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Originally published In Press as doi:10.1074/jbc.M301946200 on May 13, 2003 Originally published In Press as doi:10.1074/jbc.M301946200 on May 9, 2003

J. Biol. Chem., Vol. 278, Issue 30, 27742-27749, July 25, 2003
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Co-stimulation of mGluR5 and N-Methyl-D-aspartate Receptors Is Required for Potentiation of Excitatory Synaptic Transmission in Hippocampal Neurons*

Suhas A. Kotecha {ddagger}, Michael F. Jackson {ddagger}, Azza Al-Mahrouki {ddagger}, John C. Roder §, Beverley A. Orser {ddagger} ¶ and John F. MacDonald {ddagger} || **

From the Departments of {ddagger}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.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the central nervous system, excitatory synaptic transmission is mediated by the neurotransmitter glutamate and its receptors. Interestingly, stimulation of group I metabotropic glutamate receptors (mGluRs) can either enhance or depress synaptic transmission at CA1 hippocampal synapses. Here we report that co-activation of mGluR5, a member of the group I mGluR family, and N-methyl-D-aspartate receptors (NMDARs) potentiates NMDAR currents and induces a long lasting enhancement of excitatory synaptic transmission in primary cultured hippocampal neurons. Unexpectedly, activation of mGluR5 alone fails to enhance evoked NMDAR currents and synaptic {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) AMPAR currents. The observed potentiation requires an mGluR5-induced, inositol 1,4,5-trisphosphate receptor-mediated mobilization of intracellular Ca2+, which acts in concert with a protein kinase C, calcium-activated tyrosine kinase cascade to induce a long lasting enhancement of NMDAR and AMPAR currents.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transmitter glutamate, together with its postsynaptic receptors, mediates much of the excitatory neurotransmission in the central nervous system. Of these postsynaptic receptors, the NMDAR1 is an ionotropic receptor implicated in neuronal development (1), synaptic plasticity (2), and excitotoxicity (3). In the pyramidal cell synapses of the CA1 region of the hippocampus, the NMDAR is composed of at least one NR1 subunit as well as multiple NR2B or NR2A subunits (4). These postsynaptic NMDARs are associated with a complex of proteins (the "NMDAR complex"), which includes the scaffolding proteins PSD-95, Homer, Shank, and GKAP as well as a number of serine/threonine and tyrosine kinases and phosphatases (5). Furthermore, other receptors, including mGluR and IP3R are potentially linked to this complex (6). The presence and activity of many of these proteins is integral for LTP and LTD, which are the leading cellular and molecular models for learning and memory (7).

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{beta} 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 (17–19) and cultures (14, 20–22) bath applications of either NMDA or of a group I mGluR agonist induce LTD, but not LTP, and enhance AMPA receptor endocytosis (14, 23–25). However, in cultures selective stimulation of synaptic but not extrasynaptic NMDARs induces LTP and not LTD (22, 26–28). 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


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Isolation and Whole-cell and Perforated Patch Recordings—CA1 neurons were isolated from hippocampal slices from postnatal rats (Wistar 10–20 days) using previously described procedures (55). To control for variation in response, recordings from control and treated cells were made on the same day, unless otherwise indicated. The extracellular solution consisted of (in mM): 140 NaCl, 1.3 CaCl2, 5 KCl, 25 HEPES, 33 glucose, and 0.0005 tetrodotoxin, with pH 7.4 and osmolarity between 320 and 325 mosmol. Recordings were done at room temperature (20–22 °C). After formation of a whole-cell configuration, the recorded neurons were voltage-clamped at –60 mV and lifted into the stream of solution supplied by a computer-controlled, multibarreled perfusion system. Continuous perfusion was achieved by using a gravity-fed system. This system allows for a delay in the application of two different drugs from as little as <2 ms. To monitor access resistance, a voltage step of –10 mV was made before each application of NMDA. Recordings where series resistance varied by more than 10% were rejected. The intracellular solution contained (in mM): 140 CsF or CsCl2, 11 EGTA, 1 CaCl2, 2 MgCl2, 10 HEPES, 2 tetraethylammonium, and 2 K2ATP with pH 7.2 and osmolarity between 295 and 300 mosmol. Several recordings were also performed from HEK 293 cells expressing recombinant NMDARs under identical recording conditions. For these experiments, HEK cells were transiently transfected using a lipid transfer method (Invitrogen, Carlsbad, CA) with cDNAs encoding for the NMDA receptor subunits NR2a and a C-terminal truncated form of the NR1 subunit (NR1Stop838) previously demonstrated to abolish Ca2+-dependent inactivation of NMDARs (56). In perforated patch recordings, the antibiotic amphotericin B (0.5 µg/ml) was included in the patch electrode. Once the cell-attached configuration was established, the negative pressure was relieved and ~10 min elapsed before electrical access to the cell was achieved. Some drugs were included in the patch pipette. Data are expressed as mean ± S.E. and were analyzed using a two-way analysis of variance.

