Cloning, Transport Properties, andDifferential Localization of Two

Splice Variants of GLT-1 in the RatCNS: Implications for CNS

Glutamate HomeostasisROBERT SULLIVAN,1 THOMAS RAUEN,2 FRAUKE FISCHER,3

MICHAEL WIEßNER,4 CHRISTOF GREWER,5 ANA BICHO,5 AND DAVID V. POW1*1School of Biomedical Sciences, Department of Physiology and Pharmacology, University of

Queensland, Brisbane, Australia2Westfalische-Wilhelms-Universitat Munster, Institut fur Biochemie, Munster, Germany

3Novartis Pharma AG, Basel, Switzerland4ETH Zurich, Zurich, Switzerland

5Max-Planck-Institut fur Biophysik, Frankfurt, Germany

KEY WORDS glutamate; transporter; GLT-1; EAAT; splice variant; astrocyte; ret-ina; photoreceptor; bipolar cell; synapse

ABSTRACT At least two splice variants of GLT-1 are expressed by rat brain astro-cytes, albeit in different membrane domains. There is at present only limited dataavailable as to the spatial relationship of such variants relative to the location ofsynapses and their functional properties. We have characterized the transport proper-ties of GLT-1v in a heterologous expression system and conclude that its transportproperties are similar to those of the originally described form of GLT-1, namely GLT-1�.We demonstrate that GLT-1� is localized to glial processes, some of which are interposedbetween multiple synapse types, including GABAergic synapses, whereas GLT-1v isexpressed by astrocytic processes, at sites not interposed between synapses. Both splicevariants can be expressed by a single astrocyte, but such expression is not uniform overthe surface of the astrocytes. Neither splice variant of GLT-1 is evident in brain neurons,but both are abundantly expressed in some retinal neurons. We conclude that GLT-1vmay not be involved in shaping the kinetics of synaptic signaling in the brain, but maybe critical in preventing spillover of glutamate between adjacent synapses, therebyregulating intersynaptic glutamatergic and GABAergic transmission. Furthermore,GLT-1v may be crucial in ensuring that low levels of glutamate are maintained atextrasynaptic locations, especially in pathological conditions such as ischemia, motorneurone disease, and epilepsy. © 2003 Wiley-Liss, Inc.

INTRODUCTION

Glutamate is the major excitatory neurotransmitterin the mammalian central nervous system (Fonnum,1984; Danbolt, 2001). Glutamate transport is catalyzedby high-affinity plasma membrane transporters (Kan-ner and Sharon, 1978; Wadiche et al., 1995; Zerangueand Kavanaugh, 1996). Such transport is electrogenic,is driven by the Na�/K� ion gradient, and can be stud-ied electrophysiologically (Neher and Sakmann, 1976;Hamill et al., 1981). Glutamate transporters may alsoexhibit a substrate-induced chloride conductance not

stoichiometrically linked to glutamate transport(Wadiche et al., 1995; Otis and Jahr, 1998).

Christof Grewer and Ana Bicho’s current address is Department of Physiologyand Biophysics, University of Miami School of Medicine, Miami, FL.

*Correspondence to: Dr. David V. Pow, School of Biomedical Sciences, Depart-ment of Physiology and Pharmacology, University of Queensland, Brisbane4072, Australia. E-mail: d.pow@uq.edu.au

Received 17 April 2003; Accepted 4 August 2003

DOI 10.1002/glia.10317

GLIA 45:155–169 (2004)

© 2003 Wiley-Liss, Inc.

Within many brain regions, the dominant glutamatetransporter is GLT-1. Early immunocytochemical stud-ies (Levy et al., 1993; Rothstein et al., 1994; Lehre etal., 1995) demonstrated that GLT-1 was restricted toglial cells and normally absent from neurons, the fewexceptions being in retina, cultured neurones, and de-veloping brain (Rauen and Kanner, 1994; Euler andWassle, 1995; Yamada et al., 1998; Rauen, 2000; Reyeet al., 2002a, 2002b).

At least two splice variants of GLT-1 are expressedin the rodent brain. The original splicing was describedby Pines et al. (1992); we refer to this as GLT-1�, toavoid confusion between this and unspecified splicevariants of GLT-1. GLT-1B, described in mouse byUtsonomiya–Tate et al. (1997), is alternatively splicedat the C-terminal region and contains a short alterna-tively spliced region at its N-terminus. A hybrid of thissplicing has been described in rat by Schmitt et al.(2002) as GLT-1 variant (GLT-1v) and Chen et al.(2002) as GLT-1B. This variant retains the originalN-terminal splicing present in GLT-1�, but has a C-terminal identical to GLT-1B, suggesting that C- andN-terminal splicings are independently controlled. Asthis variant form is not the same as the GLT-1B de-scribed previously by Utsonomiya-Tate et al. (1997), wehave used the term GLT-1v in subsequent references tothis protein. Since alternate splicing might confer dif-ferent functional properties, we characterized thetransport properties and ionic fluxes associated withGLT-1v.

GLT-1 is thought to be predominantly associatedwith those parts of the glial membranes closely ap-posed to synapses (Danbolt, 2001). In view of the exis-tence of multiple splice variants, we reevaluated thisfinding using bright-field and confocal light microscopyto define the localization of GLT-1 splice variants rel-ative to synapses. We have examined specific anatomiczones (around cell bodies and dendrites of neurons)where pre- and postsynaptic elements and perisynapticglial elements are easily identified.

Cell types of different brain regions were selectedwith reference to their neurochemical phenotype andtypes of synapses they receive (inhibitory and/or exci-tatory). The aim was to ascertain if common patterns ofdistribution of GLT-1 splice variants were demonstra-ble or if distributions were dependent on the phenotypeof surrounding neuronal elements.

