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Vimentin and glial fibrillary acidic protein filaments in radial glia of the adult urodele spinal cord
0306-4522/88 $3.00+ 0.00 Pergamon Press plc ~) 1988IBRO
Neuroscience Vol. 27, No. 1, pp. 279 288, 1988
Printed in Great Britain
V I M E N T I N A N D GLIAL F I B R I L L A R Y ACIDIC P R O T E I N F I L A M E N T S IN R A D I A L GLIA OF THE A D U L T U R O D E L E SPINAL C O R D A. J. ZAMORA* and M. MUTIN Unit6 de Rechereches Neurobiologiques, INSERM U 6 and CNRS UA 634, 280, boulevard Sainte Marguerite, 13009 Marseille, France Abstract--This work, based on Golgi impregnations, transmission electron microscopy and immu-
nocytochemistry, demonstrates that the intermediate filaments found in the radial gliocytes of the adult newt spinal cord are both vimentin and glial fibrillary acidic protein (GFAP) structures. Gliocytes appeared as large, arboreous cells, with appendages extending peripherally. They were extensively immunolabelled with both anti-vimentin and anti-GFAP monoclonal antibody conjugates. Outstanding correspondence in cell configuration was found when Golgi-impregnated specimenswere compared to the distribution of immunolabels. Electron micrographs showed cytoplasmic bundles of anti-vimentin decorated intermediate filaments occupying the radial projections. The presence of GFAP confirms the astroglial character of the radial glia in urodeles; the existence of vimentin suggests that the spinal cord of the adult animal retains immature astroglia, which should express enlarged capabilities of adaptation.
The classical works by Van Gehuchten 32 and by Ramon y Caja123 have established the basic cytoarchitectural features of gliocytes in the embryonic CNS of Amphibia. One of the important points to be stressed in these studies is the discovery of macroglial cells whose somata are located in the periependymal region (central field of Ebbesson H) and send long radial cytoplasmic processes that pervade the neuropil and the fibre tracts up to the pial surface. This configuration is present throughout the whole life of the animal. Few cytological studies have been devoted to these glial cells, in spite of their outstanding size and the complexity of their cytoplasmic expansions. The spinal cord of postmetamorphic amphibians, such as toads, 29'3° frogs 24 and newts, 34 is organized upon a complex radial glial framework, which by conventional electron microscopical analysis has been postulated as astroglial in character; in fact, the cytoplasm of the radial processes contains bundles of intermediate filaments (IF) closely resembling those found in mammalian fibrous astrocytes. These filaments occupy mainly the cytoplasmic processes running toward the periphery or the perivascular basal laminae, lmmunocytochemical studies9,/9 have shown that the radial gliocytes of the adult frog spinal cord react positively with antibodies against mammalian glial fibrillary acidic protein (GFAP). On
the other hand, evidence has been presented to support the generalized concept that several kinds of intermediate filament proteins coexist in one cellular population. Concerning astroglia, some studies performed in mammalian nervous tissue have shown, in vivo and in vitro, that astrocytes coexpress both GFAP and vimentin IF. Mammalian astrocytes are vimentin-positive cells in the cerebellum, hippocampus, cerebral cortex, pons and retina. 8,13,25 Since vimentin had been considered as a "mesenchymal" IF protein, these observations are significant by showing that several kinds of cells, irrespective of their embryological parentages, express vimentin synthetizing capabilities, suggesting that vimentin IF play a more general function. The first ultrastructural description of astroglia in urodeles was made by Schoenbach26 who concluded that urodele astroglia differs little from mammalian astrocytes. We have previously described 34 the cytological features as well as the topographical and intercellular relationships of astroglial cells in the central field of the spinal cord of the adult ribbed newt. In the present study we illustrate, by the Golgi impregnation method and by transmission electron microscopy, the radial arborizations of these gliocytes. The observations have been interpreted in connection with current ideas about the function of cytoskeleton in cells displaying complex, changing cytoplasmic shape. A partial report of this work has been previously presented. 35
*To whom correspondence should be addressed at: INSERM U 6, 280, boulevard Sainte Marguerite, 13009 Marseille, France. EXPERIMENTAL PROCEDURES "Abbreviations: DAB, 3,Y-diaminobenzidine; GFAP, glial fibrillary acidic protein; HRP, horseradish peroxidase; Adult male and female ribbed newts Pleurodeles waltlii IF, intermediate filaments; NSS, normal sheep serum; Michaelles31 obtained from SEREA (Argenton l'Eglise, PBS, phosphate-buffered saline. France) were used in this study. The animals were kept in 279
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flesh water at room temperature and fed twice a week. They were aneasthetized by immersion in a 0.01% aqueous solution of tricaine methane sulphonate (Sandoz MS222) and perfused through the conus arteriosus with fixative at room temperature under hydrostatic pressure of 130 mmHg.
Light microscopy After perfusion with 2% paraformaldehyde and 2% glutaraldehyde in 0.2M phosphate buffer, segments of brachial spinal cord were dissected and double impregnated by the rapid Golgi method according to Palay and ChanPalay. 22 The specimens were dehydrated and embedded in nitrocellulose. Sections of 100 #m in thickness were done on a Leitz sliding microtome.
