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Immunocytochemical characterization of cloned cells from normal adult rat brain
Neuroscience Letters, 42 (1983) 213-218
213
Elsevier Scientific Publishers Ireland Ltd.
IMMUNOCYTOCHEMICAL CHARACTERIZATION OF CLONED CELLS FROM NORMAL ADULT RAT BRAIN
GEOFFREY J. PILKINGTON, DAVID A. SMITH and PETER L. LANTOS
Department o f Neuropathology, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF (U.K.) (Received August 4th, 1983; Accepted September 16th, 1983)
Key words: cloned cells - rat brain - glutamine synthetase - vimentin - fibronectin - glial fibrillary acidic protein
~.
A cell clone, isolated from normal adult rat brain and maintained in culture for many passages, has been previously characterized by electron microscopy, lmmunohistochemistry has now been shown that it possesses glutamine synthetase (GS) which supports its astrocytic origin. Cells failed, however, to express glial fibrillary acidic protein (GFAP) but did express the intermediate filament protein vimentin. The presence of fibronectin confirms the normal derivation of these cells and excludes the possibility of malignant transformation.
The importance of a cell line composed of normal glial cells, as a control for astrocytes treated with the chemical carcinogen N-ethyl-N-nitrosourea (ENU) and for cultures derived from ENU-induced brain tumours, has been previously reported [23]. A cell clone - ARBO C9 - obtained from the periventricular region of a normal adult BDIX rat brain has been shown in an ultrastructural study to possess astrocytic characteristics, including the presence of 9-11 nm diameter filaments [23]. In the present work antisera to the two astrocyte-specific markers, glial fibriUary acidic protein (GFAP) and glutamine synthetase (GS), to vimentin an intermediate filament protein of certain cultured cells including astrocytes - and to fibronectin, a protein reported to be reduced or lost in malignantly transformed cells [16, 26-28], have been used to characterise further the constituent cells of the ARBO C9 clone by indirect immunofluorescence. GFAP [8, 10] has, for over a decade, been successfully used as an intracellular marker specific for astrocytes in both immunoperoxidase [7, 9] and immunofluorescence [1] techniques. GS, a cytoplasmic enzyme which plays an important role in the detoxification of ammonia and in the metabolism of the putative excitatory neurotransmitter glutamate [3, 19], has also been described as an astrocytic-specific marker in the central nervous system of the rat [20]. The 57,000-58,000 molecular weight protein vimentin, first isolated from the 0304-3940/83/$ 03.00 © 1983 Elsevier Scientific Publishers ireland Ltd.
21.1 cvtoskeleton of routine mesenchymal cells [121, has subsequently been found - by immunofluoresccnce and polyacrylamide gel electrophoresis - to exist in glial cells, both in vitro and in xivo, including primary astrocyte cultures from immature rat brain 121 and astrocytes of the optic nerves of blinded rats [4]. Indeed it has been proposed that vimentin is the major cytoskeletal component of immature glia [5]. The high molecular veeight glycoprotein fibronectin, first isolated from the cell surface of fibroblasts [25], has subsequently been demonstrated on normal and neoplastic glia and fibroblasts in culture [27]. Fibronectin is localized by immunofluorescence on both the cell membrane in fibrillar striae and intracellularly as a microgranular reaction product, predominantly in the perinuclear region [27]. In the present ,qudv cells were grov, n to confluence on 13 mm round coverslips maintained in Dulbecco's modification of Eagle's medium containing 15% foetal caif scttltl'l and l°b penicillin, streptomycin ampholericin. ('ells of passage numbers 199 and 409 x~e.re used. Since dibulyrVl adenosine 3'5'-cyclic monophosphate ~dbc.