Skip to main content
SpringerLink
Log in

Gliogenesis in organotypic tissue culture of the spinal cord of the embryonic mouse. I. Immunocytochemical and ultrastructural studies

  • Published:
Journal of Neurocytology

Summary

The technique of organotypic tissue culture offers an opportunity to observein vitro complex interactions among glial cells and neurons, leading to the formation of myelin. In the present and accompanying work a combined ultrastructural, immunocytochemical and autoradiographic approach was used in a detailed study of the process of gliogenesis. Using immunocytochemical and ultrastructural criteria, differentiation along the oligodendroglia cell line is seen to be initiated a few days later than along the astroglial line. The sequence and timing of oligodendroglial differentiation both ultrastructurally and chemically follow those describedin vivo. Formation of myelin has been demonstrated only by oligodendrocytes in which there is continuity between the perikaryal plasmalemma and myelin membranes. Oligodendroglial maturation culminated with the formation of light, medium and dark oligodendrocytes. The periodic acid Schiff-positive, glial fibrillary acidic protein (GFAP)-negative processes of radial glial cells at explantation become GFAP-positive within 3 days, as describedin vivo. Many of the astrocytes appear to have been derived from radial glial cells. Large numbers of dark glial cells, similar to the so-called ‘intermediate glial cells’, were seen. These were found to be astrocytes whose appearance probably reflected reaction to explantation-induced injury.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Abney, E. R., Bartlett, P. B. &Raff, M. C. (1981) Astrocytes, ependymal cells and oligodendrocytes develop on schedule in dissociated cell cultures of embryonic rat brain.Developmental Biology 83, 301–10.

    Google Scholar 

  • Abney, E. R., Williams, B. P. &Raff, M. C. (1983) Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence activated cell sorting and cell culture.Developmental Biology 100, 166–71.

    Google Scholar 

  • Bancroft, J. D. &Stevens, A. (1982) (eds)Theory and Practice of Histological Techniques p. 33. Edinburgh: Churchill Livingstone.

    Google Scholar 

  • Berg, G. &Schachner, M. (1981) Immuno-electron-microscopic identification of O-antigen-bearing oligodendroglial cellsin vitro.Cell and Tissue Research 219, 313–25.

    Google Scholar 

  • Bhat, S., Barbarese, E. &Pfeiffer, S. E. (1981) Requirement for non-oligodendrocyte cell signals for enhanced myelogenic gene expression in long-term cultures of purified rat oligodendrocytes.Proceedings of the National Academy of Sciences USA 78, 1283–7.

    Google Scholar 

  • Bignami, A. &Dahl, D. (1974) Astrocyte-specific protein and neuroglial differentiation: An immunofluorescence study with antibodies to the glial fibrillary acidic protein.Journal of Comparative Neurology 153, 27–38.

    Google Scholar 

  • Bologa, L., Bisconte, J.-C., Joubert, R., Marangos, P. J., Derbin, C., Rioux, F. &Herschkowitz, N. (1982a) Accelerated differentiation of oligodendrocytes in neuronal-rich embryonic mouse brain cell cultures.Brain Research 252, 129–36.

    Google Scholar 

  • Bologa, L., Z'Graggen, A., Rossi, E. &Herschkowitz, N. (1982b) Differentiation and proliferation: two possible mechanisms for the regeneration of oligodendrocytes in culture.Journal of the Neurological Sciences 57, 419–34.

    Google Scholar 

  • Bologa-Sandru, L., Siegrist, H. P., Z'Graggen, A., Hofman, K., Wiesman, V., Dahl, D. &Herschkowitz, N. (1981) Expression of antigenic markers during the development of oligodendrocytes in mouse brain cultures.Brain Research 210, 217–29.

    Google Scholar 

  • Borit, A. &McIntosh, C. G. (1981) Myelin basic protein and glial fibrillary acidic protein in human fetal brain.Neuropathology and Applied Neurobiology 7, 279–87.

    Google Scholar 

  • Bornstein, M. B. (1973) Organotypic mammalian central and peripheral nerve tissue. InTissue Culture. Methods and Applications (edited byKruse, P. F., Jr &Patterson, M. K., Jr). New York: Academic Press.

    Google Scholar 

  • Bunge, M. B., Bunge, R. P. &Ris, H. (1961) Ultrastructural study of remyelination in an experimental lesion in adult cat spinal cord.Journal of Biophysical and Biochemical Cytology 10, 67–94.

