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Developmental expression of G-proteins and adenylyl cyclase in peripheral olfactory systems. Light microscopic and freeze-substitution electron microscopic immunocytochemistry

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Journal of Neurocytology

Summary

Light microscopic immunohistochemistry coupled with freeze-substitution electron microscopic immunocytochemistry was used to localize α-subunits of G-proteins and type III adenylyl cyclase in developing rat olfactory epithelia. Some cilia immunoreacted with antibodies to G and type III adenylyl cyclase as early as prenatal day 15 (E15; El=sperm-positive), but immunolabelling with antibodies to Golfα was not observed until E16. From then on numbers of receptor cells with immunolabelled cilia increased for all three probes. Immunoreactivity for antibodies to the olfactory signal-transduction proteins tended to parallel cilium development, though Golfα lags somewhat behind. Newly formed cilia labelled along their lengths, whereas mature cilia labelled predominantly along their long distal parts. Dendritic knobs and ciliary necklaces showed little or no labelling. While at E22 most multiciliate cells immunolabelled with antibodies to G, Golfα, and type III adenylyl cyclase, not all of these cells labelled with antibodies to olfactory marker protein. Olfactory axons immunoreacted more intensely than epithelial surface structures with antibodies to G at E15; the reverse occurred by about E18. Immunoreactivity with antibodies to a-subunits of the G-proteins Go, Gq/G11, and Gi was also found as early as E15. Antibodies to G labelled receptor cell dendritic knobs and cilia during development only. Antibodies to G labelled Bowman's glands, whereas those to G/G11αbound to receptor cell cilia and axons (primarily vomeronasal), and supporting cell microvilli. We propose that Gs is the predominant G protein in cilia of immature olfactory receptor cells, while Goif is predominant in cilia of mature cells. Axonal immunoreactivity for some G-protein antibodies suggests G-protein participation in processing of olfactory axon and/or axon terminal-bound signals.

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References

  • Ache, B. W. (1994) Toward a common strategy for transducing olfactory information.Seminars in Cell Biology 5, 55–63.

    Google Scholar 

  • Anholt, R. R. H. (1994) Signal integration in the nervous system: adenylate cyclases as molecular coincidence detectors.Trends in Neurosciences 17, 37–41.

    Google Scholar 

  • Anholt, R. R. H., Mumby, S. M., Stoffers, D. A., Girard, P. R., Kuo, J. F. &Snyder, S. H. (1987) Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C.Biochemistry 26, 788–95.

    Google Scholar 

  • Asanuma, N. &Nomura, H. (1991) Cytochemical localization of adenylate cyclase activity in rat olfactory receptor cell.Histochemical Journal 23, 83–90.

    Google Scholar 

  • Bakalyar, H. A. &Reed, R. R. (1990) Identification of a specialized adenylyl cyclase that may mediate odorant detection.Science 250, 1403–6.

    Google Scholar 

  • Baker, H. &Farbman, A. I. (1993) Olfactory afferent regulation of the dopamine phenotype in the fetal rat olfactory system.Neuroscience 52, 115–34.

    Google Scholar 

  • Boekhoff, I. &Breer, H. (1992) Termination of second messenger signaling in olfaction.Proceedings of the National Academy of Sciences (USA) 89, 471–4.

    Google Scholar 

  • Boekhoff, I., Michel, W. C., Breer, H. &Ache, B. W. (1994) Single odors differentially stimulate dual second messenger pathways in lobster olfactory receptor cells.Journal of Neuroscience 14, 3304–9.

    Google Scholar 

  • Breer, H. (1993) Discussion note. InThe Molecular Basis of Smell and Taste Transduction, Ciba Foundation Symposium 179 (edited byChadwick, D., Marsh, J. &Goode, J.) pp. 66. Chichester: John Wiley & Sons.

    Google Scholar 

  • Breer, H., ed. (1994)Cell Biology of Olfaction. Seminars in Cell Biology 5.

  • Buck, L. &Axel, R. (1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.Cell 65, 175–87.

    Google Scholar 

  • Carr, V. Mcm., Farbman, A. I., Lidow, M. S., Coletti, L. M., Hempstead, J. L. &Morgan, J. I. (1989) Developmental expression of reactivity to monoclonal antibodies generated against olfactory epithelia.Journal of Neuroscience 9, 1179–98.

    Google Scholar 

  • Choi, E. J., Xia, Z. &Storm, D. R. (1992) Stimulation of type III olfactory adenylyl cyclase by calcium and calmodulin.Biochemistry 31, 6492–8.

