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The Journal of Neuroscience, November 1, 1998, 18(21):8751-8757
The Number of Glutamate Transporter Subtype Molecules at
Glutamatergic Synapses: Chemical and Stereological Quantification in
Young Adult Rat Brain
Knut P.
Lehre and
Niels C.
Danbolt
Department of Anatomy, Institute of Basic Medical Sciences,
University of Oslo, N-0317 Oslo, Norway
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ABSTRACT |
The role of transporters in shaping the glutamate concentration in
the extracellular space after synaptic release is controversial because
of their slow cycling and because diffusion alone gives a rapid
removal. The transporter densities have been measured electrophysiologically, but these data are from immature brains and do
not give precise information on the concentrations of the individual
transporter subtypes. Here we show by quantitative immunoblotting that
the numbers of the astroglial glutamate transporters GLAST
(EAAT1) and GLT (EAAT2) are 3200 and 12,000 per
µm3 tissue in the stratum radiatum of adult rat
hippocampus (CA1) and 18,000 and 2800 in the cerebellar molecular
layer, respectively. The total astroglial cell surface is 1.4 and 3.8 m2/cm3 in the two regions,
respectively, implying average densities of GLAST and GLT molecules in
the membranes around 2300 and 8500 µm 2 in the
former and 4700 and 740 µm2 in the latter
region. The total concentration of glial glutamate transporters in both
regions corresponds to three to five times the estimated number of
glutamate molecules in one synaptic vesicle from each of all
glutamatergic synapses. However, the role of glial glutamate
transporters in limiting synaptic spillover is likely to vary between
the two regions because of differences in the distribution of
astroglia. Synapses are completely ensheathed and separated from each
other by astroglia in the cerebellar molecular layer. In contrast,
synapses in hippocampus (stratum radiatum) are only contacted by
astroglia and are often found side by side without intervening glial
processes.
Key words:
neurotransmitter transport; glutamate uptake; protein
purification; astroglia; cerebellum; hippocampus
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INTRODUCTION |
The glutamate uptake system (Danbolt
et al., 1998 ) consists of at least five different transporter proteins
(GLAST/EAAT1, GLT/EAAT2, EAAC/EAAT3, EAAT4, and EAAT5) and represents
the only mechanism for removal of excitatory amino acids from the
extracellular fluid in the brain. Its importance for the long-term
maintenance of low extracellular concentrations of glutamate is well
documented (for review, see Danbolt, 1994; Robinson and Dowd,
1997).
The roles of these transporters during the first millisecond after
synaptic release of glutamate, however, is currently being debated.
Mathematical models (Holmes, 1995; Clements, 1996; Kleinle et al.,
1996; Barbour and Häusser, 1997) suggest that passive diffusion
alone causes a rapid decline in the glutamate concentration in the
synaptic cleft after release. Considering the slow kinetics of the
glutamate transporters [a cycling time of 50-100 msec (Wadiche et
al., 1995)], it has been argued (Otis et al., 1996) that glutamate uptake is important only for the slow components of glutamate removal
and for the ambient glutamate levels. However, the glutamate transporters could buffer glutamate on a submillisecond time scale by
binding rather than by transport if they are present in sufficient numbers close to the release sites (Tong and Jahr, 1994). There is now
experimental evidence for a rapid effect of the transporters (Barbour
et al., 1994; Maki et al., 1994; Tong and Jahr, 1994; Takahashi et al.,
1996; Diamond and Jahr, 1997; Otis et al., 1997). Furthermore,
astroglial anion-potentiated glutamate transporter currents are
activated in <1 msec after release of glutamate (Bergles and Jahr,
1997; Bergles et al., 1997). Rusakov and Kullmann (1998) have performed
kinetic simulations of the glutamate diffusion, and they used a range
(0-0.5 mM) of values for the average glutamate transporter
density because good data were lacking. Using the highest values they
predicted that the transporters rapidly reduce the extrasynaptic
glutamate concentration after the first millisecond and that
interaction with the transporters slows down the diffusion of glutamate
away from the site of release.
The densities of the glutamate transporters have been estimated
electrophysiologically (Takahashi et al., 1996; Bergles and Jahr, 1997;
Otis et al., 1997), but these data do not discriminate precisely
between the individual transporter subtypes. In addition, the data are
from immature animals, whereas the transporter densities are known to
change dramatically during the development of the brain (Furuta et al.,
1997; Ullensvang et al., 1997). Furthermore, biochemical uptake
activity measurements have insufficient anatomical resolution and are
likely to underestimate the true Vmax value. Transport activity measurements are further hampered by the lack of
nontransportable high-affinity subtype-specific blockers.
For these reasons, determination of the concentrations of the
individual glutamate transporter proteins is required to obtain information on the transporter densities in the mature brain and on the
contributions of the individual transporter subtypes. We have measured
the concentrations of GLAST and GLT in the hippocampus CA1 (stratum
radiatum) and in the cerebellum (molecular layer) in absolute terms in
adult rats. We have also measured the astroglial surface densities in
the two regions to calculate the numbers of transporter molecules per
square micrometer of membrane.
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MATERIALS AND METHODS |
Antibodies. Antipeptide antibodies against GLT and
GLAST were prepared as described (Lehre et al., 1995). The peptides
representing parts of GLAST and GLT are referred to by capital letters
"A" and "B," respectively, followed by numbers indicating the
corresponding amino acid residues in the sequences (given in
parentheses). The sequences refer to the rat sequences (Pines et al.,
1992; Storck et al., 1992): A522-541 (PYQLIAQDNEPEKPVADSET), B12-26
(KQVEVRMHDSHLSSE), and B493-508 (YHLSKSELDTIDSQHR). The corresponding
anti-peptide antibodies are referred to as anti-A522 (rabbit 68488),
anti-B12 (rabbit 68518), and anti-B493 (rabbit 84912).
Animals. Adult male Wistar rats from Møllegaard Hansen
(Lille Skensved, Denmark) were kept in the animal facility at the
Institute of Basic Medical Sciences. All handling of animals was
according to European regulations and was under veterinary supervision. The rats were killed by stunning and decapitation. The rat used for the
estimation of surface densities and the rats in groups C and D in Table
1 were 7-8 weeks old.
