G. Roussel

Demonstration of a specific localization of carbonic anhydrase C in the glial cells of rat CNS by an immunohistochemical method

The localization of carbonic anhydrase C isoenzyme in the central nervous system (CNS) of the rat has been investigated using the indirect immunoperoxidase technique, at both optic and electron microscopic levels. Evidence is presented for a specific localization of the enzyme in the cytoplasm of the oligodendrocytes and astrocytes. Myelinated fibers show a weak staining. The positive reaction is restricted to the cytoplasmic areas of the myelin sheath and does not appear in the compact myelin. Neuronal cell bodies do not stain at all. A strong positive reaction to the antiserum was also observed in the choroid plexus.

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.

Oligodendroglia content of glial cell primary cultures, from newborn rat brain hemispheres, depends on the initial plating density

Abstract Cultures enriched in oligodendrocytes were obtained by plating dispersed unfractionated cells from newborn rat brain hemispheres at high cell density in a standard medium (Waymouth MD 705 1 supplemented with 10% fetal calf serum). In dishes seeded at low cell density only astrocytes proliferated.

Characterization of oligodendrocytes in primary cultures from brain hemispheres of newborn rats

Characterization of putative oligodendrocytes obtained in primary cultures of brain hemispheres from newborn rats is reported. Most of the oligodendrocytes are scattered in the culture dish until around 20 days after seeding, the time at which they start to form aggregates made up of one to three layers of cells upon the astrocytes. At the electron microscopic level the oligodendrocytes ultrastructure appears undifferentiated but very different from that of the underlying astrocytes. These oligodendrocytes do not react to W1 Wolfgram protein and myelin basic proteins antisera until the sixth day after seeding. On Day 8, a few oligodendrocytes give a positive reaction; after 4 weeks most of them react. These results represent a further step in the identification of oligodendrocytes in culture and in the characterization of their development in vitro.

Isolation and immunohistochemical localization of a lectin-like molecule from the rat cerebellum

A lectin with a mannose specificity was isolated from the cerebellum of young rats. The method of purification was based on the observation that during homogenization of the tissue, the lectin binds to a class of mannose-rich glycoproteins highly insoluble in Triton X-100. Sequential extractions in saline buffer devoid of, then containing, 0.5% Triton X-100 allowed the elimination of a great part of other proteins. Using the same buffer containing 0.5 M mannose, a specific class of protein can be solubilized. This fraction was enriched by affinity adsorption on insolubilized mannose-rich glycoproteins followed by specific detachment with mannose. One of the protein subunits, of molecular weight (MW) 130,000, was isolated by preparative gel electrophoresis. Upon re-electrophoresis, this compound gives two bands of MW 65,000 and 130,000, which appear to be a monomer and a dimer of a molecule called R1. Antibodies were raised against R1 which react with the monomer and the dimer and not against other proteins of the rat cerebellum. The immunohistochemical localization of this lectin was performed in cerebella of 20-day-old rats. The antigen is concentrated in endothelial cells and in large and intermediate size neurons (Purkinje, Golgi, basket and deep nuclei neurons). Granule cell bodies are lightly stained and no label at all was found in glial cells. At the level of electron microscopy, the antigen was found to be very concentrated in multivesicular bodies and lysosomes of large neurons, on parts of the endoplasmic reticulum, on some mitochondrial outer membranes and on the plasma membrane of the dendrites. The possible role of this lectin in cerebella of young rats is discussed in relation to its interaction with a specific class of mannose-rich glycoproteins.

Ultrastructural localization study of two Wolfgram proteins in rat brain tissue.

SummaryThe ultrastructural immunohistochemical localization of Wolfgram proteins W1 and W2 is described in young rat brain tissue. The labelling by the antiserum to W1 is restricted to oligodendroglial cells and myelin sheaths. The plasma membrane of the cells as well as the polysomes are positively stained whereas the mitochondria and the nuclei are always free of labelling. Glial cell processes with definite organelles, which are involved in the myelination of neighbouring axons, are also positive to the antiserum. In the myelin sheaths, the positive staining occurs predominantly at the dense period line of the innermost and outermost lamellae. The present results add further evidence for a specific local synthesis of these Wolfgram proteins in oligodendroglial cells during myelination.

