From the The mitogen-activated protein kinase (MAPK)
cascade has been shown to play an essential role in regulation of cell
proliferation and cell differentiation. Although mammalian MAPKs are
most abundantly expressed in postmitotic and terminally differentiated
neuronal cells, their function in the central nervous system is still
largely undefined. We present evidence here for a role of the MAPK
cascade in cerebellar long term depression (LTD), which is a widely
studied form of synaptic plasticity in mammalian brain. In cultured
Purkinje cells, LTD is known to be induced by iontophoretic application of glutamate and depolarization of Purkinje cells. We found that MAPK
was activated in Purkinje cells by treatment of primary cultures of rat
embryonic cerebella with glutamate and a depolarization-inducing agent,
KCl. Application of PD98059, a specific inhibitor of MAPK kinase
(MAPKK/MEK), inhibited both the activation of MAPK and the induction of
LTD in Purkinje cells. Furthermore, the induction of LTD was completely
blocked by introduction into Purkinje cells of anti-active MAPK
antibody, which was found to specifically and potently inhibit the
activity of MAPK. These results suggest that postsynaptic activation of
the MAPK cascade is essential for the induction of cerebellar LTD.
Mitogen-activated protein kinase
(MAPK,1 also known as ERK) is
a serine/threonine protein kinase that is commonly activated by various
growth factors and differentiation factors (1-3). Activation of MAPK
requires phosphorylation of both threonine and tyrosine residues in its
TEY sequence, which is catalyzed by an upstream activator MAPK kinase
(MAPKK, also known as MEK). MAPK and MAPKK constitute a functional unit
called the MAPK cascade, which has been shown to play a crucial role in
regulation of cell proliferation, cell differentiation, and early
embryonic development. The abundant expression of both MAPK and MAPKK
in postmitotic and differentiated neurons (1-5), however, suggests a
possible function of the MAPK cascade in the mammalian central nervous system.
Persistent changes in synaptic strength such as hippocampal long term
potentiation and cerebellar long term depression (LTD) are thought to
be cellular mechanisms for learning and memory (6, 7). Cerebellar LTD
is a persistent reduction of synaptic transmission between parallel
fibers and Purkinje cells (7, 8). Cerebellar LTD is elicited by the
simultaneous activation of parallel fibers and climbing fibers, which
can be replaced by iontophoretic application of glutamate and
depolarization of Purkinje cells, respectively. Although the induction
of cerebellar LTD has been reported to require an increase in
intracellular Ca2+ concentration and protein kinase C in
Purkinje cells (9-12), molecular mechanisms of the induction of
cerebellar LTD are not well defined. It has previously been reported
that treatment of cultured cortical neurons with glutamate, which
triggers Ca2+ influx through the
N-methyl-D-aspartate type of glutamate
receptors, results in activation of MAPK (13). In marine snail
Aplysia, MAPK was identified as a component of the induction
of long term facilitation in sensory-motor neuron synapse (14, 15). A
recent pharmacological experiment suggested involvement of MAPK in
hippocampal long term potentiation (16). These findings prompted us to
examine the possible involvement of the MAPK cascade in cerebellar LTD. Here we report evidence that activation of MAPK in Purkinje cells is
required for the induction of cerebellar LTD.
Cerebellar Culture--
Primary cultures of rat embryonic
cerebellum were prepared as described previously (17). Cerebella were
dissected from rat fetuses on around embryonic day 17, dissociated by
trituration with a Pasteur pipette, plated in a serum-free medium on
glass coverslips coated with 0.01% poly-D-lysine, and
cultured for 3-4 weeks. One-half of the culture medium was exchanged
once a week.
