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The Journal of Neuroscience, November 15, 2000, 20(22):8269-8278
Identification of the Kainate Receptor Subunits Underlying
Modulation of Excitatory Synaptic Transmission in the CA3 Region of the
Hippocampus
Anis
Contractor1,
Geoffrey T.
Swanson1,
Andreas
Sailer1,
Stephen
O'Gorman2, and
Stephen F.
Heinemann1
1 Molecular Neurobiology Laboratory and
2 Gene Expression Laboratory, The Salk Institute for
Biological Studies, La Jolla, California 92037
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ABSTRACT |
To understand the physiological role of kainate receptors and their
participation in seizure induction in animal models of epilepsy, it
will be necessary to develop a comprehensive description of their
action in the CA3 region of the hippocampus. Activation of presynaptic
kainate receptors depresses excitatory synaptic transmission at mossy
fiber and associational-commissural inputs to CA3 pyramidal neurons
(Vignes et al., 1998 ; Bortolotto et al., 1999; Kamiya and Ozawa, 2000).
In this study, we use gene-targeted mice lacking glutamate receptor 5 (GluR5) or GluR6 kainate receptor subunits to identify the
receptor subunits that comprise the kainate receptors responsible for
presynaptic modulation of CA3 transmission. We found that bath
application of kainate (3 µM) profoundly reduced EPSCs at mossy fiber and collateral synapses in neurons from
wild-type and GluR5 / mice but
had no effect on EPSCs in neurons from
GluR6/ mice. These results
therefore contrast with previous studies that supported a role for
GluR5-containing receptors at mossy fiber and associational-commissural
synapses (Vignes et al., 1998; Bortolotto et al., 1999). Surprisingly,
at perforant path synapses kainate receptor activation enhanced
transmission; this potentiation was abolished in both GluR5 and GluR6
knock-out mice. Kainate receptors thus play multiple and complex roles
to modulate excitatory synaptic transmission in the CA3 region of the hippocampus.
Key words:
presynaptic kainate receptors; CA3 pyramidal neurons; kainate receptor knock-out mice; hippocampus; mossy fiber; excitatory
synaptic transmission
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INTRODUCTION |
Kainate receptor subunits comprise a
family of channel-forming glutamate receptors known to be involved in
the induction of seizure behavior in the hippocampus (Ben-Ari, 1985;
Mulle et al., 1998), but whose physiological function and spectrum of
activities are just beginning to be understood. Central excitatory
synaptic transmission is mediated predominantly by ionotropic glutamate receptors composed of either NMDA or AMPA receptor subunits
(Hollmann and Heinemann, 1994; Dingledine et al., 1999). The
contribution of these receptors to brain function has been well
characterized because of their prominent roles in synaptic transmission
and the availability of selective agonists and antagonists. In
contrast, kainate receptors seem to have more subtle roles in
modulating neurotransmission and contribute modestly to some EPSCs. The
advent of pharmacological tools and the generation of knock-out mice lacking kainate receptor subunits have produced an understanding of the
neurophysiology of these receptors, but a consensus as to their primary
role in the brain has been slower to develop (Chittajallu et al., 1999;
Frerking and Nicoll, 2000).
Kainate receptors are localized to both presynaptic and postsynaptic
sites in the hippocampus. Kainate-mediated EPSCs have been
characterized at the mossy fiber (MF) synapse onto CA3 pyramidal neurons and at pyramidal cell inputs to CA1 interneurons (Castillo et
al., 1997; Vignes and Collingridge, 1997; Cossart et al., 1998; Frerking et al., 1998; Mulle et al., 1998; Bureau et al., 1999). Hippocampal kainate receptors also have presynaptic modulatory effects
on glutamate (Chittajallu et al., 1996; Kamiya and Ozawa, 1998; Vignes
et al., 1998) and GABA release in the CA1 region (Clarke et al., 1997;
Rodriguez-Moreno et al., 1997; but also see Frerking et al., 1999).
In the CA3 region of the hippocampus, activation of presynaptic kainate
receptors has been shown to depress excitatory input at mossy fibers
and associational-commissural synapses. Most recently, Kamiya and Ozawa
(2000) showed that application of low concentrations of kainate
depressed MF transmission, which likely occurred because of kainate
receptor-mediated axonal depolarization. Pharmacological experiments
with a putative selective agonist supported the interpretation that
glutamate receptor 5 (GluR5) subunits critically comprised presynaptic
mossy fiber and associational-commissural kainate receptors (Vignes et
al., 1998; Bortolotto et al., 1999). This result is surprising, because
expression of GluR5 mRNA is confined predominantly to hippocampal
interneurons and is not detectable in the dentate gyrus granule neurons
or CA3 pyramidal neurons (Bahn et al., 1994; Paternain et al., 2000).
In mouse hippocampus, granule neurons express high levels of GluR6,
GluR7, and KA-2 mRNA, whereas pyramidal neurons express GluR6, KA-1,
and KA-2 mRNA (Bureau et al., 1999).
In this study, we have used gene-targeted mice lacking GluR5 or GluR6
kainate receptor subunits to determine the identity of the subunit
comprising presynaptic CA3 kainate receptors. Additionally, we extended
our analysis of CA3 excitatory transmission to perforant path synapses,
where we found that kainate receptors enhance, rather than depress,
excitatory transmission. These studies lead us to conclude that the
GluR6 receptor subunit comprises presynaptic kainate receptors that
inhibit MF and associational-commissural (A-C) neurotransmission.
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MATERIALS AND METHODS |
Horizontal hippocampal slices (250-350 µm) were made from
postnatal day 14 (P14) to P27 mice with the following genotypes: wild
type,
GluR6/,
GluR5/,
and
GluR5//GluR6/.
