Glutamate transporter EAAC1 is expressed in neurons and glial cells in the rat nervous system

Glutamate Transporter EAAC1 IsExpressed in Neurons and Glial Cells

in the Rat Nervous SystemPETER KUGLER* AND ANGELIKA SCHMITT

Institute of Anatomy, University of Wurzburg, Wurzburg, Germany

KEY WORDS excitatory amino acid transporter; oligodendrocytes; ependyma; plexuschoroideus

ABSTRACT Oligonucleotide and cRNA probes were used for non-radioactive in situhybridization, carried out to identify the cell types in the nervous system of ratexpressing the glutamate transporter EAAC1 mRNA. The results were compared withimmunocytochemical data obtained using an antibody against a synthetic EAAC1peptide. The present data confirm that EAAC1 is expressed in neurons of the CNS.Additionally, our findings indicate the localization of EAAC1 mRNA and protein inperipheral neurons (spinal ganglia) and in glial cells, i.e., oligodendrocytes in variouswhite matter regions of the CNS, ependymal cells, and epithelial cells of the plexuschoroideus of the four ventricles, as well as in satellite cells of spinal ganglia.Immunolabeling revealed a preferentially cytoplasmic staining of neurons and glial cells.The cytoplasmic staining was frequently granular, suggesting a localization of EAAC1protein in vesicle membranes. A membrane localization of EAAC1 was also indicated byWestern blotting, which showed immunoreactivity only in the 100,000 3 g pellet of brainhomogenate. We conclude that the glutamate transporter EAAC1 is not restricted toneurons but may also play an important role in glial cells, particularly in oligodendro-cytes. GLIA 27:129–142, 1999. r 1999 Wiley-Liss, Inc.

INTRODUCTION

Glutamate reuptake from the extracellular spaceplays an important role in the CNS. It terminates thetransmitter signal and prevents a harmful receptoroverstimulation (Kanai et al., 1993; Lipton and Rosen-berg, 1994). In recent years, the cDNAs of at least fivedifferent subtypes of glutamate transporters have beencloned—GLT1 (Pines et al., 1992), GLAST (Storck etal., 1992), EAAC1 (Kanai and Hediger, 1992), EAAT4(Fairman et al., 1995), and EAAT5 (Arriza et al., 1997).GLT1, GLAST, and EAAC1 are widely distributedthroughout the CNS (Kanai and Hediger, 1992; Pines etal., 1992; Storck et al., 1992; Rothstein et al., 1994; Torpet al., 1994, 1997; Chaudry et al., 1995; Lehre et al.,1995; Schmitt et al., 1996, 1997; Velaz-Faircloth et al.,1996; Berger and Hediger, 1998). In contrast, EAAT4and EAAT5 seem to be restricted to the cerebellum(Fairman et al., 1995; Furuta et al., 1997; Dehnes et al.,1998) and retina (Arriza et al., 1997), respectively.

With respect to the cellular localization of EAAC1,immunocytochemistry using antibodies against syn-thetic peptides (Rothstein et al., 1994; Shashidharan etal., 1997) detected transporter protein primarily insomatodendritic membranes of glutamatergic and GA-BAergic neurons, while presynaptic structures werenot labeled (Rothstein et al., 1994; Coco et al., 1997).Labeling in glial cells was not reported. EAAC1 wastherefore considered to be an exclusively neuronal,postsynaptic glutamate transporter.

Using radioactive and non-radioactive in situ hybrid-ization (ISH), EAAC1 mRNA was detected in presum-ably neuronal perikarya of various CNS regions (Kanaiand Hediger, 1992; Meister et al., 1993; Kiryu et al.,

Grant sponsor: Deutsche Forschungsgemeinschaft.

*Correspondence to: Prof. Dr. Peter Kugler, Institute of Anatomy, Koelliker-strasse 6, D-97070 Wurzburg, Germany.E-mail: [email protected]

Received 18 January 1999; Accepted 25 February 1999

GLIA 27:129–142 (1999)

r 1999 Wiley-Liss, Inc.

1995; Kanai et al., 1995; Velaz-Faircloth et al., 1996;Torp et al., 1997; Berger and Hediger, 1998). In addi-tion, Kiryu et al. (1995) detected flat-labeled cells in thecorpus callosum, which they suggested were oligoden-drocytes. Because of the poor cellular resolution of theradioactive ISH method used in that study, however,the question of the precise cellular source of the EAAC1mRNA signal could not be answered conclusively. In arecent non-radioactive ISH study, Berger and Hediger(1998) reported that EAAC1 mRNA is localized in cellsof white matter tracts, e.g., in the corpus callosum, thefimbria-fornix, and in the anterior commissure. Basedon their scattered distribution, the authors suggestedthat these labeled cells are not mature oligodendrocytesbut rather represent oligodendrocyte-progenitor cells,or alternatively, neurons.

In the present study, we have addressed the questionof EAAC1 localization in glial cell populations usinghighly sensitive methods—non-radioactive ISH usingcRNA and oligonucleotide probes (Asan and Kugler,1995; Schmitt et al., 1996, 1997) in combination withcomparative immunocytochemistry using an antibodyagainst a synthetic EAAC1 peptide. Additionally, thesemethods provide clear cellular resolution of labeling.Because the expression pattern of EAAC1 in neuronshas already been extensively analysed in the rat CNS(see above), a regional mapping of neuronal labelingwas not included in this study. However, in order to beable to compare our data with previous investigations(see above), documentation of neuronal labeling wascarried out in the hippocampus and the cerebellum.Additionally, cervical spinal ganglia were studied todocument EAAC1 localization in peripheral nervoustissue.

MATERIALS AND METHODSAnimals and Tissue Sources

The brains, cervical spinal cords, and spinal gangliaof 30 male Wistar rats (of our own breeding, aged 8–12weeks) were used for RNA preparation, ISH, immuno-blotting, and immunohistochemistry, as described be-low.

