The lateral spread of ictal discharges in neocortical brain slices

Epilepsy Res., 7 (1990) 29-39

29

Elsevier EPIRES 00347

The lateral spread of ictal discharges in neocortical brain slices

B o w e n Y. W o n g * a n d D a v i d A. Prince Department of Neurology and Neurological .Sciences, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) (Revision received 16 April 1990; accepted 17 April 1990)

Key words: lctal discharges; In vitro brain slice technique; Brain slices; Neocortex

The in vitro brain slice technique was used to examine the lateral propagation of spontaneous electrographic ictal episodes across adjacent areas of guinea pig neocortex. Epileptiform activity was induced by perfusing slices with Mg-free artificial CSF. Simultaneous field potential recordings of ictal episodes were obtained from 4 micropipettes placed 1-3 mm apart across coronal slices in middle-cortical layers. Two types of lateral spread were characterized. Ictal episodes often developed focally and then spread as a slowly moving wavefront traveling at <0.3 mm/sec into adjacent, uninvolved cortex. By contrast, other episodes began nearly synchronously at all cortical sites. The individual afterdischarges that composed each ictal episode propagated rapidly across the cortex at >30 mm/sec and were triggered by multiple pacemakers. Ictal episodes always terminated abruptly across the entire slice. The NMDA-receptor antagonist, 2-amino-phosphono-valerate, applied focally between recording sites, blocked rapid propagation across treated areas and resulted in the emergence of spatially separate, independent pacemakers. Pacemaker failure is the proposed mechanism for simultaneous and generalized termination of ictal episodes in this in vitro model of epileptogenesis.

INTRODUCTION Current hypotheses regarding neocortical function are based on the concept that cortical structures are organized to process information in a parallel manner via excitatory and inhibitory interactions within and between adjacent cortical columnar units 3a. At the same time this functional architecture restricts the lateral spread of information to within highly localized cortical receptive fields22. On the other hand, during pathological *

Current address: 3300 Webster St., Suite 402, Oakland, CA 94609, U.S.A.

Correspondence to: Dr. David A. Prince, Department of Neurology, C338 Medical Center, Stanford, CA 94305, U.S.A.

states such as a seizure, abnormal activity spreads rapidly and synchronously throughout the brain 23'46. Neocortical mechanisms that restrain abnormal spread of excitation while preserving normal brain function must critically depend on the dynamic balance of inhibitory and excitatory interactions. Breakdown in these cellular mechanisms is hypothesized to be the pathophysiologicai basis for epilepsy 13a4a9.35'36'3s. Although analyses of underlying basic cellular mechanisms have provided insights into the genesis of synchronized interictal discharge in small populations of neurons (e.g., see ref. 15 for review), the events underlying the transition from focal interictal behavior to the initiation and unrestricted propagation of ictal discharges have received much less attention ~2'35.The mechanisms of

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30 seizure spread have been particularly hard to approach because of the inherent difficulties in performing detailed '3-dimensional' analyses of propagation in vivo. For example, spectral analysis of signals recorded from multiple cortical and subcortical structures would be required to adequately track the routes along which epileptic activity could be conducted tS"29m. On the other hand, use of in vitro brain slice techniques allows a more direct a~ad technically easier examination of the spread of epileptiform brain activity. With this approach, Chervin et al. '; simplified the problem by studying propagation of interictal discharges in only one spatial dimension across neocortical brain slices. They found that, over large distances, propagation appeared to be linear and smooth, confirming previous measurements of conduction ''~, however, over small distances propagation velocity tended to be variable and direction dependent. Comparable studies of the spread of ictal discharge have not been reported. Recently, several groups have described models of ictal epileptiform activity in vitro 2'20'26'3L40'45 that share many electrographic features with experimental seizures in vivo and with clinical ictal episodes observed on the electroencephalogram. In one such preparation, epileptogenesis is produced by perfusing slices with solution free of magnesium ions (0-Mg) 2'5'32, presumably due to the marked enhancement of excitatory synaptic currents that occurs when the Mg"+-related voltagedependent block of N-methyl-D-aspartate receptor-coupled synaptic transmission is removed 3~'3~. We performed pilot studies in the 0-Mg model and found that ictal epileptiform activity was not uniform across the slice but instead was highly dependent on local factors. In the experiments reported here we describe unidimensional temporal-spatial patterns of ictai spread in neocortical slices bathed in Mg-free perfusate. Portions of these results have been published in an abstract ~s. METHODS Standar i techniques for preparation and maintenance of in vitro cortical slices and conventional electrophysiological methods were used ~t. Guinea pigs were deeply anesthetized with pentobarbital

