Myelination of axons within cytosine arabinoside treated mouse cerebellar explants by cultured rat oligodendrocytes

Brain Research, 503 (1989) 111-117

111

Elsevier

BRES 14999

Myelination of axons within cytosine arabinoside treated mouse cerebellar explants by cultured rat oligodendrocytes F r e d r i c k J. Seil ~, M a r i l y n L . J o h n s o n 2, R u s s e l l P. S a n e t o 3, R o b e r t M . H e r n d o n 4 a n d Michele K. Mass 5 I'ZNeurology Research, Veterans Administration Medical Center and Departments of 1'2"4'SNeurologyand 3Cell Biology and Anatomy, Oregon Health Sciences University, Portland, OR 97201; 3Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, OR 97006; and 4NeurologicalSciences Center, Good Samaritan Hospital and Medical Center, Portland, OR 97210 (U.S.A.)

(Accepted 2 May 1989) Key words: Cerebellar explant culture; Purified oligodendrocyte;Myelination; Myelin-like membrane; Myelin spherule; Transplantation;

Cytosine arabinoside

Ceil suspensions of cultured purified rat oligodendroeytesprepared by the differential substrate adhesion method were applied to neonatal mouse cerebellar explant cultures in which myelination and oligodendrocytematuration had been irreversibly inhibited by exposure to cytosine arabinoside. Myelination of Purkinje cell axons within 92% of the host explants was observed 2-5 days after oligodendrocyte application. Ultrastrueturaily, mature oligodendroeytes and axons surrounded by compact myelin, as well as spherules of compact myelin membranes without axons, were present within the cerebellar explants. It is evident that cultured dissociated purified oligodendrocytes retain the ability to myelinate appropriate axons. Such oligodendrocytesmay be hyperreactive with regard to myelin membrane formation, as suggested by the presence of spheres of compact myelin without axons. INTRODUCTION The ability of transplanted oligodendrocytes to myelinate host axons has been demonstrated in many studies, including the grafting of tissue 7'26 or freshly dissociated cell suspensions ~° o~ cultured glial cells6 into an in vivo host, and the insertion of tissue fragments 23'39or cultured tissue ts or cells n'31 into an in vitro system containing myelin receptive axons. An example of the latter is a culture system we described in which the oligodendrocyte population was markedly reduced and oligodendrocyte maturation and myelination were !rreversibly inhibited by exposure of cerebellar explants derived from newborn mice to cytosine arabinoside (Ara-C) for the first 5 days in vitro (DIV), as determined by both light 22 and electron microscopic3 studies. When such cultures were superimposed at 9 or 16 DIV with:,.alouse cerebellar cultures that had been exposed to kainic acid for the first 5 DIV to destroy all neurons with myelin receptive axons, while leaving glia intact ~9, 78% of the host Ara-C treated explants became myelinated between 3-5 days after transplantation 2'18'21. Such myelin was easily visible by light microscopic observation in the living state, and appeared almost exclusively within the host explants, generally between cortical and intracerebellar nucleus

areas of the cultures, where Purkinje cell axons were concentrated. The same Ara-C treated mouse cerebellar explant system was used by Nishimura et al. 12 to test the myelinating capability of cultured purified rat oligodendrocytes prepared by the differential substrate adhesion method of McCarthy and de Vellis 1~. After maintenance in culture for 13-14 DIV, the rat oligodendrocytes were superimposed in the form of pellets onto the host mouse Ara-C exposed cerebellar explants. A few myelinated axons were observed by light microscopy in only 2 of 20 transplanted cultures. All of the cultures examined ultrastructurally had myelinated fibers, but these were found among the aggregates of transplanted oligodendroglia overlying the host cerebellar cultures, i.e., involving axons which had grown out of the explants, and only rarely within the cerebellar explants. While demonstrating that purified cultured oligodendrocytes prepared by the McCarthy and de Vellis method are capable of myelination and that rat oligodendroglia can myelinate mouse axons in vitro, some interesting questions were raised by this study because of the relative paucity of myelin and the apparent lack of myelination of the abundant axons within the host explants. Although cross-species myelination in vitro (lamb oligodendrocytes

Correspondence: EJ. Seil, Neurology Research (15IN), VA Medical Center, Portland, OR 97201, U.S.A.

