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Glutamine synthetase (GS, EC 6.3.1.2.) has long been considered as a protein specific for astrocytes in the brain, but recently GS immunoreactivity has been reported in oligodendrocytes both in mixed primary glial cell cultures and in vivo. We have investigated its expression and regulation in "pure" oligodendrocyte cultures. "Pure" oligodendrocyte secondary cultures were derived from newborn rat brain primary cultures enriched in oligodendrocytes as described by Besnard et al. (1987) and were grown in chemically defined medium. These cultures contain more than 90% galactocerebroside-positive oligodendrocytes and produce "myelin" membranes (Fressinaud et al., 1990) after 6-10 days in subcultures (30-35 days, total time in culture). The presence of GS in oligodendrocytes from both primary glial cell cultures and "pure" oligodendrocyte cultures was confirmed by double immunostaining with a rabbit antisheep GS and guinea pig antirat brain myelin 2', 3'-cyclic nucleotide 3'-phosphodiesterase. In "pure" oligodendrocyte cultures, about half of cells were labeled with anti-GS antibody. Furthermore, on the immunoblot performed with a rabbit antisheep GS, the GS protein in "pure" oligodendrocyte secondary cultures was visualized as a single band with an apparent molecular mass of about 43 kDa. In contrast, two protein bands for GS were observed in cultured astrocytes. On the immunoblot performed with a rabbit antichick GS, two immunopositive protein bands were observed: a major one migrating as the purified adult chick brain GS and a minor one with a lower molecular mass. Two similar immunoreactive bands were also observed in pure rat astrocyte cultures. Compared to pure rat astrocyte cultures, "pure" oligodendrocyte cultures of the same age displayed an unexpectedly high GS specific activity that could not be explained by astrocytic contamination of the cultures (less than 5%). As for cultured astrocytes, treatment of oligodendrocyte cultures with dibutyryl-adenosine 3':5'-cyclic monophosphate, triiodothyronine, or hydrocortisone increased significantly GS specific activity. Interestingly, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor that increase the GS activity in astrocytes do not affect this activity in oligodendrocytes. Thus we confirm the finding of Warringa et al. (1988) that GS is also expressed in oligodendrocytes. We show that its activity is regulated similarly in astrocytes and oligodendrocytes by hormones, but that it is regulated differently by growth factors in these two cell types.

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The effects of basic fibroblast growth factor (bFGF) on the morphology and the expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) in cultured astrocytes prepared from various areas of newborn rat brain was studied. The brain was dissected in two ways, either the telencephalon (area A) and the diencephalon (area B) were dissected out of the brain (without olfactory bulbs, mesencephalon and cerebellum) or the brain was cut transversely into 3 parts (areas 1, 2 and 3). Area 1 (the anterior part) included the frontal cortex, the olfactory nuclei, the neostriatum, the accumbens nucleus and the septum; area 2 (the medial part) included the cortex, hippocampus, amygdale, thalamus and hypothalamus, and area 3 (the posterior part) included the occipital cortex, the posterior part of hippocampus and thalamus and the mamillary bodies. Essentially two different morphological aspects were observed. Most cells from areas A, 1 and 3, were flat, large, presented an irregular shape and were loosely arranged; cells from areas B and 2 were essentially polygonal in shape and closely apposed to each other. The various control cultures showed nearly the same immunostaining pattern for GFAP, but different patterns for GS. Most astroglial cells responded to bFGF and became fibrous. The GFAP immunoreaction was intense and localized in the cell bodies and processes of most cells from area A, but essentially in the processes for cells from areas 1 and 2. The immunoreactivity was weaker in cells from areas B and 3. GS-positive cells, heavily and weakly stained, were found in all treated cultures, and very strongly stained cells were located in certain zones of cultures from area A. But GS-negative cells were also seen in these treated cultures as well as in control cultures. Measurements of GS activities revealed no differences. These results indicate that astrocytes from different regions of the brain in primary culture show differences in their responsiveness to bFGF. The astroglial cells from the cerebral cortex and from the thalamus seem to present the highest and the lowest response to bFGF, respectively.

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