Immunocytochemistry--
Primary cultures were treated with both
10 µM glutamate and a depolarization-inducing stimulus,
the addition of an iso-osmotic solution (145 mM KCl, 5 mM KOH, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM Hepes, pH 7.3) to a final concentration of 50 mM KCl (18). A control solution contained NaCl instead of KCl. The cells were fixed in 3.7% formaldehyde and permeabilized with
0.2% Triton X-100. After blocking with 10% fetal calf serum, the
cells were incubated with anti-calbindin-D antibody (Sigma) and
anti-active MAPK antibody (Promega) at dilutions of 1:50 at 37 °C
for 1 h. The cells were washed, and fluorescein
isothiocyanate-conjugated goat anti-mouse IgG antibody (Organon
Teknika) and Cy3-conjugated goat anti-rabbit IgG antibody (Amersham
Pharmacia Biotech) were applied at dilutions of 1:200 and 1:1000,
respectively, for 1 h. The cells were washed and mounted on
slides. For semiquantitation, images of Purkinje cells stained with
anti-active MAPK antibody were collected using a ZEISS Axiophot
microscope and a CCD camera. In each Purkinje cell, the staining
intensity at three different points in the cell body was measured using
NIH Image software. The staining intensity of the cell body stained
with secondary antibody alone was measured as background intensity and
subtracted from each value. In each point, the staining intensity of 20 Purkinje cells was measured. The mean ±S.D. of the subtracted values
from two independent experiments is shown in the text.
P Antibody--
We raised rabbit polyclonal anti-active MAPK
antibody (P antibody) against a synthetic phosphopeptide DHTGFLT
(PO4)EY(PO4)VA, which corresponds to residues
182-192 of a dually phosphorylated form of Xenopus
MAPK.2 The P antibody was
affinity-purified before use.
Electrophysiology--
Electrophysiological experiments were
performed as described previously (17, 19) with slight modifications.
To record glutamate-induced current, a morphologically identified
Purkinje cell was whole cell voltage-clamped at
Voltage-gated Ca2+ currents, AMPA-induced currents and
DHPG-induced currents were measured in cultured Purkinje cells under a
whole cell voltage-clamp condition. To record voltage-gated Ca2+ currents, 1 mM external Ca2+
was replaced with Mg2+, and 1 mM
4-aminopyridine (Sigma), 10 mM tetraethylammonium (Sigma), and 10 µM CNQX (Tocris) were added to the external
solution in addition to tetrodotoxin and bicuculline. 4-Aminopyridine
and tetraethylammonium suppress currents through K+
channels, and CNQX suppresses current through AMPA receptor channels. Ca2+ currents were recorded by depolarizing Purkinje cells
from Preparation of Cell Extracts and Mono Q
Chromatography--
Cultures of rat fibroblastic 3Y1 cells and
preparation of their extracts were performed as described (20). After
3Y1 cells were exposed to hyperosmotic shock with 0.7 M
NaCl for 1 h, extracts were prepared and subjected to Mono Q
chromatography. After proteins were eluted with a linear gradient of
0-0.5 M NaCl, fractions were subjected to immunoblotting
and kinase assays.
Kinase Assays--
Samples were incubated with 5 µg of myelin
basic protein, c-Jun, or ATF2 in a solution (15 µl) containing 20 mM Tris, pH 7.5, 10 mM MgCl2, and
50 µM [ Immunoblotting--
The fractions from Mono Q chromatography
were subjected to SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride membranes. After blocking with
5% skim milk, membranes were incubated with anti-MAPK antibody (21),
anti-SAPK/JNK antibody (22), or anti-p38 antibody (Santa Cruz) in 20 mM Tris, pH 7.5, 500 mM NaCl, and subsequently
with horseradish peroxidase-conjugated anti-rabbit IgG antibody or
horseradish peroxidase-conjugated anti-mouse IgG antibody.
Immunoreactive bands were detected by ECL Western blotting detection
system (Amersham Pharmacia Biotech).