MATERIALS AND METHODS

All experiments on animals were carried out in ac-cordance with the National Health and Medical Re-search Council (Australia) guidelines, which arebroadly aligned with the National Institutes of Healthguidelines, as well as with ethical permission from theGroup 1 Animal Ethics Committee, University ofQueensland.

cDNA Cloning, Sequence Analysis,and Plasmid Construction

The coding sequence of the GLT-1� and GLT-1v glu-tamate transporter was isolated from complementaryDNA (cDNA) derived from adult rat retina of bothsexes by the polymerase chain reaction (PCR). Briefly,total RNA was isolated from rat retinae by the methodof Chomczynski and Sacchi (1987). Oligo(dT)-primedfirst-strand cDNA synthesis was performed with Su-perscript II reverse transcriptase (Gibco-BRL, Eggen-stein, Germany). cDNA encoding the GLT-1� orGLT-1v gene product was amplified from retinal first-strand cDNA by PCR using the following protocol: 35cycles of denaturation (30 s, 94°C), annealing (90 s,60°C), and extension (3 min, 68°C) were performed in50 �l reactions that contained oligonucleotide primersat 1 �M and each retinal cDNA template equivalent to250 ng RNA using the Expand Long Template PCRSystem according to the manufacturer’s instructions(Roche Applied Science, Mannheim, Germany). Thesense primer sequence for GLT-1� and GLT-1v was5�-CACGCCATGGCATCAACCGAGGGTGCCAACAA-3�, corresponding to the nucleotide sequences 93–124(Pines et al., 1992). The antisense primer sequence forGLT-1� was 5�-CCCGATTCTCAGCCAATTCTCTC-GAGCGG-3�, corresponding to nucleotide sequence1824–1843 (Pines et al., 1992). For GLT-1v, the se-quence was 5�-CTGGATGCAGAGTGAGATGG-3�, cor-responding to the nucleotide sequences at positions1819–1838 (Utsunomiya-Tate et al., 1997).

The amplification products of GLT-1� and GLT-1vwere agarose gel-purified, ligated into an EcoRV-di-gested pBluescript KS-plasmid vector (Stratagene, LaJolla, CA), and transformed in E. coli XL1-Blue (Strat-agene). Inserts from positive recombinants were se-quenced on both strands (Sanger et al., 1977) and sub-cloned into the modified expression vector pBK-CMV(�[1098–1300]; Stratagene) at the BamHI and XhoIrestriction sites. The expression plasmids were termedpCMV-GLT-1� and pCMV-GLT-1v and were used forfunctional analysis in a mammalian expression sys-tem.

Transient Expression of Transporters andElectrophysiological Recording

Subconfluent human embryonic kidney cell(HEK293; ATCC number CRL 1573) cultures weretransiently transfected either with pCMV-GLT-1�(termed HEKGLT-1�), pCMV-GLT-1v (termed HEKGLT-1v), or with pBK-CMV (�[1098–1300]; control plasmid)by calcium phosphate-mediated transfection as de-scribed previously (Chen and Okayama, 1987; Greweret al., 2000). At 2 days posttransfection, membranevesicles were prepared from the two sets of transfectedcells for immunoblot analysis as described previously(Rauen et al., 1998; Grewer et al., 2000).

156 SULLIVAN ET AL.

Electrophysiological recordings were performed24–72 h after transfection with an Adams and ListEPC7 amplifier under voltage clamp conditions in thewhole-cell current recording configuration (Hamill etal., 1981). Data were recorded with a digitizer board,which was controlled by the pClamp6 software (AxonInstruments, Foster City, CA), digitized with a sam-pling rate of 1 kHz (solution exchange) or 25 kHz (la-ser-pulse photolysis), and low pass-filtered at 250 Hz or3–10 kHz, respectively. Micropipettes (pulled to 2–4 MOhm resistance) were filled with (in mM) 130 KSCN orKCl, 10 TEA-Cl, 1 MgCl2, 10 EGTA, 10 HEPES (pH7.4/KOH). Typical bath solution contained (in mM) 140NaCl or NaSCN, 2 MgCl2, 2 CaCl2, 30 HEPES (pH7.4/NaOH). All experiments were performed at roomtemperature.

Solution Exchange and Laser-Pulse Photolysis

Laser-pulse photolysis experiments were performedas described previously (Niu et al., 1996; Grewer et al.,2000). Briefly, carboxy-nitrobenzyl (�CNB)-caged glu-tamate (Molecular Probes, Eugene, OR) was applied tothe cells using a fast solution exchange device andphotolysis of caged glutamate was initiated with a lightflash (340 nm, 10-ns pulse duration, excimer laserpumped dye laser; Lambda Physik, Gottingen, Ger-many). The light was coupled into a quartz fiber (365�m in diameter) positioned at 500 �m distance fromthe cell. The laser energy (� 400 mJ/cm2) was adjustedwith neutral density filters (Andover). The releasedglutamate concentration was estimated by comparisonof the steady-state current with that generated byrapid perfusion of the same cell with 100 �M L-gluta-mate.

Data Evaluation

Steady-state currents (Iss) as a function of glutamateconcentration were fitted with a Michaelis-Menten-likeexpression. The pre-steady-state currents were fittedwith a sum of three exponential functions and a steady-state current component: I � I1 � exp(�t/_rise) � I2 �exp(�t/_decay1) � I3 � exp(�t/_decay2) � Iss. The programused for data analysis and fitting was Origin (Microcal).The fitting algorithm is based on the Levenberg-Mar-quardt algorithm.

Animals and Tissue Preparation forImmunocytochemistry

Adult rats (n 6) were euthanased by an overdose ofsodium pentobarbital (100 mg/kg, administered i.p.).Animals were fixed initially by perfusion via the heartwith 4% paraformaldehyde in 0.1 M sodium phosphatebuffer, pH 7.2, and then removed and fixed by immer-sion for a further 1 h in the same fixative.

Coronal sections of brains and spinal cords (40 �mthick) were cut using a Vibratome. Immunocytochem-istry was performed on free-floating sections from ce-rebral cortex, hippocampus, cerebellum, and spinalcord using standard immunolabeling techniques, withfluorescently labeled secondary antibodies, using alka-line phosphatase-tagged secondary antibodies or bio-tinylated antibodies, as detailed below. Isolated ratretinae, HEKGLT-1� or HEKGLT-1v cell cultures wereimmersion-fixed in 4% paraformaldehyde in 0.1 Mphosphate buffer and 150 mM NaCl, pH 7.4 (PBS), for10, 30, or 60 min at room temperature, respectively.After several washes in PBS, retinae were cryopro-tected in 30% sucrose in PBS, cut into 15 �m thickvertical sections on a cryostat, collected onto slidescoated with poly-D-lysine (1 mg/ml), air-dried, andstored at �20°C.

Antibodies and Immunocytochemistry

We have previously generated and characterizedrabbit antisera against the carboxyl terminal regions ofGLT-1� and GLT-1B (Reye at al., 2002a, 2002b, 2002c).The synthetic peptide KSADCSVEEEPWKREK corre-sponds to the carboxyl terminus of GLT-1� from rat[Genbank accession number P13596, published byPines et al. (1992)] and the synthetic peptide PFP-FLDIETCI corresponds to the carboxyl terminus ofboth GLT-1B and GLT-1v in mouse [Genbank acces-sion number NP035523, published by Utsunomiya-Tate et al. (1997)] and the same carboxyl terminalregion of the rat (Chen et al., 2002; Schmitt et al.,2002). Current antibodies do not distinguish betweenGLT-1B and GLT-1v due to the common identity of theC-terminal region. Thus, future discussions regardingimmunocytochemical localizations refer, for the sake ofbrevity, to GLT-1v alone when commenting on distri-bution of these proteins.

