Diversification of glial lineages: a novel method to clone brain cells in vitro on nitrocellulose substratum.

We have developed a novel in vitro method to analyze the diversification of glial cells during development. The primary advantage of the approach is that glial lineages are formed in discrete clones on a nitrocellulose substratum where the relationship of the progeny is strictly defined. This method facilitates the comparison of a large complement of astrocyte and oligodendrocyte lineages under controlled conditions. Clones were formed by plating a brain dissociate on nitrocellulose at very low density (5,000-40,000 cells/154 mm2). However, growth depended on diffusible factors produced by brain cells growing under the nitrocellulose support at high density (feeder layer). The cloning efficiency of cells from mouse forebrain (P0) was 1-3%. This means we can detect 100,000 to 300,000 clonal progenitors in the dissociate (10(7) cells per forebrain) using the clonal culture technique. Cell phenotypes were determined by immunocytochemical staining with anti-glial fibrillary acidic protein (GFAP) to label astrocytes and anti-galactocerebroside (GC) and anti-myelin basic protein (MBP) to label oligodendrocytes. There was a remarkable diversity of glia represented in different lineages. The number of astrocyte clones was greater than the number of oligodendrocyte clones but combined their total was 90%. Clone sizes were distributed over a wide range, which indicated that growth rates varied. Clones appeared compact or dispersed but astrocyte clones exhibited three different morphologies-fibroblast-like, stellate, and elongated. Oligodendrocytes had different morphologies distinct from astrocytes. Although there were different glial lineages the cells in most clones were homogeneous, indicating the progeny had the same fate. However, a small number of the clones, approximately 2%, were heterogeneous and contained both astrocytes and oligodendrocytes. The application of this technique to glial lineages demonstrates that intrinsic factors have a role in determining cell fate since different clones formed under the same external conditions. Finally, these results are consistent with the existence of multiple glial progenitors or the continued presence of multipotential progenitors at the time of birth.

Localized survival of ciliary ganglionic neurons identifies neuronotrophic factor bands on nitrocellulose blots.

A novel and sensitive method has been developed to identify ciliary neuronotrophic factors (CNTFs) from tissue extracts after blotting to nitrocellulose paper. The CNTF proteins are required for the in vitro survival of embryonic chick ciliary ganglionic neurons. Tissue extracts containing such CNTFs are electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Purified ciliary ganglionic neurons are seeded on the surface of the nitrocellulose blot, and the culture is incubated for 24 hr in medium lacking CNTF. CNTF can be localized on the blot because it retains its ability to support the survival of the neurons cultured on the nitrocellulose. A band of viable neurons, easily visualized by staining with a vital dye, is supported by the blotted CNTF polypeptide. The number of neurons surviving on the blotted CNTF is related to the amount of CNTF originally loaded on the electrophoretic gel. As little as 2 ng (16 trophic units) of CNTF protein contained in crude tissue extracts can be loaded on the sodium dodecyl sulfate gel and still be recognized by the cultured neurons. This method was used to identify CNTF polypeptides from extracts of adult rat nerve (24,000 and 19,000 daltons) and from tissue found near experimentally induced adult rat brain lesions (24,000 daltons). The electrophoretic mobilities of these peptides are distinct from the previously purified chick eye CNTF polypeptide (20,400 daltons).

Myelin basic protein gene expression in quaking, jimpy, and myelin synthesis-deficient mice.

Jimpy (jp), myelin synthesis-deficient (jpmsd), and quaking (qk) are mutations which affect myelination to different degrees in the mouse central nervous system (CNS). Total messenger RNA (mRNA) and myelin basic protein (MBP)-specific mRNA from brains of these three mutants have been analyzed by in vitro translation and immunoprecipitation with antibody to MBP. The results indicate that the three mutations do not affect the level of total MBP-specific mRNA in the CNS but do affect the relative proportions of the various MBP-related translation products encoded in vitro. In each case the proportions of 14K and 12K Mr MBP-related translation products are reduced and the proportions of 21.5K, 18.5K, and 17K Mr MBP-related translation products are increased relative to wild type. This effect is most pronounced in jp, less so in jpmsd, and least pronounced in qk animals. The MBP-related polypeptides that accumulate in vivo have also been analyzed in the three mutants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with antibody to MBP. The levels of all the major MBP-related polypeptides that accumulate in vivo are reduced in all three mutations. The reduction is most pronounced in jp, less in jpmsd, and least pronounced in qk animals. These results indicate that the jp, jpmsd, and qk mutations exhibit qualitatively similar phenotypic effects on MBP gene expression but the magnitude of the effect is proportional to the extent of hypomyelination in each mutant.

Radioimmunoassay for the P2 protein of bovine peripheral nerve myelin.

The P2 protein of bovine nerve root myelin was radiolabeled with 125I in homogeneous solution by the chloramine-T method and purified by gel filtration on Sephadex G-75 to obtain monomeric 125I-P2. Antigen-antibody complexes were isolated by the silica gel, double antibody, or polyethylene glycol techniques by using rabbit antibody to P2. As little as 1 ng/ml P2 could be detected. The RIA was used to measure the P2 content in nerve tissue and isolated myelin. The presence of P2 in spinal cord as well as in the peripheral nervous system was confirmed. Peptides isolated by CNBr digestion of the P2 protein were tested in the RIA. CN-1, comprising 80 to 90 residues from the interior of the molecule displayed complete immunologic cross-reactivity with intact P2. Neither CN-2, representing 18 amino acids from the COOH terminal, nor CN-3, representing 20 amino acids from the NH2 terminal, showed cross-reactivity. Since the major determinant for experimental allergic neuritis in the rabbit is located in peptide CN-2, our present data suggest that the major neuritogen and the major determinant(s) for humoral antibody response in this species may be at different locations within the P2 molecule.

Histological localization of nervous-system antigens in the cerebellum by immunoperoxidase labeling.

The indirect immunoperoxidase method was used to localize histologically on sagittal sections of mouse cerebellum antigenic determinants detected by the following antisera: anti-NS-2, anti-NS-3, anti-NS-4 rabbit anti-bovine corpus callosum, rabbit anti-mouse brain, rabbit anti-glial fibrillary acidic protein, and rabbit anti-neurofilament protein. Anti-alpha-bungarotoxin serum and normal rabbit serum were used as negative controls. The various sera showed similarities in staining pattern as well as differences. Anti-NS-2 antiserum labeled the somata of interneurons in the molecular layer, granule cell bodies, glial cells in the white matter, and along the surfaces of blood vessels. A similar pattern of staining is produced by the anti-NS-3 antiserum except that glial cells are less prominent in the white matter and the blood vessels are not visible at all. Anti-NS-4 antiserum does not label interneurons but does label glomeruli and, less intensely, granule cell bodies in the granular layer. Rabbit anti-mouse brain antiserum is similar to anti-NS-4 antiserum except that fiber tracts in the white matter are stained more intensely; Rabbit anti-bovine corpus callosum labels only white matter. Antisera to neurofilament ans astrocytes.