From the
The phosphorylation state of two identified neural-specific
protein kinase C substrates (the presynaptic protein B-50 and the
postsynaptic protein neurogranin) was monitored after the induction of
long term potentiation in the CA1 field of rat hippocampus slices by
quantitative immunoprecipitation following
Hippocampal long term potentiation (LTP)
Activation of PKC is
thought to be one of the steps in the molecular mechanisms underlying
increased synaptic efficacy (for review, see Ref. 6). This notion is
based on different lines of evidence: (i) pharmacological manipulation
of PKC activity alters
LTP
(7, 8, 9, 10, 11) ; (ii) mice
lacking the
B-50 (also known as GAP-43, F1,
or neuromodulin) (for review, see Ref. 21) is nervous tissue-specific
in adult animals, has a strict presynaptic
localization
(22, 23) , and has been implicated in
neurotransmitter release
(24, 25) . Recently, a
postsynaptic, neuron-specific substrate for PKC has been identified,
named neurogranin (also known as RC3,
BICKS)
(26, 27, 28, 29, 30) . Rat
neurogranin shares an 18-amino acid sequence with B-50 which comprises
the single PKC phosphorylation site (the only phosphorylation site
reported) and the atypical calmodulin binding
domain
(26, 27, 31) . Taking advantage of this
sequence identity in B-50 and neurogranin, we developed a quantitative
procedure to precipitate B-50 and neurogranin from a single hippocampal
slice, thus enabling us to monitor the phosphorylation state of a pre-
and postsynaptic PKC substrate simultaneously. We show that
phosphorylation of both PKC substrates is increased in a narrow time
window around 60 min after LTP induction and that the increase in B-50
phosphorylation precedes that of neurogranin phosphorylation.
Transversal hippocampal slices (450 µm) were made from
hippocampi from male Wistar rats (100-120 g) on a McIlwain
tissue-chopper, collected in Petri dishes containing constantly gassed
with 95% O
Three hippocampal slices from three different
rats were transferred to tubes containing 900 µl of phosphate-free
ACSF at 30 °C. After 30 min, 100 µCi of
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. Bipolar stimulation electrodes, stainless steel wires of 50
µm diameter (insulated except for the tip), were placed on the
radial layer of the CA1 field. Glass microelectrodes with a tip
diameter of 3-5 µm, resistance 2-5 M
The degree of B-50
phosphorylation was determined using the method described by De Graan
et al.(34) , and the degree of neurogranin
phosphorylation was determined with a slightly modified procedure (see
below). For B-50 immunoprecipitation, 10 µg of protein homogenate
was incubated overnight at 4 °C with 1:200 (final dilution)
antibody 8919 or 8420 in 400 µl of radioimmunoassay buffer of the
following composition: 200 mM NaCl, 10 mM EDTA, 10
mM NaH
Total
To assess whether neurogranin is a substrate for PKC in the
hippocampal slice, we quantified B-50 and neurogranin phosphorylation
in slices treated with either phorbol ester, PKC inhibitors, or both.
After treatment of slices for 30 min with 10
To study the temporal relationship between the
increase in phosphorylation of both pre- and postsynaptic PKC
substrates, experiments were stopped at different times after high
frequency stimulation (Fig. 5). Neurogranin phosphorylation shows
a significant increase in phosphorylation only 60 min after LTP was
induced (77.9 ± 17.5%, n = 6). At no other time
points significant differences could be detected between neurogranin
phosphorylation in slices expressing LTP and slices receiving low
frequency stimulation, although there was a robust potentiation at
these time points (see Fig. 2, A-E). B-50
phosphorylation in potentiated slices is already increased 10 min after
induction of LTP (62.6 ± 19.9%, n = 5) and
remained elevated at 30 (66 ± 13.0%, n = 6) and
60 min (31 ± 6.5%, n = 13) after LTP induction,
compared with slices receiving only low frequency stimulation. At all
time points with a significant increase in B-50 phosphorylation
(i.e. 10, 30, and 60 min after application of high frequency
stimulation) this increase is significantly correlated with the
increase in the evoked EPSP (correlation coefficient = 0.94,
p < 0.01, n = 6 10 min after LTP;
correlation coefficient = 0.81, p < 0.01, n = 6 30 min after LTP and correlation coefficient =
0.80, p < 0.001, n = 13 60 min after LTP).