Antibodies, which we have previously raised againstthe sequence KQVEVRMHDSHLSSE of GLT-1� (resi-dues 12–26) and KSELDTIDSQHR (residues 497–508),were also used. These sequences are common to allknown splice variants of rat GLT-1; to simplify discus-sions regarding these two antisera, they are referred toas Ab12 and Ab497, respectively. The specificity of eachantiserum has previously been evaluated by Westernblot analysis (Reye et al., 2002a). In this study, therabbit antisera were used in dual immunofluorescencestudies on brain tissues in conjunction with mousemonoclonal antibodies against the synaptic vesiclemarkers SV2 (Developmental Studies HybridomaBank, University of Iowa) or mouse monoclonal anti-body against synaptophysin (Sigma, Castle Hill, Aus-tralia). Alternatively, double labels for transporterswere performed in conjunction with a mouse monoclo-nal antibody to glutamic acid decarboxylase (GAD; De-velopmental Studies Hybridoma Bank, University ofIowa).

157TWO SPLICE VARIANTS OF GLT-1

The double-immunofluorescence labeling studieswere performed using each of the rabbit GLT-1 anti-bodies (at a dilution of 1:1,000) in combination with amouse monoclonal anti-SV2 or synaptophysin antibody(at dilutions of 1:50 and 1:500, respectively) or withmonoclonal GAD65 (1:20). Triple labeling was also per-formed using rabbit antisera to GLT-1� and a rat an-tiserum to GLT-1v in conjunction with a mouse mono-clonal antibody to GFAP (Sigma) or SV2.

Specific labeling was revealed using affinity-purifiedsecondary antibodies against rabbit or mouse immuno-globulins, coupled to Cy5, Texas Red, or FITC. Sectionswere viewed using a Biorad MRC 1024 confocal micro-scope. In addition, double-label studies for GFAP andeither of the GLT-1 splice variants were performedusing alkaline phosphatase-conjugated secondary anti-bodies (Sigma), labeling being revealed using Fast RedAS-MX solution (Sigma F4523) prepared and used ac-cording to the manufacturer’s instructions, and horse-radish peroxidase-labeled secondary antibodies (label-ing revealed using diaminobenzidine).

GLT-1 Immunolabeling

Immunostaining for the various regions of the splicevariants of GLT-1 was performed as follows: retinalsections or HEKGLT-1�/HEKGLT-1v cell cultures werewashed in PBS, preincubated for 1 h at room temper-ature (22°C) with 3% (v/v) normal goat serum (NGS)diluted in 0.1% (v/v) Triton X-100 and Tris-bufferedsaline (TBS; pH 7.2), and subsequently incubated over-night at 4°C with the appropriate primary antibodydiluted in TBS containing 2% (v/v) NGS. All antipep-tide antibodies were affinity-purified and characterizedpreviously as indicated.

Anti-GLT-1(B493) antibodies, directed against the C-terminal region of GLT-1 (493–508: YHL-SKSELDTIDSQHR), used at a concentration of 0.2�g/ml were generously donated by N.C. Danbolt, Oslo,Norway (Lehre et al., 1995). Anti-GLT-1(B12) antibod-ies, directed against the N-terminal region of GLT-1(12–26: KQVEVRMHDSHLSSE), used at a concentra-tion of 0.3 �g/ml were generously donated by N.C.Danbolt (Lehre et al., 1995). Anti-GLT-1(559) antibod-ies, directed against the C-terminal region of GLT-1(559–573: SADCSVEEEPWKREK), used at a concen-tration of 0.1 �g/ml were generously donated by J.D.Rothstein, Baltimore, Maryland (Rothstein et al.,1994).

Following primary antibody incubation, sections orcell cultures were rinsed (30 min) in TBS, incubated (1h) with antirabbit IgG conjugated to Cy3 (Dianova,Hamburg, Germany) diluted 1:1,000 in TBS containing2% (v/v) NGS and 0.1% (v/v) Triton X-100. After thefinal incubation, sections and cultures were rinsed (30min) in TBS and coverslipped in Aqua Poly/Mount(Polyscience, Warrington, PA)

Controls consisted of omission of the primary anti-body or substitution with nonimmune rabbit serum. No

labeling was observed in any of the controls. Retinalsections, GLT-1� and GLT-1v expressing cell cultureswere imaged with a Leica TCS laser scanning confocalmicroscope (Leica, Wetzlae, Germany). Nonconfocalimages were taken using a Zeiss Axioskop, with a Ni-kon Dx1200 digital camera operating at maximum res-olution (12 million pixels).

All digital files were imported into Adobe Photoshop7. Only minor contrast and brightness adjustmentswere made. Composite plate images of the digital fileswere generated using Macromedia Freehand MX andexported as TIFF files to the publisher.

RESULTSMolecular Cloning and Functional Expression

of GLT-1� and GLT-1v in Mammalian Cells

The glutamate transporter isoforms GLT-1� andGLT-1v characterized here were isolated as cDNA fromthe mammalian retina. The predicted amino acid se-quence for GLT-1� encodes for 573 amino acids, has atheoretical molecular mass of 62.1 kDa, and is identicalto the rat brain GLT-1� (Pines et al., 1992). GLT-1vencodes for 562 amino acids, has a theoretical molecu-lar mass of 60.9 kDa, and, up to amino acid position551, is identical to the rat brain GLT-1�. The carboxylterminus of GLT-1� contains a sequence motif of 22amino acid (552–573: TLAANGKSADCSVEEEP-WKREK), while that of GLT-1v is substituted by asequence motif of 11 amino acids (552–562: PFPFLDI-ETCI), possibly due to alternate splicing.

Functional characterization of the GLT-1 isoformswas performed by transient expression in HEK293cells. Prior to transfection, the cells were analyzed forendogenous expression of the five known glutamatetransporters subtypes. Neither biochemical tech-niques, such as PCR, immunocytochemistry, immuno-blot analysis (Fig. 1B, lane 2), nor electrophysiologicalexperiments indicated the expression of endogenousGLT-1 in HEK293 cells.

