Developmental expression of glutamate transporters and glutamate dehydrogenase in astrocytes of the postnatal rat hippocampus

Developmental Expression of Glutamate Transporters and GlutamateDehydrogenase in Astrocytes of the Postnatal Rat Hippocampus

Peter Kugler* and Verena Schleyer

ABSTRACT: Glutamate is the major excitatory transmitter in the CNSand plays distinct roles in a number of developmental events. Its extra-cellular concentration, which mediates these activities, is regulated byglutamate transporters in glial cells and neurons. In the present study, wehave used nonradioactive in situ hybridization, immunocytochemistry,and immunoblotting to show the cellular and regional expression of thehigh-affinity glutamate transporters GLAST (EAAT1) and generic GLT1(EAAT2; glial form of GLT1) in the rat hippocampus during postnataldevelopment (P1–60). The results of transporter expression were com-pared with the localization and activity pattern of glutamate dehydroge-nase (GDH), an important glutamate-metabolizing enzyme. The studyshowed that both transporters and GDH were demonstrable at P1 (day ofbirth). The expression of GLAST (detected by in situ hybridization andimmunocytochemistry) in the early postnatal development was higherthan GLT1. Thereafter, the expression of both transporters increased,showing adult levels at between P20 and P30 (detected by in situ hybrid-ization and immunoblotting). At these time points, the expression of GLT1appeared to be significantly higher than the GLAST expression. GLT1 andGLAST proteins were demonstrable only in astrocytes. The increase ofGDH activities (steepest increase from P5–P8), which were localizedpreferentially in astrocytes, was in agreement with the increase of trans-porter expression, preferentially with that of GLT1. These observationssuggest that the extent of glutamate transporter expression and of gluta-mate-metabolizing GDH activity in astrocytes is intimately correlatedwith the formation of glutamatergic synapses in the developing hippocam-pus. © 2004 Wiley-Liss, Inc.

KEY WORDS: GLT1; GLAST; in situ hybridization; immunocytochem-istry; GDH activity

INTRODUCTION

In the adult hippocampus, glutamate is the major excitatory transmitter(for review, see Storm-Mathisen and Ottersen, 1984; Frotscher et al., 1988).Most of the glutamatergic synapses are formed during the postnatal devel-opment of the hippocampus (Loy, 1980; Amaral and Dent, 1981; Gaar-skjaer, 1986; Stanfield and Cowan, 1988; Tamamaki, 1999). In recentmorphological and electrophysiological studies on the rat intact hippocam-pal formation in vitro, it is shown that glutamatergic entorhinal and com-missural afferents as well as Schaffer collaterals are detectable at birth in CA1,and that AMPA receptor-mediated excitatory postsynaptic potentials aredemonstrable in CA1 at embryonic day 19, the amplitude of which in-

creased until postnatal day 9 (P9) (Diabira et al., 1999).Furthermore, spontaneous postsynaptic currents, medi-ated by AMPA and NMDA receptors, are detected inCA1 pyramidal cells over the first postnatal week (Groc etal., 2002). Thus, glutamate plays an important role in thepostnatal hippocampal development not only in the reg-ulation of neuronal cytoarchitecture (outgrowth of neu-ronal processes; Mattson et al., 1988), but in the devel-opment of excitatory circuitry as well (Mattson et al.,1988; Hsia et al., 1998; Diabira et al., 1999; Luthi et al.,2001).

In view of the importance of glutamate for the estab-lishment of excitatory circuitry in the postnatal develop-ment of the hippocampus, it seems essential to studydevelopmental changes in the expression of glutamatetransporters and glutamate-metabolizing enzymes. Theprimary role of high-affinity glutamate transporters inplasma membranes is to remove glutamate from the ex-tracellular space and thus control receptor activation andto regulate developmental events and synaptic transmis-sion (for review, see Danbolt, 2001). To date, five high-affinity glutamate transporters have been cloned fromwhich in the adult CNS GLAST (EAAT1) and genericGLT1 (EAAT2; glial form of GLT1) are concentrated oronly localized in astrocytes, respectively (Schmitt et al.,1996, 1997; Kugler and Schmitt, 2003; for review, seeDanbolt, 2001). It is known that astrocytes have thehighest glutamate uptake activity in the adult brain (forreview, see Kugler, 1993). Glutamate taken up into as-trocytes is metabolized by various enzymes (Kugler,1993). One of the important glutamate-metabolizing en-zymes in astrocytes is glutamate dehydrogenase (GDH)(Kugler, 1993). Expression of GDH in the hippocampusof adult rats and its functional meaning in glutamatergictransmission have been analyzed and discussed previ-ously (Kugler, 1993; Kugler et al., 1995; Schmitt andKugler, 1999).

