 |
 Previous Article | Next Article 
The Journal of Neuroscience, July 1, 2002, 22(13):5442-5451
A Third Vesicular Glutamate Transporter Expressed by Cholinergic
and Serotoninergic Neurons
Christelle
Gras1, 2, *,
Etienne
Herzog1, *,
Gian
Carlo
Bellenchi2,
Véronique
Bernard3,
Philippe
Ravassard4,
Michel
Pohl5,
Bruno
Gasnier2,
Bruno
Giros1, and
Salah
El
Mestikawy1
1 Faculté de Médecine, Institut National de
la Santé et de la Recherche Médicale (INSERM)
Unité 513, 94010 Créteil Cedex, France,
2 Institut de Biologie Physico-Chimique, Centre National de
la Recherche Scientifique (CNRS) Unité Propre de Recherche 1929, 75005 Paris, France, 3 CNRS Unité Mixte de Recherche
(UMR) 5541, Université Bordeaux 2, 33076 Bordeaux Cedex, France,
4 CNRS UMR 7091, Hôpital
Pitié-Salpétrière, 75013 Paris, France, and
5 Faculté de Médecine Pitié
Salpétrière, INSERM Unité 288, 75013 Paris,
France
 |
ABSTRACT |
Two proteins previously known as Na+-dependent
phosphate transporters have been identified recently as vesicular
glutamate transporters (VGLUT1 and VGLUT2). Together, VGLUT1 and VGLUT2 are operating at most central glutamatergic synapses. In this study, we
characterized a third vesicular glutamate transporter (VGLUT3), highly
homologous to VGLUT1 and VGLUT2. Vesicles isolated from endocrine cells
expressing recombinant VGLUT3 accumulated L-glutamate with
bioenergetic and pharmacological characteristics similar, but not
identical, to those displayed by the type-1 and type-2 isoforms.
Interestingly, the distribution of VGLUT3 mRNA was restricted to a
small number of neurons scattered in the striatum, hippocampus,
cerebral cortex, and raphe nuclei, in contrast to VGLUT1 and VGLUT2
transcripts, which are massively expressed in cortical and deep
structures of the brain, respectively. At the ultrastructural level,
VGLUT3 immunoreactivity was concentrated over synaptic vesicle clusters
present in nerve endings forming asymmetrical as well as symmetrical
synapses. Finally, VGLUT3-positive neurons of the striatum and raphe
nuclei were shown to coexpress acetylcholine and serotonin
transporters, respectively. Our study reveals a novel class of
glutamatergic nerve terminals and suggests that cholinergic striatal
interneurons and serotoninergic neurons from the brainstem may store
and release glutamate.
Key words:
glutamate; VGLUT3; neurotransmitter
transporter; synaptic vesicle; excitatory
neurotransmission; brain
| |
INTRODUCTION |
Glutamate, a neurotransmitter used
by a majority of excitatory connections in the mammalian brain, has to
be loaded into synaptic vesicles by proton-dependent transporters
before its exocytotic release (Ozkan and Ueda, 1998 ; Reimer et al.,
1998; Erickson and Varoqui, 2000; Gasnier, 2000). Brain-specific
Na+-dependent inorganic phosphate
transporter (Ni et al., 1994) and differentiation-associated
Na+-dependent inorganic phosphate
transporter (Aihara et al., 2000), two members of the
Na+-dependent inorganic phosphate
transporter family, are now unambiguously established as two vesicular
glutamate transporters (VGLUT1 and VGLUT2) by an array of biochemical,
anatomical, electrophysiological, and genetic evidence (Dent et al.,
1997; Lee et al., 1999; Bellocchio et al., 2000; Takamori et al., 2000,
2001; Bai et al., 2001; Fremeau et al., 2001; Herzog et al., 2001).
Both transporters are abundantly expressed in the brain (Ni et al.,
1995; Hisano et al., 1997). VGLUT1 is massively present in excitatory
neurons from the cerebral and cerebellar cortices, as well as the
hippocampus, whereas most glutamatergic neurons from the diencephalon
and rhombencephalon preferentially use VGLUT2 (Fremeau et al., 2001;
Herzog et al., 2001; Varoqui et al., 2002). At the subcellular level,
VGLUT1 and VGLUT2 are found in synaptic vesicles located in terminals forming asymmetrical contacts (Bellocchio et al., 1998; Fremeau et al.,
2001; Fujiyama et al., 2001; Hayashi et al., 2001; Sakata-Haga et al.,
2001; Takamori et al., 2001; Varoqui et al., 2002), the hallmark of
glutamatergic terminals (Shepherd, 1998). Together, VGLUT1 and VGLUT2,
with their complementary distributions, seem to account for most of the
known glutamatergic neurons of the CNS (Fremeau et al., 2001; Varoqui
et al., 2002).
We now report the isolation of VGLUT3, an unforeseen third vesicular
glutamate transporter. Although structurally and functionally closely
related to the widely expressed VGLUT1 and VGLUT2, this novel
transporter exhibits several unique features. In particular, it is
found in all cholinergic interneurons of the striatum, as well as in
serotoninergic neurons from the raphe nuclei.
| |
MATERIALS AND METHODS |
cDNA cloning and expression in BON
cells. VGLUT3 full-length clones were obtained by screening
a  Zap II rat hippocampus cDNA library (Stratagene, La Jolla,
CA) as described previously (Herzog et al., 2001). Inserts were
sequenced and subcloned into the expression vector pcDNA3 (Invitrogen,
San Diego, CA).
The neuroendocrine cell line BON (Everest et al., 1991) (a kind gift
from B. Wiedenmann, Humboldt University, Berlin, Germany) was
cultured at 37°C under 5% CO2 in
DMEM/nutrient mix F-12 (1:1) (Invitrogen), supplemented with
7.5% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml
streptomycin. After electroporation with the pcDNA3-VGLUT3 plasmid,
stable transfectants were selected in the presence of 800 µg/ml G418
(Invitrogen), screened by immunofluorescence with a serum against
a VGLUT3 peptide (P45-1; see below) and further confirmed using a serum
raised to another peptide (P45-3; see below). BON cells stably
transfected with rat VGLUT2 or vesicular inhibitory amino acid
transporter (VIAAT) cDNAs were used as controls (Herzog et al.,
2001).
Amino acid uptake assay. For each BON transfectant, cells
from four confluent 15 cm dishes (~150 × 106 cells) were washed once with PBS,
scrapped, and recovered in 10 ml of chilled 0.32 M sucrose and 4 mM
HEPES-KOH, pH 7.4. Cells were broken using a Bioneb cell
disruption system (Glas-Gol, Terre Haute, IN), with a single cycle of
atomization under a 3.3 l/min stream of nitrogen at 2.5 bar. Organelle
intactness was assessed by measuring the latency of a lysosomal enzyme,
 -glucuronidase, using
4-methylumbelliferyl--D-glucuronide as a
substrate. Typically, ~60% of -glucuronidase activity was
protected in the absence of Triton X-100, showing that an
identical percentage of lysosomes remained intact after cell breakage.
Nuclei and cell debris were discarded by centrifugation at 10,000 × g for 5 min. Membranes were then pelleted at 200,000 × g for 20 min and resuspended in 400 µl of ice-cold 0.32 M sucrose, 4 mM KCl, 4 mM MgSO4, and 10 mM HEPES-KOH, pH 7.4 (uptake buffer).
The transport reaction was started by addition of 10 µl of membranes
(100 µg of protein) to 90 µl of uptake buffer containing 2.2 mM ATP, 1.1 mM MgSO4, and
44 µM (2 µCi)
[3H]L-glutamate or
[3H]L-aspartate (both from
Amersham Biosciences, Buckinghamshire, UK), with or without 50 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) and
other additives, as stated in Results and figure legends. After
incubation at 30°C for 10 min, [3H]
amino acid uptake was terminated by dilution with 3 ml of ice-cold 0.15 M KCl, rapid filtration through a 0.45 µm pore size
membrane filter (Millipore, Bedford, MA), and three washes with
3 ml of ice-cold 0.15 M KCl. The radioactivity retained on
the filters was measured by scintillation counting in Ready
Protein+ cocktail (Beckman, Fullerton,
CA). Each uptake measurement was performed in triplicate and is
expressed as mean ± SEM. All experiments were independently
performed three or more times on two independent BON-VGLUT3 clones. In
each experiment, membranes from VGLUT3-expressing clones were compared
with those of VGLUT2 or mock (VIAAT-expressing) transfectants.
RT-PCR. RT-PCR detection of VGLUT3 mRNA in body organs and
in different brain areas was performed as described previously (Antunes
Bras et al., 1998) with the following primers: 5'-ACCCGGGAAGA- ATGGCAGAATCTG-3' and 5'-ATGGGAAAAGCAATGGGTGTG- GAG-3'. RT-PCR generated product with glyceraldehyde-3-phosphate dehydrogenase (G3PDH)-specific probes (Clontech, Palo Alto, CA) was used to normalize
the RNA level in tissue sample extracts.
