Localization of the GLYT1 Glycine Transporter at Glutamatergic Synapses in the Rat Brain

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Cerebral Cortex April 2005;15:448--459 doi:10.1093/cercor/bhh147 Advance Access publication August 5, 2004 Localization of the GLYT1 Glycine Beatriz Cubelos, Cecilio Gimeńez and Francisco Zafra Transporter at Glutamatergic Synapses Centro de Biologıá Molecular ‘Severo Ochoa’, Facultad de in the Rat Brain Ciencias, Universidad Autońoma de Madrid, Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain

In this study, we present evidence that a glycine transporter, GLYT1, responses to NMDA both in vitro and in vivo (Chen et al., 2003; Downloaded from https://academic.oup.com/cercor/article/15/4/448/351216 by guest on 24 September 2021 is expressed in neurons and that it is associated with glutamatergic Kinney et al., 2003). Recent evidence obtained in knockout mice synapses. Despite the presence of GLYT1 mRNA in both glial cells and for glycine transporter genes confirmed the involvement of both in glutamatergic neurons, previous studies have mainly localized GLYT1 and GLYT2 in glycinergic inhibitory neurotransmission. GLYT1 immunoreactivity to glial cells in the caudal regions of the In these mutant mice, glycinergic neurotransmission was largely nervous system. However, using novel sequence specific antibodies, altered, leading to the suggestion that GLYT1 might remove we have identified GLYT1 not only in glia, but also in neurons. The glycine from the synaptic cleft, whereas GLYT2 would replenish immunostaining of neuronal elements could best be appreciated in the presynaptic pool of glycine (Gomeza et al., 2003a,b). The forebrain areas such as the neocortex or the hippocampus, and it was possible role of GLYT1 in glutamatergic neurotransmission in found in fibers, terminal boutons and in some dendrites. Double these GLYT1 deficient mice was not addressed since they died labeling confocal microscopy with the glutamatergic marker vGLUT1 shortly after birth. However, heterozygous animals that ex- revealed an enrichment of GLYT1 in a subpopulation of glutamatergic pressed only 50% of GLYT1 had an enhanced hippocampal terminals. Moreover, through electron microscopy, we observed an NMDA receptor function and memory retention. These animals enrichment of GLYT1 in both the presynaptic and the postsynaptic were also protected against an amphetamine disruption of aspects of putative glutamatergic terminals that established asym- sensory gating, suggesting that drugs which inhibit GLYT1 metric synapses. In addition, we demonstrated that GLYT1 was might have both cognitive enhancing and antipsychotic effects physically associated with the NMDA receptor in a biochemical (Tsai et al., 2004). assay. In conclusion, the close spatial association of GLYT1 and In order to fully clarify the role of GLYT1 in glutamatergic glutamatergic synapses strongly supports a role for this protein in neurotransmission, it is necessary to determine the precise neurotransmission mediated by NMDA receptors in the forebrain, and cellular and subcellular localization of this transporter. Previous perhaps in other regions of the CNS. histochemical studies revealed a discrepancy between the distribution of GLYT1 mRNA and protein. While GLYT1 mRNA Keywords: glycine, glutamate, immunohistochemistry, NMDA receptor transcripts were detected in glia and many types of neuron throughout the nervous system, the protein was only detected in glial cells (Zafra et al., 1995a,b). Introduction Here, we present detailed immunohistochemical data show- Beside its well-characterized role as an inhibitory neurotrans- ing that, in addition to the previously described expression of mitter, glycine is a co-agonist of NMDA receptors that is GLYT1 in glia, this transporter is also expressed at the pre- and necessary both for ion channel opening and probably for the postsynaptic aspects of glutamatergic synapses. This association internalization of the receptor from the cell surface (Kemp and was especially evident in the neocortex and hippocampus, areas Leeson, 1993; Nong et al., 2003). While it was initially believed where GLYT1 formed immunoprecipitable complexes with that the concentration of glycine in the synaptic cleft would be NMDA receptors. These results indicate a close relationship high enough to saturate the glycine site on the NMDA receptor, between GLYT1 and glutamatergic neurotransmission. recent pharmacological and electrophysiological evidence in- dicates that this might be not the case. Indeed, the levels of Materials and Methods glycine in the synaptic cleft could be close to the threshold levels for activation of the receptor (Supplisson and Bergman, Materials 1997; Berger et al., 1998; Bergeron et al., 1998; Kew et al., 1998, Oligonucleotides were synthesized by Isogen (Utrecht, NL). Bovine 2000). The concentration of glycine in the synaptic cleft is serum albumin (BSA), ethylene glycol-bis-aminoethyl ether N,N,N9,N9- regulated by the glycine transporters GLYT1 and GLYT2 (for tetraacetic acid, N-2-hydroxyethylpiperazine-N9-2-ethane sulfonic acid (HEPES), ovalbumin, diaminobenzidine, phenylmethanesulfonyl fluoride a review see Lo´pez-Corcuera et al., 2001). GLYT2 is predom- (PMSF), Triton X-100, Trizma base, Freund’s adjuvant and glutathione inantly found in the spinal cord and the brainstem, associated were from Sigma (St Louis, MO). Dextran-70, pGEX2, Glutathione with glycinergic neurotransmission (Luque et al., 1995; Zafra Sepharose 4B, standard proteins for sodium dodecyl sulfate--polyacryl- et al., 1995a,b; Jursky and Nelson, 1996). However, while GLYT1 amide gel electrophoresis (SDS--PAGE) (Rainbow markers), biotinylated is also expressed highly in glycinergic areas, GLYT1 mRNA was donkey anti-rabbit immunoglobulin Gs (IgGs), streptavidin-biotinylated also found in areas devoid of inhibitory glycinergic neuro- horseradish peroxidase and the enhanced chemioluminescent detection method (ECL) were from Amersham-Pharmacia (Amersham, UK). transmission where it might play a role in NMDA-mediated Isopropylthiogalactoside, EcoRI, BamHI and T4 ligase were from Roche neurotransmission (Smith et al., 1992). In support of this (Germany). Lipofectamine-PLUS was from Invitrogen and Taq poly- hypothesis, N[3-(49-fluorophenyl)-3-(49-phenylphenoxy)propyl]- merase from Perkin Elmer Cetus. Affi-Gel 15 (N-hydroxysuccinimide sarcosine (NFPS), a specific inhibitor of GLYT1, potentiates the esters of crosslinked agarose) and nitrocellulose sheets were from

