Epilepsia, m447-434, 1979.
Raven Press. New York
Effects of Electrical Stimulation and Removal of Cerebellar Structures in an Experimental Model of Generalized Epilepsy G. Testa, A. Pellegrini, and D. Giaretta Istituto di Clinica delle Malattie Nervose e Mentali dell’ Universita di Padova, Padova, Italy
Summary: Electrical stimulation of the cerebellum has recently been proposed as a valid treatment of human epilepsy. The inhibitory role of cerebellar structures in experimental epileptic seizures has been stressed by many authors in recent years. However, since most of these studies were carried out on models of focal epilepsy, we investigated the possible inhibitory effect of cerebellar structures in an experimental model of generalized corticoreticular epilepsy, reproduced in the cat by large parenteral doses of penicillin. Twelve “chronic” animals were used to study the effects of electrical paleo- or neocerebellar stimulation and of cerebellar removal on bilaterally synchronous spike- wave discharges.‘The results of our investigation can be summarized as follows: (1) removal of cerebellar structures did not modify the frequency of the epileptiform discharges; (2) similar results were found after stimulation of the cerebellar hemisphere, as well as after low frequency stimulation of the culmen; (3) a mild and inconstant inhibitory effect was observed during paleocerebellar stimulation of brief duration at high frequency; (4) prolonged high frequency stimulation of the paleocerebellum with gradual voltage increases were more effective. Many doubts still remain concerning a clear, direct inhibitory effect of cerebellar structures on epileptic discharges in this feline model of generalized epilepsy.
The ability of the cerebellum to inhibit experimentally induced seizures has been stressed by many authors in recent years. Most of these experiments, however, have been carried out on models of focal epilepsy (Steriade, 1960; Dow et al., 1962; Mutani et al., 1969; Hutton et al., 1972; Babb et al., 1974; Dow, 1974; Hablitz et al., 1975). Other studies, like those of Myers et al. (1975), Hablitz (1976), and Hablitz and Rea (1976), examined the effects of cere-
bellar electrical stimulation in generalized penicillin epilepsy of the cat (Prince and Farrel, 1969), a model closely resembling human myoclonic petit ma1 (Gloor and Testa, 1974; Testa and Gloor, 1974; Guberman et al., 1975). Data obtained from all these studies are equivocal. In fact, in some experiments, cerebellar stimulation did not exert any inhibitory effect (Myers et al., 1975), or even have a facilitatory effect on experimentally
Received September 12, 1978. Key words: Generalized epilepsy-Penicillin-Cerebellum.
447
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induced seizures (Steriade, 1960; Kreindler, 1962; Hablitz et al., 1975). However, Cooper et al. (1974) stressed the inhibitory role played. by various cerebellar areas in human patients and reported a significant decrease in seizures in subjects with intractable epilepsy through the use of chronic cerebellar stimulation. Since doubts remain about the action of these cerebellar structures and since the results reported by Myers et al. (1975) and by Hablitz (1976) in generalized penicillin epilepsy are difficult to assess, we attempted to evaluate, in this same model, the effects of different kinds of paleo- and neocerebellar stimulations, as well as the effects of removing the cerebellum. METHODS Chronic experiments were carried out in 12 adult cats weighing between 3 and 4 kg. Under aseptic conditions and thiopental anesthesia, six stainless steel screw electrodes were symmetrically implanted in the skull over the pericruciate, middle, and posterior suprasylvian gyri for EEG recordings. A screw serving as a reference electrode was inserted over the frontal sinus or in the external occipital protuberance. In 8 cats, through a posterior craniotomy (behind the occipital protuberance), bipolar silver ball electrodes (at an interelectrode distance of 1 mm), were placed on the culmen and lobulus simplex of the cerebellum. Two rectangular stainless steel plate electrodes of 10 mm2each, separated by 1.5 mm and attached to a thin plastic support, were placed over the cerebellar hemisphere. All recording and stimulating electrodes were connected to a 12 hole plug attached to the calvarium by acrylic cement. In 6 animals, after a wide posterior craniotomy, the cerebellar cortex and nuclei were almost completely removed by suction and coagulation; 2 of these animals had previously been subjected to cerebellar stimulation. After cerebellectomy a plastic
Epilepsia, Vol. 20, August 1979
wall was placed over the caudal part of the skull to avoid any infection of the CNS. Two weeks after surgery, the animals received an intramuscular injection of 300,000-400,000 I.U. of Na G penicillin. An eight-channel Grass electroencephalograph was used for EEG recording. Cerebellar Stimulation Electrical stimulation was carried out through an Alvar Phisiovar. In one series of experiments, 0.5 msec rectangular pulses with a frequency ranging from 5 to 200 c/sec and with a voltage varying from 1 to 10 V were applied for 1-2 sec; in a second series they were applied for 5 - 10 min. The voltage was adjusted so as to avoid any behavioral manifestation. Histology Histological controls were carried out in every animal at the end of the experiment. In 2 animals after cerebellectomy, the uvula, nodulus, and a small part of the paramedial lobulus were found to have been spared. In the others, only part of the uvula and nodulus were left intact. Results The effects of electrical stimulation can be divided according to the area stimulated and the electrical parameters used. Stimulation of the Vermal Area Low frequency stimulations (5-30 c/sec) at different voltages always failed to modify the typical epileptic activity induced by i.m. penicillin. Higher frequency stimulations (100-200 c/sec) at voltages which did not produce any behavioral effects (usually 6-7 V) and lasting for 1-2 sec showed inconstant effects on bilateral and synchronous spike-and-wave discharges. In fact, only 20% of the stimulations exerted an inhibitory action, which consisted in an arrest of the epileptic bursts (Fig. 1). In some of these cases, desynchronization of the background activity following electrical stimula-
CEREBELLAR ROLE IN GENERALIZED EPILEPSY
449
FIG. 1. Vermal stimulation. A shows two bursts: the first one is not affected by electrical stimulation, while the second one is arrested; this inhibitory effect is also shown in B where a desynchronized EEG background activity appears during and immediately after the stimulations; in C the burst is only partially affected by vermal stimulation. Abbreviations: RT, right; LT, left; REF (F), frontal reference; F, frontal; P, parietal; 0, occipital.
