Temporal Expression of Myelin-Specific Components in Neonatal Mouse Brain Cultures: Evidence that 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase Appears Prior to Galactocerebroside

Abstract

A range of antibodies to cell-specific markers was employed to characterize and quantify the cell types present in neonatal mouse brain cell cultures. Astrocytes were the major cell type to develop in culture and formed a bed layer by 5–7 days in vitro (DIV). A population of cells appeared on this layer which consisted of oligodendrocytes, progenitor cells and microglia. Some neurons were detected in the cultures up to at least 27 DIV. With time, the number of progenitor cells decreased in the cultures and there was a concomitant increase in the number of oligodendrocytes. Maximal numbers of cells expressing galactocerebroside (GC), 2'',3''-cyclic nucleotide 3''-phosphodiesterase (CNP) and myelin-associated glycoprotein were observed at 18 DIV. The highest number of cells expressing myelin basic protein and proteolipid protein were observed at 25–30 and 35–40 DIV, respectively. Double-label studies with antibodies to A2B5/CNP and A2B5/GC showed that several A2B5⁺, CNP⁺ cells were present at 6 DIV. In contrast, no A2B5⁺, GC⁺ cells could be seen at this age. Confirming these findings, some CNP⁺.GC^– cells were observed when cells were double-labeled with antibodies to CNP and GC. These findings suggested that the expression of CNP precedes that of GC in oligodendrocytes in these cultures.

Murine oligodendroglial cells express nerve growth factor.

“…In our laboratory, we have demonstrated that the expression and timing of appearance of the key oligodendroglial markers in mouse primary tissue culture system are similar to that observed (25) in vivo. This leads us to suspect that the observations reported in this study may not be restricted to the culture system.…”

“…One of these lines, N19, expresses the characteristics of immature oligodendroglial cells (24). As part ofa larger study to (25). Enriched oligodendroglial cultures were prepared from these at 7 days in vitro (DIV) by the method of Suzumura et al (26).…”

“…The use of this procedure has been described (29). Immunohistochemical staining for NGF, galactocerebroside (GC), and A2B5 was carried out using the same general procedure described previously (24,25). The combined procedure for in situ hybridization and immunohistochemical staining followed by confocal microscopy was carried out as described (29) except that digoxigenin antibody conjugated to alkaline phosphatase was used to detect the digoxigenin-labeled mRNAs.…”

Region-Specific Myelin Pathology in Mice Lacking the Golli Products of the Myelin Basic Protein Gene

“…The method used for preparing primary mixed glial cultures was in accordance with that described by Amur-Umarjee (1990). Briefly, mechanically dissociated cerebral hemispheres from 0-to 3-d-old mice were plated on polylysine-coated coverslips in DMEM/Ham's F12 media containing 6 g/L glucose and 10% fetal bovine serum (FBS).…”

Proliferation of differentiated glial cells in the brain stem

“…However, precocious markers are those expressed by proliferating cells or, at least, by cells that have withdrawn recently from the mitotic cycle, whereas late markers are those expressed at the final stage of myelination, i.e., at the time of myelin compaction. Among those considered as precocious markers are sulfatides, the main antigen recognized by the O4 antibody (45,46), carbonic anhydrase II (27,30 but see 31), the antigens recognized by the monoclonal antibody Rip (23 kDa and 160 kDa molecular mass) (47,48) and CNPase (20,(49)(50)(51).…”