©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Increased Phosphorylation of Ca/Calmodulin-dependent Protein Kinase II and Its Endogenous Substrates in the Induction of Long Term Potentiation (*)

(Received for publication, June 23, 1994; and in revised form, November 14, 1994)

Kohji Fukunaga (1)(§) Dominique Muller (2) Eishichi Miyamoto (1)

From the  (1)Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto 860, Japan and the (2)Department of Pharmacology, Centre Médical Universitaire, 1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Induction of long term potentiation in the CA1 region of hippocampal slices is associated with increased activity of Ca/calmodulin-dependent protein kinase II (CaM kinase II) (Fukunaga, K., Stoppini, L., Miyamoto, E., and Muller, D.(1993) J. Biol. Chem. 268, 7863-7867). Here we report that application of high but not low frequency stimulation to two groups of afferents in the CA1 region of P-labeled slices resulted in the phosphorylation of two major substrates of this enzyme, synapsin I and microtubule-associated protein 2, as well as in the autophosphorylation of CaM kinase II. Furthermore, immunoblotting analysis revealed that long term potentiation induction was associated with an increase in the amount of CaM kinase II in the same region. All these changes were prevented when high frequency stimulation was applied in the presence of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoate. These results indicate that activation of CaM kinase II is involved in the induction of synaptic potentiation in both the postsynaptic and presynaptic regions.


INTRODUCTION

Activities of several protein kinases, CaM kinase II (^1)(Fukunaga et al., 1993), protein kinase C (Lovinger et al., 1987; Klann et al., 1991), and casein kinase II (Charriaut et al., 1991), have been reported to increase during induction of LTP in the hippocampus. Among these enzymes, CaM kinase II and protein kinase C are of particular interest for contributing to LTP, because these two enzymes are present in high concentrations in the hippocampus (Ouimet et al., 1984; Erondu and Kennedy, 1985; Kitano et al., 1987; Fukunaga et al., 1988) and distributed in both presynaptic terminals and postsynaptic elements. Biochemical experiments have provided evidence for increased activity of protein kinase C for up to 60 min after induction of LTP (Klann et al., 1991, 1992). To account for these observations, it has been proposed that protein kinase C might be converted into a permanently active, Ca-independent kinase through cleavage by Ca-dependent proteases (Sacktor et al., 1993) and thus contribute to both the induction and the maintenance of LTP (Malinow et al., 1989).

CaM kinase II can also be converted into a Ca-independent enzyme by autophosphorylation (Miller et al., 1988; Schworer et al., 1988; Thiel et al., 1988). The kinase could thus function as a kind of molecular switch responsible for keeping biochemical modifications in the phosphorylation of various presynaptic and postsynaptic substrates. An interesting feature of this regulation is that the Ca-independent activity can be reversed by protein phosphatases.

In recent experiments, we have provided evidence that a transient increase in the Ca-independent activity of CaM kinase II could be observed in cultured cerebellar granule cells (Fukunaga and Soderling, 1990) and hippocampal neurons (Fukunaga et al., 1992) following stimulation of NMDA receptors. More recently, in experiments in which we have assessed the enzyme activity following LTP induction, we have observed an increase in the Ca-independent activity of CaM kinase II up to 1 h after tetanic stimulation (Fukunaga et al., 1993). These results therefore support the interpretation that modulation of the degree of enzyme autophosphorylation may contribute to synaptic plasticity. Physiological studies also support the idea that CaM kinase II may play a key role in the mechanisms of LTP, because specific inhibitory peptides for the enzyme have been shown to abolish LTP (Malenka et al., 1989; Malinow et al., 1989) and because slices prepared from mutant mice deficient in the alpha subunit of CaM kinase II exhibited impaired synaptic potentiation (Silva et al., 1993).

To confirm the possibility that activity-dependent modulation of the level of autophosphorylation of CaM kinase II plays an important role in synaptic plasticity, we have measured in the present study the in situ phosphorylation of the enzyme following induction of LTP in P-labeled slices. In addition, we have also analyzed the in situ phosphorylation of two major substrates for CaM kinase II, synapsin I and MAP2. The results add support to the concept that phosphorylation mechanisms contribute to LTP and provide evidence for another modulation involving the beta subunit of the kinase.


