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Regulation of the phosphorylation of glial fibrillary acidic protein (GFAP) by glutamate and calcium ions in slices of immature rat spinal cord: comparison with immature hippocampus
Neuroscience Letters 248 (1998) 141–143
Regulation of the phosphorylation of glial fibrillary acidic protein (GFAP) by glutamate and calcium ions in slices of immature rat spinal cord: comparison with immature hippocampus Trı´cia Kommers, Lucia Vinade´, Clarissa Pereira, Carlos A. Gonc¸alves, Susana Wofchuk, Richard Rodnight* Departamento de Bioquı´mica, UFRGS, Instituto de Cieˆncias Ba´sicas de Sau´de, Rua Ramiro Barcelos 2600-Anexo, 90.035.003 Porto Alegre, Brazil Received 6 February 1998; received in revised form 8 April 1998; accepted 14 April 1998
Abstract The effect of glutamate and lack of external Ca2+ on the phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) was studied in slices of hippocampus and thoracic spinal cord from immature (P12–16) rats. Confirming previous work with immature hippocampal slices (Wofchuk, S.T. and Rodnight, R., Neurochem. Int., 24 (1994) 517–523; Wofchuk, S.T. and Rodnight, R., Dev. Brain Res., 85 (1995) 181–186), glutamate strongly stimulated GFAP phosphorylation in media with Ca2+ and in media lacking Ca2+ a quantitatively similar stimulation of basal GFAP phosphorylation was observed. By contrast in slices of immature thoracic spinal cord, glutamate had no effect on GFAP phosphorylation and in media lacking Ca2+ phosphorylation of GFAP was inhibited. Since GFAP phosphorylation is Ca2+-dependent and is not stimulated by glutamate in slices of adult hippocampus, the present results suggest that astrocytic functions in the rat spinal cord mature more rapidly than in the hippocampus. The possibility that the difference in the control of GFAP phosphorylation in the two structures is related to differences in the control of GFAP dephosphorylation was investigated by incubating spinal cord slices with the calcineurin inhibitor FK506 in the presence of Ca2+. In contrast to results obtained with hippocampal slices FK506 had no effect on the phosphorylation state of GFAP in spinal cord slices. 1998 Published by Elsevier Science Ireland Ltd. All rights reserved
Keywords: Glial fibrillary acidic protein; Hippocampus; Spinal cord; Glutamate; Calcium ions; Protein phosphorylation
We have shown that the phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) in immature hippocampal slices (P12–16) is strongly stimulated by glutamate via a metabotropic receptor (mGluR) in a reaction dependent on external Ca2+ [11]. In further work we found that a lack of external Ca2+ increased basal phosphorylation of GFAP to the same extent as glutamate plus Ca2+ [12], suggesting that the two effects involve the same mechanism. These results contrast strikingly with the situation in adult hippocampal slices where the phosphorylation of GFAP is completely Ca2+-dependent and is independent of glutamate [11,12]. A study using tryptic phosphopeptide mapping of GFAP suggested that this change in Ca2+ sensitivity during * Corresponding author. Tel.: +55 51 316 5570; fax: +55 51 316 5535.
