antz, A. (1986) Dev. Brain Res. 26, 23-33. 18. Kellerman, 0. & Kelly, F. (1986) Differentiation 32, 74-81. 19. Alliot, F. & Pessac, B. (1984) Brain Res. 306, 283-297.
Proc. Nati. Acad. Sci. USA Vol. 87, pp. 3062-3066, April 1990 Developmental Biology
Immortalization of bipotential and plastic glio-neuronal precursor cells (differentiation/neural celis/brain/oncogenes/retrovirus)
CLAUDINE EVRARD*, ISABELLE BORDE*, PHILIPPE MARINt, ERIC GALIANA*, JOEL PRtMONTt, FRANCOIS GROS*, AND PIERRE ROUGET*t *Laboratoire de Biochimie Cellulaire, Cedex 05, France
tLaboratoire de Neuropharmacologie, College de France et Universitd Paris 6, 11, place Marcelin Berthelot, 75231 Paris
Communicated by Andre Lwoff, January 22, 1990
ABSTRACT Permanent clonal cell lines from newborn mouse striatum have been established after transfer of the simian virus 40 large tumor oncogene by means of a retroviral vector. Some of the lines obtained displayed properties of bipotential and plastic glio-neuronal precursors. Depending on the culture conditions, these cells express either the glial fibrillary acidic protein or neuroframents. In addition, the cells can display adrenergic, D1 and D2 dopaminergic, muscarinic, and 5-hydroxytryptamine type 2 serotoninergic receptors, which are coupled either to the adenylate cyclase or to the phosphatidylinositol signaling pathways. The panel of receptors for neurotransmitters exhibited by these lines closely resembles that of primary striatal neurons. Results suggest that plastic common precursors of astrocytes and neurons persist in the striatum at a late developmental stage. As these permanent cell lines constitute an unlimited source of homogenous cell material, we suggest that they should be useful for molecular and pharmacological studies on the mechanisms and regulation of signal transduction as well as the commitment, plasticity, and differentiation of neural cells.
properties, were derived from long-term primary cultures
(19).
Several laboratories have shown that the tumorigenic conversion of fibroblasts results from a multistep process. The first step, usually named immortalization, can be induced by the transfer of the polyoma virus large T gene (20, 21), the adenovirus early region 1A sequences (22), and the viral or rearranged cellular myc gene (21). Their expression confers an unlimited growth potential to the fibroblasts without leading through the other transformation steps. These observations prompted us to initiate a system, based on the transfer of defined oncogenes, that allows the establishment of phenotypically untransformed neural cells (23). Subsequently, we have described the immortalization of bipotential glial progenitors and the generation of permanent cell lines carrying the Escherichia coli lacZ reporter gene (24). Very recently, by oncogene transfer with retroviral vectors (2527), a few different types of neural precursors have been immortalized (28-30). Now we report the establishment of bipotential glioneuronal precursor permanent lines from recombinant retroviral vector-mediated transduction of the SV40 large T gene into mouse striatal cells. Depending on the culture conditions, these lines display either astrocyte properties, such as the expression of the glial fibrillary acidic protein (GFAP), or neuronal characteristics, as for example the expression of neurofilaments. Furthermore, these cell lines also showed adrenergic, dopaminergic, muscarinic and serotoninergic receptors, linked either to the adenylate cyclase or to the phosphatidylinositol signaling pathways and whose combination was very similar to that of primary striatal neurons. The results suggest that the present cells may be suitable for studying the mechanisms involved in the interactions between receptors and neurotransmitters, as well as in signal transduction. This type of permanent neural bipotential precursor cell line, which can be oriented to express astroglial or neuronal properties should also be of interest for further molecular studies on the commitment, differentiation, and interactions of neural cells.