Immunocytochemistry of pCMV-GLT-1�- or pCMV-GLT-1v-transfected HEK293 cells (HEKGLT-1�, HEK-GLT-1v) with specific antibodies to GLT-1v (Fig. 1A) andGLT-1� (not shown) revealed a membranous localiza-tion of the heterologously expressed transporter, whilenontransfected or vector-alone-transfected HEK293cells showed no staining either by immunocytochemis-try or by immunoblot analysis (Fig. 1B, lane 2). Asshown in Figure 1B (lane 3), immunoblot analysis dem-onstrated heterologous expression of the GLT-1v trans-porter protein with a comparable electrophoretic mo-bility (main band centered at 71 kDa and a oligomerband at 205 kDa) as determined for the native GLT-1�from rat retina and brain (Rauen et al., 1996; Danbolt,2001). Consistent with our results, N-linked glycosyla-tion of the native GLT-1 protein has been shown toincrease its molecular mass by about 10 kDa (Danboltet al., 1992). The functional properties of heterolo-

158 SULLIVAN ET AL.

gously expressed glutamate transporters were charac-terized for a variety of parameters.

Steady-State Currents (Iss)

Glutamate transporter function was studied by us-ing whole-cell current recording from HEK293 cellstransiently transfected with GLT-1� or GLT-1vcDNAs. In both cases, the external application of L-glutamate at 0 mV induced negative currents in thepresence of internal SCN� (Fig. 3A and C), the trans-port being saturable and showing apparent Km valuescalculated for GLT-1� and GLT-1v of 15.7 1.0 �M(n 6) and 12.6 1.0 �M (n 5), respectively (Fig.2A). These results demonstrate that there is no differ-ence of the apparent affinity of the transporters forglutamate within experimental error. The voltage de-pendence of the detected currents was measured as afunction of the applied membrane potential. Figure 2Bshows the coupled transport component of the trans-porter current (in the absence of SCN�) presentinginward rectification and not reversing at the appliedpotentials as expected from zero-trans conditions ap-plied for the substrates and ions and the stoichiometry

(two positive charges for each glutamate molecule en-tering the cell).

To study the uncoupled anion component of the cur-rent, intracellular Cl� was replaced by SCN� (a morepermeable ion). For both transporters, an inward cur-rent was observed at all the membrane potentialstested, in agreement with an infinite electrochemicaldriving force for SCN� (Fig. 2C). Finally, with intra-cellular KCl- and extracellular NaSCN-based buffers,the current-voltage relationships also showed identicalbehavior for GLT-1� and GLT-1v and reverted at �47and �52 mV, respectively (Fig. 2D). At potentials morenegative than �50 mV, the current is predominantlymediated by the coupled transport component, whereasat potentials more positive than �50 mV, it is mainlycarried by anions. Furthermore, we determined theratio between the coupled transport current and theuncoupled anion current. This ratio can be determinedby applying either SCN� or Cl� to the transporter inthe presence of intracellular KCl and by subsequentlymeasuring the respective currents in the same cell. At0 mV transmembrane potential and 100 �M gluta-mate, the ratio between the coupled transport currentand the uncoupled anion current was 0.06 0.02 (n 8) for GLT-1�, and 0.07 0.04 (n 3) for GLT-1v.

Fig. 1. Protein expression of heterologously expressed GLT-1v inHEK293 cells. A: Fluorescence micrograph of HEKGLT-1v cells immu-nolabeled for GLT-1v. GLT-1v immunoreactivity revealed intenselylabeled cell boundaries (arrow) indicating the membranous localiza-tion of the heterologously expressed transporter (bar 10 �m). B:

Immunoblot analysis. Aliquots of SDS extracts (5 �g per lane) ofmembrane protein fractions of vector-alone-transfected (lane 2) andHEKGLT-1v cells (lane 3) were subjected to SDS-polyacrylamide gelelectrophoresis (10%) and were immunoblotted with GLT-1v-specificantibodies. Molecular mass markers (lane 1) are indicated in kDa.

159TWO SPLICE VARIANTS OF GLT-1

Together, these experiments indicate that neither cou-pled transport nor the anion conductance are affectedby the C-terminal truncation of GLT-1v.

Pre-Steady-State Currents

To investigate any differences at the level of ion bind-ing and translocation steps of the glutamate transportcycle, laser-induced photolysis of �CNB-caged L-gluta-mate was used in order to generate neurotransmitterconcentration jumps within 100 �s. L-glutamate-inducedtransient currents obtained after the photolytic release ofa saturating concentration of neurotransmitter (see cali-bration of the steady-state current by using rapid solutionexchange with a known concentration of free glutamate,Fig. 3A and C) could be described with three exponentialfunctions corresponding to three distinct phases of thecurrent: a fast rising phase and two slower decayingphases to the steady-state level (Fig. 3B and D). The ratio

of the preexponential factors for the two decaying phases,I2/I3, is 3.2 0.6 for GLT-1� and 3.1 0.3 for GLT-1v.The corresponding time constants found were 0.58, 6.6,and 24 ms for GLT-1� (n 4; one cell) and 0.39 0.03,7.0 1.1, and 27.9 4.2 ms for GLT-1v (n 4).

Again, in agreement with the results obtained fromthe steady-state currents, no differences were observedbetween the kinetics of GLT-1�- and GLT-1v-mediatedpre-steady-state currents.

GLT-1v in Retinal Tissues

As indicated in the upper part of Figure 4, bothGLT-1� and GLT-1v exhibited an identical amino acidsequence between positions 1 and 551. However, incomparison to the carboxyl terminal motif of GLT-1�(552–573), that of GLT-1v (552–562) is truncated andexhibits a transformed amino acid sequence.

GLT-1�-specific antibodies (Fig. 4D) revealed severalhighly immunoreactive bands in the inner plexiform

Fig. 2. Typical behavior of the whole-cell steady-state L-glutamate-evoked currents of HEK293 cells expressing GLT-1� or GLT-1v. A:Concentration dependence at 0 mV. The solid lines represent fitting ofthe Michaelis-Menten equation to the data with apparent Km valuesof 15.3 1.0 (GLT-1�, n 6) and 12.6 1.0 (GLT-1v; n 5). Voltagedependences of (B) the coupled transport current (GLT-1�, n 8;GLT-1v, n 3), (C) the uncoupled anion current (GLT-1�, n 4;GLT-1v, n 6), and (D) both current components (GLT-1�, n 14;GLT-1v, n 11). Currents were measured with internal KSCN�- andexternal NaCl-based buffers (A and C) or with internal KCl- andexternal NaCl- (B) or NaSCN-based buffers (D). The currents werenormalized for each cell to the current amplitude at 100 �M L-glutamate (A) or at 0 mV (B–D). Values represent mean SEM.Absolute mean currents normalized to cell capacitance at 100 �Mglutamate and 0 mV transmembrane potential were as follows (inpA/pF): coupled transport current (B), 0.3 (GLT-1�) and 0.3 (GLT-1v);uncoupled anion current (SCN� internal, C), 3.7 (GLT-1�) and 4.7(GLT-1v); uncoupled anion current (SCN� external, D), 4.6 (GLT-1�)and 5.4 (GLT-1v).