Electron microscopy The tissue processing for standard transmission electron microscopy has been previously described. 34
lmmunocytochemistry The spinal cord was fixed by transcardiac perfusion with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brachial segments of the spinal cord were dissected out, wrapped in 2% agar, and mounted vertically on the chuck of a vibratome (Lancer, U.S.A.). Transverse sections, 50/~ m in thickness, were cut under PBS. Unspecific binding sites were inactivated by preincubation of tissue sections in 5% normal sheep serum (NSS) in PBS for l h at room temperature. Sections were then incubated overnight at 4°C in a PBS-0.02% sodium azide solution of monoclonal antibodies. Adjacent sections were treated with monoclonal antibodies to vimentin or to GFAP. The anti-vimentin monoclonal antibody was obtained from cultured rat astrocytes, and characterized by immunoblotting revealing a single band corresponding to vimentin (for details, see Ref. 13); anti-GFAP monoclonal antibody was purchased from Marseilles Immunotech, code No. 0161. The dilutions employed were 1/ 1000 for vimentin, and 1/1000 for GFAP. Control sections were incubated in similar conditions in 5% NSS in PBS. After four PBS washes the sections treated with anti-vimentin and antiGFAP monoclonal antibodies were incubated for 2h at room temperature in 1/100 dilution of goat anti-mouse IgG conjugated to horseradish peroxidase (HRP). After washing, sections were incubated in 0.05% diaminobenzidine (DAB, Sigma Chemical Co.) and 0.5% hydrogen peroxide for 6 min at room temperature in darkness. They were then rinsed in PBS and mounted for observation and photomicrography. Later they were post-fixed in 1% osmium tetroxide in 0.2 M phosphate buffer for 30 min, embedded in epoxy resin and processed for transmission electron microscopy; unstained 1-#m-thick plastic sections were also microphotographed, and 80 nm thin sections were observed at 60 kV without uranyl or lead contrast enhancement. RESULTS In transverse sections, the urodele spinal cord showed a typical H-shaped central gray surrounded by white matter (Fig. 1). The periependymal gray matter (substantia gliosa centralis) contained the somata of gliocytes, the neurons being placed more peripherally. In the white matter, the cell elements that we observed were only represented by the intrafascicular oligodendrocytes. A m o n g the gliocytes of the gray matter, the most striking elements corresponded to large arboreous cells. Golgi-impregnated sections showed that these cells projected, from the lateral aspect of the cell body, a single long cytoplasmic process that progressively ramified as it
approached the peripheral region after traversing the longitudinal fibre tracts of the white matter. These processes ended about the periphery of the spinal cord. The rest of the surface of the soma developed veil-like cytoplasmic sheets curling around the other components of the gray matter (Fig. 2). In this respect, these cells resembled the Golgi epithelial cells of the mammalian cerebellum. 22 The entire profile of these macrogliocytes made them resemble the so called "astroblast" observed in the spinal cord of the new-born mouse. 23 Electronmicroscopically, four major features characterized the peripheral arborization of these gliocytes, namely glycogen particles, microtubules, 8 n m IF, and tubules of smooth endoplasmic reticulum (Fig. 3). Glycogen particles were homogeneously distributed all over the cell process, including the cytoplamic end-feet surrounding both the pericapillary and the subpial basal laminae. The microtubules as well as the profiles of smooth endoplasmic reticulum were preferentially present in the main shafts of the arborizations: in fact, they were not found in the projections passing through the nerve fibres of the white matter nor in the perivascular or subpial end-feet, where I F were abundantly represented (Fig. 4). In those sites where desmosomes joined together two homologous gliocyte processes, a condition frequently observed at any level of the urodele spinal cord, the peripheral bundles of IF were intermingled to the subplasmalemmal desmosome web (Fig. 3). The only membranous compartment found at the end-foot level was represented by large polymorphic vacuoles limited by smooth membrane units easily observed among the glycogen aggregates (Fig. 4). Transverse adjacent vibratome sections of the newt spinal cord processed for the immunoperoxidase detection of both vimentin and G F A P I F showed the precipitation product in the perinuclear cytoplasm of the gliocytes of the substantia gliosa centralis (Fig. 5A and B), as well as all over the extensive radial arborization that pervaded the spinal cord from the periependymal layer to the subpial stratum (Fig. 6A and B). Extensive vimentin immunolabelling was observed in the tanycytes forming the dorsal aspect of the ependyma, and forming the septum dorsalis of K611iker, which became clearly perceptible (Fig. 5A). Contrarily, these tanycytes and their projections were consistently unlabelled in sections treated with antiG F A P monoclonal antibody (Fig. 5B). Immunostained l-/~m-thick plastic sections showed longitudinal threads running among the cell somata of the gray matter. In this region, few cells displayed perinuclear positive reaction (Fig. 7). The white matter was traversed by radial profiles of precipitation product contacting the subpial zone as typical triangular end-feet (Fig. 8). At the ultrastructural level, anti-vimentin conjugates decorated longitudinal bundles of cytoplasmic filaments, located in correspondence to radial arborizations.
Vimentin and GFAP filaments in urodele gliocytes
Fig. 1. Transverse section of newt brachial spinal cord (1-p m-thick plastic, toluidine blue staining). From the compact gray matter radial projections arise (arrows) and penetrate the white matter through the fascicles of myelinated fibres. These projections correspond in fact to dendrites and/or glial radiations when observed in the electron microscope. Asterisk, central canal, x 200.
Fig. 2. Photomicrograph of the ventrolateral region of the brachial spinal cord. Rapid Golgi method. Two astroglial cells (A) with somata located in the periependymal substantia gliosa (G). Large stems (large arrows) arise from the soma, which also displays some velate formations (small arrows); the peripheral branches of the arborizations end at the subpial level (white arrows), x 500.
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Fig. 3. Electron micrograph of the substantia gliosa centralis. A glial nucleus (N) is shown: the cytoplasm of this cell is contiguous to glial processes where tubular profiles of smooth endoplasmic reticulum, microtubules (large arrows) and intermediate filaments (small arrows) are found. A desmosome (arrowhead) joins a glial cytoplasmic process to the soma of a gliocyte. Cytoplasmic filament bundles intermingle with the desmosomal web. The slender arrows point to glycogen particles, x 31,200.