Recordings of Miniature Postsynaptic Excitatory Currents—Procedures for the preparation of primary dissociated cultures of hippocampal neurons have been previously described (57). Whole-cell recordings were made from these cultures 12–17 days after plating. Recordings were performed at room temperature (20–22 °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 Assays—CA1 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{beta} or anti-Src. Immune complexes were isolated by addition of 40 µl of protein G-Sepharose beads, followed by incubation for 1–2 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{beta} or anti-Src (1:3000 and 1:5000 dilution, respectively). The film images were digitized and imported into Corel Draw for presentation purposes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We first assessed the role of mGluR5 in the regulation of excitatory synaptic transmission in primary hippocampal cultures. Applications of CHPG (100 µM), a selective mGluR5 agonist, but relatively insensitive for mGluR1 (31), enhanced miniature excitatory postsynaptic current mediated by NMDARs (mEPSCNMDA) but not the AMPA receptor component (mEPSCAMPA). The concentration of CHPG employed was chosen to match that used recently to study mGluR5-mediated responses in cultured retinal amacrine neurons (32). The CHPG-induced enhancement of mEPSCNMDA was attenuated by co-application of the selective mGluR5 antagonist MPEP. The concentration of MPEP was just maximal (10 µM, Fig. 1a) for antagonism of mGluR5 with little or no effect on mGluR1 (33). In these recordings, and as previously described, intracellular Ca2+ was sufficiently buffered to prevent the secondary potentiation of mEPSCAMPA without preventing the Ca2+-dependent modulation of NMDARs (34, 35).



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  		FIG. 1.
mGluR5 enhances synaptic NMDAR currents in cultured hippocampal neurons and peak currents in isolated CA1 pyramidal neurons. a, CHPG (100 µM) enhanced the mEPSCNMDA component but not mEPSCAMPA, and this effect was blocked by co-application of MPEP (10 µM). Inset, current traces illustrating that CHPG enhances mEPSCNMDA, but not mEPSCAMPA, and this effect is blocked by co-application of MPEP. b, stability of NMDA-evoked currents in both whole-cell and perforated patch configurations in acutely isolated hippocampal neurons. c1, Application of CHPG (100 µM, included in the control barrel for the period indicated by the bar but not in the NMDA-containing barrel) potentiates peak NMDA-evoked currents (Ip) but not steady-state currents (Iss) when subsaturating concentrations of NMDA (30 µM) and glycine (500 nM) were applied, and this effect was blocked by co-application of the mGluR5 antagonist MPEP. c2, the CHPG effect was attenuated by co-application of MPEP (CHPG: 135 ± 12%, n = 7; CHPG plus MPEP: 95 ± 8%, n = 8, p < 0.001, data obtained at 35 min) d1 and d2, CHPG directly blocks NMDA-evoked currents in HEK293 cells expressing recombinant NMDA receptors. The onset and offset of the block were rapid, occurring in less than 1 s. e1 and e2, applications of CHPG failed to enhance Ip in mGluR5 knockout mice (–/–), but not in control (+/+: 153 ± 11%, n = 5;–/–: 97 ± 5%, n = 4, p < 0.001, data obtained at 30 min). f, in perforated patch recordings, CHPG depressed Iss with near saturating concentrations of agonist (3 µM glycine, 300 µM NMDA, Iss: 69 ± 4%, n = 8, p < 0.001, Ip: 112 ± 8%, n = 8, data obtained at 40 min). The black bars in b, c, d2, and e represent the time and duration of drug application.

