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EEG and seizure threshold in normal and lissencephalic ferrets
Brain Research, 307 (1984) 29-38 Elsevier
29
BRE 10218
EEG and Seizure Threshold in Normal and Lissencephalic Ferrets JERZY MAJKOWSKI, MOON HE LEE, PIOTR B. KOZLOWSKI and RAEF HADDAD Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, N Y 10314 (U.S.A.)
(Accepted January 3rd, 1984) Key words: EEG - - lissencephaly - - ferret - - extreme spindle - - epileptic seizure
Changes in EEG and susceptibility to electrically induced seizures were examined in the ferret with lissencephaly produced by exposure to a single injection of methylazoxymethanol acetate (MAM Ac) given to the pregnant jill on gestation day 32. Ten lissencephalic and 11 normal ferrets were chronically implanted with 14 cortical stainless steel electrodes. EEG records were sampled from various stages of the sleep/awake cycle. Six of each group were subjected to electrical stimulation for seizure threshold. Although the number of stimulations and the current intensity required to produce epileptiform afterdischarges (AD) and seizures were not different between the two groups, the lissencephalic ferrets had significantly longer AD and seizures, and a greater number of generalized seizures, indicating an enhanced seizure susceptibility. The EEG of the lissencephalic ferrets was characterized by increased slow wave activity within the low theta band range, extreme spindle activity, focal or multifocal slow and sharp waves, spikes, or spike and slow wave complexes. The differences in the EEG were more pronounced during drowsiness and sleep stages. The brains of all of the treated animals were lissencephalic and hydrocephalic, and weighed significantly less than those of the normals. The cerebral cortex was thin and flattened, with the parieto-occipital region most severely affected. Heterotopic foci were found in the cerebellum as well as in the cerebral cortex. Abnormalities in the configuration of the cerebeUar folia were also seen. Comparison between the electrophysiological and neuropathological data suggests that the extent of the extreme spindle activity, and longer AD and seizure duration depended on the degree of cerebellar dysplasia, whereas the EEG focal abnormalities were related to lesions in the cerebral hemispheres. INTRODUCTION Lissencephaly (agyria, pachygyria) is one of the developmental CNS malformations in higher animals, which usually is associated with microcephaly, hydrocephaly, and a variable degree of dysgenesis or dysplasia of various brain structures. Heterotopias and abnormal cytoarchitecture patterns are also common. While the brains of many lower animals (e.g. rats, mice, frogs) are normally lissencephalic, the degree of gyrencephaly tends to increase with phylogenetic advancement. In man, this rare syndrome of lissencephaly is characterized by mental retardation, varying degrees of neurological deficits, epilepsy and E E G abnormalities. In contrast to the neurological and neuropathological aspects, however, the E E G of the syndrome is not well characterized. Recently, H a k a m a d a et al. 7 reported on the development of E E G in 3 lissencephalic infants along
with E E G records of 16 cases from the literature. All the cases reviewed had epileptic seizures and grossly abnormal E E G s , including synchronous, generalized high-voltage spikes, delta waves, sharp and slow wave complexes, hypsarrhythmia, and extreme spindles in middle or late infancy. The extreme spindles, recognized as an interesting bioelectrical feature by Winfield et al.2a, have been claimed to be related to mental retardation4, 20. Developmental CNS malformations can be reliably produced in the offspring of various species of animals by a single intraperitoneal injection of methylazoxymethanol acetate ( M A M Ac) during pregnancy12,22. In the ferret, an injection on gestation day 32 interferes with the development of cerebral convolutions, resulting in a lissencephalic brainS. In addition to the brain pathology, which has many similarities to human lissencephaly, the lissencephalic ferret also shows other features of the syndrome,
Correspondence: M. H. Lee, NYS Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, U.S.A.
0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
30 such as learning deficitsS, 6 and E E G abnormalities 13. In our preliminary studies, we found that lissencephalic ferrets have an abnormal E E G pattern, which we interpreted as an altered sleep spindle and possibly subclinical epileptiform discharges. The purpose of the present study was (1) to examine whether the prenatally induced lissencephalic ferret has an altered seizure susceptibility as measured by afterdischarge (AD) and seizure thresholds to cortical electrical stimulation (ES), and (2) to compare its E E G patterns with those of the normal in relation to the neuropathological brain changes. The usefulness of the lissencephalic ferret for the study of epilepsy and other developmental defects has been discussed elsewhere 17.
PRES'I ANT. SI( POST. SIGIV SUPRASYLVh ECTOSYLVIAI~ LATERAL
MATERIALS AND METHODS Parent animals were 6 time-dated pregnant jills obtained from Marshall Research Animals, North Rose, NY. On gestation day 32, 3 jills were given a single intraperitoneal injection of 15 mg of MAM Ac per kg body weight, and another 3 received an equivalent volume of physiological saline. The ferrets were housed in standard stainless steel rabbit cages under a 12/12 light/dark cycle. The young were weaned at 6 weeks. Food and water were given ad libitum throughout the experiment. The number of ferrets subjected to the E E G study was 21: the experimental group consisted of 10 (3 males, 7 females) and the control group of 11 (3 males, 8 females). At about one year of age all the animals were implanted under Nembutal anesthesia (40 mg/kg, i.p.) with 14 stainless steel screws (Morris self-tapping screws with a tip diameter of 0.3 mm) for chronic E E G recordings. In the normal ferrets 12 electrodes were placed subdurally in symmetrical cortices for bipolar or monopolar recordings: 4 in the anterior sigmoid gyri, 4 in the most posterior part of the sigmoid gyri, 2 in the anterior part of the lateral gyri, and 2 in the middle part of the suprasylvian gyri (Fig. 1). In the lissencephalic ferrets the 12 electrodes were placed in corresponding areas. Another electrode was placed in the frontal sinus (reference electrode for recording or for stimulation) and one in the midline over the cerebellum (ground). The screws were attached to a 14 pin Winchester connector and affixed to the skull with acrylic cement. After a few weeks re-
Fig. 1. Placement of cortical electrodes: odd numbers designate left and even numbers right hemispheres.
covery, the E E G from each animal was recorded in an open chamber. E E G records were taken in awake and natural sleep periods and during cortical ES. The current thresholds for epileptic A D of spike activity as well as for seizures development (partial motor or generalized tonic-clonic) were established for each electrode in 6 control and 6 lissencephalic ferrets. ES was delivered by a Frederick Haer Constant Current Stimulator. The electrical stimulus was a 1-s train of 1 ms biphasic square-wave pulses at a frequency of 60 Hz. The current intensity of the initial trial was 500 /~A and it was increased by 200-500/~A steps, until an AD and then seizures were observed. Stimulations were given every 3 min if there was no AD. However, if a stimulation resulted in AD, the interval was increased to 5 min for the next electrode to be stimulated. If a stimulation resulted in seizures, no additional stimulation was given during that session. The interval between sessions was about 1-2 weeks. Each animal had at least 3 full sessions. In the second and third sessions the starting current intensity was 300-500/~A below the previously established threshold. The repeated stimulation was aimed to establish reliable thresholds for A D and seizures for a given electrode.