Fig. 3. Typical whole-cell currents induced by L-glutamate at Vm 0 mV. A and C: Steady-state currents measured in response to theapplication of 100 �M external L-glutamate (rapid solution exchange)from a cell expressing GLT-1� or GLT-1v, respectively. B and D:Typical pre-steady-state currents induced by the photolytic release ofa saturating amount of L-glutamate from the same cells. The concen-tration of caged glutamate delivered to the cells in the bath solution is1 mM. Photolysis is indicated by the arrow. The time constants �rise,�decay1, and �decay2 were obtained from fitting the data with a sum ofthree exponential functions. The fitting parameters for the tracesshown are for GLT-1�, I1 364 pA, �1 0.5 ms, I2 �90 pA, �2 6.0ms, I3 �28 pA, �3 23 ms, Iss �40 pA. For GLT-1v, theparameters are I1 556 pA, �1 0.4 ms, I2 �128 pA, �2 7.2 ms,I3 �52 pA, �3 28 ms, Iss �67 pA. All currents were measuredwith internal KSCN- and external NaCl-based buffers, and the laserenergy was approximately 400 mJ/cm2. The inwardly directed base-line current, which is caused by the anion leak and is already presentbefore glutamate application, was subtracted.

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(synaptic) layer, in accordance with previous studies(Rauen et al., 1996). By contrast, the GLT-1v expres-sion pattern was completely different (Fig. 4C). GLT-1vwas strongly expressed in cell bodies and cell processesof a subset of bipolar cells. GLT-1v immunoreactivitywas highly concentrated in the terminals of cone pho-toreceptors in the outer plexiform layer (OPL) withadditional staining of some perikarya of cone photore-ceptors.

Using antibodies that recognize common amino acidsequences of both GLT isoforms either in the N-termi-nal region (Fig. 4A) or proximal to the C-terminal re-gion (Fig. 4B), a staining pattern was revealed thatrepresents an overlap of both staining patterns ofGLT-1v (Fig. 4C) and GLT-1� (Fig. 4D), indicating thatGLT-1� and GLT-1v make up a considerable compo-nent of all GLT-1 expressed in the retina. These dataconfirm that neurons can express GLT-1� and GLT-1vand illustrate that different cells express differentsplice variants. Moreover, these splice variants may betargeted to specific membrane domains as illustratedby the lack of bipolar terminal labeling for GLT-1v andthe lack of cell body labeling for GLT-1�.

Immunocytochemistry of Brain Tissues

Immunocytochemical localization studies were suc-cessfully performed for all brain regions investigated.

As the results obtained using antibodies against syn-aptic vesicle antigens SV2 and synaptophysin wereessentially the same, we have, for brevity, made refer-ence only to SV2 in further descriptions.

Current antibodies do not distinguish betweenGLT-1B and GLT-1v due to the common identity of theC-terminal region. Thus, future discussions regardingimmunocytochemical localizations refer to bothGLT-1B and GLT-1v when commenting on distributionof these proteins in the brain, but for brevity we useonly the term GLT-1v.

Colocalization of GLT-1 SpliceVariants With GFAP

GFAP was used to mark individual astrocytes. Inaccordance with previous studies, we noted that theexpression of GFAP differed widely between differentbrain regions, with only scattered labeling in areassuch as cortex (Fig. 5A). Examination of double label-ing for GLT-1� and GFAP revealed that GLT-1� waslocalized in a punctate manner over the surface ofcortical astrocytes, and that most labeling for GLT-1�was localized to peripheral parts of the astrocyte pro-cesses rather than the somata of the astrocytes (Fig.5B). Triple labeling for GFAP and GLT-1� and GLT-1vrevealed differential labeling of GLT-1� relative to

Fig. 4. Schematic illustration of the two splice variants GLT-1� andGLT-1v investigated in this study, and representative staining ofretinal sections using antibodies directed against each of the indicatedregions. Antibodies directed against an epitope close to the aminoterminus (A) and a region proximal to the carboxyl terminus (B) arepresent in both isoforms, and the immunostaining patterns are sim-

ilar. Sequences specific to the carboxyl terminal region of GLT-1v (C)and GLT-1� (D) differ conspicuously from each other, but the com-posite staining pattern that would ensue from combining C and Dwould, as expected, resemble the staining patterns in A and B. Thissuggests that both GLT-1� and GLT-1v are expressed in the retina,albeit in different cellular compartments. Scale bar, 50 �m.

161TWO SPLICE VARIANTS OF GLT-1

Figure 5.

162 SULLIVAN ET AL.

GLT-1v, albeit with some overlap. GFAP-labeled pro-cesses appeared to overlap GLT-1�- and GLT-1v-la-beled elements, suggesting that a single astrocytes ex-pressed both splice variants of GLT-1, albeit indifferent membrane compartments. Analysis of areassuch as the granule cell layer of the cerebellum, wherethere was extensive GFAP labeling, revealed that allGFAP-immunoreactive astrocytes appeared to be im-munoreactive for GLT-1� and GLT-1v (Fig. 5D and E)and implies that these GFAP immunoreactive astro-cytes express both splice variants of GLT-1. However,there were clear differences in their localization;GLT-1� was not normally expressed in the proximalparts of the astrocyte such as the somata (Fig. 5D andF), whereas GLT-1v was routinely encountered in theastrocyte somata (Fig. 5E and G). These data suggestedthat GLT-1v was associated mainly with proximal re-gions of the astrocytes, including the somata of such,whereas GLT-1� was targeted to more distal parts ofthe astrocyte processes. GLT-1v immunoreactivity wasfrequently punctate (Fig. 5G), suggesting that it wasbeing targeted to specific locations. Similarly, in whitematter of the spinal cord, we note that GLT-1� waslocalized in a punctate manner over the surface ofGFAP-immunreactive processes, including fine sidebranches of the astrocytes, which were not GFAP-im-munoreactive (Fig. 5H).