EXPERIMENTAL PROCEDURES

Materials

The following chemicals and reagent were obtained from the indicated source: [-P]ATP and I-protein A, DuPont NEN; [P]orthophosphate, ICN Biomedical Inc.; D-AP5, Cambridge Research Biochemicals; calmidazolium and anti-microtubule-associated proteins (MAPs) antibody (M7273), Sigma. The polyclonal antibodies against brain CaM kinase II (Fukunaga et al., 1988) and synapsin I (Fukunaga et al., 1992) were prepared as described previously.

Preparation of Hippocampal Slices and Electrophysiological Recordings

Following decapitation, brains of male Sprague-Dawley rats (5-8 weeks old) were removed, the hippocampi were dissected, and hippocampal slices (400 µm thick) were prepared using a MacIlwain chopper. The slices were maintained under continuous perfusion in an interface type of a chamber with a medium that contained (in mM): NaCl, 124; KCl, 3; CaCl(2), 2.5; MgCl(2), 1.5; NaH(2)PO(4), 1.25; NaHCO(3), 26; glucose, 10; ascorbic acid, 2; pH 7.4 and was exposed to a 95% O(2), 5% CO(2) atmosphere at 34 °C.

After a 1-2-h recovery period, synaptic activity was monitored using two stimulation electrodes made of twisted Nichrome wire and placed on both sides of a glass recording pipette in the CA1 region. In all slices, LTP was induced by burst-patterned stimulation (10 bursts at 5 Hz, each composed of 4 pulses at 100 Hz) repeated 3 times consecutively at 10-20-s intervals and applied simultaneously to the two inputs. The stimulation polarity on the two electrodes was then reversed, and the same procedure was applied a second time. Control slices were stimulated by low frequency trains consisting of 100 pulses at 10 Hz to the two inputs. These trains did not induce synaptic potentiation. One hour after stimulation, slices were transferred on a glass slide at ice temperature, and the CA1 areas were dissected out and frozen in liquid nitrogen.

For immunoprecipitation experiments and analysis of the in situ phosphorylation of CaM kinase II and its substrates, slices were incubated in a phosphate-free medium containing carrier-free [P]orthophosphate (0.5-0.7 mCi/ml) after the recovery period. After a 30-min incubation, slices were stimulated by burst or low frequency stimulation protocols and maintained for 1 h in the presence of [P]orthophosphate. They were frozen as non-labeled slices.

Immunoprecipitation and Quantitation of P-labeled CaM Kinase II, P-labeled MAP2, and P-labeled Synapsin I

Immunoprecipitation with P-labeled slices was carried out as described previously (Fukunaga et al., 1992). Briefly, the two slices of the CA1 areas were combined and homogenized with a hand homogenizer in 0.8 ml of a solubilization solution containing 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 0.5% Triton X-100, 10 mM EDTA, 4 mM EGTA, 1 mM Na(3)VO(4), 30 mM sodium pyrophosphate, 50 mM NaF, 0.1% SDS, 0.1 mM leupeptin, 75 µM pepstatin A, and 0.1 mg/ml aprotinin. After sonication with a Branson sonifier 250, the insoluble materials were removed by centrifugation at 15,000 times g for 20 min. Aliquots (20 µl) of the supernatant were used to determine protein content (Bradford, 1976) and P incorporation into total proteins by trichloroacetic acid precipitation. To determine the P incorporation, Whatman 3MM filter papers, on which the aliquots (20 µl) were spotted, were washed with trichloroacetic acid, acetone, and ethanol. After drying, the radioactivity was measured by a liquid scintillation counter. The radioactivity of the P incorporation into the total proteins was not different between the control and the potentiated slices (data not shown). The supernatant fraction containing the same amount of protein was incubated at 4 °C for 4 h with the antibodies against CaM kinase II and synapsin I (5 µg of each IgG protein) and 50 µl of protein A-Sepharose CL-4B suspension (50% v/v). The immunocomplex immobilized on protein A was washed three times with the solubilization solution. After immunoprecipitation of P-labeled CaM kinase II and P-labeled synapsin I, the cell supernatant fractions were further subjected to immunoprecipitation of P-labeled MAP2 using the anti-MAPs antibody. All these immunoprecipitates were eluted from protein A-Sepharose CL-4B by treatment with the SDS sample buffer (Laemmli, 1970) and boiled for 4 min. Supernatants were subjected to SDS-PAGE (Laemmli, 1970) followed by autoradiography. A Bio-Image analyzer (BA100, Fuji Film) was used to quantify the amount of P incorporation into CaM kinase II subunits, synapsin I and MAP2. The phosphorylation site of synapsin I was determined by limited proteolysis of P-labeled synapsin I in gel pieces with Staphylococcus aureus protease V8 (5 µg/well) and one-dimensional SDS-PAGE as described (Huttner and Greengard, 1979). The radioactivity in both site I and site II in synapsin I was measured by a Bio-Image analyzer.

