LASER CONFOCAL IMAGING OF OPIOID PEPTIDES AND RECEPTORS

过去二十年来的发现证明， 神经系统产生阿片肽，以及几类 阿片受体偶联到细胞内效应器。很可能是 内源性或外源性阿片类药物与这些受体相互作用， 尽管导致入住率的生理环境 由内源性阿片类药物引起的受体还远不清楚。人们通常认为 内源性阿片类药物通过细胞-阿片受体- 类似于为古典音乐而建立的安排和机制 神经递质，也就是在突触上。然而，有几点观察到 提示经典形式的突触传递可能不适用于 一般是神经肽，特别是阿片肽。 本实验室研究的总体目标是确定 细胞水平：分子在功能上的空间组织 参与阿片类神经传递。这一目标将通过以下途径实现 旨在调查以下具体目标的实验：1) 确定含有阿片类药物的神经末梢的空间关系 针对树突状细胞树和靶细胞胞体的特定区域的多肽 2)确定阿片受体的空间分布 在“阿片感受器”树突状细胞和胞体的膜内 神经元。对于某些神经元，观察到这些受体的分布 将在生理条件下的分布情况与 在“结合”研究中通常采用的条件下的受体； 确定G蛋白的空间分布预测为中介 树突状细胞膜和胞体内的阿片类药物作用 4)确定三者之间的空间关系 上面概述的功能。特别是，阿片类神经的近似性 阿片受体的末端将被确定，关系也将被确定 阿片受体和G蛋白之间的关系。后一种关系也将 在被认为是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，我们建议合成新的， 阿片受体的高荧光配体。这些配体将是 设计不仅是为了使它们能够选择性地绑定到 阿片受体，也是为了开发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是否 神经传递遵守组织提出的存在规则 突触传递。如果阿片能神经末梢和阿片受体 不像经典突触那样被分割，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。

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.