Advances in Cellular Neurobiology: Volume 4

Index

Section 1. Cell Differentiation and Interaction

Outline

Recent Advances in the Neurobiology of Oligodendroglia

Neuronal Differentiation in Reaggregate Cell Cultures1

Locus Coeruleus

Structure of Electric Organ and Mammalian Acetylcholine Receptor Molecules

Molecular Properties of Benzodiazepine Receptors

Glial and Neuronal Na+,K+ Pump

Recent Advances in the Neurobiology of Oligodendroglia

William T. Norton,     Department of Neurology, Albert Einstein College of Medicine, New York, New York

Publisher Summary

This chapter focuses on recent advances in the neurobiology of oligodendroglia. The principal known function of the oligodendrocyte is to form and sustain the myelin sheath on axons of the central nervous system (CNS). Oligodendrocytes and astrocytes arise from cells of the subependymal layer, which are derived from the neuroectoderm. The enzyme 2′:3′-cyclic-nucleotide 3′-phosphodiesterase follows a developmental curve similar to that of myelin proteins basic protein, but it starts earlier and reaches a plateau sooner. A discovery that may have very interesting implications for remyelination processes in disease is that myelinogenesis can be inhibited by anticerebroside antibody and can be suspended for weeks. Presumably, primary dissociated cultures that contain oligodendrocytes do not myelinate because few or no neurons are present.

I Introduction

II Gliogenesis and Differentiation

III Gene Expression in Vitro

A Expiants

B Reaggregating Cultures

C Clonal Lines and Hybrid Cells

D Primary Cultures

IV Bulk-Isolated Cells

A Isolation Methods

B Properties

C Metabolism and Culture of Bulk-Isolated Cells

V The Status of Cell-Specific Markers and Immunohistochemical Results

A Lipids

B Proteins

C Enzymes

VI Neuronal–Glial Interactions

VII Concluding Remarks

References

I Introduction

The principal known function of the oligodendrocyte is to form and sustain the myelin sheath on axons of the central nervous system (CNS). That oligodendrocytes perform this function was proposed by Del Rio-Hortega (1924) and Penfield (1924) nearly 60 years ago, and the general morphological description of the myelination process has been established for 20 years. In the past decade our knowledge of the biology of oligodendroglia has accelerated rapidly. This progress has resulted largely from methodological advances in several areas. These include improved methods for tissue culture of nervous tissue, the development of procedures for isolating oligodendroglia, and the use of immunocytochemistry for identifying specific cell types and their differentiated properties. It is now possible to study the cell biology of oligodendroglia in vitro and to modify the cellular environment at will. Gene expression during differentiation can be followed both in vivo and in vitro, and direct investigations of oligodendroglial biochemistry can be made on pure cell populations. The purpose of this chapter is to review the recent progress that has been achieved in these areas. The historical aspects of the histology of oligodendroglia and the morphology of myelinogenesis, topics familiar to most neurobiologists, will not be covered. The reader is referred to several reviews of these subjects (Bunge, 1968; Davison and Peters, 1970; Raine, 1977; Norton, 1981a).

Most of what we know about the biochemistry of oligodendroglia has been derived from studies of myelin and myelination. Procedures for the isolation of myelin made it possible to obtain large quantities of the major portion of the oligodendroglial cell. Thus, studies of myelin metabolism reflect a significant proportion of the metabolism of the whole cell. Some of this work will be related to recent in vitro studies, but an extensive summary of myelin biochemistry is beyond the scope of this article. This topic continues to provide many intriguing problems and has been the subject of several recent reviews (Benjamins and Smith, 1977; Benjamins and Morell, 1978; Morell, 1977; Norton, 1981b).

II Gliogenesis and Differentiation

Oligodendrocytes and astrocytes arise from cells of the subependymal layer, which are derived from the neuroectoderm. These subventricular cells give rise to immature glia that migrate to their final location and eventually become transformed into fully differentiated glia (see, e.g., Altman, 1966; Privat and Leblond, 1972). Recent autoradiographic studies of gliogenesis in developing rats have considerably clarified the kinetics of this process of generation and differentiation of oligodendrocytes (Skoff et al., 1976a,b; Imamoto et al., 1978).

