1Department of Pharmacology and 2Department of Neurology, College of Medicine, National Cheng-Kung University, Tainan City, Taiwan 70101
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ABSTRACT |
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Hsu, Kuei-Sen, Wen-Chia Ho, Chiung-Chun Huang, and Jing-Jane Tsai. Transient Removal of Extracellular Mg2+ Elicits Persistent Suppression of LTP at Hippocampal CA1 Synapses Via PKC Activation. J. Neurophysiol. 84: 1279-1288, 2000. Previous work has shown that seizure-like activity can disrupt the induction of long-term potentiation (LTP). However, how seizure-like event disrupts the LTP induction remains unknown. To understand the cellular and molecular mechanisms underlying this process better, a set of studies was implemented in area CA1 of rat hippocampal slices using extracellular recording methods. We showed here that prior transient seizure-like activity generated by perfused slices with Mg2+-free artificial cerebrospinal fluid (ACSF) exhibited a persistent suppression of LTP induction. This effect lasted between 2 and 3 h after normal ACSF replacement and was specifically inhibited by N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphovaleric acid (D-APV) and L-type voltage-operated Ca2+ channel (VOCC) blocker nimodipine, but not by non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In addition, this suppressive effect was specifically blocked by the selective protein kinase C (PKC) inhibitor NPC-15437. However, neither Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62 nor cAMP-dependent protein kinase inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) affected this suppressive effect. This persistent suppression of LTP was not secondary to the long-lasting changes in NMDA receptor activation, because the isolated NMDA receptor-mediated responses did not show a long-term enhancement in response to a 30-min Mg2+-free ACSF application. Additionally, in prior Mg2+-free ACSF-treated slices, the entire frequency-response curve of LTP and long-term depression (LTD) is shifted systematically to favor LTD. These results suggest that the increase of Ca2+ influx through NMDA channels and L-type VOCCs in turn triggering a PKC-dependent signaling cascade is a possible cellular basis underlying this seizure-like activity-induced inhibition of LTP.
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INTRODUCTION |
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Long-term potentiation (LTP) is a
form of activity-dependent increase in synaptic efficacy that has been
proposed as the cellular and molecular bases for learning and memory in
the brain (Bliss and Collingridge 1993; Goda and
Stevens 1996
). Although a vast array of molecules and processes
has been reported to involve in LTP induction and expression, the true
nature and physiological relevance of this long-lasting form of
synaptic plasticity remain uncertain (Sanes and Lichtman
1999
). Recently, a set of experimental studies have shown that
the threshold for the induction of LTP was not fixed but could be
strongly influenced by the recent history of synaptic activity. For
example, prior activation of
N-methyl-D-aspartate (NMDA) receptors either by
synaptic stimulation or by pharmacological means could effectively
inhibit the subsequent LTP induction (Fujii et al. 1991
;
Huang et al. 1992
; Izumi et al. 1992
).
Additionally, a brief burst of stimuli that did not produce LTP, but
did cause short-term potentiation (STP) of synaptic strength, raised
the threshold for LTP induction, but lowered the threshold for
long-term depression (LTD) induction (Christie and Abraham
1992
; Holland and Wagner 1998
; Huang et
al. 1992
). Furthermore, prior transient protein kinase C (PKC)
activation has been shown to facilitate the subsequent LTD induction
but to suppress the subsequent LTP induction in hippocampus
(Stanton 1995
). These results experimentally provided independent support for the theoretical model for
synaptic plasticity proposed by Bienenstock et al.
(1982)
, who assumed that the stimulation threshold
(
M) for synaptic modification is not fixed but
rather dynamically changed as a function of overall activity levels in
the neuronal networks. The direction and magnitude of a synaptic change
evoked by a given stimulus depend on the recent history of synaptic activity.
Epileptic seizures and LTP are both models of altered neuronal
plasticity and excitability (Martinez and Derrick 1996;
Suzuki 1996
). Recently, an interesting set of
experiments has demonstrated that electrically induced seizure-like
activity consistently disrupted hippocampal LTP in both in vivo animal
seizure model (Anwyl et al. 1987
; Hesse and
Teyler 1976
) and in vitro slice model (Barr et al.
1997
; Moore et al. 1993
). However, the cellular
mechanisms underlying this process remain unclear at present time.
Since memory impairment is one of the most common consequences of
seizure and LTP has been conceptualized as a reflection of biological processes underlying memory formation, an understanding of the mechanisms involved in seizure-like activity-induced disruption of LTP
may help us to clarify the reasons for seizure-related memory
impairment observed in clinic (Halgren and Wilson 1985
; Thompson 1991
). In the present study, we sought to
address this issue at cellular and molecular levels and focused on the
effect of prior transient seizure-like activity induced by perfused
slices with Mg2+-free artificial cerebrospinal
fluid (ACSF) on the subsequent induction of LTP in area CA1 of rat
hippocampal slices using extracellular recording methods. Our results
indicate that prior Mg2+-free epileptogenesis can
effectively raise the threshold for LTP induction, and that the
suppression of LTP is possibly mediated by an increase of
Ca2+ influx through NMDA channels
and L-type voltage-operated Ca2+ channels
(VOCCs), in turn triggering the activation of PKC activity.
