Epiiepsia, 17 :177 -18 2, 1976. Raven Press, New York

Effects of Acute Cerebellectomy on Maximal Electroshock Seizures and Anticonvulsant Efficacy of Diazepam in the Rat Arthur Raines and Rebecca J. Anderson Department of Pharmacology, Georgetown University Schools of Medicine and Dentistry, 3900 Reservoir Road, NW, Washington, D.C. 20007 (Received April 12, 1976)

INTRODUCTION of cerebellar influence has long been associated with abnormal motor In addition to regulating muscle Performance, this brain site appears to influence seizure activity and has recently aroused much interest (Cooper et al., 1974; Dow, 1974; Julien, 1974). Myoclonic epilepSy has been associated with cerebellar degeneration ( mis tophe and Remond, 1951; Hammond, 1867; Hodskins and Yakovlev, 193% Hunt, 1914). Russell (1895) showed that when the convulsant material absYnth Was administered to dogs with Unilateral cerebellar ablation, COnVUlSiOnS iPSilateral to the lesions were more severe. This suggested the loss of an inhibitory cerebellar control mechanism. Simijarly, the loss of cerebellar inhibitory capacity has been reported to facilitate Seizure activity in rats and cats. Removal or Cooling (Dow, 1962a,b) of the rat cerebellum enhances and Prolongs epileptic manifestations of CObalt-induced seizures. h concordance with these observations are the reports that cerebellar activation (electrically or pharmacolof$cally) suppresses seizure activity. Cooke and Snider (1955) have shown that stimulation of the fastigial nucleus or cerebellar cortex can stop electroencephalographic manifestations O f seizure discharges produced by LOSS

excessive stimulation of exposed sensorimotor cortex. Similarly, Iwata and Snider (1959) have shown that in anesthetized cats, hippocampal seizures are stopped by cerebellar stimulation. Hutton et al. (1972) have reported that

stimulation of the cerebellar cortex suppresses penicillin-induced seizure activity in the cat. m e influence of cerebellar stimulation on cobalt-induced epilepsy in the cat is not completely settled and probably depends on the site of the seizure focus as well as the area of the cerebellum stimulated. Thus, Mutani et al. (1969) reported that stimulation of the midline paleocerebellum inhibits seizure activity in the amygdda and hippocampus, whereas Reimer et al. (1967) reported that sensorimotor cortical foci are not inhibited by contralateral stimulation of the vermis or cerebellar cortex. Cerebellar stimulation has recently been utilized t o arrest intractable epileptiform seizures in man with seemingly promising results (cooper et al., 1974). Accordingly, recent work suggests that the cerebellum may be an important site for anticonvulsant action by three diversely acting anticonvulsant mate+&: diphenylhydantoin (Julien and Halpern, 1971), phenobarbital, and diazepam (Julien, 1972). These workers have reported that the administration of these anticonvulsant drugs produces a marked enhancement of the discharge frequency of Purkinje cells in the cerebellar cortex of the cat and that these discharges inhibit EEG correlates Kev words: Cerebellum- Anticonvulsant of seizure activity in the cerebral cortex. drug - Pentylenetetruol - Diazepam - Con- Inhibition of cortical neurons by enhancing the vulsive seizures. activity of Purkinje cells was suggested as an

177

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A. RAINES A N D R.J. ANDERSON

