DUAL OPIOID MODULATION OF SPINAL CORD-GANGLION ACTIVITY

双阿片类药物对脊髓神经节活动的调节

• 批准号: 3207081
• 负责人: STANLEY M CRAIN
• 金额: $ 32.5万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-07-01 至 1992-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3207081
• 关键词: action potentials; adenylate cyclase; analgesics; calcium channel; calcium channel blockers; cyclic AMP; dorsal root; drug addiction antagonist; drug tolerance; electrophysiology; enzyme inhibitors; forskolin; laboratory mouse; morphine; naloxone; neuropharmacology; opiate alkaloid; opioid receptor; protein kinase A; radiotracer; tissue /cell culture; voltage /patch clamp

Our major objective are: 1) to analyze mechanisms underlying the dual receptor-mediated excitatory and inhibitory modulation of the action potentials (APs) of mouse dorsal-root ganglion (DRG) neurons that we have observed in explants and dissociated cell cultures; 2) to test the hypothesis that enhancement of the adenylate cyclase (AC)/cyclic AMP system is causally related to the tolerance that occurs in DRG neuron APs and dorsal-horn network response after chronic opioid treatment of DRG-cord cultures. Whereas the duration of the AP calcium component (APD) of naive DRG neurons is generally shortened by acute exposure to high (muM) concentrations of opioids, low (nM) levels of specific mu, delta and kappa opioids elicit APD prolongation in many of the same cells. Both excitatory and inhibitory opioid effects are prevented by naloxone or diprenorphine. Intracellular and whole- cell patch-clamp recording will be used to analyze this dual opioid modulation of DRG neurons in explant and dissociated-cell cultures. Excitatory vs. inhibitory opioid receptors will be characterized by electrophysiologic tests during systematic treatments with specific opioid agonists, antagonists and receptor inactivators. These physiologic studies will be coordinated with opioid binding assays and receptor autoradiography after similar treatments, including pertussis (PTX) vs. cholera toxin (CTX). To further evaluate our evidence that opioid-induced APD prolongation is mediated by receptors positively coupled via Gs to the AC/cAMP/protein kinase A(PKA) system, the opioid responsivity of DRG neurons will be tested extracellular or intracellular perfusion with CTX vs. PTX, cAMP, PKA, inhibitors of AC and PKA, specific ion channel blockers, or altered ion concentrations. Linkages of excitatory opioid receptors with specific K+ and Ca2+ conductances in DRG cells will be analyzed. Correlative biochemical assays of AC activity of these neuros will made, especially during the physiologic expression of tolerance and during the increase in opioid excitatory responsiveness in DRG cells chronically exposed to specific mu, delta, or kappa opioids. Correlative binding assays and autoradiography will be made to determine if specific subtypes of opioid receptors are up-regulated or down-regulated in tolerant DRG neurons. These studies will provide valuable insights into cellular compensatory mechanisms that mediate tolerance to opioid analgesic effects in primary afferent synaptic networks and clues to some of the addictive and euphoric effects of opioids in the CNS.

我们的主要目标是：1）分析潜在的机制 双重受体介导的兴奋性和抑制性调节 小鼠背根神经节（DRG）的动作电位（AP） 我们在外植体和分离细胞中观察到的神经元 文化； 2）检验增强的假设 腺苷酸环化酶 (AC)/环 AMP 系统与 发生在 DRG 神经元 AP 和背角的耐受性 DRG-cord 慢性阿片类药物治疗后的网络反应 文化。 而幼稚的 AP 钙成分 (APD) 的持续时间 DRG 神经元通常因急性暴露于高浓度而缩短 (μM) 浓度的阿片类药物，低 (nM) 水平的特定 mu， δ 和 kappa 阿片类药物会在许多患者中引起 APD 延长 相同的细胞。 阿片类药物具有兴奋性和抑制性作用 纳洛酮或二丙诺啡可以预防。 细胞内和整体 细胞膜片钳记录将用于分析这种双重 阿片类药物对外植体和分离细胞中 DRG 神经元的调节 文化。 兴奋性与抑制性阿片受体将 以系统性电生理测试为特征 使用特定阿片类激动剂、拮抗剂和 受体失活剂。 这些生理学研究将 与阿片类药物结合测定和受体协调 类似治疗后的放射自显影，包括百日咳 (PTX) 与霍乱毒素 (CTX)。 进一步评估我们的证据 阿片类药物诱导的 APD 延长是由受体介导的 通过 Gs 与 AC/cAMP/蛋白激酶 A(PKA) 正耦合 系统，将测试 DRG 神经元的阿片类药物反应性 细胞外或细胞内灌注 CTX 与 PTX、cAMP、 PKA、AC 和 PKA 抑制剂、特定离子通道阻滞剂，或 改变离子浓度。 兴奋性阿片类药物的联系 DRG 细胞中具有特定 K+ 和 Ca2+ 电导的受体 将被分析。 AC 活性的相关生化测定 这些神经元会产生，特别是在生理期间 耐受性的表达和阿片类药物增加期间 长期暴露于 DRG 细胞的兴奋反应 特定的 mu、delta 或 kappa 阿片类药物。 相关结合分析 并进行放射自显影以确定是否具体 阿片受体亚型上调或下调 在耐受的 DRG 神经元中。 这些研究将为细胞提供有价值的见解 介导阿片类药物耐受性的补偿机制 初级传入突触网络的镇痛作用和线索 阿片类药物的一些成瘾和欣快作用 中枢神经系统。