To date, limited data are available on the developmen-tal expression of GLT1 and GLAST at the subregionallevel in the hippocampus of men (immunohistochemis-try; Bar-Peled et al., 1997), mice (radioctive in situ hy-bridization [ISH]; Sutherland et al., 1996; Shibata et al.,1996), and rats (immunohistochemistry; Furuta et al.,1997). In the present study, we have used a highly sensi-tive method that provides clear cellular resolution,namely nonradioactive ISH, using digoxigenin (DIG)-labeled cRNA probes to detect generic GLT1 (glial formof GLT1) and GLAST in the hippocampus during post-

Institute of Anatomy and Cell Biology, University of Wurzburg, Wurz-burg, GermanyGrant sponsor: Deutsche Forschungsgemeinschaft (DFG).*Correspondence to: Peter Kugler, Institute of Anatomy and Cell Biology,Koellikerstrasse 6, D-97070 Wurzburg, Germany.E-mail: [email protected] for publication 19 February 2004DOI 10.1002/hipo.20015Published online 9 April 2004 in Wiley InterScience (www.interscience.wiley.com).

HIPPOCAMPUS 14:975–985 (2004)

© 2004 WILEY-LISS, INC.

natal development of rats (P1–60). The ISH data were comparedwith the distribution of EAAT proteins detected by immunocyto-chemistry. The content of EAAT proteins in hippocampal homog-enates at various development stages was analyzed by immunoblot-ting. Furthermore, the results of transporter expression werecompared with localization and activity of the glutamate-metabo-lizing GDH.

MATERIALS AND METHODS

Tissue Sources

Postnatal male Wistar rats (n � 185) from our own colony(housed under standard conditions) were used. From P1 (day ofbirth) on, at least 20 rats of each postnatal day (five animals in eachdetection method) were examined for GLT1- and GLAST-ISH(P1, 3, 6, 8, 10, 20, 30, 60), GLT1- and GLAST-immunoblotting(P1, 2, 3, 4, 5, 7, 8, 10, 20, 30), GLT1- and GLAST-immunocy-tochemistry (P5, 8, 15, 20), GDH immunocytochemistry (P5, 8,15, 60), and quantitative GDH histochemistry (P5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 20, 30, 60). The animals were killed by decap-itation, the brains quickly removed and further processed as de-scribed below.

In Situ Hybridization

The brains were removed and frontal blocks of the brain werefrozen in liquid nitrogen-cooled propane. Twelve-�m-thick sec-tions were cut in a cryostat (�20°C; Frigocut 2800 E, Reichert-Jung, Nussloch, Germany). The sections were mounted on pre-coated glass slides (Superfrost Plus; Menzel, Braunschweig,Germany) and thawed. The sections were fixed for 5 min in freshlyprepared 4% formaldehyde in 0.1 M phosphate-buffered saline(PBS; pH 7.4). The fixed sections were transferred to absoluteethanol and stored at 4°C for 1 to 2 days and subsequently pro-cessed for ISH.

The ISH procedure used was described previously (Schmitt andKugler, 1999; Kugler and Schmitt, 1999; Schmitt et al., 2002).Briefly, the sections were removed from ethanol, rehydrated in agraded series of ethanol, transferred to 2 � standard saline citrate(SSC) and treated with 0.05 N HCl for 5 min. After washing with2 � SSC, the sections were incubated with freshly prepared 0.25%acidic anhydride, washed again with 2 � SSC and covered with thehybridization solution, containing the digoxigenin(DIG)-labeledanti-sense (sense) cRNA probe (final concentration: GLT1 cRNA1 ng/�l, GLAST cRNA 8 ng/�l) and 550 �g/ml Salmon testesDNA (Sigma, Deisenhofen, Germany) in 4� SSC, 1� Den-hardt’s solution (Sambrook et al., 1989), 10% dextran sulfate, and50% deionized formamide at 60°C for 16–18 h. Posthybridizationwashes were done stepwise at room temperature with 2 � SSC,1 � SSC, 50% formamide and then again with 2 � SSC. Next, thesections were treated with 30 �g/ml ribonuclease A (50 Kunitz-units/mg; Boehringer, Mannheim, Germany) in a solution con-taining 500 mM NaCl, 10 mM Tris-HCl (pH 8), 1 mM EDTA at37°C for 30 min to remove unhybridized single-strand RNAs.

After this treatment, the sections were incubated with the samebuffer without RNase A at 60°C for 30 min.

Subsequently, the sections were rinsed in Tris-buffered saline(TBS; 100 mM Tris and 150 mM NaCl, pH 7.5) for 5 min,incubated with TBS containing 0.5% blocking reagent (DIG Nu-cleic Acid Detection Kit, Boehringer) for 30 min, followed by0.3% Triton X-100 in TBS for 20 min. After incubation with 1.5U/ml sheep anti-DIG-aP (anti-digoxigenin alkaline phosphatase)conjugate (Boehringer) in TBS containing 0.3% Triton X-100 for60 min, the sections were washed in TBS and transferred to a 0.1M Tris-buffer (pH 9.5), containing 100 mM NaCl and 50 mMMgCl2 for 2 min prior to the aP visualization (see below).