Fig. 4. Electron micrograph of the peripheral region of the spinal cord. Two glial processes (A1 and A2) show an area of interdigitation (arrowhead) and end in the basal lamina (arrows). The cytoplasm contains glycogen particles, polymorphic vacuoles, and bundles of intermediate filaments (IF). FT, tract of unmyelinated axons transversely sectioned, x 31,200. 282
Fig. 5. Adjacent-50 pm-thick vibratome sections, not counterstained, showing the dorsal region of the brachial spinal cord. (A) Section treated with anti-vimentin monoclonal antibody; the HRP precipitation product is localized mainly in the tanycyte (T) forming the septum dorsalis of K611iker (large arrows); the perinuclear cytoplasm and the radial arborizations of the gliocytes located in the substantia gliosa centralis are also labelled (small arrows). E, ependyma. × 500. (B) Section showing HRP precipitation product of anti-GFAP conjugate. The ependymal cells (E) are not labelled, nor the tanycytes (T) that send projections to form the septum of K611iker (large arrows). The cells of the substantia gliosa centralis are labelled in the perinuclear cytoplasm and in their peripheral radiations (small arrows). S, sulcus medianus dorsalis. × 500. 283
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Fig. 6. Same sections as shown in Fig. 5. The ventrolateral region of the brachial spinal cord displays many fine HRP-immunolabelled radiations (arrows) traversing the white matter (W) and abutting at the periphery. V, ventral horn. (A) Anti-vimentin H R P immunolabelling; (B) anti-GFAP immunolabelling. × 500.
Vimentin and GFAP filaments in urodele gliocytes
Figs 7 and 8. Plastic, 1-#m-thick unstained sections showing HRP precipitation product of anti-vimentin conjugate. Fig. 7. The cells of the substantia gliosa centralis show mild cytoplasmic perinuclear labelling, whereas some dense bundles (arrows) point radially toward the periphery of the spinal cord. The arrowheads point to lipid inclusions in the gliocyte cytoplasm (see Ref. 34). Fig. 8. The peripheral marginal plexus in which several radial labelled bundles (large arrows) cross the fiber tracts to end as triangular subpial end-feet (small arrows), x 350.
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Fig. 9. Electronmicrograph showing a high magnification of one filament bundle shown in Fig. 8. In the cytoplasm of a radial glial projection (A) parallel IF are labelled with HRP product (arrows). Some vacuoles are also labelled (arrowhead). M, myelinated axon. × 90,000. Besides the filament bundles, the marker was also associated with annular or vacuolar structures, about 80-150 nm in diameter (Fig. 9). The control sections incubated with NSS did not reveal any immunoperoxidase precipitation product. DISCUSSION
Using combined cytological and immunological techniques we have shown the astroglial character of the large radial macrogliocytes of the urodele spinal cord. Golgi impregnations allowed us to localize the cell soma in the substantia gliosa centralis and to trace out the profiles of its peripheral expansions. The ultrastructural analysis of these expansions revealed the presence of 8 nm IF and of glycogen particles which were also present in the perivascular and supbial investments. These features are considered as distinctive of astroglia. Furthermore, the arboreous cell shape closely correlated with the distribution of GFAP immunolabels. The expression of GFAP in nervous cells of a wide variety of vertebrates from fish to humans has been reported. 9 G F A P is considered as the specific IF protein of astroglial cells, s As far as we know the astroglial character of radial gliocytes of Urodela had not been immunocytochemically demonstrated in previous reports. Furthermore, the present work is the first demonstration of the presence of both GFAP and vimentin IF in radial macrogliocytes of Urodela. The reactivity of urodele IF
with antisera induced by mammalian antigens confirms the idea that IF proteins have undergone little change in evolutionJ 7 In Bergmann as well as in M/iller glia, which are considered as relatively undifferentiated types of glia, GFAP 3 and vimentin27 have been localized by immunofluorescence. It has been pointed out 28 that astroglia of adult lower vertebrates resemble embryonic astroglia of higher vertebrates. In fact, the frog 19 and the newt (this paper) astroglial cells are morphologically indistinguishable from the radial gliocytes of the new born mouse spinal cord, 14'23 and also resemble the radial glia of the 7-week-old human fetal cerebrum. 5 The morphological "immaturity" of astroglia in lower vertebrates may be associated with embryonic types of cell behaviour, such as pluripotentiality, enhanced cellular motility and migratory capabilities, reappearance of mitotic activity or cell pattern generation. Function pluripotentialitymay be related to the spatial compartition of cell constituents in the cytoplasm. In this context, the work of Miller and Liuzzi 19 is significant. These authors have shown the preferential distribution of GFAP at the more peripheral branches of the frog radial gliocytes; they have postulated that this differential distribution expresses the dual capacity of these gliocytes to combine the functions of both white matter (peripheral branches) and gray matter (central branches, somata) astrocytes of mammals. Notwithstanding, the idea of functional compartition deserves further investigation. In our
