640

Electroencephalography and Clinical Neurophysiology, 1977, 4 2 : 6 4 0 - - 6 5 5 © Elsevier/North-Holland Scientific Publishers Ltd.

P A T H O P H Y S I O L O G Y OF G E N E R A L I Z E D PENICILLIN EPILEPSY IN THE CAT: THE ROLE OF CORTICAL AND SUBCORTICAL STRUCTURES * I. SYSTEMIC APPLICATION OF PENICILLIN L.F. QUESNEY, P. GLOOR, E. KRATZENBERG and H. ZUMSTEIN **

Department of Neurology and Neurosurgery, McGill University and Montreal Neurological Institute, 3801 University Street, Montreal, Quebec H3A 2B4 (Canada) (Accepted for publication: August 24, ]976)

Generalized penicillin epilepsy of the cat presents many similarities to human generalized corticoreticular ('centrencephalic') epilepsy. This applies to its electrophysiological and behavioral manifestations, as well as to its responses to several pharmacological agents (Prince and Farrell 1969; Gloor and Testa 1974; Testa and Gloor 1974; Guberman et al. 1975). To study the physiological features of this model seems to be worthwhile, since it may clarify some of the still unanswered questions concerning the possible pathophysiological mechanism of the human condition. Such studies may in particular shed some light on the still unresolved problem of whether the main underlying disturbance responsible for the generalized epileptiform discharges resides in deep midline subcortical structures (Jasper and Droogleever-Fortuyn 1947; Hunter and Jasper 1949; Penfield and Jasper 1954; Pollen 1968; Pollen et al. 1963; Weir 1964), in the cerebral cortex (Gibbs and Gibbs 1953; Marcus and Watson 1966, 1968; Marcus et al. 1968a,b; Bancaud 1971; Goldring 1972; Bancaud et al. 1974) or in both (Gloor 1968, 1969, 1972). The present paper * A brief report on these investigations was presented in October, 1975 at the Joint Meeting of the American and Mexican EEG Societies in Mexico City. This work was supported by Operating Grant MT3140 of the Medical Research Council of Canada. ** Present address: Liebeggerweg, 5000 Aarau, Switzerland.

presents some results of an ongoing series of neurophysiological investigations carried out on this model of generalized epilepsy. It deals with some of the possible mechanisms involved in the precipitation of the bilaterally synchronous epileptic discharges observed in this model.

Materials and methods

Chronic and acute experiments were carried o u t in 41 adult cats.

Chronic experiments In 17 adult male cats weighing between 3.0 and 3.7 kg intracerebral electrodes were stereotaxically implanted into various cortical and subcortical structures according to the coordinates of the stereotaxic atlas of Jasper and Ajmone Marsan (1954). A Kopf stereotaxic frame was used. The intracerebral electrodes consisted of bipolar concentric 24gauge stainless-steel needles with an interelectrode distance of 0.1 mm and a resistance of 40 k~2. The sites of electrode implantation in chronic experiments are shown in Table I. The surface EEG was recorded from the cortical convexity by means of 10 stainlesssteel screws chronically inserted into the skull in bilaterally symmetrical positions over the frontal, central, parietal and occipital brain

641

GENERALIZED PENICILLIN EPILEPSY IN THE CAT TABLE I Sites of electrode implantation. Anatomical structure

Chronic experiments

Acute experiments

N,C.M. (nucleus centralis medialis including nucleus reuniens) N. Retic. (nucleus reticularis -- oral pole) V.A. (nucleus ventralis anterior) P.C. (nucleus paracentralis) . C.L. (nucleus centralis lateralis) L.P. (nucleus lateralis posterior) G.M. (medial geniculate body) G.L. (lateral geniculate body) V.L. (nucleus ventralis lateralis) Pulvinar M.D. (nucleus dorsomedialis) Claustrum Putamen Caudate nucleus S.S.G. (suprasylvian gyrus) Gyrus cinguli Orbito-frontal cortex Gyrus proteus Am (amygdala) Hp (hypothalamus) C.C. (corpus callosum) I.C. (internal capsule) M.R.F. (mesencephalic reticular Formation) A.T.R. (anterior thalamic radiation) I.T.P. (inferior thalamic peduncle)

5

2

1 1 0 0 2 3 1 1 l 0 1 1 1 2 0 0 0 2 1 1 1 1b 0 0

2 2 2 1 2 1 1 1 1 1 0 1 3 1 1 1 1 1 1 1 2a 2 1 1

a A generalized seizure occurred shortly after the onset of I.C. stimulation in one of the animals. b Only the effects of high frequency stimulation were studied in this animal (data obtained in this animal are not included in Fig. 7). regions corresponding to the sigmoid and the s u p r a s y l v i a n g y r i ( F i g . 1, A ) . T h e i n t e r e l e c trode distances were 5 mm. In addition, a r e f e r e n c e s c r e w e l e c t r o d e w a s i n s e r t e d in t h e external occipital protuberance. Both the intracerebral and the skull electrodes were connected to a 20-hole Winchester plug which was s c r e w e d t o t h e s k u l l a n d w a s f i r m l y f i x e d t o it w i t h d e n t a l a c r y l i c c e m e n t . All surgical procedures were carried out under aseptic conditions under pentobarbital (Nembutal 30 mg/kg intraperitoneally) anesthesia. During the operation, single shock and repetitive stimulation was carried out through t h e i n t r a c e r e b r a l e l e c t r o d e s in o r d e r t o c h e c k t h e i r p o s i t i o n in t h e l i g h t o f e l e c t r o p h y s i o l o -

gical c r i t e r i a . A c o n s t a n t c u r r e n t N u c l e a r C h i c a g o s t i m u l a t o r w a s u s e d f o r t h e s e a n d all other subsequent stimulations. Single shock A

B

4o

o9

Fig. 1. Electrode positions in chronic (A) and acute experiments (B).