The DIG-labeled anti-sense (sense) cRNA probes used weregenerated exactly as described previously (Schmitt et al., 1996,1997). The probes were complementary to nt �38 to 1776 on thegeneric GLT1 sequence (GLT1 cRNA) (Schmitt et al., 1996) andto nt 5–1710 on the GLAST sequence (GLAST cRNA) (Schmittet al., 1997).

The procedure used for detection of alkaline phosphatase wasdescribed previously (Asan and Kugler, 1995). The incubationmedia contained 0.4 mM 5-bromo-4-chloro-3-indolylphosphate(BCIP; Boehringer), 100 mM sodium chloride, 50 mM MgCl2,0.4 mM tetranitroblue tetrazolium chloride (Serva, Heidelberg,Germany) in 100 mM Tris-HCl buffer at pH 9.5.

In some experiments following aP visualization, several brainsections were used for the immunocytochemical detection of glialfibrillary protein (GFAP) and vimentin, applying the peroxidase-antiperoxidase method (PAP complex 1:100; mouse monoclonalantibody against GFAP 1:2000; DAKO, Hamburg, Germany;mouse monoclonal antibody against vimentin 1:100; Boehringer;Sternberger et al., 1990).

As a control, substitution of the antisense cRNA probe by anequivalent amount of labeled sense cRNA probe led to a completelack of staining. Omission of labeled cRNA probes from the re-spective hybridization mixtures resulted in completely unstainedsections (controls not shown).

Antibodies, Immunoblotting, andImmunostaining

Antibodies

Affinity-purified polyclonal (rabbit) peptide antibodies againstthe glutamate transporters were used. These antibodies were char-acterized elsewhere: anti-GLT1-antibody (against a peptide corre-sponding to amino acid residues 554–573; Schmitt et al., 1996)and anti-GLAST-antibody (against a peptide corresponding toamino acid residues 523–542; Schmitt et al., 1997). An affinity-purified polyclonal (rabbit) antibody against GDH was used,which has been described elsewhere (Schmitt and Kugler, 1999).

Mouse monoclonal antibodies against GFAP and glutaminesynthetase (GS) were purchased from DAKO and Sigma, respec-tively. In the CNS, GFAP is a specific marker protein of astrocytes(Bignami et al., 1972) and GS is enriched in astrocytes (for review,see Kugler, 1993).

976 KUGLER AND SCHLEYER

Immunoblotting

For immunoblotting, the hippocampi were excised and homog-enized at 4°C in 10 mM NaH2PO4 (pH 7.2) containing 2 mMMgCl2, aprotinin (5 �g/ml), leupeptin (2 �g/ml), pepstatin (2�g/ml), and phenylmethylsulfonyl fluoride (PMSF; 100 �g/ml).The homogenate was centrifuged at 1,000g for 10 min and thesupernatant was centrifuged at 100,000g for 1 h. The proteincontent of the 100,000g pellet (membrane fraction) was deter-mined by the Bio-Rad protein assay (Bio-Rad, Munich, Germany)and the pellet was used for immunoblotting. Proteins (25 or 50 �gper lane) were electrophoretically separated on 10% gels by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Subsequently, the proteins were transferred electrophoretically tonitrocellulose membranes (Burnette, 1981). Strips of the nitrocel-lulose membranes were incubated for 24 h at 4°C with the affinity-purified antibody against GLT1 or GLAST (�1.8 �g/ml). Boundimmunoglobulins were visualized using peroxidase-conjugatedgoat anti-rabbit IgG (1:3,000; Bio-Rad, Richmond, Canada; blot-ting grade) and the enhanced luminol chemiluminescence tech-nique (Amersham, Braunschweig, Germany). Antibody previouslyabsorbed with an excess of the peptides used for immunizationserved as a control. These immunoblots exhibited no labeling (notshown).

Immunostaining

Pieces of the hippocampi removed were frozen in liquid nitro-gen-cooled propane, then freeze-dried and embedded in Epoxyresin Quetol 651 (Science Services, Munich, Germany) (Kuglerand Schmitt, 2003; Kugler and Beyer, 2003). Semithin sections (1�m) were mounted on glass slides. The resin was removed byplacing the slides for 5 min in sodium methylate solution (Fluka,Neu-Ulm, Germany) (Mayor et al., 1961).