Cerebral Cortex V 15 N 4 Oxford University Press 2004; all rights reserved BioRad (Richmond, CA). Newborn calf serum was from Gibco (Paisley, Electrophoresis and Blotting UK). Guinea pig anti-vesicular glutamate transporters 1 and 2 (anti- SDS--PAGE was done in the presence of 2-mercaptoethanol. The gels vGLUT1 and anti-vGLUT2) were from Chemicon (Temecula, CA) and were run slowly (overnight) at constant current starting at 30 V. After mouse anti-NMDA from Phamingen. Gold-coupled antibodies were from electrophoresis, samples were transferred by electroblotting onto Auroprobes. Goat anti-rabbit, goat anti-guinea pig and goat anti-mouse a nitrocellulose membrane in a semidry electroblotting system (LKB) coupled to Alexa Fluor 488 or Alexa Fluor 594 were from Molecular at 1.2 mA/cm2 for 2 h. The transfer buffer consisted of 192 mM glycine Probes (Eugene, OR). TAAB 812 was from TAAB laboratories (Reading, and 25 mM Tris--HCl, pH 8.3. Nonspecific protein binding to the blot was UK) and Lowicryl HM20 from Agar Scientific Ltd (Stanstead, UK). All blocked by the incubation of the filter with 5% non-fat milk protein in other reagents were analytical grade. 10 mM Tris--HCl, pH 7.5, 150 mM NaCl for 4 h at 25C. The blot was then probed with the indicated dilutions of crude antisera or purified Methods antibodies overnight at 4C. After washing, blots were then probed with an anti-rabbit IgG peroxidase linked, and bands were visualized Preparation of Glutathione S-transferase--Glycine Transporter with ECL and quantified by densitometry (Molecular Dynamics Image Fusion Proteins Quant v. 3.0). The 111 bp cDNA fragment encoding the amino terminus of GLYT1b was synthesized by polymerase chain reaction using the full-length Downloaded from https://academic.oup.com/cercor/article/15/4/448/351216 by guest on 24 September 2021 clone as template. According to available experimental evidence, the Immunocytochemistry N-terminal domain of GLYT1 is intracellularly placed (Olivares et al., Five adult rats (3 months old) (Wistar strain) were deeply anesthetized 1997). The specific oligonucleotide primers were CGGGATC- with pentobarbital (100 mg/kg) and perfused through the left ventricle-- CATGGCTGTGGCTCACGGA and GGAATTCACTCGATCTGGTTGCCC- aorta with a fixative solution containing formaldehyde (4 or 0.5%) in CA. Each primer consisted of 18 nucleotides of the GLYT1b sequence 0.1 M sodium phosphate pH 7.4 (500 ml, room temperature), preceded plus the restriction sites either for BamHI or EcoRI in their 59 ends. The by a brief flush (10--15 s) of Dextran-70 (MW 70000; 20 mg/ml) in the BamHI site was placed such that the inserts were in the correct reading same buffer without fixative. The right atrium was cut open at the start frame after insertion in BamHI/EcoRI sites of the pGEX-2 plasmid. These of the perfusion. The perfusion liquids were delivered by a peristaltic restriction sites are located in the carboxyl terminus of the glutathione S- pump at 50 ml/min, starting within 20 s after thoracotomy. The brains transferase (GST). After ligation of the inserts to pGEX-2, Escherichia were postfixed (overnight) in the same fixative. The tissue was stored at coli XL-1 blue transformants were selected in LB (10 g/l bacto-tryptone, 4C in a storage solution consisting of 1 part fixative and 9 parts 0.1 M 5 g/l bacto-yeast extract, 10 g/l NaCl, pH 7.0)--agar plates containing sodium phosphate until it was processed for light and electron 50 lg/ml of ampicillin. Individual colonies containing the pGEX-GLYT1b- microscopic immunocytochemistry or cryoprotected with sucrose NT were identified and sequenced using standard procedures. Fusion (30%). The formaldehyde was freshly depolymerized from paraformal- protein expression was induced by isopropylthiogalactoside (IPTG) as dehyde (PFA). Vibratome or cryostat sections (40 lm thick) were cut follows: bacteria were grown overnight at 37C in 50 ml of LB medium and stored (4C, up to 3 weeks) in 0.1 M sodium phosphate with NaN3 containing 50 lg/ml of ampicillin. Afterwards, bacteria were diluted (0.2--1 mg/ml). Then the sections were rinsed in 0.1 M sodium with 200 ml of the same growth medium and incubated at 37C for 1 h. phosphate, incubated (30 min) in 1 M ethanolamine with 0.1 M sodium Induction was carried out for 5 h by adding IPTG (1 mM final phosphate, washed (3 3 1 min) in buffer A (0.135 M NaCl, 0.01 M sodium concentration). The induced bacteria were collected by centrifugation phosphate), incubated in buffer B (0.3 M NaCl, 0.1 M Tris--HCl, pH 7.4) resuspended in phosphate-buffered saline (PBS) medium (137 mM NaCl, with 10% (v/v) newborn calf serum and NaN3 (1 mg/ml), and then incubated (12--48 h, 4C at room temperature) with antibodies (5--10 lg/ml) 0.9 mM CaCl2, 2.68 mM KCl, 1.47 mM KH2PO4, 0.49 mM MgCl2, 7.37 mM diluted in buffer C (buffer B with 1% newborn calf serum). The sections Na2HPO4, pH 7.4) containing 10 mM EDTA, 0.2 mg/ml lysozyme and 1 mM PMSF, and lysed by sonication. The lysate was cleared by centrifu- were washed (3 3 1 min and 2 3 10--20 min) in buffer C, incubated gation and the supernatant was passed through a Glutathione Sepharose (1 h) with biotinylated donkey anti-rabbit Ig (1:100) in buffer C, washed 4B column. The fusion protein was eluted with 10 mM glutathione in (3 3 1 min and 2 3 15 min) in buffer C, incubated (1 h) with 50 mM Tris--HCl, pH 7.5. A BLAST search in non-redundant and EST streptavidin-biotinylated horseradish peroxidase complex in buffer C sequences using as bait the N-terminal GLYT1 sequence revealed the and washed (3 3 1 min and 2 3 15 min) in buffer C. Following this, the absence of any significative homology with other proteins in sequence sections were washed (3 3 1 min) in buffer A, incubated for 6 min in data banks, except for GLYT1 from different species. 0.1 M sodium phosphate with H2O2 (0.1 mg/ml) and diaminobenzidine (0.5 mg/ml) after 6 min of preincubation in sodium phosphate/ Immunization diaminobenzidine without H2O2, and finally the reaction was stopped Immunization was carried out in MDL New Zealand rabbits (Navarra, with 0.1 M sodium phosphate (2 3 3 min). Triton X-100 (0.1%) was Spain). The aforementioned fusion protein (200 lg) was mixed with included only when stated. For light microscopy, sections were 0.8 ml of water, emulsified with 1 ml Freund’s complete adjuvant and mounted in glycerol--gelatin. Control experiments were performed injected into two rabbits. The rabbits were reimmunized every second after immunosorption of the antibody for 4 h with the fusion protein week as above, but using Freund’s incomplete adjuvant. immobilized on agarose (as indicated above) after the antibody had been diluted to the final working concentration in buffer C. These controls Immobilization of the Fusion Protein resulted in complete suppression of staining of tissue sections with the Fusion protein (1 mg) was dissolved in Na-HEPES buffer, pH 8.0, and antibody used in the present study. Staining was also absent when coupled to Affi-Gel 15 (5 ml gel) following the manufacturer’s primary antibody was omitted. Brain areas were identified referring to instructions. A 10 mg aliquot of GST was immobilized (as above) on the atlas of Paxinos and Watson (1982). For double labeling immuno- 25 ml Affi-Gel 15. histochemistry, sections were treated as above but secondary antibodies were coupled to the fluorescent dyes Alexa Fluor 488 or Alexa Fluor 594 Antibody Purification and the immunofluorescence was visualized in an Axioscop2 (Zeiss) The purification of antibodies was performed as described (Zafra et al., coupled to a confocal Microradiance (BioRad) or to a digital camera. 1995a). The anti-fusion protein antisera were absorbed with immobilized GST, concentrated on a protein A--Sepharose column and finally affi- Pre-embedding Electron Microscopy nity purified on a column with immobilized fusion protein. BSA (1 mg/ml) Immunoperoxidase reacted sections were treated with osmium (30-- and NaN3 (1 mg/ml) were added to stabilize the antibodies. 45 min, 10 mg/ml in 0.1 M sodium phosphate), washed (3 3 1 min) in 0.1 M sodium phosphate, dehydrated in graded ethanol (50, 70, 80 and Preparation of SDS Extracts 96% for 1 3 5 min and 100% for 3 3 10 min) and propylene oxide (2 3 SDS extracts from whole rat brains or specific regions were prepared by 5 min), and embedded in TAAB 812. Ultrathin sections were cut at right homogenizing and solubilizing the tissue in PBS with SDS (10 mg/ml) angles to the thick (40 lm) ones in order to be able to study the parts of and 1 mM PMSF, and removing unsolubilized material by centrifugation the tissue that had been in immediate contact with the reagents. The (50 000 g, 10 min, 10C). sections were contrasted (with 10 mg/ml uranyl acetate for 10--15 min