The complexity of cell differentiation and interactions in the central nervous system has been emphasized during the last decade by a number of in vitro studies. Experiments on brain cells in primary culture have underlined the role of cell interactions in the development of the mammalian central nervous system (1-3). Analysis of the differentiation of cells from rat optic nerve has led to the identification of oligodendrocyte-type 2 astrocyte progenitors (4, 5). Studies on chemically defined media (6, 7), growth and trophic factors (for review, see refs. 8 and 9), and hormonal effects (10, 11) have largely improved our current knowledge of neurogenesis. New outlooks are arising from analysis of the structure and organization of genes specifically expressed in neural cells (for review, see refs. 12 and 13). However, most molecular and regulatory mechanisms concerning the expression of genes involved in neural-cell differentiation and interaction remain poorly understood. Such study would be considerably facilitated by establishing permanent cell lines that retain the main differentiation and functional properties of their normal cell counterparts. Although presenting transformed or tumoral characteristics, a few brain cell lines have been suitable for studying some facets of neural differentiation. Most of them were derived from spontaneous or chemically induced tumors (14, 15) or after transformation with oncogenic viruses (16, 17). Some neural precursor cell line were obtained by transfection of the F9 embryonic carcinoma cell line with the simian virus 40 (SV40) large tumor (T) gene (18). Other lines, with astroglial
MATERIALS AND METHODS Gene Transfer and Selection of the Immortalized Cells. Unless otherwise stated, the standard medium was Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%o fetal calf serum. The striatum was dissected from 1-day postnatal C57BL/6 mice, and the dissociated cells were plated at densities ranging between 5 x 104 to 2 x 105 cells per 100-mm dish. After 24 hr, the cells were infected for 2 hr Abbreviations: large T gene, large tumor gene; GFAP, glial fibrillary acidic protein; LTR, long terminal repeat; SV40, simian virus 40; 5HT2, 5-hydroxytryptamine type 2; mAb, monoclonal antibody. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Developmental Biology: Evrard et al. with the supernatant of a 4i2 cell line that produced a retroviral vector carrying the SV40 large T gene inserted in the pZIP-Neo-SVX construct (25). This line was a gift from C. Cepko (Harvard Medical School) (27). After infection, cells were grown in the standard medium and, 48 hr later, G418 (Geneticin, GIBCO) at 150 gg/ml was added; the selective medium was changed every 4 days. The colonies emerged 6 weeks later; they were trypsinized individually in cloning cylinders and then cloned by limiting dilution. Immunofluorescence Analysis. The cells were seeded on glass coverslips previously coated with either gelatin at 250 jig/ml or poly-DL-ornithine at 1.5 ,ug/ml. Then, according to the experiments, they were grown in the above-mentioned standard medium or in the chemically defined BottensteinSato N2 medium (6). Fixation and incubation procedures were as already described (24), with the following antibodies. Supernatant from clone 105 of the American Type Culture Collection was used as A2B5 antibody (31), attenuated tetanus toxin and corresponding antibodies were gifts from B. Bizzini (Institut Pasteur, Paris) (32). The rabbit antibodies to GFAP were purchased from Dakopatts (Sebia, Paris), whereas those antibodies against y-enolase (33) and neurofilaments (34) were provided us by L. Legault-Demare and A. Prochiantz (College de France, Paris), respectively. The monoclonal antibodies (mAbs) to the 68-kDa and the 150-kDa forms of neurofilaments were obtained from Amersham; those mAbs against the 200-kDa neurofilament protein (35) were a gift from A. Koulakoff(College de France, Paris). The mAb to the SV40 large T antigen (36) was given us by E. May (Institut Recherches/Cancer, Villejuif, France). The fluorochrome-conjugated antibodies against rabbit immunoglobulins, mouse IgM, or mouse IgG were obtained from Amersham, Cappel Laboratories, and KPL Laboratories (Gaithersburg, MD), respectively. Southern and Northern (RNA) Blot Hybridizations. Highmolecular-weight DNA was digested with restriction enzymes and subjected to electrophoresis in 0.7% agarose gel. After blotting (37), the nitrocellulose filters were hybridized as described (38) with the randomly primed 32P-labeled SV40 large T gene fragment. Cytoplasmic RNA was subjected to electrophoresis in a 1.4% agarose-formaldehyde gel (38), transferred onto GeneScreen filters, which were then hybridized according to the manufacturer's instructions with probes specific for GFAP (39) or neurofilament H (40), which were gifts of J. de Vellis (University of California, Los Angeles) and A. Dautigny (University of Paris), respectively. Immunoblotting Procedure. After removal of growth medium and extensive phosphate-buffered saline washing, the cells were lysed with 0.5% Nonidet detergent, and the extracts were enriched for intermediate-filament proteins, as described (41). After 10%6 SDS/acrylamide gel electrophoresis, the proteins were transferred electrophoretically onto nitrocellulose membranes and incubated with rabbit antibodies against neurofilaments (34). The bound antibodies were detected with donkey 35S-labeled antibodies against rabbit immunoglobulins (Amersham), and the bands were visualized by autoradiography. Responses to Neurotransmitters. The cell lines were grown in 2-cm2 wells containing 0.5 ml of either standard or chemically defined N2 medium; the primary cultures of astrocytes and neurons were as described (42). They were preincubated for 2 hr at 37°C with 2 ,tCi (74 kBq) of [2-3H]adenine (27 Ci/mmol, Amersham). The medium was then replaced with Krebs phosphate buffer containing appropriate agonists and antagonists and 1 mM of the phosphodiesterase inhibitor isobutylmethylxanthine. After 5 min at 37TC the incubation was stopped, and cAMP was isolated by the double chromatographic method (43).
