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REGULATION OF OPIOID RESPONSIVITY OF SENSORY NEURONS

感觉神经元阿片类药物反应的调节

基本信息

批准号：
3211384
负责人：
STANLEY M CRAIN
金额：
$ 14.76万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-09-01 至 1991-03-31
项目状态：
已结题

项目摘要

Our long-term objective is to analyze the mechanisms underlying the physiologic tolerance that develops during chronic opioid exposure of mouse spinal cord explants with attached dorsal root ganglia (DRGs). The marked decreases in opioid sensitivity of DRG-evoked dorsal-horn synaptic network responses in drug- treated cultures will be coordinated with biochemical assays of adenylate cyclase activity and levels of cyclic AMP and opiate receptors in DRG and cord neurons. These electrophysiologic and biochemical analyses will test the hypothesis that neurons may develop tolerance/dependence during chronic opioid exposure by a compensatory enchancement of their adenylate cyclase/cAMP system and by alterations in opiate-receptor linkages to GTP- binding regulatory proteins (e.g. Gi and Gs). Whereas the duration of the Ca++ component of the action potential (APD) of DRG cells is generally shortened by opioids, we have recently found that the APD of some DRG neurons in our cultures is prolonged by acute exposure to opioids, as observed in nodose ganglion neurons in vivo. After treatment of DRG explants with forskolin or pertussis toxin, much larger fractions (greater than 50%) of the DRG neurons show opioid-prolonged APDs. We propose to carry out systematic electrophysiologic analyses of these direct excitatory effects of opioids on DRG neurons. Intracellular and wholecell patch-clamp recording techniques will be used to analyze alterations in opioid responsivity of DRG neurons during manipulation of their ionic channels and adenylate cyclase/cAMP system by extracellular and intracellular perfusions. These tests will evaluate the functional linkages of specific subtypes of opiate receptors, via Gi, Gs and other G proteins, to adenylate cyclase and cAMP-modulated ionic channels. We will determine whether DRG neurons also show increased evidence of opioid-prolonged APDs and hyperexcitability (e.g. repetitive firing) after chronic exposure to opioids. These studies may provide valuable insights into basic cellular compensatory mechanisms that mediate the palsticity properties of opioid networks underlying tolerance and dependence. In addition, we will analyze DRG neurons co-cultured with peripheral (skin or muscle) vs. CNS target explants to determine if opiate receptors at peripheral DRG terminals mediate excitatory functions, in contrast to opioid-inhibitory functions at central DRG presynaptic terminals.

我们的长期目标是分析在慢性阿片类药物治疗过程中产生的生理耐受性小鼠脊髓外植体与附着背根的暴露神经节（DRG）。阿片类药物敏感性的显著降低 DRG诱发的背角突触网络反应处理过的培养物将与以下生物化学测定相协调：腺苷酸环化酶活性和环腺苷酸及阿片水平背根神经节和脊髓神经元中的受体。这些电生理和生物化学分析将检验神经元可能在慢性阿片类药物暴露期间，腺苷酸环化酶/cAMP的代偿性增强系统和阿片受体连接的改变，以GTP- 结合调节蛋白（例如Gi和Gs）。而钙离子的持续时间背根神经节细胞的动作电位（APD）通常被阿片类药物缩短，我们我最近发现，在我们的一些DRG神经元的APD，急性暴露于阿片类药物延长了培养时间，体内结状神经节神经元。 DRG治疗后含有毛喉素或百日咳毒素的外植体，更大的组分（大于50%）的DRG神经元显示阿片样物质延长的 APD。我们建议进行系统的电生理检查阿片类药物对DRG的这些直接兴奋作用的分析神经元细胞内和全细胞膜片钳记录技术将被用来分析阿片类药物的变化，背根神经节神经元在操纵其离子通道和腺苷酸环化酶/cAMP系统，细胞内灌注。这些测试将评估功能阿片受体的特定亚型的连接，通过Gi，Gs和其他G蛋白、腺苷酸环化酶和cAMP调节的离子渠道我们将确定DRG神经元是否也显示出阿片类药物延长APD的证据增加，慢性暴露后的过度兴奋（例如重复性放电）阿片类药物这些研究可能会提供有价值的见解，细胞代偿机制，介导的palsticity 阿片类药物网络的耐受性和依赖此外，我们还将分析DRG神经元与外周（皮肤或肌肉）与CNS靶向外植体，以确定如果外周DRG末梢的阿片受体介导兴奋功能，与阿片样物质抑制功能相反，中央DRG突触前终末。