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Immunohistochemical localization of Wolfgram proteins in nervous tissue of rat brain
Neuroscimcc,
1977. Vol.
2. pp. 307-313.
PergamonPress.Printedin Great Britain.
IMMUNOHISTOCHEMICAL LOCALIZATION OF WOLFGRAM PROTEINS IN NERVOUS TISSUE OF RAT BRAIN G. ROU~~EL,J. P. DELAUNOY,J. L. TURBAN' and P. MANDEL Centre de Neurochimie du C.N.R.S., 11 rue Humann 67085 Strasbourg, Cedex, France Abstract-The localization of Wolfgram proteins Wl and W2 has been studied with the indirect immunofluorescence and immunoperoxidase techniques on semi-thin sections. The presence of these proteins in myelinated fibers was observed, as a function of age, in the corpus callosum, cerebellum and spinal cord; Wl and W2 proteins were also localized in the oligodendroglial cells. In contrast, they could not be visualized in the sciatic nerve. The present results indicate that Wolfgram proteins Wl and W2 originate from the oligodendroglial cells, and are presumably incorporated in the myelin sheaths during the myelination process. These proteins may be used as an oligodendroglial cell marker.
A LARGE number of studies have been devoted to cellular and subcellular localization of proteins in nervous tissue. Thus, the basic en~phalitogenic protein was recognized, early, to be a myelin specific. protein (KORNGUTH, ANDERSON & SCOTT, 1966); more recently a myelin localization was also suggested for the proteolipid P7 (NUSSBAUM& MANDEL, 1973). Glial fibrillary acidic protein has been described as being selectively found in astrocytic glia (BIGNAMI, ENG, DAHL & UYEDA,1972) whereas the S-100 protein (CICERO,COWAN,MOORE& SUNTZEFF,1970) and the alpha 2 glycoprotein (WARECIO\,MILLER, VOGEL & TRIPATZIS.1972) seem to be more broadly distributed in the cells of glial origin. Neuronal cell specificity has been attributed to the 14-3-2 protein (CICERO et al., 1970) and to the GP 350 sialoglycoprotein (VAN NIEUW AMERONGEN, ROUKEMA& VAN ROSSUM, 1974). Recently, we studied the minor class of myelin proteins, the so-called Wolfgram proteins ~U~BAUM, DELAUNOY& MANDEL,1976) and have been able to isolate the two most important Wolfgram proteins Wl and W2 from an insoluble chloroform/methanol 2/l (v/v) myelin pellet. Antibodies against these two proteins were produced in rabbits. It was found that the two antigens have common antigenic determinants. The availability of these antibodies makes it now possible to answer some crucial questions such as, are these proteins an integral part of the myelin or do they derive from axolemma or other membrane structures attached to myelin during the isolation procedure. In this paper, we report initial studies on the localization of Wolfgram proteins in nervous tissue by an appropriate immunohistochemical technique.
’ Charge de Recherche au C.N.R.S. Abhdfftions: Fab. Fragment antigen binding.
MATERIAL AND METHODS Preparation of antiserum A crude Woifgr~ protein fraction was prepared from myelin of adult Wistar rat brain as described previously (NUSSBAUM et al., 1976). The purification of the two Wolfgram proteins Wl and W2 was performed on 7.5% (w/v) accylamide preparative gels in the presence of sodium dodecylsulphate. Antibodies to these two antigens were produced in rabbits and the final bleeding was performed l-2 months after the last injection (NUSSBAUM ef al., 1976). Our previous findings have shown that antiserum against WI reacts with WI and W2 produ~ng a pr~ipitin line of identity between the two antigens; antiserum against W2 behaves in the same way. In earlier work (NUSSBAUM et al., 1976) a close structural relationship between the two proteins has been shown. In this paper, the immunohistochemical studies will thus be restricted to the use of antibodies against Wl. lmmuno~uorescence and
jrnrn~o~zy~tjc
techniques
Young (6, 8, 12, 18 days old) as well as adult rats (2 months) were anesthetized by an intraperitoneal injection of sodium pentobarbitone (40 mg/kg body weight) and the tissues were fixed by intracardiac perfusion with a solution containing 4% (w/v) of paraformaldehyde and 0.1% (w/v) of glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Fragments obtained from the corpus callosum, striaturn, cerebellum, spinal cord and sciatic nerve were immersed for 2-3 h in the same fixative. Some samples were postfixed in 1% (w/v) osmic acid for electron microscopic identification of the different cell types in the investigated brain regions. After dehydration in graded ethanol, all the samples were embedded in Araldite. The immunochemical procedures were run on approx 1 m thick semi-thin sections, mounted on to fat-free glass slides. Araldite was dissolved with Na methoxide (MAYOR, HAMPTON & ROSARIO, 1961)during 1mm, and the sections were then treated with a solution of hydrogen peroxide (20 vol) for another minute. It is important to keep strictly to this timing for dissolving Araldite and treatment with H,O, in order to obtain satisfactory immunochemical results. 307
308
G. Rotssr-I
J. P. DELACSWY.J. I_. N~SS~ALIMand P. MANIXI
PLATI I