 

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 (36–38) 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 {beta} (CAK{beta}) 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{beta}-phorbol 12-myristate 13-acetate (4{beta}-PMA) (100 nM) potentiated Ip as anticipated but also occluded the mGluR5-induced potentiation (Fig. 2a) whereas an inactive phorbol ester, 4{alpha}-PMA (100 nM), did neither ({alpha}-PMA: 95 ± 5%, n = 8; {alpha}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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  		FIG. 2.
mGluR5-induced enhancement of NMDAR currents is dependent upon PKC, CAK{beta}, and Src. a, the PKC inhibitor chelerythrine (10 µM) was included in the recording electrode solution and was found to block the CHPG-induced enhancement, whereas the active phorbol ester ({beta}-PMA, 100 nM) occluded the CHPG effect ({beta}-PMA: 133 ± 6%, n = 7, data obtained at 15 min; {beta}-PMA plus CHPG: 129 ± 9%, n = 7, data obtained at 35 min; chelerythrine plus CHPG: 94 ± 5%, n = 8, p < 0.001, data obtained at 35 min). b, the functional dominant negative CAK{beta}-K457A blocked the CHPG-induced enhancement. Inclusion of recombinant CAK{beta} in the recording electrode solution enhanced NMDAR currents and occluded the CHPG response (CAK{beta}: 146 ± 13%, n = 6, data obtained at 12 min; CAK{beta} plus CHPG: 155 ± 18%, n = 6, data obtained at 30 min; CAK{beta}-K457A plus CHPG: 94 ± 8%, n = 7, data obtained at 30 min). c, recordings from primary cultured hippocampal neurons indicate that CHPG enhanced mEPSCNMDA, and this effect was attenuated with CAK{beta}-K457A in the patch electrode solution (CHPG: 21.1 ± 5.4%, n = 8; CHPG plus CAK{beta}-K457A: 7.52 ± 4.1%, n = 7, p < 0.001, data obtained after 25 min). In contrast, mEPSCAMPA was not affected in response to CHPG (CHPG: 3.8 ± 1.9%, n = 8; CHPG plus CAK{beta}-K457A: 2.4 ± 1.5%, n = 7, data obtained after 25 min). Inset, current traces illustrate that CHPG enhances mEPSCNMDA, but not mEPSCAMPA, and this effect is abrogated in the presence of CAK{beta}-K457A. d, inclusion of Src in the intracellular solution occluded the CHPG response, whereas inactive Src failed to do so (active Src: 123 ± 4.2%, n = 6, data obtained at 10 min; active Src plus CHPG: 126 ± 3.6%, n = 6, data obtained at 35 min; inactive Src: 110 ± 6%, n = 7; inactive Src plus CHPG: 123 ± 6%, n = 7, p < 0.001). e, the selective Src kinase peptide inhibitor Src40–58 blocked the CHPG response, but the scrambled peptide (inactive Src40–58) did not (CHPG plus Src40–58: 104 ± 8%, n = 6; CHPG plus sSrc40–58: 138 ± 8%, n = 6, p < 0.001, data obtained at 30 min). f, in vitro phosphorylation assays reveal that immunopurified CAK{beta} and Src become tyrosine-phosphorylated (immunoblotted with a phosphotyrosine antibody) in response to CHPG but not CHPG plus MPEP. The black bars in a, b, d, and e represent the time and duration of CHPG application.

 

There is strong evidence that PKC can activate the non-receptor tyrosine kinase CAK{beta}, and this kinase is highly expressed as an unspliced isoform in hippocampal tissue (47). CAK{beta} is also associated with NMDARs (5, 35). We therefore tested the hypothesis that mGluR5 activates PKC and then CAK{beta}. The CHPG-induced potentiation of Ip was blocked by the functional dominant negative mutant of CAK{beta} (kinase mutant, CAK{beta}-K475A, 0.05 µg/ml) (35). Furthermore, recombinant CAK{beta} (0.05 µg/ml) itself slowly enhanced Ip and subsequently occluded the CHPG-induced potentiation (Fig. 2b). Intracellular applications of CAK{beta}-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{beta} 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{beta} can relieve the autoinhibition of Src tyrosine kinases (48). In CA1 pyramidal neurons Src is downstream of a CAK{beta}, and both the induction of LTP and application of phorbol esters increase CAK{beta} 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, Src40–58 (25 µg/ml) (49) blocked the CHPG-induced enhancement, whereas its control peptide sSrc40–58 (a scrambled peptide, 25 µg/ml) failed to do so (Fig. 2e).