    Google Scholar 

  • Cunningham, A. M., Ryugo, D. K., Sharp, A. H., Reed, R. R., Snyder, S. H. &Ronnett, G. V. (1993) Neuronal inositol 1,4,5-trisphosphate receptor localized to the plasma membrane of olfactory cilia.Neuroscience 15, 339–52.

    Google Scholar 

  • Dau, B., Menco, B. P. M., Bruch, R. C., Danho, W. &Farbman, A. I. (1991) Appearance of the transduction proteins G, Golfα and adenylate cyclase in the olfactory epithelium of rats occurs on different prenatal days.Chemical Senses 16, 511–12 (abstract).

    Google Scholar 

  • Dhallan, R. S., Yau, K.-W., Schrader, K. A. &Reed, R. R. (1990) Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons.Nature 347, 184–7.

    Google Scholar 

  • Drinnan, S. L., Hope, B. T., Snutch, T. P. &Vincent, S. R. (1991) Golf in the basal ganglia.Molecular and Cellular Neurosdence 2, 66–70.

    Google Scholar 

  • Farbman, A. I. (1992)Cell Biology of Olfaction. Cambridge: Cambridge University Press.

    Google Scholar 

  • Farbman, A. I. &Margolis, F. L. (1980) Olfactory marker protein during ontogeny: immunohistochemical localization.Developmental Biology 74, 205–15.

    Google Scholar 

  • Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W.-T., Krupinski, J., Gilman, A. G. &Reed, R. R. (1991) Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain.Proceedings of the National Academy of Sciences (USA) 88, 10173–7.

    Google Scholar 

  • Gesteland, R. C., Yancey, R. A. &Farbman, A. I. (1982) Development of olfactory receptor neuron selectivity in the rat fetus.Neurosdence 7, 3127–36.

    Google Scholar 

  • Hervé, D., Lévi-Strauss, M., Marey-Semper, I., Verney, C., Tassin, J.-P., Glowinski, J. &Girault, J.-A. (1993) Golf and Gs in rat basal ganglia: possible involvement of Golf in the coupling of dopamine D1 receptor with adenylyl cyclase.Journal of Neurosdence 13, 2237–48.

    Google Scholar 

  • Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, Y., Jan, L. &Evans, L. (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.Journal of Cell Biology 81, 275–300.

    Google Scholar 

  • Hille, B. (1992) G protein-coupled mechanisms and nervous signaling.Neuron 9, 187–95.

    Google Scholar 

  • Jones, D. T. (1990) Distribution of the stimulatory GTP-binding proteins, Gs and GoIf, within olfactory neuroepithelium.Chemical Senses 15, 333–40.

    Google Scholar 

  • Jones, D. T. &Reed, R. R. (1987) Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium.Journal of Biological Chemistry 29, 14241–9.

    Google Scholar 

  • Jones, D. T. &Reed, R. R. (1989) GoIf: an olfactory neuron specific-G protein involved in odorant signal transduction.Science 244, 790–5.

    Google Scholar 

  • Jones, D. T., Barbosa, E. &Reed, R. R. (1988) Expression of G-protein a-subunits in rat olfactory neuroepithelium: candidates for olfactory signal transduction.Cold Spring Harbor Symposia on Quantitative Biology 53, 349–53.

    Google Scholar 

  • Jones, D. T., Masters, S. B., Bourne, H. R. &Reed, R. R. (1990) Biochemical characterization of three stimulatory GTP-binding proteins. The large and small forms of Gs and the olfactory-specific G-protein, Colf.Journal of Biological Chemistry 265, 2671–6.

    Google Scholar 

  • Kalinoski, D. L., Dellacorte, C., Johnson, L. C. &Restrepo, D. (1993) Immunocytochemical localization of IP3 and Gq in the olfactory neuroepithelium.Society for Neurosdence Abstracts 19, 119.

    Google Scholar 

  • Kalinoski, D. L., Dellacorte, C., Menco, B. P. M. &Restrepo, D. (1994) Localization of inositol 1,4,5-trisphosphate receptors in the olfactory neuroepithelium of the rat and channel catfish.Proceedings AChemS XVI, Sarasota, Florida, abstract 283,Chemical Senses 19, in press.

  • Koshimoto, H., Katoh, K., Yoshihara, Y., Nemoto, Y. &Mori, K. (1994) Immunohistochemical demonstration of embryonic expression of an odor receptor protein and its zonal distribution in the rat olfactory epithelium.Neurosdence Letters 169, 73–6.

    Google Scholar 

  • Krishna, N. S. R., Getchell, T. V. &Getchell, M. L. (1994) Differential expression of Alpha, Mu, and Pi classes of gluthathione S-transferases in chemosensory mucosae of rats during development.Cell and Tissue Research 275, 435–50.

