Role of Group I Metabotropic Glutamate Receptors in the Patterning of Epileptiform Activities In Vitro

Lisa R. Merlin and Robert K. S. Wong

Departments of Neurology 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. and Robert K. S. Wong. Role of group I metabotropic glutamate receptors in the patterning of epileptiform activities in vitro. J. Neurophysiol. 78: 539-544, 1997. In guinea pig hippocampal slices, picrotoxin elicited spontaneous epileptiform bursts 300-550 ms in duration. Additional application of (R,S)-3,5-dihydroxyphenylglycine or (S)-3-hydroxyphenylglycine, agonists specific for group I metabotropic glutamate receptors(mGluRs), or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylicacid, a broad-spectrum mGluR agonist, converted picrotoxin-induced interictal bursts into prolonged discharges measured on the order of seconds. The prolonged discharges induced by selective group I mGluR agonist continued to be produced for hours after agonist removal. The antagonists (S)-4-carboxyphenylglycine and (+)-alpha -methyl-4-carboxyphenylglycine had no effect on the duration of picrotoxin-induced interictal bursts. However, after agonist exposure, the persistent prolonged discharges occurring in the absence of agonist were reversibly suppressed by the antagonists, suggesting that the activity is maintained via endogenous activation of group I mGluRs by synaptically released glutamate. Our results suggest that, under some conditions, activation of group I mGluRs produces long-lasting enhancement of synaptic responses, mediated at least in part by autopotentiation of the group I mGluR response itself, which may result in the production of seizure discharges and contribute to epileptogenesis.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Electrographic recordings show that the epileptic phenomenon is associated with two types of synchronized discharges of cortical neurons: the brief interictal spikes or sharp waves (<500 ms in duration with no perceptible behavioral correlate) and the prolonged ictal discharges underlying seizures (measured on the order of s to min) (Zifkin and Cracco 1990). The role of ionotropic glutamate receptor activation in the generation of such activities has been extensively studied in vitro (Anderson et al. 1990; Dingledine et al. 1986; Miles et al. 1984; Traub and Wong 1982; Traub et al. 1996), but we are only beginning to recognize the importance of the metabotropic glutamate receptors (mGluRs) in determining the overall patterning of synchronized epileptiform discharges (Arvanov et al. 1995; Burke and Hablitz 1995; McBain et al. 1994; Merlin et al. 1995; Taschenberger et al. 1992). We have previously demonstrated that activation of group II mGluRs rapidly and reversibly elicits an increase in epileptiform burst frequency when the agonist is applied in the presence of ongoing picrotoxin-induced epileptiform activity in guinea pig hippocampal slices (Merlin et al. 1995). With the use of this same model, we now examine the effects of selective group I mGluR activation. Portions of this work have appeared in abstract form (Merlin and Wong 1996a,b).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Guinea pigs 2-4 wk of age were anesthetized with halothane before decapitation. The brain was promptly removed from the cranium and placed in icy artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 5 KCl, 1.6 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 D-glucose. A Vibratome (Technical Products International) was used to prepare 400-µm transverse hippocampal slices, which were placed on nylon mesh in an interface chamber 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 initial onset of effect.

Intracellular recordings were obtained from the CA3 stratum pyramidale with the use of thin-walled glass microelectrodes containing 2 M potassium acetate (25-75 MOmega tip resistance).Recordings were amplified and digitized (Axoclamp 2A andTL1/Labmaster DMA Interface, Axon Instruments). Drug effects were elicited via bath perfusion. Picrotoxin (50 µM) was present in all experiments to elicit baseline epileptiform activity. Picrotoxin was obtained from Sigma; mGluR agonists and antagonists were purchased from Tocris Cookson.