31 The EEGs were samples for a period of 1-3 years. The total number of resting EEGs was about 200. Each recording session lasted for 20-60 rain, and each animal had at least two sessions covering various stages of sleep. One to three years after implantation the animals were sacrificed by decapitation. The brains of 6 lissencephalic and 6 normal ferrets were removed, fixed in 10% formalin, and then 1.5-2-mm-thick coronal sections were embedded in paraffin. Paraffin sections of 6/~m thickness were stained with hematoxylin-eosine, cresyl-violet and Kliiver-Barrera techniques. RESULTS Cortical E S
Although ESs were given during the same behavioral (awake) and E E G states, considerable inter- as well as intrasubject variabilities were observed for different electrodes to produce AD and seizures. No hemispheric difference, however, was found. The most stable responses at the lowest current intensity were obtained from the suprasylvian gyri, and the effects of repeated ES on the selected sites were consistent. In general, the AD threshold correlated with the easiness of seizure production and their generalization. Results of the cortical ES are summarized in Table I. The total number of ES the lissencephalic ferrets received to establish the AD/seizure thresholds was not significantly different from that of the controls. Neither was the mean current intensity for AD significantly different between the groups. The lissencephalic animals, however, had longer ADs than did the control ferrets (P < 0.05). Similarly, the mean current intensity for seizure
production failed to discern the lissencephalics from the controls. The lissencephalic ferrets, however, had a higher number of generalized seizures. The generalized seizures were of the tonic-clonic type, occasionally associated with urination and defecation; they were often preceded by a loud cry, and followed by postseizure sleep with E E G depression. The partial seizures were of the clonic type most frequently affecting one side of the body contralateral to the stimulated cortex. For all types of seizures combined, 25 were induced in the lissencephalic group and 16 seizures in the control. Although no group difference was found for the partial seizures, the total number of generalized seizures was significantly higher for the lissencephalic than for the controls (14 vs 4, g2 = 7.63, P < 0.01). The mean seizure duration, as measured by the accompanying spike discharges, was also longer for the lissencephalic group than the control (P < 0.05), suggesting that the lissencephalic animals had an increased tendency for focal ADs to spread to the diffuse projection systems. It is interesting to note that within the lissencephalic group there was a noticeable difference in the mean duration of the AD, ranging from 2.6 to 43.6 s. Although the small sample size, as shown in Table II, does not permit one to generalize, there was a tendency that the increase in AD duration was related to the degree of cerebellar dysplasia, but not to that of cerebral hemisphere damage. E E G in normal ferrets
The typical E E G of normal ferrets in awake and different sleep stages is presented in Fig. 2. The background E E G activity in the awake state consisted of two basic rhythms, slow and fast waves
TABLE I Effects of cortical electrical stimulations in normal (N) and lissencephalic (L) ferrets
N L
Mean number of stimulations
A D threshold Mean current intensity (IzA)
Mean duration (s)
Mean current intensity (IzA)
Mean duration (s)
Generalized
Partial
42 37.6
1910 1704
7.2 22.7*
3230 2617
6.7 21.3"
4 14"*
12 11
P < 0.05. ** P < 0.01. *
Seizure threshold
Number of seizures
32 F 28
A
B
C
9 -11 10-12
3-7
4
- 8
~'~-+---'-".~,
"'++'+'-','"" +
-~ .... "+ - -
5-9 6 - 10 ,~%~,,~,'~r,.~Jv~,r+,/,~\~v~r'~+'~-~'~ 7 -11 8-I
Awake
Light
Sleep
Sleep
I sac
1oopv
Fig. 2. Typical EEG of a normal ferret (F 26). A: awake - - slow wave activity of 4.5-7 Hz and amplitude 50-100/~V with superimposed low voltage fast waves. B: quiet (light sleep) - - slowing of the background activity to 3.5-5 Hz with superimposed low voltage fast activityand trains of spindles at frequency 12-14 Hz and of amplitude up to 300/~V. C: sleep -- mixed fast and slowwaves with single high-voltage(700/zV) spikes and sharp waves•
(Fig. 2A). The slow wave activity appeared in a continuous form of irregular rhythms at 4.5-7 Hz, with a dominant frequency of 5 Hz and amplitude of 50-100 /~V. This activity was quite uniform across the different regions of the brain. Superimposed on this activity were fast waves at 30-40 Hz and of 20-30 ~V amplitude. This type of E E G pattern occurred when the animal was quiet with its head up and eyes open. If the animal was undisturbed, and there was no stimulation in the recording room, the E E G progressed in a few minutes into another pattern of slower waves with 1-2 s trains of fast activity of medium or high voltage. This occurred even with the animal's eyes open (Fig. 2B). When the animal fell into a more relaxed state, such as lying on the floor with its head down and with eyes closed or open, the E E G pattern changed into a light sleep pattern of high voltage
(100-150/~V) fast activity at a frequency of 20-40 Hz, mixed with low voltage theta waves and occasionally sleep spindles of amplitude 200-300 ~V. This activity was most pronounced in the central areas, although it could be traced in all cortical areas. In a deeper stage of sleep, as characterized by a 'rolled' body position with eyes closed and deep regular respiration, the E E G was characterized by irregular and mixed waves of different frequencies and amplitudes (Fig. 2C). Most impressive of all, however, were high voltage (300-500 ~V) potentials appearing in a single form or in trains lasting for 2-7 s. These high voltage spindles appeared at a frequency of about 4-5 Hz. The spindle trains appeared several times within a 30-min recording session. Although this type of EEG pattern was found in all animals, the amount of sleep spindles varied within the normal subjects.
33
Fig. 3. EEG of lissencephalicferret (F 46) with moderately affectedcerebral hemispheres and cerebellum. Upper part during awake phase; note focalspike activitywellpronounced in the 6-10derivation.Lower part showsalternatingfocaloccurrenceof extreme spindle activitywithina 10-minrecordingduring light sleep. A: left focalspindles. B: bilateral, and C: right focalpredominance.
