Changes in expression and distribution of the glutamate transporter EAAT4 in developing mouse Purkinje cells

Neuroscience Research 27 (1997) 191–198

Changes in expression and distribution of the glutamate transporterEAAT4 in developing mouse Purkinje cells

Keiko Yamada a, Shima Wada b, Masahiko Watanabe a,*, Kohichi Tanaka c, Keiji Wada c,Yoshiro Inoue a

a Department of Anatomy, Hokkaido Uni6ersity School of Medicine, Sapporo 060, Japanb Department of Pediatric Dentistry, Hokkaido Uni6ersity School of Dentistry, Sapporo 060, Japan

c Department of Degenerati6e Neurological Diseases, National Institute of Neuroscience, NCNP, Kodaira 187, Japan

Received 30 October 1996; accepted 9 December 1996

Abstract

EAAT4 is a Purkinje cell (PC)-specific, postsynaptic glutamate transporter in the adult mouse brain. Here, we performeddevelopmental analyses to reveal its temporal expression in relation to PC differentiation. Using in situ hybridization, EAAT4mRNA was specifically detected in the PC layer of the cerebellar primordium at embryonic day 13 (E13). During late fetal andneonatal periods, the transcripts were detected only in the PC layer in the caudal cerebellum. At postnatal day 7 (P7) and,thereafter, the prominent transcripts were found on monolayered PCs in the entire cerebellum. Using immunohistochemistry, lowlevels of EAAT4 immunoreactivity were first observed at E18 in the perikarya of PCs in the caudal cerebellum, and this patternof immunostaining was maintained at P1 and P7. At P14 and, thereafter, the molecular layer in the entire cerebellum becameimmunopositive for EAAT4, and the intense immunoreactivity was detected preferentially in PC spines synapsing with parallelfiber terminals. Therefore, the present study has clarified that the transcription of EAAT4 begins in PCs from early embryonicstages, and that the synaptic localization of EAAT4 is established during the second postnatal week. When considered in the lightof the synaptogenesis of parallel fiber-PC synapses which actively occurs in the rodent cerebellum during the second and thirdweeks of life, synaptic localization of the glutamate transporter EAAT4 may be closely associated with the synapse formation. ©1997 Elsevier Science Ireland Ltd.

Keywords: Glutamate transporter; EAAT4; Mouse; Brain; Purkinje cells; Immunohistochemistry; In situ hybridization; Develop-ment

1. Introduction

Glutamate is a major neurotransmitter involved infast excitatory synaptic transmission in the mammaliancentral nervous system (Mayer and Westbrook, 1987),and is essential for synaptic plasticity, which is thoughtto underlie development, learning, and memory (Ito,1989; McDonald and Johnston, 1990; Bliss andCollingridge, 1993). Glutamate released from presynap-tic terminals has to be removed rapidly from the synap-

tic cleft by high affinity, sodium-dependent glutamatetransporters to keep the extracellular glutamate concen-tration sufficiently low to terminate receptor activationand to protect neurons from glutamate excitotoxicity(Hertz, 1979; Choi, 1992). Recent studies have iden-tified four subtypes of the glutamate transporter withdistinct structures, functions, and expression patterns:GLAST (GluT-1, EAAT1), GLT1 (EAAT2), EAAC1(EAAT3), and EAAT4 (Storck et al., 1992; Kanai andHediger, 1992; Kanai et al., 1993; Kanner, 1993;Tanaka, 1993; Torp et al., 1992; Rothstein et al., 1994;Fairman et al., 1995; Kanai et al., 1995; Lehre et al.,1995; Shibata et al., 1996; Yamada et al., 1996).

* Corresponding author. Fax.: +81 11 706 7863; e-mail: [email protected]

0168-0102/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.

