From the
Departments of Physiology,
||Pharmacology,
Immunology,
¶Anesthesiology, Faculty of Medicine, University
of Toronto, Mt. Sinai Hospital, Toronto, Ontario M5S 1A8, Canada
Received for publication, February 24, 2003 , and in revised form, May 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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Glutamate also activates postsynaptic mGluRs, which are coupled via
G-protein activation to intracellular signaling cascades. Eight mGluRs have
been cloned, and they are classified into three groups (I, II, and III) based
upon sequence homology, similarities in signal transduction cascades, and
pharmacological profiles (8).
The group I metabotropic glutamate receptor, mGluR5, is positively coupled to
phospholipase C activity
(9), PKC, and mobilization of
intracellular calcium via IP3Rs. Of the group I mGluRs, mGluR5
expression in the CA1 hippocampus has been found to be localized to
extrasynaptic and perisynaptic sites
(10,
11) of CA1 pyramidal neurons,
whereas mGluR1 is not highly expressed in CA1 pyramidal neurons and is more
predominantly expressed in interneurons
(12). Recent evidence
demonstrates that mGluR1 and mGluR5 play separate functional roles, via
activation of distinct intracellular signaling pathways in CA1 pyramidal
neurons (13).
Group I mGluRs can either enhance or depress excitatory synapses (14). The mechanisms by which group I mGluRs act to modulate synaptic performance are not entirely clear, but post-translational modifications (15) or increases or decreases in the number of ionotropic glutamate receptors located at excitatory synapses can contribute to either LTP or LTD, respectively (16). At many hippocampal synapses low frequency afferent stimulation induces LTD, but brief high frequency stimulation leads to LTP even though both forms of synaptic plasticity require an influx of postsynaptic calcium via NMDARs. In hippocampal slices (1719) and cultures (14, 2022) bath applications of either NMDA or of a group I mGluR agonist induce LTD, but not LTP, and enhance AMPA receptor endocytosis (14, 2325). However, in cultures selective stimulation of synaptic but not extrasynaptic NMDARs induces LTP and not LTD (22, 2628). The role of mGluRs in the induction of LTP is highly controversial, and the exact role of group I mGluRs in modulating NMDARs and LTP is unclear (29, 30). In the present study we set out to delineate the mechanism by which group I mGluRs modulate NMDA channel activity
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MATERIALS AND METHODS |
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Recordings of Miniature Postsynaptic Excitatory
CurrentsProcedures for the preparation of primary dissociated
cultures of hippocampal neurons have been previously described
(57). Whole-cell recordings
were made from these cultures 1217 days after plating. Recordings were
performed at room temperature (2022 °C). Recordings from each
neuron lasted from at least 45 to 75 min. The series resistance in these
recordings varied between 6 and 8 megohms, and recordings where series
resistance varied by more than 10% were rejected. No electronic compensation
for series resistance was employed. The patch electrode solution contained the
following (mM): 140 CsCl, 2.5 EGTA, or 20 BAPTA, 2
MgCl2, 10 HEPES, 2 tetraethylammonium, and 4 K2ATP (pH
7.3), and osmolarity was between 300 and 310 mosmol. The extracellular
(perfusion or bathing) solution was of the following composition
(mM): 140 NaCl, 1.3 CaCl2, 5 KCl, 25 HEPES, 33 glucose,
0.0005 tetrodotoxin, 0.001 strychnine, and 0.02 bicuculline methiodide (pH
7.4), and osmolarity was between 325 and 335 mosmol. Each cell was
continuously superfused (1 ml/min) with this solution from a single barrel of
a computer-controlled multibarreled perfusion system. mEPSCs were recorded
using an Axopatch 1-B amplifier (Axon Instruments, Inc.), and records were
filtered at 2 kHz, stored on tape, and subsequently acquired offline with an
event detection program (Mini Analysis; Justin Lee). Cells that demonstrated a
change in "leak" current of more than 10% (usually less than 10
pA) were rejected from the analysis. The trigger level for detection of events
was set approximately three times higher than the baseline noise. Inspection
of the raw data was used to eliminate any false events, and 300 mEPSCs
were averaged for display purposes. The same number of events was used when
averaged mEPSCs were compared. The AMPAR component of mEPSC was determined by
selecting the area under the event from the start of the event to 8 ms after
the start of the event. The NMDAR component was determined by selecting the
area under the event from 8 ms onward. All population data were expressed as
mean ± S.E. The Student's paired t test or the analysis of
variance test (two-way) was employed when appropriate to examine the
statistical significance of the differences between groups of data.