Generation of a Digoxigenin-LabeledcRNA Probe

All procedures for the preparation of cRNA probeswere performed as described by Sambrook et al. (1989)and Schmitt et al. (1996) and will be described here onlyin brief. Total RNA from the whole rat brain wasisolated by acid guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi, 1987).The first-strand synthesis of the cDNA was performedfor 1 h at 42°C in a reaction volume of 20 µl containing 3µg total RNA, 1.8 pM Oligo(dT)-primer (18-mer), 1 mMof each dNTP, 20 units RNasin, 50 mM Tris/HCl (pH8.5), 8 mM MgCl2, 30 mM KCl, 1 mM dithiothreitol, and

40 units of AMV-reverse transcriptase (Boehringer,Mannheim, Germany). Two primers, EAAC1 (a): 58-GAGCTCTCGAATCTGGATAA-38 (complementary tonucleotides 247–267), and EAAC1 (b): 58-CTAAGGC-CAG-GCATCTAGAA-38 (complementary to nucleotides1687–1706; purchased from Roth, Karlsruhe, Ger-many), based on the rat EAAC1 sequence published byKiryu et al. (1995), were used to amplify a rat brainEAAC1cDNA fragment.

Ten percent of the 1:5 diluted reverse transcriptionmixture was utilized for a polymerase chain reaction ina final volume of 100 µl containing 0.4 pM of eachprimer, 0.4 mM of each dNTP, 1 unit Taq-Polymerase(MBI Fermentas, St. Leon-Rot, Germany), 50 mM KCl,1.5 mM MgCl2 and 10 mM Tris-HCl (pH 8). Foramplification, the following procedure was used: dena-turation 1 min/94°C, annealing 2 min/59°C, extension 3min/72°C, 40 cycles, and a final elongation for 10 min at72°C. The resulting cDNA was cloned into the Eco RVsite of the Bluescript vector (pBluescript II SK1, Strata-gene, La Jolla, CA) and transfected and propagated inEscherichia coli XL 1 Blue. The identity of the clonedcDNA was verified by restriction analysis and partialDNA sequencing (Sanger et al., 1977).

To produce a digoxigenin-labeled antisense (sense)probe, plasmids were linearized by Hind III (Bam HI)restriction, phenol-chloroform extracted, precipitated,and transcribed by T3 RNA polymerase (T7 RNA poly-merase) according the manufacturer’s manual (Boeh-ringer). Usually, 1.5 µg of cDNA template yielded 10 to30 µg of labeled cRNA, incorporating approximatelyone DIG-11-UTP at every 20th nucleotide. cRNA probeswere analyzed on a formaldehyde agarose gel (1%).

Northern Blotting

For Northern blotting, whole brain, cerebellum, hip-pocampus, and cervical spinal cord were dissected andthe total RNA of each preparation was isolated asdescribed above. The Northern blotting was performedessentially according to the instructions of Boehringerfor non-radioactive hybridization (The DIG systemuser’s guide for filter hybridization; Boehringer). Briefly,probes of RNA were separated in a standard formalde-hyde gel (1%) and transferred onto nylon membranes(Hybond N1; Amersham, Braunschweig, Germany).Prehybridization was performed in 5 3 standard salinecitrate (SSC) containing 50% formamide, 0.1% N-lauroyl-sarcosine, 0.02% sodium dodecyl sulfate (SDS),and 2% blocking reagent (Boehringer). Subsequently,the blots were placed in the prehybridization solutioncontaining 100 ng/µl digoxigenin-labeled antisense(sense) cRNA (see above) overnight at 68°C. The blotswere washed in 2 3 SSC and 0.1% SDS at roomtemperature and subsequently in 0.5 3 SSC and 0.1%SDS at 68°C. After equilibration in washing buffer (0.1M maleic acid, 0.15 M NaCl, 0.3% Tween 20, pH 7.5) atroom temperature, the membranes were transferredinto a blocking solution (0.1 M maleic acid, 0.15 M

130 KUGLER AND SCHMITT

NaCl, 1% blocking reagent, pH 7.5) for 30 min. Afterincubation with sheep anti-digoxigenin-alkaline phos-phatase (aP) conjugated antibody (1:5000; correspond-ing to 150 mU/ml aP activity; Boehringer) for 1 h themembranes were rinsed in the washing buffer, equili-brated in 0.1 M Tris-HCl buffer at pH 9.5 and used forthe detection of aP (see below).

In Situ Hybridization

cRNA

Frontal blocks of brains, 3–5 mm thick (approximateinteraural levels 7.0 to 4.5 mm and 21 to 23.0 accord-ing to the rat brain stereotaxic atlas of Paxinos andWatson, 1986), spinal cord, and spinal ganglia werefrozen in liquid-nitrogen-cooled isopentane. Twelve-micrometer-thick cryostat sections were mounted onprecoated glass slides (Superfrost Plus; Menzel, Braun-schweig, Germany) and thawed. The sections werefixed for 5 min in freshly prepared 4% formaldehyde in0.01 M phosphate-buffered saline (PBS; pH 7.4). Thesections were transferred to ethanol and stored for 1 to2 days.

