Morphine tolerance in live neurons

描述（由申请人提供）：吗啡是治疗疼痛最常用的阿片类药物之一。然而，长期使用往往会导致耐受性的发展，其特征是有效性丧失和剂量递增。吗啡耐受性部分是由于μ阿片受体（莫尔）信号传导的损失，而与受体水平的变化无关。尽管经过多年的研究，导致这种信号丢失的机制仍然没有解决。传统上，耐受性是在从长期治疗的动物中取出的细胞或组织中测量的，并在几个小时内清洗掉吗啡。初步实验发现，如果来自吗啡处理过的大鼠的脑切片连续保持在吗啡中，则与吗啡被冲洗掉的切片相比，吗啡反应明显较小，这表明部分耐受性可以在相对较短的冲洗内恢复。这种快速恢复后，吗啡去除是新发现的，可能有相当大的重要性，持久的耐受性。这个提议的目的是通过仔细检查经常被忽视的持续存在的吗啡维持的脱敏来更好地理解吗啡耐受。总的假设是，两种类型的耐受（持久的耐受和慢性脱敏）在长期吗啡治疗过程中发展的恢复的时间过程中区分，这有不同的机制，并取决于治疗范式。这一假设将使用来自未给药或吗啡处理的动物的脑切片中所含的蓝斑神经元进行检验。将使用全细胞电压钳电生理学来测量吗啡效应，以检测MOR介导的G蛋白偶联内向整流钾（GIRK）通道的激活。这项建议的具体目标如下：（1）鉴定大鼠和小鼠长期连续吗啡治疗所导致的持久耐受性和慢性脱敏，（2）将其与长期间歇性吗啡治疗后的耐受和脱敏进行比较，以及（3）确定2-arrestin 2在长期耐受发展中的作用（使用2-抑制蛋白2敲除小鼠）和Ser/Thr磷酸酶在慢性脱敏的发展和恢复中的作用（使用冈田酸）。总之，这一建议将提供有关长期吗啡治疗后慢性脱敏的新信息。特别关注吗啡将有助于确定对这种最常用的阿片类激动剂耐受的机制。

DESCRIPTION (provided by applicant): Morphine is one of the most commonly prescribed opioids to treat pain. However, prolonged use often leads to the development of tolerance characterized by loss of effectiveness and dose escalation. Morphine tolerance is in part due to a loss of signaling from mu opioid receptors (MOR) independent of a change in receptor levels. Despite years of study, the mechanisms causing this loss of signaling remain unsolved. Traditionally, tolerance is measured in cells or tissue removed from a chronically treated animal and washed free of morphine for several hours. Preliminary experiments have discovered that if brain slices from a morphine-treated rat are instead maintained in morphine continuously, the morphine response is significantly smaller compared to slices in which morphine was washed out, indicating that a portion of tolerance can recover within a relatively short wash. This rapid recovery following morphine removal is newly identified and may have appreciable importance to the persistence of tolerance. The goal of this proposal is to better understand morphine tolerance by carefully examining the often overlooked desensitization that is maintained by the continuous presence of morphine. The overall hypothesis is that two types of tolerance (long-lasting tolerance and chronic desensitization) develop during long-term morphine treatment distinguished by the time-course of recovery, which have different mechanisms and depend on treatment paradigm. This hypothesis will be tested using locus coeruleus neurons contained in brain slices from naove or morphine-treated animals. Morphine effects will be measured using whole-cell voltage-clamp electrophysiology to detect MOR-mediated activation of G protein-coupled inwardly rectifying potassium (GIRK) channels. The specific aims of this proposal are as follows: (1) to identify the long-lasting tolerance and chronic desensitization that results from long-term continuous morphine treatment of rats and mice, (2) to compare this to tolerance and desensitization following long-term intermittent morphine treatment and (3) to determine the role of 2-arrestin 2 in the development of long-lasting tolerance (using 2-arrestin 2 knockout mice) and the role of ser/thr phosphatases in development of and recovery from chronic desensitization (using okadaic acid). Together, this proposal will provide new information about chronic desensitization that develops after long-term morphine treatment. Focusing on morphine specifically will help identify mechanisms involved in tolerance to this most commonly used opioid agonist.