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J. Biol. Chem., Vol. 275, Issue 37, 28682-28687, September 15, 2000
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From the Rudolf Magnus Institute for Neurosciences, Department of
Medical Pharmacology, Universiteitsweg
100, 3584 CG Utrecht, The Netherlands
Received for publication, April 10, 2000, and in revised form, June 13, 2000
Induction of homosynaptic long term depression
(LTD) in the CA1 field of the hippocampus is thought to require
activation of N-methyl-D-aspartate receptors,
an elevation of postsynaptic Ca2+ levels, and a subsequent
increase in phosphatase activity. To investigate the spatial and
temporal changes in protein phosphatase activity following LTD
induction, we determined the in situ phosphorylation state
of a pre- (GAP-43/B-50) and postsynaptic (RC3) protein kinase C
substrate during N-methyl-D-aspartate
receptor-dependent LTD in the CA1 field of rat hippocampal
slices. We show that LTD is associated with a transient (<30 min) and
D-AP5-sensitive reduction in GAP-43/B-50 and RC3 phosphorylation and
that LTD is prevented by the phosphatase inhibitors okadaic acid and
cyclosporin A. Our data provide strong evidence for a transient
increase in pre- and postsynaptic phosphatase activity during LTD.
Since the in situ phosphorylation of the calmodulin-binding
proteins GAP-43/B-50 and RC3 changes during both LTD and long term
potentiation, these proteins may form part of the link between the
Ca2+ signal and
Ca2+/calmodulin-dependent processes implicated in
long term potentiation and LTD.
Several forms of activity-dependent plasticity can be
induced in the Schaffer collateral CA1 synapses of the hippocampus, including long term potentiation (LTP)1 and long term
depression (LTD) (1-3). In CA1, the
major forms of LTP and LTD depend on activation of NMDA receptors and a
subsequent elevation of the intracellular Ca2+
concentration ([Ca2+]i) (4). It has been
suggested that specific properties of the Ca2+ signal
(magnitude and temporal structure) determine the direction of the
synaptic change (5-7). During LTP, a large rise in
[Ca2+]i presumably activates protein kinases such
as calcium/calmodulin-dependent protein kinase II and/or PKC,
whereas during LTD a small rise in [Ca2+]i favors
an activation of protein phosphatases (8).
The involvement of kinases and, notably, PKC in LTP has been shown in
several ways (3, 9-11). Direct evidence for an involvement of
phosphatases (PPases) in LTD is, however, less abundant.
Pharmacological studies have shown that inhibitors of PPase 1, PPase
2A, and PPase 2B, applied intra- or extracellularly block the induction
of LTD (12, 13). In addition to this effect on LTD induction, there have been contradictory results on the effect of PPase inhibitors on
basic synaptic transmission. Figurov et al. (14) report that calyculin A, in a concentration of 1 µM, induced a
potentiation of the field excitatory postsynaptic potentials (fEPSP) of
40%. In addition, it has been shown that calyculin A increased
synaptic transmission in an activity-dependent manner (15).
In contrast, no effect on synaptic transmission in the hippocampus and
visual cortex have been reported by others (12, 16). Because of the contradictory pharmacological data and the fact that it cannot be ruled
out that the blocking effect of the PPase inhibitors on LTD induction
was due to modulation of NMDA receptor function, we used another
approach to investigate the role of PPases in LTD. We monitored
substrate phosphorylation in hippocampal slices labeled with inorganic
phosphate after the induction of LTD, a method we have exploited
successfully in LTP (17, 18).