The sections were subsequently removed from etha-nol, rehydrated in a graded series of ethanol, trans-ferred to 2 3 SSC and treated with 0.05 N HCl for 30min. After washing with 2 3 SSC, the sections wereincubated with freshly prepared 0.25% acidic anhy-dride, washed again with 2 3 SSC and covered with thehybridization solution, which contained the digoxigenin-labeled antisense (sense) cRNA probe (final concentra-tion 3–6 ng/µl) and 550 µg/ml Salmon testes DNA(Sigma, Deisenhofen, Germany) in 4 3 SSC, 1 3Denhardt’s solution (Sambrook et al., 1989), 10% dex-tran sulfate, and 50% deionized formamide at 60°C for16–18 h. Posthybridization washes were done stepwiseat room temperature with 2 3 SSC, 1 3 SSC, 50%formamide, and then again with 2 3 SSC. The sectionswere then treated with 1 µg/ml proteinase K (Boeh-ringer) in 50 mM Tris-HCl (pH 7.4) for 30 min at roomtemperature and washed with bi-distilled water for 5min. Next, the sections were treated with 40 µg/mlribonuclease A (50 Kunitz-units/mg; Boehringer) in asolution containing 500 mM NaCl, 10 mM Tris-HCl (pH8), and 1 mM EDTA at 37°C for 30 min to removeunhybridized single strand RNAs. After the treatment,the sections were incubated with the same bufferwithout 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 Nucleic Acid Detection Kit,Boehringer; 30 min), followed by 0.3% Triton X-100 inTBS (20 min). After incubation with 1.5 U/ml sheepanti-DIG-aP conjugated (Boehringer) in TBS contain-ing 0.3% Triton X-100 for 60 min, the sections werewashed in TBS, and transferred to a 0.1 M Tris-buffercontaining 100 mM NaCl and 50 mM MgCl2 (pH 9.5) for2 min prior to the aP visualization (see below).

In some experiments, following the aP visualization,several brain sections were used for the immunocyto-chemical detection of glial fibrillary acidic protein(GFAP), applying the PAP method (peroxidase-antiper-oxidase complex 1:100; mouse monoclonal antibodyagainst GFAP 1:2000; DAKO, Hamburg, Germany;Sternberger et al., 1990).

Oligonucleotide probe

ISH was carried out according to the method ofDagerlind et al. (1992), using an aP coupled 33meroligonucleotide probe complementary to part of thecoding region of EAAC1 mRNA (antisense probe to thenucleotides 1657–1689: 58-GAACTGCGAGGTCTGAGT-GAACGAGATGGTGTC-38; custom synthesized by DNATechnology, Aarhus, Denmark). Twelve µm-thick cryo-stat sections of snap-frozen frontal tissue blocks ofbrain (see above) mounted on Superfrost slides werethawed and covered with hybridization solution (seeabove) containing 6-8 fmol/ml antisense oligonucleotideprobe at 37°C for 20–40 h. Posthybridization washesconsisted of 1 3 SSC for 4 3 15 min at 55°C. Aftercooling to room temperature, the sections were trans-ferred to TBS for 30 min, followed by 100 mM Tris-HClcontaining 100 mM NaCl, 50 mM MgCl2 (pH 9.5) for 10min, prior to the aP visualization.

Detection of alkaline phosphatase

The procedure used has been described recently byAsan and Kugler (1995). The incubation media con-tained 0.4 mM 5-bromo-4-chloro-3-indolylphosphate(BCIP; Boehringer), 100 mM sodium chloride, 50 mMMgCl2, 0.4 mM tetranitroblue tetrazoliumchloride ornitroblue tetrazoliumchloride (Serva, Heidelberg, Ger-many) in 100 mM Tris-HCl buffer at pH 9.5.

Controls for ISH

Substitution of the antisense cRNA probe by anequivalent amount of labeled sense cRNA probe led to alack of staining (cf. Figs. 3f, 5c). Neither was stainingobserved in sections of unfixed tissue if a 100-foldexcess of unlabeled oligonucleotide probe was appliedtogether with the aP-labelled probe (cf. Figs. 6f, 7c),indicating a complete competitive inhibition of specificbinding of the labeled probe in these preparations.Omission of labeled cRNA or oligonucleotide probesfrom the respective hybridization mixtures resulted incompletely unstained sections. From these findings itcan be concluded that (1) the antisense probes werespecific, (2) the digoxigenin detection did not createlabeling artifacts, and (3) there was no endogenous aPactivity left in the sections.

131EAAC1 IN NEURONS AND GLIAL CELLS

Antibodies and Immunoblotting

Antibodies

A peptide corresponding to the C-terminal region480–499 (I-V-N-P-F-A-L-E-P-T-I-L-D-N-E-D-S-D-T-K) ofthe EAAC1 protein (Kiryu et al., 1995) was synthesizedby the fmoc method and purified by reverse phase highpressure liquid chromatography (Atherton et al., 1981).For immunization, the peptide was coupled to keyholelimpet hemocyanin (KLH) by glutaraldehyde, as de-scribed in detail by Drenckhahn et al. (1993). One ml ofthe peptide solution (corresponding to 500 µg peptide)was mixed with polyalphaolefin adjuvant (Ethyl S. A.,Brussels, Belgium) and injected subscapularly in rab-bits (Drenckhahn et al., 1993). At intervals of 3 weeks,animals were boosted with the same amount of antigen.Positive antisera were identified by dot-blot assay(Drenckhahn et al., 1993). The antisera were affinitypurified, using the synthetic peptide immobilized bytransfer to nitrocellulose paper (Schleicher and Schull,Darmstadt, Germany). The bound immunoglobulinswere eluted by low pH (pH 2.8) and the protein contentwas determined spectrophotometrically (Drenckhahnet al., 1993).

Mouse monoclonal antibody against GFAP and b-tu-bulin were purchased from DAKO and Sigma, respec-tively. In the CNS, GFAP is a specific marker protein ofastrocytes (Bignami et al., 1972) and b-tubulin is highlyexpressed in oligodendrocytes (as well as in neurons;Schaeren-Wiemers et al., 1995).