development is not due to a change in the balance of kinase and phosphatase activities, but rather to a change in the mechanism whereby Ca2+ controls the relative activities of the two enzymes [6]. As a working hypothesis to explain the results in immature tissue we proposed that lack of external Ca2+ or activation of an mGluR by glutamate decreases Ca2+-entry and consequently downregulates a Ca2+-dependent dephosphorylation event associated with GFAP [7]. Inhibition of Ca2+ channels by mGluR activation is a wellknown phenomenon in neurons [1]. However, since the main dephosphorylation of GFAP in hippocampal slices is catalysed by the Ca2+-independent protein phosphatase 1 (PP1) [9], this event probably involves an enzyme-cascade consisting of PP1, the phosphorylated form of a protein that inhibits PP1, inhibitor-1 (inh-1), and the Ca2+-dependent phosphatase calcineurin, which dephosphorylates and inac-
0304-3940/98/$19.00 1998 Published by Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00340- 1
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T. Kommers et al. / Neuroscience Letters 248 (1998) 141–143
Fig. 1. Representative autoradiographs showing the effect of (a) 1 mM glutamate and (b) external Ca2+, on the phosphorylation of GFAP in slices of immature hippocampus and thoracic spinal cord. Slices from the two structures were incubated in parallel with [32P]phosphate and labelled proteins analysed by NEPHGE as indicated in the text. To facilitate comparison first-dimension gels from control and test samples were mounted on one second-dimension slab gel as mirror images. Arrowheads point to GFAP previously identified by immunoblotting [2]. Note that 40 mCi of 32P was used to label hippocampal slices and 80 mCi to label spinal cord slices.
tivates inh-1. In support of this conclusion we showed that although calcineurin does not directly dephosphorylate sites in GFAP, inhibition of the enzyme by FK506 in hippocampal slices or astrocyte cultures increases the phosphorylation state of GFAP [10]. Since the expression of inh-1 in the spinal cord is either absent [3,8] or very low compared with hippocampus [4], it became of interest to compare the regulation of GFAP phosphorylation by glutamate and Ca2+ in the two structures. Slices (0.4 mm) of hippocampus and thoracic spinal cord were prepared with a McIlwain tissue chopper from immature rats (P12–16). Slices from both structures were nor-
mally incubated and analysed in parallel in the same experiment. They were first preincubated in 100 ml of Krebs/HEPES medium (124 mM NaCl; 4 mM KCl; 1.2 mM MgSO4; 1 mM CaCl2; 12 mM glucose; 25 mM HEPES/HCl, pH, 7.4) for 30 min at 30°C, followed by incubation for 1 h in 50 ml of the same medium containing 40 mCi (hippocampus) or 80 mCi (spinal cord) of [32P]phosphate. Where appropriate glutamate (1 mM) was added to the labelling medium; in experiments on the effect of Ca2+lack, EGTA (1 mM) was added to both the preincubation and incubation media. In experiments designed to test the effect of FK506, spinal cord slices were pre-incubated and incubated with 100 mM FK506 as described previously [10]. Labelled proteins were analysed by 2D electrophoresis and densitometry exactly as described previously [11]. Data were analysed statistically by a paired t-test, values from each gel being treated as one pair. Immunodetection of GFAP in extracts of hippocampus and spinal cord was carried out as described previously [2]. Confirming previous observations [11,12] GFAP phosphorylation in immature slices of hippocampus was stimulated equally by glutamate in the presence of Ca2+ and by incubation in media lacking external Ca2+. By contrast in slices of immature thoracic spinal cord glutamate had no effect on GFAP phosphorylation and in media lacking Ca2+ phosphorylation of GFAP was inhibited (Fig. 1, Table 1). Thus the effects of glutamate and Ca2+-lack in immature spinal cord slices resemble their effects on GFAP phosphorylation in adult hippocampal slices where the phosphorylation of GFAP is Ca2+-dependent and is unaffected by glutamate [11,12]. This suggests that astrocytic functions in the rat spinal cord mature more rapidly than in the hippocampus, a conclusion consistent with the general observation of a caudal-to-rostral gradient of overall maturation of the central nervous system. Indeed expression of mRNA for GFAP during development was found to follow such a gradient [5]. Our results lend indirect support to our hypothesis postulating that the effect of external Ca2+ on GFAP phosphorylation in immature hippocampal slices involves an enzyme cascade consisting of PP1, inh-1 and calcineurin. Thus, assuming that the dephosphorylation of GFAP in the spinal cord is catalysed by PP1 and that a Ca2+-dependent kinase is
Table 1 Effect of glutamate and lack of external Ca2+ on the phosphorylation of GFAP in slices of hippocampus and spinal cord Structure
Treatment
Mean normalised peak heights (control = 100)
Mean differences (%)
P-value
Hippocampus Hippocampus Spinal cord Spinal cord Spinal cord
Glutamate −Ca2+ + EGTA Glutamate Ca2+ + EGTA +Ca2+ + FK506
218 ± 21 (5) 217 ± 21 (5) 117 ± 10 (7) 45 ± 5 (14) 102 ± 10 (10)
+118 +117 +17 −55 +2
,0.005 ,0.005 n.s. ,0.001 n.s.