Because it has been reported that the expression of cerebellar LTD
is mediated postsynaptically (23, 24), we first examined whether MAPK
in cultured Purkinje cells is activated in response to glutamate
treatment plus membrane depolarization. Primary cultures of rat
embryonic cerebella were treated with simultaneous stimulation with 10 µM glutamate and 50 mM KCl for 4 min, the
latter being known to induce membrane depolarization in neuronal cells
(18). In another series of experiments, bath application of glutamate plus KCl induced sustained reduction in the amplitudes of miniature excitatory postsynaptic currents recorded from cultured Purkinje cells,
suggesting that bath application of glutamate plus KCl can induce
cerebellar LTD.3 Then we
examined activation of MAPK using indirect immunofluorescent staining
with anti-active MAPK antibody. Purkinje cells were identified by
staining with anti-calbindin-D antibody. Although Purkinje cells were
faintly stained with anti-active MAPK antibody before stimulation, they
became more intensely stained after the simultaneous stimulation (Fig.
1A). The anti-active MAPK
immunoreactivity was often observed in dendrites of the stimulated
Purkinje cells but not in those of unstimulated cells (Fig.
1A). Semiquantitation showed that the staining intensity of
the cell body in the stimulated Purkinje cells was about 1.6-fold
(1.65 ± 0.31) that of the unstimulated Purkinje cells. In another
series of experiments in which rat fibroblastic 3Y1 cells were
stimulated with 10% fetal calf serum that induced full activation of
MAPK as revealed by the mobility shift of MAPK bands in immunoblotting
(data not shown), the staining intensity with anti-active MAPK antibody
in the cytoplasm increased about 4.6-fold (4.58 ± 1.01) upon the
stimulation. Thus, the MAPK activation in the stimulated Purkinje cells
is not full but significant. When the cerebellar cultures were
pretreated with PD98059, a specific inhibitor of MAPKK (25), the
increase in the anti-active MAPK immunoreactivity in Purkinje cells in
response to 10 µM glutamate plus 50 mM KCl
was almost completely inhibited (Fig. 1B). Semiquantitation showed that the staining intensity of the PD-treated cells was 1.04 ± 0.10-fold that of the untreated cells, and that of the PD-
and glutamate plus KCl-treated cells was 0.98 ± 0.21. These results suggest that MAPK is activated in response to the LTD-producing stimulation in Purkinje cells.
To examine the involvement of the MAPK cascade in cerebellar LTD, we
carried out electrophysiological studies. Morphologically identified
Purkinje cells were whole cell voltage-clamped, and currents in
response to iontophoretic application of glutamate were recorded. Then
LTD was induced by the simultaneous stimulation with iontophoretic
glutamate application and depolarization of Purkinje cells. In control
experiments, we applied Me2SO, a vehicle for solubilizing
PD98059, to the external solution (data not shown) or to the internal
solution of the patch electrode (Fig. 2,
open squares). In both cases, stable LTD was induced in
Purkinje cells. When Me2SO was applied to the internal
solution, the reduction of the glutamate-induced current was 77% ± 2.9% (mean ±S.E., t = 25-30 min, 6 cells) of
base-line responses (Fig. 2, open squares).
Department of Biophysics,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
80 mV with a
fire-polished recording pipette of around 5 megaohms containing 150 mM CsCl, 0.5 mM EGTA, 10 mM Hepes,
titrated to pH 7.3 with CsOH. An iontophoretic glutamate pipette
containing 10 mM glutamate was placed at about 20 µm away
from a primary dendrite, and 10-ms negative current pulses were applied
to the pipette every 20 s. After stable recording of
glutamate-induced current for 10 min, the conditioning stimulation was
applied to the Purkinje cell. The conditioning stimulation consisted of
9 glutamate applications coupled with 9 depolarizations for 3 s to
0 mV at 0.05 Hz, and the depolarization onset was timed to precede the
glutamate pulse by 1 s. Input resistance (100-300 megaohms) and
series resistance (20-35 megaohms) were monitored by applying 80-ms
voltage pulses to
90 mV once every 5 min. The composition of the
external solution was 145 mM NaCl, 5 mM KOH, 2 mM CaCl2, 1 mM MgCl2,
10 mM glucose, 10 mM Hepes, pH 7.3, 1 µM tetrodotoxin, and 20 µM bicuculline.
