Influence of basic fibroblast growth factor and astroglial cells on the ultrastructure of developing rat brain neuronal precursors in vitro.

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 Niss1 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.

Proliferation of neuronal precursor cells from the central nervous system in culture.

Studies over the past ten years have revealed that neuronal precursors from the central nervous system of chick, rat and mouse embryos are able to divide in culture and that their proliferation is enhanced by several nervous tissue extracts as well as by growth factors, hormones and various other molecules. In this article we present an overview of this subject. It has been found that neuronal precursors from chick embryo cerebral hemispheres proliferate in culture during the first week and that those from 6 day-old chick embryos possess the highest proliferative activity. Neuronal precursors from fetal rat cerebral cortex and spinal cord can also proliferate in vitro. The highest proliferative activity was observed between 24 and 48 h. Brain and meningeal extracts have been shown to stimulate the proliferation of chick neuroblasts. Moreover, RNAs, purine nucleotides, purine bases and transferrin present in these extracts are able to reinduce the proliferation of these cells. Other investigations have indicated that several hormones and growth factors stimulate the proliferation of rat and mouse neuronal precursors. Acidic and basic fibroblast growth factors are potent mitogens for these cells. Nerve growth factor, epidermal growth factor and insulin-like growth factor also affect the growth of the neuroblasts. The reported in vitro observations are discussed in relation to the physiological role of these molecules during neuronal proliferation in brain development.

Influence of basic fibroblast growth factor on the development of cholinoceptive neurons from fetal rat cerebrum in culture.

Neuronal cells from cerebral hemispheres of 14-day-old rats were grown for 6 days in a serum-free, chemically defined medium. About 95-98 and 3% of these cells were neurofilament and acetylcholinesterase (AChE)-positive, respectively. The addition of basic fibroblast growth factor (bFGF) at three developmental stages, i.e. at 4 h, 2 and 4 days resulted in an increase (about 2-fold) of the number of AChE-positive neurons. The enzyme reaction was present in the cell body as well as in the fibers, which often ramified extensively under the influence of bFGF. Treatment with bFGF after the 2nd day of culture had no or only a low stimulatory effect. Our findings indicate that bFGF affects the development of AChE-containing neurons, i.e. cholinoceptive neurons from rat cerebrum.

Effects of basic fibroblast growth factor on the development of GABAergic neurons in culture.

Six-day-old neuronal cultures derived from 14-day-old embryonic rat cerebral hemispheres were highly enriched in GABAergic neurons, as was demonstrated by immunocytochemistry using an anti-glutamate decarboxylase antiserum. They contained about 64% glutamate decarboxylase-positive neurons. About 8% of these neurons proliferated, as shown by a combination of glutamate decarboxylase immunocytochemistry and [3H]thymidine incorporation into cell nuclei. The proliferative activity of GABAergic precursor cells and changes in the cellular concentrations of the non-essential amino acids, including GABA under the effect of basic fibroblast growth factor were studied. When basic fibroblast growth factor was added to the cultures 4 h after seeding, the proliferation of the GABAergic neurons was stimulated about threefold. Under this culture condition, the concentration per cell of all amino acids increased, except those of GABA and beta-alanine. When basic fibroblast growth factor was added to cultures only on day four, the proliferation of the neuronal cells was no more enhanced. Under this condition of treatment, the concentrations of all non-essential amino acids, including those of GABA and beta-alanine were enhanced. Under both basic fibroblast growth factor treatments the concentration of GABA per GABAergic cell was increased. In contrast, the specific activity of glutamate decarboxylase was not stimulated under these conditions. We hypothesize that under the effect of basic fibroblast growth factor the capabilities of the cells to store GABA are improved.

Brain basic fibroblast growth factor stimulates the proliferation of rat neuronal precursor cells in vitro.

Neuronal cells from cerebral hemispheres of 13-day-old rat embryos were grown in a serum-free culture medium for 48 h, 4 or 8 days. The neuronal precursor cells proliferate for 5 days. The addition of bovine brain basic fibroblast growth factor stimulates their proliferation as determined by measurement of [125I]-iododeoxyuridine incorporation and by autoradiographic analysis after [3H]thymidine incorporation. The proliferating responsive cells were characterized as neurons by immunostaining against neurofilament proteins. Five other growth factors tested were without effect on the proliferative activity of these neuronal cells. The present results show that bFGF is mitogenic in vitro for rat neuronal precursor cells of the central nervous system.

