Epilepthom Activity Induced by Low Extracellular Magnesium in the Humm Cortex Maintained In Vitro M. Avoli, MD, PhD, C. Drapeau, MSc, J. Louvel, PhD, R. Pumain, PhD, A. Olivier, MD, PhD, and J.-G. Villemure, MD
Extracellular field potentials and fK+}, were recorded in slices of human epileptogenic neocortex maintained in vitro during perfusion with M2+-free artificial cerebrospinal fluid (ACSF). The human neocortex was obtained during neurosurgical procedures for the relief of seizures that were resistant to medical treatment. Spontaneous epileptiform activity and episodes of spreading depression appeared within 1.5 to 2 hours of perfusion with Mgz+-freeACSF. The epileptiform discharges consisted of negative field potential shifts (amplitude, 0.8-10 mV) that lasted 2.5 to 80 seconds and recurred at intervals ranging between 4 and 160 seconds. Both duration and frequency of occurrence of epileptiform events were not significantly different when measured in slices obtained from spiking tissue compared with those gathered from nonspiking neocortical areas. Transient increases in {K+], of up to 10.5 mM were associated with each epileptiform discharge; these changes were maximal and fastest in the middle neocortical layers. Spreading depression episodes were characterized by 20 to 30-mV negative shifts that lasted up to 200 seconds and were accompanied by increases in {K+], of approximately 100 mM. Epileptiform discharges and spreading depressions did not occur during perfusion with Mg2 -free ACSF that contained either competitive or noncompetitive antagonists of the N-methyla-aspartate (NMDA) receptor subtype. ln contrast, pharmacological blockade of non-NMDA receptors did not influence the epileptiform activity observed in Mg2+-freeACSF. These findings demonstrate that decreasing IMg2+}, Ieads to the appearance of both spontaneous epileptiform discharges and spreading depression in the human epileptogenic neocortex. Both activities are solely dependent on NMDA-activated conductances, suggesting that removal of the voltage-dependent gating effect exerted by extracellular Mg2+on the NMDA ionophore is a condition sufficient for neurons in the human neocortex to generate spontaneous, synchronous epileptiform activity. We propose that this experimental paradigm might represent a model suitable for analyzing the cellular mechanisms underlying human neocortical epileptogenesis in vitro as well as for preliminary screening of antiepileptic drugs. +
Avoli M, Drapeau C, Louvel J, Pumain R, Olivier A, Villemure J-G. Epileptiform activity induced by low extracellular magnesium in the human cortex maintained in vitro. Ann Neurol 1991;30:589-596
After the demonstration that antagonists of the Nmethyla-aspartate (NMDA) receptor can reduce the occurrence of experimentally induced seizures 117, much attention has been directed to the role played in epileptogenesis by the different receptors for the excitatory amino acid transmitters. In the course of these studies, it has also been shown that the NMDA receptor is presumably involved in chronic epileptogenesis. Accordingly, an NMDA-mediated potential is recorded in the dentate gyrus of the rat hippocampus after repeated stimulation (kindling) of the amygdala or hippocampal commissures {2, 31. Furthermore, electrophysiological studies of the human epileptogenic neocortex maintained in vitro have revealed that some of the neurons located in the deep layers generate a synaptic depolarization that is blocked by the
competitive antagonist of the NMDA receptor DL-2amino-5-phosphonovalerate(APV) C4, 51. The NMDA receptor is coupled to an ionophore that is permeable to cations such as Ca2+, Na', and K+, and gated in a voltage-dependent manner by M&+ present in the extracellular space [G, 71. Accordingly cortical slices maintained in vitro generate synchronous epileptiform discharges when the extracellular M&+ is decreased by perfusing the tissue with Mg2+-free artificial cerebrospinal fluid (ACSF) [S- 151. Because these epileptiform discharges are blocked by NMDA receptor antagonists, it has been suggested that the genesis of Mg2T-freeepileptiform discharges is caused mainly by NMDA-mediated conductances that are no longer gated by M&+. Given the possible participation of NMDA-medi-
From the Montreal Neurological Institute and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada.
Received Jan 24, 1991. Accepted for pubiication Apr 19, 1991. Address correspondence to Dr Avoli, 3801 University, Room 850, Montreal, QC, Canada H3A 2B4.
Copyright 0 1991 b y the American Neurological Association 589
ated potentials in synaptic transmission in the human epileptogenic cortex 14, 57, we analyzed the effects induced by perfusing slices of human epileptogenic cortex maintained in vitro with M$+-free ACSF. In the present study, we used extracellular field potential recordings and measurements of the extracellular concentrations of K+ ([K’],) to answer two main questions. First, which are the electrophysiological characteristics of the epileptiform activity recorded in the human cortex during perfusion with M$+-free ACSF? Second, is this type of activity sensitive to NMDA receptor antagonists as reported for the epileptiform discharges seen in the rat cortex and, in addition, which are the effects elicited by non-NMDA receptor antagonists? Preliminary reports of some of these findings have appeared {IG, 177.
Methods Human Cortical Tissue: Clinicul, Electrographic and Neuroputbological Features Human neocortical tissue was obtained during neurosurgical procedures performed for the relief of seizures that were resistant to medical treatment (22 patients of either sex). In any of these patients, the tissue used for the electrophysiological experiments was part of a block of brain tissue removed for strictly therapeutical reasons. Informed consent was obtained in all patients. Most of the patients undergoing surgery for epilepsy had been maintained on a variety of antiepileptic drugs that were progressively discontinued or markedly reduced during the weeks preceding surgery. Surgical procedures were performed under local anesthesia in all but 3 patients. In these patients, general anesthetics with halogenated compounds were used. Patients who underwent surgery under local anesthesia received during the operation the analgesic drug fentanyl citrate (Sublimaze [Abbott Lab Ltd, Montreal, Quebec, Canada] 0.2 mg/kg IV) and the short-lasting barbiturate methohexital sodium (0.5 mg/kg IV). As in previous studies performed in our laboratory [ 5 , 18}, brain tissue obtained from patients receiving general anesthesia displayed electrophysiologicalcharacteristics,generated epileptiforrn activity, or both that were indistinguishable from those of samples from patients who underwent surgery under local anesthesia. This finding suggests that the short-term effects induced by the drugs present in the brain in situ are readily washed in the tissue chamber during the time allowed for recovery after slicing. Clinical neurophysiological investigations,including the intraoperative electrocorticogram, were routinely performed for diagnostic purposes. These studies revealed that the brain tissue samples used in the experiments reported here belonged to areas that either displayed interictal spiking during the intraoperative electrocorticogram (so-called epileptogenic samples) or resided near the site of spiking (so-called juxtaepileptogenic samples). These samples were obtained from the first (n = 4 surgcal procedures) or the second (n = 18 surgical procedures) temporal gyrus. Neuropathological analysis of the removed brain tissue revealed in most patients a moderate degree of gliosis and neuronal loss.
590 Annals of Neurology Vol 30 N o 4 October 1991
Preparation and Maintenance of the Slices The techniques used for preparing and maintaining the slices in vitro have already been described in a previous study 151. In brief, neocortical slices were cut 500 to 600 pm thick by using a tissue chopper. They were placed in an interphase tissue chamber where they were perfused at 35 ? 1°C with oxygenated ACSF (95% 0,, 596 CO,). The rate of perfusion (0.5-1.5 ml/min) was kept constant in each experiment. Slices were allowed 1 to 2 hours of recovery before the recording was begun. The composition of the ACSF was (in mM) as follows: NaCl 124, KCI 2, KH,P04 1.25, CaCl, 1.8-2, MgS04 2 or 0, NaHCO, 26, and glucose 10. APV (Sigma Chemical, St Louis, MO), dizocilpine (MK-801, a gift of Merck Sharp and Dohme, Fbhway, NJ), 3-[(%)-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid (CPP; Tocris Neuramin, Bristol, UK), and 6-cyano-7-nitroquinoxaline2,3-dione (CNQX; Tocris Neuramin) were added to the perfusing ACSF.