Proc. Natl. Acad. Sci. USA 87 (1990)
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For the phosphatidylinositol assay, the cells were preincubated for 24 hr at 370C with 1 uzCi of [myo-3H]inositol (17 Ci/mmol; Commissariat a l'Energie Atomique). Cells were then incubated for 20 min at 370C in Krebs phosphate buffer containing the agonists and antagonists and 10 mM LiCl. The inositol phosphates were then extracted and isolated as described (42).
RESULTS Selection and Growth Properties of the Clonal Permanent CeU Lines. After transduction of the SV40 large T gene with the retroviral vector pZIP-neo-SVX-SVLT (25, 27), the cells were selected by their resistance to G418 and their capacity to grow at low cell density. The isolated colonies were individually picked up; they were cloned and subcloned by limiting dilution. The frequency of emergence of the immortalized clones was on the order of 5 x 10-5; their generation time ranged between 24 and 48 hr, and their plating efficiency was nearly 20% when seeded at low cell density-i.e., 3 X 102 cells per 100-mm dish. We verified that they could grow >12 mo without a crisis period. Stable integration of the transferred sequences was checked by Southern blot analysis (37). Fig. 1A illustrates the results for the Str.SVLT.3.8 cell line that will represent the present cell lines throughout this report. As expected, Xba I released the 6.9-kilobase (kb) full-length proviral sequence, whereas BamHI excised the 2.2-kb SV40 large T insert. The digestions with BgI II and EcoRI yielded single fragments hybridizing with the SV40 large T gene probe. Their respective lengths, 6.4 and 10 kb, show that, in addition to the large T gene, they contain cellular sequences flanking the long terminal repeat (LTR). These data also indicate that the cells have integrated a single copy of the proviral DNA and confirm the clonality of X
Bg
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FIG. 1. Integration and expression of the SV40 large T gene. (A) Southern blot analysis. Cellular DNA (10 /ig) was digested with the following restriction enzymes: X, Xba I; B, BamHI; Bg, Bgl II; and E, EcoRI. The fragments were separated by electrophoresis through 0.7% agarose gel, transferred onto a nitrocellulose membrane, and hybridized with the 32P-labeled BstXI-Bcl I probe from SV40 DNA, specific for the large T insert. Numbers at left refer to the size of DNA markers in kb pair. (B) Immunofluorescence for SV40 large T antigen. Cells were incubated with mAb to large T antigen (36) and then with fluorescein-conjugated antibodies against mouse immunoglobulins. (x400.) (C) Schematic representation of the proviral DNA.
