GLIA 27:129–142 (1999)
Glutamate Transporter EAAC1 Is Expressed in Neurons and Glial Cells in the Rat Nervous System PETER KUGLER* AND ANGELIKA SCHMITT Institute of Anatomy, University of Wu¨rzburg, Wu¨rzburg, Germany
KEY WORDS
excitatory amino acid transporter; oligodendrocytes; ependyma; plexus choroideus
ABSTRACT Oligonucleotide and cRNA probes were used for non-radioactive in situ hybridization, carried out to identify the cell types in the nervous system of rat expressing the glutamate transporter EAAC1 mRNA. The results were compared with immunocytochemical data obtained using an antibody against a synthetic EAAC1 peptide. The present data confirm that EAAC1 is expressed in neurons of the CNS. Additionally, our findings indicate the localization of EAAC1 mRNA and protein in peripheral neurons (spinal ganglia) and in glial cells, i.e., oligodendrocytes in various white matter regions of the CNS, ependymal cells, and epithelial cells of the plexus choroideus 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 EAAC1 protein in vesicle membranes. A membrane localization of EAAC1 was also indicated by Western blotting, which showed immunoreactivity only in the 100,000 ⫻ g pellet of brain homogenate. We conclude that the glutamate transporter EAAC1 is not restricted to neurons but may also play an important role in glial cells, particularly in oligodendrocytes. GLIA 27:129–142, 1999. r 1999 Wiley-Liss, Inc.
INTRODUCTION Glutamate reuptake from the extracellular space plays an important role in the CNS. It terminates the transmitter signal and prevents a harmful receptor overstimulation (Kanai et al., 1993; Lipton and Rosenberg, 1994). In recent years, the cDNAs of at least five different subtypes of glutamate transporters have been cloned—GLT1 (Pines et al., 1992), GLAST (Storck et al., 1992), EAAC1 (Kanai and Hediger, 1992), EAAT4 (Fairman et al., 1995), and EAAT5 (Arriza et al., 1997). GLT1, GLAST, and EAAC1 are widely distributed throughout the CNS (Kanai and Hediger, 1992; Pines et al., 1992; Storck et al., 1992; Rothstein et al., 1994; Torp et 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, EAAT4 and 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. r 1999 Wiley-Liss, Inc.
With respect to the cellular localization of EAAC1, immunocytochemistry using antibodies against synthetic peptides (Rothstein et al., 1994; Shashidharan et al., 1997) detected transporter protein primarily in somatodendritic membranes of glutamatergic and GABAergic neurons, while presynaptic structures were not labeled (Rothstein et al., 1994; Coco et al., 1997). Labeling in glial cells was not reported. EAAC1 was therefore considered to be an exclusively neuronal, postsynaptic glutamate transporter. Using radioactive and non-radioactive in situ hybridization (ISH), EAAC1 mRNA was detected in presumably neuronal perikarya of various CNS regions (Kanai and Hediger, 1992; Meister et al., 1993; Kiryu et al.,
Grant sponsor: Deutsche Forschungsgemeinschaft. *Correspondence to: Prof. Dr. Peter Kugler, Institute of Anatomy, Koellikerstrasse 6, D-97070 Wu¨rzburg, Germany. E-mail:
[email protected] Received 18 January 1999; Accepted 25 February 1999
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1995; Kanai et al., 1995; Velaz-Faircloth et al., 1996; Torp et al., 1997; Berger and Hediger, 1998). In addition, Kiryu et al. (1995) detected flat-labeled cells in the corpus callosum, which they suggested were oligodendrocytes. Because of the poor cellular resolution of the radioactive ISH method used in that study, however, the question of the precise cellular source of the EAAC1 mRNA signal could not be answered conclusively. In a recent non-radioactive ISH study, Berger and Hediger (1998) reported that EAAC1 mRNA is localized in cells of white matter tracts, e.g., in the corpus callosum, the fimbria-fornix, and in the anterior commissure. Based on their scattered distribution, the authors suggested that these labeled cells are not mature oligodendrocytes but rather represent oligodendrocyte-progenitor cells, or alternatively, neurons. In the present study, we have addressed the question of EAAC1 localization in glial cell populations using highly sensitive methods—non-radioactive ISH using cRNA and oligonucleotide probes (Asan and Kugler, 1995; Schmitt et al., 1996, 1997) in combination with comparative immunocytochemistry using an antibody against a synthetic EAAC1 peptide. Additionally, these methods provide clear cellular resolution of labeling. Because the expression pattern of EAAC1 in neurons has already been extensively analysed in the rat CNS (see above), a regional mapping of neuronal labeling was not included in this study. However, in order to be able to compare our data with previous investigations (see above), documentation of neuronal labeling was carried out in the hippocampus and the cerebellum. Additionally, cervical spinal ganglia were studied to document EAAC1 localization in peripheral nervous tissue.