PII S 0 1 68 -0102 ( 96 )01148 -0

K. Yamada et al. / Neuroscience Research 27 (1997) 191–198192

Purkinje cells (PCs) in the cerebellum receive massiveglutamatergic inputs from parallel fibers, the bifurcatedaxons of granule cells (Palay and Chan-Palay, 1974;Ito, 1989). The parallel fiber-PC synapse is estimated tobe 105 per PC (Napper and Harvey, 1988), and formedon PC spines of distal dendrites. In addition, climbingfibers from the inferior olive establish strong excitatorysynapses along the proximal dendrites, which triggerCa2+ entry through voltage-gated Ca2+ channels(Sakurai, 1990; Hirano, 1990; Crepel and Jaillard, 1991;Konnerth et al., 1992). Immunohistochemical studieshave revealed that several subtypes of the glutamatetransporter are localized at discrete elements of PCsynapses. GLAST and GLT1 are localized densely onthe cell membrane of the Bergmann astroglia, whichseals the PC synapses (Rothstein et al., 1994; Lehre etal., 1995). EAAT4, on the other hand, is localizedselectively in dendritic spines of PCs (Yamada et al.,1996). In view of the importance of rapid glutamateclearance in regulating synaptic transmission and inprotecting neurons from glutamate excitotoxicity, theformation of glutamatergic synapses should be accom-panied by the establishment of the glutamate trans-porter system. In fact, recent expression studies haveshown that transcription of GLAST and GLT1 under-goes marked up-regulation during early postnatal peri-ods (Shibata et al., 1996; Sutherland et al., 1996). Toclarify developmental regulation of the gene expressionfor EAAT4 in relation to the differentiation and synap-togenesis of the PCs, we performed analyses in develop-ing mouse cerebella using in situ hybridization andimmunohistochemistry.

2. Materials and methods

2.1. In situ hybridization

Two non-overlapping antisense oligonucleotides wereused for detection of the mouse EAAT4 mRNA in thepresent study. The sequences of the oligonucleotides are5%- GCCCCCAGCTCTGAACCATTGTCTGTCCT-TACAATTGTCCTTGTCA-3% and 5%-GATGCCCC-CTTTTCTTGTGCCATGAGTGACTTATAGGGTTTCCCCA-3%, which are complementary to nucleotideresidues 611–656 (probe 1) and 1607–1652 (probe 2),respectively, of the mouse EAAT4 cDNA (Maeno-Hi-kichi et al., unpublished data). These oligonucleotideswere labeled with [33P]dATP to a specific activity of1×109 dpm/mg DNA, using terminal deoxyribonucle-otidyl transferase (BRL).

Brains of C57BL/6J mice were used at embryonicdays 13 (E13), E15, E18 and postnatal days 1 (P1), P7,P14, P21, and P120. The day following overnight mat-ing was counted as E0. Under deep pentobarbital anes-thesia, brains were immediately removed from the skull

and frozen in powdered dry ice to obtain cryostat freshfrozen sections (20 mm in thickness). For paraffin sec-tions (5 mm), embryonic brains were immersedovernight in Bouin fixative, while postnatal brains werefixed transcardially with 4% paraformaldehyde–0.1 Msodium phosphate buffer (pH 7). Sections weremounted on glass slides precoated with 3-aminopropyl-triethoxisilane (Sigma, St. Louis, MO), and processedfor in situ hybridization, as described previously(Watanabe et al., 1993). After overnight hybridizationat 42°C in the presence of 50% formamide, sectionswere washed twice at 55°C for 40 min in 0.1×SSCcontaining 0.1% sarcosyl. Fresh frozen sections wereexposed to Hyperfilm-b max. (Amersham) for 2 weeks.Paraffin sections were dipped in nuclear track emulsion(NTB-2, Kodak) and exposed for 2 months. Followingdevelopment, the paraffin sections were counterstainedwith toluidine blue.

2.2. Immunohistochemistry

Paraffin sections were incubated overnight with rab-bit anti-EAAT4 antibody (Yamada et al., 1996) or withrabbit anti-spot 35/calbindin antiserum (Yamakuni etal., 1984) at a concentration of 0.25 mg/ml or a dilutionof 1:15 000, respectively. The immunoreaction was visu-alized using the ABC kit (Histofine SAB-PO(R) Kit,Nichirei) and diaminobenzidine for light microscopy orusing fluorescine isothiocyanate (FITC)-labeled goatanti-rabbit IgG (1:200, Jackson ImmunoResearch) forconfocal laser scanning microscopy (MRC 1024, Bio-Rad).

For immunoelectron microscopy, microslicer sections(50 mm in thickness) immunostained using the ABCmethod were further treated with 0.5% osmium tetrox-ide for 15 min, stained with 1% uranyl acetate aqueoussolution for 20 min, and embedded in Epon 812. Ultra-thin sections cut with a Reichert ultramicrotome werephotographed using a Hitachi H-7100 electron micro-scope.

