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Immunocytochemical localization of nuclear 3,5,3′-triiodothyronine (l -T3) receptors in astrocyte cultures
Developrnental Brain Research, 46 (1989) 131-136 Elsevier
131
BRD50880
Immunocytochemical localization of nuclear 3,5,3'triiodothyronine (L-T3) receptors in astrocyte cultures Min Luo, Jack Puymirat and Jean H. Dussault Unitd de Recherche en Ontogdndse et GdnOtique Mol~culaire, Le Centre Hospitalier de l'Universit~ Laval, Ste-Foy, Qud. (Canada)
(Accepted 1 November 1988) Key words: Immunolocalization; Nuclear T3 receptor; Astrocyte
By means of a monoclonal antil~ody (mab) against the rat liver nuclear L-T3receptor (NT3R) and a polyclonal anti-GFAp serum, it has been possible to demonstrate nuclear thyroid hormone receptors in astrocyte cultures. On day 3, 47% of GFAp + cell nuclei were labeled by 2B3 mab. Between day 3 and day 15, the number of GFA÷ cell nuclei stained by 2B3 mab increased from 47 to 75%. Thyroid hormone nuclear receptors were present in fibrous and protoplasmic astrocytes. However, they developed asynchronously in both types of astrocytes. Indeed, 60% of fibrous astrocytes were stained by 2B3 mab on day 3 and this percentage reached 77% after 8 days in vitro. In contrast, only 30% of protoplasmic astrocytes were immunoreactive for 2B3 mab on day 3 and this percentage increased slowly reaching 47% on day 8 and around 75-80% on day 15. By immunoblotting, the monoclonal antibody recognized two bands of proteins with a molecular weight of 57 and 45 kDa respectively. These proteins have the same electrophoretic mobility as [~251]bromoacetyl-LT3rat liver nuclear L-T3 receptor. This paper presents the first immunocytochemical localization of nuclear L-T3 receptors in astrocyte cultures. Furthermore, we show that thyroid hormone receptors develop more rapidly in fibrous than in protoplasmic astrocytes. INTRODUCTION Thyroid hormones play an important role during brain development, where the absence of thyroid hormones produces multiple morphological and biochemical alterations 2'6A6. It is generally accepted that thyroid hormone action is initiated by the binding of L-triiodothyronine (L-T3) to a nuclear T3 receptor (NT3R) 9. Scatchard analyses performed in primary cultures of brain cells have clearly demonstrated the presence of L-T3 binding sites in astrocytes 4'7'1°A2. However, these biochemical studies do not tell us whether the nuclear L-T3 binding sites are homogeneously distributed in astrocytes or if these receptors are located in specific subpopulations of astrocytes. We have recently produced a monoclonal antibody (mab) (2B3) against rat liver nuclear L-T3 receptor which has been purified by using affinity labelling bromoacetyl [125I]T3 (ref. 8). The access to a specific antibody against NT3R has made it possible to study
the distribution of these receptors in specific cell populations. In the present paper, we report for the first time the immunocytochemical localization of NT3R in astrocyte cultures. MATERIALS AND METHODS Astrocyte cultures Cerebral hemispheres from 2-day-old SpragueDawley rats were dissociated in Puck's solution by passing through a nylon mesh (48 p m pore size). After centrifugation (800 g, 10 min), the cells were resuspended in Dulbecco's Modified Eagle's Medium ( D M E M ) supplemented with D-glucose (4.5 g/l), H E P E S (3.57 g/l), penicillin (100 U/ml), streptomycin (100/xg/ml), fungizone (0.225 pg/ml), horse serum (5%), and newborn calf serum (5%). Both sera were depleted of thyroid hormones by resin treatment as previously described iS. Cells were seeded at
Correspondence: J.H. Dussault, Unit~ de Recherche en Ontog~n~se et G~n6tique Mol6culaires, Le Centre Hospitalier de l'Universit6 Laval, Ste-Foy, Qu6., G1V 4G2, Canada.
0165-3806/89/$03.50 ~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)
132 a density of 4 × 105 cells/cm2 in 100 mm diameter plastic petri-dishes previously coated with poly-L-lysine 7. The cultures were incubated at 37 °C in a humidified 10% CO2, 90% air atmosphere. The medium was changed 3 days after seeding and subsequently every 3 days, for the entire culture period.
