The Journal of Neurophysiology Vol. 80 No. 2 August 1998, pp. 989-993
Copyright ©1998 by the American Physiological Society

RAPID COMMUNICATION

Requirement of Protein Synthesis for Group I mGluR-Mediated Induction of Epileptiform Discharges

Lisa R. Merlin^{1, 2}, Peter J. Bergold², and Robert K. S. Wong^{1, 2}

¹ Department of Neurology and ² Department of Physiology and Pharmacology, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Merlin, Lisa R., Peter J. Bergold, and Robert K. S. Wong. Requirement of protein synthesis for group I mGluR-mediated induction of epileptiform discharges. J. Neurophysiol. 80: 989-993, 1998. Picrotoxin (50 µM) elicited rhythmic synchronized bursting in CA3 pyramidal cells in guinea pig hippocampal slices. Addition of the selective group I metabotropic glutamate receptor (mGluR) agonist (S)-3,5-dihydroxyphenylglycine (25 µM) elicited an increase in burst frequency. This was soon followed by a slowly progressive increase in burst duration (BD), converting the brief 250-520 ms picrotoxin-induced synchronized bursts into prolonged discharges of 1-5 s in duration. BD was significantly increased within 60 min and reached a maximum after 2-2.5 h of agonist exposure. The protein synthesis inhibitors anisomycin (15 µM) or cycloheximide (25 µM) significantly impeded the mGluR-mediated development of the prolonged bursts; 90-120 min of agonist application failed to elicit the expected burst prolongation. By contrast, the mGluR-mediated enhancement of burst frequency progressed unimpeded. Furthermore, protein synthesis inhibitors had no significant effect on the frequency or duration of fully developed mGluR-induced prolonged discharges. These results suggest that the group I mGluR-mediated prolongation of synchronized bursts has a protein synthesis-dependent mechanism.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Long-term potentiation (LTP) and depression (LTD) are well-studied plasticity phenomena in central synapses. Modifications of synaptic transmission underlying LTP and LTD involve the phosphorylation and dephosphorylation of target proteins (Huang and Kandel 1994; Klann et al. 1993; Malinow et al. 1988; Mulkey et al. 1993; Nicoll et al. 1988; Sacktor et al. 1993). Recent experiments suggest that protein synthesis may also be involved in these plastic processes (Fazeli et al. 1993; Frey et al. 1988; Kang and Schuman 1996; Linden 1997; Montarolo et al. 1986; Nguyen et al. 1994; Osten et al. 1996; Otani et al. 1989; Stanton and Sarvey 1984). In general, the requirement of protein synthesis for sustaining synaptic efficacy changes is correlated with the enduring nature of the change (Bailey et al. 1996; Davis and Squire 1984).

Plasticity in the neuronal circuit can result in epileptogenesis, a persistent propensity of the cortical network to produce seizure discharges. Experiments on kindling-induced epileptogenesis reveal that this process may also be protein synthesis dependent (Cain et al. 1980; Jones et al. 1992; Ogata 1977).

Activation of metabotropic glutamate receptors (mGluRs) has been shown to have convulsant effects (McDonald et al. 1993; Sacaan and Schoepp 1992), and we have elicited group I mGluR-mediated prolonged epileptiform discharges in vitro as well (Merlin and Wong 1997b; Taylor et al. 1995). Furthermore, our data suggest that group I mGluR activation has epileptogenic properties: transient mGluR activation results in persistent epileptiform activity, with long-term autopotentiation of the group I mGluR response accompanying this modification (Merlin and Wong 1997b). In the following studies we address whether the induction and/or maintenance of the epileptiform activities produced via mGluR activation requires active protein synthesis. If the process is protein synthesis dependent, it would further support the conclusion that group I mGluR activation initiates an enduring epileptogenic process. Portions of this work have appeared in abstract form (Merlin and Wong 1997a).

	METHODS

Abstract Introduction Methods Results Discussion References

Guinea pigs 2-4 wk of age were anesthetized with halothane and decapitated in conformance with the recommendations of the Guide for the Humane Care and Use of Animals. The brain was rapidly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 26 NaHCO₃, 5 KCl, 1.6 MgCl₂, 2 CaCl₂, and 10 D-glucose. Transverse hippocampal slices (400 µm) were prepared with the use of a Vibratome (Technical Products International), placed on nylon mesh in an interface chamber (Fine Science Tools), and perfused with ACSF bubbled with 95% O₂-5% CO₂ and maintained at 35.5°C and pH 7.4. Perfusion rate and dead space accounted for a 10-15 min lag between onset of drug application and initial onset of effect.

Electrophysiological data shown were obtained via intracellular recording from the CA3 stratum pyramidale with the use of 25-60 M thin-walled glass microelectrodes filled with 2 M potassium acetate. Recordings were amplified and digitized (Axoclamp 2A and TL1/Labmaster DMA Interface, Axon Instruments) and drugs were applied via continuous bath perfusion. Picrotoxin (Sigma; 50 µM), an antagonist of GABA_A receptor-mediated inhibition, was used in all experiments to elicit baseline epileptiform activity. Cycloheximide and anisomycin were obtained from Sigma; mGluR agonists were from Tocris Cookson.

