Cloning, Expression, and Localization of a Rat Brain High-Affinity Glycine Transporter JOHN GUASTELLA*, NICHOLAS Brechat, CHRISTINE Weigmannt, HENRY A

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Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 7189-7193, August 1992 Neurobiology Cloning, expression, and localization of a rat brain high-affinity glycine transporter JOHN GUASTELLA*, NICHOLAS BRECHAt, CHRISTINE WEIGMANNt, HENRY A. LESTER*, AND NORMAN DAVIDSON* *Division of Biology, 156-29, California Institute of Technology, Pasadena, CA 91125; and tDepartments of Anatomy and Cell Biology and Medicine, Brain Research Institute and Center for Ulcer Research, University of California, Los Angeles School of Medicine and Veterans Administration Medical Center-Wadsworth, Los Angeles, CA 90073 Contributed by Norman Davidson, May 6, 1992

ABSTRACT A cDNA clone encoding a glycine transporter ranges from "-44% for the GABA transporter (GAT1) versus has been isolated from rat brain by a combined PCR and the norepinephrine transporter (NET1) to =64% for NET1 plaque-hybridization strategy. mRNA synthesized from this versus the dopamine transporter (DAT1), indicating that clone (designated GLYT1) directs the expression of sodium- subfamilies exist within the superfamily. In addition to an and chloride-dependent, high-affinity uptake of [3H]glycine by overall homology, members of this superfamily contain sev- Xenopus oocytes. [3H]Glycine transport mediated by clone eral regions of sequence identity distributed throughout the GLYT1 is blocked by sarcosine but is not blocked by methyl- length of the protein. These regions can be used to design aminoisobutyric acid or L-alanine, a substrate specificity sim- PCR primers which can be employed to clone additional ilar to that described for a previously identified glycine-uptake members of the superfamily from brain and other tissues. In system called system Gly. In situ hybridization reveals that this paper we report the results of such a cloning approach, GLYT1 is prominently expressed in the cervical spinal cord and which has yielded a rat brain cDNAt encoding a sodium- and brainstem, two regions of the central nervous system where chloride-dependent high-affinity glycine transporter. glycine is a putative neurotransmitter. GLYT1 is also strongly expressed in the cerebellum and olfactory bulb and is expressed at lower levels in other brain regions. The open reading frame MATERIALS AND METHODS of the GLYT1 cDNA predicts a protein containing 633 amino RNA Isolation and PCR. Total RNA was prepared from C6 acids with a molecular mass of =70 kDa. The primary struc- glioma cells and rat tissues by the acid guanidinium/phenol ture and hydropathicity profile of GLYT1 protein reveal that method (12). cDNA was synthesized directly from 5 Mg of this protein is a member ofthe sodium- and chloride-dependent total RNA or mRNA and amplified by the PCR and standard superfamily of transporters that utilize neurotransmitters and protocols. The primers chosen for amplification consisted of related substances as substrates. a 128-fold degenerate sense primer corresponding to bases 375-394 of GAT1 with the sequence (5'-- 3') GAG CTC GTC Termination of synaptic activity is thought to occur through GAC AAG AAC GG(TCAG) GG(TCAG) GG(TCAG) removal of neurotransmitter from the synaptic cleft by ion- GC(TC) TT and a 48-fold degenerate antisense primer cor- coupled, high-affinity neurotransmitter transport proteins responding to bases 1108-1030 ofGAT1 with the sequence (5' (neurotransmitter transporters) located in neuronal and glial -* 3') GGG CCC TCT AGA AA (GA)AT CTG (GA)GT plasma membranes. Drugs that block the action of these (GAT)GC (GA)GC (AG)TC. The upstream primer corre- transporters can modulate neural function, probably by in- sponds in the GAT1 protein to amino acids 76-82 with the creasing the duration of neurotransmitter action. Some neu- sequence KNGGGAF, and the downstream primer corre- rotransmitter transporters, such as those for the monoam- sponds to amino acids 287-293 with the sequence DAATQIF. ines, are the sites of action for clinically important drugs (for With these primers the PCR will generate a 677-base-pair (bp) example, antidepressants) (1), as well as drugs of abuse (for product from GAT1 mRNA and a 710-bp product from NET1 example, cocaine) (2). Other neurotransmitter transporters, mRNA. The amplification protocol consisted of a 1-min such as those for the inhibitory amino acid y-aminobutyric denaturation at 94°C, a 4-min annealing at 55°C, and a 2-min acid (GABA), are potential targets for drugs with anticon- extension at 72°C for 25-30 cycles on a Perkin-Elmer DNA vulsant properties (3). Therefore, an understanding of the thermal cycler. A sample of the amplification reaction was structure and function of neitrotransmitter transporters may analyzed by agarose gel electrophoresis, and the appropriate lead to a better understanding of synaptic regulation and band was gel-purified and subcloned into pBluescript SK(-). could also provide a rational basis for the development of RNA Blot. Two micrograms of mRNA was subjected to additional, more-specific anti-transporter drugs. agarose gel/formaldehyde electrophoresis and transferred to The recent isolation ofcDNA clones encoding transporters filter paper. The blot was hybridized overnight at 42°C under for GABA (4), the monoamines norepinephrine, dopamine, high-stringency conditions [50%o formamide/6x standard sa- and serotonin (5-10), and a nonneurotransmitter, betaine line phosphate/EDTA (SSPE; lx SSPE = 0.15 M NaCI/10 (11), has revealed that these carriers comprise a superfamily mM phosphate, pH 7.4/1 mM EDTA)/5x Denhardt's solu- of homologous proteins. Predictions of membrane topology tion/salmon sperm DNA at 100 tg/ml]. The final stringency suggest that these transporters all contain =12 membrane- wash was done in 0.2x standard saline citrate at 65°C for 15 spanning domains, a structural motif shared by a number of min. The filter was exposed to film for 2-4 days with two carriers that, however, share little mutual sequence homol- intensifying screens at -70°C. ogy. The amino acid-sequence identity between the known cDNA Isolation and Sequencing. A AZAP rat brain cDNA members of the neurotransmitter transporter superfamily library (13) containing inserts in the 2-4 kilobase-pair (kbp)

