Receptor variant-based changes in the role of PACAP in the nucleus accumbens during the transition to ethanol dependence

项目总结 应激相关系统在向酒精依赖的转变过程中经历了变化， 这在非依赖状态下可能不受影响或抑制摄取，而是增加消耗。这 效应逆转通常与应激相关神经肽受体基因表达上调相一致； 然而，对于应激相关神经肽，垂体腺苷环化酶激活多肽(PACAP)， 相反，变化似乎是通过受体变体表达的特定增加而发生的。我们的研究重点是 注意丘脑室旁核(PVT)，它具有显著的和选择性的致密 不太普遍的PACAP多肽亚型PACAP-27的表达，其中它在一个亚群中表达 谷氨酸能神经元。在初步研究中，我们发现用20%的乙醇间歇进入(IA) 狂饮模型：PVT中PACAP+细胞调控效应可因饮酒而改变 历史。在IA模型下饮用6周的PACAP-Cre小鼠，Cre依赖的化学发生抑制 PACAP+细胞刺激了低饮酒者的酒精饮用，但却抑制了高饮酒者的摄入。使用 初步证据表明，这些PACAP-27+PVT神经元向伏核发出密集的投射 (NAC)，我们还在大鼠身上发现，与小鼠相比，大鼠喝的酒精水平更低，那些在 持续6周的IA模型对NAc注射PACAP-27的反应是抑制乙醇摄取。此外， 饮酒史较长的大鼠(10周)在NAC中的基因表达显著增加 PACAP受体的HOP变异体(PAC1)。我们已经用定量实时(qRT-)聚合酶链式反应证实 HOP和Short变种存在于小鼠的NAC中。根据我们的初步和公布的数据，我们 假设从PVT向NAC发送传入信号的PACAP-27+细胞的激活抑制了非 依赖的、暴饮暴食的乙醇摄入量，但在依赖状态下增加摄入量(目标1)； 当PACAP系统变得失调导致PAC1的特定增加时，行为输出就会发生 HOP受体变异体表达(AIM 2)。为了测试这一点，目标1调查了激活和抑制的效果 依赖前后PVT-→-NAC-PACAP通路对酒精摄入量的影响 和CRE依赖的兴奋性和抑制性DREADDS在插管引导下注射到PVT内 向NAC壳内微量注射CNO。确定PAC1受体变异体在NAC中的参与 在酒精摄入方面，Aim 2将检测非依赖和乙醇NAC中PAC1变体mRNA的表达 并使用特异性干扰(Si)RNA敲除NAC中的PAC1受体变体 非依赖型和依赖型小鼠。这些目标加在一起将决定PACAP+PVT→NAC 影响跨州饮酒的途径以及NAC中PAC1变异群体如何随 向依赖的转变，进而影响乙醇的摄入量。

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.