Medical Research Council Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, Bristol BS8 1TD, United Kingdom
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ABSTRACT |
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Kidd, Fleur L. and John T. R. Isaac. Kinetics and Activation of Postsynaptic Kainate Receptors at Thalamocortical Synapses: Role of Glutamate Clearance. J. Neurophysiol. 86: 1139-1148, 2001. Kainate (KA) receptor-mediated excitatory postsynaptic currents (EPSCs) exhibit slow kinetics at the great majority of synapses. However, native or heterologously expressed KA receptors exhibit rapid kinetics in response to agonist application. One possibility to explain this discrepancy is that KA receptors are extrasynaptic and sense glutamate diffusing from the synaptic cleft. We investigated this by studying the effect of three manipulations that change glutamate clearance on evoked KA EPSCs at thalamocortical synapses. First, we used high-frequency stimulation to increase extrasynaptic glutamate levels. This caused an apparent increase in the relative contribution of the KA EPSC to transmission and slowed the decay kinetics. However, scaling and summing the EPSC evoked at low frequency reproduced this, demonstrating that the effect was due to postsynaptic summation of KA EPSCs. Second, we applied inhibitors of high-affinity glutamate transport. This caused a depression in both AMPA and KA EPSC amplitude due to the activation of a presynaptic glutamatergic autoreceptor. However, transport inhibitors had no selective effect on the amplitude or kinetics of the KA EPSC. Third, to increase glutamate clearance, we raised temperature during recordings. This shortened the decay of both the AMPA and KA components and increased their amplitudes, but this effect was the same for both. Therefore these data provide evidence against glutamate diffusion out of the synaptic cleft as the mechanism for the slow kinetics of KA EPSCs. Other possibilities such as interactions of KA receptors with other proteins or novel properties of native synaptic heteromeric receptors are required to explain the slow kinetics.
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INTRODUCTION |
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Fast excitatory synaptic
transmission in the CNS is mediated predominantly by the ionotropic
glutamate receptors that can be divided into three classes:
-amino-3-hydroxyl-5-methyl-isoxazolepropionic acid (AMPA),
N-methyl-D-aspartate (NMDA), and kainate
(KA) receptors (Hollmann and Heinemann 1994
;
Watkins and Evans 1981
). Although AMPA and NMDA
receptors have been extensively studied, KA receptors have received
less attention and their physiological functions remain relatively
unexplored. The recent development of pharmacological agents that
distinguish between AMPA and KA receptors (Bleakman and Lodge
1998
; Chittajallu et al. 1999
; Frerking
and Nicoll 2000
) have now enabled the roles of KA receptors in
the CNS to be studied.
KA receptors are made up of different combinations of the GluR5-7 and
KA1 and KA2 subunits, which are widely expressed throughout the CNS
(Hollmann and Heinemann 1994). The synaptic activation of KA receptors has been described in a number of brain regions (Chittajallu et al. 1999
; Frerking and Nicoll
2000
). Presynaptic KA receptors are involved in regulating
transmitter release at both excitatory and inhibitory synapses in the
hippocampus (Chittajallu et al. 1996
; Clarke et
al. 1997
; Rodríguez-Moreno et al. 1997
) and at inhibitory synapses in the hypothalamus (Lui et al.
1999
). Postsynaptic KA receptor-mediated excitatory
postsynaptic currents (EPSCs) were first described at mossy fiber
synapses in the CA3 region of the hippocampus (Castillo et al.
1997
; Vignes and Collingridge 1997
). Since these
first two reports, KA receptor-mediated EPSCs (KA EPSCs) have been
described in a variety of other preparations such as hippocampal
interneurons (Cossart et al. 1998
; Frerking et
al. 1998
), amygdala (Li and Rogawski 1998
),
retina (DeVries and Schwartz 1999
), spinal cord
(Li et al. 1999
), barrel cortex (Kidd and Isaac
1999
), and cerebellum (Bureau et al. 2000
). The precise roles of KA receptors in many of these brain regions still remain to be determined. Most is known about KA receptor function in
the hippocampus where they have been shown to be important for
regulating excitability (e.g., Chittajallu et al. 1996
;
Clarke et al. 1997
; Contractor et al.