The publication costs of this article were defrayed in part by page charge Abbreviation: GABA, y--aminobutyric acid. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M95413).

7189 Downloaded by guest on September 27, 2021 7190 Neurobiology: Guastella et al. Proc. Natl. Acad. Sci. USA 89 (1992) A B of 50 pl, and uptake assays were done as described (5). Each 1 2 3 4 5 data point represents the mean of three to five oocytes. M_40.. In Situ Hybridization. Rat brain and cervical spinal cord were fixed with 4% paraformaldehyde and processed as described (15). Twenty-five- to 30-tum frozen sections were prepared in both sagittal and horizontal planes and were incubated, free-floating, with 35S-labeled antisense RNA probes. Control sections were probed with 35S-labeled sense RNA to determine nonspecific hybridization. Sequence Analysis. DNA and amino acid sequences were FIG. 1. (A) Ethidium bromide-stained agarose gel of the DNA analyzed on a personal computer with the program PCGENE products generated with described primers and C6 glioma cell cDNA and on a DEC VAX computer with the Genetics Computer as template. Arrow denotes a major product of -700 bp. Lanes: 1 and Group package. Data bases were searched on the National 2, products with C6 glioma cell mRNA as starting material; 3 and 4, Center for Biotechnology Information computer with the products with C6 glioma cell total RNA; 2 and 4, products with a program BLAST (16). 2-fold dilution of the respective cDNA mixtures. (B) RNA blot of mRNA from C6 glioma cells (lane 1), rat liver and kidney (lanes 2 and 3), and rat forebrain and cerebellum (lanes 4 and 5) probed with the RESULTS AND DISCUSSION 32P-labeled 700-bp PCR product from C6 glioma cells. Arrow denotes a single transcript of -3.3 kb. A faint band, visible in lane 2 (liver Cloning Strategy. C6 glioma cells have been reported to mRNA) of the original autoradiogram, is difficult to see in this possess transporters for a variety of neurotransmitters, in- photograph. cluding GABA (17, 18), aspartate (19), glutamate (19), and glycine (20, 21). We, therefore, chose these cells as a poten- size range was screened with the same random primer- tial source of additional members of the neurotransmitter- labeled PCR product used to probe the RNA blot and under transporter superfamily. When RNA from C6 cells is ampli- the same hybridization conditions. Six positive clones were fied by the PCR with the degenerate primers described in identified. The phagemids were rescued from each clone and Materials and Methods, a major product of -700 bp is seen used for further DNA (Fig. 1A). The nucleotide sequence of this PCR product analysis. Dideoxynucleotide sequenc- encodes a peptide with significant homology to both GAT1 ing was done by a combination ofnested deletions and primer and NET1 proteins (data not shown). RNA blot analysis with walks. the PCR product as a probe reveals a single transcript of -3.3 Xenopus Oocyte Expression and Transport Assays. Xenopus kilobases (kb) in C6 cell mRNA and in rat forebrain and oocytes were defolliculated and maintained in ND96 medi- cerebellum mRNA (Fig. 1B). There is a barely detectable um/5% horse serum (14). mRNA was synthesized from 5 ug signal in rat liver mRNA and no detectable signal in kidney ofplasmid template and gel-purified on a Sephadex G-50 spin mRNA. This result indicated that the gene corresponding to column. Ten nanograms of mRNA was microinjected into this PCR product is expressed in brain but is not expressed each oocyte, and the samples were allowed to translate for (or is expressed at low levels) in nonneural tissue. Therefore, 4-7 days. Controls consisted of oocytes injected with 50 nl of a size-selected AZAP rat brain cDNA library was screened water. [3H]Glycine and drugs were added to a total volume with the C6 cell PCR product, and six positive clones were

