Properties of Bovine Oligodendroglia Isolated by a New Procedure Using Physiologic Conditions

A new class of procedures, previously shown to permit the isolation of pure oligodendroglia from whole rat cerebrum, has been applied with equal or greater success for the bulk isolation of this cell type from bovine white matter. Thus, the generality of this approach has been demonstrated. The bovine preparations have a purity of greater than 90% intact, phase-bright oligodendroglia and are obtained in a yield of 8 x 10(6) cells per gram of white matter. Within 1 day it is possible to obtain a preparation containing 60 mg of protein from a single cell type. These cells show a higher degree of ultrastructural preservation of all cytoplasmic constituents than previously obtained. The values for protein (33 pg/cell), DNA (5.4 pg/cell), and lipid (5-6 pg/cell) are very similar to those obtained with an earlier procedure. The cell lipids are rich in galactolipid, which comprises 20% of the total. The activity of the "myelin-specific" enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37), is 4.7 mumol/min/mg protein, similar to that obtained previously for isolated oligodendroglia and about 25-40% of that found in myelin. The activity of 5'-nucleotidase (EC 3.1.3.5) in the cells is about 10% of that in myelin or white matter.     

Absence of ganglioside GM1 in astroglial cells from 21-day old rat brain: Immunohistochemical,histochemical, and biochemical studies

A procedure was developed for the cultivation of cells derived from the cerebral hemispheres of the 21-day old rat. Approximately 98 percent of the cells in a 10 day culture are astrocytes that contain glial fibrillary acidic protein. Analysis of the extracted gangliosides by thin layer chromatography revealed that ganglioside GM1 was absent and that the predominant ganglioside was GM3. Very small amounts of the polysialogangliosides GD1a, GD1b, and GT1b were detected. The concentration of gangliosidic NeuNAc per mg protein in these astrocytes was only 3 percent that observed in the 5 day culture of a mixed cell preparation from newborn rat brain. Immunohistochemical and histochemical studies were performed on the mixed cell population of the minced tissue of 21-day old rat brain prior to cultivation. Astrocytes did not stain for hyaluronectin. These cells also did not provide a positive staining reaction for ganglioside GM1 utilizing the antiganglioside GM1 peroxidase-antiperoxidase procedure and the biotinylated choleragen-avidin-peroxidase procedure. These two histochemical methods for ganglioside GM1 also did not stain astrocytes that had been cultured for 5 days. Oligodendroglial cells, which were also present in the uncultured 21-day-old minced brain tissue, stained positively for ganglioside GM1 and hyaluronectin. Hyaluronectin had previously been shown to be a marker for oligodendroglia. Oligodendroglial cells which were present in the 5 day cultures of 21-day old brain tissue also provided a positive reaction for ganglioside GM1. It is concluded that ganglioside GM1 is absent in astroglia. The presence of small amounts of polysialogangliosides in the "pure" astrocyte preparation is discussed.     

Relative levels of hexokinase in isolated neuronal, astrocytic, and oligodendroglial fractions from rat brain

The levels of hexokinase, as well as those of the cytoplasmic glycolytic enzyme lactate dehydrogenase and the mitochondrial tricarboxylic acid cycle enzymes fumarase and citrate synthase, have been determined in whole rat brain and in neuronal, astrocytic, and oligodendroglial fractions isolated from rat brain. Compared with either whole brain or with isolated neurons or astrocytes, oligodendroglia are low in hexokinase content. This provides direct confirmation for the conclusion, based on an electron microscopic immunohistochemical method, that oligodendroglia, compared with other neural structures, contain relatively low levels of this key enzyme of glucose metabolism. Based on this confirmation, it is concluded that the electron-microscopic immunohistochemical procedure provides a valid indication of hexokinase content, and thus that other structures shown to stain weakly by the latter technique (e.g., dendritic terminals of cerebellar granule and Purkinje cells) are, indeed, low in hexokinase activity.     

