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.
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