The tissue sections were preincubated for 3 h at room temper-ature with 2% bovine serum albumin (BSA), 10% normal goatserum (NGS), and 0.05% Tween 20 (Ferrak, Berlin, Germany) inphosphate-buffered saline (PBS) pH 7.4. Then the sections wereincubated for 24–48 h at 4°C with the primary antibody diluted inthe preincubation solution (anti-GDH, 15 �g/ml; anti-GLT1 15�g/ml; anti-GLAST 15 �g/ml; anti-GFAP 1: 10,000; anti-GS1:200). After several washes with PBS, the semithin plastic sectionswere incubated for 90 min at room temperature with indocarbo-cyanin (Cy3)-labeled secondary antibody (1:300; goat anti-rabbitIgG; Dianova, Hamburg, Germany) for detection of glutamatetransporters and GDH, furthermore with carbocyanin (Cy2)-la-beled secondary antibody (1:300; goat-antimouse IgG, Dianova)for the detection of GFAP or GS. Controls were performed withprimary antibody, previously absorbed with an excess of GDHprotein or the corresponding glutamate transporter peptide usedfor immunization or without primary antibody. These sectionswere unstained (not shown). The sections were examined with aZeiss laser scanning microscope (Zeiss LSM 510) equipped with anArgon laser (488 nm) for excitation of Cy2, an HeNe1 laser (543nm) for excitation of Cy3 and appropriate filter combinations forvisualization of Cy2 and Cy3 emissions.

Quantitative GDH histochemistry

The hippocampi were excised and embedded in 8% gelatin (Kugleret al., 1988) and were frozen in liquid nitrogen-cooled propane. Ten-�m-thick coronal sections from the middle part of the right hip-pocampus each were cut in a Frigocut cryostat (�20°C) and mountedon room-temperature slides. Sections were incubated in cuvettes for10 min at 25°C using the GDH incubation medium described else-where (Kugler, 1988, 1990). In brief, the incubation medium con-sisted of 100 mM L-glutamic acid monosodium salt (Fluka, Neu-Ulm, Germany), 5 mM NAD (Boehringer), 5 mM ADP disodiumsalt (Serva), 10 mM sodium azide, 20 mM NaCl, 0.15 mM phenazinemethosulfate (Serva), 5 mM nitroblue tetrazolium chloride (Serva),22% polyvinyl alcohol (polyviol G 04/14; Wacker Chemie, Munich,Germany) in 0.05 M Hepes buffer; the final pH was 7.5. The reactionwas stopped by rinsing the sections in ice-cold water. Then the sec-tions were air dried and mounted in Karion F. The reaction productwas subjected to microphotometric analysis within 12 h of the reac-tion taking place.

From each hippocampus, five slides with two adjacent sectionseach were prepared for the test reaction and three slides with twoadjacent sections for the control reaction (without substrate in theincubation medium).

For the endpoint measurements, we employed a computer-con-trolled Leitz-MPV 3 scanning microscope photometer (Leitz,Wetzlar, Germany; for details, see Kugler, 1988; Kugler et al.,1988). The microphotometer settings and the measuring proce-dure were the same as described by Kugler et al. (1988): finalmagnification of �40; measuring diaphragm of 1.2 �m; final mea-surement field of 900 �m2; wavelength of 585 nm (Butcher,1978). The measurements were performed in the following regionsand layers of the hippocampus: the lacunosum-molecular, radia-tum, and oriens layers of the cornu ammonis (sectors CA1 andCA3) and the molecular layer of the dentate gyrus.

Somata and coarse processes of astrocytes were excluded from themeasurements. Using an appropriate program of the MPV-3 com-puter, the mean optical density (MOD) values for each measuringfield were calculated and then corrected for the corresponding controlvalues. Afterward the mean and standard deviations of the correctedMOD values were calculated. The MOD values were converted intoabsolute units of enzyme activity using the molar extinction coefficientof the formazan of nitroblue tetrazolium chloride at 585 nm (Butcher,1978; formula for calculation: c � E � ��1 � d�1; c � nmol sub-strate � cm�3 wet tissue � min�1, E � MOD � min�1, � � 0.016cm2 � nmol�1, d � section thickness in cm).

RESULTS

Expression of GLT1 and GLAST

In situ hybridization

GLAST. During the postnatal development of the hippocam-pus, GLAST mRNA could be detected only in glial cells, which

_______________________________________________ EXPRESSION OF GLUTAMATE TRANSPORTERS 977

FIGURE 1. In situ hybridization using GLAST (a–e) and GLT1cRNA probes (f–k). Developmental expression changes in the hip-pocampus are shown from P1 to P20. A cellular labeling is alreadypresent at P1, which increased in the neuropil layers, above all in thedemonstration of GLT1 (f–k). Note the changes in the labeling ofneuronal cell layers detecting GLT1 (f–k) and of the subventricular

zone (ependymal and subependymal cells; arrowheads) of the lateralventricle detecting GLAST (a–e) and GLT1 (f–k). f, fimbria; pc,plexus choroideus; p, pyramidal cell layer; g, granule cell layer; CA,cornu ammonis with sectors CA1 and CA3; m, dentate gyrus molec-ular layer; DG, dentate gyrus. Scale bar � 400 �m (applies to a–k).

appeared to be astrocytes, on the basis of shape and distribution(Figs. 1a–e and 2a–d). Double-labeling experiments usingGLAST-ISH in the first step, followed by GFAP/vimentin immu-nostaining, supported this suggestion (not shown).