(30 mg/kg i.p. injection) and decapitated. Four hundred/~m thick coronal sections from sensorimotor neocortex were cut with a vibratome. Cingulate cortex was trimmed with a scalpel blade, and neocortical slices 8-10 mm long were rapidly transferred to an interface type chamber and maintained at 37 °C. Brain slices were incubated for at least 1 h before the recordings were made. The standard Ringer's perfusate consisted of (in mM): NaCI, 124; KCI, 2.5-5; MgSO4, 1-2; CaCI2, 2; NaH2PO 4, 1.25; NaHCO 3, 26; and dextrose, 10. MgSO 4 was omitted in 0-Mg solutions. The solution was equilibrated with a gas mixture of 95% O 2 and 5% CO2; the pH was 7.4. Slice viability was checked by recording the amplitudes of field potentials evoked by stimulation of sub-cortical white matter-- layer 6. The methods used for the induction of 0-Mg ictal phenomena are identical to those previously published for both neocortical and hippocampal brain slices 2'32'47. Extracellularly recorded spontaneous activity began 15-60 rain after changing to a 0-Mg bath solution perfused at a rate of 10-20 ml/h; the deadspace of the chamber and inflow lines was less than 2 ml. Simultaneous field potential recordings of 0-Mg seizures were obtained via 4 bath solutionfilled (i.e., the 0-Mg perfusate) recording pipettes positioned at approximately 1-3 mm intervals across the length of the slice in the middle cortical layers (usually about 500-750/~m from the pial edge). Field potentials were amplified and recorded monopolarly (against an indifferent electrode in the bath) with a digital video cassette tape system. The electrode array was fabricated by fastening pairs of broken (1-10 MQ) micropipettes onto small pieces of plexiglass so that electrode tips were spaced approximately 1-3 mm apart. Two plexiglass holders were then mounted onto separate micromanipulators so that pairs of electrodes could be independently positioned on the slice, lnterelectrode distances were measured with a calibrated eyepiece through the dissecting microscope, after the electrodes were in position. Drugs were diluted with freshly mixed stock solution using the above standard medium (0-Mg medium in all 0-Mg-seizure experiments) and were applied topically as focal microdrops from a broken-tipped drug-filled pipette. Recorded data seg-

31 ments of interest were played back and analyzed using a digital oscilloscope. The terminology used in this paper to describe 0Mg-induced epileptogenesis 2'47 in field potential recordings can be summarized as follows. An 'interictal' event was defined as a single isolated electrographic epileptiform 'spike' and/or 'wave' typically having a duration of less than 0.1 sec. 'Ictal' events were marked by the occurrence of numerous individual 'spike' and/or 'wave' discharges that typically lasted many seconds and rode on a slow potential envelope. Descriptions of other types of electrographic epileptiform behaviors according to their temporal-spatial patterns are presented below. Since these neocortical slices were cut in the coronal plane, the term 'lateral' in this paper refers to the medial to lateral dimension of the cortex and 'verticar refers to the axis extending from the pia to white matter. The lateral spread of ictal discharges induced by exposure to 0-Mg perfusate was examined in a total of 24 slices. In most experiments, extracellular field potentials were continuously monitored from 4 recording sites from the time that perfusion with 0-Mg solution was begun until the end of the experiment several hours later. Two electrodes were used in some of the early experiments. In general only one slice in the chamber was selected for study on any given experimental day; however, multiple slices were occasionally examined sequentially to measure conduction velocity, to study the effects of electrical stimulation or to test pharmacological agents. Conduction velocity (mm/sec) was calculated from measurements of straightline interelectrode distances divided by the latency difference at the 2 electrodes. Any 2 electrodes were usually sufficient for velocity measurements of electrically triggered discharges; however, in order to measure the velocity of spontaneously occurring discharges, three or more electrodes were required in order to first be certain that the events were not being initiated between the sites of measurement. Pilot studies (Wong and Prince, unpublished) have shown that there are only very small latency differences for vertical propagation across different cortical layers (i.e., pia to white matter) and since the recording sites were chosen at approximately the same

depth below the pia, this potential source for error is probably negligible. RESULI'S Based on our preliminary findings 48 we were able to distinguish 2 types of ictal epileptiform behavior, i.e., episodes of generalized vs. those of focal onset. The present results characterize these 2 types of behavior in relation to their patterns of initiation, spread, and termination.

