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Stimulation-induced epileptogenesis : kindling of spontaneous motor seizures in rats Rovner, Louis Irving 1976

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STIMULATION-INDUCED EPILEPTOGENESIS: KINDLING OF SPONTANEOUS MOTOR SEIZURES IN RATS by LOUIS IRVING ROVNER B.A., U n i v e r s i t y of Nevada, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n THE FACULTY OF GRADUATE STUDIES i n the Department of Psychology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1976 (c) Louis Irving Rovner, 1976 In presenting th i s thes is in pa r t i a l fu.lf i lment. o f the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I further agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my written permission. Department of Psychology The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date August 21. 1976 i ABSTRACT When p e r i o d i c e l e c t r i c a l s t i m u l a t i o n i s a p p l i e d to any of a number of b r a i n s i t e s there can be a pro g r e s s i v e development and i n t e n s i f i c a t i o n of e l i c i t e d motor s e i z u r e s . This has been termed the k i n d l i n g e f f e c t . Although i t has been repeatedly suggested that the k i n d l i n g paradigm could provide a v a l u a b l e experimental model of c l i n i c a l e p i l e p t o g e n e s i s , i t was only r e c e n t l y found that k i n d l i n g would e v e n t u a l l y l e a d to a bona f i d e e p i l e p t i c syndrome i n r a t s , cats and baboons, c h a r a c t e r i z e d by spontaneous motor s e i z u r e s . The purpose of the present study was to s y s t e m a t i c a l l y describe the development of the e p i l e p t i c syndrome i n k i n d l e d r a t s . In the two experiments animals were s t i m u l a t e d about 15 times per week f o r s e v e r a l months. K i n d l i n g progressed as others have reported, although w i t h continued s t i m u l a t i o n s there were changes i n the e l i c i t e d s e i z u r e s which had not been p r e v i o u s l y described. Almost a l l of the animals, r e g a r d l e s s of whether they were s t i m u l a t e d i n the amygdala, hippocampus, or e n t o r h i n a l c o r t e x , e v e n t u a l l y d i s p l a y e d spontaneous motor s e i z u r e s before the te r m i n a t i o n of the experiment. These s e i z u r e s were found to p e r s i s t i n some animals f o r at l e a s t 7 months. Thus, i t appears that the k i n d l i n g paradigm may provide a v a l u a b l e a d d i t i o n to the methods a v a i l a b l e f o r the experimental i n v e s t i g a t i o n of e p i l e p s y and i t s genesis. i i TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i LIST OF FIGURES i i i INTRODUCTION 1 EXPERIMENT 1 18 EXPERIMENT 2 41 GENERAL DISCUSSION 63 REFERENCES 74 APPENDIX Analyses of Variance Tables f o r Experiment 2 78 i i i LIST OF FIGURES Page F i g . 1. Progressive changes i n the mean du r a t i o n of ADs recorded from s t i m u l a t e d and c o n t r a l a t e r a l amydgalae (Experiment 1). 24 F i g . 2. Pr o g r e s s i v e increase i n the mean c l a s s of e l i c i t e d MSs (Experiment 1). 27 F i g . 3. Pro g r e s s i v e changes i n the mean du r a t i o n of e l i c i t e d MS (Experiment 1). 32 F i g . 4. Incidence of i n t e r i c t a l discharges recorded from s t i m u l a t e d and c o n t r a l a t e r a l amygdalae (Experiment 1). 36 F i g . 5. E l e c t r o g r a p h i c and motor a c t i v i t y of a subject during a spontaneous Class 7 motor s e i z u r e (Experiment 1). 39 F i g . 6. Pro g r e s s i v e changes i n the mean du r a t i o n of ADs (Experiment 2). 47 F i g . 7. Pr o g r e s s i v e increases i n the mean c l a s s of e l i c i t e d MSs (Experiment 2). 50 F i g . 8. Pro g r e s s i v e changes i n the mean du r a t i o n of e l i c i t e d MSs (Experiment 2). 54 F i g . 9. Incidence of i n t e r i c t a l discharges (Experiment 2). 57 F i g . 10. Incidence of spontaneous MSs during poststimu-l a t i o n observations (Experiment 2). 61 -1-INTRODUCTION In cases where e p i l e p s y i s produced by s e r i o u s head i n j u r y , months or even years may elapse before the f i r s t overt c o n v u l s i o n ( P e n f i e l d , 1956). In such cases the i n i t i a l motor s e i z u r e s (MSs) may be r e l a t i v e l y minor, but as time passes, subsequent s e i z u r e s f r e q u e n t l y become more and more severe (Jackson, 1870). Although there have been numerous s t u d i e s of e p i l e p s y i t s e l f , l i t t l e a t t e n t i o n has been given i t s p r o g r e s s i v e develop-ments or e p i l e p t o g e n e s i s . Of the many l a b o r a t o r y models of e p i l e p s y , few are s u i t a b l e f o r the study of the development of the c o n d i t i o n . Recently, however, Goddard, Mc l n t y r e , and Leech (1969) suggested t h a t the k i n d l i n g paradigm could be v a l u a b l e i n ..this respect. The purpose of the present st u d i e s was to examine the v i a b i l i t y of k i n d l i n g as a l a b o r a t o r y model of c l i n i c a l e p i l e p t o g e n e s i s . Thus, t h i s I n t r o d u c t i o n i s comprised of a review of the k i n d l i n g l i t e r a t u r e f o l lowed by a statement of the major purpose. A. K i n d l i n g 1) D e f i n i t i o n . There can be a gradual development and i n t e n s i -f i c a t i o n of MSs when p e r i o d i c e l e c t r i c a l s t i m u l a t i o n s are a p p l i e d to any of a number of f o r e b r a i n s t r u c t u r e s , even at current l e v e l s which are i n i t i a l l y too low to evoke any behavioural e f f e c t s . With each successive s t i m u l a t i o n MSs grow more severe. For example, i f the r a t amygdala i s s t i m u l a t e d once a day at 50 \sA, there i s i n i t i a l l y no behavioural response; however, w i t h repeated s t i m u l a t i o n s the animal begins to d i s p l a y some minor behavioural symptoms which increase i n s e v e r i t y and d u r a t i o n w i t h -2-each successive s t i m u l a t i o n . This development and exacerbation of MSs by p e r i o d i c b r a i n s t i m u l a t i o n has been termed the k i n d l i n g e f f e c t (Goddard, et a l . , 1969). K i n d l i n g has been demonstrated i n a v a r i e t y of species even though the exact p a t t e r n of development may d i f f e r from one species to the next. Although k i n d l i n g has been s t u d i e d most o f t e n i n r a t s , i t has a l s o been observed i n monkeys (Goddard et a l . , 1969), r a b b i t s (Tanaka, 1972), mice (Leech, 1972), cats (Wada & Sato, 1974), frogs ( M o r r e l l , Tsuru, Hoeppner, & Morgan, 1975), and baboons (Wada, Osawa, & Corcoran, 1975). 2) P r o g r e s s i v e Development of Motor Seizures. The exact course of k i n d l i n g i s a f u n c t i o n of the p a r t i c u l a r n e u r a l s t r u c t u r e that i s stimu-l a t e d . Although most d e s c r i p t i o n s of k i n d l i n g have been based on obser-vat i o n s of the consequences of p e r i o d i c s t i m u l a t i o n of the r a t amygdala, recent evidence has shown that k i n d l i n g i n the r a t can take at l e a s t two general forms. Amygdaloid k i n d l i n g i s t y p i c a l of the k i n d l i n g which i s observed when s t i m u l a t i o n i s a p p l i e d to any of the s t r u c t u r e s r e l a t e d to the o l f a c t o r y - l i m b i c system, i . e . , amygdala, o l f a c t o r y bulb, hippo-campus, et c . U s u a l l y a r a t shows no c o n v u l s i v e response to the f i r s t few s t i m u l a t i o n s of any of the o l f a c t o r y - l i m b i c s t r u c t u r e s . However, w i t h continued s t i m u l a t i o n s r a t s begin to d i s p l a y rhythmic jaw and ear move-ments, and subsequent MSs grow more severe u n t i l each s t i m u l a t i o n e l i c i t s a b i l a t e r a l c l o n i c MS, c h a r a c t e r i z e d by f a c i a l and f o r e l i m b clonus, r e a r i n g , and a l o s s of e q u i l i b r i u m . Perhaps the most s u c c i n c t method of d e s c r i b i n g t h i s p rogression i s i n terms of Racine's (1972b) f i v e MS c l a s s e s : Class 1 - rhythmic jaw and ear movements; Class 2 - jaw and ear movements followed by head nodding; Class 3 - jaw, ear,and head movements -3-followed by foreli m b clonus; C l a s s 4 - jaw, ear, and head movements, f o r e -limb clonus, followed by r e a r i n g ; Class 5 - jaw, ear, and head movements, forelim b clonus, r e a r i n g , followed by l o s s of e q u i l i b r i u m ( f a l l i n g ) . Thus, the p r o g r e s s i v e development of MSs from s t i m u l a t i o n to s t i m u l a t i o n i s r e f l e c t e d i n the progression of symptoms during the course of an i n d i v i -dual MS. Although p e r i o d i c s t i m u l a t i o n of the a n t e r i o r neocortex i s a l s o a s s o c i a t e d w i t h a pro g r e s s i v e i n t e n s i f i c a t i o n of e l i c i t e d MSs, t h i s ' k i n d l i n g d i f f e r s i n s e v e r a l ways from that produced by o l f a c t o r y - l i m b i c s t i m u l a t i o n s (Racine, 1975). One major d i f f e r e n c e i s that MSs are u s u a l l y e l i c i t e d by the f i r s t s t i m u l a t i o n of a n t e r i o r neocortex. These i n i t i a l MSs c o n s i s t of m i l d mouth movements and myoclonic forel i m b c o n t r a c t i o n s , but w i t h repeated s t i m u l a t i o n s these MSs become more severe u n t i l even-t u a l l y s t i m u l a t i o n s e l i c i t MSs c h a r a c t e r i z e d by immediate l o s s of p o s t u r a l c o n t r o l and t o n i c extension of the body. In c o n t r a s t , Racine (1975) found that s t i m u l a t i o n of p o s t e r i o r p o r t i o n s of neocortex evoked no overt c o n v u l s i v e response whatsoever, even a f t e r 90 .daily s t i m u l a t i o n s . P e r i o d i c s t i m u l a t i o n of p a l e o c o r t i c a l regions i s a s s o c i a t e d w i t h a progressive development of MSs l i k e that a s s o c i a t e d w i t h o l f a c t o r y - l i m b i c s t i m u l a t i o n . The only d i f f e r e n c e i s that there i s an imm o b i l i t y of the animal f o l l o w i n g the f i r s t few s t i m u l a t i o n s . Subsequent s t i m u l a t i o n s then e l i c i t the stereotyped o l f a c t o r y - l i m b i c MS progression. There i s one exception to t h i s course of MS development produced by p a l e o c o r t i c a l s t i m u l a t i o n ; s t i m u l a t i o n s to the c i n g u l a t e or f r o n t a l - c i n g u l a t e c o r t i c e s at f i r s t e l i c i t MSs of the n e o c o r t i c a l type, which subsequently develop i n t o s u b c o r t i c a l - t y p e s e i z u r e s (Racine, 1975). -4-Although k i n d l e d MSs can be e l i c i t e d from any of a number of d i f f e r e n t s i t e s , most i n v e s t i g a t o r s have s t u d i e d amygdaloid k i n d l i n g , apparently because of the precedent e s t a b l i s h e d by Goddard et a l . (1969). In one of t h e i r e a r l y experiments Goddard et a l . found that of the many s t r u c t u r e s they t e s t e d , g e n e r a l i z e d MSs were e l i c i t e d by fewer s t i m u l a -t i o n s of the amygdala than of any other s t r u c t u r e . In the r e s t of the experiments i n t h i s i n i t i a l study they stu d i e d amygdaloid s t i m u l a t i o n . Since t h i s 1969 report-has,^. i n general;, been the most i n f l u e n t i a l paper i n the k i n d l i n g l i t e r a t u r e , i t i s not s u r p r i s i n g that other i n v e s t i g a t o r s have followed the l e a d of Goddard et a l . and have used amygdaloid stimu-l a t i o n almost e x c l u s i v e l y . 