Electrophysiologic Responses and Adenylate Cyclase Activities of Mouse Spinal Cord-Dorsal Root Ganglion Explants Rendered Tolerant by Chronic Exposure to Morphine or Pertussis Toxin

  • Stanley M. Crain
  • Maynard H. Makman
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 221)


Explant cultures of fetal mouse spinal cord with attached dorsal-root ganglia (DRGs) (Fig. 1: Crain 1976) provide a valuable in vitro system for study of neurotransmitter modulation in the CNS. We have previously developed and utilized this system extensively for analysis of the actions of opioids on the electrophysiologic responses of these neurons and for study of the development of tolerance to opioid depressant effects (see review by Crain, 1984). Exposure of fetal mouse spinal cord-DRG explants to opioid alkaloid or peptide agonists resulted in stereospecific, naloxone-reversible, dose-dependent depression of sensory-evoked dorsal-horn synaptic-network responses within a few minutes (e.g. Fig. 2A; Crain et al., 1977, 1978). After chronic exposure to opioids, e.g. 2–3 days in 1 μM morphine (at 35°C), sensory-evoked dorsal-horn responses recovered, and they could then be elicited by DRG stimuli in the presence of opioids at concentrations 10- to 100-fold higher than required to depress a naive expiant (Fig. 2B; (Crain et al., 1979). In addition, these opioid-tolerant explants developed significant cross-tolerance to serotonin (5HT) (Crain et al., 1982). The tolerant state did not develop if the explants were exposed to morphine at lower temperatures (e.g. 20°C for as long as 7 days; Fig. 2C). The data suggest that the sustained decrease in opioid sensitivity observed during chronic opioid exposure at 35°C is mediated by a temperature-dependent metabolic change in these neurons (Crain et al., 1979; Crain, 1984).