In situ hybridization. Regional in situ
hybridization was performed with a mix of six antisense
oligonucleotides (5'-GTAAGATCCCCAGCGAA- TCTCCCACGGCAT-3',
5'-CAATAGGAGAGGCACCTCAGAGCC- CTTAGC-3',
5'-ACTCAGCTCAATGGCATCTCCCTCCTCGTT-3', 5'-TTCATTCTGGTAGGATAATGGCTCCTCCCC-3', 5'-GGATTC-
TCTCTGTTGTCTCCGATCCGTCTT-3', and 5'-GACCTCACAATT-
CTGGGTGGTGGCTCCATA-3') as described previously (Davis et al., 2000;
Herzog et al., 2001). In brief, oligonucleotides were labeled with
[35S]dATP, using terminal transferase
(Amersham Biosciences), to a specific activity of 5 × 10 8 dpm/µg. Sections were covered with
100 µl of a solution containing 50% hybridization solution (Amersham
Biosciences), 40% deionized formamide (Merck Eurolab,
Strasbourg, France), 500 µg/ml poly(A) (Roche, Meylan,
France), 100 mM 4-dithiothreitol, and
3-5 × 105 dpm of each labeled
oligonucleotide. The samples were incubated overnight at 42°C,
washed, and exposed to x-ray films (Biomax; Eastman Kodak, Rochester,
NY) for 21 d.
The plasmids used to synthesize the cRNA probes for cold
in situ hybridization were obtained by PCR amplification
with the following primer couples: 5'-ACTGTTACCAAGATGCCC-3' and
5'-ATGAGCACGAACCATTCC-3' for the rat serotonin transporter (SERT)
and 5'-AAAACAGGACTGGGCTGATCC-3' and 5'-GAGACCAAGATCCATACGCCC-3' for
VGLUT3. The choline acetyltransferase (ChAT) plasmid used for cold
in situ hybridization was a generous gift from S. Berrard
(CNRS UMR7091, Paris, France). Double cold in situ
hybridization was performed with antisense riboprobes labeled with
either fluorescein-UTP (for ChAT or SERT) or digoxygenin (DIG)-UTP (for VGLUT3) (Herzog et al., 2001). Colorimetric revelations were obtained with 5-bromo-4-chloro-3-indolyl phosphate (Roche) and either nitroblue tetrazolium (Roche) for VGLUT3 or
2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyl-tetrazolium chloride
(Roche) for ChAT and SERT, to obtain the blue and red staining, respectively.
Antiserum. Two anti-VGLUT3 antisera, named P45-1 and P45-3,
were obtained by immunizing rabbits (Agro-Bio, Villeny, France) against
the following peptides, coupled via their cysteine residues to keyhole
limpet hemocyanin: CDSLGILQRKLDGTNEEGD
(Asp27-Asp44),
and CETELNHEAFVSPRKKM
(Glu531-Met547),
respectively. Both antisera were decomplemented by heating 30 min at
56°C, dialyzed, and stored in the presence of 50% glycerol at
 20°C. For immunological detection, the antisera were affinity purified on peptides linked to Affigel-15 (Bio-Rad, Richmond, CA).
Immunoautoradiography. Immunoautoradiography was performed
as described previously (Herzog et al., 2001). In brief, adult male
Sprague Dawley rats were anesthetized and perfused via the ascending
aorta with 200 ml of 0.9% NaCl containing sodium nitrite (1 gm/l).
Brains were dissected and frozen in isopentane at 30°C. Horizontal
or coronal rat brain sections were fixed with 4% paraformaldehyde and
washed with PBS containing 3% bovine serum albumin (BSA), 1% goat
serum, and 1 mM NaI (buffer A). Sections were
incubated with buffer supplemented with affinity-purified VGLUT3
antiserum (1:5000 dilution) and then with
[125I] IgG (0.25 µCi/ml; Amersham).
Sections were apposed to x-ray films (Biomax; Kodak) for 5 d.
Immunohistochemistry. Immunocytochemistry was performed as
described previously (Herzog et al., 2001). Adult male Sprague Dawley
rats were anesthetized and perfused intracardially with 300 ml of 120 mM sodium phosphate buffer (PB, pH 7.4)
supplemented with 4% paraformaldehyde. The brains were dissected,
postfixed by immersion in the same fixative, and cryoprotected in PB
containing 30% sucrose. Coronal sections were taken at 20°C and
mounted on glass slides. Sections were washed with PB containing
gelatin (2 gm/l) and Triton X-100 (0.25%) and incubated with VGLUT3
antiserum in the presence or absence of guinea pig anti-vesicular
acetylcholine transporter (VAChT) antiserum (Chemicon, Temecula, CA).
VGLUT3 alone was detected with anti-rabbit IgG coupled to Alexa Fluor 568 dye (Molecular Probes, Eugene, OR). VGLUT3 and VAChT were codetected by immunofluorescence, using goat anti-rabbit IgG coupled to
CY3 and goat anti-guinea-pig coupled to FITC, respectively, as
secondary antibodies. The sections were observed using a conventional (Axioskop 2 Plus; Zeiss, Thornwood, NY) or a confocal laser scanning (LSM 410; Zeiss) microscope.
VGLUT3 was visualized at the electron-microscopic level using the
pre-embedding immunogold method with silver intensification (Bernard et
al., 1999). Briefly, the sections were incubated in goat anti-rabbit
IgGs conjugated to gold particles (0.8 nm diameter; 1:100 in
PBS/acetylated BSA (BSA-C); Aurion, Wageningen, The
Netherlands) for 2 hr in PBS/BSA-C. The sections were then washed (3×
PBS) and postfixed in 1% glutaraldehyde in PBS for 10 min. After
washing (2× PBS; 2× sodium acetate buffer, 0.1 M, pH
7.0), the diameter of the gold immunoparticles was increased using a
silver enhancement kit (HQ silver; Nanoprobes, Yaphank, NY) for
5 min at room temperature in the dark. After treatment with 1%
osmium, dehydration, and embedding in resin, ultrathin sections were
cut, stained with lead citrate, and examined using a Philips
(Eindhoven, The Netherlands) CM10 electron microscope or a
Philips Tecnai 20. VGLUT3 and VAChT were codetected at the
electron-microscopic level by pre-embedding immunogold and
immunoperoxidase, respectively. Briefly, the sections were incubated in
a mixture of goat anti-rabbit IgGs conjugated to gold particles (0.8 nm
diameter; 1:100 in PBS/BSA-C; Aurion) and goat anti-guinea pig coupled
to biotin (1:200) for 2 hr in PBS/BSA-C. The sections were then washed
(3× PBS) and postfixed in 1% glutaraldehyde in PBS for 10 min. After
washing (2× PBS; 2× sodium acetate buffer, 0.1 M, pH
7.0), the diameter of the gold immunoparticles was increased using a
silver enhancement kit (HQ silver; Nanoprobes) for 5 min at room
temperature in the dark. The sections were finally washed in acetate
buffer and in PBS and incubated in an avidin-biotin-peroxidase
complex (1:100; Vector Laboratories, Burlingame, CA) for 1.5 hr at room
temperature. After washing (2× PBS; 1× Tris buffer, 0.05 M, pH 7.6), the immunoreactive sites for VAChT were
revealed using DAB. The sections were treated with 1% osmium,
dehydrated, and embedded in resin. Ultrathin sections were cut, stained
with lead citrate, and examined in the electron microscope.
| |
RESULTS |
Cloning and functional characterization of VGLUT3
A rat hippocampus cDNA library was screened with a combination of
VGLUT1 and VGLUT2 probes (Herzog et al., 2001). Of 6 × 105 plated phages, 55 positive clones
were isolated and analyzed. Thirty-seven were identical to VGLUT1 and
four were identical to VGLUT2. One cDNA, designated P45, encodes a 588 aa protein (calculated Mr  64,700).
P45 is highly homologous to VGLUT1 and VGLUT2 (>70% aa identity).
Most of the divergences between P45, VGLUT1, and VGLUT2 sequences are
concentrated in the N and C termini (Fig.
1).
View larger version (87K):
[in this window]
[in a new window]
|
Figure 1.
Alignment of VGLUT3, VGLUT2, and VGLUT1
amino acid sequences. The three proteins are highly conserved in their
central portion. (Black boxing indicates
identical residues.)
|
|
To analyze whether P45 represents a novel glutamate transporter, its
cDNA was stably expressed in the serotoninergic endocrine cell line BON
(Evers et al., 1991). Intracellular membrane vesicles were purified
from two independent positive clones (see Fig. 5C) and
analyzed for their capacity to take up
[3H]glutamate in the presence of ATP. In
each experiment, these vesicles were compared with membranes derived
from mock- and VGLUT2-transfected cells. P45-containing vesicles, as
VGLUT2-containing membranes, accumulated approximately twice as much
[3H]glutamate as mock vesicles (Fig.
