spontaneous sharp waves in human neocortical slices excised from epileptic patients

Brain (1998),121,1073–1087

Spontaneous sharp waves in human neocorticalslices excised from epileptic patientsRudiger Kohling,1 Anne Lucke,1 Heidrun Straub,1 Erwin-Josef Speckmann,1,2 Ingrid Tuxhorn,3

Peter Wolf,3 Hans Pannek4 and Falk Oppel4

1Institut fur Physiologie,2Institut fur Experimentelle Correspondence to: Dr Ru¨diger Kohling, Institut furEpilepsieforschung, Mu¨nster,3Epilepsiezentrum Bethel Physiologie, Robert-Koch-Str. 27a, 48149 Mu¨nster,Bielefeld and4Neurochirurgische Klinik, Bielefeld, GermanyGermany

SummaryHuman neocortical temporal lobe tissue resected fortreatment of pharmacoresistant epilepsy was investigated.In slices prepared from this tissue, field potentialssometimes superimposed by population spikes were foundto appear spontaneously. In individual slices, they weregeneralized or highly localized to a field of ~200µm indiameter. Synchronous with these potentials, hyper-polarizing and depolarizing postsynaptic potentials wererecorded from neurons in the vicinity of the field potentialelectrode. Hyperpolarizing postsynaptic potentialsappeared to be mainly chloride mediated. All potentials,i.e. sharp field potentials as well as postsynaptic potentials,were reversibly suppressed by blockade of the non-NMDA(non-N-methyl-D-aspartate) glutamate-subreceptor and of

Keywords: human brain slice; spontaneous activity; calcium channel blockers; glutamate; GABA

Abbreviations: APV 5 DL-2-amino-5-phosphonovalerate; CNQX5 6-cyano-7-nitroquinoxaline-2,3-dione; EPSP5 excitatorypostsynaptic potential; GABA5 γ-aminobutyric acid; IPSP5 inhibitory postsynaptic potential; NMDA5 N-methyl-D-aspartate

IntroductionThe investigation of surviving human brain tissue obtainedduring epilepsy surgery has been of increasing interest inrecent years (Schwartzkroin and Prince, 1976; Prince andWong, 1981; Schwartzkroin and Knowles, 1984; Avoliet al.,1987, 1994a, b, 1995; Avoli and Olivier, 1989; McCormick,1989; McCormick and Williamson, 1989; Hwaet al., 1991;Knowles et al., 1992; Straubet al., 1992b, 1996; Taskeretal., 1992; Williamsonet al., 1993, 1995; Francket al., 1995;Luckeet al., 1995; Isokawa, 1996; Isokawa and Fried, 1996).Most investigators focused on the characterization of neuronalfunctions at the cellular level and, specifically, on thedetermination of epileptogenicity and the comparison withanimal cells. In these comparisons, only a few differences inelectrophysiological characteristics such as membrane andrepetitive firing properties, postsynaptic potentials, or aheightened intrinsic bursting propensity were found(Schwartzkroin and Prince, 1976; Prince and Wong, 1981;

© Oxford University Press 1998

the GABAA (γ-aminobutyric acid) receptor, and byapplication of the organic calcium channel blockerverapamil. By contrast, all potentials remained unaffectedby blockade of the NMDA glutamate-subreceptor and theGABAB receptor. The antiepileptic drugs carbamazepineand phenytoin failed to suppress the spontaneouspotentials at therapeutic concentrations. Washout of Mg21

from the superfusate left the spontaneous potentialsunchanged or converted them to ictal-type discharges.This epileptiform activity was not suppressed, butaugmented by blockade of the GABAA receptor. As awhole, the spontaneously appearing field potentials maybe assumed to reflect a state of increased neuronalsynchronization.

Schwartzkroin and Knowles, 1984; Avoli and Olivier, 1989;McCormick 1989; McCormick and Williamson, 1989; Taskeret al., 1992; Williamsonet al., 1993, 1995; Avoliet al.,1994a; Isokawa, 1996).

The question arises, however, whether extended neuronalnetworks of human brain tissue show bioelectric potentialsgrossly different from those in non-epileptic material. As thetissue in question originates from human temporal lobe whichhas chronically been involved in seizure activity, it is ofspecial interest if the networks of such tissue displayspontaneous potentialsin vitro. Most studies have failed todemonstrate such spontaneous potentialsin vitro(Schwartzkroin and Prince, 1976; Prince and Wong, 1981;Schwartzkroin and Knowles, 1984; Avoliet al., 1987;McCormick and Williamson, 1989; Knowleset al., 1992).However, in one investigation field potential activity wasreported to occur in a few cases (McCormick, 1989), and in

1074 R. Kohling et al.

another, spontaneous postsynaptic potentials wereoccasionally recorded intracellularly (Schwartzkroin andHaglund, 1986). The fact that spontaneous activity was sorarely observed in slices could reflect that (i) spontaneousactivity appears primarilyin situ and is eventually lost ininvitro preparations or (ii) the foci of such activity are verysmall and thus difficult to detect. In the present study, wetherefore attempted a systematic search for spontaneousactivity in human neocortical tissue resected during epilepsysurgery. The result was that in fact spontaneously occurringsharp waves could be detected in field potential recordings.Furthermore, we tried to establish the role of glutamatergicand GABAergic synaptic transmission, as well as of voltage-gated calcium channels in the generation of such spontaneousactivity. A part of this work has been published in abstractform (Kohling et al., 1995b).