The genetic background of the wild-type and
GluR5/
mice was isogenic 129SvEv, whereas
GluR6/
and double knock-out mice had a hybrid 129SvEv/C57BL/6 background. Developmental compensation could be a concern in knock-out experiments; however we feel this is unlikely because levels of mRNA expression of
other kainate receptor subunits are unchanged in
GluR6/
(Mulle et al., 1998) or
GluR5/
(A. Sailer, unpublished observations) mice. Furthermore, functional replacement of whole-cell kainate receptor currents arising from either
GluR5- or GluR6-containing receptors does not occur in CA3 pyramidal
neurons (Mulle et al., 1998), cerebellar Purkinje neurons (Brickley et
al., 1999), or dorsal root ganglion neurons (G. T. Swanson,
unpublished observation). Each knock-out mouse used in the study was
genotyped by Southern blot analysis of tail DNA. Animals were
anesthetized with methoxyflurane and decapitated. The brain was removed
under ice-cold artificial CSF (ACSF) containing 125 mM
NaCl, 2.4 mM KCl, 1.2 mM
NaH2PO4, 25 mM
NaHCO3, 25 mM glucose, 1 mM CaCl2, and 4 mM
MgCl2, equilibrated with 95%
O2 and 5% CO2. In some
experiments, the Mg2+ concentration was
reduced to 2 mM, and 50 µM
D,L-2-amino-5-phosphonopentanoic acid (D,L-APV;
Research Biochemicals, Natick, MA) and 300 µM kynurenate (Tocris Cookson) were included in the slice or incubation
solution. Slices were made on ice with a DSK microslicer (Dosaka
Instruments) and transferred to a holding chamber at room temperature
for a minimum of 1 hr. Individual slices were transferred to a
recording chamber and visualized under Nomarski optics by the use of a
Zeiss Axioskop FS or FS2 (Carl Zeiss). Slices were continuously
perfused with ACSF containing 4 mM
CaCl2 and 4 mM
MgCl2. Whole-cell patch-clamp recordings were
made from visually identified pyramidal cells in the CA3 region of the
hippocampus. All experiments were done at room temperature. Glass
electrodes were pulled from borosilicate glass and had resistances of
4-5 M when filled with an internal solution containing 95 mM CsF, 25 mM CsCl, 10 mM Cs-HEPES,
10 mM Cs-EGTA, 2 mM NaCl, 2 mM
Mg-ATP, 10 mM QX-314, 5 mM
tetraethyammonium chloride, and 5 mM 4-AP, with pH
adjusted to 7.3 with CsOH.
For evoked synaptic responses, slices were bathed in ACSF with 40 µM bicuculline and 50 µM D-AP-5
(from Research Biochemicals). Synaptic currents were evoked by a
monopolar glass electrode positioned either in the dentate gyrus or
stratum lucidum for MF stimulation, in the stratum radiatum or stratum
oriens in the CA3 subfield for A-C stimulation, or in the stratum
lacunosum moleculare on the border of the subiculum and area CA1 for
perforant path (PP) stimulation (Berzhanskaya et al., 1998). The glass
electrode was filled with extracellular solution, and current pulse
width and intensity were altered to give a stable EPSC. A 40 or 50 msec paired-pulse stimulation protocol was used, and data collection and
analysis were done with pClamp 6 or 7 software (Axon Instruments, Foster City, CA). We defined a "failure" as a current record in which no detectable EPSC was observed above the baseline noise level.
The level of resolution in our experiments was ~10 pA. In experiments
using wild-type and
GluR5/
mice, in which perforant path inputs were being recorded, 2 µM baclofen was included in the external solution
in a subset of the recordings to reduce the excitability of CA3
pyramidal neurons (Xiang and Brown, 1998). Comparison with recordings
in which baclofen was omitted demonstrated that this concentration did
not impact the action of kainate on excitatory synaptic transmission.
Also, in a subset of wild-type and
GluR5/
slices, a cut was made that bisected the slice from the edge of the
stratum oriens in the proximal CA3 area to the tip of the suprapyramidal blade of the dentate gyrus [protocol according to
Berzhanskaya et al. (1998)]. Test pulses were given at 0.1 Hz for 5 min during drug application and 10-20 min for baseline recordings.
For miniature EPSC (mEPSC) recordings, 1 µM tetrodotoxin
(TTX), 40 µM bicuculline methiodide, and 50 µM D-AP-5 (all from Research Biochemicals)
were included in the external solution. Neurons were voltage-clamped at
 60 to 80 mV using an Axopatch 200B amplifier (Axon Instruments),
and series resistance was compensated at 60-70%. Access was monitored
periodically throughout the experiment. All drugs were bath-applied
under gravity. Drugs used were 3 µM kainate and 1 µM RS-2-amino-3-(3-hydroxy-5-butylisoxazol-4-yl)
propanoic acid (Research Biochemicals). Data were collected on
DAT tape (Biological) and subsequently filtered at 1-2 kHz and
acquired onto a computer using a Digidata 1200B analog-to-digital
interface and Axotape2 software (Axon Instruments). Analysis of mEPSCs
was performed by the use of the Mini Analysis Program (Synaptosoft, Leonia, NJ). All records were fitted manually by screening through and
picking events from the digitized data. The whole data file was fitted
to check for stability of the recordings. Two to 3 min stretches of
data were used for mEPSC frequency analysis. Data are presented as the
mean ± SEM. Parameters were compared by the use of the Student's
unpaired t test or the Wilcoxon signed rank test.
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RESULTS |
Presynaptic kainate receptors inhibit mossy fiber and
associational-commissuralCA3 synaptic transmission
CA3 pyramidal neurons receive excitatory inputs from three major
pathways that form synaptic contacts with different regions of the
pyramidal cell dendritic arbor: MFs from the dentate gyrus; A-C,
or collateral, inputs from other hippocampal CA3 pyramidal neurons; and
PP connections from layer II of the entorhinal cortex (Steward, 1976;
Amaral and Witter, 1989). Bath application of kainate receptor agonists
depresses excitatory synaptic transmission at MF and A-C synapses; this
effect was postulated to arise from receptors containing the GluR5
receptor subunit (Vignes et al., 1998; Bortolotto et al., 1999). We
initially sought to test this hypothesis by performing similar
experiments in gene-targeted mice that lack GluR5 and GluR6 subunit
receptors. We recorded EPSCs in hippocampal slices from wild-type and
kainate receptor mutant mice while stimulating the afferent pathways
with a glass electrode appropriately placed to activate the input of
interest (see Materials and Methods). To confirm that we were
stimulating the appropriate fibers, we used a pharmacological
criterion, i.e., inhibition by the group II metabotropic GluR
(mGluR) agonist
(2S,3S,4S)-CCG/(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-1), to distinguish MF or PP input from A-C pathways (Kamiya et
al., 1996; Macek et al., 1996). Bicuculline (40 µM) and D-APV (50 µM) were included in the external solution to
block GABAA and NMDA receptors, respectively. The
divalent cation concentration was raised to 4 mM
Ca2+ and 4 mM
Mg2+ while recording EPSCs to suppress
seizure activity of the slice preparation. In some recordings,
particularly those from wild-type and
GluR5/
mice, the GABAB agonist baclofen (2 µM) was included in the bathing solution to
reduce collateral activation when PP inputs were stimulated (Xiang and
Brown, 1998).
