Modifications of glial metabolism of glutamate after serotonergic neuron degeneration in the hippocampus of the rat

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 26 (1994) 1-8

Research Report

Modifications of glial metabolism of glutamate after serotonergic neuron degeneration in the hippocampus of the rat H. Hardin a,., A. Bernard a, F. Rajas b, M. Fevre-Montange a, E. Derrington a, M.F. Belin a, M. Didier-Bazes a a INSERM CJF 90-10, Facult~ de M~decine A. Carrel, rue G. Paradin, 69 372 Lyon Cedex 08, France b INSERM U369, Facult~ de M~decine A. Carrel, rue G. Paradin, 69 372 Lyon Cedex 08, France

Accepted 12 April 1994

Abstract

We have investigated the role of serotonergic neurons on the astrocytes catabolism of glutamate by analyzing glutamine synthetase (GS) and glutamate dehydrogenase (GDH) expression in the hippocampus after the degeneration of serotonergic neurons by a specific neurotoxin (5,7-DHT). 5,7-DHT caused reactive gliosis with hypertrophy (increase in glial fibrillary acidic protein (GFAP) expression) but not proliferation of astrocytes. Glutamate metabolism appeared preferentially regulated by a control of GDH expression rather than GS since the expression of GDH was specifically and significantly induced in the hippocampus whereas the level of GS remained unchanged. The inhibition of serotonin synthesis (by para-chlorophenylalanine (p-CPA) administration) produced no significant increase of GDH level. This suggests that serotonin is not the principal factor involved in this control of GDH expression.

Keywords: Astrocyte; Glutamate dehydrogenase; Glutamate metabolism; Glutamine synthetase; Hippocampus; Serotonin; Neuron-glia interaction 1. Introduction

In the central nervous system, complex reciprocal interactions occur between glial cells and neurons which are vital for their differentiation, development and function [13,31,39]. Astrocytes supply neurons with glucose, maintain the ionic balance, particularly by buffering extracellular K ÷, and metabolize products released by neurons such as NH~-, CO2, and amino acid neurotransmitters, especially y-aminobutyric acid (GABA) and glutamate [11]. These neurotransmitters, respectively the principal inhibitory and excitatory neurotransmitters, have a particular importance for the excitability of hippocampal neurons [22]. The levels of GABA and glutamate are largely controlled by glia since they are taken up and catabolized principally by astrocytes [35]. In neurons and glia, GABA is converted by GABA transaminase to succinate semialdehyde, which is further converted to succinate and then

* Corresponding author. Fax: (33) 78-77.86.12. 0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 9 - 3 2 8 X ( 9 4 ) 0 0 0 8 8 - V

to glutamate or oxidatively degraded. In astrocytes, glutamate, released by neurons or originating from GABA, is metabolized by glutamine synthetase or glutamate dehydrogenase [36] into neutral metabolites, which do not affect hippocampal excitability. Glutamine synthetase (GS, EC 6.3.1.2) catabolizes glutamate and ammonia into glutamine [7]. Glutamate dehydrogenase (GDH, EC 1.4.1.2) supplies astrocytes with metabolic precursors for the tricarboxylic acid cycle by converting glutamate to a-ketoglutarate [20,32]. The hippocampus is richly innervated by serotonergic terminals [18,24]. This serotonergic input could constitute a modulatory system for GABA and glutamate metabolism not only by neuron-neuron interactions [2,29] but also by neuron-glia interactions. The presence of serotonin (5-HT) uptake systems [1], and 5-HT 1 [46] or 5-HT 2 [8,33] receptors on astrocytes support this hypothesis. Hertz [16] proposed that the degeneration of serotonergic neurons could lead to an impairment of metabolic and functional interactions between neurons and astrocytes and then to a perturbation of hippocampal glutamatergic and GABAergic metabolism.

2

fL Hardin et al./Molecular Brain Research 20 (1994) 1 8

In order to investigate interactions between serotone r g i c a n d g l u t a m a t e r g i c n e u r o n s via i n t e r p o s e d a s t r o cytes, w e a n a l y z e d t h e r e s p o n s e o f h i p p o c a m p a l a s t r o c y t e s t o t h e d e g e n e r a t i o n o f s e r o t o n e r g i c n e u r o n s by 5,7-dihydroxytryptamine treatment. The subsequent o c c u r e n c e o f r e a c t i v e gliosis w a s t e s t e d b y i n v e s t i g a t i n g t h e i n d u c t i o n o f glial f i b r i l l a r y a c i d i c p r o t e i n ( G F A P ) and the proliferation of astrocytes. The consequences o n glial g l u t a m a t e m e t a b o l i s m w e r e a n a l y z e d b y s t u d y ing the expression of GS and GDH. The role of 5-HT i t s e l f o n glial g l u t a m a t e m e t a b o l i s m w a s i n v e s t i g a t e d b y comparing the effect of the inhibition of serotonin synthesis, produced by parachlorophenylalanine (pCPA).