40 units of AMV-reverse transcriptase (Boehringer, Mannheim, Germany). Two primers, EAAC1 (a): 58GAGCTCTCGAATCTGGATAA-38 (complementary to nucleotides 247–267), and EAAC1 (b): 58-CTAAGGCCAG-GCATCTAGAA-38 (complementary to nucleotides 1687–1706; purchased from Roth, Karlsruhe, Germany), based on the rat EAAC1 sequence published by Kiryu et al. (1995), were used to amplify a rat brain EAAC1cDNA fragment. Ten percent of the 1:5 diluted reverse transcription mixture was utilized for a polymerase chain reaction in a final volume of 100 µl containing 0.4 pM of each primer, 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). For amplification, the following procedure was used: denaturation 1 min/94°C, annealing 2 min/59°C, extension 3 min/72°C, 40 cycles, and a final elongation for 10 min at 72°C. The resulting cDNA was cloned into the Eco RV site of the Bluescript vector (pBluescript II SK⫹, Stratagene, La Jolla, CA) and transfected and propagated in Escherichia coli XL 1 Blue. The identity of the cloned cDNA was verified by restriction analysis and partial DNA 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 polymerase) according the manufacturer’s manual (Boehringer). Usually, 1.5 µg of cDNA template yielded 10 to 30 µg of labeled cRNA, incorporating approximately one DIG-11-UTP at every 20th nucleotide. cRNA probes were analyzed on a formaldehyde agarose gel (1%).
Northern Blotting MATERIALS AND METHODS Animals and Tissue Sources The brains, cervical spinal cords, and spinal ganglia of 30 male Wistar rats (of our own breeding, aged 8–12 weeks) were used for RNA preparation, ISH, immunoblotting, and immunohistochemistry, as described below.
Generation of a Digoxigenin-Labeled cRNA Probe All procedures for the preparation of cRNA probes were performed as described by Sambrook et al. (1989) and Schmitt et al. (1996) and will be described here only in brief. Total RNA from the whole rat brain was isolated by acid guanidinium thiocyanate-phenolchloroform extraction (Chomczynski and Sacchi, 1987). The first-strand synthesis of the cDNA was performed for 1 h at 42°C in a reaction volume of 20 µl containing 3 µg total RNA, 1.8 pM Oligo(dT)-primer (18-mer), 1 mM of each dNTP, 20 units RNasin, 50 mM Tris/HCl (pH 8.5), 8 mM MgCl2, 30 mM KCl, 1 mM dithiothreitol, and
For Northern blotting, whole brain, cerebellum, hippocampus, and cervical spinal cord were dissected and the total RNA of each preparation was isolated as described above. The Northern blotting was performed essentially according to the instructions of Boehringer for non-radioactive hybridization (The DIG system user’s guide for filter hybridization; Boehringer). Briefly, probes of RNA were separated in a standard formaldehyde gel (1%) and transferred onto nylon membranes (Hybond N⫹; Amersham, Braunschweig, Germany). Prehybridization was performed in 5 ⫻ standard saline citrate (SSC) containing 50% formamide, 0.1% Nlauroyl-sarcosine, 0.02% sodium dodecyl sulfate (SDS), and 2% blocking reagent (Boehringer). Subsequently, the blots were placed in the prehybridization solution containing 100 ng/µl digoxigenin-labeled antisense (sense) cRNA (see above) overnight at 68°C. The blots were washed in 2 ⫻ SSC and 0.1% SDS at room temperature and subsequently in 0.5 ⫻ SSC and 0.1% SDS at 68°C. After equilibration in washing buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% Tween 20, pH 7.5) at room temperature, the membranes were transferred into a blocking solution (0.1 M maleic acid, 0.15 M
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NaCl, 1% blocking reagent, pH 7.5) for 30 min. After incubation with sheep anti-digoxigenin-alkaline phosphatase (aP) conjugated antibody (1:5000; corresponding to 150 mU/ml aP activity; Boehringer) for 1 h the membranes were rinsed in the washing buffer, equilibrated in 0.1 M Tris-HCl buffer at pH 9.5 and used for the detection of aP (see below).
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In some experiments, following the aP visualization, several brain sections were used for the immunocytochemical detection of glial fibrillary acidic protein (GFAP), applying the PAP method (peroxidase-antiperoxidase complex 1:100; mouse monoclonal antibody against GFAP 1:2000; DAKO, Hamburg, Germany; Sternberger et al., 1990).