642 and low frequency (2.5--8 c/sec) stimulation of the nonspecific thalamic nuclei triggered barbiturate spindle activity or induced recruiting responses. Stimulation of specific thalamic nuclei elicited evoked potentials which were confined to the appropriate cortical projection areas. The intracerebral electrodes were fixed into their final positions after the desired electrophysiological responses had been obtained. The experiments on the epileptic condition were begun 7--10 days after surgery at a time when neither the behavior nor the EEG showed any residual effects of the surgical procedure. Bipolar and monopolar EEGs were recorded from the surface and deep intraeerebral electrodes on an 8-channel Mingograph EEG recording instrument. During the experimental sessions which lasted from 6 to 8 h, the animals were allowed to move about freely within a well ventilated wooden box (43.5 × 38.5 X 51 cm) with a clear transparent plastic front panel. Most experiments were performed in the same room and the background level of sensory stimulation was kept low in order to promote relaxation of the animals. Each experimental session consisted of three stages. In the first stage, the background EEG activity was recorded from the surface and the deep intraeerebral electrodes for approximately half an hour. In the second stage, single shock and/or repetitive stimulation was performed through the intracerebral electrodes while the EEG was being recorded simultaneously. At the beginning of the third stage, an aqueous solution of penicillin G sodium was injected intramuscularly in doses ranging from 300,000 to 400,000 I.U./kg. The EEG was then recorded uninterruptedly for 4--6 h. Approximately 2--3 h after the penicillin administration when the spontaneous generalized penicillin epiletic activity had reached its peak (Gloor and Testa 1974; Guberman and Gloor 1974; Quesney et al. 1975), various cortical and subcortical structures were stimulated in the intervals between spontaneous

L.F. QUESNEY ET AL. epileptic bursts through the intracerebral electrodes and the effects upon the EEG, particularly those on the generalized epileptic bursts, were observed. All animals were studied in repeated experimental sessions, but a period of 3 days was always allowed to elapse prior to the next experimental session. Following completion of the experiments, the animals were deeply anesthetized with pentobarbital {30 mg/kg intraperitoneally). A 2.0--2.5 mA direct current was applied to each intracerebral electrode for 30 sec and 10--12 ml of a potassium ferrocyanide solution was injected intravenously. The animals were then exsanguinated and perfused through the heart with 20--30 ml of normal saline and subsequently with 80--100 ml of a 10% formalin solution. The brain was fixed in formalin and 50 p thick histological sections stained with cresyl violet were prepared for verification of the electrode positions. A cu te e x p e r i m e n t s

Twenty-four cats of both sexes weighing between 2.6 and 3.6 kg underwent a tracheost o m y under ether anesthesia. Artificial respiration was started after connecting the tracheostomy tube to an intermittent positive pressure Bird Mark 14 respirator. Halothane at a concentration of 2--2.5% was then given as an anesthetic agent. A wide craniotomy was performed preserving the dura mater intact. Intracerebral electrodes were implanted using the same stereotaxic technique as in chronic animals. The sites of electrode implantation in acute experiments are listed in Table I. Following the insertion of the electrodes, the animals were painlessly fixed in a Kopf semichronic head holder through screws attached to the skull. There were no ear or eye bars. Halothane anesthesia was discontinued and the animals received periodic intravenous injections of small amounts of gallamine triethiodide (Flaxedill, 4 mg/ml) and of fentanyl citrate (Sublimaze, 0.015 mg/ ml), a potent narcotic analgesic which does not affect the normal or abnormal EEG (per-

G E N E R A L I Z E D P E N I C I L L I N E P I L E P S Y IN THE CAT

sonal observations). The use of this drug together with the painless fixation of the head prevented pain and anxiety. The absence of mydriasis and the abundance of epileptiform discharges after penicillin (which are known to be very sensitive to arousal stimuli) indicated that the animal suffered no discomfort. The expired CO2 was monitored with a Beckman infrared analyzer. The respiratory rate was adjusted to maintain the CO2 level at about 4%. The surface EEG was recorded with silverball electrodes applied against the dura overlying the sigmoid and syprasylvian gyri bilaterally. The electrode positions are shown in Fig. 1, B. Each experimental session consisted of three stages and various intracerebral structures were electrically stimulated, as in the chronic experiments. At the end, the animals were killed and the brains processed for histological study using the same techniques as used in the chronic experiments.

Results

I. Effect of stimulation o f cortical and subcortical structures on the EEG, prior to penicillin administration a. Stimulation of nonspecific thalamic nuclei (N.C.M., V.A., oral pole of N. Retic., P.C., C.L. *) The effects on the cortical EEG produced by stimulation of the nonspecific thalamic nuclei were consistent and similar in acute and chronic experiments. Under pentobarbital anesthesia single shock stimulation of the intralaminar nuclei triggered barbiturate spindles bilaterally as originally described by Jasper and Droogleever-Fortuyn (1947). Single shock stimulation of these nuclei in awake animals also triggered stimulus-bound spindle activity which was recorded from the cortex of both hemispheres (Fig. 2, A). In awake animals the spindle waves were fre* A key to t h e a b b r e v i a t i o n s used in this p a p e r is t o be f o u n d in Tables I and II.

