Skip to main content
Log in

Cyclic morphological changes of glial cells in long-term cultures of rat brain

  • Published:
Journal of Neurocytology

Summary

Brain cells from embryonic rats were dissociated with trypsin, cultivated under constant conditions in Falcon flasks, and studied for periods of one year or more. Antisera against glial fibrillary acidic protein (GFA) and myelin basic protein (MBP) were used to identify glial cell types. For scanning electron microscopical (SEM) observation an embedding method in resin was developed that allows good preservation of the fine ultrastructural features of the cultivated cells and precise characterization of the cell types. Under our culture conditions, after four subcultures and 8–10 weeks of cultivation, the following cell types can be distinguished. (a) Flat epitheloid cells. From an immunocytological point of view these cells form a heterogeneous population composed of GFA- and MBP-positive and negative cells. They are the precursors of the following cell types. (b) Astroglial cells. SEM observations show a characteristic network of radially orientated prolongations. 92% of these cells are GFA-positive. (c) Oligodendroglial cells with characteristic dichotomously dividing branches. Secondary and tertiary branches end in flat amoeboid prolongations. These cells are MBP-positive.

After approximately six weeks the most prominent cells are the flat epitheloid cells. The astroglial cells originate continuously from the epitheloid cells during the whole cultivation time. The formation of oligodendroglial cells, on the other hand, takes place at relatively precise intervals of time (approximately every 20–30 days) over the entire cultivation period (of more than one year).

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Akeson, R. (1979) Cell surface antigens of cultured neuronal cells.Current Topics in Developmental Biology 13, 215–36.

    PubMed  Google Scholar 

  • Antanitus, D. S., Choi, B. H. &Lapham, L. W. (1975) Immunofluorescence staining of astrocytesin vitro using antiserum to glial fibrillary acidic protein.Brain Research 89, 363–67.

    PubMed  Google Scholar 

  • Barbarese, E., Bhat, S., Bansal, R., Carson, J., Edgar, A., Pfeiffer, S. E., Singh, H. &Woodiel, F. (1983) Regulation of early myelinogenesis in primary cultures of dissociated rat brain. InNeuroscience Approached Through Cell Culture Vol. II (edited byPfeiffer, S. E.), pp. 167–76. Boca Raton, Florida: CRC Press.

    Google Scholar 

  • Benda, P. De Vitry, F., Picart, R. &Tixier-Vidal, A. (1975) Dissociated cell cultures from fetal mouse hypothalamus patterns of organization and ultrastructural features.Experimental Brain Research 23, 29–47.

    Google Scholar 

  • Bock, E., Moller, M., Nissen, C. &Sensenbrenner, M. (1977) Glial fibrillary acidic protein in primary astroglial cell cultures derived from newborn rat brain.FEBS Letters 83, 207–11.

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  • Booher, J. &Sensenbrenner, M. (1972) Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures.Neurobiology 2, 97–105.

    PubMed  Google Scholar 

  • Cam, Y., Sensenbrenner, M. &Mandel, P. (1975) A comparative study of the effects of brain extracts and mesodermal membrane extracts on nerve cell differentiation.Experientia 31, 1430–31.

    PubMed  Google Scholar 

  • Campbell, G. LéM. &Williams, M. P. (1978)In vitro growth of glial cell-enriched and depleted populations from mouse cerebellum.Brain Research 156, 227–39.

    PubMed  Google Scholar 

  • Choi, B. H. &Lapham, L. W. (1976) Interactions of neurons and astrocytes during growth and development of human fetal brainin vitro.Experimental and Molecular Pathology 24, 110–25.

    PubMed  Google Scholar 

  • Cummins, C. J. &Glover, R. A. (1978) Propagation and histological characterization of a homotypic population of astrocytes derived from neonatal rat brain.Journal of Anatomy 125, 117–25.

    PubMed  Google Scholar 

  • Debbage, P. L. (1982) Selective neuronal survival in long-term cell cultures of chick embryo spinal cord.Journal of Anatomy 134, 597 (abstract).

    Google Scholar 

  • Del Rio Hortega, P. (1921) La glia de escasas radiaciones (oligodendroglia).Boletín de la Real Sociedad Española de Historia Natural 21, 63–92.