Immunocytochemical procedures Cultured cells were studied on days 3, 8, 15 and 21. The culture medium was gently aspirated, and the cells were fixed with 3% paraformaldehyde in 0.1 M sodium phosphate buffer, 0.2% NaC1, pH 7.4, (PBS), for 15 rain at room temperature. Cultures were then washed 3 times with PBS, permeabilized 10 rain with 0.2% of Triton X-100, and the non-specific binding was blocked by incubating in 5% fatty acid-free milk for 60 min at room temperature 3. For double staining, the cells were simultaneously incubated with a polyclonal rabbit anti-GFAp serum diluted 1/200 (gift of Bignami), and the monoclonal antibody (2B3) against the nuclear L-T3 receptor (diluted 1/200) overnight at 4 °C. After washing with PBS, the cells were incubated with FITC goat antimouse immunoglobulin (1:50), and with a rhodamine goat anti-rabbit immunoglobulin (1:50) for 60 min at room temperature. The cells were examined under a Leitz microscope equipped with phase-contrast and fluorescence optics. Several control experiments were performed to ascertain the specificity of the staining observed with 2B3 mab: First, incubation of the cells with control ascites fluid was performed, secondly, with a mixture of primary antibody and partially purified nuclear LT3 receptor prepared as previously described and finally, in some experiments, nuclear L-T3 receptor was separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. Then, nitrocellulose strips were incubated successively with 2B3 mab (1/800) previously preincubated overnight with different amounts of partially purified nuclear L-T3 receptor (0-100/ag protein/ml), and with an 125I-antimouse immunoglobulin,. The complex was revealed by autoradiography. Western blots Astrocyte nuclear L-T3 receptors were prepared as previously described-7. Briefly, cells were scraped in
PBS and centrifuged at 800 g for l0 rain. Then, the cellular precipitate was resuspended in 5 ml of HPC100 buffer (0.5 M hexylene glycol, 0.5 M PIPES pH 7.0, 100 ~tM CaCI2), and homogenized by 5 passages through a syringe fitted with a 27-gauge needle. After centrifugation at 1000 g for 10 rain at 4 °C, the pellet was washed with S.M. buffer (0.32 mM sucrose, 3 mM MgC12) containing 0.5% Triton X-100 and 0.14 M NaCI, 3 mM MgCI 2, then the receptor was extracted with TEM (20 mM Tris, 1 mM EDTA, 3 mM MgC12, 5 mM mercaptoethanol, 10% glycerol (v/v)) containing 0.4 M NaCI for 45 min at 4 °C by vortexing for 15 s at 5-rain intervals. The suspension was centrifuged at 20,000 g for 20 rain. The supernatant was precipitated by 2 vols of ethanol, and after centrifugation the pellet was dissolved in electrophoresis sample buffer. Ten ~g of purified nuclear proteins were applied to a 10% SDS-polyacrylamide gel. The proteins were electrotransferred on nitrocellulose paper, and reacted alternately with 2B3 mab and with an anti-mouse immunoglobulin labelled with alkaline phosphatase. RESULTS
Characterization of astrocyte cultures Astrocyte cultures initiated from 2-day-old rats grown in SSM were characterized immunochemically7. After 8 days of growth, routine cultures were 90-95% astrocytes (GFAp-positive), no cells were stained by an anti-neurofitament antibody, and only a small percentage of the cells were stained by an anti-galactocerebroside antibody (<1%) and by an anti-fibronectin antibody (5%). Based on their form and size, it appears that two subpopulations of astrocytes were observed in culture: polygonal flat GFAp ÷ cells having a large nucleus corresponding to protoplasmic astrocytes (Fig. 1A), and GFAp + cells with long processes corresponding to fibrous astrocytes (Fig. 2). With time in culture, the number of GFAp ÷ cells significantly increased from 375 _+ 65 on day 3 to 690 + 46 on day 15 (Table I) and remained stable thereafter at least until day 21 (Table I), Fibrous astrocytes were the predominant GFAp ÷ cells early in culture where they represented around 60-65% of the GFAp + cells on day 3. With time in culture, their number decreased and after 8 days in vitro, they represented only 16% of the GFAp ÷ cells.
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O -
Fig. 1. Immunocytochemical localization of NT3R in 15-dayold astrocytes. A: immunofluorescence with an anti-GFAp antibody; most of astrocytes were GFAp-positive protoplasmic astrocytes. B: the same field showed that these GFA ÷ protoplasmic astrocytes were partially labelled with 2B3-NT3R-mab in nuclei (× 167).