To further demonstrate the activation of CAK{beta} 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{beta} and Src were immunopurified using anti-CAK{beta} and anti-Src antibodies, respectively. We then probed phosphorylation of CAK{beta} and Src using tyrosine phosphorylation-specific antibodies. CHPG, but not CHPG plus MPEP treatment, enhanced tyrosine phosphorylation indicating mGluR5 stimulation activates CAK{beta} 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{beta}/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{beta}/Src cascade, and an enhancement of NMDARs. In support of this we found that intracellular applications of chelerythrine (10 µM), Src40–58 (25 µg/ml), or CAK{beta}-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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  		FIG. 3.
IP3Rs stimulate a PKC/CAK{beta}/Src cascade and are needed for mGluR5-effect. a, intracellular administration of BAPTA (25 mM) blocked the CHPG effect as did Xe-C (CHPG: 137 ± 8%, n = 6; CHPG plus BAPTA: 106 ± 6%, n = 5, p < 0.001; CHPG plus Xe-C: 96 ± 5%, n = 6, p < 0.001, data obtained at 30 min). The black bar indicates the time and duration of CHPG application. b, thapsigargin enhanced Ip with sub-saturating concentrations of agonist (500 nM glycine, 10 µM NMDA; Ip: 153 ± 12%, n = 6, p < 0.001; Iss: 102 ± 4%, n = 6, data obtained at 25 min of recording) yet depressed Iss with near saturating concentrations of agonist (3 µM glycine, 300 µM NMDA; peak: 100 ± 5%, n = 5; steady state: 53 ± 6%, n = 5, p < 0.001, data obtained at 25 min of recording). c, the thapsigargin effect was blocked by inhibitors to IP3Rs, PKC, Src, and CAK{beta} (thapsigargin: 142 ± 8.1%, n = 7, thapsigargin plus chelerythrine: 109 ± 9%, n = 5, p < 0.001; thapsigargin plus Src40–58: 105 ± 6%, n = 8, p < 0.001; thapsigargin plus CAK{beta}-K457A: 108 ± 9%, n = 6, p < 0.001, data obtained at 25 min of recording) but not to the inactive Src kinase peptide inhibitor (thapsigargin plus sSrc40–58: 139 ± 12%, n = 5, data obtained at 25 min of recording). Thapsigargin occluded the CHPG effect (thapsigargin: 139 ± 12%, n = 6, p < 0.001, data obtained at 15 min of recording; thapsigargin plus CHPG: 144 ± 5%, n = 6, data obtained at 25 min of recording).

 

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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  		FIG. 4.
Co-activation of NMDAR and mGluR5 is required for the mGluR5-induced enhancement of NMDAR currents. a, application of CHPG in the absence of NMDAR activity failed to subsequently enhance NMDA-evoked responses (control: 100%, t = 25 min: 88 ± 9%, n = 6, data obtained at 25 min of recording). b, time intervals less than 15 s between stimulation of mGluR5 and NMDARs were required for the mGluR5-induced enhancement of NMDAR currents. All data were obtained at 20 min of recording. c, ketamine (50 µM) and magnesium (2 mM), reversible, open channel NMDA blockers were applied during CHPG to block ion flux. During subsequent wash NMDAR currents were not enhanced (for magnesium, t = 25 min: 89 ± 7%, n = 6; for ketamine, t = 35 min: 85 ± 12%, n = 7). d, the influx of extracellular calcium via NMDARs was required, because stimulation of NMDARs and mGluR5s with 0.2 mM extracellular calcium (in the presence of neomycin) failed to result in a long lasting potentiation of NMDA responses (no CHPG: 103 ± 8%, n = 6; CHPG: 108 ± 13%, n = 6, data obtained at 20 min of recording). Inset, current traces illustrate that neomycin failed to block the CHPG-induced enhancement of NMDAR currents in the recordings.

