Neuronal mechanisms mediating the effects of chronic alcohol consumption on sleep homeostasis.

酒精使用障碍(AUD)是一种慢性复发性大脑疾病，以强迫和过度为特征 饮酒是导致过早残疾和死亡的主要可预防原因。失眠与相关睡眠 骚乱是澳元最严重和最持久的症状之一。事实上，主观的和客观的 睡眠障碍的指标是复发的预测指标。因此，虽然两者之间存在直接关系 失眠和AUD，其潜在的病理生理机制还不是很清楚。有证据表明，睡眠 动态平衡机制[基底前脑(BF)的腺苷能/胆碱能机制]可能起着至关重要的作用 在调节酒精对睡眠的影响方面的作用。因此，我们假设长期饮酒直接 间接地，通过神经炎症，扰乱睡眠平衡，导致失眠和睡眠障碍。 我们将使用野生型(C57BL/6J)和转基因ChAT-cre(Cre重组酶在 胆碱能神经元)，并将它们暴露在常见的酒精消费适应不良模式中：慢性 间歇性访问两瓶选择范式(IA2BC)。一种多学科技术的新组合 包括体内钙成像、药物遗传学、病毒介导的基因过度表达、微透析和 将使用局部药物操作。我们设计了三个目标来检验我们的假设。 在目标1中，将进行结合睡眠觉醒的电生理监测的实时Ca+2成像 探讨胆碱能神经元活动、觉醒与慢性饮酒的关系。我们 预测暴露于IA2BC的小鼠将以不断升级的模式饮酒，并显示出阳性 胆碱能神经元活动、睡眠动态平衡紊乱与睡眠紊乱严重程度的关系。 而药物对BF胆碱能神经元的沉默将减弱慢性酒精和 使睡眠正常化，药物遗传激活会减弱急性酒精的促进睡眠作用。 目标2将研究长期饮酒对睡眠稳态的影响。我们预测酒精会 将下调MAPK信号级联和乙酰化组蛋白，以减少平衡蛋白的表达 核苷转运体1(ENT1)和腺苷A1受体(A1R)。2)病毒介导的过表达 BF中ENT1的表达将减弱酒精对睡眠稳态的影响，提高睡眠质量和数量 睡着了。3)局部输注曲古抑素-A(组蛋白去乙酰化酶抑制剂)将通过上调 乙酰化组蛋白以及ENT1和A1R在BF中的表达，而不影响MAPK信号转导。 目标3将研究慢性酒精诱导的BF神经炎症及其对睡眠稳态的影响。 我们的预测是，选择性阻断1)米诺环素的神经炎性改变，2)Toll样 BF中TAK-242受体和/或ABT-702受体对慢性酒精诱导的抑制作用 神经炎症；减少BF中的胶质细胞和细胞因子水平，并使睡眠稳态指数正常化 以及睡不着觉。

Alcohol use disorder (AUD), a chronic relapsing brain disorder, characterized by compulsive and excessive alcohol use, is a leading preventable cause of premature disability and death. Insomnia and associated sleep disturbances are amongst the most severe and protracted symptoms of AUD. In fact, subjective and objective indicators of sleep disturbances are predictors of relapse. Thus, while there is a direct relationship between insomnia and AUD, the underlying pathophysiology is not well understood. Evidence suggest that sleep homeostatic mechanism [adenosinergic/cholinergic mechanisms in the basal forebrain (BF)] may play a crucial role in mediating the effects of alcohol on sleep. Hence, we hypothesize that chronic alcohol consumption directly and indirectly, via neuroinflammation, disrupts sleep homeostasis leading to insomnia and sleep disturbances. We will use wild type (C57BL/6J) and transgenic ChAT-cre (expression of Cre-recombinase exclusively in cholinergic neurons) mice and expose them to the common maladaptive pattern of alcohol consumption: chronic intermittent access two-botte choice paradigm (IA2BC). A novel combination of multidisciplinary techniques including in vivo calcium imaging, pharmacogenetics, viral-mediating gene overexpression, microdialysis and local pharmacological manipulations will be used. Three aims are designed to test our hypothesis. In Aim 1, live Ca+2 imaging coupled with electrophysiological monitoring of sleep-wakefulness will be performed to examine the relationship between cholinergic neuronal activity, wakefulness and chronic alcohol intake. We predict that mice exposed to IA2BC will consume alcohol in an escalating pattern and display a positive relationship between cholinergic neuronal activity, disrupted sleep homeostasis and severity of sleep disruptions. While pharmacogenetic silencing of BF cholinergic neurons will attenuate the effects of chronic alcohol and normalize sleep, pharmacogenetic activation will attenuate sleep-promoting effects of acute alcohol. Aim 2 will examine the effects of chronic alcohol consumption on sleep homeostasis. We predict that 1) alcohol will downregulate MAPK signaling cascade and acetylated histones, to reduce the expressions of equilibrative nucleoside transporter 1 (ENT1) and adenosine A1 receptor (A1R) in the BF. 2) Virally mediated overexpression of ENT1 in the BF will attenuate the effects of alcohol on sleep homeostasis and improve the quality and quantity of sleep. 3) Local infusion of trichostatin-A (histone deacetylase inhibitor) will normalize sleep by upregulation of acetylated histones along with the expression of ENT1 and A1R in the BF, without affecting MAPK signaling. Aim 3 will examine chronic alcohol induced neuroinflammation in the BF and its effect on sleep homeostasis. Our predictions are that selective blockade of 1) neuroinflammatory changes by minocycline, 2) Toll-like receptors by TAK-242 and/or c) adenosine kinase by ABT-702, in the BF will attenuate chronic alcohol induced neuroinflammation; reduce gliosis and cytokine levels in the BF and normalize the indices of sleep homeostasis and sleep-wakefulness.