Skoff and co-workers (1976a,b) showed that in the fetal rat optic nerve, before closure of the optic canal, most of the cells in active mitosis were ventricular cells. After closure (16 days of gestation), however, the majority of cells incorporating [³H]thymidine could be identified as either differentiating astroblasts or oligodendroblasts. On average, astrocytes were generated much earlier than oligodendrocytes. A few astrocytes underwent their final cell division prenatally, but most were generated during the first postnatal week. The earliest that oligodendroglia were seen to undergo their final division (considered their birthdate) was about 5 days of postnatal life, only a day or two before the start of myelination, but most were generated in the period of 7–17 days postnatally, with the peak for the time of final cell division occurring at about 10 days postnatal.

These studies showed that the immediate precursor cells for astrocytes and oligodendrocytes are morphologically distinguishable astroblasts and oligodendroblasts, rather than a common stem cell, or undifferentiated glioblast. They also showed that not only are the blast cells partially differentiated, but that neither cell type attains its mature differentiated appearance until approximately two weeks after its final cell division.

Leblond and his colleagues (Mori and Leblond, 1970; Paterson et al., 1973) had previously shown that differentiated oligodendrocytes exist in three subtypes, having light-, medium-, and dark-staining cytoplasm, respectively. They provided evidence that the light oligodendrocytes were the first to be generated from oligodendroblasts, which were then transformed progressively into medium oligodendrocytes and then into dark cells as the brain matured. Although Skoff et al. (1976a) did not address that question specifically, the newly formed oligodendroglia seen in that study resembled light oligodendrocytes. Imamoto et al. (1978) have now provided further evidence for this progression by examining autoradiographs of [³H]thymidine uptake in the corpus callosum of 20- and 26-day-old rats. At these ages there is still significant generation of new of oligodendrocytes from oligodendroblasts, as Skoff et al. (1976b) have also shown. None of the three subtypes of oligodendrocytes present at these ages took up label, indicating that they were not further dividing. After various periods label did appear in identifiable oligodendrocytes in a sequential manner. The light cells showed labeling first, and their labeling index reached a peak 7 days after injection; then the medium cells reached a labeling peak at 14–21 days, and the last to become labeled were the dark cells with a peak at around 28 days. Good precursor-product kinetic relationships were found among these three subtypes (Nadler, 1978), and they demonstrated that oligodendroblasts give rise to light oligodendrocytes, which transform to medium cells after 4–7 days. The medium cells persist about 11–18 days and finally become dark cells that apparently persist indefinitely.

There is some evidence that in the rat oligodendroglia are still being generated at >90 days, suggesting a slow turnover of neuroglia in the adult, but only 0.07–0.08% of the total oligodendroglia were heavily labeled at that age (Kaplan and Hinds, 1980).

These studies permit the construction of a chart of gliogenesis in the rat optic nerve (Fig. 1), which as been modified from those presented by Sturrock (1976), Skoff et al. (1976a), and Imamoto et al. (1978). The nature of the glioblasts that give rise to the oligodendroblasts is not known, nor is it known whether the same glioblasts can generate astroblasts. This chart illustrates (1) that differentiation of astrocytes begins prenatally, but that, in this tissue at least, differentiation of oligodendroglia is a postnatal process; (2) that oligodendroblasts, though still dividing, are partially differentiated and morphologically distinguishable from astroblasts; and (3) that light oligodendrocytes, having a large cytoplasmic volume, rich in organelles, are the first to be formed. These light cells do not divide further, but change progressively to medium and dark cells having less cytoplasm.

Fig. 1 Gliogenesis in rat brain (modified from Sturrock, 1976; Skoff et al., 1976a; and Imamoto et al., 1978.)

Similar autoradiographic studies of the kinetics of oligodendroglial proliferation have not been performed in other regions of the CNS. Since myelination begins at different times in different parts of the nervous system, presumably oligodendroglia follow suit. For example, oligodendroglia in the spinal cord must be generated several days earlier than those in the optic nerve. Thus it would not be profitable to correlate biochemical studies of myelination in the whole brain with the development of oligodendroglia in optic nerve. Fortunately there are detailed studies of myelinogenesis and oligodendroglial gene expression in the optic nerve that may be correlated with gliogenesis.