2. Materials and methods 2.1. Pharmacological treatments

All animal experiments were performed in accordance with French legal requirements (decree 87-848). Adult male Sprague-Dawley rats of 180-200 g were obtained from Iffa-Credo (Lyon-France). The 5,7-DHT-treated or sham-operated animals received an injection of desipramine (20 mg/kg, Sigma) intraperitoneally 30 min before being anesthetized with chloral hydrate. 5,7-DHT (5,7-dihydroxytryptamine creatinine sulfate, Sigma, 345 ~,g in 20 ~1 of 0.9% NaCI) or vehicle was injected stereotaxieally into the lateral ventricle. (5,7-DHT is taken up by serotonergic and catecholaminergic terminals and pretreatment by desipramine protects the latter from degeneration.) The p-CPA-treated and control animals received 300 mg/kg of p-CPA (parachlorophenylalanine methyl ester, Sigma, diluted in water) or vehicle intraperitoneally. The treatments (5,7-DHT and p-CPA) used in this study have been described to cause a strong decrease of serotonin in the brain. Injection of 5,7-DHT in the lateral ventricle causes a progressive hydrogen peroxide mediated cytotoxic destruction of serotonergic neurons [3] particularly in the upper 2 / 3 of the dorsal raphe nucleus (DRN) [45] and in the hippocampus which becomes almost devoid of serotonergic fibers [43]. Ten days after the injection, the degeneration of serotonergic neurons is complete. 5,7-DHT treatment has the advantage to be specific for serotonergic neurons, in contrast with mechanical or other pharmacological lesions that destroy other fibers or neurons present in the lesioned area. A direct toxic effect of 5,7-DHT on astrocytes is possible since this neurotoxin acts via serotonergic uptake systems that are also present on astrocytes [1]. However, this seems unlikely since only a few astrocytes take up serotonin in vitro [1] and previous studies in our laboratory showed no cell bodies radiolabelled by tritiated serotonin in the hippocampus when injected in the same conditions as 5,7-DHT (unpublished

data). Furthermore, astrocytes could neutralize the toxicity of free radicals produced by 5,7-DHT by heme oxygenase 1 and apolipoprotein D [11]. The administration of p-CPA, which is an irreversible inhibitor of tryptophan hydroxylase activity, decreased the brain serotonin content by 80% 4 days after the treatment (data not shown). The level of serotonin returned to 60% of the control 8 days after the injection confirming the results of Koe and Weissman [23] and of Fuller [12]. 2.2. Tritiated-thymidine incorporation

In nine 5,7-DHT-treated rats and nine sham-operated rats a fixed cannula was implanted in the lateral ventricle 2 days before the desipramine injection. 3H-thymidine (20 ~zl, 740 kBq, Amersham, specific activity: 247.9 GBq/mmol) was injected via the cannula on either the 2nd, the 4th or the 7th day after 5,7-DHT or vehicle treatment (3 rats at each time). In 3 p-CPA-treated rats and in 3 controls, 20 Ixl of [3H]thymidine were injected into the lateral ventricle on the 2nd day after p-CPA or vehicle treatment. Four days after p-CPA administration or 10 days after 5,7-DHT treatment, rats were perfused under anesthesia with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4. Brains were removed, post-fixed and embedded in paraffin. Sections of 10 txm were cut and dipped in Ilford K5 emulsion. After 20-30 days of exposure, they were developed with D19 Kodak and fixed with Ilford Hypam. Sections were stained with Cresyl violet and mounted for light microscopy. The [3H]thymidine labeled cells were counted in an area of 0.16 mm 2 on each section. The value obtained for each rat was the sum of 8 sections. The data were represented as the mean percentages of the control_+ S.E.M. for 3 hippocampi. The different groups were compared using a one-way analysis of variance (ANOVA). 2.3. lmmunohistochemistry of GFAP, GS and GDH