-\~lPl has been knoven to induce GFAP in glial cells [24], this cyclic nucleotide x~a~, added to the cuhurc rhodium in some flasks at a final concentration of 1 raM. (ells on coxerslips were washed in Hanks' balanced salt solution and fixed in acid alcohol for 1() rain. After a brief wash in phosphate-buffered saline (PBS}, they were ,.torcd ill PBS at 4:'(" until required for immtlnocytochemistrv. The indirect im!!~tlllt~lltiorescencc technique ~as then carried out. (?ells were incubated for 1 h at room tcnlpcr,tturc in either ral+bil anti-boxine til:Ai' llSl, rabbit anti-ovine (}S [22], ~,tbbit ant i-tlam,:ter vimcnlin 1171, rabbit anti-iluman l'ibroncctin, or normal rabbit ,,crtttll (ncgatixc Colllrol}. lu each c,lsc the dilution was 1:50 in !% ovalbumin in I'B.";...\ttcr x~ashine for 5 rain in IJi+S lhc cells veerc incubated in swine anti-rabbit tiuore,,cein (1:1 I ( ) or rhodamine {IRIT('I, at 1:20 dilution. The coxerslips were agztitl ~ashcd in PBS then mounted in 90oo glycc,oi in PBS and examined in a Zeiss Phototllicroscope 111 incorporating re!'lected light fluorescence excitation with barricr filter,, for F I | ( " and FRII(7. .~taining for (iN x~as positive: discrete, brightly fluorescing granules and linear arrays \~ete present in the cytoplasm, particularly in the perinuclear region {Fig. 1), x~hilt nuclei remained unlabelled. ('ells treated x~ith dbcAMP and untreated cells x~ete both negatixc for (;i:AP. A fine, but intense, librillar staining for vimentin was detected in the cytoplasm of the cells (Fig. 2), ~ hilt nuclei again remained negative. l he cells ~ere also strongly positive for fibronectin (Fig. 3}, the reaction product bcillg tllO',t maTkcd at cell to cell boundaries. Staining was also apparent in a net-like pattern oxcr iudixidual cells" this resulted from the ,,tainino of closely apposed folds in t!~e cell membrane. A positixe intracellular reaction yeas also detected in the cylopla~,m which corresponded to the microgranular perinuch:'ar staining of glia described by Vaheri et al. [27]. No differences in inamunocytochemical staining were apparetlt bctv+een cells of passage 199 and passage 409. Controls incubated in norreal rabbit seturn sho~,+ed no fluorescence reaction. (+dis trom ARBO C9, a cloned line from normal adult rat brain, show the fine
~iii!!!il¸~,~
~•~~ ¸~¸I¸¸~¸¸
~ ,'~i!!ii!~I~
216
Fig. 3. Intense extracellular tluorescence is detected at cell-cell boundaries (arrows} as well as cytoplasmic microgranular staining. Rabbit anti-human fibronectin/TRITC, x 600.
structural features of astrocytes [23], and, by scanning electron microscopy, are seen to express little surface activity [28], unlike neoplastic gila. Moreover, the clone was not malignant when injected into syngeneic animals [15] (also Skidmore, C.J. and Roscoe, J.P., unpublished results) and failed to demonstrate other properties of glioma cells [23]. The present study was undertaken in order to obtain immunocytochemical data on both the astrocytic and the non-malignant nature of ARBO C9. The expression of GS antigen in-ARBO C9 cells strongly supports their astrocytic lineage. The precise localization of the discrete cytoplasmic reaction sites may be related to the organelles where the enzyme is produced; the rough endoplasmic reticulum and the polyribosomes. Fibronectin has been reported to be decreased or lost in cells transformed in vitro [16, 26, 27, 29] therefore its strong expression in the ARBO C9 clone suggests that although this normal glial line has been maintained in culture for more than 400 passages over a period of several years, it has not undergone spontaneous transformation. Although the presence of a number of glial-specific markers is invaluable in the positive identification of astrocytes, the absence of GFAP in ARBO C9 does not necessarily exclude its glial nature. Indeed cloned cell lines rarely express this antigen. Negative staining for GFAP in ABRO C9 is, moreover, entirely consistent with the observations of Paetau et ai. [21] on the mutually exclusive expression of fibronectin and GFAP in