antiseizure mechanism of these drugs. In view of the emergence of cerebellar influence as a significant factor in the development and control of convulsions (cf. review by Dow, 1974) experiments were performed ‘on the role of the cerebellum on seizure production by maximal electroshock (MES) and pentylenetetrazol (PTZ), techniques routinely used for the laboratory evaluation of potential anticonvulsants. In addition, the influence of the cerebellectomy on the anticonvulsant efficacy of diazepam was assessed. METHODS Adult male Sprague-Dawley albino rats weighing from 250-300 g were used. The animals were preshocked 48-72 hr prior to cerebellectomy using a stimulus of 300 mA with a duration of 200 msec delivered through corneal electrodes (Swinyard et al., 1952). Animals failing to exhibit typical flexor-extensor MES patterns (approximately 5% of rats) were excluded. For cerebellectomy, animals were anesthetized with sodium pentobarbital, 30-50 mg/kg, i.p. Using aseptic technique, a craniotomy was performed and the cerebellum totally removed by excision. Control animals were subjected to sham operations in which the cerebelli were exposed but not removed. In all animals overlying tissues were closed and the rats allowed to recover. Postoperatively, we essentially employed the measures of Kopeloff and Alexander (1972). L Control (sham-operated) animals were permitted only limited access to food and water to limit their weight to correspond with those of the cerebellectomized animals. Both groups of animals received 10 ml of 5% dextrose in saline and 0.1 ml of a multivitamin preparation subcutaneously daily. These animals were used 72-96 hr after surgery. For electroshock seizures, stimulus intensity was varied over the range of 150-500 mA. The criterion for occurrence of a maximal seizure was tonic extension of the hindlimbs (Swinyard et al., 1952). In experiments on the incidence of convulsions produced by PTZ, the dose was varied from 40 to 120 mg/kg and administered subcutaneously in a volume of 1.0 m1/100 g body weight. Animals were observed for 2 hr

for signs of seizure activity. The number of animals exhibiting clonic seizures, not only a single myoclonic jerk, was noted in each group and the EDs0 (dose effective in producing clonic seizures in 50% of rats) determined. The LD5 (dose effective in producing death in 50% of rats) of PTZ was also evaluated in animals with and without cerebelli over a 2-hr period. The anticonvulsant efficacy of diazepam (Valium@, Roche Laboratories, Nutley, N.J.), administered as the commercial parenteral preparation, was assessed by determining its efficacy in preventing PTZ-induced seizures. In our hands, a dose of 115 mg/kg PTZ reliably (i.e., in 95% of trials) produces clonic seizures. This dose was administered subcutaneously in a volume of 1.0 m1/100 g body weight and animals were observed for 2 hr; seizures usually terminated in death. In preliminary experiments, we determined that 1 5 min was the time of maximal anti-PTZ activity of diazepam; it was at this time that anticonvulsant efficacy was assessed and the ED5 estimated. Subsequent to use in the seizure or toxicity studies, the animals were exposed to a lethal chloroform concentration and the operation sites reexamined under a dissecting microscope. In all cases cerebellectomy was complete. Animals were not reused and each animal furnished only one datum point. Estimates of ED50 values with 95% confidence limits for PTZ and diazepam were made using the method of Litchfield and Wilcoxon (1949). Group comparisons were made using a 2 X 2 Chi’ test. RESULTS Ataxia and locomotion difficulties were apparent in the cerebellectomized rats on emergence from anesthesia; these animals also experienced difficulty in feeding, findings consistent with those of Kopeloff and Alexander (1972). Cerebellectomy markedly diminished the incidence of tonic hindlimb extension in the MES test at all current intensities evaluated (p < 0.05; 2 X 2 Chi’). Stimulus intensities of less than 300 mA failed t o produce this endpoint in cerebellectomized rats. With more intense stimulation, some animals did exhibit a maximal seizure, but the greatest incidence observed was only 12.5%. With stimulus

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CEREBELLUM A N D EXPERIMENTAL SEIZURES TABLE 1. Effect o f cerebellectomy on the incidence o f tonic hindlimb extension in rats No. exhibiting extension/ no. tested

Stimulus intensity (mA)

Controls

Cerebellectomized ~

150 200 250 300 350 400 450 500 Op

018

318 518 16/24 35/42 8/10 9/10 7 18 7 18

< 0.05: 2 X

0/8('

0/aa 3/50a 2/16a l/Ba

2 Chi'.

TABLE 2. Effect o f cerebellectomy on the incidence o f clonic seizures produced by pentylenetetrazol

Controls 40 60 80 100 120

Dose (mg/kg) Controls 40 60 80 100 120

No. dead/ no. tested 014 2j8 3/16 11/16 14/15

Cerebellectomized 40 014 -, 60 3/12 80 5/13 100 9/10 120 13/13

LD50 with 95%confidence limits

88 (77-100)