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METHODS |
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Slice preparation
Animal care was consistent with the guidelines set by the
Laboratory Animal Center of National Cheng-Kung University. All experimental procedures were approved by the Animal Research Committee of Medical College of the National Cheng-Kung University. Hippocampal slices were obtained as described previously (Hsu 1996;
Hsu and Huang 1997
). Briefly, 21- to 28-day-old male
Sprague-Dawley rats were anesthetized with ether and decapitated by
guillotine. The brain was quickly removed from the skull, and the
transverse slices (400 µm thick) were cut from a tissue block of the
brain using Vibroslice (Campden Instruments, Silbey, UK). The slices
were placed in a storage chamber of ACSF oxygenated with 95%
O2-5% CO2 and kept at room
temperature for at least 1 h before recording. The composition of
the ACSF solution was (in mM) 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 11 glucose at
pH 7.3-7.4 and equilibrated with 95% O2-5%
CO2. Mg2+-free medium was
prepared by omitting MgCl2.
Electrophysiological recordings
A single slice was then transferred to the recording chamber, in
which it was held submerged between two nylon nets and maintained at
32.0 ± 0.5°C. The chamber consisted of a circular well of a low
volume (1-2 ml) and was perfused constantly at a rate of 2-3 ml/min.
Standard extracellular field recording techniques were used.
Extracellular recordings of field excitatory postsynaptic potentials
(fEPSPs) and population spikes (PSs) were obtained from the stratum
radiatum and stratum pyramidal, respectively, using microelectrodes
filled with 1 M NaCl (resistance 2-3 M). A bipolar stainless steel
stimulating electrode was placed in stratum radiatum to activate
Schaffer collateral/commissural afferents. Stimulus-response curves
were performed at the beginning of each experiment. Pulses of an
intensity that gave 30-40% of the maximal fEPSP were given at a
frequency of 0.033 Hz for the remainder of the experiment, and a
population spike started to appear at this stimulus intensity. In all
experiments, baseline synaptic transmission was monitored for 30 min
before drug administration or delivering either high- or low-frequency
stimulation. The strength of synaptic transmission was quantified by
measuring the slope of fEPSP and the amplitude of PS. The fEPSP slopes
were measured from approximately 20-70% of the rising phase using a
least-squares regression. The PS amplitudes were measured from the peak
negativity to a tangent line drawn between the first and second maximum
positivities. The fiber volley recorded as a field potential in the
presence of 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 µM D-2-amino-5-phosphovaleric acid (D-APV).
LTP was induced by high-frequency stimulation, at the test
pulse intensity, consisting of two 1-s trains of stimuli at 100 Hz,
delivered with an interval of 20 s. The average of responses
during a 5-min period before Mg2+-free ACSF
application or LTP induction was taken as the baseline, and all values
were normalized to this baseline. All values of LTP reported here were
calculated as the changes in fEPSP slope or PS amplitude measured 40 min after tetanic stimulation. Electrical signals were collected with
an Axoclamp-2B (Axon Instruments, Foster City, CA) filtered at 1 kHz
and sampled at 10 kHz, and a personal computer with pCLAMP software
(Versions 7.0, Axon Instruments, Foster City, CA) was used to on-line
acquire and analyze the data.
Drug application
All drugs were applied by dissolving them to the desired final
concentrations in the ACSF and by switching the perfusion from control
ACSF to drug-containing ACSF. Appropriate stock solutions of drugs were
made and diluted with ACSF just before application. Rp-adenosine
3',5'-cyclic monophosphothioate (Rp-cAMPS), KN-62, and nimodipine were
dissolved in dimethylsulfoxide (DMSO) stock solutions and stored at
20°C until the day of experiment. The concentration of DMSO in the
perfusion medium was 0.05%, which alone had no effect on the induction
of either LTP or LTD in the CA1 region of rat hippocampus (Hsu
and Huang 1997
). NPC-15437, D-APV, KN-62, and
Rp-cAMPS were purchased from Research Biochemicals (Natick, MA); CNQX,
picrotoxin, and nimodipine were obtained from Sigma (St Louis, MO).
Statistical analysis
The data for each experiment were normalized relative to baseline. All figures show means ± SE. The significance difference was evaluated by two-tailed Student's t-test. Numbers of experiments are indicated by n. Probability values (P) of <0.05 were considered to be significant.