To study drug effects across slices, percent change in mean burst duration was determined for each slice; Student's t-test was used to determine statistical significance. Percent change is reported in the text as mean ± SD.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Effect of mGluR agonists on picrotoxin-induced epileptiform discharges

Hippocampal neurons in the slice preparation exhibit spontaneous synchronized bursting activity in the presence of picrotoxin, a gamma -aminobutyric acid-A receptor antagonist (Schwartzkroin and Prince 1978). These synchronized bursts rarely exceed 500 ms in duration and thus resemble interictal discharges. Figure 1 (control) shows one such synchronized burst, which typically consisted of an initial abrupt discharge 100-200 ms in duration (primary burst) followed by a series of briefer phasic discharges (secondary bursts). Addition of the broad-spectrum mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 50-100 µM) (Schoepp et al. 1991; Watkins and Collingridge 1994) or the selective group I mGluR agonists (R,S)-3,5-dihydroxyphenylglycine (DHPG, 50-100 µM) (Ito et al. 1992) or (S)-3-hydroxyphenylglycine (3HPG, 250-500 µM) (Birse et al. 1993; Hayashi et al. 1994) significantly prolonged the synchronized burst duration, primarily via a marked increase in the number of secondary bursts (Fig. 1). These prolonged discharges ranged from 1 to 7 s in duration (ACPD: 2,224 ± 674 ms, mean ± SD, a 508 ± 157% increase over control, n = 6; DHPG: 2,156 ± 617 ms, a 403 ± 199% increase, n = 7; 3HPG: 4,282 ± 1,060 ms, a 1,021 ± 247% increase, n = 7), and the secondary bursts within each discharge gradually changed in amplitude and frequency (Taylor et al. 1995); the prolonged discharges thus appeared analogous to electrographic ictal events (Zifkin and Cracco 1990).


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FIG. 1. Prolongation of epileptiform discharges induced by group I metabotropic glutamate receptor (mGluR) activation. A: summary graph demonstrating mean duration of synchronized bursts elicited by picrotoxin (control) and maximum duration of prolonged bursts during application of the selective group I mGluR agonist (S)-3-hydroxyphenylglycine (3HPG, 250-500 µM, n = 7 slices). Error bars in all figures: mean ± SE. B: intracellular recording from a CA3 pyramidal cell in a hippocampal slice before and during exposure to 250 µM 3HPG. Similar activity could be elicited with either (R,S)-3,5-dihydroxyphenylglycine (DHPG) or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 50-100 µM each).

Selective group I mGluR agonists: comparison with ACPD effect

The prolonged synchronized discharges elicited with ACPD or the selective group I agonists appeared promptly on exposure to the agonist, but continued to develop in a slowly progressive manner, requiring up to 2 h before reaching maximum (Fig. 2A). However, the actions of group I agonists and ACPD differed significantly in one way. Although the ACPD-induced prolonged bursts rapidly returned to control duration within 1 h after exposure, 1 h of washout of either group I agonist only reduced the burst prolongation by 20% and 35% for DHPG (n = 6) and 3HPG (n = 5), respectively (Fig. 2). Because ACPD activates both group I and group II mGluRs, we examined whether the simultaneous activation of group I and II receptors would suppress persistent burst prolongation. In three experiments, selective group I agonist was applied in the presence of 5-10 µM ACPD, which selectively activates group II mGluRs (Cartmell et al. 1993). The prolonged bursts induced by the mixture were no less persistent than those elicited by group I agonist alone (79 ± 9% of peak prolongation remaining after 1 h of washout). These results suggest that the reversibility of ACPD-induced effects cannot be accounted for by the simultaneous activation of group I and group II receptors. At present, our data do not reveal the reason for the difference in the effects of ACPD and specific group I agonists. It may be the result of additional activities of the agents at as yet unidentified receptors (Chung et al. 1994; Pellegrini-Giampietro et al. 1996).