~~

l/fja l/tia

intensities of 300 mA or greater, the incidence of tonic hindlimb extension was always at least 80% in sham-operated controls. The results of cerebellectomy on the incidence of tonic hindlimb extension appear in Table 1. Cerebellectomy had no influence on the capacity of PTZ to induce clonic seizures. The ED50's with 95% confidence limits for both cerebellectomized and sham-operated controls were 76 (69-83) and 87 (78-97), respectively, values which are not different. These data appear in Table 2. Two animals in the

Dose (mglkg)

TABLE 3. Effect o f cerebellectomy on the incidence o f a lethal outcome after pentylenetetrazol administration

No. convulsed/ no. tested

EDsoa with 95%confidence limits

014 -, 3/15 11/30 22/31 14/14

87 (78-97)

Cerebellectomized 40 014 60 lil2 80 15/24 100 19/22 120 616

76 (69-83)

~

80

(68-94)

sham-operated group receiving the largest PTZ dose exhibited maximal seizures (i.e., tonic hindlimb extension), an event which did not occur in any of the cerebellectomized animals. In different animals, experiments on the lethality of PTZ revealed that the LD50 values were the same in cerebellectomized and in sham-operated controls. The results of these experiments appear in Table 3. The capacity of diazepam to antagonize PTZ-induced clonic seizures was not influenced by cerebellectomy. In the sham-operated controls, a dose of 0.59 (0.41-0.86) mg/kg of diazepam was required to prevent clonic seizures in 50% of animals receiving WZ, 115 mg/kg, S.C. Under identical conditions cerebellectomized animals required 0.38 (0.30-0.49) mg/kg of diazepam. The results of these experiments appear in Table 4.

~

aDose effective in producing clonic seizures in 50% of rats.

DISCUSSION The present experiments indicate that the cerebellum is necessary for the full expression of the maximal electroshock seizure, a seemingly paradoxical finding in view of this brain site's proposed antiseizure activity. It is possible that the tonic hindlimb extension observed in a small number of cerebellectomized animals after very high intensity stimulation was due to stimulus spread to the cervical spinal cord, since the current is delivered by spread through a

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A . RAINES A N D R . J. ANDERSON

TABLE 4. E f f e c t o f cerebellecfomy on the capacity of diazepam pretreatment (15 min) to prevent pentylenetetrazol-induced clonic seizures