A Water-injected 0 8.0 M RNA-injected -

6.0 [ 0 I-U I- I.Z8 FIG. 2. ofGLYT1 4.0 Expression [ a-- in Xenopus oocytes. Assays were .0:S done 4 days after injection of is oocytes with 10 ng ofmRNA or 50 2.0 nl ofwater. Each point represents 10 the mean ± SEM of [3H]glycine a _n_ uptake by three to five oocytes. 0 All assays were done at room -4 [3HJGABA [3HlGLY [3H]GLU [3HJ3-ALA Control No Control No temperature for 1 hr. (A) Uptake Sodium Chloride of [3H]GABA, [3H]glycine, [3H]- glutamic acid, and ,B-[3H]alanine. Each amino acid was used at a final concentration of 1 ,uM. (B) Uptake of [3H]glycine in normal 8. saline and saline in which sodium was replaced by lithium (No So- dium) or saline in which chloride C- was replaced by acetate (No Chloride). (C) Inhibition of [3H]- .4 glycine uptake by three amino acids or amino acid analogs. Unla- beled L-alanine (L-ALA), methyl- aminobutyric acid (MAIB), or sarcosine (SARC) was included in the incubation mixture at a final Water-injected RNA-injected concentration of 500 ,M. Downloaded by guest on September 27, 2021 Neurobiology: Guastefla et al. Proc. Natl. Acad. Sci. USA 89 (1992) 7191 plaque-purified. Partial nucleotide sequences from these 633 amino acids with a molecular mass of 70,570 Da (Fig. 4). clones indicated that five of the six clones were clearly The protein contains four putative N-glycosylation sites related to each other and to the original PCR product, located in a region, which, for GATi, is proposed to form a whereas the sixth represented an unrelated clone. One of large extracellular loop (4). There are five putative protein these five clones (clone 9-iB) appeared to be full-length at the kinase C phosphorylation sites. A Kyte-Doolittle analysis of 5' end of the open reading frame, and this clone was chosen GLYT1 yielded a hydrophobicity profile almost identical to for further analysis and expression. that seen for GATi (4) and other members ofthis transporter Functional Expression. Oocytes injected with 9-lB mRNA superfamily, suggesting that the membrane topology of were tested for their ability to take up radiolabeled neuro- GLYT1 is similar to proposed models (4, 5, 8). Further transmitters or related compounds. Fig. 2A shows that analysis with CLUSTAL and PALIGN (PCGENE) showed that oocytes expressing clone 9-lB do not significantly accumu- GLYT1 is homologous to all known members of the neuro- late [3H]GABA, [3H]glutamate, or /[3H]alanine above back- transmitter-transporter superfamily, with identities ranging ground levels. In contrast, the accumulation of [3H]glycine is from 37% to 41% and overall homologies (identity plus enhanced markedly in oocytes injected with 9-lB RNA, similarity) of 53% to 61%. A search of the GenBank and reaching levels of -10-20 pmol after 1 hr of incubation in 2.5 Protein Identification Resource data bases with the program tkM [3H]glycine. This result indicated that clone 9-lB en- BLAST did not reveal significant homologies between GLYT1 codes a glycine transporter, and it was designated GLYT1. and any cloned genes other than the known members of this Uptake by oocytes injected with GLYT1 mRNA depends superfamily. on both extracellular sodium and extracellular chloride, Cellular Localization. In situ hybridization was used to indicating that the transporter encoded by this clone mediates study distribution of GLYT1 mRNA in the rat central ner- ion-coupled active transport of [3H]glycine (Fig. 2B). Inter- vous system (Fig. 5 A-D). There is prominent labeling in all estingly, the moderate level of [3H]glycine