tion was observed without any behavioral modification. High frequency stimulations at high voltages invariably produced a typical electrographic and behavioral arousal response, with the disappearance of the epileptic bursts. In 4 animals a quite clear inhibitory effect was found when peculiar patterns of stimulation were used consisting of longlasting electrical stimulation with progressive increases in pulse voltage (Fig. 2). These patterns of stimulation were carried out during the plateau of epileptic activity, .which occurred in this model between the
second and fourth hour after penicillin injection. It was noted that the epileptic bursts were reduced in frequency or completely suppressed for some time during continuous stimulation at a constant voltage, but reappeared after a variable delay. If, at that time, the voltage was increased the inhibitory cerebellar effect reappeared. Subsequent voltage increases produced the same phenomenon, sometimes coupled with desynchronization of the EEG activity. However, even with the highest voltages (10- 12 V), no behavioral modification was observed and the period of reduced
Epilepsie, Val. 20, August 1979
450
G . TESTA ET A L .
FIG. 2. Effects of long-lasting vermal stimulation with increasing voltages.
epileptic activity was more pronounced. On some occasions this inhibitory period lasted for several minutes after the electrical stimulation was terminated. The mean number of epileptic discharges occurring during such a stimulation was found to be significantly lower (p < 0.01, Student’s ttest) in the 4 animals when compared to the frequency of the bursts recorded for a similar length of time prior to and following the stimulation period. The diagrammatic representation of one of these experiments is shown in. Fig. 3A.
Epilepsia, Vol. 20, August 1979
Stimulation of the Cerebral Hemisphere All the above-mentioned stimulations were also applied to a single cerebellar hemisphere without producing any effect on epileptic activity. Only high voltage stimulations were able to induce postural modifications and an EEG arousal effect with disappearance of the epileptic bursts. Prolonged stimulations at increasing voltages produced an effect similar to that obtained by stimulation of the culmen (Fig.
CEREBELLAR ROLE IN GENERALIZED EPILEPSY GENERALIZED
PENICILLIN
EPILEPSY
csl
451
r, tchrMIC,
A -stimulation o i the culmcn -eiiccl o i I n d u a l increase o i voltage
B -stimulation o i lei1 cerebellar hemisphere -eiieCl of gradual increase ot v o ~ t a ~ e
n
n
FIG. 3. Diagrammatic representation of vermal (A) and hemispheral (B) stimulations with gradually increasing voltages. It can be seen that culmen stimulation (A) causes a significant reduction in the number of bursts in the overall stimulation period (Fanalysis of variance:p < 0.05), while the decrease induced by hemispheral stimulation (B) is not significant. In both situations, this effect was maximal immediately after the voltage was increased.
3B), but the effect was not statistically sig- be attributable to several causes: species differences, the various sites and paramenificant with Student's r-test. ters of stimulation used, etc. Because of several electrical and clinical Removal of Neo- and Paleocerebellar features, penicillin-induced generalized Structures epilepsy in cats (Prince and Farrell, 1969) Removal of cerebellar structures, incan be considered as a model similar cluding the stimulated areas, modified to human corticoreticular epilepsy (Gloor neither the frequency or morphology of the and Testa, 1974; Testa and Gloor, 1974; spike-and-wave bursts, nor the background Guberman et al., 1975). Many observations activity (Fig. 4). indicate that in this feline model the fundamental epileptiform disturbance mainly inDISCUSSION volves cortical neurons, even though the On the basis of the data reported in the spike-wave discharges are dependent on literature, the influence of cerebellar various impulses arising from many substructures on epilepsy is still controversial, cortical or other extracortical structures even though Cooper et al. (1974) reported (Gloor et al., 1977; Quesney et al., 1977). It has also been demonstrated that the the effectiveness of cerebellar stimulation in 'epileptic patients. In experimental mod- ascending reticular formation (ARF), els the reported contradictory effects could through its desynchronizing drive, exerts Epilepsia. Vol. 20,August 1979
G . TESTA ET A L .
452
I
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C A I ED,
L
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5 E
n
m
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a
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0 L
D u
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cerebellectoniy
a f t e r cerebellectomy
FIG. 4. Histogram representing four experiments carried out in 2 chronic animals with different amounts of penicillin. The bars correspond to the total number of epileptic bursts during the second and the fourth hour after penicillin injection.