    Google Scholar 

  • Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R. &Gilman, A. G. (1989) Adenylyl cyclase amino acid sequence: possible channel- or transporterlike structure.Science 244, 1558–64.

    Google Scholar 

  • Lamb, T. D. &Pugh, Jr, E. N. (1992) G-protein cascades; gain and kinetics.Trends in Neurosciences 15, 291–8.

    Google Scholar 

  • Lancet, D., Shafir, I., Pace, U. &Lazard, D. (1989) Receptor-activated chemosensory cascades. InChemical Senses, Volume 1. Receptor Events and Transduction in Olfaction and Taste (edited byBrand, J. G., Teeter, J. H., Cagan, R. H. &Kare, M. R.) pp. 263–81. New York: Marcel Dekker.

    Google Scholar 

  • Leunissen, J. L. M. (1990) Background suppression using Aurion BSA-C and/or Tween-20®.Aurion Newsletter 1.

  • Lidow, M. S. &Menco, B. P. M. (1984) Observations on axonemes and membranes of olfactory and respiratory cilia in frogs and rats using tannic acid-supplemented fixation and photographic rotation.Journal of Ultrastructure Research 86, 18–30.

    Google Scholar 

  • Lowe, G. &Gold, G. H. (1991) The spatial distributions of odorant sensitivity and odorant-induced currents in salamander olfactory receptor cells.Journal of Physiology 442, 147–68.

    Google Scholar 

  • Mailleux, P., Mitchell, F., Vanderhaeghen, J.-J., Milligan, G. &Erneux, C. (1992) Immunohistochemical distribution of neurons containing the G-proteins Gqa/G11α in the adult rat brain.Neurosdence 51, 311–16.

    Google Scholar 

  • Mania-Farnell, B. &Farbman, A. I. (1990) Immunohisto-chemical localization of guanine nucleotide-binding proteins in rat olfactory epithelium during development.Developmental Brain Research 51, 103–12.

    Google Scholar 

  • Mania-Farnell, B. L., Farbman, A. I. &Bruch, R. C. (1993) Bromocriptine, a dopamine D2 receptor agonist, inhibits adenylyl cyclase activity in rat olfactory epithelium.Neurosdence 57, 173–80.

    Google Scholar 

  • Margalit, T. &Lancet, D. (1993) Expression of olfactory receptor and transduction genes during rat development.Developmental Brain Research 73, 7–16.

    Google Scholar 

  • Matsuoka, I., Giuili, G., Poyard, M., Stengel, D., Parma, J., Gueliaen, G. &Hanoune, J. (1992) Localization of adenylyl and guanylyl cyclase in rat brain byin situ hybridization: comparison with calmodulin mRNA distribution.Journal of Neurosdence 12, 3350–60.

    Google Scholar 

  • Menco, B. P. M. (1980) Qualitative and quantitative freezefracture studies on olfactory and nasal respiratory epithelial surfaces of frog, ox, rat, and dog. IV. Ciliogenesis and ciliary necklaces (including high-voltage observations).Cell and Tissue Research 212, 1–16.

    Google Scholar 

  • Menco, B. P. M. (1986) A survey of ultra-rapid cryofixation methods with particular emphasis on applications to freeze-fracturing, freeze-etching, and freeze-substitution.Journal of Electron Microscopy Technique 4, 177–240.

    Google Scholar 

  • Menco, B. P. M. (1988) Pre-natal development of nasal epithelia. IV. Freeze-fracturing on apices, microvilli and primary and secondary cilia of olfactory and respiratory epithelial cells, and on olfactory axons.Anatomy and Embryology 178, 309–26.

    Google Scholar 

  • Menco, B. P. M. (1992) Ultrastructural studies on membrane, cytoskeletal, mucous and protective compartments in olfaction.Microscopy Research and Technique 22, 215–24.

    Google Scholar 

  • Menco, B. P. M. (1994a) Ultrastructural aspects of olfactory transduction and perireceptor events.Seminars in Cell Biology 5, 11–24.

    Google Scholar 

  • Menco, B. P. M. (1994b) Freeze-fracture, deep-etch and freeze-substitution studies of olfactory epithelia, with special emphasis on immunocytochemical variables. InRapid Freezing, Freeze-Fracture and Deep Etching: The State of the Art (edited bySevers, N. &Shotton, D. M.), in press. New York: Wiley-Liss.

    Google Scholar 

  • Menco, B. P. M. (1994c) Ultrastructural aspects of olfactory signal-transduction and its development. InProceedings 52nd MSA Meeting (edited byBailey, G. W. &Garrattreed, A. J.) pp. 142–3. San Francisco: San Francisco Press.