2001
; Cossart et al. 1998
; Frerking et
al. 1998
, 1999
; Kamiya and Ozawa 2000
;
Min et al. 1999
; Mulle et al. 2000
;
Rodríguez-Moreno et al. 1997
; Schmitz et
al. 2000
). They have also been shown to be important for the
induction of mossy fiber LTP (Bortolotto et al. 1999
).
At the great majority of synapses studied, KA EPSCs exhibit slow
kinetics (Bureau et al. 2000; Castillo et al.
1997
; Cossart et al. 1998
; Frerking et
al. 1998
; Kidd and Isaac 1999
; Li and Rogawski 1998
; Li et al. 1999
; Vignes and
Collingridge 1997
; but see DeVries and Schwartz
1999
). This might suggest that KA receptors are of high
affinity, similar to N-methyl-D-aspartate
receptors (NMDARs). However, this idea is not supported by studies in
which agonists were exogenously applied to either native or
heterologously expressed KA receptors. Under these conditions, KA
receptors always exhibit rapid kinetics similar to AMPA receptors even
when high-affinity subunits (KA1 and KA2) are present (e.g., Cui
and Mayer 1999
; Herb et al. 1992
;
Huettner 1990
; Lerma et al. 1993
;
Paternain et al. 1995
; Patneau et al.
1994
; Schiffer et al. 1997
; Swanson and
Heinemann 1998
; Swanson et al. 1996
). In some of
these studies, differences in kinetics were observed with certain
combinations of subunits (Cui and Mayer 1999
;
Herb et al. 1992
), and another study has shown that the
association of GluR6 and KA2 with the postsynaptic density proteins
SAP90 and SAP102 also causes alterations in kinetics (Garcia et
al. 1998
). However, these relatively small differences fall far
short of being able to explain the large discrepancy between the
kinetics of synaptically activated KA receptors and KA receptors
activated by exogenously applied agonists.
Therefore another possibility that must be considered is that it is the
time course of the application of the agonist that differs between
these types of studies. That is, while exogenously applied agonist
arrives rapidly at the receptors, synaptically released glutamate
arrives at KA receptors more slowly, and this influences the time
course and extent of their activation. Although this is not a new idea
(see: Castillo et al. 1997; Lerma 1997
; Mayer 1997
; Vignes and Collingridge
1997
), the original proposal was used to explain the slow
kinetics of the KA EPSCs at the mossy fiber-CA3 synapse, which are
only observed after high-frequency stimulation (Castillo et al.
1997
; Vignes and Collingridge 1997
), a condition
likely to favor a build up of glutamate in the synaptic cleft and its
slow diffusion to receptors. However, more recent studies have
demonstrated equally slow kinetics for KA EPSCs evoked by a single
stimulus (Bureau et al. 2000
; Cossart et al.
1998
; Frerking et al. 1998
; Kidd and
Isaac 1999
; Li and Rogawski 1998
; Li et
al. 1999
), making the kinetics much more perplexing. Figure 1 shows some possibilities for the
location of KA receptors under which conditions diffusion of glutamate
from the synaptic cleft could influence the extent of their activation
and kinetics. They could be present on the spine but on the edge of the
synapse (Fig. 1A) existing as perisynaptic receptors sensing
"spill-out" of glutamate from one synapse. They could be present in
the center of the postsynaptic density of a synapse that has a
nonfunctional presynaptic bouton and only sense glutamate released from
other synapses (Fig. 1B), thus sensing spillover of
glutamate. Another possibility is that they could be entirely
extrasynaptic and sense spillover (Fig. 1C).
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These models could explain the slow kinetics of the KA EPSC; however,
there is one problem. Data from recent studies modeling the
spatiotemporal properties of glutamate diffusion indicate that
glutamate is unlikely to remain in the extrasynaptic space for long
enough to activate KA receptors for 100 ms (Barbour and
Hausser 1997
; Rusakov and Kullman 1998
;
Wahl et al. 1996
), particularly at a concentration to
activate a low-affinity receptor. However, these studies also show that
the profile of extrasynaptic glutamate is critically dependent on a
number of unknowns, such as the glutamate diffusion coefficient, for
which there are currently no accurate estimates and which may vary from
region to region of the brain. Furthermore when synaptic transporter
currents are studied in brain slices, it is noticeable that there is a
slow component to the current that persists for >150 ms (e.g.,
Bergles and Jahr 1997
). This presumably reflects the
long-lasting presence of glutamate in the extracellular space and
indicates that modeling studies may not provide a full description of
glutamate diffusion in the brain. For example, uncertainties remain
about the location of high-affinity glutamate transporters and the
possibility that transporters act as a buffers for released glutamate
and slowly re-release glutamate over a prolonged period (Diamond
and Jahr 1997
). Furthermore these glutamate diffusion studies
have been performed using hippocampal CA1 synapses; therefore without
further studies and more detailed analysis of glutamate diffusion in
other areas of the CNS, it is unclear whether glutamate diffusion alone can account for the very slow kinetics of the KA EPSC.