Covalent immobilization of antibodies to protein
A-Sepharose. Incubations were performed at room temperature if not
stated otherwise. Antibodies to GLT (anti-B493) or GLAST (anti-A522) were covalently immobilized on protein A-Sepharose essentially as
described (Danbolt et al., 1992) using 25 mM
dimethylsuberimidate in 0.2 M triethanolamine-HCl at pH
8.3. Noncovalently attached antibodies were removed by washing with 0.2 M sodium citrate, pH 3.7.
Immunoaffinity purification of GLT and GLAST. For each
purification experiment, four forebrains (~550 mg protein) or eight cerebella (~240 mg protein) freshly dissected from Wistar rats were
homogenized in ice-cold solubilization buffer (2% cholate, 6 mM EDTA, 1 mM PMSF, 0.03%
NaN3, 60 mM NaPi, pH 7.4, and ammonium sulfate to 10% saturation) in a total volume of 32 ml. The homogenate was incubated (10 min on ice) and centrifuged (39,000 × g, 20 min, 4°C). The supernatant was mixed with 128 ml
buffer (1.05% cholate, 6 mM EDTA, 94 mM NaCl,
75 mM NaPi, pH 7.4, 4°C), and incubated end-over-end (60 min, 4°C) with the covalently immobilized antibodies (see above). The
gel was washed (3 × 6 min, 4°C) with buffer (0.3 M
NaCl, 20 mM CHAPS, 40 mM NaPi, pH 7.4),
and the bound proteins were eluted (2 × 5 min, 4°C) with low-pH
buffer (0.15 M NaCl, 20 mM
3-[(3-cholamido-propyl)dimethylammonio]-1-propanesulphonate (CHAPS),
0.2 M glycine-HCl, pH 2.5). The eluate was immediately neutralized with 2 M Tris-HCl, pH 9. Dithiothreitol (50 mM), EDTA (5 mM), and PMSF (1 mM)
and 10 µl of phenol red concentrate (as low molecular mass marker)
were added. The solution was desalted on a 35 ml Sephadex G-50 Fine
column and concentrated on a 0.5 ml DEAE-cellulose column coupled in
series. The columns were equilibrated (at 4°C) with degassed buffer
[30 mM dithiothreitol, 20 mM CHAPS, 10 mM NaPi (pH 7.4 for GLT, pH 7.8 for GLAST)]. The
DEAE-cellulose column was washed (5 min) with 7.5 ml of the same buffer
without dithiothreitol, and the protein was eluted (2 min) with 1 ml of 50 mM NaPi with 0.2 M NaCl and 20 mM CHAPS. An aliquot of the eluate (destined for SDS-PAGE)
was mixed with SDS sample buffer (Laemmli, 1970) containing 50 mM dithiothreitol and frozen. The rest of the eluate (for
protein measurement) was frozen without additions. Because the
conversion of immunoreactivities to micrograms of protein is dependent
on the amounts of transporter protein in the standards, protein was
determined both with the assay of Lowry (Lowry et al., 1951) and with
the bicinchoninic acid assay (Smith et al., 1985). The values obtained
were very similar (data not shown). Bovine serum albumin was used as
standard.
The purity of the isolated protein was analyzed by SDS-PAGE (Laemmli,
1970) followed by staining with Coomassie brilliant blue or silver
(Danbolt et al., 1990). The formation of SDS-insoluble higher molecular
mass aggregates was largely avoided, although bands representing dimers
are seen in the lanes loaded with the largest amounts of protein. No
detectable amounts of IgG were leaking from the affinity column. As
shown in Figure 1B, the IgG heavy chains gave rise to
a band just below that of GLAST. This band was not present in the
purified preparations (Fig. 1B, lane 3). Because the
immunoaffinity isolation method is expensive with regard to antibodies,
the antigen was always added in excess to ensure saturation of the
antibodies. Under these conditions, 1 ml of gel containing 1 mg of
immobilized antibodies gave ~200 µg of transporter protein after
the final purification step. The calculations assume that the proteins
were 90% pure.
Quantitative immunoblotting. The blotting was performed as
described (Towbin et al., 1979; Levy et al., 1995). The tissue was
dissolved in SDS. (SDS solubilizes brain tissue completely. It goes
into a clear solution. Thus, the SDS extracts contained all tissue
components.) After protein determination, the extracts were subjected
to SDS-PAGE and blotted onto nitrocellulose membranes. The gels
(Laemmli, 1970; Levy et al., 1995) were 0.75 mm thick, 14 cm wide, and
11 cm long and consisted of 7.5% acrylamide. Each gel had 20 lanes.
Known amounts of the purified GLT or GLAST proteins were used as
standard. The tissue homogenates were prepared from whole hippocampus,
whole cerebellum, microdissected stratum radiatum of hippocampus
(subfield CA1, ~4 mm from the temporal pole), and microdissected
molecular layer of cerebellar vermis. The glial surface density (see
below) was measured in the molecular layer of lobulus 6 only, whereas
the tissue used for the quantification of transporter protein was
collected from the molecular layer of all lobules. This was done to
obtain enough protein without using an exceedingly large number of
rats, and because in contrast to EAAT4, GLAST and GLT are evenly
distributed in the molecular layer (Lehre et al., 1995; Dehnes et al.,
1998). Differing amounts of standard and sample proteins were applied
on each gel to verify signal linearity.
The immunolabeling of the blots was performed as described (Levy et
al., 1995; Ullensvang et al., 1997). Briefly, the protein blots were
blocked with gelatin, incubated (overnight) with antibody (anti-B12,
0.2 µg/ml; anti-B493, 0.2 µg/ml; anti-A522, 0.2 µg/ml), washed,
reblocked, incubated (90 min) with iodinated protein A (600-2000
cpm/µl), washed, dried, and mounted on transparent acetate sheets.
Then the blots were autoradiographed with x-ray film. The films were
developed, put behind the transparent acetate sheets (on which the
blots were mounted), and aligned with the nitrocellulose blots
(Ullensvang et al., 1997). The nitrocellulose-film sandwiches were put
on a glass plate illuminated from behind in a dark room so that the
labeling on the films could be seen through the blots. The bands were
cut out from the blots, and the radioactivity was determined. The
background was determined on nitrocellulose membrane pieces of the same
size, cut out from the blots below the labeled bands.
Whether anti-B12 or anti-B493 antibodies were used to detect GLT on the
immunoblots did not seem to matter (data not shown), suggesting that
any variable mRNA splicing does not significantly affect the amounts of
these epitopes. The results shown from the immunoblotting are based on
anti-B12 (whereas anti-B493 has been used for the purification).
Protein measurement. Protein was determined in purified
protein and homogenates as described (Lowry et al., 1951) using bovine serum albumin as standard. To block the CHAPS interference with the
color reaction, SDS (50 mg/ml) was added. SDS, CHAPS, buffer ions, and
salt were added to give equal concentrations in samples and standards.
The protein concentrations in several of the samples were also measured
with the bicinchoninic acid assay (Smith et al., 1985), with bovine
serum albumin as standard using a kit from Pierce (Rockford, IL). The
results obtained with the two protein assays were very similar both in
the crude tissue extracts and in the purified preparations of
transporter proteins. The average values were used.
Estimation of glial surface density. This was performed in
the stratum radiatum of hippocampus (subfield CA1) ~4 mm from the temporal pole and in the stratum moleculare in vermis of cerebellum (lobulus 6).
One rat was perfusion-fixed (Lehre et al., 1995) with a mixture of
2.5% glutaraldehyde and 1% freshly depolymerized paraformaldehyde in
0.1 M NaPi. Pieces of fixed tissue were embedded in
Durcupan as described (Lehre et al., 1995). Serial sections were cut
following the vertical sectioning method of Baddeley et al. (1986) at
60-90 nm with a diamond knife and collected on nickel grids with a
2 × 1 mm slot covered by a formvar/carbon film. The sections were treated for 2 sec with xylene vapor, contrasted with uranyl acetate and
lead citrate, and observed in a Phillips CM10 electron microscope. Pictures were taken from the corresponding parts of five to six sections in series at 4600 or 6300× primary magnification and printed
at a final magnification of 40,000 or 55,000×.