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FIG. 2. Persistent burst prolongation following transient exposure to group I mGluR agonists. A: representative examples of time course of prolongation of burst duration (BD) induced by transient agonist exposure. Data points represent individual bursts recorded from a single CA3 pyramidal cell; gaps in graphs are due to intermittent sampling. A1: exposure to 500 µM 3HPG. Note slowly progressive development (100-120 min to peak); also note significant persistence of effect 3 h after removal of agonist. A2: ACPD (50 µM) had a similar time course to peak effect, but the effect rapidly returned to control duration post exposure. Time course of DHPG washout resembled that of 3HPG, not ACPD. B: summary data for postexposure persistence of potentiation, normalized relative to peak potentiated component [(BD - control BD) × 100/(peak - control BD)]. For ACPD, n = 4; for DHPG, n = 6 at 0.5-2 h, n = 3 at 3 and 4 h. 3HPG effect was tested to 3 h after exposure (n = 5); there was no significant difference between the persistence of the effect of 3HPG and that of DHPG (P > 0.05).

Effects of group I mGluR antagonists

( + ) - alpha  - M e t h y l - 4 - c a r b o x y p h e n y l g l y c i n e   ( M C P G )   a n d(S)-4-carboxyphenylglycine (4CPG) both have antagonist action at group I mGluRs (Hayashi et al. 1994). Neither MCPG (500-1000 µM) (see Merlin et al. 1995) nor 4CPG (400-600 µM, n = 6) had any effect on the duration of picrotoxin-induced interictal bursts. However, either agent could suppress the induction of burst prolongation mediated by group I mGluR agonists. As shown in Fig. 3A, transient (35 min) application of 50 µM DHPG in the presence of antagonist had no effect on burst duration. On 35 min of reexposure in the absence of antagonist, persistent burst prolongation appeared.


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FIG. 3. Group I antagonists suppress persistent bursts. A1: application of 50 µM DHPG in the presence of 600 µM (S)-4-carboxyphenylglycine (4CPG) failed to elicit any significant effect on burst duration. Failure of agonist effect during washout of antagonist reveals that agonist is not lingering in tissue to account for slow decay of potentiation seen in Fig. 2. A2: summary data, n = 3 slices. DHPG (50 µM) and 4CPG (400-600 µM) applied as shown in A1. Percent change in burst duration was measured relative to mean burst duration during 5 min immediately preceding initial drug application. DHPG/4CPG: mean burst duration during final 5 min of 1st DHPG application. Wash is measured during the final 5 min before second DHPG application. DHPG: peak effect measured at the end of the final DHPG application. Asterisk: statistically significant change from control (P < 0.05). B1: example of DHPG-induced postexposure potentiation as revealed via continuous recording from a CA3 pyramidal cell. DHPG, 100 µM; 4CPG, 400 µM. 4CPG initially fractionated (see inset), then markedly suppressed potentiated bursts. Washout of antagonist resulted in a prompt restoration of prolonged bursts, indicating that bursts are generated by synaptically released glutamate acting at group I mGluRs. Traces 1-4 correspond to circled numbers on graph. B2: summary data, n = 3 slices. Percent change in burst duration was measured relative to mean burst duration preceding agonist application. Post exposure: burst duration was measured 1 h after washout of agonist. Wash was measured at 1 h after washout of antagonist (3-4 h after agonist removal). Application of antagonist significantly attenuated prolonged bursts (P < 0.05). After antagonist washout, burst prolongation reappeared to a level that was no longer significantly different from postexposure level (P > 0.05); it was, however, significantly different from abbreviated bursts during antagonist application (P < 0.05).

Additional experiments revealed that prolonged bursts persisting after agonist washout were reversibly suppressed by group I mGluR antagonists (Fig. 3B). This suppressive effect sometimes initially manifested itself by disrupting the rhythmic patterning of the secondary bursts, fractionating the secondary bursts into clusters and resulting in a transient increase in overall discharge duration (Fig. 3B1, inset). This would be immediately followed by the rapidly progressive, reversible suppression of the secondary bursts that reduced the overall burst duration to near that of control.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Group I mGluR activation prolongs epileptiform discharge duration

We have previously demonstrated in hippocampal slices that, at low concentrations, ACPD increases epileptiform burst frequency without affecting burst duration (Merlin et al. 1995), whereas at higher concentrations it prolongs the epileptiform burst duration (Taylor et al. 1995). Here we demonstrate that selective group I mGluR agonists also prolong the burst duration, and that group I mGluR antagonists prevent the induction of this effect. Thus, although group II mGluR activation appears to modulate the frequency of interictal burst activity (Merlin et al. 1995), group I mGluR activation may play a role in converting brief interictal discharges into more prolonged ictal events.