Cerebral Cortex April 2005, V 15 N 4 449 and 3 mg/ml Pb-citrate for 1--2 min) and examined in a Jeol 1010 Borowsky et al., 1993; Zafra et al., 1995b), the antibodies electron microscope. For controls, primary antibody was substituted by currently available have only been able to detect the protein in pre-immune IgG, or immune IgG freed of specific IgG by immuno- glia (Zafra et al., 1995a). In an attempt to demonstrate that sorption with the fusion protein. Immunosorption of the antibody was GLYT1 protein is indeed present in neurons, we have generated performed as indicated above. These controls resulted in complete suppression of staining of tissue sections with the antibody used in the a novel polyclonal antiserum against a fusion protein containing present study. the 37 N-terminal amino acids of the GLYT1b isoform. This affinity-purified antibody (antibody 224) was first tested by Post-embedding Electron Microscopy immunoblotting for immunoreactivity against a variety of GST Small pieces of brain tissue were cryoprotected in increasing concen- fusion proteins. The serum reacted strongly with the GST- trations of glycerol in PB (10, 20 and 30%; 30 min in each) and store GLYT1Nt protein (Fig. 1A, lane 2), but it did not recognize GST overnight at 4C in the 30% glycerol solution, followed by 100% glycerol. whether alone (Fig. 1A, lane 1), fused to the C terminus of The sections were slam-frozen on a copper plate cooled in liquid nitrogen (MM80E, Leica Microsystems Inc.) to preserve ultrastructure, GLYT1 (Fig. 1A, lane 3) or fused to the N terminus of GLYT2 (Fig 1A, lane 4). freeze-substituted in propanol, then embedded in resin (Lowicryl Downloaded from https://academic.oup.com/cercor/article/15/4/448/351216 by guest on 24 September 2021 HM20) according to the manufacturer’s instructions. The resin blocks To test whether the antibody was also able to recognize the were polymerized under UV light, and ultrathin sections (60 nm) were full-length protein, COS cells were transfected with expression cut using an Ultracut E microtome and collected on nickel slot grids. vectors containing GLYT1 and cell extracts were again analyzed The sections were etched briefly (3 s) in a saturated solution of NaOH in by immunoblotting. The antibody recognized a smear typical of absolute ethanol, washed in distilled water and blocked in 1% fetal calf a heavily glycosylated protein of between 70 and 100 kDa, similar serum in PBST for 1 h. The sections were then incubated in primary antibodies in blocking solution overnight, washed in PBS (3 3 10 min), in size and aspect to that recognized by previously described and incubated in secondary antibodies (5 or 10 nm gold-conjugated anti- antibodies against GLYT1 (Fig. 1B). A second, 52--55 kDa, protein rabbit IgG at 1/50 dilution and 10 nm gold-conjugated anti-mouse IgG at that corresponded in size to the partially glycosylated intracel- 1/50 dilution) in blocking solution containing 0.5% polyethylene glycol lular forms of the transporter reported previously (Olivares et al., for 2 h. The sections were washed as described above, postfixed in 1% 1995) was also recognized in COS cells overexpressing GLYT1. glutaraldehyde, counterstained in 2% uranyl acetate and lead citrate Moreover, a single band of 46--50 kDa was recognized in extracts according to standard techniques, and examined as described above. Controls were performed as stated for pre-embedding technique. For from cells transfected with a construct in which the four quantitative analysis pictures of distinct and transversally cut mem- glycosylation sites located in the large extracellular loop had branes were printed at 380 000 magnification and the distance from the been mutated, this representing the unmodified transporter reference line to the centre of gold particles was measured manually peptide (Fig. 1B). None of these bands were observed in mock (Matsubara et al., 1996, Landsend et al., 1997). For analysis of the transfected COS cells. perpendicular distribution of particles (presynaptic versus postsynap- Although the antigen against which the serum was generated tic) we considered a line parallel to the postsynaptic density drawn along the synaptic cleft centre and we determined manually the shorter corresponded to the N-terminus of the GLYT1b isoform, the distance from each gold particle to this line. antibody also recognized the GLYT1a isoform both on immunoblotting (Fig. 1B) and in immunofluorescence of transfected Immunofluorescence in Cultured Cells COS cells (Fig. 1C). This cross-reactivity was not surprising, COS cells were transfected with expression vectors for GLYT1a or since both isoforms share 27 of the 37 N-terminal amino acids in GLYT1b by using lipofectamine-PLUS following the manufacturer’s GLYT1. Nevertheless, the 224 antibody reacted more strongly instructions. After 48 h the cells were rinsed with PBS and fixed for with GLYT1b than GLYT1a in immunoblots (Fig. 1B, lanes 3--4). 20 min with 4% paraformaldehyde in PBS. After washing in PBS, the cells As expected, the C-terminal antibody (Ab 176) reacted identi- were permeabilized and non-specific binding sites were blocked in PBS containing 1% BSA and 0.02% digitonin for 1 h at room temperature. Cells cally with both isoforms which share this domain of the were incubated overnight at 4 C with anti-GLYT1 antiserum in PBS transporter (Fig. 1B, lanes 7--8). No cross-reactivity was ob- containing 0.1% BSA and 0.02% digitonin. After washing three times in served with closely related transporters, such as GLYT2 or the PBS, the cells were incubated with goat anti-guinea pig conjugated with GABA transporter (GAT1) transfected in COS cells (not shown). Alexa 594 (1:250) for 1 h at room temperature. Cells were washed three In addition, the 224 antibody recognized a single broad band in times with PBS and mounted in Vectashield (Vector, Burlingame, CA). extracts from rat nervous system (Fig. 1B, lane 5), with an identical size and aspect to that recognized by the antibody 176 Immunoprecipitation (Fig.1B, lane9). Indeed, as reported previously, the protein from Cerebral neocortex and hippocampus from 3-week-old rats were solubilized in ice-cold lysis buffer (50 mM Tris--HCl, pH 8.0, 150 mM native tissue was considerably smaller than that in transfected NaCl and 2 mM EDTA, 0.1% SDS 1% NP-40 and 0.5% deoxycholate) for cells, ranging from 65 to 70 kDa. It has previously been shown 30 min at 4C. The solubilized material was centrifuged at 10 000 g for that this difference in size is caused by differential glycosylation, 20 min, and the supernatants were incubated with anti-GLYT1 anti- since enzymatic deglycosylation of either the transfected or the bodies (2 lg/ml) or with control antibodies overnight at 4C. Then, 40 ll native GLYT1 yielded the 46 kDa band (Olivares et al., 1995). of a suspension of protein A cross-linked to agarose beads (Sigma) was Neither the 224 antibody nor 176 antibody reacted with added, and the mixture was incubated for 2 h with constant rotation at 4C. The beads were washed twice with ice-cold lysis buffer and three proteins in other tissues such as the lung, liver, pancreas or times with PBS. Then, 50 ll of SDS--PAGE sample buffer was added to kidney (not shown). each sample. Bound proteins were dissociated by boiling for 5 min and resolved by SDS--PAGE on 10% gels as described above. Immunohistochemical Localization of GLYT1 in the Rat Brain Results General Regional Distribution Characterization of a Novel Anti-GLYT1 Antibody The regional distribution of GLYT1 was examined in rat brain Despite the expression of GLYT1 mRNA by both neurons and sections with the anti-GLYT1 antibody. When the immunoper- glia in glycinergic and non-glycinergic areas (Smith et al., 1992; oxidase staining was visualized by low-power light microscopy