Vimentin and GFAP filaments in urodele gliocytes material, the precipitation of H R P was homogenously distributed. This difference between Anura and Urodela gives rise to the question of whether urodele astroglia stand at a more primitive level of differentiation in comparison to their anuran homologues. In vitro and in situ coexistence of two different types of IF in cells has been firmly establishedJ 6,17,21,33 The meaning of this condition is still not completely understood. During development of mouse neuroectoderm, vimentin precedes the appearance of G F A P . 25 As astrocytes mature, there is a vimentin to G F A P transition, 7 suggesting that undifferentiated cells express vimentin as their main cytoskeletal system. Vimentin filaments form by assembling soluble precursors 4 through links established at the headpiece level of the molecule. ~2'2° In desmofibrocytes, vimentin filments attach together with cytokeratin filaments to desmosomes. ~5 Vimentin filaments can therefore be considered as elements integrating the true cytoskeleton of relatively undifferentiated, growing and proliferating cells. ~°'33The levels of vimentin correlate well with a rapid cell growth in vitro. 2'6 The complexity of cell shape is one of the most impressive features of radial gliocytes. The spreading of cytoplasm through branches, sheets, terminal investments and filopodia-like projections results in the constitution of a macrocellular system with multiple interaction possibilities. Experiences performed in cultured mammalian cells, in which the cell shape can be modified by changing the composition of the milieu, indicate that the cell configuration influences cell behaviour. In particular, it has been shown that the morphological state of the cell modifies the
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biosynthesis of vimentin. 1 In cultured epithelial cells keratin and vimentin syntheses are related to the extent of cell contacts and to the changes in cell shape, respectively. 2 An increase in the projected cell area is accompanied by an increment in the content of vimentin; for instance, the pseudopodial arborizations developed by cells treated with cytochalasin B display vimentin exclusively. 18 Conversely, cell rounding is accompanied by a depression in vimentin synthesis. 2 Thus, phenomena observed in cultured cells can lead to a better understanding of the presence of vimentin IF in embryonic types of astroglia, a condition that can be related to the arboreous shape of these cells. The immature character of the cytoskeleton could be associated to an increased capability to change the tridimensional configuration of the cytoplasm. In the case of embryos, cells are continuously modifying their shape configuration as overt ontogenetic events proceed. Does the presence of vimentin-rich radial gliocytes in adult animals imply the existence of larger remodelling capabilities? An answer to this question should be advanced through the analysis of astroglial cytoskeletal changes during regeneration of the spinal cord. In these circumstances, the process normally observed in maturation, i.e. the vimentin to G F A P shift, should be reproduced, leading to the predominence of vimentin in the astroglial cytoplasm during the early stages of the regenerative period. Acknowledgements--The authors are grateful to Dr J.
Ciesielski-Treska who kindly provided the vimentin antiserum, and to Dr O. K. Langley for helpful discussions.
REFERENCES
1. Ben-Ze'ev A. (1983) Cell configuration related control of vimentin biosynthesis and phsophorylation in cultured mammalian cells. J. Cell Biol. 97, 858-865. 2. Ben-Ze'ev A. (1984) Different control of cytokeratins and vimentin synthesis by cell-cell contact and cell spreading in cultured epithelial cells. J. Cell Biol. 99, 1424~1435. 3. Bignami A. (1984) Glial fibrillary acidic (GFA) protein in Miiller glia: immunoftuorescence study of the goldfish retina. Brain Res. 300, 175 177. 4. Blikstad I. and Lazarides E. (1983) Vimentin filaments are assembled from a soluble precursor in avian erithroid cells. J. Cell Biol. 96, 1803 1808. 5. Choi B. H. and Lapham L. W. (1978) Radial glia in the human fetal cerebrum: a combined Golgi, immunofluorescent and electron microscopic study. Brain Res. 148, 295 311. 6. Connell N. D. and Rheinwald J. G. (1983) Regulation of the cytoskeleton in mesothelial cells: reversible loss of keratin and increase in vimentin during rapid growth in culture. Cell 34, 245 253. 7. Dahl D. (1981) The vimentin~GFA protein transition in rat neuroglia cytoskeleton occurs at the time of myelination. J. Neurosci. Res. 6, 741-748. 8. Dahl D. and Bignami A. (1985) Intermediate filaments in nervous tissue. In Cell and Muscle Motility (ed. Shay J. W.), Vol. 6, pp. 75-96. Plenum Press, New York. 9. Dahl D., Crosby C. J., Sethi J. S. and Bignami A. (1985) Glial fibrillary acidic (GFA) protein in vertebrates: immunofluorescence and immunoblotting study with monoclonal and polyclonal antibodies. J. comp. Neurol. 239, 75-88. 10. Dahl D., Rueger D. C., Bignami A., Weber K. and Osborn M. (1981) Vimentin, the 57,000 dalton protein of fibroblast is the major cytoskeletal component in immature glia. Eur. J. Cell Biol. 24, 191 196. 11. Ebbesson S. O. E. (1976) Morphology of the spinal cord. In Frog Neurobiology. A Handbook (eds Llin~is R. and Precht W.), pp. 679 706. Springer, Berlin. 12. Geisler N., Plessman U. and Weber K. (1982) Related amino acid sequences in neurofilaments and non-neuronal intermediate filaments. Nature 296, 448~450.