In contrast, the n u m b e r of protoplasmic astrocytes increased with time in culture and these astrocytes b e c o m e the p r e d o m i n a n t G F A p ÷ cells after 8 days in vitro (around 80%) (Table I). Localization o f nuclear t . - T 3 receptors (NT3R) Using a double immunostaining procedure, we have studied the cellular localization of nuclear L-T3 receptors in G F A p + cells. On day 3, about 47% of G F A p + cells were labelled by 2B3 m a b (Table I). Between day 3 and 15, the n u m b e r of G F A p ÷ cells stained by 2B3 m a b increased from 47 to 75%. This percentage r e m a i n e d stable at least until day 21 (Table I). Study of the cellular localization of N T 3 R in fibrous and protoplasmic astrocytes is p r e s e n t e d in
Fig. 2. Immunoeytochemical localization of NT3R in 3-day-old astrocytes. A: most of the GFAp+ astrocytes were fibrous astrocytes with long processes. B: the double immunostaining shows that the same fibrous astrocytes nuclei have been strongly labelled with the 2B3-NT3R mab (x 167).
Table II. On day 3, 30% of protoplasmic and 60% of fibrous astrocytes were stained by 2B3 mab. These percentages reached 47 and 77% for protoplasmic TABLE I Evolution of the number of NT3R in astrocyte cultures Cells were grown for different periods and the cultures were processed for NT3R immunocytochemistry. NT3R/GFAp-positive ceils were counted on two axes of the coverslips. A mean of 20 fields per axis were counted. Results are expressed as the mean ± S.D. of 3 different experiments. Statistical analyses were performed using Student's t-test; * significantly different from 3-day-old culture, P < 0.001; (NS), not significantly different from 3-day-old cultures. Days in culture 3 Number of GFAppositive cells Number of NT3Rpositive cells
8
15
21
375 + 65 528 ± 79 690 ± 46* 645 + 48* 176 + 88 253 + 79 515 + 17" 494 + 31" (NS)
134 TABLE II
Distribution of NT3R in fibrous (FB), protoplasmic (PP) astrocytes after 3 days and 8 days in vitro Positive cells were counted on two axes of the coverslips. A mean of 8-16 fields per axis were counted. Results are expressed as the mean _+ SD of 3 different experiments.
Days in culture 3 PP Number of GFAppositive cells Number of NT3Rpositive cells
8 FB
PP
145___+20 242-+22 4 2 7 + 6 8 46.5+65
140+15 200+21
FB 82+22
cells in 8- and 15-day-old cultures (Table II), this means that GFA/NT3R-positive cells corresponded exclusively to protoplasmic astrocytes in 15-day-old cultures. In astrocytes, the staining was observed only in the nucleus (Figs. 1B, 2B). No staining was observed with normal mouse serum or with control ascites fluid (data not shown). Furthermore, the staining disappeared when the primary antibody was diluted to 1/800, and when the cells were incubated with a mixture of primary antibody and partially purified nuclear receptor (100 gg) (Fig. 3).
63+18
and fibrous astrocytes after 8 days in vitro. Since with time in culture, the number of fibrous astrocytes decreased whereas the number of protoplasmic astrocytes increased reaching 80 and 90% of the GFAp ÷
Western blots The immunoblotting techniques were used to test
-,--57 4-- 45
A
Fig. 3. Specificity of the immunocytochemical staining using NT3R mob. A: the cultured astrocytes on a 3-day-old rat were immunostained with anti-GFAp antibody. B: no staining was observed in nuclei when the cells were incubated with 2B3 mab (1/200 dilution) preadsorbed with 100 /~g partially purified NT3R (x 167).
B
C
Fig. 4. Western blotting of nuclear extracts from the astrocytes (A,B) and liver (C). This figure shows that the 2B3-NT3R mob recognised two bands of 57 and 45 kDa (B) which have the same electrophoretic mobility as the [l~I]bromoacetyl-T3 labelled rat liver nuclear T3 receptor (C). No staining was observed with control ascites fluid (A).
