Requirement for Mitogen-activated Protein Kinase in Cerebellar Long Term Depression*

Hiroshi KawasakiDagger §, Hiroaki FujiiDagger , Yukiko Gotoh, Takaya MorookaDagger , Shun Shimohama§, Eisuke NishidaDagger parallel , and Tomoo HiranoDagger **

From the Dagger  Department of Biophysics, Graduate School of Science, § Department of Neurology, Graduate School of Medicine, and  Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606 and ** CREST, Japan Science and Technology Corp., Sakyo-ku, Kyoto 606, Japan

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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.

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INTRODUCTION
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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.

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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 -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.

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 -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.

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 [gamma -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.

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).

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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.


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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.

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).


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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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Fig. 3.   Inhibition of the activity of mammalian MAPKs by P antibody. A, cell extracts, which were obtained from rat fibroblastic 3Y1 cells treated with osmotic shock (0.7 M NaCl for 60 min), were subjected to Mono Q chromatography, and fractions were subjected to immunoblotting with anti-MAPK antibody (21), anti-SAPK/JNK antibody (22), and anti-p38 antibody (32). Closed arrowheads indicate the positions of inactive forms of p44MAPK/ERK1 and p42MAPK/ERK2. Electrophoretically retarded bands, which are represented by open arrowheads, indicate active forms of MAPKs. Fractions 28 and 30 were used as active p42MAPK/ERK2 and active p44MAPK/ERK1 in Fig. 3, B and C, respectively. Upper and lower arrows indicate SAPKalpha /JNK2 and SAPKgamma /JNK1, respectively. Fractions 23 and 39 were used as active SAPK/JNK and active p38 in Fig. 3, B and C, respectively. Fractions from unstimulated 3Y1 cells were examined with immunoblotting, and inactive MAPK/ERKs, inactive SAPK/JNK, and inactive p38 fractions were obtained (data not shown). B, the active and inactive MAPK/ERK fractions were assayed for myelin basic protein-phosphorylating activity (20). The SAPK/JNK fractions and p38 fractions were assayed for c-Jun- and ATF2-phosphorylating activity, respectively (27, 33). C, active MAPK/ERKs, active SAPK/JNK, and active p38 fractions were subjected to kinase assays using myelin basic protein, c-Jun, and ATF2 as substrates, respectively, in the presence of increasing concentrations of P antibody or control rabbit IgG.

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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Fig. 4.   Inhibition of cerebellar LTD induction by P antibody. P antibody (closed circles) or control rabbit IgG (open circles) was applied to the internal solution of the whole cell patch electrode at a final concentration of 1 mg/ml. After Purkinje cells were voltage-clamped, LTD was induced by glutamate depolarization conjunctive stimulation (from 0 to 3 min). Representative results are shown. Similar results were obtained from 5 and 4 independent experiments using control IgG and P antibody, respectively. Graph points are the mean ±S.D. of about 12 currents recording. Representative current traces, which were obtained before and 30 min after the stimulation, are shown.

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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Fig. 5.   Effect of PD98059 on voltage-gated Ca2+ current, AMPA-induced current, and DHPG-induced current. Morphologically identified Purkinje cells were whole cell voltage-clamped in the presence of 0.1% Me2SO (control) or 30 µM PD98059. A, representative voltage-gated Ca2+ currents induced by depolarizing Purkinje cells from -80 mV to 10 mV for 80 ms in the presence of 1 mM 4-aminopyridine, 10 mM tetraethylammonium, and 10 µM CNQX in the external solution. B, representative AMPA-induced currents recorded by iontophoretic application of AMPA. C, representative DHPG-induced currents recorded by iontophoretic application of DHPG in the presence of 10 µM CNQX in the external solution. D, effect of 100 µM CPCCOEt on DHPG-induced current. CPCCOEt suppressed DHPG-induced current.

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.

    ACKNOWLEDGEMENT

We thank T. Harada for technical assistance.

    FOOTNOTES

* 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.

parallel 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.

    ABBREVIATIONS

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, alpha -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.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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