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

CeA 中 GluN2A 和 MMP 在 Etoh 饮酒依赖性升级中的作用

• 批准号: 10525274
• 负责人: JOHN J. WOODWARD
• 金额: $ 21.71万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-02 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10525274
• 关键词: Acute; Adolescent; Adult; Air; Alcohol consumption; Alcohol dependence; Alcohol withdrawal syndrome; Alcohols; Amino Acids; Amygdaloid structure; Animals; Anxiety; Applications Grants; Area; Bilateral; Brain; CRISPR/Cas technology; Calcium-Sensing Receptors; Cannulas; Cell Nucleus; Chronic; Clinical Research; Data; Dendritic Spines; Dependence; Effectiveness; Electrophysiology (science); Ethanol; Extracellular Matrix; FDA approved; Female; Fiber; Future; Gelatinase B; Gelatinases; Genes; Glutamates; Guide RNA; Health; Heavy Drinking; Human; Implant; Individual; Infusion procedures; Intoxication; Ion Channel Gating; Knock-in Mouse; Knock-out; Laboratories; Learning; Legal Status; Ligands; Link; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Measures; Mediating; Mediator of activation protein; Memory; Molecular; Mus; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NMDA receptor A1; Neurons; Outcome; Permeability; Pharmaceutical Preparations; Pharmacological Treatment; Phenotype; Photometry; Play; Population; Proteins; Resistance; Rewards; Role; Signal Transduction; Single Nucleotide Polymorphism; Slice; Synapses; Synaptic plasticity; Testing; Time; Transgenic Mice; Transmembrane Domain; Vertebral column; Viral; Virus; Western Blotting; Wild Type Mouse; Withdrawal; Work; Zinc; addiction; alcohol abuse therapy; alcohol exposure; alcohol measurement; alcohol sensitivity; alcohol use disorder; base; binge drinking; cell type; cohort; drinking; drinking behavior; effective therapy; endonuclease; in vivo; inhibitor; knock-down; learned behavior; male; negative affect; neurotransmission; new growth; novel; postoperative recovery; preclinical study; prevent; recruit; reduce symptoms; social

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.

项目摘要 美国人口中酒精的法律的地位和广泛使用与不良健康有关 青少年和成人的结果。近88%的美国人至少喝过一次酒。 酗酒和大量饮酒的比率仍然令人关切。药理 酒精滥用的治疗效果有限，只有三种药物被FDA批准用于 治疗酒精依赖造成这一问题的一个因素是缺乏对机制的了解 会导致过量饮酒我们之前的研究结果表明，基因敲入小鼠 表达乙醇抗性GluN 2A N-甲基-D-天冬氨酸受体（NMDAR）的小鼠与野生型相同 但与野生型小鼠不同， 慢性间歇性酒精暴露的重复循环。这些发现表明，GluN 2A NMDAR发挥着重要作用。 在酒精依赖受试者中观察到的饮酒失控中的关键作用。重要的是，类似的缺乏 在给予锌依赖性基质抑制剂后， 通过i. c. v.或直接将金属蛋白酶（MMPs）导入杏仁核中央核（CeA）。MMPs是关键 NMDA介导的突触可塑性形式的介质，其中它们重塑细胞外基质以支持 新的生长和扩大的多巴胺能树突棘。在本提案中，我们测试了 假设CeA中的MMPs和乙醇敏感的GluN 2A NMDAR关键地参与了转换 酗酒是酒精依赖的标志在目标1中，我们使用切片电生理学，在体内 酶谱分析和蛋白质印迹分析来检验CIE诱导的CeA神经信号传导变化的假设 和MMP活性在GluN 2A敲入小鼠中不存在。在目标2中，我们使用Crispr表达AAV并指导 靶向GluN 2A基因的RNA以检验CIE诱导的饮酒和MMP增加的假设。 活性需要CeA中的功能性GluN 2A NMDAR。这些新研究的发现将提供关键的 数据，以支持未来的R 01赠款申请，重点是确定哪些CeA细胞类型驱动改变 GluN 2A基因敲入小鼠的饮酒表型及其表达是否与其他关键成瘾相关 大脑区域有助于这种效果。