Tetrodotoxin and bicuculline were used to suppress action potentials
and inhibitory postsynaptic currents, respectively. In some
experiments, P antibody or control rabbit IgG was added to the internal
electrode solution at a final concentration of 1 mg/ml. A MAPKK/MEK
inhibitor PD98059 (New England Biolabs) was added to the internal
electrode solution at 100 µM or added to the external
solution at 30 µM at least 30 min before the induction of
LTD.
80 mV to 10 mV for 80 ms. Leakage and capacitative transient
currents were canceled by adding a current trace induced by the voltage pulse to
170 mV. To record AMPA-induced currents, iontophoretic pipettes containing 1 mM AMPA were placed at about 20 µm
away from the primary dendrites of Purkinje cells, and negative current pulses were applied. When DHPG-induced currents were recorded, 10 µM CNQX was added to the external solution in addition to
tetrodotoxin and bicuculline. Iontophoretic pipettes containing 2 mM DHPG (Tocris) were placed 1-2 µm away from the
primary dendrites of Purkinje cells, and positive current pulses were
applied. CPCCOEt (Tocris) was applied to the external solution at a
concentration of 100 µM. In some experiments, 30 µM PD98059 or its vehicle (0.1% Me2SO) was
applied to the external solution 30 min before electrophysiological recordings.
-32P]ATP (1 µCi) for 30 min at
30 °C. The reaction was stopped by the addition of Laemmli's sample
buffer. Phosphorylated myelin basic protein, c-Jun, or ATF2 was
resolved by SDS-polyacrylamide gel electrophoresis. In some
experiments, P antibody or control rabbit IgG was added to samples and
incubated at 0 °C for 30 min before kinase assays.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Activation of MAPK in response to glutamate
stimulation and depolarization in cultured Purkinje cells.
A, primary cultures of rat embryonic cerebellum were
simultaneously stimulated by both glutamate (10 µM)
treatment and depolarization treatment for 4 min in the presence of
0.1% Me2SO. Depolarization was induced by the addition of
an iso-osmotic solution containing 150 mM K+ to
a final concentration of 50 mM K+, and control
stimulations substituted Na+ for K+. The cells
were fixed in 3.7% formaldehyde and doubly stained with anti-active
MAPK antibody and anti-calbindin-D antibody. B, the cultures
were preincubated with 30 µM PD98059 for 30 min and
treated with the simultaneous stimulation for 4 min as above. The cells
were stained with anti-active MAPK antibody and anti-calbindin-D
antibody.
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Fig. 2.
Inhibition of cerebellar LTD induction by
PD98059 in cultured Purkinje cells. Morphologically identified
Purkinje cells were whole cell voltage-clamped, and glutamate-induced
currents were recorded. LTD was induced by the simultaneous stimulation
with iontophoretic application of glutamate and depolarization of
Purkinje cells for 3 min (from 0 min to 3 min). PD98059 was applied to
the external solution (closed circles, 30 µM)
or to the internal solution of the patch electrode (closed
squares, 100 µM) 30 min before the induction of LTD.
Control experiments using Me2SO (DMSO), which
was applied to the internal solution, are indicated by open
squares. When Me2SO was applied to the external
solution, stable LTD was also observed (data not shown). Graph
points are the mean ±S.E. of six separate experiments.
Representative current traces, which were obtained from Purkinje cells
treated with or without PD98059, are shown.
In contrast, bath application of 30 µM PD98059 completely blocked the induction of LTD; at t = 25-30 min, the current was 106% ± 8.6% (6 cells) that of before the induction of LTD (Fig. 2, closed circles). The PD98059 treatment tended to produce a small increase in the average currents rather than decrease (Fig. 2, closed circles), the phenomenon being similar to that caused by blockade of mGluR1 (17, 26).
