Desensitization and Internalization of Mu-Opioid Receptors in the Locus Coeruleus

描述（由申请人提供）：阿片类药物是广泛使用的镇痛药；然而，长期使用导致耐受性，对药物的反应性逐渐降低。u-阿片样物质受体（MOR）的脱敏和内化在阿片样物质的作用中很重要，似乎有助于耐受性的发展。脱敏和内化在幼稚细胞中已被很好地表征，但这些过程在慢性治疗期间如何在神经元中被调节尚不清楚。了解细胞对慢性激动剂暴露的适应性将有助于我们更好地理解产生耐受性的机制。本研究的目的是研究慢性吗啡治疗期间u-阿片受体脱敏和内吞作用的调节是如何改变的。急性脱敏是在持续使用高浓度激动剂期间发生的受体与g蛋白活化的解偶联。激动剂结合受体也经历内吞作用。我推测，在慢性吗啡治疗的动物中，激动剂诱导的内化发生上调，导致脱敏后的恢复减少。这一假设是基于对慢性吗啡治疗后大鼠蓝斑神经元中阿片作用改变的研究。慢性吗啡治疗大鼠LC神经元的激动剂效果降低，脱敏促进，恢复不完全。为了验证我的假设，我将从表达flag标记的u-阿片受体（FLAG-MOR）的小鼠中记录MOR偶联与蓝斑（LC）神经元中钾通道的激活。这将使我们能够在活体脑切片中同时研究MOR耦合和运输。该项目的第一个目标是利用电生理学和成像的结合来表征原生细胞中FLAG-MOR对[Met]5-脑啡肽的激活、脱敏和内化。第二个目标是用吗啡长期治疗小鼠，并确定内化在观察到的脱敏后恢复缓慢中所起的作用。了解慢性吗啡暴露期间MOR调节的细胞机制将有助于深入了解导致阿片类药物耐受性发展的过程。这一认识可能有助于开发不产生耐受性的有效镇痛药。

DESCRIPTION (provided by applicant): Opioids are widely used analgesics; however prolonged use results in tolerance, a progressively decreasing responsiveness to the drug. Desensitization and internalization of u-opioid receptors (MOR) are important in the actions of opioids and appear to contribute to the development of tolerance. Desensitization and internalization have been well characterized in naive cells, but how these processes are regulated in neurons during chronic treatment is unclear. Knowing what cellular adaptations occur in response to chronic agonist exposure will better our understanding of the mechanisms contributing to tolerance. The purpose of this proposal is to investigate how regulation of u-opioid receptor desensitization and endocytosis are altered during chronic morphine treatment. Acute desensitization is an uncoupling of the receptor from G-protein activation that occurs during a sustained application of a high concentration of agonist. Agonist-bound receptors also undergo endocytosis. I hypothesize that an upregulation of agonist-induced internalization occurs in chronic-morphine treated animals, resulting in reduced recovery from desensitization. This hypothesis is based on studies examining alterations in the actions of opioids in rat locus coeruleus neurons following chronic-morphine treatment. Agonist efficacy was decreased, desensitization was facilitated, and recovery was incomplete in LC neurons from chronic-morphine-treated rats. To test my hypothesis, I will record MOR coupling to activation of potassium channels in locus coeruleus (LC) neurons from mice expressing a FLAG-tagged u-opioid receptor (FLAG-MOR). This will allow us to study MOR coupling and trafficking simultaneously in a live brain slice. The first goal of this project is to characterize activation, desensitization, and internalization of FLAG-MOR to [Met]5-enkephalin in naTve cells using a combination of electrophysiology and imaging. The second goal is to treat mice chronically with morphine and determine what role internalization plays in the reduced recovery from desensitization that is observed. Understanding the cellular mechanisms of MOR regulation during chronic exposure to morphine will provide insight into the processes that lead to the development of tolerance to opioids. This understanding may allow for the development of effective analgesics which do not produce tolerance.