643

quently associated with some slower wave forms which made the spindles look somewhat irregular. Under pentobarbital anesthesia, repetitive low frequency stimulation of the nonspecific thalamic nuclei induced barbiturate spindle activity bilaterally (Fig. 3, A ). Similar stimulation carried out in awake animals produced a recruiting type of response which was recorded from the cortex of both hemispheres and also from thalamic and other subcortical structures (Fig. 3, B). Spindles or recruiting responses were elicited with such stimulations of the nonspecific thalamic nuclei in 80--100% of the trials. In only 2 animals was the phenomenon described by Jasper and Droogleever-Fortuyn (1947) observed, namely the occurrence of generalized bilaterally synchronous spike and wave activity in response to 2.5--3 c/sec stimulation of the intralaminar nuclei. These discharges were phase-locked to the frequency of stimulation and outlasted its end by one beat only (Fig. 4, A). b. Stimulation of specific thalamic nuclei (G.M., G.L., V.L., M.D., L.P. and Pulvinar) Single shock or repetitive stimulation of these nuclei evoked unilateral localized potentials which were confined to the cortical projection area of the nucleus which was being stimulated. Spindle activity was not elicited very often (10% or less of the time) and no recruiting responses were seen, except for stimulation of L.P. or of the Pulvinar which in some acute and chronic experiments produced a generalized or unilateral response resembling spindle activity or a recruiting response with an incidence ranging from 65 to 100%. It is unlikely that these effects were caused by spread of current to the intralaminar nuclei, since stimulation of other specific or association nuclei (V.L. and M.D.) which are much closer to the intralaminar nuclei did not elicit spindles or recruiting responses. c. Stimulation of extrathalamic sites 1. Basal ganglia (Putamen, Caudate nucleus and Claustrum). In acute and chronic experiments, stimulation of these structures trig-

644

L.F. QUESNEY ET AL.

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gered w i d e s p r e a d or generalized responses r e s e m b l i n g spindle activity or recruiting r e s p o n s e s in 85 to nearly 100% o f the trials. 2. Other extrathalamic sites. T h e r e was a low incidence o f spindle activity and t h e r e were n o recruiting responses with s t i m u l a t i o n o f the f o l l o w i n g s t r u c t u r e s : surface o f the suprasylvian gyrus (S.S.G.), o r b i t o - f r o n t a l cortex, gyrus p r o r e u s , a m y g d a l a ( A m ) , h y p o t h a l a m u s (Hp), c o r p u s c a l l o s u m (C.C.), internal capsule (I.C.), a n t e r i o r t h a l a m i c r a d i a t i o n (A.T.R.), and i n f e r i o r t h a l a m i c p e d u n c l e

(I.T.P.). Spindles were triggered f r o m these s t r u c t u r e s in 0 - - 2 0 % o f the trials. L o w freq u e n c y s t i m u l a t i o n o f the m e s e n c e p h a l i c reticular f o r m a t i o n (M.R.F.) was carried o u t in t w o a c u t e p r e p a r a t i o n s . In o n e o f these, there was n o r e s p o n s e in the E E G , while in the o t h e r in 34% o f the trials, an ill-defined spindle-like activity was elicited f r o m b o t h h e m i s p h e r e s w i t h p r e d o m i n a n c e on the side ipsilateral to the s t i m u l a t i n g e l e c t r o d e . Fig. 7, A and 8, A s h o w h o w o f t e n spindle activity or r e c r u i t i n g r e s p o n s e s were elicited b y

GENERALIZED PENICILLIN EPILEPSY IN THE CAT

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Stim NCM Caudat= Fig. 3. Low frequency repetitive stimulation of N.C.M. in the midline prior to and after intramuscular administration of penicillin (chronic experiment). A: Elicitation of barbiturate spindle activity under Nembutal anesthesia before penicillin. B: Elicitation of recruiting reponse recorded from cortex and subcortical structures before penicillin. C: Triggering of a generalized spike and wave burst following repetitive stimulation of N.C.M. after penicillin. s t i m u l a t i o n o f d i f f e r e n t brain s t r u c t u r e s in c h r o n i c and a c u t e e x p e r i m e n t s ( T h e k e y f o r the a b b r e v i a t i o n s used in Fig. 7 and 8 is s h o w n in T a b l e II.) T w o clearly segregated groups can be identified: the g r o u p on the right, c o m p r i s i n g the u n s p e c i f i c t h a l a m i c nuclei, s o m e basal ganglia, L.P. and the Pulvinar, e x h i b i t s a high incid e n c e o f elicitation o f spindle activity or

r e c r u i t i n g responses; in c o n t r a s t , the g r o u p on the left c o m p r i s i n g all the o t h e r s t r u c t u r e s shows a l o w i n c i d e n c e o f elicitation o f spindle activity and r e c r u i t i n g responses. T h e r e is a d e f i n i t e gap b e t w e e n t h e t w o g r o u p s w i t h n o overlap. O n l y the cingulate gyrus o c c u p i e s an i n t e r m e d i a t e p o s i t i o n b e t w e e n the t w o g r o u p s with a m o d e r a t e i n c i d e n c e o f spindle triggering (60%).

646

L.F. QUESNEY ET AL.

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Fig. 4. A : Triggering of generalized bilaterally synchronous spike and wave activity in cortex and in subcortical structures (left V.A., left G.L. and right L,P.) during l o w frequency stimulation of N.C.M. in the midline prior to intramuscular administration of penicillin. B: Same as A after intramuscular administration of penicillin. Note that the epileptic discharges n o w outlast the end of stimulation. A spontaneously occurring generalized epileptic burst is also s h o w n (chronic experiment).