FIGS. 1, 2. Immunofluorescence micrographs of a section of corpus callosum from an I&day-old rat incubated with antiserum to protein Wi. Note the localization of the fluorescence on all myelinated fibers identified by the Sudan Black B staining (see Fig. 5). In contrast, no Ruoresccncc is seen in unmyelinated fibers. Many oligodendroglial cells are also fluorescent; the Auorescence is much more intense at the periphery of the cells than in the perinuclear cytoplasm. Fig. 1: M~~~ni~c~itio~~ 1140 x Fig. 2 : Magnification 1900 x FIG. 3. Immunoperoxidase micrograph of a section of corpus callosum (CC) from an IX-day-old rat. incubated with antiserum to protein WI Many myelinated fibers and oligodendroglial cells are positive. Neuronal cell bodies in the striatum (ST) area clearly are protein Wi negative. Magnification 830 x
FIG 4. Immuno~roxidase micrograph of a section of corpus callosum (CC) from an IX-day-old rat. incubated with preimmunization rabbit serum. Ali the structures of corpus callosum (CC) and striatum (ST) are negative. Magnification 590 x
FIG. 5. Sudan Black B staining of a section of corpus callosum (CC) from an IX-day-old rat. Only the myelinated fibers are stained. Magnification 760 x
PLATE
2
FIG 6. Immunoperoxidase micrograph of a section of corpus callosum from an X-day-old rat, incubated with antiserum to protein Wi. Many cells are visible, some of which show a positive reaction. Some myelinated fibers are also positive to protein WI. Magnification 830 x FIG. 7. Immunoperoxidase micrograph of a section of corpus caliosum from a I ?-day-old rat. incubated with antiserum to protein W 1. Note the positive reaction of the my~linated fibers grouped in bundles: many cells in their neighbourhood are also protein WI positive. Neuronal cell bodies in the striatum (ST) area are negative. Magnification 640 x
FIG. 8. Immunoperoxidase micrograph of a section of a cerebellum folium from an X-day-old rat. incubated with antiserum to protein WI. The positive reaction is limited to some myelinated fibers of the white matter (WM) of the fohum. Magnification 530 t:
FIG. 9. immuno~roxi~se micrograph of a section of a cerebellum folium from a I’-day-old-rat incubated with antiserum to protein W 1. The myelinated fibers in the white matter (WM) zone are strongly positive. In contrast, the granular (GL) and molecular (ML) layers are clearly negative. The ‘holes’ in the granular layer represent the cells. Magnification 530 x
FIG. 10. Immunoperoxidase micrograph of a transverse section of spinal cord from a 6-day-old rat. incubated with antiserum to protein Wl. Myelinated fibers, isolated or in bundles at the periphery of the dorsal horn. are strongly positive to protein WI. Magnification 475 x. FIG. 11, Immunoperoxidase
micrograph of a transverse section of spinal cord from a &day-old rat, incubated with preimmunization rabbit serum. All the structures are negative. Magnification 475 x FIGS. 12. 13. Immunoperoxidase micrograph of a transverse section of sciatic nerve from an adult rat. incubated with antiserum to protein Wl (Fig. 12) or with preimmunization rabbit serum (Fig. 13). In both cases, al1 the structures show negative response. Magnification 475 x
311
Localization of Wolfgram proteins in brain Subsequently, the sections were incubated for 20 min at room temperature with two drops of a ten-fold dilution of the rabbit anti-W1 serum in a moist chamber. After removing the excess of antiserum, the slices were washed in slide racks with phosphate buffered saline for 30min, which was renewed every 10min. The preparations were then incubated for 10min with normal sheep serum, in order to eliminate the physicochemical. non-immunological reaction. The sections were then treated with 1% (v/v) sheep anti-(rabbit y-globulin) serum in phosphate buffered saline, the serum being labelled with fluoresceinisothiocyanate. Alternatively sections were treated with a sheep anti-(rabbit y-globulin) preparation of Fab (fragment antigen binding) fragments conjugated with peroxidase (Institut Pasteur Production Paris, France). Finally, after another 20min incubation time, the slides were washed once more with phosphate buffered saline as described above. Immunofluorescence observations were carried out on sections covered with buffered glycerol using the Leitz Orthoplan Fluorescence Microscope. The peroxidase activity was visualized by the diaminobenzidine method of GRAHAM & KARNOVSKY (1966). After exhaustive washing with phosphate buffered saline, the slices were mounted in glycerol and examined with an ordinary microscope. To control the specificity of the immunochemical techniques, tissue sections were also incubated with preimmunization rabbit serum. Ultrathin sections of osmic acid treated material were observed in a Philips EM 300 electron microscope with prior staining in uranyl acetate and lead citrate. The photomicrographs were taken at various exposure times using Kodak Tri X film for fluorescence and Agfapan 25 film for peroxidase. Phosphate buffered saline was composed of 0.82% (W/V) NaCl, 0.16% (w/v) Na2HP04.2H20 and 0.02% (w/v) NaH,P0,.2HZ0.