Material and methodsHuman neocortical tissueHuman tissue obtained was a small portion of that which isnormally excised for treatment of pharmacoresistant focalepilepsy originating in the temporal (n 5 28) or frontal (n 51) lobe. Temporal lobe tissue used in this study originatedfrom the anterior portion of the inferior temporal gyrus fromstandard partial temporal lobectomies. Frontal lobe tissuewas from the epileptogenic area as determined by anelectrocorticogram with subdural electrodes. The materialwas obtained during operations performed between 1994 and1997. The patients (n 5 29, 11 female and 18 male, aged8–45 years) had received a variety of antiepileptic drugs,which usually included carbamazepine or phenytoin, and oneor more of valproate, vigabatrin, and gabapentin. In manycases histopathological analysis of the neocortical samplesrevealed a mild degree of gliosis (n 5 4) or atrophy (n 57). In five cases cortical dysplasia was diagnosed, and in twocases a tumour. Hippocampal sclerosis was seen in 13 cases.In 11 cases, no pathological abnormalities were found.

The experiments were approved by the local ethicscommitee (Ethikkommission der A¨ rtzekammer Westfalen-lippe und der Medizinischen Faculta¨t der Westfa¨lischenWilhehus-Universita¨t Munster). Informed consent wasobtained from all patients.

Determination of Mg2F concentration in CSFand antiepileptic drug concentration in brainslicesA small quantity (2–5 ml) of CSF was obtained from eachof 10 patients for analysis of the content of free Mg21 byatomic absorbtion spectroscopy.

The concentrations of the antiepileptic drugs carbama-zepine and phenytoin, which had been administered to thepatients up to the operation, were determined in four slicesby high-pressure liquid chromatography according to the

methods described by Rambecket al. (1992) and Schnabelet al. (1994).

Slice preparationThe blocks of resected tissue consisted of the anterior portionof the temporal lobe (3–5 cm). Slices were prepared from a1 cm3 block of the inferior temporal gyrus within 5 min oftissue resection. The techniques for slice preparation havebeen described in detail elsewhere (Straubet al., 1992b,1996; Lucke et al., 1995). Briefly, neocortical slices of 400–500 µm thickness were cut perpendicular to the pial surfaceusing a vibratome. They were placed in a portable incubationchamber (Ko¨hling et al., 1996) with oxygenated (95% O2,5% CO2) artificial CSF at a temperature of 28°C and a pHof 7.4. Slices were allowed to recover for a period of 1–2 hbefore transferral into a submerged recording chamber. Fora control series of experiments performed both in paralleland before the investigations on human tissue, neocorticalslices from guinea pigs were prepared in an analogousfashion. The details of the preparation are described elsewhere(Schulze-Bonhageet al., 1994). The composition of theartificial CSF was (in mM): NaCl 124; KCl 4; CaCl2 2;NaH2PO4 1.24; MgSO4 1.3; NaHCO3 26; and glucose 10. Inthe experimental chamber the temperature was raised to33°C. In some cases, MgSO4 was omitted from the superfusateor raised to a concentration of 2 mM. During the experiments,pH, temperature and flow rate (4 ml/min; bath volume 1 ml)were continously monitored.

Drug applicationAll substances used were dissolved in artificial CSF andadded to the superfusate. Drugs applied were: the NMDA(N-methyl-D-aspartate) and AMPA (6α-amino-3-hydroxy-5-methylisoxazole-4-proprionic-acid) receptor antagonistsDL-2-amino-5-phosphono-valerate (APV, 100µM) and 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 5µM); the GABAA

and GABAB receptor antagonists bicuculline methiodide (5–10 µM) and CGP 55845A (1µM); the organic calciumchannel blocker verapamil (40µM); and the antiepilepticdrugs carbamazepine and phenytoin (25µM; dissolved indimethylsulphoxide and 1 N NaOH solution, respectively,each diluted to a final concentration of 0.1%).

Electrical stimulationSubcortical white matter was stimulated electrically by meansof a bipolar platinum electrode (60µm in diameter) placedat the border of white and grey matter adjacent to therecording electrodes. Single stimuli were delivered at amaximum frequency of 0.1 Hz.

Electrophysiological recordingsExtracellular field potential recordings were perfomed inlayers II to V (as estimated by measuring the position of the

Sharp waves in human brain slices 1075

electrode 300–2400µm below pial surface, Creutzfeldt, 1983)using pipettes (0.5–1.5 MΩ) filled with artificial CSF. In 14trials, recordings were done with two extracellular electrodessimultaneously. To search for spontaneous activity theelectrodes were periodically repositioned following animaginary position grid consisting of squares ~500µm acrosscovering the whole slice.

Intracellular recordings were performed using single sharpmicropipettes filled with 2 M potassium methyl sulfate or2 M KCl (60–180 MΩ) in the immediate vicinity of the fieldpotential electrode and up to 800µm away. Intracellular datawere obtained from 11 cells in layers II–V displaying aresting membrane potential more negative than –53 mV(–57.16 3.7, mean6 SEM) and action potential amplitudes.60 mV (646 2 mV). Signals were recorded on an inkwriterand computer system, as well as a digital oscilloscope.

Simultaneous recordings of field potentials andextracellular concentrations of potassium ([K1]o) wereperformed in 10 slices using double-barrelled ion-sensitivemicroelectrodes based on the valinomycin ion exchangerFluka 60398. Preparation of the electrodes is described indetail elsewhere (Ko¨hling et al., 1993, 1995a; Lucke et al.,1995). Briefly, one channel of a double-barrelled electrode(tip diameter 2–6µm) was backfilled with 151 mM NaClsolution as a reference channel. The ion-sensing channel wasbackfilled with 100 mM KCl solution, and its tip was silanizedand equipped with the ion exchanger. The electrodes showeda voltage change of 56–57 mV with a 10-fold increase in[K1]o against a fixed background of 151 mM NaCl. All dataare given as mean6 SEM.