In Vitro Phosphorylation AssaysCA1 tissue was treated with
CHPG (20 min CHPG plus 5 min of extracellular fluid without CHPG), CHPG plus
MPEP (25 min MPEP, 20 min CHPG plus MPEP), or no treatment (25 min in
extracellular fluid). Three CA1 regions were pooled together. The tissue was
homogenized in ice-cold lysis buffer containing (in mM): 50
Tris-HCl (pH 8.0), 150 NaCl, 2 EDTA, 0.1% SDS, 1% Nonidet P-40, 1 sodium
orthovanadate, protease inhibitors pepstatin A (20 µg/ml), leupeptin (20
µg/ml), and aprotinin (20 µg/ml), and 1 phenylmethylsulfonyl fluoride.
Insoluble material was removed by centrifugation at 14,000 x g
for 10 min at 4 °C. The protein content of soluble material was determined
by BCA protein assay. Soluble proteins (500 µg) were incubated overnight
with 2 µl of either anti-CAK or anti-Src. Immune complexes were
isolated by addition of 40 µl of protein G-Sepharose beads, followed by
incubation for 12 h at 4 °C. Immunoprecipitates were washed several
times with SDS lysis buffer. Samples were subjected to 10% SDS-PAGE. Membranes
were immunoblotted with a monoclonal antibody to phosphorylated tyrosine
(1:1000 dilution). Signals were detected with enhanced chemiluminescence (ECL,
Amersham Biosciences) and developed on x-ray film. The membrane was then
stripped and reprobed with anti-CAK
or anti-Src (1:3000 and 1:5000
dilution, respectively). The film images were digitized and imported into
Corel Draw for presentation purposes.
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RESULTS |
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In acutely isolated CA1 pyramidal neurons, taken from young rat hippocampi, peak NMDAR-mediated whole-cell currents (Ip) recorded in response to rapid applications of NMDA (Fig. 1b) were similarly enhanced by CHPG. This enhancement was similar whether 100 or 500 µM CHPG was applied. This enhancement of peak currents was also antagonized by MPEP (Fig. 1, c1 and c2). We further demonstrated the absence of a CHPG-induced modulation of NMDAR-mediated currents in CA1 neurons taken from mGluR5 knockout mice even though cells from wild type littermates demonstrated the anticipated enhancement (Fig. 1, e1 and e2). The low yield of mGluR5 knockout mice prevented our evaluation of changes in excitatory synaptic transmission using primary hippocampal cultures that lacked mGluR5 expression.
Note that during the period of simultaneous co-applications of CHPG, a
rapid but reversible depression of NMDA-evoked currents was observed
(e.g. Fig. 1,
c2, e2, and
f). This transient depression was not blocked by MPEP,
and it was also seen in cells taken from mGluR5 knockout mice providing
evidence that it is due to a direct interaction with NMDARs rather than
through mGluR5 signaling. Such a direct effect of mGluR reagents has been
previously reported
(3638)
and reflects in part an interaction of these agents with the glycine binding
site of the NMDA receptor
(38). Indeed, at the
concentrations of NMDA and glycine used by us, CHPG inhibited recombinant
NR1Stop838/NR2a currents in HEK293 cells by 60.9 ± 2.0% at
100 µM and 98.5 ± 0.8% at 1 mM (n =
4, Fig. 1, d1
and d2). For this reason the CPHG was
not included in the NMDA barrel during perfusion of isolated cells. Thus, the
limited depression of NMDA-induced currents simply reflected the rapid
recovery of NMDA responses following the washout of CHPG. One should note that
the CHPG-induced potentiation occurs long after (as much as 30 min) the
application of CHPG, whereas the inhibition recovers in less than 1 s.
NMDAR-mediated currents reversed at 0 mV before and after application of
all agents demonstrating that there was no change in driving force associated
with their use (data not shown).