This period of rapid proliferation of interfascicular oligodendroglia during early development has been called myelination gliosis (Jacobson, 1978). Until the detailed studies of Skoff et al. (1976a,b), it was generally accepted that myelination in a particular tract did not start until this proliferation had virtually ceased (Jacobson, 1978; Friede, 1966; Schonbach et al., 1968). It is now clear, however, that when myelination begins in the rat optic nerve at 5–7 days only about 5–10% of the eventual adult complement of oligodendrocytes are present (Vaughn, 1969; Hirose and Bass, 1973; Skoff et al, 1976a,b). It is true, however, that the majority of oligodendrocytes are generated before most axons are myelinated. By 14 days of age about half the final oligodendrocyte population is present, but only about 25% of the axons are being myelinated (Skoff et al., 1976b). As previously indicated, these cells are still being produced from oligodendroblasts at 30–35 days of age (Skoff et al., 1976a,b; Imamoto et al., 1978). These data indicate that even a particular tract, like the optic nerve, is really at any one time a rather heterogeneous tissue with respect to gliogenesis and myelinogenesis. Therefore, investigators who have selected optic nerve for studies of myelination, to avoid the problems of heterogeneous development encountered in whole brain, have only partially alleviated these problems. For a period of more than 3 weeks, the nerve contains a changing mixture of proliferating oligodendroblasts, oligodendrocytes in various stages of differentiation, unmyelinated axons, axons undergoing myelination, and fully myelinated fibers. This heterogeneity obviously limits our ability to make firm conclusions associating the expression of a specific gene with a particular stage of cell differentiation in vivo. Nevertheless, some useful correlations can be made.

The studies of particular interest are those of myelin components and presumptive oligodendroglial-specific markers (see Sections IV and V for discussions) in the optic nerve during development. Developmental studies of this type in brain have a very long history, and, in fact, the composition of myelin and the enzyme properties of oligodendroglia were deduced from correlations of biochemical studies with morphological studies of myelin deposition (see Norton, 1977, for a review). The markers most commonly followed are (1) the myelin galactosphingolipids, cerebroside (galactosylceramide, GC) and its sulfate ester, sulfatide; (2) the enzymes responsible for the last step of their synthesis, UDPgalactose—ceramide galactosyltransferase (CGalT) and cerebroside sulfotransferase (CST), respectively; (3) the myelin proteins basic protein (MBP) and proteolipid protein (PLP); (4) the myelin-specific enzyme 2’:3’-cyclic-nucleotide 3’-phosphodiesterase (CNP). These are all related to myelination, and can be considered differentiated properties of oligodendroglia.

Incorporation of Na2³⁵SO4 into sulfatide can be detected in the rat optic nerve at postnatal day 4, 1 day before the birthday of the first oligodendrocyte and 2 days before myelin can be detected (Tennekoon et al., 1977). Sulfatide labeling rises rapidly beginning at day 6, peaks at about day 15, and then declines. Activities of CGalT and CST are present at birth, and begin to rise rapidly at about day 8; CGalT reaching peak activity at day 12, and CST at day 16 (Tennekoon et al., 1980). The accumulation of cerebroside and sulfatide in the nerve follows similar developmental curves with the rapid rise beginning at days 5–10 and a maximum rate of accumulation at about days 15–17 (Hirose and Bass, 1973; Tennekoon et al., 1980). Thus the curves for galactosphingolipid accumulation do not parallel those for incorporation of ³⁵SO4²− into sulfatide nor the developmental activities of either CGalT or CST.

The enzyme CNP follows a developmental curve similar to that of MBP, but it starts earlier and reaches a plateau sooner (Sprinkle et al., 1978; Tennekoon et al., 1980). CNP is detectable at birth in rat optic nerve, whereas MBP is first detected at day 6. CNP begins to rise at day 8 and reaches adult levels at day 20 (Sprinkle et al., 1978), but MBP does not rise significantly until day 10 and attains about 90% of the adult level at day 20 (Tennekoon et al., 1977). The developmental curve for PLP is delayed by about 1 day relative to MBP (Tennekoon et al., 1977). Although it is difficult to compare the shapes of these developmental curves, since they have been expressed on different bases in different papers, it does seem that the curve for accumulation of MBP (and presumably CNP and PLP as well) is quite different from those for cerebroside and sulfatide. The proteins accumulate more rapidly and reach a plateau somewhat earlier (Tennekoon et al., 1980).