uptake by control the nuclei oocytes is also sodium- and chloride-dependent, indicating lamina of the cervical spinal cord, brainstem that Xenopus oocytes possess an endogenous transport system for glycine. This fact is not surprising because uptake 350 systems for metabolically important amino acids are found in both neural and nonneural tissues (22). Oocytes injected with 300 GLYT1 mRNA take up [3H]glycine by a high-affinity mech- anism (Fig. 3 A and B). Kinetic analysis revealed a Km of 33 250 ± 8 uM (mean ± SEM, n = 3), which is similar to the Km values reported previously for high-affinity glycine uptake in the central nervous system (23-25). This Km value is almost 0 200 3-fold lower than that reported for [3H]glycine uptake by C6 glioma cells (Km = 95 ,IM) (21). The Vm,, value for the cloned 4U, 150 transporter varied, depending on the batch of oocytes, from 120 to 390 pmol per oocyte per hr (mean = 246 ± 78). The V) 100 oocyte glycine transporter also appears to be a high-affinity C- transporter with a Km of =20 ,uM and a Vma. of =20 pmol per oocyte per hr (data not shown). 50 At least three uptake systems able to transport glycine, termed system Gly, system ASC, and system A, have been 0 demonstrated in a variety of tissues and cell types by using 0 20 40 60 80 100 120 amino acids and amino acid analogs as substrates and competitive inhibitors (26). Therefore, the substrate specificity of [Glycine] (JLM) GLYT1 was examined by using several of the subtype- specific compounds (at a final concentration of 500 ,uM) as competitive inhibitors of [3H]glycine uptake by mRNA- injected oocytes (Fig. 2C). Sarcosine (N-methylglycine), a system Gly substrate, inhibits [3H]glycine uptake by oocytes injected with GLYT1 mRNA by >85%. By contrast, L-alanine, a system ASC substrate, and methylaminoisobutyric acid, a system A substrate, are almost completely ineffective as inhibitors. This inhibition profile suggests that GLYT1 represents a system Gly-like transporter, in agreement with the substrate specificity defined for the native glycine transporter expressed by C6 cells (21). However, the substrate specificity of GLYT1 differs from that described in earlier work on the rat spinal cord (23), where sarcosine was reported to have no significant inhibitory effect at concen- trations as high as 1 mM. Thus, GLYT1 may represent one of several glycine-transporter subtypes expressed in brain. The oocyte endogenous glycine transporter shows an inhibition profile different from GLYT1. The former is inhibited almost 90%o by L-alanine (Fig. 2C), L-serine, and L-Cys- teine (data not shown). This transporter is not blocked by V/s methylaminoisobutyric acid and is only partially inhibited by FIG. 3. Kinetics of [3H]glycine uptake. Michaelis-Menten (A) sarcosine (Fig. 2C). Therefore, the endogenous transporter and Eadie-Hofstee (B) plots of [3H]glycine uptake by oocytes appears similar to system ASC. injected with GLYT1. V, velocity; V/S, velocity/substrate. These Sequence Analysis. Nucleotide sequencing of the GLYT1 values represent the difference between [3H]glycine uptake by cDNA revealed an open reading frame encoding a protein of mRNA-injected oocytes and water-injected control oocytes. Downloaded by guest on September 27, 2021 7192 Neurobiology: Guastella et al. Proc. Natl. Acad. Sci. USA 89 (1992)

v GLYTI M V - - - G K G A K G M L N C. A V P S E A - T K 7[D QN TI GATI MI- - A-I'DNSKVADGQI STEVS IEAPVASDKIPKTIVKrKT1VV i,,yKs NETl !M L L A R M N P Q V Q P E N N G A D I C P EQ F-L R A R K T AE Lj 1 tu K F F V i, F;i.!