The first synaptic input we examined was the MFCA3 pathway. Mossy
fiber transmission has been shown to be depressed by low concentrations
of kainate in a dose-dependent manner (Kamiya and Ozawa, 2000). In
agreement with this previous study, we found that bath application of
kainate (3 µM) reduced the MF EPSC amplitude by
85.7 ± 4.8% (n = 9 slices from 5 animals;
p < 0.05; Fig.
1A,C). This inhibition
of the synaptic response was comparable with that observed with the
mGluR2 agonist L-CGG-1, which reduced the evoked mossy fiber response
by 80.4 ± 7.4% (n = 9 slices from 6 animals; p < 0.05; Fig. 1A,C). As was
observed with L-CCG-1, activation of mossy fiber kainate receptors
enhanced the likelihood of synaptic failure, which we simply defined as
a stimulation event in which an EPSC was not detected above the
baseline noise; in our recordings, the failure rate increased from a
basal level of 1 to 43% (n = 9) in the presence of
kainate. The paired-pulse ratio for our recordings from wild-type mice
(measured with an interval of 40 msec) was 2.6 ± 0.4 (n = 9). During kainate application, the paired-pulse ratio significantly increased to 4.5 ± 1.0 (n = 9), suggesting that the release probability at the mossy fiber synapse
had decreased (Manabe et al., 1993). Kainate-mediated suppression of
the MF EPSC was not caused by indirect mechanisms, such as activation of mGluRs or GABAB receptors, because EPSC
recordings in the presence of the mGluR antagonist
(S)- -methyl-4-carboxyphenylglycine
[(S)-MCPG; 500 µM] or the
GABAB antagonists 2-hydroxysaclofen (200 µM) or (+)-(2S)-5,5-dimethyl-2-morpholineacetic acid (SCH 50911; 10 µM) were inhibited by kainate to the same
degree seen in control recordings [inhibition in
(S)-MCPG, 69.8 ± 8.9%; n = 4 slices from 2 animals; inhibition in 2-hydroxysaclofen, 98.9 ± 1.1%, and in SCH 50911, 79.1 ± 9.0%; combined
n = 6 from 2 animals]. These results confirm that
kainate receptors localized to the MF axons or terminals can
dramatically impact the efficacy of granule cellCA3 excitatory transmission and are consistent with data from previous studies (Kamiya
and Ozawa, 1998; Vignes et al., 1998).
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Figure 1.
Mossy fiberCA3 excitatory synaptic transmission
is inhibited by activation of kainate receptors containing the GluR6
subunit. A, Middle, A representative
experiment shows that the amplitudes of evoked AMPA receptor-mediated
mossy fiber EPSCs were reduced by application of 3 µM
kainate to neurons in acute slices from wild-type mice.
Top, Representative traces of EPSCs are
shown. mGluR activation with 10 µM L-CCG-1 suppressed
transmission, which served to identify the input as a mossy fiber.
EPSCs were evoked at 0.1 Hz frequency by monopolar stimulation in the
stratum lucidum. Bottom, A diagram illustrates the slice
preparation and recording configuration for mossy fiber
stimulation. B, Kainate application to CA3
neurons from GluR6/ mice did
not inhibit excitatory transmission, whereas mGluR-mediated suppression
was intact at these synapses. C, A summary of the effect
of application of kainate on mossy fiberCA3 recordings from the mice
used in this study is shown. Mossy fiber transmission was suppressed by
85.7 ± 4.8% (n = 9) in wild-type mice
and 86.8 ± 5.6% (n = 7) in
GluR5/ mice with 3 µM kainate. No inhibition was seen in either
GluR6/ mice (+4.1 ± 7.1% change; n = 5) or
GluR5//GluR6/
mice (8.5 ± 4.8% change; n = 4).
Activation of mGluRs with L-CCG-1 suppressed mossy fiber transmission
in each of the mice tested. Calibration: A,
x-axis, 30 msec; y-axis, 1500 pA;
B, x-axis, 30 msec;
y-axis, 66 pA. DG, Dentate
gyrus.
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To determine which receptor subunit(s) comprised the MF presynaptic
receptors, we evoked EPSCs in hippocampal slices from kainate receptor
knock-out mice. In neurons from mice in which the GluR6 gene had been
disrupted
(GluR6/
genotype) (Mulle et al., 1998), application of kainate had no effect on
synaptic currents (the mean current amplitude in the presence of
kainate was 4.1 ± 7.1% compared with control; n = 5 from 3 animals; p = 0.9; Fig.
1B,C). No change in the percent of failures of
transmission was observed during kainate application in
GluR6/
neurons, whereas L-CCG-1 application increased failures by 38 ± 19% (n = 5 slices from 3 animals). In contrast,
kainate inhibited MF EPSCs in neurons from mice that lack the GluR5
subunit
(GluR5/
genotype) (Mulle et al., 2000) to a degree similar to that in wild-type
neurons (86.8 ± 5.6% reduction of current amplitudes; n = 7 slices from 3 animals; p < 0.05;
Fig. 1C). MF synaptic currents were not altered by kainate
application to slices from mice in which there was a null mutation in
both the GluR5 and GluR6 genes (GluR5//GluR6/
genotype), which were generated by interbreeding of the single-subunit knock-out strains (a change of 8.5 ± 4.8% was observed;
n = 4 from 4 animals). Presynaptic mGluR activation by
L-CCG-1 suppressed MF EPSCs by >70% in all the recordings from
kainate receptor knock-out mice. Mossy fiber EPSC amplitudes in neurons
from mutant mice were not significantly different from those in
wild-type neurons (p > 0.1). These data suggest
that activation of GluR6-containing receptors, but not GluR5-containing
receptors, suppresses transmission at the MFCA3 synapse in mice.
