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Distinguishing forms of generalized epilepsy using magnetic brain stimulation
ELSEVIER
Electroencephalography and clinical Neurophysiology 98 (1996) 14-19
Distinguishing forms of generalized epilepsy using magnetic brain stimulation Maria Donatella Caramia a,b,*, Gianluigi Gigli a, Cesare Iani a, Maria Teresa Desiato a Marina Diomedi a, Maria Giuseppina Palmieri a, Giorgio Bernardi a,b a Clinical Neurophysiology, Department of Public Health, University of "'Tor Vergata," Via Orazio Raimondo 8, 00173 Rome, Italy b IRCCS S. Lucia, Rome, Italy Accepted for publication: 26 June 1995
Abstract In this study, we have used paired transcranial stimulation of the motor cortex to test the hypothesis that cortical inhibition is decreased in juvenile myoclonic epilepsy (JME). The double shock technique was adopted here because it offers a means for highlighting abnormal inhibitory mechanisms. From previous experiments performed on healthy subjects, it is known that a magnetic conditioning stimulus, of subthreshold intensity, suppresses the MEP in response to a subsequent suprathreshold stimulus delivered after 1-4 msec. JME patients were selected as a potential contrast with other forms of idiopathic generalized epilepsy, because they complain of myoclonic jerks without loss of consciousness, indicating with certainty a dysfunction of the motor cortex. Two patients with sporadic grand mal and one non-epileptic patient were also investigated. Paired stimulation was produced by a Bi-stim (Magstim) stimulator, with a figure-of-8 coil placed over the hand area of the motor cortex, and a set of interstimulus intervals (ISis) ranging from 1 to 6 msec was analyzed. In JME patients there were two indications of abn6rmality with respect to normal subjects and to the other epileptic patients: (1) the absence of MEP suppression to paired stimulation; (2) a progressive amplitude increase of MEPs to the test stimulus alone. In the two patients with the other form of epilepsy the pattern of inhibition was broadly preserved, even though there was some difference from the normal profile. The results suggest that the loss of MEP inhibition can be regarded as a marker of JME. Keywords: Generalized epilepsy; Magnetic brain stimulation; Cortical inhibition
1. Introduction Epilepsy is one of the most common neurological disorders, and yet there is still no proper understanding of the causes underlying about half of the known forms, hence their description as idiopathic generalized epilepsies (Wada and Masakazu, 1990). It is clear from EEG recordings and animal experiments that this grouping of diseases is characterized by altered states of cortical excitability, the EEG hallmark being spike and wave or polyspike and wave discharges.
* Corresponding author. Fax: 06-50510456.
Recently, magnetic transcranial stimulation (TCS) has begun to be applied in this field, as it offers a non-invasive means of stimulating the human motor cortex. The threshold for TCS was shown to be a key indicator of motor cortical excitability in 1989 (Caramia et al., 1989). Since then, this method has been used to show that patients with generalized epilepsy have a lower threshold of cortical excitability than healthy subjects, unless treated, in which case they have a higher threshold due to the suppressive action of the antiepileptic drug (Hufnagel et al., 1990; Reutens and Berkovic, 1992; Reutens et al., 1993). While the measurement of threshold simultaneously depicts excitation and inhibition (in that a low threshold represents both low inhibition and high excitability), this study introduces the clinical application of a technique which is able to identify inhibition alone.
0013-4694/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 0 1 3 - 4 6 9 4 ( 9 5 ) 0 0 1 5 0 - 6
EEG 95525
M.I). Caramia et al. / Electroencephalography and clinical Neurophysiology 98 (1996) 14-19
This latter technique is based on recent studies which have demonstrated that a raagnetic stimulus which is not strong enough to evoke an MEP may still be able to engage intracortical inhibitory circuits. The suppression of an MEP is seen when a subothreshold (conditioning) stimulus is delivered 1-4 msec before a suprathreshold (test) stimulus (Rothwell et al., 1991; Kujirai et al., 1993). By combining the measurement of threshold using single stimuli with the paired stimulation protocol it is possible to identify the excitatory and inhibitory aspects underlying epileptic behavior anti so distinguish between different forms of idiopathic generalized epilepsy, namely juvenile myoclonic epilepsy and other forms of idiopathic generalized epilepsy with grand mal seizures. Juvenile myoclonic epilepsy (JME) appears to be particularly suited for investigating altered states of inhibition, as EEG (bilateral polyspike and waves) and clinical abnormalities (myoclonic jerks without loss of consciousness) could be proposed as a natural model of motor cortical disinhibition (Janz, 1985; Durner et al., 1991). In order to verify this hypothesis, recordings from JME patients were compared with those from patients affected by grand mal seizures, who, while classified in the same family of idiopathic generalized epilepsy, show a contrasting clinical pattern (tonic-clonic generalized seizures without myoclonic jerks). Both generalized tonic-clonic seizures and myoclonic fits do occur early in the morning after awakening (Commission on Classification and Terminology of the International League Against Epilepsy, 1985). The results observed in this study may shed some light on the neurophysiological background underlying the differences between JME and other forms of idiopathic generalized epilepsy.