RESULTS corpus
callosum
Our initial step in the immunohistochemical localization of Wolfgram Wl protein was carried out on a brain region rich in myelinated fibers, i.e. corpus callosum from an 18-day-old rat. A strong immunofluorescence can be observed on sections which have been incubated successively with anti-W 1 and fluorescein-labeled sheep anti-rabbit sera (Figs. 1, 2), whereas control slices do not show any. It appears that nearly all myelinated fibers which are sectioned at different angles, are positively labeled. Glial cell fluorescence is generally more intense at the periphery of the cell than in the perinuclear cytoplasm. The nuclei constantly appear non-fluorescent. Underlying structures (striatum) show a bright fluorescence only where myelinated fibers can be detected. The neuronal cell bodies abundant in the putamen and the caudate nucleus do not give any fluorescence. Similar results have been obtained with an anti-rabbit Fab-labeled horseradish peroxidase preparation (Fig. 3). This technique was also applied to corpus callosum isolated from the brain of younger rats (6, 8, 12 daysold). It was thus possible to demonstrate a relation-
ship between the time of appearance of the antigen and the time of onset of myelination. In brain areas of 6-day-old rats where the corpus callosum will develop later, no peroxidase activity could be detected. positive reactions could be observed in the same regions, at 8 days, on some myelinated fibers and cells (Fig. 6). At 12 days, the reactions are much more pronounced, since more myelinated fibers and cells are present (Fig. 7). Finally, in adult brain, a very intense positive reaction is found in the corpus callosum. All control sections treated with preimmunization rabbit serum, run at the same time, were negative (Fig. 4). Cerebellum The cerebellum was also studied because its structure has been fully described. A section of a folium of the cerebellum of a 6-day-old brain did not show any positive reaction with the peroxidase technique. A distinct suggestion of labeling is seen at 8 days in the white matter, whereas all the other layers are negative (Fig. 8). At 12 and 18 days, and finally in the adult, the reaction becomes stronger, but was always restricted to the central white matter of the folium (Fig. 9). The control sections were negative. Spinal cord Transverse sections of spinal cord were also incubated with anti-W1 serum using the peroxidase technique. In contrast to the brain, the spinal cord from a 6-day-old rat already reacts positively to anti-W 1. This can be observed easily in the areas of white matter and on myelinated fibers dispersed among bundles of neurons, these latter being consistently negative (Figs. 10, 11). Sciatic nerve In the peripheral nervous system, the sciatic nerve of the adult rat has been investigated. Sections of this peripheral nerve, after anti-W1 serum treatment, do not show any peroxidase positive reaction (Fig. 12). In fact they cannot be distinguished from the controls (Fig. 13). Optical and electron microscopic
identijications
In parallel with the immunochemical work, some serial semi-thin sections of corpus callosum from an 18-day-old rat were stained with Sudan Black B (PEARSE, 1953, 1960). This staining enabled us to show that the myelinated fibers (Fig. 5) react positively with the anti-W1 serum. Other ultrathin sections of the same tissue areas were observed under the electron microscope. It it easy, at this stage of brain development, to identify in ultrathin sections the cells which react positively with the anti-W1 serum on semi-thin sections of equivalent areas. These young glial cells are rich in free ribosomes and in rough endoplasmic reticulum. Their Goigi apparatus is also relatively well developed. These cells can be clearly differentiated from neurons, which receive in addition axo-
dendriric synapses. The oligodendroglia~ nature of these cells is finally demonstrated by their relationship with axons; the beginning of lameilar ensheathmcnt in continuity with the plasma membrane of the glial cell was observed.