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RESULTS |
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Effect of seizure-like activity on LTP
To test the effect of the prior seizure-like activity on the
subsequent LTP induction, Mg2+-free ACSF was used
as an epileptogenic agent to induce epileptiform activity. As reported
previously (Coan and Collingridge 1985; Mody et
al. 1987
), replacement of the normal ACSF with
Mg2+-free ACSF within 10-15 min resulted in a
progressive potentiation of the field potential response recorded in
the stratum radiatum (fEPSP) and the stratum pyramidal (PS) of the area
CA1 following stimulation of stratum radiatum. By continuing perfusion
of Mg2+-free ACSF, both the stimulus-triggered
and spontaneous epileptiform discharges were appeared. These events
began in Mg2+-free ACSF after 10-22 min, with an
average of 15.1 ± 1.8 (SE) min. As a typical experiment shown in
Fig. 1A, both the
stimulus-triggered and spontaneous epileptiform discharges consisted of
a burst of repetitive population of discharges. The stimulus-triggered
epileptiform discharges consisted of four to six negative-going PSs of
declining amplitude on top of a positive-going postsynaptic field
potential of 30-40 ms duration. The spontaneous epileptiform
discharges had amplitude of 1-3 mV and duration of 30-80 ms. Washing
with normal ACSF stopped these epileptic behaviors within 20-30 min; however, the increase of field potential response was still observed after cessation of spontaneous epileptic behaviors but decreased progressively to control baseline within about 2 h and remained stable for as long as recording was continued. On average, the slope of
fEPSP and the amplitude of PS measured 30 min after
Mg2+-free ACSF application were 172.9 ± 9.8% (n = 6, P < 0.05) and 198.7 ± 11.2% (n = 6, P < 0.05) of the
baseline, respectively. After 2 h of washout with normal ACSF, the
slope of fEPSP and the amplitude of PS were returned to 101.5 ± 1.5% (n = 6, P > 0.05) and 103.2 ± 9.4% (n = 6, P > 0.05) of
baseline, respectively. However, Mg2+-free ACSF
application did not produce a significant alteration of the amplitude
of presynaptic fiber volley. On the average, the amplitude of
presynaptic fiber volley measured 30 min after Mg2+-free ACSF application was 102.8 ± 1.9% (n = 6, P > 0.05) of baseline.
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To examine the effect of prior Mg2+-free ACSF-induced seizure-like activity on the LTP induction, a standard 100-Hz tetanization protocol was used to elicit LTP. With the use of this tetanization protocol, the LTP measured 40 min after induction was 36.6 ± 5.7% (n = 6) in control slices. After a 30-min Mg2+-free ACSF application and then normal ACSF replacement for 2 h before LTP induction, a significantly depressed degree of LTP was found (7.5 ± 3.7%, n = 8, P < 0.05) in comparing with the control slices. To determine the duration of this suppressive effect, we varied the time interval between normal ACSF replacement and tetanic stimulation. As illustrated in Fig. 2B, significant inhibition of LTP was observed in slices with a 1- or 2-h interval between normal ACSF replacement and tetanic stimulation. However, after 3 h of normal ACSF replacement, the magnitude of the subsequent induction of LTP was not significantly different from the control slices. The mean magnitude of LTP measured 40 min after induction was 32.6 ± 4.6% (n = 8, P > 0.05) in comparing with the control slices (36.6 ± 5.7%, n = 6, Fig. 2A). Thus this suppressive effect was short-term and lasted at least 2-3 h after remove of Mg2+-free ACSF.
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To investigate whether this persistent suppressive effect could be overcome with a stronger LTP-inducing stimulus protocol, we assayed the effectiveness of prior Mg2+-free ACSF application on LTP induction by four trains of 100-Hz tetanic stimulation (20 s between trains). In control slices, this protocol yielded 102.8 ± 8.6% of LTP (n = 5). LTP induced by this four trains of stimulus protocol in slices exposed to Mg2+-free ACSF 2 h before tetanus was 9.8 ± 4.7% (n = 5, P < 0.05). This result suggests that the stronger multiple trains of tetanic stimulation cannot effectively overcome the persistent suppression of LTP by prior short-term Mg2+-free ACSF application.
In the above experiments, synaptic stimulation was continued during Mg2+-free ACSF application. We also examined whether the suppressive effect induced by prior Mg2+-free ACSF treatment required this synaptic activation to exert its effects. We found that having interrupted stimulation during Mg2+-free ACSF application did not significantly affect its suppressive effect on LTP induction. After a 30-min Mg2+-free ACSF application and the replacement with normal ACSF for 2 h before LTP induction, the mean magnitude of LTP measured 40 min after induction was 8.4 ± 3.2% (n = 3, P < 0.05) in comparing with the control slices (36.6 ± 5.7%, n = 6, Fig. 2A). These results indicate that the synaptic activation is not necessary for Mg2+-free ACSF application to establish this persistent suppressive effect.
