Compulsive Alcohol Drinking and Cortical Extracellular Matrix

诊断酒精使用障碍(AUD)的几个标准包括饮酒，尽管有负面后果， 意为在失业、关系受损或伤害风险增加的情况下仍决定饮酒 他自己。大脑中控制饮酒决定的一个区域是岛叶皮质。 过量饮酒引起的脑岛神经功能改变可能会导致向这种危险的过渡， 或者是抗厌恶酒精饮酒。我们的初步数据表明，特化的细胞外基质 被称为周围神经网(PNNS)的结构可能与抗厌恶饮酒有关。PNNS输入 皮质区域主要围绕着表达钙结合的快速放电的GABA能中间神经元 蛋白质小白蛋白(PV)。这些神经元对认知很重要，并严格控制 兴奋性皮质投射神经元。这项提案的首要目标是1)检查细胞和 长期酗酒后脑岛PV神经元PNNS的分子变化 以及2)确定操纵PV神经元和PNNS对PV神经元的行为后果 在岛上喝抗厌恶的酒。在具体目标1中，我们将研究PNNS的结构 用免疫细胞化学方法检测酗酒后不同时间点的变化 PNN基因和蛋白的表达，并测定其蛋白酶(MMPs和ADAMTS)的活性 金属蛋白酶家族)，调节PNNS的重构。在具体目标2中，我们将衡量以下方面的变化 狂饮后有无PNNS的PV神经元突触密度和体积 免疫细胞化学方法和超分辨显微镜。此外，我们将测量兴奋性和 用电生理学方法研究酗酒后这些神经元突触的变化。加在一起，这些 两个具体目标将提供有关PNNS中发生的变化及其 长期酗酒后的相关突触。在这项提案的第三个具体目标中， 我们将通过下调两种特定的PNN蛋白的表达来扰乱PV神经元上的PNN，这些蛋白编码于 基因acan和bcan使用病毒传递的短发夹状RNA。病毒将被直接注射到 在PV神经元中表达Cre重组酶的小鼠脑岛(PV-Cre)定位的细胞型特异性基因 击倒对手。这些小鼠将接受单独饮用乙醇和一种令人厌恶的乙醇溶液的测试 含有奎宁，以确定PNN中断对抗厌恶饮酒的影响。最后，我们会 通过设计受体的病毒传递直接操控胰岛PV神经元的活动 被特制药物(DREADD)激活以确定这些神经元在抗厌恶酒精中的作用 消费。这些研究将提供有关光伏上发生的细胞和分子变化的信息 脑岛上的神经元驱动从酗酒到高风险饮酒的转变，并可能导致新的 治疗抗厌恶饮酒的治疗策略。

Several criteria for diagnosis of alcohol use disorder (AUD) involve drinking despite negative consequences, meaning the decision to drink despite the loss of a job, damage to relationships, or increased risk of harm to oneself. One region of the brain that controls the decision to drink under conditions of risk is the insular cortex. Changes in neuronal function in the insula caused by excessive drinking may drive the transition to this risky, or aversion-resistant alcohol drinking. Our preliminary data indicates that specialized extracellular matrix structures, known as perineuronal nets (PNNs), may be involved in aversion-resistant drinking. PNNs in cortical regions primarily surround fast-spiking GABAergic interneurons that express the calcium-binding protein parvalbumin (PV). These neurons are important for cognition and tightly control the firing of excitatory cortical projection neurons. The overarching goals of this proposal are 1) to examine the cellular and molecular changes in PNNs on PV neurons in the insula after extended binge-like alcohol consumption by mice, and 2) to determine the behavioral consequences of manipulating PV neurons and PNNs on PV neurons in the insula on aversion-resistant drinking. In Specific Aim 1, we will examine the structure of PNNs at different time points after binge drinking using immunocytochemical methods, measure changes in the expression of PNN genes and proteins, and measure the activity of the proteases (MMP and ADAMTS metalloproteinase family) that regulate the remodeling of PNNs. In Specific Aim 2, we will measure changes in synaptic density and volume on PV neurons with and without PNNs after binge drinking using immunocytochemical methods and super-resolution microscopy. In addition, we will measure excitability and synaptic changes on these neurons after binge drinking using electrophysiological methods. Together, these two Specific Aims will provide important knowledge regarding the changes that occur in PNNs and their associated synapses after extended periods of binge alcohol drinking. In the third Specific Aim of this proposal, we will disrupt PNNs on PV neurons by knocking down the expression of two specific PNN proteins encoded by the genes Acan and Bcan using viral-delivered short hairpin RNAs. Viruses will be injected directly into the insula of mice expressing Cre recombinase in PV neurons (PV-Cre) for localized cell-type specific gene knockdown. These mice will be tested for consumption of ethanol alone and an aversive solution of ethanol containing quinine to determine the effect of PNN disruption on aversion-resistant drinking. Finally, we will directly manipulate the activity of insular PV neurons by viral delivery of designer receptors exclusively activated by designer drugs (DREADDs) to determine the role of these neurons in aversion-resistant ethanol consumption. These studies will provide information on the cellular and molecular changes that occur on PV neurons in the insula that drive the transition from binge drinking to high risk drinking and may lead to new therapeutic strategies to treat aversion-resistant drinking.