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13. Goestschy J. F., Ulrich G., Aunis D. and Ciesielski-Treska J. (1986) The organisation and solubility properties of intermediate filaments and microtubules of cortical astrocytes in culture. J. Neurocytol. 15, 375 387. 14. Henrikson C. K. and Vaughn J. E. (1974) Fine structural relationships between neurites and radial glial processes in developing mouse spinal cord. J. Neurocytol. 3, 659 675. 15. Kartembeck J., Schwechheimer K., Moll R. and Franke W. W. (1984) Attachment of vimentin filaments to desmosomal plaques in human meningiomal cells and arachnoidal tissue. J. Cell Biol. 98, 1072 1081. 16. Lane E. B., Hogan B. L., Kurkinen M. and Garrels J. I. (1983) Co-expression of vimentin and cytokeratins in parietal endoderm cells of early mouse embryo. Nature 303, 701--704. 17. Lazarides E. (1980) Intermediate filaments as mechanical integrators of cellular space. Nature 283, 249-256. 18. Menko A. S., Toyama Y., Boettiger D. and Holzer H. (1983) Altered cell spreading in cytochalasin B: a possible role of intermediate filaments. Molec. cell. Biol. 3, 113 125. 19. Miller R. H. and Liuzzi F. J. (1986) Regional specialization of the radial glial cells of the adult frog spinal cord. J. Neurocytol. 15, 187-196. 20. Moon R. T. and Lazarides E. (1983) Canavanine inhibits vimentin assembly but not its synthesis in chicken embryo erythroid cells. J. Cell Biol. 97, 1309 1314. 21. Osborn M., Franke W. W. and Weber K. (1980) Direct demonstration of the presence of two immunologically distinct intermediate-sized filament systems in the same cell by double immunofluorescence microscopy. Expl Cell Res. 125, 37-J,6. 22. Palay S. L. and Chan-Palay V. (1974) Cerebellar Cortex. Cytology and Organization. Springer, Berlin. 23. Ramon Y Cajal S. (1911) Histologie du systkme nerveux de l'homme et des" vertObrds, Vol. 1. Consejo superior de Investigaciones, Madrid. 24. Sasaki H. and Mannen H. (1981) Morphological analysis of astrocytes in the bullfrog (Rana castebeiana) spinal cord with special reference to the site of attachment of their processes. J. comp. Neurol. 198, 13 35. 25. Schnitzer J., Franke W. W. and Schachner M. (1981) Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system. J. Cell Biol. 90, 435M47. 26. Schonbach C. (1969) The neuroglia in the spinal cord of the newt, Triturus viridescens. J. comp. Neurol. 135, 93 120. 27. Shaw G. and Weber K. (1983) The structure and development of the rat retina: an immunofluorescence microscopical study using antibodies specific of intermediate filament proteins. Eur. J. Cell Biol. 30, 219 232. 28. Sommer I., Lagenaur C. and Schachner M. (1981) Recognition of Bergmann glial and ependymal cells in the mouse nervous system by monoclonal antibody. J. Cell Biol. 90, 448M58. 29. Stensaas L. J. and Stensaas S. S. (1968) Astrocytic neuroglial cells, oligodendrocytes and microgliacytes in the spinal cord of the toad. I. Light microscopy. Z. Zellforsh. 84, 473489. 30. Stensaas L. J. and Stensaas S. S. (1968) Astrocytic neuroglial cells, oligodendrocytes and microgliacytes in the spinal cord of the toad. II. Electron microscopy. Z. Zellforsch. 86, 184~213. 31. Thorn R. (1968) Les salamandres d'Europe, d'Asie et d'Afrique du Nord. Lechevalier, Paris. 32. Van Gehuchten A. (1898) La moelle 6pinidre des larves des batraciens (Salamandra maculosa). Arch. Biologie 15, 599 619. 33. Weber K. and Osborn M. (1981) Microtubules and intermediate filament networks in cells viewed by immunofluorescence microscopy. In Cytoskeletal elements and plasma membrane organization (eds Poste G. and Nicolson G. L.), Vol. 7, pp. 1 53. North Holland, Amsterdam. 34. Zamora A. J. (1978) The ependymal and glial configuration in the spinal cord of urodeles. Anat. Embryol. 154, 67 82. 35. Zamora A. J., Langley O. K., Ciesielski-Treska J. and Mutin M. (1986) Vimentin intermediate filaments in astroglia of Urodela. Abstracts of Tenth European Neuroscience Congress. Neurosci. Lett. (Suppl.) 26, S139. (Accepted 4 Mar~h 1988)
Neuroscience Vol. 27, No. 1, pp. 279 288, 1988
Printed in Great Britain
V I M E N T I N A N D GLIAL F I B R I L L A R Y ACIDIC P R O T E I N F I L A M E N T S IN R A D I A L GLIA OF THE A D U L T U R O D E L E SPINAL C O R D A. J. ZAMORA* and M. MUTIN Unit6 de Rechereches Neurobiologiques, INSERM U 6 and CNRS UA 634, 280, boulevard Sainte Marguerite, 13009 Marseille, France Abstract--This work, based on Golgi impregnations, transmission electron microscopy and immu-
nocytochemistry, demonstrates that the intermediate filaments found in the radial gliocytes of the adult newt spinal cord are both vimentin and glial fibrillary acidic protein (GFAP) structures. Gliocytes appeared as large, arboreous cells, with appendages extending peripherally. They were extensively immunolabelled with both anti-vimentin and anti-GFAP monoclonal antibody conjugates. Outstanding correspondence in cell configuration was found when Golgi-impregnated specimenswere compared to the distribution of immunolabels. Electron micrographs showed cytoplasmic bundles of anti-vimentin decorated intermediate filaments occupying the radial projections. The presence of GFAP confirms the astroglial character of the radial glia in urodeles; the existence of vimentin suggests that the spinal cord of the adult animal retains immature astroglia, which should express enlarged capabilities of adaptation.