[Effect of basic FGF on the proliferation of rat neuroblasts in culture]. / Effet du FGF basique sur la prolifération de neuroblastes de rat en culture.

Cultures highly enriched in neuronal cells, derived from brain hemispheres of 13-day-old rat embryo, were used. In these cultures, neuroblasts proliferate during the first 3 days in vitro. The addition of the astroglial growth factor (AGF2), also known as basic fibroblast growth factor (bFGF), 4 hrs. after seeding, induces a strong increase of [125I]-deoxyuridine incorporation. We checked by autoradiography combined with specific immunocytochemical staining of the neurones, using an anti-neurofilament antibody, that the number of proliferating neuroblasts is increased after the treatment. These results demonstrate that AGF2, or bFGF, is a mitogenic factor for rat neuroblasts in culture.

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

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.

Precursor cells of oligodendrocytes in rat primary cultures.

In monolayer primary cultures of brain from newborn rats, which contain astrocytes and oligodendrocytes, a new morphological cell type (flat black cells) was observed. Microphotographs of different areas of the monolayer, taken every 30 min, showed that these flat black cells can divide and that they undergo morphological transformation in vitro. They give rise to oligodendrocytes which were identified by their characteristics morphology but also by their content of W1 Wolfgram protein. These findings suggest that the flat black cells are precursors for oligodendrocytes, in culture.

Detection of Wolfgram W1 protein, myelin basic proteins and proteolipids in cultured oligodendrocytes by the electro-immunoblotting method.

Oligodendrocytes, mechanically obtained from primary cultures of newborn rat brain, were investigated by a sensitive electro-immunoblotting method for the presence of the characteristic myelin proteins: Wolfgram W1 protein, basic proteins and proteolipids. These three major myelin protein types were detectable from the 20th to the 40th day in culture. The present biochemical findings are in accordance with previous immunohistochemical data and provide additional evidence that oligodendrocytes in culture are capable of synthesizing every myelin and oligodendrocyte marker found in vivo, in the absence of neurones.

GABA transaminase inhibitors.

In summary, several branched-chain fatty acids appeared to be competitive inhibitors of GABA-T and non-competitive inhibitors of SSADH. These compounds produce an increase in brain GABA level, and for two of these it was shown that the increase differs among various brain areas. An increase of GABA cannot be obtained by inhibition of SSADH. The increase in brain GABA seems to correlate with the anticonvulsant activity of branched-chain fatty acids.

[Effect of sodium n-dipropylacetate (sodium valproate) on GABA level of neuronal and glial cells in culture]. / Effet du n-dipropylacétate de sodium (valproate de sodium) sur la teneur en GABA de cellules neuronales et gliales en culture.

The effect of sodium n dipropylacetate (nDPA), a competitive GABA-T inhibitor with respect to GABA, has been investigated on glial and neuronal cellular GABA level. After 1 to 4 days incubation with nDPA in the culture medium, a decrease of GABA level in M5 neuroblastoma clonal cell lines and no modification of GABA level in C6 astrocytoma cells has been observed. The combined addition of nDPA 4 micrometer with dibutyryl cyclic AMP (1 mM) to the culture medium induces the same decrease in GABA level in C6 astrocytoma cells as the addition of DB-c-AMP alone. After shorter incubation time with nDPA (5-150 min), we observed a decreased GABA level in C6 astrocytoma glial cells.

[Effect of differentiation of GABA levels in various cell cultures]. / Effet de la différenciation sur la teneur en GABA dans diverses cultures de cellules.

The effect of factors inducing somatic differentiation on cellular GABA level has been investigated. C6 glial cells from a rat astrocytoma present a significantly higher GABA level than the other glial or neuronal cells studied. A significant decrease in GABA levels in most cases parallels the somatic differentiation induced either by withdrawal of fetal serum, or by addition of dibutyryl cyclic AMP to the culture medium.

[Effects of sodium n-dipropylacetate on the GABA level in various areas of the mouse brain]. / Effets du n-dipropylacétate de sodium sur le taux de GABA de certaines zones du cerveau de la souris

Regional brain GABA distribution studies show that after administration of sodium n dipropylacetate, a competitive inhibitor of GABA transaminase, the concentration of GABA increases in some regions i.e. Olfactory Bulbs, Hypothalamus, Cortex, Cerebellum. The GABA level remains unchanged in Caudate Nucleus, Pons Medulla, Hippocampus in our experimental conditions. These variations do not correlate with the initial GABA level.