The aim of this study was to investigate whether the kainate receptors
underlying the KA EPSC at thalamocortical synapses are located outside
the synaptic cleft and whether this contributes to the slow kinetics of
the KA EPSC. In this situation, receptors would be activated by
glutamate diffusing from the synaptic cleft, and hence the extent and
kinetics of their activation should be altered by manipulations that
alter extrasynaptic glutamate clearance. In only two studies to date
has there been a systematic evaluation of an extrasynaptic diffusion
model for the activation of kainate receptors. One, on the mossy
fiber-CA3 KA EPSC, found that slowing diffusion with a
high-molecular-weight dextran caused a potentiation in the amplitude of
the KA EPSC with little effect on the kinetics (Min et al.
1998b). This suggests that the glutamate buildup during the high-frequency stimulation necessary to evoke the current determined the amplitude but not the kinetics of the response. In the
other study, the effects of glutamate transport inhibitors and a
glutamate scavenger were investigated on the KA EPSC at cerebellar
parallel fiber-Golgi cell synapses (Bureau et al.
2000
). No effect of these manipulations was observed on
transmission under physiological conditions, although it wasn't
possible to simultaneously investigate the effects on AMPA
receptor-mediated EPSCs in this preparation. In the present study, we
took advantage of the large size of the KA component to transmission at
developing thalamocortical synapses (Kidd and Isaac
1999
) to compare the effects of manipulating glutamate
diffusion on both AMPA and KA receptors simultaneously at the same
population of synapses. Because the affinity and synaptic localization
of AMPA receptors is well known, this enabled us to make stronger
conclusions on the affinity and localization of synaptically activated
KA receptors than has previously been possible (Bureau et al.
2000
; Frerking et al. 1998
). Using this
approach, we found that there was no selective effect on the size or
kinetics of the KA receptor-mediated component to transmission when
synapses were activated at high frequency, glutamate transport was
inhibited, or temperature was changed. This suggests that the synaptic
activation of KA receptors is not strongly influenced by extrasynaptic
glutamate diffusion.
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METHODS |
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Electrophysiology
Thalamocortical slices (Agmon and Connors 1991)
were prepared as previously described (Feldman et al.
1998
; Kidd and Isaac 1999
) from Wistar rat pups
aged between postnatal day 3 and 9 (day 0 is the day of birth). Slices
were bathed in an extracellular solution of the following composition
(mM): 119 NaCl, 2.5 KCl, 1.0 NaH2PO4, 26.2 NaHCO3, 2.5 CaCl2, 1.3 MgSO4, and 11 glucose, saturated with 95%
O2-5% CO2 at room
temperature (23-25°C, unless otherwise indicated). The
extracellular solution also contained D-2-amino-5-phosphonopentanoic acid (D-AP5, 100 µM) and picrotoxin (50 µM) to block NMDA receptor- and
GABAA receptor-mediated currents, respectively.
Whole cell patch-clamp recordings were made from neurons in layer IV of
the somatosensory (barrel) cortex (Feldman et al. 1998
;
Kidd and Isaac 1999
) using electrodes (3-5 M
)
containing the following solution (mM): 135 CsMeSO4, 8 NaCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP,
and 0.3 Na-GTP, pH 7.25, 285 mOsm. In some experiments, 5 mM lidocaine
N-ethyl bromide (QX-314) and 1 mM spermine were also
included. The presence of intracellular Cs+, and
QX-314, in addition, in some experiments, blocked postsynaptic GABAB receptor-mediated responses. EPSCs were
evoked by electrical stimulation (0.1-0.2 Hz) of thalamocortical
afferents in the ventrobasal complex of the thalamus or the white
matter (in most neurons both pathways were stimulated alternately). As
previously discussed, this stimulation method selectively activates
thalamocortical synapses with no detectable contamination from other
pathways (Agmon and Connors 1991
; Feldman et
al. 1998
; Kidd and Isaac 1999
). Pharmacologically isolated KA EPSCs were recorded in the presence of the AMPA receptor-selective antagonists GYKI53655 (25 µM active isomer) or GYKI52466 (50 µM). Recordings were made using an Axoptach 200-B amplifier, data were filtered at 5 kHz and digitized at 10 kHz.