GLYT1 TRG..NWGNQIETVLTSVGYAVG-LGNRF GATI G D L p DRIDTIWIKGRFDIFLMSCIVGIYIAIIGLGNVWRFP'~ CG-'KNmA.(';.,FLIIF'I FL AS lil! P N K GI G A F L I I NE I G D A Q P R G T W G KQ I D F L L S V G F A Y L A N V W R F I Cl N i;. _ F

GLYrl M T]V F FFM-'j~LSF [QFASQ[C C [G VWR S [T]MFK GVGN(,~ G NIlMVVST YITGI i, V i A V L S W L N I I GATI TI LI F A G V P l. F L L F C S L G Q Y T S G G L G V W K L A P|M| F K A K F K C A L. A G F .5c NET1 F L I I AG M EL A L Q Y N R E A A T I C F t( Y V .PLF.YM LiG [G [VW .. -s mu - u - -

H V W . D N ZSr GLYFI lN V V _ C I A FYYF F S sM7Ti LP W A Y 7 N N P N T P D Ar =F 1. V N T YY I V I I S W A I y Y Y 1 11-1T PX I N GATI L KQ|CI|DNDNP TI'R(I S 2 PN 'r F NETl Y Y N JI I A W S L Y Y. L F SL FW 1 N L DT[D C H TWNS NC ..2 V K S D D F E R I jP 1 I GLYTl T A I. S G N L S H 1 F N Y L.. Q R T S P S F W R 1. L D W M Q D C1 L P K R VI P A GATI ' M T S - - - - A Vr V |IE F N9 H1 M1 PIC;|Q G V L E C. D I Q V, QI NEll V L G N 1-i T K Y 1) K N K F T P A AL F Y E R L. G r 1

.121-Fb F V R V T G, T T GLYTI C G V S W v V V F L C L t R C V K S S G K V V Y F TIA TIF G I1.FTT\ L V A K E h4~ GATI T L|A I AWV L V Y F C I W K G V G W T G K V V Y F SIA IYL F F R ('\ I fl, L H V 'I P G A N NET! CWLM Y V V I V L Y F S L W K G V K T S G K V V W I TL L L \,' L. U - U - EKE - U UU - C A W IK- 7 1L GLYTI [G IIM YYL T P K W D K I L E A K V W G A A S Q I F G IGII I IT M A S;K7'N HN G L IA S GATl G IlL F YiI T P N F R K L S D S E V W L F) A A T Q I FI GI IL G |SGT1 L FHHNIN NET! L N AW L H I D F Y R L K E A T W IID A A|T FQI JA G F IVL I A F A.q NI KSF ID %AD GLYT1 IY R DISI I s I T C AE |V Y A|G F|V I F S I LW M A N H G V D V S R H PG AFV M F M A H V IK R S! AD A SCJ P LIAIFl L GATI Y R DI S I I v c C I N| S CI T S A1G FJV I F S I VIG AAS NETr IYR DA L L T S s I 1 |F V S GFA I FS I M A H E I K V N I E D JA T F G A G V F I u.n_ ! N GLYrl Al Y P E Al L T Ll L IP I S P LW S L L F F F M L IL LGTQFC L LFfTL-v[ A V .'v E V N F TA' 41I P L M I DSQF( TV|FIG FI1T V v E FP R 1, F. 4,ih L A L GATI A| Y P E AV T Q I |S|P L|W A I L F F M L LG V NET! LIYP E A| I S TL S G LS T F W JA V V F FV M L L A L D S S M C C ME A V I L C L A D F Q % I- V I L K GH7A V A G F L G I PL T S Q A G I Y W L L MT] NY A c F T iLV (, GLYrl Q K T Y V T L A ' L | \ GATI R N R R E L F 1 - A AIV|C I V S Y L I G L S N I T Q G G I Y V F K FIDI Y S A S M|S;LLFNI L T I L F .N V' NETI K R H R K L F T - F GWV T F S T FW L A L F IC I T K G G I Y V L T LW I F A ru S I I I GLYTI I M C V S I M Y Y G H R N Y F Q D L G F P P L F F Q I KR WN. _ T F F I 1 F T T GAT1 FE C V S I S W IY G V N R F Y D N |M|ml V G S R P C I WW K L cC'W!t; ;sIS I. 1: PI V A V FLV S F s ND m G F RLP G L Y W R L c w K F V S PlAF~I. i.:VVVVS NErl M E A I G V S W Y V D R I QIQ _m mu___ [ _u A- 1 F GLYTI V1QYRHIHYNHHfQYHYGWAVAI F L M A L S 1 V I C I r Qi L K1) PT T i W L MIAL SSI V P A Y GATI A V Q MTI P|LT|MG SYIV FI P|KIWIG Q G V i N1 \1F i LLK,j1. V V P~ V Y D A N W V W E L. S1M NETI I I N F KWP LW Y D WI FW PH |G F GLYTI L[K N A T KH S R D W G P A L L E H R T G R Y A P T I1 1 PS P P. ( .. QlR -N GATI QIR LQV M I Q P - S E - - D I V R P E N G P E Q r Q A G z < A S K F A - NET1 E| R LA Y G I TLWE NE - - -HLVAQRD I RQFQLI Q A

