Group I mGluR-Mediated Silent Induction of Long-Lasting Epileptiform Discharges

Lisa R. Merlin

Department of Neurology and Department of Physiology and Pharmacology, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203


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Merlin, Lisa R.. Group I mGluR-Mediated Silent Induction of Long-Lasting Epileptiform Discharges. J. Neurophysiol. 82: 1078-1081, 1999. Picrotoxin, an antagonist of GABAA receptor-mediated activity, elicited 320- to 475-ms synchronized bursts from the CA3 region of the guinea pig hippocampal slice. The addition of the selective group I metabotropic glutamate receptor (mGluR) agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 50 µM; 20- to 45-min application) gradually increased the burst duration to 1-4 s; this effect persisted 2-3 h after agonist removal. To determine whether the induction of this long-lasting effect required ongoing synchronized activity during mGluR activation, DHPG application in a second set of experiments took place in the presence of CNQX and (R,S)-CPP, antagonists of AMPA/kainate and NMDA receptors, respectively. In these experiments, synchronized bursting was silenced during the mGluR agonist application, yet after wash out of the DHPG and the ionotropic glutamate receptor (iGluR) blockers, epileptiform discharges 1-10 s in duration appeared and persisted at least 2 h after wash out of the mGluR agonist. The potentiated bursts were reversibly shortened by application of 500-1,000 µM (+)-alpha -methyl-4-carboxyphenylglycine (MCPG) or (S)-4-carboxyphenylglycine (4CPG), agents with group I mGluR antagonist activity. These data suggest that transient activation of group I mGluRs, even during silencing of synchronized epileptiform activity, may have an epileptogenic effect, converting brief interictal-length discharges into persistent seizure-length events. The induction process is iGluR independent, and the maintenance is largely mediated by the action of endogenous glutamate on group I mGluRs, suggesting that autopotentiation of the group I mGluR-mediated response may underlie the epileptogenesis seen here.


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Activation of group I metabotropic glutamate receptors (mGluRs) has been shown to have convulsant effects in vivo (Camón et al. 1998; McDonald et al. 1993; Sacaan and Schoepp 1992; Thomsen and Dalby 1998) and converts brief interictal bursts into prolonged (up to 8 s) synchronized discharges in the hippocampus in vitro (Merlin and Wong 1997; Merlin et al. 1998a; Taylor et al. 1995). The prolonged in vitro discharges persist for hours after removal of the mGluR agonist (Merlin and Wong 1997), and the induction but not the maintenance of these discharges is protein synthesis dependent (Merlin et al. 1998a). It thus appears that a long-term modification of neuronal properties underlies the group I mGluR-induced epileptogenesis.

Intense neuronal activity in the hippocampus has been associated with long-term changes in synaptic efficacy and can enhance excitation through processes such as long-term potentiation (LTP) and kindling (Collingridge and Bliss 1987; Lynch et al. 1990; Morrell and de Toledo-Morrell 1986; Slater et al. 1985; Swartzwelder et al. 1989). Thus one might hypothesize that the intense prolonged discharges elicited with group I mGluR activation may themselves be responsible for the persistence of the mGluR-mediated epileptiform burst potentiation. The experiments described herein address whether the expression of prolonged bursts during group I mGluR stimulation is required for the induction of the persistent epileptogenesis.


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METHODS
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Guinea pigs 2-4 wk of age were anesthetized with halothane and decapitated in conformance with the Guide for the Humane Care and Use of Animals. The brain was removed and promptly placed in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 26 NaHCO3, 5 KCl, 2 CaCl2, 1.6 MgCl2, and 10 D-glucose. The hippocampus was dissected free, and transverse slices 400 µm thick were prepared using a Vibratome (Technical Products International). Slices were placed on nylon mesh in an interface chamber (Fine Science Tools) maintained at 35.5°C and perfused with ACSF bubbled with 95% O2-5% CO2 at pH 7.4. Perfusion rate and dead space accounted for a 10- to 20-min lag between onset of drug application and onset of observed effect.

Intracellular recordings were obtained from the CA3 stratum pyramidale using 25- to 75-MOmega thin-walled glass microelectrodes filled with 2 M potassium acetate. Whenever necessary, continuous hyperpolarizing current was injected through the recording electrode to suppress spontaneous action potential firing. Recordings were amplified and digitized (Axoclamp-2B & DigiData 1200 series Interface; Axon Instruments), and data were analyzed using pClamp and SigmaPlot software. All drugs were applied via continuous bath perfusion. Picrotoxin (Sigma; 50 µM) was present in all experiments to elicit baseline epileptiform synchronized activity; (S)-3,5-dihydroxyphenylglycine (DHPG), (+)-alpha -methyl-4-carboxyphenylglycine (MCPG), (S)-4-carboxyphenylglycine (4CPG), and ionotropic glutamate receptor (iGluR) antagonists were obtained from Tocris Cookson.