To permit intracellular Ca2+ to vary in the absence of any applied exogenous Ca2+ buffers, we also made a series of recordings from isolated CA1 neurons using the perforated patch technique. Under these conditions, and with sub-saturating concentrations of agonists (10 µM NMDA and 500 nM glycine), peak currents (Ip) also demonstrated a time-dependent enhancement following application of 1S,3R-ACPD (10 µM; trans-ACPD) but not following application of its inactive analogue, 1R,3S-ACPD (10 µM) or the mGluR5-selective agonist CHPG (not shown). In contrast when near saturating concentrations of agonists (300 µM NMDA and 3 µM glycine) were employed, a long lasting depression of steady-state currents (Iss) was also revealed following application of CHPG (Fig. 1f). In all subsequent experiments 100 µM CHPG was used to activate mGluR5 responses in isolated CA1 neurons.
These results demonstrate that activation of mGluR5 can selectively enhance mEPSCNMDA and enhance peak currents as well as depress steady-state NMDA-evoked currents accentuating the apparent desensitization of evoked currents. The mGluR5-induced enhancement of NMDAR desensitization (e.g. reduced Iss/Ip) was directly dependent upon the concentration of extracellular glycine (data not shown) and is consistent with enhanced calcium-dependent inactivation of NMDAR-mediated currents (39). However, these results do not exclude the possibility that other forms of receptor desensitization occur. For instance, it is possible that there might be a change in the number and/or function of NMDAR subtypes.
Many GPCRs activate the mitogen-activated protein kinase (MAPK) cascade through the transactivation of receptor tyrosine kinases, such as epidermal growth factor receptors and platelet-derived growth factor receptors (PDGFRs) (40). GPCR-mediated transactivation of receptor tyrosine kinases has been well documented in heterologous cell systems, and recently we demonstrated that, in CA1 pyramidal neurons, D2/D4 dopamine receptors (GPCRs) transactivate PDGFRs to depress NMDA-mediated synaptic transmission (41). Moreover, in glial cells mGluR5-induced activation of MAPK is dependent upon epidermal growth factor receptor activity (42). However, inclusion of the PDGFR inhibitor tyrphostin A9 (2 µM) in the recording electrode solution, failed to block the CHPG-induced potentiation of NMDAR-mediated peak currents in isolated CA1 pyramidal neurons. In addition, applications of either epidermal growth factor (10 ng/ml) failed to modulate NMDA-evoked currents in isolated CA1 pyramidal neurons (data not shown). Therefore, mGluR5 is unlikely to modulate NMDA responses via transactivation of these growth factor receptors.
The simultaneous enhancement of Ip and depression of
Iss in pyramidal neurons is reminiscent of what is seen following
activation of protein kinase C (PKC)
(43). The enhancement results
from stimulation of the calcium-activated kinase (CAK
) or proline
rich kinase 2/Src cascade
(35), whereas the depression
results from a PKC-dependent facilitation of calcium-dependent inactivation
(39). The CHPG-induced
potentiation was also mediated via activation of PKC, because the response was
blocked by the selective inhibitor, chelerythrine (10 µM,
Fig. 2a)
(45). In separate experiments,
application of 4
-phorbol 12-myristate 13-acetate (4
-PMA) (100
nM) potentiated Ip as anticipated but also occluded the
mGluR5-induced potentiation (Fig.
2a) whereas an inactive phorbol ester, 4
-PMA (100
nM), did neither (
-PMA: 95 ± 5%, n = 8;
PMA plus CHPG: 133 ± 12%, n = 8, p <
0.001). The serine/threonine phosphatases, PP1 and PP2A, depress NMDA-evoked
currents (46), and appropriate
inhibitors enhance and facilitate the PKC-mediated potentiation of
Ip (43). We
therefore examined the ability of a phosphatase inhibitor to modulate the
mGluR5-induced potentiation. Application okadaic acid (10 nM)
slightly enhanced Ip but substantially accentuated the potentiation
induced by CHPG (okadaic acid: 112 ± 10%, n = 5; okadaic acid
plus CHPG: 168 ± 12%, p < 0.001; CHPG: 142 ± 8%;
data taken at 25 min after application of drug, data not illustrated).