135 the specificity of the monoclonal antibody 2B3.2B3 recognised two bands with molecular weights of 57 and 45 kDa. These proteins have the same electrophoretic mobility as the [125I]bromoacetyl-T3 rat liver nuclear receptor (Fig. 4). No staining was observed with control ascites fluid (Fig. 4). Furthermore, no staining was observed when the nitrocellulose strips were incubated with preadsorbed 2B3 mab (not shown). DISCUSSION Although most studies on the action of thyroid hormones in brain development have emphasized their effect on neuronal differentiation, it has been noted that they affected the number and the maturation of astrocytes as well 1'5'11. Our previous study shows that NT3R are present in significant amount in astrocytic cultures as revealed by binding assay7, and that thyroid hormones can directly affect the phosphorylation of some specific proteins in cultured astrocytes 14. These studies suggested that the effects of thyroid hormones on astrocytes could result from a direct action of the hormones in this cell type. In this paper, we reported the immunocytochemical localization of NT3R in astrocyte cultures using a mab raised against rat liver LT3 nuclear receptors. The specificity of this 2B3 mab has been demonstrated by several lines of evidence8. Furthermore, in this study, the two absorbant tests have further indicated the specificity of the staining obtained in culture. In our culture system, the number of astrocytes increased by two-fold between day 3 and 15, and remained stable thereafter. Based on their morphological appearance, two types of astrocytes were observed: fibrous and protoplasmic astrocytes which probably corresponded to type-2 and type-1 astrocytes described by Raft et al. 13. Fibrous astrocytes were the predominant type of GFAp-positive cells in the early stages of culture whereas in older cultures, most of the GFAppositive cells were protoplasmic astrocytes. These re-
REFERENCES 1 Clos, J., Legrand, C. and Legrand, J., Effects of thyroid state on the formation and early morphological development of Bergman glia in the developing rat cerebellum, Dev. Neurosci., 3 (1980) 199-202. 2 Grave, G.D., Thyroid Hormones and Brain Development,
suits may suggest that either fibrous astrocytes do not develop in cultures or that they are the progenitor of protoplasmic astrocytes. The hypothesis of an absence of development of fibrous astrocytes in culture is supported by the work of Raft et al. x3 who have also reported an absence of development of fibrous astrocytes in culture. With time in culture, the number of NT3R/GFAppositive cells increased three-fold between day 3 and day 15. This agrees with our previous result obtained by Scatchard analyses in similar cultures 7, and with results obtained by other groups working with astrocyte cultures 4'1°'12. Furthermore, our results show the asynchronous time-course development of nuclear LT3 receptors in fibrous and protoplasmic astrocytes. These results indicate that thyroid hormones may regulate the development of fibrous and protoplasmic astrocytes at different times during brain development depending on the expression of their receptors. In astrocytes, the staining is strictly limited to the nucleus and is in agreement with the absence of cytoplasmic immunoreactive proteins in dot blot analyses (data not shown). This is not in favor of the classical model of action of hormones including the concept of a cytoplasmic receptor protein which undergoes a nuclear translocation after its interaction with ligand. This paper reports for the first time the immunocytochemical localization of NT3R in astrocytes. Furthermore we show that the development of thyroid hormone receptors occurred more rapidly in fibrous than in protoplasmic astrocytes. Thus, a timecourse effect of thyroid hormone during brain development may be explained by a distinct time-course expression of their receptors in different subpopulations of brain cells.
ACKNOWLEDGEMENT Supported by MRC PG-35. Raven, New York, 1977. 3 Johnson, D.A., Gantsch, J.W., Sortsman, J.R. and Elder, J.H., Improved technique utilising non-fat dry milk for analysis of proteins and nucleicacid transferred in vitro-cellulose, GeneAnal. Tech., 1 (1984)3-8, 4 Kolodny, J.M., Leonard, J.L., Larsen, P.R. and Silva, J.E., Studies of nuclear 3,5,3'-Triiodothyroninebinding in
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6
7
8
9
10
primary cultures of rat brain, Endocrinology, 117 (1985) 1848. Legrand, J,, Effects of thyroid hormones on brain development, with particular emphasis on glial cells and myelination. In C. Di Benedetta, R. Balazs, G. Gombos and G. Porcelatti (Eds.), Multidisciplinary Approach to Brain Development, Elsevier/North-Holland Biomedical Press, Amsterdam, 1980, pp. 279-292. Legrand, J., Hormones thyroidiennes et maturation du syst6me nerveux, J. Physiol. (Paris), 78 (1982-1983) 603-652. Luo, M., Faure, R., Ruel, J. and Dussault, J.H., Ontogenesis of nuclear T3 receptors in the cultured neurons and astrocytes, Brain Res., 381 (1986) 275-280. Luo, M., Faure, R., Ruel, J. and Dussault, J.H., A monoclonal antibody to the rat nuclear T3 receptor: production and characterization, Endocrinology, 123 (1988) 180-186. Oppenheimer, J.H., Schwartz, H.L., Mariash, C.N., Kinlaw, W.B., Wong, N.C.W. and Freake, H.C., Advances in our understanding of thyroid hormone action at the cellular levels, Endocr. Rev., (1987) 288-308. Pascual, A., Aranda, A., Ferret-Sena, V., Gabellec, M.M., Rebel, G. and Sarli~ve, L.L., Triiodothyronine receptors in developing mouse neuronal and glial cell cultures and in chick-cultured neurons and astrocytes, Dev. Neuro-