    Google Scholar 

  • De Vitry, F., Picart, R., Jacque, C., Legault, L., Dupouey, P. &Tixier-Vidal, A. (1980) Presumptive common precursor for neuronal and glial cell lineages in mouse hypothalamus.Proceedings of the National Academy of Sciences USA 77, 4165–69.

    Google Scholar 

  • Fedoroff, S. (1978) The development of glial cells in primary cultures. InDynamic Properties of Glial Cells (edited bySchoffeniels, E., Frank, G., Hertz, L. &Tower, D. B.), pp. 83–92. Oxford, New York, Toronto, Sydney, Paris, Frankfurt: Pergamon Press.

    Google Scholar 

  • Fields, K. L. (1979) Cell type-specific antigens of cells of the central and peripheral nervous system.Current Topics in Developmental Biology 13, 237–57.

    PubMed  Google Scholar 

  • Fromme, H. G., Pfautsch, M., Pfefferkorn, G. &Byztricky, V. (1972) Die Kritische Punkt-Trocknung als Präparationsmethode für die Rasterelektronenmikroskopie.Microscopica Acta 73, 29–37.

    PubMed  Google Scholar 

  • Haugen, A. &Laerum, O. D. (1978) Induced glial differentiation of fetal rat brain cells in culture: an ultrastructural study.Brain Research 150, 225–38.

    PubMed  Google Scholar 

  • Hayflick, L. (1965) The limitedin vitro lifetime of human diploid cell strains.Experimental Cell Research 37, 614–36.

    PubMed  Google Scholar 

  • Haynes, L. W. &Weller, R. O. (1978) Induction of some features of glial differentiation in primary cultures of human gliomas by treatment with dibutyryl cyclic AMP.British Journal of Experimental Pathology 59, 259–76.

    PubMed  Google Scholar 

  • Hill, C. E., Chamley, J. H. &Burnstock, G. (1974) Cell surfaces and fibre relationships in sympathetic ganglion cultures: a scanning electron-microscopic study.Journal of Cell Science 14, 657–69.

    PubMed  Google Scholar 

  • Horisberger, M. (1979) Evaluation of colloidal gold as a cytochemical marker for transmission and scanning electron microscopy.Biologie Cellulaire 36, 253–58.

    Google Scholar 

  • Kao, W. W. -Y. &Prockop, D. J. (1977) Proline analogue removes fibroblasts from cultured mixed cell populations.Nature 266, 63–64.

    PubMed  Google Scholar 

  • Kimelberg, H. K., Narumi, S. &Bourke, R. S. (1978) Enzymatic and morphological properties of primary rat brain astrocyte cultures, and enzyme developmentin vivo.Brain Research 153, 55–77.

    PubMed  Google Scholar 

  • Kozak, L. P., Dahl, D. &Bignami, A. (1978) Glial fibrillary acidic protein in reaggregating and monolayer cultures of fetal mouse cerebral hemispheres.Brain Research 150, 631–37.

    PubMed  Google Scholar 

  • Landis, D. M. D. &Reese, T. S. (1981) Membrane structure in mammalian astrocytes: a review of freeze-fracture studies on adult, developing, reactive and cultured astrocytes.Journal of Experimental Biology 95, 35–48.

    PubMed  Google Scholar 

  • Levitt, P., Cooper, M. L. &Rakic, P. (1981) Coexistence of neuronal and glial precursor cells in the cerebral ventricular zone of the fetal monkey: an ultrastructural immunoperoxidase analysis.Journal of Neuroscience 1, 27–39.

    PubMed  Google Scholar 

  • Lim, R. &Mitsunobu, K. (1974) Brain cells in culture: morphological transformation by a protein.Science 185, 63–66.

    PubMed  Google Scholar 

  • Lim, R., Mitsunobu, K. &Li, W. K. P. (1973) Maturation-stimulating effect of brain extract and dibutyryl cyclic AMP on dissociated embryonic brain cells in culture.Experimental Cell Research 79, 243–46.

    PubMed  Google Scholar 

  • Lim, R., Turriff, D. E. &Troy, S. S. (1976) Response of glioblasts to a morphological transforming factor: cinematographic and chemical correlations.Brain Research 113, 165–70.