The cultures contain other cells than Purkinje cells such as granule neurons and glial cells, and in cultured cerebellar granule cells, bath applications of glutamate plus KCl also induced activation of MAPK, which was revealed by the mobility shift of MAPK bands (data not shown). Thus, bath application of PD98059 could inhibit activation of the MAPK cascade in non-Purkinje cells, which might affect the induction of LTD. To treat only Purkinje cells with this drug, we applied PD98059 to the internal solution of the whole cell patch electrode. In this case also, the induction of LTD was completely blocked; at t = 25-30 min, the current was 107% ± 8.6% (6 cells) that of base-line responses (Fig. 2, closed squares). These results therefore suggest that activation of the MAPKK/MAPK cascade in Purkinje cells is required for the induction of cerebellar LTD.
To directly examine the requirement of the activity of MAPK in the
induction of cerebellar LTD, we used a neutralizing antibody to MAPK.
We produced anti-active MAPK antibody by immunizing rabbits with a
dual-phosphorylated peptide encompassing the activation phosphorylation
site of MAPK as an antigen.2 The obtained antibody, which
we called P antibody, specifically recognized active forms of
p44MAPK/ERK1 and p42MAPK/ERK2 in extracts
obtained from serum-stimulated fibroblastic cells in immunoblotting
experiments (data not shown). P antibody was affinity-purified and
tested for its ability to inhibit kinase activities of three members of
the MAPK superfamily, MAPK/ERK, JNK/SAPK, and p38. To obtain active
forms of these kinases, rat fibroblastic 3Y1 cells were treated with
hyperosmotic shock, which is known to potently activate these MAPK
superfamily members (20, 27), and each member was partially purified by
Mono Q chromatography (Fig. 3,
A and B). In vitro kinase assays were
carried out in the presence of increasing concentrations of P antibody
or control rabbit IgG. P antibody, but not control IgG, inhibited the
kinase activities of both p44MAPK/ERK1 and
p42MAPK/ERK2 in a concentration-dependent
manner (Fig. 3C). P antibody did not inhibit the kinase
activity of JNK/SAPK or p38 (Fig. 3C). Therefore, P antibody
is a specific neutralizing antibody to MAPKs/ERKs.
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Then, to inhibit MAPK activity in Purkinje cells, we applied P antibody
to the internal solution of the whole cell patch electrode at a
concentration of 1 mg/ml. When fluorescein-conjugated rabbit IgG was
applied to the internal solution of the patch electrode, the
fluorescence became observed in both dendrites and cell bodies within a
few min after Purkinje cells were voltage-clamped (data not shown).
This indicates that IgG is rapidly introduced from the patch electrode
to Purkinje cells. In the presence of control rabbit IgG, LTD was
effectively induced by the simultaneous stimulation with glutamate and
depolarization of Purkinje cells (Fig. 4,
open circles). The average current at 20-25 min after the
depolarization was 67% ± 9.3% (5 cells) that of before the induction
of LTD. In contrast, when P antibody was applied, the induction of LTD was completely blocked (Fig. 4, closed circles). The average
value of the current at 20-25 min after the depolarization was 98% ± 0.5% (4 cells) that of base-line responses. Thus, the kinase activity of MAPK in Purkinje cells is required for the induction of cerebellar LTD.
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Because the induction of cerebellar LTD requires Ca2+
influx through voltage-gated Ca2+ channels and activation
of AMPA receptors and mGluR1, we examined the effect of inhibition of
the MAPK cascade on these processes. The inward current through
voltage-gated Ca2+ channels was recorded by depolarizing
Purkinje cells from 80 mV to 10 mV for 80 ms. Voltage-gated
Ca2+ currents were not significantly affected by a MAPKK
inhibitor PD98059, which was applied to the external solution at 30 µM (Fig. 5A).