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There is strong evidence that PKC can activate the non-receptor tyrosine
kinase CAK, and this kinase is highly expressed as an unspliced isoform
in hippocampal tissue (47).
CAK
is also associated with NMDARs
(5,
35). We therefore tested the
hypothesis that mGluR5 activates PKC and then CAK
. The CHPG-induced
potentiation of Ip was blocked by the functional dominant negative
mutant of CAK
(kinase mutant, CAK
-K475A, 0.05 µg/ml)
(35). Furthermore, recombinant
CAK
(0.05 µg/ml) itself slowly enhanced Ip and
subsequently occluded the CHPG-induced potentiation
(Fig. 2b).
Intracellular applications of CAK
-K457A also blocked the CHPG
enhancement of mEPSCNMDA (Fig.
2c).
Stimulation of PKC, and/or increases in intracellular calcium, triggers the
activation and autophosphorylation of CAK on tyrosyl residues, 579/580
and 402. The tyrosine phosphorylation of residue 402 is especially important,
because this region creates an SH2 ligand by which CAK
can relieve the
autoinhibition of Src tyrosine kinases
(48). In CA1 pyramidal neurons
Src is downstream of a CAK
, and both the induction of LTP and
application of phorbol esters increase CAK
phosphorylation
(35). Consistent with these
observations, intracellular application of recombinant c-Src (50 units/ml)
enhanced Ip and occluded the CHPG effect
(Fig. 2). In contrast,
heat-inactivated c-Src (50 units/ml) did not
(Fig. 2d).
Furthermore, a selective Src kinase inhibitory peptide,
Src4058 (25 µg/ml)
(49) blocked the CHPG-induced
enhancement, whereas its control peptide sSrc4058 (a
scrambled peptide, 25 µg/ml) failed to do so
(Fig. 2e).
To further demonstrate the activation of CAK and Src by mGluR5, we
performed in vitro phosphorylation assays from isolated CA1 region
tissue. CA1 slices were either untreated or exposed to CHPG or CHPG plus MPEP
prior to isolation. Both CAK
and Src were immunopurified using
anti-CAK
and anti-Src antibodies, respectively. We then probed
phosphorylation of CAK
and Src using tyrosine phosphorylation-specific
antibodies. CHPG, but not CHPG plus MPEP treatment, enhanced tyrosine
phosphorylation indicating mGluR5 stimulation activates CAK
in the CA1
region (35)
(Fig. 2f). Similarly
CHPG treatment enhanced the phosphorylation of immunoprecipitated Src
(Fig. 2f).
Our results demonstrate that, similar to muscarinic receptors
(34,
35), mGluR5 in CA1 neurons
stimulates a PKC/CAK/Src cascade to enhance peak NMDA-evoked currents.
Activation of this cascade may or may not require a concomitant rise of
intracellular Ca2+
(47,
48). We therefore tested
whether the modulation by CHPG depended upon an elevation in
Ca2+ by employing high concentrations of the chelator
BAPTA (20 mM) in the patch pipettes. With this enhanced level of
Ca2+ chelation CHPG failed to enhance NMDAR-mediated
currents (Fig. 3a). In
our recordings an increase in Ca2+ would result from an
influx via NMDARs and perhaps through mGluR5-induced mobilization of internal
Ca2+ stores by inositol triphosphate receptors
(IP3Rs). In support of the later hypothesis inclusion of the
selective IP3R inhibitor, xestospongin-C (Xe-C, 2 µM)
(50,
51) in the pipette also
blocked the response to CHPG (Fig.
3a). Furthermore, acute application of thapsigargin (50
nM), which acts to promote release of intracellular calcium via
IP3Rs (52,
53), also enhanced
Ip (Fig.
3b). This led us to hypothesize that activation of
IP3Rs results in release of intracellular
Ca2+, stimulation of the PKC/CAK
/Src cascade, and
an enhancement of NMDARs. In support of this we found that intracellular
applications of chelerythrine (10 µM), Src4058
(25 µg/ml), or CAK
-K475A (0.05 µg/ml) each blocked the
thapsigargin-induced potentiation (Fig.