II. Penicillin-induced generalized epilepsy In chronic as well as in acute experiments, the intramuscular injection of high doses of penicillin was followed, as reported previously, by the appearance of generalized and bilaterally synchronous bursts of epileptic activity (Prince and Farrell 1969; Gloor and Testa 1974; Guberman and Gloor 1974; Guberman et al. 1975; Quesney et al. 1975) (Fig. 5). Three main types of bilaterally syn-

chronous epileptic discharges were observed similar to those described by Gloor and Testa (1974): (i) bursts of spike and wave activity at a frequency of 3--4.5 c/sec which fulfilled all the criteria for typical spike and wave complexes as defined by Weir (1965); (ii) bursts of multiple spike and wave activity; and (iii) bursts of sharp and slow wave complexes. Each individual animal exhibited a rather consistent discharge pattern throughout one experiment, or from experiment to

G E N E R A L I Z E D PENICILLIN EPILEPSY IN THE CAT

experiment. The epileptic bursts usually lasted from 2 to 5 sec. In acute experiments, the spike c o m p o n e n t was more prominent than in chronic ones (Fig. 5), presumably because the EEG was recorded from the dura and not from the skull. In chronic experiments, generalized epileptic discharges began to appear approximately 30--45 min after the administration of penicillin; in acute experiments, they appeared only 1 or 2 h after the penicillin injection, probably because of a lingering aftereffect of

647

the halothane anesthesia. The peak of the epileptic activity in chronic animals was reached 120--180 min after the intramuscular administration of penicillin with progressive diminution of the epileptic activity thereafter. The EEG of 2 animals studied with chronically implanted electrodes was recorded for 24 h with an Oxford 4 channel cassette tape recorder {Ives and Woods 1975). No significant epileptic activity was seen 12 h after a single intramuscular administration of penicillin.

Fig. 5. Generalized epileptic activity recorded from cortical and subcortical structures. A: Chronic experiment (midline N.C.M., left V.A., left G.L., right L.P.). B: Acute experiment (right N. Retic., left P.C.).

648

L.F. QUESNEY ET AL.

onset was seen in the c o r t e x only; however, soon the subcortical s t r u c t u r e s also b e c a m e involved. We never observed i n d e p e n d e n t epileptic activity in subcortical s t r u c t u r e s witho u t cortical participation. Epileptic bursts in c h r o n i c e x p e r i m e n t s were f r e q u e n t l y associated with clinical signs such as staring, eye blinking, pupillary dilatation, licking and m y o c l o n i c jerks o f the face and neck. In a few animals, generalized tonic clonic convulsive seizures o c c u r r e d e i t h e r s p o n t a n e o u s l y or following several trains of repetitive stimulation of the nonspecific thala-

In m o s t animals, generalized p a r o x y s m a l E E G abnormalities were r e c o r d e d simult a n e o u s l y f r o m the c o r t e x and f r o m subcortical structures. T h e a b n o r m a l electrical activity r e c o r d e d f r o m subcortical structures was variable. It consisted o f r h y t h m i c slow waves and assumed a typical or atypical spike and wave f o r m in some instances (Fig. 7). On the basis o f o u r ink-written EEGs, these subcortical p a r o x y s m a l discharges a p p e a r e d to be roughly s y n c h r o n o u s with those r e c o r d e d f r o m the c o r t e x . This included their onset and end. In a few animals, epileptic activity at its

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GENERALIZED PENICILLIN EPILEPSY IN THE CAT mic nuclei. The epileptic discharges increased during drowsiness or sleep as reported earlier (Guberman and Gloor 1974). In some animals, epileptic bursts were triggered by noise or intermittent photic stimulation.

IIL Effect of stimulation of different brain structures on the elicitation of generalized penicillin-induced epileptic activity Single shock or low frequency (2.5--8 c/sec) repetitive stimulation of various cortical and subcortical structures frequently elicited the occurrence of generalized epileptic activity as a stimulus-bound phenomenon. A burst of generalized epileptiform activity was considered to have been triggered by such stimulation if its onset occurred within a 200 msec period after single or repetitive stimulation or at any time during a train of repetitive stimulation. The value of 200 msec was chosen, because it represented the upper limit of the latencies for spindle triggering to single shock thalamic stimulation in Pollen et al.'s (1963) experiments. These stimulationinduced bursts were in all respects similar to those occurring spontaneously. Not all structures were equally effective in precipitating a generalized epileptiform burst in response to stimulation. In chronic experiments (Fig. 7, B) two clearly segregated groups of brain structures can be distinguished with regard to the likelihood with which upon their stimulation generalized epileptiform discharge occurred in the EEG. A t test shows a statistically significant difference between the two groups (P < 0.001). The group on the right side shows a high percentage of triggering of epileptiform activity in response to such stimulation (55 to nearly 100% of the trials). This group comprises the nonspecific thalamic nuclei (N.C.M., V.A., oral pole of N. Retic.), some association nuclei (Pulvinar, L.P.) and some basal ganglia (Putamen, Caudate nucleus and Claustrum). Fig. 2, B and 3, C show examples of such responses. These highly effective structures share another common physiological property: prior to penicillin injec-

649 tion, they all responded to single shock or low frequency repetitive stimulation by eliciting cortical spindle activity or recruiting responses with a high degree of probability (Fig. 7, A). In the 2 animals in which low frequency stimulation of the intralaminar thalamic nuclei prior to penicillin had elicited bilaterally synchronous spike and wave discharges, the same type of stimulation after penicillin triggered generalized epileptiform bursts which were now no longer phaselocked to the stimulation and definitely outlasted its end (Fig. 4, B). The group of structures on the left side in Fig. 7, B comprises those from which in chronic experiments generalized epileptiform bursts could only be elicited with a low degree of probability (0--35% of the trials) (Fig. 6, A and B). Prior to penicillin, very little spindle activity and no recruiting responses could be obtained with stimulation of these structures (Fig. 7, A). This group includes specific thalamic nuclei (G.M., G.L., V.L.) and extrathalamic sites (Hp., Am., S.S.G., I.C. and C.C.). The results obtained in acute experiments were similar to those obtained in the chronic ones (Fig. 8, B). With regard to the probability of triggering of generalized epileptic activity by single shock or low frequency electrical stimulation, the brain structures which were explored could again be divided into at least two and possibly three separate groups. Statistically the group on the right is significantly different from that on the left (P < 0.001). The group on the right in Fig. 8, B comprises structures from which a very high percentage of single shock or low frequency repetitive stimulation resulted in triggering of generalized epileptic bursts (75 to nearly 100%). This group includes the nonspecific thalamic nuclei (N.C.M., V.A., oral pole of N. Retic., Pc and C.L.), but thalamic association nuclei (L.P. and Pulvinar) and two basal ganglia (Caudate nucleus and Putamen) are also included in this highly effective group. Prior to penicillin injection, stimulation of these

L.F. Q U E S N E Y ET AL.