217
normal cultured brain cells. This could be related to a possible dedifferentiation of the astrocytes in tissue culture. For example, the dedifferentiation of chondrocytes was found to result in increased expression of fibronectin [14]. Since immunological staining of ARBO C9 for GFAP was carried out at high passage levels (199 and 409), it is possible that this antigen has been lost with repeated subcultivation - a phenomenon previously described in normal human brain cells in vitro [13]. Alternatively, since vimentin has been reported to occur first in undifferentiated, immature astrocytes containing small groups of intermediate filaments and gradually decreases with a concomitant increase of GFAP and bundling of intermediate filaments [6, 1l], the presence of this antigen in ARBO C9 implies that the cells may be poorly differentiated or immature astrocytes. Since the original culture was taken from the subependymal plate region - an area of undifferentiated, mitotically active cells - of a BDIX rat (J.P. Roscoe, personal communication), the cloned cells, showing some astrocytic features, may be derived from an astroblastic precursor. Therefore, although the precise nature of the cells has not been established, the findings of this study nevertheless add some immunocytochemical evidence for the astrocytic lineage of the ARBO C9 clone and are in agreement with the nonmalignant characteristics described in previous reports. This study was supported by the Bethlem Royal Hospital and Maudsley Hospital Research Fund. We are indebted to Dr. J.P. Roscoe for the ARBO C9 cells. The rabbit anti-human fibronectin was the gift of Dr. D. McCormick, the rabbit antibovine GFAP was provided by Dr. B. Anderton and Dr. J. Yahn and the rabbit anti-hamster vimentin by Dr. R. Hynes. The technical assistance of Miss J. Martin and the secretarial assistance of Mrs. J. Bewry are gratefully acknowledged. ! Bignami, A., Eng, I+.F., Dahl, D. and Uyeda, C.T., Localization of the t.:lial fibrillary acidic protein in astrocytes by immunofluorescence, Brain Res., 43 (1972) 429-435. 2 Chiu, F.-C., Fields, K.L. and Norton, W.T., Cultured astrocytes contain the 58,000 MW fibroblast filament protein, Trans. Amer. Soc. Neurochem., I1 (1980) 105. 3 Curtis, D.R. and Watkins, J.C., The excitation and depression of spinal neurones by structurally related amino acids, J. Neurochem., 6 (1960) !17-141. 4 Dahl, D., Bignami, A., Weber, K. and Osborn, M., Filament proteins in rat optic nerves undergoing Wallerian degeneration: localization of vimentin, the fibroblastic IO0-A filament protein, in normal and reactive astrocytes, Exp. Neurol., 73 (1981) 496-506. 5 Dahl, D., Rueger, D.C., Bignami, A., Weber, K. and Osborn, M., Vimentin, the 57,000 molecular weight protein of fibroblast filaments, is the major cytoskeletal component in immature glia, tiurop. J. Cell Biol., 24 (1981) 191-196. 6 Dahl, D., Strocchi, P. and Bignami, A., Vimentin in the central nervous system. A study of the mesenchymal-type intermediate filament-protein in Wallerian degeneration and ip postnatal rat development by two-dimensional gel electrophoresis, Differentiation, 22 (1982) 185-190. 7 Duffy, P.E., Graf, L. and Rapport, M.M., Identification of glial fibrillary acidic protein by the immunoperoxidase method in human brain tumors, J. Neuropath. exp. Neuroi., 36 (1977)645-652. 8 Eng, L.F., Gerstl, B. and Vanderhaeghen, J.J., A study of proteins in old multiple sclerosis plaques, Trans. Amer. Soc. Neurochem.. I (1970)42.
