Electrophysiologic analyses during chronic application of opioids to organotypic explants of fetal mouse spinal cord with attached dorsal root ganglia (DRGs) demonstrate that isolated mammalian CNS tissues can develop marked tolerance after several days' exposure in vitro. The marked decreases in opioid sensitivity of DRG-evoked dorsal-horn network responses observed during chronic opioid exposure of cord-DRG explants will be closely coordinated with biochemical assays to test the hypothesis that the physiologic expression of tolerance may be mediated by significant enhancement of adenylate cyclase activity and cyclic AMP levels. These studies will be correlated with analyses of the acute tolerance-like state produced by brief exposure of cord-DRG explants to forskolin or other agents that elicit a rise in intracellular cyclic AMP. In order to clarify other factors that may regulate opioid tolerance, correlative physiologic and biochemical analyses will be made after physicochemical alterations of the culture environment during chronic opioid exposure, e.g. addition of specific metabolic or pharmacologic blocking agents. Extracellular recordings of dorsal-horn responses during chronic exposure to opioids--or acute exposure to forskolin and cyclic AMP analogs--will be correlated with intracellular recordings from the DRG and cord neurons. These analyses will clarify pre-and post-synaptic alterations in dorsal-horn network functions that may occur in tolerant explants. Factors leading to the marked upregulation of opiate receptor levels that occurs in mature cord-DRG explants after chronic exposure to naloxone will be analyzed, using specific metabolic/pharmacologic blocking agents and autoradiography. Electrophysiologic analyses, binding assays and autoradiography will be coordinated to determine if developmental redistribution, disappearance, or functional inactivation of opiate receptors may account for the spinal cord-regulated decrease in the fraction of opioid-sensitive DRG neuron perikarya observed in DRG-cord vs. isolated DRG explants. Development of opioid dorsal-horn networks and their DRG inputs will also be studied during chronic exposure of embryonic tissues to opioid agonists or antagonists at early stages in vitro. The proposed studies should provide valuable insights into mechanisms of tolerance and plasticity in opioid systems of the CNS and problems in maternal narcotic addiction.
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