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Seizure-like discharges induced by lowering [Mg2+]0 in the human epileptogenic neocortex maintained in vitro
Brain Research. 417 (1987) 199-203 Elsevier
199
BRE 22408
Seizure-like discharges induced by lowering [Mga+] o in the human epileptogenic neocortex maintained in vitro M. Avoli, J. Louvel*, R. Pumain* and A. Olivier Montreal Neurological Institute, Department of Neurology and Neurosurgerv, McGill University, Montreal, Qu& (Canada) (Accepted 28 April 1987) Key words: Seizure-like discharge; Neocortex; Epilepsy; N-methyl-D-aspartate
Seizure-like discharges were observed in slices of human epileptogenic neocortex maintained in vitro when [Mg2+]owas lowered near to zero. This type of epileptiform activity: (1) could occur spontaneously or following extracellular focal stimuli: (2) resembled the electrographic pattern associated with tonic-clonic seizures; (3) was accompanied by increases in [K+]o(maximally 6.2 mM from a baseline of 3.25 mM) and decreases in [Ca2+]o (maximally 0.23 mM from a baseline of 1.8 mM). Application of the selective antagonist of N-methyl-D-aspartate (NMDA) receptors, DL-2-amino-5-phosphonovalerate, suppressed in a reversible manner both spontaneous and stimulus-induced seizure-like discharges, suggesting that NMDA-activated conductances are important for the genesis of prolonged epileptiform discharges generated by human epileptogenic neocortical slices. The dicarboxylic amino acids L-glutamate and Laspartate, which are excitatory neurotransmitters in the mammalian central nervous system, exert their effects through the activation of at least 3 distinct receptor types named after the sensitivity to the agonists kainate, quisqualate and N-methyl-D-aspartate ( N M D A ) 25. While kainate and quisqualate receptors appear to be involved in fast excitatory transmission at cortical synapses 4"614, NMDA-activated conductances are associated with specific mechanisms of neuronal excitability such as the initiation of longterm potentiation 11'14 or experimentally induced epileptiform activity 3'5"8"1°'15"22. Furthermore, we have recently shown that the bursts of action potentials generated by human neocortical neurons in response to extracellular focal stimulations in vitro slices obtained from epileptogenic cortex are blocked by the selective antagonist of N M D A receptors, DL-2-amino-5-phosphonovalerate (APV), at concentrations which have no effect on the normal excitatory postsynaptic potential 2. N M D A receptors are coupled to an ionophore which is permeable to cations such as Ca 2÷, Na ÷ and
K + and is gated in a voltage-dependent manner by Mg 2+ normally present in the extracellular space 7'9' 16-19,21. Thus, during gradual depolarization from negative membrane potential , responses to N M D A show first an increase (as the block by Mg 2+ declines), then a decrease in amplitude (as the membrane potential approaches the equilibrium potential for the activated channel, around +2 mV). In keeping with such behavior, decreasing [Mg2+]o in rat cortical slices maintained in vitro uncovers conductances activated by N M D A receptors and causes epileptiform discharges to appear 13'24. The present experiments were designed to study the possible involvement of N M D A receptors in human epileptogenesis. Here we report that by perfusing slices of human epileptogenic neocortex with MgZ+-free artificial cerebrospinal fluid, it is possible to evoke prolonged, seizure-like discharges which are mainly associated with Ca 2+ sinks located in the middle cortical layers and are blocked by the antagonist of N M D A receptors APV. Human cortical tissue was obtained during resection of epileptogenic tissue carried out for the control
* Permanent address: INSERM U-97, Paris, F-75014, France. Correspondence: M. Avoli, Montreal Neurological Institute, 3801 University Street, Montr6al, Qu6bec, H3A 2B4, Canada. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. Spontaneous (Aa) and stimulus-induced (Ab,B) epileptiform discharges resembling tonic-clonic seizure discharges recorded in two different experiments (A and B ) performed in neocortical slices bathed in MgZ+-freeACSF for over 1 h. In both cases the extracellular recording microelectrode was placed 800/~m from the piai surface and the stimulating electrode was located within the cortex. Note in A the electrographic similarities between spontaneous (a) and stimulus-induced (b) discharges. The refractoriness of the epileptiform event is shown in Bb where extracellular focal stimuli delivered at a strength 3 times greater than in Ba failed in evoking the seizure-like discharge 20 s after the termination of the discharge shown in Ba (1 and 2 represent a continuous recording). of partial seizures refractory to anticonvulsant drug therapy (5 