In Situ Hybridization cRNA Frontal blocks of brains, 3–5 mm thick (approximate interaural levels 7.0 to 4.5 mm and ⫺1 to ⫺3.0 according to the rat brain stereotaxic atlas of Paxinos and Watson, 1986), spinal cord, and spinal ganglia were frozen in liquid-nitrogen-cooled isopentane. Twelvemicrometer-thick cryostat sections were mounted on precoated glass slides (Superfrost Plus; Menzel, Braunschweig, Germany) and thawed. The sections were fixed for 5 min in freshly prepared 4% formaldehyde in 0.01 M phosphate-buffered saline (PBS; pH 7.4). The sections were transferred to ethanol and stored for 1 to 2 days. The sections were subsequently removed from ethanol, rehydrated in a graded series of ethanol, transferred to 2 ⫻ SSC and treated with 0.05 N HCl for 30 min. After washing with 2 ⫻ SSC, the sections were incubated with freshly prepared 0.25% acidic anhydride, washed again with 2 ⫻ SSC and covered with the hybridization solution, which contained the digoxigeninlabeled antisense (sense) cRNA probe (final concentration 3–6 ng/µl) and 550 µg/ml Salmon testes DNA (Sigma, Deisenhofen, Germany) in 4 ⫻ SSC, 1 ⫻ Denhardt’s solution (Sambrook et al., 1989), 10% dextran sulfate, and 50% deionized formamide at 60°C for 16–18 h. Posthybridization washes were done stepwise at room temperature with 2 ⫻ SSC, 1 ⫻ SSC, 50% formamide, and then again with 2 ⫻ SSC. The sections were then treated with 1 µg/ml proteinase K (Boehringer) in 50 mM Tris-HCl (pH 7.4) for 30 min at room temperature and washed with bi-distilled water for 5 min. Next, the sections were treated with 40 µg/ml ribonuclease A (50 Kunitz-units/mg; Boehringer) in a solution containing 500 mM NaCl, 10 mM Tris-HCl (pH 8), and 1 mM EDTA at 37°C for 30 min to remove unhybridized single strand RNAs. After the treatment, the sections were incubated with the same buffer without Rnase A at 60°C for 30 min. Subsequently, the sections were rinsed in Trisbuffered 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 in TBS (20 min). After incubation with 1.5 U/ml sheep anti-DIG-aP conjugated (Boehringer) in TBS containing 0.3% Triton X-100 for 60 min, the sections were washed in TBS, and transferred to a 0.1 M Tris-buffer containing 100 mM NaCl and 50 mM MgCl2 (pH 9.5) for 2 min prior to the aP visualization (see below).
Oligonucleotide probe ISH was carried out according to the method of Da˚gerlind et al. (1992), using an aP coupled 33mer oligonucleotide probe complementary to part of the coding region of EAAC1 mRNA (antisense probe to the nucleotides 1657–1689: 58-GAACTGCGAGGTCTGAGTGAACGAGATGGTGTC-38; custom synthesized by DNA Technology, Aarhus, Denmark). Twelve µm-thick cryostat sections of snap-frozen frontal tissue blocks of brain (see above) mounted on Superfrost slides were thawed and covered with hybridization solution (see above) containing 6-8 fmol/ml antisense oligonucleotide probe at 37°C for 20–40 h. Posthybridization washes consisted of 1 ⫻ SSC for 4 ⫻ 15 min at 55°C. After cooling to room temperature, the sections were transferred to TBS for 30 min, followed by 100 mM Tris-HCl containing 100 mM NaCl, 50 mM MgCl2 (pH 9.5) for 10 min, prior to the aP visualization.
Detection of alkaline phosphatase The procedure used has been described recently by Asan and Kugler (1995). The incubation media contained 0.4 mM 5-bromo-4-chloro-3-indolylphosphate (BCIP; Boehringer), 100 mM sodium chloride, 50 mM MgCl2, 0.4 mM tetranitroblue tetrazoliumchloride or nitroblue tetrazoliumchloride (Serva, Heidelberg, Germany) in 100 mM Tris-HCl buffer at pH 9.5.
Controls for ISH Substitution of the antisense cRNA probe by an equivalent amount of labeled sense cRNA probe led to a lack of staining (cf. Figs. 3f, 5c). Neither was staining observed in sections of unfixed tissue if a 100-fold excess of unlabeled oligonucleotide probe was applied together with the aP-labelled probe (cf. Figs. 6f, 7c), indicating a complete competitive inhibition of specific binding of the labeled probe in these preparations. Omission of labeled cRNA or oligonucleotide probes from the respective hybridization mixtures resulted in completely unstained sections. From these findings it can be concluded that (1) the antisense probes were specific, (2) the digoxigenin detection did not create labeling artifacts, and (3) there was no endogenous aP activity left in the sections.
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Antibodies and Immunoblotting
sorbed with an excess of the peptide used for immunization served as control.
Antibodies A peptide corresponding to the C-terminal region 480–499 (I-V-N-P-F-A-L-E-P-T-I-L-D-N-E-D-S-D-T-K) of the EAAC1 protein (Kiryu et al., 1995) was synthesized by the fmoc method and purified by reverse phase high pressure liquid chromatography (Atherton et al., 1981). For immunization, the peptide was coupled to keyhole limpet hemocyanin (KLH) by glutaraldehyde, as described in detail by Drenckhahn et al. (1993). One ml of the peptide solution (corresponding to 500 µg peptide) was mixed with polyalphaolefin adjuvant (Ethyl S. A., Brussels, Belgium) and injected subscapularly in rabbits (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 affinity purified, using the synthetic peptide immobilized by transfer to nitrocellulose paper (Schleicher and Schu¨ll, Darmstadt, Germany). The bound immunoglobulins were eluted by low pH (pH 2.8) and the protein content was determined spectrophotometrically (Drenckhahn et al., 1993). Mouse monoclonal antibody against GFAP and -tubulin were purchased from DAKO and Sigma, respectively. In the CNS, GFAP is a specific marker protein of astrocytes (Bignami et al., 1972) and -tubulin is highly expressed in oligodendrocytes (as well as in neurons; Schaeren-Wiemers et al., 1995).
