M.S. Ghandour

Postnatal development of rat cerebellum: Massive and transient accumulation of concanavalin a binding glycoproteins in parallel fiber axolemma

Modifications of protein-bound sugars during postnatal development of rat cerebellum were studied. Glycoprotein-bound mannose accumulates, in the particulate fractions, at an earlier age than the bulk of glycoprotein sugar. This corresponds to a transient and massive accumulation of glycoproteins which bind to Concanavalin A (Con A). These glycoproteins were localized by using fluorescent Con A and the horseradish peroxidase-Con A method. Cerebellar white matter and the molecular layer bind massive amounts of Con A. The binding in the molecular layer is transient. It follows the same time course as the Con A-binding glycoproteins of particulate fractions, and it is largely confined to the axolemma of parallel fibers. Only growing or newly formed parallel fibers bind Con A. The disappearance of the binding is simultaneous with the maturation of parallel fibers and their synapse formation. These phenomena can be related to fiber growth and maturation and, also, to synapse formation. The possibility of a specific role of Con A-binding glycoproteins is discussed.

Astrocyte and oligodendrocyte distribution in adult rat cerebellum: An immunohistological study

SummaryThe distribution of the different types of glial cell in adult rat cerebellar cortex and in the underlying white matter was studied by immunohistology with a specific immune serum raised against form II of carbonic anhydrase, a specific marker for oligodendrocytes in rat cerebellum and an immune serum raised against glial fibrillary acidic protein, a specific astrocyte marker. The cellular specificity of each marker was confirmed by experiments in which the two antigens were revealed in the same cerebellar section. The unequivocal identification, with the optical microscope, of the different glial cell types allowed also a tentative estimation of the number of oligodendrocytes and astrocytes, in relation to Purkinje cells.

Carbonic anhydrase: An ultrastructural study in rat cerebellum

SummaryThe cellular and intracellular distribution of carbonic anhydrase isozyme II in rat cerebellum has been investigated with the electron microscope by the indirect antibody immunohistochemical technique. Unequivocal evidence is presented supporting the view that this enzyme is exclusively localized in oligodendrocytes. Myelin does not appear to contain detectable amounts of carbonic anhydrase though it is present in oligodendrocyte processes and in the layer of oligodendrocyte cytoplasm frequently seen to coat the external surface of myelinated fibres. The immune precipitate is found to be confined to the cytosol and the cytosolic surfaces of intracellular membranes. The data are discussed in relation to the possible function of the enzyme and the role of oligodendrocytes in the central nervous system.

A new brain cell surface glycoprotein identified by monoclonal antibody.

Of 207 monoclonal antibodies produced against cultured mouse cerebellar cells, 16 reacted with cerebellar cell surfaces and 4 reacted with glycoproteins. One of them, called an anti-BSP-3 (Brain cell Surface Protein-3) defines a 48,000 molecular weight protein which can be iodinated at the surface of cultured cerebellar cells. Lectin-binding and sugar incorporation studies established the glycoprotein nature of the antigen. Astroglia (glial fibrillary acidic protein-positive cells) in primary cerebellar cultures were labelled intensely for this antigen by the indirect immunofluorescence method while neuronal cells and their processes were more weakly labelled. Fibronectin-positive cells were negative for BSP-3. In cerebellar sections using the immunoperoxidase method at both the optical and electron microscope levels, the difference in staining intensity between astrocytes and neuronal cells was not significant: in Purkinje cells and in the large neurones present in the deep cerebellar nuclei the immunoperoxidase percipitate was confined to the plasma, membrane while in both astrocytes and granule cells cytoplasmic labelling was also observed. Oligodendrocytes do not appear to react with the anti-BSP-3 monoclonal antibody; neither do endothelial or leptomeningeal cells. The availability of a monoclonal antibody produced by a stable hybridoma line will be a powerful tool in attempts to purify the BSP-3 antigen and to elucidate its function.

Monoclonal antibodies as neural cell surface markers.

A comparison is made of the immunohistochemistry at the ultrastructural level of three monoclonal antibodies directed against surface components of CNS cells. Hybridomas secreting these antibodies were obtained from two cell fusions of a rat myeloma cell line and immune splenocytes derived from rats immunized either with primary mouse brain cultured cells or membrane components. In cultures one antibody, anti-BSP-2 (Brain Surface Protein-2), was preferentially directed against neurones while another, anti-BSP-3 (Brain Surface Protein-3), preferentially labeled astrocytes. In mouse cerebellar sections, both labeled the surface of Purkinje cells, granule cells and astrocytes. In addition a cytoplasm localization was apparent in granule cells and astrocytes. Another antibody anti-MESA-1 (Mouse Endothelial Surface Antigen-1) reacted exclusively with the surface of endothelial cells lining blood vessels. These data are discussed with reference to the biochemical nature of the corresponding antigens and to known glycoproteins of neural cell membranes.