To determine whether simply prior superfusing with Mg2+-free ACSF (without epileptiform activity generation) may block the LTP induction, we examined the effects of application of Mg2+-free ACSF for 8-10 min duration (prior to the development of stimulus-triggered or spontaneous epileptiform activity) on the subsequent LTP induction. In this series of experiments, only the slices, which did not express stimulus-triggered or spontaneous epileptiform activity during the period of Mg2+-free ACSF application, were selected to test the subsequent LTP induction. As shown in Fig. 3, a 8- to 10-min application of Mg2+-free ACSF produced a significant increase in the slope of fEPSP by 49.8 ± 8.7% (n = 6) and completely reversed within 1 h after replacement with normal ACSF. After 2 h of normal ACSF replacement, the mean magnitude of the subsequent induction of LTP was 46.5 ± 6.8 (n = 6), not significantly different from control slices (36.6 ± 5.7%, n = 6, Fig. 2A). These results demonstrate that superfusion of Mg2+-free ACSF for a shorter duration, which did not generate epileptiform activity, was not enough to impair the subsequent LTP induction.
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Effects of prior Mg2+-free epileptogenesis on short-term synaptic plasticity
We next examined the effects of prior
Mg2+-free ACSF-induced seizure-like activity on
paired-pulse facilitation (PPF), a form of presynaptic short-term
synaptic plasticity (Clark et al. 1994), at Schaffer
collateral-CA1 synapses of hippocampal slices. We compared the
magnitude of PPF ratio of fEPSP slope before and 2 h after removal
of Mg2+-free ACSF. If this seizure-like
activity-induced suppression of LTP induction is associated with the
manipulation of presynaptic transmitter release mechanism, then it may
result in a change in the magnitude of PPF ratio. Alternatively if this
suppressive effect involves only a postsynaptic mechanism, the PPF
ratio should be relatively unaffected. In this set of experiments,
synaptic responses to a paired stimuli were recorded with interpulse
intervals between 20 and 200 ms. In a typical example shown in Fig.
4A, the second response was
42% larger than the first response at 40-ms interpulse interval before
Mg2+-free ACSF superfusion. After a
30-min Mg2+-free ACSF application and then normal
ACSF replacement for 2 h, the second response was 38% larger than
the first response. We found that prior short-term application of
Mg2+-free ACSF produced no apparent effect on the
magnitude of PPF ratio at every interpulse interval tested after 2 h of normal ACSF replacement (Fig. 4B). No deficits in PPF
ratio suggest that presynaptic form of short-term synaptic plasticity
remains normal in slices after short-term
Mg2+-free epileptogenesis.
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Putative mechanisms of action
Several attempts were made to identify the possible cellular
mechanisms underlying this suppressive effect. Since it was reported that both the excitatory synaptic transmission and postsynaptic excitability were substantially enhanced after brief exposure to
Mg2+-free ACSF in the hippocampal slices
(Psarropoulou and Kostopoulos 1990), it was investigated
whether this suppressive effect is due to the modification of
glutamatergic receptor function in the hippocampal CA1 neurons. To
determine whether the regulation of non-NMDA receptors is involved in
the subsequent inhibition of LTP induction, we applied the non-NMDA
receptor antagonist CNQX (20 µM) during the application of
Mg2+-free ACSF. As shown in Fig.
5A, CNQX (20 µM) application
did not effectively block the Mg2+-free
ACSF-induced epileptiform bursts, and the subsequent inhibition of LTP
induction by prior Mg2+-free ACSF treatment was
still observed in all six slices tested. The result that CNQX is not
effective in blocking epileptiform bursts induced by
Mg2+-free ACSF is similar to those reported
previously by Jensen and Sheardown (1988)
and
Gean (1990)
. After normal ACSF replacement for 2 h
before LTP induction, the mean magnitude of LTP measured 40 min after
induction was 9.3 ± 5.6% (n = 6, P < 0.05) in comparing with control slices (36.6 ± 5.7%, n = 6, Fig. 2A). To test whether activation of NMDA receptors is required for the subsequent inhibition of LTP induction, the Mg2+-free ACSF was
delivered in the presence of NMDA receptor antagonist D-APV
(50 µM). This treatment completely prevented the appearance of
Mg2+-free ACSF-induced epileptiform activity and
subsequently inhibited the suppression of LTP caused by prior
Mg2+-free ACSF application. After normal ACSF
replacement for 2 h before LTP induction, the mean magnitude of
LTP in slices exposed to D-APV during the application of
Mg2+-free ACSF was 36.5 ± 6.7%
(n = 7; Fig. 5B), not significantly different from control slices (36.6 ± 5.7%, n = 6, Fig. 2A).
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Previous research demonstrates that a large increase of
Ca2+ influx through L-type VOCCs can impair LTP
induction via activation of the Ca2+-dependent
K+-mediated afterhyperpolarization (AHP)
(Moyer et al. 1992). We next examine whether the
Mg2+-free ACSF-induced suppressive effect was
mediated via an up-regulation of L-type VOCC function or an enhancing
biochemical step downstream of the activation of L-type VOCCs. To test
this possibility, the L-type VOCC blocker nimodipine was bath applied
to slices during the delivery of Mg2+-free ACSF.