an inhibitory effect on the epileptiform discharges (Gloor and Testa, 1974; Testa and Gloor, 1974). The results obtained in the present study show that the cerebellum exerted only a mild inhibitory effect on penicillin-induced epileptic bursts. In fact, only 20% of the short stimulations .were able to arrest an epileptic burst when delivered immediately after its beginning. More consistent inhibitory effects were observed when chronic stimulations with gradual voltage increases were applied to the vermal area. The results obtained with short stimulations could be explained by a direct or indirect involvement of the ARF, as suggested by the desynchronization of the background activity observed immediately after the electrical stimulation, even when no behavioral modifications were noted. A direct action on the ARF may be due to an unpredictable spreading of the electrical stimulus which can be strong enough to produce a desynchronization of the background activity without causing any behavioral arousal effect. The hypothesis of an indirect involvement of the ARF during stimulation on the cerebellar surface is in accord with recent reports by Bantli et al. (1976) indicating the existence of an anatomical substrate for this effect. In fact, this author has described a loop between the
Epilepsia. Yo/. 20, August 1979
cerebellar cortex and the ARF, so that certain inhibitory effects observed following surface stimulation could be related to ARF activation. However, the efficacy of this particular stimulation is not comparable to that obtained by direct ARF stimulation. A clear explanation of the results obtained with prolonged stimulation of the vermal area is much more difficult. In this condition, no clear desynchronizing effects were always observed in the EEG, but the inhibition of the epileptic activity was of limited duration, indicating some kind of adaptation mechanism. Hypothetically, the decrease in frequency of epileptic bursts found in these experiments could also be due to an indirect involvement of the ARF, even though an extrareticular pathway cannot be ruled out. These results are partially different from those obtained by Myers et al. (1975) and by Hablitz (1976) in the same experimental model. Lack of an inhibitory effect, as reported by Myers et al., was only observed in our study following the use of short stimulations. However, the data obtained by these authors are in some ways difficult to evaluate since the site and the patterns of stimulation are not clearly detailed. On the other hand, the different results reported by Hablitz might be due to either transcortical stimulation or to the peculiar
CEREBELLAR ROLE IN GENERALIZED EPILEPSY
electrical pattern used. Moreover, it is difficult to determine from this report whether there was any desynchronization of the background activity in the EEG during the periods of stimulation. The nonessential role played by the cerebellum in this experimental model of epilepsy is demonstrated by the results observed in ablation experiments, since cerebellectomy never modified the epileptiform pattern or the EEG background activity. In conclusion, our results cast many doubts on the notion that, at least in this experimental model, the cerebellum exerts a clear inhibitory action on epileptic discharge. REFERENCES Babb TL, Mitchell AG. and Crandall PH. Cerebellar influences on the hippocampus. In: IS Cooper, M Riklan, and RS Snider (Eds), Cerebellum, Epilepsy and Behavior, Plenum Press, New York, London, 1974, pp. 37-56. Bantli H, Bloedel JR, and Tolbert D. Activation of neurons in the cerebellar nuclei of ascending reticular formation ,by stimulation of the cerebellar 'surface. J Neurosurg 45539-554, 1976. Cooper IS, Amin I, Gilman S, and Walts JM. The effect of chronic stimulation of cerebellar cortex on epilepsy in man. In: IS Cooper, M Riklan, and RS Snider (Eds), Cerebellum. Epilepsy and Behavior. Plenum Press, New York, London, 1974, pp 119- 171. Dow RS. Experimental cobalt epilepsy and the cerebellum. In: IS Cooper, M Riklan, and RS Snider (Eds), Cerebellum, Epilepsy and Behavior. Plenum Press, New York, London, 1974, pp 57-95. Dow RS, Fernandez-Guardiola A, and Manni E. The influence of the cerebellum on experimental epilepsy. Electroencephalogr Clin Neurophysiol 14383-398, 1962. Gloor P, Quesney LF, and Zumstein H. Pathophysiology of generalized penicillin epilepsy in the cat: The role of cortical and subcortical structures. 11. Topical application of penicillin to the cerebral cortex and to subcortical structures. Electroencephalogr Clin Neurophysiol 43:79-94, 1977. Gloor P and Testa G. Generalized penicillin epilepsy in the cat: Effects of intracarotid and intravertebral pentylentetrazol and amobarbital injections. Electroencephalogr Clin Neurophysiol 36: 499-515, 1974. Guberman A, Gloor P, and Sherwin AL. Response of generalized penicillin epilepsy in the cat to ethosuximide and diphenylhydantoin. Neurology 25~758-764, 1975. Hablitz JJ. Intramuscular penicillin epilepsy in the cat: Effects of chronic cerebellar stimulation. Exp Neurol 50505-514, 1976.
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Hablitz JJ, McSherry JW, and Kellaway P. Cortical seizures following cerebellar stirnulation in primates. Electroencephalogr Clin Neurophysiol 38:423-426, 1975. Hablitz JJ and Rea G. Cerebellar nuclear stimulation in generalized penicillin epilepsy. Brain Res Bull 1:599-601, 1976. Hutton JT, Frost JD, and Foster J. The influence of the cerebellum in cat penicillin epilepsy. Epilepsia 13:401-408, 1972. Kreindler A. Active arrest mechanisms of epileptic seizures. Epilepsia 3:329-337, 1962. Mutani R, Bergamini L, and Doriguzzi T. Experimental evidence for the existence of an extrarhinencephalic control of the activity of the cobalt rhinencephalic epileptogenic focus. Part 2. Effects of paleocerebellar stimulation. Epilepsia 10: 351 -362, 1969. Myers RR, Burchiel KJ, Stockard JJ, and Bickford RG. Effects of acute and chronic paleocerebellar stimulation on experimental models of epilepsy in the cats: Studies with enflurane, pentylentetrazol, penicillin and chloralose. Epilepsia 16:257-267, 1975. Prince D and Farrell D. "Centrencephalic" spikewave discharges following parenteral penicillin injection in the cat. Neurology 19:309-310, 1%9. Quesney LF, Gloor P, Kratzenberg E, and Zumstein H. Pathophysiology of generalized penicillin epilepsy in the cat: The role of cortical and subcortical structures. I. Systemic application of penicillin. Elecrroencephalogr Clin Neurophysiol 42:640-655, 1977. Steriade M. Mechanisme de facilitare si inhibitie in epilepsia penicilinica focale corticale. Stud Crrcet Neurol 5:463-471, 1960. Testa G and Gloor P. Generalized penicillin epilepsy in the cat: Effect of midbrain cooling. Electroencephalogr Clin Neurophysiol 36517-524, 1974.