450 GLYT1 in Excitatory Synapses d Cubelos et al. associated with the deep cerebellar nuclei, whereas in the granular and the molecular layers it was only moderate to dense (Fig. 2C,D). At the midbrain level, GLYT1 staining was intense and widespread, with the strongest labeling in the inferior and superior colliculi, in the red nucleus and in the periaqueductal gray (Fig. 2E,F). In the substantia nigra staining was moderate to dense, being most intense in the compact part (Fig. 2E). At the level of the diencephalon (Fig. 2E--H), widespread GLYT1 immunostaining was found. In the thalamus, staining was in general moderate to intense, with the strongest labeling being detected in the principal sensory relay nuclei such as the ventroposteromedial, ventroposterolateral, and lateral geniculate and medial geniculate, whereas it was weaker in the Downloaded from https://academic.oup.com/cercor/article/15/4/448/351216 by guest on 24 September 2021 posterior nucleus (Fig. 2E--H). Strong GLYT1 expression was also seen in some association nuclei, especially in the anterodorsal nucleus (Fig. 2H). In the hypothalamus, immunolabeling was more intense in the periventricular and medial regions than in the lateral ones, either at the preoptic, anterior, tuberal or mammilary levels (Fig. 2H). In the epithalamus, staining was more intense in the lateral than in the medial habenula (Fig. 2G). Also at the level of the diencephalon, we found strong GLYT1 expression in the subthalamic nucleus (Fig. 2G), in the pretectal area and in the nuclei of the optic tract (Fig. 2F). In the forebrain, the septal region stained most intensely in the medial septum and in the nucleus of the diagonal band (Fig. 2I). In the hippocampus, the stratum lucidum and the hilar region of the dentate gyrus exhibited were moderately stained (Fig. 2E--G), as was the neocortex, although slightly stronger GLYT1 immunoreactivity was seen in layers 2--3 (Fig. 2E--I). In the olfactory bulb (not shown), the immunostaining was fairly dense in the periglomerular area and the external plexiform layer, while the granular layer was moderately stained.

Cellular and Subcellular Distribution of GLYT1 in the Forebrain. Light Microscopy Whereas at low resolution the distribution of GLYT1 revealed by the 224 antibody did not appear to differ significantly from that observed with previously used antibodies, important differ- ences were revealed at higher magnification, particularly in the forebrain regions. Thus, the staining with this antibody was analyzed in more detail in the neocortex, the hippocampus and associated regions. The labeling observed in the stratum lucidum of the hippocampus (Fig. 3A) was compatible with the presence of GLYT1 in mossy fiber terminals, since immunoreactivity was found in irregularly shaped structures, either Figure 1. Characterization of antibody 224. (A) The indicated GST fusion proteins (5 lg/lane) were subjected to SDS--PAGE, electroblotted and reacted with antibody delineating the dendrites of the principal CA3 pyramidal cells or 224. Bands were visualized with ECL. (B) Immunoblot of crude SDS extracts from COS scattered between their cell bodies (Fig. 3B). In addition to cells transfected with GLYT1a, GLYT1b or a glycosylation defective mutant of GLYT1b these terminals, a number of much smaller structures produced (GLYT1b-N4), or mock transfected (Mock), or an extract from rat brain (BR), were punctate staining in other areas of the hippocampus such as the subjected to SDS--PAGE (50 lg/lane) and reacted with antibodies 176 (Ab 176) or 224 (Ab 224), as indicated. (C) Immunofluorescence in mock transfected COS cells (Mock) stratum oriens and in the stratum radiatum of the CA3 area (Fig. or cells transfected with an expression vector for GLYT1a or GLYT1b and reacted with 3C). Indeed, this punctate pattern also extended to these strata antibody 224. Immunoreactive cells are pointed by arrows. in CA1 (Fig. 3D). These structures were evenly distributed in the neuropil and outlining neuronal cell bodies, suggesting that they corresponded to terminal boutons. (Fig. 2), in the nervous system the strongest immunostaining It should be noted that under milder fixation procedures was found in the gray matter of the spinal cord (Fig. 2A). Intense (0.5% PFA) and in the presence of Triton X-100, staining was staining was also observed in several nuclei of the pons and the also appreciated in fibers of the fimbria, the corpus callosum, medulla oblongata, such as the pontine, the spinal trigeminal, the alveus or the lacunosum-moleculare (data not shown), and the facial, the hypoglossal, the inferior olive, the dorsal and this staining was reduced, albeit not eliminated, when we used ventral cochlear, the lateral lemniscus, the reticular, the vestib- standard fixation in 4% PFA. However, the mild fixation protocol ular or the cuneate nuclei, as well as in the superior olivary was detrimental to high resolution microscopy, and in general complex (Fig. 2B--D). In the cerebellum, dense staining was we used 4% PFA. Some scattered cell bodies were labeled,

Cerebral Cortex April 2005, V 15 N 4 451 Downloaded from https://academic.oup.com/cercor/article/15/4/448/351216 by guest on 24 September 2021