Assuming that all these events are restricted to oligodendroglia and their product, myelin, we can deduce a temporal sequence of events in the developing optic nerve. The enzymes CGalT, CST, and CNP are present at birth, indicating that these differentiated functions are present in oligodendroblasts. Sulfatide synthesis is also an early event, preceding formation of oligodendrocytes and myelination. We know less about the initiation of cerebroside synthesis, but that obviously has to be active coincident with sulfatide synthesis. CGalT and CST exhibit their maximum rate of increase at 9 and 12 days, respectively, well before myelination has achieved its maximal rate (~17 days), and reach peak activities at 12 and 16 days. Thus the oligodendroblasts and young oligodendrocytes are synthesizing myelin lipids before myelination, but accumulation of these lipids, of course, occurs coincidentally with myelin production. Myelin basic protein, however, appears only when myelination begins, but then accumulates at a faster rate than the glycosphingolipids, with PLP following after a slight delay and at a little slower pace. CNP is interesting because it is expressed very early, but unlike the lipid synthesizing enzymes it is also being made and exported into the myelin sheath, so that its later developmental curve approximates that of myelin accumulation. But since it reaches a plateau earlier than MBP or the glycosphingolipids, its concentration must be higher in young myelin than in mature myelin. We will see later that this is true.

These studies indicate that the various differentiated properties of the oligodendrocyte that are directed toward myelin production do not arise simultaneously. (The exact temporal sequence is difficult to determine, even in a single homogeneous tract like optic nerve, because, as discussed earlier, it contains a heterogeneous population of cells in different stages of differentiation.) This deduction supports the general conclusion of Detering and Wells (1976) that myelin components are deposited in a sequential, nonsynchronous manner in the developing optic nerve. These investigators found that sulfatide was detected in the nerves before cerebroside and that MBP could be detected before PLP. The details of that study are probably not completely accurate because their methods were not as specific or sensitive as those now available, but similar conclusions have been reached from studies of whole brain. For example, in one of the few studies of the activity of myelin protein synthesis (rather than accumulation) during development, it was found that the synthesis of both large and small myelin basic proteins of mouse brain was maximal at about 18 days, but the activity of PLP synthesis developed much more slowly and did not peak until 4 days later (Campagnoni and Hunkeler, 1980). These results are consistent with the accumulation data of these two proteins (Tennekoon et al., 1977).

III Gene Expression in Vitro

Most of our knowledge of the biochemistry of oligodendroglia has been derived indirectly from in vivo studies of the expression of cell-specific functions, especially those related to myelinogenesis, as discussed in the preceding section. More recently these biochemical studies have been supplemented by immunocytochemical investigations (see Section V) which promise to become increasingly useful and informative. In the search for ways to examine the properties of oligodendroglia in less complex surroundings, investigators have turned to bulk-isolated cells (see Section IV) and to tissue culture systems, and, in some cases, to a combination of these approaches.

A variety of tissue culture systems are now available in which oligodendroglia thrive, although usually in combination with other cell types. In expiant and reaggregating cultures the full range of oligodendroglial gene expression may be studied, including myelinogenesis, whereas in primary dissociated cultures oligodendroglial differentiation occurs in the absence of myelin formation. The long-term goal of many investigators, to establish pure cultures of oligodendroglia which then can be shown to be competent to myelinate appropriate axons, has not yet been achieved.

A Expiants

In the expiant system extremely small slices of embryonic nervous tissue are placed in culture where they continue to develop and to maintain an organotypic appearance. It has been known for many years that these cultures myelinate, apparently on a normal developmental schedule (Bornstein and Murray, 1958; Murray, 1965). Such cultures do not solve the problem of complexity, since they contain all the cell types present in the original tissue. The advantages of this system are that myelinogenesis may be observed directly and that development may be experimentally manipulated by varying the biochemical or physical environment.