Recording and Stimulating Techniques Extracellular field potentials were measured with glass microelectrodes filled with 2 M NaCl or through the reference channel of the ion-selective electrode. Ion-selective microelectrodes were prepared according to the methods described by Avoli and colleagues [19]. Signals were fed to a high impedance amplifier and displayed on a Gould pen recorder. Constant current anodal stimuli (0.05-0.5 mA; 10-90 p e c ) were delivered through sharpened and insulated tungsten electrodes that were placed in the underlying white matter or within the cortical layers.
Results
Field Potentials and Changes in {K+)oRecordd in the Neocortex during Perjhion with M$ifwe ACSF Spontaneously occurring field-potential epileptiform discharges were seen in 92 of over 140 neocortical slices during perfusion with MgZf-free ACSF. In all patients, the epileptiform activity appeared after 1.5 to 2 hours of perfusion with M d i - f r e e ACSF. Some typical examples of spontaneous activity recorded in the middle layers of the human neocortex are shown in Figure 1A-C. They consisted of prolonged negative shifts with superimposed fast, negative transients. Epileptiform discharges lasting less than 10 seconds were usually characterized by a single negative shift (amplitude, 0.8-10 mV) associated at times with lowamplitude, high-frequency (up to 11 Hz) events (see Fig lA, B). In contrast, the most prolonged epileptiform discharges displayed an initial phase of sustained tonic activity followed by the gradual development of “clonic” discharges consisting of recurring, clustered bursts separated by short, silent intervals of increasing duration (see Fig 1C). Hence, they bore some resemblance to the electrographic pattern associated with tonic-clonic seizures. Simultaneous intracellular and extracellular recordings have revealed that in M$+free ACSF human neocortical cells display large-
A
a
b
1600
_I 5
10
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Duration
zo
25
30
White
matter
(5)
Fig 1. Epileptifom discharges recorded with extracellular microelectrodes pkzced in the middle cortical layers of three different slices during perjiusion wtth M$ $we artificzal cerebrospinal fluid. Experiments in A and B are from the same tissue sample; in each panel, traces (a) and (b) show spontaneous discharges that were recorded at low (10-second calibration bar) and high (1-second calibration bar) speed, mpectively. The experiment shown in C illustrates that similar shape and duration characterize spontaneous (a) and stimulus-induced {b) epileptijim discharges. (0)Plot of the mean duration {abscirsa)and mean interval between one discharge and the following one (ordinate)as calculated in 83 slices obtained from 16 patients. Discharges with duration larger than 30 seconds were not included in this plot because they occuwed spontaneously at an irregular frequency. +
amplitude depolarization with ionic firing, bursts of action potentials, or both that closely correlate with the field discharges (M. Avoli and C . Drapeau, unpublished data). The frequency of occurrence and the duration of the spontaneous epileptiform discharges varied considerably in different slices even when they were obtained from the same tissue sample (compare epileptiform activity in panels A and B of Figure 1, see above). They lasted 2.5 to 80 seconds (28 k 13 seconds; n = 89 slices) and recurred at intervals that varied between 4 and 160 seconds (46 2 20 seconds; n = 91 slices). In any given slice, however, these two parameters remained usually constant over the duration of the experiment (up to 8 hours). In the plot of Figure 1D, one can appreciate that in 83 slices obtained from 16 different patients there was a close relationship (regression coefficient, 0.955) between the mean duration of the epileptiform discharges and the mean frequency of occurrence (i.e., the interval between the onset of one discharge and that of the following one) in any given slice. Comparison of both duration and frequency of occurrence of the epileptiform discharges recorded in slices obtained from epileptogenic and juxraepileptogenic samples did not reveal any significant differences. Epileptiform discharges with shapes similar to those occurring spontaneously could also be evoked by ex-
- b
-
+
h
Fig 2. Srmultaneous {K'), (upper trace) and jiekd potential
(lower trace) recordings during spontaneous (A)atad stimulusInduced (B) epsleptifom discharge in M 2 ' - j k e art$cial c m brospinalJuid recorded at ddfferent depths. Distance fmm the pia is given on the kfi of each sample in micrometers. Note that szmzlar extracellular jield potentials and increases in {K+j0 characteraze the stimulus-induced and the spontaneous epikptiocform discharges as well as that the largest increases in {KKfIo cur at a depth of 1,200 pn.
tracellular, focal single-shock stimuli (see Figs lC, 2B). Such stimuli elicited epileptiform responses in most of the slices that did not generate any spontaneous epileptiform activity. Stimuli delivered in different regions of the same slice (e.g., pia, white matter, cortical layers) resulted in s d a r patterns of discharge. This finding suggested that the epileptiform tesponse was not dependent on the activation of any specific intracortical pathways. Both spontaneous and stimulus-induced discharges were accompanied by a transient increase in [K'], that reached a maximal value of 10.5 mM (6.7 -+ 2.8 mM; n = 12 slices) from a baseline level of 3.25 mM. As illustrated in Figure 2, the largest and fastest changes in {K 1, usually occurred in the middle layers of the human neocortex between 800 and 1,600 p,m from the pia. The largest negative field potentials were also recorded at these depths. Therefore, the most active layers of the neocortex during epileptiforrn activity Mg?+-free in ACSF appeared to reside in the middledeep cortical layers. Undershoots of the K+ signals did not follow the increase in { K'], observed during each epilepuform event, but were seen to occur after episodes of spreading depression (Fig 3, Control). Spreading depression was recorded in eight slices in which it occurred either spontaneously (Fig 4 ) or after extracellular focal stimuh (see Fig 3); it lasted 50 to 220 seconds (97 f 31 seconds; n = 6 slices), was associated with 10 ro 25-mV negative shifts in the extracellular field potential, and was followed by a period during which spontaneous epileptiform discharges failed to occur. In two slices in +
Avoli et al: Mg2+-freeEpileptogenesis in the Human Cortex 591
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Fig 3. Measurements of @?}, during spontaneous epileptifm discharges and spreading depression induced by a train of repetiti**elow-frequeng stimuli in M 2 '-free art$cial mehospinal fluid (Control),during addition of DL-2-amino-5-phosphonovalerate (APV; JO CJM) and after washout of the N-methyl+ aspartate receptor antagonist (Wash). Note that epileptiforn discharges as well as spreading depression are blocked by APV. The three trtangles in the Wash trace point at the time during whtch the train of stimuli (10 seconds long, 2 Hzj was deliuered. A
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Fig 4. Spontaneous epileptifm discharges and spreading depression (asterisk) recorded extracellulcsrly in the middle cortical layers of a slice perj5used with M 2 '$ree artificial cerebrospinal fEuid. Note the different amplitude and duration o f the spreading depression compared with the epileptiform discharges. Also note the period of depression in the occurrence o f epzleptiform discharges that follow the spreading depression. A kzpse of approximate& 20 seconds exists between recordings A and B.
which it was measured, [K'], increases during spreading depression attained values of approximately 100 mM (see Fig 3). Sensitivity of the Epdeptafrm Activity in ACSF to NMDA Receptor Antagonists
M 2 '-free
The participation of membrane conductances activated through the NMDA receptor in the generation of epileptiform activity in Mg?+-free ACSF was analyzed by studying the changes induced on spontaneous and stimulus-induced discharges by competitive and noncompetitive NMDA receptor antagonists. The competitive antagonist of the NMDA receptor, APV (20-100 pM) 1201, decreased the frequency of occurrence and eventually blocked the epileptiform discharges in a dose-dependent fashion (n = 11 slices).