ResultsOccurrence of spontaneous field potentialdischargesSharp field potentials appeared spontaneously in 60 of 92temporal neocortical human tissue slices from 28 patients,and in one frontal slice from one patient, all undergoingepilepsy surgery (Fig. 1). In each experiment, one to fourslices were tested from each patient, of which at least oneshowed spontaneous activity. In a control series of guineapig neocortical slices from sensorimotor cortex (n 5 60),under the same experimental conditions, spontaneous activitywas never observed (cf. Schulze-Bonhageet al., 1994).

When different slices originated from the same patient, allslices (n 5 10 patients) or more than half of the slices (52%from 12 patients) showed spontaneous activity. In slices notpresenting sharp waves, epileptiform activity was evoked byapplication of bicuculline (Hwaet al., 1991; Straubet al.,1996) or omission of Mg21 from the bath fluid (zero-Mg21 epilepsy, Avoli et al., 1987; Straubet al., 1992b),demonstrating viability of the slices.

Features of spontaneous field potentialdischargesThe amplitudes of spontaneous discharges varied from 20 to323 µV (72 6 13 µV, n 5 31 slices); in a given recording

Fig. 1 Spontaneous sharp waves (simultaneous recordings of fieldpotentials in FP1 and FP2) recorded from human neocortical slicepreparations which had been obtained during epilepsy surgeryfrom the inferior temporal gyrus. Recordings in A–C are fromthree different slices. Examples of synchronous (A) andasynchronous (B) field potentials; in each case the right partshows superimposition of three field potentials with the events inFP2 serving as guiding patterns for synchronization. (C) Changeof polarity of field potentials during the course of recording.Inkwriter tracing. Interruptions are indicated. GM5 grey matter;WM 5 white matter.

location, the potentials were uniform in amplitude. In mostcases (76%), the potentials were,70 µV (Fig. 2). Nearlyall potentials ranged between 20 and 200 ms in duration(151 6 18 ms total duration and 496 7 ms half-amplitudewidth; n 5 31 slices, Fig. 2) and the slope was 6.46 2.4mV/s (n 5 31 slices), again with little variability within agiven slice.

The repetition rates of the spontaneous field potentialdischarges varied from 4 to 108 per minute, and on averageattained values of 436 4 per minute (n 5 31 slices, Fig. 2)in the beginning of the experiment and then declined duringthe first hour of recording to ~70% of the initial value. Inthree cases, activity disappeared after 30–120 min.

Evaluating 100 spike intervals in each of 15 slices showedthat 69% of the intervals lasted 0.2–2 s, within a total rangeof 0.2–16 s (Fig. 2). Long intervals were very rare andusually appeared between series of closer-spaced potentials(e.g. see Figs 6A and 9 below), so that in the majority ofthe slices (12 of 15), only,10% of the spike intervals were.5 s.

Usually, the potentials were monophasic (see Fig. 1 andFigs 6–8 below) and did not represent field potentials broughtabout by summation of action potentials. On the otherhand, clear cut population spikes were superimposed on thepotentials in some cases (six out of 31 recordings, Fig. 3). The


Fig. 2 Spontaneous sharp waves: distribution of amplitudes,durations and half amplitude widths, repetition rates, and spikeintervals. The majority of the 31 slices displayed sharp waveswith amplitudes ofø70 µV, a duration ofø200 ms, halfamplitude widths ofø80 ms, and repetition rates of 20–80 perminute. Spike intervals: 100 intervals were measured in each ofthe 15 slices.

potentials could be triggered with extracellular stimulation ofthe white matter and gained in amplitude with increasingstimulus strength until reaching a maximum. Understimulation, both the polarity and shape of the signals werethe same as those of the spontaneous potentials.

Distribution of spontaneous field potentialdischargesActivity could be recorded either at only one recording siteof the slice preparation, or at multiple positions (Fig. 1A andB). In the latter experiments, the sharp waves were eithersynchronous (Fig. 1A) or asynchronous (Fig. 1B).

Fig. 3 Population spikes superimposed on spontaneous sharpwaves (FP trace) and concomitant membrane potential changes(MP trace) recorded at a distance of 400µm. Three potentialssuperimposed, the population spike served as a guiding pattern forsuperposition. Oscilloscope tracings.

The spatial distribution of the potentials was assessedin 14 slices with two-electrode recordings, as described inthe Material and methods section. Synchronous activitywas recorded with electrode separation of up to 400µm(n 5 3), 600 µm (n 5 5), 800 µm (n 5 1) and 1000µm(n 5 1). In four cases, synchronous activity was observedonly in a recording field of up to 200µm, and not beyond.When the electrodes were spaced at distances of 2000µm(n 5 2) and 4000µm (n 5 1), activity was seen in bothrecording sites in only three slices, and in these cases itwas asynchronous.

Additionally, the spontaneously active networks werepossibly not stationary as there were polarity changes (n 52) and waxing and waning (n 5 10) of the potentials(Fig. 1C). Such increases and decreases in field potentialamplitude could only be observed at irregular intervals whichusually lasted.30 min.

Intracellular recordingsIn six slices, intracellular recordings from 11 cells wereperformed along with field potential recordings. Theintracellular electrode was placed within layers II–V in theimmediate vicinity of the field potential electrode, or up to800 µm away (see Material and methods section).