EEG in lissencephalic ferrets In the awake state, the background activity of lissencephalic EEG was basically similar to that of the normals, but had more slow waves at a frequency of 3-4 Hz. The main differences in the EEG between normal and lissencephalic ferrets occurred during sleep stages. Although the low or moderate voltage background activity was usually symmetrical, depending on the degree of asymmetrical morphological changes in the hemisphere and the state of the animal, the EEG pattern varied from normal to focal spiking or slowing (Fig. 3). Focal or multifocal EEG abnormalities were seen during spindle activity when the animal was in light sleep with its head up (Fig. 3). Frequently, during light sleep the spindles were replaced by spike and
slow wave complexes at 2-4 Hz frequency and of 300-500 pV amplitude (Fig. 4A). As the sleep progressed into a deeper stage (as determined by EEG and the animal's position), more or less generalized or even focal sharp waves appeared (Fig. 4B). The sharp waves and spike discharges also occurred during REM sleep (judged by the animal's position and desynchronized activity, Fig. 4C). Frequently, the spikes were biphasic and followed by slow waves, thus forming a complex, occasionally associated with body tremors. This type of EEG activity was observed in the lissencephalic ferret (F 32) with the most severely affected cerebral hemispheres and the least cerebellar malformation (see Table II). It is interesting to observe that in the animals with severely affected cerebellum and mildly or moderately lesioned cerebral hemispheres (F 24, F 40, see
34
A
F 32
B
C
5
2 3
4 7
Sleep
Sleep
Sleep
1 sec
loo.v
Fig. 4. EEG of the lissencephalic ferret (F 32) with the most severely affected cerebral hemispheres and no cerebellar changes during different sleep stages. A: a different pattern of spike, sharp waves, and spike and slow wave complexes with some asymmetries. B: diffuse slow, sharp and fast waves. C: sharp wave discharges during paradoxical sleep. Note interhemispheric asymmetry in different patterns.
Table II), the E E G p a t t e r n did not show focal abnormalities or any laterization. H o w e v e r , e x t r e m e slow spindles and generalized spike and slow wave complex activity at a frequency of about 4 H z were most
TABLE II Relation between afterdischarges (AD) and the degree of neuropathological changes in lissencephalic ferrets
+ + +, severe; + +, moderate; +, mild; - - , no lesions. Number
A D (s)
Cerebellum
Cerebrum
F32 F5 F 18 F46 F40 F24
2.6 8.1 18.0 39.4 24.5 43.6
-+ ++ ++ +++ +++
+++ ++ ++ ++ ++ +
p r o n o u n c e d in this type of brain change. These highvoltage e x t r e m e spindles could be d i s t u r b e d for a few seconds by a hand clap. G e n e r a l l y , in comparison to n o r m a l ferrets, the E E G of lissencephalic ferrets showed (1) m o r e monorhythmic slow wave activity within the low theta b a n d range; (2) extreme spindle activity of higher amplitude and slower frequency; (3) focal a b n o r m a lities, e.g. asymmetries in spindles, sharp waves o r in slow wave activity; and (4) spikes, or spike and slow wave complexes during sleep. The resting background and spindle activities a l t e r n a t e d very frequently in a cycle ranging from seconds to a few minutes. In addition, the e x t r e m e spindles were most p r o n o u n c e d in animals with a high degree of cerebellar dysplasia while focal abnormalities or a s y m m e tries were associated with cerebral dysplasia.
35
Neuropathologicalchanges The brain weight of the lissencephalic ferrets varied from 3.5 to 5.0 g with a median of 4.0 g, while that of the normal ferrets from 5.6 to 7.3 g with a median 5.7 g. The difference was statistically significant (P < 0.01). The difference in body weight, however, was not significant (874 g for the lissencephalic vs 812 g for the control). Consequently, the brain to body weight ratio was lower in lissencephalic ferrets (P < 0.05). The weight of lissencephalic brains was about 70% of the normal. All the M A M Ac-treated ferrets were lissencephalic and hydrocephalic. Although there were dif-
ferences in the degree of lissencephaly in animals of the treated group, the thinning of cerebral cortex and involvement of the parieto-occipital region of hemispheres were the constant features. While in all other experimental animals the lissencephaly/hydrocephaly was symmetrical, one (F 32) had a marked asymmetry, with the cortex on the left side thinner than that on the right and with a larger cavity of a dilated ventricle on the left side. Grossly, in the treated animals the surface of the posterior part of the hemispheres was flattened at the suprasylvian, pseudosylvian and cruciate sulci, and the crus posterior of the lateral sulcus was absent. The anterior parts of the
O
"
Fig. 5. A: F 32. Coronal section of the lissencephalic-hydrocephalic brain, with dilated ventricles and thinned cortex. Arrow, only meninges are left as a wall of ventricle; double arrow, thin strip of cortex with a few rows of cells covered by meninges. Cresyl-violet, x 5. B: F 25. Coronal section of the lissencephalic ferret with micropolygyric, chaotic festoons and columns of Purkinje cells, cells at molecular and granular layer and white matter. HE, x 7. C: F 24. Lower part of cerebellum with numerous small, round ecotpias. HE, x 5. D: F 40. Onion-like arrangement of convolutions of cerebellar vermis. Cresyl-violet, x 5. E: F 40. Numerous small round ectopic foci of neurons of molecular layer in granular layer of cerebellum. HE, x 5. (Parasagittal sections of the cerebellum of the lissencephalic ferret may be seen in refs. 5 and 6 which also show gross specimens at other stages of development.)