2. Patients and methods
The protocols were performed with the approval of the Local Ethical Committee on 5 healthy volunteers (4 females and 1 male), aged from 30 to 41 years and on 7 JME patients (4 females and 3 males), aged from 17 to 41 years, well controlled for clinical seizures. Two patients (22-yearold female; 24-year-old male) with sporadic grand mal seizures (average frequency: one fit about 6 months-1 year, with normal or sporadic spike and wave on EEG recordings) and one non-epileptic 18-year-old girl, treated with phenobarbital, were also enrolled in the study. This last subject acted as a useful control as phenobarbital was administered to the two patients with grand real seizures. All the JME patients were analyzed when already undergoing treatment with sodium valproate. However, one of the patients had also been investigated prior to commencing therapy. Six out of 7 JME patients started to have myoclonic jerks at ages 13-17, with sporadic generalized tonic-clonic seizures developing after a further 1-3 years. In the other patients, convulsive seizures appearing at the
15
age of 13 preceded the myoclonic jerks by 4 years. EEGs were always characterized by discharges of bilateral and synchronous spike and wave and polyspike and wave complexes. Ambulatory cassette EEGs confirmed the activation of discharges on awakening. None of the patients showed photosensitivity. Drug plasma levels in all patients and in the treated non-epileptic subject were maintained within the usually recommended clinical ranges (VPA 50-100 /xg/ml; PB 15-40 /xg/ml). MEPs were recorded with surface electrodes from left hand thenar muscles, during complete muscle relaxation, with the subjects comfortably reclined, and with open eyes (Rossini et al., 1991). The active electrode was placed over the motor point and the reference electrode on the metacarpo-phalangeal joint. An acoustic feedback monitored the EMG background activity, in order to detect and avoid interference possibly introduced by any voluntary movement. Focal single shock TCS was delivered by a figure-of-8 coil (outer diameter 9 cm) connected to a Magstim 200 stimulator. For the experiments using double shocks the same coil was connected to 2 Magstim units through a Bi-stim module, which has been devised by the Magstim Company for such a purpose. The coil was placed over the right central sulcus, on the scalp region corresponding to the hand motor area. The site where MEPs with the lowest intensity (threshold) were elicited in the contralateral target muscle was carefully located (Amassian et al., 1989; Caramia et al., 1989; Brasil-Neto et al., 1992). A conditioning-test design was used in order to investigate the time course of MEP inhibition. Paired stimuli were applied over the hand motor area contralateral to the examined muscle, with conditioning pulses delivered 1-6 msec before test stimulation (Kujirai et al., 1993). The intensity of the conditioning pulses was maintained below the threshold for evoking responses in contracted muscles, while test pulses were delivered just suprathreshold for relaxed MEP elicitation, with a difference in intensity between test and conditioning stimuli of about 40%. In 4 patients the effects of different subthreshold conditioning intensities (50%, 40% and 30% of stimulator output) were explored at 2 and 3 msec interstimulus intervals (ISis). Because thresholds could fluctuate during a session, the basic stimulating parameters were randomly checked and modified when necessary in order to maintain a stable level of recording. Recordings were acquired with double amplification so that saturated responses could be measured on-line with suitable calibration. In each set of experiments, test and conditioning pulses at the different interstimulus intervals were randomly intermixed. Several blocks of trials were performed in order to achieve a complete set of interstimulus intervals. Each block consisted of 8 trials with two conditions: the MEP in response to the test shock alone and the MEP conditioned by a prior sub-threshold stimulus delivered at one of the preset intervals.
M.D. Caramia et al. / Electroencephalography and clinical Neurophysiology 98 (1996) 14-19
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Fig. 1. Recordings from a single control subject. MEPs to magnetic cortical stimulation in relaxed thenar muscles are inhibited by a prior subthreshold conditioning stimulus (ISis = 2 msec top panel and 3 msec bottom panel). The top two traces as well as the lower two traces, in each panel, show the response to the test stimulus given alone. In the remaining middle traces conditioned MEPs are dramatically suppressed.
The sequence began and ended with two "test only" trials with the conditioned MEPs occurring in between (Figs. 1 and 2).
3. Data analysis The following parameters were analyzed: 1. Threshold intensity of test shock alone (for single shock) and test and conditioning (for double shock), expressed as a percentage of the stimulator's maximal output. 2. Amplitudes of test and conditioned MEPs, measured peak-to-peak (negativity upward). 3. Differences in amplitude between test and conditioned MEPs, measured as a percentage of control size. A mixed analysis of variance (ANOVA) on the time course data was performed. Thresholds and differences in MEP amplitudes were also evaluated with paired Student's t tests.
Fig. 2. Recordings from a JME patient. Same construction as in Fig. 1. Here, at 2 msec and 3 msec ISis, the inhibition is absent, with conditioned MEPs matching the test MEPs in amplitude.
peak amplitude (mean = 286.55 /zV). Over the course of the session, the range of test MEP amplitudes varied slightly, but not significantly. The mean threshold intensities of test and conditioning stimuli were respectively 74.3% and 45.3%. When test and conditioning stimuli were separated by 1-3 msec, the size of responses to the test stimulus was significantly reduced ( P < 0.001) with a high percentage of missing responses (range 30-90%, Fig. 1). With an interval of 4 msec the amplitude began to recover, regaining values matching those for the test alone by 5 - 6 msec. The time course of this effect is shown for 5 control subjects in Fig. 3.
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4.1. Control subjects In healthy control subjects the test stimulus alone produced an EMG response of about 200-500 /xV peak-to-
Fig. 3. The average time course of MEP suppression in 5 control subjects (filled squares) and in 7 JME patients (empty squares). At each interstimulus interval, the size of the conditioned MEP is expressed as a percentage of the size of the MEP to test stimulus alone (control size).
M.D. Caramia et al. / Electroencephalography and clinicaI Neurophysiology 98 (1996) 14-19
4.2. JME patients
150
In these patients both test and conditioning pulses were delivered at higher intensit:ies than normal (mean = 84.8%, 59.8%, P < 0.001) because of the increased threshold for cortical excitability due to the ongoing pharmacological treatment (Michelucci et al., 1989; Reutens and Berkovic, 1992). Unlike normal controls, in JME patients the MEP inhibition was greatly diminished during paired stimulation, at all explored ISis. In particular, at 2, 3 and 4 msec intervals, inhibitory effects were virtually absent, with the conditioned MEPs almost matching the test MEPs in amplitude (Figs. 2 and 3). As we noted that in normal subjects the amount of inhibition decreased as the stimulus intensity was increased (K~ajirai et al., 1993), the effects of different conditioning intensities were explored in 4 patients. No inhibition was found at any of the sub-threshold intensities employed with the conditioning stimuli. Another feature of JME patients was a sensitivity to the application of TCS, in thai: after the first set of stimuli and recordings subsequent stinmli at the same intensity tended to elicit larger MEPs. This effect continued in an irregular and unpredictable manner as the number of shocks increased. As a result, the average of the MEP amplitude over the whole session was higher than that of control subjects (mean = 419.2 /zV, P < 0.001). In the patient not yet undergoing therapy, the initial threshold intensities for the test and conditioning stimuli were lower than both treated patients and healthy controls, being 67% and 54% respectively. The phenomenon of progressive amplitude increment during the session, seen for treated JME patients, was so marked here that the thresholds had to be dropped to just 45% and 217% in order to keep the level of recordings roughly comparable to those obtained at the beginning of the session. After the patient had been on VPA therapy for 2 weeks, a new set of recordings showed that the threshold had risen to 81%.