Absence of ganglioside GM1 in astroglial cells from newborn rat brain

An immunohistochemical method utilizing anti-ganglioside GM1 antiserum combined with the peroxidase-antiperoxidase technique was applied to a mixed cell population in primary cultures of newborn rat brain. Ganglioside GM1 was demonstrated to be present in neurons and oligodendroglia, but was absent in astroglia. This demonstration was confirmed using a newly developed biotinylated choleragen-avidin-peroxidase procedure. Primary cultures from newborn rat brain cells that had been subjected to a single treatment with trypsin (first passage) and then cultured for 14 days were predominately (95%) composed of astrocytes that stained positively for glial fibrillary acidic protein but were negative for GM1 ganglioside. This preparation contained only 0.34 nmol ganglioside NeuNAc per mg protein compared to 23.9 nmol gangliosidic NeuNAc/mg protein for a five day culture of newborn rat brain mixed cell culture that had not been subjected to passage. Prolongation of culture time from 5 to 21 days in the latter preparation reduced the ganglioside NeuNAc content to 4.9 nmol gangliosidic NeuNAc/mg protein as the proportion of astrocytes in the culture increased. Ganglioside GM1 could not be detected by TLC analysis of the lipid extract obtained from the “pure” astrocyte culture, although small amounts of GM3 and some polysialogangliosides were detected. About half of the label incorporated upon 24 h incubation of astrocytes in the presence of $\text{N-[}{}^3\text{H]acetylmannosammine}$ appeared in ganglioside GM3. It is concluded that astrocytes in mixed cell primary cultures from newborn rat brain, as well as astrocytes in astroglial preparations derived from such cultures, do not contain ganglioside GM1.     

Development of macroglial cells in rat cerebellum. II. An in situ immunohistochemical study of oligodendroglial lineage from precursor to mature myelinating cell

Using immunofluorescence with a panel of antibodies that recognize antigens expressed by oligodendroglia, the myelin-producing cells of the CNS, at different stages of differentiation from precursor to mature cell, we have investigated the development of cells of this lineage in cryostat sections of rat cerebellum. Our results are consistent with the view that glial precursors, identified by their expression of the ganglioside GD3, arise in the subependymal layers of the 4th ventricle and migrate to their final position in the cerebellum via the superior medullary velum, and to some extent the peduncles. As the cells reach their final destination they make the transition to recognizable galactocerebroside (GC)-expressing oligodendroglia, via a GD3+/GC+ intermediate. The myelin-associated protein 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) appears at the same time as GC, whereas myelin basic protein (MBP) is expressed 2-3 days after GC and CNP, immediately prior to myelin formation. A very clear progression of oligodendroglial differentiation was observed from the SMV into the base of the cerebellum, up into the white matter (WM) tracts of the folia, and then away from this central white matter into the granule cell and Purkinje cell layers, and finally the molecular layer. The time delay between the expression of GC, CNP and MBP was the same for oligodendroglia in all of these layers, suggesting the presence of an intrinsic clock controlling the initial expression of these myelin components. The early appearance of CNP in oligodendroglia suggests a role for this protein in the early stages of myelinogenesis.     

Isolation and some chemical properties of oligodendroglia from calf brain

Abstract— The method of Norton and Poduslo (1970) for isolating brain cells has been adapted for the isolation of oligodendroglia from the white matter of calf brain. The cells were obtained in greater than 90 per cent purity, and in a yield of 11 × 10⁶ cells/g of white matter. This number of cells represented a recovery of 11 per cent of the total cells in the tissue and therefore a considerably higher recovery of the original number of oligodendroglia. The average cell contained 5, 2 pg of DNA, 2–0 pg of RNA and 6, 7 pg of lipid. The lipid comprised cholesterol, galactolipid (both cerebroside and sulphatide) and phospholipid in the molar ratio of 1:0, 45:2, 3. Gangliosides were present in a concentration similar to that found in isolated rat neurons, The myelin-specific enzyme, 2′, 3′-cyclic nucleotide 3′-phosphohydrolase, was present at a level nearly equal to that in myelin, and eight-fold higher than the levels in rat neurons or astrocytes. The isolated oligodendroglia differed considerably from isolated astrocytes in size, morphology and chemical composition.     

Induction of cerebroside synthesis in oligodendroglia

Oligodendroglia function to produce myelin membranes which surround axons, enhancing saltatory conduction. Myelin consists of a multitude of condensed membranes which are rich in lipids with the major glycolipids, cerebrosides, being 25% of the total lipid. Thus a fully differentiated oligodendroglial cell that is producing myelin membranes would be actively synthesizing cerebrosides. Our laboratory has prepared and analyzed oligodendroglia from mature bovine brain, from neonatal rat brain, and from actively myelinating rat brain. Our studies suggest that the rat oligodendroglia in our culture systems are less differentiated than bovine cells in that they produce lower levels of cerebrosides. Addition of glucocorticoids, thyroid hormone, or retinoic acid all increased synthesis of cerebrosides in rat oligodendroglia. Ketone bodies were also somewhat stimulatory. Having no effect or causing dedifferentiation of the cells were 5-azacytidine and phorbol esters. Thus induction of cerebroside synthesis in oligodendroglia is complex and may involve many factors.     