218 9 Eng, L.F. and Rubinstein, L.F., Contribution of immunohistochemistry to diagnostic problems of human cerebral tumors, J. Histochem. Cytochem., 26 (1978) 513-522. 10 Eng, L.F., Var~derhaeghen, J.J., Bignami, A. and Gerstl, B., An acidic protein isolated from fibrous astrocytes, Brain Res., 28 (1971) 351-354. 11 Fedoroff, S., White, R., Neal, J., Subrahmanyan, L. and Kalnins, V.l., Astrocyte cell lineage. II. Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin- and GFA-containing intermediate filaments, Develop. Brain Res., 7 (1983) 303-315. 12 Franke, W.W., Schmid, E., Osborn, M. and Weber, K., Different intermediate-sized filaments distinguished by immunofluorescence microscopy, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 5034-5038. 13 Gilden, D.H., Molenaar, !., Devlin, M., Eng, L. and Wroblewska, Z., Human brain in tissue culture. Vl. Presence of glial fibrillary acidic protein in subcultivated human fetal brain cells as demonstrated by immunofluorescent and immunoperoxidase staining, Acta neuropath., 53 (1981) 327-330. 14 Hassel, J.R., Pennypacker, J.P., Yamada, K.M. and Pratt, R.M., Changes in cell surface protein during normal and vitamin A-inhibited chondrogenesis in vitro, Ann. N.Y. Acad. Sci., 312 (1978) 406-409. 15 Hince, T.A. arid Roscoe, J.P., Fibrinolytic activity of cultured cells derived during ethylnitrosoureainduced carcinogenesis of rat brain, Brit. J. Cancer, 37 (1978)424-433. 16 Hynes, R.O., Cell surface proteins and malignant transformation, Biochim. biophys. Acta, 458 (1976) 73-107. 17 Hynes, R.O. and Destree, A.T., l0 nm filaments in normal and transformed cells, Cell, 13 (197~;) 151-163. 18 Kahn, J., Lamos, P.l., Green, P.J., Wood, J.N., Dowding, A. and Anderton, B.H., Structural heterogeneity of GFAP specific filaments: an immunohistochemical study, Neuropath. appl. Neurobiol., 9 11983) 330. 19 Krnjevic, K., Actions of drugs on single neurones in the cerebral cortex, Brit. med. Bull., 21 (1965) 10-14. 2(1 Norenberg, M.D., Tile distribution of glulamine synthetase in the rat central nervous s~,'stem, J. Histochem.. ('ytochcm., 27 (1979) 756-762. 21 Paetau, A., Mcllstrom, K., Westermark, B., Dahl, D., Haltia, M. and Vaheri. A., Mutually exclusive expression of fibronectin and glial fibrillary acidic protein in cultured brain cells, Exp. Cell Res., 129 (1980) 337-344. 22 Pilkington, G.J. and l.antos, P.L., The role of glutamine syntl~'tase in the diagnosis of cerebral turnouts, Neuropath. Appl. Neurobiol., 8 (1982) .~_7-,36. ",9 23 Pilkington, G,J., t.antos, P.I.. and Roscoe, J.P., The fine structure of cloned cells from a normal adult rat brain, Experientia, 36 (1980) 194-195. 24 Raju, T.R., Bignami, A. and Dahl, D., Glial fibrillary acidic protein in monolaycr cultures of C-6 glioma ,:ells: effect of aging and dibutyryl cyclic AMP, Brain Res., 201, (1980) 225-230. 25 Ruoslahti, E., Vaheri, A., Kuusela, P. and Linder, E.,.lzibroblast surface antigen: a new serum prorein, Biochim. biophvs. "" (1973) 352-3¢,8. . Acta, 3,.,. 26 Vaheri, A. and Mosher, D.F., High molecular weight, cell surface-associated glycoprotein (fibronectin) lost in malignant transformation, Biochim. biophys. Acta, 516 (1978) 1-25. 27 Vaheri, A., Ruoslahti, E., Westermark, B. and Pont6n, J., A common cell-type specific surface antigen in cultured human glial cells and fibroblasts: loss in mali .nt cells, J. exp. Med., 143 (1976) 64-72. 28 ~Vinslow, D.P.. Roscoe, J.P. and Rowles, P.M., Changes in surface nlorphology associated with ethylnitrosourea-induccd malignant transformation of cultured rat brain cells studied by scanning electron microscopy, Brit. J. exp. Path., 59 (1978) 530-539. 29 Yamada, K.M. and Olden, K., Fibronectins - adhesive glycoproteins of cell s~-:f~cc and blood, Nature (Lond.), 275 (1978) 179-184.