The phosphoproteins of which the in situ phosphorylation
state was monitored were GAP-43/B-50 (also known as F1, neuromodulin) and RC3 (also known as neurogranin, BICKS). GAP-43/B-50 is a nervous tissue-specific protein that is highly expressed in neurons during development and nerve regeneration and has been implicated in neurite
outgrowth, long term potentiation, neuronal signal transduction, and
neurotransmitter release. In mature, non-damaged nervous tissue GAP-43/B-50 is expressed in most neurons, but at lower levels; expression levels are highest in human associative brain areas and rat
hippocampal and olfactory areas. In mature neurons, GAP-43/B-50 is
predominantly found in presynaptic plasma membranes and not in
dendrites. RC3 is a neuron-specific PKC substrate, mostly expressed in
forebrain neurons during postnatal development and maturity and in
hippocampus predominantly localized in dendritic spines (19-23). Both
GAP-43/B-50 and RC3 are soluble in 2.5% perchloric acid and bind CaM
at low Ca2+ concentrations (19, 24-27), which is prevented
by PKC-mediated phosphorylation (24, 25). Binding to CaM
stabilizes the amphiphilic To test the involvement of pre- and postsynaptic phosphatases in LTD,
we have examined changes in GAP-43/B-50 and RC3 phosphorylation at
different times after the induction of LTD in the Schaffer collateral
CA1 pathway. We show that both GAP-43/B-50 and RC3 phosphorylation are
transiently decreased shortly after the induction of LTD, a reduction
that is not observed when LTD induction is blocked by D-AP5. In
parallel experiments we showed that inhibitors of PPases potently
inhibit the induction of LTD. These data provide strong evidence for
activation of both pre- and postsynaptic phosphatases immediately
after the induction of LTD.
Hippocampal slices (450 µm) were prepared as described before
(18). Slices were stored in phosphate-free artificial cerebrospinal fluid (phosphate-free ACSF; 124 mM NaCl, 4.5 mM
KCl, 1.3 mM MgSO4, 10 mM glucose,
20 mM NaHCO3, and 2.5 mM CaCl2, pH 7.4, gassed with 95%
O2, 5% CO2) until the start of the experiment.
For electrophysiological recordings three slices were transferred to a
recording chamber and superfused (1 ml/min) for at least 60 min with
gassed phosphate-free ACSF at 30 °C. fEPSPs were recorded in the
radial layer of CA1 field in response to orthodromic stimulation as
described before (18). In all three slices the maximal fEPSP was
determined first. Only slices in which the maximal fEPSP was
bigger than 1 mV were selected. Two slices were randomly chosen
to be used for electrophysiological recordings. Stimulation intensity
was adjusted to evoke half-maximal responses, and this intensity was
kept constant throughout the experiment. Thirty min after placement of
stimulation and recording electrodes, the superfusion medium was
changed to ACSF containing 100 µCi/ml
[32Pi]orthophosphate, and after 90 min,
base-line recordings of fEPSPs were started (0.05 Hz, 30 min).
Subsequently one slice received a low frequency train of stimuli (1 Hz,
15 min) at test intensity, and recordings were continued for another
10, 30, 60, or 120 min. The other stimulated slice did not receive the
1-Hz stimulation, and the third slice served as an unstimulated
control. In some experiments slices were treated with 50 µM D-AP5 (Cambridge Research Biochemicals, Norwich, UK),
1 µM okadaic acid (Anawa, Wangen, Switzerland), or 1 µM cyclosporin A (gift from Dr. H. Boddeke, Sandoz AG,
Basel, Switzerland) 10 min before the 1-Hz stimulation was applied or
with 1 mM chelerythrine (Sigma) 45 min after the 1-Hz
stimulation. At the end of the experiments, all three slices were
removed from the recording chamber, washed, and homogenized (see
below). Evoked fEPSPs were recorded using a conventional AC-coupled
amplifier, and data were digitized using pCLAMP (Axon Instruments)
software. Data were analyzed using the same software, and the initial
slope of the fEPSPs was calculated. For graphical presentation, the
data are expressed as percentages of the average of the base-line slope
of the fEPSPs.