3c). Furthermore, thapsigargin itself occluded the
mGluR5-induced potentiation of peak NMDAR-mediated currents
(Fig. 3c).
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We next considered whether an influx of Ca2+ through NMDARs was also required to activate the cascade. To this end we initially determined the amplitude of Ip, and then CHPG was applied in the absence of NMDA. NMDA was subsequently applied to determine Ip. This experimental protocol differs from that of Fig. 1 (a and b) in that there was no NMDA present during stimulation of mGluR5. As shown in Fig. 4a, under this condition CHPG failed to enhance Ip. We further investigated the time dependence of this response. To do so we varied the time between the end of the application of CHPG and the first test application of NMDA (50 µM) and glycine (500 nM). We found that the minimal time between mGluR5 and NMDAR stimulation required for enhancement of NMDA channel activity was less than 15 s. Surprisingly, a time interval of 30 s resulted in a depression of evoked NMDA responses (Fig. 4b). Moreover, we found that NMDA channels must be gated to an open state during stimulation of mGluR5 for the enhancement to occur. For example, when CHPG and NMDA were co-applied in the presence of the reversible open-channel blockers, ketamine (50 µM) or magnesium (2 mM), no potentiation of peak NMDA-mediated currents was observed once the blockers were removed (Fig. 4c). In contrast, using cells taken from the same slices, CHPG potentiated currents when co-applied with NMDA/glycine in the absence of these channel blockers (data not shown). An influx of Ca2+ was required, because stimulation of NMDAR and mGluR5 in the presence of nominal extracellular 0.2 mM Ca2+ (plus 3 µM neomycin) failed to enhance NMDAR currents (Fig. 4d). Applications of neomycin were made to block the calcium-sensing non-selective cation current (54), and as a control we determined that neomycin failed to block the CHPG-stimulated potentiation of NMDAR currents (Fig. 4d, inset).
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A long lasting enhancement of mEPSCAMPA can be induced in primary cultures of hippocampal neurons by employing brief applications of glycine that selectively activate synaptically located NMDARs (22). We anticipated that CHPG should induce LTP in these cultures provided we simultaneously activate synaptic NMDARs. Therefore, a concentration of glycine (1 µM), at the subthreshold for inducing LTP itself, was added to all solutions (22). Under this condition of enhanced NMDAR activation, applications of CHPG induced a profound and long lasting potentiation of the amplitude of mEPSCAMPA that was prevented by co-application of the mGluR5 inhibitor, MPEP (Fig. 5). Consistent with our finding that mGluR5 and NMDARs must be co-activated, we found that application of CHPG in the absence of NMDAR stimulation (e.g. APV-treated cultures) failed to evoke LTP (Fig. 5). There was also a corresponding increase in the frequency of events in response to CHPG application but not during co-applications of CHPG and MPEP or CHPG plus APV (data not shown).
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DISCUSSION |
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It is tempting to speculate that co-stimulation of mGluR5 and NMDARs, resulting in potentiation of synaptic transmission in primary hippocampal cultures, serves as a model for events that occur during LTP induction in vivo. In support of this notion is our finding that co-stimulation of mGluR5 and NMDARs results in an enhancement of miniature excitatory postsynaptic currents mediated by AMPARs that lasts for 45 min and in several instances for more than 1 h. The degree of enhancement is consistent with other culture models of LTP (26, 27), however, the interplay of other receptor systems and presynaptic effects, as found in vivo, would likely modify the amplitude and kinetics of the response.
To investigate the precise timing requirement of this co-incident activation, we varied the interval between the application of CHPG and NMDA/glycine. If these two receptor systems were activated within seconds of one another, we noted an enhancement of NMDAR currents, yet longer intervals resulted in a modest depression of peak NMDAR currents. This timing may be consistent with LTP and LTD induction in vivo. Given the spatial orientation of NMDARs and mGluR5 (extrasynaptic and perisynaptic), it is conceivable that there exists a delay in the activation of NMDARs and mGluR5. However, one would expect that NMDARs and mGluR5 would be co-incidentally activated during glutamate spillover (i.e. during strong tetanus stimulation), yet a delay in activation of these two receptor systems may occur under circumstances that induce LTD. We anticipate that other receptor or signaling systems in addition to mGluR5 and NMDARs are likely involved in the onset of synaptic plasticity. This would account for our observation that stimulation of mGluR5 alone modestly depresses excitatory synaptic transmission. Indeed, activation of group II and III mGluR receptors on the presynaptic membrane is integral for LTD at CA1 synapses (58). It is difficult to evaluate the effects of co-incidence mGluR5 and NMDAR activation in a hippocampal slice preparation given that complete perfusion of drugs into such a preparation would take more than several seconds and there exists direct inhibitory effects of mGluR agonists on NMDA channels.