Values quoted are means ± standard error. Control incubations contained 1 mM Ca2+. The concentrations of glutamate and EGTA were 1 mM and that of FK506 was 100 mM. Number of observations is given in parentheses. Significance was determined by a paired t-test.
T. Kommers et al. / Neuroscience Letters 248 (1998) 141–143
involved either directly or indirectly in the phosphorylation stage, with the absence of inh-1 in the tissue and in media lacking Ca2+, the balance of kinase/phosphatase activities in the cord would favour dephosphorylation and a lower GFAP phosphorylation state. Addition of Ca2+ would then activate the kinase stage without affecting the dephosphorylation stage. To confirm this hypothesis, we measured GFAP phosphorylation in spinal cord slices in the presence of Ca2+ and the immunosuppressant FK506, an inhibitor of calcineurin. In hippocampal slices incubated under these conditions FK506 increased the phosphorylation state of GFAP [10], pointing to a role for calcineurin in the dephosphorylation stage in this structure. In contrast in spinal cord slices FK506 had no effect on GFAP phosphorylation (Table 1). Possibly relevant to these results is an observation derived from immunoblots of equal amounts of total protein which showed that the immunocontent of GFAP in the immature spinal cord was about five times higher than in the immature hippocampus (data not shown). Moreover, when the fact that twice as much radioactivity was used to label the spinal cord slices is taken into account, the in vitro rate of 32P-incorporation into much higher content of GFAP in spinal cord slices appeared less than into GFAP in hippocampal slices (Fig. 1). These results confirm an earlier study of the two regions in adult animals in which we observed the same difference in immunocontent of GFAP and a much lower rate of 32P-incorporation into GFAP of spinal cord tissue [2]. It is tempting to speculate that this difference in the ratio of phosphorylation rate to immunocontent might reflect a relatively high rate of dephosphorylation of GFAP in the cord as a result of lack of inhibitory control of PP1. However the possibility cannot be excluded that spinal cord astrocytes are heterogeneous and there exists a population in which GFAP is not phosphorylated.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
This work was supported by the Brazilian funding agencies CNPq, PRONEX, FINEP and FAPERGS. We are grateful to Fujisawa USA for a gift of FK506. [1] Chavis, P., Shinozaki, H., Bockaert, J. and Fagni, L., The metabotropic receptor types 2/3 inhibit L-type calcium channels via a
[12]
143
pertussis toxin-senstive G-protein in cultured cerebellar granule cells, J. Neurosci., 14 (1994) 7067–7076. Gonc¸alves, C.A. and Rodnight, R., Apparent identity of ppH-47, a protein highly phosphorylated in the hippocampus with a form of glial fibrillary acidic protein, Neurosci. Res. Commun., 11 (1992) 109–117. Gustafson, E.L., Girault, J.-A., Hemmings, H.C. Jr., Nairn, A.C. and Greengard, P., Immunocytochemical localization of phosphatase inhibitor-1 in rat brain, J. Comp. Neurol., 310 (1991) 170–188. Hemmings, H.C. Jr., Girault, J.-A., Nairn, A.C., Bertuzzi, G. and Greengard, P., Distribution of protein phosphatase inhibitor-1 in brain and peripheral tissues of various species: comparison with DARPP-32, J. Neurochem., 59 (1992) 1053–1061. Landry, C.F., Ivy, G.O. and Brown, I.R., Developmental expression of glial fibrillary acidic protein mRNA in the rat brain analyzed by in situ hybridization, J. Neurosci. Res., 25 (1990) 194– 203. Leal, R.B., Gonc¸alves, C.A. and Rodnight, R., Calcium-dependent phosphorylation of glial fibrillary acidic protein (GFAP) in the rat hippocampus: a comparison of the kinase/phosphatase balance in immature and mature slices using tryptic phosphopeptide mapping, Dev. Brain Res., 104 (1997) 1–10. Rodnight, R., Gonc¸alves, C.A., Wofchuk, S.T. and Leal, R., Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity, Brazil. J. Med. Biol. Res., 30 (1997) 