592 Annals of Neurology Vol 30 No 4 October 1991
Fig 5 . (Aj Eflects induced by increasing concentrations of the N-methyl-Daspartate receptor antagonist, DL-2-amino-5phosphonovalerate (APV),on spontaneous epiltpt2fown discharges; in this experiment, the extracellulitr field potential recordings were passed through an alternating current filtersecond-stage amplij5er. (B) Bar graph of the decrease in frequency of occumnce of spontaneous discharges (ordinate)during successive applications of increasingly higher concentrations of APV (abscissa). Data were obtained from three different slices in which the frequency of occurrence of epileptiform discbarges was computed after 30 minutes of perfusion with artificial cerebrospinalfEuid (ACSF) containing the stated concentrations of APV. (C) Changes induced by 3-{(+2-carboxypiperazin-4yl}-propyl-l-phosphonicacid (CPP; 2 pM) on spontaneous epiEept;fonnactivity recorded simultuneously with a K +-sensitive (upper trme) and field potential electrod? (lozuev trace). CPP trace was taken 12 minutes after onset of pwfusion and wash truce 40 minutes afer reperfusion with control M 2 -free ACSF. +
A typical experiment in which increasing doses of APV were sequentially applied to the slice is shown in Figure 5A; in this case, the complete blockade of the epileptiform activity occurred in the presence of 100 pM APV. Figure 5B shows a bar graph of the effects induced by different doses of APV in three different slices that were perfused with increasing concentrations of this NMDA receptor antagonist. The depressant action of APV (50-100 pM) was also observed while studying stimulus-induced epileptiform discharges (n = 6 slices; not shown). In eight experiments, we also analyzed the effects exerted by CPP (2-5 pM), which is another competi-
tive antagonist of the NMDA receptor 1211, on the M& +-free epileptiform activity. As illustrated in Figure SC, spontaneous epileptiform discharges recorded simultaneously with extracellular field potential and K+-selective electrodes disappeared in the presence of CPP. A similar action of CPP (2 pM) was also observed when analyzing the stimulus-induced epileptiform discharge (n = 5 slices). In one instance, the action of CPP on the epilepuform discharges was accompanied by the disappearance of spreading depression episodes that had occurred spontaneously in control M& +-free ACSF. In another experiment, spreading depressions induced by trains of repetitive extracellular focal stimuli (10 seconds long at 2 Hz) were not observed after perfusion with M&+-free ACSF containing APV (50 pM) (see Fig 3). The effects induced by both APV and CPP were reversible on returning to control M$+-free ACSF. Furthermore, the blockade of spontaneously occurring discharges was not accompanied by any progressive decrease in the duration of each discharge before the occurrence of blockade. This is shown in detail in Figure 5C, where it is possible to appreciate that the two epileptiform events generated before the complete disappearance of the spontaneous activity are practically indistinguishable from any of the discharges recorded in control conditions. In six slices, we also assessed the effects induced by the noncompetitive antagonist of the NMDA receptor, MK-801 [22]. MK-801 at concentrations of 2 to 5 pM also blocked the epileptiform discharges observed in M 8 +-free medium. Two differences were noted, however, when these effects were compared with those induced by APV or CPP. First, the blockade induced by MK-801 was irreversible (up to 3 hours of wash with control M&+-free ACSF). Second, the effects induced by this noncompetitive antagonist of the NMDA receptor subtype were more gradual. This is shown in the graphs and raw data illustrated in Figure 6 in which the epileptiform activity generated by the same neocortical slice was studied sequentially during perfusion with M$+-free ACSF containing, at first, CPP and, later, MK-801; in contrast to the blockade induced by CPP, MK-801 elicited in this experiment a progressive and gradual decrease in both the frequency of occurrence and duration of each single discharge.
A
MK-801.2 rM
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Time (mm)
Fig 6. Comparison of the e f f t s induced b the competitive 3{(~~-2-carboxypipwazin-4-yl)-propyl1-phosphonic acid (CPP) vld the noncompetitive dizorilpine (MK-801)N-methyl-o aJpurtateantagonists on M$+-free epikptifrm activity. (A) Shows the histograms of the frequeng of occuwence (upper panen and half-width duration (lowerpanel) of spontaneous epilegtiform discharges in control and during successive CPP and MK-801 application. (B) lllarstrates samples of discharges obtained at the times indicated ly each respective letter (a-d) in A. Note that CPP induces a block of the epileptifarmdischarges that is not preceded by a reduction in duration, whereas MK-801 induces a progressive and gradual decrease of both fequency of occurrence and duration of each epilegtifm event. (r
recorded in M&+-free ACSF. In two of four experiments, 3 pM CNQX induced a decrease in duration of both spontaneous and stimulus-induced discharges (Fig 7A). These changes were not statistically significant (t test), however, when the values obtained in all experiments were compared. CNQX was also unable to modify the pattern of the epileptiform discharge when applied at relatively high concentrations (10 p.M; n = 3 experiments). As shown in Figure 7B and C , CNQX did not influence the frequency of occurrence of spontaneous epileptiform events or the shape and duration of the stimulusinduced discharge. This lack of effect markedly contrasted with the depressant changes observed in this experiment when CPP (5 pM) was added to the M&+-free ACSF. CNQX reduced at times the amplitude of the rhythmic oscillations overriding the negative shift associated to the epileptiform discharge (see Fig 7A). This effect was seen in two of four experiments and one of three experiments in which 3 and 10 pM of C NQ X were used, respectively.
Sensitivity of the Epdeptifom Activity in MgZ'-fwe ACSF to tbe N o n - N M D A Receptor Antagonist C N Q X
Perfusion of the neocortical slices with M&'-free ACSF containing 3 p M of CNQX (n = 4 experiments), which is an antagonist of non-NMDA receptor subtypes [23}, failed to change significantly the frequency of occurrence of the epileptiform discharges
Avoli
CPP, 2 &AM
Discussion It has been shown that lowering M$+ in the extracellular space induces epileptiform activity in the neocor-
et
al: M$+-free Epileptogenesis in the Human Cortex
593
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Fig 7 . (A) Eflects induced by 3 f l 6-oano-7-nit~quinoxaline-2,3-dione (CNQX} on stimulus-induced (Induced)and spontaneousb occum'ng (Spontaneous}Md'gree epikptiform discharges. Although the frequeno of occurrence of spontaneous discharges was not mod$ed Sy CNQX in this experiment, there was a decrease in the duration of each M 2 'gree epileptfifom event as well as a reduction of the overriding fast-transients.(B, C) CNQX faih to injuence the fwquency of occurrence ofspontaneous activity (histogram in B) and the duration and shape of the stimulus-indmd discharges (raw data in C). This activity, hozuever, is blocked by the subsequent effectsof 3-{(4-2carboxypiperazin4-y&wopyl-l -phosphonicacid (CPP). The raw data in C were taken at the times indicated by the letters (a-c) in the histogram in B in which the ordinate axis gives the number of epileptiform discharges occum'ng every 60 seconds.