2A). Inhibition of this
transport by the H+ ionophore CCCP
indicated that the increased uptake observed in P45- and
VGLUT2-containing membranes was attributable to an
H+-driven transport, which was 3.07 ± 0.17 and 4.42 ± 0.28 times higher than in mock vesicles,
respectively (means ± SEM; n = 26). For clarity,
only the CCCP-sensitive component of uptake is considered hereafter.
Because the P45-mediated accumulation of
[3H]glutamate remained linear for 15 min
(Fig. 3A), we used a constant duration of 10 min throughout this study.
View larger version (30K):
[in this window]
[in a new window]
|
Figure 2.
Functional characterization of VGLUT3.
Membranes from BON cells stably expressing VGLUT3, VGLUT2, or an
unrelated VIAAT (mock) were incubated at 30°C for 10 min with 40 µM or 2 µCi of
[3H]L-glutamate and the following
additives as stated: 50 µM CCCP, 5 µM
nigericin, 20 µM valinomycin, and 5 µM
Evans Blue, as well as 10 mM (C)
L-glutamate (GLU),
L-aspartate (ASP), acetylcholine
(Ach), or GABA. Representative experiments are shown.
The error bars represent the SEM of triplicate determinations.
A, Expression of VGLUT2 or VGLUT3 induces a
CCCP-sensitive uptake of glutamate. In B-D, only the
specific (CCCP-sensitive) component of uptake is shown. In
C and D, the subtracted CCCP-resistant
component was determined in the presence of an identical concentration
of amino acid or Evans Blue. B, VGLUT3-mediated uptake
is more sensitive to nigericin than VGLUT2. C, Both
transporters prefer L-glutamate over other amino acids or
transmitters. D, VGLUT3 is less sensitive to Evans Blue
than VGLUT2. The dotted lines in C and
D represent the control level (i.e., 100%).
|
|
View larger version (20K):
[in this window]
[in a new window]
|
Figure 3.
Saturation kinetics of VGLUT2 and VGLUT3.
A, Time course of VGLUT2- and VGLUT3-mediated uptake at
a 40 µM [3H]glutamate.
B, The initial rate of uptake (at 3 min) into
VGLUT2-containing vesicles was determined with increasing
concentrations of [3H]glutamate (0.25-3
mM). Specific uptake was determined at each glutamate
concentration by subtracting the background uptake observed in the
presence of 50 µM CCCP. C, Dependence of
the initial rate of VGLUT3-mediated uptake (at 10 min) on glutamate
concentration was determined as in B. D,
A Lineweaver-Burke plot of the data shown in B and
C indicates KM values of 1.27 and 0.56 mM and Vmax values of
152 and 19 pmol · mg1
protein · min1 for VGLUT2 (closed
circles) and VGLUT3 (open circles),
respectively. Regression lines for VGLUT2 (r = 0.9993) and VGLUT3 (r = 0.8959) intersect the 1/S
axis at distinct 1/KM values
(closed and open arrowheads). The results
in B-D represent the average of three independent
paired analyses of VGLUT2 and VGLUT3, each performed in triplicate.
Error bars in A-C correspond to SEM.
|
|
The bioenergetic properties of P45- and VGLUT2-mediated processes were
compared by adding nigericin, an ionophore that collapses transmembrane
pH gradients by exchanging H+ for
K+. As illustrated in Figure
2B, although nigericin moderately inhibited VGLUT2 by
31.7 ± 2.3%, as reported previously (Bai et al., 2001; Fremeau
et al., 2001; Herzog et al., 2001; Varoqui et al., 2002), a stronger
inhibition (70.4 ± 6.1%) was reproducibly observed for P45
(paired t test: p < 0.001;
n = 6). Therefore, the P45-mediated process is more
dependent on the chemical component of the
H+ electrochemical gradient, suggesting
that it might translocate more H+ than
VGLUT2. Both uptakes were abolished by a further addition of
valinomycin, a K+ ionophore that disrupts
the remaining electrical component by exporting the
K+ ions accumulated by nigericin (Fig.
2B).
The substrate selectivity of P45 was assessed by applying unlabeled
amino acids and/or neurotransmitters simultaneously with [3H]glutamate (Fig. 2C).
L-glutamate (10 mM)
inhibited the P45-mediated transport by 92.1 ± 4.1%
(n = 6), whereas L-aspartate only
partially inhibited uptake (56.5 ± 4.2% inhibition;
n = 6). Acetylcholine (n = 5) or GABA
(n = 4) at the same concentration (Fig. 2C)
or serotonin at 0.5 mM (i.e., approximately two
orders of magnitude over its cytosolic concentration in serotoninergic
cells) (data not shown) had no effect. The selectivity for
L-glutamate over L-aspartate was confirmed by the fact that we
could not detect any CCCP-sensitive accumulation of
[3H]aspartate induced by P45 (data not
shown). Similar results were obtained for VGLUT2 (Fig. 2C),
as reported previously (Bai et al., 2001; Fremeau et al., 2001; Herzog
et al., 2001; Takamori et al., 2001; Varoqui et al., 2002). In
contrast, Evans Blue (5 µM), a compound that
competitively inhibits glutamate uptake into synaptic vesicles (Roseth
et al., 1995), discriminated the P45- and VGLUT2-mediated processes
(Fig. 2D), because inhibitions of 16.5 ± 5.6 and 55.9 ± 3.4% were observed, respectively (paired t
test: p < 0.005; n = 4). To
characterize further the interaction of P45 with glutamate, increasing
concentrations of [3H]glutamate were
tested. As illustrated in Figure 3C,D, the P45-mediated uptake followed Michaelis-Menten kinetics, with mean
Vmax and KM values of 20.3 ± 4.8 pmol · mg1
protein · min1 and
0.52 ± 0.21 mM (n = 3). A
similar analysis of VGLUT2 kinetics performed in parallel yielded a
much higher Vmax (168 ± 20 pmol · mg1
protein · min1) and a slightly
higher KM (1.46 ± 0.35 mM) (Fig. 3B,D). The slower Vmax of P45, observed with both BON
transfectants, may originate from a lower expression of the protein
and/or a slower turnover of the transport cycle. To confirm the
difference in substrate affinity, additional competition experiments
were performed with submillimolar concentrations of unlabeled amino
acids (data not shown). Indeed, we found that a 0.5 mM concentration of glutamate inhibited P45 more
efficiently (by 80.8 ± 3.6%; n = 5) than VGLUT2 (46.9 ± 3.0%). This difference was highly significant (paired t test: p < 0.005; n = 5).
In conclusion, P45 transports glutamate with characteristics very
similar, but not identical, to those displayed by VGLUT1 and VGLUT2.
Consequently, it was renamed VGLUT3.
Anatomic distribution of VGLUT3
VGLUT3 mRNA distribution in body organs was analyzed by reverse
transcription and amplification by PCR (Fig.
4A). A 398 kb amplification fragment was found in the brain, eyes, and liver (Fig.
4A). In the brain, VGLUT3 was detected in all
inspected areas except the cerebellum (Fig. 4A).
View larger version (51K):
[in this window]
[in a new window]
|
Figure 4.
Regional distribution of VGLUT3 mRNA.
A, RT-PCR detection of VGLUT3 mRNA in body organs and in
different brain areas. G3PDH-specific primers (Clontech) were used to
normalize the RNA level in tissue sample extracts. B-H,
In situ hybridization analysis of VGLUT3 transcript
distribution in the rat brain. Horizontal (B) or
coronal (C-H) brain sections were hybridized
with antisense 35S-labeled oligonucleotides
(B-E) or DIG-UTP-labeled cRNA probes
(F-H). CA3, CA3 field of the
hippocampus; CPu, caudate-putamen; DR,
dorsal raphe; DRD, dorsal part of the dorsal raphe
nucleus; DRV, ventral part of the dorsal raphe nucleus;
E, ependymal cells; Hi, hippocampus;
Hil, hilus of the dentate gyrus; Or,
oriens layer of the hippocampus; PMnR, paramedian raphe
nuclei; py, pyramidal layer of the hippocampus;
Rad, radiatum layer of the hippocampus. Scale bars:
F, H, 200 µm; G, 100 µm.
|
|
This finding was confirmed and extended by in situ
hybridization analysis, which revealed a very discrete pattern of
expression (Fig. 4B-H). No signal is detected
in the cerebellum and thalamus. High VGLUT3 mRNA expression is observed
in the dorsal and medial raphe nuclei, caudate-putamen, and accumbens
(Fig. 4B,C,E), whereas low levels are found in the
hippocampus (Fig. 4B,D) and habenula (data not
shown). A strong signal was also observed in ependymal cells (Fig.
4B,D). At the cellular level, the labeling was
concentrated over large, scattered neurons in the striatum (Fig.
4F), hippocampus (Fig. 4G), and cerebral
cortex (data not shown). A high density of positive neurons was
observed in the dorsal and ventral parts of the raphe dorsalis nucleus
(Fig. 4H). Lower amounts of VGLUT3 mRNA are also
present in the lateral parts of the dorsal raphe (Fig.