The aim of this work was to get some evidence about the specific localization of the Wolfgram proteins WI and W2 in nervous tissues of the rat. Since we have already demonstrated (N~ssBA~~ er ai., 1976) that the two proteins have antigenic similarities. we restricted this immunohistochemical work to the use of the antiserum to protein Wt. For these experiments. the anti-W1 protein fluorescent or peroxidase antibody test was carried out on semi-thin sections instead of slices prepared by the classical microtomecryostat. It may be assumed, from the negative results of the control sections. that the fluorescence or peroxidase activity. localized in my~linated fibers and cells under the conditions described in this paper, is due to the specific binding of antibody to Wolfgram protein W 1. The cells which react positively in the white matter arcas to the anti-W I serum have been identifi& ;IS (~i~~~~dcildr[~~tc~~rnder the electron microscopt. The ~~~~~rphologic~~l features of these ceils do not allow us to identify them as astrocytes or neurons. In the adult brain. positively reacting cells are much more difficult to observe. due to the complexity and the i~~extricability of mature brain structures. No positive immunochemical reactions are noted on ul~myeiinat~d axons: this is part~cuIarly weil demonstrated by the negative staining of the axons in the corpus callosum before 8 days of Iife, the time at which the myelination phenomenon starts. This is also true for the parallel fibers of the molecular layer. h~hich are known to be unmyclinated in the majority ~F”AL.AY & CF[AN-PALAY. 1974). The absence of marking of the Purkinje cells and the neurons of the granular layer leads us to conclude that the protein WI (;md probably protein MI21cannot be of neuronal origtn.
Positive immunochem~cal reaction on spinal cord hiices at 6 days of age is in accordance with the appearance of myelin in these structures early in the second postnatal day (KORNOLJTHet (II.. 1966). The Wolfgram protein Wl has not been detected in transverse sections of the sciatic nerve, taken as an example of peripheral nervous tissue. It seems to be in agreement with the biochemical data. showing that the gel electrophoresis pattern of the high moiecular weight protein fraction of peripheral nerve myelin differs from that of central nervous system myelin (GR~~N~I~L~. BRO>STC)F~-‘. EYLAH & MOKIILI.. 1973). It is in fact generally accepted that the Schwann cells. responsible for the synthesis of myelin in perjphera1 nerves. have a different embryonic origin (neural crest) than the oligodendrocytes. All these observations lead to the conclusion that the synthesis of the Wolfgram proteins probably takes place in the o~igodendrocytes of the central nervous system. This is in agreement with Waenheldt’s communication showing that ‘an osmotic shock derived membrane fraction from myelin’ contains substaI~tial amounts of Wolfgram proteins (values up to So”,, in the adult) (WAENHELIX. 1975). No positively marked cells can be observed before 8 days of life. so that we assume that no preliminary accumulation of these proteins exists in the cytoplasm and that they are synthetized only when Inyelination starts. The intense positive reaction in the periphery of the cells can be interpreted as an incorporation of these proteins in the oligodendro~lial plasma membrane. and later on in the myelin sheaths by its extension around the axons. Have these proteins some special functions during lnyeiinat~~~n’~ To follow more accurately the synthesis of these proteins and their incorporation in the myciin sheaths, the use of an immunocytochemical method at the ~l~trastr~lctural level would he helpful. Such methods are currently under investigation.
,~~kn(~w/edgernrnr---The authors wish to extend their appreciationto Dr. A. PORTEfor his helpful advice and reviewing of this manuscript.
REFERENCES L. F., DAHL D. & WYE~AC. T. (19721 Localization of the giial fibrillary acidic protein in astrocytes by immunofluorescence Brrrin Res. 43, 429-435. CLUIROT. J.. COWANW. M., MQOREB. W. & SL’NTZEFF V. (1970) The cellular local~ation of the two brain specific proteins. S 100 and 14-3-2. Brain Res. IS, 25-34. GRAMAMR. C. & KARNOVSKYM. J. (1966) The early stages of absorption of injected horseradish peroxidase in the BIGNAMI A.. Em
proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Nistochrm. Cytochonf. 14, 191. 302. GKEENFIF.I,D S,, BRO~TOFF S.. EYLARE. H. & MORELLP. (1973) Protein composition of myelin of the peripheral nervous system. J. Nrurochem. 20, 1207.-1216. KOKNGCTHS. E., ANDERSON J. W. & SCOTTG. (1966) Temporal relationship between myelinogenesis and the appearance of a basic protein in the spinal cord of the white rat. J. camp. Neural. 127, I-18. MAYORH. D., HAMPTONJ. C. & ROSARI~B. (1961) A simple method for removing the resin from epoxy embedded tissue. J. hioph,~~ himhem. Cgrol. 9, 909-9i0.