    Google Scholar 

  • Bunge, R. P., Bunge, M. B. &Peterson, E. R. (1965) An electron microscopic study of cultured rat spinal cord.Journal of Cell Biology 24, 163–91.

    Google Scholar 

  • Coleman, D. R., Kreibich, G., Grey, A. B. &Sabatini, D. D. (1982) Synthesis and incorporation of myelin polypeptides into CNS myelin.Journal of Cell Biology 95, 598–608.

    Google Scholar 

  • Federoff, S., Neal, J., Opas, M. &Kalnins, V. I. (1984) Astrocyte cell lineage. III. The morphology of differentiating mouse astrocytes in colony culture.Journal of Neurocytology 13, 1–20.

    Google Scholar 

  • Gebicke-Harter, P. S., Althaus, H. H., Schwartz, P. &Neuhoff, V. (1981) Oligodendrocytes from postnatal cat brain in cell cultures. I. Regeneration and maintenance.Developmental Brain Research 1, 497–518.

    Google Scholar 

  • Ghandour, M. S., Langley, O. K., Vincendon, G., Gombos, G., Filippi, D., Limozin, N., Daulmasso, C. &Laurent, G. (1980a) Immunochemical and immunohistochemical study of carbonic anhydrase II in adult rat cerebellum: a marker for oligodendrocytes.Neuroscience 5, 559–71.

    Google Scholar 

  • Ghandour, M. D., Vincendon, G. &Gombos, G. (1980b) Astrocyte and oligodendrocyte distribution in adult rat cerebellum: an immunohistological study.Journal of Neurocytology 9, 637–46.

    Google Scholar 

  • Gonatas, N. K., Hirayama, M., Stieber, A. &Silberberg, D. H. (1982) The ultrastructure of isolated rat oligodendroglial cell cultures.Journal of Neurocytology 11, 997–1008.

    Google Scholar 

  • Hager, H., Luh, S., Ruscakova, D. &Ruscak, M. (1967) Histochemische, elektronenmikroskopische and biochemische Untersuchungen uber Glykogenanhaufung in reaktiv veranderten Astrozyten der traumatisch ladierten Saugergroßhirnrinde.Zeitschrift für Zellforschung und mikroskopische Anatomie 83, 295–320.

    Google Scholar 

  • Hartman, B. K., Agrawal, H. C., Agrawal, D. &Kalmbach, S. (1982) Development and maturation of central nervous system myelin: comparison of immunohistochemical localization of proteolipid protein and basic protein in myelin and oligodendrocytes.Proceedings of the National Academy of Sciences USA 79, 4217–20.

    Google Scholar 

  • Hartman, B. K., Agrawal, H. C., Kalmbach, S. &Shearer, W. T. (1979) A comparative study of the immunohistochemical localization of basic protein to myelin and oligodendrocytes in rat and chicken brain.Journal of Comparative Neurology 188, 278–90.

    Google Scholar 

  • Henrickson, C. K. &Vaughn, J. E. (1974) Fine structure relationships between neurites and radial glial processes in developing mouse spinal cord.Journal of Neurocytology 3, 654–75.

    Google Scholar 

  • Hsu, S-M., Raine, L. &Fanger, H. (1981) The use of antividin antibody and avidin-biotinperoxidase complex in immunoperoxidase technics.American Journal of Clinical Pathology 75, 816–21.

    Google Scholar 

  • Imamoto, K., Paterson, J. A. &Leblond, C. P. (1978) Radioautographic investigation of gliogenesis in the corpus callosum of young rats. I. Sequential changes in oligodendrocytes.Journal of Comparative Neurology 180, 115–38.

    Google Scholar 

  • Itoyama, Y., Sternberger, N. H., Kies, M. W., Cohen, S. R., Richardson, E. P., &Webster, H. DeF (1980a) Immunocytochemical method to identify myelin basic protein in oligodendroglia and myelin sheaths of the human nervous system.Annals of Neurology 7, 157–66.

    Google Scholar 

  • Itoyama, Y., Sternberger, N. H., Webster, H. DeF., Quarles, R. H., Cohen, S. R. &Richardson, E. P. (1980b) Immunocytochemical observations on the distribution of myelin-associated glycoprotein and myelin basic protein in multiple sclerosis lesions.Annals of Neurology 7, 167–77.