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Fig. 5. For one patient with sporadic grand real seizures (empty square of Fig. 4) the inhibitory time course was longer and did not recover until 15 msec ISI.
4.3. Analysis of variance Statistical evaluation included a 2-way analysis of variance (ANOVA) with Group (controls vs. JME patients) as between-subject factor and Delay (ISI from 1 msec to 6 msec) as within-subject factor. When amplitudes are expressed as a percentage of test amplitudes the results are highly significant. The Group factor is significant ( F (1, 9) = 15.6; P = 0.003) because the mean over the 6 intervals of the conditioned amplitudes (48.4%) is much lower than that of patients (91.8%). The Delay factor is also significant ( F (5, 45) = 8.3; P < 0.001) as the percentage increases from 28.5% to 108% as the interval increases. Finally, the Group × Delay interaction is also significant ( F (5, 45) = 3.0; e = 0.02; Fig. 3).
4.4. Non-JME patients In the 2 patients affected by grand mal seizures the MEP inhibition was not affected and matched that of control subjects for ISis between 1 and 4 msec; if anything, there was an element of increased inhibition (Fig. 4). However, whereas at 5 msec the MEP amplitude recovered almost to the test size in control subjects, inhibition continued to be recorded for both these patients. At 6 msec the effect of MEP suppression suddenly disappeared for one patient but continued for the other. The anomalous behavior of responses from this patient was further illustrated by the long duration and instability of the inhibitory time course, which did not recover until an ISI of 15 msec (Fig. 5). Threshold intensities for test stimulation alone were lower than in JME patients, being in the range of the normal values (70% on average). No gradual increase in the test MEP amplitude over the recording sessions was observed in these last patients. Instead, cortical excitability was reduced as the session progressed, resulting in a difficulty in obtaining an MEP without raising the stimulus intensity; in fact, threshold intensity increments were necessary from 72% to 85% and from 68% to 70%. Even with
18
M.D. Caramia et aL /Electroencephalography and clinical Neurophysiology 98 (1996) 14-19
these higher intensities the double shock protocol produced a greater number of missing responses with respect to control subjects (95-100%). The patient under therapy with phenobarbital (100 mg/day), who was not epileptic, displayed a normal pattern of MEP inhibition to paired stimulation, with MEP amplitudes and threshold matching those of control subjects.
5. Discussion Previous studies with transcranial stimulation on epileptic patients have shown that cortical excitability is increased in untreated patients with idiopathic generalized epilepsy and decreased when treatment is given (Reutens and Berkovic, 1992; Reutens et al., 1993). These findings have been based primarily on measurements of changes in the threshold of stimulation obtained using single shocks. In most research of this kind, it is generally assumed that inhibition is the implicit counterpart of excitation, that is to say, high excitability corresponds to low inhibition and vice versa. However, the mechanisms of excitation and inhibition may be distinguished one from the other and also have a degree of independence. In this study, we have used paired transcranial magnetic stimulation of the motor cortex to test the hypothesis that cortical inhibition is decreased a n d / o r excitation is increased in juvenile myoclonic epilepsy. The double shock technique was adopted here because it offers a means for highlighting abnormal inhibitory-excitatory mechanisms. It is known from experiments on healthy subjects that a magnetic conditioning stimulus, at intensities below threshold for obtaining MEPs in relaxed hand muscles, suppresses the MEP in response to a subsequent suprathreshold stimulus (test) delivered after 1 to 4 msec. This inhibition has been shown to result from intracortical inhibitory mechanisms and can be reproduced consistently in healthy subjects (Rothwell et al., 1991; Kujirai et al., 1993). From both animal experiments and clinical observations on epileptic patients, it is known that GABAergic inhibition plays a major role in the regulation of neuronal excitability (Krnjevic et al., 1964). As the pattern of cortical inhibition in response to paired stimulation is similar to that due to GABA in animals (Krnjevic, 1983; Kujirai el al., 1993), the method is an obvious candidate for investigating the altered GABAergic inhibitory system in epileptic patients. JME patients were selected as a potential contrast with other forms of idiopathic generalized epilepsy, because they complain of myoclonic jerks without loss of consciousness, thus indicating with certainty a dysfunction related to the motor cortex (Delgado-Escueta and EnrileBacsal, 1984). In our recordings from JME patients there were two indications of abnormality with respect to normal controls and to the other idiopathic epileptic patients: (a)
the absence of MEP suppression to paired stimulation; (b) a progressive amplitude increase of motor responses to test shock alone over the course of the session. This first finding (a) has the clearest significance, since it is extremely unlikely that the increase in amplitude occurs because the amount of normal suppression happens to be balanced exactly by abnormal excitation. From this, it is reasonable to postulate that impairment of MEP suppression may be due to loss of intracortical inhibition. By contrast, no such loss of inhibition to paired stimulation was recorded in the patients with sporadic grand mal seizures or in normal subjects. The second point (b) offers a further marker for distinguishing JME patients. The increase in MEP amplitude recorded from JME patients is the opposite to the slight decrease which has been reported in about 60% of normal subjects over the course of a medium to long session (more than 50 consecutive stimuli; Rossini et al., 1991). It may be assumed that the trend observed in healthy controls is due to increasing inhibition as a reaction to the ongoing stimuli as if it were a measure of self-protection against over-stimulation of the motor cortex. In JME patients the inability to react in this way may be interpreted as an effect of hyperexcitability, or perhaps as a deficiency in inhibitory function. The fact that MEP amplitude does not just remain stable, but actually increased may be due to a progressive destabilization or weakening over time of such inhibition. However, it cannot be ruled out that the same phenomenon may be an expression of hyperexcitability alone. In JME patients being treated with sodium valproate, cortical excitability was reduced inasmuch as the threshold for transcranial stimulation was higher than in untreated and in normal subjects. Nevertheless, the administration of this drug does not produce recovery of the inhibitory pattern which is normally observed in response to double shock stimulation. The mechanism postulated for JME patients seems to be well supported by their specific interictal EEG activity. In fact, they show an EEG pattern of polyspike and wave discharges, which can be linked to loss of cortical inhibition and increased excitation when tested with brain TCS. Unlike JME patients and control subjects, in patients with grand mal seizures a contrasting pattern of inhibition was found, characterized by a prolonged time course of MEP suppression and by a progressive growth of the threshold intensity required for evoking an MEP. It seems that in this type of epilepsy the neurophysiological background is characterized by an increment of cortical inhibition. The excess of inhibition observed in these patients may be due to the pharmacological effect of the phenobarbital therapy, but it should be noted that this drug does not produce any appreciable changes in the cortical excitability in the control subject (unfortunately, this drug was not administered to JME patients). Assuming that the inhibitory pattern is independent of phenobarbital, then the
M.D. Caramia et aL / Electroencephalography and clinical Neurophysiology 98 (1996) 14-19
mechanism of the epileptic seizure in grand mal patient~ may be based on a functional background different from that underlying myoclonic epilepsy. If this is so, then the seizure event might represent a means for reducing an excess of cortical inhibition by transiently reversing the excitatory-inhibitory balance. It must be admitted that the evidence u, non-JME idiopathic generalized epilepsies is founded on recordings from a very small sample group. The process of assembling suitable subjects is hampered by 2 factors: first, the risk of inducing grand mal seizures in these patients during long TCS sessions (Hufuagel and Elger, 1991); second, the relatively small number of patients undergoing drug monotherapy. The aim is to investigate more patients with the same disease in due course. However, the results obtained seem significant enough to merit discussion at this stage. Thus far, besides the evaluation of MEP threshold, it seems that it is possible to distinguish forms of epilepsy according to the MEP inhibitory profile. In this regard it is interesting to note the preliminary findings reported by Fong et al. (1993) which show that patients with focal epilepsy display decreased inhibition of the affected hemisphere. In conclusion, magnetic: TCS evaluation of inhibition and of excitation is a potentially powerful tool for differentiating between subclasses of epilepsy.