EPSC amplitude, series resistance, and input resistance were analyzed
and displayed on-line using "LTP" data-acquisition software
(W. W. Anderson, Bristol University; http://www.ltp-program.com). Series resistance (typically 10-30 M
) was estimated by measuring the peak of the unfiltered whole-cell fast-capacitance transient in
response to a voltage step applied before each EPSC was collected.
Analysis
For exponential fitting, averaged EPSCs (average of 5-10) were
digitally filtered at 1 kHz, and the first 200 ms of the decay phase
was fitted with a single exponential or the sum of two exponentials (SigmaPlot 5.0) depending on which gave the best fit. As previously reported (Kidd and Isaac 1999), at this period in
development, EPSCs evoked at the thalamocortical input are dual
component in the majority of cells and, in a minority, are mediated by
AMPA or KA receptors alone. The charge-transfer through the AMPA and KA
receptor-mediated components was estimated as the product of the fast
time constant (
AMPA) and the peak current (at
t = 0; CAMPA) and slow
time constant and peak current (
KA × CKA), respectively, as previously
described (Kidd and Isaac 1999
). For experiments in
which temperature was changed during recordings, bath temperature was
continuously monitored by a thermistor. The response to the train of
five stimuli at 100 Hz was reconstructed from the averaged preceding
EPSC evoked by low-frequency stimulation (EPSCLF)
as follows: the EPSCLF was scaled to the peak of
the first EPSC in the train and subtracted from the train; following
subtraction the EPSCLF was then aligned and
scaled to the peak of the second response in the train; this second
scaled EPSCLF was then subtracted from the train
and added to the first scaled EPSCLF; and this process was repeated sequentially a further three times until a
reconstructed train had been produced from the
EPSCLF. For estimation of EPSC amplitude in the
absence of postsynaptic summation, amplitude was measured for each EPSC
in the train when the preceding EPSCs had been removed using the
subtraction procedure described in the preceding text. When summation
was not subtracted, EPSC amplitudes were measured relative to the
average current over a 10 ms time window immediately before the first
stimulus in the train. Paired-pulse potentiation was calculated as
amplitude of second EPSC (postsynaptic summation subtracted)/amplitude
of 1st EPSC. All numbers of observations quoted represent number of
pathways unless otherwise stated. Values are expressed as means ± SE. Statistical significance was assessed using the Student's
t-test, paired or unpaired as appropriate.
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RESULTS |
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In the presence of D-AP5 and picrotoxin, electrical
stimulation of thalamocortical axons evokes EPSCs in layer IV neurons in the barrel cortex of neonatal rats mediated by both AMPA and KA
receptors (Kidd and Isaac 1999). As previously
described, these two components are kinetically and pharmacologically
distinct; the fast component is mediated by AMPA receptors (AMPA EPSC), and the slow component by KA receptors (KA EPSC). Therefore the decay
of the EPSC is best fit by the sum of two exponentials (Fig. 2A) (Kidd and Isaac
1999
). The relatively large size of the KA EPSC and the good
kinetic separation between this and the AMPA EPSC enabled us to measure
the charge transfer and the decay kinetics of both components
simultaneously.
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High-frequency stimulation potentiates KA EPSCs due to postsynaptic summation and not a buildup of glutamate
Our first approach was to compare EPSCs evoked at low frequency
(EPSCLF) with EPSCs evoked by a train of five
stimuli at high frequency (100 Hz; Fig. 2A). The
high-frequency train caused a depression in peak EPSC amplitude
(amplitude of last EPSC in train = 66 ± 6% of first EPSC
amplitude, P < 0.005, n = 16; Fig.
2B) as has been reported for thalamocortical synapses onto
layer II/III pyramidal neurons in barrel cortex (Gil et al.
