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Report
1Neural and Behavioral Science Program, School of Graduate Studies and Departments of 2Neurology and 3Physiology and Pharmacology, State University of New York Downstate Medical Center, Brooklyn, New York 11203
Submitted 1 December 2003; accepted in final form 16 December 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The persistent expression of the prolonged epileptiform discharges is sustained by the continued activation of group I mGluRs by endogenous glutamate (Merlin 1999; Merlin and Wong 1997). Although both members of the group I mGluR family participate in both the induction and maintenance of these seizure discharges, mGluR1-selective antagonist is more effective at suppressing the expression of the prolonged bursts during the maintenance phase, whereas mGluR5 antagonist is better at preventing the induction process (Merlin 2002). Experiments analogous to some long-term potentiation (LTP) studies revealed that the induction but not the maintenance of this mGluR-mediated epileptogenesis is protein synthesis dependent (Merlin et al. 1998). However, unlike LTP, neither the induction nor the maintenance requires N-methyl-D-aspartate (NMDA) receptor activation (Galoyan and Merlin 2000) or active synchronized bursting activity (Merlin 1999). DHPG has actions at phospholipase D (PLD)-coupled mGluRs, which represent one type of a number of nonclassical mGluRs described in brain (Schoepp et al. 1999). Here we demonstrate that L-cysteine sulfinic acid (CSA), an agonist at an as-yet unnamed PLD-coupled mGluR (Boss et al. 1994), can provide protection from group I mGluR-induced epileptogenesis. Portions of this work have appeared in abstract form (Rico and Merlin 2002a,b). |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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glass microelectrodes filled with 2 M potassium acetate. Data were amplified, digitized, and later analyzed using Axon Instruments equipment and software. Baseline interictal activity was elicited in all experiments by continuous bath perfusion of 50 µM picrotoxin, an antagonist of GABAA receptor-mediated inhibition. Free volume was 20–25 ml and accounted for a 10- to 15-min lag between onset of drug application and initial onset of effect. Picrotoxin and CSA were purchased from Sigma (St. Louis, MO), DHPG from Tocris Cookson (Ellisvillle, MO), and (2R,1'S,2'R,3'S)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-13) from Alexis Biochemicals (San Diego, CA) and as a generous gift from R. Pellicciari. All agents were applied via transient bath perfusion as indicated. Data are reported as means ± SE. Statistical significance within groups was determined using the paired Student's t-test; for comparison across groups, ANOVA with post-ANOVA Newman-Keuls multiple comparison test was performed. P < 0.05 was deemed significant. Each burst duration (BD) was measured from onset of first depolarization to the peak of the last afterdischarge.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Bath application of picrotoxin (PTX) typically elicited spontaneous interictal-length (i.e., <500 ms) epileptiform bursts in CA3 pyramidal cells in guinea pig hippocampal slices. Exposure to 50 µM DHPG, a selective group I mGluR agonist, promptly elicited a transient increase in burst frequency (peaking at 0.44 ± 0.04 Hz, n = 9) accompanied by a gradual conversion of the brief interictal bursts into prolonged seizure-length discharges that persisted on agonist removal (see Merlin et al. 1998). In nine slices tested, the initial burst duration (BDinterictal) of 342 ± 8 ms was increased an average of 267 ± 49% by 40 min of DHPG perfusion (BDDHPG 1,235 ± 147 ms); this potentiation was significantly sustained 1 h after DHPG washout (BDwash 1,194 ± 111 ms; a 252 ± 34% increase; Fig. 1A). However, in nine experiments performed in the presence of 100 µM CSA, DHPG exposure for 40 min had no significant effect on the length of picrotoxin-induced interictal bursts (BDinterictal 352 ± 5 ms; BDDHPG 402 ± 35 ms, an increase of 14 ± 9%; P > 0.05; Fig. 1B) although the DHPG-induced increase in burst frequency was not significantly impeded by CSA (interictal burst frequency of 0.11 Hz rapidly accelerated to 0.35 Hz during DHPG application in the presence of CSA; n = 9). CSA had no significant effect on baseline picrotoxin-induced interictal bursts (BDinterictal 343 ± 11 ms at 0.09 ± 0.02 Hz; after 25 min CSA 338 ± 15 ms at 0.09 ± 0.02 Hz; n = 7).
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DHPG may activate PLD-coupled mGluRs (Albani-Torregrossa et al. 1999; Klein et al. 1997; Schoepp et al. 1999) (see DISCUSSION). However, DHPG application in the presence of 1 µM PCCG-13, a selective antagonist at PLD-coupled mGluRs (Pellicciari et al. 1999), elicited a DHPG response similar to that seen in controls (BDinterictal 334 ± 15 ms; BDDHPG 1,696 ± 482 ms, n = 6). Nevertheless, PCCG-13 did block the effect of CSA on the DHPG-induced burst prolongation; CSA (100 µM) could not prevent the DHPG-mediated induction of persistent prolonged bursts when applied in the presence of 1 µM PCCG-13. Interictal bursts of 366 ± 34 ms duration increased by 187 ± 61% to 1,023 ± 177 ms after 40-min exposure to DHPG in the presence of PCCG-13 and CSA (n = 4; P < 0.05; Fig. 2). PCCG-13 had no effect of its own on interictal burst duration (BDinterictal 325 ± 9 ms; BDPCCG13 325 ± 11 ms, n = 6). The burst prolongation induced by DHPG application in the presence of CSA and PCCG-13 was not significantly different from that induced in the control condition by DHPG alone but was indeed significantly different from DHPG application in the presence of CSA alone (P < 0.01; Fig. 2B).