Interestingly, the subsequent inhibition of LTP by prior
Mg2+-free application was more sensitive to
nimodipine than the Mg2+-free ACSF-induced
epileptiform activity. Thus although nimodipine (30 µM) had no effect
of the Mg2+-free ACSF-induced epileptiform
activity per se, it effectively attenuated the subsequent inhibition of
LTP by prior Mg2+-free ACSF application (Fig.
5C). After normal ACSF replacement for 2 h before LTP
induction, the mean magnitude of LTP in slices exposed to nimodipine
during the application of Mg2+-free ACSF was
22.5 ± 6.6% (n = 7), not significantly different from control slices (36.6 ± 5.7%, n = 6, Fig.
2A).
Because the persistent inhibition of LTP induction by prior Mg2+-free ACSF application is blocked by D-APV, it is possible that this suppressive effect is due to a sustained up-regulation of NMDA receptor function. To examine this possibility, NMDA receptor-mediated fEPSP was isolated with the use of a solution containing 20 µM CNQX and 50 µM picrotoxin. Once NMDA potentials (fEPSPNMDA) were isolated, Mg2+-free ACSF (with 20 µM CNQX and 50 µM picrotoxin) was bath applied for 30 min, and the responses were recorded for a further 2 h. On average, the area of fEPSPNMDA measured 30 min after Mg2+-free ACSF application was 224.8 ± 7.8% (n = 5, P < 0.05). After 2 h of washout with normal ACSF, the fEPSPNMDA area was returned to 104.8 ± 6.9% (n = 5, P > 0.05) of baseline (Fig. 6). At the end of the experiment, D-APV (50 µM) was added to the perfusing medium to confirm that the isolated field potentials were induced mediated by NMDA receptor activation. The fact that Mg2+-free ACSF only facilitates NMDA receptor-mediated responses during the period of its application but not after normal ACSF replacement indicates that the inhibition of LTP induction by prior Mg2+-free ACSF application is not due to a long-lasting change of NMDA receptor function.
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Because the suppressive effect of Mg2+-free ACSF
on LTP at Schaffer collateral- CA1 synapses was blocked by
D-APV and nimodipine, respectively, it indicated that a
rise of external Ca2+ influx through the NMDA
channel and L-type VOCCs seems to be responsible for a chain of
intracellular events resulting in this suppressive effect. We next
asked which Ca2+-dependent signaling pathway is
associated with the Mg2+-free ACSF-induced
suppressive effect. Since the activation of PKC and CaMKII are well
known to play an important role in the induction of LTP in the
hippocampal CA1 neurons (Linden and Connor 1991;
Malinow et al. 1989
), it is of interest to examine
whether the activation of PKC or CaMKII activity contributes to this
suppressive effect. Both the selective PKC inhibitor NPC-15437 and the
specific CaMKII inhibitor KN-62 were used to test this idea. The
results depicted in Fig. 7, A
and B, clearly demonstrate that the suppression of LTP by
prior Mg2+-free ACSF application was specifically
blocked by NPC-15437 but not by KN-62, indicating that the PKC but not
CaMKII activation is required for this suppressive effect. After normal
ACSF replacement for 2 h before LTP induction, the mean magnitudes
of LTP in slices exposed to NPC-15437 (20 µM) and KN-62 (3 µM)
during the application of Mg2+-free ACSF
application were 44.8 ± 8.9% (n = 7, P > 0.05) and 12.2 ± 6.7% (n = 4, P < 0.05) in comparing with control slices (36.6 ± 5.7%, n = 6, Fig. 2A),
respectively.
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There is increasing evidence that cAMP signal transduction pathways
play a pivotal role in the development of LTP at Schaffer collateral-CA1 synapses (Blitzer et al. 1995;
Frey et al. 1993
). To address whether the activation of
protein kinase A (PKA) is also involved in this suppressive effect, we
performed the experiments with the specific PKA inhibitor Rp-cAMPS.
Examination of the effect of Rp-cAMPS (50 µM) on
Mg2+-free ACSF-induced suppressive effect
indicates that PKA activation is not required for this suppressive
effect. After normal ACSF replacement for 2 h before LTP
induction, the mean magnitude of LTP measured 40 min after induction in
this group was 6.2 ± 2.5% (n = 7, P < 0.05, Fig. 7C) in comparing with
control slices (36.6 ± 5.7%, n = 6, Fig.
2A). Moreover, the lack of effect of KN-62 and Rp-cAMPS on
this suppressive effect could not be due to their inadequate
concentrations because KN-62 (3 µM) and Rp-cAMPS (50 µM) have been
shown to block LTP induction in a reversible manner when it was applied
several minutes before LTP induction (Bortolotto and
Collingridge 1998
; Huang and Kandel 1998
). Taken
together, these results clearly indicate that the activation of PKC but not CaMKII or PKA is possibly involved in the
Mg2+-free medium-induced suppressive effect on LTP.