Ten days after 5,7-DHT treatment, rats were killed under anaesthesia by intracardiac perfusion with 4% paraformaldehyde in 0.l M phosphate buffer pH 7.4 (3 rats in each group). Brains were removed, post-fixed, embedded in paraffin and 10 /xm sections were cut with a microtome. Non-specific antibody binding sites were saturated by incubation for 2 h in PBS containing 0.1% BSA and 0.1% Triton X-100. Sections were incubated 24 h at 4°C with polyclonal rabbit antibodies to GFAP (polyclonal rabbit antiserum, Dako; diluted 1/500), GS (polyclonal rabbit antiserum [5,40]; diluted 1:300) or GDH (polyclonal rabbit antibody generated by F. Rajas and B. Rousset, INSERM U369, Lyon, France; diluted 1 : 300). The biotin-streptavidinperoxidase technique was used to reveal antigen-antibody complexes. Briefly, the sections were rinsed in PBS-0.1% BSA, incubated 2 h in biotinylated anti-rabbit antibodies (diluted 1:1000 in PBS/0.1% BSA, Interchim), rinsed again and incubated 2 h in streptavidin-peroxidase complex (Interchim) diluted 1:2000. The sections were reacted in 50 mM Tris HC1 buffer pH 7.5 containing 0.075% diaminobenzidine and 0.005% hydrogen peroxide, rinsed again and mounted for light microscopy.

Fig. 1. GFAP immunohistochemistry and cell proliferation after 5,7-DHT treatment. A-D: GFAP immunohistochemistry. DRN (A,B) and hippocampus (C,D) of a sham-operated rat (A,C), and of a 5,7-DHT-treated rat (B,D). After the serotonergic degeneration, the GFAP staining extended into the periaqueductal grey matter and particularly in the upper 2 / 3 of the DRN. In the hippocampus, the increase was homogeneous, particularly in the CA4 and in DG. Bar = 150 tzm. E,F: cell proliferation was estimated by autoradiographic labeling of 3H-thymidine incorporation. E: hippocampus of a 5,7-DHT-treated rat. The [3H]thymidine-labeled cells, visualized by silver grains (arrow), were smaller and less numerous than GFAP stained cells (compare with figure 1D). Bar ~ 30 ~m. F: numeration of [3H]thymidine stained cells in 0.16 mm 2 of 8 sections of 3 rats. * P < 0.05. DRN, dorsal raphe nucleus; Aq: sylvius aqueduct; CA4, field CA4 of the Hammon's horn; DG, dentate gyrus; Gran, granular layer of the hippocampus.

"el

"1-

m

3

1

•

I

,

|

.--L

I

|

-lm=

"o

=

3H-thymidine labelled cells

0

¢¢

P~

4

tL Hardin et al. ~Molecular Brain Research 26 (1994) l - 8

2.4. Western blotting of GFAP, GS and GDH Ten days after 5,7-DHT injection or 4 or 8 days after p-CPA administration, the treated animals and their controls (5 rats at each time) were decapitated. Whole hippocampus was dissected out and quickly frozen. The dorsal raphe nucleus (DRN) was removed from 5 frozen 500 ~m serial slices using a 1 mm diameter stainless steel punch. Five DRN or hippocampi in each group were analyzed separately for quantitative analysis. The specificity and the linearity of the assays for the three proteins have been verified according to the criteria described previously by Jahn et al. [19] and O'Callaghan and Miller [34]. Tissues were homogenized in sample buffer (5 mM K2HPO4/ KH2PO 4 pH 7 containing 0.2% Triton X-100, 2 mM PMSF) (150 /xl/mg of tissue wet weight) and denatured in 250 mM Tris pH 6.8 containing 10% /3 mercaptoethanol, 2% SDS, 1% Bromophenol blue, 16% glycerol. Proteins were separated by electrophoresis (SDS-PAGE) on 9% acrylamide-bis acrylamide gel (5 p,l, 30 p.I and 50 tzl of samples were loaded for GFAP, GS and GDH analysis

respectively). They were blotted onto nitrocellulose sheets (Schleicher-Schuell, BA 85, 150 mA, 2 h). Immunodetection of GFAP, GS or GDH (same dilutions as for immunohistochemistry) were performed as described by Ikegaki et al. [17]. Bound antibodies were revealed by 1251-protein A (Amersham, spec. act. 3.6 kBq/~lt. Autoradiographic labeling of films was quantified by computerized densitometry using an image analysis system (Starvision, IMSTAR). After protein measurement in the initial homogenates by the method of Bradford [4], the data were expressed as (surface)× (optic density of labelling)/mg protein and represented as mean percentages of the control + standard error on the mean (S.E.M.). The different groups of rats were compared using a one-way analysis of variance (ANOVA).

