Our long-term objective is to analyze the mechanisms underlying the physiologic tolerance that develops during chronic opioid exposure of mouse spinal cord explants with attached dorsal root ganglia (DRGs). The marked decreases in opioid sensitivity of DRG-evoked dorsal-horn synaptic network responses in drug- treated cultures will be coordinated with biochemical assays of adenylate cyclase activity and levels of cyclic AMP and opiate receptors in DRG and cord neurons. These electrophysiologic and biochemical analyses will test the hypothesis that neurons may develop tolerance/dependence during chronic opioid exposure by a compensatory enchancement of their adenylate cyclase/cAMP system and by alterations in opiate-receptor linkages to GTP- binding regulatory proteins (e.g. Gi and Gs). Whereas the duration of the Ca++ component of the action potential (APD) of DRG cells is generally shortened by opioids, we have recently found that the APD of some DRG neurons in our cultures is prolonged by acute exposure to opioids, as observed in nodose ganglion neurons in vivo. After treatment of DRG explants with forskolin or pertussis toxin, much larger fractions (greater than 50%) of the DRG neurons show opioid-prolonged APDs. We propose to carry out systematic electrophysiologic analyses of these direct excitatory effects of opioids on DRG neurons. Intracellular and wholecell patch-clamp recording techniques will be used to analyze alterations in opioid responsivity of DRG neurons during manipulation of their ionic channels and adenylate cyclase/cAMP system by extracellular and intracellular perfusions. These tests will evaluate the functional linkages of specific subtypes of opiate receptors, via Gi, Gs and other G proteins, to adenylate cyclase and cAMP-modulated ionic channels. We will determine whether DRG neurons also show increased evidence of opioid-prolonged APDs and hyperexcitability (e.g. repetitive firing) after chronic exposure to opioids. These studies may provide valuable insights into basic cellular compensatory mechanisms that mediate the palsticity properties of opioid networks underlying tolerance and dependence. In addition, we will analyze DRG neurons co-cultured with peripheral (skin or muscle) vs. CNS target explants to determine if opiate receptors at peripheral DRG terminals mediate excitatory functions, in contrast to opioid-inhibitory functions at central DRG presynaptic terminals.
