Immunohistochemical study with an anti-myelin serum a marker for all glial cells except ‘dark’ oligodendrocytes

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2o9

Immunohistochemical Study with an Anti-myelin S~,.rum A Marker for All Gfial Cells except 'Dark' Oligo,dendrocytes G u y Roussel a n d Jean-Louis N u s s b a u m * Centre de Neurochimie du CNRS, and Unit~ 44 de r l N S E R M 5, rue Blaise Pasca~ 6~ (~84Strasbourg C~lex (France)

(Received14 December,1982) (Revised.received10 May, 1983) (Accepted30 May, 1983)

SeN.~ The usefulness of an anti-myefin antiserum as a possible marker for glial cells and related structures was investisated using rat brain. As expected, the myelin fibers were heavily stained but the neuronal cells and their processes were unreacfive. The ofigodendrocytes, identified on electron microscopy, revealed labelling of only the. light ~nd medium types, but not the dark cells. These results indicate that the su88ested morpholosical classi ficat~on of oligodendrocytes may be based on varying amounts of myelin antisen synthesis. Astrocytes from all areas, Golsi epitheli~ cells, Bersmann fibers and some subependymal cells also reacted with this anti-myelin antiserum but the staining was abolished completely by preabsorption with kidney powder, In contrast, the myelin fibers and the light and medium oligodendrocytes could still be labelled. We conclude that this anti-myelin antiserum should prove useful in studies of oligodendrocytes in the central nervous system. Key words: A n t i . m y e l i n s e r u m - A s t r o c y t e s - l m m u n o h i s t o c h e m i s t r y - M y e l i n Oligodendrocytes

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* Clmllt ~ R~hcr~ au CI~RS l~pfiat reqttestaad lZl~oof$shouldbe sent to: Dr. Guy Roussel,Centrede Neurochimiedu CIqRS. S, rtw ~ l~*~tl, F-67084StrasbomI C~¢x, France. 0165-STZI/ID/103.00© 1983 ElsevierSciencePubii~h~ B.V.

210 Introduction In the past few years, a number of publications dealing with antisera dir~...d against different purified myefin antigens has appeared. Among them, the anticerebroside antiserum has been the most studied due to the fact that the antigen is relatively easy to purify. This particular antiserum was first described by Raff et al. (1978) as a specific oligodendrocyte marker in vitro. In contrast, in brain tissue, the antiserum reacts with not only oligodendrocytes and myelin, but also epithelial cells of the ependymal layer of the ventricles and of the choroid plexus (Uchida et ai. 1981). Immunohistochemical studies with myelin basic protein (MBP) antiserum showed restricted localization of these proteins to myelin sheaths and their presence in oligodendrocytes only during the early stage of development (Sternberger et al. 1978; Hartman et al. 1979; Roussel and Nussbaum 1981). In culture, a low percentage of galactocerebroside-positive cells appear to contain basic proteins at a detectable level (Bhat et al. 1981; Bologa-Sandru et al. 1981). Identical labelling of myelin sheaths and actively mye!inating oligodendrocytes was also noted by Agrawal and Hartman (1979), who used a specific anti-proteolipid antiserum. Among the minor myelin proteins, W1 Wolfgram protein and the myelin-associated glycoprotein (MAG) have also been studied and their respective antisera produced (Nussbaum et al. 1977; Sell et aL 1981). Immunohistochemical results, with both brain sections and glial cell cultures, have led to the conclusion that the Wl protein is not only a myelin marker, but also a specific oligodendrocyte marker detectable during the entire life span of the animal (Labourdette et al. 1979; Roussel and Nussbaum 1981). In contrast, MAG antiserum stains oligodcndrocytes, Schwann cells and certain areas of the periaxonai region of central and peripheral myelin sheaths (Sternberger et al. 1979). Carbonic anhydrase antiserum, produced by using enzyme purified from rat erythrocytes, allowed us to show that this enzyme also was present in myelin, as well as in oligodendrocytes and protoplasmic astrocytes (Mandel et al. 1978; Roussel et al. 1979; unpublished data). The aim of the present investigation was to determine which myelin antigen(s), when purified myelin is injected in toto, induce(s) specific antibodies and to see to what extent such an anti-myelin antiserum is a suitable material for immunohistochemical analysis of brain structures. Anti-myelin antiserum has already been produced in the past (Greg~on et al. 1971; Poduslo and McFadin 1978; Seil and Agrawa11980), but, to our knowledge, it has not been used for systematic immunohistochemical studies. Ofigodendrocytes have been classified into light, medium and dark types on the basis of morphological features by Mori and Leblond (1970). In the present work, we have shown that the dark oligodendrocytes (as identified on electron microscopy), whatever the age of the animal, do not react with the anti-myelin antiserum. These findings can be interpreted as a reflection of the slowdown of their metabolism, compared to that of the light and medium oligodendrocytes.