Immunoblotting For immunoblotting, cerebellum, hippocampus, whole neocortex, and cervical spinal cord were dissected and homogenized 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 phenylmethylsulfonyl fluoride (100 µg/ml). The homogenate was centrifuged at 1,000 ⫻ g for 10 min and the 1,000 ⫻ g supernatant was centrifuged at 100,000 g for 1 h. The protein contents of the 100,000 g supernatant and pellet were determined by the Bio-Rad protein assay (Bio-Rad, Munich, Germany) and the supernatant and the pellet (membrane fraction) were used for immunoblotting. Proteins (5–50 µg per lane) were electrophoretically separated on 10% gels by SDS-polyacrylamide gel electrophoresis (PAGE). Subsequently, the proteins were transferred electrophoretically to nitrocellulose membranes (Burnette, 1981). Strips of the nitrocellulose membranes were incubated for 24 h at 4°C with the affinity-purified antibody (approximately 1.8 µg/ml). Bound immunoglobulins were visualized using peroxidase-conjugated goat anti-rabbit IgG (1:3,000; Bio-Rad, Richmond, Canada; blotting grade) and the enhanced luminol chemiluminescence technique (Amersham, Braunschweig, Germany). Antibody previously ab-
Immunostaining Pieces of hippocampus, cerebellum, spinal cord, plexus choroideus, and spinal ganglia were frozen in liquid nitrogen-cooled propane and were freeze-dried and embedded 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 in methanol-toluene (1:1) containing 10% sodium methoxide (prepared from metallic sodium; Major et al., 1961). The tissue sections were preincubated for 3 h at room temperature with 2% bovine serum albumin, 10% normal goat serum and 0.05% Tween 20 (Ferrak, Berlin, Germany) in PBS, pH 7.4. Then, the sections were incubated for 24–48 h at 4°C for single and double labeling with the primary antibody diluted in the preincubation solution (anti-EAAC1, 15 µg/ml; antiGFAP, 1:10,000; anti--tubulin 1:750). After several washes with PBS, the semithin plastic sections were incubated for 90 min at room temperature with carbocyanin (Cy2) labeled secondary antibody (1:300; goat anti-rabbit IgG; Dianova, Hamburg, Germany) for detection of EAAC1, and with indocarbocyanin (Cy3) labeled secondary antibody (1:600; goat anti-mouse IgG; Dianova) for the detection of GFAP or -tubulin. Controls were performed with primary antibody, previously absorbed with an excess of EAAC1 peptide used for immunization or without the primary antibody. The sections were examined with an Olympus BH-2 fluorescence microscope (Olympus, New Hyde Park, NY) equipped with Zeiss optics and an appropriate filter combination for selective visualization of Cy2 and Cy3 fluorescence (BH II DFC 6; Olympus).
RESULTS Northern and Western Blotting Northern blots of the total RNA of the whole brain and the hippocampus showed a main band at ⬃4.2 and weaker bands at ⬃2.7 and 7.5 kb (Fig. 1). In Northern blots of the cerebellum and spinal cord only the band at ⬃4.2 kb was detectable. No labeling could be seen in blots using the sense cRNA probe (not shown). In Western blots of the 100,000 ⫻ g pellet of tissue homogenates (cerebral cortex, cerebellum, hippocampus, spinal cord), the affinity-purified antibody against the EAAC1 peptide labeled a relatively broad ⬃64,000 mol. wt band (Fig. 2a). In the 100,000 g supernatant, no protein band was labeled using the affinity-purified antibody (data not shown). This indicates that the detected protein was localized in membranes. Preabsorption of the antibody with the synthetic peptide abolished binding to the protein bands (Fig. 2a). After deglycosylation with N-glycosidase F, a ⬃55,000 kDa
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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 of the mRNA size was carried out by comparison with ribosomal RNA bands (⬃2 and ⬃5 kb). Labeled bands around 2.7 kb, 4.2 kb, and 7.5 kb are clearly seen in the brain and hippocampus samples, whereas in other organ samples a significantly labeled band occurs only around 4.2 kb.
band was labeled in immunoblots of the cerebral cortex and hippocampus (Fig. 2b).