3) E l e c t r o g r a p h i c Factors i n K i n d l i n g . The increase i n the degree to which e l e c t r o g r a p h i c a f t e r - d i s c h a r g e s (ADs) g e n e r a l i z e from the s i t e of s t i m u l a t i o n to other s t r u c t u r e s seems to be the e l e c t r o g r a p h i c b a s i s f o r the k i n d l i n g phenomenon. Racine (1972b) s t i m u l a t e d the t i g h t amyg-dala of r a t s once per day. At f i r s t , ADs were u s u a l l y e l i c i t e d only at the s i t e of s t i m u l a t i o n , but subsequent s t i m u l a t i o n s evoked ADs which ge n e r a l i z e d to the c o n t r a l a t e r a l amygdala. As the AD durations and wave forms i n the c o n t r a l a t e r a l amygdala grew longer and more complex, the e l i c i t e d MSs became more intense. Racine, Gartner, and Burnham (1972) a l s o found t h i s g e n e r a l i z a t i o n of ADs to precede the i n t e n s i f i c a t i o n of e l i c i t e d MSs. In t h i s more thorough study, the i n c r e a s i n g d u r a t i o n and complexity of ADs i n response to p e r i o d i c amygdaloid s t i m u l a t i o n were noted i n s e v e r a l amygdaloid p r o j e c t i o n s i t e s , i n c l u d i n g the c o n t r a -l a t e r a l amygdala, ventromedial hypothalamus, p r e o p t i c area, hippocampus, and f r o n t a l pole of the cortex. These f i n d i n g s are c o n s i s t e n t w i t h -5-numerous c l i n i c a l r e p o r ts ( c f . Ward, Jasper, & Pope, 1969) which i n d i c a t e that unless ADs become g e n e r a l i z e d , they are f r e q u e n t l y not a s s o c i a t e d w i t h MSs. I n s o f a r as the g e n e r a l i z a t i o n of ADs appears to be the main e l e c t r o -graphic c o r r e l a t e of k i n d l i n g , i t i s not s u r p r i s i n g that the e l i c i t a t i o n of ADs at the s i t e of s t i m u l a t i o n i s a necessary p r e r e q u i s i t e f o r the k i n d l i n g of MSs. In a study by Racine (1972b), d a i l y amygdaloid s t i m u l a -t i o n was administered to r a t s at current i n t e n s i t i e s maintained j u s t below t h e i r AD t h r e s h o l d s ; no MSs were e l i c i t e d during the s i x weeks of the experiment. However, g e n e r a l i z e d MSs were k i n d l e d i n every subject s t i m u l a t e d at i n t e n s i t i e s j u s t above the t h r e s h o l d . In f a c t , no one has ever demonstrated k i n d l i n g without f i r s t e l i c i t i n g ADs. Racine (1972a) found that during the course of k i n d l i n g there was a p r o g r e s s i v e r e d u c t i o n i n the AD t h r e s h o l d ; w i t h each successive stimu-l a t i o n i t was p o s s i b l e to e l i c i t ADs at the s i t e of s t i m u l a t i o n w i t h c u r -rents of p r o g r e s s i v e l y lower i n t e n s i t y . This r a i s e d the p o s s i b i l i t y that t h i s t h r e s h o l d r e d u c t i o n might be the b a s i s f o r the k i n d l i n g pheno-menon, or at l e a s t c o n t r i b u t e to i t ; however, at l e a s t four independent l i n e s of evidence demonstrate that t h i s i s not the case. F i r s t , p e r i o d i c amygdaloid s t i m u l a t i o n maintained below the AD t h r e s h o l d reduces the AD t h r e s h o l d without l e a d i n g to the development of MSs (Racine, 1972); second, s t i m u l a t i o n of p o s t e r i o r noecortex does not k i n d l e MSs even though i t reduces the l o c a l AD t h r e s h o l d (Racine, 1975); t h i r d , the r e d u c t i o n of the AD t h r e s h o l d i n one b r a i n s i t e does not seem to a f f e c t the t h r e s h o l d i n another (Racine, 1972b), whereas k i n d l i n g of one s i t e g r e a t l y i n f l u e n c e s the degree to which other . s i t e s can be k i n d l e d (Goddard et a l . , 1969; -6-Mclntyre & Goddard, 1973); and f o u r t h l y , P i n e l , S k e l t o n , and Mucha (1976) demonstrated that when animals are k i n d l e d at high current i n t e n s i t i e s , t h e AD t h r e s h o l d i s a c t u a l l y r a i s e d during the course of k i n d l i n g . P i n e l et a l . (1976) viewed the r e d u c t i o n of AD thresholds and k i n d l i n g of MSs as independent but successive stages i n e p i l e p t o g e n e s i s . According to t h i s view a minor n e u r a l i r r i t a n t of i n s u f f i c i e n t s e v e r i t y to t r i g g e r ADs may e v e n t u a l l y reduce the AD t h r e s h o l d to a p o i n t where ADs are generated. Then each time an AD i s e l i c i t e d more and more n e u r a l s t r u c t u r e s are r e c r u i t e d , and t h i s g e n e r a l i z a t i o n i s r e f l e c t e d i n the development and i n t e n s i f i c a t i o n of MSs. 4) Permanence. Perhaps the most s t r i k i n g f e a t u r e of k i n d l i n g i s i t s permanence. Goddard et a l . (1969) r e k i n d l e d r a t s a f t e r a 12 week s t i m u l a t i o n - f r e e p e r i o d and found a savings of about 90% i n the number of s t i m u l a t i o n s r e q u i r e d to e l i c i t g e n e r a l i z e d MSs. Tress and Herberg (1972) measured k i n d l i n g and i t s permanence i n a d i f f e r e n t manner, but a l s o found the increased s e i z u r e s u s c e p t i b i l i t y to be r e l a t i v e l y enduring. Rather than record the p r o g r e s s i v e increases i n s e v e r i t y of MSs e l i c i t e d by p e r i o d i c s t i m u l a t i o n s of constant i n t e n s i t y , they measured the p r o g r e s s i v e r e d u c t i o n i n current i n t e n s i t y r e q u i r e d to produce MSs of constant s e v e r i t y . Each r a t was i n i t i a l l y s t i m u l a t e d at 10 \iA and i f no MS was e l i c i t e d the i n t e n s i t y was increased i n small increments every 10 sec u n t i l a MS was evoked. Both hypothalamic and s e p t a l s t i m u l a t i o n reduced the MS thresholds e f f e c t i v e l y over a 14-day per i o d . The thresholds were s t i l l s i g n i f i c a n t l y reduced f o l l o w i n g a 14-day s t i m u l a t i o n - f r e e p e r i o d . The p r o g r e s s i v e reductions i n AD thresholds which may occur during -7-the course of k i n d l i n g have a l s o shown to be r e l a t i v e l y permanent. Racine (1972a) found that r a t s ' AD t h r e s h o l d s , which had been s i g n i f i c a n t l y reduced by 60 d a i l y amygdaloid or hippocampal s t i m u l a t i o n s , were s t i l l s i g n i f i c a n t l y reduced a f t e r a 40-day s t i m u l a t i o n - f r e e p e r i o d although there was a s l i g h t trend toward recovery. In a s i m i l a r i n v e s t i g a t i o n P i n e l et a l . (1976) found no evidence of any recovery f o l l o w i n g a 7-day s t i m u l a t i o n - f r e e p e r i o d . 5) I n t e r - S t i m u l a t i o n I n t e r v a l . The i n t e r v a l between s t i m u l a t i o n s has proven to be a c r i t i c a l f a c t o r i n determining the r a t e of k i n d l i n g . Goddard et a l . (1969) sti m u l a t e d groups of r a t s of i n t e r v a l s of 5 min, 10 min, 20 min, 8 hr, 12 h r , 24 h r , or 7 days. The fewest number of s t i m u l a t i o n s were re q u i r e d f o r k i n d l i n g when the i n t e r v a l s were 24 hr or greater,whereas animals s t i m u l a t e d at i n t e r v a l s of 10 min or l e s s d i d not k i n d l e at a l l . Because the 24-hr i n t e r v a l was deemed most e f f e c t i v e by Goddard et a l . and because such a schedule f i t s w e l l i n t o general l a b o r a t o r y r o u t i n e , i n most k i n d l i n g experiments s t i m u l a t i o n s have been administered at 24-hr i n t e r v a l s . However, recent data published by Racine, Burnham, Gartner, and L e v i t a n (1973) i n d i c a t e that although more s t i m u l a t i o n s are r e q u i r e d , r a t s can be e f f e c t i v e l y k i n d l e d at i n t e r v a l s as short as 1 hr. 6) Current Parameters. Various current parameters have been shown to i n f l u e n c e the course of k i n d l i n g . Current i n t e n s i t y , f o r example, has proven to have an a p p r e c i a b l e e f f e c t on the r a t e of k i n d l i n g . In general, k i n d l i n g has been found to progress more r a p i d l y when subjects are s t i m u l a t e d at higher i n t e n s i t i e s . The study by Racine (1972b) sug-gested that t h i s r e l a t i o n i s l a r g e l y a r e s u l t of the f a c t that ADs are -8-e l i c i t e d more f r e q u e n t l y at higher current i n t e n s i t i e s . Racine a p p l i e d d a i l y amygdaloid s t i m u l a t i o n to three groups of r a t s , one group at a high current i n t e n s i t y (1000 UA), one at an i n t e n s i t y j u s t above the AD t h r e s h o l d , and a t h i r d at i n t e n s i t i e s that were c o n t i n u a l l y adjusted to be maintained j u s t below the p r o g r e s s i v e l y decreasing AD th r e s h o l d s . He found that animals d i d not k i n d l e i n the c o n d i t i o n where ADs were not e l i c i t e d , and that there was no s i g n i f i c a n t d i f f e r e n c e between the k i n d l i n g r a t e s of the two groups s t i m u l a t e d at i n t e n s i t i e s above the AD t h r e s h o l d . Thus, Racine concluded that current i n t e n s i t y had no e f f e c t on k i n d l i n g independent of i t s e f f e c t on the e l i c i t a t i o n of ADs. In a more recent r e p o r t , however, P i n e l et a l . , (1974) found that k i n d l i n g progressed more r a p i d l y at high i n t e n s i t i e s (500 UA) than at i n t e n s i t i e s j u s t above the AD th r e s h o l d . They a l s o found that once r a t s were k i n d l e d , there was much l e s s day-to-day v a r i a b i l i t y i n the ADs and MSs e l i c i t e d by s t i m u l a t i o n at the higher i n t e n s i t y . Thus, current i n t e n s i t y does seem to have some e f f e c t s on k i n d l i n g independent of i t s e f f e c t s on the r e l i a b i l i t y w i t h which ADs are e l i c i t e d . Goddard et a l . (1969) found that v a r i a t i o n s i n current parameters other than i n t e n s i t y had l i t t l e e f f e c t on k i n d l i n g . V a r i a t i o n s i n stimulus frequency between 25 and 150 Hz ( s i n e wave, 50 UA) had no e f f e c t on k i n d l i n g r a t e . S i m i l a r l y , they found that 1-msec, r e c t a n g u l a r pulses k i n d l e d r a t s as e f f e c t i v e l y as 60 Hz, si n e wave s t i m u l a t i o n s . They a l s o found that s t i m u l a t i o n durations of 1 sec and 60 sec (50 UA to 10 mA) were e q u a l l y e f f e c t i v e i n k i n d l i n g . 7) S t r a i n and Sex. S t r a i n and sex d i f f e r e n c e s are two v a r i a b l e s which have been shown to i n f l u e n c e k i n d l i n g . Goddard et a l . (1969) -9-found that a l b i n o r a t s of the Holtzman (Chicago) s t r a i n k i n d l e d more r a p i d l y than a l b i n o s of the Wistar s t r a i n or the Royal V i c t o r i a s t r a i n of hooded r a t s , and that the l a t t e r two s t r a i n s , once k i n d l e d , d i s p l a y e d MSs of s h o r t e r duration. However, i n the Racine, Burnham, Gartner, and L e v i t a n (1973) study,the Royal V i c t o r i a hooded r a t s and Sprague-Dawley a l b i n o s k i n d l e d f a s t e r than the Wistar a l b i n o s . P i n e i (1975), at the Vancouver Symposium on K i n d l i n g , described a study conducted i n h i s l a b o r a t o r y i n which the course of k i n d l i n g was compared i n male, and female r a t s . Although there were no d i f f e r e n c e s i n t h e i r k i n d l i n g r a t e s , the day-to-day v a r i a b i l i t y of MS durations was greater i n the female r a t s . This v a r i a b i l i t y appeared to be r e l a t e d to the estrous c y c l e ; MSs were longer during the estrogen surges of the pre-estrous p e r i o d . Ovariectomized females d i d not d i s p l a y t h i s v a r i a b i l i t y . 