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E x p e ~ t g procedares Anti-myelin antiserumpreparation Myelin was prepared from the brains of adult Wistar rats by a~minor modificztion of the differential centrifugation method with sucrose gradients reported by Kurihara et aI. (1971). The final membrane pellet was washed exhaustively with water and lyophilized. Two rabbits were inoculated intradermal~y with 10 rag of myelin emulsified in 0.25 ml of NaC1 0.95 (w/v) and 0.25 ml of incomplete Freund adjuvant, 50 ~tl per site along the back. Booster injections were performed 2 and 4 weeks after the first inoculation using the same amounts of antigen prepared as before. The animals were bled out a fortnight after the last inoculation. They showed no clinical symptoms during the experiment.

Absorption experiments Absorption of the antiserum on purified myelin was performed as follows: to 10 mg of lyophilized myelin was added 100 pl of anti-myelin antiserum and 400 pl of PBS (sodium phosphate buffer 0.01 M; pH 7.4, 0.15 M I,laCl); this mixture was incubated for 4 h at room temperature and then fox 2 days at 4°C. An additional volume of PBS was then added in order to obtain a 1 : 2 0 (v/v) dilution, and the diluted antiserum was recovered by centrifugation at 3000 )< g for 30 rain. The absorption procedure with kidney powder was similar to the procedure described above, except that 100 mg of lyophilizec[ kidney powder was used for absorp,;on of 100/~1 of antiserum. Absorption experiments were also performed in c,rder ~o attempt to characterize the different specific antibodies contained in this anti-myel~n antiserur~LThe myelin antigens used (cerebrosides, sulfatides, basic proteins, prote~olipids)were isolated and their purity tested as report~i in earlier papers (Nussbaum and Mandel 19"/2; Nussbaum eta}, 19/4; Rouss¢[ and Nussbaum 198t). Absorption of the antiserum on lipids was achieved with cholesterol particles coaled with the non-covalent bound lipid in accordance with Coulon-Morelec (1972). Briefly, 4.5 mg of cerebrosidcs or 2 mg of sulfatides, dissolved in chloroform/methanol (I : 4, v/v) were absorbed on 28 m$ of cholesterol particles f~r I h at room temperature; the cholcsterol-hapten particles were then washed with 0.9% NaCI (v/v) and recovered by centrifugation. /LI of anti-myelin antiserum were absorbed on these particles for 90 rain at room temperature and then overnight at 4°C. The absorbed antiserum was recovered by centrifus~tion. Absorption with myelin basic proteins or proteolipids was performed by incubating 200 ~I of antis':rum with 1.5 mg of purified protein in similar conditions as described before. The antigens were removed f:om the absorbed antiserum by centrifugation.

Dot.immunobinding experiments Dot-immunobinding assays were carried out in order to estimate approxLma',ely the titer of the antiserum with regard to the main myelin antigens (cerebrosides, ~dfatides, basic proteins, proteolipids and W1 Wolfgrara protein) (Nussbaum et al. 1977). Six ~t$ of lipid hapten dissolved in chloroform/methanol (2:1, v/v) or 3 tt$

212 of protein antigen dissoived in 25% (v/v) formic acid were spotted onto nitrocellulose strips (Hawkes et ,.1. 1982). Incubation of the nitrocellulos~ strips was performed with various dilations of the anti-myelin antiserum in PBS containing 3% (w/v) bovine serum albamin (BSA) and 2.5~ (v/v) normal sheep serum for 2 h. After exhaustive washi, g with PBS, the nitrocellulose strips were incubated for another 2 h period wi~h horse-radish peroxidase conjugated sheep anti-rabbit immunoglobulin G (H + L) antibodies (Institut Pasteur Production), diluted 1 : 1000 (v/v) in PBS containing PeA and normal sheep serum as before. Af:er a final washing period of 2 h, antibody binding was detected by incubation with 4-chloro1-naphthol (0.018%, w/v) in PBS containing 0.02% (v/v) hydrogen peroxide (30~, w/w).

lmmunocytochemical procedure Rats ranging in age from 15 days to adulthood were used. The immunohistochemical methods were identical to those described previously (Roussel and Nussbaum 1981). Briefly, brains of the animals were fixed by intracardiac perfusion with a solution containing 4% (w/v) paraf~rmaldehyde and 01% (w/v) of glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Fragments of different areas (,:orpus callosum, cortex, striatum, cerebellum and areas close to the ventricles) were cut in 60-/Lm thick sections on an Oxford vibratome. The tissue pieces were washed in PBS and incubated in a 200-fold dilution of the anti-myelin antiserum in PBS containing 5% normal sheep serum for 1 h at room temperature. After a thorough washing of the sections with PBS, they were treated witl. a 100-fold dilution in PBS of an anti-rabbit ~-globulin preparation of sheep Fab (fragment antigen binding) fragments coupled with horse-radish peroxidase (Institut Pasteur Production). The peroxidase activity was revealed after treatment for 3 min with a freshly prepared solution of 3,Y-diaminobenzidine tetrahydrochloride (Sigma) (0.025%, w/v) in PBS containing 0.006% (w/v) of hydrogen peroxide. In some experiments, a sheep anti-rabbit "y-globulinpreparation conjugated with fluorescein isothiocyanate (FITC) was also used (Roussel et al. 1977). Anti-myelin antiserum, preabsorbed with myelin or normal rabbit serum, at identical dilutions, were used for controls. After examination by light microscopy, the tissue vibratome sections were refixed in 5% (w/v) glutaraldehyde in 0.1 M phosphate buffet for 30 rain and post-fixed in 1% (w/v) osmic acid dissolved in the same buffer, dehydrated in graded ethanol and embedded in Spurr's resin. Ultra-thin sections were cut tangential to the surface of the sections and observed with a Philips EM 300 electron microscope without additional staining.