At P1, astrocytes of the hippocampus (gray matter and fimbria)showed a moderate to strong staining (Fig. 1a). The labeling in-tensity of astrocytes showed almost no changes in further develop-ment until P60. In contrast, the number of labeled astrocytes in-creased strongly from P1 to P20 (Fig. 2a–c). Thereafter, nosignificant changes in the density of labeled astrocytes were ob-served until P60 (not shown).

Additionally, the cRNA probe specifically labeled ependymalcells and cells of the subventricular zone of the lateral ventricleneighboring the hippocampus (Fig. 1a–e). The labeling of thesecells was strong at P1 and decreased continuously to a weak stain-ing until P20 (Fig. 1a–e). After P20 only a weak labeling ofependymal cells was detected.

GLT1. In all age groups studied, there was a neuronal and a gliallabeling showing different staining intensities (Fig. 1f–k). Thelabeling of astrocytes was weak to moderate until P6 (Figs. 1a–hand 2d,e). Thereafter, the staining intensity and the number oflabeled astrocytes increased strongly until P20 (Figs. 1i–k and 2f).

FIGURE 2. In situ hybridization applying GLAST (a–c) andGLT1 cRNA probes (d–f) to the dentate gyrus at P3, P6, and P20.(a–c) GLAST: The cellular (glial cell) labeling is already moderate tostrong at P3. There is an increased number of strongly labeled cells atP6 and P20. Granule cells in the granule cell layer (g) are unstained.

(d–f) GLT1: The cellular (glial cell) labeling (arrowheads) is weak tomoderate at P3 and P6 and strong at P20. Note the differential label-ing of granule cells in the granule cell layer (g). h, hilum of thedentate. Scale bar � 45 �m (applies to a–f).

FIGURE 3. Immunoblot analysis (10% SDS-PAGE) of the devel-oping hippocampus (P1–P30) using affinity-purified antibodiesagainst GLAST (a) and GLT1 (b). For probing, the 100,000g pelletwas used (the hippocampi from at least five animals per developmen-tal day were pooled for probing). The amount of protein loaded perlane was 50 �g in the detection of GLAST (a) from P1 to P30 and ofGLT1 (b) from P1 to P10. At P20 and P30 25�g protein were loadedper lane in the detection of GLT1 (b). GLAST antibody labeled a�65-kDa band (a) and GLT1 antibody a �70-kDa band and aggre-gates of that polypeptide (b). (a: GLAST: From P1 to P5 the bands aremoderately stained. From P7 to P30, the staining intensity of bandsincreases. b: GLT1: The staining intensity of bands is very low at P1and increases thereafter significantly until P10 and is very strong atP20 and P30.


FIGURE 4


Almost the same labeling pattern was observed at P20, 30, and 60(not shown).

At P1–P3, GLT1 mRNA could be detected in all pyramidal cellsof the cornu ammonis and in granule cells of the dentate gyrus (Fig.1f). Labeling of CA1/2-pyramidal cells disappeared until P6, thatof granule cells until P10 (Figs. 1f–k and 2d–f). In contrast, thestaining intensity of CA3-pyramidal cells increased form P6 on-ward showing a strong staining intensity at P20 (Fig. 1k), 30, and60.

Labeling of ependymal cells and cells of the subventricular zone(lateral ventricle) was observed only until P10 (Fig. 1f–k).

Immunoblotting

GLAST. In immunoblots of the 100,000g pellet of hippocampiat all the stages studied (P1–P30), the affinity-purified antibodyagainst the GLAST peptide labeled a �65-kDa band (Fig. 3a).This molecular mass corresponded with the reported one bySchmitt et al. (1997) for the adult hippocampus. From P1 to P5,this band was moderately stained (Fig. 3a). From P7 to P30, therewas increased staining intensity (Fig. 3a), at P30 showing a nearlyadult level of labeling intensity.

GLT1. In immunoblots of the 100,000g pellet of the hip-pocampi at all stages studied (P1– P30) the affinity-purified anti-body against the GLT1 peptide labeled a band at �70 kDa and aband of lower electrophoretic mobility, presumably aggregates ofthe 70-kDa protein (Fig. 3b). This molecular masses correspondedwith those reported by Schmitt et al. (1996, 2002) for the adulthippocampus. The staining intensity of the 70-kDa band was verylow at P1 and increased thereafter significantly until P10. At P20and 30, a very strong staining of the bands of both molecularmasses was demonstrable (Fig. 3b), showing nearly adult levels ofstaining intensity.