At 90 and 120 min after high frequency stimulation, no difference could
be detected between B-50 phosphorylation in potentiated and low
frequency stimulated slices (correlation coefficient = 0.19 and
0.21, not significant for 90 and 120 min after LTP, respectively),
despite the robust potentiation of the fEPSP induced in these slices
(see Fig. 2, D and E).
Our data demonstrate that in hippocampus slices neurogranin
is an endogenous PKC substrate. Its phosphorylation is increased by the
phorbol ester PDB, and this increase can be antagonized by kinase
inhibitors. Furthermore, we show that 60 min after LTP induction in the
CA1 field of the hippocampus the in situ phosphorylation state
of presynaptic protein B-50 as well as postsynaptic protein neurogranin
is significantly increased in potentiated as compared with
unpotentiated slices. This increase in B-50 and neurogranin
phosphorylation is dependent on LTP expression, because inhibition of
LTP induction by the NMDA receptor antagonist D-APV also
prevents the increase in phosphorylation of both proteins. The fact
that slices in which tetanic stimulation failed to induce LTP do not
exhibit increased phosphorylation shows that the increase in
phosphorylation is related to the expression of LTP, rather than to the
delivery of the high frequency stimulation. Moreover, the magnitude of
the increase in B-50 and neurogranin phosphorylation measured in a
single slice is highly correlated with the increase in the slope of the
EPSPs measured in that slice.
Our experimental approach, using
in situ phosphorylation in hippocampal slices combined with
quantitative immunoprecipitation of B-50 and neurogranin, allows to
distinguish between pre- and postsynaptic PKC activity and resolve
temporal differences in their activation. Presynaptic B-50
phosphorylation is increased from 10 to 60 min, but no longer at 90 and
120 min after tetanic stimulation. These results are in line with data
from previous experiments
(19) obtained after static labeling of
slices in an interface chamber, a setup limiting measurements to 60 min
post-tetanus. Recently, an increase in B-50 phosphorylation (+26
± 9%) at the PKC phosphorylation site 10 min post-tetanus was
found
(16) . This increase was measured by Western blotting,
using monoclonal B-50 antibodies directed against the single PKC
phosphorylation site (Ser-41). Since the increase in B-50
phosphorylation is strictly dependent upon NMDA receptor activation, a
retrograde signal appears to be involved, the nature of which is at
present unknown. Arachidonic acid
(35) as well as nitric oxide
(36) have been suggested to act as retrograde messengers during
LTP. Recently, it has been shown that activation of PKC by arachidonic
acid selectively enhances the phosphorylation of B-50 in nerve terminal
membranes
(37) , but the specific role of arachidonic acid and
nitric oxide in stimulating B-50 phosphorylation during LTP remains to
be investigated. The significance of increased presynaptic PKC
activation and B-50 phosphorylation following LTP is not yet clear.
Both PKC (38-40) and
B-50
(25, 41, 42, 43, 44) have
been implicated in the regulation of neurotransmitter release. Studies
measuring glutamate release after LTP induction indicate an increased
transmitter release until at least 60 min after tetanic
stimulation
(45, 46, 47) , a time window similar
to that found for increased B-50 phosphorylation following tetanic
stimulation. Although the use of quantal analysis for central synapses
is still a matter of debate, studies using quantal analysis also
indicate an increase in neurotransmitter release after LTP induction
(48-50). However, changes in postsynaptic parameters have also
been reported using this technique
(51, 52, 53) .