Seizure-like activity caused a frequency-dependent shift in synaptic plasticity
A question of neurobiological interest that emerged from this work
is whether there is a shift in the stimulation threshold for the
induction of LTP after a prior seizure-like activity. To explore this
question, we stimulated the Schaffer collateral afferent fibers at four
additional frequencies (1, 5, and 10 Hz) and examined the consequent
changes in the synaptic strength. Our results experimentally confirmed
the theoretical model of synaptic plasticity postulated by Bienenstock,
Cooper, and Munro (BCM) (Bienenstock et al. 1982);
high-frequency stimulation leads to LTP, intermediate frequency
produces no change in synaptic strength, and low frequencies produce
LTD. In control slices, 900 pulses of 1 and 5 Hz induced LTD of fEPSP.
The mean magnitude of LTD measured 40 min after induction were
27.5 ± 5.9% (n = 5) and 33.6 ± 6.9%
(n = 8), respectively, whereas LTD measured from prior
Mg2+-free ACSF treated slices were 39.5 ± 4.5% (n = 4, P < 0.05) and 48.7 ± 5.4% (n = 5, P < 0.05),
respectively. With 10-Hz stimulation (900 pulses), control slices show
only a very slight depression (3.8 ± 3.3%; n = 7) measured 40 min after induction. In response to this stimulation
protocol, slices from prior Mg2+-free ACSF
treatment show a significant LTD (19.6 ± 5.6%; n = 6, P < 0.05). As summarized in Fig.
8A, these results indicate that the frequency sensitivity of LTP and LTD induction at Schaffer collateral-CA1 synapses is altered by prior transient seizure-like activity. In this case, the crossovers point between LTD and LTP (
M) in the BCM model shifts to right
(Fig. 8B). In other words, the stimulation threshold for the
LTP induction is elevated following a prior seizure-like activity.
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DISCUSSION |
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"Hebbian" synaptic modification, in which the synapse will be
strengthened when activity in the pre- and postsynaptic elements are
synchronously active, is a useful model to account for the aspects of
associative learning, memory, and neuronal development (Hebb
1949). Recently, a theoretical extension of this rule called the BCM rule proposed that the history of synaptic activity could influence the stimulation threshold for the induction of
activity-dependent plasticity of synaptic communication
(Bienenstock et al. 1982
). Prior to the present study,
there has been a considerable evidence experimentally confirming this
hypothesis, indicating that the threshold for LTP and LTD induction was
not fixed but could be strongly influenced by the prior manipulations
of synaptic activity (Cohen and Abraham 1996
;
Holland and Wagner 1998
; Hsu et al. 1999
; Huang et al. 1992
; Stanton 1995
;
Wexler and Stanton 1993
). Such change in synaptic
responsiveness to plasticity-inducing stimulation is termed
"metaplasticity" (Bear 1995
; Tompa and
Friedrich 1998
). In this paper, we have specifically provided
another evidence that prior seizure-like activity induced by
Mg2+-free ACSF suppresses the subsequent LTP
induction. This suppressive effect lasted 2 h, but not 3 h,
after replacement with normal ACSF and did not require synaptic
activation during the application of Mg2+-free
ACSF. It was dependent on the NMDA receptor activation and was
specifically antagonized by L-type VOCC blocker nimodipine and PKC
inhibitor NPC-15437, but not by the CaMKII inhibitors KN-62 or by PKA
inhibitor Rp-cAMPS. In addition, the entire frequency-response curve is
shifted systemically to favor LTD after prior
Mg2+-free ACSF epileptogenesis. These results
suggest that the stimulation threshold for LTP induction at Schaffer
collateral-CA1 synapses could be elevated by prior seizure-like
activity and a NMDA receptor-dependent activation of PKC-related
signaling pathways is possibly involved in this suppressive effect.
Although the data presented here elucidate a role of
Mg2+-free-induced seizure-like activity in
regulating bidirectional long-term synaptic plasticity, they do not
indicate the site of action involved. It seems less likely a result of
action on the presynaptic site to change the probability of presynaptic
Ca2+ mechanisms in controlling the transmitter
release, because no deficits in PPF, a phenomenon generally accepted to
be presynaptic, was observed at 2 h later after normal ACSF
replacement (Fig. 4). As a consequence, this long-term suppression of
LTP induction by prior short-term Mg2+-free ACSF
application is not due to the sustained alterations in the presynaptic
transmitter release mechanisms. Although the lack of change in PPF has
been used to support the hypothesis of postsynaptic involvement in this
persistent suppressive effect, our findings do not exclude the other
presynaptic mechanism involvement because the lack of PPF changes have
been observed from other forms of synaptic plasticity (e.g., LTP) that
are nevertheless thought to be presynaptic (Malinow and Tsien
1990; Muller and Lynch 1989
; Schulz et
al. 1994
). Alternatively, it is probable that this persistent
suppressive effect is mediated by a postsynaptic mechanism. This
concept is supported by the previous observation that a short-term
Mg2+-free ACSF application may cause a clear
leftward shift of the EPSP/PS curve following
Mg2+-free ACSF treatment, indicative of E/S
potentiation, and such an effect was still observed at 1 and 2 h
following return to normal ACSF (Psarropoulou and Kostopoulos
1990
). Because the E/S potentiation was characterized by the
fact that the PS amplitude increased more than could be accounted for
by changes in the slope of fEPSP, the E/S potentiation produced by
Mg2+-free ACSF application may be explained by an
increase of the excitability of the postsynaptic neurons. Given that
the duration of the long-term suppression of LTP observed in this study
correlated extremely well that of the
Mg2+-free-induced E/S potentiation
(Psarropoulou and Kostopoulos 1990
), it is therefore
possible that the sustained increase of postsynaptic neuronal
excitability is associated with the inhibition of LTP induction by
prior short-term Mg2+-free ACSF application.