patients). Informed consent was obtained from all patients. Electrophysiological studies were performed on tissue which was removed for strictly therapeutical reasons. Surgery was performed under local anesthesia. Patients were treated with a variety of antiepileptic drugs which were reduced or discontinued several days before surgery. The samples studied in the present experiments were from the 1st or 2nd temporal gyrus and in one case from the cingulate cortex. Electrocorticography, performed during the surgical procedure for better defining the epileptic focus, indicated that the samples were obtained from regions displaying interictal electrocorticographic spiking either spontaneously or following i.v. injection of the short-lasting barbiturate methohexital (0.5 mg/kg). The procedures for slicing the cortical tissue were similar to those described by Prince and W o n g 2° and Schwartzkroin et al. 23, Slices (500-600 ~ m thick) were maintained at 35 __. 1 °C in an interface tissue chamber and were perfused with oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) (in mM): NaCI. 124; KC1, 2: KH2POa, 1.25; MgSO4, 2 or 0; CaCI2, 1.8, N a H C O s , 26; glucose, 10.
Double barrelled K*- or Ca2+-selective electrodes were made and treated in accordance to the methods described by Heinemann et al. 12. In most cases, the ion-selective electrode for K* was glued to the one for Ca 2+ so that the tips of the two electrodes were less than 10/~m apart This allowed us to record simultaneously K ÷ and Ca 2÷ signals. Extracell~tar field potentials were recorded through either 4 M NaCl-filled microelectrodes or the reference channel of the ion-selective electrodes. T h e signals were fed to a Meyer and Renz (Frankfurt) amplifier and displayed on a Gould pen recorder. Extracellular constant current stimuli were delivered through sharpened monopolar tungsten cathodes placed in the white matter or within the cortex. A P V (Sigma) was applied by perfusion. Extracellular recordings from different layers of human neocortical slices bathed in control A C S F did not reveal any type of spontaneous activity, while focal extracellular stimuli evoked a field potential response which lasted 30-150 ms and was not associated with the occurrence o f synchronous population spikes (not shown). However. switching from the control A C S F to one free of Mg 2+ induced, within 50-100 min. the appearance of spontaneous, syn-
201
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2400 2800 Fig. 2. Laminar profile of electrical field, Ca 2+ and K ÷ signals recorded stimultaneously during seizure discharges generated by 3 different neocortical slices bathed in Mg2+-free ACSF. Measurements were made at the peak of the extracellularly recorded epileptiform discharge and are indicated as percentage of the maximal change i.e. the most negative field potential as well as the highest decrease and increase of Ca 2+ and K + signals respectively. The ion-selective electrodes were moved by steps of 200 y m in an axis normal to the pial surface.
chronous epileptiform discharges which lasted 20-80 s, recurred at intervals of 150-650 s and were characterized in the middle cortical layers by a prolonged negative potential shift with superimposed fast negative transients representing synchronous population spikes (Fig. 1Aa). An epileptiform discharge with similar morphology could also be evoked by single shocks (Figs. lAb and 3) or by short trains of repetitive (1-20 Hz) stimulation (Fig. 1Ba). As shown in the experiments of Fig. 1, each synchronous discharge recorded in the human neocortical slice after lowering [Mg2+]o consisted of an initial phase of 'tonic' firing followed by the gradual development of recurring clustered bursts separated by short silent intervals of increasing duration. Thus, these epileptiform events closely resembled the electrographic pattern associated with tonic-clonic seizures. A refractory period of several tens of seconds followed each seizure-like discharge (Fig. 1Bb). Spontaneous and stimulus-evoked seizure-like discharges were accompanied by increases in [K+]o (maximally 6.2 mM from a baseline level of 3.25 raM) and decreases in [Ca2+]o (maximally 0.23 mM from a baseline level of 1.8 mM) (see Fig. 3, control). Undershoots of the K + signals did not follow the increases in [K+]o . Laminar analysis performed in 3 neocortical slices revealed that the largest and fastest
changes in [Ca2+]o and [K+]o occurred at depths between 800 and 1200 #m under the pial surface (Fig. 2). Furthermore, these were also the sites from which the largest negative field potentials were recorded. Bath application of the selective antagonist of NMDA receptors APV (50-100 pM, n = 4 slices) suppressed in a reversible manner both spontaneous and stimulus-induced seizure-like discharges (Fig.