3. Results

3.1. In situ hybridization

Using in situ hybridization with 33P-radiolabeledoligonucleotide probes, expression of EAAT4 mRNAwas examined in developing mouse brains from E13 toP120 (Figs. 1 and 2). EAAT4 mRNA was expressed inthe cerebellum at E13, when the transcripts were ob-served as a thin layer running parallel to the roof of thefourth ventricle (Fig. 1A). At E15, EAAT4 mRNA wasconcentrated in the mantle zone of the dorsal cerebel-lum (Fig. 1B and Fig. 2A). At this stage, cells express-ing EAAT4 mRNA were virtually absent in the

K. Yamada et al. / Neuroscience Research 27 (1997) 191–198 193

Fig. 1. X-ray film macroautoradiography showing developmental changes in EAAT4 mRNA expression in the brain. Parasagittal fresh frozensections through mouse brains at E13 (A), E15 (B), P1 (C), P7 (D), P14 (E), P21 (F), and P120 (G). Arrows indicate the cerebellum. Allphotographs were taken directly from a single X-ray film at the same magnification. Rostral is to the right and dorsal is to the top. CC, cerebralcortex; CP, caudate-putamen; Di, diencephalon; Hi, hippocampus; Mb, midbrain; Me, medulla oblongata; Po, pons. Scale bar, 1 mm.

proliferative zone of the cerebellum, including the ven-tricular zone, external granular layer, and germinaltrigone (Fig. 2A). At birth when the cerebellar foliabecame distinct, EAAT4 mRNA was restricted to theperikarya of PCs in the caudal cerebellum (Fig. 1C andFig. 2B). At P7 and, thereafter, EAAT4 mRNA wasdetected in the perikarya of monolayered PCs in theentire cerebellum (Figs. 1D–G, 2C, 2D). No significantsignals were detected in other cerebellar cells (granulecells, Bergmann astrocytes, and cerebellar nucleus neu-rons) nor in other brain regions at any developmentalstage examined. Only after longer exposure, faint sig-nals, the intensity of which was slightly above thebackground level were detectable in the gray matter ofvarious brain regions (data not shown).

Specificity of the hybridization was confirmed byobtaining identical expression patterns using anothernon-overlapping oligonucleotide probe and by the dis-appearance of the characteristic signals when hybridiza-tion was carried out in the presence of a 20-fold excessamount of unlabeled oligonucleotide (data not shown).

3.2. Immunohistochemistry

Intracerebellar distribution of EAAT4 was examinedin developing cerebella, using immunohistochemistrywith affinity-purified anti-EAAT4 antibody, the specifi-

city of which has been reported previously (Yamada etal., 1996). No significant degree of staining was de-tected in the cerebellum at E13 and E15 (data notshown). Weak immunoreactivity first appeared in thecerebellum at E18 (Fig. 3A). When compared with theadjacent sections stained with antibody against spot35/calbindin (Fig. 3B) (a cytosolic protein localized indendrites, perikarya, and axons of PCs (Yamakuni etal., 1984)) and with toluidine blue (Fig. 3C), the distri-bution of EAAT4 immunoreactivity was found to beconfined to the PC layer in the caudal cerebellum. Thispattern of immunostaining was maintained until P7,when PCs in the caudal region of lobule VIII and inlobules IX and X were selectively immunostained forEAAT4 (Fig. 3D–F). At P14 and P21, the molecularlayer in the entire cerebellum was strongly labeled forEAAT4 (data not shown), as previously shown at P120(Yamada et al., 1996).

Postnatal changes in the intracellular distribution ofEAAT4 were examined by confocal laser scanning mi-croscopy (Fig. 4A–D) and by electron microscopy (Fig.4E,F). At birth, faint immunoreactivity of EAAT4 wasdiscerned in the perikarya of PCs (Fig. 4A). At P7,EAAT4 immunoreactivity was detected in the apicalperikarya of PCs, and in a few dendritic structures inthe thin molecular layer (Fig. 4B). At P14, the level ofimmunoreactivity in the molecular layer was markedly


Fig. 2. Bright-field micrographs showing the distribution of EAAT4 mRNA in the developing cerebellum. A, E15; B, P1; C, P14; D, P21. At E15,the EAAT4 mRNA-positive cells (arrows) are distributed in the mantle zone of the cerebellum, but not in the proliferative zone, including theventricular zone (asterisks) surrounding the fourth ventricle (4V) and the external granular layer (arrowheads). Rostral is to the right, and dorsalis to the top. At P1, signals were distributed in a thin zone between the external and internal granular layers. At P14 and P21, EAAT4 mRNAis clearly detected in the perikarya of the Purkinje cells. All these photographs are from paraffin sections counterstained with toluidine blue. Cb,cerebellum; EG, external granular layer; Gr, granular layer; GT, germinal trigone; IG, internal granular layer; ME, medulla oblongata; Mo,molecular layer. Scale bars, A, 0.1 mm; B–D, 10 mm.

increased than at previous stages, and observed asnumerous punctate stainings (Fig. 4C). The intensity ofthese punctate stainings was higher in the basal andmiddle regions of the molecular layer than in the su-perficial region. At this stage, the intensity in theperikarya of PCs was lowered, compared with P7. AtP21, intense punctate stainings were present in themolecular layer at high density (Fig. 4D). Immunoelec-tron microscopic analysis showed that the perikaryaand dendritic shafts of PCs were immunostained at P7(Fig. 4E), while the immunoreactivity at P14 was moreconcentrated in the dendritic spines synapsing with

parallel fiber terminals than in the dendritic shafts (Fig.4F).