Statistical significance of results across slices was determined using the Student's t-test. A P value of <0.05 was considered significant. Data are reported as means ± SE.


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DHPG elicits long-lasting burst prolongation

Continuous bath perfusion of picrotoxin, an antagonist of GABAA receptor-mediated inhibition, elicited synchronized discharges (epileptiform bursts) in the CA3 region of the guinea pig hippocampal slice. Burst duration (BD) was 321 ± 19 ms (mean ± SE; n = 5). Addition of the selective group I mGluR agonist DHPG (50 µM) (Ito et al. 1992) for 20-45 min resulted in a gradual increase in burst length (Fig. 1). Mean BD at the end of the agonist application was 1,059 ± 173 ms (n = 5), representing an increase of 227 ± 41%. The potentiation of the bursts significantly persisted after 1 h of agonist washout (BD 1,005 ± 143 ms, a 211 ± 33% increase), and at 2 h after removal of the mGluR agonist, bursts remained significantly prolonged (BD 868 ± 201 ms, 165 ± 51% potentiated over control; n = 5).



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Fig. 1. mGluR-mediated potentiation of epileptiform bursts. Representative example of time course of burst prolongation and persistence during and after (S)-3,5-dihydroxyphenylglycine (DHPG) exposure. Intracellular recording from a CA3 pyramidal cell. Each filled circle represents the occurrence of a synchronized burst. Picrotoxin was present throughout the experiment and induced the control baseline epileptiform activity (Ctrl). A 40-min application of the group I mGluR agonist DHPG (50 µM) progressively increased the burst duration; this effect persisted after a 2-h washout period. Times of traces shown below graph correspond with times indicated on x-axis of graph.

mGluR-mediated induction of burst prolongation in the presence of iGluR blockade

To determine whether the potentiated bursts could be induced in the absence of ongoing synchronized bursting activity and accompanying iGluR activation, a second set of experiments was performed (Fig. 2). In these experiments, picrotoxin-induced discharges were silenced with the iGluR antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM) and (R, S)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 40 µM). No rhythmic synchronized activity appeared during the 20- to 45-min application of DHPG in the presence of iGluR blockers. To ensure adequate washout of DHPG before iGluR recovery, the iGluR antagonists were continued for 10-60 min beyond the end of the DHPG application.



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Fig. 2. Ionotropic glutamate receptor (iGluR)-independent autopotentiation of mGluR responses underlies potentiation of synchronized bursts. Continuous intracellular recording from a CA3 pyramidal cell. Picrotoxin elicited control bursting activity and was present throughout the experiment. Continuous hyperpolarizing current was injected through the recording electrode to suppress intrinsically generated activity and allow only for the expression of synaptically driven bursts. A1: example of time course of induction of potentiated bursts by DHPG application in the presence of iGluR blockers. No bursts occurred during 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and (R, S)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) application. A2: traces correspond with times shown on graphs in A1 and B1. Toward end of middle trace ("CNQX, CPP, and DHPG"), hyperpolarizing current was transiently released to allow the expression of intrinsically generated action potentials, thus establishing the quality of the recording. B1: continuation of experiment shown in A, with time scale on x-axis allowing clearer view of antagonist application period. Traces in B2 correspond to times on graph in B1. (S)-4-carboxyphenylglycine (4CPG; 1 mM) shortened the potentiated bursts; partial recovery was observed on washout.

The initial picrotoxin-induced bursts were 381 ± 19 ms long (n = 8); BD of the recovered spontaneous synchronized discharges following iGluR antagonist washout (first bursts to reappear; at least 1 h after end of DHPG application) was 4,528 ± 817 ms, a mean increase of 1,093 ± 230% (n = 8). At 2 h of DHPG washout, BD was 4,332 ± 1,192 ms (1,078 ± 361% potentiated, n = 7; Fig. 2A), demonstrating no significant additional potentiation induced by the ongoing bursting activity.