1997). When the last EPSC of the train was compared with the
preceding averaged EPSCLF, there was a slowing in
the decay of the KA component (EPSCLF
decay = 138.6 ± 19.1, last EPSC of train
decay = 211.6 ± 34.4 ms,
P < 0.05, n = 16; Fig.
2C). An increase in the proportion of charge transfer
through the KA component of the dual EPSC in response to the train was
also observed (KA EPSC charge contribution as percentage of total EPSC
charge, EPSCLF = 60 ± 7%, last EPSC of
train = 85 ± 4%, P < 0.005, n = 16; Fig. 2D). Using pharmacologically isolated KA EPSCs, similar results were obtained; the decay time constant for the isolated KA EPSC under these conditions was very similar to that estimated from the dual component EPSC, and a similar
increase in the decay time constant was also observed in response to
the 100-Hz trains (isolated KA EPSCLF
decay = 149.6 ± 18.4, isolated KA EPSC
train
decay = 248.2 ± 43.1 ms, P < 0.05, n = 9; Fig.
2C). This demonstrates that the double-exponential fitting procedure reliably reports changes in the kinetics of the KA
component of the dual EPSC.
To investigate whether the increase in charge transfer and the slowing
of the KA EPSC were due to a buildup of extrasynaptic glutamate during
the stimulus train or simply an effect of postsynaptic summation, we
reconstructed the response to the burst using the preceding averaged
EPSCLF (Fig.
3A). The response
reconstructed from the EPSCLF was very similar to
the response to the high-frequency train in all cells
(decay values for KA receptor-mediated
component: train = 211.6 ± 34.4 ms, reconstructed train = 202.5 ± 34.0 ms, P = 0.8, n = 16; KA EPSC charge contribution: train = 85 ± 4%, reconstructed train = 83 ± 5%, P = 0.7;
Fig. 3, B and C). Therefore the
slowing of the decay kinetics and the increased contribution of the KA
EPSC at the end of the train were due to the summation of the EPSCs
evoked during the train rather than increased activation of KA
receptors by a buildup of glutamate.
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We also compared the summation of the AMPA receptor and KA receptor mediated components of transmission during trains of stimuli by evoking dual component EPSCs or pharmacologically isolated KA EPSCs with trains of five stimuli at 33 Hz. Due to its slow kinetics, the pharmacologically isolated KA EPSC exhibited much greater postsynaptic summation during the trains than the AMPA receptor-dominated dual EPSC (amplitude of last isolated KA EPSC in train = 293 ± 26% of 1st EPSC, n = 3; amplitude of dual EPSC = 83 ± 11% of 1st, n = 9; Fig. 3D). A similar small amount of paired-pulse facilitation (PPF) was observed for both types of EPSCs (isolated KA EPSC = 113 ± 19%, n = 3; dual EPSC = 109 ± 14%, n = 9; P = 0.9). This indicates that both had a similar probability of release, and the lack of significant PPF also indicates that probability of release at these synapses is high. However, subtraction of postsynaptic summation revealed that the KA EPSCs did not depress during the train (last isolated KA EPSC = 128 ± 32% of 1st, n = 3). This suggests that sufficient receptors at the same population of synapses were available during repetitive stimulation to produce multiple KA EPSCs of similar sizes even though a significant proportion of receptors was still activated by the preceding release of glutamate. This indicates that only a fraction of the available KA receptors are occupied in response to a single synaptic release of glutamate.
Glutamate transport inhibitors do not have a selective effect on KA EPSCs
Another approach we used was to slow extrasynaptic glutamate
clearance by inhibiting high-affinity glutamate transport. We used a
combination of the transport inhibitors,
D,L-threo--hydroxyaspartate (THA; 300 µM)
(Balcar et al. 1977
) and dihydrokainate (DHK; 300 µM)
(Johnston et al. 1978
) to assess the effect of blocking
glutamate uptake on the KA EPSC. However, in contrast to studies using
these transport inhibitors on hippocampal slices (Bergles and
Jahr 1997
; Lüscher et al. 1998
), we
subsequently found that THA (300 µM) alone or another transport
inhibitor, L-trans-pyrrolidine-2,4-dicarboxylate (tPDC; 300 µM) (Bridges et al. 1991
), alone had very similar
effects to the combination of inhibitors. Therefore all these data were pooled. Application of the transport inhibitors caused a rapid depression in the amplitude of the dual component EPSC (EPSC
amplitude = 14 ± 7% of baseline, P < 0.001, n = 21) that was reversible on wash-out (Fig.