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Proc. Natl. Acad Sci. USA 87 (1990)
the line. Expression of the SV40 large T gene was verified by immunofluorescence; as shown in Fig. 1B, the SV40 large T antigen was clearly detectable in cell nuclei. Astroglial Differentiation ofthe Str.SVLT.3.8 Cell Line. The cells were examined with a combination of antibodies. A2B5 mAb interacts with Gq gangliosides on subpopulations of glial cells (4) and neurons (31). The tetanus toxin receptors, involving gangliosides too, have been reported to be displayed by neurons (32) and, to a lesser extent, by astrocytes (4). Vimentin is preferentially expressed in various precursor cells (13), whereas the antibodies to GFAP recognize the astrocytes (44). y-Enolase (33) and neurofilaments (34, 40, 45) have been reported to be specifically expressed in neurons. After being seeded in the standard 10% fetal calf serumsupplemented medium, the cells interacted with the A2B5 mAb, interacted faintly but unambiguously with the tetanus toxin (Fig. 2 A and B), and expressed vimentin (data not shown). At confluence the GFAP became detectable (Fig. 2C) at first in rare cells (2-4%), but this proportion increased dramatically within a few days to reach -90% of the cells 6 days later. It should be specified that the neurofilaments were undetectable in these conditions. As shown in Fig. 2D, GFAP expression was confirmed by Northern blot analysis of cytoplasmic RNA with a GFAP-specific probe (39). Neuronal Differentiation of the Str.SVLT.3.8 Cell Line. Cells were seeded at intermediate densities (2 x 103-104 cells per cm2) in the chemically defined N2 medium on polyornithine-coated coverslips. As soon as 48 hr after seeding, the cells began to extend some processes. After 6-day culture, nearly all cells interacted with A2B5 mAb (Fig. 3A) and displayed increased reactivity to tetanus toxin (compare Fig.
1
FIG. 3. Neuronal differentiation of the Str.SVLT.3.8 cells, grown for 6 days in the chemically defined N2 medium. Immunofluorescence assays with A2B5 mAb (A), tetanus toxin and corresponding antibodies (B), rabbit antibodies against neurofilament triplet proteins (C), and mAbs to the 150-kDa and 200-kDa forms of neurofilaments, respectively (D and E); and rabbit antibodies against yenolase (F). (x200.)
2
4.3 3.5
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_1 .6 FIG. 2. Astroglial differentiation of Str.SVLT.3.8 cells grown in serum-supplemented medium. (A-C) Immunofluorescence assays at 2 days after confluence. (A) Interaction with A2B5 mAb (31). (B) Binding of tetanus toxin (32). (C) Detection of GFAP. (x200.) (D) Northern (RNA) blot hybridization: 6 days (lane 1) or 10 days (lane 2) after confluence, the cytoplasmic RNA (20 ,ug) was submitted to formaldehyde-agarose gel electrophoresis, blotted onto GeneScreen membrane, and hybridized with the 32P-labeled HindIII insert of the GFAP-specific pG1 clone (39); size scale in kb is indicated at right.
3B with 2B). Only 10-20% of the cells were stained with the antibodies against GFAP (data not shown). More than 70% of the cells expressed the 150-kDa (neurofilament M) and 200kDa (neurofilament H) forms and the y-enolase, as shown by immunofluorescence assays (Fig. 3 C-F). The immunoblotting analysis indicated that the 68-kDa (neurofilament L) form was also synthetized (Fig. 4A). Synthesis of the neurofilament H mRNA was confirmed by Northern blot experiments; Fig. 4B shows that the cytoplasmic RNA hybridized with the neurofilament H-specific probe (40). Receptors for Neurotransmitters. Str.SVLT.3.8 cell line was examined for the expression of several receptors for neurotransmitters, coupled to adenylate cyclase (46, 47), or to phospholipase C (42, 48). As shown in Table 1, the 1adrenergic receptors were revealed by stimulation of the cAMP generating system with the 83-adrenergic agonist isoproterenol (49). In addition, the cells displayed D1 dopaminergic receptors because the dopamine-stimulating effect was blocked by a selective D1 antagonist, Schering 23390
Developmental Biology: Evrard et al. 1
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Proc. Natl. Acad. Sci. USA 87 (1990) B
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FIG. 4. Expression of the neurofilaments L and H. (A) Immunoblot: after electrophoresis and transfer onto nitrocellulose, the proteins (10 ,ug) were incubated with rabbit antibodies against neurofilament triplet proteins and revealed with 35S-labeled antibodies against rabbit immunoglobulins. Lanes: 1, mouse brain; 2, Str.SVLT.3.8 cell line grown for 6 days in N2 medium; 3 and 4, the same cell line in serum-supplemented medium, growing exponentially and confluent, respectively. Numbers at left refer to molecular masses in kDa. (B) Northern blot: After electrophoresis and transfer onto GeneScreen membrane, cytoplasmic RNA (20 ,ug) was hybridized with the EcoRI insert of pNF-H1 clone (40). Lanes: 1, RNA from mouse brain; 2 and 3, RNA from the cell line, grown in the N2 medium for 6 and 10 days, respectively. The size scale in kb is at right.