 

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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  		FIG. 5.
Co-activation of mGluR5 and NMDARs induces LTP in primary cultured hippocampal neurons. a and b, administration of CHPG under low intracellular calcium buffering conditions (0.1 mM EGTA/0 mM CaCl2) potentiates mEPSCAMPA (CHPG: 1.38 ± 0.09, n = 14, data obtained at 45 min of recording). This effect was blocked by co-application of MPEP (CHPG plus MPEP: 1.09 ± 0.11, n = 15, data obtained at 45 min of recording), and a modest depression of mEPSCAMPA was observed when CHPG was applied in the presence of APV (CHPG plus APV: 09.1 ± 0.17, n = 11, data obtained at 45 min of recording) or magnesium (not shown).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The objective of the present study was to elucidate the mechanism by which mGluR5 modulates NMDARs and excitatory synaptic transmission. An understanding of the mechanism by which mGluR5 modulates ionotropic glutamate receptors is of particular interest, because Group I mGluRs have been reported to both potentiate and, paradoxically, depress excitatory synaptic transmission in the CA1 hippocampus (30). We tested the novel hypothesis that co-stimulation of NMDARs and mGluR5, and the timing requirements for the activation of these two receptors, can potentiate or depress excitatory synaptic transmission. We found that co-activation of NMDARs and mGluR5 results in a profound enhancement of peak NMDAR currents and mEPSCAMPA. If, however, mGluR5 was activated in the absence of NMDAR stimulation, the resulting activity of NMDARs and mEPSCAMPA demonstrated a modest depression. Calcium influx via NMDARs was especially important for the mGluR5-induced enhancement of NMDAR currents, because blockade of calcium flux (by application of the NMDA open channel blockers ketamine or magnesium) or removal of extracellular calcium during simultaneous NMDAR and mGluR5 stimulation failed to subsequently enhance NMDAR currents. Finally, recordings from primary cultured hippocampal neurons revealed that mEPSCAMPA was enhanced in response to CHPG application but not when co-applications of CHPG and the NMDAR antagonist APV were made. Collectively, these results demonstrate that co-activation of NMDA receptors and mGluR5 is required for the enhancement of NMDAR currents and a long lasting potentiation of excitatory synaptic transmission in hippocampal cultures.

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{beta}-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{beta} in cell lines (47, 59) and in CA1 pyramidal neurons (35), the role of CAK{beta} was also examined. We determined that pre-applications of CAK{beta} occluded the mGluR5 response of NMDAR currents and intracellular perfusion of a functional dominant negative, CAK{beta}-K457A, inhibited the response to CHPG. Moreover, CHPG induced the tyrosine phosphorylation of CAK{beta} 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{beta} (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{beta} 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{beta}, 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{beta}-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{beta}/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{beta}/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{beta}/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{beta}/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{beta}/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{beta} and Src (34, 35).

Administration of catalytically active CAK{beta} 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{beta}-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{beta}/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).