Effects of Thyroid State and Undernutrition on S100 Protein and Astroglia Development in Rat Cerebellum

The developmental pattern of S100 protein, a specific marker of astroglia, was studied by radioimmunoassay in the cerebellum of thyroid-deficient, thyroxine-treated and undernourished rats during development. In the control animals, the S100 protein content of the cerebellum increased maximally after the 3rd postnatal week, i.e., after cell multiplication had stopped and when the cerebellum had acquired more than 70% of its adult weight and protein content. This developmental pattern of S100 protein reflected essentially the maturation of astroglia. In the thyroxine-treated rats the total amount and the concentration of S100 protein were higher than in controls during the first 3 weeks of postnatal life and returned to normal values thereafter. In the thyroid-deficient rats both the amount and concentration were lower than in controls throughout development. In the undernourished animals the amount of S100 protein per organ was also lower than in controls during the whole experimental period studied; the S100 protein concentration was higher during the first 2 postnatal weeks and became lower thereafter. These results are discussed taking into consideration previous histological and ultrastructural observations on the effects of altered thyroid state and undernutrition on the formation and maturation of cerebellar astrocytes.

Immunohistochemical and biochemical approaches to the development of neuroglia in the CNS, with special reference to cerebellum

Immunocytochemical methods have in recent years played a more important role in investigations of the development and function of glial cells in the nervous system because of their potential to distinguish between different cell populations. This short review attempts to highlight the value of this approach and summarizes the major cell‐type markers currently available. These include, for the astrocyte, GFA protein, S‐100 protein, vimentin, αα‐enolase and α‐2 glycoprotein. For the oligodendrocyte, myelin basic protein, the Wolfgram proteins, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase, myelin associated glycoprotein, proteolipid protein, galactocerebroside, carbonic anhydrase and glycerol 3‐phosphate dehydrogenase and other glial cell markers recognized by monoclonal antibodies are discussed. The application of these techniques to the study of the developing brain (and in particular the rodent cerebellum) are reviewed. It has proved possible to follow the development of distinct populations of astrocytes and oligodendrocytes from a very precocious age to the adult situation, thus providing new insight on the relationship between glial cells and neurons during normal and abnormal histogenesis.

ISOLATION AND CHARACTERIZATION OF DEFECTIVE JIMPY OLIGODENDROCYTES IN CULTURE

SummaryThis study characterizes jimpy oligodendrocyte-enriched secondary cultures isolated from 10–12 daysin vitro primary glial cell cultures derived from 1–2-day-old jimpy mouse brains. Proliferation of defective oligodendrocytes was carefully investigated with regard to the expression of myelin basic protein and proteolipid protein and their respective mRNAs. Less than 5% of contaminating astrocytes (GFAP+ cells) were usually present. The identity of jimpy oligodendrocytes was confirmed using an antibody directed against a peptide from the wild type proteolipid protein C-terminal sequence for immunocytochernistry and an oligonucleotide complementary to mRNA derived from exon 5 of the proteolipid protein gene forin situ hybridization. Both the antibody and the probe recognize only normal oligondendrocytes while jimpy oligodendrocytes always remain unstained. Proteolipid protein in normal and jimpy oligodendrocytes was detected with antibody recognizing normal and mutated forms. Between 80 and 95% of the cells in normal and jimpy cultures at 2 and 4 daysin vitro in secondary cultures express myelin basic protein and proteolipid protein and their respective mRNAs. The percentage of oligodendrocytes (PLP+ or MBP+) in S phase of the cell cycle was 7–10% for both normal and jimpy oligodendrocytes. This contrasts with thein vivo situation where the proliferation rate of oligodendrocytes in jimpy brains is higher than in normal brains. In addition, jimpy oligodendrocytes remain unresponsive to basic fibroblast growth factor treatment while a similar treatment stimulates the proliferation of normal oligodendrocytes.

Characterization of ependymal cells in hypothalamic and choroidal primary cultures

Long-term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus were obtained in serum-supplemented and chemically defined media. In order to identify and characterize cell types growing in our cultures, we used morphological features provided by phase-contrast, scanning and transmission electron microscopy. Immunological criteria were recognized, using antibodies against intermediate filament proteins (vimentin, gliofibrillar acid protein, cytokeratin, desmin, neurofilament proteins), actin, myosin, ciliary rootlets, laminin and fibronectin in single or double immunostaining, and monoclonal antibodies known to detect epitopes of ependymal or endothelial cells. Minor cell types such as astrocytes, fibroblasts and endothelial cells were distinguished. Ependymal cells, which exceeded 75% of the cultured cells, were identified by their cell shape and epithelial organization revealed by phase-contrast and transmission electron microscopy, by their apical differentiation evidenced by scanning and transmission electron microscopy, and by certain molecular markers (e.g. gliofibrillar acid or ciliary rootlet proteins) detected by immunofluorescence. Four ependymal cell types were recognized: choroidal ependymocytes, ciliated and unciliated ependymal cells, and tanycytes. All these cultured ependymal cell types showed a remarkable resemblance to in vivo ependymocytes, in terms of marker expression and ultrastructural features.

Monoclonal antibodies specific for glial and neuronal antigens in the young rat cerebellum.

Monoclonal antibodies from 15 different hybridomas derived from the fusion of mouse spleen cells with a myeloma cell line were selected. Mice were immunized with the particulate fraction from 10-13-day-old rat cerebella. Hybridoma secreting antibodies were screened simultaneously by both immunocytochemistry and binding assay. Each antibody reacts with specific cerebellar neuronal or glial cells and structures.