Ictal events of generalized onset In approximately 50% of slices, the initial type of epileptiform activity consisted of spontaneous ictal events that appeared to involve the entire slice as judged by their nearly simultaneous onset at 4 recording sites spanning 3-7 mm (e.g., Fig. 1, Fast spread). However, upon closer examination, slight, but variable latency differences (ca. 10-200 msec depending on the interelectrode distance) could be measured between the individual recording sites in all cases (e.g., note latency difference of the initial event between electrodes 1 and 4 in Fig. 1, Fast spread), indicating that events were initiated focally and then propagated rapidly away from the site of initiation. Even though initiation was distinctly focal, for the purposes of classification we considered these ictal episodes to be generalized, because individual discharges were propagated rapidly without restriction along the entire length of the slice from the onset of the episode. In the remaining slices the 'fast spread' pattern developed only some time after ictal events of focal onset with a 'slow spread' pattern (see below) had been recorded. Once the 'fast spread' pattern appeared, it persisted for the remainder of the experiment (hours). We chose the very first discharge of an ictus (e.g., arrows in Fig. 1, Fast spread) for most of the measurements to avoid any ambiguities in identifying the event as recorded at each electrode. The conduction velocity for the lateral spread of individual spikes or waves averaged 40 mm/sec _+ 8.9 S.D., as calculated from latency differences measured in 16 slices. This value is slow compared to conduction velocities of bicuculline-induced interictal discharges in neocortica! sJices (60 mm/sec) 9

32 and in the CA3 pyramidal region of hippocampal slices (130 mm/sec) 25'44, and is an order of magnitude slower than the action potential conduction velocities in central axons (mossy fiber conduction velocities = 400-500 mrn/sec) ~'~5. Although these events were generally stereotyped from episode to episode, a comparison of the recordings from different channels revealed significant site-to-site variability in the morphology and rhythmicity cf activities despite the nearly simultaneous onset and termination. Fig. 1 (Fast spread) illustrates these electrographic differences. Occasional isolated spikes (arrow labeled A) propagated across the length of the slice (electrodes 1-4), whereas higher frequency repetitive discharges (e.g., segment labeled B, electrodes 1 and 2) tended to have a more focal distribution. This lack of uniformity in the electrographic features of the ictal episode across the slice was also apparent when slow potential changes at different electrodes were compared. For example, in Fig. 1, Fast spread, a slow negative (downward) DC potential shift lasted for the duration of the ictal episode at all sites, whereas an initial slow positive DC component was present only at electrode 1. These local site-to-site electrographic differences were observable in all slices and appear to be related more to lateral electrode position than to small changes in sites of recording relative to the depth from the pial surface. All slices chosen for study were 'healthy' as judged by the amplitudes of evoked responses of 0-Mg-induced activity, making local injury an unlikely cause for non-uniformity of ictai activity at different electrodes.

morphosis of the ictal pattern is similar to that seen during the spontaneous evolution of 0-Mg ictal behavior in hippocampal slices 2. The mean velocity of the slow 'march' of focal ictal episodes was 0.16 mm/sec + 0.1 S.D. (n = 4; measurements were made from 4 recording sites) as calculated from the onset latencies of the slow potential envelopes that marked the beginning of an ictal episode. Although the site of onset could not be determined in 2 electrode recordings, a comparable delay lasting several seconds was measured from the time discharges were first seen at one electrode to the time they arrived at the second. Low voltage discharges were frequently ob-

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Ictal events of focal onset Ictal epileptiform activity which began at a single electrode and then slowly spread or 'marched' into neighboring sites was observed transiently in about 50% of all the slices examined. These focal ictal events were seen only at the beginning of the experiment when the epileptiform activity first began to occur. After a mean duration of 28 rain + 13.8 S.D. (range 6-45 rain, n = 6), all slices abruptly switched to a pattern of generalized ictal activity of the type described above, where epileptiform activity propagated without restriction from the onset of the episode. This sudden recta-