112 and rat axons) had been reported previously 3°, might the xenografting have reduced the efficiency of myelination? Might only a small proportion of the oligodendrocytes that survived the dissociation p r o c e d u r e have been able to myelinate available axons? Might the oligodendrocyte preparation p r o c e d u r e or the application to host cultures in the form o f pellets have c o m p r o m i s e d the dem-. onstrated j'-'8 ability of transplanted oligodendroglia to migrate? Was the purity of the cultured oligodendrocytes a factor, i.e., could greater success be achieved by a mixed glial population with regard to migration a n d / o r penetration o f the host explant and subsequent myelination? To address these questions, we transplanted m o u s e A r a - C treated cerebellar explants with three different rat derived preparations for comparison with our previous results 2"m~'2~ with m o u s e - m o u s e in vitro transplants. T h e preparations include: (1) rat cerebellar explants exposed to kainic acid for the first 5 D I V ; (2) rat dissociated o!igodendrocyte cultures that were 94% pure (6% astro,'ytes); and (3) rat dissociated oligodendrocyte cultures ttlat were 99% pure. We also applied our cultured oligodendrocytes as cell suspensions to the host cerebelfar explants, rather than as pellets. This report describes our results with these various modifications. MATERIALS AND METHODS

Culture preparation Newborn Swiss-Webster mouse and Sprague-Dawley rat ccrebellar cultures were prepared as described previously 4'~'. Parasaginally oriented explants 0.5-1.0 mm thick were placed on collagen co'~ted glass coverslips with a drop of nutrient medium, sealed in Maximow chambers, and incubated at 35.5-36.0 °C in the lying-drop position. Ara-C was incorporated into the nutrient medium of some mouse ccrcbcllar cultures at a concentration of 5.0-7.5/~g/ml for the first 5 DIV, after which they were maintained in normal medium. L-Aminoadipic acid (2-3 l0 -4 M) was incorporated along with Ara-C for application to some of the cultures in an attempt to reduce the number of astrocytes in the mouse explants X. Kainic acid, 10-4 M, was included in the nutrient medium for rat cultures during the first 5 DIV, following which they also were maintained in normal medium. The normal nutrient medium, which was exchanged twice weekly, consisted of 2 parts 3 IU/ml low-zinc insulin (courtesy of Squibb Institute for Medical Research), I part 20% dextrose, 8 parts Eagle's minimum essential medium with Hanks' base and added t.-glutamine, 7 parts Simms' X-7 balanced salt solution (BSS) with sufficient incorporated HEPES buffer to make its concentration 10--" M in the fully constituted medium, and 12 parts fetal calf serum. Purified, dissociated oligodendroglial cultures were prepared from neonatal Spraguc-Dawley rats by a modification I~ of the method of McCarthy and de Vellis ~. in brief, brain tissue was mechanically dissociated by passage through a series of meshes and sieves, followed by plating in 75 cm plastic tissue culture flasks with 50% Dulbccco's minimum essential medium (DMEM) with high glucose (450 mg%)/50% Ham's FI2 medium buffered with bicarbonate and HEPES (I.5 x 10--~ M) buffer. After 7-9 days of incubation at 37 °C in a moist 5% CO,, 95% air atmosphere (with medium changes cvery 48 h after the initial 12 h of culture), the cells were placed in DMEM/FI2 medium containing 10% fetal calf serum

and agitated on an orbital shaker at 250 rpm (1.5 inch stroke diameter) at 36 °C for 18-24 h. Cells suspended in the medium were passed through a sieve (Cellector #500) and plated onto plastic culture flasks. After 72 h, the medium was exchanged and the flasks were hand shaken at least twice, each time followed by passage through a sieve. The cells were centrifuged into a pellet and resuspended in fresh medium. The oligodendrocytes were maintained in tissue culture flasks for 16-34 DIV, after which they were collected, centrifuged, and resuspended in medium used for explants at concentrations ranging from 16 x 10~ to 20 x 106 cells/ml medium.