Figure 2. Low magniﬁcation of coronal sections immunolabeled with antibody 224. Sections (40 lm) obtained with a Vibratome from tissue ﬁxed with 4% PFA were reacted with 10 lg/ml of antibody 224 in the presence of 0.2% Triton X-100. Abbreviations are: 3, oculomotor nucleus; 7, facial nucleus; 12, hypoglossal nucleus; AD, anterodorsal thalamic nucleus; AH, anterior hypothalamic area; APT, anterior pretectal nucleus; CPu, caudate--putamen; Cx, cerebral neocortex; DB, diagonal band; DC, dorsal cochlear nucleus; DG, dentate gyrus; DH, dorsal horn of the spinal cord; DLG, dorsal lateral geniculate nucleus; DpMe, deep mesencephalic nucleus; ECu, external cuneate nucleus; Int, interposed cerebellar nucleus; IO, inferior olive; Lat, lateral cerebellar nucleus; LHb, lateral habenula; LSO, lateral superior olive; Med, medial cerebellar nucleus; MGM, medial geniculate nucleus; MS, medial septum; MVe, medial vestibular nucleus; OT, nucleus of the optic tract; PAG, periaqueductal gray; Po, posterior thalamic nucleus; PrV, principal sensory trrigeminal nucleus; R, red nucleus; SN, substantia nigra; Sp5I, spinal trigeminal nucleus, interpolar part; Sp5O, spinal trigeminal nucleus, oral part; STh, subthalamic nucleus; SuC, superior colliculus; VC, ventral cochlear nucleus; VH, ventral horn of the spinal cord; VP, ventral pallidum. VPL, ventral posterolateral thalamic nucleus. Scale bar: 0.5 mm for (A, B), 1 mm for (C--I). dispersed throughout all the hippocampus. In general, these boutons were labeled in the neuropil and around cell bodies cells had an appearance of non-pyramidal neurons (arrows in (Fig. 3F--I). The highest concentration of boutons was observed Fig. 3A). The immunostaining of the dentate gyrus was similar to in layer 1 (Fig. 3F) and the lowest in layer 6 (Fig. 3I). Pyramidal that described for the stratum oriens and radiatum of the and non-pyramidal cells were contacted by immunostained hippocampus, i.e. small punctate staining in the neuropil and boutons in all cortical layers. Some dendrites that were cut delineating the neuronal cell bodies in the hilus, and in the along their longitudinal and transversal axis presented a diffuse granular and molecular layers, with a higher density of boutons intracellular staining (Fig. 3G) and in addition, some stained in the hilar region (Fig. 3E). astrocytes were seen, especially in layer 6 (Fig. 3I). This pattern In the neocortex, the distribution of GLYT1 staining was of staining was repeated in the basal ganglia, the thalamus, and similar to that in the hippocampal formation: in all layers small the hypothalamus: abundant stained boutons in the neuropil

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Figure 3. Localization of GLYT1 in the telencephalon and diencephalon. Sections (40 lm) from tissue fixed with 4% PFA were incubated with 10 lg/ml of antibody 224 in the absence of Triton X-100 and the immunoreactivity was visualized by the immunoperoxidase method. (A) Hippocampus. Immunoreactivity is denser in the stratum lucidum (sl). Few immunoreactive cell bodies are dispersed through the hippocampus (arrows). (B--E) High magnification photomicrographs of the areas labeled in (A). (B) Stratum lucidum at the CA3 level. (C) Stratum oriens (so), stratum pyramidale (pyr) and stratum lucidum (sl) in CA3. (D) Stratum oriens (so), pyramidale (pyr) and radiatum (sr) in CA1. (E) Hilus. (F) Border of layers 1 (I) and 2 (II) of the neocortex. (G) Layer 3 of the neocortex. (H) Layer 5 of the neocortex. (I) Layer 6 of the neocortex. (J) Ventromedial hypothalamic nucleus. Punctate immunoreactive structures correspond to terminals distributed through the neuropil and around unlabeled cell bodies (*). Diffuse staining in longitudinally cut dendrites can be appreciated in (arrows in G). An immunoreactive astrocyte (a) is observed in (I). Scale bar: 0.35 mm for (A); 20 lm for (B--E); 15 lm for (F--J). and delineating the cell bodies. An example, corresponding to quently formed clusters that coincided with the vGLUT1 pre- the hypothalamus is presented in the Figure 3J. However, in the synaptic marker, a number of smaller puncta were labeled for brainstem and spinal cord strong glial staining was evident in GLYT1 alone. On occasions, these small puncta were localized the neuropil and around cell bodies that was similar to that on the surface of transversally cut dendrites (arrowheads in Fig. previously described for C-terminal antibodies (data not shown, 4A--C), compatible with them being postsynaptic clusters of Zafra et al., 1995a). This glial staining made it difficult to detect GLYT1 (see below). Other puncta were intracellular and might any punctate immunoreactivity. Also in keeping with other correspond to vesicular GLYT1 being transported or recycled. antibodies, we observed specific staining of amacrine cells in In the neocortex, co-localization of both proteins was less the retina (not shown, Zafra et al., 1995a). frequent than in the hippocampus or the amygdala, yet it was detected as white puncta in triple labeling experiments [some Neuronal GLYT1 Localizes to Glutamatergic but not marked with arrowheads in layer 1 of the neocortex (Fig. 4G--J) GABAergic Terminals in the Forebrain or in layer 3 (Fig. 4K--N)]. There were a number of structures Because the staining pattern described above was in certain that were recognized by the anti-GLYT1 but not by the anti- areas reminiscent of the distribution of glutamatergic terminals, vGLUT1 antibody. However, most of these structures were we compared the labeling of anti-GLYT1 antibodies with that of not synaptic boutons since they were not labeled with anti- anti-vGLUT1 or anti-vGLUT2 antibodies. These two proteins synaptophysin (blue channel in Fig. 4G--N). Consequently they define two subgroups of glutamatergic terminals and, together, appeared as green structures in the merged images obtained in label most of the glutamatergic terminals in the brain. It has these triple labeling experiments. Of these, the larger puncta been reported that vGLUT1 terminals are generally more might correspond to axons cut transversally or glial cell pro- abundant in the forebrain and vGLUT2 terminals more so in cesses (small arrows in Fig. 4K--N). While others were intra- the caudal brain (FremEau et al., 2001; Herzog et al., 2001; cellular, a number of small puncta were also dispersed through Kaneko et al., 2002). When visualized in the same sections, an the neuropil. Interestingly, it was unusual to find synaptic overlap was observed in the labeling for GLYT1 and vGLUT1 in boutons containing GLYT1 and synaptophysin but not vGLUT1 forebrain regions such as the hippocampus (Fig. 4A--C) and in the neocortex. Notably, synaptophysin-containing puncta amygdala (Fig. 4D--F). This co-localization could be appreciated, outlining pyramidal cells (large arrows in Fig. 4K--N), which for example, in large mossy fiber terminals in the stratum probably corresponded to GABAergic terminals (they lacked lucidum of the hippocampus. Nevertheless, while GLYT1 fre- vGLUT1), were not stained for GLYT1. The inverse was not true,