In spite of the minute quantities of material available in a single culture, several oligodendrocyte-specific properties have been studied as a function of development. These include assays of sulfatide synthesis (Silberberg et al., 1972; Fry et al., 1972, 1974), galactosphingolipid content (Latovitski and Silberberg, 1973), the activity of ceramide glycosyltransferases (Latovitski and Silberberg, 1975), and the activity of CNP (Fry et al., 1973; Pleasure and Kim, 1976). These parameters all showed good correlations with myelination in the cultures and, together with activities of various enzymes of carbohydrate metabolism (Lehrer et al., 1970a,b), developed on a schedule similar to that found in vivo at similar stages of development.

These cultures have been employed extensively for the study of demyelination and inhibition of myelination in vitro. It has been known for some time that myelinated cultures will demyelinate if treated with serum from animals with experimental allergic encephalomyelitis (EAE). If treated with EAE serum before myelin is formed, myelination is inhibited. In both inhibited and demyelinated cultures, myelination proceeds when the EAE serum is removed (Bornstein and Raine, 1970). Thus it appears that in the inhibited cultures oligodendroglial differentiation is delayed, and even after periods as long as 60 days can start up again and myelination proceed normally. The active component in the EAE serum that causes demyelination or myelin inhibition appears to be an antibody to cerebroside (galactosylceramide) (Fry et al., 1974; Raine et al., 1981).

These processes can be studied biochemically as well as morphologically. Thus EAE serum applied to cultures before they myelinated inhibited sulfatide synthesis (Fry et al., 1972) and the development of CNP (Fry et al., 1973), whereas enzymes concerned with carbohydrate metabolism were not affected. A similar inhibition of sulfatide synthesis was seen with cultures treated with cerebroside antibody (Fry et al., 1974). Sulfatide synthesis and development of CNP activity was promptly restored when the antisera were removed. If each oligodendrocyte is preprogrammed to differentiate on a certain schedule, these data imply that there is a pool of oligodendroblasts available that are programmed to differentiate sequentially over a long period. This appears to be true in situ, as we have discussed in Section II. In that case, one would predict that if the inhibition were carried out long enough, the pool of undifferentiated cells would eventually be depleted and myelination would not commence upon disinhibition of the cultures. Alternatively one could argue that the antiserum halts the cells indefinitely at a particular stage of differentiation without untoward effects.

Some information about these problems of oligodendrocyte differentiation in vitro has come from a series of studies by Silberberg and colleagues on the effects of 5-bromodeoxyuridine (BUdR) on myelination (Younkin and Silberberg, 1976; Dorfman et al., 1976; Latovitski and Silberberg, 1977). The thymidine analog, BUdR, apparently gets incorporated into DNA during cell division and is known to prevent the expression of differentiated functions in several cells types if present during certain critical mitoses prior to the time of expression. They found that myelination in expiants is delayed several days by BUdR pulses given around the time of maximum DNA synthesis, i.e., 6 days in vitro (DIV), and completely inhibited if BUdR is kept in the cultures continuously (Younkin and Silberberg, 1976). When myelination in expiants was inhibited with EAE serum until 15 DIV, when control cultures were well myelinated, the disinhibited cultures then began to myelinate after 2–5 days regardless of whether BUdR or cytosine arabinoside (a mitotic inhibitor) was present from 15 DIV (Dorfman et al., 1976). In addition, when BUdR was added to the antiserum-inhibited cultures at 5–7 DIV, and then the cultures were disinhibited, they did not form myelin at 15 DIV. These experiments indicated that the oligodendrocytes that formed myelin after disinhibition did not divide before myelination and had already undergone the necessary cell division and differentiation in the presence of EAE antiserum, even though differentiation would not be detected by the presence of markers such as CNP or sulfatide synthesis. An extension of these studies, where CGalT and CNP activities were measured in cultures treated with BUdR beginning at various DIV, showed that when BUdR is added beginning at 9–10 DIV it has no effect on myelination occurring at 16 DIV, but when added before 9 DIV partially inhibits myelination, the effect being more pronounced the earlier the age. These data imply that the oligodendrocytes that make myelin at 15 DIV have completed their last cell division at 9–10 DIV, but that some of them were fully differentiated from 1 DIV.