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Fig. 1. Top: fast generalized spread. Oeneralized ictal event appears first at electrode I and at short but increasing latency at electrodes 2-4. A conduction velocity of 55 mm/sec was measured between electrodes 2 and 4. Arrow at A over trace 1 points to single spike, late in the brief ictal event, which propagates from 1 to 4, whereas succeeding spikes under B (bar) are seen only at electrodes 1 and 2. Bottom: slow focal spread. Focal onset of un ictal event seen at electrode 1 with successive delays of several seconds as the electrographic seizure propagates to each of the other recording sites. Thirteen seconds were required for the ictai activity to 'march' across the entire electrode array (3.9 mm). Estimated velocity of spread was 0.29 ram/see, assuming that the event began between electrode 1 and the cut edge of the cortex. DC recordings, positivity up in this and subsequent figures.

33 served just prior to the onset of the slow negative shift (to the left of the arrows in Fig. 1, Slow spread). When these discharges were examined using a faster timebase, they appeared to correlate with some of the individual spikes occurring at the adjacent recording site. The frequency of discharge for individual events was always highest at the onset of the ictus and would then slowly wane with time. In spatial terms, this finding indicates that it is at the moving wavefront of the ictus where the most intense and rapidly discharging epileptiform activity is occurring, a finding consistent with previous observations 3~. Although the advancing front of focal ictal activity spread slowly, the individual afterdischarges that propagated within the area of cortex involved in the ictus conducted at velocities comparable to those seen in ictal events of generalized onset. Usually this was most evident toward the end of the ictus when only slow rhythmic spiking remained after the high frequency afterdischarge had dissipated. For example, the individual afterdischarge waves at the end of the episode in Fig. 1, Slow spread, appear to originate near electrode 1 and are rapidly conducted to electrodes 2 and 3 (dots under trace 3 ).

Local determinants of propagation Multifocal initiator sites. Close examination of individual propagating afterdischarges revealed that these events arose spontaneously from different areas across the slice. In the example of Fig. 2,

spontaneous discharges were recorded from 4 channels anti displayed at a fast timebase. Slight differences in onset latency for individual events are seen a: each electrode. The first event spread from electrode I to electrode 4 (downward arrowheads), while the second event apparently propagated in the opposite direction (upward arrowheads).

Focal conduction block by DL-2-amino-5-phosphono-valerate (APV). Since we previously found that 0-Mg-induced epileptiform discharges can be blocked by antagonists to the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor 47, we made focal applications of the competitive NMDA antagonist, DL-2-amino-5-phosphono-valerate (APV) to determine whether this compound would block propagation of generalized ictal discharges. APV (200-800 ~M) was applied to small areas of cortex between the 2 middle electrodes of the 4-channel array (Fig. 3). Within seconds of application, the lateral propagation of 0-Mg ictal events was blocked, resulting in emergence of spatially distinct, independent pacemakers in each of the 2 pharmacologically 'disconnected' halves of the slice (see Fig. 3A, below bar 'APV'; cf., Fig. 3B and 3C). After several minutes of washout the ictal events resynchronized across the drug-exposed area (Fig. 3D). We were able to repeatedly and reversibly block lateral propagation in all slices tested (n = 4). Similar applications of atropine (17-200/~M) or normal Mg-containing bath solution (2 mM Mg) did not produce any effect (data not shown).

Conduction block induced by high frequency electrical stimulation. We were also able ~c demont ':1 ~

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Fig. 2. Bidirectional propagation. Four-channel ~ecording of 0Mg-induced epileptiform discharges shown at a fast timebase. The initial wave of the first spontaneous discharge propagates from electrode 1 to electrode 4 (downward pointing arrows). The next event is initiated at the opposite end of the slice and propagates in the reverse direction from electrode 4 to 1 (upward pointing arrows).

strate another type of conduction block evoked by electrical stimulation of the cortex. Propagating discharges and ictal events were easily triggered by direct stimulation of the cortical gray matter using 1-3 sec trains of 0.5-10 Hz, 50 /~sec, 100-500/tA pulses in slices that displayed spontaneous ictal episodes of generalized onset. At the lower stimulus frequencies (i.e., usually 1 Hz or less) each stimulus of the train could evoke an epileptiform event that propagated across the slice. For example, in the recordings of Fig. 4 discharges that propagated from electrodes 1-4 were triggered by low frequency (1 Hz) stimuli delivered