Instantaneous burst frequency (Hz) represents the reciprocal of the interburst interval (seconds). Statistical significance was determined by the paired Student's t-test. For analyses across experimental protocols, data were normalized and percent change was compared. Data are reported as means ± SE.

	RESULTS

Abstract Introduction Methods Results Discussion References

Effect of group I mGluR activation on picrotoxin-induced discharges

Bath application of 50 µM picrotoxin produced epileptiform synchronized bursting that occurred in a spontaneously recurring pattern (0.138 ± 0.004 Hz, mean ± SE; n = 14). Each burst was no longer than 520 ms (375 ± 15 ms, n = 14). Introduction of the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 25 µM) rapidly elicited a marked increase in the burst frequency (peak frequency 0.285 ± 0.011 Hz, n = 14; Fig. 1). This was accompanied by a slowly progressive increase in burst duration (BD), which often became evident within 30 min of agonist introduction and reached statistical significance by 60 min (BD at 60 min, 592 ± 67 ms, n = 14). As BD increased, there was a parallel decrease in burst frequency. The enhanced epileptiform activity fully developed within 120-150 min of agonist application, after which time there was no significant further alteration in the frequency or duration of epileptiform bursts (peak BD, 2,022 ± 267 ms, n = 14; see Fig. 1). Similar results were observed with another group I mGluR agonist, (S)-3-hydroxyphenylglycine (data not shown) (see Merlin and Wong 1997b).

View larger version (40K): [in this window] [in a new window]   FIG. 1. Group I metabotropic glutamate receptor (mGluR)-mediated epileptogenesis. A: example of (S)-3,5-dihydroxyphenylglycine (DHPG) effect on picrotoxin-induced epileptiform activity. Continuous intracellular recording from a CA3 pyramidal neuron. In all graphs, each epileptiform discharge is represented by 2 symbols: an open circle, indicating instantaneous burst frequency (left y-axis), and a filled circle, indicating the duration of the burst (right y-axis). Picrotoxin was present throughout all experiments. Time 0 represents onset of DHPG application. Time indicated in A1 corresponds to time displayed in A2. B: summary data, n = 14 slices. Error bars = SE.

Protein synthesis inhibitors impede mGluR-mediated induction of burst prolongation

In 14 slices undergoing picrotoxin-induced epileptiform bursting, anisomycin (15 µM; n = 8) or cycloheximide (25 µM; n = 6) was applied 15-100 min before the introduction of the mGluR agonist DHPG. The picrotoxin-induced epileptiform activity was not significantly modified by the presence of these agents (BD_control, 419 ± 25 ms; after protein synthesis inhibitors, 394 ± 19 ms; n = 9; P > 0.05).

Under these conditions, the mGluR agonist-induced increase in burst frequency was rapidly elicited and was sustained longer than in the control (peak burst frequency in anisomycin, 0.389 ± 0.018 Hz, n = 8, Fig. 2; in cycloheximide, 0.346 ± 0.019 Hz, n = 6). Coinciding with the significant enhancement of burst frequency was a modest reduction in BD in the first 30-60 min of agonist application (BD_anisomycin at 30 min, 256 ± 19 ms, n = 8, Fig. 2; in cycloheximide, 350 ± 28 ms, n = 6).

View larger version (49K): [in this window] [in a new window]   FIG. 2. Anisomycin impedes group I mGluR-mediated epileptogenesis. A: example of anisomycin effect on DHPG-mediated prolongation of epileptiform bursts. Continuous recording from a CA3 pyramidal neuron. Time 0 represents onset of DHPG application; time in A1 correlates with that indicated next to traces in A2. At 2 h of agonist application there is an increase in burst duration variability, with short bursts predominating. B: summary data, n = 6, which includes only those slices in which no long bursts developed at 90-min application. C: direct comparison of normalized data from slices represented in Fig. 1B vs. those represented in Fig. 2B. Asterisks over individual bars indicate statistically significant change from activity recorded just before agonist application. * P < 0.05; ** P < 0.01; *** P < 0.001. All bar pairs were significantly different from each other.

In 9 of the 14 slices, 90-120 min of DHPG application in the presence of protein synthesis inhibitor failed to elicit the expected prolongation of BD (BD_anisomycin at 90 min, 227 ± 17 ms, n = 6, Fig. 2, B and C; in cycloheximide 336 ± 44 ms, n = 3). In the five remaining slices, burst lengthening did eventually occur, but this was usually no sooner than 1-2 h into the agonist application period. The longer bursts in these experiments were sporadic and interspersed among a predominance of brief (i.e., <500 ms) bursts (e.g., Fig. 2A at 120 min), and they remained intermittent 1 h after their initial appearance.