The classical works by Van Gehuchten 32 and by Ramon y Caja123 have established the basic cytoarchitectural features of gliocytes in the embryonic CNS of Amphibia. One of the important points to be stressed in these studies is the discovery of macroglial cells whose somata are located in the periependymal region (central field of Ebbesson H) and send long radial cytoplasmic processes that pervade the neuropil and the fibre tracts up to the pial surface. This configuration is present throughout the whole life of the animal. Few cytological studies have been devoted to these glial cells, in spite of their outstanding size and the complexity of their cytoplasmic expansions. The spinal cord of postmetamorphic amphibians, such as toads, 29'3° frogs 24 and newts, 34 is organized upon a complex radial glial framework, which by conventional electron microscopical analysis has been postulated as astroglial in character; in fact, the cytoplasm of the radial processes contains bundles of intermediate filaments (IF) closely resembling those found in mammalian fibrous astrocytes. These filaments occupy mainly the cytoplasmic processes running toward the periphery or the perivascular basal laminae, lmmunocytochemical studies9,/9 have shown that the radial gliocytes of the adult frog spinal cord react positively with antibodies against mammalian glial fibrillary acidic protein (GFAP). On
the other hand, evidence has been presented to support the generalized concept that several kinds of intermediate filament proteins coexist in one cellular population. Concerning astroglia, some studies performed in mammalian nervous tissue have shown, in vivo and in vitro, that astrocytes coexpress both GFAP and vimentin IF. Mammalian astrocytes are vimentin-positive cells in the cerebellum, hippocampus, cerebral cortex, pons and retina. 8,13,25 Since vimentin had been considered as a "mesenchymal" IF protein, these observations are significant by showing that several kinds of cells, irrespective of their embryological parentages, express vimentin synthetizing capabilities, suggesting that vimentin IF play a more general function. The first ultrastructural description of astroglia in urodeles was made by Schoenbach26 who concluded that urodele astroglia differs little from mammalian astrocytes. We have previously described 34 the cytological features as well as the topographical and intercellular relationships of astroglial cells in the central field of the spinal cord of the adult ribbed newt. In the present study we illustrate, by the Golgi impregnation method and by transmission electron microscopy, the radial arborizations of these gliocytes. The observations have been interpreted in connection with current ideas about the function of cytoskeleton in cells displaying complex, changing cytoplasmic shape. A partial report of this work has been previously presented. 35
*To whom correspondence should be addressed at: INSERM U 6, 280, boulevard Sainte Marguerite, 13009 Marseille, France. EXPERIMENTAL PROCEDURES "Abbreviations: DAB, 3,Y-diaminobenzidine; GFAP, glial fibrillary acidic protein; HRP, horseradish peroxidase; Adult male and female ribbed newts Pleurodeles waltlii IF, intermediate filaments; NSS, normal sheep serum; Michaelles31 obtained from SEREA (Argenton l'Eglise, PBS, phosphate-buffered saline. France) were used in this study. The animals were kept in 279
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flesh water at room temperature and fed twice a week. They were aneasthetized by immersion in a 0.01% aqueous solution of tricaine methane sulphonate (Sandoz MS222) and perfused through the conus arteriosus with fixative at room temperature under hydrostatic pressure of 130 mmHg.
Light microscopy After perfusion with 2% paraformaldehyde and 2% glutaraldehyde in 0.2M phosphate buffer, segments of brachial spinal cord were dissected and double impregnated by the rapid Golgi method according to Palay and ChanPalay. 22 The specimens were dehydrated and embedded in nitrocellulose. Sections of 100 #m in thickness were done on a Leitz sliding microtome.
Electron microscopy The tissue processing for standard transmission electron microscopy has been previously described. 34
lmmunocytochemistry The spinal cord was fixed by transcardiac perfusion with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brachial segments of the spinal cord were dissected out, wrapped in 2% agar, and mounted vertically on the chuck of a vibratome (Lancer, U.S.A.). Transverse sections, 50/~ m in thickness, were cut under PBS. Unspecific binding sites were inactivated by preincubation of tissue sections in 5% normal sheep serum (NSS) in PBS for l h at room temperature. Sections were then incubated overnight at 4°C in a PBS-0.02% sodium azide solution of monoclonal antibodies. Adjacent sections were treated with monoclonal antibodies to vimentin or to GFAP. The anti-vimentin monoclonal antibody was obtained from cultured rat astrocytes, and characterized by immunoblotting revealing a single band corresponding to vimentin (for details, see Ref. 13); anti-GFAP monoclonal antibody was purchased from Marseilles Immunotech, code No. 0161. The dilutions employed were 1/ 1000 for vimentin, and 1/1000 for GFAP. Control sections were incubated in similar conditions in 5% NSS in PBS. After four PBS washes the sections treated with anti-vimentin and antiGFAP monoclonal antibodies were incubated for 2h at room temperature in 1/100 dilution of goat anti-mouse IgG conjugated to horseradish peroxidase (HRP). After washing, sections were incubated in 0.05% diaminobenzidine (DAB, Sigma Chemical Co.) and 0.5% hydrogen peroxide for 6 min at room temperature in darkness. They were then rinsed in PBS and mounted for observation and photomicrography. Later they were post-fixed in 1% osmium tetroxide in 0.2 M phosphate buffer for 30 min, embedded in epoxy resin and processed for transmission electron microscopy; unstained 1-#m-thick plastic sections were also microphotographed, and 80 nm thin sections were observed at 60 kV without uranyl or lead contrast enhancement. RESULTS In transverse sections, the urodele spinal cord showed a typical H-shaped central gray surrounded by white matter (Fig. 1). The periependymal gray matter (substantia gliosa centralis) contained the somata of gliocytes, the neurons being placed more peripherally. In the white matter, the cell elements that we observed were only represented by the intrafascicular oligodendrocytes. A m o n g the gliocytes of the gray matter, the most striking elements corresponded to large arboreous cells. Golgi-impregnated sections showed that these cells projected, from the lateral aspect of the cell body, a single long cytoplasmic process that progressively ramified as it