On each picture, tissue components were identified according to Peters
et al. (1991) and Palay and Chan-Palay (1974). Photographs from serial
sections were obtained because it is not always possible to identify
all of the cellular processes in a single picture and because this
study required identification of all astrocytic processes to obtain a
measure for the total astroglial cell surface. The serial photographs
greatly helped the identification because the individual components
could be followed through several sections. After glial cell membranes
were identified, the surface densities were calculated using a
stereological method (Baddeley et al., 1986) based on an overlay screen
with points and 2 cm cycloid arcs. The surface densities were estimated
according to the formula S(V) = 2 × (p/l) × (I/P), where p/l is the
ratio of test points to test curve length on the overlay screen and
I/P is the ratio of test curve intersection
counts to point counts. For hippocampus, the tangent to the
septotemporal axis ~4 mm from the temporal pole was chosen as
vertical axis. For cerebellum, the direction of the parallel fibers was
chosen as vertical axis. Because the distribution of the fine
astroglial processes appears relatively isotropic (Spacek, 1985), only
two sectioning angles perpendicular to each other were used. Surface
densities were estimated from each angle separately so that the results
from the two angles could be compared, and the mean value was
calculated. For hippocampus, the two sectioning angles from one rat
gave 2 × (5.48/µm) × (48/381) = 1.38 µm2/µm3 and 2 × (4.00/µm) × (122/732) = 1.33 µm2/µm3, respectively, from a
total of 234 µm2 electron micrographs. For
cerebellum from the same rat, the two angles gave 2 × (5.48/µm) × (149/508) = 3.21 µm2/µm3
and 2 × (5.48/µm) × (305/780) = 4.29 µm2/µm3, respectively, from
171 µm2 electron micrographs.
Estimation of volume changes during tissue processing and
sectioning. The tissue volume after perfusion fixation was taken as reference volume. This was done both because of the difficulty (Harvey and Napper, 1991) in determining changes in tissue volume during tissue fixation for electron microscopy and because the volume
changes during the perfusion fixation appeared to be small enough to be
ignored in the present study.
Less than 3% change in lengths, corresponding to <10% change in
volume, was observed in fixed tissue blocks from cortex cerebri during
osmication, dehydration, and embedding in Durcupan. Sectioning resulted
in a 10% compression of the side oriented perpendicular to the knife
edge. Thus, the 10% compression roughly counterbalanced the volume
increase during embedding reported previously (Harvey and Napper,
1991). Xylene vapor treatment (removed wrinkles, but) did not change
the lengths significantly. Magnification (4600 and 6300 ×) was within
2%, according to the Agar Scientific S106 magnification calibration
grid photographed in the electron microscope.
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RESULTS |
Quantification of GLT and GLAST in fresh tissue
GLT and GLAST were quantified (by immunoblotting) by comparing the
immunoreactivity (per micrograms of protein) of whole tissue solubilized in SDS with the immunoreactivity (per micrograms of protein) of purified protein standards. To obtain sufficient amounts of
standard GLT and GLAST, an immunoaffinity purification procedure was
developed (see Materials and Methods), and highly purified transporter
protein was obtained (Fig. 1). The
immunoreactivities in the whole-tissue protein extracts were very high
(Table 1). GLAST and GLT represent as
much as 0.32 ± 0.01 and 1.3 ± 0.03% of total tissue
protein in the hippocampal (CA1) stratum radiatum, and 1.8 ± 0.02 and 0.30 ± 0.01% in the cerebellar molecular layer, respectively. The concentrations of GLAST and GLT in whole forebrain (minus hypophysis and olfactory bulbs) homogenates were ~85 and 70%,
respectively, of the concentrations in the hippocampus (data not
shown), corresponding to approximately 0.2 and 0.8% of the total
tissue protein.
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Figure 1.
Demonstration of the purity of the isolated
glutamate transporter proteins and the lack of contamination with IgG.
A, Purified GLT and GLAST proteins were separated by
SDS-PAGE (10% acrylamide) and visualized by silver staining as
described in Materials and Methods. Lanes 1-6 contain
30, 60, 100, 300, 600, and 1000 ng of GLT, respectively, whereas
lanes 7-12 contain 1000, 600, 300, 100, 60, and 30 ng
of GLAST, respectively. The positions of monomer (a, b)
and dimer (arrowheads) bands of GLAST
(a) and GLT (b) are marked.
B, Purified GLAST protein and preimmune IgG (rabbit
68488) were run on 10% SDS-PAGE and stained with silver. The IgG has
been treated with the low pH elution buffer and dithiothreitol, like
the GLAST protein during isolation (see Materials and Methods).
Lane 1, IgG alone (500 ng); lane 2, IgG
and GLAST (500 and 300 ng, respectively); lane 3, GLAST
alone (300 ng).
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From Table 2, it can be calculated that
the total number of GLAST and GLT molecules in the stratum radiatum
(hippocampus) and the total number of GLAST, GLT, and EAAT4 molecules
in the molecular layer (cerebellum) are 15,000 and 23,000 per
µm3 tissue in the two regions, respectively.
Estimation of glial surface density
For hippocampus stratum radiatum CA1, the mean estimated
astroglial surface density was 1.4 µm2/µm3
(m2/cm3). This value appears
reasonable when compared with the astroglial surface density in the rat
visual cortex, which has been estimated (Jones and Greenough, 1996) to
be in the range of 1.34-1.64
µm2/µm3 depending on the
cortical layer and the complexity of the environment the rats were
raised in.
For the molecular layer of cerebellum (vermis, lobulus 6), the mean
surface density of astroglia (mainly Bergmann glia) was 3.8 µm2/µm3. This finding of a
2.7 times higher glial surface density in cerebellum than in
hippocampus is also reasonable as judged from the available literature.
Spacek (1985) found that 74% of the circumference of longitudinal
dendritic spine profiles in the cerebellar cortex was covered by glial
sheaths (i.e., all of the surface not contacted by the afferent axon
terminals), whereas glial sheaths covered only 29% of spines in the
visual cortex of the mouse. Furthermore, the surface density of
Purkinje cell spines (Dehnes et al., 1998), which was estimated in the
present material, was virtually identical to the value that could be
calculated from previously published data on the number of Purkinje
cells and the number of spines per cell (Harvey and Napper, 1988, 1991; Napper and Harvey, 1988).
The total area of all cell membranes was estimated to be ~14
µm2/µm3 in both regions. This
is in excellent agreement with Rusakov and Kullmann (1998) who arrived
at 14.2 µm2/µm3 in the
stratum oriens of the adult rat hippocampus CA1.
Densities of GLT and GLAST in glial cell membranes
In a previous study (Chaudhry et al., 1995) of ultrastructural
distributions of GLT and GLAST in hippocampus and cerebellum, virtually
all of the immunoreactivity appeared to be related to the astrocytic
plasma membranes. Furthermore, all astrocytes (in the two regions)
appeared to express these proteins, and no concentration differences
were noted between cell bodies and processes. For the calculations
(Table 2) it is therefore assumed that all of the GLAST and GLT
proteins are evenly distributed in the astrocytic plasma membranes.