Activation of kainate receptors in CA3 pyramidal neurons produces a
robust whole-cell current and reduces the input resistance, which could
contribute to the apparent depression of the mossy fiber EPSC after
application of kainate. Shunting of IPSCs was shown recently in part to
underlie the action of kainate on inhibitory synaptic transmission in
the CA1 region (Frerking et al., 1999). Application of 3 µM kainate to slices from wild-type and
GluR5/
mice elicited whole-cell currents in CA3 pyramidal neurons that were of
similar amplitude (wild type, 491 ± 58 pA; n = 27;
GluR5/,
527 ± 59 pA; n = 23), whereas little or no
current was observed in neurons from
GluR6/
(11 ± 5 pA; n = 30) or
GluR5//GluR6/
(7 ± 3 pA; n = 20) mice. To test whether a
reduction in input resistance accounted for the kainate-mediated
depression of mossy fiber EPSCs, we measured the time course and
correlation of the input resistance and EPSC amplitude for each
stimulus in recordings from five neurons from wild-type animals (Fig.
2). We first determined that the input
resistance of CA3 pyramidal neurons decreases during kainate
application, as indicated by the increased holding current during a
10 mV step from the command voltage of 60 mV when kainate was present in the bathing solution (Fig. 2A,
top). Concurrently, the EPSC is profoundly depressed or
eliminated (Fig. 2A, bottom). However, the
input resistance increases to the original prekainate value when the
drug was removed from the bath (Fig. 2B). In
contrast, the mossy fiber EPSC remains depressed for a considerable
period after drug application (Fig. 2B). Although the
input resistance recovered to normal levels within ~7 min after
washout of kainate, EPSCs were still depressed by ~40% after 15 min.
Additionally, normalized EPSC amplitudes do not correlate with the
input resistance; depression of the EPSC by nearly 70% can occur
without significant reductions in the input resistance (Fig.
2C). It should be noted that we cannot exclude the
possibility that, in the presence of the whole-cell current, when the
input resistance is reduced, shunting may contribute to the apparent
depression of EPSC at more distal synapses. However, this is
unlikely to contribute significantly to the long-lasting
kainate-mediated depression of mossy synapses, because of the proximity
of these synapses to the cell soma.
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Figure 2.
Depression of the mossy fiber EPSC is not caused
by a reduction in the input resistance of the pyramidal neuron.
A, Top, The increased holding current
during a 10 mV step applied before the evoked EPSC demonstrates that
the input resistance of the cell is reduced after application of
kainate. Bottom, During kainate application, mossy fiber
EPSCs failed or were greatly reduced in amplitude. B,
The mean input resistance and normalized current plotted against the
time of the recording show that both are reduced during kainate
application. However, input resistance recovers fully by ~7 min after
kainate application, while the EPSC amplitude is still significantly
depressed. Input resistances and EPSC amplitudes were normalized to the
respective means of the control periods before application of kainate.
C, The mean input resistance is not correlated with the
mean normalized EPSC (data from 5 neurons). Calibration:
A, top, x-axis, 15.5 msec;
y-axis, 1.2 nA; inset,
x-axis, 14 msec; y-axis, 0.3 nA;
bottom; x-axis, 10 msec;
y-axis, 300 pA.
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We next explored kainate receptor modulation of
associational-commissural inputs to CA3 neurons, which are formed from
axon collaterals of other pyramidal neurons in the CA3 region (Fig. 3A, bottom). ATPA,
a putative GluR5-selective agonist, has been reported to reduce A-C
EPSC amplitude (Bortolotto et al., 1999). Application of 3 µM kainate to slices from wild-type mice
suppressed transmission to a degree similar to that seen at the
MFCA3 synapse (90.0 ± 4.0%; n = 12 slices
from 7 animals; p < 0.01; Fig. 3A,C). In
addition, the number of failures of transmission increased by 77 ± 8%. The group II mGluR agonist L-CCG-1 did not depress synaptic
currents (the percent change of EPSC in L-CCG-1 was +4.8 ± 13.3%; n = 8 slices from 7 animals), verifying that
the currents were not significantly contaminated by either MF or PP
inputs. Application of kainate to neurons in slices from
GluR6/
mice did not reduce A-C synaptic currents (the relative amplitude of
EPSCs in kainate was 8.7 ± 2.8%; n = 8 slices
from 5 animals; Fig. 3B,C). Similar to the effect seen in
wild-type mice, A-C EPSCs in neurons from the
GluR5/
mice were suppressed by 80.5 ± 11.9% after application of
kainate (n = 5 slices from 4 animals; p < 0.05; Fig. 3C). Finally, there was no kainate-mediated
inhibition of A-C EPSCs in the
GluR5//GluR6/
mice (2.8 ± 4.8%; n = 3 slices from 1 animal;
Fig. 3C). A-C inputs in neurons from the knock-out mice were
relatively insensitive to L-CCG-1 (inhibition of EPSCs by L-CCG-1 in
all the genotypes tested was <14%). These data suggest that in CA3
pyramidal neurons kainate receptors containing the GluR6 subunit are
located both postsynaptically at MF synapses (Castillo et al., 1997;
Vignes and Collingridge, 1997; Mulle et al., 1998) and presynaptically at collateral synapses on other CA3 pyramidal neurons, where they can
inhibit A-C excitatory transmission.
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Figure 3.
Associational-commissural synaptic transmission is
suppressed by activation of kainate receptors containing the GluR6
subunit. A, Middle,
Associational-commissural EPSC amplitudes from wild-type CA3 neurons
were suppressed by kainate application (n = 9).
Amplitudes of the EPSCs were normalized to the mean EPSC amplitude
during the control period before application of kainate.
Top, Representative EPSCs from one wild-type recording
are shown. EPSCs were evoked at 0.1 Hz frequency by stimulation in the
stratum radiatum. Bottom, A diagram illustrates the
slice preparation and recording configuration for
associational-commissural stimulation. The stimulating electrode is
shown in the stratum radiatum; EPSCs were also evoked by stimulation in
the stratum oriens. B, Bottom,
Associational-commissural synaptic transmission is not inhibited by 3 µM kainate in CA3 pyramidal neurons from
GluR6/ mice
(n = 7). Amplitudes of the EPSCs were normalized to
the mean EPSC amplitude during the control period before application of
kainate. Top, Representative EPSCs from one recording
are shown. C, A summary of the effect of application of
kainate on associational-commissuralCA3 recordings from the mice
used in this study is shown. Collateral transmission was suppressed by
90.0 ± 4.0% (n = 12) in wild-type mice and
80.5 ± 11.9% (n = 5) in
GluR5/ mice with 3 µM kainate. No inhibition was seen in either
GluR6/ mice (8.7 ± 2.8% change; n = 8) or
GluR5//GluR6/
mice (2.8 ± 4.8% change; n = 3). mGluR
activation with L-CCG-1 did not suppress associational-commissural
transmission in these recordings. Calibration: A,
x-axis, 30 msec; y-axis; 200 pA;
B, x-axis, 30 msec;
y-axis, 75 pA.
