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The Journal of Neuroscience, March 15, 2003, 23(6):2040
Neuronal Glutamate Uptake Contributes to GABA Synthesis and
Inhibitory Synaptic Strength
Gregory C.
Mathews1, 2 and
Jeffrey S.
Diamond1
1 Synaptic Physiology Unit, National Institute of
Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, Maryland 20892-4066, and 2 Department of
Neurology, Johns Hopkins University School of Medicine, Baltimore,
Maryland 21287
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ABSTRACT |
Neurons must maintain a supply of neurotransmitter in their
presynaptic terminals to fill synaptic vesicles. GABA is taken up into
inhibitory terminals by transporters or is synthesized from glutamate
by glutamic acid decarboxylase. Here we report that glutamate
transporters supply GABAergic terminals in the hippocampus with
glutamate, which is then used to synthesize GABA for filling synaptic
vesicles. Glutamate transporter antagonists reduced IPSC and miniature
IPSC (mIPSC) amplitudes, consistent with a reduction in the amount of
GABA packaged into each synaptic vesicle. This reduction occurred
rapidly and independently of synaptic activity, suggesting that
modulation of vesicular GABA content does not require vesicle release
and refilling. Raising extracellular glutamate levels increased mIPSC
amplitudes by enhancing glutamate uptake and, consequently, GABA
synthesis. These results indicate that neuronal glutamate transporters
strengthen inhibitory synapses in response to extracellular glutamate.
This modulation appears to occur under normal conditions and may
constitute a negative feedback mechanism to combat hyperexcitability.
Key words:
glutamate transporters; synaptic vesicles; GABA; metabolism; inhibition; hippocampus
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Introduction |
Glutamate is an essential
component of synaptic transmission in the CNS, both at excitatory
synapses, where it is the neurotransmitter, and at inhibitory synapses,
where it serves as the substrate for GABA synthesis. In excitatory
neurons, glutamate is synthesized either from glutamine or from glucose
metabolites. Both pathways rely on astrocytes, which supply neurons
with essential substrates (for review, see Hertz et al., 1999 ).
Glutamate is also taken up into excitatory presynaptic terminals by
high-affinity transporters (for review, see Danbolt, 2001). In
inhibitory neurons, all GABA, except that taken up through
transporters, is synthesized by decarboxylation of glutamate by
glutamic acid decarboxylase (GAD; for review, see Martin and Tobin,
2000). It is not known whether glutamate metabolism in inhibitory
terminals mirrors that in excitatory terminals, although it has been
proposed that glutamate may be taken up into inhibitory terminals by
presynaptic transporters (Rothstein et al., 1994; Sepkuty et al.,
2002).
High-affinity glutamate transporters function primarily to maintain a
low concentration of glutamate in the extracellular space. Although
some subtypes [glutamate transporter 1 (GLT-1) and
glutamate-aspartate transporter] are localized predominantly to astrocytes, others [excitatory amino acid carrier 1 (EAAC1) and
excitatory amino acid transporter 4] are expressed mostly by
neurons (Rothstein et al., 1994). Both glial and neuronal transporters may be localized to play specific homeostatic roles, although a lack of
selective inhibitors (except for GLT-1) has hindered investigation of
unique physiological roles for these transporters. Perisynaptic
transporters may be positioned to prevent excitotoxicity (Rothstein et
al., 1996), to limit diffusion of glutamate between excitatory synapses
(Asztely et al., 1997; Lozovaya et al., 1999; Carter and Regehr, 2000;
Diamond, 2001; Arnth-Jensen et al., 2002), or to supply the terminal
with neurotransmitter (Gundersen et al., 1993). Transporters used by
excitatory terminals have not been identified but may be a GLT-1-like
subtype (Danbolt, 2001; Chen et al., 2002). Anatomical data indicate
that the neuronal subtype EAAC1 colocalizes with GAD (Conti et al.,
1998) and is located at inhibitory terminals (Rothstein et al., 1994;
He et al., 2000). Reducing expression of EAAC1 is associated with a decrease in both tissue GABA levels and de novo synthesis
from glutamate, which may lead to neuronal hyperexcitability and
epilepsy (Sepkuty et al., 2002).
We show here that presynaptic glutamate transporters are involved in
the bidirectional regulation of inhibitory synaptic transmission. Inhibiting neuronal glutamate transporters reduced
GABAA receptor (GABAAR)-mediated IPSCs and miniature IPSCs
(mIPSCs), suggesting that GABA synthesis and, consequently, vesicular
filling were reduced. These effects did not depend on astrocytic
glutamate metabolism. They also occurred independently of synaptic
activity, suggesting that transmitter concentration can be modulated in vesicles that are ready to be released. In addition, enhancing glutamate uptake increased the amplitude of miniature IPSCs. The bidirectional regulation of GABAergic transmission was prevented by an
inhibitor of GAD, verifying that the uptake-dependent effects required
GABA synthesis. Our findings that glutamate transporters on inhibitory
terminals contribute to GABA release suggest that a compensatory or
protective mechanism allows extracellular glutamate to strengthen
inhibitory synapses.