Localization of Wolfgram proteins in brain
313
NUSSBAUM J. L. & MANDEL P. (1973) Brain proteolipids in neurological mutant mice. Bruin Res. 61, 295-310. NUF,BAUM J. L., DELAUNOY J. P. & MANDEL P. (1977) Some immunochemical characteristics of Wl and W2 Wolfgram
proteins isolated from rat brain myelin. .I. Neurochem. 28, 183-191. PALAY S. L. & CHAN-PALAY U. (1974) Cerebellar Cortex: Cytology and Orgunizatiorz. Springer, Berlin. PEARSE A. G. E. (1953, 1960) Histochemistry. Theoretical and Applied. lst, 2nd Edns. Little Brown, Boston. VAN NIELJW AMERONGEN A., ROUKEMA P. A. & VAN Rossu~ A. L. (1974) Immunofluorescence study in the cellular localization of GP350, a sialoglycoprotein from brain. Brain Res. 81. l-19. WAENHELDT T. (1975) Ontogenic study of a myelin-derived fraction with 2’ 3’ cyclic nucleotide 3’-phosphohydrolase activity higher than that of myelin. Biochem. J. 151, 435437. WARECKA K., MILLER H. J., VOGEL H. M. & TRIPATZIS I. (1972) Human brain specific alpha 2-glycoprotein: purification by affinity chromatography and detection of a new component; localization in nervous cells. J. Neurochem. 19, 719725. (Accepted 20 November 1976)
1977. Vol.
2. pp. 307-313.
PergamonPress.Printedin Great Britain.
IMMUNOHISTOCHEMICAL LOCALIZATION OF WOLFGRAM PROTEINS IN NERVOUS TISSUE OF RAT BRAIN G. ROU~~EL,J. P. DELAUNOY,J. L. TURBAN' and P. MANDEL Centre de Neurochimie du C.N.R.S., 11 rue Humann 67085 Strasbourg, Cedex, France Abstract-The localization of Wolfgram proteins Wl and W2 has been studied with the indirect immunofluorescence and immunoperoxidase techniques on semi-thin sections. The presence of these proteins in myelinated fibers was observed, as a function of age, in the corpus callosum, cerebellum and spinal cord; Wl and W2 proteins were also localized in the oligodendroglial cells. In contrast, they could not be visualized in the sciatic nerve. The present results indicate that Wolfgram proteins Wl and W2 originate from the oligodendroglial cells, and are presumably incorporated in the myelin sheaths during the myelination process. These proteins may be used as an oligodendroglial cell marker.
A LARGE number of studies have been devoted to cellular and subcellular localization of proteins in nervous tissue. Thus, the basic en~phalitogenic protein was recognized, early, to be a myelin specific. protein (KORNGUTH, ANDERSON & SCOTT, 1966); more recently a myelin localization was also suggested for the proteolipid P7 (NUSSBAUM& MANDEL, 1973). Glial fibrillary acidic protein has been described as being selectively found in astrocytic glia (BIGNAMI, ENG, DAHL & UYEDA,1972) whereas the S-100 protein (CICERO,COWAN,MOORE& SUNTZEFF,1970) and the alpha 2 glycoprotein (WARECIO\,MILLER, VOGEL & TRIPATZIS.1972) seem to be more broadly distributed in the cells of glial origin. Neuronal cell specificity has been attributed to the 14-3-2 protein (CICERO et al., 1970) and to the GP 350 sialoglycoprotein (VAN NIEUW AMERONGEN, ROUKEMA& VAN ROSSUM, 1974). Recently, we studied the minor class of myelin proteins, the so-called Wolfgram proteins ~U~BAUM, DELAUNOY& MANDEL,1976) and have been able to isolate the two most important Wolfgram proteins Wl and W2 from an insoluble chloroform/methanol 2/l (v/v) myelin pellet. Antibodies against these two proteins were produced in rabbits. It was found that the two antigens have common antigenic determinants. The availability of these antibodies makes it now possible to answer some crucial questions such as, are these proteins an integral part of the myelin or do they derive from axolemma or other membrane structures attached to myelin during the isolation procedure. In this paper, we report initial studies on the localization of Wolfgram proteins in nervous tissue by an appropriate immunohistochemical technique.
’ Charge de Recherche au C.N.R.S. Abhdfftions: Fab. Fragment antigen binding.