GLYTI I V G S N G S S R L Q D s R _ &13 GATI 599 NErl -_ 617

FIG. 4. Amino acid sequence of GLYT1 aligned with the amino acid sequences of GAT1 (4) and NET1 (5). Residues identical among all three proteins are boxed; residues representing conservative substitutions are shaded. Putative transmembrane regions are denoted by bars, potential N-glycosylation sites are marked with asterisks, and potential protein kinase C phosphorylation sites are marked by arrowheads. Alignment was done with the program CLUSTAL in PCGENE.

(including the spinal trigeminal nucleus, Fig. 5A), the cere- bridization to glial cells cannot be ruled out. Cerebellar label- bellum (Fig. 5B), and the olfactory bulb (Fig. SC). Lighter ingis confined to the Purkinje cell layer, where the silvergrains labeling is present in other medullary, pontine, midbrain, form a continuous band (Fig. 5B). The Purkinje cells them- thalamic, and hypothalamic nuclei (data not shown). Label- selves are not labeled, and labeling does not appear to be ing above background could not be detected in the cerebral associated with basket, Golgi, or Lugaro cells, which may be cortex, caudate-putamen and globus pallidus, hippocampal located near the Purkinje cell layer. Instead, the labeling formation, or septal nuclei. Furthermore, there is no specific pattern indicates that GLYT1 is expressed in Bergmann glia, labeling in white-matter fiber tracts. No specific labeling was which are located in this layer. The absence of GLYT1 mRNA observed in control sections incubated with sense RNA in the cerebellar Golgi neurons contrasts with previous reports probes (Fig. SD). Thus, GLYT1 is not expressed in all regions that, in the rat, these neurons take up [3H]glycine (27) and of the central nervous system. Instead, its expression is contain glycine immunoreactivity (28, 29); thus, this finding limited to those areas where glycine is thought to function as provides further evidence for the possibility of multiple gly- a neurotransmitter and to other specific regions where glycine-transporter subtypes. In the olfactory bulb, there is cine might play a neurotransmitter role. prominent labeling of many cells located in the glomerular Labeling in the brain stem (Fig. SA) and cervical spinal cord layer (Fig. 5C). Labeled cells also occur in the external is clearly associated with neuronal cell bodies, although hy- plexiform layer, the mitral cell body layer, and the granule cell Downloaded by guest on September 27, 2021 Neurobiology: Guastefla et al. Proc. Natl. Acad. Sci. USA 89 (1992) 7193 Note Added in Proof. After this paper was submitted, Smith et al. (31) reported the isolation of a rat brain glycine transporter cDNA clone whose sequence and distribution is similar, but not identical, to that of GLYT1. We thank M. Quick for oocyte preparation, L. Czyzyk for assistance with nucleotide sequencing, and A. Figl for assistance with oligonucleotide synthesis. This work was supported by National Institutes of Health Grant EY04067, Neuroendocrine Anatomy Core Grant DK4130, and Veterans Administration Medical Research Funds to N.B.; National Institutes of Health Grant NS-11756 to H.A.L. and N.D.; and a National Institutes of Health Postdoctoral Fellowship to J.G. 1. Glowinski, J. & Axelrod, J. (1964) Nature (London) 204, 1318-1319. 