Transplantation procedures After 9 DIV, rat cerebellar cultures exposed to 10 -4 M kainic acid for the first 5 DIV were dissected from their collagen substrates and superimposed upon 9 DIV mouse cerebellar cultures treated with Ara-C for the first 5 DIV. Sucit cultures were observed for myclination in the living state until 16 or 20 DIV, after which they were fixed for thionine or Holmes staining, or for electron microscopy. Rat oligodendrocyte suspensions were applied to 9 DIV mouse cerebellar explants treated with Ara-C and observed for myelination until 14-23 DIV, followed by fixation for thionine, Holmes or luxoi fast blue/cresyl violet (Kliiver-Barrera) staining, for immunocytochemical reaction with antibodies to glial fibdllary acidic protein (GFAP), galactocerebroside (GC), and myelin basic protein (MBP), or for processing for ultrastructural examination. Purity of the oligodeudrocyte cultures was determined by observation in the living state with phase contrast optics, and by counts of GFAP and GC stained cells from samples taken at the time of application of cell suspensions to explant cultures.

Morphological and immunocytochemical methods Cultures destined for luxol fast blue/cresyl violet staining for myelin or for a modified Holmes silver stain 27 were fixed as whole mount preparations in 10% formalin in buffered BSS, while cultures for thionine staining for Nissl substance were fixed in 80% ethanol 2°. Cultures for immunocytochemistry were fixed in 10% formalin in phosphate buffered saline, incubated at room temperature with 0.2% Triton X-100 (except for cultures reacted with antibody to MBP), ,'lnd exposed to polyclonal antibodies to either GFAP (courtesy of Dr. Lawrence F. Eng) or GC (courtesy of Dr. Jean de Vellis), or to a monoclonal antibody to human MBP (Interferon Sciences, Inc.). After rinsing with phosphate buffered saline, the cultures were incubated with a secondary antibody (anti-lgG) and subsequently processed by the peroxidase-antiperoxidase method 24. Cultures for ultrastructural examination were fixed in cold cacodylate or phosphate buffered glutaraldehyde (1.5%)/paraformaldehyde (1.5%), postfixcd in 2% osmium tetroxide in cacodylate or phosphate buffer, deaydrated in a series of cold graded ethanol and two changes of propylene oxide, and polymerized in Epon, simii,,r to previously described methods 3. Thick sections were stained with methylene blue for localization of myelin,~ted fibers within the explants. Thin sections were stained with uranyl acetate and lead citrate and examined with a JEOL electron microscope.

RESULTS Eighteen o f 21 m o u s e cerebellar A r a - C treated cultures (86%) myelinated after transplantation with rat cerebellar explants exposed to kainic acid. T h e myelin was visible by light microscopy, a p p e a r e d within the host explants, and was as a b u n d a n t as myelination in mousemouse transplants in vitro in o u r previous studies 18"2t. Mouse cerebellar explants e x p o s e d to A r a - C also myelinated very well following the application o f cell

113 suspensions of rat oligodendrocytes from either the enriched (94% pure) group <~r the purified (99% pure) group. Twenty-nine ot 34 explants (85%) overlain with 94% pure oligodendrocytes became myelinated. This group of explants included those exposed to Ara-C plus L-aminoadipic acid, which did not induce a significant reduction of astrocytes in the cultures, as determined by GFAP staining of non-transplanted explants. Thirty-two of 32 mouse cerebellar cultures (100%) to which 99% pure rat oligodendrocyte suspensions were added became myelinated. The combined total of the two groups receiving oligodendrocyte suspensions is 61 of 66 myelinated (92%). Myelin was slightly more abundant in the cultures overlain with 99% pure oligodendrocytes, but the amount of myelin in both groups was comparable to that observed in our earlier studies with mouse-mouse in vitro transplants 18"2~. Myelination was not as profuse in Ara-C treated cultures transplanted with rat or mouse oligodendrocytes from any source, however, as in normal, untreated cultures. The appearance of myelin in cerebellar explants was noted as early as 2 days after application of oligodendrocyte suspensions, but most preparations became myelinated 3-5 days after oligodendrocyte addition. Long segments of myelinated axons were easily observed

