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Cerebral hemispheres of 14- to 15-day-old mouse embryos were dissociated mechanically into single cells and cultured in polylysine-coated plastic flasks. The succession of changes and the identification of cell populations have been studied by transmission electron microscopy (TEM). Four distinct cell types can be clearly defined. Neuroblasts, oligodendroblasts, and astroblasts were used to denote embryonic states of neurons, oligodendrocytes, and astrocytes. Cell populations were: (1) neuroblasts; (2) astroblasts, either growing on the surface of the flasks and forming a basal layer of epithelioid cells or scattered at the surface of the bed layer; (3) oligodendroblasts, characterized by a high nucleocytoplasmic ratio and resting on top of the bed layer; (4) macrophages. Incorporation of [3H]thymidine into cells at different days in culture (DIC) delineated at least three periods of DNA synthesis (from 1 to 6, from 10 to 20, and from 25 to 60 DIC). High cell RNA/DNA and protein/DNA ratios indicated an increase in the cytoplasmic mass. Guanylate cyclase (EC 4.6.1.2; GC) and adenylate cyclase (EC 4.6.1.1; AC) activities and the ratio AC/GC also agreed with the existence of different periods in culture, but the yin yang hypothesis (postulated by Goldberg et al, 1975) was only partially confirmed. Concerning membrane constituents, the evolution of the pattern of the major gangliosides showed a phase from 1 to 20 DIC which was characterized during the first half of this period by a high percentage of tri- and tetra-sialogangliosides, thereby confirming the presence of well differentiated neurons. Choline acetyltransferase (EC 2.3.1.6; ChAc), acetylcholinesterase (EC 3.1.1.7; AChE), and [3H]muscimol binding (indicative of GABA-receptors) supported this conclusion. Another phase from 20 to 60 DIC showed a relatively higher content of GM3 and GD3 of the disialogangliosides GD1a and GD1b. These data favor a glial origin of the cells, also demonstrated by TEM and by immunohistochemistry with anti-glial fibrillary acidic protein (anti-GFAP) and anti-carbonic anhydrase isoenzyme II (anti-CAII; EC 4.2.1.1) sera. Ultrastructural, biochemical, and immunohistochemical data are in agreement with the existence of successive and different periods of cell proliferation and differentiation which have been defined as: (1) mainly neuronal from 1 to 12 DIC; (2) astroglial and oligodendroglial between 10 and 20 DIC, and (3) possibly microglial from 25 to 40 DIC. This surface-adhering primary culture system should significantly enhance efforts to examine the roles played by hormones or other effectors in the regulation of myelination. |
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