Postsynaptic PKC activity, as monitored by in situ neurogranin phosphorylation, was increased only in a short time
window around 60 min after tetanic stimulation. No effects on
neurogranin phosphorylation were found 10, 30, and 120 min after LTP
induction. Studies in which PKC inhibitors were introduced into CA1
neurons support an involvement of postsynaptic PKC in the induction and
early phases (<5 min) of
LTP
(9, 54, 55, 56) . Due to experimental
limitations, we were unable to measure neurogranin and B-50
phosphorylation at time points shorter than 10 min. Therefore we have
no information on a possible activation of postsynaptic PKC within the
first 10 min following tetanic stimulation. Our data clearly show an
increase in in situ neurogranin phosphorylation 60 min
post-tetanus, indicating postsynaptic PKC involvement in a narrow time
window overlapping with the tail of the time window of presynaptic PKC
activation, as observed by the increase in B-50 phosphorylation. This
is in line with the findings of Wang and Feng
(56) , who show
that postsynaptic PKC is important for the maintenance of LTP. However,
their data and ours are not supported by those of Huang et
al.(54) , who reported that micropressure injection of the
inhibitors H-7 or polymyxin B in CA1 pyramidal cells, 10 min prior to
tetanic stimulation, caused the potentiated EPSP slope to decay back to
base line within 30-50 min after LTP induction, whereas injection
5 min post-tetanus was ineffective
(54) . The most likely
explanation for this apparent discrepancy is that a microinjection 5
min post-tetanus, as used by Huang et al.(54) , is no
longer effective 60 min after tetanic stimulation (see also, Ref. 56).
Alternatively, the increased phosphorylation state of neurogranin could
be caused by a decrease in phosphatase activity. Phosphatases have
recently been implicated in long term depression, another form of
synaptic plasticity (57). Phosphorylation of neurogranin by other
kinases than PKC seems unlikely, because it does not contain consensus
phosphorylation sites for such kinases.
The functional significance
of neurogranin in LTP has recently been demonstrated: microinjection of
functionally interfering antibodies into CA1 pyramidal cells inhibited
the induction of LTP
(58) . Its precise function in the dendritic
spine is not yet known. In a recent study
(59) evidence was
presented that PKC-mediated phosphorylation of neurogranin expressed in
Xenopus oocytes increases the mobilization of intracellular
Ca
In conclusion, we present data showing
that during expression of LTP in the CA1 field of the hippocampus, the
in situ phosphorylation state of both a pre- and postsynaptic
PKC substrate is increased, in narrow time windows within the first
hour after LTP induction. Presynaptic PKC activation, as evidenced by
B-50 phosphorylation, precedes postsynaptic PKC activation measured by
neurogranin phosphorylation. Our data suggest that the increase in B-50
as well as neurogranin phosphorylation are part of the physiological
changes occurring during the maintenance phase of LTP, thus implicating
both pre- and postsynaptic PKC activation during the maintenance phase
of LTP. Our finding strongly supports the idea that both pre- and
postsynaptic changes occur during the expression of LTP.
We are grateful to Dr. H. Zwiers (University of
Calgary, Canada) for supplying the purified neurogranin (BICKS).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
P
labeling in the recording chamber. B-50 phosphorylation was
increased from 10 to 60 min, but no longer at 90 min after long term
potentiation had been induced, neurogranin phosphorylation only at 60
min. Increased phosphorylation was not found when long term
potentiation was blocked with the
N-methyl-D-aspartate receptor antagonist
D-2-amino-5-phosphonovalerate, when only low frequency
stimulation was applied or tetanic stimulation failed to induce long
term potentiation. Our data show that both B-50 and neurogranin
phosphorylation are increased following the induction of long term
potentiation, thus providing strong evidence for pre- and postsynaptic
protein kinase C activation during narrow, partially overlapping, time
windows after the induction of long term potentiation.
(
)
is the most extensively studied model of activity-dependent
synaptic plasticity and is proposed to be a model for certain forms of
memory
(1) . The major form of LTP in the CA1 subfield of the
hippocampus can be induced by application of a brief train of high
frequency pulses to afferent fibers
(2) and requires activation
of N-methyl-D-aspartate (NMDA) receptors (3) and an
elevation of the internal Ca
concentration
(4, 5) .