RESUME La stimulation electrique du cervelet a ete recemment proposee pour le traitement de I'epilepsie chez I'homme. Le rBle inhibiteur des structures cerebelleuses sur les crises epileptiques experimentales a ete rbcemment dbmontd, mais sur des modkles d'epilepsie focale principalement. C'est pourquoi nous avons entrepris d'etudier ce rBle inhibiteur du cervelet sur un modele experimental d'epilepsie generalisee corticoreticulaire par injection parenterale chez le chat de fortes doses de penicilline. Nous avons utilise 12 animaux "chroniques" pour apprecier les effets de la stimulation paleo et neocerebelleuse et de I'ablation cerebelleuse sur les decharges bilaterales et synchrones de pointe-onde. Les resultats ont ete les suivants: (1) I'ablation du cervelet ne modifie pas la frequence des decharges; (2) la stimulation d'un hemisphere cerebelleux, aussi bien que la stimulation a basse frequence du culmen, sont egalement sans effet; (3) un effet inhibiteur leger et inconstant a ete observe pendant une breve stimulation paleo-cerebelleuse a haute frequence; (4) la meme stimulation, plus prolongbe avec augmentation progressive du voltage est plus efkace. Aussi, de nombreux doutes subsistent en
Epilepsia, Vol. 20, August 1979
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ce qui concerne un effet inhibiteur clair et direct des structures cerebelleuses sur les decharges epileptiques dans ce modele d’epilepsie generalisee feline.
(J.-L. Gastaut, Marseilles)
RESUMEN Recientemente se ha propuesto que la estimulacion electrica del cerebelo constituye un tratamiento valido de la epilepsia humana. En alios recientes diversos autores han resaltado la propiedad inhibitoria de las estructuras cerebelosas en la epilepsia experimental. Sin embargo, puesto que la mayoria de estos estudios se llevaron a cab0 en modelos de epilepsia focal, nosotros hemos investigado el posible efecto inhibitorio de las estructuras cerebelosas en un modelo experimental de epilepsia cortico-reticular generalizada, realizado en el gato mediante dosis elevadas de penicilina parenteral. Para el estudio de 10s efectos de la estimulacion electrica del pdeo o neocerebelo, y de la excision del cerebelo, sobre las descargas de punta-onda bilaterales y sincronas, se han utilizado 12 animales cronicos. Los resultados de nuestra investigacion se resumen como sigue: (1) la exicision de estructuras cerebelosas no modifico la frecuencia de las descargas epileptiformes; (2) resultados semejantes se obtuvieron tras la estimulacion de un hemisferio cerebeloso; (3) se observo un ligero e inconstante efecto inhibitorio durante breves estimulaciones de alta frecuencia en el paleocerebelo; (4) la estimulacion prolongada de aka frecuencia del paleocerebelo, con incrementos graduales, de 10s voltajes, fue mas efectiva; Todavh quedan muchas dudas por aclarar en lo que concierne a 10s efectos inhibitorios, que Sean claros y directos, de las estructuras cerebelosas sobre las descargas epilepticas en este modelo felino de epilepsia generalizada. (A. Portera Sanchez, Madrid)
Epllepsia, Vol. 20,August 1979
ZUSAMMENFASSUNG Die elektrische Stimulation des Kleinhirns wurde kiirzlich als giiltige Therapie der menschlichen Epilepsie vorgeschlagen. Die inhibitorische Rolle cerebellarer Strukturen bei experimentellen epileptischen Krampfen wurde in den jiingsten Jahren von vielen Autoren betont. Da jedoch die meisten dieser Untersuchungen am Modell der fokalen Epiiepsie durchgefihrt wurden, untersuchten wir die mogliche inhibitorische Wirkung cerebellarer Strukturen an einem experimentellen Modell der generalisierten corticoreticul&en Epilepsie, die bei der Katze durch grok parenterale Dosen von Penicillin hervorgerufen wurde. An 12 ”chronischen” Tieren wurde die Wirkung der elektrischen paleo-oder neocerebellaren Stimulation und der Entfernung cerebellarer Strukturen auf bilateral synchrone spike-wave Entladungen gepmft. Die Ergebnisse unserer Untersuchung sind folgende: Die Entfernung cerebellarer Strukturen modifizierte n.icht die Frequenz der epileptiformen Entladungen. Ahnliche Resultate wurden nach Stimulation cerebellarer Hemispharen wie nach niederfrequenter Stimulation des Culmen gefunden. Ein milder und inkonstanter inhibitorischer Effekt wurde wahrend einer kurzdauernden paleo-cerebellaren Stimulation mit hoher Frequenz beobachtet. Wirkungsvoller war eine verlangerte hochfrequente Stimulation des Paleocerebellums mit allmahlichem Spannungsanstieg. Es bestehen weiterhin viele Zweifel iiber eine eindeutige direkt inhibitorische Wirkung cerebeU&er Strukturen auf die epileptischen Entladungen in diesem Katzenmodell der generalisierten Epilepsie. (D. Scheffner, Heidelberg)
Raven Press. New York
Effects of Electrical Stimulation and Removal of Cerebellar Structures in an Experimental Model of Generalized Epilepsy G. Testa, A. Pellegrini, and D. Giaretta Istituto di Clinica delle Malattie Nervose e Mentali dell’ Universita di Padova, Padova, Italy
Summary: Electrical stimulation of the cerebellum has recently been proposed as a valid treatment of human epilepsy. The inhibitory role of cerebellar structures in experimental epileptic seizures has been stressed by many authors in recent years. However, since most of these studies were carried out on models of focal epilepsy, we investigated the possible inhibitory effect of cerebellar structures in an experimental model of generalized corticoreticular epilepsy, reproduced in the cat by large parenteral doses of penicillin. Twelve “chronic” animals were used to study the effects of electrical paleo- or neocerebellar stimulation and of cerebellar removal on bilaterally synchronous spike- wave discharges.‘The results of our investigation can be summarized as follows: (1) removal of cerebellar structures did not modify the frequency of the epileptiform discharges; (2) similar results were found after stimulation of the cerebellar hemisphere, as well as after low frequency stimulation of the culmen; (3) a mild and inconstant inhibitory effect was observed during paleocerebellar stimulation of brief duration at high frequency; (4) prolonged high frequency stimulation of the paleocerebellum with gradual voltage increases were more effective. Many doubts still remain concerning a clear, direct inhibitory effect of cerebellar structures on epileptic discharges in this feline model of generalized epilepsy.