Group I mGluR antagonists suppress potentiated bursts

The group I mGluR-mediated prolongation of epileptiform bursts was significantly enhanced when induced in the presence of iGluR blockers. Previously we have shown that mGluR-induced potentiated epileptiform bursts can be reversibly suppressed with group I mGluR antagonists (Merlin and Wong 1997). To determine whether the bursts elicited during iGluR blockade had a similar maintenance mechanism, the same group I antagonists were tested on the potentiated bursts induced in this protocol. The addition of either 4CPG or MCPG (500-1,000 µM each), agents with antagonist action at group I mGluRs (Hayashi et al. 1994), reduced the length of the persistent prolonged bursts by 69 ± 10% (n = 4; Fig. 2B).


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Induction of group I mGluR-mediated potentiation of epileptiform activity is iGluR activation independent

Each prolonged synchronized discharge produced during group I mGluR activation provides a tetanic excitatory stimulus to the hippocampal network and could potentially thereby evoke long-lasting changes in the cellular and synaptic properties of the network. The iGluR antagonists silenced the expression of these discharges during the mGluR agonist application, thus suppressing activity-dependent modification of receptor systems. The results demonstrate that group I mGluR activation, in the absence of any ongoing synchronized bursting activity, is sufficient to induce the modification underlying the persistent production of potentiated epileptiform bursts. The data further indicate that N-methyl-D-aspartate (NMDA) receptor activation is not a critical part of the induction process, a finding that has also been established in group I mGluR-induced LTP (Bortolotto and Collingridge 1993; O'Leary and O'Connor 1997; Petrozzino and Connor 1994).

It was noted that the prolonged epileptiform bursts induced in the presence of iGluR antagonists were significantly longer than those induced in control conditions. This increase in burst prolongation may represent an enhancement of epileptiform events, the mechanism of which remains unknown. Alternatively, any weakening of synchronization could potentially result in dispersion of the oscillatory discharges within each burst, making the bursts longer (McIntyre and Wong 1986); thus the longer bursts induced during iGluR blockade may actually be less robust than the control bursts. The current data do not distinguish between these two possibilities.

Autopotentiation of synaptically activated group I mGluR-mediated responses underlies epileptogenic effects of mGluR activation

Group I mGluR activation can potentiate both NMDA and alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated responses (Bortolotto and Collingridge 1995; Fitzjohn et al. 1996; O'Connor et al. 1994; Pisani et al. 1997; Ugolini et al. 1997). Preliminary studies show that NMDA receptor activation is not required for the maintenance of the prolonged mGluR-induced discharges (Merlin et al. 1998b), suggesting that NMDA potentiation is not the critical mechanism underlying the burst prolongation seen here. Although potentiation of AMPA responses may still occur, the data demonstrate that group I mGluR antagonists significantly shorten the potentiated epileptiform bursts, suggesting that the major mechanism underlying the maintenance of the prolonged synchronized bursts involves autopotentiation of the group I mGluR-mediated responses. This may occur via enhanced receptor sensitivity (Aronica et al. 1991; Holmes et al. 1996) or through an increase in the number of available group I mGlu receptors (Akbar et al. 1996; Al-Ghoul et al. 1998). Alternatively, it may involve a presynaptic increase in glutamate release (Manahan-Vaughan and Reymann 1997; Moroni et al. 1998; Strasser et al. 1998). Because group I mGluRs have been identified perisynaptically (Baude et al. 1993; Luján et al. 1996), increased glutamate release may, via a spillover effect, allow more group I mGluRs to be synaptically activated by the ongoing pulsatile release of glutamate in the presence of picrotoxin. Further experiments are necessary to determine which of these mechanisms participates in sustaining the autopotentiation of the group I mGluR-mediated response.

Thus transient activation of group I mGluRs, even during silencing of synchronized epileptiform activity, is sufficient to induce a long-term autopotentiation, allowing the maintained production of prolonged seizurelike discharges. These results suggest that the development of the epileptic condition may take place in the absence of any observable synchronous hyperactivity in the neuronal network, the only requirement being an enhanced activation of group I mGluRs, and thereby implicates group I mGluRs as potential mediators of epileptogenesis.


    ACKNOWLEDGMENTS

The author thanks R. Bianchi, K. L. Perkins, and R.K.S. Wong for critical review of the manuscript.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-01901.


    FOOTNOTES

Address for reprint requests: State University of New York Health Science Center at Brooklyn, 450 Clarkson Ave., Box 29, Brooklyn, NY 11203.

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 19 March 1999; accepted in final form 6 April 1999.


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