4. Discussion

EAAT4 is a member of the family of glutamatetransporters, which play an important role in rapidclearing of extracellular glutamate (Kanai and Hediger,1992; Pines et al., 1992; Storck et al., 1992; Tanaka,1993; Fairman et al., 1995). From the selective expres-sion and localization, we have previously concluded


Fig. 3. Selective distribution of EAAT4 immunoreactivity in the caudal cerebellum at E18 (A–C) and P7 (D–F). Adjacent parasagittal paraffinsections were processed for staining with anti-EAAT4 antibody (A and D), anti-spot 35/calbindin antibody (B and E), and toluidine blue (C andF). Note a wider distribution of spot 35/calbindin compared with the EAAT4 immunoreactivity. Arrow heads indicate the Purkinje cell layer.Rostral is to the right and dorsal is to the top. CN, cerebellar nuclei; EG, external granular layer; IG internal granular layer; VIII, IX and X,lobules VIII, IX and X. Scale bar, F, 1 mm.

that EAAT4 is a PC-specific, postsynaptic transporterin the adult mouse brain (Yamada et al., 1996). In thepresent study, we performed developmental analyses,and elucidated characteristic temporal changes in theexpression and distribution of EAAT4.

4.1. Purkinje cell-specific transcription begins in theearly stages of de6elopment

In mice, precursors of PCs undergo their final mitoticdivisions between E11 and E13, (Miale and Sidman,1961). With the cessation of PC proliferation, the firstphase in the cerebellar development comes to an end(Altman, 1982). In the second phase, PCs migrate to-ward the pial surface to form the PC layer beneath theexternal granular layer. The mouse cerebella at E13 andE15 thus correspond to that in the end of the first phaseor in the beginning of the second phase, respectively. Atthese early stages, we detected almost selective expres-sion of EAAT4 mRNA, in particular, cerebellar re-gions, known as the PC layer (Altman, 1982). At P7and, thereafter, the transcripts were detected clearly inthe perikarya of monolayered PCs. These findings sug-

gest that the gene encoding the glutamate transporterEAAT4 undergoes PC-specific transcriptional controlfrom the early stages of brain development.

4.2. Synaptic localization is established concomitantwith PC synapse formation

In comparison with the early detection of EAAT4mRNA by in situ hybridization, the first immunohisto-chemical detection of the protein products was ratherlate at E18. From E18 to P7, the level of EAAT4immunoreactivity was consistently low, and localizedmainly in the perikarya of PCs located in the caudalcerebellum. In contrast, the molecular layer of thewhole cerebellum exhibited intense EAAT4 immunore-activity at P14 and, thereafter. Moreover, its preferen-tial localization in PC spines was clearly observed atP14, as seen in adult stages (Yamada et al., 1996).These results indicate that both translational regulationand synaptic localization of EAAT4 mature during thesecond postnatal week in the mouse cerebellum.

Parallel fibers (PFs), the granule cell axons, formglutamatergic synapses with the spines of PC dendrites


Fig. 4. Postnatal changes in intracellular localization of EAAT4 immunoreactivity in mouse cerebella at P1 (A), P7 (B, E), P14 (C, F) and P21(D). A–D, confocal laser scanning microscopy; E and F, immunoelectron microscopy. Note that EAAT4 immunoreactivity is distributed diffuselyin the perikarya and the dendritic processes (arrows) of Purkinje cells (PC) at P7. At P14, the strong immunoreactivity is concentrated in PC spines(arrowheads) in contact with parallel fiber terminals. Scale bars, D, 10 mm; F, 1 mm.