Fig. 1. Myelinated axons in a 14 DIV Ara-C treated mouse cerebellar explant that had been transplanted at 9 DIV with 6 x 10~ 23 DIV rat oligodendrecytes (99% pure) per ml medium. Photographed in the living state with brightfield optics, x570. B

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Fig. 2. Ara-C treated mouse cerebellar explant, 15 DIV, showing cortical (C) and intracerebellar nucleus (N) regions. The culture had been transplanted at 9 DIV with purified rat oligodendrocytes cultured for 23 DIV, as in Fig. 1. The arrow points to the region between cortex and intracerebellar nucleus shown at higher magnification in the inset, which illustrates myelinated Purkinje cell axons. Luxoi fast blue/cresyl violet stain, x60; inset, x615.

114 JO

Fig. 3. Clusters of purified rat oligodendrocytes in the outgrowth zones of two 15 DIV Ara-C treated mouse cerebellar cultures that had been transplanted as in Fig. 1. Cells in the clusters stain positively with antibodies to galactocerebroside (a) and myelin basic protein (b). Peroxidase-antiperoxidase method, x310.

by light microscopy of living cultures (Fig. 1), and were concentrated within the explants, especially in the area between the cortex and intracerebellar nuclei of the host cultures (Fig. 2). This area is packed with axons, most of Purkinje cell origin, and is comparable to the in vivo

white matter zone 1~. Non-transplanted Ara-C treated cerebellar cultures from both groups observed for equal periods of time failed to show myelination. Most of the surviving oligodendrocytes applied to the cerebellar cultures were present as isolated cells or small

Fig. 4. This electron micrograph from a 15 DIV Ara-C treated cerebellar culture transplanted at 9 DIV with 20 x 106 16 DIV rat oligodendrocytes (99% pure) per ml medium illustrates the appearance of a typical mature oligodendrocyte (Ol) which has migrated into the culture and is surrounded by neuropil. Two myelinated axons (a) are seen at the lower right, x7000. The inset shows a section of a myelinated fiber cut longitudinally, with paranodal loops seen at the right. × 11,000.

115

Fig. 5. Three myelinated axons (a) and a myelin spherule (s) cut in cross-section are seen in this micrograph of a 15 DIV Ara-C treated cerebellar culture transplanted with oligodendrocytes as described in Fig. 4. x l 1,700.

aggregate groups of cells in the outgrowth zone, well away from the explants (Fig. 3). These cells were positive for both GC (Fig. 3a) and MBP (Fig. 3b). Myelin was infrequently observed in the outgrowth zone, as is also the case with normal cerebellar cultures ~7. Ultrastructural examination revealed the presence of mature oligodendrocytes (Fig. 4) and myelinated fibers with occasional nodes of Ranvier (Figs. 4, 5) well below the surface of Ara-C treated mouse cerebellar explants to which rat oligodendrocyte suspensions had been added. The myelin was compact and demonstrated normal periodicity. There wel/e no evident differences between this myelin and that observed in mouse cerebeilar cultures exposed to Ara-C and subsequently transplanted with mouse cerebellar cultures exposed to kainic acid z. Also noted within the explants was the presence of spherules of compact myelin without axons (Fig. 5), similar to the myelin-like' membranes reported in cultures

of isolated oligodendrocytes5,9't3-L~'2"~. These structures were interpreted as spherules because they always appeared as cross-sections, and never as longitudinal sections, whereas myelinated axons appeared in both cross-sectional and longitudinal planes (Figs. 4, 5). Such empty myelin spherules occurred frequently in the explants, in the neighborhood of both myelinated and unmyelinated axons. While the thickness of the membranes was variable, the numbers of wraps of the thickest of the spherules was comparable to the thickness of myelin sheaths around axons (Fig. 5). DISCUSSION

The abundant myelination of Ara-C treated host mouse cerebellar explants by rat cerebellar cultures exposed to kainic acid dispelled any notion that xenografting might compromise myelination in vitro. The