These BUdR inhibition experiments in vitro are difficult to interpret, and it may not be possible to formulate a unique explanation. They do suggest, however, that oligodendrocytes are continually reaching their final differentiated state over a period of about 10 days in vitro. If the end point for detecting these events were moved up from 16 DIV to a longer time, perhaps it would show that these cells are being generated over an even longer period. It does seem clear that inhibition of gene expression by the EAE or anticerebroside sera is operating quite differently from BUdR or cytosine arabinoside. The antisera inhibit myelinogenesis without inhibiting cell division or differentiation, although, apparently, differentiated properties such as sulfatide synthesis and CGalT are inhibited. This conclusion seems to be at odds with recent studies in primary dissociated cell cultures (see below) where differentiated properties of oligodendrocytes develop even though myelination does not occur. This discrepancy may be more apparent than real because one must distinguish between the activity of a differentiated property in a cell from accumulation of such markers in myelin. Sulfatide synthesis (as opposed to CST activity), and CGalT and CNP (which are both present in myelin) are all a reflection of myelin production, and their levels in an expiant culture would be expected to be related to myelin concentration. However, the serum-inhibited oligodendrocytes in that culture may well be expressing levels of these markers, and others, that are similar to those seen in oligodendrocytes in dissociated cultures that are not generating myelin.

B Reaggregating Cultures

If brains of fetal rats are dissociated and the cells put in culture flasks under constant rotation, the cells reaggregate into spherical clusters. These reaggregates continue morphological and biochemical development similar to that in vivo (Seeds, 1973), and eventually form myelin (Seeds and Vatter, 1971). Myelination in these cultures as determined biochemically appeared to start at an age corresponding to about 11–12 days postnatal, thus apparently on schedule (Sheppard et al., 1978; Matthieu et al., 1978a). However, myelin was not detected morphologically until about 26 DIV, corresponding to about 20 days postnatal. This is the only model system besides the expiant cultures in which CNS myelination can be obtained reliably in vitro.

Sheppard et al. (1978) found that CNP began to increase at about 12–15 DIV and reached rather high activity levels (6 μmol/min/mg protein) by 35 DIV. The cultures of Matthieu et al. (1978a) had levels of CNP approximately 10% as high, but followed similar developmental curves. The CST activity began to rise between 16 and 20 DIV and reached a maximum at about 24 DIV (Sheppard et al., 1978). CNP activity was present before myelination and began to rise before the rise in synthesis of sulfatide or the accumulation of MBP, thus resembling the sequence of events found in vivo (Matthieu et al., 1978a). The amount of myelin found in these cultures, however, is much less than that found in vivo at corresponding ages.

Matthieu et al. (1979) have actually isolated myelin from such cultures and found that its composition resembled immature myelin. The ratio of MBP to PLP and the amount of high-molecular-weight protein were both high, and the cerebroside content was low. An abnormally low cerebroside content was also found in myelin isolated from cerebellar expiant cultures (Bradbury and Lumsden, 1979). Thus, although oligodendroglia and myelin have a normal ultrastructural appearance in the myelinating cultures, the maturation of myelin is delayed or halted in vitro.

C Clonal Lines and Hybrid Cells

Until very recently it was not possible to obtain pure primary cultures of oligodendroglia, and even now it is difficult to obtain large quantities of such cells in culture (McCarthy and deVellis, 1980). Therefore, considerable effort has gone into the study of cell lines derived from experimentally induced gliomas and of clonal lines of these cells (reviewed by Pfeiffer et al., 1977; Benda, 1978; Pfeiffer et al., 1981a). The potential virtue of this approach may be best described by the following quotation from Pfeiffer et al. (1981a): The main attraction to the development of clonal lines of biochemically differentiated cells growing in culture resides in the possibility of providing permanently established, functionally and genetically homogeneous, viable populations of normal specified cell types which can be produced in quantities sufficient for biochemical studies. It does not appear that all of these requirements have been met by any of the many clonal lines thus far examined. The research on such cell lines is extensive and beyond the province of this chapter to summarize adequately.

