Transient Removal of Extracellular Mg2+ Elicits Persistent Suppression of LTP at Hippocampal CA1 Synapses Via PKC Activation

Kuei-Sen Hsu,1 Wen-Chia Ho,1 Chiung-Chun Huang,1 and Jing-Jane Tsai2

 1Department of Pharmacology and  2Department of Neurology, College of Medicine, National Cheng-Kung University, Tainan City, Taiwan 70101


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (theta 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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INTRODUCTION
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 MOmega ). 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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INTRODUCTION
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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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Fig. 1. Development of Mg2+-free artificial cerebrospinal fluid (ACSF)-induced epileptiform activity in area CA1 of hippocampal slices. A: a typical example to illustrate the general sequence of changes in field excitatory postsynaptic potential (fEPSP) and population spike (PS) due to Mg2+-free ACSF application. Superfusion of the hippocampal slice with Mg2+-free ACSF induced multiple PSs of larger amplitude than in control. However, the amplitude of presynaptic fiber volley was not significantly altered. Note that this hyperexcitability stopped by perfusion with normal ACSF. B: summary quantitation of all experiments comparing the presynaptic fiber volley amplitude, fEPSP slope, and PS amplitude (1st spike) before, during, and at different times after washout of a 30-min application of Mg2+-free ACSF. Data are presented as means ± SE from 6 slices. The asterisk denotes a significant difference compared with the control values (P < 0.05).

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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Fig. 2. Prior Mg2+-free ACSF application suppresses the induction of long-term potentiation (LTP). A: a 30-min bath application of Mg2+-free ACSF resulted in a facilitation of basal fEPSP and caused a substantial inhibition of subsequent LTP induction by high-frequency tetanic stimulation (TS). Insets are superimposed traces taken at different time, as indicated. B: the inhibitory effect of Mg2+-free ACSF-induced priming effect on LTP is time dependent. At 1 or 2 h after replacement with normal ACSF, TS could not effectively elicit a LTP. However, when the interval between normal ACSF replacement and TS was lengthened to 3 h, the inhibitory effect had completely decayed. Data are presented as means ± SE from 6-8 slices. The horizontal bar marks the time of Mg2+-free ACSF application; the large upward arrow indicates the moment of TS. Values in parentheses are the numbers of slices tested. The asterisk denotes a significant difference compared with the control values (P < 0.05). Calibration: 0.5 mV, 10 ms.

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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Fig. 3. Epileptiform activity is necessary for the Mg2+-free ACSF-induced suppression of LTP induction. Prior application of Mg2+-free ACSF for a shorter duration (8-10 min), which is not enough to generate epileptiform activity, did not affect the subsequent induction of LTP. The horizontal bar marks the time of Mg2+-free ACSF application. Insets are superimposed traces taken at different time, as indicated. Data are presented as means ± SE from 6 slices. Calibration: 0.5 mV, 10 ms.

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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Fig. 4. Short-term synaptic plasticity measured by paired-pulse facilitation (PPF) remains normal and unaffected at Schaffer collateral-CA1 synapses after prior Mg2+-free ACSF treatment. A: average of 5 consecutive fEPSP by paired stimuli with an interstimulus interval of 40 ms at control and 2 h later after replacement of normal ACSF. B: plot depicting the percent ratio of the slope of the 2nd fEPSP relative to the 1st fEPSP. Pairs of pulses were delivered at interstimulus interval of 20-200 ms. It is clear that there was no significant change in the PPF ratio after prior transient treatment with Mg2+-free ACSF. Data are presented as means ± SE from 6 slices.

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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Fig. 5. Mg2+-free ACSF-induced suppressive effect is dependent on the activation of N-methyl-D-aspartate (NMDA) receptors and L-type voltage-operated Ca2+ channels (VOCCs). A: the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 µM, present during Mg2+-free ACSF application) did not affect the subsequent inhibition of LTP induction by prior Mg2+-free ACSF application. B: this suppressive effect is NMDA receptor-dependent, because co-application of NMDA receptor antagonist D-2-amino-5-phosphovaleric acid (D-APV; 50 µM) and Mg2+-free ACSF completely blocked the Mg2+-free ACSF-induced suppression of LTP induction. C: the L-type VOCC antagonist nimodipine (30 µM, present during Mg2+-free ACSF application) also effectively prevented the suppression of LTP induction generated by prior Mg2+-free ACSF application. Insets are superimposed traces taken at different time, as indicated. Bars denote the delivery of Mg2+-free ACSF or the drugs as indicated. The control data shown in A-C is being reproduced from Fig. 2A. Calibration: 0.5 mV, 10 ms.

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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Fig. 6. Mg2+-free ACSF application does not cause a long-lasting change in the NMDA receptor-mediated synaptic response. A bath application of Mg2+-free ACSF for 30 min caused a significant increase in the area of the NMDA receptor field potential (fEPSPNMDA). After replacement with normal ACSF, the fEPSPNMDA was progressively returned to baseline level. The area was calculated by measuring the area under the curve between 2 cursors, the 1st set at the beginning the fEPSP and the 2nd set 80 ms later. D-APV (50 µM) was applied at the end of the experiment to ascertain that the potential measured was in fact due to the activation of NMDA receptors. The horizontal bars mark the time of Mg2+-free ACSF or D-APV application. Insets are superimposed traces taken at different time, as indicated. Data are presented as means ± SE from 5 slices. Calibration: 1 mV, 20 ms.

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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Fig. 7. The activation of PKC-dependent signaling pathway is involved in Mg2+-free ACSF-induced inhibition of LTP. A: co-application of a reversible protein kinase C (PKC) inhibitor NPC-15437 (20 µM) with Mg2+-free ACSF significantly antagonizing the Mg2+-free ACSF-induced suppression of LTP induction. B and C: the inhibition of LTP induction by prior Mg2+-free ACSF application was not affected by the CaMKII inhibitor KN-62 (3 µM) or protein kinase A (PKA) inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; 50 µM), when applied during Mg2+-free ACSF application, respectively. Insets are superimposed traces taken at different time, as indicated. Bars denote the delivery of Mg2+-free ACSF or the drugs as indicated. The control data shown in A-C are being reproduced from Fig. 2A. Calibration: 0.5 mV, 10 ms.

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 (theta 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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Fig. 8. Regulation of synaptic plasticity threshold by Mg2+-free ACSF-induced epileptiform activity. A: summary of the ability of different frequencies of synaptic stimulation to induce persistent changes in synaptic strength in the control and the prior short-term Mg2+-free ACSF treatment slices. The percentage change in synaptic strength from baseline at hippocampal CA1 synapses was measured at 40 min after conditioning stimulation delivered at various frequencies, as indicated. In prior Mg2+-free ACSF treatment group, conditioning stimulation was applied 2 h later after the normal ACSF replacement. Note that prior Mg2+-free ACSF application causes the shift of the frequency-response function to favor LTD. Data are presented as means ± SE from 4-7 slices. B: according to the Bienenstock, Cooper, and Munro (BCM) theory, the dynamic range of depression and potentiation is linked and the direction of the change in synaptic weight is a function of postsynaptic activity. Prior short-term seizure-like activity may shift the modification threshold (theta M) toward the right so that subsequent synaptic plasticity favors depression over potentiation.


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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 (phi ) 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 (theta M). Moreover, theta M is not fixed, but varies with the prior history of the postsynaptic activity. Consequently, after a period of increased activity, theta M shifts to higher values, requiring a greater degree of stimulation to strengthen synapses. Conversely, after a period of decreased activity, theta 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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