8) E f f e c t s of Pharmacological Agents on K i n d l e d Seizures and K i n d l i n g . The k i n d l i n g paradigm has been f r e q u e n t l y used to assess the co n v u l s i v e and a n t i - c o n v u l s i v e p r o p e r t i e s of drugs. These kinds of st u d i e s are of two b a s i c types: those which measure the e f f e c t s of pharmacological agents on k i n d l e d MSs and those which examine the e f f e c t s of the agents on the k i n d l i n g process i t s e l f . The most systematic study of the former type was r e c e n t l y published by Babington and Wedeking (1973). They determined the e f f e c t s of a number of c e n t r a l l y - a c t i v e drugs on MSs e l i c i t e d by s t i m u l a t i o n i n k i n d l e d r a t s . They found that a n t i d e p r e s s i v e drugs had a s e l e c t i v e e f f e c t , b l o c k i n g MSs e l i c i t e d from the amygdala more e f f e c t i v e l y than those e l i c i t e d by c o r t i c a l s t i m u l a t i o n . N onselective blockade of s e i z u r e s e l i c i t e d from these s i t e s was exerted by both a n t i -a n x i e t y and a n t i e p i l e p t i c drugs, w i t h a n t i a n x i e t y agents proving to be -10-more potent. The i n h i b i t o r y and f a c i l i t a t o r y e f f e c t s of n e u r o l e p t i c drugs and s t i m u l a n t s , r e s p e c t i v e l y , were s l i g h t . Two recent s t u d i e s of t h i s same type have demonstrated the a b i l i t y A 9 of A -tetrahydrocannabinol (THC) to block k i n d l e d s e i z u r e s . Corcoran, McCaughran, and Wada (1973) found that i n t r a p e r i t o n e a l i n j e c t i o n s of A 9-THC suppressed the ADs and MSs t y p i c a l l y produced by amygdaloid s t i m u l a -t i o n s i n k i n d l e d r a t s . Both A9-THC and i t s isomer, A8-THC, were found to have s i m i l a r e f f e c t s on the ADs and MSs e l i c i t e d by amygdaloid s t i m u l a -t i o n i n k i n d l e d Senegalese baboons, Papio papio (Wada, Osawa, & Corcoran, 1975). However, F r i e d and Mclntyre (1973) demonstrated that r a t s can become t o l e r a n t to the a n t i - c o n v u l s i v e e f f e c t s of A9-THC on k i n d l e d s e i z u r e s . Other i n v e s t i g a t o r s have st u d i e d the e f f e c t s of drugs on the pro-g r e s s i v e i n t e n s i f i c a t i o n of s e i z u r e s from s t i m u l a t i o n to s t i m u l a t i o n . In t h i s type of i n v e s t i g a t i o n drugs are u s u a l l y administered each day p r i o r to the s t i m u l a t i o n and the degree to which the drugs i n h i b i t or f a c i l i t a t e k i n d l i n g i s then determined. Wise and Chinerman (1974) demonstrated that diazepam and pheno b a r b i t o l , but not diphenylhydantoin, block the k i n d l i n g of MSs but not the decrease i n the l o c a l AD t h r e s h o l d produced by p e r i o d i c amygdaloid s t i m u l a t i o n . Arnold, Racine, and Wise (1973) found that a t r o p i n e , a c h o l i n e r g i c b l o c k i n g agent, produced s i m i l a r e f f e c t s , but that two drugs w i t h a n t i - c a t e c h o l a m i n e r g i c e f f e c t s , r e s e r p i n e and 6-hydroxydopamine, f a c i l i t a t e d k i n d l i n g . Wada, Wake, Sato, and Corcoran (1975) reported that i n t r a p e r i t o n e a l i n j e c t i o n s of e i t h e r A9-THC or A8-THC administered before each of 25 d a i l y amygdaloid s t i m u l a t i o n s r e -tarded k i n d l i n g i n c a t s . -11-Perhaps the most thorough i n v e s t i g a t i o n i n t o the e f f e c t s of drugs on k i n d l i n g was r e c e n t l y reported by Racine, L i v i n g s t o n e , and Joaquin (1975). In t h i s study they found that the e f f e c t s of procaine HC1, d i -phenylhydantoin, and diazepam on k i n d l i n g depended on the s i t e of e l e c -t r i c a l s t i m u l a t i o n . I n t r a p e r i t o n e a l i n j e c t i o n s of procaine HC1 and d i -phenylhydantoin administered before each s t i m u l a t i o n increased the r a t e of amygdaloid k i n d l i n g , whereas diazepam had the o p p o s i t e - e f f e c t . However, the e f f e c t s of procaine HC1 and diphenylhydantoin on k i n d l i n g from neo-c o r t i c a l s i t e s were q u i t e d i f f e r e n t . Both of these drugs e f f e c t i v e l y blocked the ADs and MSs t y p i c a l l y e l i c i t e d by n e o c o r t i c a l s t i m u l a t i o n , even when the r a t s were st i m u l a t e d at current i n t e n s i t i e s as high as 2000 uA. Diazepam, on the other hand, had no e f f e c t at a l l on the course of k i n d l i n g from n e o c o r t i c a l s i t e s . Thus, the e f f e c t s of drugs on k i n d -l i n g are a f u n c t i o n of the s i t e of s t i m u l a t i o n . 9) Mechanisms. I t i s now widely b e l i e v e d that the p r o g r e s s i v e increase i n the degree to which l o c a l ADs w i l l g e n e r a l i z e to other areas of the b r a i n w i t h repeated s t i m u l a t i o n i s the b a s i s of the k i n d l i n g phenomenon (Racine, 1972b; Racine, Okujava, & C h i p a s h v i l i , 1972; Mclntyre & Goddard, 1973). Thus,attempts to e x p l a i n the k i n d l i n g process have centred _~ around the increases i n the e f f i c i e n c y of the synaptic transmis-s i o n which are presumed to underlie t h i s increase i n g e n e r a l i z a t i o n . Racine, Okujava, and C h i p a s h v i l i (1972), f o r example, have suggested that k i n d l i n g may be the r e s u l t of an increase i n the e f f i c i e n c y of s y n a p t i c t r a n s m i s s i o n between l i m b i c s t r u c t u r e s . They found that r a t s which were simultaneously s t i m u l a t e d i n both amygdalae or both hippocampi k i n d l e d more r a p i d l y than subjects who were st i m u l a t e d at a s i n g l e s i t e . Animals -12-which were sti m u l a t e d i n the l e f t and r i g h t amygdalae on a l t e r n a t e days a l s o k i n d l e d more q u i c k l y than those r e c e i v i n g an equal number of stimu-l a t i o n s r e s t r i c t e d to a s i n g l e s i t e . Subjects s t i m u l a t e d i n the hippo-campal commissure had the f a s t e s t k i n d l i n g r a t e of a l l . I t appeared that k i n d l i n g progressed at a r a t e p r o p o r t i o n a t e to the number of l i m b i c con-nectio n s that were p o t e n t i a t e d . Further support f o r t h i s assumption of Racine et a l . was provided by Douglas and Goddard (1975). They found that the monosynaptic pe r f o r a n t - p a t h EPSP i n the hippocampus produced by s t i m u l a t i o n of the i p s i l a t e r a l e n t o r h i n a l cortex was l a s t i n g l y p o t e n t i a t e d by d a i l y e n t o r h i n a l s t i m u l a t i o n . Racine, Gartner, and Burnham (1972) found that k i n d l i n g r a t s w i t h amygdaloid s t i m u l a t i o n produced ap p r e c i a b l e increases i n the amplitudes of l a t e components of responses recorded i n amygdaloid p r o j e c t i o n pathways evoked by s i n g l e pulses or pulse t r a i n s a p p l i e d to the amygdala. Since the l a r g e s t increases i n amplitude were seen i n l a t e components presumably t r a n s m i t t e d over m u l t i s y n a p t i c pathways, they suggested that the change produced by k i n d l i n g i n v o l v e s a f a c i l i t a t i o n of synaptic, t r a n s m i s s i o n which i s widespread. This view that widespread syna p t i c changes form.the b a s i s of k i n d l i n g r e c e i v e s a d d i t i o n a l support from s t u d i e s which demonstrated that f o l l o w i n g the k i n d l i n g of one s i t e , MSs are more r e a d i l y e l i c i t e d from other s i t e s (Racine, 1972b), even a f t e r the o r i g i n a l s i t e has been l e s i o n e d (Mclntyre & Goddard, 1973). The neuroanatomical c o r r e l a t e s of k i n d l i n g remain unknown. Two recent attempts to i d e n t i f y these c o r r e l a t e s were uns u c c e s s f u l . A thorough electron-microscope examination of amygdaloid t i s s u e revealed no s i g n i f i c a n t d i f f e r e n c e s between animals k i n d l e d w i t h amygdaloid s t i m u l a t i o n -13-and unstimulated c o n t r o l s (Goddard & Douglas, 1975). Racine, T u f f , and Zaide (1975) k i n d l e d r a t s w i t h a n t e r i o r n e o c o r t i c a l s t i m u l a t i o n and pre-pared the t i s s u e around the e l e c t r o d e t i p s f o r h i s t o l o g i c a l examination w i t h a m o d i f i c a t i o n of the Golgi-Cox s t a i n i n g technique. They found no s i g n i f i c a n t d i f f e r e n c e s between t h i s t i s s u e and comparable t i s s u e taken from unstimulated c o n t r o l s . The f a c t that the r a t e of k i n d l i n g seems to be p r i m a r i l y determined by the frequency at which ADs are e l i c i t e d , r a t h e r than the passage of time per se, has r u l e d out the p o s s i b i l i t y that gross p h y s i o l o g i c a l changes (such as g l i o s i s , edema, and v a s c u l a r changes) produced by the mere presence of the e l e c t r o d e could be an important f a c t o r i n k i n d l i n g . Moreover, k i n d l i n g does not seem to r e s u l t from the stimulation-produced accumulation of e p i l e p t o g e n i c metal ions at the e l e c t r o d e s i t e . Goddard et a l . (1969), i n a d d i t i o n to employing the s t a i n l e s s s t e e l e l e c t r o d e s which are commonly used and known to despoit metal i o n s , s t i m u l a t e d other r a t s w i t h nichrome or platinum e l e c t r o d e s and found that the r a t e s of k i n d l i n g produced by s t i m u l a t i o n through these d i f f e r e n t e l e c t r o d e s were i n d i s t i n g u i s h a b l e . B. R a t i o n a l e From the outset i n v e s t i g a t o r s studying the k i n d l i n g phenomenon have emphasized i t s p o t e n t i a l as a l a b o r a t o r y model of c l i n i c a l e p i l e p -togenesis (Goddard et a l . , 1969;. M o r r e l l , 1973). There are s t r i k i n g s i m i l a r i t i e s between the k i n d l i n g phenomenon and the p r o g r e s s i v e develop-ment and subsequent i n t e n s i f i c a t i o n of e p i l e p t i c symptoms i n humans; i n untreated c l i n i c a l cases, as i n k i n d l e d animals, there i s o f t e n a delay -14-before the i n i t i a l m a n i f e s t a t i o n of overt e p i l e p t i c symptoms, followed by a progressive i n t e n s i f i c a t i o n of symptoms w i t h i n a p a r t i c u l a r a t t a c k as w e l l as from one f i t to the next (Jackson, 1870). U n t i l r e c e n t l y , however, there appeared to be reasons f o r doubting the s u i t a b i l i t y of k i n d l i n g as a model of e p i l e p t o g e n e s i s . Spontaneous MSs, those not e l i c i t e d by the s t i m u l a t i o n , were not mentioned i n any reports of k i n d l i n g p r i o r to 1974. In f a c t , Goddard et a l . e x p l i c i t l y s t a t e d that they had never observed a spontaneous MS to occur i n any of the r a t s , c a t s , or monkeys k i n d l e d during the course of the experiments published i n t h e i r 1969 paper. This f a i l u r e to observe spontaneous s e i z u r e s i s of c r i t i c a l importance since e p i l e p s y , by d e f i n i t i o n , i s a spontaneously-recurring, s e l f - s u s