Acknowledgements We gratefully acknowledge Franco Lavaroni and Bruna Maffini for their technical assistance. We thank Mark Wilson Jones for reviewing the English style of the manuscript and Augusto Carlesimo for his help in the statistical analysis.
References Amassian, V.E., Cracco, R.Q. and Maccabee, P.J. Focal stimulation of human cerebral cortex with the magnetic coil: a comparison with electrical stimulation. Electroenceph. clin. Neurophysiol., 1989, 74: 401-416. Brasil-Neto, J.P., McShane, L.M.. Fuhr, P., Hallett, M. and Cohen, L.G. Topographic mapping of the human motor cortex with magnetic
19
stimulation: factors affecting accuracy and reproducubility. Electroenceph. clin. Neurophysiol., 1992, 85: 9-16. Caramia, M.D., Pardal, A.M., Zarola, F. and Rossini, P.M. Electric vs. magnetic transcranial stimulation of the brain in healthy humans: a comparative study of central motor tract conductivity and excitability. Brain Res., 1989, 479: 98-104. Commission on Classification and Terminology of the International League Against Epilepsy. Proposal for classification of epilepsies and epileptic syndromes. Epilepsia, 1985, 26: 268-278. Delgado-Escueta, A.V. and Enrile-Bacsal, F. Juvenile myoclonic epilepsy of Janz. Neurology, 1984, 34: 285-294. Durner, M., Sander, T., Greenberg, D.A., Johnson, K., Beck-Mannagetta, G. and Janz, D. Localization of idiopathic generalized epilepsy on chromosome 6p in families of juvenile myoclonic epilepsy patients. Neurology, 1991, 41: 1651-1655. Fong, J.K.Y., Werhahn, K.J., Rothwell, J.C., Shorvon, S.D., Thompson, P.D., Day, B.L. and Marsden, C.D. Motor cortex excitability in focal and generalized epilepsy. J. Physiol. (Lond.), 1993, 459: 468P. Hufnagel, A. and Elger, E. Induction of seizures by transcranial stimulation in epileptic patients. J. Neurol., 1991, 238: 109-110. Hufnagel, A., Elger, C.E., Marx, W. and Ising, A. Magnetic motor-evoked potentials in epilepsy: effects of the disease and of anticonvulsant medications. Ann. Neurot., 1990, 28: 680-686. Janz, D. Epilepsy with impulsive petit mal (juvenile myoclonic epilepsy). Acta Neurol. Scand., 1985, 72: 449-459. Krnjevic, K. GABA-mediated inhibitory mechanisms in relation to epileptic discharges. In: Basic Mechanisms of Neuronal Hyperexcitability. Alan R. Liss, New York, 1983: 249-280. Krnjevic, K., Randic, M. and Straughan, D.W. Cortical inhibition. Nature, 1964, 201: 1294-1296. Kujirai, T., Caramia, M.D., Rothwell, J.C., Day, B.L., Thompson, P.D., Ferbert, A., Wroe, S., Asselman, P. and Marsden, C.D. Corticocortical inhibition in human motor cortex. J. Physiol. (Lond.), 1993, 471: 501-519. Michelucci, R., Rubboli, G., Plasmati, R. et al. Transcranial magnetic stimulation of the cerebral cortex in epilepsy. Neurology, 1989, 39 (Suppl. 1): 414. Reutens, D.C. and Berkovic, S.F. Increased cortical excitability in generalised epilepsy demonstrated with transcranial magnetic stimulation. Lancet, 1992, 339: 362-363. Reutens, D.C., Berkovic, S.F., Macdonell, R.A.L. and Bladin, P.F. Magnetic stimulation of the brain in generalized epilepsy: reversal of cortical hyperexcitability by anticonvulsivants. Ann. Neurol., 1993, 34: 351-355. Rossini, P.M., Desiato, M.T., Lavaroni, F. and Caramia, M.D. Brain excitability and electroencephalografic activation: non-invasive evaluation in healthy humans via transcranial magnetic stimulation. Brain Res., 1991, 567: 111-119. Rothwell, J.C., Ferbert, A., Caramia, M.D., Kujirai, T., Day, B.L. and Thompson, P.D. Intracortical inhibitory circuits studied in humans. Neurology, 1991, 41: 192. Wada, J.A. and Masakazu, S. Recent classification of seizure and epilepsies. In: J.A. Wada and R. Ellingson (Eds.), Clinical Neurophysiology of Epilepsy. EEG Handbook (revised series, Vol. 4). Elsevier Science, Amsterdam, 1990: 3-36.