Immunoblotting

For immunoblotting, cerebellum, hippocampus, wholeneocortex, and cervical spinal cord were dissected andhomogenized at 4°C in 10 mM NaH2PO4 (pH 7.2)containing 2 mM MgCl2, aprotinin (5 µg/ml), leupeptin(2 µg/ml), pepstatin (2 µg/ml), and phenylmethylsulfo-nyl fluoride (100 µg/ml). The homogenate was centri-fuged at 1,000 3 g for 10 min and the 1,000 3 gsupernatant was centrifuged at 100,000 g for 1 h. Theprotein contents of the 100,000 g supernatant andpellet were determined by the Bio-Rad protein assay(Bio-Rad, Munich, Germany) and the supernatant andthe pellet (membrane fraction) were used for immuno-blotting. Proteins (5–50 µg per lane) were electrophoreti-cally separated on 10% gels by SDS-polyacrylamide gelelectrophoresis (PAGE). Subsequently, the proteins weretransferred electrophoretically to nitrocellulose mem-branes (Burnette, 1981). Strips of the nitrocellulosemembranes were incubated for 24 h at 4°C with theaffinity-purified antibody (approximately 1.8 µg/ml).Bound immunoglobulins were visualized using peroxi-dase-conjugated goat anti-rabbit IgG (1:3,000; Bio-Rad,Richmond, Canada; blotting grade) and the enhancedluminol chemiluminescence technique (Amersham,Braunschweig, Germany). Antibody previously ab-

sorbed with an excess of the peptide used for immuniza-tion served as control.

Immunostaining

Pieces of hippocampus, cerebellum, spinal cord, plexuschoroideus, and spinal ganglia were frozen in liquidnitrogen-cooled propane and were freeze-dried andembedded in Epon (Drenckhahn and Franz, 1986).Semithin sections (1 µm) were mounted on glass slides.The resin was removed by placing the slides for 5 min inmethanol-toluene (1:1) containing 10% sodium methox-ide (prepared from metallic sodium; Major et al., 1961).

The tissue sections were preincubated for 3 h at roomtemperature with 2% bovine serum albumin, 10%normal goat serum and 0.05% Tween 20 (Ferrak,Berlin, Germany) in PBS, pH 7.4. Then, the sectionswere incubated for 24–48 h at 4°C for single and doublelabeling with the primary antibody diluted in thepreincubation solution (anti-EAAC1, 15 µg/ml; anti-GFAP, 1:10,000; anti-b-tubulin 1:750). After severalwashes with PBS, the semithin plastic sections wereincubated for 90 min at room temperature with carbo-cyanin (Cy2) labeled secondary antibody (1:300; goatanti-rabbit IgG; Dianova, Hamburg, Germany) for detec-tion of EAAC1, and with indocarbocyanin (Cy3) labeledsecondary antibody (1:600; goat anti-mouse IgG; Di-anova) for the detection of GFAP or b-tubulin. Controlswere performed with primary antibody, previously ab-sorbed with an excess of EAAC1 peptide used forimmunization or without the primary antibody. Thesections were examined with an Olympus BH-2 fluores-cence microscope (Olympus, New Hyde Park, NY)equipped with Zeiss optics and an appropriate filtercombination for selective visualization of Cy2 and Cy3fluorescence (BH II DFC 6; Olympus).

RESULTSNorthern and Western Blotting

Northern blots of the total RNA of the whole brainand the hippocampus showed a main band at ,4.2 andweaker bands at ,2.7 and 7.5 kb (Fig. 1). In Northernblots of the cerebellum and spinal cord only the band at,4.2 kb was detectable. No labeling could be seen inblots using the sense cRNA probe (not shown).

In Western blots of the 100,000 3 g pellet of tissuehomogenates (cerebral cortex, cerebellum, hippocam-pus, spinal cord), the affinity-purified antibody againstthe EAAC1 peptide labeled a relatively broad ,64,000mol. wt band (Fig. 2a). In the 100,000 g supernatant, noprotein band was labeled using the affinity-purifiedantibody (data not shown). This indicates that thedetected protein was localized in membranes. Preab-sorption of the antibody with the synthetic peptideabolished binding to the protein bands (Fig. 2a). Afterdeglycosylation with N-glycosidase F, a ,55,000 kDa


band was labeled in immunoblots of the cerebral cortexand hippocampus (Fig. 2b).

In Situ Hybridization andImmunocytochemistry

In situ hybridization

Application of the cRNA or oligonucleotide probes tocryostat sections resulted in identical patterns of cellu-lar and regional distribution of EAAC1 mRNA in therat nervous system, both methods showing a highcellular resolution. However, the DIG-labelled cRNAprobe provided a much higher signal intensity than theaP-labelled oligonucleotide probe (Figs. 6a–f, 7a–c).Therefore, ISH results were preferentially documentedusing sections hybridized with the cRNA probe.

In accordance with previous findings (Kanai andHediger, 1992; Meister et al., 1993; Kiryu et al., 1995;Kanai et al., 1995; Velaz-Faircloth et al., 1996; Torp etal., 1997; Berger and Hediger, 1998), ISH reactionproduct was detected in neuronal cell bodies. Addition-ally, using our non-radioactive ISH protocol, we ob-served a labeling of different glial cell types. We wereable to detect labeled glial cells in the white matter ofvarious CNS regions. These cells were typically ar-ranged like pearls on a string, a morphological charac-teristic of oligodendrocyte localization. This typicalarrangement of labeled cells (oligodendrocytes) was

observed particularly in the white matter of the cerebel-lum (Fig. 3b), in cerebellar peduncles (Fig. 3a), and inthe hippocampal alveus. In other white matter tracts(e.g., fimbria hippocampi, corpus callosum, internalcapsule, optic tract), strongly labeled glial cells showeda scattered distribution (Fig. 3c–e). Double-labelingwith GFAP immunocytochemistry showed that thesescattered cells were negative for GFAP-immunostain-ing (Fig. 3d,e), indicating that they were not astrocytes.In the white matter of the spinal cord, again only someindividual glial cells were ISH-labeled (not shown).