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Perforant path transmission to CA3 neurons is potentiated by
kainate receptor activation
In our final set of experiments we examined EPSCs arising from
stimulation of the PP, which projects to the CA3 from layer II of the
entorhinal cortex. The action of kainate receptors on transmission at
PP synapses has not been reported previously. The stimulating electrode
was placed in the stratum lacunosum moleculare, on the border of the
subiculum and area CA1 (Fig. 4E, left),
and in some slices a cut was made between the DG and CA3 to reduce
contamination of the MF input (Berzhanskaya et al., 1998). We also used
2 µM baclofen, a GABAB
agonist, to hyperpolarize CA3 neurons and thereby eliminate collateral
activation of A-C inputs subsequent to perforant path EPSCs (Xiang and
Brown, 1998). PP inputs were identified by the rapid rise times,
relatively long latencies, and suppression of the EPSC by the mGluR
agonist L-CCG-1.
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Figure 4.
Perforant path synaptic transmission is augmented
by activation of kainate receptors. A,
Bottom, A representative recording from a wild-type CA3
neuron demonstrating that amplitudes of evoked perforant path EPSCs
were increased and failures were decreased reversibly during kainate
application. Top, Representative EPSCs. EPSCs were
evoked at 0.1 Hz frequency by monopolar stimulation in the stratum
lacunosum moleculare. B, A representative recording from
a GluR6/ CA3 neuron
demonstrating that amplitudes of evoked perforant path EPSCs were
decreased during kainate application. C, A recording
from a GluR5/ CA3 neuron that
showed a reduction in EPSC amplitude during kainate application.
Perforant path transmission in
GluR5/ neurons showed
variable responses to 3 µM kainate; four of six synaptic
responses were reduced, whereas EPSC amplitudes increased in the
remaining two of six recordings. D, A representative
recording from a
GluR5//GluR6/
CA3 neuron demonstrating that amplitudes of evoked perforant path EPSCs
were unchanged during kainate application. E,
Left, A diagram illustrating the slice preparation and
recording configuration for perforant path stimulation.
Right, Summary of the effect of application of kainate
on perforant pathCA3 EPSCs. PP transmission was enhanced by
+170 ± 74% in wild-type mice.
GluR5/ neurons had variable
responses to kainate in four of six recordings kainate reduced
EPSC amplitudes by 38.9 ± 3.9%, but in two neurons the
amplitudes were increased by +44.9 and +113.3%. The mean for all
six recordings, 5.2 ± 23.2%, is shown in the
histogram. The mean change in
GluR6/ EPSC amplitudes was
38.1 ± 8.1%. Finally,
GluR5//GluR6/
mice showed little change in amplitude during kainate application
(+1.2 ± 6.6%). mGluRs activation with L-CCG-1 did not reduce
associational-commissural transmission in these recordings.
Calibration: A, x-axis, 32.5 msec;
y-axis, 300 pA; B, x-axis,
32.5 msec; y-axis, 150 pA; C,
x-axis, 32.5 msec; y-axis, 150 pA;
D, x-axis, 32.5 msec;
y-axis, 300 pA.
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In contrast to the effect seen at MF and A-C synaptic inputs, an
enhanced PP synaptic response was observed after application of 3 µM kainate to wild-type mice (Fig.
4A,E). Kainate application produced a reversible
increase in EPSC amplitude (+170 ± 74%; n = 7 slices from 5 animals; p < 0.05). The paired-pulse
ratio was similar for EPSCs before and during kainate application
(control, 2.1 ± 0.2, vs kainate, 2.1 ± 0.3). Synaptic
failures decreased by fourfold, from 37 ± 13 to 9 ± 6%.
In neurons from
GluR5/
mice, kainate application caused either a decrease (38.9 ± 3.9%; n = 5 slices from 4 animals) or an increase
(+44.9 and +113.3%; n = 2 slices from 2 animals) in
the amplitude of the synaptic currents (total mean change of all
recordings is 5.2 ± 23.2%; Fig. 4C,E). There was no
difference in evoked amplitude, synaptic failure rate, or
kainate-mediated whole-cell current amplitude between the PP inputs
that were inhibited compared with those that potentiated. However, the
two synaptic inputs that potentiated showed marked decreases in the
paired-pulse facilitation (reduction of 52 and 43%) and synaptic
failure rate (reduction of 40 and 60%) in the presence of kainate.
In contrast, paired-pulse facilitation was unaffected by drug
application in the subpopulation of synaptic currents that were reduced
by kainate (control paired-pulse ratio of 2.3 ± 0.3 vs the
kainate paired-pulse ratio of 2.2 ± 0.4). In addition, synaptic
failures increased slightly in this subset of responses (control,
22 ± 15%, vs kainate, 32 ± 15%; p > 0.05).
In neurons from
GluR6/
mice, kainate application consistently caused a significant reduction
in the amplitude of PP synaptic currents (38.1 ± 8.1%;
n = 7 slices from 5 animals; p < 0.05; Fig. 4B,E). No PP EPSCs in neurons from
GluR6/
mice showed an increase in amplitude similar to that observed in the
wild-type neurons or in the two potentiated neurons from GluR5/
mice. Furthermore, paired-pulse facilitation ratios were similar in
GluR6/
neurons before (2.0 ± 0.2) and during (2.1 ± 0.2)
application of kainate. Synaptic failures increased from 3 ± 3 to
22 ± 9% when kainate was applied.
Finally, we tested the effect of kainate on PP inputs in
GluR5// GluR6/
mutant mice. These knock-out mice showed no significant alteration in
EPSCs when kainate was applied (amplitude change of 1.2 ± 6.6%; n = 4 slices from 2 animals; Fig.
4D,E). Similarly, there were no differences in either
the paired-pulse ratio (2.1 ± 0.1 in control and 1.8 ± 0.3 in kainate) or synaptic failure rate (12 ± 10% during control
and 16 ± 16% in kainate) in the
GluR5//GluR6/
mutant mice. These results suggest that both GluR5 and GluR6 receptor
subunits contribute to kainate receptor-mediated potentiation of PP
synaptic transmission at synapses on CA3 neurons.