(47). Moreover, when grown in N2 medium, the cells expressed D2 dopaminergic receptors, negatively coupled to adenylate cyclase: indeed, when the cAMP pathway was stimulated in the presence of isoproterenol, the inhibitory effect of dopamine was clearly observed and could be suppressed by the addition of a D2 dopaminergic antagonist, L-sulpiride (47). Further results (Table 2) show that, when mediated by norepinephrine or carbamoylcholine, stimulation of phospholipase C was reversed by the antagonists prazosin (50) or atropine (51), respectively. These facts indicate the presence of a1-adrenergic and muscarinic receptors positively coupled to phospholipase C. In addition, the expression of 5-HT2 serotoninergic receptors was revealed by the inhibitory effect of a 5-HT2 selective antagonist, ritanserin (52), on the response to 5-hydroxytryptamine. It can be observed that most of the examined receptors were expressed by the cells cultured in both the standard and the N2 media (Tables 1 and 2). Nevertheless, the responses involving D1 dopaminergic, muscarinic, and serotoninergic Table 1. Neurotransmitter receptors coupled to
adenylate cyclase Increase in [3H]cAMP, % control Str.SVLT.3.8 N2 neurons astrocytes Standard 858 109* 1388 120* 380 41* 905 80* 875 87* 819 58*t 272 30*t 986 52* Striatal
Striatal
Drugs Iso Iso + Da Iso + Da + Sul 88* 425 37* 923 80* 835 50* 1409 14t NS Da 56 165 22* 250 18* NS Da + Sch 20 12§ 52 28t§ 23 19§ NS Da + Sul 60 6t 180 10* 272 25* The Str.SVLT.3.8 cell line, primary cultures of striatal neurons in N2 medium and primary cultures of astrocytes in standard medium, were assayed for formation of cAMP in response to neurotransmitters, agonists, and antagonists, which were absent from controls. Drugs were used at the following concentrations (M): isoproterenol (Iso),
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10-5; dopamine (Da), 3 x 10-5; L-sulpiride (Sul), 5
x
10-6;
Schering 23390 (Sch), 2 x 10-7. NS, not significantly different from controls. The indicated values are the means of triplicated determinations from two independent cultures. Student's t test: *, P < 0.01, t, P < 0.05, as compared to controls; t, P < 0.05, as compared to values determined in the presence either of isoproterenol alone or of isoproterenol plus dopamine plus L-sulpiride; §, P < 0.05, as compared to results obtained with dopamine alone.
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Table 2. Neurotransmitter receptors coupled to phospholipase C Increase in [3H]inositol phosphates, % control Str.SVLT.3.8 Striatal Striatal astrocytes neurons N2 Standard Drugs 388 ± 22* 270 ± 10* 203 ± 35* 440 ± 50* Ne 12 ± 5t 30 ± 15* Ne + Pz 65 ± 25t 125 ± 12t NS 480 ± 50* 796 90* 700 ± 55* Carb 3 ± 12§ 153 35§ NS 20 ± 15§ Carb + At 980 ± 35* 450 ± 30* ND Ne + Carb 892 ± 84* NS 5-HT 90 ± 26t 455 ± 40* 70 ± 12t NS 5 ± 7¶ 8 ± 91 296 401 5-HT + Rit The Str.SVLT.3.8 cell line, primary cultures of striatal neurons in N2 medium and of astrocytes in standard medium, were assayed for formation of inositol phosphates in response to neurotransmitters, agonists, and antagonists, which were absent from controls. Drugs were used at the following concentrations (M): norepinephrine (Ne), 10-4; prazosin (Pz), 10-5; carbamoylcholine (Carb), 10-3; atropine (At), 10-4; 5-hydroxytryptamine (5-HT), 10-5 ; ritanserin (Rit), 10-5. NS, not significantly different from controls; ND, not determined. The indicated values are the means of triplicated determinations from two independent cultures. Student's t test: *, P < 0.01, t, P < 0.05, as compared to controls; *, P < 0.01, as compared to the result obtained with norepinephrine alone; §, P < 0.01, as compared to the value determined with carbamoylcholine alone; ¶, P < 0.01, as compared to the data with 5-HT alone.
receptors, were higher in the cells maintained in the N2 medium. Furthermore, the D2 dopaminergic receptors were only detected when the cells were grown in the defined medium (Table 1).