36 lateral and presylvian sulci were preserved but shallow, and the presylvian, posterior and anterior sigmoid gyri were flattened. Histological examination showed that the cortex was most markedly thinned, almost to the point of translucency, in the areas corresponding to the posterior part of the ectosylvian gyrus and to a lesser degree in the anterior part of the ectosylvian and suprasylvian gyri, while the cortex of the lateral cingulate gyri, although flattened and thinner than in control brains, showed a normal cytoarchitecture (Fig. 5A). The white matter underlying the thinned cortex was reduced to a thin ribbon of myelin with extensive gliosis seen within and with a lack of ependymal cells on ventricular surface. The basal ganglia, thalamus, internal capsule and brainstem, although smaller than in control animals, appeared normal in microscopic examinations. The size of cerebellum in the lissencephalic ferrets was essentially normal, but there was a widening of the fourth ventricle and the cytoarchitecture was abnormal with changes ranging from a few heterotopic loci in folia cerebelli and a few irregularities in the arrangement of convolutions to almost complete disorganization of the convolutional pattern with numerous heterotopias and polymicrogyria in the vermis and/or in the cerebellar hemispheres (Fig. 5B-E). A normal cerebellum was found in only one lissencephalic ferret which, however, had the most severe changes in the cerebral hemispheres (F 32; see Table II). DISCUSSION Lissencephaly is a rare malformation and there are only about 40 human cases described in the literature. Dicker et al. 3 gave a review of 20 cases found in the literature and all but a few were severely retarded. The neuropathological features in those cases were similar, which included a smooth, agyric cortex with the parietal and occipital lobes most severely affected. There also was hydrocephaly which is particularly pronounced in the posterior horns of the lateral ventricle, and numerous islands of heterotopic neural cells which were most commonly found in the cerebral hemisphere, but seen in the cerebellum, as well n. In our lissencephalic ferrets all of these features were also seen: injection on gestation day 32 produced lesions in both the cerebrum and
cerebellum, thus giving a close resemblance to human lissencephaly. However, the severity of the lesions, especially the number and location of the heterotopic foci varied among the animals. The difference in the severity of cerebral and cerebellar lesions may possibly be attributed to the variation in developmental age of the individual fetuses at the time of treatment with MAM Ac. In addition, it has been reported that MAM Ac requires bioactivation to exert antimitotic effects20. This could well contribute to the variability of the effects, since the individual animals of a non-inbred stock are likely to show genetic variation in the enzymes required. Despite such severe structural alterations, the adult lissencephalic ferrets did not show a substantial change in the E E G background activity in the awake state; there was only an increase of slow theta waves. Although these findings appear to contradict those of the grossly abnormal E E G found in lissencephalic childrenT, the discrepancy may be attributed to the age and maturational compensation of the brain bioelectrical activity, since certain abnormal E E G patterns, such as hypsarrhythmia, are age-related. The lack of gross E E G abnormalities in the adult lissencephalic ferrets may also be compared to relatively minor E E G changes in the background activity of severely mentally retarded adult patients with epilepsy 16. The major electrophysiological differences between the normal and lissencephalic animals emerged (1) during sleep and (2) in the AD and seizure susceptibility as measured by the cortical ES. The E E G of the lissencephalic ferrets during sleep showed two types of bioelectrical abnormalities. The first one was focal abnormalities, including slow and sharp waves, spikes and focal extreme spindles which could be related to the asymmetric damage in the cerebral hemisphere. The second type of change was characterized by generalized fast and slow extreme spindle activity which was widely distributed. The origin of mechanism of extreme spindle activity is more complex to understand. Gibbs and Gibbs 4 found extreme spindles in a certain type of mental retardation associated with athetosis, and suggested that they may be related to lesions in the basal ganglia. Under light microscopic examinations, we did not find evidence of basal ganglia involvement in these animals. Neither did we find lesions in the thai-
37 amus or brainstem reticular formation. In the ferret fetuses, however, acute lesions have been found in these brain structures following an intrauterine exposure to M A M A c on gestation day 325. This suggests that subtle residual changes in these structures could contribute to extreme spindle activity. Alternatively, spindles could appear without participation of gross neuropathological changes, but as a result of metabolic disturbances or defects. In M A M Ac-induced lissencephalic ferrets a number of neurochemical changes has been found, including an overall increase and different distribution in the concentration of markers in various cortical areas for catecholaminergic and cholinergic terminals 9. A significant rise in monoamine concentration has also been found in M A M Ac-treated ratsS, w. The relation of the neurochemical changes to the altered sleep-wakefulness cycle and to the increasing amount of spindle activity, which was also found in M A M A c treated rats, requires further clarification. Our data also suggest that the lissencephalic ferret has an increased level of seizure susceptibility. In the lissencephalic ferret the excess of fast and slow spin-
REFERENCES 1 Cooper, I. S., Chronic stimulation of plaeo-cerebellar cortex in man, Lancet, 1 (1973) 206. 2 Davis, R., Engle, H., Kuszma, Y., Gray, E., Ryan, T. and Dusnak, A., Update of chronic cerebellar stimulation for spasticity, Appl. Neurophysiol., 45 (1982) 44-50. 3 Dieker, H., Edwards, R. H., ZuRhein, G., Chou, S. M., Hartman, H. A. and Opitz, J. M., The Lissencephaly syndrome. In D. Bergsma (Ed.), The First Conference on the Clinical Delineation of Birth Defects, Part H Malformation Syndromes, May 20-25, 1968. The Johns Hopkins Medical Institutions and The National Foundation - - March of Dimes, Birth Defects: Original Article Series, 5 (1959) 53-64. 4 Gibbs, E. L. and Gibbs, R. A., Extreme spindles: correlation of electroencephalographic sleep pattern with mental retardation, Science, 138 (1962) 1106-1107. 5 Haddad, R. and Rabe, A., Use of the ferret in experimental neuroteratology: cerebral, cerebellar, and retinal dysplasias induced by methylazoxymethanol acetate. In T. V. N. Persaud (Ed.), Neural and Behavioral Teratology: Advances in the Study of Birth Defects, Vol. 4, University Park Press, Baltimore, 1980, pp. 45-62. 6 Haddad, R., Rabe, A. and Dumas, R., Neuroteratogenicity of methylazoxymethanol acetate: behavioral deficits of ferrets with transplacentally induced iissencephaly, Neurotoxicology, 1 (1979) 171-189. 7 Hakameda, S., Watanahe, K., Hara, K. and Miyaszaki, S., The evolution of electroencephalographic features in lis-
dies, spike potentials, and spike and slow wave complex activity was associated with lower A D and seizure thresholds. This, in turn, appeared to be related to the severity of cerebellar dysplasia. The data seem to indicate that lesions in the cerebral hemisphere of the lissencephalic ferret are responsible for focal sharp waves and possibly epileptic focal activity. On the other hand, the cerebellar dysplasia appears to be associated with a longer duration of the A D and seizures. Moreover, the fact that the lissencephalic ferret had a significantly higher n u m b e r of generalized tonic-clonic seizures while no group difference was shown for partial seizures, suggests a decrease in inhibitory processes in the lissencephalic ferret. This observation seems to support the controversial concept of the inhibitory effect of the cerebellar cortex on epileptic discharges in the cerebral hemispheres l, 14,15,1s. The lack of inhibitory cerebellar processes in the lissencephalic ferret might also be responsible for the extreme spindle synchronous activity. However, the relationship between the slow spindle potentials and the epileptiform E E G pattern requires further study.