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Figure 4. Confocal micrographs of rat brain sections that were double-labeled for GLYT1 and vGLUT1, or triple-labeled for GLYT1, vGLUT1 and synaptophysin. Green images correspond to GLYT1 (A, D, G, K, O, R), red images to vGLUT1 (B, E, H, L, P), vGLUT2 (P) or VIAAT (S) and blue images to synaptophysin (I, M). Right panels (C, F, J, N, Q, T) are merged images. Images correspond to the stratum lucidum of the hippocampus (sl) (A--C), the amygdala (am) (D--F, O--Q), layer I of the neocortex (cx1) (G--J), layer III of the neocortex (cx3) (K--N) and layer II of the neocortex (cx2) (R--T). Note that in the stratum lucidum, GLYT1 and vGLUT1 co-localize on mossy ﬁbres terminals (arrows in A--C). Some small puncta on the surface of transversally cut dendrites only contain GLYT1 (arrowheads in A--C). In neocortex (G--N), some terminals (synaptophysin immunoreactive) contain both GLYT1 and vGLUT1 (white puncta, arrowheads in G--N). There are GLYT1 immunoreactive structures lacking vGLUT1 and synaptophysin (green in the merged image, small arrows). Large arrows point to some terminals that lack both GLYT1 and vGLUT1 outlining a pyramidal neuron (pyr). Note the presence of terminals containing vGLUT1 but not GLYT1 (purple puncta in the merged image). Scale bar: 12 lm for (A--C); 15 lm for (D--F); 20 lm for (G--J); 14 lm for (K--N); 15 lm for( O--Q); 6 lm for (R--T). and in the neocortex there were many vGLUT1 labeled terminals localized with vGLUT2 in the forebrain. For instance, terminals with low levels of GLYT1, indicating that GLYT1 is associated containing vGLUT2 (Fig. 4O--Q) in the amygdala were devoid of only with a subpopulation of glutamatergic terminals. GLYT1 immunoreactivity. These observations again indicate Consistent with a preferential association of GLYT1 with that GLYT1 is not ubiquitously expressed in glutamatergic ter- terminals containing vGLUT1, GLYT1 only occasionally co- minals but rather that it is associated to speciﬁc subpopulations

454 GLYT1 in Excitatory Synapses d Cubelos et al. of excitatory terminals. Nevertheless, in caudal areas of the observation consistent with the intracellular localization of the nervous system, like the spinal cord, vGLUT1-containing termi- N-terminal epitope (Olivares et al., 1997). In the stratum nals were less strongly labeled by the GLYT1 antibody, whereas lucidum of the hippocampus, both unmyelinated axons and vGLUT2 terminals were abundantly immunolabeled (data not terminals contained GLYT1. The labeled axons were observed shown). within fasciculi of tightly packed fibers that probably corre- To investigate whether GLYT1 was also present in inhibitory spond to the fasciculated grouping of the mossy fibers (arrow- terminals we compared labeling with the 224 antibody with heads in Fig. 6A). Nevertheless, the strongest labeling in the that of an antibody raised against the vesicular transporter of stratum lucidum was found within the complex and multi- inhibitory amino acids (VIAAT). In accordance with the afore- lobulated presynaptic expansions of the mossy fibers that were mentioned triple labeling experiments, GLYT1 only occasionally filled with synaptic vesicles and made synaptic contacts with co-localized with VIAAT. Indeed, it was rare to find structures the complex spines of the CA3 pyramidal cells — the thorny labeled by both antibodies in the neocortex (Fig. 4R--T). Similarly, excrescences — (Fig. 6B). On occasions, peroxidase staining GLYT1 did not appear to co-localize with the vesicular acetyl- was observed within filopodial expansions of the mossy fiber Downloaded from https://academic.oup.com/cercor/article/15/4/448/351216 by guest on 24 September 2021 choline transporter (vAChT), a marker of cholinergic terminals terminals (not shown). In the CA1 region we also detected (data not shown). labeling within terminals making asymmetric synapses with To investigate whether antibody 224 was able to recognize simple dendritic spines, in both the stratum oriens (not shown) the glial GLYT1 in the neocortex, we performed co-localization and radiatum (Fig. 6C). Similarly, in the neocortex we found experiments with the glial marker GFAP. As was the case with labeling of terminals that established asymmetric synapses (Fig. C-terminal antibodies, GLYT1 was detected on cell bodies and 6D). GLYT1 labeling was also associated with dendrites and processes of GFAP immunoreactive cells, where the transporter spines in both the hippocampus (not shown) and the neocortex formed large clusters (Fig. 5A--F). Clusters of GLYT1 were also (Fig. 6D,E), although the staining was in general weaker than in appreciated on astrocytic profiles around blood vessels (Fig. the terminals. Moreover, some occasional staining of glial 5G--I). profiles was observed in the hippocampus and neocortex (not shown; Zafra et al., 1995a). This contrasted with the strong Ultrastructural Localization of GLYT1 labeling of glial profiles found in caudal regions such as the To study the ultrastructural localization of GLYT1, we per- spinal cord (Fig. 6F). Nevertheless, neuronal elements such as formed pre-embedding immunoperoxidase and post-embed- dendrites (Fig. 6F) and terminals (not shown) were also labeled ding electron microscopy of hippocampal and cortical tissue. in the spinal cord. Staining was absent from control material Pre-embedding studies revealed the presence of clumps of (see Material and Methods; data not shown). the peroxidase end-product precipitated on the cytosolic side Due to the electron dense nature of the postsynaptic densities of plasma membranes and on internal cellular structures, an (PSDs), pre-embedding studies could not clarify to what extent

Figure 5. Confocal micrographs of a rat neocortex section that was double-labeled for GLYT1 and GFAP. Green images correspond to GLYT1 and red images to GFAP. Note the presence of large GLYT1 clusters in an astrocytic cell body (a) (A--C), on glial processes (arrows in D--F) and lining the glial proﬁle around a blood vessel (arrows in G--I). The vessel lumen is labeled with asterisks. Scale Bar: 10 lm for (A--C); 11 lm for (D--F); 18 lm for (G--I).

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Figure 6. Electron micrographs of GLYT1 immunoreactivity. (A--F) Pre-embedding immunohistochemistry of GLYT1 in the hippocampus (A--C), the neocortex (D--E) and the spinal cord (F). (G--K) Post-embedding immunohistochemistry of GLYT1 in the dentate gyrus (G--J) and the molecular layer of the cerebellum (K). (A) Transversally cut immunoreactive axons (arrowheads) in a fasciculated group of mossy fibers in the stratum lucidum of CA3. (B) Immunoreactive mossy fiber terminal (MF) apposed to CA3 pyramidal cells dendritic spines (sp). Postsynaptic densities can be seen in one of the spines (arrows). (C) Immunoreactive presynaptic profile (T) contacting a dendritic spine (sp) through an asymmetric synapse in CA1 stratum radiatum. (D) Labeled terminal (T) apposed to a spine through an asymmetric synapse (small arrow) in the neocortex. The spine emerges from a dendrite that also contains peroxidase reaction product (large arrows). (E) Terminal (T) apposed to a dendritic spine at an asymmetric synapse. Close to the postsynaptic density (small arrow) there is a deposit of peroxidase reaction product. (F) Immunoreactive dendrite and glial profiles (arrows) in the ventral horn of the spinal cord. (G--I) Immunoreactive synapses in the dentate gyrus. Note theaccumulation of particles on postsynaptic densities (large arrows in G and I) and in the presynaptic active zone of a terminal (T) (small arrow in H). Particles within this terminal are also over intracellular structures (arrowheads in H). (J) Double post-embedding immunogold labeling with anti-GLYT1 (5 nm gold particles, small arrows) and anti-NMDAR1 (10 nm gold particles, large arrows) at asymmetric synapses in the hippocampus. Note the simultaneous labeling of the postsynaptic density. (K) Immunoreactive synapse in the molecular layer of the cerebellum. Note accumulation of gold particles in the flanking region of the active zone in parallel fiber terminals (T) (large arrows). Gold particles were found either close to the plasma membrane or at some distance in the adjacent cytoplasm. A lower density of particles was found at postsynaptic densities (arrowhead in K) and at extrasynaptic membranes, like membrane of parallel fibers (small arrow in K). Scale bar: 0.5 lm for (A, B); 0.4 lm for (C, D); 0.25 lm for (E); 0.6 lmin(F); 325 nm for (G--I, K); 125 nm for (J).