The ability of the cerebellum to inhibit experimentally induced seizures has been stressed by many authors in recent years. Most of these experiments, however, have been carried out on models of focal epilepsy (Steriade, 1960; Dow et al., 1962; Mutani et al., 1969; Hutton et al., 1972; Babb et al., 1974; Dow, 1974; Hablitz et al., 1975). Other studies, like those of Myers et al. (1975), Hablitz (1976), and Hablitz and Rea (1976), examined the effects of cere-
bellar electrical stimulation in generalized penicillin epilepsy of the cat (Prince and Farrel, 1969), a model closely resembling human myoclonic petit ma1 (Gloor and Testa, 1974; Testa and Gloor, 1974; Guberman et al., 1975). Data obtained from all these studies are equivocal. In fact, in some experiments, cerebellar stimulation did not exert any inhibitory effect (Myers et al., 1975), or even have a facilitatory effect on experimentally
Received September 12, 1978. Key words: Generalized epilepsy-Penicillin-Cerebellum.
447
448
G . TESTA ET AL.
induced seizures (Steriade, 1960; Kreindler, 1962; Hablitz et al., 1975). However, Cooper et al. (1974) stressed the inhibitory role played. by various cerebellar areas in human patients and reported a significant decrease in seizures in subjects with intractable epilepsy through the use of chronic cerebellar stimulation. Since doubts remain about the action of these cerebellar structures and since the results reported by Myers et al. (1975) and by Hablitz (1976) in generalized penicillin epilepsy are difficult to assess, we attempted to evaluate, in this same model, the effects of different kinds of paleo- and neocerebellar stimulations, as well as the effects of removing the cerebellum. METHODS Chronic experiments were carried out in 12 adult cats weighing between 3 and 4 kg. Under aseptic conditions and thiopental anesthesia, six stainless steel screw electrodes were symmetrically implanted in the skull over the pericruciate, middle, and posterior suprasylvian gyri for EEG recordings. A screw serving as a reference electrode was inserted over the frontal sinus or in the external occipital protuberance. In 8 cats, through a posterior craniotomy (behind the occipital protuberance), bipolar silver ball electrodes (at an interelectrode distance of 1 mm), were placed on the culmen and lobulus simplex of the cerebellum. Two rectangular stainless steel plate electrodes of 10 mm2each, separated by 1.5 mm and attached to a thin plastic support, were placed over the cerebellar hemisphere. All recording and stimulating electrodes were connected to a 12 hole plug attached to the calvarium by acrylic cement. In 6 animals, after a wide posterior craniotomy, the cerebellar cortex and nuclei were almost completely removed by suction and coagulation; 2 of these animals had previously been subjected to cerebellar stimulation. After cerebellectomy a plastic
Epilepsia, Vol. 20, August 1979
wall was placed over the caudal part of the skull to avoid any infection of the CNS. Two weeks after surgery, the animals received an intramuscular injection of 300,000-400,000 I.U. of Na G penicillin. An eight-channel Grass electroencephalograph was used for EEG recording. Cerebellar Stimulation Electrical stimulation was carried out through an Alvar Phisiovar. In one series of experiments, 0.5 msec rectangular pulses with a frequency ranging from 5 to 200 c/sec and with a voltage varying from 1 to 10 V were applied for 1-2 sec; in a second series they were applied for 5 - 10 min. The voltage was adjusted so as to avoid any behavioral manifestation. Histology Histological controls were carried out in every animal at the end of the experiment. In 2 animals after cerebellectomy, the uvula, nodulus, and a small part of the paramedial lobulus were found to have been spared. In the others, only part of the uvula and nodulus were left intact. Results The effects of electrical stimulation can be divided according to the area stimulated and the electrical parameters used. Stimulation of the Vermal Area Low frequency stimulations (5-30 c/sec) at different voltages always failed to modify the typical epileptic activity induced by i.m. penicillin. Higher frequency stimulations (100-200 c/sec) at voltages which did not produce any behavioral effects (usually 6-7 V) and lasting for 1-2 sec showed inconstant effects on bilateral and synchronous spike-and-wave discharges. In fact, only 20% of the stimulations exerted an inhibitory action, which consisted in an arrest of the epileptic bursts (Fig. 1). In some of these cases, desynchronization of the background activity following electrical stimula-
CEREBELLAR ROLE IN GENERALIZED EPILEPSY
449
FIG. 1. Vermal stimulation. A shows two bursts: the first one is not affected by electrical stimulation, while the second one is arrested; this inhibitory effect is also shown in B where a desynchronized EEG background activity appears during and immediately after the stimulations; in C the burst is only partially affected by vermal stimulation. Abbreviations: RT, right; LT, left; REF (F), frontal reference; F, frontal; P, parietal; 0, occipital.