116 extent of myelination within the host explants by enriched or purified dissociated cell cultures of oligodendrocytes indicated that such oligodendrocytes retained the reported capacity of transplanted oligodendrocytes to migrate L28, and that inclusion of astrocytes in the population of cells applied ~o the host explants offered no advantage. As most of the oligodendrocytes applied to cerel~ellar cultures settled in the outgrowth zone, the myelination of axons within the explants was accomplished by only a fraction of the applied cells, suggesting that more than a small proportion of the oligodendrocytes that survive the dissociation procedure are capable of myelination. The myelin formed by such oligodendroglia was morphologically normal by light and electron microscopic criteria. Thus we can conclude from our results that cultured dissociated purified oligodendrocytes prepared by the differential substrate adhesion method of McCarthy and de Veilis ~l retain the ability to migrate and to myelinate appropriate axons. The differences between our results and those of Nishimura et al. iz may well be due to application of the oligodendrocytes to the host explants as cell suspensions rather than as pellets. It is quite possible that oligodendrocytes compacted into a pellet are not able to migrate away, as suggested by the appearance of myelin around axons within pellets, and only rarely within explants, in the study of Nishimura et al. There appeared to be a boundary between explants and pellets in their preparations, with axons within the oligodendrocyte pellets representing fibers grown out from the eerebellar explants. There was evidently little or no penetration of their explants by applied oligodendroeytes, in contrast to our preparations, in which mature oligodendrocytes were present within the host explants. Fast s),tdies of transplantatiol: of Ara-C treated cerebellar cultures with oligodendrocytes from different tissue sources suggested that the myelin within the host explants was formed by oligodendrocytes originating from the transplanted tissue, rather than by residual host oligodendrocytes whose maturation was induced by factors secreted by the added tissue. Evidence for this includes (1) the myelination of host Ara-C treated cultures only when directly superimposed with kainate exposed explants, and not when the two explants were placed adjacent to each other zS, and (2) the formation of shiverer myelin around normal axons when Ara-C treated cultures derived from normal mice were transplanted with optic nerve fragments from shiverer mutant micez~. This concept is further supported by the lack of myelin in the host Ara-C treated explants of Nishimura et ai. ~2, which were overlain with pellets of mature oligodendrocytes but not invaded by applied cells, compared with the presence of mature oligodendrocytes and

myelin in our host cultures transplanted with cell suspensions of oligodendrocytes. The expression of GC and MBP by applied oligodendrocytes which had settled in the outgrowth zones of our cultures, where the density of myelin receptive axons is considerably reduced, is not surprising in view of previous reports ~f GC and MBP expression by isolated oligodendrocytes cultured without axons 9a6. It appears that these maturational steps are intrinsic to oligodendroglia, and do not require induction by axons. Of especial interest in the present study is the formation of empty spherules of myelin membranes within the cerebellar explants, in addition to the expected axonal ensheathment. Previous reports of such membranes in cultures of isolated oligodendrocytes5'9aa-15`z5 have led to speculations that formation of myelin-like membranes may also be an intrinsic property of oligodendrocytes. However, we have not observed empty myelin spherules in normal cerebellar explants 17, nor in Ara-C treated cerebellar cultures transplanted with cerebellar explants exposed to kainic acid 2. The only neurons present in kainate treated cerebellar cultures were granule cells, whose axons did not myelinate, and the only myelin present was in the form of infreqaent myelinated granuie cell somata w. Oligodendrocytes irora such preparations, when superimposed upon Ara-C treated cerebellar cultures, myelinated axons in the host explants, plus a more than usual number of granule cell somata and occasional dendrites 2. While appearing somewhat hyperreactive, the oligodendroeytes did not form myelin membranes in the absence of apposed neuronal surfaces. The appearance of empty spherules of myelin membranes in an identical host cerebellar explant system transplanted with cultured isolated oligodendrocytes suggests that such myelin formation may represent a more extreme form of oligodendrocyte hyperreactivity that is induced by the conditions of dissociated cell culture. Evidently this acquired hyperreactivity is not entirely reversed upon exposure of the previously isolated oligodendrocytes to myelin receptive axons. Formation of myelin or myelin-like membranes may indeed be a property intrinsic to oligodendrocytes, but is probably expressed only under special circumstances, and not in conditions in which normal axon-glia interactions prevail. We had suggested a number of years ago that the Ara-C treated cerebellar explant model might be useful to test the myelinating capacity of dissociated oligodendrocytes, especially since it allowed presentation of central nervous system axons to the oligodendroglia ~s. The results of Nishimura et al. 12 raised some doubts about the feasibility of this bioassay system, as ultrastructural analysis seemed to be required to demonstrate the