3). These findings demonstrate that in slices obtained from human epileptogenic neocortex, spontaneous or stimulus-induced seizure-like discharges are generated when [Mg2+]o is lowered near to zero. Furthermore such discharges are readily blocked by the application of APV and therefore their origin is very probably related to the activation of NMDA receptors. Experiments performed in rat slices of hippocampus I and enthorinal cortex 24 have recently shown that, in both preparations, prolonged and sustained epileptiform discharges can be induced by lowering [Mg2+]o. Furthermore, in the enthorinal cortex, pharmacological evidence for a mechanism mediated through NMDA receptors has been provided 24. Some differences, however, are evident when the data gathered in animal brain slices are compared with those observed in the human neocortex. First, in
202 Control
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30 m m
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[Ca ++] °
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.
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.
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20 sec Fig. 3. Effects evoked by bath applications of the NMDA antagonist APV upon stimulus-induced seizure-like discharges generated by a neocortical slice bathed in Mg2+-free ACSF. Electrical field. Ca 2+ and K + signals were recorded simultaneously in the middle cortical layers. Single-shock stimulation was applied in the white matter. the rat enthorinal cortex 24. the largest decreases in Ca 2+ are located in the d e e p layers and not in the middle one as in the case o f our experiments. Second, spontaneous p r o l o n g e d seizure-like discharges (i.e. longer than 10 s) are not o b s e r v e d in the rat isocortex in low [Mg2+] 24. (Louvel and Pumain, in p r e p a r a tion). Finally, the seizure-like discharges g e n e r a t e d by h u m a n neocortical n e u r o n s remain stable for hours, whereas in the rat h i p p o e a m p u s such activity disappears over time to be r e p l a c e d by short interictal-type discharges 1. While it is impossible at present to d e t e r m i n e whether these differences are caused by the chronic epileptogenicity of the h u m a n tissue or rather by species differences, our e x p e r i m e n t s indicate that
1 Anderson, W.W., Lewis. D.V.. Swartzwelder. H.S. and Wilson. W.A.. Magnesium-free medium activates seizurelike events in the rat hippocampal slice, Brain Research. 398 (1986) 215-219. 2 Avoli. A. and Olivier. A.. Bursting in human epileptogemc neocortex is depressed by a N-methyl-D-aspartate antagonist. Neurosci. Lett., in press. 3 Brady, R.J. and Swann. J.W., Ketamine selectively suppresses synchronized afterdischarges in immature hippocampus, Neurosci. Len.. 69 (1986) 143-148, 4 Collingridge, G,L.. Kehl. S.S. and McLennan, H.S., The antagonism of amino acid-induced excitation of rat hippocampal CA 1 neurons in vitro. J. Physiol. (London), 334 (1986) 165-170. 5 Croucher, M.S., Collins, J.F. and Meldrum. B.S.. Anticonvulsant action of excitatory amino acid antagonists. Science, 216 (1982) 899-901. 6 CruneUi. V.. Forda. S. andKelly. J.S., Blockade of aminoacid-induced depolarizations and inhibition of excitatory postsynaptic potentials in rat dentate gyrus, J. Physiol. (London), 341 (1983) 627-640. 7 Dingledine. R.. N-methyl-D-aspartate activates voltage-
N M D A conductances are i m p o r t a n t for the genesis of seizure-like discharges. The question remains open as to whether, in t h e epileptic tissue, changes in [Mg2+]o large enough to p r o m o t e unblocking of the excitatory animo a c i d - o p e r a t e d channels are p r e s e n t during seizures. Preliminary studies using Mga+-se lective electrodes show that decreases in the [Mg2+]o occurs in the n e o c o r t e x during applications of excitatory amino acids (Pumain and Louvel, in p r e p a r a tion). This suggests that alterations in [MgZ+]o might play a m o d u l a t o r y role in seizure activity. This work was s u p p o r t e d by the Medical Research Council of C a n a d a (MA-8109), by I N S E R M and N A T O . M . A . is an F . R . S . Q . Scholar.