4A). This depression was not
due to postsynaptic desensitization because cyclothiazide (100 µM),
which blocks AMPA receptor desensitization (Yamada and Tang
1993
), did not prevent the depression (Kidd and Isaac
2000
). However, the depression was significantly antagonized by
the selective KA receptor antagonist LY382884 (10 µM)
(Bortolotto et al. 1999
), indicating that it was due to
the activation of an inhibitory presynaptic kainate autoreceptor (EPSC
amplitude in presence of transport inhibitors and LY382884 = 47 ± 12% of baseline, n = 8; P < 0.05 vs. EPSC amplitude with transport inhibitors alone). This
activation of the presynaptic autoreceptor suggests that the transport
inhibitors alter the profile of glutamate in the synaptic cleft and
hence also very likely have an effect on the extrasynaptic profile of released glutamate. This is in agreement with other studies
demonstrating that transport inhibitors can cause the activation of
presynaptic receptors due to increased ambient levels of glutamate
(Maki et al. 1994
; Zorumski et al. 1996
).
In addition it has been shown that altering glutamate clearance with
transport blockers, glutamate scavengers, or a high-molecular-weight
dextran has direct effects on the level of activation of postsynaptic
receptors (Bureau et al. 2000
; Min et al.
1998b
).
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Due to the presynaptic inhibition, glutamate transport inhibitors are
likely to actually reduce the amount of spillover of glutamate from
neighboring thalamocortical synapses (Fig. 1, B and
C). However, this manipulation is useful for
evaluating whether KA receptors are perisynaptic because it would be
predicted to enhance the spill-out of glutamate from the synaptic cleft
when transmitter is released (see Fig. 1A). The transport
inhibitors had no effect on the decay of the KA EPSC (Fig.
4C) or the relative contribution of KA receptors to
the dual component EPSC (KA EPSC charge transfer values: baseline = 875.1 ± 214.4 fC, inhibitors = 199.3 ± 54.2 fC,
P < 0.005, n = 20; KA EPSC charge
percentage total EPSC charge: baseline = 58.5 ± 7.1%,
inhibitors = 53.2 ± 7.5%, P = 0.5, n = 20; decay values:
baseline = 162.5 ± 29.1 ms, n = 17, inhibitors = 154.7 ± 28.3 ms, n = 16, P = 1.0; Fig. 4, D and E).
Therefore inhibition of high-affinity glutamate transport, although
having an effect on the peak amplitude of the dual component EPSC due
to raised ambient levels of glutamate, had no selective effect on the
KA EPSC. This provides evidence against a perisynaptic location for KA receptors.
Increasing temperature does not selectively affect KA EPSCs
Having used two approaches to increase the amount of extrasynaptic
glutamate and observed no change in the KA EPSC, we were concerned that
the lack of a detectable effect could reflect the fact that KA
receptors are fully saturated during basal conditions. Under these
conditions, reducing extrasynaptic glutamate clearance would be
expected to have no additional effect. Therefore we studied the effect
of raising temperature during recordings. This manipulation increases
the rate of glutamate uptake because transport is temperature sensitive (van der Kloot and Molgo 1994). For
example, it produces effects on glutamatergic synaptic transmission
in hippocampal slices that have been attributed to a reduced
extrasynaptic glutamate concentration (e.g., Asztely et al.
