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Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick and human)

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Summary

Explants of embryonic and fetal spinal cords (rat, chick, and human) develop and maintain in vitro many cytologic and bioelectric properties characteristic of central nervous tissues in situ. Despite the thickness of the cord explants, a condition which appears to be necessary for differentiation, considerable neuronal development becomes visible to light microscopic examination.

The mature expiant consists of large concentrations of small neurons interspersed with large neurons and neuroglia, bounded by a broad tract of nerve fibers and capped by a neuropil. Myelination in rat cultures usually begins 2–3 weeks after explantation in both the explant and outgrowth zone. Myelin is of the central glial type unless the cord explants are grown with their meningeal covering. In the latter case the myelination pattern abruptly changes to the peripheral Schwannian type as the axons penetrate the meninges.

Bouton-like endings are observed in the neuropil of rat cord explants (whole-mounts) impregnated with silver; but most neurons are only partially blackened. In 20 μ sections, neuronal somas and dendrites are identified in negative image with blackened bouton-like endings suggesting synapses.

Chick spinal cord, when grown in Rose chambers, becomes more thinly spread so that more detailed interrelationships can be visualized in the living neuronal somas, neuritic processes and termini. Bouton-like endings on neuronal somas have been selectively stained, vitally, with methylene blue.

Complex bioelectric activity can be evoked in these long-term spinal cord explants by electric stimuli localized to various regions of the cord tissues as well as to attached dorsal-root ganglia. The long-lasting “after-discharge” patterns and the neuropharmacologic sensitivity of the responses show remarkable similarity to the activity of synaptic networks of the central nervous system in situ. These functions develop gradually during the first week after explantation of fetal rat cord tissues — more slowly in cultures explanted before the establishment of reflex arcs in utero, and more rapidly in cord explants from older fetuses. Reference is made to the companion electron microscopy study of older fetuses, which shows that characteristic synaptic structures, although extremely rare at the time of explantation, are abundant in later stages of the culture's development. This confirms the functional evidence that synapses are able to develop in organized culture conditions.

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Literature

  • Bodian, D.: The staining of paraffin sections of nervous tissues with activated protargol. The role of fixatives. Anat. Rec. 69, 153–162 (1937).

    Google Scholar 

  • Bornstein, M. B.: Reconstituted rat-tail collagen used as substrate for tissue culture on coverslips in Maximow slides and roller tubes. Lab. Invest. 7, 134–140 (1958).

    Google Scholar 

  • —: Morphological development of neonatal mouse cerebral cortex in tissue culture. In Symposium: Neurological and electroencephalographic correlative studies in infancy, p. 1–11, edit. by P. Kellaway and I. Petersén. New York: Grune & Stratton 1964.

    Google Scholar 

  • —, and L. M. Breitbart: Anatomical studies of mouse embryo spinal cord-skeletal muscle in long-term tissue culture. Anat. Rec. 148, 362 (1964).

    Google Scholar 

  • —, and M. R. Murray: Serial observation on patterns of growth, myelin formation, maintenance and degeneration in cultures of new-born rat and kitten cerebellum. J. biophys. biochem. Cytol. 4, 499–504 (1958).

    Google Scholar 

  • Bunge, R. P., M. B. Bunge, and E. R. Peterson: An electron microscopic study of cultured rat spinal cord. J. Cell Biol. 24, 163–191 (1965).

    Google Scholar 

  • Crain, S. M.: Resting and action potentials of cultured chick embryo spinal ganglion cells. J. comp. Neurol. 104, 285–330 (1956).

    Google Scholar 

  • —: Electrophysiological studies of cord-innervated skeletal muscle in long-term cultures of mouse embryo myotomes. Anat. Rec. 148, 273–274 (1964).

    Google Scholar 

  • —: Development of bioelectric activity during growth of neonatal mouse cerebral cortex in tissue culture. In Symposium: Neurological and electroenecphalographic correlative studies in infancy, p. 12–26, edit. by P. Kellaway and I. Petersén. New York: Grune & Stratton 1964.

    Google Scholar 

  • —, and M. B. Bornstein: Bioelectric activity of neonatal mouse cerebral cortex during growth and differentiation in tissue culture. Exp. Neurol. 10, 425–450 (1964).

    Google Scholar 

  • —, R.P. Bunge, M.B. Bunge, and E. R. Peterson: Bioelectric and electron microscopic evidence for development of synapses in spinal cord cultures. J. Cell Biol. 23, 114–115 A (1964).

    Google Scholar 

  • -, and E. R. Peterson: Complex electrical activity evoked in long-term cultures of mammalian and avian CNS tissue. Amer. Soc. for Cell Biol., 2nd Annual Mtg., San Francisco. Abstr., 1962, p. 36.

  • —: Bioelectric activity in long-term cultures of spinal cord tissues. Science 141, 427–429 (1963).

    Google Scholar 

  • —: Complex bioelectric activity in organized tissue cultures of spinal cord (human, rat and chick). J. cell. comp. Physiol. 64, 1–13 (1964).

    Google Scholar 

  • - - Onset and development of functional relations within and between explants of mammalian spinal cord-ganglia during differentiation in vitro. Anat. Rec. 151 (2), (in press) (1965).