2.5. Purification and analysis of the mRNA of GFAP, GS, GDH and G3PDH Ten days after 5,7-DHT injections, the treated animals and their controls (5 rats at each time) were decapitated. Whole hippocampus

Fig. 2. GS and GDH distribution in the hippocampus. GS (A,B) and GDH (C,D) immunohistochemistry in the hippocampus of a 5,7-DHT treated rat. The GS stained astrocytes were preferentially located in CA4 and the dentate gyrus (A) whereas the GDH positive cells particularly appeared in the stratum lacunosum moleculare (C). B,D: higher magnification of GS- and GDH-stained cells. CA4, field CA4 of the Hammon's horn; DG, dentate gyrus. Bar = 55/zm (A), 140/xm (B), 70 ~zm (C), 170/zm (D).

H. Hardin et al. / Molecular Brain Research 26 (1994) 1-8

were dissected out, homogeneized in 2 ml of RNAzolTMB (Bioprobe). Ten ~.g of RNA were electrophoresed in 1% agarose-l.28% formaldehyde/MOPS 1 × gels according to Lehrach [26] and transferred overnight to a nitrocellulose sheet (BA 585, Schleicher-Schuell) with 20 × SSC (1 × = 0.i5 M NaCI, 0.015 M sodium citrate) according to Thomas [42]. Hybridization was performed for 48 h with a 32p-random primed-eDNA for GFAP, GS (kindly given by M. Tardy, INSERM U282, France), GDH (kindly given by A.T. Das, University of Amsterdam, Netherlands) or G3PDH (as constitutively expressed gene). Signals corresponding to GFAP, GS, GDH and G3PDH mRNA were visualized by autoradiography and were expressed as (surface) x (optic density of labelling). Data were represented as mean percentages of the control+S.E.M. The different groups of rats were compared using a one-way analysis of variance (ANOVA).

3. Results

3.1. Effects of the serotonergic denervation on the glial morphology Ten days after 5,7-DHT injection in the lateral ventricle of the rats, the major features of a reactive

A

GFAP

B

Go.

C

Gs

5

gliosis (GFAP induction, cell proliferation) were investigated in the hippocampus. The DRN, which contains the highest density of serotonergic cell bodies was observed as control for treatment efficacity. The response of GFAP to 5,7-DHT treatment was analyzed by immunohistochemistry, Western blotting and Northern blotting. Immunohistochemistry of GFAP was performed with a polyclonal antiserum. In sham operated rats, a slight GFAP staining was observed mainly in the dorsal portion of the DRN (Fig. 1A) and in all structures of the hippocampus, more particularly in CA4 (Fig. 1C). After 5,7-DHT treatment, a large increase in the number of GFAP cells was observed mainly in the periaqueductal grey matter and in the upper 2/3 of the DRN (Fig. 1B). In the hippocampus, the number of GFAP stained cells was increased particularly in CA4 and in the dentate gyrus (Fig. 1D). The GFAP-positive cells in 5,7-DHT-treated rats showed more numerous and larger processes than in control rats. The increase in GFAP expression was

I/ G3PDH a

a

b

b

Fig. 3. Effect of 5,7-DHT and p-CPA treatment on GFAP, GDH and GS expression. Representative Western blots and Northern blots of GFAP (A), GDH (B) and GS (C) in the hippocampus. For Western blots analysis, 2.5/xg of total proteins were loaded in each lane for GFAP, 25/zg for GDH and 15 g.g for GS detection. The Western blots were revealed by 125I-protein A. For Northern blot analysis, 10/zg of total RNA were loaded on a denaturating agarose gel. Hybridizations were performed with [a-32p]dCTP random-primed cDNA. The Northern blot of G3PDH demonstrates that equivalent amounts of RNA were loaded in each lane. a: sham-operated hippocampus, b: hippocampus after 5,7-DHT treatment, c: control hippocampus, d: hippocampus 4 days after the p-CPA treatment, e: hippocampus 8 days after the p-CPA treatment. Arrow: 46 kDa molecular weight.