MATERIAL AND METHODS Preparation of antiserum A crude Woifgr~ protein fraction was prepared from myelin of adult Wistar rat brain as described previously (NUSSBAUM et al., 1976). The purification of the two Wolfgram proteins Wl and W2 was performed on 7.5% (w/v) accylamide preparative gels in the presence of sodium dodecylsulphate. Antibodies to these two antigens were produced in rabbits and the final bleeding was performed l-2 months after the last injection (NUSSBAUM ef al., 1976). Our previous findings have shown that antiserum against WI reacts with WI and W2 produ~ng a pr~ipitin line of identity between the two antigens; antiserum against W2 behaves in the same way. In earlier work (NUSSBAUM et al., 1976) a close structural relationship between the two proteins has been shown. In this paper, the immunohistochemical studies will thus be restricted to the use of antibodies against Wl. lmmuno~uorescence and
jrnrn~o~zy~tjc
techniques
Young (6, 8, 12, 18 days old) as well as adult rats (2 months) were anesthetized by an intraperitoneal injection of sodium pentobarbitone (40 mg/kg body weight) and the tissues were fixed by intracardiac perfusion with a solution containing 4% (w/v) of paraformaldehyde and 0.1% (w/v) of glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Fragments obtained from the corpus callosum, striaturn, cerebellum, spinal cord and sciatic nerve were immersed for 2-3 h in the same fixative. Some samples were postfixed in 1% (w/v) osmic acid for electron microscopic identification of the different cell types in the investigated brain regions. After dehydration in graded ethanol, all the samples were embedded in Araldite. The immunochemical procedures were run on approx 1 m thick semi-thin sections, mounted on to fat-free glass slides. Araldite was dissolved with Na methoxide (MAYOR, HAMPTON & ROSARIO, 1961)during 1mm, and the sections were then treated with a solution of hydrogen peroxide (20 vol) for another minute. It is important to keep strictly to this timing for dissolving Araldite and treatment with H,O, in order to obtain satisfactory immunochemical results. 307
308
G. Rotssr-I
J. P. DELACSWY.J. I_. N~SS~ALIMand P. MANIXI
PLATI I
FIGS. 1, 2. Immunofluorescence micrographs of a section of corpus callosum from an I&day-old rat incubated with antiserum to protein Wi. Note the localization of the fluorescence on all myelinated fibers identified by the Sudan Black B staining (see Fig. 5). In contrast, no Ruoresccncc is seen in unmyelinated fibers. Many oligodendroglial cells are also fluorescent; the Auorescence is much more intense at the periphery of the cells than in the perinuclear cytoplasm. Fig. 1: M~~~ni~c~itio~~ 1140 x Fig. 2 : Magnification 1900 x FIG. 3. Immunoperoxidase micrograph of a section of corpus callosum (CC) from an IX-day-old rat. incubated with antiserum to protein WI Many myelinated fibers and oligodendroglial cells are positive. Neuronal cell bodies in the striatum (ST) area clearly are protein Wi negative. Magnification 830 x
FIG 4. Immuno~roxidase micrograph of a section of corpus callosum (CC) from an IX-day-old rat. incubated with preimmunization rabbit serum. Ali the structures of corpus callosum (CC) and striatum (ST) are negative. Magnification 590 x
FIG. 5. Sudan Black B staining of a section of corpus callosum (CC) from an IX-day-old rat. Only the myelinated fibers are stained. Magnification 760 x
PLATE
2
FIG 6. Immunoperoxidase micrograph of a section of corpus callosum from an X-day-old rat, incubated with antiserum to protein Wi. Many cells are visible, some of which show a positive reaction. Some myelinated fibers are also positive to protein WI. Magnification 830 x FIG. 7. Immunoperoxidase micrograph of a section of corpus caliosum from a I ?-day-old rat. incubated with antiserum to protein W 1. Note the positive reaction of the my~linated fibers grouped in bundles: many cells in their neighbourhood are also protein WI positive. Neuronal cell bodies in the striatum (ST) area are negative. Magnification 640 x
FIG. 8. Immunoperoxidase micrograph of a section of a cerebellum folium from an X-day-old rat. incubated with antiserum to protein WI. The positive reaction is limited to some myelinated fibers of the white matter (WM) of the fohum. Magnification 530 t:
FIG. 9. immuno~roxi~se micrograph of a section of a cerebellum folium from a I’-day-old-rat incubated with antiserum to protein W 1. The myelinated fibers in the white matter (WM) zone are strongly positive. In contrast, the granular (GL) and molecular (ML) layers are clearly negative. The ‘holes’ in the granular layer represent the cells. Magnification 530 x
FIG. 10. Immunoperoxidase micrograph of a transverse section of spinal cord from a 6-day-old rat. incubated with antiserum to protein Wl. Myelinated fibers, isolated or in bundles at the periphery of the dorsal horn. are strongly positive to protein WI. Magnification 475 x. FIG. 11, Immunoperoxidase
micrograph of a transverse section of spinal cord from a &day-old rat, incubated with preimmunization rabbit serum. All the structures are negative. Magnification 475 x FIGS. 12. 13. Immunoperoxidase micrograph of a transverse section of sciatic nerve from an adult rat. incubated with antiserum to protein Wl (Fig. 12) or with preimmunization rabbit serum (Fig. 13). In both cases, al1 the structures show negative response. Magnification 475 x
311