213
Elsevier Scientific Publishers Ireland Ltd.
IMMUNOCYTOCHEMICAL CHARACTERIZATION OF CLONED CELLS FROM NORMAL ADULT RAT BRAIN
GEOFFREY J. PILKINGTON, DAVID A. SMITH and PETER L. LANTOS
Department o f Neuropathology, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF (U.K.) (Received August 4th, 1983; Accepted September 16th, 1983)
Key words: cloned cells - rat brain - glutamine synthetase - vimentin - fibronectin - glial fibrillary acidic protein
~.
A cell clone, isolated from normal adult rat brain and maintained in culture for many passages, has been previously characterized by electron microscopy, lmmunohistochemistry has now been shown that it possesses glutamine synthetase (GS) which supports its astrocytic origin. Cells failed, however, to express glial fibrillary acidic protein (GFAP) but did express the intermediate filament protein vimentin. The presence of fibronectin confirms the normal derivation of these cells and excludes the possibility of malignant transformation.
The importance of a cell line composed of normal glial cells, as a control for astrocytes treated with the chemical carcinogen N-ethyl-N-nitrosourea (ENU) and for cultures derived from ENU-induced brain tumours, has been previously reported [23]. A cell clone - ARBO C9 - obtained from the periventricular region of a normal adult BDIX rat brain has been shown in an ultrastructural study to possess astrocytic characteristics, including the presence of 9-11 nm diameter filaments [23]. In the present work antisera to the two astrocyte-specific markers, glial fibriUary acidic protein (GFAP) and glutamine synthetase (GS), to vimentin an intermediate filament protein of certain cultured cells including astrocytes - and to fibronectin, a protein reported to be reduced or lost in malignantly transformed cells [16, 26-28], have been used to characterise further the constituent cells of the ARBO C9 clone by indirect immunofluorescence. GFAP [8, 10] has, for over a decade, been successfully used as an intracellular marker specific for astrocytes in both immunoperoxidase [7, 9] and immunofluorescence [1] techniques. GS, a cytoplasmic enzyme which plays an important role in the detoxification of ammonia and in the metabolism of the putative excitatory neurotransmitter glutamate [3, 19], has also been described as an astrocytic-specific marker in the central nervous system of the rat [20]. The 57,000-58,000 molecular weight protein vimentin, first isolated from the 0304-3940/83/$ 03.00 © 1983 Elsevier Scientific Publishers ireland Ltd.
21.1 cvtoskeleton of routine mesenchymal cells [121, has subsequently been found - by immunofluoresccnce and polyacrylamide gel electrophoresis - to exist in glial cells, both in vitro and in xivo, including primary astrocyte cultures from immature rat brain 121 and astrocytes of the optic nerves of blinded rats [4]. Indeed it has been proposed that vimentin is the major cytoskeletal component of immature glia [5]. The high molecular veeight glycoprotein fibronectin, first isolated from the cell surface of fibroblasts [25], has subsequently been demonstrated on normal and neoplastic glia and fibroblasts in culture [27]. Fibronectin is localized by immunofluorescence on both the cell membrane in fibrillar striae and intracellularly as a microgranular reaction product, predominantly in the perinuclear region [27]. In the present ,qudv cells were grov, n to confluence on 13 mm round coverslips maintained in Dulbecco's modification of Eagle's medium containing 15% foetal caif scttltl'l and l°b penicillin, streptomycin ampholericin. ('ells of passage numbers 199 and 409 x~e.re used. Since dibulyrVl adenosine 3'5'-cyclic monophosphate ~dbc.