dependent calcium conductance in rat hippocampal pyramidal cells. J. Physiol. (London), 343 (1983) 385-405. 8 Dingledine, R., Hynes. M.A~ and King. G.L., Involvement of N-methyl-D-aspartate receptors in epileptiform bursting in the rat hippocampal slice. 3. Physiol. (London), 380 (1986) 175-189. 9 Flatman, J.A.. Schwindt, P.C. and CrilL W.E.. The induction and modification of voltage-sensitive responses in cat neocortical neurons by N-methyl-D-aspartate. Brain Research. 363 (t986) 62-77. 10 Hablitz. J.J. and Langmoen, I.A.. N-rnethyl-o-aspartate receptor antagonists reduce synaptic excitation in the hippocampns, J. Neurosci., 6 (t986~ 102-106. 11 Harris. E.W.. Ganong, A.H. and Cotman. C.W., Longterm potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors, Brain Research. 323 (1984) 132-137. 12 Heinemann. U.. Lux, H.D. and Gumiek. M.J.. Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat, Exp. Brain Res.. 27 (1977) 237-243. 13 Herron. C.E.. Lester. R.A.J.. Coan. E..I. and Collingridge,
203 G.L., Intracellular demonstration of an N-methyl-D-aspartate receptor-mediated component of synaptic transmission in the rat hippocampus, Neurosci. Lett., 60 (1985) 19-23. 14 Herron, C.E., Lester, R.A.J., Coan, E.J. and Collingridge, G.L., Frequency-dependent involvement of NMDA receptors in the hippocampus: a novel synaptic mechanism, Nature (London), 322 (1986) 265-268. 15 Herron, C.E., Williamson, R. and Collingridge, G.L., A selective N-methyl-D-aspartate antagonist depresses epileptiform activity in rat hippocampal slices, Neurosci. Lett.. 61 (1985) 255-260. 16 MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature (London), 321 (1986) 519-522. 17 MacDonald, J.F. and Wojtowicz, J.M., The effects of Lglutamate and its analogues upon the membrane conductance of central murine neurones in culture, Can. J. Physiol. Pharmacol., 60 (1982) 282-296. 18 Mayer, M.L., Westbrook, G.L. and Guthrie, P.B., Voltage-dependent block by Mg ++ of NMDA responses in spinal cord neurons, Nature (London), 309 (1984) 261-263. 19 Novak, L., Bregestovski, P., Ascher, P., Herbert, A. and
20 21
22
23
24
25
Prochiantz, A., Magnesium gates glutamate-activated channels in mouse central neurons, Nature (London), 307 (1984) 462-465. Prince, D.A. and Wong, R.K.S., Human epileptic neurons studied in vitro, Brain Research, 210 (1981) 323-333. Pumain, R., Kurcewicz, I. and Louvel, J., Ionic changes induced through excitatory amino acids in the rat cerebral cortex, Can. J. Physiol. Pharmacol., in press. Ryan, G.P., Hackman, J.C. and Davidoff, R.A., Spinal seizures and excitatory amino acids-mediated synaptic transmission, Neurosci. Lett., 44 (1984) 161-166. Schwartzkroin, P.A., Turner, D.A., Knowles, W.D. and Wexler, A.R., Study on human and monkey 'epileptic' neocortex in the in vitro slice preparation, Ann. Neurol., 13 (1983) 249-257. Walther, H., Lambert, J.D.C., Jones, R.S.G., Heinemann, H. and Hammon, B., Epileptiform activity in combined slices of the hippocampus, subiculum and enthorinal cortex during perfusion with low Mg *+ medium, Neurosci. Lett., 69 (1986) 156-161. Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Annu. Rev, Pharmacol. Toxicol., 21 (1981) 165-206.