Most of the research has focused on the C6 rat astrocytoma (Benda et al., 1968), cell line RN22 from a rat Schwannoma (Pfeiffer and Wechsler, 1972), and cell lines derived from mouse glioma G26 induced by methylcholanthrene (Sundarraj et al., 1975). Many of these lines express CNP (Zanetta et al., 1972; Pfeiffer and Wechsler, 1972; Sundarraj et al., 1975) and cerebroside and sulfatide synthesis (Dawson et al., 1977; Dawson, 1979), but none appear to contain significant levels of MBP (Pfeiffer et al., 1977, 1981a). Thus, so far there are no cell lines available which express more than a few of the known differentiated markers of oligodendroglia (Pfeiffer et al., 1977; Benda, 1978). Moreover, the most-studied cell line, C6, has been shown to have properties characteristic of both oligodendroglia and astroglia (Pfeiffer et al., 1977). These lines have been useful for the studies of hormonal control of enzyme induction (Dawson, 1979; Dawson and Kernes, 1978, 1979; McGinnis and deVellis, 1974; McMorris, 1977).

An approach closely related to the study of glioma lines, but one that may have more promise, is the production of cell hybrids between normal oligodendroglia and glioma cells (McMorris et al., 1981). Bulk-isolated bovine oligodendrocytes were fused with C6 glioma clones that lacked either thymidine kinase (TK) or hypoxanthine phosphoribosyltransferase (HPRT). Hybrid cells were selected for by culture in HAT selective medium. They were cloned and their biochemical properties studied. The most promising hybrids were generated from C6(TK−) cells. One of these, hybrid CO-13-7, expressed all six of the oligodendroglial properties tested: CNP, glycerol-3-phosphate dehydrogenase (GPDH), induction of GPDH by hydrocortisone, cerebroside, sulfatide, and MBP. The CNP level was about half that in bovine cells but much higher than in C6 cells; GPDH was higher in the hybrid than in either precursor cell and was increased two- to fivefold by hydrocortisone. Cerebroside and sulfatide levels in CO-13–7 hybrids were 15–20% of those in bovine oligodendrocytes; whereas the C6(TK−) cells had no sulfatide and barely detectable levels of cerebroside. The basic protein level in CO-13-7 cells was 120 ng/mg protein vs 0 in C6(TK−) cells and 75 in isolated oligodendrocytes. This hybrid is the first cell line that expresses such a wide range of oligodendroglial-specific properties.

D Primary Cultures

Cells dissociated from newborn rat or mouse brain and put into culture eventually form a confluent layer of flat cells that consist of 80–85% astrocytes (Booher and Sensenbrenner, 1972). Many investigators have noted that there are small round cells with thin processes growing on top of the astrocytes, which prove to be oligodendroglia (Raff et al., 1978; Labourdette et al., 1979; McCarthy and deVellis, 1980). The properties of these cultures vary depending on whether the cells are taken from newborn or embryonic brain. Both systems have been more useful for the study of oligodendroglial differentiation than the other culture systems described here. The results obtained with the neonatal cultures will be described first.

Several attempts have been made to find conditions to maximize the number of oligodendrocytes in the cultures and minimize the number of astrocytes. Labourdette et al. (1979, 1980) obtained cultures that had 40–60% oligodendroglia by using calf serum instead of fetal calf serum in the medium and plating at high density. Although no mitogens have yet been found for oligodendrocytes (Pruss et al., 1982), numbers of oligodendroglia may be increased in primary dissociated cultures by adding buffer-soluble extracts of whole adult brain (Pettmann et al., 1980). Pruss et al. (1982) have also found that if the oligodendroglia that are sometimes found in suspension in these cultures are replated on monolayers of irradiated 3T3 cells, they spread out processes and undergo some cell division.

The most successful attempt to obtain enriched cultures of oligodendroglia is that of McCarthy and deVellis (1980) who have found that the oligodendroglia are more loosely attached to the astrocytes than the astrocytes are to the substrate. When the cultures are shaken vigorously, the oligodendroglia come free in suspension and can be removed and cultured separately. A few astrocytes contaminate these subcultures and multiply, whereas the oligodendroglia do not, but by repeating the shaking and subculture process several times relatively pure cultures of oligodendroglia can be obtained. The original cultures, from which the oligodendrocytes have been removed, continue to develop as essentially pure astrocyte cultures. Thus both glial types can be obtained from newborn brain in nearly homogeneous cultures.

