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在戒酒后努力保持清醒。 在早期禁欲期间出现的症状。尽管如此，潜在症状的神经机制 人们对撤军还没有很好的理解。被盖旋转内侧核(RMTg)是GABA能区 在回避行为、厌恶信号、疼痛以及更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急性戒断期间RMTg过度活动的证据 间歇性乙醇(CIE)暴露。此外，我们还证明了RMTg的药理抑制作用 减轻戒断引起的焦虑样行为。驱动RMTg介导的戒断反应的传入 症状目前尚不清楚。然而，之前发表的研究表明，外侧缰核与 (LHB)-一个为RMTg-in戒断诱导的负面影响提供密集输入的区域，类似于我们的 在RMTG中工作。此外，我们的初步数据显示，在RMTg投射的LHb中显著诱导CFO 与空气对照组相比，暴露于CIE的大鼠在急性戒断过程中的神经元。有趣的是，LHB的负担很重 富含MU-阿片受体(MORS)，已知它既能调节疼痛，又能调节情感。OPRM1，基因 编码MOR的基因在患有AUD的个体中被高度甲基化，这提示了一种潜在的机制 慢性酒精暴露会降低OPRM1的表达。然而，戒断对MORS的影响 LHB尚不清楚。总而言之，这些数据使我们假设LHb传入RMTG 与慢性酒精戒断和OPRM1基因表观遗传失调有关 可能对这一机制做出贡献。三个具体的目标将被用来检验这一假设。在AIM 1中，体内 化学遗传学将被用来确定选择性抑制RMTg投射的LHb神经元是否逆转 戒断症状。OPRM1的表达和甲基化水平将在RMTg投射的LHb中进行评估 用荧光原位杂交和甲基米纳检测AIM 2中的神经元。痕迹追踪将与EX相结合 Aim 3体内全细胞膜片钳切片电生理学研究戒断后大鼠脑内神经递质的变化 RMTg投射的LHb神经元中MOR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方面的新培训，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.