    Google Scholar 

  • Labourdette, G., Roussel, G. &Nussbaum, J. L. (1980) Oligodendroglial content of glial cell primary cultures from newborn rat brain hemispheres depends on the initial plating density.Neuroscience Letters 18, 203–9.

    Google Scholar 

  • Lenk, R., Rauson, L., Kaufman, Y. &Penman, S. (1977) A cytoskeletal structure with associated polyribosomes obtained from Hela cells.Cell 10, 67–78.

    Google Scholar 

  • Ling, E. A., Paterson, J. A., Privat, A., Mori, S. &Leblond, C. P. (1973) Investigation of glial cells in semithin sections. I. Identification of glial cells in the brain of young rats.Journal of Comparative Neurology 149, 43–72.

    Google Scholar 

  • Lisak, R. P., Pleasure, D. W., Silberberg, D. H., Manning, M. C. &Saida, T. (1981) Long-term culture of bovine oligodendroglia isolated with a percoll gradient.Brain Research 223, 107–22.

    Google Scholar 

  • Ludwin, S. K. (1979) The perineuronal satellite oligodendrocyte — a possible role in myelination.Acta Neuropathologica (Berlin) 47, 49–53.

    Google Scholar 

  • Ludwin, S. K. (1984) The function of perineuronal satellite oligodendrocytes: an immunohistochemical study.Neuropathology and Applied Neurobiology 10, 143–9.

    Google Scholar 

  • Ludwin, S. K. &Sternberger, N. H. (1984) An immunohistochemical study of myelin proteins during remyelination in the central nervous system.Acta Neuropathologica (Berlin) 63, 240–8.

    Google Scholar 

  • Lumsden, C. E. (1968) Nervous tissue in culture. InThe Structure and Function of Nervous Tissue (edited byBourne, G. H.), Vol. 1, pp. 67–140. New York: Academic Press.

    Google Scholar 

  • Manuelidis, L. &Manuelidis, E. (1971) An autoradiographic study of the proliferation and differentiation of glial cellsin vitro.Acta neuropathologica (Berlin) 18, 193–213.

    Google Scholar 

  • Meier, D. &Schachner, M. (1982) Immunoselection of oligodendrocytes by magnetic beads. II.In vivo maintenance of immunoselected oligodendrocytes.Journal of Neuroscience Research 7, 135–45.

    Google Scholar 

  • Mirsky, R., Winter, J., Abney, E. R., Pruss, R. M., Gavrilovic, J. &Raff, M. C. (1980) Myelin specific proteins and glycoproteins in rat Schwann cells and oligodendrocytes in culture.Journal of Cell Biology 84, 483–94.

    Google Scholar 

  • Mithen, F. A., Wood, P. M., Agrawal, H. C. &Bunge, R. P. (1983) Immunocytochemical study of myelin sheaths formed by oligodendrocytes interacting with dissociated dorsal root ganglion cells in culture.Brain Research 262, 63–9.

    Google Scholar 

  • Mori, S. &Leblond, C. P. (1969) Electron microscopic features and proliferation of astrocytes in the corpus callosum of the rat.Journal of Comparative Neurology 137, 197–226.

    Google Scholar 

  • Mori, S. &Leblond, C. P. (1970) Electron microscopic identification of three classes of oligodendrocytes and a prelimary study of their proliferative activity in the corpus callosum of young rats.Journal of Comparative Neurology 139, 1–30.

    Google Scholar 

  • Munoz-Garcia, D. &Ludwin, S. K. (1986) Gliogenesis in organotypic tissue culture of the spinal cord of the embryonic mouse. II. Autoradiographic studies.Journal of Neurocytology 15, 291–302.

    Google Scholar 

  • Nagashima, K. (1979) Ultrastructural study of myelinating cells and subpial astrocytes in developing rat spinal cord.Journal of the Neurological Sciences 44, 1–12.

    Google Scholar 

  • Oster-Granite, M. L. &Herndon, R. M. (1976) The development of the cerebellar cortex of the Syrian hamster, Mesocricetus auratus: foliation, cytoarchitectonic, Golgi and electron microscopic studies.Journal of Comparative Neurology 169, 443–80.