It should be noted, however, that the transporter concentrations are
lower (but not zero) in the parts of the astrocytic
membranes facing other astrocytes, cell bodies, large dendrites, pia
mater, and vascular epithelium than in the parts facing neuronal
processes in the neuropil (Chaudhry et al., 1995). To decide how much
these differences affect the calculations (Table 2), we estimated the surface density of astrocytic membranes contacting other astrocytic membranes in the cerebellar molecular layer and arrived at 0.46 µm2/µm3, or ~12% of the
total astrocytic surface area. The corresponding value for the stratum
radiatum was not determined because astrocyte-to-astrocyte contacts are
seen less frequently here. The surface density of astrocytic membranes
facing large dendrites in the molecular layer was 0.13 µm2/µm3, or ~3% of the
total. The percentages of the astrocytic area contacting vascular
epithelium or pia mater were not determined either, but they appeared
to be orders of magnitude smaller. Thus, ignoring the differences in
transporter densities between neuropil- and non-neuropil-facing parts
of the astrocytic membrane introduces an error that is <10% in the
cerebellum and even less in the hippocampus.
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DISCUSSION |
The total tissue concentration of glutamate transporters
Available data (Haugeto et al., 1996; Rothstein et al., 1996)
suggest that GLT and GLAST are the quantitatively dominating glutamate
transporters and that the contribution of EAAC to the total uptake is
small. This conclusion is also in line with the observations that
genetically EAAC-deficient mice (Peghini et al., 1997) do not have
elevated levels of extracellular glutamate and do not develop
neurodegeneration. In contrast, mice deficient in GLT develop both
epilepsy and neurodegeneration (Tanaka et al., 1997). The concentration
of EAAT4 is quite high in the cerebellar cortex (Table 2) but very low
in the forebrain (Dehnes et al., 1998), whereas EAAT5 seems to be a
retinal protein (Arriza et al., 1997). The astroglial localization of
GLT and GLAST (Levy et al., 1993; Lehre et al., 1995) is in agreement
with the notion that astrocytes have the largest glutamate uptake
activity (Schousboe, 1981). Postsynaptic uptake may be of functional
significance in the cerebellum at postnatal day 12 (Takahashi et al.,
1996), but because the total transporter densities as well as the
relative contribution of the glial transporters to the total uptake are much higher in the adult (Furuta et al., 1997; Ullensvang et al., 1997), we do not know how important postsynaptic uptake is in the adult
cerebellum. Nevertheless, it is likely that postsynaptic uptake plays a
functional role in the parts of adult cerebellum with high levels of
EAAT4 (see Table 2 legend). The importance of EAAC for postsynaptic
uptake is unclear because of the low levels and lack of quantitative
data.
The glutamate uptake in nerve endings (Divac et al., 1977) is still a
puzzle because the nerve terminal glutamate transporter has not yet
been identified by molecular cloning. Still, its existence is difficult
to dispute (Gundersen et al., 1993). Like GLT (Arriza et al., 1994) it
appears to be sensitive to dihydrokainic acid, and it is legitimate to
speculate about whether it is a variant of GLT (Eliasof et al., 1998)
in accordance with the apparent presence of GLT mRNA in some neurons
(Torp et al., 1994, 1997). Recent data (Asztely et al., 1997) based on
hippocampal slices from 4- to 5-week-old guinea pigs suggest that
dihydrokainate-sensitive transporters are of great physiological
significance in reducing cross-talk between neighboring synapses, but
at present, we cannot tell whether this is mainly attributable to
astrocytic GLT or to the elusive nerve terminal transporter.
Thus, assuming that the concentration of the nerve terminal transporter
is low, the total concentrations of glutamate transporters in the
hippocampus stratum radiatum and in the cerebellar molecular layer are
almost 30 and 40 µM, respectively (Table 2). The
effective concentrations, however, are higher because these proteins
are accessible from the extracellular fluid and are mostly associated with astrocytes (see below and Table 2). The extracellular fluid represents 0.12-0.22% of the tissue volume in the normal adult rat
hippocampus (McBain et al., 1990; Nicholson and Syková, 1998; Rusakov and Kullmann, 1998) and about the same in the cerebellar molecular layer (Nicholson and Syková, 1998). This implies
average effective concentrations of 0.14-0.25 and 0.18-0.33
mM, respectively, depending on the extracellular volume.
From the data presented here (see Results and Table 2), it can be
calculated that the fraction of the extracellular space being enclosed
between one astroglial and one nonastroglial membrane is ~20 and 50%
in the hippocampus (CA1 stratum radiatum) and cerebellum (molecular
layer), respectively. Thus, the effective concentrations of the
transporters in the vicinity of astrocytes facing neuropil
are on the order of 0.7-1.3 and 0.36-0.66 mM in the two
regions, respectively, depending on the extracellular volume.
Comparison of the present findings with recent
electrophysiological data
It should be kept in mind that the numbers presented here
represent the total number of glutamate transporter molecules and do
not give information on transport activity. The activities of the
proteins are subject to regulation (for review, see Danbolt et al.,
1998), and the percentages of the transporters in the various activity
states are unknown. Furthermore, transporter molecules (with the
expected molecular mass) both in plasma membranes and in intracellular
membranes are included in our measurements; however, the latter is
minor compared with the former (Chaudhry et al., 1995; Dehnes et al.,
1998). Nevertheless, our data are in good agreement with recent
electrophysiological observations. Bergles and Jahr (1997) estimated
the density of glutamate transporters to be >2500
µm2 in the somatic membrane of astrocytes from
14-d-old rats (hippocampus CA1 stratum radiatum). With the caveat that
the glial surface density is unknown at this age, this value is in good
agreement with our value of 10,800 µm2 in adult
rats (Table 2: 8500 GLT + 2300 GLAST). First, the uptake activity at
60 d (adult) is 3-5 times higher than that at 14 d (Furuta
et al., 1997; Ullensvang et al., 1997). Second, no differences in GLT
and GLAST densities have been detected between astroglial bodies and
processes (Chaudhry et al., 1995). Furthermore, the glial membranes
also contain some EAAC molecules (Conti et al., 1998), although this
value is probably low, as explained above, compared with the values for
GLT and GLAST. Takahashi and co-workers (1996) estimated the
transporter densities on Purkinje cells from 12-d-old rats to be
between 1315 and 13,150, depending on which transporter (EAAT4 or EAAC)
is the more abundant. Otis and coworkers (1997) predicted that a
postsynaptic transporter (presumably EAAT4) binds at least 880 glutamate molecules per release site (implying that the number of
transporters must be higher because saturation cannot be expected).
Binding and transport capacities compared with
release capacity
Stevens and Tsujimoto (1995) estimated that each average central
synapse has approximately 20 release sites, each of which needs ~10
sec to refill. Thus, each terminal can release a total of approximately
20 vesicles within a 10 sec period. This implies a maximum average
release rate of two vesicles/sec. The average densities of
glutamatergic synapses in the stratum radiatum of hippocampus CA1 and
the cerebellar molecular layer are 0.9-1.3 µm3
(Woolley and McEwen, 1992) and 0.8 µm3 (Harvey
and Napper, 1991), respectively. If one synaptic vesicle contains
4000-5000 molecules (Clements, 1996; Barbour and Häusser, 1997),
it follows that the binding capacity of the known transporters (15,000 and 23,000 µm3) is significant compared with the
release capacity. A transporter cycling time of 70 msec implies that
the theoretical Vmax of 20,000 glutamate
transporters is 290,000 glutamate molecules/sec.