   FOOTNOTES
 
* This work was supported by Canadian Institutes of Health Research and by the Heart and Stroke Foundation of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** 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, {alpha}-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{beta}, calcium-activated kinase {beta}; 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. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Constantine-Paton, M. (2000) Neurobiol. Dis. 7, 515–519[CrossRef][Medline] [Order article via Infotrieve]
  2. Collingridge, G. L., and Bliss, T. V. (1995) Trends. Neurosci. 18, 54–56[CrossRef][Medline] [Order article via Infotrieve]
  3. Hardingham, G. E., and Bading, H. (2003) Trends Neurosci. 26, 81–89[CrossRef][Medline] [Order article via Infotrieve]
  4. Monyer, H., Burnashev, N., Laurie, D. J., Sakmann, B., and Seeburg, P. H. (1994) Neuron 12, 529–540[CrossRef][Medline] [Order article via Infotrieve]
  5. Husi, H., Ward, M. A., Choudhary, J. S., Blackstock, W. P., and Grant, S. G. (2000) Nat. Neurosci. 3, 661–669[CrossRef][Medline] [Order article via Infotrieve]
  6. Sheng, M., and Pak, D. T. (2000) Annu. Rev. Physiol. 62, 755–778[CrossRef][Medline] [Order article via Infotrieve]
  7. Bliss, T. V., and Collingridge, G. L. (1993) Nature 361, 31–39[CrossRef][Medline] [Order article via Infotrieve]
  8. Conn, P. J., and Pin, J. P. (1997) Annu. Rev. Pharmacol. Toxicol. 37, 205–237[CrossRef][Medline] [Order article via Infotrieve]
  9. Hannan, A. J., Blakemore, C., Katsnelson, A., Vitalis, T., Huber, K. M., Bear, M., Roder, J., Kim, D., Shin, H. S., and Kind, P. C. (2001) Nat. Neurosci. 4, 282–288[CrossRef][Medline] [Order article via Infotrieve]
  10. Baude, A., Nusser, Z., Roberts, J. D., Mulvihill, E., McIlhinney, R. A., and Somogyi, P. (1993) Neuron 11, 771–787[CrossRef][Medline] [Order article via Infotrieve]
  11. Lujan, R., Nusser, Z., Roberts, J. D., Shigemoto, R., and Somogyi, P. (1996) Eur. J. Neurosci. 8, 1488–1500[CrossRef][Medline] [Order article via Infotrieve]
  12. Ferraguti, F., Conquet, F., Corti, C., Grandes, P., Kuhn, R., and Knopfel, T. (1998) J. Comp Neurol. 400, 391–407[CrossRef][Medline] [Order article via Infotrieve]
  13. Marino, M. J., Wittmann, M., Bradley, S. R., Hubert, G. W., Smith, Y., and Conn, P. J. (2001) J. Neurosci. 21, 7001–7012[Abstract/Free Full Text]
  14. Snyder, E. M., Philpot, B. D., Huber, K. M., Dong, X., Fallon, J. R., and Bear, M. F. (2001) Nat. Neurosci. 4, 1079–1085[CrossRef][Medline] [Order article via Infotrieve]
  15. Soderling, T. R., and Derkach, V. A. (2000) Trends. Neurosci. 23, 75–80[CrossRef][Medline] [Order article via Infotrieve]
  16. Luscher, C., and Frerking, M. (2001) Trends Neurosci. 24, 665–670[CrossRef][Medline] [Order article via Infotrieve]
  17. Lee, H. K., Kameyama, K., Huganir, R. L., and Bear, M. F. (1998) Neuron 21, 1151–1162[CrossRef][Medline] [Order article via Infotrieve]
  18. Huber, K. M., Kayser, M. S., and Bear, M. F. (2000) Science 288, 1254–1257[Abstract/Free Full Text]
  19. Huber, K. M., Roder, J. C., and Bear, M. F. (2001) J. Neurophysiol. 86, 321–325[Abstract/Free Full Text]
  20. Carroll, R. C., Lissin, D. V., von Zastrow, M., Nicoll, R. A., and Malenka, R. C. (1999) Nat. Neurosci. 2, 454–460[CrossRef][Medline] [Order article via Infotrieve]
  21. Carroll, R. C., Beattie, E. C., Xia, H., Luscher, C., Altschuler, Y., Nicoll, R. A., Malenka, R. C., and von Zastrow, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 14112–14117[Abstract/Free Full Text]
  22. Lu, W., Man, H., Ju, W., Trimble, W. S., MacDonald, J. F., and Wang, Y. T. (2001) Neuron 29, 243–254[CrossRef][Medline] [Order article via Infotrieve]