tex 110, 141, the entorhinal cortex [S, 111, and the hippocampus 18, 9, 13, 151. Our experiments extend these observations to the neocortical tissue of humans obtained from epileptic patients undergoing surgery for the relief of seizures resistant to pharmacological therapy. We have characterized some electrophysiological properties of the epileptiform activity studied with extracellular field recordings and measurement of EK'], in the human brain tissue perfused with Mg2+free ACSF. Furthermore, we demonstrated that this activity is caused by the selective activation of conductances that are mediated through the NMDA receptor subtype. Finally, we reported the occurrence of spreading depression in the human neocortex perfused with W f - f r e e ACSF. The electrophysiologicalfeatures of the epileptiform discharges described in the present study are very similar to those recorded in slices of entorhinal cortex [S, 111 and neocortex [lo, 143 obtained from rats during perfusion with MgZ+-free ACSF. These similarities include the spontaneous occurrence of the discharges,
594 Annals of Neurology Vol 30 No 4 October 1991
the regular rate of occurrence, the relative long duration of each single discharge, and the resemblance of the most prolonged ones to the tonic-clonic electrographic seizures recorded in situ. All these characteristics indicate that the epileptiform discharges described here r e p r e s e a good experimental model for studying the cellular mechanisms underlying ictal-like epileptiform events in the neocortex. Furthermore, the fact that the epileptiform discharges observed in our experiments occur spontaneously indicates that this type of epileptiform activity might represent a suitable in vitro model for analyzing the effects induced by anticonvulsant agents, including preliminary screening of antiepileptic drugs. Interestingly, in the human neocortex maintained in vitro, epileptiform discharges do not occur spontaneously after blockade of the y-aminobutyric acid, receptor [181. Epileptiform discharges in Mg2+-free ACSF observed in this study are readily blocked by antagonists of the NMDA receptor, indicating that conductances activated through this receptor subtype participate in the generation of this type of epileptiform activity. Hence, a major mechanism in this model of acure epileptogenesis is due to the removal of the voltagedependent gating effect exerted by extracellular Mg2+ on the NMDA ionophore [G, 71. We cannot exclude, however, that an additional contribution to this type of epileptiform activity might arise from nonspecific effects due to the removal of M d f from the extracellular space. These effects include the antagonistic action of M d + on Ca" conductances [24) and the reduced membrane surface screening 1251. Both effects are well known to lead to an increase in neuronal excitability. Both competitive and noncompetitive antagonists of the NMDA receptor subtype were effective in blocking epileptiform discharges in M8+-free ACSF. The effects induced by these two classes of NMDA receptor antagonists, however, were not similar. When the competitive antagonists APV or CPP were used, Mg2 -free epileptiform discharges decreased in their frequency of occurrence, but not in their duration or amplitude. Therefore, when the blockade of the spontaneous activity occurred, the last epileptiform event to be observed did not differ from those seen in the control situation. Conversely, during application of MK-801, the progressive decrease in rate of occurrence was paralleled by a gradual reduction of the duration of each single discharge. Similar differences between the effects induced by competitive (APV) and noncompetitive (phenocyclidine) NMDA receptor antagonists on M$+-free epileptiform activity have been reported in the CA3 subfield of the rat hippocampal slice [131. Hence, the differences observed here might be due to the use-dependent mode of action of noncompetitive antagonists such as MK-801. +
A rather puzzling finding obtained in our experiments is the lack of effects induced on the M2+-free epileptiform discharges by the antagonist of nonNMDA receptor, CNQX [23]. Given the fact that the fast excitatory postsynaptic potential in the human neocortex is presumably due to the activation of nonNMDA receptor subtypes (IS}; G. Hwa and M. Avoli, unpublished data), the present data are unexpected. Indeed, in the light of these findings, one must conclude that not only the triggering mechanism responsible for the occurrence of epileptiform discharges in Mg2+-freeACSF, but also the synaptic events that underlie the self-sustained character of each epileptiform discharge, are solely caused by the activation of the NMDA receptor. It should be noted that, in some experiments, CNQX was capable of reducing the rhythmic, fast oscillations that occur during the negative epileptiform shift. The mechanism responsible for this effect is as yet unknown. We are inclined to exclude the possibility that this effect of CNQX was mediated through the glycine site of the NMDA receptor, however, because preliminary experiments in our laboratory have indicated that perfusion with Mg2+-free ACSF containing glycine does not modify this type of epileptiform activity in the human neocortex (C. Drapeau and M. Avoli, unpublished data). An interesting observation reported in our study is the occurrence of spreading depression in the human neocortex perfused with Mgz’-free ACSF. Although we did not analyze the velocity of propagation of this type of event, both the amplitude and the duration of the associated field potential as well as the value attained by [K’], suggest that it represented spreading depression as originally described by Lea0 [26}. Failure to observe spreading depression in the cortex during surgery where electrocorticograms are recorded under conditions that are likely to trigger spreading depression has suggested that this phenomenon might be absent in humans {27]. Our data, however, show that spreading depression occurs in the human neocortex maintained in vitro when perfused with Md+-free medium and, thus, suggest that this experimental condition might represent a critical factor in causing human tissue to undergo spreading depression. This conclusion is supported by the finding that, in our experiments, the occurrence of spreading depression was blocked by antagonists of the NMDA receptor along with epileptiform discharges. The spreading depression observed in rat hippocampal slices bathed in M&+-free medium is not seen after application of APV [9]. Furthermore, Schneiderman and MacDonald [I 31 have reported that spreading depression is recorded in the in vitro hippocampal slice during perfusion with Mg2 free ACSF, but not in the presence of the convulsant drug penicillin. Therefore, NMDA-activated conduc+
-
tances might play a critical role in the generation of spreading depression at least in the mammalian cortex. This study was supported by grants from the Medical Research Council of Canada (MA-8190) and NATO. M.A. and R.P. were recipients of INSERM-FRSQ Exchange Programs. We thank D r V. Tancredi and Ms C. Tzeng for participating in some experiments.