After the electrophysiological recordings had ended, the slices
were rapidly washed twice in 2 ml of ice-old H2O containing 100 mM NaF, 1.2 mM
NaH2PO4, 10 mM EDTA, 5 mM EGTA and homogenized in 100 µl of H2O
containing 100 mM NaF, 10 mM EDTA, and 5 mM EGTA. Of the total homogenate, 60 µl was added to 40 µl of stop-mix solution containing (final concentration) 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol (v/v), 5%
In the present experiments we monitored the phosphorylation state
of GAP-43/B-50 and RC3 in 32P-labeled hippocampal slices
from young rats (less than 2 weeks old) at different times after the
induction of LTD. Fig. 1 shows that
prolonged (15 min) low frequency (1 Hz) stimulation of the Schaffer
collaterals induced a robust and long-lasting decrease in the slope of
the fEPSP, showing that LTD can be induced in 32P-labeled
slices. Ten minutes after the 1-Hz stimulation was stopped, the slope
of the fEPSP was reduced to 57.6 ± 4.8% (n = 6),
and this reduction in the slope of the fEPSP remained at 30 min
(58.0 ± 7.8%, n = 6), 60 min (50.1 ± 4.6%, n = 6), and 120 min (38.1 ± 2.3%). The
normally stimulated slices, placed in the same recording chamber, did
not show any run-down in synaptic responses (95.3 ± 3.7%,
107.2 ± 4.1%, 90.0 ± 5.4%, 98.8 ± 6.5% at 10, 30, 60 and 120 min after the LTD slice received the 1-Hz stimulation). The competitive NMDA receptor antagonist D-AP5 completely blocked the induction of LTD (data not shown). Therefore, we can conclude that
the stimulation paradigm used in the present study induces a
reproducible, robust, and NMDA receptor-dependent LTD in
the CA1 field of 32P-labeled hippocampal slices.
To examine the effect of LTD on the in situ GAP-43/B-50 and
RC3 phosphorylation, GAP-43/B-50 and RC3 phosphorylation was determined 10 min after LTD induction. At this time point there is a decrease in
the in situ phosphorylation of both GAP-43/B-50 and RC3
(Fig. 2). GAP-43/B-50 phosphorylation is
reduced to 61.8 ± 8.4% (Fig. 2A, n = 6), and RC3 phosphorylation is reduced to 69.9 ± 6.8% (Fig.
2B, n = 6). When LTD induction is blocked by
50 µM D-AP5, there is no change in the phosphorylation of
both GAP-43/B-50 (96.4 ± 4.3%, n = 5) and RC3
(95.9 ± 4.9%, n = 5) compared with the normal
stimulated slices (97.4 ± 8.3% and 107.8 ± 12.6% for GAP-43/B-50 and RC3, respectively). Typical examples of
immunoprecipitates of a control slice, a slice expressing LTD for 10 min, and a slice only receiving normal stimulation are shown as insets
in Fig. 2, A and C. Ten minutes after LTD
induction there is a significant correlation between the decrease in
the in situ GAP-43/B-50 and RC3 phosphorylation and the
decrease in the slope of the fEPSP (correlation coefficient = 0.97, p < 0.001, n = 6 for GAP-43/B-50 and correlation coefficient = 0.89, p < 0.001, n = 6 for RC3; Fig. 2, B and D).
We also performed experiments on slices from older animals (4 to 6 weeks) in which the likelihood of LTD induction is reduced (33).
Prolonged (15 min) low frequency (1 Hz) stimulation failed to induce
significant LTD in these slices; the slope of the fEPSP returned to
93.8 ± 7.4% (n = 4) of the base-line value within 10 min after the end of the low frequency stimulation. In these
slices we determined the phosphorylation of GAP-43/B-50 and RC3. There
was no reduction of GAP-43/B-50 (101.2 ± 4.3%, n = 4) and RC3 (98.5 ± 6.3%, n = 4)
phosphorylation in these slices. The decrease in GAP-43/B-50 and RC3
phosphorylation seems to be specific, since the in situ
phosphorylation of another well defined PKC substrate, the
myristoylated alanine-rich protein kinase C substrate, MARCKS (34),
does not change after the induction to LTD (35).