We have previously shown that Gq-linked GPCRs enhance NMDAR
currents and this effect is mediated via a PKC-CAK-Src cascade
(35,
43). Our finding that mGluR5
stimulates the serine/threonine kinase PKC and then non-receptor tyrosine
kinase Src to enhance NMDARs and long term synaptic transmission is entirely
consistent with these reports. Given that PKC activates Src kinase via the
intermediate tyrosine kinase CAK
in cell lines
(47,
59) and in CA1 pyramidal
neurons (35), the role of
CAK
was also examined. We determined that pre-applications of CAK
occluded the mGluR5 response of NMDAR currents and intracellular perfusion of
a functional dominant negative, CAK
-K457A, inhibited the response to
CHPG. Moreover, CHPG induced the tyrosine phosphorylation of CAK
and Src
isolated from the CA1 region, and this effect was blocked by co-applications
with MPEP.
A rise in intracellular Ca2+ resulting from either
release from intracellular stores or from an influx, as well as via
stimulation of PKC, can lead to activation of CAK
(47). In CA1 neurons,
inclusion of a strong Ca2+ buffer in the patch pipettes
prevented the CHPG-induced potentiation of NMDA currents, indicating that
activation of CAK
requires both stimulation of PKC and elevated
Ca2+. It also required mobilization of intracellular
calcium, via IP3Rs, because the selective blockade of
IP3Rs blocked the mGluR5 effect. Acute application of thapsigargin,
which promotes calcium release, closely mimicked activation of mGluR5 and
occluded the CHPG-induced potentiation. Moreover, the thapsigargin-induced
enhancement of NMDA currents was blocked by inhibitors of PKC, CAK
, and
Src. These results collectively suggest that release of calcium from
IP3R-dependent stores serves as an upstream signal to the
activation of the PKC-CAK
-Src cascade. However, this signal was not
sufficient on its own to activate the cascade, because an influx of
Ca2+ via NMDARs was also required.
The co-incidence of an influx of Ca2+ and its release
from intracellular stores under our experimental conditions had to occur on
the order of seconds, because longer intervals result in depression of
NMDA-evoked responses, a finding consistent with observations of a depression
of these currents when mGluR5s are stimulated on their own
(18,
19) or when the influx of
Ca2+ via NMDARs is impaired (e.g. cells are
depolarized) (14). Our results
imply that a threshold concentration of intracellular
Ca2+ must be achieved in the vicinity of synaptic NMDARs
for the activation of the PKC/CAK/Src cascade and the resulting
potentiation of excitatory synaptic transmission. Alternatively, an influx of
Ca2+ through NMDARs may be required to
"load" intracellular stores such that, upon the subsequent
stimulation of IP3Rs (via mGluR5), sufficient
Ca2+ is mobilized to activate or facilitate the
PKC/CAK
/Src cascade. Indeed, in hippocampal neurons, the
Ca2+ influx through NMDARs or voltage-gated
Ca2+ channels can act to load intracellular
Ca2+ stores
(60), and stores are only
partially loaded or functionally empty at rest
(61,
62). Another possible
explanation is that NMDARs may be required to "sensitize" mGluR5
sufficiently to permit activation of the PKC/CAK
/Src cascade. For
example, stimulation of NMDARs reverses the desensitization of mGluR5 via a
PKC-dependent pathway (63).
Also, the scaffolding protein Homer may retain mGluR5 in an inactive state,
because upon dissociation mGluR5 demonstrates constitutive activity
(64,
65).
