Glutamine synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors.

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.

[Immunocytochemical characteristics of organotypic cerebellar culture using the avidin-biotin complex method (ABC)]. / Immunocytochemiczna charakterystyka organotypowej hodowli mózdzku przy uzyciu metody kompleksu awidyna-biotyna (ABC).

The occurrence and cellular localization of some CNS antigenic markers were studied in organotypic cultures of newborn rat cerebellum grown for up to 5 weeks. The avidin-biotin-complex method was employed using polyclonal immune sera against glial fibrillary acidic protein (GFAP), S-100 protein and vimentin, and monoclonal antibodies against neurofilaments. In all cultures GFAP was selectively localized in astrocytes, both in perikarya and in cellular processes. The difference in immunoreactivity between particular cells and culture "zones", and considerable morphological polymorphism of the astrocytes were noted. In majority of cells the immunostaining for vimentin was very intensive, whereas reaction with antiserum against S-100 protein was weak or negative. Neurofilament antigen was localized only in neuronal processes lying in explants of the younger--one- to two-week cultures.

Glial fibrillary acidic protein and differentiation of neonatal rat pituicytes in vitro.

The appearance and intracellular localisation of glial fibrillary acidic protein (GFAP) in pituicytes in neural lobe cultures of newborn rats aged 7 to 30 days were investigated by use of the indirect immunofluorescence method. GFAP-immunoreactive cells were observed mostly in the outgrowth zone. GFAP was localised in the perikaryal cytoplasm as well as in pituicyte processes. GFAP-positive pituicytes showed considerable morphological polymorphism. The presence of GFAP - astrocytic marker - in pituicytes in vitro and the evident morphological similarity to cultured astrocytes suggest the astroglial character of these cells.

Glia-specific antigen in the intracranial tumors. Immunofluorescence study.

26 gliomas and 14 non-glial tumors were examined for the presence of nervous system specific antigen (CGSA) to assess the antigenic properties of neoplastic tissue in relation to histogenesis and degree of differentiation of tumors. Double layer immunofluorescence (IMF) technique was used for the cellular localization of the antigen. CGSA was found in the cytoplasm of normal, reactive and neoplastic neuroglial cells. Well differentiated astrocytomas showed the strongest IMF reactions and largest number of IMF-positive cells. Tumors with histological signs of anaplasia displayed foci of IMF-negative cells irregularly distributed in the sections. There were no completely negative astrocytomas owing to a marked affinity of the specific astisera to the astrocytic cell line. In the oligodendrogliomas a smaller amount of the antigen was found than in the astrocytomas. Histological evidence of malignancy in these tumors was accompanied by strikingly small number of positive cells and weaker IMF reactions as compared to the well differentiated oligodendrogliomas. Anaplastic gliomas showed only traces of CGSA and non-glial tumors were entirely negative. The results suggest a deficiency of normal antigenic material in the neoplastic glia, particularly of oligodendrogliomas and anaplastic gliomas.