In Situ Hybridization and Immunocytochemistry In situ hybridization Application of the cRNA or oligonucleotide probes to cryostat sections resulted in identical patterns of cellular and regional distribution of EAAC1 mRNA in the rat nervous system, both methods showing a high cellular resolution. However, the DIG-labelled cRNA probe provided a much higher signal intensity than the aP-labelled oligonucleotide probe (Figs. 6a–f, 7a–c). Therefore, ISH results were preferentially documented using sections hybridized with the cRNA probe. In accordance with previous findings (Kanai and Hediger, 1992; Meister et al., 1993; Kiryu et al., 1995; Kanai et al., 1995; Velaz-Faircloth et al., 1996; Torp et al., 1997; Berger and Hediger, 1998), ISH reaction product was detected in neuronal cell bodies. Additionally, using our non-radioactive ISH protocol, we observed a labeling of different glial cell types. We were able to detect labeled glial cells in the white matter of various CNS regions. These cells were typically arranged like pearls on a string, a morphological characteristic of oligodendrocyte localization. This typical arrangement of labeled cells (oligodendrocytes) was
Fig. 2. Western blot analysis (10% SDS-PAGE) of cerebral cortex (cc), hippocampus (h), cerebellum (c), and cervical spinal cord (s) using the affinity purified EAAC1 antibody. For probing, the 100,000 ⫻ g pellet was used. The amount of protein loaded per lane was 50 µg. a: In the various CNS regions the EAAC1 antibody labeled a ⬃64 kDa band. Immunoblotting of hippocampal proteins, using antibody previously absorbed with an excess of the EAAC1 peptide used for immunization, served as control (co). b: Deglycosylation experiment. For probing, the 100,000 ⫻ g pellet of cerebral cortex (cc) and hippocampus (h) was used with (cc⫹, h⫹) and without (cc⫺, h⫺) treatment with N-glycosidase F, as described by Schmitt et al. (1977). After enzyme treatment, the molecular mass of the protein was reduced by ⬃9 kDa.
observed particularly in the white matter of the cerebellum (Fig. 3b), in cerebellar peduncles (Fig. 3a), and in the hippocampal alveus. In other white matter tracts (e.g., fimbria hippocampi, corpus callosum, internal capsule, optic tract), strongly labeled glial cells showed a scattered distribution (Fig. 3c–e). Double-labeling with GFAP immunocytochemistry showed that these scattered cells were negative for GFAP-immunostaining (Fig. 3d,e), indicating that they were not astrocytes. In the white matter of the spinal cord, again only some individual glial cells were ISH-labeled (not shown). Additionally, ISH reaction product was localized in other glial cell types, i.e., in ependymal cells (Fig. 5a,b) and epithelial cells of the choroid plexus (Fig. 5b,c) of all
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Fig. 3. Cellular distribution of EAAC1 mRNA in the CNS white matter using the cRNA probe. White matter of the inferior cerebellar peduncle (a) and of the cerebellum (b). ISH reaction product is detected in glial cells, which are typically arranged in rows (arrows) and therefore appear to be oligodendrocytes. bv, blood vessel. c: Corpus callosum. Strongly labeled cells are scattered throughout the white matter. d,e: A micrograph pair showing a section of the corpus callosum after ISH, using the antisense cRNA probe in the first, and
after additional GFAP immunostaining, in the second figure. No GFAP-immunoreactivity is observed in glial cells labeled for EAAC1 mRNA (arrows). GFAP-immunolabeled astrocytes and astrocytic processes (arrowheads) shown in (e) are scattered throughout the white matter. f: Control. Applying the sense cRNA, no reaction product is observed in the inferior cerebellar peduncle. For (a, c–f), scale bars ⫽ 30 µm; for (b), scale bar ⫽ 15 µm.
four ventricles, and in satellite cells of spinal ganglia (Fig. 7e). Astrocytes did not show any labeling. Neuronal EAAC1 mRNA labeling was observed in the hippocampus (pyramidal and granule cells; interneurons scattered throughout the oriens layer of the hippocampus proper; Fig. 6a–f) and in the cerebellar cortex (Fig. 7a–c). In the cerebellar cortex, very weak
labeling was observed in granule cells, whereas Purkinje cells, interneurons of the molecular layer, and Golgi cells (identified by their localization and distribution in the granule cell layer) were moderately to strongly labelled (Fig. 7a,b). In addition to neurons of the CNS, EAAC1 mRNA was detected in peripheral neurons, i.e., in neurons of
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spinal ganglia. Labeling of these neurons ranged from weak to strong (Fig. 7e).
Immunocytochemistry When the affinity-purified antibody against the EAAC1 peptide was applied to thin plastic sections (1 µm thick), a finely granular cytoplasmic staining of neurons and glial cells predominated (Figs. 4c,g,i, 5e, 7d,f). A delineation of labeled cell membranes was almost impossible. No staining was observed with the EAAC1 affinity-purified antibody previously absorbed to the synthetic peptide (not shown). In the white matter of the cerebellum and hippocampus (fimbria, alveus) and in white matter tracts (spinal cord, corpus callosum) we observed a moderate to strong staining of glial cells (Fig. 4a–c,e,g,i). These glial cells often showed an arrangement in rows, which is typical for oligodendrocytes (Fig. 4a; cf. ISH). Furthermore, we identified these glial cells not only by their distributional pattern but also by double-labeling experiments: the EAAC1 immunolabeled glial cells of the white matter (Fig. 4c) were not labeled by GFAPantibody (Fig. 4d) but were intensely stained using a monoclonal antibody against -tubulin (Fig. 4e–h). It is known that -tubulin is highly expressed in oligodendrocytes and neurons (Schaeren-Wiemers et al., 1995). As the white matter areas studied are free of neurons, the -tubulin positive cells were most probably oligodendrocytes. Especially in white matter tracts of the spinal cord, the EAAC1 immunostained glial cells displayed the typical shape of oligodendrocytes—angular cell bodies, frequently with trunk-like primary processes from which fine secondary processes arose (Fig. 4e,i). Such fine processes partly surrounded cross-sectioned fibers with (Fig. 4e) or without immunostained axoplasm (Fig. 4i). The myelin of fibers showed no labeling (Fig. 4i). Sometimes, labeled rims were seen, which surrounded axons (Fig. 4i). From light microscopic observation it was not possible to decide if this labeling belonged to axons (axolemma) or to processes of oligodendrocytes (inner loop). In agreement with the ISH findings, we detected EAAC1 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, as well as scattered cells in the neuropil layers, displayed immunolabeling; in the cerebellar cortex, granule cells and 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 cerebellares displayed labeling, which was intense in granular profiles and somewhat lighter throughout the glomerular area (Fig. 7d).