References 1. Croucher MJ, CollinsJF, Meldrum BS. Anticonvulsant action of excitatory amino acid antagonists. Science 1982;216:899-901 2. Mody I, Heinemann U. NMDA receptors of dentate gym granule cells participate in synaptic transmission following kindling. Nature 1987;326:701-704 3. Mody I, Stanton PK, Heinemann U. Activation of N-methyl+aspanate receptors parallels changes in cellular and synaptic properties of dentate gyms granule cells after kindling.J Neurophysiol 1988;59:1033-1054 4. Avoli M, Olivier A. Bursting in human epileptogenic neocortex is depressed by an N-methyld-aspartate antagonist. Neurosci Lett 1987;76:249-254 5. Avoli M, Olivier A. Electrophysiologicalproperties and synaptic responses in the deep layers of the human epileptogenic neocortex maintained “in vitro.” J Neurophysiol 1989;61:589-606 6. Mayer ML, Westbrook GL, Guthrie PB. Voltage-dependent block of Mg2+ of NMDA responses in spinal cord neurones. Nature 1984;309:261-263 7. Nowak L, Bregestovski P, Ascher P, et al. Magnesium gates glutamic activated channels in mouse central neurons. Nature 1984;307:462-465 8. Walther H, Lambert JDC, Jones RSG, et al. Epileptiforrn activity in combined slices of the hippocampus, subiculum and entorhinal cortex during perfusion of low magnesium medium. Neurosci Lett 1986;69:156-161 9. Mody I, Lambert JDC, Heinemann U. Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices. J Neurophysiol 1987;57:869-888 10. Aram JA, Lodge D. Validation of a neocortical slice preparation for the study of epileptiform activity. J Neurosci Methods 1988;23:211-224 11. Jones RSG, Heinemann U. Synaptic and intrinsic responses of medial entorhinal cortical cells in normal and magnesium-free medium in vitro. J Neurophysiol 1988;59:1476-1496 12. Wilson WA, Swanmelder HS, Anderson WW, Lewis DV. Seizure activity in vitro: a dual focus model. Epilepsy Res 1988; 2:289-293 13. Schneiderman JH, MacDonald JF. Excitatory amino acid blockers differentially affect bursting of in vitro hippocampal neurons in two pharmacological models of epilepsy. Neuroscience 1989;31:593-603 14. Sutor B, Hablitz JJ. EPSPs in rat neocortical neurons in vitro. 11. Involvement of N-methyl-mupartate receptors in the generation of EPSPs. J Neurophysiol 1989;61:621-634 15. Tancredi V, Hwa GGC, Zona C , et al. Low magnesium epileptogenesis in the rat hippocampal slice: electrophysiological and pharmacological features. Brain Res 1990;511:280-290 16. Drapeau C, Avoli M, Olivier A, Villemure JG. Pharmacological features of the Mg++-free epileptiform bursts in the human neocortex maintained “in vitro.” Neurosci Lett 1988;14:572 (Abstract) 17. Avoli M, Louvel J, Pumain R, Olivier A. Seizure-like discharges induced by lowering [Md+],in the human epileptogenic neocortex mantained “in vitro.” Brain Res 1987;417:199-203
Avoli et al: Md+-free Epileptogenesis in the Human Cortex 595
18. Hwa GGC, Avoli M, Olivier A, Villemure JG. Bicucullineinduced epileptogenesis in the human neocortex maintained in vitro. Exp Brain Res 1991 (in press) 19. Avoli M, Drapeau C, Perreault P, Louvel J, Pumain R. Epileptiform activity induced by low chloride medium in the CA1 sub field of the hippocampal slice. J Neurophysiol 1990;64:17471757 20. Colkngridge GL, Kehl SJ, McLennan H. The antagonism of amino acid-induced excitations of rat hippocampal CA1 neumnes in v i m . J Physiol (Lond) 1983;334:19-31 21. Harris EW, Ganong AH, Monaghan DT, et al. Action of 3-(( ?)-2-carboxypiperazin-4-yl)-propyl-l-phosphonicacid (CPP): a new and h a y potent antagonist of N-methyl-* aspartate receptors in the hippocampus. Brain Res 1986;382: 174-177
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22. Wong EHF, Kemp JA, Priestley T, et al. The anticonvulsant MK-801 is a potent N-methyl-Daspartate antagonist. Proc Natl Acad Sci USA 1986;83:7104-7108 23. Honore T, Davies SN, Drejer J, et al. Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists. Science 1988;24 1:70 1-704 24. Kacz B, Miledi R. Tetrodotoxin-resistant electric activity in presynaptic terminals. J Physiol (Lond) 1969;203:459-487 25. Frankenhaeuser B, Hodgkin AL. The action of calcium on the electrical properties of squid axons. J Physiol (Lond) 1957; 137:2 18-244 26. Lea0 AAP. Spreading depression of activity in the cerebral cortex. J Neurophysiol 1944;7:359-390 27. Gloor P. Migraine and regional cerebral blood flow. Trends Neurosci 1986;9:2 1 (Letter)
Extracellular field potentials and fK+}, were recorded in slices of human epileptogenic neocortex maintained in vitro during perfusion with M2+-free artificial cerebrospinal fluid (ACSF). The human neocortex was obtained during neurosurgical procedures for the relief of seizures that were resistant to medical treatment. Spontaneous epileptiform activity and episodes of spreading depression appeared within 1.5 to 2 hours of perfusion with Mgz+-freeACSF. The epileptiform discharges consisted of negative field potential shifts (amplitude, 0.8-10 mV) that lasted 2.5 to 80 seconds and recurred at intervals ranging between 4 and 160 seconds. Both duration and frequency of occurrence of epileptiform events were not significantly different when measured in slices obtained from spiking tissue compared with those gathered from nonspiking neocortical areas. Transient increases in {K+], of up to 10.5 mM were associated with each epileptiform discharge; these changes were maximal and fastest in the middle neocortical layers. Spreading depression episodes were characterized by 20 to 30-mV negative shifts that lasted up to 200 seconds and were accompanied by increases in {K+], of approximately 100 mM. Epileptiform discharges and spreading depressions did not occur during perfusion with Mg2 -free ACSF that contained either competitive or noncompetitive antagonists of the N-methyla-aspartate (NMDA) receptor subtype. ln contrast, pharmacological blockade of non-NMDA receptors did not influence the epileptiform activity observed in Mg2+-freeACSF. These findings demonstrate that decreasing IMg2+}, Ieads to the appearance of both spontaneous epileptiform discharges and spreading depression in the human epileptogenic neocortex. Both activities are solely dependent on NMDA-activated conductances, suggesting that removal of the voltage-dependent gating effect exerted by extracellular Mg2+on the NMDA ionophore is a condition sufficient for neurons in the human neocortex to generate spontaneous, synchronous epileptiform activity. We propose that this experimental paradigm might represent a model suitable for analyzing the cellular mechanisms underlying human neocortical epileptogenesis in vitro as well as for preliminary screening of antiepileptic drugs. +
Avoli M, Drapeau C, Louvel J, Pumain R, Olivier A, Villemure J-G. Epileptiform activity induced by low extracellular magnesium in the human cortex maintained in vitro. Ann Neurol 1991;30:589-596
After the demonstration that antagonists of the Nmethyla-aspartate (NMDA) receptor can reduce the occurrence of experimentally induced seizures 117, much attention has been directed to the role played in epileptogenesis by the different receptors for the excitatory amino acid transmitters. In the course of these studies, it has also been shown that the NMDA receptor is presumably involved in chronic epileptogenesis. Accordingly, an NMDA-mediated potential is recorded in the dentate gyrus of the rat hippocampus after repeated stimulation (kindling) of the amygdala or hippocampal commissures {2, 31. Furthermore, electrophysiological studies of the human epileptogenic neocortex maintained in vitro have revealed that some of the neurons located in the deep layers generate a synaptic depolarization that is blocked by the
competitive antagonist of the NMDA receptor DL-2amino-5-phosphonovalerate(APV) C4, 51. The NMDA receptor is coupled to an ionophore that is permeable to cations such as Ca2+, Na', and K+, and gated in a voltage-dependent manner by M&+ present in the extracellular space [G, 71. Accordingly cortical slices maintained in vitro generate synchronous epileptiform discharges when the extracellular M&+ is decreased by perfusing the tissue with Mg2+-free artificial cerebrospinal fluid (ACSF) [S- 151. Because these epileptiform discharges are blocked by NMDA receptor antagonists, it has been suggested that the genesis of Mg2T-freeepileptiform discharges is caused mainly by NMDA-mediated conductances that are no longer gated by M&+. Given the possible participation of NMDA-medi-
From the Montreal Neurological Institute and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada.
Received Jan 24, 1991. Accepted for pubiication Apr 19, 1991. Address correspondence to Dr Avoli, 3801 University, Room 850, Montreal, QC, Canada H3A 2B4.
Copyright 0 1991 b y the American Neurological Association 589
ated potentials in synaptic transmission in the human epileptogenic cortex 14, 57, we analyzed the effects induced by perfusing slices of human epileptogenic cortex maintained in vitro with M$+-free ACSF. In the present study, we used extracellular field potential recordings and measurements of the extracellular concentrations of K+ ([K’],) to answer two main questions. First, which are the electrophysiological characteristics of the epileptiform activity recorded in the human cortex during perfusion with M$+-free ACSF? Second, is this type of activity sensitive to NMDA receptor antagonists as reported for the epileptiform discharges seen in the rat cortex and, in addition, which are the effects elicited by non-NMDA receptor antagonists? Preliminary reports of some of these findings have appeared {IG, 177.