Dorsal Root Ganglion Adenylate Cyclase Pertussis Toxin Adenylate Cyclase Activity Opiate Receptor 
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  1. Berridge, M. J. and Irvine, R. F., Inositol trisphosphate, a novel second messenger in cellular signal transduction, Nature, 312: 315–321 (1984).CrossRefGoogle Scholar
  2. Blume, A. J., Licktenstein, D. and Boone, G., Coupling of opiate receptors to adenylate cyclase: requirement for Na+ and GTP, Proc. Natl. Acad.Sci. USA, 76: 5626–5630 (1979).CrossRefGoogle Scholar
  3. Bogoch, G. M., Katada, T., Northup, J. K., Ui, M. and Gilman, A. G., Purification and properties of the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase, J. Biol. Chem., 259: 3560–3567 (1984).Google Scholar
  4. Chalazonitis, A., Groth, J., Simon, E. J. and Crain, S. M., Development of met-enkephalin immunoreactivity in organotypic explants of fetal mouse spinal cord and attached dorsal root ganglia, Devel. Brain Res. 12: 183–189 (1984).CrossRefGoogle Scholar
  5. Collier, H. O. J., Cellular site of opiate dependence, Nature (London), 283: 625–629 (1980).CrossRefGoogle Scholar
  6. Cooper, D. F. M., Londos, C., Gill, D. L. and Rodbell, M., Opiate receptormediated inhibition of adenylate cyclase in rat striatal membranes, J. Neurochem., 38: 1164–1167 (1982).CrossRefGoogle Scholar
  7. Crain, S. M., Neurophysologic Studies in Tissue Culture, Raven Press, New York (1976).Google Scholar
  8. Crain, S. M., Role of CNS target cues in formation of specific afferent synaptic connections in organotypic cultures. In: Neuroscience Approached Through Cell Culture, Vol. II, S. E. Pfeiffer, ed., CRC Press, Florida, pp. 1–32 (1882).Google Scholar
  9. Crain, S. M., Spinal cord tissue culture models for analyses of opioid analgesia, tolerance and plasticity, in Mechanisms of Tolerance and Dependence, C. Sharp, Ed., National Institute on Drug Abuse Research Monograph, U.S. Govt. Printing Office (ADM 84-1330), Washington, D.C., pp. 260–292 (1984).Google Scholar
  10. Crain, S. M. and Peterson, E. R., Enhanced afferent synaptic functions in fetal mouse spinal cord-sensory ganglion explants following NGF-induced ganglion hypertrophy, Brain Research, 79: 145–152 (1974).CrossRefGoogle Scholar
  11. Crain, S. M., Crain, B., Finnigan, T. and Simon, E. J., Development of tolerance to opiates and opioid peptides in organotypic cultures of mouse spinal cord, Life Sci., 25: 1797–1802 (1979).CrossRefGoogle Scholar
  12. Crain, S. M., Crain, B. and Makman, M. H., Pertussis toxin blocks depressant effects of opioid, monoaminergic and muscarinic agonists on dorsal-horn network responses in spinal cord-ganglion cultures, (subm. for publ.) (1986b).Google Scholar
  13. Crain, S. M., Crain, B., Peterson, E. R. and Simon, E. J., Selective depression by opioid peptides of sensory-evoked dorsal-horn network responses in organized spinal cord cultures, Brain Research, 157: 191–201 (1978).CrossRefGoogle Scholar
  14. Crain, S. M., Crain, B. and Peterson, E. R., Development of cross-tolerance to 5-hydroxytryptamine in organotypic cultures of mouse spinal cordganglia during chronic exposure to morphine, Life Sci., 31: 241–247 (1982).CrossRefGoogle Scholar
  15. Crain, S. M., Crain, B. and Peterson, E. R., Cyclic AMP or forskolin produces rapid “tolerance” to the depressant effects of opiates on sensory-evoked dorsal-horn responses in spinal cord-dorsal root ganglion (DRG) explants, Soc. Neurosci. Abstr., 10: 111 (1984).Google Scholar
  16. Crain, S. M., Crain, B. and Peterson, E. R., Cyclic AMP or forskolin rapidly attenuates the depressant effects of opioids on sensory-evoked dorsalhorn responses in mouse spinal cord-ganglion explants, Brain Res., 370: 61–72 (1986a).CrossRefGoogle Scholar
  17. Crain, S. M., Peterson, E. R., Crain, B. and Simon, E. J., Selective opiate depression of sensory-evoked synaptic networks in dorsal-horn regions of spinal cord cultures, Brain Research, 133: 162–166 (1977).CrossRefGoogle Scholar