Results

Dot-immunobindin 8 assays The sensitive dot-immunobinding technique was used in order to est~tblish approximately the titer of the anti-myelin antiserum versus the main myelin anti-

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gens. This antiserum, up to a dilution of 1:2000, reacts with cerebro~des and sulfatides under our experimental condition~. The titer of tl~is antiserum is very high ~ith regard to the basic proteins (1 : 50000) and proteofipids (1 : 20000), but it d o ~ not contain anti-W1 protein antibodies.

Light-microscopic immunohistochemical studie When vibratome sections of 1S-day-old r~,t brain hemispheres are incubated with anti.myelin antiserum, a great number of cells appears labelled amon 8 the stained myelin fiber tracts. A careful examination of the sections showed that these cells are distributed uniformly from the area of the corpses callosum to the cortex (Fig. la). These findings are in contrast with the pictur~ obtained with specific anti-W1 and anti-MBP sera which show stained cells l~resent mainly in myelin fiber-rich areas (goussel and Nussbaum 1981). In flgt, it appears that two different cell types are stained with this antiserum: (1) the first eel! type is recog6zed easily by the presence of immunoreactivity localized exclusively to their cytoplasm and the neighbouring thin processes; the nuclei are distinguishal:le and do not stain. The cells are observed more frequently in the corpus callosum between the myelin fiber tracts, hut are also present in the cortex and striatum although in limited number. These cells are probably oligodendrocytes (Figs. 1 and 2); (2) the second cell type shows a more evenly distributed labelling over the whole cell body and the nucleus cannot be distinguished. They appear smaller than the other cell types and their processes are generally broader. The distribution of this second cell type does not parallel the presence of the myelin tracts and they are frequently present in the neighbourhood of the capillaries; they are probably astrocytes (Figs. 1 and 2). Cerebellar sections of rat; of the same age were also exan"-~n-ed (Fi8. 3). In the white matter, oHgodendrocytes appear to be aligned in numerous rows alcmg the stained myelin fibers and are heavily labelled. In the granular layer, the same cell types as observed previously in the hemispheres are noticed; moreover, a very faint staining is detected around the granule cells. Similarly, there is also immunoreaoivity at the periphery of the Purkinjc cell bodies and along their respective dendrites. The Golgi epithelial cells in the neighbourhood of the Purkinje cells are labelled, as well u their corresponding Bergmann fibers as they cross the molecular layer. Obvious immunostalning was always observed in some of the cells in the subependymal layer of the lateral ventricles and of the dorsal part of the third ventricle; in contrast there was no labelling of the subependymal cells situated in the ventral part of this latter area (Figs. 2e-f). The neurons present in the hemispheres and the endothelial cells of the vessels were always negative after anti.myefin antiserum treatment (Figs. 1 and 2). The specificity of the immunolabelling by the anti-myelin antiserum was checked by substituting it with normal rabbit serum or with anti-myelin antiserum previously ebsorbed with lyophUized myefin. In both cases, no brain structure was found to be stained (not illustrated). Light.microscopic immunohistochemical studies with antiserum preabsorbed with kidney i~¢der In order to find out the nature of the antigen~ corresponding to the antibodies

Fig. 1. Vibratome sections through the corpus callosum and cortex areas from a 15-day-old rat brain incubated with 1/200 anti-myelin ant serum (a, b. c). Neurons (arrow head) are unstained by this antiserum. Many immunoreactive myeli 1 fibers are seen in the corpus callosum (CC). Putative' labelled ,.,ligodendrocytes (dark arrow) Idc~se immure,reactivity in the pc ipberal cytoplasm, unstained nucleus. tllil~ labelled processes) are more nmler ~us in the corpus caliosum than in the c~rlex (Co). in contrast. putative labelled astrocytes (open arrow, (labelling evenly distributed over the whole ~:¢11 body. nucleus undb.tinguishable, thicker stained processes) are more frequently present in the cortex. Some reactivity can be seen around the blood vessels. The labelling of the astrocytes and labellin~g around the vessels are st;ppres,,ed by using anti-myelin antiserum preabsorbed with kidney powder: only oligodendrocytes and m~,elin fibers remain immunoreactive under this condition (d). a: Section through corpus callosum and c~rtex: t, aml d: Sections slwwing a cortex area close to the corpus callosum; c: Section corresponding to the middle part of the ct~rlex. (a) x750- (b), tcL ( d ) x850.