199
BRE 22408
Seizure-like discharges induced by lowering [Mga+] o in the human epileptogenic neocortex maintained in vitro M. Avoli, J. Louvel*, R. Pumain* and A. Olivier Montreal Neurological Institute, Department of Neurology and Neurosurgerv, McGill University, Montreal, Qu& (Canada) (Accepted 28 April 1987) Key words: Seizure-like discharge; Neocortex; Epilepsy; N-methyl-D-aspartate
Seizure-like discharges were observed in slices of human epileptogenic neocortex maintained in vitro when [Mg2+]owas lowered near to zero. This type of epileptiform activity: (1) could occur spontaneously or following extracellular focal stimuli: (2) resembled the electrographic pattern associated with tonic-clonic seizures; (3) was accompanied by increases in [K+]o(maximally 6.2 mM from a baseline of 3.25 mM) and decreases in [Ca2+]o (maximally 0.23 mM from a baseline of 1.8 mM). Application of the selective antagonist of N-methyl-D-aspartate (NMDA) receptors, DL-2-amino-5-phosphonovalerate, suppressed in a reversible manner both spontaneous and stimulus-induced seizure-like discharges, suggesting that NMDA-activated conductances are important for the genesis of prolonged epileptiform discharges generated by human epileptogenic neocortical slices. The dicarboxylic amino acids L-glutamate and Laspartate, which are excitatory neurotransmitters in the mammalian central nervous system, exert their effects through the activation of at least 3 distinct receptor types named after the sensitivity to the agonists kainate, quisqualate and N-methyl-D-aspartate ( N M D A ) 25. While kainate and quisqualate receptors appear to be involved in fast excitatory transmission at cortical synapses 4"614, NMDA-activated conductances are associated with specific mechanisms of neuronal excitability such as the initiation of longterm potentiation 11'14 or experimentally induced epileptiform activity 3'5"8"1°'15"22. Furthermore, we have recently shown that the bursts of action potentials generated by human neocortical neurons in response to extracellular focal stimulations in vitro slices obtained from epileptogenic cortex are blocked by the selective antagonist of N M D A receptors, DL-2-amino-5-phosphonovalerate (APV), at concentrations which have no effect on the normal excitatory postsynaptic potential 2. N M D A receptors are coupled to an ionophore which is permeable to cations such as Ca 2÷, Na ÷ and
K + and is gated in a voltage-dependent manner by Mg 2+ normally present in the extracellular space 7'9' 16-19,21. Thus, during gradual depolarization from negative membrane potential , responses to N M D A show first an increase (as the block by Mg 2+ declines), then a decrease in amplitude (as the membrane potential approaches the equilibrium potential for the activated channel, around +2 mV). In keeping with such behavior, decreasing [Mg2+]o in rat cortical slices maintained in vitro uncovers conductances activated by N M D A receptors and causes epileptiform discharges to appear 13'24. The present experiments were designed to study the possible involvement of N M D A receptors in human epileptogenesis. Here we report that by perfusing slices of human epileptogenic neocortex with MgZ+-free artificial cerebrospinal fluid, it is possible to evoke prolonged, seizure-like discharges which are mainly associated with Ca 2+ sinks located in the middle cortical layers and are blocked by the antagonist of N M D A receptors APV. Human cortical tissue was obtained during resection of epileptogenic tissue carried out for the control
* Permanent address: INSERM U-97, Paris, F-75014, France. Correspondence: M. Avoli, Montreal Neurological Institute, 3801 University Street, Montr6al, Qu6bec, H3A 2B4, Canada. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
200
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•
2
sec
B a 1 ....~
.