~

H. Hardin et aL /Molecular Brain Research26 11q94) l -8

quantified by Western blots on 5 hippocampi of 5,7D H T treated or sham-operated rats. The Western blots showed a band for G F A P at 51 kDa [10] (Fig. 3A) and the 5,7-DHT treatment caused a significant increase in G F A P both in the D R N (data not shown) and in the hippocampus (Table 1A). The Northern blots analysis indicated an increase of G F A P m R N A in the hippocampus after 5,7-DHT treatment (Fig. 3A and Table IB). This suggests a transcriptional control of G F A P induction. In order to detect an astrocytic proliferation possibly induced by 5,7-DHT treatment, an injection of [3H]thymidine was performed in the lateral ventricle. In the control there were very few cells which incorporated [3H]thymidine in the hippocampus. After 5,7D H T treatment, the number of [3H]thymidine cells was increased in the hippocampus (Fig. 1E,F). The staining was essentially observed in the nucleus of small cells ( < 10 txm) which contrast with larger G F A P positive cells and probably correspond to microglia (Fig. 1E). No difference was detected as a result of the different injection delays after 5,7-DHT or [3H]thymidine administration. Moreover, in all cases, the increase in the number of [3H]thymidine cells was of far lower magnitude than the increase in the number of anti-GFAP stained cells.

treated and control animals. G D H - s t a i n e d astrocytes could be observed in all structures of the hippocampus but particularly in the stratum lacunosum moleculare, usually close to blood vessels (Fig. 2C,D). No apparent difference of staining was detected between treated and control animals (data not shown). The Western bIots showed a main band for GS at 43 kDa [7] (Fig. 3C) and a main band for G D H at 56 kDa [6] (Fig. 3B). The GS content did not change after thc treatment (Fig. 3C) whereas G D H concentration was significantly increased in the hippocampus (Table 1A) but not in the D R N (data not shown). The apparent discrepancy between results obtained using immunohistochemistry and Western blots is due to the fact that the immunohistochemistry protocol is qualitative while Western blot technique using ~25I-protein A is quantitative and have a high sensitivity. The absence of modification in the pattern of G D H immunoreactivity in the hippocampus after 5,7-DHT treatment suggests an increase in the cellular content of G D H rather than in the number of astrocytes expressing G D H . Northern blot analysis showed no change in GS m R N A level (Fig. 3C and Table 1B). The level of G D H m R N A remained unchanged suggesting a post-transcriptional control of G D H induction (Fig. 3B and Table I B).

3.2. Effects o f serotonergic denervation on glial glutamate metabolism in the hippocampus

3.3. Effect of the inhibition of serotonin synthesis on the astrocytic response

We investigated the response to 5,7-DHT treatment of the two astrocytic key enzymes for G A B A and glutamate metabolism. GS and G D H expression were studied by immunohistochemistry and Western blotting and their m R N A s by Northern blotting. In treated rats and their controls, GS-positive astrocytes were principally visualized in CA4 and the dentate gyrus (Fig. 2A,B). They showed long and thin processes but no apparent difference was detected in the intensity and density of labelled astrocytes between

The involvement of serotonin in the regulation of the reactive gliosis ( G F A P expression and cell proliferation) and in the control of GS and G D H expression in hippocampal astrocytes was investigated 4 or 8 days after the p - C P A treatment by immunohistochemistry and Western blots. No significant changes of any of these markers were observed by immunohistochemistry or Western blots either 4 or 8 days after p - C P A treatment despite the fact that serotonin level was reduced by 80% (Fig. 3 and Table 1A).

Table 1 Quantified variations of GFA, GDH and GS expression Sham 5,7-DHT

Control

p-CPA 4 days

p-CPA 8 days

100± 9 100 + 23 100± 7

144+14 138 +_21 1385:16

131 5:15 105 5:21 105±10

A.

GFAP level GDH level GS level

72 ± 14 131 ___18 120 ± 15

220 5:28 * 215 ± 28 * 121 ± 17

100 ± 6 100 ± 16 100 5:18

244 ± 37 * 103 5:13 87 ± 17

B.

GFAP mRNA GDH mRNA GS mRNA

Autoradiographic labelling of Western blots or Northern blots of 5 isolated hippocampi were quantified by computerized densitometry using an image analysis system. Data were expressed as (surface) × (optic density)/mg protein for Western blots, and as (surface) × (optic density) for Northern blots. They were represented as mean percentage of control + S.E.M. for 5 isolated hippocampi. * P < 0.05 (as compared to the control value using a one-way ANOVA).