Expression of GLT-1 Splice Variants inAstrocytes Surrounding Different

Neuronal Types

We have examined the distribution of GLT-1 splicevariants around specific neuronal types to determine ifthe specific phenotype of the postsynaptic neurone in-

fluenced the expression of either of the splice variantsof GLT-1

Motor Neurons

Examination of large somata and dendrites of motorneurons in the cervical and thoracic spinal cords (Fig.6) revealed consistently strong labeling with each ofthe GLT-1 antibodies in the glial cells surrounding themotor neurones but a complete absence of staining inthe somata or processes of the cholinergic motor neu-rones. In all cases, strong punctate labeling for SV2was observed, which we interpret as representing thesites of individual synapses. Examination of GLT-1�labeling (Fig. 6A and C) revealed strong labeling,which was present both in glial membranes that inter-digitated between the sites of SV2 labeling as well asbeing present at sites more distal to the synapses. Asimilar pattern of labeling was also observed using theAb12 and Ab497 antisera against common GLT-1epitopes (Fig. 6B and D). Immunolabeling with theGLT-1v antiserum (Fig. 6E and F) revealed a signifi-cantly different labeling pattern; GLT-1v immunoreactiv-ity was absent from the interstices between SV2 or syn-aptophysin-immunoreactive puncta. Instead, GLT-1vwas preferentially localized to regions of glial membraneand cytoplasm behind the layer of synapses that directlyapposed the motor neurone dendrites and somata. Thesedata suggest that GLT-1v is preferentially excluded fromthe tips of the glial cell processes that normally interdig-itate between synaptic terminals. Further examination oflabeling for each of the antibodies used revealed thatGLT-1 immunoreactivity was always absent in locationsdirectly between labeled synapses and the postsynapticneurons, indicating that GLT-1 was not expressed eitherin regions of the motor neurone plasmalemma apposed toinput synapses, or the portion of each synaptic terminalthat directly apposes the postsynaptic neuron.

Deep Cerebellar Nuclei

The fastigial nucleus (one of the deep cerebellar nu-clei) contains populations of readily identifiable largeexcitatory long projection neurons (principal neurons)that are part of the output pathway of the cerebellum.The vast majority of the inputs to these neurones areinhibitory, yet these neurones were enshrouded byglial cells that were intensely immunoreactive for bothisoforms of GLT-1.

Again, GLT-1� and common epitopes of GLT-1were present at sites interposed between most if notall synapses onto somata and proximal dendrites ofthe principal neurons (Fig. 7A and B), whereasGLT-1v was excluded from the final terminal portionof the astrocyte processes that interdigitated be-tween adjacent synaptic terminals on the somata anddendrites of these cells (Fig. 7C). Again, no evidencewas obtained to suggest that either splice variant

Fig. 5. Colocalization of GLT-1 splice variants and GFAP in grayand white matter regions of brain and spinal cord. A illustrates thelimited number of GFAP immunofluorescence-labeled astrocytes(green labeling) in rat cortex, such reactive astrocytes being associ-ated mainly with blood vessels (arrow), and the more extensive im-munolabeling of glial cells for GLT-1� (red). Where there is colocal-ization of labeling for GFAP (green) and GLT-1� (red), yellow labelingis evident. B illustrates brown peroxidase labeling of acortical astro-cyte (a) for GFAP, and punctate immunolabeling (Fast Red) forGLT-1� (arrows), which is mainly localized to sites distal to the cellbody. C illustrates triple labeling for GFAP (green), GLT-1� (red), andGLT-1v (blue) in processes of a cortical astrocyte. The GFAP-labeledprocess traverses patches of GLT-1� and GLT-v labeling. GLT-1� andGLT-1v may often be coincident (pink labeling) but may also beindependently expressed. D and E illustrate the cerebellar granulecell layer, immunolabeled for GFAP (brown peroxidase reaction prod-uct; blue arrows) and GLT-1� (D and F) or GLT-1v (E and G), labeledwith Fast Red (black arrows). The labeling of the cell surface trans-porters is often just out of the optical plane occupied by GFAP, whichlabels the structural core of the astrocyte. Nevertheless, the illustra-tions reveal that labeling for GLT-1� is not normally associated withastrocyte somata and proximal parts of the astrocytes. Conversely,GLT-1v is normally associated with the somata and proximal parts ofthese GFAP-immunoreactive astrocytes. H illustrates spinal cordwhite matter astrocytes labeled for GFAP (green) and GLT-1� (red).While there is often coincidence between the punctate GLT-1� label-ing and GFAP (white arrows), some lateral processes of the astrocyteslack the GFAP core but do express GLT-1� (blue arrows). Scale bars:A, 100 �m; B, 10 �m; C, F, G, and H, 5 �m; D and E, 25 �m.

163TWO SPLICE VARIANTS OF GLT-1

was colocalized within synapses, the postsynapticcells, or interposed between the pre- and postsynap-tic cells, suggesting that GLT-1 is not acting as aneuronal transporter.

Hippocampal CA1 Pyramidal Neurons

In accordance with previous studies, we show thatinterposed between the densely packed somata of

pyramidal cells there was a fine plexus of GLT-1�-immunoreactive glial processes (Fig. 7D). Such label-ing was more intense than that in the adjacent strataradiens and oriens, despite the lack of glutamatergicsynapses innervating the somata of these neurons(these neuronal somata receive only GABAergic syn-apses). High-magnification analysis revealed that ingeneral, each soma was usually separated from itsneighbor by a single unbranched GLT-1�- or GLT-1v-immunoreactive process, which passed around in-

Fig. 6. Spinal cords immunolabeled for SV2 (green) and, in red,either C-terminal of GLT-1� (A and C), Ab12 (B), Ab497 (D), orGLT-1v (E and F). In all cases, somata of motor neurones (M) areunlabeled. Arrows indicate glial processes interdigitating betweensynaptic terminals apposed to motor neurone somata. Antibodies

against common domains or the C-terminal of GLT-1� all label glialprocesses (arrows) that interdigitate between synaptic boutons (ar-rowheads, B–D), whereas GLT-1v antisera heavily label glial pro-cesses except those parts (arrows) that interdigitate between syn-apses (F). Scale bars, 10 �m.

164 SULLIVAN ET AL.

tervening synaptic terminals without sending off anyfurther finger-like interdigitating processes (Fig. 7Eand F). We suggest this simple arrangement is aconsequence of the very tight packing of the neuronalsomata, which prevents the elaboration of more com-plex glial architectures in the narrow spaces betweenneuronal somata.

Cerebral Cortex

Somata and apical dendrites of cortical pyramidalcells were examined in layers 3 and 5 of rat somato-sensory cortex. Strong labeling was observed with eachof the transporter antibodies in many of the surround-ing glial cells, though as previously reported, some

Fig. 7. Deep cerebellar nucleus (A–C) and hippocampus CA1 region(D–F) immunolabeled for SV2 (green) and, in red, either C-terminusof GLT-1� (A and D), Ab12 (B and E), or GLT-1v (C and F). In allcases, somata of neurones are unlabeled. In B, arrows indicate glialprocesses interdigitating between SV2-immunoreactive synaptic ter-minals apposed to neurone somata, whereas GLT-1v immunoreactiv-ity (C) is not present in such interdigitating processes (arrows). In the

hippocampus, strong GLT-1� labeling is especially evident aroundsomata of CA1 pyramidal cells (P; D). High-magnification observa-tions reveal only simple unbranched processes of glial cells in theregions interposed between the pyramidal neuronal somata, with noobvious specializations or differential immunoreactivity in the prox-imity of synapses (E and F). Scale bars, A–C, E, and F, 10 �m; D,50 �m.