  23. Luscher, C., Xia, H., Beattie, E. C., Carroll, R. C., von Zastrow, M., Malenka, R. C., and Nicoll, R. A. (1999) Neuron 24, 649–658[CrossRef][Medline] [Order article via Infotrieve]
  24. Man, H.-Y., Lin, J. W., Ju, W. H., Ahmadian, G., Liu, L., Becker, L. E., Sheng, M., and Wang, Y. T. (2000) Neuron 25, 649–662[CrossRef][Medline] [Order article via Infotrieve]
  25. Wang, Y. T., and Linden, D. J. (2000) Neuron 25, 635–647[CrossRef][Medline] [Order article via Infotrieve]
  26. Pickard, L., Noel, J., Duckworth, J. K., Fitzjohn, S. M., Henley, J. M., Collingridge, G. L., and Molnar, E. (2001) Neuropharmacology 41, 700–713[CrossRef][Medline] [Order article via Infotrieve]
  27. Fitzjohn, S. M., Pickard, L., Duckworth, J. K., Molnar, E., Henley, J. M., Collingridge, G. L., and Noel, J. (2001) Neuropharmacology 41, 693–699[CrossRef][Medline] [Order article via Infotrieve]
  28. Musleh, W., Bi, X., Tocco, G., Yaghoubi, S., and Baudry, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9451–9456[Abstract/Free Full Text]
  29. Anwyl, R. (1999) Brain Res. Brain Res. Rev. 29, 83–120[CrossRef][Medline] [Order article via Infotrieve]
  30. Bortolotto, Z. A., Fitzjohn, S. M., and Collingridge, G. L. (1999) Curr. Opin. Neurobiol. 9, 299–304[CrossRef][Medline] [Order article via Infotrieve]
  31. Doherty, A. J., Palmer, M. J., Henley, J. M., Collingridge, G. L., and Jane, D. E. (1997) Neuropharmacology 36, 265–267[CrossRef][Medline] [Order article via Infotrieve]
  32. Sosa, R., Hoffpauir, B., Rankin, M. L., Bruch, R. C., and Gleason, E. L. (2002) J. Neurochem. 81, 973–983[CrossRef][Medline] [Order article via Infotrieve]
  33. Gasparini, F., Lingenhohl, K., Stoehr, N., Flor, P. J., Heinrich, M., Vranesic, I., Biollaz, M., Allgeier, H., Heckendorn, R., Urwyler, S., Varney, M. A., Johnson, E. C., Hess, S. D., Rao, S. P., Sacaan, A. I., Santori, E. M., Velicelebi, G., and Kuhn, R. (1999) Neuropharmacology 38, 1493–1503[CrossRef][Medline] [Order article via Infotrieve]
  34. Lu, Y. M., Roder, J. C., Davidow, J., and Salter, M. W. (1998) Science 279, 1363–1367[Abstract/Free Full Text]
  35. Huang, Y. Q., Lu, W. Y., Ali, D. W., Pelkey, K. A., Pitcher, G. M., Lu, Y. M., Aoto, H., Roder, J. C., Sasaki, T., Salter, M. W., and MacDonald, J. F. (2001) Neuron 29, 485–496[CrossRef][Medline] [Order article via Infotrieve]
  36. Movsesyan, V. A., O'Leary, D. M., Fan, L., Bao, W., Mullins, P. G., Knoblach, S. M., and Faden, A. I. (2001) J. Pharmacol. Exp. Ther. 296, 41–47[Abstract/Free Full Text]
  37. O'Leary, D. M., Movsesyan, V., Vicini, S., and Faden, A. I. (2000) Br. J. Pharmacol. 131, 1429–1437[CrossRef][Medline] [Order article via Infotrieve]
  38. Contractor, A., Gereau, R. W., Green, T., and Heinemann, S. F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8969–8974[Abstract/Free Full Text]
  39. Lu, W. Y., Jackson, M. F., Bai, D., Orser, B. A., and MacDonald, J. F. (2000) J. Neurosci. 20, 4452–4461[Abstract/Free Full Text]
  40. Luttrell, L. M., Daaka, Y., and Lefkowitz, R. J. (1999) Curr. Opin. Cell Biol. 11, 177–183[CrossRef][Medline] [Order article via Infotrieve]
  41. Kotecha, S. A., Oak, J. N., Jackson, M. F., Perez, Y., Orser, B. A., Van Tol, H. H. M., and MacDonald, J. F. (2002) Neuron 35, 1111–1122[CrossRef][Medline] [Order article via Infotrieve]
  42. Peavy, R. D., Chang, M. S., Sanders-Bush, E., and Conn, P. J. (2001) J. Neurosci. 21, 9619–9628[Abstract/Free Full Text]
  43. Lu, W. Y., Xiong, Z. G., Lei, S., Orser, B. A., Dudek, E., Browning, M. D., and MacDonald, J. F. (1999) Nat. Neurosci 2, 331–338[CrossRef][Medline] [Order article via Infotrieve]
  44. Lu, Y. M., Jia, Z., Janus, C., Henderson, J. T., Gerlai, R., Wojtowicz, J. M., and Roder, J. C. (1997) J. Neurosci. 17, 5196–5205[Abstract/Free Full Text]