Using potassium methyl sulfate filled electrodes, differenttypes of potentials, i.e. depolarizing (Fig. 4Aa and Bd; 37%of 178 events recorded in seven cells; see Fig. 4Ca and Cb)or hyperpolarizing (Fig. 4Ab and Bb; 44% of all events; seeFig. 4Ca and Cb) or sequences of hyperpolarizing anddepolarizing potentials (Fig. 4Bc, 19% of all events; Fig. 4Caand Cb) could be observed in all neurons synchronous withthe field potentials. A paroxysmal depolarization was neverseen. Within a resting membrane potential range of –50 to


Fig. 4 Spontaneous sharp waves (FP trace) and concomitant membrane potential changes (MP trace). Membrane potentials recordedfrom neurons 200–800µm away from the field potential electrode. Sharp waves in the FP trace served as guiding patterns forsuperposition. (A) Depolarizing (a) or hyperpolarizing (b) membrane potential changes, five potentials superimposed. Asterisk: Note theintermediate depolarization within the hyperpolarizing envelope is synchronous with the population spike riding on the sharp wave. (B)The field potential recordings (a) and, at the same resting membrane potential, corresponding hyperpolarizing (b), sequence of de- andhyperpolarizing (c) and depolarizing potentials (d) in one neuron. The field potential (e) and accompanying paroxysmal depolarizationshift (f) after 20 min washout of 5µM bicuculline. (C) Summary of depolarizing (DE), hyperpolarizing (HYPER) and depolarizing/hyperpolarizing membrane potential changes (resting membrane potential: –50 to –60 mV) concomitant with sharp waves. a: number ofcells showing the aforementioned membrane potential changes, b: fraction of all events. (D) Membrane potential changes concomitantwith postsynaptic potentials during sharp wave generation at different resting membrane potential in one neuron. Note reversal at ~–68mV.

–60 mV, most of the neurons displayed two or all three typesof potentials (Fig. 4Ca,n 5 7).

To analyse the ionic nature of the membrane potentialchanges of a given neuron, the resting membrane potentialwas altered by passing a current over the bridge circuit, orKCl was used in the recording pipette (see Material andmethods section). In experiments in which the membranepotential of a given neuron was altered, purely hyper-polarizing potentials consisted of monophasic smoothpotentials at low membrane potentials which reversed at~ –68 mV, near the putative GABA equilibrium potential (cf.Staley et al., 1995), to a depolarization of similar shape(Fig. 4D). In three recordings, pipettes filled with 2 MKCl were used. In such recordings, all potentials weredepolarizing. Action potential generation was shunted by the

occurrence of a spontaneous potential even when the neuronwas firing tonically due to strong depolarization to –40 mVvia current injection.

Factors possibly contributing to the generationof spontaneous activityClinical dataThe appearance of spontaneous activity was not correlatedwith sex, age, seizure history or antiepileptic medication ofthe patients, nor with histopathological findings as mentionedin the Material and methods section. Specifically, there wasalso no correlation with hippocampal sclerosis or corticaldysplasia.


Table 1 Extracellular K1 concentrations in 500µm slice preparations

[K1]o (mM) at different depths in the slice

0 µm 100µm 200µm

Spontaneously active 4.0926 0.04 4.1746 0.06 4.3236 0.09slices (n 5 5) (n 5 27) (n 5 22) (n 5 20)

Slices without spontaneous 4.1656 0.05 4.396 0.136 4.686 0.193activity (n 5 5) (n 5 14) (n 5 13) (n 5 14)

No significant differences were found, i.e. allP . 0.05.

Tissue transportIn order to exclude the possibility that spontaneous activitywas induced by the transport process over a period of ~90 minin a portable incubation bath (Ko¨hling et al., 1996), twoexperiments were conducted on site next to the operatingroom. In these two experiments, spontaneous field potentials,showing the same characteristics as those found in materialwhich had been transported, were observed in 7 of 16 slices,as little as 30 min after excision of the tissue.

Furthermore, the potentials could be recorded immediatelyupon electrode placement independent of the duration of thepre-incubation period (30–240 min).

Concentration of Mg21 in the artificial CSFThe omission of Mg21 from the bath fluid is known to induceepileptiform activity in human brain slicesin vitro (Avoliet al., 1987; Straubet al., 1992b). Furthermore, abnormallylow Mg21 concentrations may cause seizures in humans(Morris, 1992), and the CSF Mg21 concentration was foundto be decreased in children with seizures (Becker, 1965). Toexclude the possibility that low Mg21 concentration inthe artificial CSF could be involved in the appearance ofspontaneous activity in the slice preparations, theconcentration of free Mg21 in the CSF was determined in10 patients. The mean concentration was 1.016 0.048 mM(n 5 10) and thus lower than the concentration in the artificialCSF (i.e. 1.3 mM, see Material and methods section).

Baseline extracellular potassium concentrationTo elucidate the role of baseline extracellular potassiumconcentration ([K1]o) in the generation of spontaneouspotentials, [K1]o was measured in layers II–V at differentpenetration depths normal to the cut surface of the slices.Both spontaneously active and silent slices showed similar[K1]o baseline levels which ranged from 3.8 to 4.3 mM(spontaneously active slices,n 5 5) and from 3.8 to 4.7 mM(slices without spontaneous activity,n 5 5; Table 1) whenthe electrode was lowered from the surface to the centre ofthe slice. No statistical difference could be seen between thetwo groups.

Fig. 5 The conventional antiepileptic drugs carbamazepine (CBZ,A) and phenytoin (PHT,B) fail to suppress spontaneous sharpwaves (FP trace) close to therapeutic concentrations (25µM).Inkwriter tracings, with recordings before application of thesubstances (CTRL1) and 30 min after washout (CTRL2).

Withdrawal of antiepileptic medicationAntiepileptic drugs such as carbamazepine and phenytoinwere administered to the patients and were present in theirbrain tissue up to the operation. These drugs were washedout completely during incubation (90 min), as in high-pressure liquid chromatography analyses no quantifiableconcentrations of carbamazepine and phenytoin could bedetected in the slices. Such a ‘withdrawal’ of antiepilepticdrugs could be responsible for the appearance of spontaneoussharp waves in the slices. Therefore, the effects of theconventional antiepileptic drugs phenytoin and carbama-zepine on spontaneous activity were tested. Phenytoin andcarbamazepine were given close to or slightly abovetherapeutic concentrations (25µM, n 5 3 for both phenytoinand carbamazepine; Troupin, 1993). Apart from a slightreduction of the repetition rate to 60–70% of the initial value,the drugs failed to suppress spontaneous activity within 60min of the application (Fig. 5).