165TWO SPLICE VARIANTS OF GLT-1

small patches of cortex, especially around blood ves-sels, did not exhibit GLT-1v labeling. No labeling wasobserved in the somata or processes of the pyramidalcells with any of the antibodies used in this study.Strong labeling for GLT-1� was present in glial mem-branes that interdigitated between all the sites of syn-aptic vesicle antigen labeling on the neuronal somata

and dendrites (Fig. 8A), and similar labeling was ob-served with Ab12 (Fig. 8B).

Immunocytochemistry with the GLT-1v antiserumagain revealed a different labeling pattern in thatGLT-1v immunoreactivity was absent from the glialprocesses that extended between SV2 or synaptophy-sin-immunoreactive puncta apposed to pyramidal cell

Fig. 8. Somatosensory cortex immunolabeled for SV2 (green; A–D)or the GABA-synthesizing enzyme glutamate-decarboxylase (GAD65;green; E). Red denotes labeling for either C-terminal of GLT-1� (Aand E), Ab12 (B), or GLT-1v (C and D). In all cases, somata ofpyramidal neurones (P) and their dendrites (d) are unlabeled. Anti-bodies against the C-terminus of GLT-1� or Ab12 label glial processes

that interdigitate between synaptic boutons that are labeled not onlyfor SV2 (A and B) but also between adjacent GAD-immunoreactivesynaptic terminals (arrows, E). By contrast, GLT-1v immunoreactiv-ity is absent from regions interposed between synaptic terminals thatinnervate the soma (C) or dendrites (D) of pyramidal cells. Scale bars:A and C–E, 10 �m; B, 5�m.

166 SULLIVAN ET AL.

somata or dendrites (Fig. 8C and D) and was insteadpreferentially localized to nonperisynaptic regions ofglial processes.

Our initial observations suggested that GLT-1� orcommon epitope labeling was present in glial processesthat interleaved between all synapses apposed to thecortical pyramidal cells. To verify if glutamate trans-porters were expressed on glial processes between ad-jacent GABAergic terminals, double labeling was per-formed on cerebral cortex and other brain regions usingGAD65 (glutamic acid decarboxylase) in place of SV2 orsynaptophysin. Such labeling (Fig. 8E) revealed inter-digitation of GLT-1�-immunoreactive processes be-tween GAD65-immunoreactive synapses. Conversely,examination of the narrow region between the SV2 orsynaptophysin-immunoreactive synapses innervatingthe pyramidal cells and the somata and dendrites ofthe pyramidal cells revealed that GLT-1 immunoreac-tivity was always absent, confirming that GLT-1 ingeneral is not expressed in regions of the pyramidal cellplasmalemma apposed to input synapses or the synap-tic terminals of the presynaptic neurons.

DISCUSSION

We have characterized the primary functional char-acteristics of GLT-1v and determine that they are com-parable to those observed for GLT-1�. Both proteinsexhibited distinct cellular and subcellular distributionpatterns in retinal neurons as well as in brain astro-cytes. We demonstrate differential targeting of GLT-1vto specific membrane domains in single identifiableglial cells away from the sites of synaptic specializationand we note that synapse neurochemical phenotypemay not be the driving factor in determining if aninterdigitating astrocyte process expresses a glutamatetransporter.

Targeting of GLT-1 Splice Variants

The concept and implications of proximal and distalregions of an astrocyte process have not been exten-sively explored. Astrocytes may be demonstrably polar-ized with respect to extension of processes toward bloodvessels and that features such as intramembranousparticle distributions change depending on locationwithin the process (Gotow, 1984), suggesting that dis-tal parts of astrocyte processes may be biologicallydistinguishable from more proximal parts. We haveobserved that along a single GFAP-immunoreactiveastrocyte process, punctate regions of labeling forGLT-1� or GLT-1v may be observed, and that in somecases the labeling for one splice variant did not overlapthe labeling for another splice variant, suggesting thateach of the splice variants of the transporters are ei-ther simply segregated into small rafts containingmany molecules of each splice variant, and/or that each

splice variant is targeted to distinct membrane do-mains.

We construe those parts of an astrocyte process in-terdigitating between synapses as distal parts of theastrocyte process. Accordingly, we propose thatGLT-1� appears to be targeted to the distal parts of theastrocyte, whereas GLT-1v is targeted to proximalparts of astrocyte processes and not the distal tips thatinterdigitate between synapses. The exclusion ofGLT-1v from the tips of astrocyte processes irrespec-tive of whether the synapses are glutamatergic or con-tain other transmitters such as GABA suggests thatsynapses do not drive the targeting of GLT-1v. Instead,the differential expression might arise because of C-terminal-directed targeting of the GLT-1 splice vari-ants. Intriguingly, in the retina, we show that themajority of the GLT-1v protein in bipolar cells is tar-geted to areas of the plasma membrane other than thebipolar synaptic terminals, that is, to membrane do-mains that may be analogous to the proximal parts ofthe processes in astrocytes. This suggests that similarmechanisms governing proximal and distal compart-mentalization operate in both neurons and astrocytes.

Our results accord with those of Danbolt (2001); weagree that GLT-1�, which was the form studied by thisauthor, is strongly expressed in glial processes adja-cent to synapses sites. We extend this by suggestingthat GLT-1v is expressed in nonsynaptic locations, pre-sumably to mediate recovery of general extracellularpools of glutamate.