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Materials and Methods |
Electrophysiology. Whole-cell patch-clamp recordings
from CA1 pyramidal neurons were made in transverse hippocampal slices (400 µm thick) made from postnatal day 10-14 d rats as described previously (Sakmann and Stuart, 1995). Slices were stored in and perfused with a solution containing (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose, bubbled with 95%
O2 and 5% CO2 at room
temperature. All solutions also contained 10 µM
6,7-dinitroquinoxaline-2,3-dione (DNQX) and 5 µM MK-801 (dizocilpine maleate) to block
glutamatergic responses (20 µM DNQX was used
when exogenous glutamate was applied). Patch pipettes (resistance, 2-4
M ) were filled with (in mM): 130 CsCl, 10 EGTA, 10 HEPES, 2 Mg-ATP, 0.2 Na-GTP, and 1 QX-314 (lidocaine N-ethyl bromide), titrated to pH 7.4 with CsOH. Using
an upright fixed stage microscope (Zeiss, Thornwood, NY),
pyramidal neurons were identified under video observation using
infrared differential interference contrast imaging. Unless indicated
otherwise (see Figs. 4C, 5D), voltage-clamp
recordings (Vhold =  60 mV, uncorrected for junction potential) were made at room temperature using an Axopatch
1D amplifier (Axon Instruments); currents were filtered at
2 kHz, sampled at 5 kHz, and digitally stored for off-line analysis
(IGOR Pro software). Access resistance was monitored throughout the
experiment, and data were discarded when >10% change occurred.
IPSCs were evoked at 8-15 sec intervals with a bipolar stainless steel
electrode placed in the stratum oriens (constant current stimulation,
25-150 µA; 100 µsec duration). When required, D,L-threo- -hydroxyaspartic acid (THA, 300 µM) and dihydrokainic acid (DHK, 300 µM; Ocean Produce International) were dissolved in the recording solutions just before the experiment;
D,L-threo--benzyloxyaspartic acid (TBOA, 30 µM) was diluted from a stock solution. Sucrose (500 mM) and GABA (10 µM)
were added to the control solution and were pressure-applied (2-3 sec
puffs, 4-5 psi) through a patch pipette. When effects of THA on puffed
GABA responses were tested, THA was included in the perfusion and
pipette solutions. In hyperosmotic stimulation experiments, 1 min was
allowed between applications to ensure refilling of the vesicular pool,
and responses were quantitated by integrating the current over the
duration of the sucrose application. For studies involving inhibition
of GAD or glutamine synthetase, mercaptopropionic acid (MPA, 250 µM) or methionine sulfoximine (MSO, 1.5 mM) was included in solutions used for storage
(3-7 hr before recording) and perfusion of slices.
mIPSC recording and analysis. Electrically evoked mIPSCs
were obtained as described above, except that calcium in the
extracellular solution was replaced with strontium, giving rise to
numerous asynchronous events after each stimulus (Goda and Stevens,
1994). Stimuli were delivered every 8-10 sec, and events were
collected in the 718 msec after each stimulus, excluding the initial
100 msec. Raw traces were differentiated and smoothed; events exceeding a slope threshold (chosen to detect even the smallest and slowest events) were then visually examined. Only single events occurring from
a level baseline were selected for analysis. Responses to 60-70
stimuli, resulting in ~200 events, were elicited in each condition in
each cell. When drug effects were measured, 5 min of wash-in was
allowed before mIPSCs were collected. To compare between cells,
individual event amplitudes in each cell were normalized to the mean
amplitude in a control. Decay time constants were estimated by fitting
a single exponential to the decay phase of the averaged mIPSC in each cell.
Unless indicated otherwise, data are expressed as mean ± SD, and
p values were derived from paired t tests, with
significance concluded at p < 0.05. For pooled mIPSC
amplitudes, which were not normally distributed, the
Mann-Whitney U test was used. In graphs illustrating
responses over time from a collection of experiments (see Figs.
1C, 3D) and in cumulative probability histograms
(see Figs. 4-6), error bars indicate SEM.
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Results |
Glutamate transporter blockade reduces electrically
evoked IPSCs
Inhibitory interneurons are abundant in stratum oriens (SO) of
area CA1, where distinct subtypes make either axosomatic or axodendritic synapses onto excitatory pyramidal neurons (McBain and
Fisahn, 2001). In juvenile hippocampal slices (postnatal days 10-14),
electrical stimulation in SO elicited monosynaptic
GABAAR-mediated IPSCs, which were recorded under
whole-cell voltage-clamp conditions (Vhold = 60 mV) in the presence of glutamate receptor antagonists (see
Materials and Methods). Inward IPSCs (attributable to symmetrical chloride concentrations) could be blocked completely by SR95531 (gabazine) (10 µM), a selective
GABAAR antagonist (Fig.
1A). To examine the
effects of glutamate uptake on IPSCs, we applied THA (300 µM), a competitive inhibitor of both neuronal
and astrocytic glutamate transporters (Arriza et al., 1994). THA (300 µM) does not completely block glutamate uptake
in hippocampal slices (Bergles and Jahr, 1997), because it is also
transported, thereby reducing its effective concentration. THA reduced
evoked IPSC amplitudes to 74 ± 22% of control
(p < 0.001; n = 16 cells; Fig.
1B,C,H); this effect recovered only partially
on washout. A nontransported inhibitor, TBOA (30 µM; Shimamoto et al., 1998), caused a similar reduction in (and incomplete recovery of) IPSC amplitude (to 71 ± 10% of control; p = 0.005; n = 4 cells; Fig. 1D,H), suggesting that THA did not
exert its effect intracellularly (e.g., by inhibiting vesicular
transporters). To test whether the effects of glutamate transporter
inhibition on IPSCs were mediated by the GABA synthetic pathway, THA
was applied to slices that had been preincubated for 3-7 hr with the
GAD inhibitor MPA (250 µM; Lamar, 1970). IPSC amplitudes were gradually reduced after application of MPA without reaching a plateau in 30 min (data not shown); after several hours of
preincubation with MPA, stronger stimuli were required to evoke IPSCs
that were smaller on average than those of controls (380 ± 177 pA in MPA vs 711 ± 447 pA). In MPA-treated slices, THA had no
significant effect on IPSC amplitudes (110 ± 20% of control; p = 0.17; n = 5; Fig.
1E,H), suggesting that transported glutamate acts as a substrate for GABA synthesis.
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Figure 1.