Levels of sulfatide synthesis distinguish oligodendroglia in different stages of maturation

In the central nervous system, oligodendroglia elaborate extensive quantities of membranes to form the multilamellar myelin sheath. Whether the production of extensive networks of processes by oligodendroglia in culture is a similar type of phenomenon as the formation of myelin is an unanswered question. Rat oligodendroglia, prepared by a modification of a differential shaking and plating method, elaborate extensive processes in culture. In contrast, bovine oligodendroglia, obtained by a bulk-isolation method, produce whorls of membrane lamellae, adjacent to the cell soma. The incorporation of various radiolabeled substrates into specific lipids was compared with the two cell types. It was found that rat oligodendroglia do produce myelin specific lipids, but at a lower level than bovine oligodendroglia which are actively synthesizing myelin lipids, especially cerebrosides, from a variety of substrates. Interestingly sulfatides are produced at a higher level in the cells not producing myelin, rat oligodendroglia. Other lipids that are associated with myelination (cerebrosides with -hydroxy fatty acids and phosphatidylinositides) are produced at higher levels in bovine oligodendroglia. Thus it appears that the extension of processes by oligodendroglia in culture is a different phenomenon than the production of myelin membranes and requires lower levels of myelin lipids.     

UDP-Galactose:Ceramide Galactosyl Transferase of Isolated Oligodendroglia

Abstract: The activity of UDP-galactose:ceramide galactosyl transferase (CGalT) has been studied in isolated oligodendroglia from bovine brain white matter and myelinating rat brain. The specific activity and activity per mg DNA are 4- and 10-fold higher in rat oligodendroglia compared with neuronal perikarya from rat brain, and is higher in oligodendroglia from myelinating rat brain compared with bovine oligodendroglia. In membranes isolated from oligodendroglia, the specific activity decreased in the order endoplasmic reticulum > plasma membrane > myelin.     

Proliferation and differentiation potential of rat forebrain oligodendroglial progenitors both in vitro and in vivo

We have followed the development of the O-2A progenitor cell from the neonatal rat forebrain, both in dissociated cell culture and in cryostat sections, using immunocytochemical techniques employing a panel of antibodies that recognise the cells at different stages of their development. This included the monoclonal antibody LB1, which binds to the surface ganglioside GD3 expressed on O-2A progenitor cells. In secondary cultures enriched for O-2A progenitors maintained in a serum-free chemically defined medium, a large proportion of the cells are primed to differentiate into oligodendroglia and go on to express the oligodendroglial specific surface glycolipid galactocerebroside (GC) and then the myelin proteins CNP and MBP. However, a significant proportion of immature bipolar GD3+ cells remained after 6 days in secondary culture. It appears that not all the O-2A progenitors in our cultures differentiate immediately and some cells remain in an undifferentiated state and divide to replenish progenitor numbers. We have also identified in our cultures a small apolar GD3- cell, which when isolated differentiated into a GD3+ bipolar O-2A progenitor cell. We have termed this cell type a preprogenitor. The differentiation of this cell type into O-2A progenitors may be the source of the immature GD3+ cells present at the later stages of our secondary cultures. The proliferative profile of the cultures was studied using 5'bromo-2-deoxyuridine (BrdU) incorporation as an index of mitosis. Only the immature, bipolar O-2A progenitors were seen to divide at any time in serum-free culture. Neither the more mature multipolar O-2A cells nor the oligodendroglia were seen to divide. The developmental profile of the O-2A cells in the rat forebrain in vivo showed a largely similar progression to that in culture, with a time lag of at least 6 days between GD3 expression and the onset of myelination. BrdU incorporation studies in vivo also showed that the GD3+ progenitor cell is mitotic whereas the GC(+)-expressing oligodendroglia is not. We have shown that there are several significant alterations in the timing of antigen expression in both O-2A progenitors and oligodendroglia in vitro compared to that seen in vivo.     