2. Horn, A. S. (1990) Prog. Neurobiol. 34, 387-400. 3. Krogsgaard-Larsen, P., Labouta, I. M., Meldrum, B., Croucher, M. & Schousboe, A. (1981) in Neurotransmitter, Seizures and Epilepsy, eds. Morselli, P. L., Loscher, W., Lloyd, K. G., Meldrum, B. & Reynolds, E. H. (Raven, New York), pp. 23-33. 4. Guastella, J., Nelson, N., Nelson, H., Czyzyk, L., Keynan, S., Miedel, M. C., Davidson, N., Lester, H. A. & Kanner, B. I. (1990) Science 249, 1303-1306. 5. Pacholcyzk, T., Blakely, R. D. & Amara, S. G. (1991) Nature (London) 350, 350-353. 6. Kilty, J. E., Lorange, D. & Amara, S. G. (1991) Science 254, 578-579. 7. Shimada, S., Kitayama, S., Lin, C.-L., Patel, A., Nanthaku- mar, E., Gregor, P., Kuhar, M. & Uhl, G. (1991) Science 254, 576-578. 8. Usdin, T. B., Mezey, E., Chen, C., Brownstein, M. J. & Hoff- man, B. J. (1991) Proc. Natl. Acad. Sci. USA 88, 11168-11171. 9. Blakely, R. D., Berson, H. E., Fremeau, R. T., Caron, M. G., Peek, M. M., Prince, H. 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T., Werrbach, K., Vance, C. & Haber, B. (1974) Horizontal section through cerebellum showing a band of labeling in Brain Res. 66, 265-274. the Purkinje cell layer. ML, molecular cell layer; PCL, Purkinje cell 18. Schrier, B. K. & Thompson, E. J. (1974) J. Biol. Chem. 249, layer; GCL, granule cell layer; WM, white matter. (C) Horizontal 1769-1780. section through the main olfactory bulb. (D) Control section. Same 19. Erecinska, M., Troeger, M. B., Wilson, D. F. & Silver, I. A. as C, except section was incubated with sense mRNA probe. GL, (1986) Brain Res. 369, 203-214. glomerular layer; EPL, external plexiform layer; MCL, mitral cell 20. Zafra, F. & Gimenez, C. (1986) Brain Res. 397, 108-116. layer; GRL, granule cell layer. (Bar = 200 pm for A and 100 ,m for 21. Zafra, F. & Gimenez, C. (1989) Biochem. J. 258, 403-408. B-D.) 22. Schultz, S. G. & Curran, P. F. (1970) Physiol. Rev. 50, 637-710. 23. Johnston, G. A. R. & Iversen, L. L. (1971) J. Neurochem. 18, layer (Fig. 5C). Although localization of [3H]glycine uptake in 1951-1961. the 24. Staatz-Benson, C. & Potashner, S. J. (1987) J. Neurochem. 49, olfactory bulb is equivocal (30), recent immunocytochem- 128-137. ical studies suggest that the periglomerular neurons and some 25. Debler, E. A. & Lajtha, A. (1987) J. Neurochem. 48,1851-1856. cells in the granule cell layer contain glycine (29). As in the 26. Christensen, H. N. (1975) Curr. Top. Membr. Transp. 6, 227- other labeled regions, we cannot rule out the possibility that 258. some glia cells in the olfactory bulb express GLYT1. 27. Wilkin, G. P., Csillag, A., Balazs, R., Kingsbury, A. E., Wil- Availability of the GLYT1 cDNA should facilitate our son, J. E. & Johnson, A. L. (1981) Brain Res. 216, 11-33. understanding of glycinergic metabolism in the central ner- 28. Ottersen, 0. P., Davanger, S. & Storm-Mathisen, J. (1987) Brain Res. 211-221. vous studies Exp. 66, system. In addition, further in situ hybridization 29. Pourcho, R. G., Goegel, D. 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