Inhibition of glutamate transporters reduces
electrically evoked IPSCs. A, Evoked IPSC recorded from
a CA1 pyramidal cell (Vhold = 60 mV) in
control solution (gray) and in the presence of
the GABAAR antagonist SR95531 (black).
B, Evoked IPSCs in control solution
(gray) and in the presence of 300 µM THA (black), a nonselective glutamate
transporter antagonist. C, Normalized IPSC amplitudes
[Amp. (norm)] during experiments testing the effects
of THA (n = 16 cells) with stimulation either
continued (filled circles) or suspended
(open circles) during wash-in. Error bars indicate SEM.
D, IPSC in control solution
(gray) and in the presence of 30 µM TBOA (black), a nonselective,
nonsubstrate inhibitor of glutamate uptake. E, IPSC in
control solution (gray) and in the presence of
300 µM THA (black) after inhibition of
GABA synthesis with 250 µM MPA, a GAD inhibitor.
F, IPSC in control solution
(gray) and in the presence of 300 µM THA (black) after inhibition of
astrocytic glutamine synthetase with 1.5 mM MSO.
G, IPSCs recorded from a CA1 pyramidal cell after
stimulation in stratum radiatum (s. radiatum) in control
solution (gray) and in the presence of 300 µM THA (black). H, Summary
of effects of glutamate transporter antagonists on IPSC amplitude in
various conditions. White numbers in bars
indicate the numbers of cells tested. Error bars indicate SD.
Asterisks denote a statistically significant difference
compared with control (p < 0.05).
s. rad., Stratum radiatum.
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Glutamate taken up by astrocytes may be converted to glutamine and
returned to neurons, a cycle requiring the glial-specific enzyme
glutamine synthetase (Martinez-Hernandez et al., 1977). It is possible
that THA, a blocker of astrocytic and neuronal glutamate transporters,
reduces IPSC amplitudes by disrupting this intermediary metabolism. We
tested this possibility with MSO, an inhibitor of glutamine synthetase
(Sellinger, 1967). MSO (1.5 mM) prevents conversion of
glutamate to glutamine, thereby trapping glutamate in glial cells and
decreasing glutamate levels in synaptic terminals twofold (Laake et
al., 1995). In slices that had been preincubated for 3-7 hr with 1.5 mM MSO, field EPSPs were reduced by 35% compared with
responses elicited under identical conditions in control slices
(control, 0.51 ± 0.21 mV; n = 12; MSO, 0.33 ± 0.16 mV; n = 12; p = 0.02, unpaired
t test), indicating that glutamate metabolism was
compromised. In MSO-treated slices, however, THA still reduced IPSC
amplitudes to 71 ± 17% of control (p = 0.03; n = 7 cells; Fig. 1F,H),
indicating that glutamate transporter antagonists do not reduce IPSCs
through actions on astrocytic glutamate uptake.
Interneurons in stratum radiatum (SR) express higher levels of the GABA
transporter GAT-1 than interneurons in SO (Engel et al., 1998) and
therefore may rely less than SO interneurons on GABA synthesis to fill
synaptic vesicles. Nonetheless, THA also reduced IPSCs elicited by SR
stimulation (55 ± 27% of control; p = 0.014;
n = 5 cells; Fig. 1G,H), indicating
that a role for presynaptic glutamate uptake is not unique to SO
interneurons. Responses in all subsequent experiments were elicited by
stimulation in SO.
These results suggest that glutamate uptake into inhibitory synaptic
terminals is an important source of GABA for filling synaptic vesicles.
If vesicles remain filled to a "set point" until being released,
one would expect that the effects of transporter blockade would not be
evident until the pool of filled vesicles was released and recycled.
Evidence suggests, however, that the vesicular transmitter
concentration reflects a dynamic equilibrium between the vesicle and
the synaptic terminal (Williams, 1997), allowing transmitter levels in
vesicles to be modulated before release. To distinguish between these
possibilities, in a separate set of experiments stimulation was
suspended for the first 5 min of THA application to test whether the
effect of THA required synaptic activity. When stimulation was resumed,
a similar reduction in IPSC amplitude was observed (70 ± 16% of
control; p = 0.03; n = 5 cells; Fig.
1C) as when stimulation was not interrupted, suggesting that
the effects of blocking glutamate transporters do not require release
and subsequent recycling of synaptic vesicles.
THA does not affect release probability or
postsynaptic GABAARs
Activation of presynaptic metabotropic glutamate receptors
(mGluRs) inhibits GABA release at some synapses (Desai et al., 1994;
Gereau and Conn, 1995; Mitchell and Silver, 2000); blocking glutamate
transporters enhances mGluR activation at both excitatory and
inhibitory synapses (Fitzsimonds and Dichter, 1996; Brasnjo and Otis,
2001; Reichelt and Knopfel, 2002). To test whether the decreased IPSC
amplitude in the presence of THA involved mGluR activation, THA was
applied in the presence of group I and II and group III mGluR
antagonists [(RS)- -methyl-4-carboxyphenylglycine (MCPG),
500 µM; and
(RS)--cyclopropyl-4-phosphonophenylglycine (CPPG), 200 µM, respectively]. THA nonetheless reduced the
IPSC amplitude (to 81 ± 3% of control; p = 0.02;
n = 4; Fig.
2A). The effect in the
presence of mGluR antagonists was not significantly different from that
in their absence (p = 0.5, unpaired t
test; Fig. 2B), suggesting that mGluRs do not mediate
the IPSC reduction. THA affected neither paired pulse depression
(observed in 2.5 mM calcium: 105 ± 13% of
control; n = 6; p = 0.37) nor paired pulse facilitation (observed in 1 mM external
calcium: 99 ± 26% of control; n = 5;
p = 0.99) of IPSCs (Fig. 2C,D), suggesting that the reduction of IPSC amplitude was not attributable to a change
in release probability. Moreover, THA did not reduce the response to
exogenously applied GABA (puff responses in THA were 102 ± 9% of
control; p = 0.9; n = 3; Fig.