Electroencephalography and clinical Neurophysiology 98 (1996) 14-19
Distinguishing forms of generalized epilepsy using magnetic brain stimulation Maria Donatella Caramia a,b,*, Gianluigi Gigli a, Cesare Iani a, Maria Teresa Desiato a Marina Diomedi a, Maria Giuseppina Palmieri a, Giorgio Bernardi a,b a Clinical Neurophysiology, Department of Public Health, University of "'Tor Vergata," Via Orazio Raimondo 8, 00173 Rome, Italy b IRCCS S. Lucia, Rome, Italy Accepted for publication: 26 June 1995
Abstract In this study, we have used paired transcranial stimulation of the motor cortex to test the hypothesis that cortical inhibition is decreased in juvenile myoclonic epilepsy (JME). The double shock technique was adopted here because it offers a means for highlighting abnormal inhibitory mechanisms. From previous experiments performed on healthy subjects, it is known that a magnetic conditioning stimulus, of subthreshold intensity, suppresses the MEP in response to a subsequent suprathreshold stimulus delivered after 1-4 msec. JME patients were selected as a potential contrast with other forms of idiopathic generalized epilepsy, because they complain of myoclonic jerks without loss of consciousness, indicating with certainty a dysfunction of the motor cortex. Two patients with sporadic grand mal and one non-epileptic patient were also investigated. Paired stimulation was produced by a Bi-stim (Magstim) stimulator, with a figure-of-8 coil placed over the hand area of the motor cortex, and a set of interstimulus intervals (ISis) ranging from 1 to 6 msec was analyzed. In JME patients there were two indications of abn6rmality with respect to normal subjects and to the other epileptic patients: (1) the absence of MEP suppression to paired stimulation; (2) a progressive amplitude increase of MEPs to the test stimulus alone. In the two patients with the other form of epilepsy the pattern of inhibition was broadly preserved, even though there was some difference from the normal profile. The results suggest that the loss of MEP inhibition can be regarded as a marker of JME. Keywords: Generalized epilepsy; Magnetic brain stimulation; Cortical inhibition
1. Introduction Epilepsy is one of the most common neurological disorders, and yet there is still no proper understanding of the causes underlying about half of the known forms, hence their description as idiopathic generalized epilepsies (Wada and Masakazu, 1990). It is clear from EEG recordings and animal experiments that this grouping of diseases is characterized by altered states of cortical excitability, the EEG hallmark being spike and wave or polyspike and wave discharges.
* Corresponding author. Fax: 06-50510456.
Recently, magnetic transcranial stimulation (TCS) has begun to be applied in this field, as it offers a non-invasive means of stimulating the human motor cortex. The threshold for TCS was shown to be a key indicator of motor cortical excitability in 1989 (Caramia et al., 1989). Since then, this method has been used to show that patients with generalized epilepsy have a lower threshold of cortical excitability than healthy subjects, unless treated, in which case they have a higher threshold due to the suppressive action of the antiepileptic drug (Hufnagel et al., 1990; Reutens and Berkovic, 1992; Reutens et al., 1993). While the measurement of threshold simultaneously depicts excitation and inhibition (in that a low threshold represents both low inhibition and high excitability), this study introduces the clinical application of a technique which is able to identify inhibition alone.
0013-4694/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 0 1 3 - 4 6 9 4 ( 9 5 ) 0 0 1 5 0 - 6
EEG 95525
M.I). Caramia et al. / Electroencephalography and clinical Neurophysiology 98 (1996) 14-19
This latter technique is based on recent studies which have demonstrated that a raagnetic stimulus which is not strong enough to evoke an MEP may still be able to engage intracortical inhibitory circuits. The suppression of an MEP is seen when a subothreshold (conditioning) stimulus is delivered 1-4 msec before a suprathreshold (test) stimulus (Rothwell et al., 1991; Kujirai et al., 1993). By combining the measurement of threshold using single stimuli with the paired stimulation protocol it is possible to identify the excitatory and inhibitory aspects underlying epileptic behavior anti so distinguish between different forms of idiopathic generalized epilepsy, namely juvenile myoclonic epilepsy and other forms of idiopathic generalized epilepsy with grand mal seizures. Juvenile myoclonic epilepsy (JME) appears to be particularly suited for investigating altered states of inhibition, as EEG (bilateral polyspike and waves) and clinical abnormalities (myoclonic jerks without loss of consciousness) could be proposed as a natural model of motor cortical disinhibition (Janz, 1985; Durner et al., 1991). In order to verify this hypothesis, recordings from JME patients were compared with those from patients affected by grand mal seizures, who, while classified in the same family of idiopathic generalized epilepsy, show a contrasting clinical pattern (tonic-clonic generalized seizures without myoclonic jerks). Both generalized tonic-clonic seizures and myoclonic fits do occur early in the morning after awakening (Commission on Classification and Terminology of the International League Against Epilepsy, 1985). The results observed in this study may shed some light on the neurophysiological background underlying the differences between JME and other forms of idiopathic generalized epilepsy.
