Distinctive structural and cytoskeletal properties of the long-surviving neurons in cell cultures of embryonic spinal cord

Debbage, Paul

doi:10.1016/0306-4522(85)90174-5

Cited by 5 publications

(5 citation statements)

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“…The neuron-like cells remained scattered over the entire surface of the dish. These cultures showed striking differences from most of those previously described for spinal neurons of rat (Debbage, 1985;Digby et al, 1985;Gilad et al, 1988;Bignami and Dahl, 1989), in which large neuronal aggregates were seen connected by thick fiber bundles, as also observed by us when cells were seeded in dishes precoated with polylysine prepared in distilled water. The difference between the effects of polylysine dissolved in borate buffer and distilled water might be due to the fact that at pH 8.4 (in the borate buffer) the polypeptidic chain probably has a different helicoidale structure and possibly a more homogeneous coating layer is formed under this condition, which allows to the seeded cells to remain dispersed.…”

Section: Discussionmentioning

confidence: 64%

“…A number of methods have been described for the growth of neurons from avian (Fishbach and Dichter, 1974;Barald and Berg, 1979;Debbage, 1985;Pierce et al, 1988) and mammalian (Koenig et al, 1982;Smith and Appel, 1983;Debbage, 1985;McManaman et al, 1985;Bignami and Dahl, 1989) spinal cords. Such spinal cord cell primary cultures contain several types of neurons and have been used for morphological, biochemical, and electrophysiological studies.…”

Section: Introductionmentioning

confidence: 99%

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Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor

Deloulme

Baudier

Sensenbrenner

1991

J of Neuroscience Research

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A primary culture system of nearly pure neuronal cells from 14-day-old fetal rat spinal cord has been developed by combining a preplating step, the use of a chemically defined serum-free medium, and borated polylysine-coated dishes that prevented the formation of cell aggregates. About 98% of the cells were found to be immunostained with neuron-specific enolase antibodies, confirming their neuronal nature. The cultures are composed essentially of a population of non-motoneurons and contain few motoneurons, characterized by their large size and multipolar aspect, the presence of acetylcholinesterase (AChE), and the intense immunoreaction for growth-associated protein GAP-43. Neuronal precursor cells are also present in these cultures and proliferate during the first 3 days. The addition of bovine brain basic fibroblast growth factor (bFGF) stimulates their proliferation over a period of 2 days, as determined by measurement of [125I]iododeoxyuridine incorporation and by immunocytochemical reaction after bromodeoxyuridine incorporation into nuclei. The proliferating cells were characterized as neurons by immunostaining against neuron-specific enolase. Recombinant human bFGF and bovine brain acidic FGF (aFGF) exerted similar effects. Other growth factors, including epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-beta 1), and thrombin, were without effect on the proliferative activity of these neuronal cells. bFGF has no effect on the survival of motoneurons and on the fiber outgrowth of the whole neuronal population. However, bFGF affects the development of bipolar AChE-positive neurons, probably belonging to the non-motoneuron population. The data indicate that bFGF and aFGF are mitogens for neuroblasts from rat spinal cord in culture and that bFGF influences the development of a subpopulation of spinal neurons that are AChE-positive.

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Section: Discussionmentioning

confidence: 64%

Section: Introductionmentioning

confidence: 99%

Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor

Deloulme

Baudier

Sensenbrenner

1991

J of Neuroscience Research

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“…The microtubules are structural elements essential for neurite growth and maintenance [Yamada et al, 1970], We also observed microfilaments, another cytoskeletal structure, which are localized beneath the cell membrane in contact with the culture support (not shown). The presence of high amounts of microtubules within the neuronal cells in culture, but derived from older embryonic brain, has also been mentioned in previ ous electron microscopic studies [Bird and James, 1977;Trenkner and Sidman, 1977;Banker, 1984a, 1984b;Debbage, 1985].…”