Immunoblotting and Quantitation of CaM Kinase II Subunits, MAP2 and Synapsin I

In experiments with non-labeled slices, the CA1 areas of the hippocampal slices were frozen 1 h after the stimulation. The slices were homogenized in 0.2 ml of the solubilization solution. Sonication and centrifugation were performed as described above. Immunoblotting of the supernatants (20 or 25 µg of protein) was performed with anti-CaM kinase II (5 µg of IgG/ml), anti-synapsin I (2 µg of IgG/ml), or anti-MAPs antibodies (200-fold dilution), which were detected using I-protein A as described previously (Yano et al., 1994). A Bio-Image analyzer was used to quantify the activity of I-protein A bound to the immunocomplex.

Other Methods

Protein concentration was determined by the method of Bradford(1976) using bovine serum albumin as a standard.


RESULTS

In Situ Phosphorylation of CaM Kinase II Subunits and Synapsin I

In recent work (Fukunaga et al., 1993), we have provided evidence that LTP induction in the CA1 area of the hippocampus was associated with an increase in the Ca-independent activity of CaM kinase II. To further assess that this increased Ca-independent activity was due to autophosphorylation of the enzyme, we measured here the in situ incorporation of [P]phosphate into the subunits of the enzyme. To achieve this, hippocampal slices were incubated and stimulated in the presence of [P]phosphate to label the ATP pool. A group of the slices then received burst patterned stimulation to induce LTP, whereas others received low frequency trains that did not produce any long lasting modification of synaptic efficacy. As an additional control, the same experiments were also carried out in the presence of 50 µMD-AP5, an NMDA receptor antagonist that prevents induction of synaptic potentiation.

In a first set of experiments, P-labeled CaM kinase II and one of its major presynaptic substrates, synapsin I, were immunoprecipitated using the extracts of control and potentiated CA1 areas and the antibodies against brain CaM kinase II and synapsin I. The samples were then subjected to SDS-PAGE followed by autoradiography (Fig. 1). The total amount of radioactivity incorporated into both the alpha and beta subunits of CaM kinase II was determined using a Bio-Image analyzer. As shown in Fig. 1, high frequency stimulation resulted in a significant increase in the incorporation of P into the alpha and beta subunits of the kinase, thereby indicating autophosphorylation of the enzyme. When compared with control conditions, the levels of autophosphorylation of the alpha and beta subunits were found to increase by 29 ± 5 and 35 ± 5%, respectively.


Figure 1: Increased phosphorylation of CaM kinase II and synapsin I following induction of LTP. Hippocampal slices were prelabeled with P (0.5-0.7 mCi/ml) for 30 min. Then slices received either burst-patterned stimulation (10 bursts at 5 Hz, each composed of 4 pulses at 100 Hz) to induce LTP (HFS) or low frequency stimulation (100 pulses at 10 Hz) that did not produce long lasting changes in synaptic efficacy (Control). 60 min after stimulation, the CA1 areas were dissected out and frozen in liquid nitrogen. When indicated, D-AP5 (50 µM) was added during the whole experiment. A, CaM kinase II (indicated by alpha and beta subunits) and synapsin I were immunoprecipitated with specific antibodies and analyzed by SDS-PAGE followed by autoradiography. B, the incorporation of P into the alpha and beta subunits of CaM kinase II was determined in potentiated (hatchedbars) and control (blackbars) slices using a Bio-Image analyzer. Results were obtained from 21 potentiated and 21 control slices in the absence of D-AP5 and 6 slices in each condition in the presence of D-AP5. The treatment of slices with 50 µM D-AP5 prevented LTP induction and abolished the increase in the autophosphorylation of CaM kinase II. Values are means ± S.E. and are expressed as percentage of control. *, statistically significant difference between potentiated and control slices (p < 0.01).