4H).
Thus, in contrast to VGLUT1 and VGLUT2 (Ni et al., 1994, 1995; Hisano
et al., 2000; Fremeau et al., 2001; Herzog et al., 2001), only a few
neurons of the brain express VGLUT3.
To examine the localization of the protein, two independent polyclonal
antiserums were raised by immunizing rabbits against two different
peptides of the VGLUT3 N and C terminus. These two anti-peptide
antiserums, named P45-1 and P45-3, gave very similar, if not identical,
results in all tested conditions. Both antiserums were able to detect a
strong intracellular and punctiform signal on BON cells permanently
transfected with pcDNA3-VGLUT3, but not with plasmids coding for
VGLUT1 or VGLUT2 (as shown in Fig.
5A-C with P45-3; data not
shown for P45-1). On rat brain sections, the same discrete
immunoautoradiographic labeling pattern was observed with the two
antiserums (Fig. 5D,E). Finally, the immunolabeling of brain
sections was not detected with a preimmune serum (data not shown) or
antiserums saturated with their cognate peptides (Fig. 5F;
data not shown). Together, these experiments demonstrate the
specificity of the antiserums.
View larger version (80K):
[in this window]
[in a new window]
|
Figure 5.
Antiserum specificity and regional distribution of
VGLUT3. Two polyclonal antisera directed against distinct peptides were
generated. A-C, Immunofluorescence detection of VGLUT3
in BON stable transfectants (white puncta) with the P45-1
antiserum at a dilution of 1:5000. The serum recognizes VGLUT3 but not
VGLUT1 or VGLUT2 in BON stable transfectants. The same result was
obtained with the P45-3 antiserum (data not shown).
D-I, Localization of VGLUT3 protein by
immunoautoradiography. In D and G-I the
P45-3 antiserum was used. In E and F, the
P45-1 antiserum was used in the presence (F) or
absence (E) of its cognate peptide (0.1 mg/ml).
Acb, accumbens; CPu,
caudate-putamen; DR, dorsal raphe; Ent
Cx, entorhinal cortex; Hi, hippocampus;
MnR, median raphe nucleus; S,
septum; SNC, substantia nigra pars compacta;
Tu, olfactory tubercles; VTA, ventral
tegmental area. Scale bars: J, L, N, 200 µm;
K, M, O, 50 µm.
|
|
In contrast to its transcript, the VGLUT3 protein was relatively
broadly distributed in the gray matter of the CNS (Fig.
5D-I). No signal was detected in the white matter. A
strong labeling was observed in the caudate-putamen, accumbens
(shell > core), olfactory tubercles, hippocampus (pyramidal and
granular cell layers), ventral tegmental area, substantia nigra (pars
compacta), and raphe nuclei (Fig. 5G-I). Thus,
VGLUT3 mRNA (Fig. 4) and protein (Fig. 5) are present in the same brain
regions, but their distribution patterns are not similar. These
differences imply that the protein VGLUT3 is not addressed exclusively
to cell soma. Furthermore, the regional colocalization of VGLUT3
transcripts and protein suggests that VGLUT3 is expressed by striatal
and hippocampal interneurons. However, VGLUT3 is also abundant in
regions in which its mRNA is absent, such as the substantia nigra (pars
compacta) and ventral tegmental area (Fig. 5H). The
partial mismatch between the locations of the transcript and the
protein suggests that VGLUT3 is also expressed at the terminals of
principal neurons.
At the light microscopic level, the caudate-putamen, hippocampus, and
raphe nuclei exhibited an intense punctiform labeling of their neuropil
(Fig. 6). VGLUT3 is evenly distributed in
the entire striatal neuropil but not in fiber track patches (Fig. 6A,B). At higher magnification, this labeling appears
as multiple small puncta (Fig. 6B). In the
hippocampus, VGLUT3-positive neurons, although very scattered (Fig.
4B,D,G), produce a dense network surrounding the soma
of pyramidal and granular cells (Fig. 6C,D). VGLUT3 puncta
are widespread in the raphe dorsalis (Fig.
6E,F). In the hippocampal formation and raphe
nucleus, the puncta have a somewhat larger size than in the striatum
(Fig. 6B,D,F).
View larger version (96K):
[in this window]
[in a new window]
|
Figure 6.
Immunofluorescent detection of VGLUT3 in the
brain. VGLUT3 immunoreactivity appears as red puncta in the
caudate-putamen (A, B), hippocampus (C,
D), and dorsal raphe (E, F).
CA1, CA1 field of the hippocampus; CA3,
CA3 field of the hippocampus; CPu, caudate-putamen;
DRD, dorsal part of the dorsal raphe nucleus;
DRV, ventral part of the dorsal raphe nucleus;
Hi, hippocampus; Or, oriens layer of the
hippocampus; py, pyramidal layer of the hippocampus;
Rad, radiatum layer of the hippocampus;
WM, white matter. Scale bars: A, C, E,
200 µm; B, D, F, 50 µm.
|
|
Localization of VGLUT3 over synaptic vesicle clusters
At the electron-microscopic level, the above-described puncta were
found to correspond to nerve endings in the striatum, hippocampus, and
raphe nucleus (Fig. 7A-E), as
well as all other inspected brain areas (data not shown). The terminals
were identified by the presence of synaptic vesicles. Immunoparticles
for VGLUT3 accumulated over vesicle clusters in terminals making
classical asymmetrical synapses in the hippocampus (Fig. 7C,
arrows) and raphe nucleus (Fig. 7E,
arrows). However, in both structures a large number of
VGLUT3-positive terminals are also forming symmetrical synapses (Fig.
7B,D, arrowheads). The VGLUT3-immunoreactive
terminals made close appositions or synaptic contacts with different
parts of the neurons. In the CA3 field of the hippocampus, labeled
terminals made symmetrical and asymmetrical contacts with dendrites
and symmetrical contacts with perikarya of pyramidal
neurons. In the dorsal raphe, VGLUT3-positive terminals made
symmetrical and asymmetrical synapses with dendrites. In the striatum,
VGLUT3 immunolabeled terminals made close appositions and symmetrical
contacts with dendrites.
View larger version (159K):
[in this window]
[in a new window]
|
Figure 7.
VGLUT3 is localized in nerve endings in the
striatum (A), hippocampus (B, C),
and dorsal raphe (D, E). In all three areas,
immunoparticles for VGLUT3 are localized in terminals
(t). In the striatum, dorsal raphe, and
hippocampus (C), the labeled terminals are in
close apposition with dendrites (d). In the
hippocampus and dorsal raphe, the immunoreactive terminal makes a
symmetrical synapse (arrowheads) with a pyramidal cell
(Pyr Cell) in B and
D; one terminal makes an asymmetrical synapse
(arrow) with a dendrite (d) in
C and E. Scale bar, 250 nm.
|
|
Because this finding does not fit with the conventional view that
glutamate synapses make asymmetrical contacts, we investigated the
nature of the VGLUT3-positive neurons in more detail.
VGLUT3 is expressed in cholinergic and serotoninergic neurons
The sparse VGLUT3-positive giant cells in the caudate-putamen
nucleus are reminiscent of cholinergic neurons, as detected with a
probe for the ChAT transcript (Fig.
8A,C). We thus compared the expression of the VGLUT3 and ChAT mRNAs, using in situ
hybridization with double colorimetric detection. As illustrated in
Figure 8B,D, we found in the caudate-putamen that
every ChAT-positive neuron (detected by a red precipitate) also
expressed VGLUT3 (labeled in blue).
View larger version (106K):
[in this window]
[in a new window]
|
Figure 8.
VGLUT3 is colocalized with ChAT and
VAChT in the striatum. A, C, Cold in situ
hybridization with the ChAT cRNA probe alone (in
red). D, Enlargement of the neuron
indicated in A by the red arrow.
B, D, Double-labeling in situ
hybridization (ChAT in red and VGLUT3 in
blue). D, The double-labeled neuron
indicated by a blue arrow in B has been
enlarged. E, F, Confocal laser detection of
double-immunofluorescence for VGLUT3 (CY3, red
fluorescence) and VAChT (fluorescein, green
fluorescence) in the striatum (E) or the
hippocampus (F). In false colors, overlapping
signals appear prominently as yellow-orange.
G, double detection of VGLUT3 (immunogold) and VAChT
(immunoperoxidase) with the electron microscope in the striatum. Scale
bars: A, B, E, F, 50 µm; C, D, 10 µm;
G, 0.25 µm.
|
|
Laser confocal microscopy was then used to investigate the potential
colocalization of VGLUT3 with another specific marker of cholinergic
nerve endings, the VAChT, at the protein level (Gilmor et al., 1996;
Roghani et al., 1996; Weihe et al., 1996). In the hippocampus (Fig.