  18. Crain, S. M., Shen, K. E. and Chalazonitis, A., Altered pharmacologic sensitivities of opioid-sensitive dorsal root ganglion (DRG) neurons rendered hyperexcitable by exposure of DRG-cord explants to forskolin or pertussis toxin. In N. Chalazonitis (ed.), Inactivation of Hypersensitive Neurones, in press (1986c).Google Scholar
  19. Dvorkin, B., Crain, S. M. and Makman, M. H., Increased adenylate cyclase activity in mouse spinal cord-dorsal root ganglion (DRG) explants rendered tolerant by chronic exposure to morphine, Soc. Neurosci. Abstr., 11: 1198 (1985).Google Scholar
  20. Gentleman, S., Parenti, M., Neff, N. H. and Pert, C. B., Inhibition of dopamine-activated adenylate cyclase and dopamine binding by opiate receptors in rat striatum, Cell. Mol. Neurobiol., 3: 17–26 (1983).CrossRefGoogle Scholar
  21. Gilman, A. G., G proteins and dual control of adenylate cyclase, Cell, 36: 577–579 (1984).CrossRefGoogle Scholar
  22. Hiller, J. M., Simon, E. J., Crain, S. M. and Peterson, E. R., Opiate receptors in cultures of fetal mouse dorsal root ganglia (DRG) and spinal cord: predominance in DRG neurites, Brain Research, 145: 396–400 (1978).CrossRefGoogle Scholar
  23. Holz, G. G., Rane, S. G. and Dunlap, K., GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels, Nature, 319: 670–672 (1986).CrossRefGoogle Scholar
  24. Hsia, J., Moss, J., Hewlett, E. L. and Vaughan, M., ADP-ribosylation of adenylate cyclase by pertussis toxin, J. Biol. Chem., 259: 1086–1090 (1984).Google Scholar
  25. Jakobs, K. H., Aktories, K. and Schultz, G., Inhibition of adenylate cyclase by hormones and neurotransmitters, Adv. Cyclic Nucleotide Res., 14: 173–187 (1981).Google Scholar
  26. Katada, T. and Ui, M., Direct modification of the membrane adenylate cyclase system by islet activating protein due to ADP-ribosylation of a membrane protein, Proc. Nat. Acad. Sci. USA, 79: 3129–3133 (1982).CrossRefGoogle Scholar
  27. Klee, W. A., Milligan, G., Simonds, W. F. and Tocque, B., The role of adenyl cyclase in opiate tolerance and dependence, in Mechanisms of Tolerance and Dependence, C. Sharp, ed., U. S. Govt. Printing Office (ADM 84-1330). Washington, D.C., pp. 109–118 (1984).Google Scholar
  28. Kurose, H., Katada, T., Amano, T. and Ui, M., Specific uncoupling by isletactivating protein, pertussis toxin, of negative signal transduction via α-adrenergic, cholinergic, and opiate receptors in neuroblastoma x glioma hybrid cells, J. Biol. Chem., 258: 4870–4875 (1983).Google Scholar
  29. Law, P. Y., Wu, J., Koehler, J. E. and Loh, H. H., Demonstration and characterization of opiate inhibition of the striatal adenylate cyclase, J. Neurochem., 36: 1834–1846 (1981).CrossRefGoogle Scholar
  30. Lichtenstein, D., Boone, G. and Blume, A., Muscarinic receptor regulation of NG108-15 adenylate cyclase: requirement for Na and GTP, J. Cyclic Nucleotide Res., 5: 367–375 (1979).Google Scholar
  31. Lujan, M., Lopez, E. Ramirez, R., Aguilar, H., Martinez-Olmedo, M. A. and Garcia-Sainz, J. A., Pertussis toxin blocks the action of morphine, norepinephrine and Clonidine on isolated guinea-pig ileum, Eur. J. Pharmacol., 100: 377–380 (1984).CrossRefGoogle Scholar
  32. McLawhon, R. W., Schoon, G. S. and Dawson, G., Possible role of cyclic AMP in the receptor-mediated regulation of glycosyltransferase activated in neurotumor cell lines, J. Neurochem., 37: 132–139 (1981a).CrossRefGoogle Scholar
  33. McLawhon, R. W., West Jr., R. E., Miller, R. J. and Dawson, G., Distinct high-affinity binding sites for benzomorphan drugs and enkephalin in a neuroblastoma-brain hybrid cell line, Proc. Natl. Acad. Sci., USA, 78: 4309–4313 (1981b).CrossRefGoogle Scholar
  34. Makman, M. H., Dvorkin, B. and Klein, P. N., Sodium ion modulates D2 receptor characteristics of dopamine agonist and antagonist binding sites in striatum and retina, Proc. Natl. Acad. Sci. USA, 79: 4212–4216 (1982).CrossRefGoogle Scholar