2. Patients and methods
The protocols were performed with the approval of the Local Ethical Committee on 5 healthy volunteers (4 females and 1 male), aged from 30 to 41 years and on 7 JME patients (4 females and 3 males), aged from 17 to 41 years, well controlled for clinical seizures. Two patients (22-yearold female; 24-year-old male) with sporadic grand mal seizures (average frequency: one fit about 6 months-1 year, with normal or sporadic spike and wave on EEG recordings) and one non-epileptic 18-year-old girl, treated with phenobarbital, were also enrolled in the study. This last subject acted as a useful control as phenobarbital was administered to the two patients with grand real seizures. All the JME patients were analyzed when already undergoing treatment with sodium valproate. However, one of the patients had also been investigated prior to commencing therapy. Six out of 7 JME patients started to have myoclonic jerks at ages 13-17, with sporadic generalized tonic-clonic seizures developing after a further 1-3 years. In the other patients, convulsive seizures appearing at the
15
age of 13 preceded the myoclonic jerks by 4 years. EEGs were always characterized by discharges of bilateral and synchronous spike and wave and polyspike and wave complexes. Ambulatory cassette EEGs confirmed the activation of discharges on awakening. None of the patients showed photosensitivity. Drug plasma levels in all patients and in the treated non-epileptic subject were maintained within the usually recommended clinical ranges (VPA 50-100 /xg/ml; PB 15-40 /xg/ml). MEPs were recorded with surface electrodes from left hand thenar muscles, during complete muscle relaxation, with the subjects comfortably reclined, and with open eyes (Rossini et al., 1991). The active electrode was placed over the motor point and the reference electrode on the metacarpo-phalangeal joint. An acoustic feedback monitored the EMG background activity, in order to detect and avoid interference possibly introduced by any voluntary movement. Focal single shock TCS was delivered by a figure-of-8 coil (outer diameter 9 cm) connected to a Magstim 200 stimulator. For the experiments using double shocks the same coil was connected to 2 Magstim units through a Bi-stim module, which has been devised by the Magstim Company for such a purpose. The coil was placed over the right central sulcus, on the scalp region corresponding to the hand motor area. The site where MEPs with the lowest intensity (threshold) were elicited in the contralateral target muscle was carefully located (Amassian et al., 1989; Caramia et al., 1989; Brasil-Neto et al., 1992). A conditioning-test design was used in order to investigate the time course of MEP inhibition. Paired stimuli were applied over the hand motor area contralateral to the examined muscle, with conditioning pulses delivered 1-6 msec before test stimulation (Kujirai et al., 1993). The intensity of the conditioning pulses was maintained below the threshold for evoking responses in contracted muscles, while test pulses were delivered just suprathreshold for relaxed MEP elicitation, with a difference in intensity between test and conditioning stimuli of about 40%. In 4 patients the effects of different subthreshold conditioning intensities (50%, 40% and 30% of stimulator output) were explored at 2 and 3 msec interstimulus intervals (ISis). Because thresholds could fluctuate during a session, the basic stimulating parameters were randomly checked and modified when necessary in order to maintain a stable level of recording. Recordings were acquired with double amplification so that saturated responses could be measured on-line with suitable calibration. In each set of experiments, test and conditioning pulses at the different interstimulus intervals were randomly intermixed. Several blocks of trials were performed in order to achieve a complete set of interstimulus intervals. Each block consisted of 8 trials with two conditions: the MEP in response to the test shock alone and the MEP conditioned by a prior sub-threshold stimulus delivered at one of the preset intervals.
M.D. Caramia et al. / Electroencephalography and clinical Neurophysiology 98 (1996) 14-19
16
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Fig. 1. Recordings from a single control subject. MEPs to magnetic cortical stimulation in relaxed thenar muscles are inhibited by a prior subthreshold conditioning stimulus (ISis = 2 msec top panel and 3 msec bottom panel). The top two traces as well as the lower two traces, in each panel, show the response to the test stimulus given alone. In the remaining middle traces conditioned MEPs are dramatically suppressed.
The sequence began and ended with two "test only" trials with the conditioned MEPs occurring in between (Figs. 1 and 2).
3. Data analysis The following parameters were analyzed: 1. Threshold intensity of test shock alone (for single shock) and test and conditioning (for double shock), expressed as a percentage of the stimulator's maximal output. 2. Amplitudes of test and conditioned MEPs, measured peak-to-peak (negativity upward). 3. Differences in amplitude between test and conditioned MEPs, measured as a percentage of control size. A mixed analysis of variance (ANOVA) on the time course data was performed. Thresholds and differences in MEP amplitudes were also evaluated with paired Student's t tests.
Fig. 2. Recordings from a JME patient. Same construction as in Fig. 1. Here, at 2 msec and 3 msec ISis, the inhibition is absent, with conditioned MEPs matching the test MEPs in amplitude.
peak amplitude (mean = 286.55 /zV). Over the course of the session, the range of test MEP amplitudes varied slightly, but not significantly. The mean threshold intensities of test and conditioning stimuli were respectively 74.3% and 45.3%. When test and conditioning stimuli were separated by 1-3 msec, the size of responses to the test stimulus was significantly reduced ( P < 0.001) with a high percentage of missing responses (range 30-90%, Fig. 1). With an interval of 4 msec the amplitude began to recover, regaining values matching those for the test alone by 5 - 6 msec. The time course of this effect is shown for 5 control subjects in Fig. 3.
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4.1. Control subjects In healthy control subjects the test stimulus alone produced an EMG response of about 200-500 /xV peak-to-
Fig. 3. The average time course of MEP suppression in 5 control subjects (filled squares) and in 7 JME patients (empty squares). At each interstimulus interval, the size of the conditioned MEP is expressed as a percentage of the size of the MEP to test stimulus alone (control size).
M.D. Caramia et al. / Electroencephalography and clinicaI Neurophysiology 98 (1996) 14-19
4.2. JME patients
150
In these patients both test and conditioning pulses were delivered at higher intensit:ies than normal (mean = 84.8%, 59.8%, P < 0.001) because of the increased threshold for cortical excitability due to the ongoing pharmacological treatment (Michelucci et al., 1989; Reutens and Berkovic, 1992). Unlike normal controls, in JME patients the MEP inhibition was greatly diminished during paired stimulation, at all explored ISis. In particular, at 2, 3 and 4 msec intervals, inhibitory effects were virtually absent, with the conditioned MEPs almost matching the test MEPs in amplitude (Figs. 2 and 3). As we noted that in normal subjects the amount of inhibition decreased as the stimulus intensity was increased (K~ajirai et al., 1993), the effects of different conditioning intensities were explored in 4 patients. No inhibition was found at any of the sub-threshold intensities employed with the conditioning stimuli. Another feature of JME patients was a sensitivity to the application of TCS, in thai: after the first set of stimuli and recordings subsequent stinmli at the same intensity tended to elicit larger MEPs. This effect continued in an irregular and unpredictable manner as the number of shocks increased. As a result, the average of the MEP amplitude over the whole session was higher than that of control subjects (mean = 419.2 /zV, P < 0.001). In the patient not yet undergoing therapy, the initial threshold intensities for the test and conditioning stimuli were lower than both treated patients and healthy controls, being 67% and 54% respectively. The phenomenon of progressive amplitude increment during the session, seen for treated JME patients, was so marked here that the thresholds had to be dropped to just 45% and 217% in order to keep the level of recordings roughly comparable to those obtained at the beginning of the session. After the patient had been on VPA therapy for 2 weeks, a new set of recordings showed that the threshold had risen to 81%.