sencephaly syndrome, Brain Develop., 1 (1979) 277-283. 8 Johnston, M. V. and Coyle, J. T., Histological and neurochemical effect of fetal treatment with methylazoxymethanol on rat neocortex in adulthood, Brain Research, 170 (1979) 135-155. 9 Johnston, M. V., Haddad, R., Carman-Young, A. and Coyle, J. T., Neurotransmitter chemistry of lissencephalic cortex induced in ferrets by fetal treatment with methylazoxymethanol acetate, Develop. Brain Res., 4 (1982) 285-291. 10 Kyono, S., Seo, M. and Shibagaki, M., Sleep-waking cycle in microencephalic rats induced by prenatal methylazoxymethanol application, Electroenceph. clin. Neurophysiol., 48 (1980) 73-79. 11 Larroche, J.-C., Lissencephaly-pachygyria (agyria). In P. J. Vinken and G.W. Bruyn (Eds.), Neurogenetic Directory, Vol. 42, Elsevier/North-Holland Biomedical Press, Amsterdam, 1981, pp. 39--41. 12 Laqueur, G. L., Oncogenicity of cycades and its implications. In H. F. Kraybill and M. A. Mehlman (Eds.), Advances in Modern Toxicology: Environmental Cancer, Vol. 3, Wiley, New York, 1977, pp. 231-261. 13 Lee, M. H., Majkowski, J. and Haddad, R., EEG abnormalities in ferrets with transplacentally induced lissencephaly, Teratology, 24 (1981) 13A. 14 Levy, L. F. and Auchtedonie, W. C., Chronic cerebellar stimulation in the treatment of epilepsy, Epilepsia, 20 (1979) 235-245. 15 Lockard, J. S., Ojeman, G. A., Congdon, W. C. and Ducharme, L. L., Cerebellar stimulation in alumina-gel mon-
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key model: inverse relationship between clinical seizures and EEG inter-ictal burst, Epilepsia, 20 (1979) 223-234. Majkowski, J., Electrophysiological studies in mental retardation. In J. Reyner and B. Lindstrom (Eds.), Mental Retardation Symposium, May 13-14, 1982, Ciba-Geigy, Gothenburg, 1982, pp. 145-161. Majkowski, J., Drug effects on afterdischarges and seizure thresholds in lissencephalic ferrets: an epilepsy model for drug evaluation, Epilepsia, 24 (1983) 678--685. Majkowski, J., Karlinski, A. and Klimowicz-Mlodzik, I., Effect of cerebellar stimulation on hippocampal epileptic discharges in kindling preparation. In J. Majkowski (Ed.), Epilepsy. A Clinical and Experimental Research, Monogr. neural Sci. Vol. 5, Karger, Basel, 1980, pp. 40-45. Matsutani, T., Nagayoshi, M., Tamaru, M. and Tsukada, Y., Elevated monoamine levels in the cerebral hemi-
20 21
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spheres of microcephalic rats treated prenatally with mcthylazoxymethanol or cytosine arabinoside, J. Neurochem., 34 (1980) 950-956. Oka, E., An electroencephalographic study of mentally retarded children, Psychiatr. Neurol. (Jap.), 72 (1971)) 555-578. Poynter, R. W., Ball, C. R., Goodban, J. and Thackrah, T., The influence of physostigmine on the activation of methylazoxymethanol acetate, Chem. Biol. Interactions, 4 (1971) 139. Spatz, M. and Laqueur, G. L., Transplacental chemical induction of microencephaly in two strains of rats I. (33404). Proc. Soc. exp. Biol. Med., 129 (1967) 705-710. Winfield, D. L., Hughes, J. G. and Sayle, W. E., Electroencephalography sleep findings in cerebral palsy, Pediatrics, 16 (1955) 88-92.
29
BRE 10218
EEG and Seizure Threshold in Normal and Lissencephalic Ferrets JERZY MAJKOWSKI, MOON HE LEE, PIOTR B. KOZLOWSKI and RAEF HADDAD Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, N Y 10314 (U.S.A.)
(Accepted January 3rd, 1984) Key words: EEG - - lissencephaly - - ferret - - extreme spindle - - epileptic seizure
Changes in EEG and susceptibility to electrically induced seizures were examined in the ferret with lissencephaly produced by exposure to a single injection of methylazoxymethanol acetate (MAM Ac) given to the pregnant jill on gestation day 32. Ten lissencephalic and 11 normal ferrets were chronically implanted with 14 cortical stainless steel electrodes. EEG records were sampled from various stages of the sleep/awake cycle. Six of each group were subjected to electrical stimulation for seizure threshold. Although the number of stimulations and the current intensity required to produce epileptiform afterdischarges (AD) and seizures were not different between the two groups, the lissencephalic ferrets had significantly longer AD and seizures, and a greater number of generalized seizures, indicating an enhanced seizure susceptibility. The EEG of the lissencephalic ferrets was characterized by increased slow wave activity within the low theta band range, extreme spindle activity, focal or multifocal slow and sharp waves, spikes, or spike and slow wave complexes. The differences in the EEG were more pronounced during drowsiness and sleep stages. The brains of all of the treated animals were lissencephalic and hydrocephalic, and weighed significantly less than those of the normals. The cerebral cortex was thin and flattened, with the parieto-occipital region most severely affected. Heterotopic foci were found in the cerebellum as well as in the cerebral cortex. Abnormalities in the configuration of the cerebeUar folia were also seen. Comparison between the electrophysiological and neuropathological data suggests that the extent of the extreme spindle activity, and longer AD and seizure duration depended on the degree of cerebellar dysplasia, whereas the EEG focal abnormalities were related to lesions in the cerebral hemispheres. INTRODUCTION Lissencephaly (agyria, pachygyria) is one of the developmental CNS malformations in higher animals, which usually is associated with microcephaly, hydrocephaly, and a variable degree of dysgenesis or dysplasia of various brain structures. Heterotopias and abnormal cytoarchitecture patterns are also common. While the brains of many lower animals (e.g. rats, mice, frogs) are normally lissencephalic, the degree of gyrencephaly tends to increase with phylogenetic advancement. In man, this rare syndrome of lissencephaly is characterized by mental retardation, varying degrees of neurological deficits, epilepsy and E E G abnormalities. In contrast to the neurological and neuropathological aspects, however, the E E G of the syndrome is not well characterized. Recently, H a k a m a d a et al. 7 reported on the development of E E G in 3 lissencephalic infants along
with E E G records of 16 cases from the literature. All the cases reviewed had epileptic seizures and grossly abnormal E E G s , including synchronous, generalized high-voltage spikes, delta waves, sharp and slow wave complexes, hypsarrhythmia, and extreme spindles in middle or late infancy. The extreme spindles, recognized as an interesting bioelectrical feature by Winfield et al.2a, have been claimed to be related to mental retardation4, 20. Developmental CNS malformations can be reliably produced in the offspring of various species of animals by a single intraperitoneal injection of methylazoxymethanol acetate ( M A M Ac) during pregnancy12,22. In the ferret, an injection on gestation day 32 interferes with the development of cerebral convolutions, resulting in a lissencephalic brainS. In addition to the brain pathology, which has many similarities to human lissencephaly, the lissencephalic ferret also shows other features of the syndrome,
Correspondence: M. H. Lee, NYS Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, U.S.A.
0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
30 such as learning deficitsS, 6 and E E G abnormalities 13. In our preliminary studies, we found that lissencephalic ferrets have an abnormal E E G pattern, which we interpreted as an altered sleep spindle and possibly subclinical epileptiform discharges. The purpose of the present study was (1) to examine whether the prenatally induced lissencephalic ferret has an altered seizure susceptibility as measured by afterdischarge (AD) and seizure thresholds to cortical electrical stimulation (ES), and (2) to compare its E E G patterns with those of the normal in relation to the neuropathological brain changes. The usefulness of the lissencephalic ferret for the study of epilepsy and other developmental defects has been discussed elsewhere 17.