2E,F), indicating that the drug did not affect
postsynaptic GABAA receptors.
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Figure 2.
THA does not affect release probability or
postsynaptic GABAA receptors. A, In the
presence of group I and II and group III metabotropic glutamate
receptor antagonists (MCPG, 500 µM; and CPPG, 200 µM, respectively), THA still reduced evoked IPSCs.
B, Bar graph comparing the effects of THA alone and in
the presence of mGluR antagonists. amp, Amplitude.
C, Effects of 300 µM THA on IPSCs elicited
by a pair of stimuli (50 msec interval). Paired pulse depression was
observed in normal (2.5 mM) calcium (top),
and paired pulse facilitation was observed in low (1 mM)
calcium (bottom). THA (black) reduced
IPSC amplitudes compared with control (gray) but
did not affect the paired pulse ratio. D, Summary bar
graph showing that THA (black bars) affected neither
paired pulse depression nor facilitation. E, Currents
elicited in a pyramidal cell by puff application of GABA in control
solution (gray) and in the presence of 300 µM THA (black; THA was included in the
bath and puffer solutions). F, Summary graph showing
that THA did not affect GABAA receptors on pyramidal cells.
In all bar graphs, white numbers indicate
the numbers of cells tested, and error bars indicate SD.
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THA reduces hyperosmotically evoked IPSCs
The above results suggest that THA acts presynaptically to reduce
IPSC amplitude without altering release probability. If glutamate
uptake contributes to the process by which presynaptic vesicles are
filled with GABA, one would predict that blocking transporters with THA
would reduce vesicular filling and diminish the postsynaptic response
to release of the entire pool of synaptic vesicles. Application of
hyperosmolar extracellular solution elicits release of the "readily
releasable pool" (RRP) of vesicles through mechanisms that are
independent of action potentials and calcium influx (Rosenmund and
Stevens, 1996). Puff application of control extracellular solution
supplemented with 500 mM sucrose evoked a barrage of
synaptic events, giving rise to a postsynaptic response that could be
blocked by the GABAAR antagonist SR95531 (Fig.
3A). The sucrose-evoked
response decayed back toward baseline during the puff, and immediately
subsequent puffs elicited greatly diminished responses (Fig.
3B), suggesting that most of the RRP was depleted during a
single application. The depletion gradually recovered such that
consistent responses could be elicited at 1 min intervals (Fig.
3D). THA reduced the postsynaptic response to hyperosmotic stimulation (to 76 ± 9% of control; n = 9;
p < 0.0001; Fig. 3C,D), consistent with a
reduction in the total amount of GABA packaged into the vesicles
composing the RRP. The onset and extent of the effect of THA on
electrically and sucrose-evoked IPSCs were similar (compare Figs.
1C, 3D), even though each sucrose application
depleted the RRP, and electrical stimulation did not. These results,
together with those showing that the effect of THA on IPSCs is not
activity-dependent (Fig. 1C), are consistent with the idea
that modulation by glutamate uptake of vesicular GABA content does not
require release and recycling of synaptic vesicles.
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Figure 3.
THA reduces hyperosmotically stimulated release of
GABA. A, Postsynaptic current elicited by puff
application of control solution with 500 mM sucrose
(gray). The puffer was directed toward the
pyramidal cell body. The response was almost completely abolished when
the GABAA antagonist SR95531 (10 µM) was
added to the bath (black). B, The
responses to a second puff application of sucrose 1 sec after the first
indicated that the releasable pool only partially recovered from
depletion by the first puff. C, Response to hyperosmotic
stimulation in control solution (gray) and in
the presence of 300 µM THA (black).
D, Normalized charge transfer during sucrose-evoked
IPSCs in the presence of 300 µM THA
(n = 9 cells). THA was not included in the sucrose
solution. Error bars indicate SEM.
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Blocking glutamate uptake reduces miniature IPSC amplitudes
Although the results presented thus far are consistent with
blockade of glutamate uptake reducing GABA synthesis and, consequently, the amount of transmitter packaged into each vesicle, the effects of
THA also could be explained by a reduction in the total number of
synaptic vesicles available for release. In other systems, manipulations that increase or decrease vesicle filling cause commensurate changes in the amplitude of miniature synaptic events (Song et al., 1997; Pothos et al., 1998; Van der Kloot et al., 2000),
suggesting that vesicles need not contain a prescribed amount of
transmitter to be released. Moreover, mIPSCs recorded from hippocampal
pyramidal cells in rats in which EAAC1 had been knocked down with
chronic exposure to antisense oligonucleotides were smaller than those
recorded in control animals (Sepkuty et al., 2002). Conversely,
knocking out the synaptically localized GAD isoform GAD65 did not
affect amplitude but decreased the frequency of
GABAAR-mediated mIPSCs (Tian et al., 1999),
possibly because only the "full" vesicles were released. To
distinguish between these alternatives, we tested the effects of
glutamate transporter blockade on postsynaptic responses to the release
of single vesicles. Extracellular calcium was replaced with strontium
to desynchronize electrically evoked release and thereby to resolve
postsynaptic responses to individual release events occurring at
stimulated synapses (Goda and Stevens, 1994) (Fig.