Methods Human Cortical Tissue: Clinicul, Electrographic and Neuroputbological Features Human neocortical tissue was obtained during neurosurgical procedures performed for the relief of seizures that were resistant to medical treatment (22 patients of either sex). In any of these patients, the tissue used for the electrophysiological experiments was part of a block of brain tissue removed for strictly therapeutical reasons. Informed consent was obtained in all patients. Most of the patients undergoing surgery for epilepsy had been maintained on a variety of antiepileptic drugs that were progressively discontinued or markedly reduced during the weeks preceding surgery. Surgical procedures were performed under local anesthesia in all but 3 patients. In these patients, general anesthetics with halogenated compounds were used. Patients who underwent surgery under local anesthesia received during the operation the analgesic drug fentanyl citrate (Sublimaze [Abbott Lab Ltd, Montreal, Quebec, Canada] 0.2 mg/kg IV) and the short-lasting barbiturate methohexital sodium (0.5 mg/kg IV). As in previous studies performed in our laboratory [ 5 , 18}, brain tissue obtained from patients receiving general anesthesia displayed electrophysiologicalcharacteristics,generated epileptiforrn activity, or both that were indistinguishable from those of samples from patients who underwent surgery under local anesthesia. This finding suggests that the short-term effects induced by the drugs present in the brain in situ are readily washed in the tissue chamber during the time allowed for recovery after slicing. Clinical neurophysiological investigations,including the intraoperative electrocorticogram, were routinely performed for diagnostic purposes. These studies revealed that the brain tissue samples used in the experiments reported here belonged to areas that either displayed interictal spiking during the intraoperative electrocorticogram (so-called epileptogenic samples) or resided near the site of spiking (so-called juxtaepileptogenic samples). These samples were obtained from the first (n = 4 surgcal procedures) or the second (n = 18 surgical procedures) temporal gyrus. Neuropathological analysis of the removed brain tissue revealed in most patients a moderate degree of gliosis and neuronal loss.
590 Annals of Neurology Vol 30 N o 4 October 1991
Preparation and Maintenance of the Slices The techniques used for preparing and maintaining the slices in vitro have already been described in a previous study 151. In brief, neocortical slices were cut 500 to 600 pm thick by using a tissue chopper. They were placed in an interphase tissue chamber where they were perfused at 35 ? 1°C with oxygenated ACSF (95% 0,, 596 CO,). The rate of perfusion (0.5-1.5 ml/min) was kept constant in each experiment. Slices were allowed 1 to 2 hours of recovery before the recording was begun. The composition of the ACSF was (in mM) as follows: NaCl 124, KCI 2, KH,P04 1.25, CaCl, 1.8-2, MgS04 2 or 0, NaHCO, 26, and glucose 10. APV (Sigma Chemical, St Louis, MO), dizocilpine (MK-801, a gift of Merck Sharp and Dohme, Fbhway, NJ), 3-[(%)-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid (CPP; Tocris Neuramin, Bristol, UK), and 6-cyano-7-nitroquinoxaline2,3-dione (CNQX; Tocris Neuramin) were added to the perfusing ACSF.
Recording and Stimulating Techniques Extracellular field potentials were measured with glass microelectrodes filled with 2 M NaCl or through the reference channel of the ion-selective electrode. Ion-selective microelectrodes were prepared according to the methods described by Avoli and colleagues [19]. Signals were fed to a high impedance amplifier and displayed on a Gould pen recorder. Constant current anodal stimuli (0.05-0.5 mA; 10-90 p e c ) were delivered through sharpened and insulated tungsten electrodes that were placed in the underlying white matter or within the cortical layers.
Results
Field Potentials and Changes in {K+)oRecordd in the Neocortex during Perjhion with M$ifwe ACSF Spontaneously occurring field-potential epileptiform discharges were seen in 92 of over 140 neocortical slices during perfusion with MgZf-free ACSF. In all patients, the epileptiform activity appeared after 1.5 to 2 hours of perfusion with M d i - f r e e ACSF. Some typical examples of spontaneous activity recorded in the middle layers of the human neocortex are shown in Figure 1A-C. They consisted of prolonged negative shifts with superimposed fast, negative transients. Epileptiform discharges lasting less than 10 seconds were usually characterized by a single negative shift (amplitude, 0.8-10 mV) associated at times with lowamplitude, high-frequency (up to 11 Hz) events (see Fig lA, B). In contrast, the most prolonged epileptiform discharges displayed an initial phase of sustained tonic activity followed by the gradual development of “clonic” discharges consisting of recurring, clustered bursts separated by short, silent intervals of increasing duration (see Fig 1C). Hence, they bore some resemblance to the electrographic pattern associated with tonic-clonic seizures. Simultaneous intracellular and extracellular recordings have revealed that in M$+free ACSF human neocortical cells display large-
A
a
b
1600
_I 5
10
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Duration
zo
25
30
White
matter
(5)
Fig 1. Epileptifom discharges recorded with extracellular microelectrodes pkzced in the middle cortical layers of three different slices during perjiusion wtth M$ $we artificzal cerebrospinal fluid. Experiments in A and B are from the same tissue sample; in each panel, traces (a) and (b) show spontaneous discharges that were recorded at low (10-second calibration bar) and high (1-second calibration bar) speed, mpectively. The experiment shown in C illustrates that similar shape and duration characterize spontaneous (a) and stimulus-induced {b) epileptijim discharges. (0)Plot of the mean duration {abscirsa)and mean interval between one discharge and the following one (ordinate)as calculated in 83 slices obtained from 16 patients. Discharges with duration larger than 30 seconds were not included in this plot because they occuwed spontaneously at an irregular frequency. +
amplitude depolarization with ionic firing, bursts of action potentials, or both that closely correlate with the field discharges (M. Avoli and C . Drapeau, unpublished data). The frequency of occurrence and the duration of the spontaneous epileptiform discharges varied considerably in different slices even when they were obtained from the same tissue sample (compare epileptiform activity in panels A and B of Figure 1, see above). They lasted 2.5 to 80 seconds (28 k 13 seconds; n = 89 slices) and recurred at intervals that varied between 4 and 160 seconds (46 2 20 seconds; n = 91 slices). In any given slice, however, these two parameters remained usually constant over the duration of the experiment (up to 8 hours). In the plot of Figure 1D, one can appreciate that in 83 slices obtained from 16 different patients there was a close relationship (regression coefficient, 0.955) between the mean duration of the epileptiform discharges and the mean frequency of occurrence (i.e., the interval between the onset of one discharge and that of the following one) in any given slice. Comparison of both duration and frequency of occurrence of the epileptiform discharges recorded in slices obtained from epileptogenic and juxraepileptogenic samples did not reveal any significant differences. Epileptiform discharges with shapes similar to those occurring spontaneously could also be evoked by ex-
- b
-
+
h
Fig 2. Srmultaneous {K'), (upper trace) and jiekd potential
(lower trace) recordings during spontaneous (A)atad stimulusInduced (B) epsleptifom discharge in M 2 ' - j k e art$cial c m brospinalJuid recorded at ddfferent depths. Distance fmm the pia is given on the kfi of each sample in micrometers. Note that szmzlar extracellular jield potentials and increases in {K+j0 characteraze the stimulus-induced and the spontaneous epikptiocform discharges as well as that the largest increases in {KKfIo cur at a depth of 1,200 pn.