Long Term Depression in the CA1 Field Is Associated with a
Transient Decrease in Pre- and Postsynaptic PKC Substrate
Phosphorylation*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-helix within RC3 and GAP-43/B-50
only when Ca2+ is absent (28). Purified,
PKC-phosphorylated RC3 and GAP-43/B-50 can be dephosphorylated by
purified PPase 1, PPase 2A, and PPase 2B (29, 30).
EXPERIMENTAL PROCEDURES
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-mercaptoethanol (v/v), 0.001% bromphenol blue, and after boiling
for 10 min, samples were stored at 
20 °C. The remainder of the
homogenate was used to determine protein content (31) with BSA as a
standard. The degree of GAP-43/B-50 and RC3 phosphorylation was
determined using quantitative immunoprecipitation as described earlier
(18, 32). Briefly, for GAP-43/B-50 immunoprecipitation, 10 µg of the
protein homogenate was incubated overnight at 4 °C with antibody
9527 (1:200 final dilution), and for RC3 immunoprecipitation, 0.33 µg
of protein homogenate was incubated overnight at 4 °C with antibody
8420 (1:100 final dilution). Antigen complexes were precipitated with
Pansorbin (Calbiochem) and solubilized, and immunoprecipitates
were separated using 11% (GAP-43/B-50) or 15% (RC3)
SDS-polyacrylamide gel electrophoresis. 32P incorporation
into proteins was detected using a Fuji BAS1000 imaging system
(Raytest, Germany) and quantified using TINA analysis software. The
total 32P incorporation into proteins was determined by
trichloroacetic acid precipitation as described previously, and
32P incorporation into GAP-43/B-50 and RC3 was normalized
accordingly (18). Statistical analysis were carried out using a
Student's t test for paired or unpaired samples.
RESULTS
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View larger version (26K):
[in a new window]
Fig. 1.
LTD induction in the CA1 field of
32P-labeled hippocampal slices. Three slices were
placed in an electrophysiological recording chamber, and in two of
these slices fEPSP were recorded in the dendritical layer of the CA1 in
response to stimulation of the Schaffer collateral/commisural fibers.
After the slices were labeled with 32P, LTD was induced in
one slice (filled circles) by repetitive low frequency
stimulation (1 Hz, 15 min; indicated by the black bar) of
the afferent fibers, whereas the other slice did not receive the
conditioning stimuli (open circles). Experiments were
stopped 10 (A), 30 (B), 60 (C), and
120 (D) min after LTD had been induced. At all time points,
there was a significant reduction in the slope of the fEPSP in the
slices receiving the conditioning stimulation, indicating that LTD had
been induced in these slices. The slope of the fEPSP of the normal
stimulated slices did not change. Typical traces of fEPSP in a slice in
which LTD was induced are shown at the top corresponding to the time
points indicated in D. Calibration, 2 mV, 5 ms.
View larger version (32K):
[in a new window]
Fig. 2.
Reduced in situ
phosphorylation of GAP-43/B-50 and RC3 10 min after LTD
induction. A and C, 10 min after LTD induction,
the 32P-labeled hippocampal slices were removed from the
recording chamber, washed, and homogenized, and the in situ
phosphorylation of GAP-43/B-50 (A) and RC3 (C)
was determined using a quantitative immunoprecipitation. GAP-43/B-50
and RC3 phosphorylation is significantly reduced in slices expressing
LTD for 10 min (LTD) compared with the unstimulated control
(CON; p 
0.05, n = 6).
GAP-43/B-50 and RC3 phosphorylation was not changed in slices receiving
only normal stimulation (normal stimulated slice (NSS); not
significant, n = 6) or slices in which LTD
induction had been blocked by the NMDA receptor antagonist D-AP5
(LTD/AP5; not significant, n = 5).