  35. Michael, T., Hoffman, B. B. and Lefkowitz, R. J., Differential regulation of the α2-adrenergic receptor by Na+ and guanine nucleotides, Nature (Lond.), 288: 709–711 (1983).CrossRefGoogle Scholar
  36. Miller, R. J., Second messengers, phosphorylation and neurotransmitter release, Trends in Neurosci., 8: 462–465 (1985).Google Scholar
  37. Pfaffinger, P. J., Martin, J. M., Hunter, D. D., Nathanson, N. M. and Hille, B., GTP-binding proteins couple cardiac muscarinic receptors to a K channel, Nature, 317: 536–538 (1985).CrossRefGoogle Scholar
  38. Rodbell, M., Structure-function problems with the adenylate cyclase system, Adv. Cyclic Nucleotide Res., 17: 207–214 (1984).Google Scholar
  39. Seamon, K. B. and Daly, J. W., Forskolin: a unique diterpene activator of cyclic AMP-generating systems, J. Cyclic Nucleot. Res., 7: 201–224 (1981).Google Scholar
  40. Sharma, S. K., Klee, W. A. and Nirenberg, M., Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance, Proc. Natl. Acad. Sci. USA, 72: 3092–3096 (1975a).CrossRefGoogle Scholar
  41. Sharma, S. K., Nirenberg, M. and Klee, W. A., Morphine receptors as regulators of adenylate cyclase activity, Proc. Natl. Acad. Sci. USA, 72: 590–594 (1975b).CrossRefGoogle Scholar
  42. Sharma, S. K., Klee, W. A. and Nirenberg, M., Opiate-dependent modulation of adenylate cyclase, Proc. Natl. Acad. Sci. USA, 74: 3365–3369 (1977).CrossRefGoogle Scholar
  43. Stefano, G. B., Catapano, E. J. and Kream, R. M., Characterization of the dopamine stimulated adenylate cyclase in the pedal ganglia of Mytilus edulis: interactions with etorphine, β-endorphins, D-ala-and methionine-enkephalin, Cell Mol. Neurobiol., 1: 57–68 (1981).CrossRefGoogle Scholar
  44. Sternweis, P. C. and Robishaw, J. D., Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain, J. Biol. Chem., 259: 13806–13813 (1984).Google Scholar
  45. Tucker, J. F., Effect of pertussis toxin on normorphine-dependence and on acute inhibitory effects of normorphine and Clonidine in guinea-pig isolated ileum, Br. J. Pharmacol., 83: 326–328 (1984).CrossRefGoogle Scholar
  46. Walczak, S. A., Wilkening, D. and Makman, M. H., Interaction of morphine, etorphine and enkephalins with dopamine-stimulated adenylate cyclase of monkey amygdala, Brain Res., 160: 105–116 (1979).CrossRefGoogle Scholar
  47. Walczak, S. A., Makman, M. H. and Gardner, E. L., Acetyl-methadol metabolites influence opiate receptors and adenylate cyclase in amygdala, Eur. J. Pharmacol., 72: 343–349 (1981).CrossRefGoogle Scholar
  48. Yajima, Y., Akita, Y. and Saito, T., Pertussis toxin blocks the inhibitory effects of somatostatin on cAMP-dependent vasoactive intestinal peptide and cAMP-independent thyrotropin releasing hormone-stimulated prolactin secretion of GH3 cells, J. Biol. Chem., 261: 2684–2689 (1986).Google Scholar

Supplementary References

  1. Crain, S. M., Shen, K.-F. and A. Chalazonitis, Opioids excite rather than inhibit sensory neurons after chronic opioid exposure of mouse dorsal root ganglion-spinal cord explants. Soc. Neurosci. Abstr., 13 (1987) in press.Google Scholar
  2. Makman, M. H., Dvorkin, B. and Crain, S. M., Modulation of adenylate cyclase activity of mouse spinal cord-ganglion explants by opioids, serotonin and pertussis toxin. Brain Res. (1987) in press.Google Scholar
  3. Qiu, X.-C., Crain, S. M. and Makman, M. H., Serotonin receptor systems in spinal cord and sensory ganglia: relation to opioid action, tolerance and cross tolerance. Soc. Neurosci. Abstr. 13 (1987) in press.Google Scholar

Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • Stanley M. Crain
    • 1
  • Maynard H. Makman
    • 2
  1. 1.Department of NeuroscienceYeshiva UniversityBronxUSA
  2. 2.Departments of Biochemistry and Molecular Pharmacology, Albert Einstein College of MedicineYeshiva UniversityBronxUSA

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