4A). In some cells,
relatively high spontaneous release rates made it difficult to ensure
that all events occurring within 700 msec of stimulation arose at
stimulated synapses (the first 100 msec of the response, comprising
primarily the synchronous IPSC, was excluded). However, the size and
shape of presumptive evoked events in these cells, as well as the
effects of various experimental manipulations described below, were
indistinguishable from those obtained in cells with lower spontaneous
activity, so the results were pooled, and the events will be referred
to as mIPSCs. THA (300 µM) reduced the mean
mIPSC amplitudes obtained in 11 cells from 32 ± 5 to 28 ± 3 pA, or by ~13% (Fig. 4B, inset). Individual mIPSCs
normalized to the mean for each cell were then pooled, and cumulative
probability histograms were generated, demonstrating a significant
shift in the amplitude distribution (p < 0.0001, Mann-Whitney U test; n = 1491 in
control and 1624 in THA; Fig. 4B). Using identical
selection criteria, similar numbers of events were obtained in each
condition, suggesting that a change in mIPSC frequency did not occur in
THA. The decay time constants of the average mIPSCs from individual
cells were not altered by THA (24.7 ± 9.6 msec in control vs
23.8 ± 4.8 msec in THA; p = 0.8;
n = 11 cells), indicating that altered membrane properties did not account for the amplitude changes.
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Figure 4.
THA reduces mIPSC amplitudes. A,
Electrically evoked IPSCs in normal calcium
(gray) and in the same cell after calcium was
replaced with strontium (black). In the presence of
strontium, a diminished synchronous component of the response was
followed by asynchronously released events, which were collected and
pooled for each cell under each experimental condition.
B, mIPSC amplitudes in control (strontium) solution
(gray symbols; n = 1624 events) and in the additional presence of 300 µM THA
(black symbols; n = 1491 events).
Events in each of 11 cells were normalized (norm) to the
mean mIPSC amplitude in control, and cumulative probability histograms
were placed into 25 equal bins (p between 0 and
1), allowing events in different cells to be pooled. Error bars
indicate cell-to-cell variability (SEM) of the normalized histograms.
Inset, Average mIPSC waveforms from a representative
cell in control solution (gray) and in the
presence of THA (black). C, mIPSC
amplitudes in control (strontium) solution (gray
symbols; n = 909 events) and in the
additional presence of 300 µM THA (black
symbols; n = 958 events) recorded at
34°C. Inset, Average mIPSC waveforms from a
representative cell in control solution (gray)
and in the presence of THA (black).
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Neurotransmitter uptake and synthesis involve a series of
temperature-dependent enzymatic reactions. The experiments presented thus far were performed at room temperature; at physiological temperatures, the increased capacity of other processes may reduce the
impact of glutamate uptake on GABA synthesis, even though glutamate
uptake itself is steeply temperature-dependent (Wadiche and Kavanaugh,
1998). At 34°C, however, THA reduced mIPSC amplitudes to 84 ± 8% of control in five cells (pooled data, p < 0.0001; n = 909 in control and 958 in THA; Fig. 4C),
similar to the effects at room temperature, suggesting that glutamate
uptake contributes to GABA synthesis at more physiological
temperatures. Consistent with recent studies using molecular approaches
to knock down neuronal glutamate transporters (Sepkuty et al., 2002),
these results suggest that reducing glutamate uptake leads to a
decrease in intraterminal glutamate levels and, as a result, a lower
concentration of GABA in synaptic vesicles.
If filling of synaptic vesicles depends in part on newly synthesized
GABA, and glutamate transporters provide the substrate for GABA
synthesis, one would expect that incubation with MPA, which reduced
IPSC amplitudes, would also reduce mIPSC amplitudes. The mean amplitude
of pooled mIPSCs recorded in nine cells that had been preincubated with
the GAD inhibitor MPA was 16% smaller than the mean from 19 pooled
controls, and their distribution was significantly changed
(p < 0.0001, Mann-Whitney U test;
n = 3483 for control and 1758 for MPA; data not shown).
Exogenous glutamate application increases mIPSC amplitudes
The data presented in Figure 4 indicate that reducing glutamate
uptake into inhibitory terminals reduces GABA synthesis and, consequently, the amount of GABA released from synaptic vesicles. These
results raise the possibility that increasing glutamate uptake may
enhance GABAergic transmission, provided that postsynaptic GABAARs are not normally saturated during a
synaptic event (Frerking et al., 1995; Nusser et al., 1997; Hajos et
al., 2000; Perrais and Ropert, 2000). Such a mechanism could increase
inhibition and thereby offer protection against hyperexcitability
associated with pathological increases in extracellular glutamate
levels. To test this idea, glutamate was added to the superfusion
solution while mIPSCs were evoked in the presence of
Sr2+ (Fig.
5). Addition of 100 µM
glutamate increased mean mIPSC amplitudes in 10 cells to 120 ± 25% of control (pooled data, p < 0.0001, Mann-Whitney U test; n = 1990 in control
and 1975 in glutamate; Fig. 5A). A higher concentration (500 µM) increased mIPSC amplitudes in nine cells
even more (156 ± 41% of control; pooled data, p < 0.0001; n = 1493 in control and 1477 in glutamate;
Fig. 5A). These increases appeared to reflect enhanced GABA
synthesis, because 500 µM glutamate exerted a
much smaller effect in slices preincubated with 250 µM MPA (mean mIPSCs from 10 cells, 117 ± 27% of control; Fig. 5B). Moreover, the effect of 100 µM glutamate was abolished when it was applied
together with THA (300 µM) in five cells (300 µM; 102 ± 23% of control; pooled data,
p = 0.95; n = 1088 in control and 1418 in glutamate plus THA; Fig. 5C). Applied alone, glutamate (100 µM) also caused an amplitude increase in
mIPSCs recorded at 34°C (116 ± 22% of control in six cells;
pooled data, p < 0.0001; n = 973 in
control and 1177 in glutamate; Fig. 5D).
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Figure 5.
Exogenously applied glutamate increases mIPSC
amplitudes. A, Addition of 100 µM
(black, closed circles; n = 1975 events) or 500 µM (black, open circles;
n = 1477 events) glutamate
(glu) increased mIPSC amplitude compared with
control (gray, closed circles;
n = 1990; gray, open circles;
n = 1493, respectively). Cumulative probability
histograms were constructed as described in the Figure
4B legend. Inset, Average mIPSC
waveforms from two representative cells in control solution
(gray) and in the presence of either 100 µM (black, left) or 500 µM
(black, right) glutamate. B,
Preincubation with MPA, a GAD inhibitor, reduced the effect of 500 µM glutamate on mIPSC amplitude. The
data for 500 µM glutamate from A
(open circles) are included for comparison.
