Receptor variant-based changes in the role of PACAP in the nucleus accumbens during the transition to ethanol dependence - PROJECT SUMMARY Stress-related systems undergo a change during the transition to ethanol dependence, such that activation, which in a non-dependent state may leave unaffected or suppress intake, instead increases consumption. This effect reversal often coincides with an upregulation in receptor gene expression for stress-related neuropeptides; however, for the stress-related neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), the changes instead appear to occur via a specific increase in receptor variant expression. Our study focuses attention on the paraventricular nucleus of the thalamus (PVT), which has notably and selectively dense expression of the less ubiquitous PACAP peptide isoform, PACAP-27, where it is expressed in a subpopulation of glutamatergic neurons. In preliminary studies, we have found with a 20% ethanol intermittent access (IA) model of binge drinking, that effects of PACAP+ cell manipulation in the PVT can change based on drinking history. In PACAP-Cre mice drinking under the IA model for 6 weeks, Cre-dependent chemogenetic inhibition of PACAP+ cells stimulated ethanol drinking in low drinkers but instead inhibited intake in high drinkers. With preliminary evidence that these PACAP-27+ PVT neurons send dense projections to the nucleus accumbens (NAc), we have also found in rats, which drink lower levels of ethanol than mice, that those drinking under the IA model for 6 weeks respond to PACAP-27 injection into the NAc with a suppression of ethanol intake. Further, rats with a longer IA drinking history (10 weeks) show a specific increase in gene expression in the NAc of the HOP variant of the PACAP receptor (PAC1). We have confirmed with quantitative real-time (qRT-)PCR that the HOP and SHORT variants are present in the NAc of mice. Building on our preliminary and published data, we hypothesize that activation of PACAP-27+ cells that send afferents from the PVT to the NAc suppresses non- dependent, binge-like ethanol intake, but increases intake in a dependent state (Aim 1); and this shift in behavioral output occurs as the PACAP system becomes dysregulated resulting in a specific increase in PAC1 HOP receptor variant expression (Aim 2). To test this, Aim 1 investigates the effect of activation and inhibition of the PVT→NAc PACAP pathway on ethanol intake before and after dependence, by using PACAP-Cre mice and Cre-dependent excitatory and inhibitory DREADDs injected into the PVT paired with cannula guided microinjections of CNO into the NAc shell. To determine the involvement of PAC1 receptor variants in the NAc on ethanol intake, Aim 2 will measure PAC1 variant mRNA expression in the NAc of non-dependent and ethanol dependent mice and also use specific interfering (si)RNA to knock down the PAC1 receptor variants in the NAc of non-dependent and dependent mice. Together, these Aims will determine how the PACAP+ PVT→NAc pathway affects ethanol drinking across states and how PAC1 variant populations in the NAc are changed with the transition to dependence and, in turn, affect ethanol intake.