-PKC gene have altered LTP
(12) ; (iii)
measurement of total PKC activity after tetanic stimulation either
using exogenous artificial or neuron-specific substrates shows
increased activity (13-15); (iv) tetanic stimulation is
paralleled by a PKC translocation as measured by Western blotting
(16) or an increase in the amount of a persistent active form of
PKC
(17) . Limited information is available about the temporal
and spatial distribution of PKC activation during LTP. Interestingly,
phorbol ester- and tetanus-induced LTP were shown to be accompanied by
an increased phosphorylation of protein B-50 (16, 18, 19), confirming
previous post-hoc phosphorylation data implying an involvement of
presynaptic PKC in LTP
(20) .
, 5% CO
phosphate-free artificial
cerebrospinal fluid (phosphate-free ACSF: NaCl, 124 mM; KCl,
4.5 mM; MgSO
, 1.3 mM; glucose, 10
mM; NaHCO
, 20 mM; and CaCl
,
2.5 mM, pH 7.4) and stored at room temperature until the start
of the experiments.
P
(40 mCi/ml, Amersham, Buckinghamshire, United Kingdom) was added,
and slices were labeled for 90 min. Slices were treated for 30 min with
4-
-phorbol 12,13-dibutyrate (PDB, 10
M; Sigma), 4-
-phorbol 12,13-didecanoate (PDD,
10
M; Sigma), or a combination of PDB
(10
M) with
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 10
M; Sigma), polymyxin B (PMXB, 10,000 IU/ml; Sigma),
chelerythrine (10
M; Sigma), calphostin C
(5
10
M; gift from H. Boddeke,
Sandoz AG, Basel, Switzerland) or staurosporine (10
M; Sigma). The PKC antagonists were added 15 min prior
to PDB. Phorbol esters, chelerythrine, calphostin C, and staurosporine
were dissolved in Me
SO (0.01%), H-7, and PMXB in
phosphate-free ACSF. Me
SO did not affect B-50 or
neurogranin phosphorylation. After treatment the slices were washed
twice in 2 ml of ice-cold ACSF buffer containing 100 mM NaF,
1.2 mM NaHPO
, 10 mM EDTA, 5 mM
EGTA, and homogenized in 100 µl of H
O containing 100
mM NaF, 10 mM EDTA, and 5 mM EGTA. Of the
total homogenate 75 µl was added to 50 µl stop-mix solution
containing (final concentration) 62.5 mM Tris-HCl (pH 6.8), 2%
SDS, 10% glycerol (v/v), 5%
-mercaptoethanol (v/v), 0.001%
bromphenol blue, and samples were stored at -20 °C until
trichloroacetic acid precipitation and immunoprecipitation. The
remainder of the homogenate was used to determine protein content
(32) with bovin serum albumin as a standard. To determine
quantitative conditions for neurogranin immunoprecipitation slices were
P
labeled, washed, and homogenized. A linear
relationship between the amount of hippocampal tissue added to the
precipitation mixture and the amount of immunoprecipitated neurogranin
was found in the range of 0.15-2.5 µg of hippocampal tissue
(data not shown). Therefore all immunoprecipitations to determine
neurogranin phosphorylation were performed with 0.625 µg slice
homogenate protein. Purified neurogranin was phosphorylated with PKC as
described earlier
(33) .
, filled with
phosphate-free ACSF were used to record field excitatory postsynaptic
potentials (fEPSPs) in the radial layer of CA1 field in response to
orthodromic stimulation. 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 was kept constant throughout the
experiment. Thirty min after placement of the electrodes the
superfusion medium was changed to ACSF containing 100 µCi/ml
P
, and after 90 min, base-line recordings of
fEPSPs were started (0.05 Hz, 25 min). Subsequently, one slice received
a short train of high frequency stimulation (100 Hz, 1 s, test
intensity), and recordings were continued for another 10, 30, 60, 90,
or 120 min. The other stimulated slice did not receive high frequency
stimulation and the third slice served as an unstimulated control. In
some experiments slices were treated with 50 µMD-2-amino-5-phosphonovalerate (D-APV; Cambridge
Research Biochemicals, Norwich, UK) 10 min before high frequency
stimulation was applied. At the end of the experiments, all three
slices were removed from the recording chamber, washed, and
homogenized. Evoked fEPSPs were recorded using a conventional
AC-coupled amplifier, stored on magnetic tape, and 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.