DOW,1962a,b; Mutani et al., 1969; Julien and Halpern, 1971; Hutton et al., 1972; Julien, 1972; Julien and Halpem, 1972; Kopeloff and Alexander, 1972; Cooper et al., 1974; Dow, Dose diazeNo. exhibiting EDSOwith 1974; Julien, 1974), the differences are clonic seizures/ no. Pam 95% 'Onfiprobably due to the types of seizures under dence limits (mg/kg) tested study. Most other workers have utilized focally triggered seizures produced by cobalt, penicilControls 0.25 718 lin, or focal electrical stimulation. The ad0.50 6/12 0.59 ministration of PTZ via the systemic circulation 0.75 6112 (0'41-0*86) as presently administered delivers the drug to 1.00 0112 all brain sites. The drug distributes in total Cerebellectomized body water and apparently encounters no 0.25 518 barriers (Toman and Taylor, 1952). Straw and 0.50 5/12 0.38 Mitchell (1967a,b) have shown that seizures 0.75 5/13 (0.30-0.49) produced by PTZ originate simultaneously at 1.00 0111 multiple brain sites. Furthermore, spinal (Lewin and -Esplin, 1961) and peripheral nerve (Eyzaguirre and Lillienthal, 1949) neuroexvolume conductor. In this regard, stimulation citatory effects of PTZ have been described. of the cervical spinal cord produced typical Thus, PTZ appears to excite many neural MES patterns in cats (Esplin, 1959; Esplin and elements at several levels. These data suggest Freston, 1960). The reasons for blockade of that the seizures are generalized from the start tonic extension by cerebellectomy are not and not focal in origin. Accordingly, Essig evident from the experimental design em- (1964) reported that cerebellar lesions did not ployed. influence convulsions produced by barbiturate The present experiments indicate that withdrawal. Perhaps cerebellar antiseizure incerebellectomy does not influence the efficacy fluences are manifest in the suppression of focal of PTZ t o produce clonic convulsions. The dose rather than generalized types of seizure activity. in sham-operated controls was the same as that It may very well be that the interaction of required in the cerebellectomized rats. Thus, at diazepam with PTZ is purely a drug interaction least under the present experimental circum- and that the cerebellum plays an inconsequenstances, no anti-PTZ effect could be attributed tial role under these circumstances. It is also t o any cerebellar activity. Similarly, no possible that the seizures produced by this PTZ differences existed between the lethal doses of dose are too severe a test to exhibit an PTZ for both of these groups. The similarity in antiseizure influence of the cerebellum. dose observed in the present group of animals Nevertheless, under the present experimental for the above two criteria are a reflection of the circumstances, the cerebellum did not appear to steep dose-response curve of PTZ for CNS be a significant site for anticonvulsant activity stimulation. These data appear t o be consistent for diazepam; the anticonvulsant efficacy of with those of Kopeloff and Alexander (1972) diazepam was not influenced by acute cerebelwho reported that the thresholds for PTZ-in- lectomy. It is likely, as in the case of PTZ, that duced seizures in sham-operated and in the actions of diazepam are rather widespread, cerebellectomized rats are not different. These involving several central and peripheral sites data are also consistent with those of Schlosser (Guerrero-Fiqueroa et al., 1972; Schallek et al., et al. (1975) who demonstrated that decerebel- 1972). lation did not influence inhibition of supraIn the present experiments, the system spinal reflexes by diazepam or by diphenylhy- under evaluation was that of acute cerebeldantoin in the cat. lectomy. The animals were utilized 3 to 4 days Although these data appear t o be at variance after surgery, and at this time local changes with those of others who have attributed associated with the consequences of surgery are antiseizure functions to the cerebellum (Cooke certain to be occurring. However, efforts to and Snider, 1955; Iwata and Snider, 1959; keep the animals viable for longer periods led to

CEREBELLUM A N D EXPERIMENTAL SEIZURES withholding substantial amounts of food from the sham-operated controls. Woodbury (1969) has pointed out that starvation increases seizure susceptibility and we wished to avoid this complication. Thus, the data of the present report reflect acute cerebellar loss and not changes which may occur at later times. In view of the emergence of the cerebellum as a determinant of seizure production or arrest, it seems important to delineate those conditions under which cerebellar activation or inhibition might favorably modify convulsive activity. Furthermore, it is necessary to evaluate cerebellar influences in different experimental settings.

REFERENCES Christophe J and Remond A. Dyssnergia cerebellarias myoclonica de Ramsay-Hunt. Rev Neurol84:256-262, 1951. Cooke PM and Snider RS. Some cerebellar influences on electrically induced cerebral seizures. Epilepsia 4:19-28, 1955. Cooper IS, Amin I, Gilman S, and Waltz JM. The effect of chronic stimulation of cerebellar cortex on epilepsy in man. In: IS Cooper, M Riklan, and RS Snider (Eds), The Cerebellum, Epilepsy and Behavior, Plenum Press, New York, pp 119-171, 1974. Dow RS, Femandez-Guardiolo A, and Manni E. The influence of the cerebellum on experimental evilevsv. EEG Clin NeuroDhvsiol - 14:383-398, i9620. Dow RS. Fernandez-Guardiola A. and Manni E. The production of cobalt ' experimental epilepsy in the rat. EEG Clin Neurophysiol 1 4 ~ 3 9 9 - 4 0 719626. , Dow RS, Some novel concepts ot cerebellar physiology, M t Sinai J Med 41:103-119, 1974. Esplin DW. Spinal cord convulsions. Arch Neurol 1:485-490, 1959. Esplin DW and Freston JW. Physiological and pharmacological analysis of spinal cord Exp Ther convulsions. J Pharmacol 130:68-80, 1960. Essig CF. Withdrawal convulsions in decerebrate dogs. Int J Neuropharmacol 3:453, 1964. Eyzaguirre C and Lillienthal JL, Jr. Veritrinic effects of p e n tamethylenethetrazol (Metrazol) and 2,2-Bis (p-chlorophenyl) l,l,l-trichloroethane (DDT) in mammalian neuromuscular function.' Proc soc 70:272-275. 1949. Guerrero-Figue'roa R; Gallant DM, GuerreroFigueroa C, and Gallant J. Electrophysiological analysis of the action of four benzodiazepine derivatives on the central nervous system. In: S Garattini, S Mussini,