Localization of Wolfgram proteins in brain Subsequently, the sections were incubated for 20 min at room temperature with two drops of a ten-fold dilution of the rabbit anti-W1 serum in a moist chamber. After removing the excess of antiserum, the slices were washed in slide racks with phosphate buffered saline for 30min, which was renewed every 10min. The preparations were then incubated for 10min with normal sheep serum, in order to eliminate the physicochemical. non-immunological reaction. The sections were then treated with 1% (v/v) sheep anti-(rabbit y-globulin) serum in phosphate buffered saline, the serum being labelled with fluoresceinisothiocyanate. Alternatively sections were treated with a sheep anti-(rabbit y-globulin) preparation of Fab (fragment antigen binding) fragments conjugated with peroxidase (Institut Pasteur Production Paris, France). Finally, after another 20min incubation time, the slides were washed once more with phosphate buffered saline as described above. Immunofluorescence observations were carried out on sections covered with buffered glycerol using the Leitz Orthoplan Fluorescence Microscope. The peroxidase activity was visualized by the diaminobenzidine method of GRAHAM & KARNOVSKY (1966). After exhaustive washing with phosphate buffered saline, the slices were mounted in glycerol and examined with an ordinary microscope. To control the specificity of the immunochemical techniques, tissue sections were also incubated with preimmunization rabbit serum. Ultrathin sections of osmic acid treated material were observed in a Philips EM 300 electron microscope with prior staining in uranyl acetate and lead citrate. The photomicrographs were taken at various exposure times using Kodak Tri X film for fluorescence and Agfapan 25 film for peroxidase. Phosphate buffered saline was composed of 0.82% (W/V) NaCl, 0.16% (w/v) Na2HP04.2H20 and 0.02% (w/v) NaH,P0,.2HZ0.
RESULTS corpus
callosum
Our initial step in the immunohistochemical localization of Wolfgram Wl protein was carried out on a brain region rich in myelinated fibers, i.e. corpus callosum from an 18-day-old rat. A strong immunofluorescence can be observed on sections which have been incubated successively with anti-W 1 and fluorescein-labeled sheep anti-rabbit sera (Figs. 1, 2), whereas control slices do not show any. It appears that nearly all myelinated fibers which are sectioned at different angles, are positively labeled. Glial cell fluorescence is generally more intense at the periphery of the cell than in the perinuclear cytoplasm. The nuclei constantly appear non-fluorescent. Underlying structures (striatum) show a bright fluorescence only where myelinated fibers can be detected. The neuronal cell bodies abundant in the putamen and the caudate nucleus do not give any fluorescence. Similar results have been obtained with an anti-rabbit Fab-labeled horseradish peroxidase preparation (Fig. 3). This technique was also applied to corpus callosum isolated from the brain of younger rats (6, 8, 12 daysold). It was thus possible to demonstrate a relation-
ship between the time of appearance of the antigen and the time of onset of myelination. In brain areas of 6-day-old rats where the corpus callosum will develop later, no peroxidase activity could be detected. positive reactions could be observed in the same regions, at 8 days, on some myelinated fibers and cells (Fig. 6). At 12 days, the reactions are much more pronounced, since more myelinated fibers and cells are present (Fig. 7). Finally, in adult brain, a very intense positive reaction is found in the corpus callosum. All control sections treated with preimmunization rabbit serum, run at the same time, were negative (Fig. 4). Cerebellum The cerebellum was also studied because its structure has been fully described. A section of a folium of the cerebellum of a 6-day-old brain did not show any positive reaction with the peroxidase technique. A distinct suggestion of labeling is seen at 8 days in the white matter, whereas all the other layers are negative (Fig. 8). At 12 and 18 days, and finally in the adult, the reaction becomes stronger, but was always restricted to the central white matter of the folium (Fig. 9). The control sections were negative. Spinal cord Transverse sections of spinal cord were also incubated with anti-W1 serum using the peroxidase technique. In contrast to the brain, the spinal cord from a 6-day-old rat already reacts positively to anti-W 1. This can be observed easily in the areas of white matter and on myelinated fibers dispersed among bundles of neurons, these latter being consistently negative (Figs. 10, 11). Sciatic nerve In the peripheral nervous system, the sciatic nerve of the adult rat has been investigated. Sections of this peripheral nerve, after anti-W1 serum treatment, do not show any peroxidase positive reaction (Fig. 12). In fact they cannot be distinguished from the controls (Fig. 13). Optical and electron microscopic
identijications
In parallel with the immunochemical work, some serial semi-thin sections of corpus callosum from an 18-day-old rat were stained with Sudan Black B (PEARSE, 1953, 1960). This staining enabled us to show that the myelinated fibers (Fig. 5) react positively with the anti-W1 serum. Other ultrathin sections of the same tissue areas were observed under the electron microscope. It it easy, at this stage of brain development, to identify in ultrathin sections the cells which react positively with the anti-W1 serum on semi-thin sections of equivalent areas. These young glial cells are rich in free ribosomes and in rough endoplasmic reticulum. Their Goigi apparatus is also relatively well developed. These cells can be clearly differentiated from neurons, which receive in addition axo-
dendriric synapses. The oligodendroglia~ nature of these cells is finally demonstrated by their relationship with axons; the beginning of lameilar ensheathmcnt in continuity with the plasma membrane of the glial cell was observed.