Fig. 2. Vibratome sections through the corpus callosum (a), the striatum (b, c) and the upper (e) and lower ( f ) parts of the 3rd ventricle from a 15-day-old rat brain incubated with 1/200 anti-myelin antiserum. A distinct labelfing in putative oligodendrocytes and myelin fibers is observed by both the immanofluorescence (a, b) and immunoperoxidase (c) techniques. Moreover, by immanofluorescence. myelinated axons cut in cross-sections show a fluorescent myelin ring with non-reacting axoplasm in the center, c: Note also that there are numerous labelled putative astrocytes (open arrow) close to a blood vessel (V) of which the periphery shows also some immunoreactivity, d: Absorbed ~,nti-myelinantiserum with kidney powder stains only putative oligodendrocytes and the myelin fibers in striatum. Some cells present in the suhependymal ce!l layer of the upper part of the 3rd ventricle are labelled by the anti-myelin antiserum (e), whereas no stained cell could be detected in the lower p~.ct(f), the staining of th©s¢ few subependymal cells is lost by using a peabsorbed anti-myelin antiserum (g). (a) xgo0; (b) xlSO0; (c) xl100; (d) x850; (e), (f), (g) x750.

Fig. 3. Transverse sections of the cerebellum from a 16-day.olcl rat after incubation with 1/200 anti-myelin anti~;erum (a-c). The white matter (WM) appears heavily stained: a number of rows of labelled oligodendrocytes can be distinguished between the labelled myelin fibers. A very faint immm~o-staining is present around t~le non-imr,mnoreactive granule cells (G). b; The perimeters of Purkinje eel) somata and dendrites are labelled and this is probably due to stained glial processes on their smf~ce (see also Fig. 7 and Discussion~; Golgi epithelia] cells (white arrow) in the neighbourhood of the Purkirtje cells and their respective Bergmann fibers (open white arrow) crossing the molec.Jlar layer (ML) are als~J labelled, d: Only the white matter and putative oligodendrocytes and some myelin fibers in the granular layer are immunoreactive ~o the anti-myelin antiserum after preabsorgtion with kidney powder; all othe~ immunoreactivity seen in other ,~ell structures is eliminated. ( a ) x240; (b), (c) ×950; ( d ) x65~J.

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present in this antiserum, different experiments with preabsorbed antiserum were carried out. Absorption of the and-myelin antiserum ~'ith either pure cerebrosides, sulfatides, myelin proteolipid or basic pre*.eins, or in combination reduced slightly the labelling of the structures when compared to non-treated serum. By the dot-immunobinding technique, however, we demonstrated the presence -n this anti-myelin antiserum of all the 4 corresponding antibodies. In contrast, preabsorption of the antiserum on kidney powder led to the complete disappearance of the staining of the astrocytes in both hemispheres and the cerebellum, and the I~.belling frequently observed around vesgeIs was also abolished (Figs. ld~ 2d, 3d). In the same way. the ~taining of the Golgi epithelial cells and their respective Bergmann fibers was suppressed, as well as the immunoreactivity noticed over the surface of the Purkinje cells and their dendrites (Fig. 3d). Finallv, labelling was also absent in the cells of the subependymal layer under identical conditions (Fig. 2g). In contrast to this, the oligodendrocytes and the myelin fiber tracts were still dist[nctly stained in the hemispheres and cerebellum (Figs. ld, 2d, 3d). Control experiments with anti-myelin antiserum, performed on kidney sections, showed an intense labelling restricted to the compact tubular basement membranes in the medulla; the e~ithelia! cells of the tubules were always devoid of staining (not shown). This specific staining of the basement membranes is abolished when preabsorbed anti-myelin antiserum (using either lyophilized myelin or kidney powder) is used.

Electron.microscopic immunocytochemical studies Immunohistochemical studies at the optical level have limits: they do not allo~" us to determine whether or not all the oligodendrocyte sub-classes are labelled, or if the staining is restricted to only one type: light, medium or dark. Discrimination between fibrous and protoplasmic astrocytes is also impossible under t l ~ e con&tions. For these reasons, we extended the study to the electron-microscopic level Th~ identification of the different oligodendroglial cell types were based on the morphological features defined by Mori and Leblond (1970), i.e. the size of the ceiL the density of the nucleus, the development of the Golgi apparatus and of the rough endoplasmic reticulum. The de~e~ of prominence of the Golgi saccules and the cisternae of the endoplasmic reticulum, and the extent of stacking of these cistern~e we,-e also useful criteria for identification. Astrocytes were compared to ol;godendrocytes on the same sections. At ~ e earlier stages (10-15 days after birth), astroc~tes are generally smaller than the neighbouring oligoder~drocytes (most of them being of the light and medium t,~l~es}; their ~,ytoplasm is also less abundant and their nucleoplasm more homogeneous. In addition, the presence of gliofilaments is helpful. In the adult brain, ~strocv~es appear larger than the numerous dark oligodendrocytes which are preser, t at this stage. In 15-day-old rat brain hemispheres, light and medium oligodendlocytes are numerous. After anti-myelin antiserum treatment, both of these cell ~1~s are immunoreactive. The staining is identical in both, with more stain concentrated consistently at the peripl'.ery of the cell cytoplasm (Fig. 4). A staining grat~ient from