tion was observed without any behavioral modification. High frequency stimulations at high voltages invariably produced a typical electrographic and behavioral arousal response, with the disappearance of the epileptic bursts. In 4 animals a quite clear inhibitory effect was found when peculiar patterns of stimulation were used consisting of longlasting electrical stimulation with progressive increases in pulse voltage (Fig. 2). These patterns of stimulation were carried out during the plateau of epileptic activity, .which occurred in this model between the
second and fourth hour after penicillin injection. It was noted that the epileptic bursts were reduced in frequency or completely suppressed for some time during continuous stimulation at a constant voltage, but reappeared after a variable delay. If, at that time, the voltage was increased the inhibitory cerebellar effect reappeared. Subsequent voltage increases produced the same phenomenon, sometimes coupled with desynchronization of the EEG activity. However, even with the highest voltages (10- 12 V), no behavioral modification was observed and the period of reduced
Epilepsie, Val. 20, August 1979
450
G . TESTA ET A L .
FIG. 2. Effects of long-lasting vermal stimulation with increasing voltages.
epileptic activity was more pronounced. On some occasions this inhibitory period lasted for several minutes after the electrical stimulation was terminated. The mean number of epileptic discharges occurring during such a stimulation was found to be significantly lower (p < 0.01, Student’s ttest) in the 4 animals when compared to the frequency of the bursts recorded for a similar length of time prior to and following the stimulation period. The diagrammatic representation of one of these experiments is shown in. Fig. 3A.
Epilepsia, Vol. 20, August 1979
Stimulation of the Cerebral Hemisphere All the above-mentioned stimulations were also applied to a single cerebellar hemisphere without producing any effect on epileptic activity. Only high voltage stimulations were able to induce postural modifications and an EEG arousal effect with disappearance of the epileptic bursts. Prolonged stimulations at increasing voltages produced an effect similar to that obtained by stimulation of the culmen (Fig.
CEREBELLAR ROLE IN GENERALIZED EPILEPSY GENERALIZED
PENICILLIN
EPILEPSY
csl
451
r, tchrMIC,
A -stimulation o i the culmcn -eiiccl o i I n d u a l increase o i voltage
B -stimulation o i lei1 cerebellar hemisphere -eiieCl of gradual increase ot v o ~ t a ~ e
n
n
FIG. 3. Diagrammatic representation of vermal (A) and hemispheral (B) stimulations with gradually increasing voltages. It can be seen that culmen stimulation (A) causes a significant reduction in the number of bursts in the overall stimulation period (Fanalysis of variance:p < 0.05), while the decrease induced by hemispheral stimulation (B) is not significant. In both situations, this effect was maximal immediately after the voltage was increased.
3B), but the effect was not statistically sig- be attributable to several causes: species differences, the various sites and paramenificant with Student's r-test. ters of stimulation used, etc. Because of several electrical and clinical Removal of Neo- and Paleocerebellar features, penicillin-induced generalized Structures epilepsy in cats (Prince and Farrell, 1969) Removal of cerebellar structures, incan be considered as a model similar cluding the stimulated areas, modified to human corticoreticular epilepsy (Gloor neither the frequency or morphology of the and Testa, 1974; Testa and Gloor, 1974; spike-and-wave bursts, nor the background Guberman et al., 1975). Many observations activity (Fig. 4). indicate that in this feline model the fundamental epileptiform disturbance mainly inDISCUSSION volves cortical neurons, even though the On the basis of the data reported in the spike-wave discharges are dependent on literature, the influence of cerebellar various impulses arising from many substructures on epilepsy is still controversial, cortical or other extracortical structures even though Cooper et al. (1974) reported (Gloor et al., 1977; Quesney et al., 1977). It has also been demonstrated that the the effectiveness of cerebellar stimulation in 'epileptic patients. In experimental mod- ascending reticular formation (ARF), els the reported contradictory effects could through its desynchronizing drive, exerts Epilepsia. Vol. 20,August 1979
G . TESTA ET A L .
452
I
C A I E 04
C A I ED,
L
700
5 E
n
m
500 L
a
D L
300
0 L
D u
0before
cerebellectoniy
a f t e r cerebellectomy
FIG. 4. Histogram representing four experiments carried out in 2 chronic animals with different amounts of penicillin. The bars correspond to the total number of epileptic bursts during the second and the fourth hour after penicillin injection.