117 myelin. If the dissociated cells are applied as cell suspensions, however, rather than as pellets, the results are easily readable by light m i c r o s c o p y within a few days after application o f the cells, thus confirming o u r original impression o f the potential value o f this culture system as an assay for myelination.

Acknowledgements. This work was supported by the Veterans Administration (EJ.S.), the Medical Research Foundation of Oregon (R.P.S.), and by NIH Grants NS 17493 (EJ.S.), 1R01PK41035-01 (R.P.S.), 5P01 HD-0998812 (R.P.S.) and NS 21759 (R.M.H.). We are indebted to Daniel R. Austin and Beth Anne Healey for technical assistance.

REFERENCES

(1986) 49-65. 16 Saneto, R.P. and de Vellis, J., Characterization of cultured rat oliggdendrocytes proliferating in a serum-free, chemically defil~e~i medium, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3509-3513. 17 Seil, EJ., Cerebellum in tissue culture. In D.M. Schneider (Ed.), Reviews ofNeuroscience, Vol. 4, Raven, New York, 1979, pp. 105-177. 18 Seil, EJ. and Blank, N.K., Myelination of cerebral nervou~ system axons in tissue culture by transplanted oligodendrocytes, Science, 212 (1981) 1407-1408. 19 Seil, EJ., Blank, N.K. and Leiman, A.L., Toxic effects of kainie acid on mouse cerebellum in tissue culture, Brain Research, 161 (1979) 253-265. 20 Seii, EJ. and Herndon, R.M., Cerebellar granule cells in vitro: a light and electron microscopic study, J. Cell Biol., 45 (1970) 212-220. 21 Seil, EJ., Leiman, A.L. and Blank, N.K., Reorganization in granuioprival cerebellar cultures after transplantation of granule cells and glia. I. Light microscopic and electrophysiological studies, J. Comp. Neurol., 214 (1983) 258-266. 22 Seil, EJ., Leiman, A.L. and Woodward, W.R., Cytosine arabinoside effects on developing cerebellum in tissue culture, Brain Research, 186 (1980) 393-408. 23 Stanhope, G.B., Wolf, M.K. and Billings-Gagliardi, S., Genotype-specific myelin formation around normal axons in cytosine arabinoside-treated organotypic cultures injected with normal or shiverer optic nerves, Dev. Brain Res., 24 (1986) 109-116. 24 Steraberger, L.A., Hardy, P.H., Cuculis, J.J. and Meyer, H.G., The unlabeled antibody enzyme method of immunohistochemistry, J. Histochem. Cytochem., 18 (1970) 315-333. 25 Szuchet, S., Polak, P.E. and Yim, S.H., Mature oligodendrocytes cultured in the absence of neurons recapitulate the ontogenic development of myelin membranes, Dev. Neurosci.. 8 (1986) 2(18-221. 26 Weinberg, E.L. and Spencer, P.S., Studies on the control of myelinogenesis. 3. Signalling of oligodendroeyte myelination by regenerating peripheral axons, Brain Research, 162 (1979) 273-279. 27 Wolf, M.K., Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum, J. Cell Biol., 22 (1964) 259-279. 28 Wolf, M.K., Brandenburg, M.C. and Billings-Gagliardi, S., Migration and myelination by adult gliai cells: reconstructive analysis of tissue culture experiments, J. Neurosci., 6 (1986) 3731-3738. 29 Wood, P., Okada, E. and Bunge, R., The use of networks of dissociated rat dorsal root ganglion neurons to induce myelination by oligodendrocytes in culture, Brain Research, 196 (1980) 247-252. 30 Wood, P., Szuchet, S., Williams, A.K., Bunge, R.P. and Arnason, B.G.W., CNS myelin formation in co-cultures of rat neurons and lamb oligodendrocytes, Am. Soc. Neurochem. Abstr., AI4 (1983) 212. 31 Wood, P.M. and Williams, A.K., Oiigodendrocyte proliferation and CNS myelination in cultures containing dissociated embryonic neuroglia and dorsal root ganglia neurons, Dev. Brain Res., 12 (1984) 225-241.