8F) or the septum (data not shown), VGLUT3 (red
fluorescence) and VAChT (in green) are clearly present in two distinct
sets of nerve terminals. In contrast, in the striatum, numerous thin
varicose fibers are labeled in yellow fluorescence, very few are in
red, and green terminals are absent (Fig. 8E). This
experiment suggests that the vast majority of striatal cholinergic terminals contain VGLUT3. Some noncholinergic VGLUT3-positive nerve
endings are observed that may originate from brain regions sending
projections to the striatum.
In the experiment depicted in Figure 8G, VAChT is detected
by immunoperoxidase and VGLUT3 is detected by immunogold at the ultrastructural level. As shown in Figure 8G, two small
terminals, containing both the peroxidase precipitate and immunogold
particles, are VAChT- as well as VGLUT3-positive, respectively. Thus,
the colocalization of VGLUT3 and VAChT in striatal nerve terminals is
also confirmed at the electron-microscopic level.
Together, these results clearly show that almost all cholinergic
striatal interneurons express VGLUT3 at their nerve endings.
As shown by in situ hybridization and immunoautoradiography,
VGLUT3 is also strongly expressed in the serotoninergic raphe nuclei
(Figs. 4E,H,
9A,B). We thus analyzed by
double in situ hybridization whether VGLUT3 is present in
serotoninergic neurons. In Figure 9, VGLUT3-positive neurons are in
blue and serotoninergic neurons, which labeled the plasma membrane SERT
transcript, are detected by a red precipitate. As demonstrated
by this experiment, all of the SERT-positive neurons also expressed
VGLUT3 in the dorsal and median raphe (Fig. 9A,B). A
red/blue neuron is shown at a higher magnification on Figure
9C. Interestingly, numerous neurons from the dorsal raphe
are expressing VGLUT3 but not SERT. Accordingly, nonserotoninergic
neurons from the raphe dorsalis, which are VGLUT3-positive, could be
glutamatergic. Because serotoninergic neurons are known to send
projections to the substantia nigra, ventral tegmental area,
hippocampus, striatum, and cerebral cortex, the VGLUT3 protein present
in these regions might thus originate from the raphe nuclei. However,
such an hypothesis needs to be investigated further.
View larger version (192K):
[in this window]
[in a new window]
|
Figure 9.
VGLUT3 is colocalized with SERT in the dorsal and
medial raphe. The SERT probe is shown in red, and the
VGLUT3 probe is shown in blue. A shows
the dorsal and medial raphe. B, Higher magnification of
the dorsal raphe. C, Enlargement of double-labeled
neurons (indicated in B by a blue arrow).
A and B are taken from different
sections. DR, Dorsal raphe; MnR, medial
raphe. Scale bars: A, 300 µm; B, 100 µm; C, 10 µm.
|
|
| |
DISCUSSION |
VGLUT3 is a novel vesicular glutamate transporter
Two subtypes of vesicular glutamate transporters, named VGLUT1 and
VGLUT2, have been characterized recently. In this study we isolated and
functionally characterized VGLUT3, a third member of the family. VGLUT3
represents a novel H+-dependent glutamate
transporter that is both structurally (75% aa identity) and
functionally very similar to VGLUT1 and VGLUT2. Because VGLUT3
localizes to synaptic vesicles, as do VGLUT1 and VGLUT2, we concluded
that it is a novel vesicular glutamate transporter. Our study of its
uptake activity revealed subtle differences with VGLUT2, including a
slightly increased affinity for glutamate, a lesser sensitivity to
Evans Blue, and a higher dependence on the  pH component of the
proton electrochemical gradient. This last property might represent an
intrinsic difference between the two transporters, such as the coupling
of the VGLUT3-mediated glutamate uptake to the export of more protons
(implying a higher loading of vesicles with glutamate). However, we
cannot exclude extrinsic factors, such as the localization of VGLUT2
and VGLUT3 in distinct compartments of the BON cells, differing by
their lumenal pH. Additional studies are needed to discriminate between these possibilities. A major (eightfold) difference was also observed for the uptake capacity (Vmax) of
VGLUT3- and VGLUT2-containing vesicles. However, in the absence of a
common ligand enabling us to compare the amounts of both transporters,
the issue of whether the slower Vmax
of VGLUT3 reflects a slower turnover or, merely, a lower expression
level will remain open.
VGLUT3 has discrete localization
Despite the functional similarity to VGLUT1 and VGLUT2, VGLUT3
differs from these isoforms by several unique anatomical features. First, it is expressed in a few scattered neurons of the brain, in
contrast to VGLUT1 and VGLUT2, which are massively expressed throughout
large areas of the brain. Neurons expressing VGLUT3 are found in brain
regions that also contain the VGLUT1 and VGLUT2 mRNAs, such as the
cerebral cortex, hippocampus, and brainstem. However, VGLUT3 is
the only vesicular glutamate transporter synthesized by striatal
neurons. The regional colocalization of VGLUT3 mRNA and protein,
together with the double-labeling experiments, demonstrates that VGLUT3
is expressed by striatal and hippocampal interneurons. Nonetheless,
VGLUT3 also seems to be present in neurons from the raphe region
sending long projections (for example to the substantia nigra and the
ventral tegmental area). Tracing methods as well as lesion studies
should help to clarify this point.
VGLUT3 is present in terminals forming symmetrical and
asymmetrical synapses
Second, at the ultrastructural level, while VGLUT1 and VGLUT2 are
exclusively present in terminals forming asymmetrical synapses (Bellocchio et al., 1998; Fremeau et al., 2001; Fujiyama et al., 2001;
Herzog et al., 2001; Sakata-Haga et al., 2001), VGLUT3 is found in
asymmetrical as well as symmetrical synapses. Therefore, if VGLUT3 is
fully functional in vivo, the population of excitatory synapses can no longer be identified by a morphological criterion.
It is tantalizing to consider that VGLUT3-positive symmetrical synapses
are cholinergic and serotoninergic while asymmetrical synaptic contacts
containing VGLUT3 are glutamatergic. According to Fremeau et al.
(2001), all asymmetrical synapses express either VGLUT1 or VGLUT2. It
can thus be anticipated that VGLUT3 and VGLUT1 or VGLUT2 are
coexpressed in some of these glutamatergic terminals. Such a
colocalization is already suspected for VGLUT1 and VGLUT2 (Herzog et
al., 2001).
VGLUT3 is present in cholinergic and serotoninergic neurons
Finally, we report here, for the first time, that
cholinergic and serotoninergic neurons express a vesicular glutamate
transporter. In this study we have solidly documented the presence
VGLUT3 and VAChT in the same terminals. A similar demonstration remains
to be established for VGLUT3 and vesicular monoamine transporter type-2
(VMAT2). Whether or not VGLUT3 and VAChT or VMAT2 are found on the same
synaptic vesicle is still an open question. If this is the case, then
cholinergic and serotoninergic terminals have the potential to store,
and thus to release (Takamori et al., 2000, 2001), glutamate in
addition to acetylcholine or serotonin. Indeed, concentrations of
glutamate above VGLUT3 KM are achieved in the cytosol of monoaminergic neurons (Kaneko et al., 1990; Danbolt,
2001). Furthermore, the codetection of the excitatory amino acid on one
hand and acetylcholine or serotonin on the other hand in the same
terminals has already been reported in other brain areas (Clements and
Grant, 1990; Waerhaug and Ottersen, 1993; Lavoie and Parent, 1994a,b;
Clarke et al., 1996, 1997; Lebrand et al., 1996, 1998; Cases et al.,
1998; Hökfelt et al., 2000). The coexpression of the
corresponding vesicular transporters thus represents a step further.
However, the synaptic corelease of glutamate simultaneously with
serotonin or acetylcholine, and its physiological relevance, remains to
be formally established. Interestingly, a recent study has reported
that dopaminergic neurons can also form asymmetric synaptic
specialization and release glutamate in vitro (Sulzer et
al., 1998). This study supports the working hypothesis that the
corelease of glutamate with other transmitters may be more general than
currently suspected.
However, it cannot be ruled out that VGLUT3 is addressed to a different
set of functional synaptic vesicles than VAChT/VMAT2. Alternatively,
VGLUT3 could be present in vesicles that do not belong to a readily
releasable pool or are in the reserve pool. Moreover, to our knowledge,
the presence of glutamate receptor in postsynaptic specialization of
the synaptic cleft of cholinergic or serotoninergic neurons has not
been documented. If VGLUT3 is in the reserve pool of vesicles or if
glutamate ion channel receptors are not present, the physiological
function of VGLUT3 will have to be elucidated. Consequently the main
challenge for the forthcoming studies on VGLUT3 will be to clearly
establish its functionality in monoaminergic neurons.
Cholinergic and serotoninergic neurons are involved in a wide variety
of neurological and psychiatric diseases (Kawaguchi et al., 1995;
Calabresi et al., 1997; Pollack, 2001). Because of its discrete
localization in these neurons, VGLUT3 represents a promising
therapeutic target for these pathologies.
| |
FOOTNOTES |
Received Feb. 12, 2002; revised April 15, 2002; accepted April 18, 2002.
*
C.G. and E.H. contributed equally to this work.