Inset, Average mIPSC waveforms from a representative
cell preincubated in MPA in control solution
(gray) and in the presence of 500 µM glutamate (black). C,
Three hundred micromolar THA blocked the effect of glutamate on mIPSCs.
mIPSCs recorded during coapplication of 300 µM THA and
100 µM glutamate (black symbols;
n = 1418 events) were not different compared with
control (gray symbols; n = 1088 events). Inset, Average mIPSC waveforms from a
representative cell in control solution (gray)
and in the presence of 300 µM THA and 100 µM glutamate (black). D,
Recorded at 34°C, addition of 100 µM glutamate
(black symbols; n = 1177 events)
increased mIPSC amplitude compared with control (gray
symbols; n = 973 events).
Inset, Average mIPSC waveforms from a representative
cell in control solution (gray) and in the
presence of 100 µM glutamate (black).
norm, Normalized.
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Presynaptic uptake does not exhibit GLT-1 pharmacology
A selective blocker of GLT-1, DHK (Arriza et al., 1994) inhibits
glutamate uptake into excitatory synaptic terminals (Gundersen et al.,
1993; Danbolt, 2001). Recently, a splice variant of GLT-1 that may be localized presynaptically on neurons was
identified (Chen et al., 2002), suggesting a role in supplying
excitatory terminals with glutamate. We used DHK to test whether the
presynaptic transporter on inhibitory terminals is GLT-1-like. If it
were, DHK would be expected to exert effects similar to those of THA. Alternatively, if DHK blocked only astrocytic transporters, it might be
expected to raise extracellular glutamate (Muñoz et al., 1987) and to
mimic exogenous glutamate.
The effects of DHK (300 µM) on evoked IPSCs were complex
(data not shown), possibly confounded by effects on release probability through activation of presynaptic kainate receptors (Kamboj et al.,
1992; Rodriguez-Moreno et al., 1997). Because mIPSC amplitudes should
not be affected by changes in release probability, we examined the
effect of DHK on mIPSCs. DHK increased mIPSC amplitudes to 132 ± 20% of control in eight cells (pooled data, p < 0.0001; n = 1455 in control and 1409 in DHK; Fig.
6A). The effect of DHK on mIPSC amplitudes was attributable to increased GABA synthesis, because no changes were observed in slices preincubated in 250 µM MPA (100 ± 9% of control in five
cells; pooled data, p = 0.6; n = 790 in
control and 1096 in DHK; Fig. 6B). The striking
difference between the effects of DHK and THA indicates that the
presynaptic transporter on inhibitory terminals is not DHK-sensitive
and therefore not a GLT-1 isoform. Furthermore, the effect of DHK is
consistent with the hypothesis that elevating extracellular glutamate
levels by blocking glial transport (Muñoz et al., 1987) enhances
glutamate uptake into neurons and, consequently, increases GABA
synthesis. These opposing effects of THA and DHK on mIPSC amplitudes
(Figs. 4, 6) suggest that alterations of glutamate uptake into
inhibitory presynaptic terminals may downregulate or upregulate the
amount of GABA contained in synaptic vesicles and thereby regulate
inhibitory synaptic strength.
View larger version (25K):
[in this window]
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|
Figure 6.
Presynaptic glutamate transporters do not exhibit
GLT-1 pharmacology. A, Addition of 300 µM
DHK (black symbols; n = 1409 events), a selective GLT-1 blocker, increased mIPSC amplitudes compared
with control (gray symbols; n = 1455 events). Inset, Average mIPSC waveforms from a
representative cell in control solution (gray)
and in the presence of DHK (black). B,
After preincubation with MPA, 300 µM DHK
(black; n = 1096 events) no longer
increased mIPSC amplitudes compared with control
(gray; n = 790 events).
Inset, Average mIPSC waveforms from a representative
cell in control solution (gray) and in the
presence of DHK (black). norm,
Normalized.
|
|
| |
Discussion |
The findings presented here indicate that glutamate transporters
regulate the synaptic pool of GABA in hippocampal CA1 interneurons. Reducing glutamate uptake diminished evoked IPSCs and mIPSCs without affecting postsynaptic receptors, suggesting that vesicular GABA content was reduced. These effects required GABA synthesis but not
glial glutamate metabolism, suggesting that glutamate is taken up
directly into inhibitory terminals and converted to GABA, which is then
packaged into synaptic vesicles. Enhancement of mIPSCs by exogenous
glutamate also required glutamate uptake and GABA synthesis. Previous
work has shown that modulating GABA metabolism influences mIPSC
amplitudes (Engel et al., 2001); our data point to glutamate uptake as
an endogenous mechanism for bidirectional modulation of GABA synthesis
and, consequently, inhibitory synaptic strength. GABA synthesis
in vivo is decreased after knockdown of EAAC1 expression
(Sepkuty et al., 2002), arguing that this modulation is not limited to
the slice preparation. Manipulations of glutamate uptake exerted their
effects on GABA release quickly and independently of synaptic activity,
suggesting that presynaptic modulation of quantal size does not require
vesicle release and recycling and that inhibition may be regulated
rapidly. Reducing glutamate transport or GABA synthesis reduced mIPSC
amplitudes only partially, however, indicating that other sources of
GABA (e.g., reuptake) maintain the majority of the synaptic pool of transmitter. The primary role of glutamate transporters on inhibitory terminals, then, may be to strengthen inhibitory synapses in response to local increases in extracellular glutamate.