In the same experiments we also investigated the possibility that LTP induction resulted in modifications in the phosphorylation of synapsin I, a major presynaptic substrate of CaM kinase II. Following immunoprecipitation, SDS-PAGE, and autoradiography, synapsin I appeared as a doublet of bands of Ia and Ib with 80 and 78 kDa, respectively. As illustrated in Fig. 1A and quantified in Fig. 2B, the synapsin I phosphorylation increased by 37 ± 7% following high frequency stimulation. The change was thus comparable with that of the CaM kinase II autophosphorylation.


Figure 2: Increased phosphorylation of MAP2 and synapsin I following induction of LTP. A, P-labeled hippocampal slices were stimulated in the presence or absence of D-AP5 (50 µM) as in Fig. 1A. After dissection of the CA1 areas of control and potentiated (HFS) slices, the samples were subjected to immunoprecipitation using the anti-MAPs polyclonal antibody and analyzed by SDS-PAGE followed by autoradiography. B, incorporation of P into synapsin I (Fig. 1) and MAP2 was analyzed using a Bio-Image analyzer. For synapsin I, results were obtained from 23 control and 23 potentiated slices without D-AP5 and 6 slices in each condition with D-AP5. For MAP2, results were obtained from 13 control and 13 potentiated slices without D-AP5 and 6 slices in each condition with D-AP5. Values represent means ± S.E. and are expressed as percentage of control. *, statistically significant difference between potentiated and control slices (p < 0.01).



We then tested whether these changes in phosphorylation were dependent upon NMDA receptor activation as in LTP. Slices were thus incubated in the presence of 50 µMD-AP5, an NMDA receptor antagonist, during the application of the stimulation protocols. This treatment completely prevented the induction of synaptic potentiation (data not shown) and abolished the increases in phosphorylation of both CaM kinase II subunits and synapsin I ( Fig. 1and Fig. 2).

CaM kinase II is a major component of postsynaptic densities, but it is also present in presynaptic terminals. Because synapsin I is exclusively a presynaptic protein, the implication of these results was that LTP induction resulted in a possible activation and autophosphorylation of CaM kinase II in presynaptic terminals. To further examine this possibility, we used phosphopeptide mapping of P-labeled synapsin I to determine the site of phosphorylation of the protein. Peptide mapping was done by limited digestion of synapsin I with V8 protease followed by one-dimensional SDS-PAGE. In intact neurons, the major site of phosphorylation of synapsin I was site II (more than 60%), a site that is phosphorylated by CaM kinase II (Czernik et al., 1987). Following high frequency stimulation, the phosphorylation of site II increased by 26 ± 10% (p < 0.05, n = 8) (Fig. 3). In addition, after LTP induction, there was a small but significant increase in the phosphorylation of site I, a site phosphorylated by cAMP-dependent protein kinase and/or CaM kinase I (12 ± 5% increase; p < 0.05, n = 8).


Figure 3: Phosphorylation sites of synapsin I following induction of LTP. Following immunoprecipitation and SDS-PAGE as in Fig. 1A, the 78-kDa synapsin I phosphorylated in the CA1 areas of control (control, lanes3 and 4) and potentiated (HFS, lanes5 and 6) slices were cut out from the gel, and the phosphorylation sites of synapsin Ib were determined as described (Huttner and Greengard, 1979) after limited proteolysis with S. aureus V8 protease (3 µg/well). The P-labeled peptides were separated by SDS-PAGE in 15% polyacrylamide, followed by autoradiography. As a control, purified brain synapsin I phosphorylated with the purified catalytic subunit of cAMP-dependent protein kinase (lane1) or CaM kinase II (lane2) was subjected to treatment with the protease and analyzed on the same gel. B, incorporation of P into sites I and II of synapsin I was analyzed using a Bio-Image analyzer. Results were obtained from 8 control and 8 potentiated slices. Values are means ± S.E. and are expressed as percentage of site II of control. *, statistically significant difference between potentiated and control slices (p < 0.05).