This research was supported by grants from Hoescht Marion Roussel and
Institut National de la Santé et de la Recherche Médicale (B. Giros) and Centre National de la Recherche Scientifique. G.C.B was
supported by the European Community Training and Mobility for
Researchers Program (contract no. FMRX-CT98-0228). We thank B. Wiedenmann for giving the BON cell line and E. Doudnikoff for technical
support on electronic microscopy. We are also very grateful to P. Ascher, J. P. Henry, and C. Mulle for their helpful advice and discussion.
Correspondence should be addressed to Dr. Salah El Mestikawy,
Faculté de Médecine, Institut National de la Santé et
de la Recherche Médicale Unité 513, 8 rue du
Général Sarrail, 94010 Créteil Cedex, France. E-mail:
salah.elmestikawy{at}im3.inserm.fr.
| |
REFERENCES |
-
Aihara Y,
Mashima H,
Onda H,
Hisano S,
Kasuya H,
Hori T,
Yamada S,
Tomura H,
Yamada Y,
Inoue I,
Kojima I,
Takeda J
(2000)
Molecular cloning of a novel brain-type Na+-dependent inorganic phosphate cotransporter.
J Neurochem
74:2622-2625[Web of Science][Medline].
-
Antunes Bras JM,
Epstein AL,
Bourgoin S,
Hamon M,
Cesselin F,
Pohl M
(1998)
Herpes simplex virus 1-mediated transfer of preproenkephalin A in rat dorsal root ganglia.
J Neurochem
70:1299-1303[Medline].
-
Bai L,
Xu H,
Collins JF,
Ghishan FK
(2001)
Molecular and functional analysis of a novel neuronal vesicular glutamate transporter.
J Biol Chem
276:36764-36769[Abstract/Free Full Text].
-
Bellocchio EE,
Hu H,
Pohorille A,
Chan J,
Pickel VM,
Edwards RH
(1998)
The localization of the brain-specific inorganic phosphate transporter suggests a specific presynaptic role in glutamatergic transmission.
J Neurosci
18:8648-8659[Abstract/Free Full Text].
-
Bellocchio EE,
Reimer RJ,
Fremeau Jr RT,
Edwards RH
(2000)
Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter.
Science
289:957-960[Abstract/Free Full Text].
-
Bernard V,
Levey AI,
Bloch B
(1999)
Regulation of the subcellular distribution of m4 muscarinic acetylcholine receptors in striatal neurons in vivo by the cholinergic environment: evidence for regulation of cell surface receptors by endogenous and exogenous stimulation.
J Neurosci
19:10237-10249[Abstract/Free Full Text].
-
Calabresi P,
De Murtas M,
Bernardi G
(1997)
The neostriatum beyond the motor function: experimental and clinical evidence.
Neuroscience
78:39-60[Web of Science][Medline].
-
Cases O,
Lebrand C,
Giros B,
Vitalis T,
De Maeyer E,
Caron MG,
Price DJ,
Gaspar P,
Seif I
(1998)
Plasma membrane transporters of serotonin, dopamine, and norepinephrine mediate serotonin accumulation in atypical locations in the developing brain of monoamine oxidase A knock-outs.
J Neurosci
18:6914-6927[Abstract/Free Full Text].
-
Clarke NP,
Bolam JP,
Bevan MD
(1996)
Glutamate-enriched inputs from the mesopontine tegmentum to the entopeduncular nucleus in the rat.
Eur J Neurosci
8:1363-1376[Web of Science][Medline].
-
Clarke NP,
Bevan MD,
Cozzari C,
Hartman BK,
Bolam JP
(1997)
Glutamate-enriched cholinergic synaptic terminals in the entopeduncular nucleus and subthalamic nucleus of the rat.
Neuroscience
81:371-385[Medline].
-
Clements JR,
Grant S
(1990)
Glutamate-like immunoreactivity in neurons of the laterodorsal tegmental and pedunculopontine nuclei in the rat.
Neurosci Lett
120:70-73[Web of Science][Medline].
-
Danbolt NC
(2001)
Glutamate uptake.
Prog Neurobiol
65:1-105[Web of Science][Medline].
-
Davis S,
Salin H,
Helme-Guizon A,
Dumas S,
Stephan A,
Corbex M,
Mallet J,
Laroche S
(2000)
Dysfunctional regulation of
 CaMKII and syntaxin 1B transcription after induction of LTP in the aged rat.
Eur J Neurosci
12:3276-3282[Web of Science][Medline]. -
Dent JA,
Davis MW,
Avery L
(1997)
avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans.
EMBO J
16:5867-5879[Web of Science][Medline].
-
Erickson JD,
Varoqui H
(2000)
Molecular analysis of vesicular amine transporter function and targeting to secretory organelles.
FASEB J
14:2450-2458[Abstract/Free Full Text].
-
Evers BM,
Townsend Jr CM,
Upp JR,
Allen E,
Hurlbut SC,
Kim SW,
Rajaraman S,
Singh P,
Reubi JC,
Thompson JC
(1991)
Establishment and characterization of a human carcinoid in nude mice and effect of various agents on tumor growth.
Gastroenterology
101:303-311[Web of Science][Medline].
-
Fremeau RT,
Troyer MD,
Pahner I,
Nygaard GO,
Tran CH,
Reimer RJ,
Bellocchio EE,
Fortin D,
Storm-Mathisen J,
Edwards RH
(2001)
The expression of vesicular glutamate transporters defines two classes of excitatory synapse.
Neuron
31:247-260[Web of Science][Medline].
-
Fujiyama F,
Furuta T,
Kaneko T
(2001)
Immunocytochemical localization of candidates for vesicular glutamate transporters in the rat cerebral cortex.
J Comp Neurol
435:379-387[Web of Science][Medline].
-
Gasnier B
(2000)
The loading of neurotransmitters into synaptic vesicles.
Biochimie
82:327-337[Medline].
-
Gilmor ML,
Nash NR,
Roghani A,
Edwards RH,
Yi H,
Hersch SM,
Levey AI
(1996)
Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles.
J Neurosci
16:2179-2190[Abstract/Free Full Text].
-
Hayashi M,
Otsuka M,
Morimoto R,
Hirota S,
Yatsushiro S,
Takeda J,
Yamamoto A,
Moriyama Y
(2001)
Differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI) is a vesicular glutamate transporter in endocrine glutamatergic systems.
J Biol Chem
276:43400-43406[Abstract/Free Full Text].
-
Herzog E,
Bellenchi GC,
Gras C,
Bernard V,
Ravassard P,
Bedet C,
Gasnier B,
Giros B,
El Mestikawy S
(2001)
The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons.
J Neurosci
21:RC181[Abstract/Free Full Text]:1-6.
-
Hisano S,
Haga H,
Li Z,
Tatsumi S,
Miyamoto KI,
Takeda E,
Fukui Y
(1997)
Immunohistochemical and RT-PCR detection of Na+-dependent inorganic phosphate cotransporter (NaPi-2) in rat brain.
Brain Res
772:149-155[Web of Science][Medline].
-
Hisano S,
Hoshi K,
Ikeda Y,
Maruyama D,
Kanemoto M,
Ichijo H,
Kojima I,
Takeda J,
Nogami H
(2000)
Regional expression of a gene encoding a neuron-specific Na+-dependent inorganic phosphate cotransporter (DNPI) in the rat forebrain.
Brain Res Mol Brain Res
83:34-43[Medline].
-
Hökfelt T,
Arvidsson U,
Cullheim S,
Millhorn D,
Nicholas AP,
Pieribone V,
Seroogy K,
Ulfhake B
(2000)
Multiple messengers in descending serotonin neurons: localization and functional implications.
J Chem Neuroanat
18:75-86[Web of Science][Medline].
-
Kaneko T,
Akiyama H,
Nagatsu I,
Mizuno N
(1990)
Immunohistochemical demonstration of glutaminase in catecholaminergic and serotoninergic neurons of rat brain.
Brain Res
507:151-154[Web of Science][Medline].
-
Kawaguchi Y,
Wilson CJ,
Augood SJ,
Emson PC
(1995)
Striatal interneurones: chemical, physiological, and morphological characterization.
Trends Neurosci
18:527-535[Web of Science][Medline].
-
Lavoie B,
Parent A
(1994a)
Pedunculopontine nucleus in the squirrel monkey: distribution of cholinergic and monoaminergic neurons in the mesopontine tegmentum with evidence for the presence of glutamate in cholinergic neurons.
J Comp Neurol
344:190-209[Web of Science][Medline].
-
Lavoie B,
Parent A
(1994b)
Pedunculopontine nucleus in the squirrel monkey: cholinergic and glutamatergic projections to the substantia nigra.
J Comp Neurol
344:232-241[Web of Science][Medline].
-
Lebrand C,
Cases O,
Adelbrecht C,
Doye A,
Alvarez C,
El Mestikawy S,
Seif I,
Gaspar P
(1996)
Transient uptake and storage of serotonin in developing thalamic neurons.