Typical examples of phosphoimaging analysis of
immunoprecipitated, 32P-labeled GAP-43/B-50 and RC3 are
shown as insets. B and D, the decrease
in the individual slices in situ phosphorylation of
GAP-43/B-50 (B) and RC3 (D) was plotted against
the decrease in the slope of the fEPSP in each individual slice (data
from Fig. 1A). The decrease in the phosphorylation of
GAP-43/B-50 and RC3 was strongly and significantly correlated with the
change in the evoked fEPSP (correlation coefficient = 0.97, p = 0.001, n = 6 for GAP-43/B-50 and
correlation coefficient = 0.89, p 0.001, n = 6 for RC3).
To study the temporal relationship between the decrease in
phosphorylation of pre- and postsynaptic PKC substrates, experiments were stopped at different times after low frequency stimulation (Fig.
3). The in situ
phosphorylation state of postsynaptic protein RC3 is significantly
reduced 10 min after LTD induction (see also Fig. 2) and is back
to control levels at 30 min after LTD induction (98.3 ± 10.0%)
and remains unaltered at 60 and 120 min (100.2 ± 11.6% and
92.4 ± 8.3%, n = 6 and 5, respectively).
At the presynaptic site, GAP-43/B-50 phosphorylation, which is
decreased at 10 min after LTD induction (see also Fig. 2), is
still reduced at 30 min (74.0 ± 4.9%, n = 6).
One hour after LTP induction GAP-43/B-50 phosphorylation is
increased (132.7 ± 8.8%, n = 6) and returns to
control levels 120 min after LTD induction (102.3 ± 6.7%,
n = 5).
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To investigate the involvement of phosphatases in the transient
decrease in RC3 and GAP-43/B-50 phosphorylation, we determined the
effect of two different phosphatase inhibitors known to increase basal
RC3 and GAP-43/B-50 phosphorylation in hippocampal slices on LTD
induction (Fig. 4). The addition of
1 µM okadaic acid (Fig. 4A) or 1 µM cyclosporin A (Fig. 4B) starting 10 min before and ending 5 min after the 15-min 1-Hz stimulation paradigm
completely blocked the induction of LTD. The average slope of the fEPSP
60 min after the 1-Hz stimulation in the presence of okadaic acid or
cyclosporin A was 100.5 ± 5.4% and 96.0 ± 4.8%,
respectively (n = 5), whereas the 1-Hz stimulation in
phosphate-free ACSF resulted in a long-lasting decrease in the slope of
the fEPSP (see Fig. 1).
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The increase in GAP-43/B-50 1 h after LTD induction (see Fig. 3)
could be caused by either decreased PPase activity or increased kinase
activity. Since it is at present not possible to pharmacologically activate phosphatases, we decided to assess if increased PKC activity was causing the increase in GAP-43/B-50 phosphorylation. We treated slices with chelerythrine, a specific PKC inhibitor known to block a
phorbol ester-induced increase in GAP-43/B-50 phosphorylation (18). The
addition of 1 µM chelerythrine to the phosphate-free ACSF
starting 30 min after LTD induction for 30 min did not affect the
expression of LTD as compared with untreated control slices (Fig.
5). The slope of the fEPSP was still
depressed to 43.7 ± 8.1% (n = 5) of the base
line in chelerythrine-treated slices as compared with the depression to
38.1 ± 2.3% in control slices (see Fig. 1).
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ABSTRACT
INTRODUCTION
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DISCUSSION
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In this study we show that LTD induction in the CA1 field of the
hippocampus is accompanied by a transient decrease in GAP-43/B-50 and
RC3 phosphorylation. This reduction in phosphorylation is firmly linked
to LTD, because (i) it is NMDA receptor-dependent, (ii) it
is significantly correlated with the degree of LTD, (iii) it is not
elicited by test stimulation, (iv) it is not observed in slices from
older animals in which low frequency stimulation fails to induce LTD,
and (v) it is specific for certain PKC substrates since no effect was
found on myristoylated alanine-rich protein kinase C substrate (MARCKS)
phosphorylation (35). The transient reduction in the phosphorylation of
GAP-43/B-50 and RC3 we find after LTD induction most likely reflects an
increased phosphatase activity. Our pharmacological studies confirm the
involvement of phosphatases (PPase 1, 2A, and 2B) in LTD, as already
indicated by others, since okadaic acid and cyclosporin A block
LTD. However, these pharmacological studies do not address the temporal
and spatial aspects of PPase involvement in LTD. Our findings clearly show an NMDA receptor-dependent decrease in the in
situ phosphorylation state of two identified pre- and postsynaptic
phosphoproteins, providing independent evidence for an increased pre-
and postsynaptic PPase activity following NMDA receptor activation.