456 GLYT1 in Excitatory Synapses d Cubelos et al. GLYT1 localized to PSDs. Thus, to improve the resolution and to fibers, GLYT1 was enriched in the presynaptic element (Fig. quantify GLYT1, post-embedding immunogold staining on ultra- 6K). A quantitative analysis of the gold particles along the thin sections of hippocampal tissue was performed. Consistent perpendicular axis of the parallel fiber--Purkinje cell synapses with the pre-embedding experiments, gold particles were not- indicated that of the 327 particles counted in these synapses ably enriched at asymmetric synapses, identified by their dense (n = 62), 226 (69%) were presynaptic (Fig. 7B). These particles postsynaptic density (Fig. 6G). Indeed, particles were observed were found within a stretch of ~70 nm, both in the presynaptic both at the pre- (Fig. 6H) and postsynaptic side of these synapses membrane and the adjacent cytoplasm. These experiments (Fig. 6I), and in the dentate gyrus, the density of grains was higher indicated that the high concentration of GLYT1 present in the in the postsynaptic element than in the presynaptic one. A PSDs of hippocampal synapses cannot be attributed to non- quantitative analysis of the distribution of gold particles along the specific binding of the antibody, a common phenomenon due to perpendicular axis of the synapses in the dentate gyrus revealed the high protein concentration at the PSDs. that 61% of the gold particles (187 out of 305, n = 75 synapses) were localized in the postsynaptic element whereas only 39%

GLYT1 Interacts with the NMDA Receptor Downloaded from https://academic.oup.com/cercor/article/15/4/448/351216 by guest on 24 September 2021 were presynaptic (Fig. 7A). Additionally, some labeling was In view of the close association of GLYT1 with glutamatergic localized close to the synapse and intracellular structures in synapses, we adopted a biochemical approach to determine the presynaptic element were frequently labeled (Fig. 6H). It whether GLYT1 was physically associated with the NMDA re- remains to be determined whether these intracellular structures ceptor. Native rat cortical and hippocampal tissue was obtained are synaptic vesicles or recycling endosomes, as shown for other and solubilized in a buffer containing a mixture of ionic and non- transporters (Geerlings et al., 2001; Deken et al., 2003). Double ionic detergents (0.1% SDS, 1% NP-40 and 0.5% deoxycholate). labeling of ultrathin sections for GLYT1 and the NMDA receptor The material resistant to solubilization was removed by centri- revealed the co-existence of these proteins in PSDs within the fugation and the solubilized proteins were immunoprecipitated dentate gyrus (Fig. 6J). with the anti-GLYT1 antibody or with preimmune serum. The presynaptic/postsynaptic ratio of GLYT1 in the dentate Immunoblot analysis of the precipitated material revealed that gyrus contrasted with that found in other regions analyzed for anti-GLYT1 antibody but not the preimmune serum, precipitated comparative and control purposes. Thus, in the cerebellum, the NR2A subunit of the NMDA receptor (Fig. 8). However, where GLYT1 was abundantly expressed in terminals of parallel antibodies against the closely related transporter GLYT2, which is expressed exclusively in glycinergic neurons, were unable to immunoprecipitate NR2A from solubilized proteins obtained from the brainstem, which were processed in parallel to the forebrain tissue. Together, these data indicate that GLYT1 is associated with the NMDA receptor in the forebrain of the rat.

Discussion Through the generation of a novel polyclonal antibody against the amino terminal region of the GLYT1b isoform, we have high- lighted the close association between the glycine transporter

Figure 8. Co-immunoprecipitation of GLYT1 and NMDA receptor. Lysates were prepared from rat forebrain or from brainstem as described under Materials and Figure 7. Distribution of gold particles in synapses following immunolabeling for Methods. The forebrain lysate was used for immunoprecipitation with anti-GLYT1 GLYT1. Histograms showing the distribution of gold particles along the perpendicular antibodies or with control preimmune serum (lanes 1 and 2). The brainstem lysate was axis dentate gyrus synapses (A) or parallel fiber--PC synapses (B). The distances used for immunoprecipitation with anti-GLYT2 antibodies or with control preimmune between the centers of the 10 nm gold particles and the center of the synaptic cleft serum (lanes 3 and 4). Immunoprecipitated material was analyzed by immunoblotting, were grouped into bins of 6 nm wide. Negative values in the abscissa indicate the and the blots were reacted with anti NR2A (upper panel). The input lane corresponds direction of the presynaptic element. The data were pooled from 75 (A) and 62 (B) to 50 lg of forebrain extract. To control the efficiency of anti GLYT1 and anti-GLYT2 synapses, respectively. Synapses were selected for analysis only if the radius of the antibodies to immunoprecipitate, lanes 1 and 2 of the blot were reprobed with anti- postsynaptic density profile exceeded 150 nm. GLYT1 and lanes 3 and 4 with anti-GLYT2 (lower panels).