In Situ Phosphorylation of MAP2

We investigated the effect of high frequency stimulation on the phosphorylation of MAP2, a protein essentially present in postsynaptic spines and dendrites. Following immunoprecipitation of MAP2 with the specific polyclonal antibody, we observed a 27 ± 5% (p < 0.05, n = 13) increase in the phosphorylation of MAP2 following LTP induction (Fig. 2, A and B). This increase in phosphorylation was completely prevented by pretreatment with 50 µMD-AP5, as was also observed in the autophosphorylation of CaM kinase II subunits and the phosphorylation of synapsin I.

Effect of Calmidazolium on the Increased Phosphorylation of CaM Kinase II, Synapsin I, and MAP2 during LTP

To further evaluate involvement of CaM kinase II in the increased phosphorylation of synapsin I and MAP2 during LTP, we tested the effects of a calmodulin inhibitor, calmidazolium, on the increased phosphorylation as well as on LTP induction. As reported by Reymann et al.(1988), inclusion of calmidazolium (50 µM) in the perfusion medium prevented the induction of LTP without modifications of basal synaptic transmission (Fig. 4B). Under the same conditions, the increases in autophosphorylation of CaM kinase II and phosphorylation of MAP2 associated with LTP induction were totally abolished (Fig. 4C). It is also interesting to note that the increased phosphorylation of synapsin I, although greatly inhibited in the presence of calmidazolium, was not completely prevented (compare Fig. 2to Fig. 4C) (p < 0.05, n = 10).


Figure 4: Effects of calmidazolium on LTP induction and the increased phosphorylation of CaM kinase II, synapsin I, and MAP2. A, summary of the changes in excitatory postsynaptic potential (EPSP) slope measured in four control experiments following application of burst stimulation (TBS) to a group of afferents in the CA1 regions of hippocampal slices. B, addition of calmidazolium (50 µM, blackbar) to the perfusion medium prevented induction of LTP without modifications of basal synaptic transmission. Data are means ± S.E. of the changes in excitatory postsynaptic potential slope measured in four experiments following application of burst stimulation (TBS). C, under the same conditions as in B, we determined the incorporation of P into the alpha and beta subunits of CaM kinase II, synapsin I, or MAP2. Results were obtained from 6 control and 10 burst-stimulated slices in the presence of 50 µM calmidazolium. The treatment of slices with calmidazolium totally abolished the increases in autophosphorylation of CaM kinase II and phosphorylation of MAP2, whereas a small but significant increase in the phosphorylation of synapsin I was still observed. Values are means ± S.E. and are expressed as percentage of control. *, statistically significant difference between potentiated and control slices (p < 0.05).



Increase in the Amount of the beta Subunit of CaM Kinase II with LTP

In previous experiments, we observed not only an increase in the Ca-independent activity of CaM kinase II but also a small but significant increase in the total activity of CaM kinase II 30-60 min after LTP induction. Among possible explanations for this observation, there was the possibility of translocation of the enzyme, unmasking of cryptic kinase activity, changes in phosphatase activities, or expression of newly synthesized enzyme. Comparisons of the total kinase activity in Triton X-100 insoluble and soluble fractions revealed no detectable decrease in activity in the insoluble fraction that could have explained the increase observed in the soluble one (data not shown). We therefore analyzed whether any regulation of CaM kinase II activity could occur through post-translational modifications, such as proteolysis by calpain. To address this issue, immunoblot analysis was carried out using the anti-CaM kinase II antibody. The results showed a small but significant increase in the amount of the beta subunit of CaM kinase II following LTP induction. No changes in the alpha subunit were detected. There was no evidence for the presence of any proteolytic fragments by immunoblot analysis (Fig. 5A). To evaluate the specificity of the increase in the beta subunit of the kinase, immunoblotting experiments using the anti-synapsin I and MAPs antibodies were also carried out with the same samples. As shown in Fig. 5B, where each lane corresponds to the same sample as in Fig. 5A, no significant changes in the amount of synapsin I were detected following high frequency stimulation. In the experiments with the anti-MAPs antibody, a small but not statistically significant decrease was observed in the potentiated slices. All these results are summarized in Table 1and Fig. 5C. Fig. 5C also shows that, when immunoblotting experiments were carried out with slices preincubated in the presence of D-AP5, the increase in the beta subunit of CaM kinase II was no longer detected.