Immunostaining

In semithin sections (1 �m thick) of the hippocampus, we ob-served a labeling for GLT1 and GLAST only in glial cells (cellmembranes of somata and processes), which we identified by dou-ble-immunolabeling with anti-GFAP (not shown) and anti-GSantibodies to be astrocytes (Fig. 4a–i). Immunostained membraneprofiles in the neuropil seemed to represent fine astrocytic pro-cesses. This cellular labeling pattern in the postnatal developmentof hippocampus corresponded with that of adult hippocampus(Schmitt et al., 1996). GLT1 and GLAST proteins could not bedetected in neurons of the hippocampus during the postnatal de-velopment (Fig. 4a–i).

In agreement with the ISH and immunoblot findings, GLASTimmunostaining of the hippocampus neuropil was moderate tostrong at P5 (Fig. 4a–c) and further increased until P20. By con-trast, GLT1 labeling was low at P5 and increased strongly untilP20.

Distribution and Activity of GDH

Distribution

The distribution of the mitochondrial GDH was studied insemithin plastic sections of the hippocampus at P5, 8, 15 and 60,using a polyclonal affinity-purified antibody characterized else-where (Schmitt and Kugler, 1999). As already shown for the adulthippocampus (Schmitt and Kugler, 1999) a mitochondrial immu-noreaction was detected predominantly in glial cells, and to a sig-nificantly smaller extent in neurons at the developmental stagesstudied. Double-immunolabeling with anti-GFAP (Fig. 4q–s) andanti-GS antibodies (Fig. 4n–p) allowed identification of the la-beled glial cells as astrocytes. GDH was detected in puncta-likeoval and elongated profiles of astroglial processes (Fig. 4n,q). Theseprofiles are identified in a previous study as mitochondria (Schmittand Kugler, 1999). The density of labeled profiles increasedstrongly from P5 to P8 and moderately from P15 to P60 in all theneuropil layers of the hippocampus.

Quantitative GDH histochemistry

GDH activities at the various developmental stages studied weredetermined by densitometric measurements in the neuropil layersof the hippocampus proper (sectors CA1 and CA3) and of thedentate gyrus. In the evaluation of measuring data the molecularlayer of the dentate gyrus and the laculosum-molecular layer ofcornu ammonis (CA1 and CA3) (both are termination fields of theglutamatergic perforant path) were taken together and comparedwith the oriens and radiatum layers of the cornu ammonis (CA1and CA3). GDH activities in the termination field of the perforantpath were higher at all developmental stages studied (at P5, 1.5�and at P20, 1.7� ) than in the other layers (Fig. 5). There wasincreased GDH activity from P5 to P20 in all layers examined. Thesteepest increase of GDH activities was between P5 and P8 in thetermination field of the perforant path (Fig. 5).

FIGURE 4. Cellular distribution of GLAST (a,d), GLT1 (g), andglutamate dehydrogenase (GDH) (k,n,q) and of glutamine synthetase(GS) (b,e,h,l,o) and GFAP (r) (both are used as marker proteins ofastrocytes) detected by immunofluorescence staining in 1-�m-thickplastic sections of the dentate gyrus during development (upper rightcorner). Superimposed (merged) images of corresponding micro-graphs (a and b merged in c; d and e merged in f; g and h merged in i;k and l merged in m; n and o merged in p; q and r merged in s) provethe localization of glutamate transporters and GDH in astrocytes. Inthe detection of glutamate transporters (a–i), no labeling is observedin granule cells localized in the granule cell layer (g). As GLAST andGLT1 are localized in plasma membranes (arrowheads in a,d,g) andGS is contained in the cytoplasm (arrowheads in b,e,h), the greenfluorescent cell bodies of astrocytes are outlined by red fluorescentplasma membranes (arrowheads in c,f,i). The puncta-like and elon-gated staining profiles in the detection of GDH (k,n,q) correspond toimmunolabeled mitochondria. Arrowheads in k–m and q–s point tolabeled astrocytic processes. Note the strong increase of GDH immu-nostained profiles from P5 (k) to P8 (n). m, dentate gyrus molecularlayer. Scale bars � 25 �m (in a, applies to a–p); 10 �m (in q, appliesto q–s). [Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com.]


DISCUSSION

In the present study, we examined the expression of the gluta-mate transporters GLT1 and GLAST as well as the glutamate-metabolizing enzyme GDH in the rat hippocampus during post-natal development. The expression of both transporters and ofGDH was detected the day of birth (P1) and thereafter a differen-tial increase in their expression was shown reaching adult levels atP20–P30.