PO
, 0.5% Nonidet P-40 (v/v). For
neurogranin immunoprecipitation 0.625 µg of protein homogenate was
incubated overnight at 4 °C with 1:66.7 (final dilution) antibody
8420 in 400 µl of radioimmunoassay buffer. Antigen complexes were
precipitated with washed pansorbin (Calbiochem), solubilized in
stop-mix solution, and boiled for 10 min. Immunoprecipitates were
analyzed by 11% (B-50) or 18% (neurogranin) SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) in duplicate or triplicate. Incorporation of
P into proteins was detected using a Fuji BAS1000 imaging
system (Raytest, Straubenhardt, Germany) and quantified using PDI
Quantity One software.
P incorporation into
proteins was determined by trichloroacetic acid precipitation. In
short, 2.5 µg of protein homogenate was spotted on 3MM Whatmann
paper, the filters were washed three times with ice-cold 10%
trichloroacetic acid solution and thereafter with ethanol,
ethanol/acetone (1:1), and acetone alone. Filters were dried and
counted in a Packard 2000 CA liquid scintillation counter. The analyzed
amounts of
P incorporation into B-50 and neurogranin were
normalized for total
P incorporation into proteins as
determined by trichloroacetic acid precipitation and are expressed as
percentages of the unstimulated controls. Statistical analysis were
performed using Student's t test for unpaired or paired
samples as appropriate.
M PDB neurogranin as well as B-50 phosphorylation was
significantly increased compared with buffer controls (Fig. 1):
134 ± 44% (mean ± S.E.) and 182 ± 36%,
respectively. The PDB-induced increase in neurogranin and B-50
phosphorylation could be antagonized by the kinase inhibitors H-7
(10
M), PMXB (10,000 IU/ml), calphostin C
(5
10
M), chelerythrine
(10
M), or staurosporine (10
M). Neither antagonists had an effect on basal
neurogranin or B-50 phosphorylation (data not shown). The phorbol ester
PDD (10
M), inactive with respect to PKC
activation, affected neither B-50 nor neurogranin phosphorylation.
These results show that it is possible to quantify changes in the
in situ phosphorylation state of neurogranin and B-50 in a
single hippocampal slice and that neurogranin is an in situ PKC substrate.
Figure 1:
Phorbol ester-induced increase in
neurogranin and B-50 phosphorylation can be inhibited by kinase
inhibitors. Hippocampal slices were P
-labeled
and treated with PDB (10
M), PDB
(10
M) + H-7 (10
M), PDB (10
M) + PMXB
(10,000 IU/ml), PDB (10
M) +
chelerythrine (Chel; 10
M), PDB
(10
M) + calphostin C (Cal C;
5
10
M), PDB (10
M) + staurosporine (Stau; 10
M) or PDD (10
M) for 30 min,
and B-50 (A) and neurogranin (B) phosphorylation was
determined (n = 2 or 4, for details see
``Experimental Procedures''). Antagonists were applied 15 min
prior to phorbol ester treatment. PDB induces an increase in both
neurogranin (134 ± 44%) and B-50 (182 ± 36%)
phosphorylation, which could be antagonized by all kinase inhibitors.
The inactive phorbol ester PDD and the inhibitors alone did not
influence B-50 or neurogranin phosphorylation. Asterisk, p < 0.05 (n = 4, comparing PDB with PDB +
inhibitor.
Application of a short train of high frequency
stimulation (100 Hz, 1 s, test intensity) induced an increase in the
slope of the fEPSPs in the CA1 field of the hippocampus (Fig. 2).
