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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ABSTRACT |
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Galoyan, Samvel M. and Lisa R. Merlin. Long-Lasting Potentiation of Epileptiform Bursts by Group I mGluRs Is NMDA Receptor Independent. J. Neurophysiol. 83: 2463-2467, 2000. In CA3 pyramidal cells of guinea pig hippocampal slices, picrotoxin (50 µM) elicited spontaneous, rhythmically recurring epileptiform bursts 285-435 ms in duration. The addition of (S)-3,5-dihydroxyphenylglycine (DHPG, 50 µM, 90 min application), a selective group I metabotropic glutamate receptor (mGluR) agonist, resulted in a rapid-onset transient increase in burst frequency. This was followed by a slowly progressive increase in burst duration, with bursts reaching 1.5-3.8 s in duration at 90 min of DHPG application. The potentiation of epileptiform burst duration persisted at least 2 h after agonist removal. To determine whether N-methyl-D-aspartate (NMDA) receptor activation participates in the mGluR-induced potentiation of epileptiform bursts, experiments were carried out in the presence of D-2-amino-5-phosphonovaleric acid (APV, 50-100 µM), an NMDA receptor antagonist. Application of DHPG in the presence of APV resulted in a significantly enhanced transient increase in burst frequency. Nevertheless, when compared with the control described above, there was no significant alteration in the rate of development of the burst prolongation nor its persistence after washout. In other experiments, application of APV in the presence of fully developed mGluR-induced potentiated bursts (after 90 min washout of DHPG) resulted in no significant change in either burst frequency or duration. The data reveal that both induction and maintenance of group I mGluR-mediated potentiation of epileptiform discharges are NMDA receptor-independent processes, suggesting that epileptogenesis, when induced by group I mGluR activation, may occur and be sustained in the absence of NMDA receptor activation.
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INTRODUCTION |
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Group I metabotropic glutamate receptor (mGluR)
activation produces two effects on ongoing interictal activity in
vitro: a rapid-onset, transient acceleration of interictal bursting and a slowly progressive, persistent prolongation of individual
synchronized bursts into 1-12 s events (Merlin 1999;
Merlin and Wong 1997
; Merlin et al.
1998a
; Taylor et al. 1995
). The persistent
increase in burst duration has a protein synthesis-dependent induction mechanism (Merlin et al. 1998a
), which supports the
conclusion that a long-term modification of cortical network properties
has occurred. Such group I mGluR-mediated network plasticity may
contribute to epileptogenesis, the process in which the network
develops a long-lasting propensity for the production of seizure discharges.
Experiments examining the mechanisms for synaptic and network
plasticity in the hippocampus frequently demonstrate a requirement of
N-methyl-D-aspartate (NMDA) receptor
activation for induction to occur (Collingridge and Bliss
1987; Lynch et al. 1990
; Morris et al.
1986
; Slater et al. 1985
; Stasheff et al.
1993
; Swartzwelder et al. 1989
; Young and
Dragunow 1994
). Potentiated NMDA receptor-mediated responses
may also participate in sustaining the enhancement of network events
(Traub et al. 1994
, 1996
). The studies presented herein
address the role of NMDA receptor activation in the induction and
maintenance of group I mGluR-mediated epileptogenesis. Portions of this
work have appeared in abstract form (Merlin et al.
1998b
).
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METHODS |
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Guinea pigs (2-4 wk old) were anesthetized with 2-bromo-2-chloro-1,1,1-trifluoroethane and decapitated. Brains were promptly placed in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 5 KCl, 1.6 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 D-glucose. Hippocampi were 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 and perfused with ACSF bubbled with 95% O2-5% CO2 at pH 7.4. Temperature was maintained at 35.5-36.0°C.
Intracellular recordings were obtained from the CA3 stratum
pyramidale using thin-walled glass microelectrodes with tip resistance of 30-80 M and filled with 2 M potassium acetate. Recordings were
amplified and digitized (Axoclamp 2A and TL-1 DMA Interface, Axon
Instruments). Drugs were applied via continuous bath perfusion. Picrotoxin (50 µM) was present throughout all experiments to elicit baseline epileptiform activity. (S)-3,5-dihydroxyphenylglycine (DHPG)
and D-2-amino-5-phosphonovaleric acid (APV) were obtained from Tocris Cookson (Ballwin, MO). All other chemicals were obtained from Sigma (St. Louis, MO).