t a i n e d , paroxysmal dysfunction of the b r a i n . I t i s d i f f i c u l t to see how an ex-perimental paradigm which had not been shown to produce an e p i l e p t i c s t a t e could s e r i o u s l y be considered as a model of c l i n i c a l e p i l e p t o g e n e s i s . However, there have been s e v e r a l recent r e p o r t s of spontaneous MSs i n k i n d l e d r a t s ( P i n e l , Mucha, & P h i l l i p s , 1975; P i n e l , P h i l l i p s , Mucha, & Deol, 1973), cats (Wada, Sato, & Corcoran, 1974), and baboons (Wada & Osawa, 1976; Wada, Osawa, & Mizoguchi, 1975). In the Wada et a l . (1974) study the cats were s t i m u l a t e d d a i l y u n t i l g e n e r a l i z e d MSs were e l i c i t e d (15 to 36 s t i m u l a t i o n s , X = -25.5) and then each subject r e c e i v e d 13 to 60 a d d i t i o n a l s t i m u l a t i o n s . Of the f i v e cats i n the study, three e v e n t u a l l y d i s p l a y e d spontaneous MSs during the course of s t i m u l a t i o n s . During the course of the experiment they n o t i c e d , as others had before (cf. Wada & Sato, 1974), that between s t i m u l a t i o n s the ongoing e l e c t r o -graphic a c t i v i t y was f r e q u e n t l y punctuated by i n t e r i c t a l discharges (IIDs). There appeared to be a marked increase i n the prevalence of these IIDs i n -15-the p e r i o d before the f i r s t spontaneous MSs were observed. Although they reported that one subject experienced spontaneous s e i z u r e s f o r 53 days a f t e r the s t i m u l a t i o n s were c u r t a i l e d and then developed s t a t u s e p i l e p -t i c u s , they provided no data to i l l u s t r a t e the time course of spontaneity i n the other c a t s . In view of the number of i n v e s t i g a t o r s who had k i n d l e d r a t s and had never observed spontaneous MSs i n those k i n d l e d animals, the r e p o r t s of P i n e l et a l . (1973, 1975) were p a r t i c u l a r l y s u r p r i s i n g . The assumption inherent i n a l l of the e a r l i e r k i n d l i n g s t u d i e s employing r a t s was that the k i n d l i n g process was complete once g e n e r a l i z e d MSs (Class 5) could be r e l i a b l y e l i c i t e d ; thus, most of the s t u d i e s were c u r t a i l e d at t h i s p o i n t . However, i n the P i n e l et a l . i n v e s t i g a t i o n the r a t s were s t i m u l a t e d a t l e a s t once a day f o r s e v e r a l months. This l e d P i n e l et a l . to conclude that spontaneity i s the u l t i m a t e m a n i f e s t a t i o n of k i n d l i n g , and that pre-vious i n v e s t i g a t o r s had i n f a c t been r e s t r i c t i n g t h e i r i n v e s t i g a t i o n s to the e a r l y phases of e p i l e p t o g e n e s i s . P i n e l et a l . observed three changes i n t h e i r animals i n the days before they f i r s t d i s p l a y e d spontaneous MSs. F j i r s t , they observed the same increase i n the r a t e of IIDs that had been observed to precede spontaneity i n the cat (Wada et a l . , 1975). Secondly, although s t i m u l a t i o n s had r e l i a b l y e l i c i t e d g e n e r a l i z e d MSs p r e v i o u s l y , the animals o c c a s i o n a l l y showed an attenuated response, or even no r e s -ponse at a l l , to the s t i m u l a t i o n . And t h i r d l y , the r a t s began to d i s p l a y myoclonic j e r k s i n conjunction w i t h IIDS of p a r t i c u l a r l y h i g h amplitude. Wada et a l . (1976) s t i m u l a t e d the l e f t amygdalae of Sengalese baboons (Papio papio) each day u n t i l g e n e r a l i z e d MSs were e l i c i t e d (100 to 135 s t i m u l a t i o n s ) . While subsequently s t i m u l a t i n g the c o n t r a l a t e r a l -16-amygdalae of these animals, spontaneous recurrent MSs were observed i n two of the four subjects. Abrupt increases i n the incidence of IIDs were seen throughout the b r a i n about 24 hr before the occurrence of spontaneous MSs. The p e r s i s t e n c e of the e p i l e p t i c syndrome was not s t u d i e d , as the animals were employed i n a p o s t k i n d l i n g p h o t o s e n s i t i v i t y experiment and then s a c r i f i c e d . Spontaneous MSs have only been observed i n a s i n g l e k i n d l e d animal who was sti m u l a t e d at a s i t e other than the amygdala. Wada et a l . (1975) k i n d l e d two Sengalese baboons (Papio papio) w i t h e l e c t r i c a l s t i m u l a t i o n of the p r e f r o n t a l cortex administered twice a day. Although one of these animals died of an i l l n e s s apparently u n r e l a t e d to k i n d l i n g , the other subject d i s p l a y e d 12 spontaneous MSs during the course of the experiment a f t e r about 280 s t i m u l a t i o n s had been administered. Fewer IIDs were ob-served i n t h i s animal than had been seen i n baboons r e c e i v i n g amygdaloid s t i m u l a t i o n (Wada et a l . , 1976) and IIDs were more numerous i n the c o n t r a -l a t e r a l p r e f r o n t a l cortex than at the s i t e of s t i m u l a t i o n . Information was not provided concerning IID increases before the onset of spontaneous MSs or the permanence of the e p i l e p t i c syndrome. The observations of Wada et a l . and P i n e l et a l . of spontaneous s e i z u r e s i n k i n d l e d animals are of obvious c r i t i c a l importance to ... researchers studying the k i n d l i n g process as a model of c l i n i c a l e p i l e p -togenesis. In none of these s t u d i e s , though, were the spontaneous s e i -zures or the events which l e d up to them described or i n v e s t i g a t e d sys-t e m a t i c a l l y . However, now that i t i s c l e a r that k i n d l i n g w i l l e v e n t u a l l y culminate i n an e p i l e p t i c syndrome i t i s p o s s i b l e to explore t h i s develop-ment i n a more systematic f a s h i o n . -17-Thus, the general purpose of t h i s t h e s i s was to confirm the reports of P i n e l et a l . (1973, 1975) that p e r i o d i c e l e c t r i c a l s t i m u l a t i o n of the b r a i n can e v e n t u a l l y l e a d to the development of spontaneous s e i z u r e s i n r a t s , and to provide a systematic d e s c r i p t i o n of the e n t i r e course of k i n d l i n g from the f i r s t s t i m u l a t i o n to the eventual d i s p l a y of spontaneous MSs, w i t h a p a r t i c u l a r emphasis on the events l a t e r i n the course of k i n d l i n g which..have not been described by other i n v e s t i g a t o r s . The i n t e n -t i o n of the t h e s i s was to deal w i t h some b a s i c i s s u e s r e l a t e d to the e p i l e p t o g e n e t i c e f f e c t s of repeated e l e c t r i c a l b r a i n s t i m u l a t i o n , and thus to provide a data base f o r those researchers who want to use the k i n d l i n g paradigm as a model of c l i n i c a l e p i l e p t o g e n e s i s . -18-EXPERIMENT 1 In Experiment 1 the development of spontaneous MSs i n r a t s k i n d l e d w i t h amygdaloid s t i m u l a t i o n was i n v e s t i g a t e d . In many respects t h i s study was s i m i l a r to the study of spontaneous MSs i n k i n d l e d r a t s by P i n e l et a l . (1975); however, i t i n c l u d e d three important methodological innovations. F i r s t , s i n c e only four animals completed the P i n e l et a l . experiment and only two of those d i s p l a y e d spontaneous MSs, i t was d i f f i c u l t to estimate the consistency w i t h which t h i s method can produce an e p i l e p t i c syndrome. In Experiment 1 the sample s i z e s were s u f f i c i e n t l y l a r g e so that i t was p o s s i b l e to estimate the p r o p o r t i o n of k i n d l e d animals that would even-t u a l l y become "spontaneous." Such in f o r m a t i o n should make a p r a c t i c a l c o n t r i b u t i o n to the design of fu t u r e i n v e s t i g a t i o n s of spontaneous k i n d l e d s e i z u r e s by p r o v i d i n g researchers w i t h a b a s i s f o r determining the most appropriate sample s i z e s . From a more t h e o r e t i c a l viewpoint, determining whether spontaneity i s the i n e v i t a b l e r e s u l t of k i n d l i n g or whether i t i s simply an anomaly which occurs i n a few animals should prove to be an important step i n developing an understanding f o r the k i n d l i n g phenomenon. Secondly, i n the present experiment e l e c t r o g r a p h i c a c t i v i t y was monitored from the c o n t r a l a t e r a l amygdala as w e l l as from the s i t e of s t i m u l a t i o n , whereas P i n e l et a l . recorded from only, the l a t t e r s i t e . Wada et a l . have r o u t i n e l y recorded from m u l t i p l e s i t e s i n t h e i r e x p e r i -ments on amygdaloical k i n d l i n g i n cats and baboons and have found that the development of spontaneous e l e c t r o g r a p h i c s p i k i n g i s p a r t i c u l a r l y s t r i k i n g i n the c o n t r a l a t e r a l amygdala. Thus, i t was p o s s i b l e f o r the f i r s t time to record the e l e c t r o g r a p h i c antecedents of spontaneous k i n d l e d -19-s e i z u r e s i n the r a t from the c o n t r a l a t e r a l amydgala. The t h i r d methodological improvement was that the p r o g r e s s i v e i n -t e n s i f i c a t i o n of e l i c i t e d MSs was . c a r e f u l l y documented. Most i n v e s t i -gators have assumed that the p r o g r e s s i v e i n t e n s i f i c a t i o n of e l i c i t e d MSs was complete i n the r a t once Class 5 s e i z u r e s were r e l i a b l y e l i c i t e d ; i n f a c t , Class 5 MSs have f r e q u e n t l y been r e f e r r e d to as " f u l l " MSs ( c f . Racine, 1972b). Thus, i n the P i n e l et a l . study there was no systematic attempt to document change i n the MS p a t t e r n once the s t i m u l a t i o n was r e l i a b l y e l i c i t i n g Class 5 MSs. Although they became aware during the course of t h e i r i n v e s t i g a t i o n that there were changes i n the MSs which occurred a f t e r the development of Class 5 MSs, they were unable to pro-v i d e accurate r e t r o s p e c t i v e d e s c r i p t i o n s ( P i n e l , P h i l l i p s , Mucha, & Deol, 1973). Thus, i n Experiment 1, the p a t t e r n of each MS was c a r e f u l l y observed and recorded. METHODS Two b i p o l a r e l e c t r o d e s constructed of i n s u l a t e d nichrome wire (diameter = 0.01 i n ) were implanted i n each of the 45 male, 350 to 450. g hooded r a t s (Canadian Breeding L a b o r a t o r i e s , La P r a i r i e , Quebec) which served as sub j e c t s . The e l e c t r o d e s were aimed at the r i g h t and l e f t amyg-dalae of each subject (coordinates: 1.5 mm p o s t e r i o r to Bregma, 4.2 mm on e i t h e r side of the s a g i t t a l suture, and 8.8 mm v e n t r a l to the dura). The e l e c t r o d e connectors were housed i n pedestal caps (MS363, P l a s t i c Products Co., Roanoke, V i r g i n i a ) which were i n t u r n secured to the s k u l l i n the standard f a s h i o n w i t h s t a i n l e s s s t e e l screws and dental a c r y l i c . ^20-A f t e r . 