Ten min after the tetanus was applied the increase was 71.3 ±
17.3%; at 30 min, 56.2 ± 12.8%; at 60 min, 60.8 ± 14.1%;
at 90 min, 59.7 ± 7%; and at 120 min, 53 ± 6.2%. Typical
examples of evoked responses are shown in Fig. 2F.
Slices not receiving high frequency stimulation showed stable responses
throughout the experiments (fig. 2 , A-E, open circles).
Figure 2:
Induction of LTP by high frequency
stimulation. A-E, to examine the development of LTP
after application of the conditioning train electrophysiological
recordings were stopped 10 (A), 30 (B), 60
(C), 90 (D), or 120 (E) min after induction
of LTP. Presented are means ± S.E. from 5 (E), 6
(A, B, and D), or 13 (C) experiments.
Filled circles represent slices receiving high frequency
stimulation at t = 0 min, and open circles represent slices only receiving low frequency stimulation.
F, typical examples of evoked responses in the dendritic
region of area CA1 of the hippocampus. The upper plot is a response 5
min before induction of LTP, and the middle plot is a response 55 min
after induction of LTP. Both responses are plotted together in the
lower panel. Calibration: 2 mV and 10
ms.
A typical example of phosphorimage analysis of immunoprecipitates
from hippocampal slices 60 min after tetanic stimulation is shown in
Fig. 3
. B-50 immunoprecipitation reveals a single phospho band at
48 kDa on 11% SDS-PAGE (Fig. 3A). Phosphorylated
neurogranin is not seen under these conditions, because it runs of the
gel in the dye front. Neurogranin immunoprecipitation
(Fig. 3B) reveals a single band at 17 kDa on 18%
SDS-PAGE. Under these conditions B-50 phosphorylation is not
detectable, because it is much less prominent than neurogranin
phosphorylation. The 17-kDa phospho-band was identified as neurogranin
by three independent techniques. 1) The 17-kDa band comigrated with PKC
phosphorylated purified neurogranin on 18% SDS-PAGE. 2) Unlabeled
hippocampal tissue displaced PKC-phosphorylated purified neurogranin in
the immunoprecipitation in a concentration-dependent manner. 3) No
17-kDa phosphoprotein was detectable when the immunoprecipitation was
performed after preabsorption of the antibodies with purified
neurogranin (data not shown). Quantification of these phosphorimages
revealed that neurogranin phosphorylation (Fig. 3B) in
the LTP slice was 168%, in the slice receiving only low frequency
stimulation 98.7% compared with the unstimulated control slice (100%).
For B-50 phosphorylation (Fig. 3A), these data were
166.3 and 94.3%, respectively. Total P incorporation as
determined with trichloroacetic acid precipitation was not different
between the three slices in this experiment.
Figure 3:
Increase
in B-50 and neurogranin phosphorylation 60 min after induction of LTP.
A, typical example of phosphorimage analysis of B-50
immunoprecipitation after 11% SDS-PAGE, showing the amount of
P incorporation into B-50 in an unstimulated control slice
(CON), a slice in which LTP is induced with high frequency
stimulation (LTP), and a slice only receiving low frequency
stimulation (LFS). B, typical example of
phosphorimage analysis of neurogranin immunoprecipitation after 18%
SDS-PAGE (same experiment as for A). Arrows indicate
the position of phosphorylated B-50 and neurogranin,
respectively.
One hour after LTP
induction neurogranin phosphorylation was increased by 77.9 ±
17.5% in slices showing LTP (n = 6, p <
0.01) and B-50 phosphorylation was increased by 31 ± 6.5% (n = 13, p < 0.001) (Fig. 4, A and
C). To test if this increase was specific for LTP expression,
induction of LTP was prevented by application of 50 µMD-APV (electrophysiological data not shown). Slices in which
LTP was blocked with D-APV did not show an increase in
neurogranin or B-50 phosphorylation 60 min after high frequency
stimulation (Fig. 4, A and C). D-APV
itself had no effect on B-50 or neurogranin phosphorylation in low
frequency stimulated slices. In four slices, the tetanic stimulation
induced only a short (less than 5 min) increase in the evoked responses
(data not shown). In these four slices, recordings were continued for
60 min and than B-50 and neurogranin phosphorylation were determined.