Will synaptic spill-over cause cross-talk?
In an extreme situation with the simultaneous release of many
vesicles, it is clear that the transporters cannot absorb all of the
released glutamate without going through several transport cycles, but
in moderate cases with only a few vesicles released each second, the
concentration of glutamate reaching a neighboring synapse will depend
on the geometry of the extracellular space with its diffusion barriers
and on the location of the transporters. Because most of the
transporters are on astrocytes (GLAST and GLT as well as some of the
EAAC) or on neuronal membranes facing astrocytes (EAAT4), the question
of whether the transporters contribute significantly to preventing
glutamate from reaching neighboring synapses is more or less the same
as asking where the astrocytic processes are in relation to the release
sites and the diffusion barriers (unless the nerve terminal glutamate
transporter or novel postsynaptic transporters, as explained above,
contribute significantly).
In the cerebellum, glutamatergic synapses are often almost completely
ensheathed by glia (Fig.
2B). Neighboring
synapses are usually separated by astrocytic processes, which express
high densities of GLAST and GLT (Table 2). The situation is very
different in the stratum radiatum of hippocampus CA1. Although most of
the spines are contacted by astrocytes, only a fraction of the spine surface is covered with glia. This is illustrated in Figure
2A, which also shows an example of two neighboring
synapses in close proximity to each other without separating glial
processes, a very common sight in this region. In cases such as that
depicted in Figure 2A, the glutamate diffusing out of
the synaptic cleft opposite to the glial process will not be hindered
by the glial glutamate transporters on its way toward the cleft of the
neighboring synapse. Thus, at these sites (on the short time scale)
glutamate is inactivated by diffusion unless EAAC or novel transporters are present in high concentrations. Note that astroglial processes are
close to the synaptic clefts in both regions, in agreement with the
observed activation of glial transporter currents shortly after
glutamate release (Bergles and Jahr, 1997; Bergles et al., 1997).
View larger version (179K):
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|
Figure 2.
Electron micrographs of neighboring synapses in
stratum radiatum of the hippocampus CA1 (A) and
of the cerebellar molecular layer (B). The
synapses shown have the typical morphology of glutamatergic synapses in
the two regions: synapses with asymmetric postsynaptic specializations
between boutons (b) and spines
(s). Note that the (GLAST- and GLT-expressing)
glial processes (asterisks) in hippocampus
(A) surround the group of synapses and axons
(a), and that one of the boutons shown is in
close contact with the neighboring synaptic cleft
(arrow). In contrast, the three cerebellar synapses
(B) between parallel fibers and Purkinje cell
spines are almost completely ensheathed and thereby separated from each
other by (GLAST- and GLT-expressing) glial processes
(asterisks). Scale bar, 400 nm.
|
|
Although detailed three-dimensional models of the tissue would be
valuable to simulate glutamate diffusion and inactivation, not even
such models would give the complete picture because all of the
structures are dynamic. Both the dendritic spines (Fifkova, 1985;
Fischer et al., 1998) and astrocytic processes (Wenzel et al., 1991)
are able to change their forms by contraction and distension. Furthermore, the glutamate transporter (Gegelashvilli et al., 1997) and
receptor (Rao and Craig, 1997) densities are subject to various kinds
of regulation.
In conclusion, glutamate transporters are present at sufficiently high
average densities to support the notion (Tong and Jahr, 1994; Diamond
and Jahr, 1997) that they can contribute to glutamate inactivation on
the short time scale by binding rather than by transport. However,
their importance in the control of extrasynaptic and intersynaptic
glutamate diffusion is likely to vary considerably between different
synapses because the transporters are predominantly associated with
astrocytes and thereby not evenly distributed in the extracellular
space. Mathematical models of the spatiotemporal transmitter profile
after synaptic release should therefore take into account the
localizations of astrocytic processes in relation to the transmitter
release sites.
| |
FOOTNOTES |
Received June 26, 1998; revised Aug. 17, 1998; accepted Aug. 21, 1998.
We thank Jon Storm-Mathisen, Ole Petter Ottersen, and Theodor Blackstad
for discussions. This work was supported by European Union BIOMED II
(contract BMH4-CT95-0571), Schreiners fond, Bruuns fond, Nansenfondet,
and the Norwegian Research Council.
Correspondence should be addressed to Dr. Niels C. Danbolt, Department
of Anatomy, Institute of Basic Medical Sciences, University of Oslo,
P.O. Box 1105 Blindern, N-0317 Oslo, Norway.
| |
REFERENCES |
-
Arriza JL,
Fairman WA,
Wadiche JI,
Murdoch GH,
Kavanaugh MP,
Amara SG
(1994)
Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex.
J Neurosci
14:5559-5569[Abstract].
-
Arriza JL,
Eliasof S,
Kavanaugh MP,
Amara SG
(1997)
Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance.
Proc Natl Acad Sci USA
94:4155-4160[Abstract/Free Full Text].
-
Asztely F,
Erdemli G,
Kullmann DM
(1997)
Extrasynaptic glutamate spillover in the hippocampus: dependence on temperature and the role of active glutamate uptake.
Neuron
18:281-293[Web of Science][Medline].
-
Baddeley AJ,
Gundersen HJG,
Cruz-Orive LM
(1986)
Estimation of surface area from vertical sections.
J Microsc
142:259-276[Web of Science][Medline].
-
Barbour B,
Häusser M
(1997)
Intersynaptic diffusion of neurotransmitter.
Trends Neurosci
20:377-384[Web of Science][Medline].
-
Barbour B,
Keller BU,
Llano I,
Marty A
(1994)
Prolonged presence of glutamate during excitatory synaptic transmission to cerebellar Purkinje cells.
Neuron
12:1331-1343[Web of Science][Medline].
-
Bergles DE,
Jahr CE
(1997)
Synaptic activation of glutamate transporters in hippocampal astrocytes.
Neuron
19:1297-1308[Web of Science][Medline].
-
Bergles DE,
Dzubay JA,
Jahr CE
(1997)
Glutamate transporter currents in bergmann glial cells follow the time course of extrasynaptic glutamate.
Proc Natl Acad Sci USA
94:14821-14825[Abstract/Free Full Text].
-
Chaudhry FA,
Lehre KP,
Campagne MV,
Ottersen OP,
Danbolt NC,
Storm-Mathisen J
(1995)
Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry.
Neuron
15:711-720[Web of Science][Medline].
-
Clements JD
(1996)
Transmitter timecourse in the synaptic cleft: its role in central synaptic function.
Trends Neurosci
19:163-171[Web of Science][Medline].
-
Conti F,
Debiasi S,
Minelli A,
Rothstein JD,
Melone M
(1998)
EAAC1, a high-affinity glutamate transporter, is localized to astrocytes and GABAergic neurons besides pyramidal cells in the rat cerebral cortex.
Cereb Cortex
8:108-116[Abstract/Free Full Text].
-
Danbolt NC
(1994)
The high affinity uptake system for excitatory amino acids in the brain.
Prog Neurobiol
44:377-396[Web of Science][Medline].