    Google Scholar 

  • Parnavelas, J. G., Luder, R., Pollard, S. G., Sullivan, K. &Lieberman, A. R. (1983) A qualitative and quantitative, ultrastructural study of glial cells in the developing visual cortex of the rat.Philosophical Transactions of the Royal Society of London, Series B 301, 55–84.

    Google Scholar 

  • Paterson, J. A., Privat, A., Ling, E. A. &Leblond, C. P. (1973) Investigation of glial cells in semithin sections. III. Transformation of subependymal cells into glial cells, as shown by autoradiography after H3 thymidine injection into the lateral ventricle of the young rat.Journal of Comparative Neurology 146, 277–302.

    Google Scholar 

  • Peterson, E. R., Crain, S. M. &Murray, M. R. (1965) Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick, and human).Zeitschrift für Zellforschung und mikroskopische Anatomie 66, 130–54.

    Google Scholar 

  • Pettmann, B., Delounoy, J-P., Couragest, S., Devilliers, G. &Sensenbrenner, M. (1980) Rat brain cells in culture: effects of brain extracts on the development of oligodendroglia-like cells.Developmental Biology 75, 278–87.

    Google Scholar 

  • Phillips, E. E. (1973) An electron microscopic study of macroglia and microglia in the lateral funiculus of the developing spinal cord in the fetal monkey.Zeitschrift für Zellforschung und mikroskopische Anatomie 140, 145–67.

    Google Scholar 

  • Privat, A. &Fulcrand, J. (1977) Neuroglia: from the subventricular precursors to the mature cell. InCell, Tissue and Organ Cultures in Neurobiology (edited byFederoff, S. &Hertz, L.), pp. 11–38. New York: Academic Press.

    Google Scholar 

  • Privat, A. &Leblond, C. P. (1972) The subependymal layer and neighboring region in the brain of the young rat.Journal of Comparative Neurology 146, 277–302.

    Google Scholar 

  • Raff, M., Fields, K. L., Hakomori, S-I., Mirsky, R., Pruss, R. M. &Winter, J. (1979) Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture.Brain Research 174, 283–308.

    Google Scholar 

  • Raff, M., Miller, R. H. &Noble, M. (1983) A glial progenitor cell that developsin vitro into an astrocyte or an oligodendrocyte depending on culture medium.Nature 303, 390–6.

    Google Scholar 

  • Raine, C. (1973) Ultrastructural applications of cultured nervous system tissue to neuropathology.Progress in Neuropathology 2, 27–68.

    Google Scholar 

  • Raine, C. &Bornstein, M. B. (1970a) Experimental allergic encephalomyelitis: an ultrastructural study of experimental demyelinationin vitro.Journal of Neuropathology and Experimental Neurology 29, 177–91.

    Google Scholar 

  • Raine, C. &Bornstein, M. B. (1970b) Experimental allergic encephalomyelitis: a light and electron microscopic study of remyelination and sclerosisin vitro.Journal of Neuropathology and Experimental Neurology 29, 552–74.

    Google Scholar 

  • Raine, C. S. &Wisniewski, H. (1970) On the occurrence of microtubules within mature astrocytes.Anatomical Record 167, 303–8.

    Google Scholar 

  • Rioux, F., Derbin, C., Marqules, S., Joubert, R. &Bisconte, J-C. (1980) Kinetics of oligodendrocyte-like cells in primary culture of mouse embryonic brain.Developmental Biology 26, 87–99.

    Google Scholar 

  • Rio Hortega, P. del (1928) Tecera aportacion al conocimiento morfologico e interpretacion functional de la oligodendroglia.Memorias de la Real Sociedad Espanola de Historia Natural 14, 5–122.

    Google Scholar 

  • Ross, L. L., Bornstein, M. B. &Lehrer, G. M. (1962) Electron microscopic observation of rat and mouse cerebellum in tissue culture.Journal of Cell Biology 14, 19–30.

    Google Scholar 

  • Roussel, G. &Nussbaum, J. L. (1981) Comparative localization of Wolfgram protein and myelin basic protein in the rat brain during ontongenesis.Histochemical Journal 13, 1029–47.

    Google Scholar 

  • Sensenbrenner, M., Delaunoy, J-P., Labourdette, G. &Pettmann, B. (1982) Effects of brain extracts on the proliferation and the maturation of astroglial and oligodendroglial cells in culture.Biochemical Society Transactions 10, 424–6.