Neuron
17:823-835[Web of Science][Medline].
-
Lebrand C,
Cases O,
Wehrle R,
Blakely RD,
Edwards RH,
Gaspar P
(1998)
Transient developmental expression of monoamine transporters in the rodent forebrain.
J Comp Neurol
401:506-524[Web of Science][Medline].
-
Lee RY,
Sawin ER,
Chalfie M,
Horvitz HR,
Avery L
(1999)
EAT-4, a homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is necessary for glutamatergic neurotransmission in Caenorhabditis elegans.
J Neurosci
19:159-167[Abstract/Free Full Text].
-
Ni B,
Rosteck Jr PR,
Nadi NS,
Paul SM
(1994)
Cloning and expression of a cDNA encoding a brain-specific Na+-dependent inorganic phosphate cotransporter.
Proc Natl Acad Sci USA
91:5607-5611[Abstract/Free Full Text].
-
Ni B,
Wu X,
Yan GM,
Wang J,
Paul SM
(1995)
Regional expression and cellular localization of the Na+-dependent inorganic phosphate cotransporter of rat brain.
J Neurosci
15:5789-5799[Abstract].
-
Ozkan ED,
Ueda T
(1998)
Glutamate transport and storage in synaptic vesicles.
Jpn J Pharmacol
77:1-10[Medline].
-
Pollack AE
(2001)
Anatomy, physiology, and pharmacology of the basal ganglia.
Neurol Clin
19:523-534[Medline].
-
Reimer RJ,
Fon EA,
Edwards RH
(1998)
Vesicular neurotransmitter transport and the presynaptic regulation of quantal size.
Curr Opin Neurobiol
8:405-412[Web of Science][Medline].
-
Roghani A,
Shirzadi A,
Kohan SA,
Edwards RH,
Butcher LL
(1996)
Differential distribution of the putative vesicular transporter for acetylcholine in the rat central nervous system.
Brain Res Mol Brain Res
43:65-76[Medline].
-
Roseth S,
Fykse EM,
Fonnum F
(1995)
Uptake of L-glutamate into rat brain synaptic vesicles: effect of inhibitors that bind specifically to the glutamate transporter.
J Neurochem
65:96-103[Web of Science][Medline].
-
Sakata-Haga H,
Kanemoto M,
Maruyama D,
Hoshi K,
Mogi K,
Narita M,
Okado N,
Ikeda Y,
Nogami H,
Fukui Y,
Kojima I,
Takeda J,
Hisano S
(2001)
Differential localization and colocalization of two neuron-types of sodium-dependent inorganic phosphate cotransporters in rat forebrain.
Brain Res
902:143-155[Web of Science][Medline].
-
Shepherd GM
(1998)
In: The synaptic organization of the brain, Ed 4. New York: Oxford UP.
-
Sulzer D,
Joyce MP,
Lin L,
Geldwert D,
Haber SN,
Hattori T,
Rayport S
(1998)
Dopamine neurons make glutamatergic synapses in vitro.
J Neurosci
18:4588-4602[Abstract/Free Full Text].
-
Takamori S,
Rhee JS,
Rosenmund C,
Jahn R
(2000)
Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons.
Nature
407:189-194[Medline].
-
Takamori S,
Rhee JS,
Rosenmund C,
Jahn R
(2001)
Identification of differentiation-associated brain-specific phosphate transporter as a second vesicular glutamate transporter (VGLUT2).
J Neurosci
21:RC182[Abstract/Free Full Text]:1-6.
-
Varoqui H,
Schafer MK,
Zhu H,
Weihe E,
Erickson JD
(2002)
Identification of the differentiation-associated Na+/PI transporter as a novel vesicular glutamate transporter expressed in a distinct set of glutamatergic synapses.
J Neurosci
22:142-155[Abstract/Free Full Text].
-
Waerhaug O,
Ottersen OP
(1993)
Demonstration of glutamate-like immunoreactivity at rat neuromuscular junctions by quantitative electron microscopic immunocytochemistry.
Anat Embryol (Berl)
188:501-513[Medline].
-
Weihe E,
Tao-Cheng JH,
Schafer MK,
Erickson JD,
Eiden LE
(1996)
Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles.
Proc Natl Acad Sci USA
93:3547-3552[Abstract/Free Full Text].
Copyright © 2002 Society for Neuroscience 0270-6474/02/22135442-10$05.00/0
This article has been cited by other articles:
|
|
|
|
 
V. Varga, A. Losonczy, B. V. Zemelman, Z. Borhegyi, G. Nyiri, A. Domonkos, B. Hangya, N. Holderith, J. C. Magee, and T. F. Freund
Fast Synaptic Subcortical Control of Hippocampal Circuits
Science,
October 16, 2009;
326(5951):
449 - 453.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Heidrych, U. Zimmermann, S. Kuhn, C. Franz, J. Engel, S. V. Duncker, B. Hirt, C. M. Pusch, P. Ruth, M. Pfister, et al.
Otoferlin interacts with myosin VI: implications for maintenance of the basolateral synaptic structure of the inner hair cell
Hum. Mol. Genet.,
August 1, 2009;
18(15):
2779 - 2790.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Balschun, D. Moechars, Z. Callaerts-Vegh, B. Vermaercke, N. Van Acker, L. Andries, and R. D'Hooge
Vesicular Glutamate Transporter VGLUT1 Has a Role in Hippocampal Long-Term Potentiation and Spatial Reversal Learning
Cereb Cortex,
July 2, 2009;
(2009)
bhp133v1.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Wallen-Mackenzie, K. Nordenankar, K. Fejgin, M. C. Lagerstrom, L. Emilsson, R. Fredriksson, C. Wass, D. Andersson, E. Egecioglu, M. Andersson, et al.
Restricted Cortical and Amygdaloid Removal of Vesicular Glutamate Transporter 2 in Preadolescent Mice Impacts Dopaminergic Activity and Neuronal Circuitry of Higher Brain Function
J. Neurosci.,
February 18, 2009;
29(7):
2238 - 2251.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-L. Boulland, M. Jenstad, A. J. Boekel, F. G. Wouterlood, R. H. Edwards, J. Storm-Mathisen, and F. A. Chaudhry
Vesicular Glutamate and GABA Transporters Sort to Distinct Sets of Vesicles in a Population of Presynaptic Terminals
Cereb Cortex,
January 1, 2009;
19(1):
241 - 248.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Dehmel, Y. L. Cui, and S. E. Shore
Cross-Modal Interactions of Auditory and Somatic Inputs in the Brainstem and Midbrain and Their Imbalance in Tinnitus and Deafness
Am J Audiol,
December 1, 2008;
17(2):
S193 - S209.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. A. Parra, T. Baust, S. El Mestikawy, M. Quiroz, B. Hoffman, J. M. Haflett, J. K. Yao, and G. E. Torres
The Orphan Transporter Rxt1/NTT4 (SLC6A17) Functions as a Synaptic Vesicle Amino Acid Transporter Selective for Proline, Glycine, Leucine, and Alanine
Mol. Pharmacol.,
December 1, 2008;
74(6):
1521 - 1532.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Mendez, M.-J. Bourque, G. D. Bo, M. L. Bourdeau, M. Danik, S. Williams, J.-C. Lacaille, and L.-E. Trudeau
Developmental and Target-Dependent Regulation of Vesicular Glutamate Transporter Expression by Dopamine Neurons
J. Neurosci.,
June 18, 2008;
28(25):
6309 - 6318.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. J. Maher and G. L. Westbrook
Co-Transmission of Dopamine and GABA in Periglomerular Cells
J Neurophysiol,
March 1, 2008;
99(3):
1559 - 1564.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. R. Aubrey, F. M. Rossi, R. Ruivo, S. Alboni, G. C. Bellenchi, A. Le Goff, B. Gasnier, and S. Supplisson
The Transporters GlyT2 and VIAAT Cooperate to Determine the Vesicular Glycinergic Phenotype
J. Neurosci.,
June 6, 2007;
27(23):
6273 - 6281.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L.-E. Trudeau and R. Gutierrez
On Cotransmission & Neurotransmitter Phenotype Plasticity
Mol. Interv.,
June 1, 2007;
7(3):
138 - 146.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Geisler, C. Derst, R. W. Veh, and D. S. Zahm
Glutamatergic Afferents of the Ventral Tegmental Area in the Rat
J. Neurosci.,
May 23, 2007;
27(21):
5730 - 5743.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Juge, Y. Yoshida, S. Yatsushiro, H. Omote, and Y. Moriyama
Vesicular Glutamate Transporter Contains Two Independent Transport Machineries
J. Biol. Chem.,
December 22, 2006;
281(51):
39499 - 39506.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Moechars, M. C. Weston, S. Leo, Z. Callaerts-Vegh, I. Goris, G. Daneels, A. Buist, M. Cik, P. van der Spek, S. Kass, et al.
Vesicular Glutamate Transporter VGLUT2 Expression Levels Control Quantal Size and Neuropathic Pain.