-
Danbolt NC,
Pines G,
Kanner BI
(1990)
Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain.
Biochemistry
29:6734-6740[Medline].
-
Danbolt NC,
Storm-Mathisen J,
Kanner BI
(1992)
A [Na++K+]coupled L-glutamate transporter purified from rat brain is located in glial cell processes.
Neuroscience
51:295-310[Web of Science][Medline].
-
Danbolt NC,
Chaudhry FA,
Dehnes Y,
Lehre KP,
Levy LM,
Ullensvang K,
Storm-Mathisen J
(1998)
Properties and localization of glutamate transporters.
Prog Brain Res
116:21-41.
-
Dehnes Y,
Chaudhry FA,
Ullensvang K,
Lehre KP,
Storm-Mathisen J,
Danbolt NC
(1998)
The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia.
J Neurosci
18:3606-3619[Abstract/Free Full Text].
-
Diamond JS,
Jahr CE
(1997)
Transporters buffer synaptically released glutamate on a submillisecond time scale.
J Neurosci
17:4672-4687[Abstract/Free Full Text].
-
Divac I,
Fonnum F,
Storm-Mathisen J
(1977)
High affinity uptake of glutamate in terminals of corticostriatal axons.
Nature
266:377-378[Medline].
-
Eliasof S,
Arriza JL,
Leighton BH,
Kavanaugh MP
(1998)
Excitatory amino acid transporters of the salamander retina: identification, localization, and function.
J Neurosci
18:698-712[Abstract/Free Full Text].
-
Fifkova E
(1985)
A possible mechanism of morphometric changes in dendritic spines induced by stimulation.
Cell Mol Neurobiol
5:47-63[Web of Science][Medline].
-
Fischer M,
Kaech S,
Knutti D,
Matus A
(1998)
Rapid actin-based plasticity in dendritic spines.
Neuron
20:847-854[Web of Science][Medline].
-
Furuta A,
Rothstein JD,
Martin LJ
(1997)
Glutamate transporter protein subtypes are expressed differentially during rat CNS development.
J Neurosci
17:8363-8375[Abstract/Free Full Text].
-
Gegelashvili G,
Danbolt NC,
Schousboe A
(1997)
Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia.
J Neurochem
69:2612-2615[Web of Science][Medline].
-
Gundersen V,
Danbolt NC,
Ottersen OP,
Storm-Mathisen J
(1993)
Demonstration of glutamate/aspartate uptake activity in nerve endings by use of antibodies recognizing exogenous D-aspartate.
Neuroscience
57:97-111[Web of Science][Medline].
-
Harvey RJ,
Napper RM
(1988)
Quantitative study of granule and Purkinje cells in the cerebellar cortex of the rat.
J Comp Neurol
274:151-157[Web of Science][Medline].
-
Harvey RJ,
Napper RM
(1991)
Quantitative studies on the mammalian cerebellum.
Prog Neurobiol
36:437-463[Web of Science][Medline].
-
Haugeto Ø,
Ullensvang K,
Levy LM,
Chaudhry FA,
Honoré T,
Nielsen M,
Lehre KP,
Danbolt NC
(1996)
Brain glutamate transporter proteins form homomultimers.
J Biol Chem
271:27715-27722[Abstract/Free Full Text].
-
Holmes WR
(1995)
Modeling the effect of glutamate diffusion and uptake on NMDA and non-NMDA receptor saturation.
Biophys J
69:1734-1747[Web of Science][Medline].
-
Jones TA,
Greenough WT
(1996)
Ultrastructural evidence for increased contact between astrocytes and synapses in rats reared in a complex environment.
Neurobiol Learn Mem
65:48-56[Web of Science][Medline].
-
Kleinle J,
Vogt K,
Luscher HR,
Muller L,
Senn W,
Wyler K,
Streit J
(1996)
Transmitter concentration profiles in the synaptic cleft: an analytical model of release and diffusion.
Biophys J
71:2413-2426[Web of Science][Medline].
-
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[Medline].
-
Lehre KP,
Levy LM,
Ottersen OP,
Storm-Mathisen J,
Danbolt NC
(1995)
Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations.
J Neurosci
15:1835-1853[Abstract].
-
Levy LM,
Lehre KP,
Rolstad B,
Danbolt NC
(1993)
A monoclonal antibody raised against an [Na++K+]coupled L-glutamate transporter purified from rat brain confirms glial cell localization.
FEBS Lett
317:79-84[Web of Science][Medline].
-
Levy LM,
Lehre KP,
Walaas SI,
Storm-Mathisen J,
Danbolt NC
(1995)
Down-regulation of glial glutamate transporters after glutamatergic denervation in the rat brain.
Eur J Neurosci
7:2036-2041[Web of Science][Medline].
-
Lowry OH
(1953)
The quantitative histochemistry of the brain.
J Histochem Cytochem
1:420-428[Abstract].
-
Lowry OH,
Rosebrough NJ,
Farr AL,
Randall RJ
(1951)
Protein measurement with the Folin phenol reagent.
J Biol Chem
193:265-275[Free Full Text].
-
Lowry OH,
Roberts NR,
Leiner KY,
Wu ML,
Farr AL,
Albers RW
(1954)
The quantitative histochemistry of brain, III. Ammon's horn.
J Biol Chem
207:39-49[Free Full Text].
-
Maki R,
Robinson MB,
Dichter MA
(1994)
The glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate depresses excitatory synaptic transmission via a presynaptic mechanism in cultured hippocampal neurons.
J Neurosci
14:6754-6762[Abstract].
-
McBain CJ,
Traynelis SF,
Dingledine R
(1990)
Regional variation of extracellular space in the hippocampus.
Science
249:674-677[Abstract/Free Full Text].
-
Napper RM,
Harvey RJ
(1988)
Quantitative study of the Purkinje cell dendritic spines in the rat cerebellum.
J Comp Neurol
274:158-167[Web of Science][Medline].
-
Nicholson C,
Syková E
(1998)
Extracellular space structure revealed by diffusion analysis.
Trends Neurosci
21:207-215[Web of Science][Medline].
-
Otis TS,
Wu YC,
Trussell LO
(1996)
Delayed clearance of transmitter and the role of glutamate transporters at synapses with multiple release sites.
J Neurosci
16:1634-1644[Abstract/Free Full Text].
-
Otis TS,
Kavanaugh MP,
Jahr CE
(1997)
Postsynaptic glutamate transport at the climbing fiber purkinje cell synapse.
Science
277:1515-1518[Abstract/Free Full Text].
-
Palay SL,
Chan-Palay V
(1974)
In: Cerebellar cortex: cytology and organization. New York: Springer.
-
Peghini P,
Janzen J,
Stoffel W
(1997)
Glutamate transporter EAAC-1-deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration.
EMBO J
16:3822-3832[Web of Science][Medline].
-
Peters A,
Palay SL,
Webster HF
(1991)
In: The fine structure of the nervous system. Neurons and their supporting cells. New York: Oxford UP.
-
Pines G,
Danbolt NC,
Bjørås M,
Zhang Y,
Bendahan A,
Eide L,
Koepsell H,
Storm-Mathisen J,
Seeberg EK,
Kanner BI
(1992)
Cloning and expression of a rat brain L-glutamate transporter.