Neuronal glutamate transporters provide a substrate for
GABA synthesis
Although astrocytic glutamate transporters (GLT-1 and GLAST)
mediate the large majority of uptake in brain tissue (Rothstein et al.,
1996; Tanaka et al., 1997; Diamond and Jahr, 2000), evidence also
suggests that glutamate is taken up directly into nerve terminals (Gundersen et al., 1993; Danbolt, 2001). EAAC1 is expressed at inhibitory presynaptic terminals (Rothstein et al., 1994; He et al.,
2000), where it could supply a substrate for GABA synthesis (Rothstein
et al., 1994). In vivo studies demonstrate that infusion of
EAAC1 antisense oligonucleotides into rat brain reduces both GABA
content and de novo GABA synthesis in the hippocampus
(Sepkuty et al., 2002). Because no selective inhibitor for EAAC1
exists, its physiological role in inhibitory transmission has been
difficult to determine. THA and TBOA inhibit uptake by all of the
transporter subtypes, whereas DHK (at the concentration used here)
blocks only GLT-1. Together with the biochemical and anatomical studies mentioned above, our results showing that THA but not DHK reduces the
synaptic GABA pool suggest that uptake into inhibitory terminals is
mediated by EAAC1 or a transporter with similar pharmacology.
Glutamate taken up by astrocytes is converted by glutamine synthetase
into glutamine, which is released and taken up into axon terminals,
most likely by system A transporters (Chaudhry et al., 2002). In
neurons, glutamate is produced from glutamine by phosphate-activated
glutaminase (PAG). Some GABAergic neurons express PAG (Manns et al.,
2001), whereas others do not (Kaneko and Mizuno, 1994), and it is
unclear whether GABAergic neurons use this pathway to produce glutamate
for GABA synthesis. Several of our findings argue against the
possibility that THA indirectly reduced the supply of glutamate to
inhibitory neurons by inhibiting astrocytic uptake. First, the
glutamine synthetase inhibitor MSO, which disrupts astrocytic glutamate
metabolism (Laake et al., 1995) and the supply of glutamine to
presynaptic terminals (Rothstein and Tabakoff, 1984; Laake et al.,
1995), did not diminish the effect of THA. Second, although in rats at
this age, DHK and THA, at the concentrations used, exert comparable
effects on glial transporter currents (Bergles and Jahr, 1997), DHK did
not reduce mIPSC amplitudes (Fig. 6). The increase in mIPSC amplitudes
in the presence of DHK argues that blocking astrocytic uptake led to
higher glutamate concentrations near inhibitory terminals (Muñoz et
al., 1987), thereby increasing neuronal uptake and GABA synthesis, in
agreement with biochemical studies (Sepkuty et al., 2002). Although it
is possible that glutamine may serve as a precursor for glutamate in
inhibitory terminals, it does not appear to mediate the effects of
glutamate transporter antagonists described here.
Inhibiting GAD with MPA reduced mIPSC amplitudes, but even prolonged
incubation in MPA did not completely deplete GABA in synaptic
terminals. The lack of detectable GABA levels in GAD 65/67 double
knock-out mice (Ji et al., 1999) indicates that nearly all GABA is
synthesized by GAD; the incomplete effects of MPA suggest that, once
synthesized, GABA is recycled effectively for an extended period,
presumably by GABA reuptake.
Vesicular GABA content is dynamically regulated
We have shown that the GABA content of vesicles is downregulated
or upregulated by glutamate uptake in a way that is linked to GABA
synthesis, and that incompletely filled vesicles, or "overfilled" vesicles, can be released. This is in agreement with studies showing that modulation of vesicular content can increase or decrease miniature
postsynaptic event amplitudes (Song et al., 1997; Williams, 1997; Zhou
et al., 2000). Enhancing glutamate uptake increased mIPSC amplitudes
(Fig. 5), suggesting that CA1 synapses normally may not be saturated by
synaptically released transmitter (Hajos et al., 2000). Our results
showing that the releasable pool of synaptic vesicles did not have to
be recycled before an effect of THA was observed (Fig. 1C)
support the view that the vesicular transmitter concentration reflects
an equilibrium between the vesicle lumen and the terminal cytoplasm
(Williams, 1997). As a result, changes in GABA synthetic rate may be
expressed very quickly in the vesicular transmitter concentration.
In light of these conclusions, it is surprising that we were unable to
wash out the effects of THA on inhibitory synaptic responses, and,
although glutamate-induced increases in mIPSC amplitude usually
recovered, subsequent glutamate applications caused inconsistent
results (data not shown). Modulating the extent of glutamate uptake may
cause secondary metabolic effects that recover much more slowly. Such
actions, however, may be specific to inhibitory terminals; the effects
of transporter blockade on glutamatergic synaptic responses in
astrocytes and pyramidal cells appear to wash out more readily (e.g.,
Bergles and Jahr, 1997; Diamond, 2001).
A new protective role for glutamate transporters
The broad clinical utility of benzodiazapines, which exert only
modest effects on GABAergic transmission (Perrais and Ropert, 2000),
indicates that even small changes in inhibitory strength have a
significant physiological impact. Data reported here and elsewhere
(Sepkuty et al., 2002) suggest that suppression of neuronal transporters, perhaps by modulatory proteins (Lin et al., 2001), could
reduce inhibition. Our results showing that exogenous glutamate enhances mIPSCs by way of neuronal transporters (Fig. 5) argue that
glutamate uptake into inhibitory terminals may function as a mechanism
to upregulate inhibition under certain conditions. For example,
transient increases in extracellular glutamate levels that occur during
hypoxia or seizures (for review, see Danbolt, 2001) may result in a
compensatory or protective increase in inhibitory synaptic strength.
