LASER CONFOCAL IMAGING OF OPIOID PEPTIDES AND RECEPTORS

在过去的二十年里，发现已经确定， 神经系统产生阿片肽，以及几类 与细胞内效应物偶联的阿片受体。很可能 内源性或外源性阿片样物质与这些受体相互作用， 虽然生理环境，导致占用的 内源性阿片类药物的受体还远不清楚。一般认为 内源性阿片类物质通过细胞传递给阿片受体， 类似于传统的安排和机制， 神经递质，也就是说，在突触上。然而，几项意见表明， 表明突触传递的经典形式可能不适用于 神经肽，特别是阿片肽。 本实验室研究的总体目标是确定 细胞水平分子功能上的空间组织 参与阿片类神经传递。这一目标将通过 实验旨在研究以下具体目标：1） 确定含有阿片样物质的神经末梢的空间关系 肽的树突状树和靶细胞体的特定区域 2）确定阿片受体的空间分布 在树突树和“阿片感受”细胞体的膜内， 神经元对于某些神经元，观察到这些受体的分布 在生理条件下，将与 在“结合”研究中通常采用的条件下， 确定G蛋白的空间分布预测介导 阿片样物质的作用在树突树和细胞体的膜内， 4）确定三者之间的空间关系 上面概述的特征。尤其是阿片神经 将确定阿片受体的终末，以及 阿片受体和G蛋白之间的联系后一种关系也将 在被认为增加或减少的条件下进行检查 G蛋白/受体偶联。 为了实现这些目标中的一些，我们提出合成新的， 阿片受体的高荧光配体。这些配体将是 设计的目的不仅在于它们能够选择性地与 阿片受体，但也利用扫描激光的成像能力 共聚焦显微镜 从这些研究中获得的知识将确定阿片类药物 神经传递服从组织的“规则”提出存在， 突触传递如果阿片能神经末梢和阿片受体 不像经典的突触那样被划分，这些研究将 确定阿片类药物作用于其细胞的细胞排列， 目标的

Discoveries made over the last two decades have established that the nervous system produces opioid peptides, as well as several classes of opioid receptors coupled to intracellular effectors. It is likely that either endogenous or exogenous opioids interact with these receptors, although the physiological circumstances which lead to occupancy of receptors by endogenous opioids is far from clear. It is generally assumed that endogenous opioids are delivered to opioid receptors by cellular - arrangements and mechanisms similar to those established for classical neurotransmitters, that is, at a synapse. However, several observations suggest that the classical form of synaptic transmission may not hold for neuropeptides in general, and opioid peptides in particular. The overall goal of studies in this laboratory is to determine at the cellular level the spatial organization of the molecules functionally involved in opioid neurotransmission. This goal will be approached through experiments designed to investigate the following specific aims: 1) To determine the spatial relationship of nerve terminals which contain opioid peptides to specific regions of the dendritic tree and cell body of target neurons; 2) To determine the spatial distribution of opioid receptors within the membrane of the dendritic tree and cell body of "opioceptive" neurons. For certain neurons, the distribution of these receptors observed under physiological conditions will be compared with the distribution of receptors under the conditions usually employed in "binding" studies; 3) To determine the spatial distribution of G-proteins predicted to mediate opioid action within the membrane of the dendritic tree and cell body of opioceptive neurons; 4) To determine the spatial relations among the three features outlined above. In particular, the proximity of opioid nerve terminals to opioid receptors will be determined, as will the relationship between opioid receptors and G-proteins. The latter relationship will also be examined under conditions thought to increase or decrease G-protein/receptor coupling. In order to accomplish some of these aims, we propose the synthesis of new, highly fluorescent ligands for opioid receptors. These ligands will be designed not only for their ability to bind selectively to classes of opioid receptors, but also to exploit the imaging power of scanning laser confocal microscopy. Knowledge gained from these studies will determine if opioidergic neurotransmission obeys the organizational 'rules' proposed to exist for synaptic transmission. If opioidergic nerve terminals and opioid receptors are not compartmentalized as in a classical synapse, these studies will determine the cellular arrangements through which opioids act upon their targets.