325–338. Sakagami, H., Ebina, K. and Kondo, H., Localization of phosphatase inhibitor-1 mRNA in the developing and adult rat brain in comparison with that of protein phosphatase-1 mRNAs, Mol. Brain Res., 25 (1994) 7–18. Vinade´, L. and Rodnight, R., The dephosphorylation of glial fibrillary acidic protein (GFAP) in the immature rat hippocampus is catalyzed mainly by a type 1 protein phosphatase, Brain Res., 732 (1996) 195–200. Vinade´, L., Gonc¸alves, C.A., Wofchuk, S., Gottfried, C. and Rodnight, R., Evidence for a role for calcium ions in the dephosphorylation of glial fibrillary acidic protein (GFAP) in immature hippocampal slices and in astrocyte cultures from the rat, Dev. Brain Res., 104 (1997) 11–17. Wofchuk, S.T. and Rodnight, R., Glutamate stimulates the phosphorylation of glial fibrillary acidic protein in slices of immature hippocampus via a metabotropic receptor, Neurochem. Int., 24 (1994) 517–523. Wofchuk, S.T. and Rodnight, R., Age-dependent changes in the regulation by external calcium ions of the phosphorylation of glial fibrillary acidic protein in slices of rat hippocampus, Dev. Brain Res., 85 (1995) 181–186.
Regulation of the phosphorylation of glial fibrillary acidic protein (GFAP) by glutamate and calcium ions in slices of immature rat spinal cord: comparison with immature hippocampus Trı´cia Kommers, Lucia Vinade´, Clarissa Pereira, Carlos A. Gonc¸alves, Susana Wofchuk, Richard Rodnight* Departamento de Bioquı´mica, UFRGS, Instituto de Cieˆncias Ba´sicas de Sau´de, Rua Ramiro Barcelos 2600-Anexo, 90.035.003 Porto Alegre, Brazil Received 6 February 1998; received in revised form 8 April 1998; accepted 14 April 1998
Abstract The effect of glutamate and lack of external Ca2+ on the phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) was studied in slices of hippocampus and thoracic spinal cord from immature (P12–16) rats. Confirming previous work with immature hippocampal slices (Wofchuk, S.T. and Rodnight, R., Neurochem. Int., 24 (1994) 517–523; Wofchuk, S.T. and Rodnight, R., Dev. Brain Res., 85 (1995) 181–186), glutamate strongly stimulated GFAP phosphorylation in media with Ca2+ and in media lacking Ca2+ a quantitatively similar stimulation of basal GFAP phosphorylation was observed. By contrast in slices of immature thoracic spinal cord, glutamate had no effect on GFAP phosphorylation and in media lacking Ca2+ phosphorylation of GFAP was inhibited. Since GFAP phosphorylation is Ca2+-dependent and is not stimulated by glutamate in slices of adult hippocampus, the present results suggest that astrocytic functions in the rat spinal cord mature more rapidly than in the hippocampus. The possibility that the difference in the control of GFAP phosphorylation in the two structures is related to differences in the control of GFAP dephosphorylation was investigated by incubating spinal cord slices with the calcineurin inhibitor FK506 in the presence of Ca2+. In contrast to results obtained with hippocampal slices FK506 had no effect on the phosphorylation state of GFAP in spinal cord slices. 1998 Published by Elsevier Science Ireland Ltd. All rights reserved
Keywords: Glial fibrillary acidic protein; Hippocampus; Spinal cord; Glutamate; Calcium ions; Protein phosphorylation
We have shown that the phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) in immature hippocampal slices (P12–16) is strongly stimulated by glutamate via a metabotropic receptor (mGluR) in a reaction dependent on external Ca2+ [11]. In further work we found that a lack of external Ca2+ increased basal phosphorylation of GFAP to the same extent as glutamate plus Ca2+ [12], suggesting that the two effects involve the same mechanism. These results contrast strikingly with the situation in adult hippocampal slices where the phosphorylation of GFAP is completely Ca2+-dependent and is independent of glutamate [11,12]. A study using tryptic phosphopeptide mapping of GFAP suggested that this change in Ca2+ sensitivity during * Corresponding author. Tel.: +55 51 316 5570; fax: +55 51 316 5535.