H. Hardin et al. / Molecular Brain Research 26 (1994) 1-8

4. Discussion Serotonergic neurons may modulate hippocampal glutamatergic and GABAergic transmission directly by synaptic contacts [2,29] or indirectly by interposed aso trocytes [15]. The degeneration of serotonergic neurons could perturbate the functional interactions between neurons and astrocytes and impair hippocampal glutamatergic and GABAergic transmission [16]. This possibility was investigated by studying the in vivo regulation of glutamate catabolism by hippocampal astrocytes after serotonergic denervation by 5,7-DHT treatment. The disappearance of the serotonergic perikarya in the DRN and their terminals in the hippocampus [3,43,45] was accompagnied by a strong hypertrophy of astrocytes assessed by morphological changes and induction of GFAP expression. The reactive astrocytes are not proliferative since only a small number of ceils, with a small size compared to reactive astrocytes, incorporated [3H]thymidine. These radiolabelled cells probably are microglia [25,30]. The effect of the degeneration of the serotonergic neurons on glial glutamate metabolism were investigated by studying the regulation of GS and GDH expression in the hippocampus. The serotonergic degeneration produced a large increase in GDH expression that was mediated at the post-transcriptional level. This induction appears specific to functional activity of hippocampal astrocytes since, in the DRN, GDH expression was not apparently modified in spite of an extensive gliosis (unpublished results). This confirms the preferential metabolism of glutamate towards the TCA cycle demonstrated in vitro [36]. The stability of GS levels in the hippocampus is in contrast with previous results which showed a fall of GS activity after glutamatergic degeneration [44]. This stability of GS in response to lesion of the serotonergic inputs to the hippocampus may be to avoid perturbation of local concentrations of glutamine and NH~-. Indeed, an increase of glutamine level causes cerebral edema [13,37]. A fall in the NH~- level and subsequent acidosis could lead to an impairment of neuronal excitability, to cell swelling and even to cell death [21]. Moreover, only a tiny part of glutamine returns to neurons and is converted into glutamate, the majority is eliminated in the blood stream or oxidized into CO 2 [15]. Alternatively, a-ketoglutarate is taken up by glutamatergic neurons as a precursor for the neurotransmitter glutamate and also supplies neurons with metabolic precursors, particularly glucose, through anaplerotic pathways [16]. Various factors could be involved in the regulation of GDH expression. Serotonin appeared to be a likely candidate since receptors for this neurotransmitter are present on astrocytes [1,8,33,46] and serotonin has been shown to influence GFAP expression in vitro [27]. Moreover, serotonin has previously been shown to in-

7

hibit glutamate release by cerebellar neurons [28,14]. Our results demonstrate that serotonergic innervation may also modulate glutamate metabolism by astrocytes. However the contrast between the increase in GDH and GFAP levels produced by 5,7-DHT treatment and the slight and transitory effect after p-CPA administration, despite the decrease in serotonin level, indicates either that the remaining 20% of serotonin is sufficient to maintain GFAP and GDH at control levels or that serotonin is not the main factor involved in the increase in GFAP and GDH expressions. Other signalling molecules present in serotonergic terminals or in their environment, such as trophic factors, cytokines or components of the extracellular matrix [9] may be modified after serotonergic neurons degeneration. They are presumed to be involved in astroglial activation [41] and could modulate hippocampal activity [38]. Acknowledgements The authors are very grateful to Dr M. Tardy, INSERM U282, Cr6teil, France, for the gift of glutamine synthetase cDNA. Drs. A.T. Das and W.H. Lamers, University of Amsterdam, Netherlands, are warmly acknowledged for their sending of glutamate dehydrogenase cDNA. This research was supported by INSERM CJF 90-10 and CNRS URA 1195. References [1] Anderson, E.J., McFarland, D. and Kimelberg, H.K., Serotonin uptake by astrocytes in situ, Glia, 6 (1992) 154-158. [2] Andrade, R. and Nicoll, R.A., Novel anxiolytics discriminate between postsynaptic serotonin receptors mediating different physiological response on single neurons of the rat hippocampus, Naunyn-Schrniedeberg's Arch. Pharmacol., 336 (1987) 5-10. [3] Bjfrklund, A., Baumgarten, H.G. and Rensch, A., 5,7-Dihydroxytryptamine: improvement of its selectivity for serotonin neurons in the CNS by pretreatment with desipramine, J. Neurochem., 24 (1975) 833-835. [4] Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem., 72 (1976) 248-254. [5] Caldani, M., Rolland, B., Fages, C. and Tardy, M., Glutamine synthetase activity during mouse brain development, Experientia, 38 (1982) 1199-1202. [6] Chee, P.Y., Dahl, J.L. and Fahien, L.A., The purification and properties of rat brain glutamate dehydrogenase, J. Neurochem., 33 (1979) 53-60. [7] De Vellis, J., Wu, D.K. and Kumar, S., Enzyme induction and regulation of protein synthesis. In FedorofL S. and Vernadakis, A. (Eds.), Astrocytes, Iiol. 2, Academic Press, London, 1986, pp. 209-237. [8] Deecher, D.C., Wilcox, B.D., Dave, V., Rossman, P.A. and Kimelberg, H., Detection of 5-hydroxytryptamine 2 receptors by radioligand binding, Northern blot analysis, and Ca 2+ responses in rat primary astrocyte cultures, J. Neurosci. Res., 35 (1993) 246-256. [9] Eddleston, M. and Mucke, L., Molecular profile of reactive astrocytes - implications for their role in neurologic disease, Neuroscience, 54 (1993), 15-36.