Immunohistochemical localization of a lectin-like molecule, R1, during the postnatal development of the rat cerebellum

The immunohistochemical localization of an endogenous lectin R1 isolated from the rat cerebellum was studied during its postnatal development. The lectin is present in the cerebellum from birth to adulthood, essentially in lysosomes, multivesicular bodies, and parts of the endoplasmic reticulum, principally of large and intermediate size neurons. During the period of massive synaptogenesis in the molecular layer, there is a sprouting of R1 in some distal dendrites of Purkinje cells. The lectin appears to be particularly concentrated on their plasma membranes, in coated pits, in coated vesicles, multivesicular bodies and lysosomes. At the same period, in cerebella of rats treated with chloroquine (an inhibitor of lysosomal function), both the lectin and mannose-rich glycoproteins of newly formed parallel fibres (able to bind specifically this lectin) are found in the same non-functional lysosomes of Purkinje cells. It is thus suggested that both this lectin (with a high-affinity for the glycans of the mannose-rich glycoproteins of the membrane of the newly formed parallel fibres) and these glycoproteins could be the recognition molecules allowing a specific contact between parallel fibres and Purkinje cells at the period of synaptogenesis.

Immunological Investigation of a 21-Kilodalton Cytosolic Basic Protein in Rat Brain

A gamma-globulin fraction was isolated from the antiserum raised against a 21-kilodalton (kDa) basic protein which was purified from bovine brain cytosol. This fraction was employed to study the immunocytochemical localization of the 21-kDa protein during the development of rat brain. Immunostaining was observed on oligodendrocytes and their processes at all stages of development investigated. This immunostaining was less prominent in very young and adult brains. Myelin fibers were always moderately stained; neurons and astrocytes were not immunolabelled. The electron microscopic study revealed that the labelling covers the entire cytoplasm of the oligodendrocytes, being more dense along the membranes of the rough endoplasmic reticulum and the plasma membrane. Other cytoplasmic organelles were unstained. The present report emphasizes that 21-kDa protein may serve as a specific marker for oligodendroglial cells in the central nervous system despite its presence in peripheral organs.

Effects of dl-α-amino adipic acid on Müller cells in frog and chicken retinae in vivo: Relation to ERG b wave, ganglion cell discharge and tectal evoked potentials

In both frog and chicken, an intravitreal injection of DL-alpha-amino-adipic acid, (DL-alpha aaa) provoked a progressive depression and eventually the disappearance of the ERG b wave that was concomitant with severe damage to the Müller cells without any apparent damage to retinal neurons. Ganglion cell discharges as well as tectal evoked potentials were still recorded, i.e. a visual message was still generated in the retina and transmitted to the optic tectum, when the Müller cells had been damaged to as to provoke an abolition of the ERG b-wave. The whole of the drug-induced effects proved to be reversible.

Biochemical and immunohistochemical studies with specific polyclonal antibodies directed against bovine myelin/oligodendrocyte glycoprotein

Bovine myelin/oligodendrocyte glycoprotein (MOG) was purified from a Wolfgram protein fraction of brain myelin by molecular sieving and preparative gel electrophoresis. The N-terminal sequence of this wheat germ agglutinin reacting glycoprotein was determined. Antibodies against purified MOG and synthetic N-terminal octapeptide of MOG were produced in rabbits. Respective affinity purified antibody preparations gave identical results on Western blots. Treatment with specific glycosidases indicated that the oligosaccharide chains of MOG are only of N-chain type. This glycoprotein seems to be restricted to mammalian species since it was not detected in other animal species, ranging from fish up to reptiles. Immunohistochemical investigations on rat brain sections revealed that MOG is restricted to myelin sheaths and oligodendrocytes, thus corroborating previous results obtained with the MOG 8-18C5 monoclonal antibody. Decreased staining pattern in Jimpy brain further attested its specific localization in myelin-related structures. The octapeptide site-specific antibodies were not reactive on brain sections which may be attributed to the burying of this N-terminal sequence in the membrane. These MOG polyclonal antibodies appear to be valuable tools for further studies concerning this minor glycoprotein.