Pharmacological characterizationGlutamate receptorsIn order to establish the possible role of excitatorytransmission mediated by glutamate in the generation of thespontaneous potentials, the non-NMDA receptor antagonistCNQX (5 µM, 30 min) or the NMDA receptor antagonistAPV (100 µM, 30–60 min) was added to the superfusate.

Upon application of CNQX, the spontaneous sharp waveswere abolished along with the synchronous postsynaptic


Fig. 6 Action of glutamate subreceptor antagonists CNQX (5µM, A) and APV (100µM, B) on spontaneous sharp waves (FP trace) andaccompanying membrane potential changes (MP trace). Inkwriter (left panel) and oscilloscope tracings on an expanded time scale (rightpanel), with recordings before application of the substances (CTRL1) and 30 min after washout (CTRL2).

potentials within 5–15 min in all trials (n 5 14, Fig. 6A).This effect was reversible, as potentials reappeared uponwashout of CNQX, within 45 min. After washout of thesubstance, the frequency of occurrence returned to 946 8%of the initial value.

The NMDA antagonist APV had no appreciable effect onthe observed potentials (n 5 11, Fig. 6B). However, theelevation of the concentration of MgSO4 in the superfusatefrom 1.3 to 2 mM, a manipulation which would usuallyaccentuate the voltage dependent Mg21-block of the NMDAreceptor (Collingridge and Lester, 1989) and thus also blocksynaptic transmission mediated by NMDA receptors, partiallyreduced spontaneous activity within 20–60 min in two slices,and suppressed them in one slice. Upon reduction of theMg21 to 1.3 mM, spontaneous potentials reappeared, or theirfrequency of occurrence increased again, within 5 min.

GABA receptorsTo test for the functional significance of inhibitory synaptictransmission for the appearance of spontaneous activity, theGABAA receptor antagonists, bicuculline (5µM, 30 min,Fig. 7A) or picrotoxin (100µM, 15 min) or the GABAB

receptor antagonist CGP 55845A (1µM, 30 min, Fig. 7B)were superfused. Immediately after application of bicuculline,the amplitudes of the field potentials were reduced and theaccompanying hyperpolarizing potentials became depolari-zing (Fig. 7A, bicuculline). After 10–15 min, however, thepotentials disappeared completely (n 5 8). During this

period of suppression of spontaneous potentials, electricalstimulation of the subcortical white matter could trigger largefield potentials, demonstrating that evoked events were notblocked by bicuculline (results not shown). Upon wash-outof bicuculline, spontaneous potentials reappeared. In two outof eight slices, the field potentials observed showed a transientincrease in amplitude (for a period of 5–10 min; Fig. 7A,CTRL2), and intracellular recordings revealed that they wereaccompanied by epileptiform paroxysmal depolarizations(Fig. 4Be and Bf, Fig. 7A, CTRL2) synchronous with thefield potential changes. After this transient accentuation ofspontaneous activity, and the appearance of epilepticpotentials, the spontaneous activity returned to its originalamplitude, and the paroxysmal depolarizations disappearedwithin 60 min.

Like bicuculline, picrotoxin suppressed the spontaneousfield potentials within 5 min (n 5 2), and upon wash-out, inboth cases, grouped activity transiently appeared, resemblingdischarges found with tonic–clonic activity (data not shown).

The GABAB antagonist CGP 55845A (n 5 2) did notinfluence the spontaneous activity, although in one experimentaction potentials were generated more frequently than beforesuperfusion of the substance (five versus 57 action potentialsin 5 min) and the neuron was slightly depolarized by 2 mV(Fig. 7B, CGP 55845A).

L-type voltage-gated calcium channelsThe organic calcium channel blocker verapamil (40µM) wasused to assess the involvement ofL-type transmembrane


Fig. 7 Action of (A) GABAA receptor antagonist bicuculline (5µM) and (B) GABAB receptor antagonist CGP 55845A (1µM) onspontaneous sharp waves (FP trace) and accompanying membrane potential shifts (MP trace). Inkwriter (left panel) and oscilloscopetracings on an expanded time scale (right panel), with recordings before application of the substances (CTRL1), and 30 min afterwashout (CTRL2), as well as 60 min after washout inA (lower panel). Note that during bicuculline application membrane potentialchanges transiently become depolarizing before all potentials are abolished. With washout of bicuculline, paroxysmal depolarizationshifts are generated transiently, before membrane potential changes become hyperpolarizing again. Note, too, that during application ofCGP 55845A action potentials are being generated more frequently than under control conditions.

calcium currents in the generation of spontaneous discharges.In all experiments, spontaneous potentials were suppressedreversibly within 60–120 min (Fig. 8,n 5 6). Specifically,a suppression by 50% and 90% of the initial repetition ratewas reached within 25.86 3.7 and 63.36 14.3 min(n 5 6). Usually, the potentials did not recover completelywhen verapamil was washed out, and the frequency ofoccurrence returned to 4167% of the value before theverapamil superfusion.

Effect of raised epileptogenicityThe spontaneous sharp waves might be the result of anincreased epileptogenicity of the tissue. Therfore, both typesof slice (with and without spontaneous activity) were testedunder epileptogenic conditions, i.e. omission of Mg21 fromthe superfusate (zero-Mg21 epilepsy; Avoli et al., 1987;Straubet al., 1992b).

Spontaneously active slicesWith superfusion of nominally Mg21-free artificial CSF, ineight out of 13 slices, the spontaneous potentials were

unaltered in shape and amplitude but showed anapproximately two-fold (2246 50%,n 5 8) increase in therepetition rate (Fig. 9). In another five slices, ictal-typetonic–clonic type discharges (duration 2–120 s, occurring atintervals of 2–300 s) appeared under these conditions inaddition to interictal-type potentials (duration,2 s).