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Fig. 4. Electron micrographs of sections of the corpus callosum from a 15-day-old rat brain incubated v.'i,h 1/2t~O anti-m~,elin anti,.erum. The labelled cell depicted in a shows the features of a me,clium oligodendrocyte. Not,~ :he intense imm:moreactivity in the peripheral cytoplasm and the absence of staining in the nucleus. This cell shows ;~lso labelled processes (double arr,)w,, extending toward neighbouri,,g myelinated axons. Many m)e!in sheaths are labelled (arrow). b shows another lat~elled alcdium oligodendrocyte :it a higher magnification. Mitochondria and (.;olgi apparatus vesicles, are unstained, but in contrast, free ribosomes close to the plasma membrane and the ribosomes o ¢ the endoplasmic reticulum are alwa~¢s dense. The cisternae of the endoplasmic reticulum are free of staining. ~ ) ×4(}00: (b) ×15{}{)0.

Fig. 5. Electron micrographs of sections of the corpus callosum from a 15-day-old tat b~ain incabated with 1/200 anti-myelin antiserum. These labelled cells could be recc,gnized as astrocytes I:ocause ¢~f t.he presence of gliofilaments (arrow). The cytoplasm staining of the astrocytes appears more e ~ e ~ distributed throughout the cytoplasm than in the oligodendrocytes; the nucleus, the mitochc~ndria and the Golgi apparatus vesicles are free of labelling; the ghofilan~ents are denser than norer~al. A s ~ processes could also be identified by the presence of gliofilar:lents, a: The labelled astroc-jte is in ckr~e contact with a non-immunoreactive dark oligodcndrocyte (DO); b: Another stained astrocyte g/t~s processes in tight contact with an endothelial cell of a blood vessel; this endothelial cell as wee as the basal membrane of the vessel (V) are unstained. (a) x 18 800; (b) × 20000.

Fig. 6. Electron micrographs of sections of corpus callosum from 15-day-rid (a. d) and adult (b. c. e) rat after incubation with I/2(i0 antl-myelin antiserum, a shows a ~ibelled astrocytic foot (pres~.'nceof giiofilaments) (arrow) runn:,ng between and i'm close contact with an unlabelled dark oligodendrocyte (DO) and a neighbouring blood vessel (V). b and c: Additional dark oligoder~drocytes(DO) w~,~ch are non-immunoreactive to the anti-myelin arLtiserum and in close proximity to eith~,'ra labelled astrocylc (b) or a labelledmedium oligodendrocyte (c) are also shown, d: In young rat brain, when the myelin sheath~ have a restricted number of lamellae, the immunoreactivity is detected in both the innermost and outermost cytoplasmic loops. The axoplasm ~'Ax) is free of staining, e: In contrast, in the adult ~v;met, the myelin sheaths sh,~w a high number of larnellae, the !abeiling can be observed o¢ly on the surfac,': of the n,ost external lamella. (a) x22600: (b) x 17600; (c) ×13000; {d) x43200; {¢) x6g000.

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the nucleus to the ,:ell membrane can be observed. The nucleus is free of staining and its nucleolus can be distinguished in tl~e der~sc nucleoplasm. The mitochondda and the: Golgi apparatus vesicles are un~,tained. In comrasL the free ribosomes close to the membrane, and the ribosomes of the rough endoplasmic reticulum always appear dense, but the cisternae of the endoplasmic reticulum are not stained. The dark oligodendrocytes (dark nuclei and dark cytoplasm) occur in limit.ed numbers at 15 days; after immunolabe'ling, they are always unstained (Figs. 5a, 6a-c). As reported by Mori and Leblond (1970), we also noticed that, during brain maturation, the number of dark oligodendro,:ytes increases at the same time as the light and, to a lesser degree, the medium oligodendrocytes disappear. During this period, the light and medium oligodendrocytes react with this antiserum. In contrast, the dark oligodendrocy~es were unstained at all developmental stages. In .order to exclude tile possibility that the latter cells at e not labelled because of unfavorable local experimental conditions, we show diffex ent micxographs of dark oligodendracytes in close contact with either labelled medium oligodendrocytes, labelled astrocytes or their respective processes, after incubation with the anti-myelin antiserum (Figs. 5a, 6a-c). These figures show that the fact that the dark oligodendrocytes are not labelled is probably not due to the non.accessibility of an antigen but to a biological change in these oligodendrocytes, i.e. a loss of, or a dramatic decrease in, the specific :mtigeas which are present in the ligh~ and medium cells. The immunoreactivity shown by cells referred to as astrocytes at the optica~l level could be confirmed by electron micrograph anaiysis (Figs. 5, 6a, b). Fibrous astrocytes are easily recognized by the occurrence of bundles of gliofilaments in their cytoplasm; in addition the large processes which extend from these cell l~xiies contain numerous gliofdaments. A number of other cells which were labell,~ by the anti-myelin antiserum could not be recognized with certainty, but they did not have the appearance of either oligodendrocytes or neurons. We assumed~ since these unidentifiable cells were mor,:, numerous in the ccrtical areas, that they were probably protoplasmic astrocytes. The staining shown by both, these cell ~.yprs and their respective processes appeared more homogeneous than that of the oligodendrocytes. The nfitochondria and the Golsi apparatus vesicles were free of labeliing and the gliofilaments were denser than normal (Fig. 5). Immunostaining at the electron-microscopic level confirms that there is a labelling of the Golgi epithelial cells and of the Bergmann fibers (not shown). Moreover, the faint immunoreactivity around the granule cells appears to be dt~e to the m.'my thin, labelled, glial processes present (Fig. 7c). Finally, we also deduced from electron micrographs that the labelling presen,,, along the Purldnje cell bodies; and their characteristic dendrites is in fact due to 'very thin glial processes sticking on these neuronal structures (Figs. 7a and b). A strong positive immunostain.ng on the myelin sheaths was noticed without any pretreatment of the tissue sections. In young rat brain (15-day-old) the labelling appeared very strong in the inner and outer cytoplasmic loops of the myelin sheaths, contrasting strongly with the negative staining of the axoplasm (Fig. 6d). In adult rat brain, wher~ the myelin sheaths show a high number of lamellae, the stainir~g is observed only at the periphery of the outer lamella (Fig. 6e).

