O.K. Langley

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.

An ultrastructural immunocytochemical study of nerve-specific protein in rat cerebellum

SummaryThe ultrastructural localization of the neuron-specific enolase (14-3-2 protein) has been investigated in the cerebellum of the adult rat using the indirect antibody immunohistochemical method. The protein was found exclusively in neurons: perikaryal cytoplasm, axons and dendrites were labelled while nuclei were not. Reaction product was found to be attached to intracytoplasmic membranes, the surface membranes of mitochondria and microtubules in addition to its dispersion as a flocculent material throughout the cytoplasm. All classes of cerebellar neurons were found to be labelled though large variations in the level of labelling between different types of neuron were noted. Purkinje cells appeared to have a much lower cytoplasmic concentration of this protein than other neurons.

Immunocytology of carbonic anhydrase II in the central nervous system of jimpy mutant mice

In agreement with previous observations in rats, carbonic anhydrase II (CAII) demonstrated immunocytochemically with a specific immune serum, is exclusively localized in oligodendrocytes in the central nervous system of both normal and dysmyelinating Jimpy mutant mice. In spite of the lack of myelin, the CAII-containing cells in Jimpy mice are abundant. These data suggest that oligodendrocytes are formed in Jimpy mice but they are not able to reach an advanced stage of maturation as defined by their capacity for producing myelin.

Cellular localization of the brain specificα2-glycoprotein in rat cerebellum: An immunohistological study

The cellular localization of the brain-specific, soluble, acidic alpha 2-glycoprotein was studied in rat cerebellum by using the immunoperoxidase technique at the light-and electron-microscopy levels with monospecific immune serum directed against this glycoprotein. Only astrocytes, their processes, and their end feet (subpial or perivascular) contained heavy immunoperoxidase reaction product. Cerebellar neurones, oligodendrocytes, myelin and blood vessel endothelia did not stain. Thus it appears that alpha 2-glycoprotein is an astrocyte marker.

A monoclonal antibody recognizing subpopulations of neurones in mouse brain

A monoclonal antibody, designated C138, was raised against a particulate fraction from young postnatal mouse cerebellum. In sections from adult mouse brain and cerebellum, this antibody stained a sub-population of neurons. Cell bodies of large neurones and fibre tracts were negative in all brain regions examined, whereas neuropil was heavily labelled. Immune-electron microscopy confirmed the specificity of the antibody for certain types of neurones and indicated that the antigen may be associated with microtubular structures. Immunofluorescence studies on dissociated cerebellar cultures showed that the antigen was expressed inside all tetanus toxin-positive neurones but not by non-neuronal cells.

Immunoelectron microscopy of α2-glycoprotein: An astrocyte-specific antigen

Abstract The cellular and fine structural localization of the soluble brain-specific acidic α 2 -glycoprotein was investigated using the indirect immunohistochemical method. The electron microscope was used to unambiguously identify cells containing the antigen. A single type of cell, the astrocyte, was found to be labelled with specific antisera directed against α 2 -glycoprotein. Immunoperoxidase reaction product was found in astrocyte perikarya, their processes and perivascular end feet. It was found to be apparently associated with the cytoplasmic surface of mitochondria, reticular membranes and the plasma membrane. No specific labelling of neurones, oligodendrocytes, myelin or capillary endothelial cells was observed. The data is discussed in relation to the immunological properties of α 2 -glycoprotein already reported.

AN IMMUNOHISTOCHEMICAL STUDY OF CEREBELLAR NEURONAL AND GLIAL ENOLASES

Publisher Summary This chapter elaborates an immunohistochemical study of cerebellar neuronal and glial enolases. Two isoenzymes of the glycolytic enzyme enolase were isolated in pure form from mouse brain. A comparison was made between the cellular distribution of these two isoenzymes using immunocytochemical methods with the light and electron microscopes. Granule cells, Golgi neurons, and neurons of deep cerebellar nuclei were particularly well labeled, while Purkinje cells appeared to contain relatively low levels. The apparent absence of the enolase in oligodendrocytes was unexpected. It is possible that either a different isoenzyme is present in these cells, or the levels are below the limit of detection of the methods employed.

Cellular specificity in rat cerebellum monitored by monoclonal antibodies

Monoclonal antibodies (MAbs) against molecules of cerebellar structures were obtained by injecting, as immunogens, into mice, cerebellar “membrane” fractions from 12 day old rats; the mice immunocytes were fused with cells from a mouse myeloma line and the hybridomae were cloned. Immunocytology with the MAbs produced by each hybridoma cell line and differential binding tests of these !4Abs to 12 day old and to adult membrane were used for screening and for selecting developmentally regulated antigens. Several of the obtained MAbs showed specificity for a single cell type (either neurones or astrocytes) or subtype (i.e. different neuronal types) or structures (i.e. the pinceau of basket cells, the parallel fibres, the glomeruli). Few MAbs reacted with developmentally regulated antigens. According to GJestern blots of SDS-PAGE electrophoresis of proteins derived from cerebellar membranes, from cytosol and from cytosqueleton fractions none of the epitopes revealed by our MAbs are localized in known CNS antigens.