DISCUSSION Striatal cell lines have been generated by transduction with a retroviral vector (27) carrying the SV40 large T gene and the NeoR sequence. After selection for G418 resistance, the cells were cloned and subcloned by limiting dilution. The stable integration of the transduced sequences was verified by Southern blot analysis (37), the patterns of which confirmed clonality of the lines. The frequency of emergence of the immortalized clones was in the order of 5 x 10-5, without significant variation whether the supernatant from the if2 producing cells was used directly or concentrated up to 50-fold. This could mean that the limiting factor in the present immortalization experiments may be essentially the proportion of the cells that (i) carry receptors for the recombinant retrovirus, (ii) have kept a potential for at least one division cycle, and (iii) also synthetize nuclear factors compatible with the U3 control region of the retroviral LTR. As some of the obtained lines showed strikingly morphological differences when grown either in serum-supplemented or in chemically defined medium, we decided to investigate a few of their differentiation and functional properties, the results being illustrated for the representative Str.SVLT.3.8 line. The data show that, in serum-supplemented medium, nearly all cells differentiate towards mature astrocytes expressing GFAP, whereas after seeding in the N2 chemically defined medium, the increased reactivity to tetanus toxin and, more especially, the expression of neurofilaments by >70%o of the cells show that the cells have acquired neuronal characteristics. When taken together, the clonality of the cells and their differentiation properties strongly suggest that these lines could correspond to glio-neuronal bipotential precursors, or to cells that have already entered into one of the differentiation pathways, but only initially and still retain plastic differentiation properties. It may seem somewhat surprising to immortalize bipotential precursors from postnatal striatum. Indeed, the few cell lines displaying neuronal precursor properties described thus far were derived either from postnatal cerebel-
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lum, the neurogenesis of which is late (29), from early neuroepithelium (28), or from the embryonic carcinoma F9 line (18). Nevertheless, the late persistence of common glioneuronal progenitors have been reported in postnatal retina (53) and optic nerve (54). As differentiation of the present cell lines toward either direction occurs under different culture conditions well-known for favoring the development of astrocytes or neurons (6, 28, 29), it appears unlikely that this bipotentiality results from a deregulating effect of the SV40 large T antigen, although this possibility cannot be formally excluded. A more rational hypothesis would be that, even though corresponding to a rare cell population, precursor cells that have retained the potential for at least one division have much more readily integrated the transduced sequences and thus have been preferentially immortalized. Another interesting property is that, in addition to expressingl-adreneric receptors, the present cells can express D1 and D2 dopaminergic receptors linked to adenylate cyclase, as well as a1-adrenergic, muscarinic, and 5-HT2 serotoninergic receptors positively coupled to the phosphatidylinositol signaling pathway. Most of these receptors are found in cells grown in both the standard and the defined N2 media. However, the D1 dopaminergic, muscarinic, and 5-HT2 serotoninergic responses are higher in the N2 medium than in the standard medium. A possible explanation is that this combination of receptors occurred earlier than the expression of neurofilaments in the course of differentiation of neural cells, although this hypothesis needs additional experiments for confimation. Furthermore, in the N2 medium, the panel of receptors'of the Str.SVLT.3.8 cell line includes D2 dopaminergic receptors and is very similar to that of striatal neurons in primary culture (46, 48). To our knowledge, 'such a pattern of receptors has not been described in any other permanent cell lines. We are grateful to Drs. F. Cuzin and J. Glowinski for their constant interest and encouragement and to Drs. C. Cepko, A. Dautigny, J. de Vellis, and J. P. Julien for gifts of vectors and probes. This work was supported by the Institut National de la Sante et de la Recherche Mddicale (Grant 871014) and by the Fondation pour la Recherche Medicale. I.B. and E.G. were supported by fellowships from the Ligue Nationale Frangaise contre le Cancer and the Association pour la Recherche sur la Cancer, respectively.
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