-\~lPl has been knoven to induce GFAP in glial cells [24], this cyclic nucleotide x~a~, added to the cuhurc rhodium in some flasks at a final concentration of 1 raM. (ells on coxerslips were washed in Hanks' balanced salt solution and fixed in acid alcohol for 1() rain. After a brief wash in phosphate-buffered saline (PBS}, they were ,.torcd ill PBS at 4:'(" until required for immtlnocytochemistrv. The indirect im!!~tlllt~lltiorescencc technique ~as then carried out. (?ells were incubated for 1 h at room tcnlpcr,tturc in either ral+bil anti-boxine til:Ai' llSl, rabbit anti-ovine (}S [22], ~,tbbit ant i-tlam,:ter vimcnlin 1171, rabbit anti-iluman l'ibroncctin, or normal rabbit ,,crtttll (ncgatixc Colllrol}. lu each c,lsc the dilution was 1:50 in !% ovalbumin in I'B.";...\ttcr x~ashine for 5 rain in IJi+S lhc cells veerc incubated in swine anti-rabbit tiuore,,cein (1:1 I ( ) or rhodamine {IRIT('I, at 1:20 dilution. The coxerslips were agztitl ~ashcd in PBS then mounted in 90oo glycc,oi in PBS and examined in a Zeiss Phototllicroscope 111 incorporating re!'lected light fluorescence excitation with barricr filter,, for F I | ( " and FRII(7. .~taining for (iN x~as positive: discrete, brightly fluorescing granules and linear arrays \~ete present in the cytoplasm, particularly in the perinuclear region {Fig. 1), x~hilt nuclei remained unlabelled. ('ells treated x~ith dbcAMP and untreated cells x~ete both negatixc for (;i:AP. A fine, but intense, librillar staining for vimentin was detected in the cytoplasm of the cells (Fig. 2), ~ hilt nuclei again remained negative. l he cells ~ere also strongly positive for fibronectin (Fig. 3}, the reaction product bcillg tllO',t maTkcd at cell to cell boundaries. Staining was also apparent in a net-like pattern oxcr iudixidual cells" this resulted from the ,,tainino of closely apposed folds in t!~e cell membrane. A positixe intracellular reaction yeas also detected in the cylopla~,m which corresponded to the microgranular perinuch:'ar staining of glia described by Vaheri et al. [27]. No differences in inamunocytochemical staining were apparetlt bctv+een cells of passage 199 and passage 409. Controls incubated in norreal rabbit seturn sho~,+ed no fluorescence reaction. (+dis trom ARBO C9, a cloned line from normal adult rat brain, show the fine
~iii!!!il¸~,~
~•~~ ¸~¸I¸¸~¸¸
~ ,'~i!!ii!~I~
216
Fig. 3. Intense extracellular tluorescence is detected at cell-cell boundaries (arrows} as well as cytoplasmic microgranular staining. Rabbit anti-human fibronectin/TRITC, x 600.
structural features of astrocytes [23], and, by scanning electron microscopy, are seen to express little surface activity [28], unlike neoplastic gila. Moreover, the clone was not malignant when injected into syngeneic animals [15] (also Skidmore, C.J. and Roscoe, J.P., unpublished results) and failed to demonstrate other properties of glioma cells [23]. The present study was undertaken in order to obtain immunocytochemical data on both the astrocytic and the non-malignant nature of ARBO C9. The expression of GS antigen in-ARBO C9 cells strongly supports their astrocytic lineage. The precise localization of the discrete cytoplasmic reaction sites may be related to the organelles where the enzyme is produced; the rough endoplasmic reticulum and the polyribosomes. Fibronectin has been reported to be decreased or lost in cells transformed in vitro [16, 26, 27, 29] therefore its strong expression in the ARBO C9 clone suggests that although this normal glial line has been maintained in culture for more than 400 passages over a period of several years, it has not undergone spontaneous transformation. Although the presence of a number of glial-specific markers is invaluable in the positive identification of astrocytes, the absence of GFAP in ARBO C9 does not necessarily exclude its glial nature. Indeed cloned cell lines rarely express this antigen. Negative staining for GFAP in ABRO C9 is, moreover, entirely consistent with the observations of Paetau et ai. [21] on the mutually exclusive expression of fibronectin and GFAP in