PRES'I ANT. SI( POST. SIGIV SUPRASYLVh ECTOSYLVIAI~ LATERAL
MATERIALS AND METHODS Parent animals were 6 time-dated pregnant jills obtained from Marshall Research Animals, North Rose, NY. On gestation day 32, 3 jills were given a single intraperitoneal injection of 15 mg of MAM Ac per kg body weight, and another 3 received an equivalent volume of physiological saline. The ferrets were housed in standard stainless steel rabbit cages under a 12/12 light/dark cycle. The young were weaned at 6 weeks. Food and water were given ad libitum throughout the experiment. The number of ferrets subjected to the E E G study was 21: the experimental group consisted of 10 (3 males, 7 females) and the control group of 11 (3 males, 8 females). At about one year of age all the animals were implanted under Nembutal anesthesia (40 mg/kg, i.p.) with 14 stainless steel screws (Morris self-tapping screws with a tip diameter of 0.3 mm) for chronic E E G recordings. In the normal ferrets 12 electrodes were placed subdurally in symmetrical cortices for bipolar or monopolar recordings: 4 in the anterior sigmoid gyri, 4 in the most posterior part of the sigmoid gyri, 2 in the anterior part of the lateral gyri, and 2 in the middle part of the suprasylvian gyri (Fig. 1). In the lissencephalic ferrets the 12 electrodes were placed in corresponding areas. Another electrode was placed in the frontal sinus (reference electrode for recording or for stimulation) and one in the midline over the cerebellum (ground). The screws were attached to a 14 pin Winchester connector and affixed to the skull with acrylic cement. After a few weeks re-
Fig. 1. Placement of cortical electrodes: odd numbers designate left and even numbers right hemispheres.
covery, the E E G from each animal was recorded in an open chamber. E E G records were taken in awake and natural sleep periods and during cortical ES. The current thresholds for epileptic A D of spike activity as well as for seizures development (partial motor or generalized tonic-clonic) were established for each electrode in 6 control and 6 lissencephalic ferrets. ES was delivered by a Frederick Haer Constant Current Stimulator. The electrical stimulus was a 1-s train of 1 ms biphasic square-wave pulses at a frequency of 60 Hz. The current intensity of the initial trial was 500 /~A and it was increased by 200-500/~A steps, until an AD and then seizures were observed. Stimulations were given every 3 min if there was no AD. However, if a stimulation resulted in AD, the interval was increased to 5 min for the next electrode to be stimulated. If a stimulation resulted in seizures, no additional stimulation was given during that session. The interval between sessions was about 1-2 weeks. Each animal had at least 3 full sessions. In the second and third sessions the starting current intensity was 300-500/~A below the previously established threshold. The repeated stimulation was aimed to establish reliable thresholds for A D and seizures for a given electrode.
31 The EEGs were samples for a period of 1-3 years. The total number of resting EEGs was about 200. Each recording session lasted for 20-60 rain, and each animal had at least two sessions covering various stages of sleep. One to three years after implantation the animals were sacrificed by decapitation. The brains of 6 lissencephalic and 6 normal ferrets were removed, fixed in 10% formalin, and then 1.5-2-mm-thick coronal sections were embedded in paraffin. Paraffin sections of 6/~m thickness were stained with hematoxylin-eosine, cresyl-violet and Kliiver-Barrera techniques. RESULTS Cortical E S
Although ESs were given during the same behavioral (awake) and E E G states, considerable inter- as well as intrasubject variabilities were observed for different electrodes to produce AD and seizures. No hemispheric difference, however, was found. The most stable responses at the lowest current intensity were obtained from the suprasylvian gyri, and the effects of repeated ES on the selected sites were consistent. In general, the AD threshold correlated with the easiness of seizure production and their generalization. Results of the cortical ES are summarized in Table I. The total number of ES the lissencephalic ferrets received to establish the AD/seizure thresholds was not significantly different from that of the controls. Neither was the mean current intensity for AD significantly different between the groups. The lissencephalic animals, however, had longer ADs than did the control ferrets (P < 0.05). Similarly, the mean current intensity for seizure
production failed to discern the lissencephalics from the controls. The lissencephalic ferrets, however, had a higher number of generalized seizures. The generalized seizures were of the tonic-clonic type, occasionally associated with urination and defecation; they were often preceded by a loud cry, and followed by postseizure sleep with E E G depression. The partial seizures were of the clonic type most frequently affecting one side of the body contralateral to the stimulated cortex. For all types of seizures combined, 25 were induced in the lissencephalic group and 16 seizures in the control. Although no group difference was found for the partial seizures, the total number of generalized seizures was significantly higher for the lissencephalic than for the controls (14 vs 4, g2 = 7.63, P < 0.01). The mean seizure duration, as measured by the accompanying spike discharges, was also longer for the lissencephalic group than the control (P < 0.05), suggesting that the lissencephalic animals had an increased tendency for focal ADs to spread to the diffuse projection systems. It is interesting to note that within the lissencephalic group there was a noticeable difference in the mean duration of the AD, ranging from 2.6 to 43.6 s. Although the small sample size, as shown in Table II, does not permit one to generalize, there was a tendency that the increase in AD duration was related to the degree of cerebellar dysplasia, but not to that of cerebral hemisphere damage. E E G in normal ferrets
The typical E E G of normal ferrets in awake and different sleep stages is presented in Fig. 2. The background E E G activity in the awake state consisted of two basic rhythms, slow and fast waves
TABLE I Effects of cortical electrical stimulations in normal (N) and lissencephalic (L) ferrets
N L
Mean number of stimulations
A D threshold Mean current intensity (IzA)
Mean duration (s)
Mean current intensity (IzA)
Mean duration (s)
Generalized
Partial
42 37.6
1910 1704
7.2 22.7*
3230 2617
6.7 21.3"
4 14"*
12 11
P < 0.05. ** P < 0.01. *
Seizure threshold
Number of seizures
32 F 28
A
B
C
9 -11 10-12
3-7
4
- 8
~'~-+---'-".~,
"'++'+'-','"" +
-~ .... "+ - -
5-9 6 - 10 ,~%~,,~,'~r,.~Jv~,r+,/,~\~v~r'~+'~-~'~ 7 -11 8-I
Awake
Light
Sleep
Sleep
I sac
1oopv
Fig. 2. Typical EEG of a normal ferret (F 26). A: awake - - slow wave activity of 4.5-7 Hz and amplitude 50-100/~V with superimposed low voltage fast waves. B: quiet (light sleep) - - slowing of the background activity to 3.5-5 Hz with superimposed low voltage fast activityand trains of spindles at frequency 12-14 Hz and of amplitude up to 300/~V. C: sleep -- mixed fast and slowwaves with single high-voltage(700/zV) spikes and sharp waves•
(Fig. 2A). The slow wave activity appeared in a continuous form of irregular rhythms at 4.5-7 Hz, with a dominant frequency of 5 Hz and amplitude of 50-100 /~V. This activity was quite uniform across the different regions of the brain. Superimposed on this activity were fast waves at 30-40 Hz and of 20-30 ~V amplitude. This type of E E G pattern occurred when the animal was quiet with its head up and eyes open. If the animal was undisturbed, and there was no stimulation in the recording room, the E E G progressed in a few minutes into another pattern of slower waves with 1-2 s trains of fast activity of medium or high voltage. This occurred even with the animal's eyes open (Fig. 2B). When the animal fell into a more relaxed state, such as lying on the floor with its head down and with eyes closed or open, the E E G pattern changed into a light sleep pattern of high voltage
(100-150/~V) fast activity at a frequency of 20-40 Hz, mixed with low voltage theta waves and occasionally sleep spindles of amplitude 200-300 ~V. This activity was most pronounced in the central areas, although it could be traced in all cortical areas. In a deeper stage of sleep, as characterized by a 'rolled' body position with eyes closed and deep regular respiration, the E E G was characterized by irregular and mixed waves of different frequencies and amplitudes (Fig. 2C). Most impressive of all, however, were high voltage (300-500 ~V) potentials appearing in a single form or in trains lasting for 2-7 s. These high voltage spindles appeared at a frequency of about 4-5 Hz. The spindle trains appeared several times within a 30-min recording session. Although this type of EEG pattern was found in all animals, the amount of sleep spindles varied within the normal subjects.