sci., 8 (1986) 89-101. 11 Pesetsky, I., The development of abnormal cerebellar astrocytes in young hypothyroid rats, Brain Res., 63 (1983) 456-460. 12 Puymirat, J. and Faivre-Bauman, J., Evolution of triiodothyronine nuclear binding sites in hypothalamic serum-free cultures: evidence for their presence in neurons and astrocytes, Neurosci. Lett., 68 (1986) 299-304. 13 Raft, M.C., Miler, R.H. and Nobel, M., A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium, Nature (Lond.), 303 (1983) 390-396. 14 Ruel, J., Gavaret, J.M., Luo, M. and Dussault, J.H., Regulation of protein phosphorylation by triiodothyronine (T3) in neural cell cultures. Part I. Astrocytes, Mol. Cell. Endocrinol., 45 (1986) 223-232. 15 Samuels, H.H., Stanley, F. and Casanova, J., Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone, Endocrinology, 105 (1979) 80-85. 16 Sokoloff, L. and Kennedy, C., The action of thyroid hormones and their influences on brain development and function. In G.E. Ganl (Ed.), Biology of Brain Function, Vol. 2, Plenum, New York, 1973, pp. 295-332.
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BRD50880
Immunocytochemical localization of nuclear 3,5,3'triiodothyronine (L-T3) receptors in astrocyte cultures Min Luo, Jack Puymirat and Jean H. Dussault Unitd de Recherche en Ontogdndse et GdnOtique Mol~culaire, Le Centre Hospitalier de l'Universit~ Laval, Ste-Foy, Qud. (Canada)
(Accepted 1 November 1988) Key words: Immunolocalization; Nuclear T3 receptor; Astrocyte
By means of a monoclonal antil~ody (mab) against the rat liver nuclear L-T3receptor (NT3R) and a polyclonal anti-GFAp serum, it has been possible to demonstrate nuclear thyroid hormone receptors in astrocyte cultures. On day 3, 47% of GFAp + cell nuclei were labeled by 2B3 mab. Between day 3 and day 15, the number of GFA÷ cell nuclei stained by 2B3 mab increased from 47 to 75%. Thyroid hormone nuclear receptors were present in fibrous and protoplasmic astrocytes. However, they developed asynchronously in both types of astrocytes. Indeed, 60% of fibrous astrocytes were stained by 2B3 mab on day 3 and this percentage reached 77% after 8 days in vitro. In contrast, only 30% of protoplasmic astrocytes were immunoreactive for 2B3 mab on day 3 and this percentage increased slowly reaching 47% on day 8 and around 75-80% on day 15. By immunoblotting, the monoclonal antibody recognized two bands of proteins with a molecular weight of 57 and 45 kDa respectively. These proteins have the same electrophoretic mobility as [~251]bromoacetyl-LT3rat liver nuclear L-T3 receptor. This paper presents the first immunocytochemical localization of nuclear L-T3 receptors in astrocyte cultures. Furthermore, we show that thyroid hormone receptors develop more rapidly in fibrous than in protoplasmic astrocytes. INTRODUCTION Thyroid hormones play an important role during brain development, where the absence of thyroid hormones produces multiple morphological and biochemical alterations 2'6A6. It is generally accepted that thyroid hormone action is initiated by the binding of L-triiodothyronine (L-T3) to a nuclear T3 receptor (NT3R) 9. Scatchard analyses performed in primary cultures of brain cells have clearly demonstrated the presence of L-T3 binding sites in astrocytes 4'7'1°A2. However, these biochemical studies do not tell us whether the nuclear L-T3 binding sites are homogeneously distributed in astrocytes or if these receptors are located in specific subpopulations of astrocytes. We have recently produced a monoclonal antibody (mab) (2B3) against rat liver nuclear L-T3 receptor which has been purified by using affinity labelling bromoacetyl [125I]T3 (ref. 8). The access to a specific antibody against NT3R has made it possible to study
the distribution of these receptors in specific cell populations. In the present paper, we report for the first time the immunocytochemical localization of NT3R in astrocyte cultures. MATERIALS AND METHODS Astrocyte cultures Cerebral hemispheres from 2-day-old SpragueDawley rats were dissociated in Puck's solution by passing through a nylon mesh (48 p m pore size). After centrifugation (800 g, 10 min), the cells were resuspended in Dulbecco's Modified Eagle's Medium ( D M E M ) supplemented with D-glucose (4.5 g/l), H E P E S (3.57 g/l), penicillin (100 U/ml), streptomycin (100/xg/ml), fungizone (0.225 pg/ml), horse serum (5%), and newborn calf serum (5%). Both sera were depleted of thyroid hormones by resin treatment as previously described iS. Cells were seeded at
Correspondence: J.H. Dussault, Unit~ de Recherche en Ontog~n~se et G~n6tique Mol6culaires, Le Centre Hospitalier de l'Universit6 Laval, Ste-Foy, Qu6., G1V 4G2, Canada.