650 T A B L E II Key to figs. 7 a n d 8.

Unspecific thalamic nuclei

Extrathalamic sites

Ai A2 A3 A4 As

Basal ganglia: B1 = C l a u s t r u m B2 = P u t a m e n B3 = C a u d a t e nucleus

= = = = =

nucleus nucleus nucleus nucleus nucleus

centralis medialis (N.C.M) reticularis (N. Retic. - oral pole) ventralis a n t e r i o r (V.A.) paracentralis (P.C.) centralis lateralis (C.L.)

Others Specific and association thalarnic nuclei C1 C2 C3 C4 C5 C6

D1 = suprasylvian gyrus (S.S.G.) D2 = gyrus cinguli D3 = o r b i t o - f r o n t a l c o r t e x D4 = gyrus p r o r e u s Ds = a m y g d a l a ( A m ) D5 = h y p o t h a l a m u s (Hp) D7 = c o r p u s c a l l o s u m (C.C.) Ds = i n t e r n a l capsule (I.C.) D9 = m e s e n c e p h a l i c reticular f o r m a t i o n ( M . R . F . ) D O = anterior thalamic radiation (A.T.R.) D x = inferior t h a l a m i c p e d u n c l e (I.T.P.)

= n u c l e u s lateralis p o s t e r i o r (L.P.) = medial geniculate b o d y (G.M.) = lateral geniculate b o d y (G.L.) = nucleus ventralis lateralis (V.L.) = Pulvinar = n u c l e u s d o r s o m e d i a l i s (M.D.)

Elicitatlon of Spindle Activity or Recruiting Response Following Stimulation of Cortical and Subcortical Slructures Prior to Penicillin (Chronic Experimenlsl

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Fig. 7. P e r c e n t a g e of trials of single s h o c k a n d l o w f r e q u e n c y r e p e t i t i v e s t i m u l a t i o n o f various brain s t r u c t u r e s in c h r o n i c e x p e r i m e n t s w h i c h elicited spindle activity a n d / o r r e c r u i t i n g r e s p o n s e s before penicillin (A), a n d generalized e p i l e p t i c b u r s t s after penicillin (B). T h e various s t r u c t u r e s are labelled w i t h letters a n d s u b s c r i p t e d n u m b e r s w h i c h can be i d e n t i f i e d b y referring to t h e key in T a b l e II. Each s y m b o l r e p r e s e n t s the average p e r c e n t a g e value of several e x p e r i m e n t s carried o u t for a single brain s t r u c t u r e in a given animal. T h e vertical a r r o w indicates t h e level of c h a n c e at w h i c h b r a i n s t i m u l a t i o n is associated with o c c u r r e n c e o f a s p o n t a n e o u s b u r s t of epileptic activity.

D6 Da D5 DI I 0

Do I }0

I 20

D9 I 30

D2 I 40

t 50 Percent

:0

A5 t 70

I 80

1 90

i 100

Effect of Stimulation of Di|ferent Brain Structures on the Triggering of Generalized Penicillin Epileptic Activity in the Cat (Acute Experiments)

B

B2 C5

D4 D9 D5 D6 C4C6De D1 D3 DxC3 C2Oo

D2 D7

D~ Percent

A4As B3

C~ B3 A3 A~A3

CI A2A2B3 AIA I 80

90

I00

Fig. 8. T h e same as Fig. 7 for acute e x p e r i m e n t s , e x c e p t t h a t each s y m b o l r e p r e s e n t s t h e p e r c e n t a g e value f o u n d in a single e x p e r i m e n t a l session for each s t r u c t u r e in a given animal.

GENERALIZED PENICILLIN EPILEPSY IN THE CAT elicited only a low percentage of stimulationinduced generalized epileptic activity (approximately 0--30%). This group includes the specific thalamic nuclei (G.M., G.L., V.L., M.D.) and many extrathalamic structures such as Hp, Am, S.S.G., orbito-frontal cortex, gyrus proreus, I.T.P., I.C., M.R.F., A.T.R. and C.C. Stimulation of these structures prior to penicillin injection elicited little spindle activity and failed to induce recruiting responses (Fig. 8, A). In the acute experiments, two brain structures assumed an intermediate position between the two main groups and may be considered as a separate third group. They included the cingulate gyrus and the M.R.F. The incidence of elicitation of epileptiform activity by stimulation of these two structures was o f the order of 50--55%, but there was no overlap with either the high or the low probability group. Prior to penicillin, at least one of these structures, the cingulate gyrus, showed an intermediate percentage of triggering of spindle activity upon stimulation (Fig. 8, A). It should be noted that high frequency stimulation at 30--60 c/sec of the M.R.F. reduced the incidence of epileptic discharges in one chronic and two acute preparations, a finding which was entirely expected in view of the earlier observations made by Gloor and Testa (1974) and by Testa and Gloor (1974). The question arises to what e x t e n t chance application o f electrical stimulation at the time when a spontaneous burst was about to occur may provide spurious evidence for a stimulation-induced response, or in o t her words, what are the percentage values in Fig. 7, B and 8, B which would correspond to a chance association of stimulation and occurrence of a spontaneous epileptic burst. The probability of such a chance association was established in two ways: it was first calculated on a theoretical basis taking into consideration the following features: stimulations were always carried o u t in between spontaneous epileptic bursts and at a time when the generalized penicillin epilepsy had reached its peak. At that time, there was an