    Google Scholar 

  • Menco, B. P. M. (1995) Scanning- and transmission electron microscopy of olfaction. InExperimental Cell Biology of Taste and Smell (edited bySpielman, A. I. &Brand, J. G.) in press. Boca Raton, FL: CRC Press.

    Google Scholar 

  • Menco, B. P. M. &Farbman, A. I. (1985) Genesis of cilia and microvilli of rat nasal epithelia during pre-natal development. II. Olfactory epithelium, a morphometric analysis.Journal of Cell Science 78, 311–36.

    Google Scholar 

  • Menco, B. P. M. &Farbman, A. I. (1987) Genesis of cilia and microvilli of rat nasal epithelia during pre-natal development. III. Respiratory epithelium surface, including a comparison with the surface of the olfactory epithelium.Journal of Anatomy 152, 145–60.

    Google Scholar 

  • Menco, B. P. M., Bruch, R. C., Dau, B. &Danho, W. (1992) Ultrastructural localization of olfactory transduction components: the G protein subunit Golfα and type III adenylyl cyclase.Neuron 8, 441–53.

    Google Scholar 

  • Menco, B. P. M., Dellacorte, C., Kalinoski, D. L. &Restrepo, D. (1993) IP3 receptors may occur in supporting cell microvilli of rat olfactory epithelia.Chemical Senses 18, 599 (abstract).

    Google Scholar 

  • Menco, B. P. M., Tekula, F. D., Farbman, A. I. &Danho, W. (1994) G-proteins and type III adenylyl cyclase during rat olfactory epithelium ontogeny. InOlfaction and Taste XI (edited byKurihara, K. &Suzuki, N.) pp. 141–4. Berlin: Springer Verlag.

    Google Scholar 

  • Milligan, G. (1993) Regional distribution and quantitative measurement of the phosphoinositidase C-linked guanine nucleotide binding proteins G11α and G.Journal of Neurochemistry 61, 845–51.

    Google Scholar 

  • Miyamoto, T., Restrepo, D., Cragoe, E. J., Jr &Teeter, J. H. (1992) IP3- and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish.Journal of Membrane Biology 127, 173–83.

    Google Scholar 

  • Mollner, S., Simmoteit, R., Palm, D. &Pfeuffer, T. (1991) Monoclonal antibodies against various forms of the adenylyl cyclase catalytic subunit and associated proteins.European Journal of Biochemistry 195, 281–6.

    Google Scholar 

  • Pfeuffer, E., Mollner, S., Lancet, D. &Pfeuffer, T. (1989) Olfactory adenylyl cyclase. Identification and purification of a novel enzyme form.Journal of Biological Chemistry 264, 18803–7.

    Google Scholar 

  • Phillips, T. E. &Boyne, A. F. (1984) Liquid nitrogen-based quick freezing: experiences with bounce-free delivery of cholinergic nerve terminals to a metal surface.Journal of Electron Microscopy Technique 1, 9–29.

    Google Scholar 

  • Phillips, G. W. &Bridgman, P. C. (1991) Immunoelectron microscopy of acetylcholine receptors and 43 kD protein after rapid freezing, free-substitution, and low-temperature embedding in Lowicryl K11M.Journal of Histochemistry and Cytochemistry 39, 625–34.

    Google Scholar 

  • Pieroni, J. P., Jacobowitz, O., Chen, J. &Iyengar, R. (1993) Signal recognition and integration by Gsstimulated adenylyl cyclases.Current Opinion in Neurobiology 3, 345–51.

    Google Scholar 

  • Reed, R. R., Bakalyar, H. A., Cunningham, A. M. &Levy, N. S. (1992) The molecular basis of signal transduction in olfactory sensory neurons. InSensory Transduction (edited byCorey, D. P. &Roper, S. D.) pp. 53–60. New York: Rockefeller University Press.

    Google Scholar 

  • Ressler, K. J., Sullivan, S. L. &Buck, L. B. (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium.Cell 73, 597–609.

    Google Scholar 

  • Restrepo, D., Teeter, J. H., Honda, E., Boyle, A. G., Marecek, J. F., Prestwich, G. D. &Kalinoski, L. D. (1992) Evidence for an InsP3-gated channel protein in isolated rat olfactory cilia.American Journal of Physiology 263, C667–73.

    Google Scholar 

  • Rius, R. A., Mollner, S., Pfeuffer, T. &Loh, Y. P. (1994) Developmental changes in Gs and Golf proteins and adenylyl cyclases in mouse brain membranes.Brain Research 643, 50–8.