Additionally, ISH reaction product was localized inother glial cell types, i.e., in ependymal cells (Fig. 5a,b)and epithelial cells of the choroid plexus (Fig. 5b,c) of all

Fig. 1. Northern blot analysis of RNA from brain (b), hippocampus(h), cerebellum (c), and cervical spinal cord (s) using the cRNA probe.The amount of total RNA loaded per lane was 25 µg. Determination ofthe mRNA size was carried out by comparison with ribosomal RNAbands (,2 and ,5 kb). Labeled bands around 2.7 kb, 4.2 kb, and 7.5 kbare clearly seen in the brain and hippocampus samples, whereas inother organ samples a significantly labeled band occurs only around4.2 kb.

Fig. 2. Western blot analysis (10% SDS-PAGE) of cerebral cortex(cc), hippocampus (h), cerebellum (c), and cervical spinal cord (s) usingthe affinity purified EAAC1 antibody. For probing, the 100,000 3 gpellet was used. The amount of protein loaded per lane was 50 µg. a: Inthe various CNS regions the EAAC1 antibody labeled a ,64 kDa band.Immunoblotting of hippocampal proteins, using antibody previouslyabsorbed with an excess of the EAAC1 peptide used for immunization,served as control (co). b: Deglycosylation experiment. For probing, the100,000 3 g pellet of cerebral cortex (cc) and hippocampus (h) was usedwith (cc1, h1) and without (cc2, h2) treatment with N-glycosidase F,as described by Schmitt et al. (1977). After enzyme treatment, themolecular mass of the protein was reduced by ,9 kDa.


four ventricles, and in satellite cells of spinal ganglia(Fig. 7e). Astrocytes did not show any labeling.

Neuronal EAAC1 mRNA labeling was observed inthe hippocampus (pyramidal and granule cells; inter-neurons scattered throughout the oriens layer of thehippocampus proper; Fig. 6a–f) and in the cerebellarcortex (Fig. 7a–c). In the cerebellar cortex, very weak

labeling was observed in granule cells, whereas Pur-kinje cells, interneurons of the molecular layer, andGolgi cells (identified by their localization and distribu-tion in the granule cell layer) were moderately tostrongly labelled (Fig. 7a,b).

In addition to neurons of the CNS, EAAC1 mRNAwas detected in peripheral neurons, i.e., in neurons of

Fig. 3. Cellular distribution of EAAC1 mRNA in the CNS whitematter using the cRNA probe. White matter of the inferior cerebellarpeduncle (a) and of the cerebellum (b). ISH reaction product isdetected in glial cells, which are typically arranged in rows (arrows)and therefore appear to be oligodendrocytes. bv, blood vessel. c: Corpuscallosum. Strongly labeled cells are scattered throughout the whitematter. d,e: A micrograph pair showing a section of the corpuscallosum after ISH, using the antisense cRNA probe in the first, and

after additional GFAP immunostaining, in the second figure. NoGFAP-immunoreactivity is observed in glial cells labeled for EAAC1mRNA (arrows). GFAP-immunolabeled astrocytes and astrocytic pro-cesses (arrowheads) shown in (e) are scattered throughout the whitematter. f: Control. Applying the sense cRNA, no reaction product isobserved in the inferior cerebellar peduncle. For (a, c–f), scale bars 530 µm; for (b), scale bar 5 15 µm.


spinal ganglia. Labeling of these neurons ranged fromweak to strong (Fig. 7e).

Immunocytochemistry

When the affinity-purified antibody against theEAAC1 peptide was applied to thin plastic sections (1µm thick), a finely granular cytoplasmic staining ofneurons and glial cells predominated (Figs. 4c,g,i, 5e,7d,f). A delineation of labeled cell membranes wasalmost impossible. No staining was observed with theEAAC1 affinity-purified antibody previously absorbedto the synthetic peptide (not shown).

In the white matter of the cerebellum and hippocam-pus (fimbria, alveus) and in white matter tracts (spinalcord, corpus callosum) we observed a moderate tostrong staining of glial cells (Fig. 4a–c,e,g,i). These glialcells often showed an arrangement in rows, which istypical for oligodendrocytes (Fig. 4a; cf. ISH). Further-more, we identified these glial cells not only by theirdistributional pattern but also by double-labeling experi-ments: the EAAC1 immunolabeled glial cells of thewhite matter (Fig. 4c) were not labeled by GFAP-antibody (Fig. 4d) but were intensely stained using amonoclonal antibody against b-tubulin (Fig. 4e–h). It isknown that b-tubulin is highly expressed in oligodendro-cytes and neurons (Schaeren-Wiemers et al., 1995). Asthe white matter areas studied are free of neurons, theb-tubulin positive cells were most probably oligodendro-cytes. Especially in white matter tracts of the spinalcord, the EAAC1 immunostained glial cells displayedthe typical shape of oligodendrocytes—angular cellbodies, frequently with trunk-like primary processesfrom which fine secondary processes arose (Fig. 4e,i).Such fine processes partly surrounded cross-sectionedfibers with (Fig. 4e) or without immunostained axo-plasm (Fig. 4i). The myelin of fibers showed no labeling(Fig. 4i). Sometimes, labeled rims were seen, whichsurrounded axons (Fig. 4i). From light microscopicobservation it was not possible to decide if this labelingbelonged to axons (axolemma) or to processes of oligo-dendrocytes (inner loop).

In agreement with the ISH findings, we detectedEAAC1 protein in ependymal cells of all ventricles (Fig.5d), and in epithelial cells of the plexus choriodeus (Fig.5e). Staining of these cell types was moderate to strong.