Effect of protein synthesis inhibitors on fully developed mGluR-mediated epileptiform activity

To determine whether active protein synthesis was required for the expression of the mGluR-mediated prolonged epileptiform discharges, a protein synthesis inhibitor was introduced to the perfusing solution of 11 slices in which DHPG was present for 2 h. Anisomycin (15 µM) for 90-120 min failed to affect significantly either the duration or the frequency of the fully developed prolonged discharges (BD_{90 min}, 2,237 ± 393 ms at frequency of 0.100 ± 0.008 Hz, compared with BD_{pre-anisomycin}, 1,825 ± 242 ms at frequency of 0.113 ± 0.007 Hz, n = 7, P > 0.05; Fig. 3). Similar results were seen with 25 µM cycloheximide (BD_{90 min}, 2,027 ± 128 ms, frequency 0.107 ± 0.009 Hz, compared with BD_{pre-cycloheximide}, 1,825 ± 299, frequency 0.118 ± 0.016, n = 4, P > 0.05).

View larger version (15K): [in this window] [in a new window]   FIG. 3. Failure of protein synthesis inhibitors to affect either frequency or duration of prolonged epileptiform bursts. A: example of anisomycin effect on DHPG-induced fully developed prolonged discharges. B: summary data, n = 7 slices. Time 0 refers to onset of anisomycin application, which was begun 2 h after onset of DHPG application to allow adequate time for near-full development of DHPG effect.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Group I mGluR-mediated enhancement of synchronized burst frequency

Our data reveal that selective group I mGluR activation elicits a dramatic increase in the frequency of synchronized bursts. The transience of this effect may be secondary to the progressive increase in BD, and indeed the period of enhancement of burst frequency is lengthened when the burst prolongation is suppressed (see Fig. 2). We have previously shown that group II mGluR activation also increases burst frequency (Merlin et al. 1995). The group II mediated effect is more modest, plateaus with sustained agonist application, and is readily reversible upon the removal of agonist. Whether these two receptor systems with different signal transduction mechanisms ultimately converge on a common path to elicit this shared effect remains to be explored.

Group I mGluR-mediated epileptogenesis

Selective group I mGluR activation elicits a slowly progressive prolongation of BD, suggesting a role for these receptors in the interictal-to-ictal transition. This burst prolongation persists on removal of agonist (Merlin and Wong 1997b), implicating a long-term modification underlying the mGluR-mediated ictogenesis. Group I mGluR activation is known to activate generation of IP3, leading to an increase of intracellular free calcium (Abe et al. 1992; Houamed et al. 1991; Masu et al. 1991), which could contribute to a progressive, lasting modification of cellular properties enhancing excitability and leading to long-term epileptogenesis.

Role of protein synthesis in mGluR-mediated epileptogenesis

Anisomycin suppressed the group I mGluR-mediated prolongation of synchronized bursts without affecting the agonist's ability to enhance burst frequency. In fact it appears that the agonist effect on burst frequency is normally masked by the concomitant increase in BD, which secondarily reduces the frequency. The two effects thus may be independently generated, perhaps revealing separate effects of mGluR1 versus mGluR5 activation. Whatever the process mediating the burst prolongation (as opposed to that increasing the burst frequency), it appears to be protein synthesis dependent.

In 36% of slices in which induction of prolonged bursts was attempted in the presence of protein synthesis inhibitors, sporadic prolonged bursts did eventually appear. This may have been the result of incomplete inhibition of protein synthesis. Studies in hippocampal slices have revealed that 10 µM anisomycin inhibits incorporation of [³⁵S]methionine into polypeptides by 96 ± 4%, and 30-60 µM cycloheximide inhibits total protein synthesis by 82 ± 3% (Osten et al. 1996).

If basal proteins present could serve as a substrate to support epileptogenesis, one would expect mGluR activation to elicit a submaximal level of burst prolongation in the presence of protein synthesis inhibitors. The absence of such an effect suggests that the basal level of the required protein at the time of agonist introduction is insufficient for the expression of the response. Thus new protein synthesis is required. Indeed, activation of group I mGluRs was shown to activate protein synthesis in synaptoneurosomes (Weiler and Greenough 1993). These data therefore suggest that group I mGluR-mediated activation of protein synthesis may contribute to the long-term changes underlying epileptogenesis.

	ACKNOWLEDGEMENTS

   The authors thank S. Galoyan for assistance with this manuscript.

  This work was funded in part by the Pharmaceutical Research and Manufacturers of America Foundation (to L. R. Merlin) and by the National Institute of Neurological Disorders and Stroke.

	FOOTNOTES

  Address for reprint requests: L. R. Merlin, SUNY Health Science Center at Brooklyn, 450 Clarkson Ave., Box 29, Brooklyn, NY 11203.

  Received 11 March 1998; accepted in final form 21 April 1998.

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