Nature
360:464-467[Medline].
-
Rao A,
Craig AM
(1997)
Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons.
Neuron
19:801-812[Web of Science][Medline].
-
Robinson MB,
Dowd LA
(1997)
Heterogeneity and functional properties of subtypes of sodium-dependent glutamate transporters in the mammalian central nervous system.
Adv Pharmacol
37:69-115.
-
Rothstein JD,
Dykes-Hoberg M,
Pardo CA,
Bristol LA,
Jin L,
Kuncl RW,
Kanai Y,
Hediger MA,
Wang Y,
Schielke JP,
Welty DF
(1996)
Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.
Neuron
16:675-686[Web of Science][Medline].
-
Rusakov DA,
Kullmann DM
(1998)
Extrasynaptic glutamate diffusion in the hippocampus: ultrastructural constraints, uptake, and receptor activation.
J Neurosci
18:3158-3170[Abstract/Free Full Text].
-
Schousboe A
(1981)
Transport and metabolism of glutamate and GABA in neurons and glial cells.
Int Rev Neurobiol
22:1-45[Medline].
-
Smith PK,
Krohn RI,
Hermanson GT,
Mallia AK,
Gartner FH,
Provenzano MD,
Fujimoto EK,
Goeke NM,
Olson BJ,
Klenk DC
(1985)
Measurement of protein using bicinchoninic acid.
Anal Biochem
150:76-85[Web of Science][Medline].
-
Spacek J
(1985)
Three-dimensional analysis of dendritic spines. III. Glial sheath.
Anat Embryol
171:245-252[Medline].
-
Stevens CF,
Tsujimoto T
(1995)
Estimates for the pool size of releasable quanta at a single central synapse and for the time required to refill the pool.
Proc Natl Acad Sci USA
92:846-849[Abstract/Free Full Text].
-
Storck T,
Schulte S,
Hofmann K,
Stoffel W
(1992)
Structure, expression, and functional analysis of a Na+-dependent glutamate/aspartate transporter from rat brain.
Proc Natl Acad Sci USA
89:10955-10959[Abstract/Free Full Text].
-
Takahashi M,
Sarantis M,
Attwell D
(1996)
Postsynaptic glutamate uptake in rat cerebellar Purkinje cells.
J Physiol (Lond)
497:523-530[Abstract/Free Full Text].
-
Tanaka K,
Watase K,
Manabe T,
Yamada K,
Watanabe M,
Takahashi K,
Iwama H,
Nishikawa T,
Ichihara N,
Kikuchi T,
Okuyama S,
Kawashima N,
Hori S,
Takimoto M,
Wada K
(1997)
Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1.
Science
276:1699-1702[Abstract/Free Full Text].
-
Tong G,
Jahr CE
(1994)
Block of glutamate transporters potentiates postsynaptic excitation.
Neuron
13:1195-1203[Web of Science][Medline].
-
Torp R,
Danbolt NC,
Babaie E,
Bjørås M,
Seeberg E,
Storm-Mathisen J,
Ottersen OP
(1994)
Differential expression of two glial glutamate transporters in the rat brain: an in situ hybridization study.
Eur J Neurosci
6:936-942[Web of Science][Medline].
-
Torp R,
Hoover F,
Danbolt NC,
Storm-Mathisen J,
Ottersen OP
(1997)
Differential distribution of the glutamate transporters GLT1 and rEAAC1 in rat cerebral cortex and thalamus: an in situ hybridization analysis.
Anat Embryol
195:317-326[Medline].
-
Towbin H,
Staehelin T,
Gordon J
(1979)
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci USA
76:4350-4354[Abstract/Free Full Text].
-
Ullensvang K,
Lehre KP,
Storm-Mathisen J,
Danbolt NC
(1997)
Differential developmental expression of the two rat brain glutamate transporters GLAST and GLT.
Eur J Neurosci
9:1646-1655[Web of Science][Medline].
-
Wadiche JI,
Arriza JL,
Amara SG,
Kavanaugh MP
(1995)
Kinetics of a human glutamate transporter.
Neuron
14:1019-1027[Web of Science][Medline].
-
Wenzel J,
Lammert G,
Meyer U,
Krug M
(1991)
The influence of long-term potentiation on the spatial relationship between astrocyte processes and potentiated synapses in the dentate gyrus neuropil of rat brain.
Brain Res
560:122-131[Web of Science][Medline].
-
Woolley CS,
McEwen BS
(1992)
Estradiol mediates fluctuation in hippocampal synapse density during the estrous cycle in the adult rat.
J Neurosci
12:2549-2554[Abstract].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18218751-07$05.00/0
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[Full Text]
[PDF]
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Tonic release of glutamate by a DIDS-sensitive mechanism in rat hippocampal slices
J. Physiol.,
April 15, 2005;
564(2):
397 - 410.
[Abstract]
[Full Text]
[PDF]
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J. S. Diamond
Deriving the Glutamate Clearance Time Course from Transporter Currents in CA1 Hippocampal Astrocytes: Transmitter Uptake Gets Faster during Development
J. Neurosci.,
March 16, 2005;
25(11):
2906 - 2916.
[Abstract]
[Full Text]
[PDF]
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C.-Y. Chen and A. C. Bonham
Glutamate suppresses GABA release via presynaptic metabotropic glutamate receptors at baroreceptor neurones in rats
J. Physiol.,
January 15, 2005;
562(2):
535 - 551.
[Abstract]
[Full Text]
[PDF]
|
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S. Raghavachari and J. E. Lisman
Properties of Quantal Transmission at CA1 Synapses
J Neurophysiol,
October 1, 2004;
92(4):
2456 - 2467.
[Abstract]
[Full Text]
[PDF]
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H. Huang and A. Bordey
Glial Glutamate Transporters Limit Spillover Activation of Presynaptic NMDA Receptors and Influence Synaptic Inhibition of Purkinje Neurons
J. Neurosci.,
June 23, 2004;
24(25):
5659 - 5669.
[Abstract]
[Full Text]
[PDF]
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A. Scimemi, A. Fine, D. M. Kullmann, and D. A. Rusakov
NR2B-Containing Receptors Mediate Cross Talk among Hippocampal Synapses
J. Neurosci.,
May 19, 2004;
24(20):
4767 - 4777.
[Abstract]
[Full Text]
[PDF]
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Y. H. Huang, S. R. Sinha, K. Tanaka, J. D. Rothstein, and D. E. Bergles
Astrocyte Glutamate Transporters Regulate Metabotropic Glutamate Receptor-Mediated Excitation of Hippocampal Interneurons
J. Neurosci.,
May 12, 2004;
24(19):
4551 - 4559.
[Abstract]
[Full Text]
[PDF]
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R. Schniepp, K. Kohler, T. Ladewig, E. Guenther, G. Henke, M. Palmada, C. Boehmer, J. D. Rothstein, S. Broer, and F. Lang
Retinal Colocalization and In Vitro Interaction of the Glutamate Receptor EAAT3 and the Serum- and Glucocorticoid-Inducible Kinase SGK1
Invest. Ophthalmol. Vis. Sci.,
May 1, 2004;
45(5):
1442 - 1449.