651 average of 4 bursts/min (Quesney et al. 1975). The duration of each epileptic burst varied from 2 to 5 sec. Stimulation was not delivered earlier than 1 sec after a burst. Therefore, the real time during which stimulation was delivered varied from 36 to 48 sec/min (60 sec -(4 X 6) sec = 36 sec, 60 s e c - - (4 X 3) sec = 48 sec). Since the burst frequency was 4/min, the average available time for stimulation between bursts varied from (36 : 4) = 9 sec to (48 : 4) = 12 sec. We had made the asumption that epileptic bursts occurring during repetitive stimulation or within 200 msec following a single shock or the end of a train of repetitive stimulation had been triggered by such a stimulation, the average duration of a train of repetitive stimulation being 650 msec in both chronic and acute experiments. The probability t hat an epileptic burst could be recorded merely by chance during or within a 200 msec interval after a period of stimulation can thus be calculated according to the following formula for bursts of 5 or 2 sec duration respectively: (200 msec + 650 msec)/9,000 msec = 0.094 (9.4%) and (200 msec + 650 msec)/12,000 msec = 0.071 (7.1%). On the basis of these calculations, any percentage of association of brain stimulation with the occurrence of a generalized epileptic burst which exceeded 10% was suggestive evidence that at least some of the bursts were stimulation-induced and did not occur by chance. The probability of chance association of stimulation and epileptic bursts was also established experimentally by the following m e t h o d : m o c k stimulations were delivered in the same m anner as in the actual stimulation experiments. (A t test showed that there was no statistical difference in the durations between actual and m o c k stimulations.) In these experiments the o u t p u t of the stimulator was connected with the recording instrument in such a way as to produce a stimulus artifact on the EEG record, but not stimulation was actually delivered to the brain. The association between m ock stimulations and the occurrence of bursts was determined in the same manner as in the ot her experiments.

652

Using this method, we established that the probability that stimulation and onset of a burst of generalized epileptic activity would coincide by chance was 8.5%, a figure which is almost identical with the values derived from actual stimulation experiments. The theoretically and experimentally established probabilities are sufficiently close to allow one to conclude that all values of association of stimulation with a burst which are above 10% of the total number of stimulations given could not be attributed to chance. On this basis, stimulation of the following brain structures were considered to be at least at times ineffective in precipitating generalized epileptiform discharges: Hp, V.L., G.M., G.L., orbito-frontal cortex, S.S.G., C.C., and Am (Fig. 7, B and 8, B). In all other instances, we must conclude that the various brain structures stimulated must have been capable of triggering generalized epileptiform bursts. This includes some structures (V.L., G.M., G.L. and S.S.G.) which at times proved to be ineffective.

Discussion

In our laboratory, we have undertaken a series of experimental studies designed to clarify the interaction between cortical and subcortical structures in feline generalized penicillin epilepsy on the assumption that the results of such studies might clarify the mechanism of generalized corticoreticular epilepsy of man. Our earlier studies delineated the role of the ascending reticular formation in feline generalized penicillin epilepsy. We demonstrated that the midbrain reticular formation exerted, presumably through ascending cholinergic reticulocortical pathways a powerful inhibitory action upon the generalized epileptiform discharges in this model. (Gloor and Testa 1974; Guberman and Gloor 1974; Testa and Gloor 1974). The origin of the abnormal generalized epileptiform discharges and the mechanisms of their precipitation, however, still remained unclarified,

L.F. QUESNEY ET AL.

although it seemed probable that structures above the midbrain level, presumably the cortex or thalamo-cortical circuits were involved. The present experiments demonstrated that generalized epileptiform discharges in feline generalized penicillin epilepsy can be triggered from many parts of the cerebrum, but that the most potent trigger zones are those from which in the normal animal spindles can be elicited, particularly under barbiturate anesthesia, or from which bilateral recruiting responses can be induced by low frequency stimulation. These structures comprise the midline and intralaminar nuclei of the thalamus (the 'thalamic reticular system' of Jasper (1949)), portions of the basal ganglia (essentially the neostriatum and the Claustrum) and some posterior thalamic association nuclei (Pulvinar and L.P.) (Jasper 1949, 1960; Jasper and Droogleever-Fortuyn 1947; Starzl and Magoun 1951). In their 'classical' study of 1947, Jasper and Droogleever-Fortuyn had, of course, shown that 3 c/sec stimulation of the intralaminar and midline thalamic nuclei sometimes induced 3 c/sec bilaterally synchronous spike and wave discharges which were always phase-locked to the stimuli and never significantly outlasted the end of stimulation. Our observations confirm their observations, b u t also reemphasize the fact that such responses are not easily reproducible in the normal cat (Ingvar 1955). They also show that such stimulation-induced spike and wave discharges seen in normal animals become more vigorous after penicillin application: they then outlast the end of stimulation and are no longer phase-locked to the stimuli. The present study thus confirms Jasper's and Droogleever-Fortuyn's (1947) conclusion that the intralaminar and midline thalamic nuclei are structures which in some way are probably involved in the genesis of generalized bilaterally synchronous spike and wave discharges. The evidence presented in the present study is highly suggestive that thalamo-cortical volleys, which normally induce spindle activity, after penicillin injection frequenctly precipitate generalized spike