Kainate application increases mEPSC frequency in CA3 neurons
Because of the striking effect of kainate receptor activation on
evoked EPSCs and the changes in the paired-pulse ratio in MF EPSCs, we
examined whether kainate application altered mEPSCs frequencies under
our recording conditions. A previous study reported that mEPSC
frequencies were not altered when kainate was applied to CA3 pyramidal
neurons (Castillo et al., 1997). mEPSCs were recorded in the presence
of 1 µM TTX, 50 µM
D-APV, and 40 µM bicuculline. Bath
application of 3 µM kainate significantly increased mEPSC frequency by 2.2-fold, from a basal rate of 0.65 ± 0.14 to
1.41 ± 0.26 Hz (n = 16 slices from 9 animals;
p < 0.05; Fig. 5). We observed increases in frequency >1.2-fold over basal levels in 11 of
the 16 neurons recorded. The mEPSC frequency in 2 of the 16 neurons
decreased after application of kainate, whereas rates were unchanged in
the remaining 3 recordings. These data suggest that presynaptic kainate
receptors can modulate action potential-independent glutamate release
at a subset of excitatory synapses onto CA3 pyramidal neurons.
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Figure 5.
mEPSC frequency is increased by application of
kainate to CA3 pyramidal neurons from wild-type mice. A,
Sample traces of mEPSC recordings from a CA3 pyramidal
neuron in a hippocampal slice preparation from a wild-type mouse.
Kainate was bath-applied at a concentration of 3 µM. The
baseline noise is higher during kainate application because of the
kainate receptor-mediated whole-cell current. B,
Histogram from the same cell shown in A showing the
mEPSC frequency in 30 sec bins. Kainate caused the mEPSC frequency to
increase by ~1.7-fold in this cell. The gap in the
third bin resulted from checking the series resistance
of the recording. The cell was whole-cell patch clamped at 60 mV for
the duration of the recording. C, D, Cumulative
probability graphs of mEPSC interevent intervals
(C) and amplitudes (D).
Interevent intervals were significantly decreased in the presence of
kainate, whereas amplitudes were unchanged. Calibration:
A, x-axis, 400 msec;
y-axis, 40 pA.
|
|
To test which kainate receptors might be mediating this effect, we
again made recordings from the kainate receptor knock-out mice. We
first measured mEPSC frequencies in CA3 neurons from GluR5/
mice. In contrast to neurons in wild-type mice, in CA3 neurons from
GluR5/
mice the mEPSC frequency did not change after application of kainate
(control, 0.61 ± 0.07 Hz; kainate, 0.51 ± 0.13 Hz;
n = 10 slices from 6 animals; Fig.
6A). mEPSC frequencies
in CA3 neurons from
GluR6/
mice increased by 1.9 (± 0.4)-fold, from 0.55 ± 0.09 to
1.15 ± 0.37 Hz (n = 12 slices from 5 animals;
p < 0.05), with no change in mEPSC amplitude (Fig.
6B). The mean fold increase in mEPSC frequencies
after application of kainate to
GluR6/
neurons was not significantly different from that in wild-type neurons
(p > 0.1). Finally, in neurons from
GluR5//GluR6/
double knock-out mice, kainate did not cause any significant change in
mEPSC frequency (control, 0.79 ± 0.16 Hz, vs kainate, 0.69 ± 0.13 Hz; p = 0.1; n = 12 slices from
5 animals; Fig. 6C). Therefore, kainate had little effect on
mEPSC frequency in
GluR5//GluR6/
neurons, similar to the
GluR5/
recordings, and both genotypes differed significantly from the increase
observed in wild-type neurons (p < 0.05). Taken
together, our mEPSC recordings from the different genotypes provide
evidence that kainate receptors mediate enhancement of action
potential-independent glutamate release onto CA3 pyramidal neurons via
GluR5-containing receptors. These data suggest that the GluR5 subunit
is located presynaptically at one or more types of excitatory inputs to
CA3 pyramidal neurons and that GluR6 subunit-containing kainate
receptors are unlikely to underlie the kainate-mediated increase in
mEPSC frequency.
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Figure 6.
GluR5 subunit-containing kainate receptors
underlie the kainate-mediated increase in mEPSC frequency.
A, Left, A mEPSC frequency histogram for
a CA3 neuron from a GluR5/
mouse shows a small decrease in frequency during kainate application.
Middle, Right, No significant difference
in the interevent interval (middle) or amplitude
(right) cumulative distributions was detected.
B, An mEPSC frequency histogram for a CA3 neuron from a
GluR6/ mouse shows an
increase in frequency during kainate application similar to that
observed in wild-type neurons. The cumulative probability histograms
show that the interevent intervals are significantly briefer in the
presence of kainate but that amplitudes are unchanged.
C, The mEPSC frequency for a CA3 neuron from a
GluR5//GluR6/
mouse does not change after application of kainate. No
significant differences in the interevent interval or amplitude
cumulative distributions were observed. D, A summary of
all experiments for kainate-mediated changes in mEPSC frequency for
recordings from wild-type and kainate receptor subunit knock-out mice
is shown. mEPSCs from wild-type and
GluR6/ neurons increased
after application of kainate. This increase in frequency is absent in
GluR5 knock-out mice, and instead a small decrease was observed. mEPSC
frequencies in recordings from
GluR5//GluR6/
double knock-out mice show no change in mEPSC frequency in the presence
of kainate. E, A summary of kainate-mediated changes in
mEPSC frequency in wild-type and knock-out mice recorded in the
presence of the nonselective Ca2+ channel blocker
Cd2+ is shown. The mEPSC was depressed in wild-type
and GluR5/ mice, whereas no
change in frequency was observed in
GluR6/ mice or the double
knock-outs.
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|
To determine whether the GluR5 receptor-mediated potentiation of mEPSC
frequency involved activation of voltage-gated
Ca2+ channels, we repeated the experiments
in the presence of the nonselective Ca2+
channel blocker cadmium (Cd2+). Bath
application of cadmium-containing external solution alone did not alter
the mEPSC frequency significantly (p > 0.1 for
all genotypes, paired Student's t test). Surprisingly,
kainate application in the presence of 200 µM
CdCl2 decreased mEPSC frequency in wild-type mice
from 1.15 ± 0.47 to 0.54 ± 0.16 Hz, a reduction of
0.53-fold (n = 5 slices from 4 animals;
p < 0.05, Wilcoxon signed rank test; Fig.