    PubMed  Google Scholar 

  • Lim, R., Troy, S. S. &Turriff, D. E. (1977b) Fine structure of cultured glioblasts before and after stimulation by a glia maturation factor.Experimental Cell Research 106, 357–72.

    PubMed  Google Scholar 

  • Lim, R., Turriff, D. E., Troy, S. S., Moore, B. W. &Eng, L. F. (1977a) Glia maturation factor: effect on chemical differentiation of glioblasts in culture.Science 195, 195–96.

    PubMed  Google Scholar 

  • Lodin, Z., Korinkova, P., Faltin, J. &Fleischmannova, V. (1978) Structure and ultrastructure of cultivated glial cells from corpus callosum.Acta Histochemica 61, 165–83.

    PubMed  Google Scholar 

  • Lumsden, C. E. &Pomerat, C. M. (1951) Normal oligodendrocytes in tissue culture. A preliminary report on the pulsatile glial cells in tissue cultures from the corpus callosum of the normal adult rat brain.Experimental Cell Research 2, 103–14.

    Google Scholar 

  • Manthorpe, M., Adler, R. &Varon, S. (1979) Development, reactivity and GFA immunofluorescence of astroglia-containing monolayer cultures from rat cerebrum.Journal of Neurocytology 8, 605–21.

    PubMed  Google Scholar 

  • Meller, K. (1979) Scanning electron microscopic studies on the development of the nervous systemin vivo andin vitro.International Review of Cytology 56, 23–55.

    PubMed  Google Scholar 

  • Meller, K. (1981) General methods in scanning electron microscopy of the nervous system. InTechniques in Neuroanatomical Research (edited byHeym, Ch. &Forssmann, W. -G.), pp. 55–70. Berlin, Heidelberg, New York: Springer.

    Google Scholar 

  • Meller, K., Breipohl, W., Wagner, H. H. &Knuth, A. (1969) Die Differenzierung isolierter Nerven- und Gliazellen aus trypsiniertem Rückenmark von Hühnerembryonen in Gewebekulturen. Eine licht- und elektronenmikroskopische Studie.Zeitschrift für Zellforschung 101, 135–51.

    Google Scholar 

  • Mirsky, R., Wendon, L. M. B., Black, P., Stolkin, C. &Bray, D. (1978) Tetanus toxin: a cell surface marker for neurones in culture.Brain Research 148, 251–59.

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  • Moonen, G., Cam, Y., Sensenbrenner, M. &Mandel, P. (1975) Variability of the effects of serum-free medium, dibutyryl-cyclic AMP or Theophylline on the morphology of cultured new-born rat astroblasts.Cell and Tissue Research 163, 365–72.

    PubMed  Google Scholar 

  • Moonen, G., Meinen, E. &Goessens, G. (1976) Comparative ultrastructural study of the effects of serum-free medium and dibutyryl-cyclic AMP on newborn rat astroblasts.Cell and Tissue Research 167, 221–27.

    PubMed  Google Scholar 

  • Morrison, R. S. &De Vellis, J. (1981) Regulation of proliferation and differentiation of astrocytes in a defined medium.Journal of Cell Biology 91, 91a.

    Google Scholar 

  • Nakai, J. &Okamoto, M. (1963) Identification of neuroglial cells in tissue culture. InMorphology of Neuroglia (edited byNakai, J.), pp. 65–102. Tokyo: Igaku Shoin Ltd.

    Google Scholar 

  • Peterson, G. R., Webster, G. W. &Shuster, L. (1973) Characteristics of choline acetyltransferase and cholinesterases in two types of cultured cells from embryonic chick brain.Developmental Biology 34, 119–34.

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  • Raff, M. C., 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.

    PubMed  Google Scholar 

  • Roussel, G., Labourdette, G. &Nussbaum, J. L. (1981) Characterization of oligodendrocytes in primary cultures from brain hemispheres of newborn rats.Developmental Biology 81, 372–78.

    PubMed  Google Scholar 

  • Schachner, M., Kim, S. K. &Zehnle, R. (1981) Developmental expression in central and peripheral nervous system of oligodendrocyte cell surface antigens (O antigens) recognized by monoclonal antibodies.Developmental Biology 83, 328–38.

    PubMed  Google Scholar 

  • Sensenbrenner, M., Springer, N., Booher, J. &Mandel, P. (1972) Histochemical Studies during the differentiation of dissociated nerve cells cultivated in the presence of brain extracts.Neurobiology 2, 49–60.