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Fig. 7. Electron micrographs of cerebeilar sections from a 16-day.old rat incubated with 1/200 anti-myelin antiserum. The staining on the surface of the Purkinje cells (P) and on their dendrit~ (Ped) corresponds to stained astroglial processes, a: Note the very faint staininlt of these processes running, on one side, between the unlabelled dendrite and an adjacent blood vessel (V). b: At the sites of gynaptic contacts, the labelling is absent, c: The staining around the granule cells (O) also appears to be due to thin-labelled astrocytic processes, that show no immunoreactivity when anti-myelin antiserum that has been preabsorbed with kidney powder is used. Note also the absence of stoning o~" the granule cei: cytoplasm and plasma membrane (arrow). (¢~),(b) × 15000; (c) )<29500.

Discussion Injection of pure antigens leads to the production of antibodies directed against specific sites of the molecules. Success or failure of immunohistochemical investigations with such antibodies are therefore largely dependent on tile ease o f access oJ~ these specific antibodies tc the sites in the structures under study. Treatment is frequently required to render the anliBenic sites accessible. W e a.~umed that the injection of a whole membrane structure would have the advantage of producing some antibodies to sites recognizable without pretreatment. T o ~:est this hypothesis,

223 we produced an anti-myelin antiserum zlnd studied the reactivity of the different cells of the brain to this antiserum. As expected, myefin fibers react with this antiserum. Since the sections were not pretreated with either Triton X-100 (Roussel et al. 1978) or digitonin (Roussel and Nussbaum 1981), we observed the staining only in the external and interned cytoplasm loops of young, myelin (as has been noted ~n previous studies with anti-myelin basic proteins and anti-W1 Wolfgram protein sera), or at the peripbery of the external myelin lamella on multila~uellar myelin. No labelling was observed under these conditions at the dense or intraperi~d lines. The neurons and neuronal pro~esses structures were always negative. Therefore~ we concluded that the myelin preparation used for preparation of this antisern~,0awas either not contaminated or was only weakly so, by the neuronal structure:;. If there was some contamination, it was not antigenic under the present condition~;. Mori and Leblond (1970) described light, medium and dari~ oligodendrocytes on the basis of morphological criteria; by thymidine labelling c~xperiment$ they also deduced that the rate of division must be high in the light, ann' nearly absent in the dark oligodendrocytes. Sturrock (1976) detern~ined the relative numbers of the three oligodendrocytic types in the corpus callosum during brain ontogenesis. It appears that the light oligodendrocytes predominate in the brair~ of 14-18-day-old rats~ thereafter, their number decreases and dark olig~endrocytes become more numerous in mature brain (around 80~). Since during brain maturation there is a¢ apparent shift in number from light to medium and finally to d~,.rkofigodendrocyt~ (these latter showing no mitosis), it may be a,.~sumed that these morphological changes correspond to some metabolic alterations. Our present finding that the dark oligodendrocytes are not labelled with ant~-m:telin antiserum, whatever the age of ~he animal, are in agreement with this assumption. Indeed, we assumed that, after having actively participated in myelin sheatl~ production under l~revious states (fight and medium), the production of specific myelin components in the dark oligodendrocytes is dramatically reduced, to such a low level that they are undetectable by our immu~lohistochemical pro~edure. These last results agree with the finding of an absence of labelled oligodendrocytes after 20-25 days, using both anti-MBP (Sternberger et at. 1978; Hartman eta-:. 1979; Roussel and Nussbaum 1981) and anti-proteolipid antisera (Agrawal and Hartman 1979). No distinction was made between the different oligodendrocyte types. Our recent experiments show that the dark oligodendrocytes found in young brain are also unstained by MBP antiserum (unpublished observations). In contrast, W1 antiserum labels the different oligodendrocyte types, what,s.vet the age, up to adulthood (Roussel and Nussbaum 1981). Our results on the characterization of the present anti-myelin anti-serum show that the serum contains anti-MBP and anti-proteolipid antibodies; anti-W1 antibodies could not be detected. These results are in full concordance with the im~unohistochemical findings discussed above. Anti-cerebroside awd anti-sulfat'de antibodies have also been found in this serum by using the sensitive dot-L-mnunobinding technique; the anti-cerebroside antibodies are likely to be responsible for the labelfing of the myelin sheaths at their periphery, as others have also reported (Dupouey et al. 1979).