33
Fig. 3. EEG of lissencephalicferret (F 46) with moderately affectedcerebral hemispheres and cerebellum. Upper part during awake phase; note focalspike activitywellpronounced in the 6-10derivation.Lower part showsalternatingfocaloccurrenceof extreme spindle activitywithina 10-minrecordingduring light sleep. A: left focalspindles. B: bilateral, and C: right focalpredominance.
EEG in lissencephalic ferrets In the awake state, the background activity of lissencephalic EEG was basically similar to that of the normals, but had more slow waves at a frequency of 3-4 Hz. The main differences in the EEG between normal and lissencephalic ferrets occurred during sleep stages. Although the low or moderate voltage background activity was usually symmetrical, depending on the degree of asymmetrical morphological changes in the hemisphere and the state of the animal, the EEG pattern varied from normal to focal spiking or slowing (Fig. 3). Focal or multifocal EEG abnormalities were seen during spindle activity when the animal was in light sleep with its head up (Fig. 3). Frequently, during light sleep the spindles were replaced by spike and
slow wave complexes at 2-4 Hz frequency and of 300-500 pV amplitude (Fig. 4A). As the sleep progressed into a deeper stage (as determined by EEG and the animal's position), more or less generalized or even focal sharp waves appeared (Fig. 4B). The sharp waves and spike discharges also occurred during REM sleep (judged by the animal's position and desynchronized activity, Fig. 4C). Frequently, the spikes were biphasic and followed by slow waves, thus forming a complex, occasionally associated with body tremors. This type of EEG activity was observed in the lissencephalic ferret (F 32) with the most severely affected cerebral hemispheres and the least cerebellar malformation (see Table II). It is interesting to observe that in the animals with severely affected cerebellum and mildly or moderately lesioned cerebral hemispheres (F 24, F 40, see
34
A
F 32
B
C
5
2 3
4 7
Sleep
Sleep
Sleep
1 sec
loo.v
Fig. 4. EEG of the lissencephalic ferret (F 32) with the most severely affected cerebral hemispheres and no cerebellar changes during different sleep stages. A: a different pattern of spike, sharp waves, and spike and slow wave complexes with some asymmetries. B: diffuse slow, sharp and fast waves. C: sharp wave discharges during paradoxical sleep. Note interhemispheric asymmetry in different patterns.
Table II), the E E G p a t t e r n did not show focal abnormalities or any laterization. H o w e v e r , e x t r e m e slow spindles and generalized spike and slow wave complex activity at a frequency of about 4 H z were most
TABLE II Relation between afterdischarges (AD) and the degree of neuropathological changes in lissencephalic ferrets
+ + +, severe; + +, moderate; +, mild; - - , no lesions. Number
A D (s)
Cerebellum
Cerebrum
F32 F5 F 18 F46 F40 F24
2.6 8.1 18.0 39.4 24.5 43.6
-+ ++ ++ +++ +++
+++ ++ ++ ++ ++ +
p r o n o u n c e d in this type of brain change. These highvoltage e x t r e m e spindles could be d i s t u r b e d for a few seconds by a hand clap. G e n e r a l l y , in comparison to n o r m a l ferrets, the E E G of lissencephalic ferrets showed (1) m o r e monorhythmic slow wave activity within the low theta b a n d range; (2) extreme spindle activity of higher amplitude and slower frequency; (3) focal a b n o r m a lities, e.g. asymmetries in spindles, sharp waves o r in slow wave activity; and (4) spikes, or spike and slow wave complexes during sleep. The resting background and spindle activities a l t e r n a t e d very frequently in a cycle ranging from seconds to a few minutes. In addition, the e x t r e m e spindles were most p r o n o u n c e d in animals with a high degree of cerebellar dysplasia while focal abnormalities or a s y m m e tries were associated with cerebral dysplasia.
35
Neuropathologicalchanges The brain weight of the lissencephalic ferrets varied from 3.5 to 5.0 g with a median of 4.0 g, while that of the normal ferrets from 5.6 to 7.3 g with a median 5.7 g. The difference was statistically significant (P < 0.01). The difference in body weight, however, was not significant (874 g for the lissencephalic vs 812 g for the control). Consequently, the brain to body weight ratio was lower in lissencephalic ferrets (P < 0.05). The weight of lissencephalic brains was about 70% of the normal. All the M A M Ac-treated ferrets were lissencephalic and hydrocephalic. Although there were dif-
ferences in the degree of lissencephaly in animals of the treated group, the thinning of cerebral cortex and involvement of the parieto-occipital region of hemispheres were the constant features. While in all other experimental animals the lissencephaly/hydrocephaly was symmetrical, one (F 32) had a marked asymmetry, with the cortex on the left side thinner than that on the right and with a larger cavity of a dilated ventricle on the left side. Grossly, in the treated animals the surface of the posterior part of the hemispheres was flattened at the suprasylvian, pseudosylvian and cruciate sulci, and the crus posterior of the lateral sulcus was absent. The anterior parts of the
O
"
Fig. 5. A: F 32. Coronal section of the lissencephalic-hydrocephalic brain, with dilated ventricles and thinned cortex. Arrow, only meninges are left as a wall of ventricle; double arrow, thin strip of cortex with a few rows of cells covered by meninges. Cresyl-violet, x 5. B: F 25. Coronal section of the lissencephalic ferret with micropolygyric, chaotic festoons and columns of Purkinje cells, cells at molecular and granular layer and white matter. HE, x 7. C: F 24. Lower part of cerebellum with numerous small, round ecotpias. HE, x 5. D: F 40. Onion-like arrangement of convolutions of cerebellar vermis. Cresyl-violet, x 5. E: F 40. Numerous small round ectopic foci of neurons of molecular layer in granular layer of cerebellum. HE, x 5. (Parasagittal sections of the cerebellum of the lissencephalic ferret may be seen in refs. 5 and 6 which also show gross specimens at other stages of development.)