  • Ehrmann, R. L., and G. O. Gey: The growth of cells on a transparent gel of reconstituted rat-tail collagen. J. Nat. Cancer Inst. 16, 1375–1404 (1956).

    Google Scholar 

  • Fraser Rowell, C. H.: A general method for silvering invertebrate central nervous systems. Quart. J. micr. Sci. 104, 81–87 (1963).

    Google Scholar 

  • Geiger, R. S.: The behavior of adult mammalian brain cells in culture. In: International review of neurobiology, vol. 5, p. 1–52, edit. by C. C. Pfeiffer and J. R. Smythies. New York: Academic Press 1963.

    Google Scholar 

  • Hild, W.: Myelinogenesis in cultures of mammalian central nervous tissue. Z. Zellforsch. 46, 71–95 (1957).

    Google Scholar 

  • —: Das Neuron. In: Handbuch der mikroskopischen Anatomie des Menschen, vol. IV/4, p. 1–184, edit. by W. Bargmann. Berlin-Göttingen-Heidelberg: Springer 1959.

    Google Scholar 

  • Hörstadius, S.: The neural crest. Oxford: University Press 1950.

    Google Scholar 

  • Holmes, W.: The peripheral nerve biopsy. In: Recent advances in clinical pathology, 1st ed., p. 402–417, edit. by S. C. Dyke. Philadelphia: P. Blakiston Son & Co. 1957.

    Google Scholar 

  • Levi, G., et H. Meyer: Nouvelles recherches sur le tissu nerveux cultivé in vitro. Morphologie, croissance et relations réciproques des neurones. Arch. Biol. (Liège) 52, 133–278 (1941).

    Google Scholar 

  • Long, J. A., and P. L. Burlingame: The development of the external form of the rat with observations on the origin of the extraembryonic coelom and fetal membranes. Univ. Calif. Publ. Zool. 43, 143–184 (1938).

    Google Scholar 

  • Murray, M. R.: Nervous tissues in vitro. In: The biology of cells and tissues in culture, vol. 2, chap. 9, edit. by E. N. Willmer. London: Academic Press 1965.

    Google Scholar 

  • —, and G. Kopech: A bibliography of the research in tissue culture: 1884–1950, 2 vols. New York: Academic Press 1953.

    Google Scholar 

  • Parker, R. C.: The races that constitute the group of common fibroblasts. II. The effect of blood serum. J. exp. Med. 58, 97–114 (1933).

    Google Scholar 

  • —: Methods of tissue culture, 3rd edit. New York: Hoeber 1961.

    Google Scholar 

  • Pearse, A. G. E.: Histochemistry, theoretical and applied, 2nd edit. Boston: Brown & Co. 1960.

    Google Scholar 

  • Peterson, E. R., M. B. Bornstein, and S. M. Crain: Development, myelination and function in cultures of mammalian spinal cord. Excerpta med. (Amst.), Sect. 1. 16, 813 (1962).

    Google Scholar 

  • —, and M. R. Murray: Myelin sheath formation in cultures of avian spinal ganglia. Amer. J. Anat. 96, 319–356 (1955).

    Google Scholar 

  • Pomerat, C. M., and I. Costero: Tissue cultures of cat cerebellum. Amer. J. Anat. 99, 211–247 (1956).

    Google Scholar 

  • Rose, G. G., C. M. Pomerat, T. O. Schindler, and J. B. Trunnell: A cellophane-strip technique for culturing in multipurpose culture chambers. J. biophys. biochem. Cytol. 4, 761–764 (1958).

    Google Scholar 

  • Windle, W. F., and R. E. Baxter: Development of reflex mechanisms in the spinal cord of albino rat embryos. Correlation between structure and function, and comparisons with the cat and the chick. J. comp. Neurol. 63, 189–210 (1936).

    Google Scholar 

  • —, and J. E. Fitzgerald: Development of the spinal reflex mechanism in human embryos. J. comp. Neurol. 67, 493–509 (1937).

    Google Scholar 

  • Wolf, M. K.: Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum. J. Cell. Biol. 22, 259–279 (1964).

    Google Scholar 

  • Yonezawa, T.: Myelinogenesis. In: Manual of tissue culture, edit. by J. Nakai and M. Okamoto (in press). Tokyo: Asakura 1965.

    Google Scholar 

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Authors and Affiliations

  1. Departments of Surgery, Anatomy and Neurology, College of Physicians and Surgeons, Columbia University, New York, N.Y., USA

    Edith R. Peterson, Stanley M. Crain & Margaret R. Murray

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  1. Edith R. Peterson
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  2. Stanley M. Crain
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  3. Margaret R. Murray
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Additional information

This study was supported by grants NB-00858 and NB-03814 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service.

Research Career Development Fellow (Award NB-K3-2904 from the NINDB, USPHS).

Research Career Award 5K6-GM-15,372 from U.S. National Institutes of Health.

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Peterson, E.R., Crain, S.M. & Murray, M.R. Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick and human). Zeitschrift für Zellforschung 66, 130–154 (1965). https://doi.org/10.1007/BF00339322

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  • DOI: https://doi.org/10.1007/BF00339322

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