8

H. Hardin et al./Molecular Brain Research 26 (19941 l - 8

[10] Eng, L.F. and Shiurba, R.A., Glial fibrillary acidic protein: a review of structure, function, and clinical application. In Marangos P.J., Campbell I.C., Cohen R.M. (Eds.), Neuronal and Glial Proteins: Structure, Function, and Clinical Application, Academic Press, New York, 1988, pp. 339-359. [11] Fedoroff, S. and Vernadakis, A., Astrocytes, Academic Press, London, 1986. [12] Fuller, R.W., Drugs affecting serotonin neurons, Prog. Drug Res., 35 (199(I) 85-108. [13] Hawkins, R.A., Jessy, J., Mans, A.M. and DeJoseph, M.R., Effect of reducing brain glutamine synthesis on metabolic symptoms of hepatic encephalopathy, J. Neurochem., 60 (1993) 10001006. [14] Hertz, L., Peng, L., Hertz, E., Juurlink, B.H.J. and Yu, P.H., Glutamate release and monoamine oxidase activity in developping cerebellar granule cells, Neuroehem. Res., 10 (1989) 10391096. [15] Hertz, L., Autonomic control of neuronal-astrocytic interactions regulating metabolic activities and ion fluxes in the CNS, Brain Res. Bull., 29 (1992) 303-313. [16] Hertz, L., Is Alzheimer's disease an anterograde degeneration, originating in the brainstem, and disrupting metabolic and functional interactions between neurons and glial cells?, Brain Res. Rel~., 14 (1989) 335-353. [17] Ikegaki, N. and Kennett, R.H., Glutaraldehyde fixation of the primary antibody-antigen complex on nitrocellulose paper increases the overall sensitivity of immunoblot assay, Z ImmunoL Methods, 124 (19891 205-210. [18] Jacobs, B.L. and Azmitia, E.C., Structure and function of brain serotonin system, Physiol. Rev., 72 (1992) 165-229. [19] Jahn, R., Schiebler, W. and Greengard, P., A quantitative dot-immunobinding assay for proteins using nitrocellulose membrane filters, Proc. Natl. Acad. Sci. USA, 81 (1984) 1684-1687. [20] Kaneko, T., Shigemoto, R. and Mizuno, N., Metabolism of glutamate and ammonia in astrocyte: an immunocytochemical study, Brain Res., 457 (1988) 160-164. [21] Kempski, O., Staub, F., Jansen, M. and Baethmann, A., Molecular mechanisms of glial cell swelling in acidosis. In D.M. long (Ed.), Adl~ances in Neurology, Vol. 52, Brain Edema, Raven, New York, 1990, pp. 39-45. [22] Knowles, W.D., Normal anatomy and neurophysiology of the hippocampal formation, Z Clin. Neurophysiol., 9 (1992) 252-263. [23] Koe, K.B. and Weissman, A., p-Chlorophelylalanine: a specific depletor of brain serotonin, J. Pharmacol. Exp. Ther., 154 (1966) 499-516. [24] K6hler, C., On the serotonergic innervation of the hippocampal region: an analysis employing immunohistochemistry and retrograde fluorescent tracing in the rat brain. In Cytochemical Methods" in Neuroanatomy, A.R. Liss, New York, 1982, pp 387-405. [25] Kreutzberg, G.W., Graeber, M.B., Raivich, G. and Streit, W.J., Neuron-glial relationship during regeneration of motoneurons. In Regulation of Gene Expression in the Nen~ous System, WileyLiss, New York, 1990, pp. 333-341. [26] Lehrach, H., Diamond, D., Wozney, J.M. and Boedker, H., RNA molecular weight determination by gel electrophoresis under denaturing conditions, a critical reexamination, Biochemistry, 16 (1977) 4743. [27] LePrince, G., Copin, M.C., Hardin, H., Belin, M.F., Bouilloux, J.P. and Tardy, M., Neuron-glial interactions: effects of serotonin on the astroglial expression of GFAP and of its encoding message, Exp. Brain Res., 51 (1990) 295-298. [28] Maura, G., Roccatagliata, E., Uliviv, M. and Raiteri, M., Serotonin-glutamate interactions in rat cerebellum: involvement of 5HT1 and 5HT2 receptors, Eur. J. Pharmacol., 145 (1989) 31-38.

