Role of GluN2A and MMPs in the CeA in Dependence-Induced Escalation of Etoh Drinking

项目摘要 美国人的合法地位和普遍饮酒与有害健康有关 对青少年和成年人都有影响。近88%的美国人至少饮酒一次 在他们的一生中，酗酒和酗酒的比率仍然令人担忧。药理作用 酒精滥用的治疗效果有限，FDA只批准了三种药物用于 治疗酒精依赖。这个问题背后的一个因素是缺乏对机制的了解 这会导致过度饮酒。我们之前研究的最新结果表明，敲入小鼠 表达乙醇抗性GluN2AN-甲基-D-天冬氨酸受体(NMDAR)的饮料与野生型相同 小鼠处于基线状态，但与野生型小鼠不同，小鼠在以下情况下没有增加饮酒 反复的慢性间歇性酒精暴露。这些发现表明，GluN2ANMDAR在 在酒精依赖受试者中观察到的饮酒失控的关键作用。重要的是，类似的缺失 在服用锌依赖基质抑制剂后，观察到饮酒增加 金属蛋白酶(MMPs)无论是I.C.V.或直接进入杏仁中央核(CEA)。MMP是关键 NMDA介导的突触可塑性的介体，它们重塑细胞外基质以支持 谷氨酸能树突棘的新生长和扩大。在本提案中，我们测试了最重要的 假设CEA中的MMPs和乙醇敏感的GluN2ANMDARs在转变过程中起关键作用 大量饮酒是酒精依赖的一个标志。在目标1中，我们使用活体切片电生理学 酶谱和蛋白质印迹分析验证CIE诱导CEA神经信号通路改变的假说 而GluN2a基因敲除小鼠的脑组织中则缺乏基质金属蛋白酶的活性。在目标2中，我们使用表达AAVs和GUIDE的Crispr 靶向GluN2A基因的RNA验证CIE导致饮酒和基质金属蛋白酶增加的假设 活动需要CEA中具有功能的GluN2ANMDAR。来自这些新颖研究的发现将提供关键的 支持未来R01拨款申请的数据侧重于确定哪些CEA细胞类型推动了改变 GluN2A敲入小鼠的饮酒表型及其在其他关键成瘾中的表达 大脑区域对这种效应也有贡献。

Project Summary The legal status and widespread use of alcohol among the US population is associated with adverse health outcomes in both adolescents and adults. Nearly 88% of the US population have used alcohol at least once during their lifetime and rates of binge drinking and heavy alcohol use continue to be of concern. Pharmacological treatments for alcohol abuse show limited effectiveness and only three medications are FDA approved for treating alcohol dependence. One factor that underlies this problem is a lack of understanding of the mechanisms that contribute to excessive drinking. Recent results from our previous studies show that knock-in mice expressing ethanol resistant GluN2A N-methyl-D-aspartate receptors (NMDARs) drink the same as wild-type mice under baseline conditions but, unlike their wild-type counterparts, fail to escalate their drinking following repeated cycles of chronic intermittent ethanol exposure. These findings suggest that GluN2A NMDARs play a key role for in the loss of control over drinking observed in alcohol dependent subjects. Importantly, a similar lack of escalated drinking is observed following administration of inhibitors of zinc-dependent matrix metalloproteinases (MMPs) either i.c.v. or directly into the central nucleus of the amygdala (CeA). MMPs are key mediators of NMDA-mediated forms of synaptic plasticity where they remodel the extracellular matrix to support new growth and enlargement of glutamatergic dendritic spines. In this proposal we test the overarching hypothesis that MMPs and ethanol-sensitive GluN2A NMDARs in the CeA are critically involved in the transition to heavy drinking that is a hallmark of alcohol dependence. In Aim 1, we use slice electrophysiology, in vivo zymography and western blot analysis to test the hypothesis that CIE-induced changes in CeA neural signaling and MMP activity are absent in the GluN2A knock-in mice. In Aim 2, we use Crispr expressing AAVs and guide RNAs targeting the GluN2A gene to test the hypothesis that the CIE-induced increases in drinking and MMP activity requires functional GluN2A NMDARs in the CeA. The findings from these novel studies will provide critical data to support a future R01 grant application focused on determining which CeA cell types drive the altered drinking phenotype of the GluN2A knock-in mice and whether their expression in other key addiction-related brain areas contribute to this effect.