224 Absorption of the anti-myelin serum with isolated pure myelin haptens or antigens (cerebrosides, sulfatides, b~i:c proteins, and proteolipids)failed to reduce the immunoreactivity of this antiserum towards the brain structures to any degree; in contrast, absorption with myelin was effective. These re~lts may indicate that additional specificantibodies again.~t other myelin components are present, or that only antigens w i t h their original conforn)ation in myelin are able to remove thdr respective antibodies. The finding of astrocytic labelling at the optical level appeared at first ve:ry surprising. The staining was similar to that obtained with an S-100 antiserum (generous gift of Dr. Labourdette). The anti-myelin slaining was confirmed by electron-microscopic studies by which well characterized astrocytes were found in the corpus callosum or in adjacent ~reas, as well as in the cerebellum. The labelling of the Golgi epithelial cells, the characteristic fibrous astroglia cells and their respective Bergmann fibers, was unambiguous. The staining of the astrocytes, Golgi epithelial cells and subependymal cells and their respective extensions and processes disappeared when anti-myelin antiserum previously absorbed with kidney powder was used. In contrast, the immunoreactivity of the oligodendrocytes and the myelin sheaths remained. We assumed, therefore, that the staining of the astrocytes was due to the presence of antibodies which recognized some eammon antigens present in both astrocytes and kidney basement membranes. The nature of these antigens remains to he discovered; basement membrane-a~sociated antigens may be good candi~,ates (Courtoy et al. '~982; Liesi et ai. 1983). The anti-myelin antiserum that we used in this study has permitted us to confirm that the morphological classification of the oligodendrocytes has some metabolic significance. Moreover, an anti-myzlir, antiserum is relatively easy to produce and, after appropriate absorption with kidney tissue powder, is specific for light and medium oligodendrocytes, as well as for myelin.

Acknowledgement The authors wish to thank Mrs. F. Nussbaum for her valuable technical assistance.

References Agrawal, H.C. and B.K. Hartman, lmmunohi:;tochemicallocalizationof myelin proteolipid.~rotein oligodendrocytesin developingbrain, Trans. Amer. Soc.Neurochem.,10 (1979) 87 (Abstrac0. Bhat, S., E. Barbareseand S.E. Pfeiffer,Requirementfor nonoligodendtocI.ttecell sifjtals for enhanced myelinogenic8eneexpressi,m in IonS-termculturesof purifiedrat oli$cdcndrocytes,Proc. Nat. Acad. Sci. (USA),78 (1981) 1283-1287. BologaSandru, L., H.P. Siesri~t,A. Z'Graggen,K. Hofnumn,U. Wiesmana, D. Dahi and N. l-kerschkowitz, Expressionof antigenicmarkersdtttingthe developmentof oligodeudro~es in mousebrain cell cultures, Brain Res.. 210 (1981) 217-229. Coulon-Morelec,M.J,, Purificationdes anticorps anti-hap~ne lipidique, Aim. inst. Pasteur, 123 (lt972) 619-640.

