Our major objective are: 1) to analyze mechanisms underlying the dual receptor-mediated excitatory and inhibitory modulation of the action potentials (APs) of mouse dorsal-root ganglion (DRG) neurons that we have observed in explants and dissociated cell cultures; 2) to test the hypothesis that enhancement of the adenylate cyclase (AC)/cyclic AMP system is causally related to the tolerance that occurs in DRG neuron APs and dorsal-horn network response after chronic opioid treatment of DRG-cord cultures. Whereas the duration of the AP calcium component (APD) of naive DRG neurons is generally shortened by acute exposure to high (muM) concentrations of opioids, low (nM) levels of specific mu, delta and kappa opioids elicit APD prolongation in many of the same cells. Both excitatory and inhibitory opioid effects are prevented by naloxone or diprenorphine. Intracellular and whole- cell patch-clamp recording will be used to analyze this dual opioid modulation of DRG neurons in explant and dissociated-cell cultures. Excitatory vs. inhibitory opioid receptors will be characterized by electrophysiologic tests during systematic treatments with specific opioid agonists, antagonists and receptor inactivators. These physiologic studies will be coordinated with opioid binding assays and receptor autoradiography after similar treatments, including pertussis (PTX) vs. cholera toxin (CTX). To further evaluate our evidence that opioid-induced APD prolongation is mediated by receptors positively coupled via Gs to the AC/cAMP/protein kinase A(PKA) system, the opioid responsivity of DRG neurons will be tested extracellular or intracellular perfusion with CTX vs. PTX, cAMP, PKA, inhibitors of AC and PKA, specific ion channel blockers, or altered ion concentrations. Linkages of excitatory opioid receptors with specific K+ and Ca2+ conductances in DRG cells will be analyzed. Correlative biochemical assays of AC activity of these neuros will made, especially during the physiologic expression of tolerance and during the increase in opioid excitatory responsiveness in DRG cells chronically exposed to specific mu, delta, or kappa opioids. Correlative binding assays and autoradiography will be made to determine if specific subtypes of opioid receptors are up-regulated or down-regulated in tolerant DRG neurons. These studies will provide valuable insights into cellular compensatory mechanisms that mediate tolerance to opioid analgesic effects in primary afferent synaptic networks and clues to some of the addictive and euphoric effects of opioids in the CNS.
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