Functional Properties of GLT-1v

Our data reveal that the alternate C-terminal splic-ing of GLT-1 does not have significant effects on itsglutamate transport properties. This accords entirelywith the earlier work of Utsunomiya-Tate et al. (1997)and the subsequent data from Chen et al. (2002). Thus,differential targeting of GLT-1� and GLT-1v is not forthe purpose of creating different glutamate clearanceproperties in different brain microdomains such as inareas immediately around synapses compared with ar-eas distal to such. We suggest instead that differentialsplicing and the subsequent differential targeting ofGLT-1 in distinct membrane domains may be a mech-anism that facilitates independent regulation of theimmediate perisynaptic glutamate level and the levelof glutamate in the extracellular space in general. Insupport of this, we have recently noted that in a murinemodel of ALS/parkinsonism, GLT-1v expression isdownregulated independently of GLT-1� (Wilson et al.,2003). We speculate that the differential expression ofthese splice variants may be a key factor in the excito-toxic disease process in this model. One issue that isnot resolved is the possibility of modification of prop-erties such as the chloride conductance. The chlorideconductance is speculated to be dependent on the for-mation of glutamate transporter oligomers, which al-low gated chloride flux through a central pore (Eskan-

167TWO SPLICE VARIANTS OF GLT-1

dari et al., 2000). While oligomers composed of GLT-1�or GLT-1v alone show a similar chloride conductance,we do not know at this stage if GLT-1� and GLT-1vhave the capacity to form hetero-oligomers, and if sowhat the effect would be on the chloride conductance.Previous descriptions of the formation of GLT-1B/GLT-1� hetero-oligomers by Utsunomiya-Tate et al.(1997) render this a real possibility. However, the spa-tial segregation of GLT-1� and GLT-1v into differentneurons or different cellular compartments of glialcells may restrict the opportunity to form mixed oli-gomers in vivo.

Neuronal Expression of GLT-1

Several groups of workers have observed that neu-rons in brain can express messenger RNA for GLT-1�(Schmitt et al., 1996; Berger and Hediger, 1998; Dan-bolt, 2001). However as noted by Danbolt (2001), ap-parent low levels of immunolabeling over neurons havesubsequently been shown to be comparable to nonspe-cific background labeling levels. Accordingly, a currentand widely held view is that antibodies to many com-mon regions of GLT-1, which should detect all knownsplice variants of GLT-1, do not detect GLT-1 protein inneurons in normal adult brains at levels statisticallyabove background signal levels (Danbolt, 2001). Ourresults are consistent with this view. In the brain,synaptic vesicle proteins and GLT-1 did not exhibitoverlapping distributions. The spatial localization ofGLT-1v at sites away from sites of synaptic specializa-tion made this distinction even more apparent. Ourstudies suggest that GLT-1 proteins do not appear tolocalize to regions interposed between the synaptic ves-icle clusters and the targets of such synapses. Con-versely, GLT-1 can readily be identified in pre- andpostsynaptic retinal neurons (Rauen and Kanner,1994; Reye et al., 2002a, 2002b), suggesting that theexpression of this protein is not always restricted toastroglial cells.

In contrast to the results presented here, Schmitt etal. (2002) and Chen et al. (2002) have suggested on thebasis of immunocytochemistry that GLT-1v might be aneuronal glutamate transporter. This view accordswith the earlier suggestions by Gundersen et al. (1993),who identified a dihydrokainate-sensitive accumula-tion of D-aspartate in brain nerve terminals, suggest-ing that GLT-1 is present and functional in such neu-rons. Recent studies by Suchak et al. (2003) have alsosuggested that synaptosomes may express a proteinwith GLT-1-like properties based on pharmacologicalblockade of transport, but this study used impure syn-aptosomal fractions with GFAP contamination. AsGFAP only marks a small subset of glial cells in areassuch as cortex, this may lead to an underestimation ofthe extent of glial contamination and thus influenceinterpretation of data.

The differences between our results and those ofSchmitt et al. (2002) and Chen et al. (2002) with re-

spect to labeling of neuronal elements in the brain maydepend on many variables such as specificity and af-finity of antisera, duration of tissue fixation types offixatives used, and the use of pre- vs. postembeddingtechniques. While our data do not support a neuronallocalization of GLT-1v, we cannot exclude it at thisstage; in particular, we acknowledge the possibilitythat GLT-1v might well be present in brain neurons atlevels below our threshold for immunocytochemical de-tection.

Does Astrocyte or Nerve Terminal DriveAstrocyte Transporter Expression?

Signals such as the presence of elevated levels ofglutamate probably increase the expression of gluta-mate transporters on the surface of astrocytes (Levy etal., 1995; Poitry-Yamate, et al., 2002). However, anysingle astrocyte may contact and envelop many syn-apses, including GABAergic, glutamatergic, and gly-cinergic types. We investigated whether the neuro-chemical makeup of individual synapses governs thetargeting of specific transporters into astrocyte pro-cesses adjacent to such synapses. Our data refute thisidea since GLT-1� can be expressed at sites interposedbetween adjacent GABAergic synapses. Accordingly,we suggest that astrocytes promiscuously express thegamut of transporters required by the neurones withintheir individual territories. Transporters positioned inirrelevant areas of astrocyte processes where the trans-mitter is not released would have no significant biolog-ical impact. In support of this view, Minelli et al. (2001)have suggested that the vesicular GABA transporter (amarker of GABA or glycine synapses) is closely associ-ated with GLT-1 in cerebral cortex. Our data showingthe heavy expression of GLT-1 in glial processesaround the somata of CA1 pyramidal cells, despite thepresence of only GABA synapses in this region, indi-cate that local synapse phenotype is not the overridingfactor in determining localization of GLT-1.

We conclude that alternate splicing of GLT-1 is sig-nificant, not because it confers distinct glutamatetransport properties, but because it encodes the capac-ity for differential targeting of the transporter. Thisdifferential targeting may also be associated with dif-ferential mechanisms for regulation of expression sinceour early work on retinal neurons (Reye et al., 2002b)indicated that GLT-1v (B is not expressed in retina)could be independently expressed in distinct neuronalsubsets and at markedly different times.

Possible Roles of GLT-1v

Our suggestion that GLT-1v may be a general regu-lator of extracellular glutamate at sites distal to syn-apses accords with recent studies (Diamond and Jahr,2000), which have suggested that high-affinity gluta-mate transporters not only play a direct role in synap-

168 SULLIVAN ET AL.

tic transmission by speeding the clearance of gluta-mate from the synaptic cleft but also exert more globalactions on synaptic transmission by and limiting theextent to which transmitter spills over between syn-apses.

Clearly, dysfunctional GLT-1v expression could po-tentially lead to aberrations in cross-talk between syn-apses. Moreover, abnormal regulation of extracellularlevels of glutamate at nonsynaptic sites may be impor-tant in human disease states such as epilepsy andischemia, where global changes in extracellular gluta-mate levels may be anticipated.

ACKNOWLEDGMENTS

Supported by the Fundacao para a Ciencia e a Tec-nologia, Portugal (grant PRAXIS XXI/BD/18095/98 toA.B.), the National Health and Medical ResearchCouncil (Australia) grants 210127 and 102448 and aSenior Research Fellowship (to D.V.P.), and the Deut-sche Forschungsgemeinschaft (grants GR 1393/2-2 toC.G. and RA 753/1-1 to T.R.).

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