1997
). Increasing temperature from room temperature (22.8 ± 0.3°C, range = 22-24°C, n = 8 cells) to
close to physiological temperature (35.4 ± 1.1°C, range = 29-38°C, n = 8 cells) caused an increase in peak
EPSC amplitude (191.0 ± 25.6% of baseline, P < 0.005; n = 14) that was reversible on returning to room
temperature (Fig. 5A). The
increase in temperature also caused a decrease in
decay of both the AMPA and KA EPSCs (AMPA EPSC
decay values: 23°C = 7.1 ± 1.1 ms, 35°C = 3.9 ± 0.4 ms, P < 0.005, n = 14; KA EPSC
decay values:
23°C = 130.0 ± 15.2 ms, n = 13, 35°C = 60.4 ± 7.9 ms, n = 12, P < 0.005; Fig. 5, B and
C). However, temperature had no effect on the charge
transfer through the two components (AMPA EPSC charge values:
23°C = 330.2 ± 53.3 fC, 35°C = 402.7 ± 137.7 fC, P = 0.5, n = 14; KA EPSC charge values: 23°C = 1,077.7 ± 267.7 fC, 35°C = 1,122.1 ± 298.1 fC, P = 0.7, n = 14; Fig. 5D) and therefore did not affect the relative contribution of the KA EPSC to transmission (KA EPSC charge percentage of total EPSC charge: 23°C = 63.2 ± 7.3%, 35°C = 56.8 ± 8.6, P = 0.1, n = 14; Fig.
5E). The lack of change in charge transfer in either
component therefore indicated that the same relative numbers of KA and
AMPA channels contributed to the EPSC at both temperatures. The changes
in peak EPSC amplitude and decay were very likely due to the strong
temperature dependence of channel kinetics (Hestrin et al.
1990
; Magleby and Stevens 1972
). However, the
increase in temperature did not decrease the charge transfer of the KA
EPSC as would be expected for the glutamate diffusion model.
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DISCUSSION |
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In this study we have investigated whether the KA receptors underlying thalamocortical KA EPSCs are activated by glutamate diffusing from the synaptic cleft to receptors located peri- or extrasynaptically. To investigate this, we used three approaches to alter the rate of extrasynaptic glutamate clearance: high-frequency stimulation, glutamate transport blockade, and raising temperature. We found that none of these manipulations caused any change in the relative contribution of the KA component to synaptic transmission and had no selective effect on the kinetics of the KA EPSC. These data suggest that synaptically activated KA receptors are present within the synapse close to the release site and do not sense glutamate diffusing from distant release sites.
Modulation of glutamate clearance by high-frequency stimulation, glutamate transport blockade, and alteration of temperature
One possibility is that the manipulations used in this study did
not have significant effects on glutamate clearance and therefore did
not cause a change in the KA EPSC. We think this is unlikely for three
reasons: 1) glutamate transport inhibitors had a marked effect on EPSC amplitude due to increased ambient glutamate levels activating an inhibitory presynaptic glutamate receptor as has been reported in other studies (Asztely et al. 1997;
Maki et al. 1994
; Min et al. 1998
;
Scanziani et al. 1997
; Zorumski et al. 1996
). There are other possible explanations for the effect of transport inhibitors on EPSC amplitude, but we think these are less
likely. Desensitization of postsynaptic receptors is unlikely because
cyclothiazide did not block the effect of the inhibitors (Kidd
and Isaac 2000
). THA is also an NMDA receptor agonist
(Tong and Jahr 1994
); however, direct activation of NMDA
receptors can be ruled out because all recordings were performed in 100 µM D-AP5. Furthermore tPDC, which had a similar effect,
is not an NMDA receptor agonist. It is possible that both tPDC and THA
are agonists at a presynaptic metabotropic glutamate (mGlu) receptor.
However, while there is some evidence for tPDC as an agonist at one
mGlu receptor subtype in astrocytes (Miller et al.
1994
), other studies provide evidence against such an action
for both tPDC and THA (Fitzsimonds and Dichter 1996
;
Thomsen et al. 1994
). An alternative possibility is the
direct activation of the presynaptic kainate receptor by the transport
inhibitors; however, binding studies show that both THA and tPDC do not
displace the kainate receptor ligands,
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or KA (Balcar et al.
1995
). 2) The use of increasing temperature as a
method to speed up the clearance of glutamate has been shown to have effects on glutamate diffusion in hippocampal culture (Tong and Jahr 1994
) and has been reported to reduce spillover of
glutamate at synapses in hippocampal slices (Asztely et al.
1997
; Min et al. 1998a
). 3) A recent
study of synaptically activated transporter currents (STCs) in
hippocampal astrocytes (Diamond and Jahr 2000
) showed a
progressive change in the rise and decay kinetics of STCs during a
100-Hz train at room temperature with effects observed by the fourth
stimulus. This indicates that brief trains of 100-Hz stimulation alter
the clearance of glutamate.