Slices without spontaneous activityIn another set of experiments on the slices withoutspontaneous activity, wash-out of Mg21 resulted in theappearance of interictal-type discharges only in the majorityof slices (six out of nine). In the remaining experiments, ictal-type activity appeared as in the spontaneously active slices.

To analyse zero-Mg21 induced activity in both silent slicesand preparations displaying spontaneous sharp potentialsfurther, bicuculline (5–10µM) was added to the bath solution.With these zero-Mg21 epileptiform discharges, additionalapplication of bicuculline led to the development of ictal-type activity in slices previously not showing this type ofdischarge (Fig. 9) both in spontaneously active slices(n 5 4) and in slices without spontaneous activity (n 5 1).


Fig. 8 Suppression of spontaneous sharp waves (FP trace) by the organic calcium channel blocker verapamil (40µM). Inkwriter (lowertraces) and oscilloscope tracings at expanded time scale (upper traces), with recordings before application of verapamil (CTRL1) andafter washout (CTRL2). Examples are taken from three time points within the different experimental protocols, as indicated.

DiscussionOccurence of spontaneous field potentialsMost studies on human slice preparations from epileptogenictissue resected during epilepsy surgery, so far, have reportedno spontaneous field potential activityin vitro (Schwartzkroinand Prince, 1976; Prince and Wong, 1981; Avoliet al.,1987; McCormick and Williamson, 1989) and no increasedexcitability as reflected in an increased bursting propensityof neurons (Schwartzkroin and Prince, 1976; Schwartzkroinand Knowles, 1984; Avoli and Olivier, 1989; Taskeret al.,1996; cf. also Williamsonet al., 1995; Isokawa, 1996).One study (McCormick, 1989) reported the occurrence ofspontaneous field potentials, but in only four out of the 29human neocortical slices investigated. The presentinvestigation shows that spontaneous sharp waves can beobserved in 60–70% of brain slices of neocortex fromepileptic patients, demonstrating that the general ability ofsuch tissue to produce spontaneous field potentials is not lost

in vitro. Interestingly, this seems to hold true regardless ofthe underlying pathology of the tissue, although findings byMattia et al. (1995) suggest that dysplastic tissue displays agreater epileptogenicity.

The present results exclude the possibilities that thespontaneous activity was artificially induced due to: (i) thetransport of the slices; (ii) raised levels of baseline [K1]o; or(iii) washout of antiepileptic medication, as (i) activity wasseen also in slices which had not been transported; (ii) [K1]o

was higher in slices without spontaneous activity than inslices presenting spontaneous field potentials, and overall didnot attain excessively high levels in any of the slices butremained at or below 4.7 mM; and (iii) supplying the sliceswith therapeutic concentrations of antiepileptic drugs whichshould have reversed any withdrawal effect did not suppresssuch activity. The last assumption is supported by theobservations that the withdrawal of antiepileptic drugs didnot exceed 2 h and that, in some slices, spontaneous activity


Fig. 9 Effect of withdrawal of Mg21 (0Mg21) from thesuperfusate and addition of bicuculline in Mg21-free superfusate(0Mg21 1 BIC) on spontaneous sharp waves (FP trace).Inkwriter tracings, with recordings before withdrawal of Mg21

(CTRL1) and 30 min after return to control conditions (CTRL2).Note that during Mg21 withdrawal, the amplitude and repetitionrate of the field potentials are slightly increased. In contrast to thefield potential under control conditions (cf. Fig. 7), addition ofbicuculline during Mg21 withdrawal does not block the fieldpotential, but converts activity to ictal-type discharges.

failed to occur while slices adjacent to them showedspontaneous sharp waves. In this context, it should bementioned that the final brain tissue concentrations ofphenytoin and carbamazepine after 30-min superfusion inconcentrations of 25µM would be four- to five-fold higher(i.e. 100 to 125µM) and thus would be well within aneffective antiepileptic concentration range (Rambecket al.,1992; Schnabelet al., 1994). Neither can low levels of Mg21

in the artificial CSF account for the appearance of spontaneouspotentials, as the Mg21 level of 1.3 mM in our superfusateis above the actual CSF values reported for healthy adults(1.11–1.15 mM; Hunter and Smith, 1960; Heipertzet al.,1979) and those found for patients in this study (i.e. 1.01 mM)and thus well above critical ‘epileptogenic’ concentrations,especially when considering that actual brain concentrationscan be expected to be lower than the CSF concentration(Morris, 1992). In this context it should be mentioned thatwith the same Mg21 levels in the superfusate, spontaneousactivity was never observed in guinea pig neocortical slices(cf. Schulze-Bonhageet al., 1994).

Epileptic or physiological nature of spontaneouspotentials?The observation of sharp waves in brain tissue from epilepticpatients fosters the question whether these bioelectricphenomena are epileptic in nature or not. Although the datapresented here do not allow a final conclusion on this issue,the following considerations may help.