[Abstract]
[Full Text]
[PDF]
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Y. H. Huang, M. Dykes-Hoberg, K. Tanaka, J. D. Rothstein, and D. E. Bergles
Climbing Fiber Activation of EAAT4 Transporters and Kainate Receptors in Cerebellar Purkinje Cells
J. Neurosci.,
January 7, 2004;
24(1):
103 - 111.
[Abstract]
[Full Text]
[PDF]
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D. N. Furness and D. M. Lawton
Comparative Distribution of Glutamate Transporters and Receptors in Relation to Afferent Innervation Density in the Mammalian Cochlea
J. Neurosci.,
December 10, 2003;
23(36):
11296 - 11304.
[Abstract]
[Full Text]
[PDF]
|
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B. Voutsinos-Porche, G. Knott, K. Tanaka, C. Quairiaux, E. Welker, and G. Bonvento
Glial Glutamate Transporters and Maturation of the Mouse Somatosensory Cortex
Cereb Cortex,
October 1, 2003;
13(10):
1110 - 1121.
[Abstract]
[Full Text]
[PDF]
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P. Marcaggi, D. Billups, and D. Attwell
The Role of Glial Glutamate Transporters in Maintaining the Independent Operation of Juvenile Mouse Cerebellar Parallel Fibre Synapses
J. Physiol.,
October 1, 2003;
552(1):
89 - 107.
[Abstract]
[Full Text]
[PDF]
|
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A. Mitani and K. Tanaka
Functional Changes of Glial Glutamate Transporter GLT-1 during Ischemia: An In Vivo Study in the Hippocampal CA1 of Normal Mice and Mutant Mice Lacking GLT-1
J. Neurosci.,
August 6, 2003;
23(18):
7176 - 7182.
[Abstract]
[Full Text]
[PDF]
|
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|
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|
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K. M. Franks, C. F. Stevens, and T. J. Sejnowski
Independent Sources of Quantal Variability at Single Glutamatergic Synapses
J. Neurosci.,
April 15, 2003;
23(8):
3186 - 3195.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-S. Chiu, K. Jensen, I. Sokolova, D. Wang, M. Li, P. Deshpande, N. Davidson, I. Mody, M. W. Quick, S. R. Quake, et al.
Number, Density, and Surface/Cytoplasmic Distribution of GABA Transporters at Presynaptic Structures of Knock-In Mice Carrying GABA Transporter Subtype 1-Green Fluorescent Protein Fusions
J. Neurosci.,
December 1, 2002;
22(23):
10251 - 10266.
[Abstract]
[Full Text]
[PDF]
|
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|
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N. Cholet, L. Pellerin, P. J. Magistretti, and E. Hamel
Similar Perisynaptic Glial Localization for the Na+,K+-ATPase {alpha}2 Subunit and the Glutamate Transporters GLAST and GLT-1 in the Rat Somatosensory Cortex
Cereb Cortex,
May 1, 2002;
12(5):
515 - 525.
[Abstract]
[Full Text]
[PDF]
|
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W. Reichelt and T. Knopfel
Glutamate Uptake Controls Expression of a Slow Postsynaptic Current Mediated by mGluRs in Cerebellar Purkinje Cells
J Neurophysiol,
April 1, 2002;
87(4):
1974 - 1980.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Chen, C. Aoki, V. Mahadomrongkul, C. E. Gruber, G. J. Wang, R. Blitzblau, N. Irwin, and P. A. Rosenberg
Expression of a Variant Form of the Glutamate Transporter GLT1 in Neuronal Cultures and in Neurons and Astrocytes in the Rat Brain
J. Neurosci.,
March 15, 2002;
22(6):
2142 - 2152.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
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J. S. Diamond
Neuronal Glutamate Transporters Limit Activation of NMDA Receptors by Neurotransmitter Spillover on CA1 Pyramidal Cells
J. Neurosci.,
November 1, 2001;
21(21):
8328 - 8338.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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M. Zhou and H. K. Kimelberg
Freshly Isolated Hippocampal CA1 Astrocytes Comprise Two Populations Differing in Glutamate Transporter and AMPA Receptor Expression
J. Neurosci.,
October 15, 2001;
21(20):
7901 - 7908.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Barbour
An Evaluation of Synapse Independence
J. Neurosci.,
October 15, 2001;
21(20):
7969 - 7984.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. S. Diamond and C. E. Jahr
Synaptically Released Glutamate Does Not Overwhelm Transporters on Hippocampal Astrocytes During High-Frequency Stimulation
J Neurophysiol,
May 1, 2000;
83(5):
2835 - 2843.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. S. Otis and M. P. Kavanaugh
Isolation of Current Components and Partial Reaction Cycles in the Glial Glutamate Transporter EAAT2
J. Neurosci.,
April 15, 2000;
20(8):
2749 - 2757.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. S. Meldrum
Glutamate as a Neurotransmitter in the Brain: Review of Physiology and Pathology
J. Nutr.,
April 1, 2000;
130(4):
1007 - 1007.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
Z.-C. Ye, J. D. Rothstein, and H. Sontheimer
Compromised Glutamate Transport in Human Glioma Cells: Reduction-Mislocalization of Sodium-Dependent Glutamate Transporters and Enhanced Activity of Cystine-Glutamate Exchange
J. Neurosci.,
December 15, 1999;
19(24):
10767 - 10777.
[Abstract]
[Full Text]
[PDF]
|
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|
|
|
|
M. J. Schwei, P. Honore, S. D. Rogers, J. L. Salak-Johnson, M. P. Finke, M. L. Ramnaraine, D. R. Clohisy, and P. W. Mantyh
Neurochemical and Cellular Reorganization of the Spinal Cord in a Murine Model of Bone Cancer Pain
J. Neurosci.,
December 15, 1999;
19(24):
10886 - 10897.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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S. Mennerick, W. Shen, W. Xu, A. Benz, K. Tanaka, K. Shimamoto, K. E. Isenberg, J. E. Krause, and C. F. Zorumski
Substrate Turnover by Transporters Curtails Synaptic Glutamate Transients
J. Neurosci.,
November 1, 1999;
19(21):
9242 - 9251.
[Abstract]
[Full Text]
[PDF]
|
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|
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R. Ventura and K. M. Harris
Three-Dimensional Relationships between Hippocampal Synapses and Astrocytes
J. Neurosci.,
August 15, 1999;
19(16):
6897 - 6906.
[Abstract]
[Full Text]
[PDF]
|
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D. Trotti, M. Aoki, P. Pasinelli, U. V. Berger, N. C. Danbolt, R. H. Brown Jr., and M. A. Hediger
Amyotrophic Lateral Sclerosis-linked Glutamate Transporter Mutant Has Impaired Glutamate Clearance Capacity
J. Biol. Chem.,
January 5, 2001;
276(1):
576 - 582.
[Abstract]
[Full Text]
[PDF]
|
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A. D. Mitrovic, F. Plesko, and R. J. Vandenberg
Zn2+ Inhibits the Anion Conductance of the Glutamate Transporter EAAT4
J. Biol. Chem.,
July 6, 2001;
276(28):
26071 - 26076.
[Abstract]
[Full Text]
[PDF]
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Top
Abstract
Introduction