Burst duration (BD) was measured from the onset of primary burst depolarization until the end of the final secondary burst and did not include the postburst afterhyperpolarization. Instantaneous burst frequencies (Hz) represent the reciprocal of the interburst intervals (in seconds). Statistical significance was determined using Student's t-test (paired t-test was used to evaluate changes in given slices over time; unpaired was used for differences between treatment groups). A P value <0.05 was considered significant. Data are reported as mean ± SE.
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RESULTS |
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Effects of group I mGluR activation on picrotoxin-induced epileptiform activity
In the presence of picrotoxin, an antagonist of GABAA receptor-mediated inhibition, CA3 pyramidal cells of guinea pig hippocampal slices expressed spontaneous synchronized bursts 321 ± 10 ms in duration recurring regularly at 0.13 ± 0.01 Hz (n = 6). The addition of 50 µM DHPG, a selective group I mGluR agonist, resulted in a rapid-onset increase in the frequency of bursts (peak frequency 0.48 ± 0.02 Hz 10 min after introduction of agonist). This was accompanied by a mild decrease in burst duration (BD10 min 250 ± 18 ms, P < 0.01, n = 6; Fig. 1A). Continued exposure to the agonist elicited a gradual increase in BD and a concurrent return of burst frequency to baseline (BD90 min 2264 ± 370 ms at 0.12 ± 0.02 Hz; n = 5). At 2 h of agonist removal, BD was still significantly enhanced over the initial picrotoxin-induced BD (BD2-h wash 1826 ± 269 ms at 0.12 ± 0.01 Hz, 475 ± 80% potentiated, n = 5; Fig. 1A).
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Effect of NMDA receptor antagonist on the induction of group I mGluR-mediated enhancement of epileptiform activity
To investigate the role of NMDA receptors in the induction of group I mGluR-mediated prolongation of epileptiform bursts, experiments were performed in the presence of 50-100 µM APV, an NMDA receptor antagonist (Fig. 1B). APV caused a significant reduction in the duration of picrotoxin-induced synchronized bursts from 343 ± 13 to 284 ± 22 ms (P < 0.005) without modifying the burst frequency (0.15 ± 0.01 Hz, n = 5). The subsequent application of the group I mGluR agonist DHPG in the presence of APV resulted in a markedly enhanced increase in burst frequency (peak frequency 0.66 ± 0.07 Hz at 10 min, P < 0.05 vs. slices exposed to DHPG alone for 10 min; Fig. 1, B and C) with no initial significant change in BD (208 ± 22 ms, n = 5). Nevertheless, at 90 min of DHPG exposure, the burst frequency returned to 0.11 ± 0.01 Hz and BD reached 2354 ± 389 ms (similar to that seen in the absence of APV, P > 0.05, n = 5). The BD remained significantly potentiated throughout a 2-h washout period (BD2-h wash 2326 ± 403 ms at 0.11 ± 0.01 Hz, 589 ± 132% potentiated, n = 5; Fig. 1, B and D). Comparison of these data with control experiments revealed that the presence of NMDA receptor antagonist during DHPG application did not impede the development of burst prolongation (Fig. 1C), nor did it affect the persistence of the epileptiform burst potentiation during 2 h of washout of the mGluR agonist (Fig. 1D).
Effect of NMDA receptor antagonist on the maintenance of persistent prolonged epileptiform bursts
A second set of APV experiments (n = 4) was designed to determine whether the activation of NMDA receptors is necessary for the maintenance of mGluR-induced prolonged discharges. In these experiments, NMDA receptor antagonist was introduced after 90 min of DHPG washout (Fig. 2). Baseline picrotoxin bursts were induced as usual (BD 390 ± 8 ms at 0.11 ± 0.02 Hz); 90 min of DHPG application prolonged the BD to 2453 ± 314 ms at 0.08 ± 0.01 Hz. At 90 min of agonist washout, there was no significant change in BD or burst frequency (1990 ± 332 ms and 0.10 ± 0.02 Hz, respectively, n = 4). On the addition of APV for 30 min, BD remained significantly potentiated as compared with the initial picrotoxin-induced bursts (1924 ± 285 ms at 0.10 ± 0.01 Hz, P < 0.01). Comparison of the persistent burst potentiation at 120 min of washout of DHPG in control slices with that seen at an equivalent time with 30 min of APV application revealed no significant difference between the two groups (Fig. 2B).
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DISCUSSION |
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Enhancement of interictal burst frequency during NMDA receptor blockade is a transient effect
The depolarization that underlies the picrotoxin-induced
interictal burst is largely carried by synaptic excitatory events mediated by -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptors (Dingledine et al. 1986
;
Miles et al. 1984
; Traub and Wong 1982
;
Traub et al. 1993
). On blockade of NMDA receptors, the
picrotoxin-induced interictal discharge was therefore significantly
abbreviated. This phenomenon was a useful indicator of effective block
of NMDA receptors.