In principle, the spontaneous field potentials can bedescribed as sharp waves in the EEG as they generallydisplayed pointed peaks and lasted,200 ms (InternationalFederation of Society for Electroencephalography andClinical Neurophysiology, 1974). In most cases, however,they did not resemble typical epileptiform field potentialselicited in in vitro models. Except for the washout phase ofbicuculline, we were not able, in any of our intracellularrecordings, to show any spontaneous paroxysmaldepolarization shift, considered to be characteristic forepileptic activity (Goldensohn and Purpura, 1963).Nevertheless, the findings presented here might still beindicative of epileptic activity, e.g. activity surrounding anepileptic focus. Concomitant with the sharp field potentials,postsynaptic potentials were recorded in surrounding neurons.The occurrence of such spontaneous membrane potentialfluctuations in human tissue has also been demonstrated bySchwartzkroin and Knowles (1984). They reportedspontaneous depolarizing or hyperpolarizing potentials inhuman hippocampal slices, however, without the appearanceof field potential changes. They also found them in humanneocortical slices, albeit more rarely than in hippocampaltissue (Schwartzkroin and Haglund, 1986). As in the study ofSchwartzkroin and Haglund (1986), the membrane potentialchanges seen in the present investigation appeared to bemediated by both excitatory and inhibitory inputs. In casesof purely hyperpolarizing potentials, these seemed to beGABA-mediated. Depolarizing transient peaks within thehyperpolarizing envelope of these postsynaptic potentialswere similar to phenomena observed in neurons surroundingepileptiform penicillin foci in a field of vertical and surroundinhibition (Prince and Wilder, 1967; Elger and Speckmann,1983). Taking these observations together, the spontaneouspotentials could arguably reflect activity surrounding anepileptic focus. The observed field potentials could thus beseen to represent either interictal discharges of a miniaturefocus in the slices or synchronous activity of primary non-epileptic neurons being driven by such miniature foci. Suchan interpretation is supported by the finding that distinctpopulation spikes are sometimes superimposed on the sharpwaves although the accompanying membrane potentialchanges merely show inhibitory postsynaptic potentials(IPSPs) or excitatory postsynaptic potentials (EPSPs).

Alternatively, rather than genuine epileptic activity,phenomena secondary to epileptogenesis might also accountfor the appearance of spontaneous discharges. Thus, inhippocampal slice preparations exposed to epileptogenicconditions, ectopic action potentials have been reported to


appear. These were blocked by bicuculline, but not by APV,and thus showed similar pharmacological characteristics tothe potentials described in this investigation (Stasheffet al.,1993a, b). However, whether such ectopic spikes are able togenerate field potentials and potentials like surround inhibitionremains debatable.

Lastly, the spontaneous potentials could reflect a heightenedpropensity of network synchronization. Thus, as mentionedabove, Schwartzkroin and Knowles (1984) and Schwartzkroinand Haglund (1986) found rhythmic postsynaptic activity inhuman hippocampal and neocortical slices. Similarpostsynaptic potentials were also observed under epilepto-genic conditions in zero-Mg21-bathed human neocorticalslices (Avoli et al., 1995). It seems likely that theseobservations represent different facets of the samephenomenon, i.e. spontaneous activity such as described inthe present study. Schwartzkroin and Knowles (1984)attributed the lack of field potential changes in their study toinsufficient synchronization among neurons as revealed inpaired intracellular recordings. In fact, the small amplitudesof the potentials seen in the present study point to very smallneuronal populations generating this type of activity, althoughin a few cases neuronal populations were synchronouslyactive in locations as far as 1000µm apart. It might bespeculated thatin vivo, under certain circumstances, hithertoindependent foci might synchronize and thereby pave theway for an epileptic seizure.

Interestingly, Schwartzkroin and Haglund (1986) foundindications of raised synchronicity in the form of synchronousrhythmic postsynaptic potentials in ‘healthy’ monkeyhippocampus (a structure known for its epileptogenicity), butnot in ‘healthy’ monkey neocortex. We did not observespontanous field potentials in a control series of guinea pigneocortical slices, and they have not been reported to occurin neocortical slices of this species or of rats by othergroups either. If spontaneous field potential activity may bephysiological by itself, it could nevertheless facilitate epilepticdischarges in neocortical tissue, and possibly be a markerfor epileptogenicity.

Role of synaptic transmissionGlutamate receptorsSpontaneous potentials were suppressed by blockade of non-NMDA glutamate receptors. The same has been reportedfor epileptiform activity induced by substances blockingGABAergic transmission, such as bicuculline and picrotoxinin human (Kim et al., 1993) and animal slices (Lee andHablitz, 1991; Williamson and Wheal, 1992).

However, blockade of NMDA receptors showed no effecton the spontaneous discharges. On the other hand, it iseffective in reducing epileptiform activity in the above modelsand in the so-called zero-Mg21 model (Avoli et al., 1987,1995; Modyet al., 1987; Avoli and Olivier, 1989; Wuarinet al., 1990; Hwaet al., 1991; Lee and Hablitz, 1991; Kim

et al., 1993). The mechanism by which the elevated Mg21

concentration reduced spontaneous potentials thus seems tobe independent of the NMDA receptor and may involve aninfluence on surface charges, transmembraneous calciumcurrents or on the Na1/K1 ATPase (Modyet al., 1987).