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Fig. 5. For one patient with sporadic grand real seizures (empty square of Fig. 4) the inhibitory time course was longer and did not recover until 15 msec ISI.
4.3. Analysis of variance Statistical evaluation included a 2-way analysis of variance (ANOVA) with Group (controls vs. JME patients) as between-subject factor and Delay (ISI from 1 msec to 6 msec) as within-subject factor. When amplitudes are expressed as a percentage of test amplitudes the results are highly significant. The Group factor is significant ( F (1, 9) = 15.6; P = 0.003) because the mean over the 6 intervals of the conditioned amplitudes (48.4%) is much lower than that of patients (91.8%). The Delay factor is also significant ( F (5, 45) = 8.3; P < 0.001) as the percentage increases from 28.5% to 108% as the interval increases. Finally, the Group × Delay interaction is also significant ( F (5, 45) = 3.0; e = 0.02; Fig. 3).
4.4. Non-JME patients In the 2 patients affected by grand mal seizures the MEP inhibition was not affected and matched that of control subjects for ISis between 1 and 4 msec; if anything, there was an element of increased inhibition (Fig. 4). However, whereas at 5 msec the MEP amplitude recovered almost to the test size in control subjects, inhibition continued to be recorded for both these patients. At 6 msec the effect of MEP suppression suddenly disappeared for one patient but continued for the other. The anomalous behavior of responses from this patient was further illustrated by the long duration and instability of the inhibitory time course, which did not recover until an ISI of 15 msec (Fig. 5). Threshold intensities for test stimulation alone were lower than in JME patients, being in the range of the normal values (70% on average). No gradual increase in the test MEP amplitude over the recording sessions was observed in these last patients. Instead, cortical excitability was reduced as the session progressed, resulting in a difficulty in obtaining an MEP without raising the stimulus intensity; in fact, threshold intensity increments were necessary from 72% to 85% and from 68% to 70%. Even with
18
M.D. Caramia et aL /Electroencephalography and clinical Neurophysiology 98 (1996) 14-19
these higher intensities the double shock protocol produced a greater number of missing responses with respect to control subjects (95-100%). The patient under therapy with phenobarbital (100 mg/day), who was not epileptic, displayed a normal pattern of MEP inhibition to paired stimulation, with MEP amplitudes and threshold matching those of control subjects.
5. Discussion Previous studies with transcranial stimulation on epileptic patients have shown that cortical excitability is increased in untreated patients with idiopathic generalized epilepsy and decreased when treatment is given (Reutens and Berkovic, 1992; Reutens et al., 1993). These findings have been based primarily on measurements of changes in the threshold of stimulation obtained using single shocks. In most research of this kind, it is generally assumed that inhibition is the implicit counterpart of excitation, that is to say, high excitability corresponds to low inhibition and vice versa. However, the mechanisms of excitation and inhibition may be distinguished one from the other and also have a degree of independence. In this study, we have used paired transcranial magnetic stimulation of the motor cortex to test the hypothesis that cortical inhibition is decreased a n d / o r excitation is increased in juvenile myoclonic epilepsy. The double shock technique was adopted here because it offers a means for highlighting abnormal inhibitory-excitatory mechanisms. It is known from experiments on healthy subjects that a magnetic conditioning stimulus, at intensities below threshold for obtaining MEPs in relaxed hand muscles, suppresses the MEP in response to a subsequent suprathreshold stimulus (test) delivered after 1 to 4 msec. This inhibition has been shown to result from intracortical inhibitory mechanisms and can be reproduced consistently in healthy subjects (Rothwell et al., 1991; Kujirai et al., 1993). From both animal experiments and clinical observations on epileptic patients, it is known that GABAergic inhibition plays a major role in the regulation of neuronal excitability (Krnjevic et al., 1964). As the pattern of cortical inhibition in response to paired stimulation is similar to that due to GABA in animals (Krnjevic, 1983; Kujirai el al., 1993), the method is an obvious candidate for investigating the altered GABAergic inhibitory system in epileptic patients. JME patients were selected as a potential contrast with other forms of idiopathic generalized epilepsy, because they complain of myoclonic jerks without loss of consciousness, thus indicating with certainty a dysfunction related to the motor cortex (Delgado-Escueta and EnrileBacsal, 1984). In our recordings from JME patients there were two indications of abnormality with respect to normal controls and to the other idiopathic epileptic patients: (a)
the absence of MEP suppression to paired stimulation; (b) a progressive amplitude increase of motor responses to test shock alone over the course of the session. This first finding (a) has the clearest significance, since it is extremely unlikely that the increase in amplitude occurs because the amount of normal suppression happens to be balanced exactly by abnormal excitation. From this, it is reasonable to postulate that impairment of MEP suppression may be due to loss of intracortical inhibition. By contrast, no such loss of inhibition to paired stimulation was recorded in the patients with sporadic grand mal seizures or in normal subjects. The second point (b) offers a further marker for distinguishing JME patients. The increase in MEP amplitude recorded from JME patients is the opposite to the slight decrease which has been reported in about 60% of normal subjects over the course of a medium to long session (more than 50 consecutive stimuli; Rossini et al., 1991). It may be assumed that the trend observed in healthy controls is due to increasing inhibition as a reaction to the ongoing stimuli as if it were a measure of self-protection against over-stimulation of the motor cortex. In JME patients the inability to react in this way may be interpreted as an effect of hyperexcitability, or perhaps as a deficiency in inhibitory function. The fact that MEP amplitude does not just remain stable, but actually increased may be due to a progressive destabilization or weakening over time of such inhibition. However, it cannot be ruled out that the same phenomenon may be an expression of hyperexcitability alone. In JME patients being treated with sodium valproate, cortical excitability was reduced inasmuch as the threshold for transcranial stimulation was higher than in untreated and in normal subjects. Nevertheless, the administration of this drug does not produce recovery of the inhibitory pattern which is normally observed in response to double shock stimulation. The mechanism postulated for JME patients seems to be well supported by their specific interictal EEG activity. In fact, they show an EEG pattern of polyspike and wave discharges, which can be linked to loss of cortical inhibition and increased excitation when tested with brain TCS. Unlike JME patients and control subjects, in patients with grand mal seizures a contrasting pattern of inhibition was found, characterized by a prolonged time course of MEP suppression and by a progressive growth of the threshold intensity required for evoking an MEP. It seems that in this type of epilepsy the neurophysiological background is characterized by an increment of cortical inhibition. The excess of inhibition observed in these patients may be due to the pharmacological effect of the phenobarbital therapy, but it should be noted that this drug does not produce any appreciable changes in the cortical excitability in the control subject (unfortunately, this drug was not administered to JME patients). Assuming that the inhibitory pattern is independent of phenobarbital, then the
M.D. Caramia et aL / Electroencephalography and clinical Neurophysiology 98 (1996) 14-19
mechanism of the epileptic seizure in grand mal patient~ may be based on a functional background different from that underlying myoclonic epilepsy. If this is so, then the seizure event might represent a means for reducing an excess of cortical inhibition by transiently reversing the excitatory-inhibitory balance. It must be admitted that the evidence u, non-JME idiopathic generalized epilepsies is founded on recordings from a very small sample group. The process of assembling suitable subjects is hampered by 2 factors: first, the risk of inducing grand mal seizures in these patients during long TCS sessions (Hufuagel and Elger, 1991); second, the relatively small number of patients undergoing drug monotherapy. The aim is to investigate more patients with the same disease in due course. However, the results obtained seem significant enough to merit discussion at this stage. Thus far, besides the evaluation of MEP threshold, it seems that it is possible to distinguish forms of epilepsy according to the MEP inhibitory profile. In this regard it is interesting to note the preliminary findings reported by Fong et al. (1993) which show that patients with focal epilepsy display decreased inhibition of the affected hemisphere. In conclusion, magnetic: TCS evaluation of inhibition and of excitation is a potentially powerful tool for differentiating between subclasses of epilepsy.