In agreement with ISH findings, spinal ganglia neurons and satellite cells showed a moderate to strong immunostaining for EAAC1 protein (Fig. 7f).
DISCUSSION The present study extends previous investigations into the expression and synthesis of the high-affinity glutamate transporter EAAC1 in the rat nervous system. Using high-resolution ISH in combination with specific immunodetection, we were able to confirm findings reported by other groups, namely that EAAC1 mRNA and protein are localized in CNS neurons (see Introduction). Additionally, our results provide the first conclusive evidence that EAAC1 is the glutamate transporter expressed by peripheral neurons (spinal ganglia neurons) and by various glial cells, such as oligodendrocytes, ependymal cells, epithelial cells of the plexus choroideus, 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 by Northern blot analysis. In RNA preparations from various CNS sources a band at ⬃ 4.2 kb was labeled. Additionally, in RNA preparations from whole brain and hippocampus two bands at ⬃ 2.7 and 7.0 kb were weakly labeled. Similar banding has been described by Kanai et al. (1995). ISH using the DIG-labelled cRNA probe (detected by an aP-labeled antibody against DIG) to formaldehyde and alcohol-fixed cryostat sections resulted in specific labeling of high sensitivity and cellular resolution, and it displayed extremely low background. Both in Northern blots and in ISH, no specific labeling was detected when the cRNA sense probe was used for hybridization. Further proof for the specificity of the detection was that an oligonucleotide probe, complementary to part of the EAAC1 cDNA sequence (nt 1657–1689), showed the same labeling pattern in ISH as did the cRNA probe. Again, control hybridization using the aP-labelled oligonucleotide probe in the presence of an excess of unlabeled oligonucleotide probe (competition experiment) yielded no labelling whatsoever. Preliminary immunocytochemical experiments using the affinity-purified peptide antibody generated against EAAC1 resulted in staining patterns similar to those described previously, with strong EAAC1 staining of CNS neurons. This observation indicates that the antibody faithfully detected EAAC1 protein in immunocytochemistry. Pre-adsorption of the antibody with the antigen peptide resulted in a lack of specific immunostaining. Further evidence for the specificity of the antibody was given by the fact that it detected a ⬃64 kDA band in Western blot analyses from several CNS regions, which was reduced in size to ⬃55 kDA by
Fig. 4. Cellular distribution of EAAC1 protein detected by immunofluorescence staining in semithin plastic sections of freeze-dried tissue. a: Alveus of the hippocampus. Immunostaining of different intensity is observed in glial cells, which are typically arranged in a row and therefore seem to be oligodendrocytes. The oligodendrocytes are flanked by bundles of stained nerve fibers. The precise localization of reaction product in the nerve fibers cannot be established. b: White matter of the cerebellum. Two strongly labeled glial cells (oligodendrocytes) are surrounded by nerve fibers, which show a labeling of membranes. c,d: A micrograph pair taken from the cerebellar white matter double labeled for EAAC1 (c) and the astrocytic marker GFAP (d). n, nucleus; arrowheads point to astrocytic profiles. The strongly labeled glial cell (c) is GFAP negative (d). e,f: A micrograph pair taken from neuron-free white matter (funiculus anterior) of the cervical
spinal cord, double labeled for EAAC1 (e) and -tubulin (f), which is a marker for neurons and oligodendrocytes. The glial cells (arrows) are labeled for EAAC1 and -tubulin and therefore seem to be oligodendrocytes. Note that labeled processes of oligodendrocytes partially surround the unstained myelin of nerve fibers. The axoplasm of most nerve fibers (arrowheads) are labeled for EAAC1 (e) and -tubulin (f). g,h: A micrograph pair of an EAAC1/-tubulin positive oligodendrocyte (spinal cord, funiculus anterior) at high magnification. n, nucleus. i: An EAAC1-positive oligodendrocyte is shown (spinal cord, funiculus anterior). Labeled processes of the oligodendrocyte extend between and surround (arrow) nerve fibers. Arrowheads point to labeled rims bordering the inner surface of the unlabeled myelin sheaths. Note the finely granular cytoplasmic staining of oligodendrocytes in (c), (g), and (i). For (a–d, g–i), scale bars ⫽ 10 µm; for (e,f), scale bar ⫽ 20 µm.