development is not due to a change in the balance of kinase and phosphatase activities, but rather to a change in the mechanism whereby Ca2+ controls the relative activities of the two enzymes [6]. As a working hypothesis to explain the results in immature tissue we proposed that lack of external Ca2+ or activation of an mGluR by glutamate decreases Ca2+-entry and consequently downregulates a Ca2+-dependent dephosphorylation event associated with GFAP [7]. Inhibition of Ca2+ channels by mGluR activation is a wellknown phenomenon in neurons [1]. However, since the main dephosphorylation of GFAP in hippocampal slices is catalysed by the Ca2+-independent protein phosphatase 1 (PP1) [9], this event probably involves an enzyme-cascade consisting of PP1, the phosphorylated form of a protein that inhibits PP1, inhibitor-1 (inh-1), and the Ca2+-dependent phosphatase calcineurin, which dephosphorylates and inac-
0304-3940/98/$19.00 1998 Published by Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00340- 1
142
T. Kommers et al. / Neuroscience Letters 248 (1998) 141–143
Fig. 1. Representative autoradiographs showing the effect of (a) 1 mM glutamate and (b) external Ca2+, on the phosphorylation of GFAP in slices of immature hippocampus and thoracic spinal cord. Slices from the two structures were incubated in parallel with [32P]phosphate and labelled proteins analysed by NEPHGE as indicated in the text. To facilitate comparison first-dimension gels from control and test samples were mounted on one second-dimension slab gel as mirror images. Arrowheads point to GFAP previously identified by immunoblotting [2]. Note that 40 mCi of 32P was used to label hippocampal slices and 80 mCi to label spinal cord slices.
tivates inh-1. In support of this conclusion we showed that although calcineurin does not directly dephosphorylate sites in GFAP, inhibition of the enzyme by FK506 in hippocampal slices or astrocyte cultures increases the phosphorylation state of GFAP [10]. Since the expression of inh-1 in the spinal cord is either absent [3,8] or very low compared with hippocampus [4], it became of interest to compare the regulation of GFAP phosphorylation by glutamate and Ca2+ in the two structures. Slices (0.4 mm) of hippocampus and thoracic spinal cord were prepared with a McIlwain tissue chopper from immature rats (P12–16). Slices from both structures were nor-
mally incubated and analysed in parallel in the same experiment. They were first preincubated in 100 ml of Krebs/HEPES medium (124 mM NaCl; 4 mM KCl; 1.2 mM MgSO4; 1 mM CaCl2; 12 mM glucose; 25 mM HEPES/HCl, pH, 7.4) for 30 min at 30°C, followed by incubation for 1 h in 50 ml of the same medium containing 40 mCi (hippocampus) or 80 mCi (spinal cord) of [32P]phosphate. Where appropriate glutamate (1 mM) was added to the labelling medium; in experiments on the effect of Ca2+lack, EGTA (1 mM) was added to both the preincubation and incubation media. In experiments designed to test the effect of FK506, spinal cord slices were pre-incubated and incubated with 100 mM FK506 as described previously [10]. Labelled proteins were analysed by 2D electrophoresis and densitometry exactly as described previously [11]. Data were analysed statistically by a paired t-test, values from each gel being treated as one pair. Immunodetection of GFAP in extracts of hippocampus and spinal cord was carried out as described previously [2]. Confirming previous observations [11,12] GFAP phosphorylation in immature slices of hippocampus was stimulated equally by glutamate in the presence of Ca2+ and by incubation in media lacking external Ca2+. By contrast in slices of immature thoracic spinal cord glutamate had no effect on GFAP phosphorylation and in media lacking Ca2+ phosphorylation of GFAP was inhibited (Fig. 1, Table 1). Thus the effects of glutamate and Ca2+-lack in immature spinal cord slices resemble their effects on GFAP phosphorylation in adult hippocampal slices where the phosphorylation of GFAP is Ca2+-dependent and is unaffected by glutamate [11,12]. This suggests that astrocytic functions in the rat spinal cord mature more rapidly than in the hippocampus, a conclusion consistent with the general observation of a caudal-to-rostral gradient of overall maturation of the central nervous system. Indeed expression of mRNA for GFAP during development was found to follow such a gradient [5]. Our results lend indirect support to our hypothesis postulating that the effect of external Ca2+ on GFAP phosphorylation in immature hippocampal slices involves an enzyme cascade consisting of PP1, inh-1 and calcineurin. Thus, assuming that the dephosphorylation of GFAP in the spinal cord is catalysed by PP1 and that a Ca2+-dependent kinase is
Table 1 Effect of glutamate and lack of external Ca2+ on the phosphorylation of GFAP in slices of hippocampus and spinal cord Structure
Treatment
Mean normalised peak heights (control = 100)
Mean differences (%)
P-value
Hippocampus Hippocampus Spinal cord Spinal cord Spinal cord
Glutamate −Ca2+ + EGTA Glutamate Ca2+ + EGTA +Ca2+ + FK506
218 ± 21 (5) 217 ± 21 (5) 117 ± 10 (7) 45 ± 5 (14) 102 ± 10 (10)
+118 +117 +17 −55 +2
,0.005 ,0.005 n.s. ,0.001 n.s.
Values quoted are means ± standard error. Control incubations contained 1 mM Ca2+. The concentrations of glutamate and EGTA were 1 mM and that of FK506 was 100 mM. Number of observations is given in parentheses. Significance was determined by a paired t-test.
T. Kommers et al. / Neuroscience Letters 248 (1998) 141–143
involved either directly or indirectly in the phosphorylation stage, with the absence of inh-1 in the tissue and in media lacking Ca2+, the balance of kinase/phosphatase activities in the cord would favour dephosphorylation and a lower GFAP phosphorylation state. Addition of Ca2+ would then activate the kinase stage without affecting the dephosphorylation stage. To confirm this hypothesis, we measured GFAP phosphorylation in spinal cord slices in the presence of Ca2+ and the immunosuppressant FK506, an inhibitor of calcineurin. In hippocampal slices incubated under these conditions FK506 increased the phosphorylation state of GFAP [10], pointing to a role for calcineurin in the dephosphorylation stage in this structure. In contrast in spinal cord slices FK506 had no effect on GFAP phosphorylation (Table 1). Possibly relevant to these results is an observation derived from immunoblots of equal amounts of total protein which showed that the immunocontent of GFAP in the immature spinal cord was about five times higher than in the immature hippocampus (data not shown). Moreover, when the fact that twice as much radioactivity was used to label the spinal cord slices is taken into account, the in vitro rate of 32P-incorporation into much higher content of GFAP in spinal cord slices appeared less than into GFAP in hippocampal slices (Fig. 1). These results confirm an earlier study