In the hippocampus, pyramidal and granule cells, aswell as scattered cells in the neuropil layers, displayedimmunolabeling; in the cerebellar cortex, granule cellsand Purkinje cells were more or less intensely labeled(Figs. 6g–i, 7d). The neuropil layers of the hippocampus(Figs 6g–i) and cerebellar cortex (molecular layer)showed moderate, homogeneous staining. Additionally,in the cerebellar cortex, the glomeruli cerebellaresdisplayed labeling, which was intense in granularprofiles and somewhat lighter throughout the glomeru-lar area (Fig. 7d).

In agreement with ISH findings, spinal ganglia neu-rons and satellite cells showed a moderate to strongimmunostaining for EAAC1 protein (Fig. 7f).

DISCUSSION

The present study extends previous investigationsinto the expression and synthesis of the high-affinityglutamate transporter EAAC1 in the rat nervous sys-tem. Using high-resolution ISH in combination withspecific immunodetection, we were able to confirmfindings reported by other groups, namely that EAAC1mRNA and protein are localized in CNS neurons (seeIntroduction). Additionally, our results provide the firstconclusive evidence that EAAC1 is the glutamate trans-porter expressed by peripheral neurons (spinal ganglianeurons) and by various glial cells, such as oligodendro-cytes, ependymal cells, epithelial cells of the plexuschoroideus, and satellite cells of spinal ganglia.

Specificity

Based on the rat EAAC1 sequence (Kiryu et al.,1995), an antisense cRNA probe (nucleotides 247–1706)was generated. Specificity of the probe was ensured byNorthern blot analysis. In RNA preparations fromvarious CNS sources a band at , 4.2 kb was labeled.Additionally, in RNA preparations from whole brainand hippocampus two bands at , 2.7 and 7.0 kb wereweakly labeled. Similar banding has been described byKanai et al. (1995). ISH using the DIG-labelled cRNAprobe (detected by an aP-labeled antibody against DIG)to formaldehyde and alcohol-fixed cryostat sectionsresulted in specific labeling of high sensitivity andcellular resolution, and it displayed extremely lowbackground. Both in Northern blots and in ISH, nospecific labeling was detected when the cRNA senseprobe was used for hybridization.

Further proof for the specificity of the detection wasthat an oligonucleotide probe, complementary to part ofthe EAAC1 cDNA sequence (nt 1657–1689), showed thesame labeling pattern in ISH as did the cRNA probe.Again, control hybridization using the aP-labelled oligo-nucleotide probe in the presence of an excess of unla-beled oligonucleotide probe (competition experiment)yielded no labelling whatsoever.

Preliminary immunocytochemical experiments usingthe affinity-purified peptide antibody generated againstEAAC1 resulted in staining patterns similar to thosedescribed previously, with strong EAAC1 staining ofCNS neurons. This observation indicates that the anti-body faithfully detected EAAC1 protein in immunocyto-chemistry. Pre-adsorption of the antibody with theantigen peptide resulted in a lack of specific immuno-staining. Further evidence for the specificity of theantibody was given by the fact that it detected a ,64kDA band in Western blot analyses from several CNSregions, which was reduced in size to ,55 kDA by


Fig. 4. Cellular distribution of EAAC1 protein detected by immuno-fluorescence staining in semithin plastic sections of freeze-driedtissue. a: Alveus of the hippocampus. Immunostaining of differentintensity is observed in glial cells, which are typically arranged in arow and therefore seem to be oligodendrocytes. The oligodendrocytesare flanked by bundles of stained nerve fibers. The precise localizationof reaction product in the nerve fibers cannot be established. b: Whitematter of the cerebellum. Two strongly labeled glial cells (oligodendro-cytes) are surrounded by nerve fibers, which show a labeling ofmembranes. c,d: A micrograph pair taken from the cerebellar whitematter double labeled for EAAC1 (c) and the astrocytic marker GFAP(d). n, nucleus; arrowheads point to astrocytic profiles. The stronglylabeled glial cell (c) is GFAP negative (d). e,f: A micrograph pair takenfrom neuron-free white matter (funiculus anterior) of the cervical

spinal cord, double labeled for EAAC1 (e) and b-tubulin (f), which is amarker for neurons and oligodendrocytes. The glial cells (arrows) arelabeled for EAAC1 and b-tubulin and therefore seem to be oligodendro-cytes. Note that labeled processes of oligodendrocytes partially sur-round the unstained myelin of nerve fibers. The axoplasm of mostnerve fibers (arrowheads) are labeled for EAAC1 (e) and b-tubulin (f).g,h: A micrograph pair of an EAAC1/b-tubulin positive oligodendro-cyte (spinal cord, funiculus anterior) at high magnification. n, nucleus.i: An EAAC1-positive oligodendrocyte is shown (spinal cord, funiculusanterior). Labeled processes of the oligodendrocyte extend betweenand surround (arrow) nerve fibers. Arrowheads point to labeled rimsbordering the inner surface of the unlabeled myelin sheaths. Note thefinely granular cytoplasmic staining of oligodendrocytes in (c), (g), and(i). For (a–d, g–i), scale bars 5 10 µm; for (e,f), scale bar 5 20 µm.

deglycosylation. These molecular masses are in goodaccord with those found for EAAC1 by other authors(,69–70 kDA; Rothstein et al., 1994; Coco et al., 1997;Shashidharan et al., 1997) and with the predictedmolecular mass of the non-glycosylated protein (57kDa; Kanai and Hediger, 1992).

