(Received for publication, June 23, 1994; and in revised form, November 14, 1994)
From the
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.
Activities of several protein kinases, CaM kinase II ()(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
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
subunit of the kinase.
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.
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
and
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
and
subunits of the kinase, thereby
indicating autophosphorylation of the enzyme. When compared with
control conditions, the levels of autophosphorylation of the
and
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
and
subunits) and synapsin I were immunoprecipitated with
specific antibodies and analyzed by SDS-PAGE followed by
autoradiography. B, the incorporation of
P into
the
and
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).
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
and
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).
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
subunit of CaM kinase II but not of the
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
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).
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)--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 subunit in immunoblotting experiments but no changes in
the
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
subunit of the
enzyme was activated, because the amounts of the
subunit and
other proteins, including synapsin I and MAP2, remained unchanged. In
this context, gene expression of the
and
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
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
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
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.