Figure 5: Immunoblot analysis of CaM kinase II and synapsin I following induction of LTP. Immunoblot analysis with the anti-CaM kinase II and anti-synapsin I antibodies was carried out in the CA1 areas of potentiated (HFS) and control slices. The Triton X-100-soluble fractions (24 µg) of the CA1 areas were subjected to SDS-PAGE and electrophoretically transferred to a Durapore membrane (Millipore). The membranes were incubated with 5 µg of IgG of the anti-CaM kinase II antibody per ml (A) or 2 µg of IgG of the anti-synapsin I antibody per ml (B). Bound antibodies were detected with I-protein A. A and B, autoradiograms showing an increased detection of the beta subunit of CaM kinase II but not of the alpha subunit or of synapsin I. C, quantitation of the radioactivity of I-protein A-bound immunocomplex against CaM kinase II subunits obtained using a Bio-Image analyzer. Results were obtained from 15 potentiated and 13 controlled slices in the experiment without D-AP5. In the experiment with 50 µM D-AP5, results were obtained from 6 slices in each condition. Inclusion of D-AP5 in the medium prevented the increase in the amount of the beta subunit of CaM kinase II. Values are means ± S.E. and are expressed as the percentage of control. *, statistically significant difference between potentiated and control slices (p < 0.01).






DISCUSSION

The changes in phosphorylation of CaM kinase II and of its substrates, synapsin I and MAP2, were measured by comparing the in situ incorporation of [P]phosphate into the protein in control and potentiated slices following immunoprecipitation. The stimulation-induced increases in the phosphorylation were found to require NMDA receptor activation and were not detected in slices stimulated with low frequency trains that did not affect synaptic efficacy. It seems reasonable therefore to conclude that the changes reported here are related to the induction of LTP and not to non-selective effects of the stimulation. The changes in phosphorylation were measured in P-labeled slices 1 h after stimulation, thereby indicating that a stable increase in the phosphorylation had been induced by high frequency stimulation. In addition, experiments carried out in the presence of calmidazolium further support the idea that Ca/calmodulin-dependent protein kinases are involved in the increased phosphorylation of synapsin I and MAP2 associated with LTP induction. Taken together with the results of the previous study (Fukunaga et al., 1993), these data provide strong support to the idea that LTP induction is associated with a stable and long lasting increase in the degree of autophosphorylation of CaM kinase II. This would account for the changes in the Ca-independent activity reported previously and for the increased phosphorylation of substrates for CaM kinase II reported here.

As the increase in autophosphorylation of the kinase was NMDA receptor-dependent and associated with an increase in in situ phosphorylation of MAP2, a protein exclusively present in postsynaptic spines and dendrites, the changes in CaM kinase II activity reported here must at least in part take place in the postsynaptic compartment. This is consistent with several results indicating a possible contribution of postsynaptic activation of kinases in LTP. For example, intracellular injection of inhibitors of CaM kinase II prevented the induction of synaptic potentiation (Malenka et al., 1989; Malinow et al., 1989). Evidence has been provided for a CaM kinase II-dependent phosphorylation of postsynaptic (s)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (McGlade-McCulloh et al., 1993). This phosphorylation would result in the enhancement of postsynaptic sensitivity, and the idea has been proposed that this mechanism may represent one possible postsynaptic contribution to LTP (Figurov et al., 1993). Because MAP2 is a component of microtubule-associated proteins and a cytoskeletal protein in postsynaptic densities, the in situ phosphorylation of MAP2 reported here may also be important for the structural reorganization of synapses that has been proposed to accompany LTP induction (Geinisman et al., 1992). An increased and sustained postsynaptic autophosphorylation of CaM kinase II may thus contribute in several ways to the mechanisms underlying synaptic plasticity in the CA1 area.

An interesting result obtained in the present study is the observation that LTP induction was also associated with an increased phosphorylation of synapsin I, a presynaptic substrate of CaM kinase II. Although it is difficult at present to attribute this increased phosphorylation of synapsin I to an increased activity of CaM kinase II in presynaptic terminals, the results of a phosphopeptide mapping experiment suggest that the protein was indeed phosphorylated on the CaM kinase II-dependent site. However, the only partial inhibition produced by calmidazolium treatment of the synapsin I phosphorylation suggests a possible activation of other presynaptic protein kinases that may not be directly related to the induction of LTP. Also of interest is the observation that the phosphorylation of synapsin I was prevented by an antagonist of postsynaptic NMDA receptor. This could be interpreted as supporting the hypothesis of a retrograde messenger released following LTP induction to modify presynaptic parameters (Williams et al., 1989; Schuman and Madison, 1991; O'Dell et al., 1991). In this respect, it must be noted that phosphorylation of synapsin I in the squid giant presynaptic terminal has been proposed to participate in the modulation of neurotransmitter release and vesicle exocytosis (Lin et al., 1990). A sustained increase in the degree of autophosphorylation of CaM kinase II in presynaptic terminals associated with an increased phosphorylation of synapsin I may thus be relevant for contributing to LTP maintenance (Malgaroli and Tsien, 1992).