Expression of EAAC1 in Glial Cells

Most important seems to be the detection of EAAC1mRNA and protein in oligodendrocytes of CNS whitematter regions. In previous immunocytochemical stud-ies, the localization of EAAC1 in oligodendrocytes was

Fig. 5. Cellular distribution of EAAC1 mRNA using the cRNA probe(a–c) and of EAAC1 protein by immunofluorescence staining (d,e). a,b:Ependymal cells (arrows) of the third (III; a) and fourth ventricle (IV;b) and epithelial cells of the plexus choroideus (b) display an almostmoderate ISH labeling. Arrowheads point to hypothalamic neurons. c:Control. Applying the sense cRNA, no reaction product is observed in

the plexus choroideus of the fourth ventricle. d,e: A moderate to strongimmunofluorescent staining is shown in ependymal cells (d) and inepithelial cells of the plexus choroideus of the third ventricle (e). Notethe fine granular cytoplasmic staining of epithelial cells of the plexuschoroideus. For (a,b), scale bar 5 30 µm; for (c), scale bar 5 50 µm; for(d,e), scale bar 5 10 µm.


Fig. 6. Distribution of EAAC1 mRNA in the hippocampus using thecRNA probe (a, c) and the oligonucleotide probe (b, d–f), and of EAAC1protein detected by immunofluorescence staining (g–i). ISH using thecRNA probe results in a more intense labeling (a,c) than the oligo-nucleotide probe (b,d,e). With both probes EAAC1 mRNA is preferen-tially detected in neurons, e.g., pyramidal cells in the pyramidal celllayer (py), granule cells in the granule cell layer (gr), neurons of themultiform layer (mu), and scattered neurons in the oriens layer (or),and further hippocampal regions (a-e). f: Control for ISH using the

oligonucleotide probe. No labeling is observed using the oligonucleo-tide probe in the presence of a 100-fold excess of unlabeled oligonucleo-tide probe. g–i: A moderate, partly finely-granular immunostaining isobserved in the neuropil of the radiatum layer (ra), of the cornuammonis sectors CA1 (g) and CA3 (h), and of the molecular layer (mo)of the dentate gyrus (i). Pyramidal cells (py) of CA1 (g) and CA3 (h) andgranule cells (gr) display weak to moderate labeling. For (a,b), scalebar 5 400 µm; for (c,g,i), scale bars 5 30 µm; for (d–f), scale bar 5 70µm; for (h), scale bar 5 20 µm.


Fig. 7. Distribution of EAAC1 mRNA and protein in the cerebellarcortex (a-d) and spinal ganglion (e, f). a,b: ISH using the cRNA proberesults in a more intense labeling (a) than the oligonucleotide probe(b). With both probes, EAAC1 mRNA is preferentially detected inneurons. Purkinje cells (P) and Golgi cells (arrowheads) are moreintensely labeled than interneurons (arrows) in the molecular layer(mo) and granule cells in the granule cell layer (gr). c: Control for ISHusing the oligonucleotide probe. No specific labeling is observed usingthe oligonucleotide probe in the presence of a 100-fold excess ofunlabeled oligonucleotide probe. mo, molecular layer; gr, granule celllayer. d: Immunofluorescence staining of a semithin plastic section offreeze-dried cerebellar cortex. A strong granular staining is observed

in the glomeruli cerebellares (arrows) and in the granule cell layer(gr), whereas granule cells (g) and Purkinje cells (P) are moderatelylabeled. Note the granular labeling of the Purkinje cell cytoplasm. e:Spinal ganglion showing ISH labeling using the cRNA probe. Neuro-nal perikarya of different size are moderately to strongly labeled. Anarrowhead points to the nucleus of a satellite cell, the staining ofwhich is difficult to distinguish from the neuronal labeling. nf, nervefibers. f: Immunofluorescence staining of a semithin plastic section of afreeze-dried spinal ganglion. A moderate, granular staining is ob-served in neuronal perikarya. Note the strong staining of satellite cells(arrowheads). For (a,c), scale bars 5 30 µm; for (b), scale bar 5 70 µm;for (e), scale bar 5 15 µm; for (d,f), scale bars 5 10 µm.


not observed (Rothstein et al., 1994; Furuta et al., 1997;Shashidharan et al., 1997). Kiryu et al. (1995) detectedEAAC1 mRNA in flat cells in the corpus callosum usingradioactive ISH and suggested that these cells werelabeled oligodendrocytes. Using non-radioactive ISH,Berger and Hediger (1998) demonstrated scattered,strongly-labeled cells in the corpus callosum, fimbria-fornix, and anterior commissure. The authors sug-gested that these cells represented neurons or oligoden-drocyte-progenitor cells. The present ISH data extendthe previous observations, demonstrating the occur-rence of scattered strongly-labeled cells in the internalcapsule, optic tract, and white matter of spinal cord.Additionally, we observed short rows of closely-spacedlabeled cells in the white matter of the cerebellum, incerebellar peduncles, and in the hippocampal alveus.

An arrangement in rows is a typical morphologicalcharacteristic of oligodendrocytes in white matter tracts.Using double labeling with antibodies against EAAC1and b-tubulin, which is enriched in oligodendrocytes(Schaeren-Wiemers et al., 1995), we were able to un-equivocally identify EAAC1-synthesizing cells in thewhite matter tracts as oligodendrocytes. Systematicanalyses showed that virtually all oligodendrocytes inall white matter tracts were EAAC1-immunoreactive.The discrepancy between ISH- and immunocytochemi-cal results (scattered cells versus all oligodendrocytes)in some areas (corpus callosum, fimbria hippocampi,internal capsule, optic tract, spinal cord white matter)may be caused by methodological problems. Thus, it ispossible that the EAAC1 mRNA level in some oligoden-drocytes in these areas is below the detection limit ofthe non-radioactive ISH used by Berger and Hediger(1998) and in this study. This suggestion is supportedby the fact that radioactive ISH, which may be some-what more sensitive, shows a more ubiquitous labelingof glial cells in the corpus callosum (Kiryu et al., 1995).