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To establish whether PLD-coupled receptor activation had any effect on the maintenance (or expression) of DHPG-induced prolonged epileptiform discharges, CSA was introduced into the perfusing solution of six slices in which persistent prolonged bursts had been induced by DHPG (50 µM, 40 min). CSA (introduced at 60 min of DHPG washout) had no significant effect on the maintenance of the DHPG-induced prolonged bursts (DHPGpeak BD 1,092 ± 191 ms, a 219 ± 63% increase; BD at 30 min CSA (i.e., 90 min DHPG washout) 1,072 ± 133 ms, a 207 ± 35% increase, n = 6; Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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CSA is an endogenously occurring compound that is released in the hippocampus during bursts of activity and may participate in the induction of LTP (Klancnik et al. 1992). The mechanisms for this excitatory effect may include activation of group I mGluRs (Croucher et al. 2001; Kingston et al. 1998; Porter and Roberts 1993) accompanied by enhancement of synaptic glutamate release (Croucher et al. 2001). Nevertheless, we report herein that exogenously applied CSA at 100 µM did not significantly affect the frequency or duration of interictal bursts in the in vitro hippocampus, suggesting that at this concentration these excitatory effects are not powerful enough to enhance the network activities. Alternatively, CSA reuptake or degradation mechanisms, which are likely to be increased during epileptiform bursting (Do and Tappaz 1996; Grieve et al. 1991;Wu et al. 1998), may be suppressing the predicted excitatory effects. Additional action of CSA at group II mGluRs (Maione et al. 1998) may compensate even further, reducing the net excitatory effect by enhancing long-term depression (Otani et al. 2002). CSA is also reported to suppress GABAA receptor-mediated inhibition (Morishita and Alger 1999); this excitatory effect, however, does not come into play here because we used a GABAA antagonist to elicit the baseline interictal activity in our experiments.
CSA-mediated activation of the PLD-coupled mGluR prevents group I mGluR-induced epileptogenesis
Another reported effect of CSA is activation of a PLD-coupled metabotropic receptor (Boss et al. 1994), an action that can be competitively antagonized by PCCG-13 (Pellicciari et al. 1999). It should be noted that DHPG itself can noncompetitively antagonize the PLD-coupled metabotropic receptor in adult rat hippocampal slices with an IC50 of 70 µM (Albani-Torregrossa et al. 1999), while inducing full agonist PLD responses in 8-day-old rats (Klein et al. 1997). In our experiments, PCCG-13 successfully suppressed the CSA effect (see Fig. 2), suggesting that the site of CSA action is the PLD-coupled receptor. Yet if CSA is activating this receptor, it implies that DHPG is not effectively blocking this receptor in our system. Furthermore, given that PCCG-13 had no effect on DHPG-induced epileptiform activities, DHPG does not appear to be acting as an agonist at this receptor. This lack of agonist or antagonist effect of DHPG may be due to the intermediate age of the animals (2–4 wk, which is supposed to be neurologically mature in guinea pigs) or interspecies variability.
Hence it is the action of CSA at the PLD-coupled metabotropic receptor that enables it to be effective against the induction of epileptogenesis, while it has no impact on either interictal (Fig. 1) or fully developed ictaform bursts (Fig. 3). These findings are directly parallel to experiments with protein synthesis inhibitors, which also reveal an ability for these agents to prevent the induction of long-lasting ictaform bursts without affecting interictal bursts or fully developed ictaform bursts and without blocking the DHPG-induced increase in burst frequency (see Merlin et al. 1998). It is possible that PLD activation prevents the synthesis of a protein that is critical to the induction of persistent ictaform events. The identity of this critical protein remains to be determined. Nevertheless, the absence of effect of CSA on interictal or ictal bursting activities suggests that there may be a clinically useful concentration of CSA that will allow for normal neuronal network functioning while preventing pathological mGluR-induced long-term modifications such as epileptogenesis.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This work was supported by National Institutes of Neurological Disorders and Stroke Grant NS-40387 to L. R. Merlin.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. R. Merlin, SUNY Downstate Medical Center, 450 Clarkson Ave., Box 29, Brooklyn, NY 11203 (E-mail: lisa.merlin{at}downstate.edu).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Boss V, Nutt KM, and Conn PJ. L-Cysteine sulfinic acid as an endogenous agonist of a novel metabotropic receptor coupled to stimulation of phospholipase D activity. Mol Pharmacol 45: 1177-1182, 1994.[Abstract]
Croucher MJ, Thomas LS, Ahmadi H, Lawrence V, and Harris JR. Endogenous sulphur-containing amino acids: potent agonists at presynaptic metabotropic glutamate autoreceptors in the rat central nervous system. Br J Pharmacol 133: 815-824, 2001.[Web of Science][Medline]
Do KQ and Tappaz ML. Specificity of cysteine sulfinate decarboxylase (CSD) for sulfur-containing amino acids. Neurochem Int 28: 363-371, 1996.[CrossRef][Web of Science][Medline]
Galoyan SM and Merlin LR. Long-lasting potentiation of epileptiform bursts by group I mGluRs is NMDA receptor independent. J Neurophysiol 83: 2463-2467, 2000.