an inhibitory effect on the epileptiform discharges (Gloor and Testa, 1974; Testa and Gloor, 1974). The results obtained in the present study show that the cerebellum exerted only a mild inhibitory effect on penicillin-induced epileptic bursts. In fact, only 20% of the short stimulations .were able to arrest an epileptic burst when delivered immediately after its beginning. More consistent inhibitory effects were observed when chronic stimulations with gradual voltage increases were applied to the vermal area. The results obtained with short stimulations could be explained by a direct or indirect involvement of the ARF, as suggested by the desynchronization of the background activity observed immediately after the electrical stimulation, even when no behavioral modifications were noted. A direct action on the ARF may be due to an unpredictable spreading of the electrical stimulus which can be strong enough to produce a desynchronization of the background activity without causing any behavioral arousal effect. The hypothesis of an indirect involvement of the ARF during stimulation on the cerebellar surface is in accord with recent reports by Bantli et al. (1976) indicating the existence of an anatomical substrate for this effect. In fact, this author has described a loop between the
Epilepsia. Yo/. 20, August 1979
cerebellar cortex and the ARF, so that certain inhibitory effects observed following surface stimulation could be related to ARF activation. However, the efficacy of this particular stimulation is not comparable to that obtained by direct ARF stimulation. A clear explanation of the results obtained with prolonged stimulation of the vermal area is much more difficult. In this condition, no clear desynchronizing effects were always observed in the EEG, but the inhibition of the epileptic activity was of limited duration, indicating some kind of adaptation mechanism. Hypothetically, the decrease in frequency of epileptic bursts found in these experiments could also be due to an indirect involvement of the ARF, even though an extrareticular pathway cannot be ruled out. These results are partially different from those obtained by Myers et al. (1975) and by Hablitz (1976) in the same experimental model. Lack of an inhibitory effect, as reported by Myers et al., was only observed in our study following the use of short stimulations. However, the data obtained by these authors are in some ways difficult to evaluate since the site and the patterns of stimulation are not clearly detailed. On the other hand, the different results reported by Hablitz might be due to either transcortical stimulation or to the peculiar
CEREBELLAR ROLE IN GENERALIZED EPILEPSY
electrical pattern used. Moreover, it is difficult to determine from this report whether there was any desynchronization of the background activity in the EEG during the periods of stimulation. The nonessential role played by the cerebellum in this experimental model of epilepsy is demonstrated by the results observed in ablation experiments, since cerebellectomy never modified the epileptiform pattern or the EEG background activity. In conclusion, our results cast many doubts on the notion that, at least in this experimental model, the cerebellum exerts a clear inhibitory action on epileptic discharge. REFERENCES Babb TL, Mitchell AG. and Crandall PH. Cerebellar influences on the hippocampus. In: IS Cooper, M Riklan, and RS Snider (Eds), Cerebellum, Epilepsy and Behavior, Plenum Press, New York, London, 1974, pp. 37-56. Bantli H, Bloedel JR, and Tolbert D. Activation of neurons in the cerebellar nuclei of ascending reticular formation ,by stimulation of the cerebellar 'surface. J Neurosurg 45539-554, 1976. Cooper IS, Amin I, Gilman S, and Walts JM. The effect of chronic stimulation of cerebellar cortex on epilepsy in man. In: IS Cooper, M Riklan, and RS Snider (Eds), Cerebellum. Epilepsy and Behavior. Plenum Press, New York, London, 1974, pp 119- 171. Dow RS. Experimental cobalt epilepsy and the cerebellum. In: IS Cooper, M Riklan, and RS Snider (Eds), Cerebellum, Epilepsy and Behavior. Plenum Press, New York, London, 1974, pp 57-95. Dow RS, Fernandez-Guardiola A, and Manni E. The influence of the cerebellum on experimental epilepsy. Electroencephalogr Clin Neurophysiol 14383-398, 1962. Gloor P, Quesney LF, and Zumstein H. Pathophysiology of generalized penicillin epilepsy in the cat: The role of cortical and subcortical structures. 11. Topical application of penicillin to the cerebral cortex and to subcortical structures. Electroencephalogr Clin Neurophysiol 43:79-94, 1977. Gloor P and Testa G. Generalized penicillin epilepsy in the cat: Effects of intracarotid and intravertebral pentylentetrazol and amobarbital injections. Electroencephalogr Clin Neurophysiol 36: 499-515, 1974. Guberman A, Gloor P, and Sherwin AL. Response of generalized penicillin epilepsy in the cat to ethosuximide and diphenylhydantoin. Neurology 25~758-764, 1975. Hablitz JJ. Intramuscular penicillin epilepsy in the cat: Effects of chronic cerebellar stimulation. Exp Neurol 50505-514, 1976.
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Hablitz JJ, McSherry JW, and Kellaway P. Cortical seizures following cerebellar stirnulation in primates. Electroencephalogr Clin Neurophysiol 38:423-426, 1975. Hablitz JJ and Rea G. Cerebellar nuclear stimulation in generalized penicillin epilepsy. Brain Res Bull 1:599-601, 1976. Hutton JT, Frost JD, and Foster J. The influence of the cerebellum in cat penicillin epilepsy. Epilepsia 13:401-408, 1972. Kreindler A. Active arrest mechanisms of epileptic seizures. Epilepsia 3:329-337, 1962. Mutani R, Bergamini L, and Doriguzzi T. Experimental evidence for the existence of an extrarhinencephalic control of the activity of the cobalt rhinencephalic epileptogenic focus. Part 2. Effects of paleocerebellar stimulation. Epilepsia 10: 351 -362, 1969. Myers RR, Burchiel KJ, Stockard JJ, and Bickford RG. Effects of acute and chronic paleocerebellar stimulation on experimental models of epilepsy in the cats: Studies with enflurane, pentylentetrazol, penicillin and chloralose. Epilepsia 16:257-267, 1975. Prince D and Farrell D. "Centrencephalic" spikewave discharges following parenteral penicillin injection in the cat. Neurology 19:309-310, 1%9. Quesney LF, Gloor P, Kratzenberg E, and Zumstein H. Pathophysiology of generalized penicillin epilepsy in the cat: The role of cortical and subcortical structures. I. Systemic application of penicillin. Elecrroencephalogr Clin Neurophysiol 42:640-655, 1977. Steriade M. Mechanisme de facilitare si inhibitie in epilepsia penicilinica focale corticale. Stud Crrcet Neurol 5:463-471, 1960. Testa G and Gloor P. Generalized penicillin epilepsy in the cat: Effect of midbrain cooling. Electroencephalogr Clin Neurophysiol 36517-524, 1974.