GENERALIZED PENICILLIN EPILEPSY IN THE CAT and wave discharges. Whether this transformation from a spindle response to an epileptic burst is primarily dependent upon altered neuronal and/or synaptic mechanisms at the thalamic, at the cortical or at both levels still remains undetermined. One of the interesting findings in the present study, however, is that the triggering of generalized epileptiform bursts in feline generalized penicillin epilepsy can also be obtained by electrical stimulation of structures other than those likely to induce spindles or recruiting responses in normal animals. The probability that electrical stimulation applied to these structures is followed by a bilateral burst of generalized epileptic activity is, however, significantly lower than for those parts of the brain from which spindle triggering or recruiting responses can be elicited with a high degree of probability. Such sites are widely distributed throughout the brain and comprise thalamic, brain stem, limbic and neocortical structures. In some of these the association between stimulation and the appearance of a generalized epileptiform occurs above change level, b u t in others the association seems to be fortuitous. These observations indicate that the generalized discharges in this model do not depend on a single fixed 'pacemaker', b u t that bilaterally synchronous epileptiform discharges can be triggered from many sites, with the intralaminar and midline thalamic nuclei, the neostriatum and a few thalamic association nuclei being significantly more effective than others. Whether spontaneous bursts are also primarily triggered from these structures remains unproven. However, it is likely that spike and wave discharges represent cortical postsynaptic potentials. Since no sites were found which are more effective in precipitating generalized epileptic bursts, it is quite conceivable that spontaneous bursts are often, although not necessarily always, triggered by thalamo-cortical volleys originating in the 'thalamic reticular system' or in structures physiologically closely associated with it (basal ganglia and some thalamic association nuclei}.

653 At first glance, our findings seem to suggest that the primary abnormality in feline generalized penicillin epilepsy is in fact subcortical and that the cortex responds to abnormal afferent input. Since recordings from subcortical structures show paroxysmal discharges which occur at the time of the cortical bursts, this conclusion appears quite plausible. However, it is also possible to assume that it is the cortex that responds abnormally to afferent impulses, whether they be normal or abnormal, and that the secondary reflection of this abnormal cortical activity which is projected back to subcortical structures is what appears in the EEG records obtained from subcortical nuclei. The observation that cortical epileptiform discharges were sometimes seen before any paroxysmal abnormality appeared in subcortical recordings is somewhat in favor of this explanation. A subsequent study (Gloor et al. in preparation) will address itself more fully to this problem and will present evidence suggesting that the alteration of neuronal activity responsible for the epileptic nature of the discharges in fact primarily resides in the cerebral cortex.

Summary The mechanism of precipitation of generalized epileptiform discharges in feline generalized penicillin epilepsy, a model of human generalized corticoreticular ('centrencephalic') epilepsy, was studied in acute and chronic experiments in cats with implanted skull and intracerebral electrodes. Single shock and low frequency repetitive stimulation of subcortical sites from which prior to penicillin administration spindle activity and recruiting responses could be elicited, readily triggered epileptiform discharges in the same animals after penicillin. These structures comprised the intralaminar and midline thalamic nuclei, the neostriatum, and some posterior thalamic association nuclei (Pulvinar and nucleus lateralis posterior). Subcortical and cortical structures which prior to penicillin

654 elicited neither spindle activity nor recruiting responses were significantly less effective in triggering generalized epileptic bursts after penicillin injection. The probability with which such bursts were elicited from these structures was still, however, in m any instances above chance level. It is concluded that the generalized epileptiform discharges in feline generalized penicillin epilepsy can be triggered from a large n u m b e r of brain sites, but most reliably so from subcortical nuclei involved in spindle generation and recruiting responses. The experimental evidence presented still does not allow one to determine whether epileptic alteration of neuronal function in this form of epilepsy primarily resides in cortical or subcortical nerve cells or in both.

R~sum~

Physiopathologie de l'epilepsie generalisee par penicilline chez le chat: role des structures corticales et sous corticales. 1. Application systemique de penicilline Le m~canisme de precipitation de d~charges ~pileptiformes g~n~ralis~es dans l'~pilepsie g~n~ralis~e oar p~nicilline du chat, module de l'~pilepsie g~n~ralis~e cortico-r~ticulaire ("centrencephalique") de l ' h o m m e , a ~t~ ~tudi~e en e x p e r i m e n t a t i o n aigu~e et chronique chez des chats avec ~lectrodes implant~es dans le cr~ne et dans le cerveau. Une stimulation par chocs isol~s et par stimulation r~p~titive ~ basse fr~quence de structures sous-corticales au niveau desquelles, avant l'administration de p~nicilline, on pouvait enregistrer une activit~ de t y p e spindles et des r~ponses par recrutement, d~clenche r ap i dem e nt des d~charges apr~s p~nicilline chez les m~mes animaux. Ces structures c o m p r e n n e n t les n o y a u x thalamiques intralaminaires et de la ligne m~diane, le n~ostriatum, et certains n o y a u x d'association thalamique post~rieure (Pulvinar et nucleus lat& ralis posterior). Les structures sous corticales et corticales qui, avant p~nicilline, ne montra-

L.F. QUESNEY ET AL. ient ni activit~ de spindles ni r~ponses par recrutement, sont de fa~on significative moins efficaces dans le d~clenchement de bouff~es ~pileptiques g~n~ralis6es apr~s injection de p~nicilline. La probabilit~ suivant laquelle de telles bouff~es sont provoqu~es ~ partit de ces structures demeure, toutefois, sup~rieure cells due au hasard dans beaucoup de cas. Les auteurs concluent que les d~charges ~pileptiformes g~n~ralis~es dans l'~pilepsie g~n~ralis~e par penicilline du chat peuvent 6tre d~clench~es ~ partir d'un grand n o m b r e de localisations c~r~brales, mais de fa~on plus nette ~ partit des n o y a u x sous-corticaux impliqu~s par la product i on des spindles et des r~ponses par recrutement. Les donn~es exp~rimentales pr~sent~es ne p e r m e t t e n t toujours pas de d~terminer si l'alt~ration ~pileptique de la f o n c t i o n nerveuse dans cette d'~pilepsie r~side essentiellement dans les cellules nerreuses corticales, dans les cellules nerveuses sous-corticales, ou dans les deux. References Bancaud, J. R6le du cortex c~r~bral dans les ~pilepsies 'g~n~ralis~es' d'origine organique. Rapport des investigations st~r~oenc~phalographiques (SEEG) la discussion de la conception 'centrenc~phalique'. Presse m~d., 1971, 79: 669--673. Bancaud, J., Talairach, J., Morel, P., Bresson, M., Bonis, A., Geier, S., Hereon, E. and Buser, P. 'Generalized' epileptic seizures elicited by electrical stimulation of the frontal lobe in man. Electroenceph, clin. Neurophysiol., 1974, 37: 275--282. Gibbs, F.A. and Gibbs, E.L. Atlas of electroencephaiography, Vol. 2: Epilepsy. Addison--Wesley Press, Inc., Cambridge, Mass., 1953, 422 p. Gloor, P. Generalized cortico-reticular epilepsies. Some considerations on the pathophysoplogy of generalized bilaterally synchronous spike and wave discharge. Epilepsia (Amst.), 1968, 9: 249--263. Gloor, P. Neurophysiological bases of generalized seizures termed centrencephalic. In H. Gastaut et al. (Eds.), The physiopathogenesis of the epilepsies. Thomas, Springfield, Ill., 1969: 209--236. GIoor, P. Generalized spike and wave discharges: a consideration of cortical and subcortical mechanisms of their genesis and synchronization. In H. Petsche and M.A.B. Brazier (Eds.), Synchronization of EEG activities in epilepsies. SpringerVerlag, New York, Wien, 1972: 382--402.