In these slices where high frequency stimulation failed to produce LTP,
there was also no change in B-50 or neurogranin phosphorylation
(Fig. 4, A and C).
Figure 4:
Increase in B-50 and neurogranin
phosphorylation only occurs if LTP is induced. A and C, hippocampal
slices received only low frequency test stimulation (open
bars) or test stimulation and high frequency stimulation
(filled bars). Sixty minutes after the tetanus slices were
analyzed for B-50 (A) and neurogranin (C)
phosphorylation. Slices expressing LTP (LTP) showed increased
B-50 (31 ± 6.5%, p < 0.001, n = 13)
and neurogranin (77.9 ± 17.5%, p < 0.01, n = 6) phosphorylation. After blockade of LTP by 50
µMD-APV (APV), there was no difference
in B-50 and neurogranin phosphorylation between high frequency
stimulated slices and slices only receiving low frequency stimulation
(p > 0.1, n = 6 for both B-50 and
neurogranin). In those slices in which high frequency stimulation
failed to induce LTP (No LTP) no difference in B-50 or
neurogranin phosphorylation was found between the two groups of slices.
B and D, the increase in B-50 (B) and
neurogranin (D) phosphorylation found 60 min after high
frequency stimulation was plotted against the increase in the slope of
the fEPSP of the same slice. The plot for B-50 as well as for
neurogranin revealed a significant correlation (correlation coefficient
= 0.80, p < 0.001, n = 13 for B-50
and correlation coefficient = 0.97, p < 0.001, n = 6 for neurogranin).
Subsequently, we
determined whether there is a correlation between the increase in B-50
and neurogranin phosphorylation and the increase in the slope of the
EPSPs. Sixty min after LTP induction (Fig. 4, B and
D), the increase in B-50 as well as in neurogranin
phosphorylation was significantly correlated with the increase in the
slope of the EPSP (correlation coefficient = 0.80, p < 0.001, n = 13 for B-50 and correlation
coefficient = 0.97, p < 0.001, n = 6
for neurogranin).
Figure 5:
Increases in B-50 and neurogranin
phosphorylation show temporal differences during LTP. A,
slices expressing LTP (filled circles) show a significant
increase in the amount of phosphorylated B-50 compared with the matched
low frequency stimulated slices at 10 (p < 0.01), 30 (p < 0.01), and 60 min (p < 0.001) after application
of high frequency stimulation. Ninety and 120 min after LTP induction
there is no difference between the two groups of slices (p > 0.1). Slices only receiving low frequency stimulation for all
the time points studied show no change in B-50 phosphorylation
(open circles). B, neurogranin phosphorylation is
only increased 60 min after application of high frequency stimulation
in slices expressing LTP (filled circles) compared with slices
showing no potentiation (p < 0.01). At the other time
points there is no difference between the two groups of slices (p > 0.1). Slices only receiving low frequency stimulation for all
the time points studied show no change in neurogranin phosphorylation
(open circles).
, probably by an interaction with the inositol
triphosphate/diacylglycerol signal transduction pathway. Interestingly,
B-50 and neurogranin share an 18-amino acid sequence containing the
unique PKC phosphorylation site (Ser-41) within the atypical calmodulin
binding domain (residues 39-51; Refs. 25, 31, 60, and 61). Since
both a rise in intracellular Ca
and PKC
phosphorylation reduce calmodulin binding, B-50 as well as neurogranin
may serve as local calmodulin stores, thus triggering local
Ca
/calmodulin-dependent processes such as
calcium/calmodulin-dependent protein kinase II, which has been
implicated in both pre- and postsynaptic mechanisms underlying
LTP
(9, 62) .
-phorbol
12,13-dibutyrate; PDD, 4-
-phorbol 12,13-didecanoate; PKC, protein
kinase C; PMXB, polymyxin B; PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.