However, it is possible that not all conditions may require co-incident
activation of mGluR5 and NMDARs to potentiate excitatory synaptic
transmission. For instance, strong depolarization of the postsynaptic neuron
may allow sufficient calcium entry via NMDARs to trigger the release of
calcium via internal stores and initiation of the PKC/CAK/Src cascade to
enhance NMDAR activity. Under this scheme, the calcium influx via NMDARs may
override the necessity of calcium mobilization in response to mGluR5
stimulation (66). One must
also consider the possibility that strong stimulation of mGluR5 may initiate
the IP3R/PKC/CAK
/Src cascade independently of NMDAR activity.
Such a mechanism seems less likely given that mGluR5 is spatially localized at
perisynaptic and extrasynaptic sites and as such their activation would
require glutamate spillover, which is likely to activate NMDARs.
The induction of LTP at CA1 synapses requires an elevation of intracellular
calcium, most likely through calcium influx via NMDARs, which results in an
increase in AMPAR currents
(15). This increase in
postsynaptic function may be the result of increased AMPAR gating, an increase
in AMPARs at the membrane surface, or a combination of both
(15,
67). There is convincing
evidence that demonstrates that at least one mechanism for the induction of
CA1-LTP requires an up-regulation of NMDAR activity
(34,
35) and NMDAR surface
expression (68) and that this
amplification is achieved in part through the sequential stimulation of
CAK and Src (34,
35).
Administration of catalytically active CAK or a peptide activator of
Src has been shown to enhance AMPAR activity. However, this enhancement is
indirect, because it is abrogated by blocking NMDARs or by buffering
intracellular Ca2+ using a relatively slow buffer such
as EGTA (34,
35). In contrast, buffering
with EGTA fails to block the Src-induced potentiation of NMDA channel activity
(34,
35). Similarly, the
enhancement of NMDA currents in response to activation of both mGluR5 and
muscarinic receptors can be observed when EGTA is employed
(35,
43) but is blocked with the
rapid buffer BAPTA. These results suggest that the Ca2+
signal responsible for activation of the PKC-CAK
-Src cascade is more
spatially restricted to the vicinity of NMDARs than that required for
up-regulation of AMPARs.
High frequency stimulation depolarizes CA1 neurons and relieves the
voltage-dependent blockade of NMDA channels by Mg2+,
which acts to promote Ca2+ entry and induces LTP. This
strong stimulation may also enhance glutamate "spillover" to the
perisynaptic sites of metabotropic glutamate receptors. Activation of NMDARs
and mGluR5 will then give rise to a Ca2+- and
PKC-dependent CAK/Src cascade, which feeds back to further enhance NMDAR
responses. This conclusion fits with observations that mGluR5 is highly
expressed in CA1 pyramidal neurons at perisynaptic as well as extrasynaptic
sites (11,
69) and with the demonstration
that mGluR5-deficient mice display impaired LTP of NMDAR-mediated transmission
(44,
70).
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FOOTNOTES |
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** To whom correspondence should be addressed: Dept. of Physiology, University of Toronto, Medical Sciences Bldg., 1 King's College, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-0711; Fax: 416-978-4940; E-mail: j.macdonald{at}utoronto.ca.
1 The abbreviations used are: NMDAR, N-methyl-D-aspartate
receptor; IP3, inositol triphosphate; IP3R,
IP3 receptor; mGluR, metabotropic glutamate receptor; PKC, protein
kinase C; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionate; AMPAR,
AMPA receptor; mEPSC, miniature excitatory postsynaptic current; GPCRs,
G-protein-coupled receptors; MAPK, mitogen-activated protein kinase; PDGFR,
platelet-derived growth factor receptor; CAK
, calcium-activated kinase
; PMA, phorbol 12-myristate 13-acetate; Xe-C, xestospongin-C; APV, 2
amino-5-phosphopentanoic acid; CHPG,
(RS)-2-chloro-5-hydroxy-phenylglycine; MPEP,
2-methyl-6-(phenylethynyl)pyridine; ACPD,
1-aminocyclopentane-trans-1,3-phosphonobutyric acid; LTP, long term
potentiation; LTD, long term depression; BAPTA,
1,2-bis(O-aminophenoxy)ethane-N,N,N'-N'-tetraacetic
acid.
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REFERENCES |
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