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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 almost moderate 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 strong immunofluorescent staining is shown in ependymal cells (d) and in epithelial cells of the plexus choroideus of the third ventricle (e). Note the fine granular cytoplasmic staining of epithelial cells of the plexus choroideus. For (a,b), scale bar ⫽ 30 µm; for (c), scale bar ⫽ 50 µm; for (d,e), scale bar ⫽ 10 µm.
deglycosylation. These molecular masses are in good accord 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 predicted molecular mass of the non-glycosylated protein (57 kDa; Kanai and Hediger, 1992).
Expression of EAAC1 in Glial Cells Most important seems to be the detection of EAAC1 mRNA and protein in oligodendrocytes of CNS white matter regions. In previous immunocytochemical studies, the localization of EAAC1 in oligodendrocytes was
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Fig. 6. Distribution of EAAC1 mRNA in the hippocampus using the cRNA probe (a, c) and the oligonucleotide probe (b, d–f), and of EAAC1 protein detected by immunofluorescence staining (g–i). ISH using the cRNA probe results in a more intense labeling (a,c) than the oligonucleotide probe (b,d,e). With both probes EAAC1 mRNA is preferentially detected in neurons, e.g., pyramidal cells in the pyramidal cell layer (py), granule cells in the granule cell layer (gr), neurons of the multiform 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 oligonucleotide probe in the presence of a 100-fold excess of unlabeled oligonucleotide probe. g–i: A moderate, partly finely-granular immunostaining is observed in the neuropil of the radiatum layer (ra), of the cornu ammonis 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) and granule cells (gr) display weak to moderate labeling. For (a,b), scale bar ⫽ 400 µm; for (c,g,i), scale bars ⫽ 30 µm; for (d–f), scale bar ⫽ 70 µm; for (h), scale bar ⫽ 20 µm.
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Fig. 7. Distribution of EAAC1 mRNA and protein in the cerebellar cortex (a-d) and spinal ganglion (e, f). a,b: ISH using the cRNA probe results in a more intense labeling (a) than the oligonucleotide probe (b). With both probes, EAAC1 mRNA is preferentially detected in neurons. Purkinje cells (P) and Golgi cells (arrowheads) are more intensely labeled than interneurons (arrows) in the molecular layer (mo) and granule cells in the granule cell layer (gr). c: Control for ISH using the oligonucleotide probe. No specific labeling is observed using the oligonucleotide probe in the presence of a 100-fold excess of unlabeled oligonucleotide probe. mo, molecular layer; gr, granule cell layer. d: Immunofluorescence staining of a semithin plastic section of freeze-dried cerebellar cortex. A strong granular staining is observed
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in the glomeruli cerebellares (arrows) and in the granule cell layer (gr), whereas granule cells (g) and Purkinje cells (P) are moderately labeled. Note the granular labeling of the Purkinje cell cytoplasm. e: Spinal ganglion showing ISH labeling using the cRNA probe. Neuronal perikarya of different size are moderately to strongly labeled. An arrowhead points to the nucleus of a satellite cell, the staining of which is difficult to distinguish from the neuronal labeling. nf, nerve fibers. f: Immunofluorescence staining of a semithin plastic section of a freeze-dried spinal ganglion. A moderate, granular staining is observed in neuronal perikarya. Note the strong staining of satellite cells (arrowheads). For (a,c), scale bars ⫽ 30 µm; for (b), scale bar ⫽ 70 µm; for (e), scale bar ⫽ 15 µm; for (d,f), scale bars ⫽ 10 µm.
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not observed (Rothstein et al., 1994; Furuta et al., 1997; Shashidharan et al., 1997). Kiryu et al. (1995) detected EAAC1 mRNA in flat cells in the corpus callosum using radioactive ISH and suggested that these cells were labeled oligodendrocytes. Using non-radioactive ISH, Berger and Hediger (1998) demonstrated scattered, strongly-labeled cells in the corpus callosum, fimbriafornix, and anterior commissure. The authors suggested that these cells represented neurons or oligodendrocyte-progenitor cells. The present ISH data extend the previous observations, demonstrating the occurrence of scattered strongly-labeled cells in the internal capsule, optic tract, and white matter of spinal cord. Additionally, we observed short rows of closely-spaced labeled cells in the white matter of the cerebellum, in cerebellar peduncles, and in the hippocampal alveus. An arrangement in rows is a typical morphological characteristic of oligodendrocytes in white matter tracts. Using double labeling with antibodies against EAAC1 and -tubulin, which is enriched in oligodendrocytes (Schaeren-Wiemers et al., 1995), we were able to unequivocally identify EAAC1-synthesizing cells in the white matter tracts as oligodendrocytes. Systematic analyses showed that virtually all oligodendrocytes in all white matter tracts were EAAC1-immunoreactive. The discrepancy between ISH- and immunocytochemical 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 is possible that the EAAC1 mRNA level in some oligodendrocytes in these areas is below the detection limit of the non-radioactive ISH used by Berger and Hediger (1998) and in this study. This suggestion is supported by the fact that radioactive ISH, which may be somewhat more sensitive, shows a more ubiquitous labeling of glial cells in the corpus callosum (Kiryu et al., 1995). Our results leave little doubt that EAAC1 is the glutamate