tracellular, focal single-shock stimuli (see Figs lC, 2B). Such stimuli elicited epileptiform responses in most of the slices that did not generate any spontaneous epileptiform activity. Stimuli delivered in different regions of the same slice (e.g., pia, white matter, cortical layers) resulted in s d a r patterns of discharge. This finding suggested that the epileptiform tesponse was not dependent on the activation of any specific intracortical pathways. Both spontaneous and stimulus-induced discharges were accompanied by a transient increase in [K'], that reached a maximal value of 10.5 mM (6.7 -+ 2.8 mM; n = 12 slices) from a baseline level of 3.25 mM. As illustrated in Figure 2, the largest and fastest changes in {K 1, usually occurred in the middle layers of the human neocortex between 800 and 1,600 p,m from the pia. The largest negative field potentials were also recorded at these depths. Therefore, the most active layers of the neocortex during epileptiforrn activity Mg?+-free in ACSF appeared to reside in the middledeep cortical layers. Undershoots of the K+ signals did not follow the increase in { K'], observed during each epilepuform event, but were seen to occur after episodes of spreading depression (Fig 3, Control). Spreading depression was recorded in eight slices in which it occurred either spontaneously (Fig 4 ) or after extracellular focal stimuh (see Fig 3); it lasted 50 to 220 seconds (97 f 31 seconds; n = 6 slices), was associated with 10 ro 25-mV negative shifts in the extracellular field potential, and was followed by a period during which spontaneous epileptiform discharges failed to occur. In two slices in +
Avoli et al: Mg2+-freeEpileptogenesis in the Human Cortex 591
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Fig 3. Measurements of @?}, during spontaneous epileptifm discharges and spreading depression induced by a train of repetiti**elow-frequeng stimuli in M 2 '-free art$cial mehospinal fluid (Control),during addition of DL-2-amino-5-phosphonovalerate (APV; JO CJM) and after washout of the N-methyl+ aspartate receptor antagonist (Wash). Note that epileptiforn discharges as well as spreading depression are blocked by APV. The three trtangles in the Wash trace point at the time during whtch the train of stimuli (10 seconds long, 2 Hzj was deliuered. A
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Fig 4. Spontaneous epileptifm discharges and spreading depression (asterisk) recorded extracellulcsrly in the middle cortical layers of a slice perj5used with M 2 '$ree artificial cerebrospinal fEuid. Note the different amplitude and duration o f the spreading depression compared with the epileptiform discharges. Also note the period of depression in the occurrence o f epzleptiform discharges that follow the spreading depression. A kzpse of approximate& 20 seconds exists between recordings A and B.
which it was measured, [K'], increases during spreading depression attained values of approximately 100 mM (see Fig 3). Sensitivity of the Epdeptafrm Activity in ACSF to NMDA Receptor Antagonists
M 2 '-free
The participation of membrane conductances activated through the NMDA receptor in the generation of epileptiform activity in Mg?+-free ACSF was analyzed by studying the changes induced on spontaneous and stimulus-induced discharges by competitive and noncompetitive NMDA receptor antagonists. The competitive antagonist of the NMDA receptor, APV (20-100 pM) 1201, decreased the frequency of occurrence and eventually blocked the epileptiform discharges in a dose-dependent fashion (n = 11 slices).
592 Annals of Neurology Vol 30 No 4 October 1991
Fig 5 . (Aj Eflects induced by increasing concentrations of the N-methyl-Daspartate receptor antagonist, DL-2-amino-5phosphonovalerate (APV),on spontaneous epiltpt2fown discharges; in this experiment, the extracellulitr field potential recordings were passed through an alternating current filtersecond-stage amplij5er. (B) Bar graph of the decrease in frequency of occumnce of spontaneous discharges (ordinate)during successive applications of increasingly higher concentrations of APV (abscissa). Data were obtained from three different slices in which the frequency of occurrence of epileptiform discbarges was computed after 30 minutes of perfusion with artificial cerebrospinalfEuid (ACSF) containing the stated concentrations of APV. (C) Changes induced by 3-{(+2-carboxypiperazin-4yl}-propyl-l-phosphonicacid (CPP; 2 pM) on spontaneous epiEept;fonnactivity recorded simultuneously with a K +-sensitive (upper trme) and field potential electrod? (lozuev trace). CPP trace was taken 12 minutes after onset of pwfusion and wash truce 40 minutes afer reperfusion with control M 2 -free ACSF. +
A typical experiment in which increasing doses of APV were sequentially applied to the slice is shown in Figure 5A; in this case, the complete blockade of the epileptiform activity occurred in the presence of 100 pM APV. Figure 5B shows a bar graph of the effects induced by different doses of APV in three different slices that were perfused with increasing concentrations of this NMDA receptor antagonist. The depressant action of APV (50-100 pM) was also observed while studying stimulus-induced epileptiform discharges (n = 6 slices; not shown). In eight experiments, we also analyzed the effects exerted by CPP (2-5 pM), which is another competi-
tive antagonist of the NMDA receptor 1211, on the M& +-free epileptiform activity. As illustrated in Figure SC, spontaneous epileptiform discharges recorded simultaneously with extracellular field potential and K+-selective electrodes disappeared in the presence of CPP. A similar action of CPP (2 pM) was also observed when analyzing the stimulus-induced epileptiform discharge (n = 5 slices). In one instance, the action of CPP on the epilepuform discharges was accompanied by the disappearance of spreading depression episodes that had occurred spontaneously in control M& +-free ACSF. In another experiment, spreading depressions induced by trains of repetitive extracellular focal stimuli (10 seconds long at 2 Hz) were not observed after perfusion with M&+-free ACSF containing APV (50 pM) (see Fig 3). The effects induced by both APV and CPP were reversible on returning to control M$+-free ACSF. Furthermore, the blockade of spontaneously occurring discharges was not accompanied by any progressive decrease in the duration of each discharge before the occurrence of blockade. This is shown in detail in Figure 5C, where it is possible to appreciate that the two epileptiform events generated before the complete disappearance of the spontaneous activity are practically indistinguishable from any of the discharges recorded in control conditions. In six slices, we also assessed the effects induced by the noncompetitive antagonist of the NMDA receptor, MK-801 [22]. MK-801 at concentrations of 2 to 5 pM also blocked the epileptiform discharges observed in M 8 +-free medium. Two differences were noted, however, when these effects were compared with those induced by APV or CPP. First, the blockade induced by MK-801 was irreversible (up to 3 hours of wash with control M&+-free ACSF). Second, the effects induced by this noncompetitive antagonist of the NMDA receptor subtype were more gradual. This is shown in the graphs and raw data illustrated in Figure 6 in which the epileptiform activity generated by the same neocortical slice was studied sequentially during perfusion with M$+-free ACSF containing, at first, CPP and, later, MK-801; in contrast to the blockade induced by CPP, MK-801 elicited in this experiment a progressive and gradual decrease in both the frequency of occurrence and duration of each single discharge.
A
MK-801.2 rM
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Time (mm)
Fig 6. Comparison of the e f f t s induced b the competitive 3{(~~-2-carboxypipwazin-4-yl)-propyl1-phosphonic acid (CPP) vld the noncompetitive dizorilpine (MK-801)N-methyl-o aJpurtateantagonists on M$+-free epikptifrm activity. (A) Shows the histograms of the frequeng of occuwence (upper panen and half-width duration (lowerpanel) of spontaneous epilegtiform discharges in control and during successive CPP and MK-801 application. (B) lllarstrates samples of discharges obtained at the times indicated ly each respective letter (a-d) in A. Note that CPP induces a block of the epileptifarmdischarges that is not preceded by a reduction in duration, whereas MK-801 induces a progressive and gradual decrease of both fequency of occurrence and duration of each epilegtifm event. (r
recorded in M&+-free ACSF. In two of four experiments, 3 pM CNQX induced a decrease in duration of both spontaneous and stimulus-induced discharges (Fig 7A). These changes were not statistically significant (t test), however, when the values obtained in all experiments were compared. CNQX was also unable to modify the pattern of the epileptiform discharge when applied at relatively high concentrations (10 p.M; n = 3 experiments). As shown in Figure 7B and C , CNQX did not influence the frequency of occurrence of spontaneous epileptiform events or the shape and duration of the stimulusinduced discharge. This lack of effect markedly contrasted with the depressant changes observed in this experiment when CPP (5 pM) was added to the M&+-free ACSF. CNQX reduced at times the amplitude of the rhythmic oscillations overriding the negative shift associated to the epileptiform discharge (see Fig 7A). This effect was seen in two of four experiments and one of three experiments in which 3 and 10 pM of C NQ X were used, respectively.