1 Baulac, M., Lachapelle, E, Gout, O., Berger, B., Baumann, N. and Gumpel, M., Transplantation of oligodendrocytes in the newborn mouse brain: extension of myelination by transplanted cells. Anatomical study, Brain Research, 420 (1987) 39-47. 2 Blank, N.K. and Seii, EJ., Reorganization in granuloprival cerebellar cultures after transplantation of granule cells and glia. II. Uitrastructural studies, J. Cutup. Neurol., 214 (1983) 267278. 3 Blank, N.K., Seil, EJ. and Herndon, R.M., An ultrastructural study of cortical remodeling in cytosine arabinoside induced granuloprival cerebellum in tissue culture, Neuroscience, 7 (1982) 1509-1531. 4 Bornstein, M.B. and Murray, M.R., Serial observations on patterns of growth, myelin formation, maintenance and degeneration in cultures of newborn rat and kitten cerebellum, J. Biophys. Biochem. Cytol., 4 (1958) 499-5134. 5 Bradel, E.J. and Prince, EP., Cultured neonatal rat oligodendrocytes elaborate myelin membrane in the absence of neurons, J. Neurosci. Res., 9 (1953) 38!-392. 6 Doering, L.C. and Fedoroff, S.~ Isolation and transplantation of oligodendrocyte precursor cells, J. Neuroi. Sci., 63 (1984) 183-196. 7 Gumpel, M., Baumann, N., Raoul, M. and Jaeque, C., Survival and differentiation of oligodendroeytes from neural tissue transplanted into newborn mouse brain, Neurosci. Lett,, 37 (1983) 307-312. 8 Huck, S., Grass, E and HaRem M.E., Gliotoxie effects of a-aminoadipie acid on monolayer cultures of dissociated postnatal mouse cerebellum, Neuroscience. 12 (1984) 783-791. 9 Knapp, I.E., Bartlett, W.P. and Skoff, R.P., Cultured oligodendrocytes mimic in vivo phenotypic characteristics: cell shape, expression of myelin-specific antigens, and membrane production, Dev. Biol., 120 (1987) 356-365. 10 Kohsaka, S., Yoshida, K., Inoue, Y., Shinozaki, T., Takayama, H., Inoue, H., Mikoshiba, K., Takamatsu, K., Otani, M. and Tsukada, Y., Transplantation of bulk-separated oligodendrocytes into the brains of shiverer mutant mice: immunohistochemical and electron microscopic studies on the myelination, Brain Research, 372 (1986) 137-!42. 11 McCarthy, K.D. and de Vellis, J., Preparation of separate astroglial and oligudendroglial cell cultures from rat ~.erebral tissue, J. Cell. BioL, 85 (1980) 890-902. 12 Nishimura, R.N., Blank, N.K., Tiekotter, K.L., Cole, R. and de Vellis, J., Myelination of mouse cerebellar explants by rat cultured oligodendrocytes, Brain Research, 337 (1985) 159-162. 13 Nussbaum, J.L., Espinosa de los Monteros, A., Pari, EM., Doerr-Schott, J., Roussel, G. and Neskovic, N.M., A morphological and biochemical study of the myelin-like membrane structures formed in cultures of pure oligodendrocytes, Int. J. Dev. Neurosci., 6 (1988) 395-408. 14 Poduslo, S.E., Miller, K. and Wolinsky, J.S., The production of a membrane by purified oligodendroglia maintained in culture, Exp. Cell Res., 137 (1982) 203-215. 15 Rome, L.H., Bullock, P.N., Chiapelli, E, Cardwell, M., Adinolfi, A.M. and Swanson, D., Synthesis of a myelin-like membrane by oligodendrocytes in culture, J. Neurosci. Res., 15