34

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Fig. 3. Propagation blocked by APV. A: repetitive brief spontaneous ictal events of generalized onset, each lasting about 3-4 sec, are recorded from 4 sites as diagrammed. The bars above the first trace represent segments shown at an expanded time base in panels labeled Pre (B), APV (C), and Wash (D). Contro| recording (PRE) shows spontaneous ictal events occurring across the entire slice. A drop of APV (800pM) is topically applied between recording sites 2 and 3 (see diagram) at time noted by triangle event marker. Segment D recorded during APV washout ('wash').

near electrode 1. When higher frequencies were used (i.e., > 1 Hz), epileptiform events could still be evoked near the stimulating electrode (Fig. 4B and C), but would either completely fail to propagate (Fig. 4C) or would propagate only intermittently during the stimulus train (Fig. 4B). At times, an 'ectopic' discharge would arise from the opposite end of the slice and propagate in the reverse direction towards the portion of cortex that was closest to the stimulating electrode (arrows in Fig. 4C).

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Fig. 4. Frequency-dependent conduction block. Recordings of stimulus-induc~:d generalized discharges are shown at several rates of stimulation. Stimuli applied near electrode 1. A: each 1 Hz stimulus triggers a slow event seen at progressively lower ~:.,.plitude and longer latency at 2-4. With 2 Hz stimulation in B, a variable failure of evoked events is seen at 2-4 on every 4th or 5th stimulus. C: 5 Hz stimulation at the same intensity fails to evoke events at sites 2-4 following the first stimulus of the train. An 'ectopic' pacemaker triggers a propagating event from the opposite end of the slice indicated by the arrows, e\ ~'~ though stimuli at 1 fail to do so.

Changes in excitability occur at the onset but not termination of ictal episodes Cessation of ictal afterdischarge. Although the onset of an ictal episode could be either focal or generalized as noted above, the termination of the electrical 'seizure' was almost always abrupt, i.e., the cessation of each ictal episode was nearly simultaneous and generalized across the entire slice (e.g., Fig. 1). In only one instance was there persistence of focal discharges after cessation of the generalized event, perhaps due to unrecognized damage or discontinuity in that particular slice

35 (data not shown). We considered 2 possible explanations for arrest of ictal episodes in our model (see also ref. 46). In one hypothesis, cessation of ictal behavior could be caused by a generalized and simultaneous failure of propagation due to suppression of cortical excitability• Alternatively, cessation could be caused by the sudden arrest of pacemaker activity. In this case, propagating waves could emanate from active pacemaker sites• Sudden cessation of activity at these sites would then be followed by an abrupt and generalized electrical 'silence•' B

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A: typical 0-Mg-induced 'jacksonian march' was triggered by applying a 0.5 Hz stimulus train near site I. Stimulus was on for

entire recorded segment except for a 14 sec pause (STM OFF above top trace). High frequency spontaneous discharges appeared at the onset of the ictal episode at each recording site (below bar 'B') and eventually ended synchronously at sites 1-4 (below bar 'C'). Each subsequent stimulus evoked a single epileptiform wave that propagated from 1 to 4 (segment marked by dashed line under A4). When the stimulator was turned off, all epileptiform activity ceased synchronously across the slice. Portions of record marked by bars 'B' and 'C' are expanded in panels B and C to show differences in propagation of activities during various stagesof the ictal episode. B: prior to the spread of the focal ictal event from 1 to 2, both stimulus.evoked and spontaneous potentials are seen only in 1 and minimally in 2, but not at 3 or 4. C: at the termination of the ictel episode, triggered discharges (dots) propagate from 1 to 4 after the last generalized spontaneous event. Arrowheads in C4 mark spontaneous rapid spikes. Dots in B and C indicate stimuli applied at 0.5 Hz. Note different time calibrations in A-C.