    Google Scholar 

  • Silberberg, D. H., Dorfmann, S. A., Latovitzki, N. &Yoonkin, L. H. (1980) Oligodendrocyte differentiation in myelinating cultures. InTissue Culture in Neurobiology (edited byGiacobini, E., Vernadakis, A. &Shahar, A.), pp. 489–500. New York: Raven Press.

    Google Scholar 

  • Skoff, R. P., Price, D. L. &Stocks, A. (1976a) Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. I. Cell proliferation.Journal of Comparative Neurology 169, 291–311.

    Google Scholar 

  • Skoff, R. P., Price, D. L. &Stocks, A. (1976b) Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. II. Time of origin.Journal of Comparative Neurology 169, 313–33.

    Google Scholar 

  • Sternberger, N. H., Itoyama, Y., Kies, M. W. &Webster, H. DeF. (1978) Myelin basic protein demonstrated immunohistochemically in oligodendroglia prior to myelin sheath formation.Proceedings of the National Academy of Sciences USA 75, 2521–4.

    Google Scholar 

  • Sternberger, N. H., Quarles, R. H., Itoyama, Y. &Webster, H. DeF. (1979) Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin forming cells of developing rat.Proceedings of the National Academy of Sciences USA 76, 1510–4.

    Google Scholar 

  • Sturrock, R. R. (1974) Histogenesis of the anterior limb of the anterior commissure of the mouse brain. III. An electron microscopic study of gliogenesis.Journal of Anatomy 117, 37–53.

    Google Scholar 

  • Sturrock, R. R. (1982) Gliogenesis in the prenatal rabbit spinal cord.Journal of Anatomy 134, 771–93.

    Google Scholar 

  • Szuchet, S. &Dumas, M. (1983) Anin vitro approach to the study of oligodendrocytes and their involvement in multiple sclerosis.Neurology Clinics 1, 729–55.

    Google Scholar 

  • Szuchet, S., Stefansson, K. R. L., Dawson, G. &Arnason, B. G. W. (1980) Maintenance of two subpopulations of oligodendrocytes in long-term culture.Brain Research 200, 151–64.

    Google Scholar 

  • Trapp, B. P., Itoyama, Y., MacIntosh, T. P. &Quarles, R. H. (1983) P2 in oligodendrocytes and myelin of the rabbit central nervous system.Journal of Neurochemistry 40, 47–54.

    Google Scholar 

  • Ulrich, J., Kasper, M., Jancer, H., Basler, V. &Heitz, U. (1982) Glial protein in myelinating tissue cultures visualized by postembedding immunocytochemistry.Brain Research 240, 199–202.

    Google Scholar 

  • Valat, J., Privat, A. &Fulcrand, J. (1983) Multiplication and differentiation of glial cells in the optic nerve of the postnatal rat.Anatomy and Embryology 167, 335–46.

    Google Scholar 

  • Vaughn, J. E. (1969) An electron microscopic analysis of gliogenesis in rat optic nerve.Zeitschrift für Zellforschung und mikroskopische Anatomie 94, 294–324.

    Google Scholar 

  • Wollman, R. L., Szuchet, S., Barlow, J. &Jerkovic, M. (1981) Ultrastructural changes accompanying the growth of isolated oligodendrocytes.Journal of Neuroscience Research 6, 757–69.

    Google Scholar 

  • Wood, P., Okada, E. &Bunge, R. (1980) The use of networks of dissociated rat dorsal root ganglion nerves to induce myelination of oligodendrocytes in cultures.Brain Research 196, 247–52.

    Google Scholar 

Download references

Author information

Author notes

  1. D. Munoz-Garcia

    Present address: Department of Pathology (Neuropathology), University of Vermont, 05405, Burlington, VT, USA

Authors and Affiliations

  1. Departments of Pathology (Neuropathology), Queen's University and Kingston General Hospital, K7L 3N6, Kingston, Ontario, Canada

    D. Munoz-Garcia & S. K. Ludwin

Authors
  1. D. Munoz-Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. K. Ludwin
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Munoz-Garcia, D., Ludwin, S.K. Gliogenesis in organotypic tissue culture of the spinal cord of the embryonic mouse. I. Immunocytochemical and ultrastructural studies. J Neurocytol 15, 273–290 (1986). https://doi.org/10.1007/BF01611431

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01611431

Keywords

Search

Navigation