10 days of p o s t - s u r g i c a l recovery, 33 of the subjects were stimu-l a t e d (1-sec, 60-Hz, 400-UA, RMS, sine-wave current) through the l e f t e l e c t r o d e at i n t e r v a l s of no l e s s than 2 hr and no greater than 48, about 15 times per week. A l l experimental subjects were maintained on t h i s s t i m u l a t i o n schedule f o r 134 days. Of the o r i g i n a l 33 experimental sub-j e c t s , one animal was found to have a f a u l t y e l e c t r o d e and 14 e i t h e r died or dislodged t h e i r e l e c t r o d e s during the course of the experiment, and thus none of the data of these 15 r a t s were subjected to a n a l y s i s . The 12 unstimulated c o n t r o l s were handled as o f t e n as the 18 s u r v i v i n g e x p e r i -mental s u b j e c t s . On Day 135 a l l of the animals were s a c r i f i c e d and per-fused w i t h b u f f e r e d f o r m a l i n . Several of the b r a i n s were shipped f o r G o l g i a n a l y s i s to Madge and Arnold Scheibel of the U n i v e r s i t y of C a l i f o r n i a , Los Angeles, School of Medicine. The other b r a i n s were sectioned and st a i n e d w i t h c r e s y l v i o l e t i n order to v e r i f y the e l e c t r o d e placements. A l l of the el e c t r o d e s were found to terminate i n the amygdaloid complex. Pre- and p o s t - s t i m u l a t i o n e l e c t r o g r a p h i c a c t i v i t y was monitored from both e l e c t r o d e s i t e s about three times per week through #363-open "L" connectors ( P l a s t i c Products Co.) which conducted the s i g n a l s to a Grass model 78B polygraph. E l e c t r o g r a p h i c a c t i v i t y was monitored f o r 60 sec p r i o r to the s t i m u l a t i o n and f o r 30 sec f o l l o w i n g the c e s s a t i o n of the AD. Switching from the recording to the s t i m u l a t i o n mode and back again was done a u t o m a t i c a l l y by a device which a l s o i s o l a t e d the polygraph a m p l i f i e r s during current d e l i v e r y to reduce p o s t - s t i m u l a t i o n i n t e r f e r e n c e i n the recording channels to about 1 sec. A permanent record of motor a c t i v i t y was obtained during recording sessions by d i s p l a y i n g the movement a r t i -f a c t generated i n a s i n g l e i n s u l a t e d w i r e , housed i n the connector but -21-not connected to the animal, on a channel of the polygraph. Movement of the wire by the subjects produced changes i n p o t e n t i a l p r o p o r t i o n a l to the magnitude of the motor a c t i v i t y . On those occasions when e l e c t r o g r a p h i c a c t i v i t y was monitored, the durations of AD from both amydgalae were recorded, as w e l l as the dura-t i o n and i n t e n s i t y of MSs. The i n t e n s i t y of each MS was r a t e d according to the aforementioned f i v e - c l a s s s c a l e of Racine (1972b): (1) f a c i a l movements only, (2) f a c i a l movements and head nodding, (3) f a c i a l move-ments, head nodding, and f o r e l i m b clonus, (4) f a c i a l movements, head nodding, fore l i m b clonus, and r e a r i n g , (5) f a c i a l movements, head nodding, forelimb clonus, r e a r i n g , and f a l l i n g . On occasions when e l e c t r o g r a p h i c responses to s t i m u l a t i o n were not monitored only the MS c l a s s was recorded. Once a spontaneous MS was observed i n an animal, that subject was observed i r r e g u l a r l y f o r 2 or 3 hr per day, i n a d d i t i o n to the r o u t i n e observation during the s t i m u l a t i o n sessions. The incidence and patterns of MSs were recorded during these a d d i t i o n a l s e s s i o n s , and e l e c t r o g r a p h i c a c t i v i t y was o c c a s i o n a l l y monitored. RESULTS AND DISCUSSION .. During the course of the experiment both the e l e c t r o g r a p h i c and behavioural aspects of the e l i c i t e d s e i z u r e s increased p r o g r e s s i v e l y i n s e v e r i t y , and 16 of the 18 experimental subjects that completed the experiment e v e n t u a l l y d i s p l a y e d spontaneous MSs. The progressive i n t e n s i f i c a t i o n of e l i c i t e d s e i z u r e s i s summarized i n Figures 1, 2, and 3 which i l l u s t r a t e the progressive increases i n AD -22-d u r a t i o n , MS c l a s s , and MS d u r a t i o n , r e s p e c t i v e l y . The mean responses to s t i m u l a t i o n administered during the f i r s t 60 days of the experiment are i l l u s t r a t e d i n the l e f t panel of each f i g u r e . The r i g h t panel of both Figures 1 and 3 i l l u s t r a t e s the mean responses of subjects during the recording sessions i n the 3- or 4-week p e r i o d before each d i s p l a y e d i t s f i r s t spontaneous MS. For example, the sub j e c t s ' average response, observed during the recording s e s s i o n immediately preceding each animal's f i r s t spontaneous MS, i s p l o t t e d i n both f i g u r e s above "Day -1-" Figure 2 . d i f f e r s i n that the r i g h t panel i l l u s t r a t e s the subjects' average c l a s s of e l i c i t e d MSs f o r the l a s t 10 days before each animal d i s p l a y e d spontaneity. The averages were determined and p l o t t e d i n t h i s f a s h i o n so that any systematic changes o c c u r r i n g j u s t before the development of spontaneity would be evident. AD Duration. Figure 1 i l l u s t r a t e s the progressive changes i n the durations of ADs recorded from the s i t e of s t i m u l a t i o n and from the c o n t r a -l a t e r a l amygdala i n the 16 experimental animals that e v e n t u a l l y d i s p l a y e d spontaneous MSs. There was a sharp increase i n the d u r a t i o n of ADs r e -corded from both s i t e s i n the f i r s t 8 days (18 s t i m u l a t i o n s ) of the e x p e r i -ment (both s i g n t e s t s , x=0, N=16, p<0.001). However, there was a subse-quent p a r t i a l , b u t s i g n i f i c a n t decrease i n the dur a t i o n of ADs recorded from the s i t e of s t i m u l a t i o n ( s i g n t e s t , x=2, N=16, p=0.002) and from the c o n t r a l a t e r a l amygdala ( s i g n t e s t , x=5, M=16, p=0.105) between Days 8 and 28. Thereafter,there were no systematic changes i n AD du r a t i o n s ; thus, on Day 60 the ADs recorded from both s i t e s were s i g n i f i c a n t l y longer than the ADs e l i c i t e d by the f i r s t s t i m u l a t i o n (both s i g n t e s t s , x=0, N=16, -23-F i g . 1. Pro g r e s s i v e changes i n the mean du r a t i o n of ADs recorded from s t i m u l a t e d and c o n t r a l a t e r a l amygdalae (Experiment 1), The l e f t panel i l l u s t r a t e s the changes in.AD d u r a t i o n over the f i r s t 60 days of the experiment. The r i g h t panel i l l u s t r a t e s the mean dura t i o n of ADs e l i c i t e d during the ten rec o r d i n g sessions before each animal d i s p l a y e d i t s f i r s t spontaneous MS. Thus, the p o i n t above Day -1 i n d i c a t e s the mean dur a t i o n of ADs e l i c i t e d during the l a s t recording session before each animal d i s p l a y e d i t s f i r s t spontaneous MS. There were 2 or 3 days between each of these l a s t ten recording sessions. -25-p<0.001). Many i n v e s t i g a t o r s have reported that the durations of ADs recorded from the s i t e of s t i m u l a t i o n increase monotonically during the course of k i n d l i n g ( c f . Mclntyre & Goddard, 1973). However, the marked increase and subsequent p a r t i a l d e c l i n e observed i n the present experiment has been reported p r e v i o u s l y (Racine, 1972b) and i s almost i d e n t i c a l to the f u n c t i o n reported by P i n e l , P h i l l i p s , and Deol (1974), who used methods comparable to those employed here. In response to> the f i r s t s t i m u l a t i o n , ADs were recorded from the s i t e of s t i m u l a t i o n i n a l l 18 experimental subjects and from the contra -l a t e r a l amygdala i n 7. By Day 7, however, ADs were i n v a r i a b l y recorded from both amygdalae i n a l l of the experimental subjects. In none of the seven cases i n which c o n t r a l a t e r a l ADs were e l i c i t e d by the f i r s t stimu-l a t i o n were they longer than those recorded from the s i t e of s t i m u l a t i o n , but by Day 13 (27 s t i m u l a t i o n s ) the mean ADs recorded from the co n t r a -l a t e r a l amygdala were s l i g h t l y , but c o n s i s t e n t l y , longer and remained so through Day 60 (except f o r Day 29). On an average day during t h i s p e r i o d (Day 13 to Day 60), s i x of the animals d i s p l a y e d ADs which were longer on the c o n t r a l a t e r a l s i d e , i n four the durations were longer at the s i t e of s t i m u l a t i o n , and i n s i x cases the durations were equal. Thus, although the mean durations of the ADs from the c o n t r a l a t e r a l s i d e were c o n s i s t e n t l y longer than the means from the s i t e of s t i m u l a t i o n , on no day was t h i s d i f f e r e n c e s i g n i f i c a n t . ( s i g n t e s t , x=4, N=10, p=0.377). The r i g h t panel of Figure 1 c l e a r l y i n d i c a t e s that there were no systematic changes i n AD durations during the ten recording sessions before each animal f i r s t d i s p l a y e d spontaneity. For example, the durations of a l l ADs recorded during the l a s t s e s s i o n before each i n d i v i d u a l animal -26-d i s p l a y e d a spontaneous MS (Day -1) were not s i g n i f i c a n t l y d i f f e r e n t from those recorded on Day 60 ( s t i m u l a t e d s i d e , s i g n t e s t , x=7, N=14, p=0.605; c o n t r a l a t e r a l s i d e , s i g n t e s t , x=6, N=14, p=0.395). Thus, the development of spontaneity does not seem to be r e l a t e d i n any obvious way to changes i n the d u r a t i o n of e l i c i t e d ADs. Class of E l i c i t e d MSs. Figure 2 i l l u s t r a t e s the p r o g r e s s i v e increase in the s e v e r i t y of the c l a s s of e l i c i t e d MSs. Each poi n t i n - F i g u r e 2 represents the mean MS c l a s s on a par-t i c u l a r day. For a day when more than one s t i m u l a t i o n was administered to each s u b j e c t , the score f o r each subject was the average MS c l a s s observed during that day. The f i r s t s t i m u l a t i o n of the experiment g e n e r a l l y e l i c i t e d no response, however s t a r t l e responses were observed i n s e v e r a l subjects and one animal d i s p l a y e d m i l d c l o n i c f a c i a l movements. By the t h i r d or f o u r t h s t i m u l a t i o n m i l d overt c o n v u l s i v e responses were e l i c i t e d i n most animals and these increased i n s e v e r i t y w i t h each successive s t i m u l a t i o n . Class 5 MSs had been e l i c i t e d i n a l l of the subjects by the end of the second week. This i s the p o i n t at which most i n v e s t i g a t o r s have assumed the i n t e n s i f i c a t i o n of e l i c i t e d MSs to be complete. However, the l e f t panel of Figure 2 c l e a r l y shows that the MSs continued to grow more intense even though the r a t e of change was not as great as that a s s o c i a t e d w i t h the e a r l i e r phases of k i n d l i n g . No systematic changes i n the c l a s s of e l i c i t e d MSs were observed a f t e r Day 37 (80 s t i m u l a t i o n s ) . In order to i l l u s t r a t e the p r o g r e s s i v e i n t e n s i f i c a t i o n of e l i c i t e d MSs during the l a t e r stages of the experiment i t was necessary to add s e v e r a l c l a s s e s to Racine's f i v e - c l a s s s c a l e . The p a t t e r n of MSs e l i c i t e d -27-F i g . 