Section: Discussionmentioning

confidence: 99%

“…In a previous study we mentioned briefly that under similar culture conditions the neuronal precursor cells reach only a lim ited stage of maturation and that the fine ultrastructural aspect corresponds to typical young neurons [Gens burger et al, 1986]. Other authors, using dispersed cell cultures from various brain regions and maintained under serum-free condition, but derived from more advanced embryonic stages in which neuroblasts were already postmitotic, reported similar results [Yavin and Yavin, 1980;Romijn et al, 1982;Ahmed el al., 1983;Bartlett and Banker, 1984a;Debbage, 1985;TixierVidal et al, 1986;Weiss el al., 1986;Massacrier et al, 1988].…”

Section: Discussionmentioning

confidence: 99%

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Influence of Basic Fibroblast Growth Factor and Astroglial Cells on the infrastructure of Developing Rat Brain Neuronal Precursors in vitro

Miehe¹,

Leterrier

Deloulme³

et al. 1996

Dev Neurosci

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We have examined the ultrastructural aspect of neuronal precursors derived from 14-day-old rat embryos during their development under various culture conditions. Cells maintained in serum-free medium which have developed for 1 week in vitro present ultrastructural features of young neurons. They contain many free ribosomes and microtubules, but few other organelles and incompletely developed Golgi apparatus. In the presence of basic fibroblast growth factor (bFGF), besides cells remaining in aggregates and displaying morphological features of undifferentiated cells, dispersed neuroblasts underwent accelerated ultrastructural maturation. They present well-developed Golgi apparatus, axodendritic synapses and dense-core vesicles already after 3 days in culture. By contrast, in the presence of astroglial-conditioned medium a more homogeneous population developed showing ultrastructural features of relatively mature neurons. However, the neuronal precursors acquired the most mature ultrastructural aspect when they were cocultured with astroglial cells. The neuronal cell bodies contain highly developed Golgi complexes, well-differentiated ergastoplasm and Nissl body formations, while in the complex neurite network much more numerous mature synapses with clear and dense-core vesicles are visible. These observations indicate that a combination of soluble factors and membrane-bound factors is essential for extensive ultrastructural development of neuronal precursors in vitro. Another finding was that in these cultured neurons neurofilaments (NF) were never seen, while NF protein subunits were found. These data suggest that the polymerization of the three NF subunits into intermediate filaments might need particular cellular factors which probably do not exist under our in vitro conditions.

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Influence of meningeal cells on the proliferation and maturation of rat neuroblasts in culture

1986

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Neuronal cells were obtained by dissociating cells from the cerebral hemispheres of rat embryos (10 to 17-day-old), either cleaned entirely or only partially of their meningeal membranes. These cells were seeded on poly-lysine-coated Petri dishes in serum-containing medium. The cultures most enriched in neuronal cells were obtained from brains of 13- to 15-day-old embryos and after 2 h, the culture medium was switched to Dulbecco's modified Eagle's medium, without serum, supplemented with the N1 supplements as described by Bottenstein et al. (1980). The proliferation of neuroblasts from 13-day-old embryos in the presence or absence of meningeal cells was studied by using a combination of tritiated thymidine autoradiography and immuno-staining against neurofilament proteins. The neuroblasts seem to proliferate during the first 3 days. The proliferative activity was further enhanced in the presence of meningeal cells. The glioblasts multiply only after a period of one week in culture conditions as observed here. The subsequent development of the neuroblasts was followed over a period of 4 weeks and the ultrastructural appearance of these cells was investigated at 2 and 3 weeks. In the presence of meningeal cells, many neurons, intensely stained for neurofilament proteins, survived for 21 days, while in control cultures they underwent massive degeneration after 2 weeks. Synapses with numerous clear vesicles were abundant in cultures grown under the influence of meningeal cells; they were rare and possessed few vesicles in control cultures. The data indicate that meningeal cells affect the proliferation and maturation of rat neuroblasts in culture.

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Distinctive structural and cytoskeletal properties of the long-surviving neurons in cell cultures of embryonic spinal cord

Cited by 5 publications

References 54 publications

Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor

Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor

Influence of Basic Fibroblast Growth Factor and Astroglial Cells on the infrastructure of Developing Rat Brain Neuronal Precursors in vitro

Influence of meningeal cells on the proliferation and maturation of rat neuroblasts in culture

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