Isolation of oligodendroglial cells from young and adult whole rat brains using an in situ generated percoll density gradient

A method for the isolation of oligodendroglial cells from young and adult whole rat brains, using a Percoll density gradient is presented. The minced tissue, incubated in a balanced salt solution containing 0.1% trypsin is further dissociated by forcing it through nylon screens to 145 and 74 μm pore size. The crude suspension is then mixed with an isosmotic Percoll solution and centrifuged for 15 min. An in situ generated density gradient allows the separation of five bands, only one of which (Band C) lying between δ1.050 and δ1.062 contains cellular elements. The isolated cells show the typical morphological characteristics of oligodendroglia.A detailed morphological study of the cells isolated from whole brains of 10-, 30- and 120-day old rats is presented for the first time in the literature and immunocytochemical characterization is carried out using specific (antigalactocerebroside) and non specific (anti-glial fibrillary acidic protein) anti-sera.The method is simple and rapid and isosmotic conditions are maintained throughout, resulting in a better preservation of cell integrity. It represents an improvement over the two previous methods described in the literature and will be useful for studying different developmental events (biochemical and morphological) occurring in oligodendroglial cells at early stages of myelin formation.     

A simple method for purification of chymase from rat tongue and rat peritoneal cells

Chymase was purified from rat tongue and rat peritoneal cells by a simple new method involving hydrophobic chromatography on octyl-Sepharose 4B and hydroxylapatite column chromatography. This procedure can be completed in 1 or 2 days and the recovery is 45-60% from rat tongue and 32-47% from rat peritoneal cells. The specific activity of the purified enzyme is higher than that of crystallized enzyme previously reported (Y. Sanada, N. Yasogawa, and N. Katunuma (1978) Biochem. Biophys. Res. Commun. 82, 108-113). This procedure should be particularly useful for purifying chymase on a large scale from tissues in which it is present in relatively low concentrations.     

Dystrophin: a sensitive and reliable immunochemical assay in tissue and cell culture homogenates.

A modified polyacrylamide gel electrophoresis system is described which provides excellent resolution of very high molecular weight proteins. This system has been successfully applied to the immunochemical detection of dystrophin in mouse and rat skeletal muscle, mouse myotubes in cell culture, and in human muscle-biopsy specimens. The mass of total homogenate protein (3-12 micrograms) and the relative quantity of dystrophin detected immunologically were found to be strongly correlated (r = 0.970 - 0.995). The method described here requires minute quantities of tissue or cells to accurately evaluate the relative amount of dystrophin present. The entire procedure for the detection of dystrophin is simple, rapid and cost efficient compared to other available techniques.     

Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit brain

Adenosine deaminase and adenosine deaminase complexing protein have been localized in rabbit brain. Brains fixed in paraformaldehyde or in Clarke's solution were blocked coronally. Blocks from brains fixed in paraformaldehyde were either frozen in liquid nitrogen or embedded in paraffin. Tissue fixed in Clarke's solution was embedded in paraffin. Sections from each block were stained by the peroxidase-antiperoxidase method for adenosine deaminase or complexing protein using affinity-purified goat antibodies. Adenosine deaminase and complexing protein did not co-localize. Adenosine deaminase was detected in oligodendroglia and in endothelial cells lining blood vessels, whereas complexing protein was concentrated in neurons. The subcellular location and appearance of the peroxidase reaction product associated with individual cells was also quite distinctive. The cell bodies of adenosine deaminase-positive oligodendroglia were filled with intense deposits of peroxidase reaction product. In contrast to oligodendroglia, the reaction product associated with most neurons stained for complexing protein was concentrated in granular-appearing cytoplasmic deposits. In some instances, these deposits were clustered about the nuclear membrane. Staining of neurons in the granular layer of cerebellum was an exception. Granule cells were lightly outlined by peroxidase reaction product. Cerebellar islands, also referred to as glomeruli, were stained an intense uniform brown. These results raise the possibility that oligodendroglia and blood vessel endothelia, through the action of adenosine deaminase, might play a role in controlling the concentration of extracellular adenosine in brain. They do not, however, support the suggestion that complexing protein aids in adenosine metabolism by positioning adenosine deaminase on the plasma membrane.     

Direct assay of membrane-associated protein kinase C activity in B lymphocytes in the presence of Brij 58.

This paper describes a simple and direct procedure for assaying Ca(2+)-dependent protein kinase C (PKC) activity in membrane fractions isolated from purified murine B lymphocytes (B cells) treated with phorbol 12-myristate 13-acetate (PMA). The results indicate that membrane-bound PKC in B cells, treated with PMA, can be measured directly in the presence of 0.5% Brij 58 by assaying the transfer of 32P from [gamma-32P]ATP to histone type III-S. This method obviates the need for partial purification of the protein kinase by ion-exchange chromatography prior to assaying PKC activity. The properties of membrane-associated PKC activity in B cells have been characterized, and the kinetics of PMA-induced translocation of PKC in cultured murine B cells, the rat glial tumor clone C6, and primary neonatal osteoblastic cells have been defined by this direct assay. The results obtained with B cells and the other cell lines indicate that this direct assay procedure could be useful for studies on the factors controlling PKC translocation in a variety of cultured mammalian cells.     