2. Progressive increase i n the mean c l a s s of e l i c i t e d MSs (Experiment 1). The l e f t panel i l l u s t r a t e s the increase i n the mean c l a s s of e l i c i t e d MSs over the f i r s t 60 days of the experiment. The r i g h t panel i l l u s t r a t e s the mean c l a s s of MSs e l i c i t e d during the ten r e c o r d i n g sessions before each animal d i s p l a y e d i t s f i r s t spontaneous MS. Thus, the poi n t above Day -1 i n d i c a t e s the mean c l a s s of MSs e l i c i t e d during the l a s t recording s e s s i o n before each animal d i s p l a y e d i t s f i r s t spontaneous MS. There were 2 or 3 days between each of these l a s t ten recording sessions. -29-i n each animal seemed to progress through three a d d i t i o n a l c l a s s e s . When the r e a r i n g and l o s s of e q u i l i b r i u m c h a r a c t e r i s t i c of a Cla s s 5 MS occurred more than once the MS was l a b e l l e d Class 6. Thus, a Class MS was com-p r i s e d of jaw and ear movements, head nodding, f o r e l i m b clonus, and a m u l t i p l e sequence of r e a r i n g and f a l l i n g . The next p a t t e r n of MS that emerged (Class 7) was s t r i k i n g l y ; d i f f e r e n t from any of the MSs that had preceded i t . Up to t h i s p o i n t each new MS p a t t e r n could be c h a r a c t e r i z e d simply by adding an a d d i t i o n a l behaviour p a t t e r n to the end of the p a t t e r n at the previous l e v e l . However, i n t h i s case the p a t t e r n was completely d i f f e r e n t and seemed to be i d e n t i c a l to the running f i t s t y p i c a l l y e l i c i t e d i n r a t s by audiogenic s t i m u l a t i o n ( C o l l i n s , 1972). Immediately upon d e l i v e r y of the current the animals ran r a p i d l y i n c i r c l e s , v i o l e n t l y jumped around, or r o l l e d over repeatedly. These three behaviours occurred e i t h e r s e p a r a t e l y or i n combination and were almost always accompanied by loud squealing. These Class 7 MSs u s u a l l y l a s t e d f o r about 30 sec. A Class 8 MS was e s s e n t i a l l y the same as a Cla s s 7, but i t culminated i n a curious p a t t e r n of body tonus: each animal reared and supported himself against a w a l l of the s t i m u l a t i o n chambre, balancing on i t s t a i l and one hindlimb. The MS ended a f t e r t h i s 5-sec p e r i o d of tonus. Three animals i n which Class 8 MSs had been e l i c i t e d e x h i b i t e d a stereotyped r e a c t i o n to the s t i m u l a t i o n on s e v e r a l occasions. Following s t i m u l a t i o n to the l e f t amygdala they walked i n slow clockwise c i r c l e s and were hy p e r r e a c t i v e to handling f o r s e v e r a l min. However, s i n c e t h i s response was observed i n only three animals, i t was not inc l u d e d i n the o v e r a l c l a s s i f i c a t i o n system. The r i g h t panel of Figure 2 i l l u s t r a t e s the mean c l a s s of MSs e l i c i t e d during the 10 days before spontaneous MSs were f i r s t observed -30-i n each i n d i v i d u a l subject. There d i d not appear to be any s t r i k i n g change i n the e l i c i t e d MSs during t h i s p e r i o d . Although the mean c l a s s of MSs on the l a s t day before spontaneity was higher than i t had been at any other p e r i o d of the experiment, the MSs e l i c i t e d on t h i s day were not s i g n i f i c a n t l y more intense than those e l i c i t e d on Day 60 ( s i g n t e s t , x=5, N = l l , p=0.50). Over the l a s t 10 days before spontaneity the s t i m u l a t i o n s e l i c i t e d MSs that were remarkably stereotyped i n i n d i v i d u a l s u b j e c t s . This ob-s e r v a t i o n i s i n c o n s i s t e n t w i t h the report of P i n e l et a l . (1975) that t h e i r two e p i l e p t i c subjects o c c a s i o n a l l y f a i l e d to respond to the stimu-l a t i o n , or responded w i t h MSs of low i n t e n s i t y and unusually short d u r a t i o n , im'Fithe p e r i o d j u s t before t h e i r f i r s t spontaneous--MSs were observed. I t i s p o s s i b l e that those two subjects were e x c e p t i o n a l cases; however, the present data suggest a more parsimonious i n t e r p r e t a t i o n . In the present study, although the subjects were very c o n s i s t e n t i n t h e i r responses to the s t i m u l a t i o n s p r i o r to spontaneity, the responses to s t i m u l a t i o n s i n the p e r i o d when animals were d i s p l a y i n g spontaneous MSs were f r e q u e n t l y extremely v a r i a b l e . Thus, the i n s t a b i l i t y of the p a t t e r n of e l i c i t e d MSs may be a feat u r e of the p e r i o d of spontaneity i t s e l f , r a t h e r than being an antecedent. J u s t such an hypothesis has been proposed p r e v i o u s l y by the authors of the o r i g i n a l report ( P i n e l et a l . , 1973). Duration of E l i c i t e d MSs. The pr o g r e s s i v e increases i n the dura-t i o n of e l i c i t e d MSs are i l l u s t r a t e d i n Figure 3. Although the f i r s t s t i m u l a t i o n e l i c i t e d a MS i n only one animal, by Day 11 MSs were e l i -c i t e d i n a l l subjects and the MS durations d i d not appear to change -31-F i g . 3. Progressive changes i n the mean du r a t i o n of e l i c i t e d MS ( E x p e r i -ment 1). The l e f t panel i l l u s t r a t e s the changes i n the du r a t i o n of e l i c i t e d MSs over the f i r s t 60 days of the experiment. The r i g h t panel i l l u s t r a t e s the mean du r a t i o n of e l i c i t e d MSs e l i c i t e d during the ten recording sessions before each animal d i s p l a y e d i t s f i r s t spontaneous MS. Thus, the poin t above Day -1 i n d i c a t e s the mean dur a t i o n of MSs e l i c i t e d during the l a s t r e c ording s e s s i o n before each animal d i s p l a y e d i t s f i r s t spontaneous MS. There were 2 or 3 days between each of these l a s t ten recording sessions. - z e --33-s y s t e m a t i c a l l y a f t e r t h i s p o i n t . This s t r i k i n g increase i n the durations of e l i c i t e d MSs during the e a r l y phase of k i n d l i n g has been f r e q u e n t l y reported ( c f . P i n e l et a l . , 1974). The r i g h t panel of Figure 3 i l l u s t r a t e s the mean durations of MSs e l i c i t e d during the ten recording sessions before each animal's f i r s t observed spontaneous MS. I t i s obvious that there were no systematic changes i n the durations of e l i c i t e d MSs during t h i s time. For example, during the s e s s i o n before the f i r s t spontaneous MS was observed i n each animal (Day -1) the e l i c i t e d MSs d i d not d i f f e r s i g n i f i c a n t l y i n d u r a t i o n from those e l i c i t e d on Day 60 ( s i g n t e s t , x=6, N=16, p=0.227). Thus, changes i n the d u r a t i o n of e l i c i t e d MSs do not seem to be r e l a t e d i n any obvious way to the development of spontaneity. I n t e r i c t a l Discharges. The development and p r o l i f e r a t i o n of IIDs (spike-shaped discharges w i t h more than twice the amplitude of the back-ground EEG) was i n d i c a t i v e of the imminent development of spontaneous MSs. Because e l e c t r o g r a p h i c a c t i v i t y was not monitored every d a y , i t i s d i f f i -c u l t to s p e c i f y e x a c t l y when IIDs developed i n each animal, but by the record-ing s e s s i o n on Day 29, IIDs were f i r s t observed i n 8 of the 16 experimental animals who e v e n t u a l l y d i s p l a y e d spontaneity. Although IIDs were observed i n some of these animals as e a r l y as Day 19 and i n others as l a t e as Day 43 (X=59.6 s t i m u l a t i o n s , range = 41 to 93 s t i m u l a t i o n s ) , they were observed i n each of these 16 s u b j e c t s . IIDs were f i r s t observed at the s i t e of s t i m u l a t i o n i n three animals, whereas they were f i r s t observed i n the c o n t r a l a t e r a l amygdala i n seven. In the remaining s i x experimental subjects which e v e n t u a l l y became spontaneous, the development of IIDs -34-seemed to progress at the same r a t e i n both amygdalae. IIDs e v e n t u a l l y developed i n both amygdalae i n a l l animals. Figure 4 i l l u s t r a t e s the mean incidence of the IIDs observed during the 60-sec p r e s t i m u l a t i o n p e r i o d before each of the l a s t ten re c o r d i n g sessions preceding each animal's f i r s t observed spontaneous..MS. I t i s c l e a r from Figure 4 that IIDs were observed i n both amygdalae during t h i s p e r i o d and that there was a s t r i k i n g increase i n the incidence of IIDs i n the animals' c o n t r a l a t e r a l amygdalae i n the l a s t three of these sessions ( s i g n t e s t , x=0, N=7, p=0.008). Thus, these f i n d i n g s are con-s i s t e n t w i t h the observations of both P i n e l et a l . (1975) and Wada et  a l . (1974); the present r e s u l t s agree w i t h those of P i n e l et a l . that IIDs can be recorded from the s i t e of s t i m u l a t i o n before spontaneity, and they al s o agree w i t h the report of Wada et a l . i n that the increase i n IIDs may be more s t r i k i n g at the c o n t r a l a t e r a l s i t e s . In f a c t , i n the present experiment t h i s increase on the c o n t r a l a t e r a l s i d e seemed to be a par-t i c u l a r l y r e l i a b l e s i g n that an animal would soon d i s p l a y spontaneous MSs. The two animals which s u r v i v e d the e n t i r e experiment, but d i d not ev e n t u a l l y d i s p l a y spontaneous MSs, d i s p l a y e d spontaneous IIDs r e l a t i v e l y e a r l y i n the experiment (Days 19 and 22). These IIDs were f i r s t observed i n the l e f t amygdala of one r a t and i n the r i g h t amygdala of the other, and ev e n t u a l l y became b i l a t e r a l i n both animals. Thus, i t does not appear that the e a r l y development of IIDs n e c e s s a r i l y s i g n a l s the e a r l y develop-ment of spontaneous MSs. In the past many i n v e s t i g a t o r s have r e f e r r e d to the focus estab-l i s h e d at the s i t e of s t i m u l a t i o n as the "primary focus" ( c f . A r n o l d et a l . , 1973). I n s o f a r as seven of the subjects which e v e n t u a l l y became -35-F i g . 4. Incidence of i n t e r i c t a l discharges recorded from s t i m u l a t e d and c o n t r a l a t e r a l amygdalae (Experiment 1). F i g . 4 i l l u s t r a t e s the mean incidence of i n t e r i c t a l discharges observed i n both amygdalae during the 60-sec p r e s t i m u l a t i o n p e r i o d preceding each of the l a s t ten s t i m u l a t i o n s before each animal d i s p l a y e d i t s f i r s t spontaneous MS. Thus, the poin t above Day -1 i n -d i c a t e s the mean incidence of i n t e r i c t a l discharges observed during the l a s t r e c ording s e s s i o n before each animal d i s p l a y e d i t s f i r s t spontaneous MS. There were 2 or 3 days between each of these l a s t ten recording sessions. -36-1—1 1 1 l L_ • • . i -10 -8 -6 -4 -2 RECORDING SESSIONS PRECEEDING SPONTANEOUS MSs -37-spontaneous i n the present experiment f i r s t d i s p l a y e d spontaneous IIDs i n the c o n t r a l a t e r a l amygdala, i t i s c l e a r that e p i l e p t o g e n i c f o c i may be i n i t i a l l y e s t a b l i s h e d i n areas other than the s i t e of s t i m u l a t i o n . Spontaneous MSs. P e r i o d i c s t i m u l a t i o n produced the e p i l e p t i c syndrome w i t h considerable r e l i a b i l i t y ; spontaneous MSs were e v e n t u a l l y d i s p l a y e d by 16 of the 18 experimental subjects although none were ever observed i n any of the unstimulated c o n t r o l s . The animals v a r i e d con-s i d e r a b l y i n the r a t e at which these spontaneous MSs developed during the course of the experiment. Spontaneous MSs were f i r s t observed i n each subject a f t e r an average of 79 days (170 s t i m u l a t i o n s ) ; however, t h i s number ranged f rom 41 days (88 s t i m u l a t i o n s ) to 132 days (293 s t i m u l a -t i o n s ) . The spontaneous MSs were s i m i l a r i n both p a t t e r n and dur a t i o n to -those e l i c i t e d by the s t i m u l a t i o n s . The c l a s s e s of these spontaneous MSs ranged from 1 to 7, and although some were abbreviated, i n many cases t h e i r durations were the same as those e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n . Even though the animals were not observed continuously, most subjects were observed to have at l e a s t three spontaneous MSs. Although only one spontaneous MS was observed i n each of three s u b j e c t s , 30 were observed i n one r a t . The e l e c t r o g r a p h i c a c t i v i t y a s s o c i a t e d w i t h a spontaneous Class 7 MS i s i l l u s t r a t e d i n Figure^ 5. The f a c t that the MS began s e v e r a l seconds before there was any obvious s p i k i n g recorded from e i t h e r of the two amygdalae suggests that the abnormal discharges r e s p o n s i b l e f o r t r i g g e r i n g t h i s p a r t i c u a l r s e i z u r e o r i g i n a t e d i n some other s t r u c t u r e . This confirms -38-F i g . 