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and LO Randall (Eds), The Benzodiazepines. Raven Press, New York, p p 489-511, 1972. Hammond WA. On convulsive tremor. N Y Med J5:185-198, 1867. Hodskins MB and Yakovlev PI. Anatomicoclinical observations on myoclonus in epileptics and on related symptom complexes. A m JPsychiatry 9:827-848, 1930. Hunt JR. Dyssnergia cerebellaris progressivaa chronic progressive form of cerebellar tremor. Brain 37:247-268, 1914. Hutton JT, Frost JD, and Foster J. The influence of the cerebellum in cat penicillin epilepsy. Epilepsia 13:401-408, 197 2. Iwata K and Snider R. Cerebello-hippocampal influences on the electroencephalogram. EEG Clin Neurophysiol 11:439-446, 1959. Julien RM. Cerebellar involvement in the antiepileptic action of diazepam. Neuropharmacology 11:683-691, 1972. Julien RM. Experimental epilepsy: Cerebrocerebellar interaction and antiepileptic drugs. In: IS Cooper, M Riklan, and RS Snider (Eds), The Cerebellum, Epilepsy and Behavior. Plenum Press, New York, pp 97-118, 1974. Julien RM and Halpern LM. Diphenylhydantoin: Evidence for a central action. Life Sci 10:575-582, 1971. Julien RM and Halpern LM. Effects of diphenylhydantoin and other antiepileptic drugs on epileptiform activity and Purkinje cell discharge rate. Epilepsia 13:387-400, 1972. Kopeloff LM and Alexander GJ. Mechanism of p-chlorophenyl-alanine-mediatedincrease in seizure susceptibility: Inhibition by cerebellar ablation. Proc SOC E x p Biol Med 139:647-651, 1972. Lewin J and Esplin DW. Analysis of the spinal excitatory action of pentylenetetrazol. J Pharmacol E x p Ther 132:245-250, 1961. Litchfield JT and Wilcoxon F. A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther 96:99-113, 1949. Mutani R, Bergamini L, and Doriguzzi T. Experimental evidence for an existence of an extrarhinencephalic control of the activity of the cobalt rhinencephdic epileptogenic focus. Epilepsia 10:351-362, 1969. Reimer GR, Grimm RJ, and Dow RS. Effects of cerebellar stimulation on cobalt-induced epilepsy in the cat. EEG Clin Neurophysiol 23:456-562, 1967. Russell JSR. Experimental researches into the functions of the cerebellum. Philos Trans R Soc Lond 185:819-861, 1895. Schallek W, Schlosser W, and Randall LO. Recent developments in the pharmacology of the benzodiazepines. A d v Pharmacol Chemother 10:119-183, 1972. Schlosser W, Zavatsky E, Franco S, and Sigg EG. Analysis of the action of CNS