    Google Scholar 

  • Roof, D. J., Applebury, M. L. &Sternweis, P. C. (1985) Relationships within the family of GTP-binding proteins isolated from bovine central nervous system.Journal of Biological Chemistry 260, 16242–9.

    Google Scholar 

  • Schulze, W., Kössler, A., Hinsch, K. D., Rosenthal, W., Will-Shahab, L., Küttner, L., Rada, T., Vannauer, M. &Breter, H. (1991) Immunocytochemical localization of G-proteins (α subunits) in rat heart tissue.European Heart Journal 12 (Supplement F), 132–4.

    Google Scholar 

  • Seifert, K. (1970) Die Ultrastruktur des Riechepithels beim Makrosmatiker. Eine elektronenmikroskopische Untersuchung. InNormale und pathologische Anatomie, Heft 21 (edited byBargmann, W. &Doerr, E.) pp. 000–000. Stuttgart: Georg Thieme Verlag.

    Google Scholar 

  • Shinohara, H., Kato, K. &Asano, T. (1992) Differential localization of G proteins, Gi and Go in the olfactory epithelium and the olfactory bulb of the rat.Ada Anatomica 144, 167–71.

    Google Scholar 

  • Shirsat, N. &Siddiqi, O. (1993) Olfaction in invertebrates.Current Opinion in Neurobiology 3, 553–7.

    Google Scholar 

  • Simonds, W. F., Goldsmith, P. K., Codina, J., Unson, C. G. &Spiegel, A. M. (1989) G12 mediates α2-adrenergic inhibition of adenylyl cyclase in platelet membranes:in situ identification with Gα C-terminal antibodies.Proceedings of the National Academy of Sciences (USA) 86, 7809–13.

    Google Scholar 

  • Sklar, P. B., Anholt, R. R. H. &Snyder, S. H. (1986) The odorant-sensitive adenylate cyclase of olfactory receptor cells.Journal of Biological Chemistry 261, 15538–43.

    Google Scholar 

  • Smrcka, A. V., Hepler, J. R., Brown, K. O. &Sternweis, P. C. (1991) Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq.Science 251, 804–7.

    Google Scholar 

  • Strittmatter, S. M., Valenzuela, D., Kennedy, T. E., Neer, E. J. &Fishman, M. C. (1990) Go is a major growth cone protein subject to regulation by GAP-43.Nature 344, 836–42.

    Google Scholar 

  • Suzuki, N. (1994) IP3-activated ion channel activities in olfactory receptor cells from different vertebrate species. InOlfaction and Taste XI (edited byKurihara, K. &Suzuki, N.) pp. 173–7. Berlin: Springer Verlag.

    Google Scholar 

  • Tang, W.-J. &Gilman, A. G. (1992) Adenylyl cyclases.Cell 70, 869–72.

    Google Scholar 

  • Van Lockeren Campagne, M. B., Obstreicher, B., Van Der Krift, T. P., Gispen, W. H. &Verkleij, A. J. (1991) Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.Journal of Histochemistry and Cytochemistry 39, 1267–79.

    Google Scholar 

  • Vassar, R., Ngai, J. &Axel, R. (1993) Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium.Cell 74, 309–18.

    Google Scholar 

  • Verhaagen, J., Obstreicher, A. B., Gispen, W. H. &Margolis, F. L. (1989) The expression of the growth associated protein B50/GAP43 in the olfactory system of neonatal and adult rats.Journal of Neuroscience 9, 683–91.

    Google Scholar 

  • Villiger, W. (1991) Lowicryl resins. InColloidal Gold. Principles, Methods, and Application, Vol. 3 (edited byHayat, M. A.) pp. 59–71. San Diego: Academic Press, Inc.

    Google Scholar 

  • Xia, Z., Choi, E.-J., Wang, F. &Storm, D. R. (1992) The type III calcium/calmodulin-sensitive adenylyl cyclase is not specific to olfactory sensory neurons.Neuroscience Letters 144, 169–73.

    Google Scholar 

  • Zigman, J. M., Westermark, G. T., Lamendola, J., Boel, E. &Steiner, D. F. (1993) Human G(olfa)complementary deoxyribonucleic acid structure and expression in pancreatic islets and other tissues outside the olfactory neuroepithelium and central nervous system.Endocrinology 133, 2508–14.

    Google Scholar 

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Menco, B.P.M., Tekula, F.D., Farbman, A.I. et al. Developmental expression of G-proteins and adenylyl cyclase in peripheral olfactory systems. Light microscopic and freeze-substitution electron microscopic immunocytochemistry. J Neurocytol 23, 708–727 (1994). https://doi.org/10.1007/BF01181645

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