RESUME La stimulation electrique du cervelet a ete recemment proposee pour le traitement de I'epilepsie chez I'homme. Le rBle inhibiteur des structures cerebelleuses sur les crises epileptiques experimentales a ete rbcemment dbmontd, mais sur des modkles d'epilepsie focale principalement. C'est pourquoi nous avons entrepris d'etudier ce rBle inhibiteur du cervelet sur un modele experimental d'epilepsie generalisee corticoreticulaire par injection parenterale chez le chat de fortes doses de penicilline. Nous avons utilise 12 animaux "chroniques" pour apprecier les effets de la stimulation paleo et neocerebelleuse et de I'ablation cerebelleuse sur les decharges bilaterales et synchrones de pointe-onde. Les resultats ont ete les suivants: (1) I'ablation du cervelet ne modifie pas la frequence des decharges; (2) la stimulation d'un hemisphere cerebelleux, aussi bien que la stimulation a basse frequence du culmen, sont egalement sans effet; (3) un effet inhibiteur leger et inconstant a ete observe pendant une breve stimulation paleo-cerebelleuse a haute frequence; (4) la meme stimulation, plus prolongbe avec augmentation progressive du voltage est plus efkace. Aussi, de nombreux doutes subsistent en
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ce qui concerne un effet inhibiteur clair et direct des structures cerebelleuses sur les decharges epileptiques dans ce modele d’epilepsie generalisee feline.
(J.-L. Gastaut, Marseilles)
RESUMEN Recientemente se ha propuesto que la estimulacion electrica del cerebelo constituye un tratamiento valido de la epilepsia humana. En alios recientes diversos autores han resaltado la propiedad inhibitoria de las estructuras cerebelosas en la epilepsia experimental. Sin embargo, puesto que la mayoria de estos estudios se llevaron a cab0 en modelos de epilepsia focal, nosotros hemos investigado el posible efecto inhibitorio de las estructuras cerebelosas en un modelo experimental de epilepsia cortico-reticular generalizada, realizado en el gato mediante dosis elevadas de penicilina parenteral. Para el estudio de 10s efectos de la estimulacion electrica del pdeo o neocerebelo, y de la excision del cerebelo, sobre las descargas de punta-onda bilaterales y sincronas, se han utilizado 12 animales cronicos. Los resultados de nuestra investigacion se resumen como sigue: (1) la exicision de estructuras cerebelosas no modifico la frecuencia de las descargas epileptiformes; (2) resultados semejantes se obtuvieron tras la estimulacion de un hemisferio cerebeloso; (3) se observo un ligero e inconstante efecto inhibitorio durante breves estimulaciones de alta frecuencia en el paleocerebelo; (4) la estimulacion prolongada de aka frecuencia del paleocerebelo, con incrementos graduales, de 10s voltajes, fue mas efectiva; Todavh quedan muchas dudas por aclarar en lo que concierne a 10s efectos inhibitorios, que Sean claros y directos, de las estructuras cerebelosas sobre las descargas epilepticas en este modelo felino de epilepsia generalizada. (A. Portera Sanchez, Madrid)
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ZUSAMMENFASSUNG Die elektrische Stimulation des Kleinhirns wurde kiirzlich als giiltige Therapie der menschlichen Epilepsie vorgeschlagen. Die inhibitorische Rolle cerebellarer Strukturen bei experimentellen epileptischen Krampfen wurde in den jiingsten Jahren von vielen Autoren betont. Da jedoch die meisten dieser Untersuchungen am Modell der fokalen Epiiepsie durchgefihrt wurden, untersuchten wir die mogliche inhibitorische Wirkung cerebellarer Strukturen an einem experimentellen Modell der generalisierten corticoreticul&en Epilepsie, die bei der Katze durch grok parenterale Dosen von Penicillin hervorgerufen wurde. An 12 ”chronischen” Tieren wurde die Wirkung der elektrischen paleo-oder neocerebellaren Stimulation und der Entfernung cerebellarer Strukturen auf bilateral synchrone spike-wave Entladungen gepmft. Die Ergebnisse unserer Untersuchung sind folgende: Die Entfernung cerebellarer Strukturen modifizierte n.icht die Frequenz der epileptiformen Entladungen. Ahnliche Resultate wurden nach Stimulation cerebellarer Hemispharen wie nach niederfrequenter Stimulation des Culmen gefunden. Ein milder und inkonstanter inhibitorischer Effekt wurde wahrend einer kurzdauernden paleo-cerebellaren Stimulation mit hoher Frequenz beobachtet. Wirkungsvoller war eine verlangerte hochfrequente Stimulation des Paleocerebellums mit allmahlichem Spannungsanstieg. Es bestehen weiterhin viele Zweifel iiber eine eindeutige direkt inhibitorische Wirkung cerebeU&er Strukturen auf die epileptischen Entladungen in diesem Katzenmodell der generalisierten Epilepsie. (D. Scheffner, Heidelberg)