  45. Hemmings, H. C. J. (ed) (1997) in Regulatory Protein Modification: Techniques and Protocols, pp. 147–148, Humana Press Inc., Totowa, NJ
  46. Wang, L. Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. (1994) Nature 369, 230–232[CrossRef][Medline] [Order article via Infotrieve]
  47. Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376, 737–745[CrossRef][Medline] [Order article via Infotrieve]
  48. Ali, D. W., and Salter, M. W. (2001) Curr. Opin. Neurobiol. 11, 336–342[CrossRef][Medline] [Order article via Infotrieve]
  49. Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233–235[CrossRef][Medline] [Order article via Infotrieve]
  50. Gafni, J., Munsch, J. A., Lam, T. H., Catlin, M. C., Costa, L. G., Molinski, T. F., and Pessah, I. N. (1997) Neuron 19, 723–733[CrossRef][Medline] [Order article via Infotrieve]
  51. Miyamoto, S., Izumi, M., Hori, M., Kobayashi, M., Ozaki, H., and Karaki, H. (2000) Br. J. Pharmacol. 130, 650–654[CrossRef][Medline] [Order article via Infotrieve]
  52. Lytton, J., Westlin, M., and Hanley, M. R. (1991) J. Biol. Chem. 266, 17067–17071[Abstract/Free Full Text]
  53. Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R., and Dawson, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2466–2470[Abstract/Free Full Text]
  54. Xiong, Z. G., and MacDonald, J. F. (1999) Can. J. Physiol. Pharmacol. 77, 715–721[CrossRef][Medline] [Order article via Infotrieve]
  55. Wang, L. Y., and MacDonald, J. F. (1995) J. Physiol. (Lond.) 486, 83–95[Abstract/Free Full Text]
  56. Krupp, J. J., Vissel, B., Thomas, C. G., Heinemann, S. F., and Westbrook, G. L. (1999) J. Neurosci. 19, 1165–1178[Abstract/Free Full Text]
  57. MacDonald, J. F., Mody, I., and Salter, M. W. (1989) J. Physiol. (Lond.) 414, 17–34,[Abstract/Free Full Text]
  58. Kemp, N., and Bashir, Z. I. (2001) Prog. Neurobiol. 65, 339–365[CrossRef][Medline] [Order article via Infotrieve]
  59. Dikic, I., Tokiwa, G., Lev, S., Courtneidge, S. A., and Schlessinger, J. (1996) Nature 383, 547–550[CrossRef][Medline] [Order article via Infotrieve]
  60. Rae, M. G., Martin, D. J., Collingridge, G. L., and Irving, A. J. (2000) J. Neurosci. 20, 8628–8636[Abstract/Free Full Text]
  61. Shmigol, A., Svichar, N., Kostyuk, P., and Verkhratsky, A. (1996) Neuroscience 73, 1061–1067[CrossRef][Medline] [Order article via Infotrieve]
  62. Koizumi, S., Bootman, M. D., Bobanovic, L. K., Schell, M. J., Berridge, M. J., and Lipp, P. (1999) Neuron 22, 125–137[CrossRef][Medline] [Order article via Infotrieve]
  63. Alagarsamy, S., Sorensen, S. D., and Conn, P. J. (2001) Curr. Opin. Neurobiol. 11, 357–362[CrossRef][Medline] [Order article via Infotrieve]
  64. Ango, F., Pin, J. P., Tu, J. C., Xiao, B., Worley, P. F., Bockaert, J., and Fagni, L. (2000) J. Neurosci. 20, 8710–8716[Abstract/Free Full Text]
  65. Ango, F., Prezeau, L., Muller, T., Tu, J. C., Xiao, B., Worley, P. F., Pin, J. P., Bockaert, J., and Fagni, L. (2001) Nature 411, 962–965[CrossRef][Medline] [Order article via Infotrieve]
  66. Wilsch, V. W., Behnisch, T., Jager, T., Reymann, K. G., and Balschun, D. (1998) J. Neurosci. 18, 6071–6080[Abstract/Free Full Text]
  67. Lisman, J., Schulman, H., and Cline, H. (2002) Nat. Rev. Neurosci. 3, 175–190[CrossRef][Medline] [Order article via Infotrieve]
  68. Grosshans, D. R., and Browning, M. D. (2001) J. Neurochem. 76, 737–744[CrossRef][Medline] [Order article via Infotrieve]
  69. Romano, C., Sesma, M. A., McDonald, C. T., O'Malley, K., Van den Pol, A. N., and Olney, J. W. (1995) J. Comp. Neurol. 355, 455–469[CrossRef][Medline] [Order article via Infotrieve]
  70. Jia, Z., Lu, Y., Henderson, J., Taverna, F., Romano, C., Abramow-Newerly, W., Wojtowicz, J. M., and Roder, J. (1998) Learn. Mem. 5, 331–343[Abstract/Free Full Text]

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