approached the peripheral region after traversing the longitudinal fibre tracts of the white matter. These processes ended about the periphery of the spinal cord. The rest of the surface of the soma developed veil-like cytoplasmic sheets curling around the other components of the gray matter (Fig. 2). In this respect, these cells resembled the Golgi epithelial cells of the mammalian cerebellum. 22 The entire profile of these macrogliocytes made them resemble the so called "astroblast" observed in the spinal cord of the new-born mouse. 23 Electronmicroscopically, four major features characterized the peripheral arborization of these gliocytes, namely glycogen particles, microtubules, 8 n m IF, and tubules of smooth endoplasmic reticulum (Fig. 3). Glycogen particles were homogeneously distributed all over the cell process, including the cytoplamic end-feet surrounding both the pericapillary and the subpial basal laminae. The microtubules as well as the profiles of smooth endoplasmic reticulum were preferentially present in the main shafts of the arborizations: in fact, they were not found in the projections passing through the nerve fibres of the white matter nor in the perivascular or subpial end-feet, where I F were abundantly represented (Fig. 4). In those sites where desmosomes joined together two homologous gliocyte processes, a condition frequently observed at any level of the urodele spinal cord, the peripheral bundles of IF were intermingled to the subplasmalemmal desmosome web (Fig. 3). The only membranous compartment found at the end-foot level was represented by large polymorphic vacuoles limited by smooth membrane units easily observed among the glycogen aggregates (Fig. 4). Transverse adjacent vibratome sections of the newt spinal cord processed for the immunoperoxidase detection of both vimentin and G F A P I F showed the precipitation product in the perinuclear cytoplasm of the gliocytes of the substantia gliosa centralis (Fig. 5A and B), as well as all over the extensive radial arborization that pervaded the spinal cord from the periependymal layer to the subpial stratum (Fig. 6A and B). Extensive vimentin immunolabelling was observed in the tanycytes forming the dorsal aspect of the ependyma, and forming the septum dorsalis of K611iker, which became clearly perceptible (Fig. 5A). Contrarily, these tanycytes and their projections were consistently unlabelled in sections treated with antiG F A P monoclonal antibody (Fig. 5B). Immunostained l-/~m-thick plastic sections showed longitudinal threads running among the cell somata of the gray matter. In this region, few cells displayed perinuclear positive reaction (Fig. 7). The white matter was traversed by radial profiles of precipitation product contacting the subpial zone as typical triangular end-feet (Fig. 8). At the ultrastructural level, anti-vimentin conjugates decorated longitudinal bundles of cytoplasmic filaments, located in correspondence to radial arborizations.
Vimentin and GFAP filaments in urodele gliocytes
Fig. 1. Transverse section of newt brachial spinal cord (1-p m-thick plastic, toluidine blue staining). From the compact gray matter radial projections arise (arrows) and penetrate the white matter through the fascicles of myelinated fibres. These projections correspond in fact to dendrites and/or glial radiations when observed in the electron microscope. Asterisk, central canal, x 200.
Fig. 2. Photomicrograph of the ventrolateral region of the brachial spinal cord. Rapid Golgi method. Two astroglial cells (A) with somata located in the periependymal substantia gliosa (G). Large stems (large arrows) arise from the soma, which also displays some velate formations (small arrows); the peripheral branches of the arborizations end at the subpial level (white arrows), x 500.
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Fig. 3. Electron micrograph of the substantia gliosa centralis. A glial nucleus (N) is shown: the cytoplasm of this cell is contiguous to glial processes where tubular profiles of smooth endoplasmic reticulum, microtubules (large arrows) and intermediate filaments (small arrows) are found. A desmosome (arrowhead) joins a glial cytoplasmic process to the soma of a gliocyte. Cytoplasmic filament bundles intermingle with the desmosomal web. The slender arrows point to glycogen particles, x 31,200.
Fig. 4. Electron micrograph of the peripheral region of the spinal cord. Two glial processes (A1 and A2) show an area of interdigitation (arrowhead) and end in the basal lamina (arrows). The cytoplasm contains glycogen particles, polymorphic vacuoles, and bundles of intermediate filaments (IF). FT, tract of unmyelinated axons transversely sectioned, x 31,200. 282
Fig. 5. Adjacent-50 pm-thick vibratome sections, not counterstained, showing the dorsal region of the brachial spinal cord. (A) Section treated with anti-vimentin monoclonal antibody; the HRP precipitation product is localized mainly in the tanycyte (T) forming the septum dorsalis of K611iker (large arrows); the perinuclear cytoplasm and the radial arborizations of the gliocytes located in the substantia gliosa centralis are also labelled (small arrows). E, ependyma. × 500. (B) Section showing HRP precipitation product of anti-GFAP conjugate. The ependymal cells (E) are not labelled, nor the tanycytes (T) that send projections to form the septum of K611iker (large arrows). The cells of the substantia gliosa centralis are labelled in the perinuclear cytoplasm and in their peripheral radiations (small arrows). S, sulcus medianus dorsalis. × 500. 283
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Fig. 6. Same sections as shown in Fig. 5. The ventrolateral region of the brachial spinal cord displays many fine HRP-immunolabelled radiations (arrows) traversing the white matter (W) and abutting at the periphery. V, ventral horn. (A) Anti-vimentin H R P immunolabelling; (B) anti-GFAP immunolabelling. × 500.
Vimentin and GFAP filaments in urodele gliocytes
Figs 7 and 8. Plastic, 1-#m-thick unstained sections showing HRP precipitation product of anti-vimentin conjugate. Fig. 7. The cells of the substantia gliosa centralis show mild cytoplasmic perinuclear labelling, whereas some dense bundles (arrows) point radially toward the periphery of the spinal cord. The arrowheads point to lipid inclusions in the gliocyte cytoplasm (see Ref. 34). Fig. 8. The peripheral marginal plexus in which several radial labelled bundles (large arrows) cross the fiber tracts to end as triangular subpial end-feet (small arrows), x 350.