All this suggests that the manipulations used in the present study
alter extrasynaptic glutamate clearance. Indeed similar manipulations
have been used at mossy fiber-CA3 synapses, CA1 synapses and cerebellar
synapses (Asztely et al. 1997; Bureau et al.
2000
; Min et al. 1998
; Scanziani et al.
1997
). These manipulations have been shown to change the
activation of both presynaptic and postsynaptic receptors,
demonstrating that glutamate transport can play an important role in
determining the level of activation of glutamate receptors at synapses.
KA receptors appear to be far from fully occupied following a single
synaptic release of glutamate, an observation also reported for
cerebellar parallel fiber-Golgi cell synapses (Bureau et al. 2000). This suggests that the receptors are of low affinity or are located far from the site of release. The lack of effects on the KA
EPSC of alterations in glutamate clearance reported in the present
study provides evidence against this latter possibility. The lack of a
selective effect of manipulating glutamate clearance also suggests that
AMPA receptors have a similar low level of occupancy. This would be
consistent with both AMPA and KA receptors having similar affinities
for glutamate and being at similar locations in the synapse.
Mechanisms for the slow kinetics of the KA EPSC
The lack of effect of manipulating glutamate clearance on the KA
EPSC provides evidence against glutamate diffusion as the mechanism for
the slow kinetics of the KA EPSC. Therefore what other mechanisms might
explain the slow kinetics? One possibility is that native KA receptors
interact with proteins at the synapse and this causes a change in their
kinetic properties. A recent study has provided evidence for this by
showing that interactions of GluR6 homomers or GluR6/KA2 heteromers
with the postsynaptic density protein SAP 90 produced receptors that
did not fully desensitize in response to glutamate (Garcia et
al. 1998). Another possibility is that synaptic KA receptors
are heteromers that have novel properties and that these combinations
of subunits are not found for the native extrasynaptic or
heterologously expressed KA receptors so far studied. For
thalamocortical synapses, although the subunit composition of the
native receptors is not known, a prediction can be made from in situ
hybridization studies. During the first postnatal week GluR5 is
expressed at high levels in layers II/III and IV of the rat barrel
cortex, GluR7 is expressed in layers IV-VI, and KA2 is expressed
throughout the neocortex (Bahn et al. 1994
). KA
receptors at thalamocortical synapses during this developmental period
also exhibit a strongly rectifying I-V relationship (Kidd and Isaac 1999
), suggesting they contain a
substantial proportion of unedited subunits. Therefore the native KA
receptors at thalamocortical synapses in neonatal tissue are likely to
be heteromeric combinations of unedited GluR5 [GluR5(Q)], with GluR7
and KA2. The exact stoichiometry will depend on the relative levels of
expression of these subunits and their targeting to synapses. The
properties of GluR5 homomers are well known; they exhibit rapid
kinetics similar to AMPA receptors (Herb et al. 1992
;
Swanson and Heinemann 1998
; Swanson et al. 1996
). While there is no information on GluR5(Q)/GluR7/KA2
heteromers, it is known that GluR5(Q)/KA2 also exhibits rapid kinetics
(Herb et al. 1992
). However, co-assembly of different
subunits can cause alterations in the properties of the receptor
complex (Cui and Mayer 1999
; Herb et al.
1992
), and GluR5-containing receptors exhibit variations in
kinetics, for example, in the rate of recovery from desensitization
(Swanson and Heinemann 1998
). These properties could
provide potential mechanisms for the slow kinetics of synaptic KA receptors.
In summary we have found that manipulations that affect the clearance of glutamate have no selective effect on the KA EPSC at thalamocortical synapses. This provides evidence against glutamate diffusing out of the synaptic cleft and activating peri- or extrasynaptic kainate receptors. Further work is necessary to determine if single-channel properties or interactions with synaptic proteins can explain the slow kinetics of the KA EPSC.
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ACKNOWLEDGMENTS |
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We are grateful to G. Collingridge and A. Lüthi for comments.
This work was supported by The Wellcome Trust.
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FOOTNOTES |
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Address for reprint requests: J.T.R. Isaac, Dept. of Anatomy, University of Bristol, Bristol BS8 1TD, UK (E-mail: j.t.r.isaac{at}bristol.ac.uk).
Received 16 February 2001; accepted in final form 2 May 2001.
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REFERENCES |
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