6E). This concentration of
Cd2+ does not reduce peak or steady-state
kainate receptor currents in cultured hippocampal or dorsal root
ganglion neurons or recombinant GluR5 receptors (G. T. Swanson and
A. Ghetti, unpublished observations). These data suggest that the
GluR5-dependent increase in mEPSC frequency involves activation of
presynaptic Ca2+ channels.
To determine the subunits comprising receptors underlying the decrease
in mEPSC frequency in the presence of cadmium, we recorded mEPSCs in
the presence of cadmium in the mutant mice. The kainate-mediated decrease in mEPSC frequency in the presence of
Cd2+ was also observed in the
GluR5/
mice [0.70 (± 0.09)-fold change; n = 5;
p < 0.05], whereas there was no change in mEPSC
frequency in the
GluR6/
mice [0.92 (± 0.15)-fold change; n = 6;
p > 0.05; Fig. 6E]. As expected,
there was also no decrease in the mEPSC frequency in GluR5//GluR6/
double knock-out mice in the presence of
Cd2+ after application of kainate [1.12
(± 0.13)-fold change; n = 7; p > 0.05; Fig. 6E]. These results suggest that the
depression of mEPSC frequency uncovered by blockade of voltage-gated
calcium channels was mediated by GluR6-containing receptors. In total, these results suggest that presynaptic kainate receptors can have heterogeneous and complex actions on mEPSC frequencies.
Analysis of the basic characteristics of mEPSCs in CA3 neurons from the
kainate receptor knock-out mice revealed that there were no significant
differences in any of the genotypes compared with wild-type neurons.
Basal frequencies of mEPSCs were comparable between genotypes (mean
control mEPSC frequencies, wild type, 0.65 ± 0.14 Hz;
GluR5/,
0.61 ± 0.07 Hz;
GluR6/,
0.55 ± 0.09 Hz;
GluR5//GluR6/,
0.79 ± 0.16 Hz). Similarly, mean amplitudes were not
significantly different (wild type, 25.0 ± 1.6 pA;
GluR5/,
29.5 ± 3.1 pA;
GluR6/,
21.1 ± 1.7 pA;
GluR5//GluR6/,
24.1 ± 2.4 pA), nor were they altered in the presence of kainate (wild type, 26.6 ± 1.9 pA;
GluR5/,
34.5 ± 4.3 pA;
GluR6/,
22.0 ± 2.4 pA;
GluR5//GluR6/,
23.0 ± 2.3 pA). This would suggest that there is little or no tonic activation of the presynaptic kainate receptors that modulate mEPSC frequencies in the slice preparation.
| |
DISCUSSION |
We have shown that kainate receptors comprised of GluR5 and GluR6
subunits play a role in modulating excitatory synaptic transmission to
hippocampal CA3 pyramidal neurons. Activation of kainate receptors can
either decrease synaptic strength, at mossy fibers and
associational-commissural inputs, or increase synaptic strength at
perforant path inputs. In contrast to previous reports, which relied on
pharmacological characterization of kainate receptors, we used kainate
receptor knock-out mice to identify receptor subunits that comprise
presynaptic kainate receptors. In this way we determined that the GluR6
subunit is critically involved in inhibiting transmission at both the MF and A-C pathways, whereas both GluR5 and GluR6 subunits are involved
in increasing transmission at the perforant path. These results support
the hypothesis that kainate receptors act as one of the major signaling
systems for modulating excitatory transmission in the CA3 region of the hippocampus.
Kainate receptor-mediated depression of synaptic transmission
Several groups have reported that kainate receptors can act
presynaptically to reduce the strength of hippocampal excitatory transmission at mossy fiber and collateral synapses (Vignes et al.,
1998; Bortolotto et al., 1999; Kamiya and Ozawa, 2000). We compared the
effect of kainate on CA3 synaptic transmission in wild-type and kainate
receptor knock-out mice to determine which receptor subunits are
involved in this action. Kainate profoundly reduced EPSCs evoked by
stimulation of MF and A-C collaterals in neurons from wild-type and
GluR5/
mice; in contrast, kainate had no significant effect on EPSC amplitudes
recorded from neurons from
GluR6/
mice. The increase in paired-pulse facilitation at MF synapses that we
observed during kainate receptor activation is consistent with a
reduction in release probability (Manabe et al., 1993). This action of
kainate suggests that in addition to axonal depolarization reported by
Kamiya and Ozawa (2000), kainate receptors may also mediate their
effect by a direct reduction in release probability at MFCA3
synapses. Definitive localization of the receptor protein, which would
facilitate interpretation of functional data, awaits the development of
specific and sensitive antibodies to kainate receptor subunits.
Our data from gene-targeted mice contrast with pharmacological studies
that explored the contribution of GluR5-containing kainate receptors to
presynaptic actions on MF transmission. Vignes et al. (1998) found that
ATPA, a putative GluR5-selective agonist, depressed transmission at
mossy fiber synapses. Because of the apparent divergence between our
data and that of Vignes et al. (1998), we also looked for ATPA-mediated
depression of MF EPSCs; however, in our experiments ATPA (1 µM) had no significant effect on amplitudes of MF EPSCs
in wild-type mice (data not shown). This absence of ATPA-mediated
depression therefore precludes a direct test of the selectivity of the
compound in
GluR5/
mice. We are unsure why this discrepancy exists between our current results and those from Vignes and coworkers. It is possible that the
pharmacological action of putative GluR5-selective compounds may differ
between native and recombinant kainate receptors. Also, one potential
explanation that may reconcile the pharmacological and genetic
observations is formation of heteromeric neuronal kainate receptors
composed of both GluR5 and GluR6 subunits (and potentially KA-1 and
KA-2 subunits), which can coassemble to form receptors with distinct
characteristics (Cui and Mayer, 1999). Indeed, ATPA has been shown to
activate heteromeric GluR5/GluR6 and GluR6/KA-2 receptors (Paternain et
al., 2000). Although formally possible, our results would require that
GluR5/
subunits be essentially nonfunctional in the absence of a GluR6 subunit; furthermore, the absence of GluR5 subunits from such a
heteromeric assembly would make little difference in the action of
presynaptic kainate receptors. Our observation that kainate reduced MF
EPSC amplitudes in
GluR5/
mice, but not
GluR6/ or
GluR5//GluR6/
mice, supports the interpretation that in mice GluR6 is the critical subunit that comprises the presynaptic MF kainate receptor and is
consistent with the high levels of expression of GluR6 mRNA in the
dentate gyrus (Bahn et al., 1994; Bureau et al., 1999).