The aim of this work was to get some evidence about the specific localization of the Wolfgram proteins WI and W2 in nervous tissues of the rat. Since we have already demonstrated (N~ssBA~~ er ai., 1976) that the two proteins have antigenic similarities. we restricted this immunohistochemical work to the use of the antiserum to protein Wt. For these experiments. the anti-W1 protein fluorescent or peroxidase antibody test was carried out on semi-thin sections instead of slices prepared by the classical microtomecryostat. It may be assumed, from the negative results of the control sections. that the fluorescence or peroxidase activity. localized in my~linated fibers and cells under the conditions described in this paper, is due to the specific binding of antibody to Wolfgram protein W 1. The cells which react positively in the white matter arcas to the anti-W I serum have been identifi& ;IS (~i~~~~dcildr[~~tc~~rnder the electron microscopt. The ~~~~~rphologic~~l features of these ceils do not allow us to identify them as astrocytes or neurons. In the adult brain. positively reacting cells are much more difficult to observe. due to the complexity and the i~~extricability of mature brain structures. No positive immunochemical reactions are noted on ul~myeiinat~d axons: this is part~cuIarly weil demonstrated by the negative staining of the axons in the corpus callosum before 8 days of Iife, the time at which the myelination phenomenon starts. This is also true for the parallel fibers of the molecular layer. h~hich are known to be unmyclinated in the majority ~F”AL.AY & CF[AN-PALAY. 1974). The absence of marking of the Purkinje cells and the neurons of the granular layer leads us to conclude that the protein WI (;md probably protein MI21cannot be of neuronal origtn.
Positive immunochem~cal reaction on spinal cord hiices at 6 days of age is in accordance with the appearance of myelin in these structures early in the second postnatal day (KORNOLJTHet (II.. 1966). The Wolfgram protein Wl has not been detected in transverse sections of the sciatic nerve, taken as an example of peripheral nervous tissue. It seems to be in agreement with the biochemical data. showing that the gel electrophoresis pattern of the high moiecular weight protein fraction of peripheral nerve myelin differs from that of central nervous system myelin (GR~~N~I~L~. BRO>STC)F~-‘. EYLAH & MOKIILI.. 1973). It is in fact generally accepted that the Schwann cells. responsible for the synthesis of myelin in perjphera1 nerves. have a different embryonic origin (neural crest) than the oligodendrocytes. All these observations lead to the conclusion that the synthesis of the Wolfgram proteins probably takes place in the o~igodendrocytes of the central nervous system. This is in agreement with Waenheldt’s communication showing that ‘an osmotic shock derived membrane fraction from myelin’ contains substaI~tial amounts of Wolfgram proteins (values up to So”,, in the adult) (WAENHELIX. 1975). No positively marked cells can be observed before 8 days of life. so that we assume that no preliminary accumulation of these proteins exists in the cytoplasm and that they are synthetized only when Inyelination starts. The intense positive reaction in the periphery of the cells can be interpreted as an incorporation of these proteins in the oligodendro~lial plasma membrane. and later on in the myelin sheaths by its extension around the axons. Have these proteins some special functions during lnyeiinat~~~n’~ To follow more accurately the synthesis of these proteins and their incorporation in the myciin sheaths, the use of an immunocytochemical method at the ~l~trastr~lctural level would he helpful. Such methods are currently under investigation.
,~~kn(~w/edgernrnr---The authors wish to extend their appreciationto Dr. A. PORTEfor his helpful advice and reviewing of this manuscript.
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