Cellular Distribution

We showed that GLAST was expressed postnatally (P1–P60)only in astroglial cells, namely in astrocytes of gray and whitematter and in ependymal (and subependymal) cells of the subven-tricular zone (lateral ventricle). An exclusive glial localization is alsodemonstrated in previous immunocytochemical studies dealingwith rat brain development (Ullensvang et al., 1997). This astro-glial expression pattern of GLAST corresponds to that detected inadult CNS, including the hippocampus (Torp et al., 1994;Chaudhry et al., 1995; Lehre et al., 1995; Schmitt et al., 1997;Berger and Hediger, 1998; Kugler and Schmitt, 2003) and indi-cating that there was no change in the cellular expression ofGLAST during postnatal development until adulthood. Like theadult hippocampus, GLAST was only detected in plasma mem-branes (cf. Fig. 4a–c). A transient neuronal expression of GLASThas been reported from hippocampal neurons in primary culture(Plachez et al., 2000), a localization which has never been shown invivo (for review, see Danbolt, 2001).

In contrast, the cellular GLT1 expression was differential at themRNA and protein level. In addition to astroglial cells, we detected

in the early postnatal development (P1–P3) GLT1 mRNA in py-ramidal cells of CA1–CA3 and in granule cells of the dentate gyrus.Thereafter, the neuronal signal disappeared gradually in CA1/2and in the dentate gyrus, whereas in CA3 increased from P6 on-ward. Conversely, generic GLT1 protein (glial form of GLT1) wasfound only in astroglial cells in the postnatal development. A sim-ilar expression pattern at the cellular level is shown in the adultbrain, including the hippocampus (Schmitt et al., 1996; Kuglerand Schmitt, 2003; for review, see Danbolt, 2001). It should benoted that the peptide antibody used in the present study for thedetection of GLT1 is specific for the generic form of GLT1 (glialform of GLT1) and does not detect a recently cloned splice variantof GLT1 (GLT1v), which is mainly localized in neurons (Schmittet al., 2002; Kugler and Schmitt, 2003). The reason generic GLT1mRNA but not protein is detectable in postnatal brain neuronsremains unknown (Kugler and Schmitt, 2003). It is reported thatin the mouse hippocampus two populations of GFAP-positiveastrocytes are present, one expressing the glutamate transportersGLT1 and GLAST and the other expressing ionotropic glutamatereceptors (Matthias et al., 2003). A differentiation of both astro-cytic types was not attempted in the present study.

It has been shown that in the prenatal development (not in-cluded in this study) GLT1 is transiently expressed at the proteinlevel in subsets of CNS neurons of rat (Furuta et al., 1997), sheep(Northington et al., 1999), mouse (Yamada et al., 1998), and man(Bar-Peled et al., 1997). Possibly reminiscent to this developmen-tal in situ situation is the expression of GLT1 protein in culturedhippocampal neurons (Brooks-Kayal et al., 1998; Mennerick et al.,1998; Plachez et al., 2000). This indicates that neurons have theability to express GLT1 protein under certain conditions, e.g., inthe prenatal development or after ischemic brain injury (Martin etal., 1997).

The glutamate-metabolizing enzyme GDH is expressed in theadult hippocampus at the mRNA level in neurons and glial cells atalmost equal extent. At the protein level, however, GDH is con-tained predominantly in astrocytes, and only to a very small extentin neurons (Kugler, 1993; Schmitt and Kugler, 1999). In thisstudy, we showed that also in the postnatal development GDHprotein was present above all in astroglial cells.

Changes in Glutamate Transporter Expression

It is generally accepted that the expression of high-affinity glu-tamate transporters is differential and very low at early develop-mental stages and increases in the postnatal development reachingat various time points adult levels (for review, see Danbolt, 2001).In a recent study it is shown that at birth GLT1 is not detectable,but GLAST is present at significant concentrations in the rat fore-brain. GLT1 is first detected in the forebrain in the second post-natal week and both transporters reach adult levels by postnatal day35 (Ullensvang et al., 1997). We can show that after the applica-tion of ISH, immunocytochemistry, and immunoblotting to thehippocampus, both GLAST and GLT1 were clearly detectable atP1 (day of birth), reaching adult levels at P20–P30. Furthermore,we detected in the early postnatal development (until �P4) a highexpression of GLAST. Thereafter, the increase of GLAST expres-

FIGURE 5. Developmental changes of glutamate dehydrogenase(GDH) activities (expressed in �mol glutamate � cm�3 � min�1;means and standard deviations) in the hippocampus. At each devel-opmental day the hippocampi from at least five animals were used formicrophotometric measurements. GDH activities in the terminationfield of the glutamatergic perforant path are shown in the upper curveand those in the oriens and radiatum layers in the lower curve. Notethe steep increase of GDH activities between P5 and P8 in the termi-nation field of the perforant path (upper curve).


sion was almost continuous reaching nearly adult levels at P30 (atthe protein level), whereas GLT1 expression increased significantlyuntil P10 and showed high protein levels at P20 and P30 (adultlevels). Our findings in the hippocampus are supported by studiesapplying immunoblot analyses and immunohistochemistry toother brain regions (Furuta et al., 1997), inasmuch as these showthat GLT1 and GLAST proteins are detectable at birth and reachnearly adult levels at P26. In mouse brain, the levels of GLAST andGLT1 mRNAs increase postnatally reaching maximal levels at P14(Shibata et al., 1996), somewhat earlier as in the rat hippocampus(this study).