V'
b ..... f
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Fig. 1. Spontaneous (Aa) and stimulus-induced (Ab,B) epileptiform discharges resembling tonic-clonic seizure discharges recorded in two different experiments (A and B ) performed in neocortical slices bathed in MgZ+-freeACSF for over 1 h. In both cases the extracellular recording microelectrode was placed 800/~m from the piai surface and the stimulating electrode was located within the cortex. Note in A the electrographic similarities between spontaneous (a) and stimulus-induced (b) discharges. The refractoriness of the epileptiform event is shown in Bb where extracellular focal stimuli delivered at a strength 3 times greater than in Ba failed in evoking the seizure-like discharge 20 s after the termination of the discharge shown in Ba (1 and 2 represent a continuous recording). of partial seizures refractory to anticonvulsant drug therapy (5 patients). Informed consent was obtained from all patients. Electrophysiological studies were performed on tissue which was removed for strictly therapeutical reasons. Surgery was performed under local anesthesia. Patients were treated with a variety of antiepileptic drugs which were reduced or discontinued several days before surgery. The samples studied in the present experiments were from the 1st or 2nd temporal gyrus and in one case from the cingulate cortex. Electrocorticography, performed during the surgical procedure for better defining the epileptic focus, indicated that the samples were obtained from regions displaying interictal electrocorticographic spiking either spontaneously or following i.v. injection of the short-lasting barbiturate methohexital (0.5 mg/kg). The procedures for slicing the cortical tissue were similar to those described by Prince and W o n g 2° and Schwartzkroin et al. 23, Slices (500-600 ~ m thick) were maintained at 35 __. 1 °C in an interface tissue chamber and were perfused with oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) (in mM): NaCI. 124; KC1, 2: KH2POa, 1.25; MgSO4, 2 or 0; CaCI2, 1.8, N a H C O s , 26; glucose, 10.
Double barrelled K*- or Ca2+-selective electrodes were made and treated in accordance to the methods described by Heinemann et al. 12. In most cases, the ion-selective electrode for K* was glued to the one for Ca 2+ so that the tips of the two electrodes were less than 10/~m apart This allowed us to record simultaneously K ÷ and Ca 2÷ signals. Extracell~tar field potentials were recorded through either 4 M NaCl-filled microelectrodes or the reference channel of the ion-selective electrodes. T h e signals were fed to a Meyer and Renz (Frankfurt) amplifier and displayed on a Gould pen recorder. Extracellular constant current stimuli were delivered through sharpened monopolar tungsten cathodes placed in the white matter or within the cortex. A P V (Sigma) was applied by perfusion. Extracellular recordings from different layers of human neocortical slices bathed in control A C S F did not reveal any type of spontaneous activity, while focal extracellular stimuli evoked a field potential response which lasted 30-150 ms and was not associated with the occurrence o f synchronous population spikes (not shown). However. switching from the control A C S F to one free of Mg 2+ induced, within 50-100 min. the appearance of spontaneous, syn-
201
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2400 2800 Fig. 2. Laminar profile of electrical field, Ca 2+ and K ÷ signals recorded stimultaneously during seizure discharges generated by 3 different neocortical slices bathed in Mg2+-free ACSF. Measurements were made at the peak of the extracellularly recorded epileptiform discharge and are indicated as percentage of the maximal change i.e. the most negative field potential as well as the highest decrease and increase of Ca 2+ and K + signals respectively. The ion-selective electrodes were moved by steps of 200 y m in an axis normal to the pial surface.
chronous epileptiform discharges which lasted 20-80 s, recurred at intervals of 150-650 s and were characterized in the middle cortical layers by a prolonged negative potential shift with superimposed fast negative transients representing synchronous population spikes (Fig. 1Aa). An epileptiform discharge with similar morphology could also be evoked by single shocks (Figs. lAb and 3) or by short trains of repetitive (1-20 Hz) stimulation (Fig. 1Ba). As shown in the experiments of Fig. 1, each synchronous discharge recorded in the human neocortical slice after lowering [Mg2+]o consisted of an initial phase of 'tonic' firing followed by the gradual development of recurring clustered bursts separated by short silent intervals of increasing duration. Thus, these epileptiform events closely resembled the electrographic pattern associated with tonic-clonic seizures. A refractory period of several tens of seconds followed each seizure-like discharge (Fig. 1Bb). Spontaneous and stimulus-evoked seizure-like discharges were accompanied by increases in [K+]o (maximally 6.2 mM from a baseline level of 3.25 raM) and decreases in [Ca2+]o (maximally 0.23 mM from a baseline level of 1.8 mM) (see Fig. 3, control). Undershoots of the K + signals did not follow the increases in [K+]o . Laminar analysis performed in 3 neocortical slices revealed that the largest and fastest
changes in [Ca2+]o and [K+]o occurred at depths between 800 and 1200 #m under the pial surface (Fig. 2). Furthermore, these were also the sites from which the largest negative field potentials were recorded. Bath application of the selective antagonist of NMDA receptors APV (50-100 pM, n = 4 slices) suppressed in a reversible manner both spontaneous and stimulus-induced seizure-like discharges (Fig.