It also seems probable that such modulation occurs even in
nonpathological conditions: Knockdown of EAAC1 expression in rats with
antisense DNA induces epilepsy (Rothstein et al., 1996; Sepkuty et al.,
2002). This may indicate that the loss of the protective mechanism
proposed here may itself be epileptogenic, although EAAC1 also may
limit excitability in other ways (Diamond, 2001). Evidence from human
studies indicates that extracellular glutamate rises in the hippocampus
before seizure onset (During and Spencer, 1993). If this were generally
true in epilepsy, then glutamate transporters could play an important
role in preventing seizures. In addition to changes in extracellular
glutamate, changes in the surface expression and capacity of
transporters on terminals could play a role in regulating inhibitory synapses.
| |
FOOTNOTES |
Received Oct. 11, 2002; revised Dec. 4, 2002; accepted Dec. 20, 2002.
This work was supported by the National Institute of Neurological
Disorders and Stroke Intramural Research Program. We thank the members
of our laboratory for valuable discussions.
Correspondence should be addressed to Dr. Jeffrey S. Diamond, Synaptic
Physiology Unit, National Institute of Neurological Disorders and
Stroke, National Institutes of Health, 36 Convent Drive, Room 2C09,
Bethesda, MD 20892-4066. E-mail: diamondj{at}ninds.nih.gov.
| |
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38133 - 38138.
[Abstract]
[Full Text]
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Y.-X. Yu, L. Shen, P. Xia, Y.-W. Tang, L. Bao, and G. Pei
Syntaxin 1A promotes the endocytic sorting of EAAC1 leading to inhibition of glutamate transport.
J. Cell Sci.,
September 15, 2006;
119(Pt 18):
3776 - 3787.
[Abstract]
[Full Text]
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S.-L. Liang, G. C. Carlson, and D. A. Coulter
Dynamic Regulation of Synaptic GABA Release by the Glutamate-Glutamine Cycle in Hippocampal Area CA1.
J. Neurosci.,
August 15, 2006;
26(33):
8537 - 8548.
[Abstract]
[Full Text]
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K. Ma, S. Zheng, and Z. Zuo
The Transcription Factor Regulatory Factor X1 Increases the Expression of Neuronal Glutamate Transporter Type 3
J. Biol. Chem.,
July 28, 2006;
281(30):
21250 - 21255.
[Abstract]
[Full Text]
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L. C. R. Tafoya, M. Mameli, T. Miyashita, J. F. Guzowski, C. F. Valenzuela, and M. C. Wilson
Expression and function of SNAP-25 as a universal SNARE component in GABAergic neurons.
J. Neurosci.,
July 26, 2006;
26(30):
7826 - 7838.
[Abstract]
[Full Text]
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P. D. Arnold, T. Sicard, E. Burroughs, M. A. Richter, and J. L. Kennedy
Glutamate Transporter Gene SLC1A1 Associated With Obsessive-compulsive Disorder.
Arch Gen Psychiatry,
July 1, 2006;
63(7):
769 - 776.
[Abstract]
[Full Text]
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D. E. Dickel, J. Veenstra-VanderWeele, N. J. Cox, X. Wu, D. J. Fischer, M. Van Etten-Lee, J. A. Himle, B. L. Leventhal, E. H. Cook Jr, and G. L. Hanna
Association Testing of the Positional and Functional Candidate Gene SLC1A1/EAAC1 in Early-Onset Obsessive-compulsive Disorder.
Arch Gen Psychiatry,
July 1, 2006;
63(7):
778 - 785.
[Abstract]
[Full Text]
[PDF]
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H. Fang, Y. Huang, and Z. Zuo
Enhancement of substrate-gated Cl- currents via rat glutamate transporter EAAT4 by PMA
Am J Physiol Cell Physiol,
May 1, 2006;
290(5):
C1334 - C1340.
[Abstract]
[Full Text]
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A. L. Sheldon, M. I. Gonzalez, and M. B. Robinson
A Carboxyl-terminal Determinant of the Neuronal Glutamate Transporter, EAAC1, Is Required for Platelet-derived Growth Factor-dependent Trafficking
J. Biol. Chem.,
February 24, 2006;
281(8):
4876 - 4886.
[Abstract]
[Full Text]
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A. Williamson, P. R. Patrylo, J. Pan, D. D. Spencer, and H. Hetherington
Correlations between granule cell physiology and bioenergetics in human temporal lobe epilepsy
Brain,
May 1, 2005;
128(5):
1199 - 1208.
[Abstract]
[Full Text]
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Y. Huang and Z. Zuo
Isoflurane Induces a Protein Kinase C {alpha}-Dependent Increase in Cell-Surface Protein Level and Activity of Glutamate Transporter Type 3
Mol. Pharmacol.,
May 1, 2005;
67(5):
1522 - 1533.
[Abstract]
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P. Cavelier and D. Attwell
Tonic release of glutamate by a DIDS-sensitive mechanism in rat hippocampal slices
J. Physiol.,
April 15, 2005;
564(2):
397 - 410.
[Abstract]
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K. M. Fournier, M. I. Gonzalez, and M. B. Robinson
Rapid Trafficking of the Neuronal Glutamate Transporter, EAAC1: EVIDENCE FOR DISTINCT TRAFFICKING PATHWAYS DIFFERENTIALLY REGULATED BY PROTEIN KINASE C AND PLATELET-DERIVED GROWTH FACTOR
J. Biol. Chem.,
August 13, 2004;
279(33):
34505 - 34513.
[Abstract]
[Full Text]
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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]
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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]
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M. Demarque, N. Villeneuve, J.-B. Manent, H. Becq, A. Represa, Y. Ben-Ari, and L. Aniksztejn
Glutamate Transporters Prevent the Generation of Seizures in the Developing Rat Neocortex
J. Neurosci.,
March 31, 2004;
24(13):
3289 - 3294.
[Abstract]
[Full Text]
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TOP
ABSTRACT
Introduction
Compound via MeSH