The results of the present study demonstrated not only an increased activity of CaM kinase II via autophosphorylation but also an increased amount of the enzyme in the solubilized extract of the CA1 area following LTP induction. One hour after application of high frequency stimulation, we could detect an increased amount in CaM kinase II beta subunit in immunoblotting experiments but no changes in the alpha subunit and in synapsin I. This may contribute to the increase in the total activity reported in the previous study (Fukunaga et al., 1993). Although other possibilities, such as translocation and unmasking of the kinase activity, may still occur, one may assume that protein synthesis of the beta subunit of the enzyme was activated, because the amounts of the alpha subunit and other proteins, including synapsin I and MAP2, remained unchanged. In this context, gene expression of the alpha and beta subunits may be differently regulated as previously reported (Burgin et al., 1990). Furthermore, it should be noted that protein synthesis is required for the maintenance of LTP (Frey et al., 1989; Otani et al., 1989), probably not via transcription of new mRNAs but possibly by synthesis from pre-existing messengers. The mRNAs for both the alpha subunit of CaM kinase II and MAP2 have been demonstrated in the dendrites of hippocampal neurons in the CA1 area and in hippocampal cultures (Kleiman et al., 1990; Benson et al., 1992; Chicurel et al., 1993). On the other hand, the mRNA for the beta subunit of the kinase is predominantly localized in the cell body (Burgin et al., 1990). Interestingly, Frey et al.(1989) suggested that de novo protein synthesis in postsynaptic cell bodies rather than in dendrites was more important for the maintenance of late phases of LTP. In addition, Otani et al.(1989) observed that anisomycin, an inhibitor of the translation step of protein synthesis, could produce decay of LTP when injected immediately after tetanization but that the drug had no effect when injected after a 15-min delay and thus concluded that the protein synthesis required for LTP maintenance is mostly completed within 15 min after tetanization. The increased protein level of CaM kinase II was observed within 1 h after tetanization in the present study, and the increased total activity was detected at least within 30 min in the previous study (Fukunaga et al., 1993). Mackler et al.(1992) observed 3-fold increase in the CaM kinase II mRNA level as well as a transcriptional factor, Zif-268 mRNA, between 30 min and 3 h after tetanization. They did not address which subtypes of CaM kinase II mRNA were induced in the LTP. In this respect, specific inhibitors that prevent transcriptional or translational processes of protein synthesis should be tested on the changes in the beta subunit reported here.

In conclusion, the results obtained here indicate that LTP induction in the CA1 regions of the hippocampus is associated with an increased and sustained in situ autophosphorylation of CaM kinase II as well as with an increased phosphorylation of two major substrates of this enzyme, synapsin I and MAP2. Together with the recent results showing that LTP is also associated with increased phosphorylation of protein kinase C substrates, GAP-43 and neurogranin (Klann et al., 1991; 1992; Gianotti et al., 1992; Sacktor et al., 1993), these observations support the idea that activation of protein kinases contributes to modifications of synaptic efficacy.


FOOTNOTES

*
This work was supported in part by grants-in-aid for scientific research and for scientific research on priority areas from the Ministry of Education, Science, and Culture (to E. M. and K. F.) and by the Swiss National Research Foundation, (FNRS 31-30980.91 to D. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel: 81-96-373-5076; Fax: 81-96-373-5078.

(^1)
The abbreviations used are: CaM kinase II, Ca/calmodulin-dependent protein kinase II; D-AP5, D-2-amino-5-phosphonopentanoate; LTP, long term potentiation; MAP2, microtubule-associated protein 2; MAPs, microtubule-associated proteins; NMDA, N-methyl-D-aspartate; PAGE, polyacrylamide gel electrophoresis.


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