    PubMed  Google Scholar 

  • Sensenbrenner, M., Wittendorp, E., Barakat, I. &Rechenmann, R. V. (1980a) Autoradiographic study of proliferating brain cells in culture.Developmental Biology 75, 268–77.

    PubMed  Google Scholar 

  • Sensenbrenner, M., Devilliers, G., Bock, E. &Porte, A. (1980b) Biochemical and ultrastructural studies of cultured rat astroglial cells. Effect of brain extract and dibutyryl cyclic AMP on glial fibrillary acidic protein and glial filaments.Differentiation 17, 51–61.

    PubMed  Google Scholar 

  • Seymour, R. M. &Berry, M. (1979) The nervous system. InBiomedical Research Applications of Scanning Electron Microscopy Vol. 1 (edited byHodges, G. M. &Hallowes, R. C.), pp. 127–76. London, New York, San Francisco: Academic Press.

    Google Scholar 

  • Shapiro, D. L. (1973) Morphological and biochemical alterations in foetal rat brain cells cultured in the presence of monobutyryl cyclic AMP.Nature 241, 203–4.

    PubMed  Google Scholar 

  • Shapiro, D. L. &Schrier, B. K., (1973) Cell cultures of fetal rat brain. Growth and marker enzyme development.Experimental Cell Research 77, 239–47.

    PubMed  Google Scholar 

  • Sommer, I. &Schachner, M. (1981) Monoclonal antibodies (01 to 04) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system.Developmental Biology 83, 311–27.

    PubMed  Google Scholar 

  • Stallcup, W. B. (1981) The NG2 antigen, a putative lineage marker: immunofluorescent localization in primary cultures of rat brain.Developmental Biology 83, 154–65.

    PubMed  Google Scholar 

  • Sternberger, N. H., Itoyama, Y., Kies, M. W. &Webster, H. De F. (1978) Myelin basic protein demonstrated immunocytochemically in oligodendroglia prior to myelin sheath formation.Proceedings of the National Academy of Sciences USA 75, 2521–24.

    Google Scholar 

  • Varon, S. (1978) Macromolecular glial cell markers. InDynamic Properties of Glial Cells (edited bySchoffeniels, E., Franck, G., Tower, D. B. &Hertz, L.), pp. 93–103. Oxford, New York, Toronto, Sydney, Paris, Frankfurt: Pergamon Press.

    Google Scholar 

  • Varon, S. &Raiborn, C. W. (1969) Dissociation, fractionation, and culture of embryonic brain cells.Brain Research 12, 180–99.

    PubMed  Google Scholar 

  • Venable, J. H. &Coggeshall, R. (1964) A simplified lead citrate stain for use in electron microscopy.Journal of Cell Biology 25, 407–8.

    Google Scholar 

  • Wilson, S. H., Schrier, B. K., Farber, J. L., Thompson, E. J., Rosenberg, R. N., Blume, A. J. &Nirenberg, M. W. (1972) Markers for gene expression in cultured cells from the nervous system.Journal of Biological Chemistry 247, 3159–69.

    PubMed  Google Scholar 

  • Wilson, S. -S., Baetge, E. E. &Stallcup, W. B. (1981) Antisera specific for cell lines with mixed neuronal and glial properties.Developmental Biology 83, 146–53.

    PubMed  Google Scholar 

  • Yavin, E. &Menkes, J. H. (1973) The culture of dissociated cells from rat cerebral cortex.Journal of Cell Biology 57, 232–37.

    PubMed  Google Scholar 

  • Yavin, E. &Yavin, Z. (1974) Attachment and culture of dissociated cells from rat embryo cerebral hemispheres on polylysine-coated surface.Journal of Cell Biology 62, 540–46.

    PubMed  Google Scholar 

  • Yavin, Z. &Yavin, E. (1977) Synaptogenesis and myelinogenesis in dissociated cerebral cells from rat embryo on polylysine coated surface.Experimental Brain Research 29, 137–47.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Meller, K., Waelsch, M. Cyclic morphological changes of glial cells in long-term cultures of rat brain. J Neurocytol 13, 29–47 (1984). https://doi.org/10.1007/BF01148317

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01148317

Keywords

Navigation