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Fig. 9. Electronmicrograph showing a high magnification of one filament bundle shown in Fig. 8. In the cytoplasm of a radial glial projection (A) parallel IF are labelled with HRP product (arrows). Some vacuoles are also labelled (arrowhead). M, myelinated axon. × 90,000. Besides the filament bundles, the marker was also associated with annular or vacuolar structures, about 80-150 nm in diameter (Fig. 9). The control sections incubated with NSS did not reveal any immunoperoxidase precipitation product. DISCUSSION
Using combined cytological and immunological techniques we have shown the astroglial character of the large radial macrogliocytes of the urodele spinal cord. Golgi impregnations allowed us to localize the cell soma in the substantia gliosa centralis and to trace out the profiles of its peripheral expansions. The ultrastructural analysis of these expansions revealed the presence of 8 nm IF and of glycogen particles which were also present in the perivascular and supbial investments. These features are considered as distinctive of astroglia. Furthermore, the arboreous cell shape closely correlated with the distribution of GFAP immunolabels. The expression of GFAP in nervous cells of a wide variety of vertebrates from fish to humans has been reported. 9 G F A P is considered as the specific IF protein of astroglial cells, s As far as we know the astroglial character of radial gliocytes of Urodela had not been immunocytochemically demonstrated in previous reports. Furthermore, the present work is the first demonstration of the presence of both GFAP and vimentin IF in radial macrogliocytes of Urodela. The reactivity of urodele IF
with antisera induced by mammalian antigens confirms the idea that IF proteins have undergone little change in evolutionJ 7 In Bergmann as well as in M/iller glia, which are considered as relatively undifferentiated types of glia, GFAP 3 and vimentin27 have been localized by immunofluorescence. It has been pointed out 28 that astroglia of adult lower vertebrates resemble embryonic astroglia of higher vertebrates. In fact, the frog 19 and the newt (this paper) astroglial cells are morphologically indistinguishable from the radial gliocytes of the new born mouse spinal cord, 14'23 and also resemble the radial glia of the 7-week-old human fetal cerebrum. 5 The morphological "immaturity" of astroglia in lower vertebrates may be associated with embryonic types of cell behaviour, such as pluripotentiality, enhanced cellular motility and migratory capabilities, reappearance of mitotic activity or cell pattern generation. Function pluripotentialitymay be related to the spatial compartition of cell constituents in the cytoplasm. In this context, the work of Miller and Liuzzi 19 is significant. These authors have shown the preferential distribution of GFAP at the more peripheral branches of the frog radial gliocytes; they have postulated that this differential distribution expresses the dual capacity of these gliocytes to combine the functions of both white matter (peripheral branches) and gray matter (central branches, somata) astrocytes of mammals. Notwithstanding, the idea of functional compartition deserves further investigation. In our
Vimentin and GFAP filaments in urodele gliocytes material, the precipitation of H R P was homogenously distributed. This difference between Anura and Urodela gives rise to the question of whether urodele astroglia stand at a more primitive level of differentiation in comparison to their anuran homologues. In vitro and in situ coexistence of two different types of IF in cells has been firmly establishedJ 6,17,21,33 The meaning of this condition is still not completely understood. During development of mouse neuroectoderm, vimentin precedes the appearance of G F A P . 25 As astrocytes mature, there is a vimentin to G F A P transition, 7 suggesting that undifferentiated cells express vimentin as their main cytoskeletal system. Vimentin filaments form by assembling soluble precursors 4 through links established at the headpiece level of the molecule. ~2'2° In desmofibrocytes, vimentin filments attach together with cytokeratin filaments to desmosomes. ~5 Vimentin filaments can therefore be considered as elements integrating the true cytoskeleton of relatively undifferentiated, growing and proliferating cells. ~°'33The levels of vimentin correlate well with a rapid cell growth in vitro. 2'6 The complexity of cell shape is one of the most impressive features of radial gliocytes. The spreading of cytoplasm through branches, sheets, terminal investments and filopodia-like projections results in the constitution of a macrocellular system with multiple interaction possibilities. Experiences performed in cultured mammalian cells, in which the cell shape can be modified by changing the composition of the milieu, indicate that the cell configuration influences cell behaviour. In particular, it has been shown that the morphological state of the cell modifies the
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biosynthesis of vimentin. 1 In cultured epithelial cells keratin and vimentin syntheses are related to the extent of cell contacts and to the changes in cell shape, respectively. 2 An increase in the projected cell area is accompanied by an increment in the content of vimentin; for instance, the pseudopodial arborizations developed by cells treated with cytochalasin B display vimentin exclusively. 18 Conversely, cell rounding is accompanied by a depression in vimentin synthesis. 2 Thus, phenomena observed in cultured cells can lead to a better understanding of the presence of vimentin IF in embryonic types of astroglia, a condition that can be related to the arboreous shape of these cells. The immature character of the cytoskeleton could be associated to an increased capability to change the tridimensional configuration of the cytoplasm. In the case of embryos, cells are continuously modifying their shape configuration as overt ontogenetic events proceed. Does the presence of vimentin-rich radial gliocytes in adult animals imply the existence of larger remodelling capabilities? An answer to this question should be advanced through the analysis of astroglial cytoskeletal changes during regeneration of the spinal cord. In these circumstances, the process normally observed in maturation, i.e. the vimentin to G F A P shift, should be reproduced, leading to the predominence of vimentin in the astroglial cytoplasm during the early stages of the regenerative period. Acknowledgements--The authors are grateful to Dr J.
Ciesielski-Treska who kindly provided the vimentin antiserum, and to Dr O. K. Langley for helpful discussions.
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