We tested this latter hypothesis in one experiment by using a continuous constant intensity stimulus train to compete with endogenous pacemakers present in the slice. We estimated cortical excitability grossly by recording the amplitude and spread of evoked responses before, during, and after development of a focal ictal event (see Fig. 5). As expected, prior to generalization of the focal event, stimuli failed to elicit any propagating spikes (see Fig. 5, segments before bar 'B' in first panel, and panel B). As ictal activity spread throughout the slice, stimuli elicited single spikes that propagated to all recording sites, indicating that widespread cortical areas had become excitable (Fig. 5C). Note the spontaneous cessation of endogenously initiated discharges (under bar C and in panel C) at a time when the applied stimulus continued to evoke epileptiform events. The external stimulus train at this time is effectively the last remaining 'pacemaker' that is driving the 'rhythmic ictal activity.' Halting an externally applied stimtdus train (see Fig. 5, 'STM off') effectively simulates the nearly simultaneous and generalized arrest of 'ictal' behavior. These results suggest that simultaneous termination is due to a failure of initiation (i.e., refractoriness of automaticity and initiation, or 'pacemaker failure') and not to a failure of propagation.

'Use-dependent' changes in excitability are involved in slow spread. Comparison of ictal events of focal versus generalized onset revealed that, in the former, ictal onset and propagation of individual discharges were initially restricted to an area of excitable cortex, while in the latter, spread was unrestricted from the outset (compare 'f~+st spread' and 'slow spread' in Fig. 1). Significant changes in the spatial limits of conduction of stimulus-evoked discharges were demonstrated in the experiment described above (see Fig. 5). Before the onset of the ictus, low frequency, 0.5 Hz, stimuli evoked regular interictal spikes that were seen only in the electrode nearest to the site of stimulation, although very low amplitude potentials were seen at the adjacent site as well (Fig. 5A1.2 and B). As the ictal episode ev¢lved, the stimulus train evoked discharges that readily propagated across the entire slice (traces above dashed line in Fig. 5A). When the stimulus train was reapplied after it

36 was briefly turned off, propagation of evoked discharges was again restricted to a focal area of the slice (Fig. 5A, STM OFF and following). Only with continued stimulation did propagation begin to extend across the slice to electrode 3 (near end of the segment) and later to electrode 4 (segment not shown). Note that the stimulus train was reapplied during the refractory period of the endogenous pacemakers. DISCUSSION Two types of electrographic ictal episode can be provoked by perfusion of neocortical slices with solutions containing 0 - M g - those of focal and those of generalized onset. Slow spread of focal ictal discharge only occurred early in the 0-Mg perfusion and always evolved into ictai episodes that were generalized from the onset. We assume that the major difference underlying these 2 patterns of propagation is the degree of facilitation of NMDA receptor-mediated synaptic transmission at a given time. It is interesting to speculate as to whether minor degrees of a generalized disturbance in brain excitability in man can lead to seizures with a focal or multifocal onset through mechanisms similar to those described here, while quantitatively more severe degrees of the same disturbance might lead to seizures that are generalized from the onset. Our observations provide information about the spread of such ictal activities. In the focal type, ictal episodes begin in a localized area of cortex before slowly spreading into adjacent uninvolved areas. The speed of this type of spread closely resembles that seen in jacksonian epilepsy in humans. Cortical spreading depression (SD) 27'28, which has similar rates of propagation (2-4 mm/min), has been invoked as a possible mechanism underlying the 'jacksonian march '23'-'~. However. the slowly spreading focal ictal events recorded in these experiments differ from SD in that they are not associated with electrical silence or with large DC shifts (e.g., as in ref. 17), and they terminate nearly simultaneously across all areas of the neocortical slice. Generalized ictal events are characterized by unrestricted propagation of discharges at the onset

of the episode, whereas discharge propagation in focal events is restricted to a small, slowly enlarging zone. The rate of recruitment of adjacent 'inactive' areas of cortex will therefore determine the velocity of the focal slow spread. By contrast, the 'fast spread' of individual discharges appears to be related to a different phenomenon and probably involves conduction mechanisms similar to those underlying the rapid propagation of paroxysmal discharges observed in neocortical slices bathed in bicuculline 9. The horizontal conduction of seizure activity in both 0-M~ ~nduced ictal and bicuculline-induced interictal paroxysmal activity is most likely mediated by laterally projecting excitatory synaptic connections present in the cortex 4'a9. According to Chervin et al. 9, lateral propagation of interictal epileptiform activity may involve sequential excitation of adjacent columns of neurons which results in generation of a synchronized wave of activity that travels from one end of the slice to the other. In a 400pro thick neocortical slice, these adjacent columns effectively line up side by side in a one-dimensional array so that the spread of ictal activity can be interrupted by focal application of drug. Focal application of APV blocks seizure conduction, suggesting that lateral excitation of the NMDA subtype of glutamate receptor is critical for seizure propagation in the 0-Mg model 2'32'43. This finding is not surprising since augmentation of NMDA receptor-mediated excitation is the presumed mechanism for seizure generation in this model. However, Swarm and colleagues have shown that NMDA receptor antagonists can also block the initiation of ictai discharges induced by bicuculline in immature hippocampal slices7. We have been unable to block propagation of interictal discharges with NMDA-receptor antagonists in bicuculline-treated mature neocortical slices (Wong, Coulter and Prince, unpublished observations), although focal applications of GABA are effective in this preparation s. In the hippocampus, only the latter portions of the depolarization shifts that characterize neuronal activities during interictal discharges are eliminated by APV ~6. These findings suggest that excitation mediated by both NMDA and non-NMDA-activated receptors plays a rn!c in the spread of epileptiform activity.