transporter of oligodendrocytes. This observation is supported by a number of other findings. Thus, cultured rat CNS oligodendrocytes possess selective glutamate uptake mechanisms (Reynolds and Herschkowitz, 1986), and express EAAC1 (Wang et al., 1997). There is evidence from several physiological studies that oligodendrocytes possess AMPA/kainate-type glutamate receptors (Steinha¨user, 1993; Pende et al., 1994; Gallo et al., 1994; Garcia-Barcina and Matute, 1996; McDonald et al., 1998). These receptors may be activated by the glutamate released from nerve fibers (Weinrich and Hammerschlag, 1975). Furthermore, glutamate application results in a depolarization of oligodendrocytes (Kettenmann et al., 1984; Butt and Tutton, 1992) and regulates, via AMPA receptors, immediate early gene expression by increasing intracellular calcium (Pende et al., 1994; Gallo et al., 1994). It has been shown that neuronal contact and neuronal activity contributes to the maintenance of functional neurotransmitter-activated signaling pathways coupled to mobilization of intracellular calcium in oligodendro-
cytes (He et al., 1996). On the other hand, recent studies revealed (Matute et al., 1997; McDonald et al., 1998) that oligodendrocytes were selectively destroyed by low concentrations of AMPA, kainate, or glutamate. These findings suggest that oligodendrocytes share with neurons a high vulnerability to AMPA/kainate receptor-mediated death. This may contribute to white matter injury in CNS disease (McDonald et al., 1998). Therefore, it is reasonable to assume that EAAC1 in oligodendrocytes and GLT1 and GLAST in white matter astrocytes (Schmitt et al., 1996, 1997) lower the extracellular glutamate concentration and serve to protect oligodendrocytes from the toxic action of glutamate. Glutamate taken up into oligodendrocytes can be further metabolized via the action of glutamate dehydrogenase (Schmitt and Kugler, 1999) and/or glutamine synthetase (Tansey et al., 1991). In previous studies it has been shown that cultured oligodendrocytes possess not only transport mechanisms for glutamate but also rapidly metabolize it and release the metabolites (Reynolds and Herschkowitz, 1986). In addition to the occurrence of EAAC1 in oligodendrocytes, we observed ISH signal and immunolabeling in ependymal cells and epithelial cells of plexus choroideus in the ventricular system and satellite cells of the spinal ganglia. In ependymal cells we had previously detected a further glutamate transporter, GLAST (Schmitt et al., 1997). This finding has been corroborated by Berger and Hediger (1998) using non-radioactive ISH. It can be supposed that the two glutamate transporters in ependymal cells prevent the diffusion of synaptically-released glutamate from the intercellular space into the cerebro-spinal fluid. In a previous study it has been shown that circulating glutamate is accumulated in the plexus choroideus and does not enter the cerebro-spinal fluid (Hawkins et al., 1995), although the choroidal epithelium has been classified as ‘‘leaky’’ (Rapoport, 1976). It can be supposed that EAAC1 in epithelial cells of the plexus choroideus could prevent the passage of glutamate from the blood stream into the cerebro-spinal fluid, where the glutamate concentration seems to be very low. In ependymal cells, as well as in epithelial cells of the plexus choroideus, glutamate may be further metabolized via glutamate dehydrogenase, which we have detected by ISH and immunostaining in both localizations (Schmitt and Kugler, 1999). Concerning the subcellular localization of EAAC1 protein, cytoplasmic staining of neurons and glial cells predominated. A delineation of labeled cell membranes was almost impossible at the light microscopic level. Cytoplasmic labeling of neurons has also been described in previous studies (Rothstein et al., 1994; Shashidharan et al., 1997), but detailed electron microscopic studies are not available. Frequently, we have observed in thin plastic sections a finely granular cytoplasmic staining of neurons and glial cells. Together with the immunoblot finding that EAAC1 is only detectable in the membrane fraction (100,000 ⫻ g pellet) of brain homogenates, this indicates a vesicular
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localization of EAAC1 protein. A similar vesicular distribution is also found for other transporter molecules, e.g., the dopamine transporter (Nirenberg et al., 1996), the Na⫹-dependent glucose transporter (Dele´zay et 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 translocation from vesicles into the cell membrane is induced by the action of protein kinase C (Dele´zay et al., 1995; Qian et al., 1997; Quick et al., 1997). Therefore, it is likely that the EAAC1 glutamate transport rate is modulated not only by factors regulating the amount of protein expressed (Gegelashvili and Schousboe, 1997), but also by recruiting the transporter from cytoplasmic vesicles to the cytoplasmic membrane. We have clearly demonstrated that EAAC1, the major neuronal glutamate transporter, is also expressed in various glial cells. EAAC1 in neurons seems not to be intimately related to glutamatergic transmission because EAAC1-deficient mice develop only significantly reduced spontaneous locomotor activity but no neurodegeneration (Peghini et al., 1997). Thus, the functional importance of EAAC1 is not quite clear. Further studies on EAAC1 should also focus on oligodendrocytes to elucidate a possible involvement of EAAC1 in oligodendrocytes in white matter diseases.
ACKNOWLEDGMENTS We thank Erna Kleinschroth and Heike Fella for their excellent technical assistance.
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