Previous research also supports the existence of presynaptic
kainate receptors on (or near) CA3 axon terminals, because kainate receptor activation was shown to inhibit excitatory transmission at
A-CCA3 synapses (Bortolotto et al., 1999). As with MF transmission, we found that kainate receptors containing GluR6 subunits mediate this
suppression of transmission at collateral synapses.
Kainate receptor-mediated potentiation of
synaptic transmission
In contrast to MF and A-C inputs, perforant path EPSC amplitudes
were increased and failures were decreased after activation of kainate
receptors. We found that both GluR5 and GluR6 subunits contributed to
the increase in PP stimulus-evoked transmission. In addition to an
increase in EPSC amplitude at the PP, we also observed a decrease in
synaptic failures. This is suggestive of a presynaptic change in
release probability; conversely, the perforant path paired-pulse ratio,
which would be predicted to decrease with an increased release
probability, did not change during kainate application. However, in a
number of studies the paired-pulse ratio was shown not to be correlated
with changes in vesicular release probability (Alger et al., 1996;
Glitsch and Marty, 1999). Furthermore, we cannot exclude the
possibility that activation of kainate receptors potentiates perforant
path transmission via a postsynaptic mechanism. Perforant path EPSPs
were shown to be amplified by voltage-gated calcium and sodium
conductances (Urban et al., 1998). Although voltage-dependent sodium
channels were blocked by inclusion of QX-314 in our internal
solution, it is conceivable that postsynaptic calcium channels involved
in perforant path EPSC propagation to the soma were modulated by
kainate receptor activation. Therefore, kainate may affect perforant
path transmission via multiple mechanisms at the nerve terminal, axons,
or the postsynaptic dendrites.
Presynaptic kainate receptors increase action potential-independent
glutamate release
Although a previous report did not find a kainate-mediated change
in mEPSC frequency in guinea pigs (Castillo et al., 1997), because of
the effects of kainate receptor activation on evoked EPSCs we tested
whether mEPSC frequencies might be affected in our recordings. We found
that in a subset of neurons mEPSC frequencies were increased after
activation of kainate receptors; this increase arose from activation of
receptors incorporating the GluR5 subunit. Although it is not possible
to identify the synaptic source of the mEPSCs with any degree of
certainty, the observed increase in frequency and the dependence on
GluR5 subunits supports the hypothesis that kainate effects on mEPSC
frequency occurred primarily at perforant path synapses. The GluR5
receptor-dependent increase in mEPSC frequency was entirely occluded in
the presence of the nonselective Ca2+
channel blocker cadmium. Rather, in the presence of cadmium we observed
a small depression of mEPSC frequency in neurons from wild-type and
GluR5/
mice that was not observed in the
GluR6/ or
GluR5//GluR6/
knock-out mice, suggesting that the GluR6 receptor subunit underlay this effect. The fact that these kainate receptors mediate opposing actions on mEPSC frequency suggests that they are coupled to different signaling systems within the presynaptic nerve terminals. The GluR5
receptor-mediated increase in frequency may be explained by a
receptor-mediated presynaptic depolarization of the nerve terminal that
results in activation of voltage-sensitive,
Cd2+-sensitive calcium channels. After
block of this pathway, a GluR6-mediated depression of release is
observed that is independent of Ca2+
channel activation.
The objective of this study was to use gene-targeted mice to determine
the receptor subunits that underlie kainate-mediated modulation of CA3
excitatory transmission; consequently, we have not explored the
physiological conditions that might activate presynaptic kainate
receptors. However, these are questions that are integral to
understanding the physiological and pathological significance of
kainate receptors. It is unlikely that these receptors will be
activated to a degree equivalent to bath application of kainate during
normal brain function, and it is probable that activation of terminal
or axonal kainate receptors by endogenous glutamate will have less
dramatic effects on synaptic transmission. Studies comparing the
characteristics of excitatory transmission in wild-type and kainate
receptor knock-out mice, particularly during short- and long-term
plasticity paradigms, will further clarify the function of these receptors.
Recently it was shown that kainate receptors are necessary for
induction of long-term potentiation; these studies again relied on an
antagonist presumed selective for GluR5 kainate receptors (Bortolotto
et al., 1999). Regardless of the receptor composition, however, the
results from Bortolotto et al. (1999) suggest that kainate receptors
play an important role in the physiology of the mossy fiber synapse.
Whether the kainate receptors involved in long-term potentiation (LTP)
are located presynaptically or in fact mediate induction of LTP from a
postsynaptic locus remains to be established (Zalutsky and Nicoll,
1990; Yeckel et al., 1999). Our data and those of previous studies have
established that kainate receptors are present on axons or terminals
and could modulate synaptic transmission onto CA3 neurons.
In this study we show that kainate receptors act via heterogeneous
mechanisms to modulate excitatory transmission to CA3 pyramidal neurons. We have made use of kainate receptor knockout mice
to reveal which subunits are involved in these effects. Our data support the hypothesis that kainate receptors act by regulating excitatory synaptic transmission in the CA3 region and thus may be of
critical importance in hippocampal function.
| |
FOOTNOTES |
Received July 17, 2000; revised Aug. 23, 2000; accepted Aug. 29, 2000.
This research was supported by an International Prize Fellowship from
the Wellcome Trust (A.C.), a Young Investigator Award from the National
Alliance for Schizophrenia and Depression (G.T.S.), the Schweizerische
Nationalfond and the Deutsche Forschungsgemeinschaft (A.S.), and the
National Institutes of Health and the McKnight Foundation (S.F.H.). We
would like to acknowledge the technical efforts of Conny Maron and Lora
O'Leary. We also would like to thank Drs. Robert Gereau, Jane
Sullivan, and Charles F. Stevens for critical reading of this manuscript.
A.C. and G.T.S. contributed equally to this work.
Correspondence should be addressed to Dr. Anis Contractor, Molecular
Neurobiology Laboratory, The Salk Institute for Biological Studies,
10010 North Torrey Pines Road, La Jolla, CA 92037. E-mail: contractor{at}salk.edu.
Dr. Sailer's present address: Merck and Company, Inc., P.O. Box
R80M-213, Rahway, NJ 07065.
| |
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ABSTRACT
INTRODUCTION