Group I agonist effects are long lasting

The persistent burst prolongation elicited by selective group I mGluR agonists (Fig. 2) suggests that a long-term modification has been induced; however, slow clearance of an agent from the tissue during washout could result in the apparent persistence of its effects. For example, the agent may have a high lipid solubility and be absorbed into the cell membrane during exposure, then slowly rereleased into the extracellular solution during washout (Furchgott 1964). Figure 3A demonstrates that this mechanism is unlikely to account for the persistence of effect seen here. In this series of experiments, group I mGluR agonist and antagonist were coapplied. No burst prolongation was observed, suggesting that the antagonist blocked the receptor-mediated effects of the agonist. Antagonist, however, would not be expected to affect processes such as absorption of agents into cell membranes; thus, if group I mGluR agonist were accumulating in the tissue, this process should proceed uninhibited. Available data also suggest that the antagonist action is readily reversible (see for example Fig. 3B). Therefore, if indeed agonist were accumulating or lingering in the system, one would expect the agonist effect (i.e., burst prolongation) to emerge on removal of the antagonist during washout of both agonist and antagonist. However, the data revealed no significant change in burst duration in the 2-h period following simultaneous exposure to both agonist and antagonist (Fig. 3A). These results suggest that the persistent burst prolongation cannot be easily explained by slow clearance of the agonist during washout.

Persistence of agonist effects could also be caused by a slow rate of dissociation of the agonist-receptor complex. In this case, the decay rate of the potentiated response would represent the rate at which receptors become unbound from the agonist. If so, addition of antagonist during washout should not have a significant effect on the decay of the potentiated responses, assuming the usual action of antagonists in that they can bind to free receptors but cannot displace agonists from the agonist-receptor complex (Furchgott 1964). The data here (Fig. 3B) show that the suppression of prolonged bursts by 4CPG occurred at a significantly faster rate than the decay of the potentiated response during washout. Moreover, we observed a recovery of the potentiated response on washout of the antagonist (Fig. 3B), a finding that is inconsistent with the hypothesis that the persistent effect of DHPG and 3HPG is due to a slow dissociation rate of the agonist-receptor complex.

Enhancement of the group I mGluR response sustains the persistent burst prolongation

The results shown in Fig. 3 suggest that potentiated bursts during postexposure cannot be explained by a slow clearance of the agonist during the washout period. And yet, in the absence of exogenous agonist, potentiated responses are still suppressed by group I antagonists (Fig. 3B), suggesting that group I mGluR-mediated responses sustain the prolonged bursts. Because the only source of agonist available is the synaptically released glutamate, our data suggest that the prolonged synchronized discharge is maintained by a long-lasting potentiation of the group I mGluR-mediated response, now allowing endogenous glutamate to elicit the response. Recent studies have shown that activation of mGluRs in the hippocampus produces a persistent enhancement of neuronal responses mediated by ionotropic glutamate receptors, which contributes to a long-lasting potentiation of the synaptic responses (Aniksztejn et al. 1992; Bortolotto and Collingridge 1995; Chinestra et al. 1993; Harvey and Collingridge 1993; Mannaioni et al. 1996). Our results suggest that, under some conditions, activation of group I mGluRs can also potentiate synaptic responses mediated by the mGluR itself, allowing the generation of seizure-length synchronized discharges. This modifiability of the group I mGluR response may constitute one of the processes responsible for epileptogenesis.

    ACKNOWLEDGEMENTS

  We thank G. W. Taylor and R. F. Furchgott for helpful discussions.

  This work was supported by grants from the National Institutes of Health, the Epilepsy Foundation of America (L. R. Merlin), and the PhRMA Foundation (L. R. Merlin).

    FOOTNOTES

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

  Received 26 December 1996; accepted in final form 19 March 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society