAP27 from the pPVT, (2) determine the effects on ethanol intake of PACAP agonists and antagonists in the major projection region(s), and (3) establish if the effects of PACAP27 on ethanol intake are due to direct projections from the pPVT. Together, these proposed studies should benefit public health by offering insight into an understudied peptide isoform with few known behavioral effects, which could ultimately lead to innovative drug therapies for treating alcohol use disorder.