37 Blockade of NMDA receptors by APV can eliminate ictal discharges 5,7 and portions of interictal discharges ~6whereas blockade of non-NMDA receptors by CNQX ,.ppears to eliminate interictal events 3. This is not unexpected, since the slow membrane depolarization seen in neurons during the transition between interictal and ictal states 6 would facilitate activation of voltage dependent NMDA-operated channels I°'3m4. Site-to-site comparisons of the morphology of ictal events reveal prominent variability in shapes, amplitudes, and frequencies of local discharge. This suggests that neocortex is not a 'homogenous' tissue in terms of its capacity to generate epileptiform events; rather significant local or regional differences are present. For example, differences in cortical circuitry and distribution of cell types and transmitter systems are known features of cortical organization u which could affect the local activities that underlie epileptogenesis. Local factors may therefore be involved in determining the sites of spontaneous initiation of synchronized activity and can also account for the presence of multifocal initiators. The spread of excitation from one vertical column to the next may depend on the number and strength of lateral excitatory or inhibitory synaptic connections between regions. High frequency stimulation at one site might effectively reduce the size of the afferent volley to the adjacent strip, or trigger local inhibitory circuitry, and account for the frequency-dependent conduction block (e.g., Fig. 4). Local factors as described above are thought to account for the directionality and periodicity of propagation seen in bicuculline-induced discharges as well 9. A number of mechanisms could be involved in termination of an ictal episode, including hyperpolarization of participating neurons through activation of the Na-K pump 6'42, rundown of transmitter release 2~, receptor desensitization 3° or other factors that would lead to depressed postsynaptic excitability, failure of conduction in polysynaptic circuits, blockade of impulse propagation in fine terminals conditioned by large increases in extracellular potassium, etc. The simultaneous cessation of ictal activity in widespread areas of O-Mg-

treated cortical slices could be due to another factor, namely failure of multifocal pacemakers to initiate epileptiform activity. Some of our data indirectly support this conclusion. The slice can remain excitable to exogenous stimuli at the time the ictal episode ends (Fig. 5), suggesting that factors like conduction blockade or failure of transmitter release are not important. Also, multifocal pacemakers can be demonstrated in slices 'chemically divided' by APV applications (Fig. 3) and may compete to initiate discharges, as judged by the finding that propagation may occur in different directions even during a single brief paroxysm, i.e., a given site may lead or lag the onset of the discharge (Figs. 2 and 4). We assume that pacemaker regions gradually become 'refractory' after repetitive excitation and that electrical silence occurs when the last initiator site suddenly fails to trigger another discharge, although the ability to propagate remains unchanged as shown in Fig. 5. The next round of activity might begin when the initiator region with the shortest refractory period begins to trigger the synchronized activity again. Depending on the state of the slice, focal or generalized ictal activity will be triggered once pacemaker activity resumes. The rapid conduction of synchronized activity to all parts of the slice is essential in organizing the group behavior of multifocal pacemakers that in turn determine the onset and termination of individual ictal episodes. We recognize that this explanation merely shifts the site of loss of excitability from the epileptogenic cortex as a whole to a smaller subset of neurons and circuits without providing a specific mechanism for pacemaker 'failure.' Nonetheless, this proposal explains much of the above phenomenology and makes it easier to understand how focal decreases in excitability, such as might occur following excessive discharge at one site 6'42might cause abrupt seizure termination in widespread areas. ACKNOWLEDGEMENTS Supported by NIH Grants NS01179, NS06477, and NS12151, and the Morris Research Fund.

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