However, we cannot rule out the possibility that the decrease in
GAP-43/B-50 and RC3 phosphorylation is caused by reduced kinase
activity. Application of the kinase inhibitor H7 or the specific PKC
inhibitor chelerythrine to naive slices mimics and occludes the
maintenance of homosynaptic LTD in the CA1 field of the hippocampus. In
addition, it was shown that LTD is accompanied with a reduction in the
levels of protein kinase M
(the constitutively active
catalytic fragment of PKC) and the PKC isoforms PKC
and PKC
in
homogenates (mixed pre- and postsynaptic compartments) 30 min after LTD
induction and a concomitant reduction in constitutive PKC activity
(36). At present it is not known whether GAP-43/B-50 or RC3 are
substrates for these forms of PKC.
Repetitive low frequency stimulation (15 min, 1 Hz) of the afferent fibers in the CA1 field of the hippocampus of young rats induced a robust and long-lasting decrease in synaptic transmission, which is comparable to that found by others (37). The stimulation and recording electrodes were separated as far as possible to guarantee the biggest possible area in which LTD was induced. The reduction in in situ GAP-43/B-50 and RC3 phosphorylation we observed probably is an underestimation of that in individual affected synapses, since our experimental approach averages phosphorylation in the whole slice. Although in the hippocampus GAP-43/B-50 and RC3 are found in high concentrations in the CA1 area, they are also present in other parts of the hippocampus (21, 38, 39). The use of subslices to increase the number of synapses expressing LTD was not successful because of the increase in signal to noise ratio (data not shown). Recently, a protocol to chemically induce LTD in the whole slice has been described (40-42). This form of LTD is accompanied by a reduction in GluR1 phosphorylation (41).
Our data show that LTD in the CA1 field of the hippocampus is associated with pre- and postsynaptic reduction in the phosphorylation state of two identified PKC substrates. Since LTD induction is thought to be postsynaptic, the reduction in GAP-43/B-50 phosphorylation indicates an involvement of a retrograde messenger. The identity of the retrograde signal in LTD is still unknown (43-47).
The reduction in RC3 phosphorylation is transient and maximal 10 min
after LTD induction. This indicates that there is a transient activation of protein phosphatase activity following the elevation in
[Ca2+]i due to NMDA receptor activation and
presumably secondary release of Ca2+ from intracellular
Ca2+ stores (48). Due to technical limitations, time points
shorter than 10 min could not be measured; this is longer than the
Ca2+ signal (49). The mechanism of this transient PPase
activation and the nature of the PPase involved remain unknown. The
calcium calmodulin-dependent phosphatase calcineurin has
been implicated in the mechanisms underlying LTD (13, 50) and is known
to dephosphorylate RC3 and GAP-43/B-50(29, 30). The recent finding that
mice lacking the A isoform of calcineurin express normal LTD
indicates that the A isoform is required for LTD (51).
GAP-43/B-50 phosphorylation was increased 1 h after LTD induction. This finding can reflect either a decreased PPase activity or increased PKC activity. Because no potent and specific PPase activators are available at the moment, we tested the possibility of increased PKC activity using chelerythrine (52). Chelerythrine, applied 30 min after LTD induction, did not affect the maintenance of LTD, consistent with previous reports (36). Thus is seems unlikely that increased presynaptic PKC activity around 1 h after the induction of LTD is required for its maintenance. Therefore, we suggest that the increase in GAP-43/B-50 phosphorylation is due to an overshoot in inhibition of PPase activity after its activation following LTD induction.