0165-3806/89/$03.50 ~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)
132 a density of 4 × 105 cells/cm2 in 100 mm diameter plastic petri-dishes previously coated with poly-L-lysine 7. The cultures were incubated at 37 °C in a humidified 10% CO2, 90% air atmosphere. The medium was changed 3 days after seeding and subsequently every 3 days, for the entire culture period.
Immunocytochemical procedures Cultured cells were studied on days 3, 8, 15 and 21. The culture medium was gently aspirated, and the cells were fixed with 3% paraformaldehyde in 0.1 M sodium phosphate buffer, 0.2% NaC1, pH 7.4, (PBS), for 15 rain at room temperature. Cultures were then washed 3 times with PBS, permeabilized 10 rain with 0.2% of Triton X-100, and the non-specific binding was blocked by incubating in 5% fatty acid-free milk for 60 min at room temperature 3. For double staining, the cells were simultaneously incubated with a polyclonal rabbit anti-GFAp serum diluted 1/200 (gift of Bignami), and the monoclonal antibody (2B3) against the nuclear L-T3 receptor (diluted 1/200) overnight at 4 °C. After washing with PBS, the cells were incubated with FITC goat antimouse immunoglobulin (1:50), and with a rhodamine goat anti-rabbit immunoglobulin (1:50) for 60 min at room temperature. The cells were examined under a Leitz microscope equipped with phase-contrast and fluorescence optics. Several control experiments were performed to ascertain the specificity of the staining observed with 2B3 mab: First, incubation of the cells with control ascites fluid was performed, secondly, with a mixture of primary antibody and partially purified nuclear LT3 receptor prepared as previously described and finally, in some experiments, nuclear L-T3 receptor was separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. Then, nitrocellulose strips were incubated successively with 2B3 mab (1/800) previously preincubated overnight with different amounts of partially purified nuclear L-T3 receptor (0-100/ag protein/ml), and with an 125I-antimouse immunoglobulin,. The complex was revealed by autoradiography. Western blots Astrocyte nuclear L-T3 receptors were prepared as previously described-7. Briefly, cells were scraped in
PBS and centrifuged at 800 g for l0 rain. Then, the cellular precipitate was resuspended in 5 ml of HPC100 buffer (0.5 M hexylene glycol, 0.5 M PIPES pH 7.0, 100 ~tM CaCI2), and homogenized by 5 passages through a syringe fitted with a 27-gauge needle. After centrifugation at 1000 g for 10 rain at 4 °C, the pellet was washed with S.M. buffer (0.32 mM sucrose, 3 mM MgC12) containing 0.5% Triton X-100 and 0.14 M NaCI, 3 mM MgCI 2, then the receptor was extracted with TEM (20 mM Tris, 1 mM EDTA, 3 mM MgC12, 5 mM mercaptoethanol, 10% glycerol (v/v)) containing 0.4 M NaCI for 45 min at 4 °C by vortexing for 15 s at 5-rain intervals. The suspension was centrifuged at 20,000 g for 20 rain. The supernatant was precipitated by 2 vols of ethanol, and after centrifugation the pellet was dissolved in electrophoresis sample buffer. Ten ~g of purified nuclear proteins were applied to a 10% SDS-polyacrylamide gel. The proteins were electrotransferred on nitrocellulose paper, and reacted alternately with 2B3 mab and with an anti-mouse immunoglobulin labelled with alkaline phosphatase. RESULTS
Characterization of astrocyte cultures Astrocyte cultures initiated from 2-day-old rats grown in SSM were characterized immunochemically7. After 8 days of growth, routine cultures were 90-95% astrocytes (GFAp-positive), no cells were stained by an anti-neurofitament antibody, and only a small percentage of the cells were stained by an anti-galactocerebroside antibody (<1%) and by an anti-fibronectin antibody (5%). Based on their form and size, it appears that two subpopulations of astrocytes were observed in culture: polygonal flat GFAp ÷ cells having a large nucleus corresponding to protoplasmic astrocytes (Fig. 1A), and GFAp + cells with long processes corresponding to fibrous astrocytes (Fig. 2). With time in culture, the number of GFAp ÷ cells significantly increased from 375 _+ 65 on day 3 to 690 + 46 on day 15 (Table I) and remained stable thereafter at least until day 21 (Table I), Fibrous astrocytes were the predominant GFAp ÷ cells early in culture where they represented around 60-65% of the GFAp + cells on day 3. With time in culture, their number decreased and after 8 days in vitro, they represented only 16% of the GFAp ÷ cells.