of the two regions in adult animals in which we observed the same difference in immunocontent of GFAP and a much lower rate of 32P-incorporation into GFAP of spinal cord tissue [2]. It is tempting to speculate that this difference in the ratio of phosphorylation rate to immunocontent might reflect a relatively high rate of dephosphorylation of GFAP in the cord as a result of lack of inhibitory control of PP1. However the possibility cannot be excluded that spinal cord astrocytes are heterogeneous and there exists a population in which GFAP is not phosphorylated.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
This work was supported by the Brazilian funding agencies CNPq, PRONEX, FINEP and FAPERGS. We are grateful to Fujisawa USA for a gift of FK506. [1] Chavis, P., Shinozaki, H., Bockaert, J. and Fagni, L., The metabotropic receptor types 2/3 inhibit L-type calcium channels via a
[12]
143
pertussis toxin-senstive G-protein in cultured cerebellar granule cells, J. Neurosci., 14 (1994) 7067–7076. Gonc¸alves, C.A. and Rodnight, R., Apparent identity of ppH-47, a protein highly phosphorylated in the hippocampus with a form of glial fibrillary acidic protein, Neurosci. Res. Commun., 11 (1992) 109–117. Gustafson, E.L., Girault, J.-A., Hemmings, H.C. Jr., Nairn, A.C. and Greengard, P., Immunocytochemical localization of phosphatase inhibitor-1 in rat brain, J. Comp. Neurol., 310 (1991) 170–188. Hemmings, H.C. Jr., Girault, J.-A., Nairn, A.C., Bertuzzi, G. and Greengard, P., Distribution of protein phosphatase inhibitor-1 in brain and peripheral tissues of various species: comparison with DARPP-32, J. Neurochem., 59 (1992) 1053–1061. Landry, C.F., Ivy, G.O. and Brown, I.R., Developmental expression of glial fibrillary acidic protein mRNA in the rat brain analyzed by in situ hybridization, J. Neurosci. Res., 25 (1990) 194– 203. Leal, R.B., Gonc¸alves, C.A. and Rodnight, R., Calcium-dependent phosphorylation of glial fibrillary acidic protein (GFAP) in the rat hippocampus: a comparison of the kinase/phosphatase balance in immature and mature slices using tryptic phosphopeptide mapping, Dev. Brain Res., 104 (1997) 1–10. Rodnight, R., Gonc¸alves, C.A., Wofchuk, S.T. and Leal, R., Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity, Brazil. J. Med. Biol. Res., 30 (1997) 325–338. Sakagami, H., Ebina, K. and Kondo, H., Localization of phosphatase inhibitor-1 mRNA in the developing and adult rat brain in comparison with that of protein phosphatase-1 mRNAs, Mol. Brain Res., 25 (1994) 7–18. Vinade´, L. and Rodnight, R., The dephosphorylation of glial fibrillary acidic protein (GFAP) in the immature rat hippocampus is catalyzed mainly by a type 1 protein phosphatase, Brain Res., 732 (1996) 195–200. Vinade´, L., Gonc¸alves, C.A., Wofchuk, S., Gottfried, C. and Rodnight, R., Evidence for a role for calcium ions in the dephosphorylation of glial fibrillary acidic protein (GFAP) in immature hippocampal slices and in astrocyte cultures from the rat, Dev. Brain Res., 104 (1997) 11–17. Wofchuk, S.T. and Rodnight, R., Glutamate stimulates the phosphorylation of glial fibrillary acidic protein in slices of immature hippocampus via a metabotropic receptor, Neurochem. Int., 24 (1994) 517–523. Wofchuk, S.T. and Rodnight, R., Age-dependent changes in the regulation by external calcium ions of the phosphorylation of glial fibrillary acidic protein in slices of rat hippocampus, Dev. Brain Res., 85 (1995) 181–186.