36 lateral and presylvian sulci were preserved but shallow, and the presylvian, posterior and anterior sigmoid gyri were flattened. Histological examination showed that the cortex was most markedly thinned, almost to the point of translucency, in the areas corresponding to the posterior part of the ectosylvian gyrus and to a lesser degree in the anterior part of the ectosylvian and suprasylvian gyri, while the cortex of the lateral cingulate gyri, although flattened and thinner than in control brains, showed a normal cytoarchitecture (Fig. 5A). The white matter underlying the thinned cortex was reduced to a thin ribbon of myelin with extensive gliosis seen within and with a lack of ependymal cells on ventricular surface. The basal ganglia, thalamus, internal capsule and brainstem, although smaller than in control animals, appeared normal in microscopic examinations. The size of cerebellum in the lissencephalic ferrets was essentially normal, but there was a widening of the fourth ventricle and the cytoarchitecture was abnormal with changes ranging from a few heterotopic loci in folia cerebelli and a few irregularities in the arrangement of convolutions to almost complete disorganization of the convolutional pattern with numerous heterotopias and polymicrogyria in the vermis and/or in the cerebellar hemispheres (Fig. 5B-E). A normal cerebellum was found in only one lissencephalic ferret which, however, had the most severe changes in the cerebral hemispheres (F 32; see Table II). DISCUSSION Lissencephaly is a rare malformation and there are only about 40 human cases described in the literature. Dicker et al. 3 gave a review of 20 cases found in the literature and all but a few were severely retarded. The neuropathological features in those cases were similar, which included a smooth, agyric cortex with the parietal and occipital lobes most severely affected. There also was hydrocephaly which is particularly pronounced in the posterior horns of the lateral ventricle, and numerous islands of heterotopic neural cells which were most commonly found in the cerebral hemisphere, but seen in the cerebellum, as well n. In our lissencephalic ferrets all of these features were also seen: injection on gestation day 32 produced lesions in both the cerebrum and
cerebellum, thus giving a close resemblance to human lissencephaly. However, the severity of the lesions, especially the number and location of the heterotopic foci varied among the animals. The difference in the severity of cerebral and cerebellar lesions may possibly be attributed to the variation in developmental age of the individual fetuses at the time of treatment with MAM Ac. In addition, it has been reported that MAM Ac requires bioactivation to exert antimitotic effects20. This could well contribute to the variability of the effects, since the individual animals of a non-inbred stock are likely to show genetic variation in the enzymes required. Despite such severe structural alterations, the adult lissencephalic ferrets did not show a substantial change in the E E G background activity in the awake state; there was only an increase of slow theta waves. Although these findings appear to contradict those of the grossly abnormal E E G found in lissencephalic childrenT, the discrepancy may be attributed to the age and maturational compensation of the brain bioelectrical activity, since certain abnormal E E G patterns, such as hypsarrhythmia, are age-related. The lack of gross E E G abnormalities in the adult lissencephalic ferrets may also be compared to relatively minor E E G changes in the background activity of severely mentally retarded adult patients with epilepsy 16. The major electrophysiological differences between the normal and lissencephalic animals emerged (1) during sleep and (2) in the AD and seizure susceptibility as measured by the cortical ES. The E E G of the lissencephalic ferrets during sleep showed two types of bioelectrical abnormalities. The first one was focal abnormalities, including slow and sharp waves, spikes and focal extreme spindles which could be related to the asymmetric damage in the cerebral hemisphere. The second type of change was characterized by generalized fast and slow extreme spindle activity which was widely distributed. The origin of mechanism of extreme spindle activity is more complex to understand. Gibbs and Gibbs 4 found extreme spindles in a certain type of mental retardation associated with athetosis, and suggested that they may be related to lesions in the basal ganglia. Under light microscopic examinations, we did not find evidence of basal ganglia involvement in these animals. Neither did we find lesions in the thai-
37 amus or brainstem reticular formation. In the ferret fetuses, however, acute lesions have been found in these brain structures following an intrauterine exposure to M A M A c on gestation day 325. This suggests that subtle residual changes in these structures could contribute to extreme spindle activity. Alternatively, spindles could appear without participation of gross neuropathological changes, but as a result of metabolic disturbances or defects. In M A M Ac-induced lissencephalic ferrets a number of neurochemical changes has been found, including an overall increase and different distribution in the concentration of markers in various cortical areas for catecholaminergic and cholinergic terminals 9. A significant rise in monoamine concentration has also been found in M A M Ac-treated ratsS, w. The relation of the neurochemical changes to the altered sleep-wakefulness cycle and to the increasing amount of spindle activity, which was also found in M A M A c treated rats, requires further clarification. Our data also suggest that the lissencephalic ferret has an increased level of seizure susceptibility. In the lissencephalic ferret the excess of fast and slow spin-
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dies, spike potentials, and spike and slow wave complex activity was associated with lower A D and seizure thresholds. This, in turn, appeared to be related to the severity of cerebellar dysplasia. The data seem to indicate that lesions in the cerebral hemisphere of the lissencephalic ferret are responsible for focal sharp waves and possibly epileptic focal activity. On the other hand, the cerebellar dysplasia appears to be associated with a longer duration of the A D and seizures. Moreover, the fact that the lissencephalic ferret had a significantly higher n u m b e r of generalized tonic-clonic seizures while no group difference was shown for partial seizures, suggests a decrease in inhibitory processes in the lissencephalic ferret. This observation seems to support the controversial concept of the inhibitory effect of the cerebellar cortex on epileptic discharges in the cerebral hemispheres l, 14,15,1s. The lack of inhibitory cerebellar processes in the lissencephalic ferret might also be responsible for the extreme spindle synchronous activity. However, the relationship between the slow spindle potentials and the epileptiform E E G pattern requires further study.
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