217
normal cultured brain cells. This could be related to a possible dedifferentiation of the astrocytes in tissue culture. For example, the dedifferentiation of chondrocytes was found to result in increased expression of fibronectin [14]. Since immunological staining of ARBO C9 for GFAP was carried out at high passage levels (199 and 409), it is possible that this antigen has been lost with repeated subcultivation - a phenomenon previously described in normal human brain cells in vitro [13]. Alternatively, since vimentin has been reported to occur first in undifferentiated, immature astrocytes containing small groups of intermediate filaments and gradually decreases with a concomitant increase of GFAP and bundling of intermediate filaments [6, 1l], the presence of this antigen in ARBO C9 implies that the cells may be poorly differentiated or immature astrocytes. Since the original culture was taken from the subependymal plate region - an area of undifferentiated, mitotically active cells - of a BDIX rat (J.P. Roscoe, personal communication), the cloned cells, showing some astrocytic features, may be derived from an astroblastic precursor. Therefore, although the precise nature of the cells has not been established, the findings of this study nevertheless add some immunocytochemical evidence for the astrocytic lineage of the ARBO C9 clone and are in agreement with the nonmalignant characteristics described in previous reports. This study was supported by the Bethlem Royal Hospital and Maudsley Hospital Research Fund. We are indebted to Dr. J.P. Roscoe for the ARBO C9 cells. The rabbit anti-human fibronectin was the gift of Dr. D. McCormick, the rabbit antibovine GFAP was provided by Dr. B. Anderton and Dr. J. Yahn and the rabbit anti-hamster vimentin by Dr. R. Hynes. The technical assistance of Miss J. Martin and the secretarial assistance of Mrs. J. Bewry are gratefully acknowledged. ! Bignami, A., Eng, I+.F., Dahl, D. and Uyeda, C.T., Localization of the t.:lial fibrillary acidic protein in astrocytes by immunofluorescence, Brain Res., 43 (1972) 429-435. 2 Chiu, F.-C., Fields, K.L. and Norton, W.T., Cultured astrocytes contain the 58,000 MW fibroblast filament protein, Trans. Amer. Soc. Neurochem., I1 (1980) 105. 3 Curtis, D.R. and Watkins, J.C., The excitation and depression of spinal neurones by structurally related amino acids, J. Neurochem., 6 (1960) !17-141. 4 Dahl, D., Bignami, A., Weber, K. and Osborn, M., Filament proteins in rat optic nerves undergoing Wallerian degeneration: localization of vimentin, the fibroblastic IO0-A filament protein, in normal and reactive astrocytes, Exp. Neurol., 73 (1981) 496-506. 5 Dahl, D., Rueger, D.C., Bignami, A., Weber, K. and Osborn, M., Vimentin, the 57,000 molecular weight protein of fibroblast filaments, is the major cytoskeletal component in immature glia, tiurop. J. Cell Biol., 24 (1981) 191-196. 6 Dahl, D., Strocchi, P. and Bignami, A., Vimentin in the central nervous system. A study of the mesenchymal-type intermediate filament-protein in Wallerian degeneration and ip postnatal rat development by two-dimensional gel electrophoresis, Differentiation, 22 (1982) 185-190. 7 Duffy, P.E., Graf, L. and Rapport, M.M., Identification of glial fibrillary acidic protein by the immunoperoxidase method in human brain tumors, J. Neuropath. exp. Neuroi., 36 (1977)645-652. 8 Eng, L.F., Gerstl, B. and Vanderhaeghen, J.J., A study of proteins in old multiple sclerosis plaques, Trans. Amer. Soc. Neurochem.. I (1970)42.