Isolation of sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function

Much of what is known about the molecular regulation and function of adult Sertoli cells has been inferred from in vitro studies of immature Sertoli cells. However, adult and immature cells differ in significant ways and, moreover, many Sertoli cell functions are regulated by conditions that are difficult to replicate in vitro. Our objective was to develop a procedure to isolate Sertoli cells rapidly and in sufficient number and purity to make it possible to assess Sertoli cell function immediately after the isolation of the cells. The isolation procedure described herein takes less than 4 h and does not require culturing the cells. From a single 4-mo-old adult rat, we routinely obtain 7.0 +/- 0.4 x 10(6) Sertoli cells per testis, and from a 21-mo-old rat, 7.2 +/- 0.4 x 10(6) Sertoli cells per testis. The purity, determined by morphologic analyses of plastic-embedded cells or after staining for tyrosine-tubulin or vimentin, averaged 80%. The contaminants typically included germ cells (10%) and myoid cells (10%). The germ cell-expressed genes protamine-2 and hemiferrin were not detected in the Sertoli cell preparations by Northern blot analyses, but the Sertoli cell-expressed genes clusterin, cathepsin L, and transferrin were highly expressed. Transferrin mRNA levels were greater in Sertoli cells isolated from aged than from young adult rats, consistent with previous analyses of whole testes; and cathepsin L mRNA levels were far more highly expressed in Sertoli cells isolated from stages VI-VII than from other stages of the cycle of the seminiferous epithelium, also consistent with previous analyses of whole testes and isolated tubules. These studies indicate that the freshly isolated cells retain differentiated function, and thus it should be possible to assess the in vivo function of adult Sertoli cells by isolating the Sertoli cells and immediately assessing their function.     

A Method for Silver Impregnation of Mammary Myoepithelia

Histochemical methods for microscopic visualization of nummary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

A method for silver impregnation of mammary myoepithelia

Histochemical methods for microscopic visualization of mammary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

Oligodendroglial Signal Transduction Systems Are Developmentally Regulated

Abstract: Studies from several laboratories indicate that oligodendroglia exhibit signal transduction systems that can be activated by classical neurotransmitters. Previous studies from this laboratory indicate that oligodendroglia express neuroligand receptors linked to the regulation of Ca²⁺_i. Experiments presented in this article were designed to determine if developmental processes that influence the ability of oligodendroglia to respond to neuroligands with an increase in Ca²⁺_i proceed either in vitro or in vivo. Findings support the view that developmental processes markedly affected the sensitivity of these cells to both purinergic and cholinergic receptor agonists, whereas their responsiveness to either histamine or bradykinin appeared relatively stable over time. Approximately 90 and 75% of oligodendroglia responded to ATP or carbachol, respectively, after 4 days in vitro, whereas <10% of these cells responded to either of these neuroligands after 8 days in vitro. The decrease in the percentage of oligodendroglia responding to ATP, but not carbachol, could be prevented by including dibutyryl cyclic AMP in the culture medium during the final 4 days in vitro. However, once the loss in responsiveness to ATP had occurred, it could not be reversed by exposure to dibutyryl cyclic AMP. Developmental changes in the ATP sensitivity of oligodendroglia occurred in cells expressing galactocerebroside and myelin basic protein. The neuroligand sensitivity of oligodendroglia isolated from either neonatal, 2-, 3-, or 5-week-old spinal cord was examined to determine if developmental changes in oligodendroglial Ca²⁺ regulation occurred in vivo. The results of these experiments indicate that the percentage of oligodendroglia responding to either ATP or carbachol markedly decreased as a function of the age of the animal used to prepare the cultures; this was not the case for the stimulation of Ca²⁺_i by histamine. The decreased sensitivity of oligodendroglia isolated from older animals could not be reversed through the addition of dibutyryl cyclic AMP. Overall, the results of these experiments indicate that developmental processes selectively influence the sensitivity of oligodendroglia to specific neuroligands and suggest that oligodendroglial processes unrelated to myelin formation may be regulated by neuroligands in vivo.