Group I mGluR activation produces a transient acceleration of
interictal epileptiform bursts (Merlin and Wong 1997;
Merlin et al. 1998a
). Similar mGluR-induced burst
acceleration was observed in spinal cord locomotor burst frequency in
the lamprey (Krieger et al. 1998
) and was attributed to
potentiation of NMDA-induced depolarizations. However, in our system,
the acceleration of interictal bursting was markedly enhanced when
induced in the presence of NMDA receptor antagonist (Fig.
1C), making it unlikely that potentiation of NMDA
receptor-mediated responses accounts for the burst acceleration. Furthermore, the lack of a concurrent significant shortening of burst
duration at the time of maximal acceleration (compared with control;
Fig. 1C) makes the shortening of interictal bursts in APV an
inadequate explanation for the enhancement of burst frequency. The
mechanism for the frequency modification remains unclear. Nevertheless,
the transient enhancement of interictal burst frequency had no enduring
effect on the ultimate frequency or duration of the potentiated
epileptiform bursts.
Induction of mGluR-mediated burst prolongation is not NMDA receptor dependent
The group I metabotropic receptor mGluR5 can be desensitized by
phosphorylation (Gereau and Heinemann 1998). This
desensitization can be reversed by an NMDA receptor-dependent
dephosphorylation process (Alagarsamy et al. 1999
). One
might therefore expect NMDA receptor activation to be necessary for the
prolongation of epileptiform bursts during group I mGluR activation.
However, our data demonstrate that group I mGluR-mediated
epileptogenesis will proceed unimpeded in the absence of concurrent
NMDA receptor activation.
We previously demonstrated that transient group I mGluR activation
during suppression of ionotropic glutamate receptor (iGluR)-mediated activity results in the appearance of persistent potentiated
epileptiform bursts upon washout of the iGluR antagonists
(Merlin 1999). Because the experiments previously
reported were performed in the presence of both NMDA and AMPA receptor
antagonists, all spontaneous synchronized bursting activity was
suppressed during the DHPG application period, leaving no way to gauge
the efficacy of the NMDA blockade. The current data, in contrast, allow
the confirmation of effective NMDA antagonism (through the observation
of shortened interictal bursts), thereby strengthening the conclusion
that NMDA receptor activation is not necessary for group I
mGluR-mediated induction of epileptogenesis. Furthermore, the design of
the current experiments allows for assessment of the rate of
development of the mGluR-induced modifications. The data reveal no
delay in the development of the mGluR-mediated burst prolongation when
induced in the absence of NMDA receptor activation (Fig.
1C), suggesting that activation of NMDA receptors does not
significantly contribute to the potentiation induction process.
Group I mGluR-mediated epileptogenesis is not sustained by potentiation of NMDA responses
Enhancement of NMDA receptor-mediated responses will lengthen
synchronized discharges, thereby encouraging the production of seizures
(Traub et al. 1994, 1996
). Because group
I mGluR activation in the hippocampus can induce a long-lasting
potentiation of NMDA receptor-mediated responses (Jia et al.
1998
; O'Connor et al. 1994
, 1995
), one might
hypothesize that potentiation of NMDA responses underlies the
maintenance of mGluR-induced prolonged bursts. Here we show that NMDA
receptor antagonists fail to suppress the prolonged bursts,
demonstrating that NMDA potentiation does not significantly contribute
to sustaining the persistent burst prolongation. In contrast, our
previous data showed that group I mGluR antagonists are effective at
reversibly shortening the persistent bursts (Merlin 1999
; Merlin and Wong 1997
). Taken together, the
data support the conclusion that transient activation of group I mGluRs
results in autopotentiation of group I mGluR-mediated responses,
allowing endogenous glutamate to stimulate group I mGluRs, which
results in the persistent expression of potentiated epileptiform
discharges. This autopotentiation of group I mGluR-mediated responses
is NMDA receptor independent and appears to be the major process
underlying mGluR-mediated epileptogenesis.
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ACKNOWLEDGMENTS |
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This work was funded by National Institute of Neurological Disorders and Stroke Grant NS-01901 to L. R. Merlin.
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FOOTNOTES |
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Address for reprint requests: L. R. Merlin, 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 4 November 1999; accepted in final form 30 December 1999.
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REFERENCES |
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