GENERALIZED PENICILLIN EPILEPSY IN THE CAT Gloor, P. and Testa, G. Generalized penicillin epilepsy in the cat: effects of intracarotid and intravertebral pentylenetetrazol and amobarbital injections. Electroenceph. clin. Neurophysiol., 1974, 36: 499--515. Goldring, S. The role of prefrontal cortex in grand mal convulsions. Arch. Neurol. (Chic.), 1972, 26: 109--119. Guberman, A. and Gloor, P. Cholinergic drug studies of generalized penicillin epilepsy in the cat. Brain Res., 1974, 78: 203--222. Guberman, A., Gloor, P. and Sherwin, A.L. Response of generalized penicillin epilepsy in the cat to ethosuximide and diphenylhydantoin. Neurology (Minneap.), 1975, 25: 758--764. Hunter, J. and Jasper, H.H. Effects of thalamic stimulation in unanaesthetized animals. Electroenceph. clin. Neurophysiol., 1949, 1: 305--324. Ingvar, D.H. Reproduction of the 3 per second spike and wave EEG pattern by subcortical electrical stimulation in cats. Acta physiol, scand., 1955, 33: 137--150. Ives, J.R. and Woods, J.F. 4-channel 24-hour cassette recorder for long-term EEG monitoring of ambulatory patients. Electroenceph. clin. Neurophysiol., 1975, 39: 88--92, Jasper, H.H. Diffuse projection systems: the integrative action of the thalamic reticular system. Electroenceph, clin. Neurophysiol., 1949, 1: 405--420. Jasper, H.H. Unspecific thalamocortical relations. In J. Field (Ed.), Handbook of physiology, Section I: Neurophysiology, Vol. 11. American Physiological Society, Washington, D.C., 1960: 1307--1321. Jasper, H.H. and Ajmone Marsan, C. A stereotaxic atlas of the diencephalon of the cat. National Research Council of Canada, Ottawa, 1954, 15 p. Jasper, H.H. and Droogleever-Fortuyn, J. Experimental studies on the functional anatomy of petit mal epilepsy. Res. Publ. Ass. nerv. ment. Dis., 1947, 26: 272--298. Marcus, E.M. and Watson, C.W. Bilateral synchronous spike wave electrographic patterns in the cat. (Interaction of bilateral cortical foci in the intact, the

655 bilateral cortico-callosal and adiencephalic preparation). Arch Neurol. (Chic.), 1966, 14: 601--610. Marcus, E.M. and Watson, C.W. Symmetrical epileptogenic foci in monkey cerebral cortex. Arch. Neurol. (Chic.), 1968, 19: 99--116. Marcus, E.M., Watson, C.W. and Simon, S.A. An experimental model of some varieties of petit mal epilepsy. Electrical-behavioral correlations of acute bilateral epileptogenic loci in cerebral cortex. Epilepsia (Amst.), 1968a, 9: 233--248. Marcus, E.M., Watson, C.W. and Simon, S.A. Behavioral correlates of acute bilateral symmetrical epileptogenic loci in monkey cerebral cortex. Brain Res., 1968b, 9: 370--373. Penfield, W. and Jasper, H.H. Epilepsy and the functional anatomy of the human brain. Little, Brown & Co., Boston, Mass., 1954, 896 p. Pollen, D.A. Experimental spike and wave responses and petit mal epilepsy. Epilepsia (Amst.), 1968, 9: 221--232. Pollen, D.A., Perot, P. and Reid, K.H. Experimental bilateral wave and spike from thalamic stimulation in relation to level of arousal. Electroenceph. clin. Neurophysiol., 1963, 15: 1017--1028. Prince, D.A. and Farrell, D. 'Centrencephalic' spike wave discharges following parenteral penicillin injection in the cat. Neurology (Minneap.), 1969, 19: 309--310. Quesney, L.F., Gloor, P., Wolfe, L.S. and Jozsef, S. Effect of PGF2 and 15(S)-15-methyl PGE2 methyl ester on feline generalized penicillin epilepsy. Prostaglandins, 1975, 10: 383--393. Starzl, T.E. and Magoun, H.W. Organization of the diffuse thalamic projection system. J. Neurophysiol., 1951, 14: 133--146. Testa, G. and Gloor, P. Generalized penicillin epilepsy in the cat: effect of midbrain cooling. Electroenceph, clin. Neurophysiol., 1974, 36: 517--524. Weir, B. Spikes-wave from stimulation of reticular core. Arch. Neurol. (Chic.), 1964, 11: 209--218. Weir, B. The morphology of the spike-wave complex. Electroenceph. clin. Neurophysiol., 1965, 19: 284--290.