GABA receptorsBlockade of inhibitory transmission with the epileptogenicdrug bicuculline also suppressed all spontaneous activity.During bicuculline application, hyperpolarizing potentialsdisappeared transiently unmasking depolarizing ones. Inaddition, during bicuculline superfusion electrical stimuliwere able to trigger field discharges. With wash-out ofbicuculline, paroxysmal depolarizations transiently appeared.Thus, excitability was probably raised rather than reduced,but synchronization was apparently lost. Rather than loweringepileptogenicity in epileptic tissue, we propose thatGABAergic transmission seems necessary for the generationof discharges. Indeed, inhibiton is strongly preserved inanimal models of epilepsy (Prince and Wilder, 1967; Elgerand Speckmann, 1983; Perreault and Avoli, 1992; Witte,1994; Westerhoffet al., 1995) and application of GABA-mimetic substances can induce discharges in rodents (Farielloand Golden, 1987). Furthermore, Kostopoulos and Antoniadis(1992) proposed a synchronizing and accentuating role ofGABAergic inhibition in a feline model of spike-and-wavedischarges. Such a synchronizing role for GABAergictransmission was also proposed for human temporal lobeepilepsy by Babbet al. (1989), who found indicationsof GABAergic hyperinnervation of sclerotic epileptogenichuman hippocampus. The synchronization among neuronsrather than the ability to generate dischargesper se alsoseemed to be diminished by bicuculline administration in thepresent study since potentials could still be evoked byelectrical stimulation, thus suggesting that GABAergictransmission provides a ‘reset mechanism’ crucial forsynchronization. Blockade of glutamatergic transmissionwould in this context reduce synchronization amonginterneurons and thus lead to a collapse of activity. Anotherinterpretation may be that inverted (depolarizing) GABA-IPSPs might have been blocked by bicuculline application,and such inverted IPSPs could possibly provide, besides thenon-NMDA EPSP, an excitatory synaptic drive (cf. Michelsonand Wong, 1991; Perrault and Avoli, 1992). In fact, excitatoryGABAergic activity has been reported by Michelson andWong (1991) in the hippocampus. In their study, applicationof 4-aminopyridine elicited spontaneous GABA-mediatedexcitatory coupling between hippocampal interneurons which,in turn, evoked large IPSPs in principal neurons reminiscentof potentials seen in the present study. Thus, a reduction ofdepolarizing and hyperpolarizing GABA-IPSP may exert itseffect in the same direction, i.e. by reducing synchronizationand excitation.


Role of transmembraneous calcium currentsThe depression of spontaneous activity by the organic calciumchannel blocker verapamil corresponds to the antiepilepticaction of this drug in various experimental models of epilepsy(Bingmann and Speckmann, 1989; Aicardi andSchwartzkroin, 1990; Straubet al., 1992a, 1994; Kohling etal., 1994) including human tissue (Straubet al., 1992b, 1996)and in the first clinical trials with other organic calciumchannel blockers (Overweget al., 1984). In line with theaforementioned results are the findings that elevation of theMg21 concentration from 1.3 to 2 mM reduces thespontaneous potentials and the application of APV fails todo so. This points to a calcium-antagonistic effect of Mg21

in this case (Saftet al., 1997). Calcium currents thusapparently play a crucial role in the generation of spontaneousactivity observed in this study, and epileptiform activityinduced in experimental models and in seizures in epilepsypatients.

Effects of raised epileptogenicityIn most slices showing spontaneous sharp waves, withwash-out of Mg21 from the superfusate, interictal-type fieldpotential activity remained more or less unchanged, so thatno clear distinction between spontaneous potentials anddischarges under epileptogenic conditions could be made. Inthe rest of the slices, however, ictal-type activity appeared inzero-Mg21 medium, usually along with intercalated interictal-type discharges.

In slices which did not present with spontaneous sharpwaves (n 5 9), with washout of Mg21 interictal-typepotentials appeared in all cases. Ictal-type activity wasobserved in only one slice for the entire duration of theexperiment, and transiently in two other slices. The proportionof ictal to interictal activity was thus smaller in the groupwithout spontaneous activity. This finding corresponds withobservations of another study on human neocortical tissueresected during tumour surgery, where no spontaneous activityhad been seen in any of the slices tested (Straubet al.,1992b). In none of these preparations was ictal-type activityobserved with wash-out of Mg21. The appearance of ictal-type activity might thus be seen to reflect the underlyingincreased epileptogenicity of the spontaneously active slices.Nevertheless, ictal-type activity was observed relatively rarelyin comparison with other studies (Avoliet al., 1987, 1995),regardless of the generation of spontaneous sharp waves. Inthe cited investigations, ictal-type potentials were found toappear in all slices with washout of Mg21. In none of theseslices, however, was spontaneous activity reported to occur.As a whole, the type of ictal activity found in the presentstudy with omission of Mg21 resembles that found by othergroups, but appears only rarely. A possible explanation forthis discrepancy may be seen in the fact that, in the presentinvestigation, a submerged-type chamber was used, whereasin the cited studies interface-type chambers were utilized, in

which, in general, certain types of epilepsy models are knownto be generated far more readily than in submerged-typechambers.

In those slices generating interictal-type zero-Mg21-induced activity, application of bicuculline transformed themto ictal-type discharges. This is in line with findings inrat hippocampal slices, where bicuculline administrationenlarged and prolonged zero-Mg21-induced discharges(Tancrediet al., 1990; Traubet al., 1994; Whittingtonet al.,1995). This effect was attributed to the lability of inhibitionin the low-magnesium model of epilepsy (Whittingtonet al.,1995). However, both in another study in guinea pighippocampal slices (Lu¨cke et al., 1996) and in entorhinalcortex slices, bicuculline was found not to influence, or evento shorten epileptiform field potentials, which could convertthem into recurrent discharges in the latter case (Pfeifferetal., 1996). To summarize, on the one hand, spontaneoussharp waves were suppressed by bicuculline and, on theother hand, discharges elicited by omission of Mg21 wereaccentuated by the same drug. This suggests that differentelementary mechanisms are responsible for the generation ofthe two types of discharges.

In the present investigation, sharp waves have been foundin brain slices from epileptic patients which cannot beobserved under similar conditions in animal neocortical slices.Since healthy human neocortical tissue cannot be investigated,it remains uncertain whether these potentials are epileptic innature or not. Regardless of this, they may reflect a state ofincreased neuronal synchronization which could facilitateinitiation or spread of seizures under certain conditions.

AcknowledgementsThe authors wish to thank Dr B. Rambeck, Gesellschaft fu¨rEpilepsieforschung, Bielefeld, for determination of phenytoinand carbamazepine concentrations, and Dr K. Kisters, Klinikund Poliklinik fur Innere Medizin, Mu¨nster, for determinationof magnesium concentrations.

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Received September 11, 1997. Revised December 9, 1997.Accepted February 2, 1998

spontaneous sharp waves in human neocortical slices excised from epileptic patients

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