Grieve A, Dunlop J, Schousboe A, and Griffiths R. Kinetic characterization of sulphur-containing excitatory amino acid uptake in primary cultures of neurons and astrocytes. Neurochem Int 19: 467-474, 1991.[CrossRef]
Kingston AE, Lowndes J, Evans N, Clark B, Tomlinson R, Burnett JP, Mayne NG, Cockerham SL, and Lodge D. Sulphur-containing amino acids are agonists for group I metabotropic receptors expressed in clonal RGT cell lines. Neuropharmacology 37: 277-287, 1998.[CrossRef][Web of Science][Medline]
Klancnik JM, Cuénod M, Gähwiler BH, Jiang ZP, and Do KQ. Release of endogenous amino acids, including homocysteic acid and cysteine sulphinic acid, from rat hippocampal slices evoked by electrical stimulation of Schaffer collateral-commissural fibers. Neuroscience 49: 557-570, 1992.[CrossRef][Web of Science][Medline]
Klein J, Iovino M, Vakil M, Shinozaki H, and Löffelholz K. Ontogenetic and pharmacological studies on metabotropic glutamate receptors coupled to phospholipase D activation. Neuropharmacology 36: 305-311, 1997.[CrossRef][Web of Science][Medline]
Maione S, Leyva J, Palazzo E, Stella L, and Rossi F. L-Cysteinesulfinic acid modulates cardiovascular function in the periaqueductal gray area of rat. J Cardiovasc Pharmacol 32: 650-653, 1998.[CrossRef][Web of Science][Medline]
Merlin LR. Group I mGluR-mediated silent induction of long-lasting epileptiform discharges. J Neurophysiol 82: 1078-1081, 1999.
Merlin LR. Differential roles for mGluR1 and mGluR5 in the persistent prolongation of epileptiform bursts. J Neurophysiol 87: 621-625, 2002.
Merlin LR, Bergold PJ, and Wong RKS. Requirement of protein synthesis for group I mGluR-mediated induction of epileptiform discharges. J Neurophysiol 80: 989-993, 1998.
Merlin LR and Wong RKS. Role of group I metabotropic glutamate receptors in the patterning of epileptiform activities in vitro. J Neurophysiol 78: 539-544, 1997.
Morishita W and Alger BE. Evidence for endogenous excitatory amino acids as mediators in DSI of GABA(A)ergic transmission in hippocampal CA1. J Neurophysiol 82: 2556-2564, 1999.
Otani S, Daniel H, Takita M, and Crépel F. Long-term depression induced by postsynaptic group II metabotropic glutamate receptors linked to phospholipase C and intracellular calcium rises in rat prefrontal cortex. J Neurosci 22: 3434-3444, 2002.
Pellicciari R, Marinozzi M, Costantino G, Natalini B, Moroni F, and Pellegrini-Giampietro D. (2R,1'S,2'R,3'S)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-13), the first potent and selective competitive antagonist of phospholipase D-coupled metabotropic glutamate receptors: asymmetric synthesis and preliminary biological properties. J Med Chem 42: 2716-2720, 1999.[CrossRef][Web of Science][Medline]
Porter RHP and Roberts PJ. Glutamate metabotropic receptor activation in neonatal rat cerebral cortex by sulphur-containing excitatory amino acids. Neurosci Lett 154: 78-80, 1993.[CrossRef][Web of Science][Medline]
Rico MJ and Merlin LR. Activation of PLD-coupled mGluRs blocks group I mGluR-induced epileptogenesis. Ann Neurol 52, Suppl 1: S89, 2002a.[CrossRef]
Rico MJ and Merlin LR. L-Cysteine sulfinic acid, a potential novel antiepileptogenic agent. Epilepsia 43: 131-132, 2002b.
Schoepp DD, Jane DE, and Monn JA. Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38: 1431-1476, 1999.[CrossRef][Web of Science][Medline]
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  J. C. Cuellar, E. L. Griffith, and L. R. Merlin Contrasting Roles of Protein Kinase C in Induction Versus Suppression of Group I mGluR-Mediated Epileptogenesis In Vitro J Neurophysiol, November 1, 2005; 94(5): 3643 - 3647. [Abstract] [Full Text] [PDF]
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, an epileptiform burst. Plots of time course of change in burst duration shown for DHPG application in the absence (A1) or presence (B1) of 100 µM CSA. Traces in A2 and B2 correspond to bursts occurring in A1 and B1 prior to DHPG application (PTX), at 40 min of DHPG exposure (DHPG), and after 1 h DHPG washout (1 h wash).
in 2nd group); *P < 0.01; **P < 0.001.