Sensitivity of the Epdeptifom Activity in MgZ'-fwe ACSF to tbe N o n - N M D A Receptor Antagonist C N Q X
Perfusion of the neocortical slices with M&'-free ACSF containing 3 p M of CNQX (n = 4 experiments), which is an antagonist of non-NMDA receptor subtypes [23}, failed to change significantly the frequency of occurrence of the epileptiform discharges
Avoli
CPP, 2 &AM
Discussion It has been shown that lowering M$+ in the extracellular space induces epileptiform activity in the neocor-
et
al: M$+-free Epileptogenesis in the Human Cortex
593
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Fig 7 . (A) Eflects induced by 3 f l 6-oano-7-nit~quinoxaline-2,3-dione (CNQX} on stimulus-induced (Induced)and spontaneousb occum'ng (Spontaneous}Md'gree epikptiform discharges. Although the frequeno of occurrence of spontaneous discharges was not mod$ed Sy CNQX in this experiment, there was a decrease in the duration of each M 2 'gree epileptfifom event as well as a reduction of the overriding fast-transients.(B, C) CNQX faih to injuence the fwquency of occurrence ofspontaneous activity (histogram in B) and the duration and shape of the stimulus-indmd discharges (raw data in C). This activity, hozuever, is blocked by the subsequent effectsof 3-{(4-2carboxypiperazin4-y&wopyl-l -phosphonicacid (CPP). The raw data in C were taken at the times indicated by the letters (a-c) in the histogram in B in which the ordinate axis gives the number of epileptiform discharges occum'ng every 60 seconds.
tex 110, 141, the entorhinal cortex [S, 111, and the hippocampus 18, 9, 13, 151. Our experiments extend these observations to the neocortical tissue of humans obtained from epileptic patients undergoing surgery for the relief of seizures resistant to pharmacological therapy. We have characterized some electrophysiological properties of the epileptiform activity studied with extracellular field recordings and measurement of EK'], in the human brain tissue perfused with Mg2+free ACSF. Furthermore, we demonstrated that this activity is caused by the selective activation of conductances that are mediated through the NMDA receptor subtype. Finally, we reported the occurrence of spreading depression in the human neocortex perfused with W f - f r e e ACSF. The electrophysiologicalfeatures of the epileptiform discharges described in the present study are very similar to those recorded in slices of entorhinal cortex [S, 111 and neocortex [lo, 143 obtained from rats during perfusion with MgZ+-free ACSF. These similarities include the spontaneous occurrence of the discharges,
594 Annals of Neurology Vol 30 No 4 October 1991
the regular rate of occurrence, the relative long duration of each single discharge, and the resemblance of the most prolonged ones to the tonic-clonic electrographic seizures recorded in situ. All these characteristics indicate that the epileptiform discharges described here r e p r e s e a good experimental model for studying the cellular mechanisms underlying ictal-like epileptiform events in the neocortex. Furthermore, the fact that the epileptiform discharges observed in our experiments occur spontaneously indicates that this type of epileptiform activity might represent a suitable in vitro model for analyzing the effects induced by anticonvulsant agents, including preliminary screening of antiepileptic drugs. Interestingly, in the human neocortex maintained in vitro, epileptiform discharges do not occur spontaneously after blockade of the y-aminobutyric acid, receptor [181. Epileptiform discharges in Mg2+-free ACSF observed in this study are readily blocked by antagonists of the NMDA receptor, indicating that conductances activated through this receptor subtype participate in the generation of this type of epileptiform activity. Hence, a major mechanism in this model of acure epileptogenesis is due to the removal of the voltagedependent gating effect exerted by extracellular Mg2+ on the NMDA ionophore [G, 71. We cannot exclude, however, that an additional contribution to this type of epileptiform activity might arise from nonspecific effects due to the removal of M d f from the extracellular space. These effects include the antagonistic action of M d + on Ca" conductances [24) and the reduced membrane surface screening 1251. Both effects are well known to lead to an increase in neuronal excitability. Both competitive and noncompetitive antagonists of the NMDA receptor subtype were effective in blocking epileptiform discharges in M8+-free ACSF. The effects induced by these two classes of NMDA receptor antagonists, however, were not similar. When the competitive antagonists APV or CPP were used, Mg2 -free epileptiform discharges decreased in their frequency of occurrence, but not in their duration or amplitude. Therefore, when the blockade of the spontaneous activity occurred, the last epileptiform event to be observed did not differ from those seen in the control situation. Conversely, during application of MK-801, the progressive decrease in rate of occurrence was paralleled by a gradual reduction of the duration of each single discharge. Similar differences between the effects induced by competitive (APV) and noncompetitive (phenocyclidine) NMDA receptor antagonists on M$+-free epileptiform activity have been reported in the CA3 subfield of the rat hippocampal slice [131. Hence, the differences observed here might be due to the use-dependent mode of action of noncompetitive antagonists such as MK-801. +
A rather puzzling finding obtained in our experiments is the lack of effects induced on the M2+-free epileptiform discharges by the antagonist of nonNMDA receptor, CNQX [23]. Given the fact that the fast excitatory postsynaptic potential in the human neocortex is presumably due to the activation of nonNMDA receptor subtypes (IS}; G. Hwa and M. Avoli, unpublished data), the present data are unexpected. Indeed, in the light of these findings, one must conclude that not only the triggering mechanism responsible for the occurrence of epileptiform discharges in Mg2+-freeACSF, but also the synaptic events that underlie the self-sustained character of each epileptiform discharge, are solely caused by the activation of the NMDA receptor. It should be noted that, in some experiments, CNQX was capable of reducing the rhythmic, fast oscillations that occur during the negative epileptiform shift. The mechanism responsible for this effect is as yet unknown. We are inclined to exclude the possibility that this effect of CNQX was mediated through the glycine site of the NMDA receptor, however, because preliminary experiments in our laboratory have indicated that perfusion with Mg2+-free ACSF containing glycine does not modify this type of epileptiform activity in the human neocortex (C. Drapeau and M. Avoli, unpublished data). An interesting observation reported in our study is the occurrence of spreading depression in the human neocortex perfused with Mgz’-free ACSF. Although we did not analyze the velocity of propagation of this type of event, both the amplitude and the duration of the associated field potential as well as the value attained by [K’], suggest that it represented spreading depression as originally described by Lea0 [26}. Failure to observe spreading depression in the cortex during surgery where electrocorticograms are recorded under conditions that are likely to trigger spreading depression has suggested that this phenomenon might be absent in humans {27]. Our data, however, show that spreading depression occurs in the human neocortex maintained in vitro when perfused with Md+-free medium and, thus, suggest that this experimental condition might represent a critical factor in causing human tissue to undergo spreading depression. This conclusion is supported by the finding that, in our experiments, the occurrence of spreading depression was blocked by antagonists of the NMDA receptor along with epileptiform discharges. The spreading depression observed in rat hippocampal slices bathed in M&+-free medium is not seen after application of APV [9]. Furthermore, Schneiderman and MacDonald [I 31 have reported that spreading depression is recorded in the in vitro hippocampal slice during perfusion with Mg2 free ACSF, but not in the presence of the convulsant drug penicillin. Therefore, NMDA-activated conduc+
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tances might play a critical role in the generation of spreading depression at least in the mammalian cortex. This study was supported by grants from the Medical Research Council of Canada (MA-8190) and NATO. M.A. and R.P. were recipients of INSERM-FRSQ Exchange Programs. We thank D r V. Tancredi and Ms C. Tzeng for participating in some experiments.
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