225 Courtoy, PJ., R. Timpl and M.G. Farquhar, Comparative distribution of laminin, type IV collagea, and fibronectin m the rat glomerulus, J. Hist~hem. Cytochem., 30 (1982) 874--886. Dupouey, P. B. Zalc, M. Lefroit-Joly and D. Gomes. Localization of galactosyloeramide and suifatide ~t the surface of the myelin sheath - - An immunofluol'escence study in fiquid nu:dium, Cell. MOf. BioL, 25 (1979) 269-272. Gregson, N,A., M,C. Kennedy and S. Leibowitz, Inunulaological reactions with lysolecithin-~lubilized myefin, Immnnology, 21 (1971) 501-512. Hartman, B.K., H,C. Agrawal, S. Kalmbach and W.T. Shearer, A comparative study of the immunoi~tochemicai Ioeafization of basic protein to myelin a t d ofigodendrcgytes in rat and chicken brain, J. Cutup. Neurol., 188 (1~79) 273-290. Hawkes, R., E. Niday and .L Gordon, A dot-immunobin Jing assay for monoclonal and mhef antibodies, Annlyt. Biochem., 119 (1982) 142-147. Kurihara, T., J.l,. Nussbaum and P. Mandel, 2',3'-Cyclic nuclcotide 3'-phosphohydrolase in purilied myefin from brain of Jimpy and normal young mice, Life Sci. Part 11,10 (1971) 421--429. Lab0urdettc, G., G. Roussel, M.S. Ghandour and J.L. Nnssbaum, Cultures from rat brain hemispheres enriched in ol;godendrocyte-like cells, Brain Res., 179 CI979) 199-203. Liesi, P., D. Dahi and A. Vaheri, Laminin is produced by early rat ~trocytes in primary cuhure~ J. Cell Biol., 96 (1983) 920-924. Mandel, P., G. Roussel, J.P. Delannoy and J.L. Nussbaum, Wolfgram proteins, oligodendroglial cell am/ myelin markers, carbonic anhydrase, a glial marker. In: E. Schoffeniels, L. Hertz and D.B. T o ~ (Eds.), Dynamic Properties of G|ia Cells, Pergamon Press, Oxford, 1978, pp. 267-274. Mori, S. and C.P. Leblond, Electron microscopic identification of ~hree classes of oligo~endrocytea "and~a preliminary study of their profiferative activity in the corpus caliosum of young rats, i. Cutup. NeuroL 139 (1970) 1-30. Nussbanm, J.L. and P. Mandei, Enzymic synthesis of psychosir..e sulphate, J. Neurochem., 19 (1972) 1789-1802. Nussbanm, J.L., J.F. Rouayrenc, P. Mandel, J. Joll~ and P. J~ll~s, Isolation and terminal sequence determination of the major rat brain myefin proteolipid It! apoprotein, Biochetn. Biophys. Re~ Commun., 57 (1974) 1240-1247. Nuubanm, J.L., J.P. Delaunoy and P. Mandel, Some immunot:hemicai characteristics of Wl and W2 Wolfgram proteins isolated from rat brain myelin, J. Neurochem., 28 (1977) 183-191. Podualo, J.F. and D.E. McFarlin, lmmunogenicity of a membrane surface glycoprolxdn ~ t e d with central nervous system myelin, Brain Res., 159 (1978) 234-238. Raft, M.C., R. Mirsky, K.L. Fields, R.P. Lisak, S.H. Dorfman, D.H. Silberberg, N.A. Gregson. S. Leibowitz and M.C. Kennedy, Gaiactocerebroside is a specific cell-surface antigenic marker for ofigodendrocytes in culture, Nature (Lon~ .), 270 (1978) 813-:~16. Rouge.I, G. and J.L. Nussbanm, Comparativ~ localization of Wolfgram WI and myelin basic proteins i_ the rat brain during ontogenesis, Histoche ~n. J., 13 (1981) 1029-1047. Roussel, G., J.P. Delannoy, J.L. Nussbanm and P. Mandel, lmmunohistochemical localization o -~ Wolfgram proteins in nervous tissue of rat brain, Neuroscience, 2 (1977) 307-313. Roussei, G,, J.P. Delaunoy, P. Mandel and J.L. Nussbaum, Ultrastructural localization study of tw~3 Wolfgram proteins in rat brain tissue, J. Neurocytol., 7 (1978) 155-163. Roussel, G., J.P. Delaunoy, J.L. Nussbanm and P. Mande], Demonstration of a specific Iocaltuttion of carbonic anhydrase C in the 81iai cells of rat CNS by an immunohistochemical method, Brain Res., 160 (1979) 47-55. Roussel, G., G. Labourdette and J.L. Nnssbaum, Characterization of ofigodendrncytes in pzim~xy cultures from brain hemispheres of newborn rats, Develop. Biol., 81 (1981) 372-378. Seil, FJ. and H.C, A grawai, Myelin-proteolipid protein does not induce demyelinating or myelination-inhibiting antibodies, Brain Res., 194 (1980) 273-277. Sell, F.J., R.H. Quarles, D. Johnson and R.O. Brady, hr~aanization with purified myelin-associated glycoprotein does not evoke myehnation.inhibiting or demyelinating antibodies, Brain Res., 209 (1981) 470-475. Sternbe~ger, N.H., Y. ltoyama, M.W. Kies ~md H. deF. Webster, Immanocytochemictl method to identify basic protein in myelin-forming nligodendrocytes of newborn rat central nervot:$ system, J. Neuroeytol., 7 (1978) 251-263.

226 Sternberser, N.H., R.H. Quades, Y. ltoyama and H. deF. Webst~, M y e t i n . ~ t e d 81yooprotein demonstrated immuno~ytochemicaUyin myeUnand n~,elin-formln8 tells.of developin$ rat, Proc. Nat. Acad. Sei. (USA), 76 (1979) 1510-1514. Sturroek, I~R., Lisht m i ~ o ~ identification of immature glial ~ds in semith~ sections of the developi~j8 mouse corpm eallowm, J. Anat. (Lond.), l~Z2(1976) 521-537. Uchida, T., K. Takahashi, H. Yamagucld ~ d Y. Nasai, Localization of plactoeerebrofdde in olisodendrocytes, myelin sheath and choroid :i~lexus,Jap. J. Exp. Med., 51 (1981) 29-35.