[29] Miettinen, R. and Freund, T.F., Convergence and segregation of septal and median raphe inputs onto different subsets of hippocampal inhibitory interneurons, Brain Res., 594 (1992) 263-272. [30] Morshead, C.M. and van der Kooy, D., Separate blood and brain origins of proliferating cells during gliosis in adult brains, Brain Res., 535 (1990) 237-244. [31] Murphy, F., Astrocytes, Pharmacology and Function, Academic Press, New York, 1993. [32] Nicklas, W.J., Amino acid metabolism in the central nervous system role of glutamate dehydrogenase. In R.C. Duvoisin and A. Plaitakis (Eds.), The Olit,opontocerebellar Atrophies, Raven, New York, 1984, pp. 245-253. [33] Nilsson, M., Hansson, E. and R6nnb~ick, L., Adrenergic and 5HT 2 receptors on the same astroglial cell. A microspectrofluorimetric study on cytosolic Ca 2+ response in single cells in primary culture, Det,. Brain. Res., 63 (1991) 33-41. [34] O'Callaghan, J.P. and Miller, D.B., Cerebellar hypoplasia in the Gunn rat is associated with quantitative changes in neurotypic and gliotypic proteins, J. Parmacol. Exp. Ther., 234 (19851 522533. [35] Shousboe, A., Westergaard, N. and Hertz, L., Neuronal-astrocytic interactions in glutamate metabolism, Excitatory Amino Acids, (1993) 49-53. [36] Sonnewald, U., Westergaard, N., Petersen, S.B., Unsgard, G. and Schousboe, A., Metabolism of [U-13C]glutamate in astrocytes studied by ~3C NMR spectroscopy: incorporation of more label into lactate than into glutamine demonstrates the importance of the tricarboxylic acid cycle, J. Neurochem., 61 (1993) 1179-1182. [37] Takahashi, H., Koehler, C., Brusilow, S.W. and Traystman, R.J., Inhibition of brain glutamine accumulation prevents cerebral edema in hyperammonemic rats, Z Am. Physiol. Soc., (1991) H825-H829. [38] Tancredi, V., D'Angelo, G., Grassi, F., Tarroni, P., Palmieri, G., Santoni, A. and Eusebi, F., Tumor necrosis factor alters synaptic transmission in rat hippocampal slices, Neurosci. Lett., in press. [39] Tardy, M. and LePrince, G., Glial neuronal interactions, Ann. NYAcad. Sci., 663 (1991) 630-632. [40] Tardy, M., Rolland, B., Fages, C. and Caldani, M., Astroglial cells: glucocorticoid target cells in the brain, Clin. Neuropharmacol., 7 (1984) 296-302. [41] Tchelingerian, J.L., Quinonero, J., Booss, J. and Jacque, C., Localization of TNFo~ and I L l a immunoreactivities in striatal neurons after surgical injury to the hippocampus, Neuron, 10 (1993) 213-224. [42] Thomas, P., Hybridization of denatured RNA and small DNA fragment transferred to nitrocellulose, Proc. Natl. Acad. Sci. USA, 77 (1980) 5201. [43] van Luijtelaar, M.G.P.A., Tonnaer, J.A.D.M., Frankhuijzen, A.L., Dijkstra, H., Hagan, J.J. and Steinbusch, H.W.M., Morphological, neurochemical, and behavioral studies on serotonergic denervation and graft-induced reinnervation of the rat hippocampus, Neuroscience, 42 (1991) 365-377. [44] Waniewski, R.A. and McFarland, D., Intrahippocampal kainic acid reduces glutamine synthetase, Neuroscience, 34 (1990) 305310. [45] Weissmann, D., Belin, M.F., Aguera, M., Meunier, C., Maitre, M., Cash, C.D., Ehret, M., Mandel, P. and Pujol, J.F., Immunohistochemistry of tryptophan hydroxylase in the rat brain, Neuroscience, 23 (19871 291-304. [46] Whitaker-Azmitia, P.M., Clarke, C. and Azmitia, E.C., Localization of 5HTIA receptors to astroglial cells in adult rats: implications for neuronal-glial interactions and psychoactive drug mechanism of action, Synapse, 14 (1993) 201-205.