Acknowledgements We gratefully acknowledge Franco Lavaroni and Bruna Maffini for their technical assistance. We thank Mark Wilson Jones for reviewing the English style of the manuscript and Augusto Carlesimo for his help in the statistical analysis.
References Amassian, V.E., Cracco, R.Q. and Maccabee, P.J. Focal stimulation of human cerebral cortex with the magnetic coil: a comparison with electrical stimulation. Electroenceph. clin. Neurophysiol., 1989, 74: 401-416. Brasil-Neto, J.P., McShane, L.M.. Fuhr, P., Hallett, M. and Cohen, L.G. Topographic mapping of the human motor cortex with magnetic
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stimulation: factors affecting accuracy and reproducubility. Electroenceph. clin. Neurophysiol., 1992, 85: 9-16. Caramia, M.D., Pardal, A.M., Zarola, F. and Rossini, P.M. Electric vs. magnetic transcranial stimulation of the brain in healthy humans: a comparative study of central motor tract conductivity and excitability. Brain Res., 1989, 479: 98-104. Commission on Classification and Terminology of the International League Against Epilepsy. Proposal for classification of epilepsies and epileptic syndromes. Epilepsia, 1985, 26: 268-278. Delgado-Escueta, A.V. and Enrile-Bacsal, F. Juvenile myoclonic epilepsy of Janz. Neurology, 1984, 34: 285-294. Durner, M., Sander, T., Greenberg, D.A., Johnson, K., Beck-Mannagetta, G. and Janz, D. Localization of idiopathic generalized epilepsy on chromosome 6p in families of juvenile myoclonic epilepsy patients. Neurology, 1991, 41: 1651-1655. Fong, J.K.Y., Werhahn, K.J., Rothwell, J.C., Shorvon, S.D., Thompson, P.D., Day, B.L. and Marsden, C.D. Motor cortex excitability in focal and generalized epilepsy. J. Physiol. (Lond.), 1993, 459: 468P. Hufnagel, A. and Elger, E. Induction of seizures by transcranial stimulation in epileptic patients. J. Neurol., 1991, 238: 109-110. Hufnagel, A., Elger, C.E., Marx, W. and Ising, A. Magnetic motor-evoked potentials in epilepsy: effects of the disease and of anticonvulsant medications. Ann. Neurot., 1990, 28: 680-686. Janz, D. Epilepsy with impulsive petit mal (juvenile myoclonic epilepsy). Acta Neurol. Scand., 1985, 72: 449-459. Krnjevic, K. GABA-mediated inhibitory mechanisms in relation to epileptic discharges. In: Basic Mechanisms of Neuronal Hyperexcitability. Alan R. Liss, New York, 1983: 249-280. Krnjevic, K., Randic, M. and Straughan, D.W. Cortical inhibition. Nature, 1964, 201: 1294-1296. Kujirai, T., Caramia, M.D., Rothwell, J.C., Day, B.L., Thompson, P.D., Ferbert, A., Wroe, S., Asselman, P. and Marsden, C.D. Corticocortical inhibition in human motor cortex. J. Physiol. (Lond.), 1993, 471: 501-519. Michelucci, R., Rubboli, G., Plasmati, R. et al. Transcranial magnetic stimulation of the cerebral cortex in epilepsy. Neurology, 1989, 39 (Suppl. 1): 414. Reutens, D.C. and Berkovic, S.F. Increased cortical excitability in generalised epilepsy demonstrated with transcranial magnetic stimulation. Lancet, 1992, 339: 362-363. Reutens, D.C., Berkovic, S.F., Macdonell, R.A.L. and Bladin, P.F. Magnetic stimulation of the brain in generalized epilepsy: reversal of cortical hyperexcitability by anticonvulsivants. Ann. Neurol., 1993, 34: 351-355. Rossini, P.M., Desiato, M.T., Lavaroni, F. and Caramia, M.D. Brain excitability and electroencephalografic activation: non-invasive evaluation in healthy humans via transcranial magnetic stimulation. Brain Res., 1991, 567: 111-119. Rothwell, J.C., Ferbert, A., Caramia, M.D., Kujirai, T., Day, B.L. and Thompson, P.D. Intracortical inhibitory circuits studied in humans. Neurology, 1991, 41: 192. Wada, J.A. and Masakazu, S. Recent classification of seizure and epilepsies. In: J.A. Wada and R. Ellingson (Eds.), Clinical Neurophysiology of Epilepsy. EEG Handbook (revised series, Vol. 4). Elsevier Science, Amsterdam, 1990: 3-36.