However, further experiments are required to test this possibility.
Many studies indicate that the genesis of synchronous epileptiform
discharges when perfused hippocampal slices with
Mg2+-free ACSF is due to the activation of NMDA
receptors, because the NMDA receptor antagonist D-APV could
reduce and eventually abolish this epileptiform activity (Herron
et al. 1985; Mody et al. 1987
).
D-APV was also found in the present study to inhibit the
Mg2+-free ACSF-induced suppression on the LTP
induction, indicating that the activation of NMDA receptors is
essential for the induction of this suppressive effect. Our findings
are consistent with those of Huang et al. (1992)
, who
showed that the repetitive activation of NMDA receptors by weak tetanus
(30 Hz, 0.15 s) or applying NMDA directly inhibited the subsequent
LTP induction in area CA1 of hippocampus. Taken together, these
findings suggest that prior transient activation of NMDA receptors may
induce a convert synaptic change that elevated the threshold for LTP
induction or actively suppressed its induction. It is interesting to
note that the magnitude of pharmacologically isolated NMDA
receptor-mediated component of synaptic response was not significantly
changed 2 h after normal ACSF replacement (Fig. 6), thus
demonstrating that a prior short-term change in NMDA receptor function
seems to be sufficient to elicit a persistent suppression of LTP induction.
The next question derived from our observation is what cellular
mechanism is responsible for the persistent inhibition of LTP induction
by prior short-term NMDA receptor activation. It seems clear that the
alteration of some biochemical steps downstream of the activation of
NMDA receptors implicated in LTP induction processes should be
involved. Our experiments demonstrate that LTP occurred normally when
PKC inhibitor NPC-15437 was present during the application of
Mg2+-free ACSF to the slices (Fig.
7A), suggesting that the activation of PKC is a possible
cellular mechanism underlying the Mg2+-free
ACSF-induced inhibition of LTP and PKC is an important mediator to
dynamically regulate the stimulation threshold for the induction of the
activity-dependent synaptic plasticity. This conclusion is consistent
with previous findings of Stanton (1995), showing that
prior transient perfusion of the hippocampal slices with PKC activator
phorbol 12,13-diacetate exhibits a facilitation of homosynaptic LTD
induction and a suppression of LTP induction. The finding that PKC
contributes to this persistent suppressive effect raises the following
question: How does it produce its effect? The effect of PKC might be
mediated through phosphorylation of some target proteins participating
in preventing the induction of LTP. However, the identity of target
proteins of PKC contributing to suppress LTP induction remains unknown.
Interestingly, a recent study has shown that a
transcription-independent de novo protein synthesis process is involved
in the promotion of LTP persistence in area CA1 of the hippocampus by
previous pharmacological or synaptic activation of group I metabotropic
glutamate receptors (Raymond et al. 2000
). Because a
newly synthesized protein may act to augment and prolong the action of
constitutively expressed enzymes (Sonenburg 1996
), it
remains to be seen whether the PKC-dependent suppressive effect of LTP
induction observed in this study is mediated through PKC-mediated
phosphorylation of translation initiation factors, in turn triggering
protein synthesis from existing mRNA.
There is a possibility that this long-term suppressive effect of LTP
induction is a result of alterations of CaMKII-mediated signaling
processes, since it has been proposed that CaMKII may act as a
molecular record of prior synaptic activity and plays a fundamental
role in the control of frequency-response function (Mayford et
al. 1995). This possibility seems unlikely, because the
specific CaMKII blocker KN-62 had no effect on the inhibition of LTP
induction by prior short-term Mg2+-free ACSF
application in the present experiments. As the
Ca2+ and Ca2+/CaM signaling
pathways may activate both CaMKII and PKC, the previous and present
results strongly support the hypothesis that prior transient activation
of these two signaling pathways may elicit a dual modulation in the
subsequent induction of long-term synaptic plasticity, i.e., prior
transient activation of CaMKII may facilitate LTP induction, whereas
prior increased PKC activities suppress subsequent LTP induction.