The average amplitude of the currents without or with PD98059 was
1.2 ± 0.4 nA (mean ± S.D., n = 15) or
1.4 ± 0.3 nA (n = 15), respectively. To measure
AMPA-induced currents, AMPA was iontophoretically applied to the
dendrites of cultured Purkinje cells under a whole cell voltage-clamp
condition. AMPA-induced currents were not significantly affected by 30 µM PD98059 (Fig. 5B). The average amplitude of AMPA-induced currents without or with PD98059 treatment was 204 ± 179 pA (n = 16) or 187 ± 162 pA
(n = 16), respectively. Because an inward current is
induced by mGluR1 stimulation (28), we treated whole cell
voltage-clamped Purkinje cells with DHPG, an agonist of group I mGluR
(mGluR1 and mGluR5, only mGluR1 is expressed in Purkinje cells).
Iontophoretic DHPG application induced inward currents, which were
completely blocked by bath application of 100 µM CPCCOEt,
an antagonist of group I mGluR (Fig. 5D). Interestingly, when Purkinje cells were preincubated with PD98059, DHPG-induced currents were significantly attenuated (Fig. 5C). The
average amplitude of DHPG-induced currents without or with PD98059 was 81.8 ± 78.0 pA (n = 22) or 18.1 ± 16.5 pA
(n = 22), respectively. These results suggest that
inhibition of the MAPK cascade might result in reduction of mGluR1
activity.
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Our results in this study indicate that activation of the MAPK cascade in Purkinje cells is essential for the induction of cerebellar LTD. This clearly reveals a role of the MAPK cascade in post-mitotic and differentiated mammalian neurons. Because the previous pharmacological study has shown that PD98059 inhibits long term potentiation in rat hippocampal slices (16), the MAPK cascade may play an essential role in both long term potentiation and LTD, two widely studied forms of synaptic plasticity in mammalian brain. Previously, involvement of presynaptic MAPK in long term facilitation in Aplysia was reported (14, 15). In contrast, our results here reveal the involvement of postsynaptic MAPK in synaptic plasticity.
Previously, an increase in intracellular Ca2+ was reported to induce activation of the MAPK cascade, which might involve tyrosine kinase PYK2, Src kinase, Ras, and Ras-guanine nucleotide-releasing factor (29). Protein kinase C activation by phorbol ester also results in activation of the MAPK cascade. These molecules might be involved in activation of the MAPK cascade in Purkinje cells.
Molecular mechanisms downstream of postsynaptic MAPK in cerebellar LTD
are not known at present. Because mGluR1 activity was found to be
attenuated by the inhibition of MAPKK in Purkinje cells, it is possible
that the MAPK cascade affects the activity of mGluR1 to induce
cerebellar LTD. Another possibility is that phosphorylation of the AMPA
type of glutamate receptors in Purkinje cells is involved in the
induction of cerebellar LTD. It was previously reported that the
function of AMPA receptors can be regulated by phosphorylation (30).
Moreover, the initial phase of cerebellar LTD does not require protein
synthesis in Purkinje cells (31). Interestingly, subunits of AMPA
receptors have Ser-Pro sequences in their cytoplasmic regions, which
are conserved among chicken, rat, and human. Thus, MAPK and/or its
downstream kinases might phosphorylate AMPA receptors to regulate their
function. Identification of downstream targets of MAPK in Purkinje
cells is an important step in understanding the molecular mechanisms of
synaptic plasticity.
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ACKNOWLEDGEMENT |
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We thank T. Harada for technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan (to E. N. and T. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku,
Kyoto 606-8502, Japan. Tel.: 81-75-753-4230; Fax:
81-75-753-4235; E-mail: L50174{at}sakura.kudpc.kyoto-u.ac.jp.
2 F. Itoh, M. Fukuda, and E. Nishida, manuscript in preparation.
3 M. Murashima and T. Hirano, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are:
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated
kinase;
MAPKK, MAPK kinase;
MEK, MAPK/ERK kinase;
SAPK, stress-activated protein kinase;
JNK, c-Jun N-terminal kinase;
LTD, long term depression;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropimate;
DHPG, dihydroxyphenylglycine;
CPCCOEt, 7-(hydroxyimino)cyclopropa[b]
chromen-1a-carboxylate ethyl ester;
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione disodium;
mGluR, metabotropic
glutamate receptor.
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