5. E l e c t r o g r a p h i c and motor a c t i v i t y of a subject during a spon-taneous Class 7 motor s e i z u r e (Experiment 1). -39-LEFT AMYGDALA' RIGHT AMYGDALA ACTIVITY ..^„.,..<v.^,..^.**tlMM^<«HUV*J»HM+W *ft 'VVi-^ 'h'" Isec -40-the view s t a t e d e a r l i e r that the focus f o r the e p i l e p t i c discharges i s not n e c e s s a r i l y at the s i t e of s t i m u l a t i o n . Thus, the present r e s u l t s confirm the report of P i n e l et a l . (1975) that an e p i l e p t i c syndrome can be generated i n r a t s w i t h long-term, p e r i o d i c e l e c t r i c a l s t i m u l a t i o n of the amygdala; however, these r e s u l t s extend or q u a l i f y those of P i n e l et a l . i n s e v e r a l important ways. The f a c t that 16 of the 18 experimental subjects d i s p l a y e d spontaneous MSs before the a r b i t r a r y t e r m i n a t i o n of the experiment suggests that spontane-i t y may be the i n e v i t a b l e r e s u l t of repeated amygdaloid s t i m u l a t i o n of the r a t r a t h e r than being an anomaly observed i n a small p o r t i o n of k i n d l e d animals. The impression of P i n e l et a l . that the development and p r o l i f e r a t i o n of IIDs preceded the f i r s t m a n i f e s t a t i o n of spontaneous MSs was a l s o confirmed. However, as Wada et a l . (1975) had observed i n the baboon, the p r o l i f e r a t i o n of IIDs before spontaneity was found to be more s t r i k i n g on the c o n t r a l a t e r a l s i d e . The p a t t e r n of development of e l i c i t e d MSs reported by P i n e l et a l . and many other i n v e s t i g a t o r s was confirmed i n the present experiment, but there were s e v e r a l important changes i n these e l i c i t e d MSs which occurred i n the l a t e r stages of k i n d l i n g which were documented here f o r the f i r s t time. The observation of systematic and stereotyped changes i n the form of e l i c i t e d MSs l e d to the a d d i t i o n of three c l a s s e s to Racine's o r i g i n a l f i v e - c l a s s s c a l e of kindled-MS development. EXPERIMENT 2 The primary purpose of Experiment 2 was to determine whether p e r i o d i c s t i m u l a t i o n of s t r u c t u r e s other than the amygdala would e v e n t u a l l y r e s u l t i n an e p i l e p t i c syndrome i n r a t s . Previous demonstrations that k i n d l i n g r e s u l t s i n spontaneous MSs employed only amygdaloid s t i m u l a t i o n ( P i n e l et a l . , 1973, 1975; Wada et a l . , 1974, 1976), w i t h the exception of a s i n g l e baboon who became spontaneous a f t e r p e r i o d i c s t i m u l a t i o n of the p r e f r o n t a l cortex (Wada et a l . , 1975). Thus, i n Experiment 2 the develop-ment of spontaneous MSs was s t u d i e d i n r a t s r e c e i v i n g amygdaloid, hippo-campal, caudate, or e n t o r h i n a l s t i m u l a t i o n . Goddard et a l . (1969) found that p e r i o d i c e l e c t r i c a l s t i m u l a t i o n a p p l i e d to any one of these four s t r u c t u r e s would l e a d to the development and pr o g r e s s i v e i n t e n s i f i c a t i o n of e l i c i t e d MSs. I f the development of an e p i l e p t i c syndrome can be viewed as a n a t u r a l extension of the k i n d l i n g of e l i c i t e d MSs, then stimu-l a t i o n to any of these four s i t e s should e v e n t u a l l y l e a d to the develop-ment of spontaneous MSs. Although Goddard et a l . found that the ra t e s of the development of e l i c i t e d MSs were d i f f e r e n t depending upon which of the above s t r u c t u r e s was s t i m u l a t e d , e l i c i t e d MSs seemed to develop i n the same fas h i o n regardless of the s i t e of s t i m u l a t i o n . The second purpose of Experiment 2 was to determine how long the e p i l e p t i c syndrome, once induced, would p e r s i s t . I f t h i s syndrome i n k i n d l e d animals i s comparable to c l i n i c a l cases of e p i l e p s y , one would expect i t to be r e l a t i v e l y enduring. From a p r a c t i c a l p o i n t of view, the longer the syndrome l a s t s , the more opportunity f o r i n v e s t i g a t i o n i t -42-a f f o r d s . Wada et a l . (1974) reported that one of t h e i r cats s e i z e d spontaneously f o r 53 days before developing status e p i l e p t i c u s , but d i d not provide s i m i l a r data f o r t h e i r other s u b j e c t s . P i n e l et a l . (1975) found that..the e p i l e p t i c syndrome p e r s i s t e d f o r 2 months i n one of t h e i r r a t s , and f o r only 1 week i n the other. In the present experiment, when subjects d i s p l a y e d spontaneous MSs they were no longer s t i m u l a t e d and changes i n the incidence of spontaneous MSs were s y s t e m a t i c a l l y assesse METHODS One b i p o l a r e l e c t r o d e was implanted i n each of the 72 male, b l a c k -hooded subjects (Canadian Breeding L a b o r a t o r i e s , La P r a i r i e , Quebec) which weighed between 285 and 530 g at the time of surgery. The s u r g i c a l pro-cedures were the same as those employed i n Experiment 1, except f o r the e l e c t r o d e placements. Equal numbers of subjects (n=18) had e l e c t r o d e s implanted at the f o l l o w i n g coordinates: amygdala, P 1.5, L -4.2, V 8.8; hippocampus, P 4.0, L -4.9, V 4.1; e n t o r h i n a l c o r t e x , P 3.7, L -4.0, V 8.5 (17°); caudate, A 1.9, L -3.2, V 4.7. F o l l o w i n g a 10-day p e r i o d of post-s u r g i c a l recovery, s t i m u l a t i o n s commenced f o r the experimental animals i n each group. As i n Experiment 1, the experimental subjects were s t i m u l a t e d (1-sec, 400-uA, 60-Hz, sine-wave current) about 15 times per week at i n t e r v a l s of no l e s s than 2 hr and no greater than 48; however, pre- and p o s t - s t i m u l a t i o n e l e c t r o g r a p h i c a c t i v i t y was monitored l e s s f r e q u e n t l y . Since the development of e l i c i t e d s e i z u r e s had been s t u d i e d i n d e t a i l i n Experiment 1, and the e a r l y phases of k i n d l i n g have been subjected to reasonably i n t e n s i v e i n v e s t i g a t i o n by other authors, e l e c t r o g r a p h i c -43-a c t i v i t y was monitored only every 2 or 3 weeks. On these occasions, pre-s t i m u l a t i o n e l e c t r o g r a p h i c a c t i v i t y was monitored f o r 2 min r a t h e r than 1 min i n order to provide a more r e l i a b l e assessment of the incidence of IIDs. Since the development of IIDs was the most obvious antecedent of spontaneity i n Experiment 1, when IIDs began to punctuate the records of an i n d i v i d u a l animal i n the present experiment, the animal was observed f o r 1 or 2 hr each day i n a d d i t i o n to the observation which r o u t i n e l y occurred during s t i m u l a t i o n sessions. When three spontaneous MSs of Class 5 i n t e n s i t y or greater were observed i n a given animal, 10 a d d i t i o n a l s t i m u l a t i o n s were administered to that animal at the usual i n t e r v a l s . Then that animal was observed f o r 1 hr per day f o r the next 35 days, during which the incidence and c l a s s of i t s spontaneous MSs were recorded. The remaining 12 r a t s served as unstimulated c o n t r o l s . Their e l e c t r o g r a p h i c a c t i v i t y was monitored p r i o r to the beginning of the experiment i n order to ensure that i t was normal and t h e r e a f t e r they were p e r i o d i c a l l y observed i n order to determine whether any were d i s p l a y i n g spontaneous MSs. Although a l l of the c o n t r o l animals s u r v i v e d the e x p e r i -ment, 33 of the experimental animals e i t h e r died or dislodged t h e i r e l e c t r o d e s during the course of the experiment and none of t h e i r data were subjected to a n a l y s i s . Of the 27 experimental animals which s u r v i v e d the experiment, 8 had el e c t r o d e s i n the amygdala, 5 had hippocampal e l e c t r o d e s , and there were 7 i n each of the e n t o r h i n a l and caudate groups. At the end of the experiment s e v e r a l experimental and c o n t r o l subjects were s a c r i f i c e d , perfused w i t h 8% buf f e r e d f o r m a l i n , and t h e i r -b r a i n s were removed and shipped to UCLA f o r G o l g i a n a l y s i s by Madge and Arnold S c h e i b e l . The other animals were kept for^6 a d d i t i o n a l -44-months of unsystematic ob s e r v a t i o n , a f t e r which they were s a c r i f i c e d and perfused w i t h 8% buf f e r e d f o r m a l i n . Their b r a i n s were then removed, sectioned, and s t a i n e d w i t h c r e s y l v i o l e t i n order to confirm the e l e c t r o d e placements. A l l of the e l e c t r o d e t i p s were found to be p o s i t i o n e d i n the target s t r u c t u r e s . RESULTS AND DISCUSSION P e r i o d i c s t i m u l a t i o n of each of the el e c t r o d e s i t e s l e d to a pro-g r e s s i v e increase i n the s e v e r i t y of the e l e c t r o g r a p h i c and behavioural responses s i m i l a r to that observed i n Experiment 1. A l l of the e x p e r i -mental animals that completed the experiment e v e n t u a l l y d i s p l a y e d spon-taneous MSs and continued to d i s p l a y them 35 days f o l l o w i n g the c e s s a t i o n of s t i m u l a t i o n s . Some subjects continued to d i s p l a y spontaneous MSs u n t i l they were s a c r i f i c e d 7 months a f t e r t h e i r l a s t s t i m u l a t i o n . As i n Experiment 1, IIDs were recorded from the s i t e of s t i m u l a t i o n i n the p e r i o d before each animal i n the amygdala group di s p l a y e d i t s f i r s t spontaneous MS, and the same was tr u e of subjects i n the hippocampus group. However, spontaneous IIDs were recorded i n only a few subjects from the e n t o r h i n a l cortex and were never recorded from the caudate, although a l l of the subjects i n each of these two groups e v e n t u a l l y d i s p l a y e d spontaneous MSs. The progressive i n t e n s i f i c a t i o n of e l i c i t e d s e i z u r e s i s i l l u s t r a t e d i n Figures 6,7, and 8 i n a manner s i m i l a r to the way that t h i s i n f o r m a t i o n was presented i n Experiment 1. In each of these f i g u r e s , Panel A i l l u s -t r a t e s the mean responses to s t i m u l a t i o n s e l i c i t e d during the f i r s t 64 -45-day s of the experiment, and Panel B i l l u s t r a t e s the mean responses e l i c i t e d i n the weeks p r i o r to each subject's f i r s t observed spontaneous MS. AD Duration. Although there were a few exceptions e a r l y i n the experiment, the s t i m u l a t i o n s c o n s i s t e n t l y e l i c i t e d ADs i n each animal. Panel A of Figure 6 i l l u s t r a t e s the progressive increases i n the dura-t i o n s of ADs observed i n the four experimental groups over the f i r s t 64 days of the experiment. The ADs e l i c i t e d on Day 64 were s i g n i f i c a n t l y longer than those e l i c i t e d on Day 1 (F(3,69) = 13.98, p<0.001). However, the o v e r a l l d i f f e r e n c e i n AD durations between the groups f o r the same pe r i o d was not s i g n i f i c a n t (F(3,23)=2.31, p=0.104). Although the mean hippocampal ADs tended to be longer than the others f o r the f i r s t 64 days, the l a t e r d i f f e r e n c e s were l e s s s t r i k i n g . Racine (1972b) al s o found that the ADs e l i c i t e d by hippocampal s t i m u l a t i o n were i n i t i a l l y longer than those e l i c i t e d by amygdaloid s t i m u l a t i o n s ; however, i n h i s experiment he found that by the time Class 5 MSs were e l i c i t e d i n h i s r a t s amygdaloid ADs were of f a r greater duration. There seems to be one major d i f f e r e n c e i n the course of development of ADs between the animals i n Experiment 1 and those r e c e i v i n g amygdaloid s t i m u l a t i o n i n the present experiment, whereas the durations of the ADs e l i c i t e d i n the subjects i n Experiment 1 were asymptotic by about Day 11 and then d e c l i n e d somewhat, the durations of amygdaloid ADs i n E x p e r i -ment 2 increased monotonically u n t i l Day 64. An a n a l y s i s of the data summarized i n Panel B of Figure 6 i n d i c a t e d that there were no systematic changes i n the durations of ADs e l i c i t e d over the l a s t four recording sessions before each animal f i r s t d i s p l a y e d -46-F i g . 