Cerebral Cortex April 2005, V 15 N 4 457 GLYT1 and glutamatergic synapses. Both conventional and (raised against the C-terminus), even using any of the different confocal microscopy, together with ultrastructural pre- and protocols applied here. This may be due to the inaccessibility of post-embedding electron microscopy, provided anatomical the neuronal C-terminal epitopes, a problem that does not seem evidence in support of this association. The specificity of the to exist for N-terminal epitopes. Nevertheless, the accessibility antibody was similar to that of antibodies raised against the of this epitope seems to be crucial to observe immunoreactivity carboxyl terminal sequences of GLYT1, at least when evaluated in axonal fibers, since this was only optimally detected under by immunoblotting or immunocytochemistry of transfected COS mild fixation protocols. cells. However, in tissue sections, this new antibody revealed the The presence of a neuronal form of GLYT1 has been presence of GLYT1 in locations where it has not been previously suggested on the basis of pharmacological experiments in detected. Specifically, the protein was now found in a large synaptosomes derived from the cortex, cerebellum and spinal number of neuronal elements as well as in glia. cord (Herdon et al., 2001; Raiteri et al., 2001). Our observations The staining of neuronal profiles was best appreciated in provide direct anatomical evidence for the existence of this forebrain regions, since in the brainstem and spinal cord, the neuronal form of the transporter and substantiate previous Downloaded from https://academic.oup.com/cercor/article/15/4/448/351216 by guest on 24 September 2021 intense glial staining masked the detection of neuronal ele- observations of a tight association between GLYT1 and gluta- ments. Double labeling experiments with the glutamatergic matergic neurotransmission at the mRNA level (Smith et al., marker vGLUT1 and the morphological analysis of cortical and 1992; Borowsky et al., 1993; Zafra et al., 1995b). Indeed, mRNA hippocampal tissue by EM revealed that many of the labeled for GLYT1 was observed in many neuronal types throughout the structures correspond to synaptic boutons that probably use brain, including, among others, the granular and pyramidal glutamate as a neurotransmitter. However, the association of neurons of the hippocampus and pyramidal neurons in the GLYT1 with glutamatergic markers was not ubiquitous and neocortex that are probably the source of the glutamatergic showed regional specificity. Thus, in the forebrain there was terminals containing GLYT1 detected in the present study. a preferential association with vGLUT1, while it was only The localization of GLYT1 in excitatory synapses that prob- sparsely associated with vGLUT2 in this tissue. However, in ably use glutamate as a neurotransmitter is of particular interest caudal regions there was an enrichment of GLYT1 in vGLUT2- given the controversial role of glycine in NMDA receptor labeled terminals. In the telencephalon, we did not find evi- function. The low micromolar levels of glycine measured in dence for the co-localization of GLYT1 with inhibitory termi- the extracellular and cerebrospinal fluids initially led to the nals, identified with antibodies against VIAAT, even in terminals conclusion that they would saturate the glycine sites on NMDA outlining cell bodies in the neocortex. These findings are receptors (Westergren et al., 1994). However, glycine trans- compatible with a recent immunohistochemical study that porters might reduce the glycine concentration in the local demonstrated the presence vGLUT1 in terminals outlining environment of the NMDA receptor to below 1 lM (Attwell, pyramidal and non-pyramidal neurons in the neocortex and in 1993; Supplisson and Bergman 1997; Berger et al., 1998; the neuropilar region (Alonso-Nanclares et al., 2004). Bergeron et al., 1998; Roux and Supplisson, 2000; Billups and The association of GLYT1 with glutamatergic neurotransmis- Attwell, 2003). Moreover, NMDA populations with low affinity sion was further supported by ultrastructural data, indicating for glycine have been described (Kew et al., 1998). Indeed, that GLYT1 concentrates not only in the presynaptic aspect of recent pharmacological and electrophysiological evidence sup- asymmetric glutamatergic synapses, but also in postsynaptic port a regulatory role for glycine transporters on NMDA re- densities, where it co-localized with NMDA receptors. The tight ceptor. Thus, the potent and selective inhibition of GLYT1 with association of GLYT1 with NMDA receptors was reinforced by NFPS has implicated this transporter in long-term potentiation the immunoprecipitation of GLYT1-NMDA receptor detergent in the dentate gyrus induced by the stimulation of the perforant insoluble complexes with anti-GLYT1 antibodies. It remains to path (Kinney et al., 2003). Furthermore, the administration of be determined whether this association is mediated by a direct NFPS in vivo produced physiological responses consistent with interaction between the transporter and the receptor or in- those expected after potentiation of NMDA receptor function volves other interposed proteins. Interestingly, both the NR2A (Kinney et al., 2003). Moreover, experiments performed in subunit and the C-terminal sequence of GLYT1 contain a PDZ- mutant mice that expressed only 50% of GLYT1 (heterozygous interacting motif that could tether GLYT1 to the postsynaptic Glyt1+/–) revealed an enhanced hippocampal NMDA receptor density, where PDZ proteins form a complex scaffold that links function. In the water maze, these mice exhibited better spatial NMDA receptors to regulatory proteins. retention. These animals were also protected against an am- In addition, and as shown in our previous studies (Zafra et al., phetamine disruption of sensory gating, suggesting that drugs 1995a), GLYT1 was also detected in glia, particularly in the which inhibit GLYT1 might have both cognitive enhancing and caudal CNS, as well as in amacrine cells of the retina. The high antipsychotic effects (Tsai et al., 2004). concentration of GLYT1 in glial processes in glycinergic areas Finally, a novel role for glycine in regulating NMDA receptor suggests that the glial transporter is responsible for the removal internalization has been recently suggested. Apparently this of glycine from inhibitory glycinergic synapses. Indeed, recent novel function requires higher concentrations of glycine than experiments in GLYT1 deficient mice revealed a large increase those necessary to operate the ion channel (Nong et al., 2003). of glycinergic inhibition in hypoglossal motoneurons that Thus, the transporter might regulate the turnover of the NMDA contributed to respiratory failure and the premature death of receptor at the cell surface, rather than the channel function. pups (Gomeza et al., 2003a). Since motoneurons are abundantly In summary, the anatomical and biochemical data reported in decorated with GLYT1 immunoreactive glia, our data are this study reveal the presence of GLYT1 in subpopulations of consistent with glial GLYT1 being essential for the regulation glutamatergic synapses of the telencephalon, in addition to of glycinergic inhibition of motoneurons. previously reported glial transporter. These data explain the To date, it has not been possible to visualize the distribution partially contradictory distribution of mRNA and protein re- of GLYT1 in neurons with the antibodies previously available ported previously, and they support an active role of GLYT1 in

458 GLYT1 in Excitatory Synapses d Cubelos et al. regulating the glycine concentrations in the local environment Kemp JA, Leeson PD (1993) The glycine site of the NMDA receptor five of the NMDA receptor. years on. Trends Pharmacol Sci 14:20--25. Kew JNC, Richards JG, Mutel V, Kemp JA (1998) Developmental Notes changes in NMDA receptor glycine affinity and ifenprodil sensitivity reveal three distinct populations of NMDA receptors in individual rat We thank the expert technical help of E. Nuń˜ez, and Prof. Andrew Todd cortical neurons. J Neurosci 18:1935--1943. for comments on the manuscript. Anti-VIAAT antibodies were kindly Kew JNC, Koester A, Moreau JL, Jenck F, Ouagazzal AM, Mutel V, Richads provided by Dr B. Gasnier. This work was supported by the Spanish JG, Trube G, Fisher G, Montkowski A, Hundt W, Reinscheid RK, Direccioń General de Investigacioń Cientı´fica y Tećnica (BMC2002- Pauly-Evers M, Kemp JA, Bluethmann H (2000) Functional conse- 03502), Fondo de Investigaciones Sanitarias, Comunidad Autońoma de quences of reduction in NMDA receptor glycine affinity in mice Madrid and an institutional grant from the Fundacioń Ramoń Areces. carrying targeted point mutations in the glycine binding site. Address correspondence to Dr F. Zafra, Centro de Biologıá Molecular J Neurosci 20:4037--4049. ‘Severo Ochoa’, Facultad de Ciencias, Universidad Autońoma de Madrid, Kinney GG, Sur C, Burno M, Malorga PJ, Williams JB, Figueroa DJ, 28049-Madrid, Spain. Email: [email protected]. Wittmann M, Lemaira W, Conn PJ (2003) The glycine transporter

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