It is reasonable to assume that the increase of glutamate trans-porter expression, above all that of GLT1 in the postnatal devel-opment, is closely associated with development and establishmentof excitatory synaptic transmission in the hippocampus, which isaccompanied by the expression of glutamate transporters in syn-aptic vesicles and postsynaptic glutamate receptors. In recent stud-ies using immunoblot analysis and immunocytochemistry, it isreported that the vesicular glutamate transporter VGLUT1, a pro-tein necessary for glutamate loading of synaptic vesicles, is detect-able in neuropil layers of the hippocampus at P0––P2, reachingthe mature protein level between P10 and P20 (Minelli et al.,2003). Earlier biochemical studies revealed that vesicular uptakeand uptake by plasma membrane particles of glutamate increasestrongly in the first 2 weeks after birth to reach or exceed adultvalues (Christensen and Fonnum, 1992).

There is increasing evidence that newly formed glutamatergicsynapses express both NMDA and AMPA receptors (Friedman etal., 2000; Renger et al., 2001; Groc et al., 2002). Furthermore,spontaneous postsynaptic currents, mediated by AMPA andNMDA receptors, are recorded from CA1 pyramidal cells over thefirst postnatal week (Groc et al., 2002). AMPA receptor-mediatedexcitatory postsynaptic potentials are demonstrated in CA1 alreadyat embryonic day 19, the amplitude of which increased until post-natal day 9 (Diabira et al., 1999), and also in CA3 at early postnatalstages (Gasparini et al., 2000).

On the whole, the increase of expression of the high-affinityglutamate transporter GLT1 in the hippocampus parallels withthat of vesicular glutamate transporter VGLUT1, plasma mem-brane-associated glutamate uptake, and AMPA receptor-mediatedexcitatory postsynaptic potentials. As high-affinity glutamatetransporters like GLT1 remove extracellular glutamate, they candirectly regulate the level of extracellular glutamate and control inthis way neuronal receptor activation and the development of ex-citatory circuitry. Comparing GLAST and GLT1 it is also shownfrom lesion studies that expression of GLT1 appears to be moreclosely related to glutamatergic transmission than to that ofGLAST (Hein et al., 2001).

Changes in GDH Activities

In addiiton to aspartate aminotransferase, glutamine synthetase,and phosphate-activated glutaminase, GDH is an important en-zyme involved in glutamate metabolism in the CNS (for review,see Kugler, 1993). There is evidence from lesion studies that GDHlocalized in astrocytes is not only involved in the general glutamate

metabolism in the hippocampus but also in the metabolism (deg-radation) of transmitter glutamate (Kugler et al., 1995). In earlierbiochemical studies it was shown that GDH activities in hip-pocampal homogenates increase continuously during the first 3postnatal weeks, whereas almost no change is detected in the dorsalroot ganglia, where glutamatergic transmission does not take place(Rothe et al., 1983). The authors conclude that the increase ofGDH activities is a consequence of the maturation of glutamater-gic structures (Rothe et al,. 1983).

Our quantitative evaluation of GDH activities in hippocampalsections showed a steep increase of GDH activities from P5–P8preferentially in the terminal field of the glutamatergic perforantpath (molecular layer of the dentate gyrus and lacunosum-molec-ular layer of the cornu ammonis) and thereafter a smaller increaseuntil P20 reaching nearly adult levels. This increase in GDH ac-tivities was in good accordance with the expression of glutamatetransporter GLT1 and further parameters of glutamatergic trans-mission as discussed above. Thus, the increasing transmitter glu-tamate release in the hippocampus during postnatal developmentwas accompanied by an increasing expression of glutamate trans-porters and GDH activities for uptake and degradation of gluta-mate in astrocytes, respectively.

CONCLUSIONS

This study shows a tight relationship between developmentalexpression of high-affinity glutamate transporters and GDH activ-ities in astrocytes and the establishment of glutamatergic transmis-sion in the hippocampus during postnatal development (cf. liter-ature cited above). Herewith, the expression of glutamatetransporter GLT1 appears to be of special interest. It is shown inthe adult mouse brain that without the action of GLT1, glutamatelevels rise enough to cause epilepsy and neuronal death (Tanaka etal., 1997). Furthermore, these homozygous mice deficient inGLT1 tended to die prematurely (50% survival after 6 weeks)(Tanaka et al., 1997). This fact underlines the important meaningof astrocytes in the glutamate transmitter metabolism not only inthe adult (for review, see Danbolt, 2001) but also in the developingCNS (Voutsinos-Porche et al., 2003).

Acknowledgments

The authors are indebted to Erna Kleinschroth and Julia Med-vedev for their excellent technical assistance.

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Developmental expression of glutamate transporters and glutamate dehydrogenase in astrocytes of the postnatal rat hippocampus