GAP-43/B-50 and its PKC-mediated phosphorylation have been implicated in the regulation of neurite outgrowth and neurotransmitter release (53, 54), two processes that are important in activity-dependent forms of synaptic plasticity like LTP and LTD. It is tempting to speculate that reduced phosphorylation of GAP-43/B-50 would reduce synaptic efficacy during LTD by synaptic remodeling and/or reducing release. Indeed, some experimental evidence using quantal analysis indicates that LTD is accompanied by a decrease in neurotransmitter release (55). However, more recently no changes in the release probability were found (56). Interestingly, it has been shown that one of the phosphatases implicated in LTD, calcineurin, is very important in the process of neurotransmitter release (29, 57). RC3 is thought be involved in regulating release of Ca2+ from internal stores (58), a process important for LTP and LTD (48).
GAP-43/B-50 and RC3 contain an IQ domain, an amino acid sequence that
is found in a growing subset of CaM-binding proteins and that contains
a unique PKC phosphorylation site (59). Since both proteins bind CaM at
resting [Ca2+]i, they may function as a local CaM
sink. Upon a rise in [Ca2+]i GAP-43/B-50 and RC3
will release CaM. The CaM binding capacity of both proteins is
regulated by PKC phosphorylation; phosphorylation prohibits CaM
binding. Therefore, increased phosphorylation of GAP-43/B-50 and RC3,
as occurring during LTP, would result in higher free CaM levels,
activating calcium/calmodulin-dependent protein kinase II
(phosphorylation of -amino-3-hydroxy-5-methyl-4-isoxazole (AMPA)-type glutamate receptors (60), nitric-oxide synthase (47), and adenylate cyclase (higher cAMP levels and protein kinase A
activation) (61, 62). Reduced phosphorylation of GAP-43/B-50 and RC3,
as observed during LTD, would result in lower free CaM levels, thereby
favoring activation of the phosphatase calcineurin (dephosphorylation
of e.g. AMPA-type glutamate receptors (41)) or
activating phosphodiesterases (resulting in lower cAMP levels and
reduced protein kinase A activity). Thus, the phosphorylation state of
the two proteins could co-determine whether LTD-favorable CaM-dependent or LTP-favorable CaM-dependent
processes would be activated.
In conclusion, we show for the first time changes in the in
situ phosphorylation state of identified pre- and postsynaptic PKC
substrates during LTD. This supports the idea that both pre- and
postsynaptic changes occur during the expression of LTD. Our findings
indicate that the decrease in GAP-43/B-50 as well as RC3
phosphorylation are part of the physiological changes occurring during
LTD. The finding that two opposite forms of synaptic plasticity (LTP
and LTD) are both accompanied by changes in the phosphorylation state
of two neuronal phosphoproteins (GAP-43/B-50 and RC3) support the
hypothesis that LTP and LTD might converge at the level of specific
phosphoproteins (50).
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ACKNOWLEDGEMENTS |
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We thank Drs. R. H. Lenox and R. K. McNamara for the generous gift of myristoylated alanine-rich protein kinase C substrate (MARCKS) antibody and Dr. D. D. Gerendasy for critically reading the manuscript.
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FOOTNOTES |
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* 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.
Supported by Dutch Science Organization (NWO-MW) Grant
910-20-901. To whom correspondence should be addressed. Tel.: 31- 2538413; Fax: 31-2539032; E-mail: g.m.j.ramakers@med.uu.nl.
Published, JBC Papers in Press, June 23, 2000, DOI 10.1074/jbc.M003068200
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ABBREVIATIONS |
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The abbreviations used are:
LTP, long term
potentiation;
LTD, long term depression;
PKC, protein kinase C;
PPase, phosphatase;
fEPSP, field excitatory postsynaptic potentials;
NMDA, N-methyl-D-aspartate;
ACSF, artificial
cerebrospinal fluid;
CaM, calmodulin;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole.
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ABSTRACT
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DISCUSSION
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