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depressant drugs on somato-somatic reflexes in the cat. Neuropharmacology 14:517-523, 1975. Straw RN and Mitchell CL. Study on the paroxysmal bioelectrical activity and overt seizure patterns elicited by pentylenetetrazol in the cat. Arch Int Pharmacodyn Ther 170:22.30, 1 9 6 7 ~ . Straw RN and Mitchell CL. The effect of pentylenetetrazol on bioelectrical activity recorded from cat brain. Arch Int Pharmacodyn Ther 168:456-466, 19676. Swinyard EA, Brown WC, and Goodman LS. Comparative assays of antiepileptic drugs in mice and rats. J Pharrnacol Exp Ther 106~319-330, 1952. Toman JEP and Taylor JD. Mechanism of action and metabolism of anticonvulsants. Epilepsia, Third Series, 31-48, 1952. Woodbury DM. Role of pharmacological factors in the evaluation of anticonvulsant drugs. Epilepsia 10 :121-144, 1969.

ACKNOWLEDGMENT This work was supported by grant no. lROl NS 10667 from the U.S. Public Health Service.

SUMMARY Rats were cerebellectomized 72-96 hr prior to evaluation (1)during maximum electroshock seizures and (2) for their capacity to respond to pentylenetetrazol-induced clonic seizures. Cerebellectomized rats failed to exhibit tonic hindlimb extension, an endpoint characteristic of maximal electroshock seizures. The dose of pentylenetetrazol required to produce clonic seizures or death was not different in cerebellectomized and sham-operated controls. The anticonvulsant efficacy of diazepam, when assessed as a pentylenetetrazol antagonist, was not influenced by removal of the cerebellum. These data indicate that whereas cerebellar influences may suppress seizure activity which is largely focal, seizures of more diffuse origin are not markedly influenced by cerebellar activity. It is, therefore, essential that the role of the cerebellum in suppressing seizures be characterized for each kind of experimentally induced seizure process.

RESUMEN Varias ratas fueron cerebelectomizadas 72-96 horas antes de la evaluacidn de las convulsiones a1 electroshock m6ximo y de su capacidad para responder a la induccidn de convulsiones cldnicas con el pentilentetrazol. Las ratas cerebelectomizadas no demostraron extensidn tbnica de las patas traseras, dato fundamental y caracteristico d e las convulsiones al electroshock mlximo. La dosis de pentilentetrazol necesaria para producir convulsiones cldnicas o la muerte no fub distinta en 10s animales cerebelectomizados o en 10s operados-control. La eficacia anticonvulsiva del diazepan, determinada como antagonista del pentilentetrazol, no se modificd a1 extirpar el cerebelo. Estos datos indican que mientras que el cerebelo puede influenciar suprimiendo la actividad convulsivdgena fundamentalmente focal, 10s ataques de origen mls difuso no se modifican sustancialmente por la actividad cerebelosa. Resulta, pu&, esencial que la funcidn del cerebelo como supresor de convulsiones debe ser caracterizada para cada tipo de proceso epileptic0 inducido experimentalmente. (A. Portera, Madrid)

ZUSAMMENFASSUNG Cerebellektomierte Ratten wurden 72-96 Stunden nach dem Eingriff getestet wie sie sich im maximalen Elektroschockkrampf verhielten und wie stark sie auf Pentylentetrazol mit klonischen Krampfen reagierten. Cerebellektomierte Ratten zeigten keine tonische Extension der Hinterpfoten, ein charakteristisches Endzeichen der maximalen Elektroschockkrampfe. Die Dosis Pentylentetrazol, die notwendig war, klonische Krampfe oder den Tod hervorzurufen, war bei cerebellektomierten und scheinoperierten Kontrollen gleich hoch . Die antikonvulsive Wirkung des Diazepam als Antagonist des Pentylentetrazol wurde durch die Entfernung des Kleinhirns nicht beeinflusst. Diese Ergebnisse weisen daraufhin das Anfalle eher diffusen Ursprungs durch die cerebellare Aktivitat nicht wesentlich beeinflusst werden. obwohl cerebelltre EinflGsse die Gberwiegend fokale Krampfaktivitat unterdrucken konnen. Es ist deshalb notwendig, dass die Bedeutung des Cerebellums in der Anfallsverhutung fur jede Art der experimentell induzierten Krampfe bestimmt werden muss. (D. Scheffner, Heidelberg)