J. Neurosci.,
November 15, 2006;
26(46):
12055 - 12066.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Hrabovszky, I. Kallo, G. F. Turi, K. May, G. Wittmann, C. Fekete, and Z. Liposits
Expression of Vesicular Glutamate Transporter-2 in Gonadotrope and Thyrotrope Cells of the Rat Pituitary. Regulation by Estrogen and Thyroid Hormone Status
Endocrinology,
August 1, 2006;
147(8):
3818 - 3825.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Masson, M. Darmon, A. Conjard, N. Chuhma, N. Ropert, M. Thoby-Brisson, A. S. Foutz, S. Parrot, G. M. Miller, R. Jorisch, et al.
Mice lacking brain/kidney phosphate-activated glutaminase have impaired glutamatergic synaptic transmission, altered breathing, disorganized goal-directed behavior and die shortly after birth.
J. Neurosci.,
April 26, 2006;
26(17):
4660 - 4671.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zilberter, T. Harkany, and C. D. Holmgren
Dendritic Release of Retrograde Messengers Controls Synaptic Transmission in Local Neocortical Networks
Neuroscientist,
August 1, 2005;
11(4):
334 - 344.
[Abstract]
[PDF]
|
|
|
|
|
|
|
A. Lavin, L. Nogueira, C. C. Lapish, R. M. Wightman, P. E. M. Phillips, and J. K. Seamans
Mesocortical Dopamine Neurons Operate in Distinct Temporal Domains Using Multimodal Signaling
J. Neurosci.,
May 18, 2005;
25(20):
5013 - 5023.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Winter, I. Brunk, D. J. Walther, M. Holtje, M. Jiang, J.-U. Peter, S. Takamori, R. Jahn, L. Birnbaumer, and G. Ahnert-Hilger
G{alpha}o2 Regulates Vesicular Glutamate Transporter Activity by Changing Its Chloride Dependence
J. Neurosci.,
May 4, 2005;
25(18):
4672 - 4680.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. V. Puig, F. Artigas, and P. Celada
Modulation of the Activity of Pyramidal Neurons in Rat Prefrontal Cortex by Raphe Stimulation In Vivo: Involvement of Serotonin and GABA
Cereb Cortex,
January 1, 2005;
15(1):
1 - 14.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Hrabovszky, G. Wittmann, G. F. Turi, Z. Liposits, and C. Fekete
Hypophysiotropic Thyrotropin-Releasing Hormone and Corticotropin-Releasing Hormone Neurons of the Rat Contain Vesicular Glutamate Transporter-2
Endocrinology,
January 1, 2005;
146(1):
341 - 347.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. G. Croft, G. D. Fortin, A. T. Corera, R. H. Edwards, A. Beaudet, L.-E. Trudeau, and E. A. Fon
Normal Biogenesis and Cycling of Empty Synaptic Vesicles in Dopamine Neurons of Vesicular Monoamine Transporter 2 Knockout Mice
Mol. Biol. Cell,
January 1, 2005;
16(1):
306 - 315.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. V. Puig, N. Santana, P. Celada, G. Mengod, and F. Artigas
In Vivo Excitation of GABA Interneurons in the Medial Prefrontal Cortex through 5-HT3 Receptors
Cereb Cortex,
December 1, 2004;
14(12):
1365 - 1375.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Hioki, F. Fujiyama, K. Nakamura, S.-X. Wu, W. Matsuda, and T. Kaneko
Chemically Specific Circuit Composed of Vesicular Glutamate Transporter 3- and Preprotachykinin B-producing Interneurons in the Rat Neocortex
Cereb Cortex,
November 1, 2004;
14(11):
1266 - 1275.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W.-C. Li, S. R. Soffe, and A. Roberts
From The Cover: Glutamate and acetylcholine corelease at developing synapses
PNAS,
October 26, 2004;
101(43):
15488 - 15493.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Santana, A. Bortolozzi, J. Serrats, G. Mengod, and F. Artigas
Expression of Serotonin1A and Serotonin2A Receptors in Pyramidal and GABAergic Neurons of the Rat Prefrontal Cortex
Cereb Cortex,
October 1, 2004;
14(10):
1100 - 1109.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. T. Fremeau Jr., K. Kam, T. Qureshi, J. Johnson, D. R. Copenhagen, J. Storm-Mathisen, F. A. Chaudhry, R. A. Nicoll, and R. H. Edwards
Vesicular Glutamate Transporters 1 and 2 Target to Functionally Distinct Synaptic Release Sites
Science,
June 18, 2004;
304(5678):
1815 - 1819.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Nakamura, K. Matsumura, T. Hubschle, Y. Nakamura, H. Hioki, F. Fujiyama, Z. Boldogkoi, M. Konig, H.-J. Thiel, R. Gerstberger, et al.
Identification of Sympathetic Premotor Neurons in Medullary Raphe Regions Mediating Fever and Other Thermoregulatory Functions
J. Neurosci.,
June 9, 2004;
24(23):
5370 - 5380.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Harkany, C. Holmgren, W. Hartig, T. Qureshi, F. A. Chaudhry, J. Storm-Mathisen, M. B. Dobszay, P. Berghuis, G. Schulte, K. M. Sousa, et al.
Endocannabinoid-Independent Retrograde Signaling at Inhibitory Synapses in Layer 2/3 of Neocortex: Involvement of Vesicular Glutamate Transporter 3
J. Neurosci.,
May 26, 2004;
24(21):
4978 - 4988.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Faundez and H. C. Hartzell
Intracellular Chloride Channels: Determinants of Function in the Endosomal Pathway
Sci. Signal.,
May 18, 2004;
2004(233):
re8 - re8.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Wojcik, J. S. Rhee, E. Herzog, A. Sigler, R. Jahn, S. Takamori, N. Brose, and C. Rosenmund
An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size
PNAS,
May 4, 2004;
101(18):
7158 - 7163.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Montana, Y. Ni, V. Sunjara, X. Hua, and V. Parpura
Vesicular Glutamate Transporter-Dependent Glutamate Release from Astrocytes
J. Neurosci.,
March 17, 2004;
24(11):
2633 - 2642.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Moriyama and A. Yamamoto
Glutamatergic Chemical Transmission: Look! Here, There, and Anywhere
J. Biochem.,
February 1, 2004;
135(2):
155 - 163.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Chuhma, H. Zhang, J. Masson, X. Zhuang, D. Sulzer, R. Hen, and S. Rayport
Dopamine Neurons Mediate a Fast Excitatory Signal via Their Glutamatergic Synapses
J. Neurosci.,
January 28, 2004;
24(4):
972 - 981.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. L. Rosin, B. D. Hettinger, A. Lee, and J. Linden
Anatomy of adenosine A2A receptors in brain: Morphological substrates for integration of striatal function
Neurology,
December 9, 2003;
61(90116):
S12 - 18.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. L. Petersen, E. N. Ottem, and C. D. Carpenter
Direct and Indirect Regulation of Gonadotropin-Releasing Hormone Neurons by Estradiol
Biol Reprod,
December 1, 2003;
69(6):
1771 - 1778.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Cheng, C.-L. Chen, P. Luo, M. Tan, M. Qiu, R. Johnson, and Q. Ma
Lmx1b, Pet-1, and Nkx2.2 Coordinately Specify Serotonergic Neurotransmitter Phenotype
J. Neurosci.,
November 5, 2003;
23(31):
9961 - 9967.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Hayashi, R. Morimoto, A. Yamamoto, and Y. Moriyama
Expression and Localization of Vesicular Glutamate Transporters in Pancreatic Islets, Upper Gastrointestinal Tract, and Testis
J. Histochem. Cytochem.,
October 1, 2003;
51(10):
1375 - 1390.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. V. Puig, P. Celada, L. Diaz-Mataix, and F. Artigas
In Vivo Modulation of the Activity of Pyramidal Neurons in the Rat Medial Prefrontal Cortex by 5-HT2A Receptors: Relationship to Thalamocortical Afferents
Cereb Cortex,
August 1, 2003;
13(8):
870 - 882.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Lin, K. McKinney, L. Liu, S. Lakhlani, and L. Jennes
Distribution of Vesicular Glutamate Transporter-2 Messenger Ribonucleic Acid and Protein in the Septum-Hypothalamus of the Rat
Endocrinology,
February 1, 2003;
144(2):
662 - 670.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. K.-H. Schafer, H. Varoqui, N. Defamie, E. Weihe, and J. D. Erickson
Molecular Cloning and Functional Identification of Mouse Vesicular Glutamate Transporter 3 and Its Expression in Subsets of Novel Excitatory Neurons
J. Biol. Chem.,
December 20, 2002;
277(52):
50734 - 50748.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. T. Fremeau Jr., J. Burman, T. Qureshi, C. H. Tran, J. Proctor, J. Johnson, H. Zhang, D. Sulzer, D. R. Copenhagen, J. Storm-Mathisen, et al.
The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate
PNAS,
October 29, 2002;
99(22):
14488 - 14493.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