An interesting finding obtained in the present study is the observation
that L-type VOCC blocker nimodipine also effectively attenuated the
seizure-like activity-induced disruption of LTP induction, although it
does not exhibit a significant reduction of the
Mg2+-free ACSF-induced epileptiform activity
(Fig. 5C). These results indicate that the mechanisms
resulting from an activation of L-type VOCCs is also involved in the
inhibition of LTP induction by prior short-term
Mg2+-free ACSF application. Thus it appears that
L-type VOCCs can work cooperatively with NMDA receptors to underlie
this suppressive effect. A related question with this speculation is
that how L-type VOCCs work cooperatively with NMDA receptors. One
possibility is that L-type VOCC activation might potentiate NMDA
receptor-mediated response by activation of
Ca2+-dependent protein kinases or phosphatases
that regulate the NMDA receptor function (Coussens et al.
1997). Alternatively, it is possible that L-type VOCC
activation might add to the NMDA receptor-mediated Ca2+ transients or promote NMDA receptor
activation by elevating membrane depolarization, which leads to
demasking of NMDA receptor-mediated response occurring during synaptic
activation. These possibilities remain to be explored.
It could be argued that this Mg2+-free
ACSF-induced inhibition of LTP is simply an effect of the superfusion
of Mg2+-free ACSF and is not due to the prior
generation of epileptiform activity. However, when
Mg2+-free ACSF was applied for a shorter
duration, which did not induce epileptiform activity, LTP occurred
normally at 2 h later after normal ACSF application (Fig. 3).
These results support the notion that the persistent suppression of LTP
caused by prior short-term Mg2+-free ACSF
application is a consequence of the seizure-like activity generation
rather than that of activation of NMDA receptor alone. Previous
experiments have shown that the induction of LTP in area CA1 of the rat
hippocampal slices was strongly inhibited if the LTP-inducing stimulus
was delivered during the phase of postseizure depression (the
"postictal phase") following an evoked electrographic seizures
(EGS) in area CA3 (Barr et al. 1997; Moore et al.
1993
). This inhibition of LTP following epileptiform activity
is short-term, lasting for several minutes (5-10 min) after the
seizures. We now demonstrate a new long-term suppressive effect of
seizures on synaptic plasticity; i.e., the suppressive effect of prior Mg2+-free ACSF-induced seizure-like activity on
the LTP induction lasts for 2-3 h (Fig. 2B). This may be a
crucial aspect of seizure-induced memory loss, because seizure-induced
amnesia observed in the in vivo studies has been shown to last for
hours in rodent models of EGS (Squire and Spanis
1984
).
The BCM model of synaptic plasticity suggests that synaptic
modification depends on the values taken by a theoretical function () of the postsynaptic activity (Bienenstock et al.
1982
). According to this theory, synaptic modification
toward depression or potentiation depends on instantaneous activity
that is lower or greater than a modification threshold
(
M). Moreover,
M is
not fixed, but varies with the prior history of the postsynaptic
activity. Consequently, after a period of increased activity,
M shifts to higher values, requiring a greater
degree of stimulation to strengthen synapses. Conversely, after a
period of decreased activity,
M shifts to lower values, making it easier to potentate synapses. Our experiments indicate that prior transient seizure-like activity elevates PKC activity via a NMDA receptor-dependent manner and then causes the
shift of the frequency-response function to favor LTD. Our data
complement and extend the findings of Stanton (1995)
,
who showed that PKC might serve as the sensor of the level of neuronal activity and in turn control the frequency-response function.
Various lines of evidence have indicated that the molecular mechanisms
underlying the generation of seizure-like activity and
activity-dependent synaptic plasticity share several similarities. For
example, both require the enhancement of the synaptic excitation and
the postsynaptic neuronal excitability (Bliss and Collingridge 1993; McNamara 1994
). These results have led to
a general belief that the prior induction of one phenomenon may also
strongly affect the subsequent induction of the other. This study
provides a firm support to this hypothesis that prior seizure-like
activity generated by Mg2+-free ACSF could
suppress the subsequent LTP induction at Schaffer collateral-CA1
synapses. Our results not only confirm the theoretical predictions that
the degree and direction of synaptic plasticity induced by a particular
pattern of conditioning stimulation cannot be predicted unless the
previous stimulatory history of the synaptic activity is known
(Abraham and Bear 1996
; Bienenstock et al.
1982
; Gold and Bear 1994
) but also provide
important information concerning the mechanism by which seizure can
modulate the threshold for long-term synaptic plasticity, in a
PKC-dependent manner, by activating similar physiological mechanisms.
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ACKNOWLEDGMENTS |
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This work was financially supported by research grants from the Department of Health and the National Health Research Institute, Taiwan, Republic of China (NHRI-GT-EX89S830P and NHRI-GT-EX89S837C) to J.-J. Tsai and K.-S. Hsu.
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FOOTNOTES |
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Address for reprint requests: K.-S. Hsu (E-mail: richard{at}mail.ncku.edu.tw).
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.
Received 14 February 2000; accepted in final form 25 May 2000.
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REFERENCES |
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