218 9 Eng, L.F. and Rubinstein, L.F., Contribution of immunohistochemistry to diagnostic problems of human cerebral tumors, J. Histochem. Cytochem., 26 (1978) 513-522. 10 Eng, L.F., Var~derhaeghen, J.J., Bignami, A. and Gerstl, B., An acidic protein isolated from fibrous astrocytes, Brain Res., 28 (1971) 351-354. 11 Fedoroff, S., White, R., Neal, J., Subrahmanyan, L. and Kalnins, V.l., Astrocyte cell lineage. II. Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin- and GFA-containing intermediate filaments, Develop. Brain Res., 7 (1983) 303-315. 12 Franke, W.W., Schmid, E., Osborn, M. and Weber, K., Different intermediate-sized filaments distinguished by immunofluorescence microscopy, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 5034-5038. 13 Gilden, D.H., Molenaar, !., Devlin, M., Eng, L. and Wroblewska, Z., Human brain in tissue culture. Vl. Presence of glial fibrillary acidic protein in subcultivated human fetal brain cells as demonstrated by immunofluorescent and immunoperoxidase staining, Acta neuropath., 53 (1981) 327-330. 14 Hassel, J.R., Pennypacker, J.P., Yamada, K.M. and Pratt, R.M., Changes in cell surface protein during normal and vitamin A-inhibited chondrogenesis in vitro, Ann. N.Y. Acad. Sci., 312 (1978) 406-409. 15 Hince, T.A. arid Roscoe, J.P., Fibrinolytic activity of cultured cells derived during ethylnitrosoureainduced carcinogenesis of rat brain, Brit. J. Cancer, 37 (1978)424-433. 16 Hynes, R.O., Cell surface proteins and malignant transformation, Biochim. biophys. Acta, 458 (1976) 73-107. 17 Hynes, R.O. and Destree, A.T., l0 nm filaments in normal and transformed cells, Cell, 13 (197~;) 151-163. 18 Kahn, J., Lamos, P.l., Green, P.J., Wood, J.N., Dowding, A. and Anderton, B.H., Structural heterogeneity of GFAP specific filaments: an immunohistochemical study, Neuropath. appl. Neurobiol., 9 11983) 330. 19 Krnjevic, K., Actions of drugs on single neurones in the cerebral cortex, Brit. med. Bull., 21 (1965) 10-14. 2(1 Norenberg, M.D., Tile distribution of glulamine synthetase in the rat central nervous s~,'stem, J. Histochem.. ('ytochcm., 27 (1979) 756-762. 21 Paetau, A., Mcllstrom, K., Westermark, B., Dahl, D., Haltia, M. and Vaheri. A., Mutually exclusive expression of fibronectin and glial fibrillary acidic protein in cultured brain cells, Exp. Cell Res., 129 (1980) 337-344. 22 Pilkington, G.J. and l.antos, P.L., The role of glutamine syntl~'tase in the diagnosis of cerebral turnouts, Neuropath. Appl. Neurobiol., 8 (1982) .~_7-,36. ",9 23 Pilkington, G,J., t.antos, P.I.. and Roscoe, J.P., The fine structure of cloned cells from a normal adult rat brain, Experientia, 36 (1980) 194-195. 24 Raju, T.R., Bignami, A. and Dahl, D., Glial fibrillary acidic protein in monolaycr cultures of C-6 glioma ,:ells: effect of aging and dibutyryl cyclic AMP, Brain Res., 201, (1980) 225-230. 25 Ruoslahti, E., Vaheri, A., Kuusela, P. and Linder, E.,.lzibroblast surface antigen: a new serum prorein, Biochim. biophvs. "" (1973) 352-3¢,8. . Acta, 3,.,. 26 Vaheri, A. and Mosher, D.F., High molecular weight, cell surface-associated glycoprotein (fibronectin) lost in malignant transformation, Biochim. biophys. Acta, 516 (1978) 1-25. 27 Vaheri, A., Ruoslahti, E., Westermark, B. and Pont6n, J., A common cell-type specific surface antigen in cultured human glial cells and fibroblasts: loss in mali .nt cells, J. exp. Med., 143 (1976) 64-72. 28 ~Vinslow, D.P.. Roscoe, J.P. and Rowles, P.M., Changes in surface nlorphology associated with ethylnitrosourea-induccd malignant transformation of cultured rat brain cells studied by scanning electron microscopy, Brit. J. exp. Path., 59 (1978) 530-539. 29 Yamada, K.M. and Olden, K., Fibronectins - adhesive glycoproteins of cell s~-:f~cc and blood, Nature (Lond.), 275 (1978) 179-184.