Role of afferents to the rostromedial tegmental nucleus in symptoms of withdrawal from chronic ethanol exposure

摘要 相当数量的酒精使用障碍患者由于戒断而难以保持清醒 早期禁欲时出现的症状尽管如此，神经机制的潜在症状， 退出并不容易理解。头内侧被盖核（RMTg）是GABA能区域， 在回避行为，厌恶信号，疼痛，以及重要的酒精相关行为中起着关键作用。 我们实验室最近的工作提供了在慢性戒断反应急性戒断期间RMTg活动过度的证据。 间歇性乙醇（CIE）暴露。此外，我们发现，RMTg的药理学抑制作用 减弱戒断引起的焦虑样行为。驱动RMTg介导的戒断的传入神经 目前症状不明。然而，先前发表的研究表明， (LHb)- 一个向RMTg提供密集输入的区域-在戒断诱导的负面影响中，类似于我们的 在RMTG工作。此外，我们的初步数据显示，在RMTg投射的LHb中， 与AIR对照相比，CIE暴露大鼠急性戒断期间的神经元。有趣的是，LHb严重 富含μ-阿片受体（MORs），已知其调节疼痛和情感。Oprm 1基因 编码莫尔的基因在患有AUD的个体中被高甲基化，这提示了一种潜在的机制， 慢性乙醇暴露导致Oprm 1表达降低。然而，退出对 LHb不清楚。总之，这些数据使我们假设，LHb传入RMTg是 机械地参与慢性乙醇戒断和Oprm 1基因的表观遗传失调 可能有助于这种机制。三个具体目标将被用来测试这一假设。在目标1中，体内 化学遗传学将用于确定是否选择性抑制RMTg投射LHb神经元逆转 戒断症状。将在RMTg投射LHb中评估Oprm 1表达和甲基化水平 用荧光原位杂交和MethylMiner.道追踪将与前 体内全细胞膜片钳切片电生理目的3研究戒断诱导的变化， RMTg投射LHb神经元中莫尔受体介导的放电的表观遗传调节。这些研究将 为申请人提供尖端神经科学技术的新培训，同时提供新的见解 LHb-RMTg回路在乙醇戒断中的作用。

ABSTRACT A significant number of individuals with alcohol use disorder struggle to maintain sobriety as a result of withdrawal symptoms that emerge during early abstinence. Despite this, the neural mechanisms underlying symptoms of withdrawal are not well understood. The rostromedial tegmental nucleus (RMTg) is a GABAergic region that plays a critical role in avoidance behavior, aversive signaling, pain, and importantly, alcohol-related behaviors. Recent work from our lab provides evidence of RMTg hyperactivity during acute withdrawal from chronic intermittent ethanol (CIE) exposure. In addition, we showed that pharmacological inhibition of the RMTg attenuates withdrawal-induced anxiety-like behavior. The afferents that drive RMTg-mediated withdrawal symptoms are currently unknown. However, previously published studies have implicated the lateral habenula (LHb) – a region that provides dense input to the RMTg – in withdrawal-induced negative affect similar to our work in the RMTg. In addition, our preliminary data revealed significant cFos induction in RMTg-projecting LHb neurons during acute withdrawal in CIE-exposed rats compared to AIR controls. Interestingly, the LHb is heavily enriched in mu-opioid receptors (MORs), which are known to regulate both pain and affect. Oprm1, the gene that encodes MOR, is hypermethylated in individuals with AUD suggestive of a potential mechanism by which chronic ethanol exposure drives decreases in Oprm1 expression. However, the effect of withdrawal on MORs in the LHb is unclear. Together, these data lead us to hypothesize that LHb afferents to the RMTg are mechanistically involved in withdrawal from chronic ethanol and that epigenetic dysregulation of the Oprm1 gene may contribute to this mechanism. Three specific aims will be used to test this hypothesis. In Aim 1, in vivo chemogenetics will be used to determine whether selective inhibition of RMTg-projecting LHb neurons reverses symptoms of withdrawal. Oprm1 expression and methylation levels will be assessed in RMTg-projecting LHb neurons in Aim 2 using fluorescent in situ hybridization and MethylMiner. Tract tracing will be combined with ex vivo whole-cell patch-clamp slice electrophysiology in Aim 3 to investigate withdrawal-induced changes in epigenetic regulation of MOR receptor-mediated firing in RMTg-projecting LHb neurons. These studies will provide the applicant with new training in cutting-edge neuroscience techniques while also providing new insight into the role of the LHb-RMTg circuit in ethanol withdrawal.