The oligodendrocytes in the primary cultures have a distinctive morphology and can usually be easily identified (see Fig. 4). However, in all of the studies discussed above they have been identified by some positive immunochemical marker; either galactocerebroside (Raff et al., 1978; Pruss et al., 1982), myelin basic protein (Pruss et al., 1982), Wolfgram Wl protein (Labourdette et al., 1979, 1980), carbonic anhydrase II (Pettmann et al., 1980), or glycerol phosphate dehydrogenase (McCarthy and deVellis, 1980). Neurons fail to survive in these cultures from neonatal rodents; thus there are no axons for the oligodendroglia to myelinate. The presence of these markers, however, shows that the oligodendroglia differentiate and that the production of myelin-specific components is dissociated from the process of myelination. This phenomenon is not observed in cultures of Schwann cells, which stop making myelin-specific molecules in cultures where myelination is not proceeding (Mirsky et al., 1980).

Fig. 4 Phase-contrast light micrographs of oligodendrocytes isolated from adult bovine white matter by the method of Farooq et al. (1981) and kept in tissue culture for 4 weeks. These cultures have been treated with cytosine arabinoside to suppress growth of contaminating cells. Note the homogeneity of the cultures and the extensive network of processes. All cells of the morphology illustrated are positive for galactosylceramide and myelin basic protein. A, ×260; B, ×520.

The timing of the differentiation process in the mixed primary glial cultures has been studied by several groups by both biochemical assay and immunocytochemical methods. While the results are generally consistent from one group to another, they vary considerably in detail. Mirsky et al. (1980) reported that in cultures of newborn rat optic nerve there were some galactocerebroside-positive (GC+) and sulfatide-positive cells at 1 day in vitro, but no myelin basic protein-positive (MBP+) cells until 5 DIV. By 10 days all GC+ cells were also MBP+. In their cultures 2–5% of the total cells were oligodendroglia, and the amount did not change over an 8-week period. Bologa-Sandru et al. (1981b) also found that GC+ cells were present (7 DIV) before MBP+ cells (14 DIV), but that even after 27 DIV there were still only 3 MBP+ cells for every 100 GC+ cells. The GC+ cells increased 10-fold between 7 and 14 DIV, and then leveled off, but the numbers of MBP+ cells increased continually over a 28-day period. Two studies by Pfeiffer and colleagues (Barbarese and Pfeiffer, 1981; Pfeiffer et al., 1981b) gave different results for the timing of MBP expression. The difference appeared to be a function of culture technique. In the first study (one of the few to combine quantitative assay of MBP with counts of MBP+ cells) both the amount of MBP and the number of MBP+ cells increased 20-fold between 15 and 34 DIV and then decreased (Barbarese and Pfeiffer, 1981). MBP+ cells were detected at 10 DIV, but their numbers did not increase rapidly until 20 DIV. They reported, as do others, that not all GC+ cells were MBP+. The MBP+ cells were about 0.1% of the total cells at 15 DIV and 2% at 34 DIV. Thus at this time most, if not all, of the oligodendroglia in the culture must have contained MBP. These data indicate that the increase of MBP in the culture is not an increase in the amount of MBP per oligodendrocyte, but rather reflects an increase in the number of oligodendrocytes which have differentiated to the point of expressing MBP. In a subsequent paper (Pfeiffer et al., 1981b) they found that the rapid accumulation of MBP began a week earlier but then reached a maximum at about the same DIV as they had reported previously. Sulfatide synthetic rate and CNP accumulation both began to increase at 8 DIV and reached a maximum at 20 DIV, much earlier than MBP. In similar cultures the Wl Wolfgram protein was found to be absent at 6 DIV, a few positive cells were present at 8 DIV, about half the small cells were Wolfgram-positive at 16 DIV, and by 4 weeks all cells with an oligodendroglial morphology were positive for both Wolfgram antigen and MBP (Roussel et al.,