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Fig. 1. Immunocytochemical localization of NT3R in 15-dayold astrocytes. A: immunofluorescence with an anti-GFAp antibody; most of astrocytes were GFAp-positive protoplasmic astrocytes. B: the same field showed that these GFA ÷ protoplasmic astrocytes were partially labelled with 2B3-NT3R-mab in nuclei (× 167).
In contrast, the n u m b e r of protoplasmic astrocytes increased with time in culture and these astrocytes b e c o m e the p r e d o m i n a n t G F A p ÷ cells after 8 days in vitro (around 80%) (Table I). Localization o f nuclear t . - T 3 receptors (NT3R) Using a double immunostaining procedure, we have studied the cellular localization of nuclear L-T3 receptors in G F A p + cells. On day 3, about 47% of G F A p + cells were labelled by 2B3 m a b (Table I). Between day 3 and 15, the n u m b e r of G F A p ÷ cells stained by 2B3 m a b increased from 47 to 75%. This percentage r e m a i n e d stable at least until day 21 (Table I). Study of the cellular localization of N T 3 R in fibrous and protoplasmic astrocytes is p r e s e n t e d in
Fig. 2. Immunoeytochemical localization of NT3R in 3-day-old astrocytes. A: most of the GFAp+ astrocytes were fibrous astrocytes with long processes. B: the double immunostaining shows that the same fibrous astrocytes nuclei have been strongly labelled with the 2B3-NT3R mab (x 167).
Table II. On day 3, 30% of protoplasmic and 60% of fibrous astrocytes were stained by 2B3 mab. These percentages reached 47 and 77% for protoplasmic TABLE I Evolution of the number of NT3R in astrocyte cultures Cells were grown for different periods and the cultures were processed for NT3R immunocytochemistry. NT3R/GFAp-positive ceils were counted on two axes of the coverslips. A mean of 20 fields per axis were counted. Results are expressed as the mean ± S.D. of 3 different experiments. Statistical analyses were performed using Student's t-test; * significantly different from 3-day-old culture, P < 0.001; (NS), not significantly different from 3-day-old cultures. Days in culture 3 Number of GFAppositive cells Number of NT3Rpositive cells
8
15
21
375 + 65 528 ± 79 690 ± 46* 645 + 48* 176 + 88 253 + 79 515 + 17" 494 + 31" (NS)
134 TABLE II
Distribution of NT3R in fibrous (FB), protoplasmic (PP) astrocytes after 3 days and 8 days in vitro Positive cells were counted on two axes of the coverslips. A mean of 8-16 fields per axis were counted. Results are expressed as the mean _+ SD of 3 different experiments.
Days in culture 3 PP Number of GFAppositive cells Number of NT3Rpositive cells
8 FB
PP
145___+20 242-+22 4 2 7 + 6 8 46.5+65
140+15 200+21
FB 82+22
cells in 8- and 15-day-old cultures (Table II), this means that GFA/NT3R-positive cells corresponded exclusively to protoplasmic astrocytes in 15-day-old cultures. In astrocytes, the staining was observed only in the nucleus (Figs. 1B, 2B). No staining was observed with normal mouse serum or with control ascites fluid (data not shown). Furthermore, the staining disappeared when the primary antibody was diluted to 1/800, and when the cells were incubated with a mixture of primary antibody and partially purified nuclear receptor (100 gg) (Fig. 3).
63+18
and fibrous astrocytes after 8 days in vitro. Since with time in culture, the number of fibrous astrocytes decreased whereas the number of protoplasmic astrocytes increased reaching 80 and 90% of the GFAp ÷
Western blots The immunoblotting techniques were used to test
-,--57 4-- 45
A
Fig. 3. Specificity of the immunocytochemical staining using NT3R mob. A: the cultured astrocytes on a 3-day-old rat were immunostained with anti-GFAp antibody. B: no staining was observed in nuclei when the cells were incubated with 2B3 mab (1/200 dilution) preadsorbed with 100 /~g partially purified NT3R (x 167).
