Electrophysiologic analyses during chronic application of opioids to organotypic explants of fetal mouse spinal cord with attached dorsal root ganglia (DRGs) have provided the first demonstration that isolated mammalian CNS tissues can, in fact, develop a marked tolerance after several days' exposure in vitro.* Attempts will be made to clarify cellular mechanisms regulating development of tolerance and dependence in these unique CNS tissue cultures by introduction of specific metabolic and pharmacologic blocking agents and by other selective manipulations of the rigorously controlled physicochemical environment of these cultures. Extracellular recordings of the changes in DRG-evoked dorsal-horn network responses during chronic exposure to opioids will be correlated with intracellular recordings from the DRG neurons to detect possible functional alterations in presynaptic DRG terminals during development of tolerance, e.g. decreased opioid sensitivity, increased calcium conductance. Factors leading to the marked increase in opiate binding that occurs in our cord-DRG explants after chronic exposure to naloxone will be analyzed and similar methods will be used to detect possible changes in opiate receptor levels in tolerant cultures. Further studies of cross-tolerance between morphine and opioid peptides will be made to clarify the degree to which tolerance selective for receptor subtypes may develop in the dorsal-horn networks of cord-DRG explants. Development of cross-tolerance to the acute depressant effects of serotonin, and possibly to other monoamines, in opioid-tolerant cord-DRG explants will be analyzed with specific transmitter antagonists. Development of physiologic properties of opioid dorsal-horn networks and their DRG inputs will be studied in normal culture media and following chronic exposure to opioid agonists and antagonists introduced at early stages of maturation of fetal cord-DRG, and isolated DRG, explants in vitro. Correlative studies will be made with cord-DRG tissues explanted from pregnant rodents after chronic exposure to opioids in utero. The proposed studies should provide valuable insights into mechanisms of tolerance and plasticity in opioid systems of the CNS and to problems in maternal narcotic addiction. Crain et al., Life Sciences 25:1797-1802, 1979: 31:241-247, 1982.
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