Our results leave little doubt that EAAC1 is theglutamate transporter of oligodendrocytes. This obser-vation is supported by a number of other findings.Thus, cultured rat CNS oligodendrocytes possess selec-tive glutamate uptake mechanisms (Reynolds and Her-schkowitz, 1986), and express EAAC1 (Wang et al.,1997).

There is evidence from several physiological studiesthat oligodendrocytes possess AMPA/kainate-type glu-tamate receptors (Steinhauser, 1993; Pende et al.,1994; Gallo et al., 1994; Garcia-Barcina and Matute,1996; McDonald et al., 1998). These receptors may beactivated by the glutamate released from nerve fibers(Weinrich and Hammerschlag, 1975). Furthermore,glutamate application results in a depolarization ofoligodendrocytes (Kettenmann et al., 1984; Butt andTutton, 1992) and regulates, via AMPA receptors, imme-diate early gene expression by increasing intracellularcalcium (Pende et al., 1994; Gallo et al., 1994). It hasbeen shown that neuronal contact and neuronal activ-ity contributes to the maintenance of functional neuro-transmitter-activated signaling pathways coupled tomobilization of intracellular calcium in oligodendro-

cytes (He et al., 1996). On the other hand, recentstudies revealed (Matute et al., 1997; McDonald et al.,1998) that oligodendrocytes were selectively destroyedby low concentrations of AMPA, kainate, or glutamate.These findings suggest that oligodendrocytes sharewith neurons a high vulnerability to AMPA/kainatereceptor-mediated death. This may contribute to whitematter injury in CNS disease (McDonald et al., 1998).

Therefore, it is reasonable to assume that EAAC1 inoligodendrocytes and GLT1 and GLAST in white mat-ter astrocytes (Schmitt et al., 1996, 1997) lower theextracellular glutamate concentration and serve toprotect oligodendrocytes from the toxic action of gluta-mate. Glutamate taken up into oligodendrocytes can befurther metabolized via the action of glutamate dehydro-genase (Schmitt and Kugler, 1999) and/or glutaminesynthetase (Tansey et al., 1991). In previous studies ithas been shown that cultured oligodendrocytes possessnot only transport mechanisms for glutamate but alsorapidly metabolize it and release the metabolites (Rey-nolds and Herschkowitz, 1986).

In addition to the occurrence of EAAC1 in oligodendro-cytes, we observed ISH signal and immunolabeling inependymal cells and epithelial cells of plexus choroi-deus in the ventricular system and satellite cells of thespinal ganglia. In ependymal cells we had previouslydetected a further glutamate transporter, GLAST(Schmitt et al., 1997). This finding has been corrobo-rated by Berger and Hediger (1998) using non-radioac-tive ISH. It can be supposed that the two glutamatetransporters in ependymal cells prevent the diffusion ofsynaptically-released glutamate from the intercellularspace into the cerebro-spinal fluid.

In a previous study it has been shown that circulat-ing glutamate is accumulated in the plexus choroideusand does not enter the cerebro-spinal fluid (Hawkins etal., 1995), although the choroidal epithelium has beenclassified as ‘‘leaky’’ (Rapoport, 1976). It can be sup-posed that EAAC1 in epithelial cells of the plexuschoroideus could prevent the passage of glutamate fromthe blood stream into the cerebro-spinal fluid, wherethe glutamate concentration seems to be very low. Inependymal cells, as well as in epithelial cells of theplexus choroideus, glutamate may be further metabo-lized via glutamate dehydrogenase, which we havedetected by ISH and immunostaining in both localiza-tions (Schmitt and Kugler, 1999).

Concerning the subcellular localization of EAAC1protein, cytoplasmic staining of neurons and glial cellspredominated. A delineation of labeled cell membraneswas almost impossible at the light microscopic level.Cytoplasmic labeling of neurons has also been de-scribed in previous studies (Rothstein et al., 1994;Shashidharan et al., 1997), but detailed electron micro-scopic studies are not available. Frequently, we haveobserved in thin plastic sections a finely granularcytoplasmic staining of neurons and glial cells. To-gether with the immunoblot finding that EAAC1 is onlydetectable in the membrane fraction (100,000 3 gpellet) of brain homogenates, this indicates a vesicular


localization of EAAC1 protein. A similar vesiculardistribution is also found for other transporter mol-ecules, e.g., the dopamine transporter (Nirenberg et al.,1996), the Na1-dependent glucose transporter (Delezayet al., 1995), the serotonin transporter (Qian et al.,1997), and the GABA transporter, GAT1 (Quick et al.,1997). It has been shown that the transporter transloca-tion from vesicles into the cell membrane is induced bythe action of protein kinase C (Delezay et al., 1995;Qian et al., 1997; Quick et al., 1997). Therefore, it islikely that the EAAC1 glutamate transport rate ismodulated not only by factors regulating the amount ofprotein expressed (Gegelashvili and Schousboe, 1997),but also by recruiting the transporter from cytoplasmicvesicles to the cytoplasmic membrane.

We have clearly demonstrated that EAAC1, themajor neuronal glutamate transporter, is also ex-pressed in various glial cells. EAAC1 in neurons seemsnot to be intimately related to glutamatergic transmis-sion because EAAC1-deficient mice develop only signifi-cantly reduced spontaneous locomotor activity but noneurodegeneration (Peghini et al., 1997). Thus, thefunctional importance of EAAC1 is not quite clear.Further studies on EAAC1 should also focus on oligoden-drocytes to elucidate a possible involvement of EAAC1in oligodendrocytes in white matter diseases.

ACKNOWLEDGMENTS

We thank Erna Kleinschroth and Heike Fella fortheir excellent technical assistance.

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Glutamate transporter EAAC1 is expressed in neurons and glial cells in the rat nervous system