(Ito, 1989). This synapse accounts for more than 95%of the PC synapses (Sotelo, 1978), and is formed post-natally (Altman, 1972). During the first week of life, therate of PF–PC synapse formation is very slow, and thesynapse retains immature structural features, such as afew synaptic vesicles, small synaptic contacts, and ab-sence of astroglial enclosure (Larramendi, 1969). Anenormous number of PF–PC synapses having devel-oped structure are formed during the second and thirdpostnatal weeks (Altman, 1972; Larramendi, 1969;Robain et al., 1981). The d2 subunit of the glutamatereceptor channel (GluRd2) is another PC-specificmolecule (Araki et al., 1993; Lomeli et al., 1993),playing roles in the PC synapse formation, long-termdepression, and motor coordination (Kashiwabuchi etal., 1995). Synaptic localization of GluRd2 is alsoestablished during the second postnatal week(Takayama et al., 1996). These findings suggest that theestablishment of synaptic localization at such postnatalstages correlates with the formation and structural mat-uration of PF–PC synapses, which would play animportant role in regulating glutamatergic synaptictransmission.

Dendritic shafts of cerebellar interneurons also re-ceive excitatory inputs from the PFs. Despite the samesource of inputs, the glutamate receptor-mediated PF

excitatory postsynaptic current (EPSC) decays muchmore slowly in PF–PC synapses than in PF-interneu-ron synapses (Konnerth et al., 1990; Perkel et al., 1990).The use of glutamate uptake blocker further slows theEPSC decay in PF–PC synapses, whereas it only causesminor effects on PF-interneuron synapses (Barbour etal., 1994). These findings suggest a prolonged presenceof glutamate in the PF–PC synapse, presumably due toslower glutamate removal by diffusion from axo-spinous synapses than from axo-dendritic or axo-so-matic synapses (Barbour et al., 1994). Therefore, theprolonged glutamate presence as well as the massiveglutamatergic innervation will increase demands forglutamate uptake machinery in excitatory PC synapses.The GLAST is a glutamate transporter subtype and ishighly concentrated in the Bergmann glia, cerebellarastroglia associated with PCs in adult stages (Storck etal., 1992; Torp et al., 1992; Rothstein et al., 1994). Animmunoelectron microscopic study by Chaudhry et al.(1995) has shown that the PF–PC synapse is sur-rounded by astroglial membrane with higher density ofGLAST immunoreactivity than the PF-interneuronsynapse. From all these results, it is reasonable toassume that postsynaptic EAAT4 and astroglialGLAST play a major role in terminating glutamatergictransmission at PF–PC synapses. In future studies, it


will be of great interest to clarify whether these trans-porter molecules are involved in the synapse develop-ment and synaptic plasticity, in which glutamatergictransmission through the PF–PC synapse plays anessential role (Kano et al., 1995; Kashiwabuchi et al.,1995).

4.3. Different histochemical detection between EAAT4transcripts and proteins

A time-lag for several days was noticed in the presenthistochemical detection between EAAT4 mRNA andprotein. On the other hand, GluRd2 mRNA andprotein are both detected in the mouse cerebellum asearly as E15, and they exhibit similar spatial expressionand distribution at a given developmental stage(Takayama et al., 1996). This implies that translation ofEAAT4 mRNA in developing PCs is not so efficient asin mature cells, or is regulated separately from thetranscription during PC development. However, differ-ent detection sensitivities between the in situ hybridiza-tion and immunohistochemistry should also be takeninto consideration.

4.4. Transient rostro-caudal and medio-lateralheterogeneities in the cerebellum

From late embryonic to neonatal stages, PCs in thecaudal cerebellum preferentially express EAAT4mRNA and protein. Rostro-caudal differences in thecerebellum are also known for the proliferation, migra-tion, and gene expression of PCs and granule cells(Altman and Bayer, 1985; Yuasa et al., 1991; Watanabeet al., 1994; Takayama et al., 1996). When examiningmedio-lateral differences, heterogeneous immunostain-ing is found on horizontal cerebellar sections at P7(unpublished data). Regarding the medio-lateral hetero-geneity, various biochemical markers for PCs display acompartmentalized histochemical staining within thecerebellum (Hawkes and Gravel, 1991; Sotelo andWassef, 1991; Edwards et al., 1994). These rostro-cau-dal and medio-lateral differences of EAAT4 becomeless distinct at P14 and later stages. Thus, it seemslikely that EAAT4 expression in developing PCs ismore susceptible than in mature PCs to certain factorswhich affect cellular differentiation and compartmental-ization in the cerebellum.

Acknowledgements

We thank Hideo Umeda and Yoshihiko Ogawa fortheir technical assistance. This investigation was sup-ported in part by research grants from the Ministry ofEducation, Science, Sports and Culture, the Ministry ofHealth and Welfare and the Science and TechnologyAgency of Japan.

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Changes in expression and distribution of the glutamate transporter EAAT4 in developing mouse Purkinje cells