3). These findings demonstrate that in slices obtained from human epileptogenic neocortex, spontaneous or stimulus-induced seizure-like discharges are generated when [Mg2+]o is lowered near to zero. Furthermore such discharges are readily blocked by the application of APV and therefore their origin is very probably related to the activation of NMDA receptors. Experiments performed in rat slices of hippocampus I and enthorinal cortex 24 have recently shown that, in both preparations, prolonged and sustained epileptiform discharges can be induced by lowering [Mg2+]o. Furthermore, in the enthorinal cortex, pharmacological evidence for a mechanism mediated through NMDA receptors has been provided 24. Some differences, however, are evident when the data gathered in animal brain slices are compared with those observed in the human neocortex. First, in
202 Control
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100 ~M
30 m m
W a s h , 55 rntn l mV
[Ca ++] °
~
f
,
.
_
.
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.8
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20 sec Fig. 3. Effects evoked by bath applications of the NMDA antagonist APV upon stimulus-induced seizure-like discharges generated by a neocortical slice bathed in Mg2+-free ACSF. Electrical field. Ca 2+ and K + signals were recorded simultaneously in the middle cortical layers. Single-shock stimulation was applied in the white matter. the rat enthorinal cortex 24. the largest decreases in Ca 2+ are located in the d e e p layers and not in the middle one as in the case o f our experiments. Second, spontaneous p r o l o n g e d seizure-like discharges (i.e. longer than 10 s) are not o b s e r v e d in the rat isocortex in low [Mg2+] 24. (Louvel and Pumain, in p r e p a r a tion). Finally, the seizure-like discharges g e n e r a t e d by h u m a n neocortical n e u r o n s remain stable for hours, whereas in the rat h i p p o e a m p u s such activity disappears over time to be r e p l a c e d by short interictal-type discharges 1. While it is impossible at present to d e t e r m i n e whether these differences are caused by the chronic epileptogenicity of the h u m a n tissue or rather by species differences, our e x p e r i m e n t s indicate that
1 Anderson, W.W., Lewis. D.V.. Swartzwelder. H.S. and Wilson. W.A.. Magnesium-free medium activates seizurelike events in the rat hippocampal slice, Brain Research. 398 (1986) 215-219. 2 Avoli. A. and Olivier. A.. Bursting in human epileptogemc neocortex is depressed by a N-methyl-D-aspartate antagonist. Neurosci. Lett., in press. 3 Brady, R.J. and Swann. J.W., Ketamine selectively suppresses synchronized afterdischarges in immature hippocampus, Neurosci. Len.. 69 (1986) 143-148, 4 Collingridge, G,L.. Kehl. S.S. and McLennan, H.S., The antagonism of amino acid-induced excitation of rat hippocampal CA 1 neurons in vitro. J. Physiol. (London), 334 (1986) 165-170. 5 Croucher, M.S., Collins, J.F. and Meldrum. B.S.. Anticonvulsant action of excitatory amino acid antagonists. Science, 216 (1982) 899-901. 6 CruneUi. V.. Forda. S. andKelly. J.S., Blockade of aminoacid-induced depolarizations and inhibition of excitatory postsynaptic potentials in rat dentate gyrus, J. Physiol. (London), 341 (1983) 627-640. 7 Dingledine. R.. N-methyl-D-aspartate activates voltage-
N M D A conductances are i m p o r t a n t for the genesis of seizure-like discharges. The question remains open as to whether, in t h e epileptic tissue, changes in [Mg2+]o large enough to p r o m o t e unblocking of the excitatory animo a c i d - o p e r a t e d channels are p r e s e n t during seizures. Preliminary studies using Mga+-se lective electrodes show that decreases in the [Mg2+]o occurs in the n e o c o r t e x during applications of excitatory amino acids (Pumain and Louvel, in p r e p a r a tion). This suggests that alterations in [MgZ+]o might play a m o d u l a t o r y role in seizure activity. This work was s u p p o r t e d by the Medical Research Council of C a n a d a (MA-8109), by I N S E R M and N A T O . M . A . is an F . R . S . Q . Scholar.
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