B
C
Fig. 4. Western blotting of nuclear extracts from the astrocytes (A,B) and liver (C). This figure shows that the 2B3-NT3R mob recognised two bands of 57 and 45 kDa (B) which have the same electrophoretic mobility as the [l~I]bromoacetyl-T3 labelled rat liver nuclear T3 receptor (C). No staining was observed with control ascites fluid (A).
135 the specificity of the monoclonal antibody 2B3.2B3 recognised two bands with molecular weights of 57 and 45 kDa. These proteins have the same electrophoretic mobility as the [125I]bromoacetyl-T3 rat liver nuclear receptor (Fig. 4). No staining was observed with control ascites fluid (Fig. 4). Furthermore, no staining was observed when the nitrocellulose strips were incubated with preadsorbed 2B3 mab (not shown). DISCUSSION Although most studies on the action of thyroid hormones in brain development have emphasized their effect on neuronal differentiation, it has been noted that they affected the number and the maturation of astrocytes as well 1'5'11. Our previous study shows that NT3R are present in significant amount in astrocytic cultures as revealed by binding assay7, and that thyroid hormones can directly affect the phosphorylation of some specific proteins in cultured astrocytes 14. These studies suggested that the effects of thyroid hormones on astrocytes could result from a direct action of the hormones in this cell type. In this paper, we reported the immunocytochemical localization of NT3R in astrocyte cultures using a mab raised against rat liver LT3 nuclear receptors. The specificity of this 2B3 mab has been demonstrated by several lines of evidence8. Furthermore, in this study, the two absorbant tests have further indicated the specificity of the staining obtained in culture. In our culture system, the number of astrocytes increased by two-fold between day 3 and 15, and remained stable thereafter. Based on their morphological appearance, two types of astrocytes were observed: fibrous and protoplasmic astrocytes which probably corresponded to type-2 and type-1 astrocytes described by Raft et al. 13. Fibrous astrocytes were the predominant type of GFAp-positive cells in the early stages of culture whereas in older cultures, most of the GFAppositive cells were protoplasmic astrocytes. These re-
REFERENCES 1 Clos, J., Legrand, C. and Legrand, J., Effects of thyroid state on the formation and early morphological development of Bergman glia in the developing rat cerebellum, Dev. Neurosci., 3 (1980) 199-202. 2 Grave, G.D., Thyroid Hormones and Brain Development,
suits may suggest that either fibrous astrocytes do not develop in cultures or that they are the progenitor of protoplasmic astrocytes. The hypothesis of an absence of development of fibrous astrocytes in culture is supported by the work of Raft et al. x3 who have also reported an absence of development of fibrous astrocytes in culture. With time in culture, the number of NT3R/GFAppositive cells increased three-fold between day 3 and day 15. This agrees with our previous result obtained by Scatchard analyses in similar cultures 7, and with results obtained by other groups working with astrocyte cultures 4'1°'12. Furthermore, our results show the asynchronous time-course development of nuclear LT3 receptors in fibrous and protoplasmic astrocytes. These results indicate that thyroid hormones may regulate the development of fibrous and protoplasmic astrocytes at different times during brain development depending on the expression of their receptors. In astrocytes, the staining is strictly limited to the nucleus and is in agreement with the absence of cytoplasmic immunoreactive proteins in dot blot analyses (data not shown). This is not in favor of the classical model of action of hormones including the concept of a cytoplasmic receptor protein which undergoes a nuclear translocation after its interaction with ligand. This paper reports for the first time the immunocytochemical localization of NT3R in astrocytes. Furthermore we show that the development of thyroid hormone receptors occurred more rapidly in fibrous than in protoplasmic astrocytes. Thus, a timecourse effect of thyroid hormone during brain development may be explained by a distinct time-course expression of their receptors in different subpopulations of brain cells.
ACKNOWLEDGEMENT Supported by MRC PG-35. Raven, New York, 1977. 3 Johnson, D.A., Gantsch, J.W., Sortsman, J.R. and Elder, J.H., Improved technique utilising non-fat dry milk for analysis of proteins and nucleicacid transferred in vitro-cellulose, GeneAnal. Tech., 1 (1984)3-8, 4 Kolodny, J.M., Leonard, J.L., Larsen, P.R. and Silva, J.E., Studies of nuclear 3,5,3'-Triiodothyroninebinding in
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