6. Pro g r e s s i v e changes i n the mean du r a t i o n of ADs (Experiment 2). Panel A i l l u s t r a t e s the changes i n AD d u r a t i o n of the four groups over the f i r s t 64 days of the experiment. Panel B i l l u s t r a t e s the mean du r a t i o n of ADs e l i c i t e d during the four r e c o r d i n g ses-sions before each animal d i s p l a y e d i t s f i r s t spontaneous MS. Thus, the p o i n t s above Recording Session -1 i n d i c a t e the mean durations of ADs of each group e l i c i t e d during the l a s t r e c ording session before each animal d i s p l a y e d i t s f i r s t spontaneous MS. There were 2 or 3.weeks between each of these l a s t f o u r recording sessions. A O D U R A T I O N (SEC) ro O) co q o o o o o -48-spontaneous MSs. Although the durations of ADs e l i c i t e d i n the subjects i n the four groups d i f f e r e d s i g n i f i c a n t l y (F(3,23)=6.73, p=0.002), l a r g e l y because of the d i f f e r e n c e s between the hippocampal and caadate animals, there were no s i g n i f i c a n t changes during t h i s p e r i o d (E(.3,69)=0.11, p=0.95). Thus, as i n Experiment 1, the development of spontaneity does not seem to be r e l a t e d i n any obvious f a s h i o n to changes i n the durations of ADs. Class of E l i c i t e d MSs. The f i r s t s t i m u l a t i o n of the amygdala, hippocampus, or e n t o r h i n a l cortex produced no obvious motor response other than the o c c a s i o n a l C l a s s 1 MS; whereas the f i r s t caudate s t i m u l a t i o n i n v a r i a b l y e l i c i t e d MSs i n : a l l s u b j e c t s , and i n a l l but one case they were of Class 3 i n t e n s i t y or greater. However, t h e r e a f t e r a s i m i l a r progres-s i v e increase i n MS c l a s s was observed i n a l l groups (Panel A of Figure 7). Thus, by Day 64 the c l a s s e s of e l i c i t e d MSs were s i g n i f i c a n t l y higher o v e r a l l than they were on Day 1 (F(3,69)=53.96, p<0.001). Although MSs e l i c i t e d i n most of the subjects which were k i n d l e d w i t h amygdaloid, hippocampal, or e n t o r h i n a l s t i m u l a t i o n progressed through the e i g h t - c l a s s s c a l e devised i n Experiment 1, none of the animals i n the caudate group ever experienced an e l i c i t e d MS of greater s e v e r i t y than Class 6. A l -though the progressive increase i n the c l a s s of MSs e l i c i t e d by amygdaloid s t i m u l a t i o n was s i m i l a r to that observed i n Experiment 1, the " c i r c l i n g behaviour" d i s p l a y e d by three animals i n Experiment 1 was never observed i n the present experiment. There were no s i g n i f i c a n t d i f f e r e n c e s i n the r a t e of k i n d l i n g that were a t t r i b u t a b l e to the s i t e of s t i m u l a t i o n (F(3,23)=0.53, p=0.66). This i s s u r p r i s i n g indeed i n view of the report of Goddard et a l . (1969) that -49-F i g . 7. Progressive increases i n the mean c l a s s of e l i c i t e d MSs ( E x p e r i -ment 2). Panel A i l l u s t r a t e s the increases i n MS c l a s s of the four groups over the f i r s t 64 days of the experiment. Panel B i l l u s t r a t e s the mean c l a s s of MSs e l i c i t e d during the fou r recording sessions before each animal d i s p l a y e d i t s f i r s t spon-taneous MS. Thus, the p o i n t s above Recording Session -1 i n -d i c a t e the mean c l a s s e s of MSs of each group e l i c i t e d during the l a s t recording s e s s i o n before each animal d i s p l a y e d i t s f i r s t spontaneous MS. There were 2 or 3 weeks between each of these l a s t four recording sessions. 8 • 7 • 6 • 5 \-• A M Y G D A L A Q H I P P O C A M P U S A E N T O R H I N A L C O R T E X ^ C A U D A T E CO CO < _ i 4 CS CO I 1 I 1 10 32 D A Y S -51-there were l a r g e d i f f e r e n c e s i n the r a t e s of k i n d l i n g produced by p e r i o d i c s t i m u l a t i o n of the four s i t e s s t i m u l a t e d i n the present experiment. A l -though there i s other evidence which suggests that some l i m b i c s t r u c t u r e s d i f f e r i n t h e i r s u s c e p t i b i l t y to k i n d l i n g (Racine, 1972b; Racine, Okujava, & C h i p a s h v i l i , 1972), the d i f f e r e n c e s found i n those s t u d i e s were not n e a r l y as l a r g e as those reported by Goddard et a l . Since k i n d l i n g i s a f u n c t i o n of the number of ADs e l i c i t e d r a t h e r than of the number of s t i m u l a t i o n s administered (Racine, 1972b),the d i f f e r e n t r a t e s of k i n d l i n g i n the Goddard et a l . study may p r i m a r i l y r e f l e c t the d i f f e r e n t AD thresholds of the s t r u c t u r e s . This view i s q u i t e p l a u s i b l e i n l i g h t of the f i n d i n g that at l e a s t three l i m b i c s t r u c t u r e s (the amygdala, hippocampus, and s e p t a l area) have d i f f e r e n t AD thresholds (Racine, 1972a). Racine, Burnham, Gartner, and L e v i t a n (1973) have p r e v i o u s l y suggested that the low current i n t e n s i t y (50 PA) employed by Goddard et a l . may have f a i l e d to r e l i a b l y t r i g g e r ADs i n a l l of t h e i r s u b j e c t s ; however, t h i s i s im-p o s s i b l e to determine, s i n c e Goddard et a l . d i d not monitor the e l e c t r o -graphic e f f e c t s . Thus, previous observations that the s t i m u l a t i o n of various s t r u c t u r e s r e s u l t s i n d i f f e r e n t r a t e s of k i n d l i n g may be due to e i t h e r d i f f e r e n t AD thresholds i n some s t r u c t u r e s or to t h e i r d i f f e r e n t i a l s u s c e p t i b i l i t y to k i n d l i n g , or perhaps a combination of both. However, i n the present experiment i t was c l e a r that the current i n t e n s i t y employed here (400 uA) r e l i a b l y e l i c i t e d ADs at a l l four s i t e s , and there was no evidence that these s i t e s were d i f f e r e n t i a l l y s e n s i t i v e to k i n d l i n g . The p r o g r e s s i v e changes i n the patterns of MSs e l i c i t e d by p e r i o d i c s t i m u l a t i o n s of the amygdala, hippocampus, and e n t o r h i n a l c o r t e x were i n -d i s t i n g u i s h a b l e . However, the p a t t e r n s of MSs a s s o c i a t e d w i t h p e r i o d i c -52-caudate s t i m u l a t i o n s d i f f e r e d i n three important respects. F i r s t , as mentioned p r e v i o u s l y , a l l but one of the seven caudate animals d i s p l a y e d a Class 3 or 4 MS at the f i r s t s t i m u l a t i o n . Secondly, t h e i r e l i c i t e d MSs were never of greater i n t e n s i t y than Class 6. T h i r d l y , i n a form s i m i l a r to MSs e l i c i t e d by s t i m u l a t i o n of the c i n g u l a t e cortex (Racine, 1975), they immediately f e l l over when st i m u l a t e d and remained immobile f o r about 5 sec before d i s p l a y i n g the stereotyped l i m b i c MS p a t t e r n . Panel B of Figure 7 i l l u s t r a t e s the mean c l a s s of MSs e l i c i t e d during each of the four recording sessions j u s t before spontaneous MSs were f i r s t observed i n the i n d i v i d u a l animals. Subjects i n the four groups d i f f e r e d s i g n i f i c a n t l y i n t h e i r mean c l a s s e s of e l i c i t e d MSs during t h i s p e r i o d (F(3,23)=3.41, p=0.04), apparently because caudate s t i m u l a t i o n never e l i c i t e d Class 7 or 8 MSs. However, there were no systematic changes i n the c l a s s e s of e l i c i t e d MSs i n the 6- to 8-week pe r i o d before spontaneity (F(3,69)=0.58, p=0.63). Durat i o n of E l i c i t e d MSs. The pro g r e s s i v e increase i n the dura-t i o n s of e l i c i t e d MSs over the f i r s t 64 days of the experiment i s i l l u s -s t r a t e d i n Panel A of Figure 8. In those cases where the f i r s t s t i m u l a -l a t i o n e l i c i t e d a MS, i t was of r e l a t i v e l y short d u r a t i o n ; however, by the e i g h t h s t i m u l a t i o n a l l 27 of the experimental animals were d i s p l a y i n g e l i c i t e d MSs which monotonically increased i n dur a t i o n over the f i r s t 64 days of the experiment. Thus, on Day 64 the mean durations of e l i c i t e d MSs were s i g n i f i c a n t l y g reater o v e r a l l than on Day 1 (F(3,69)=28.73, p<0.001) although the s i t e of s t i m u l a t i o n had no s i g n i f i c a n t e f f e c t on the dur a t i o n of MSs e l i c i t e d during t h i s p e r i o d (F(3,23)=l.42, p=0.26. Panel B of Figure 8 i l l u s t r a t e s that the s i t e of s t i m u l a t i o n had -53-F i g . 8. Progressive changes i n the mean d u r a t i o n of e l i c i t e d MSs ( E x p e r i -ment 2). Panel A i l l u s t r a t e s the increases i n MS d u r a t i o n of the four groups over the f i r s t 64 days of the experiment. Panel B i l l u s t r a t e s the mean du r a t i o n of MSs e l i c i t e d during the four recording sessions before each animal d i s p l a y e d i t s f i r s t spon-taneous MS. Thus, the p o i n t s above Recording Session -1 i n d i c a t e the mean durations of MSs e l i c i t e d during the l a s t r e c o r d i n g s e s s i o n before each animal d i s p l a y e d i t s f i r s t spontaneous MS. There were 2 or 3 weeks between each of these l a s t four recording sessions. -55-no appreciable e f f e c t on the durations of MSs e l i c i t e d i n the p e r i o d before spontaneity (F(3,23)=0.15, p=0.93). Moreover, there were no systematic changes i n the durations of e l i c i t e d MSs that occurred over t h i s time (F(3,69)=0.88, p=0.46). Thus, the development of spontaneity appears to be u n r e l a t e d to any changes that occur i n the durations of e l i c i t e d MS. I n t e r i c t a l Discharges. As i n Experiment 1, the increases i n the incidence of IIDs was the only event which seemed to i n d i c a t e the imminent development of spontaneous MSs. However, t h i s was the case only f o r the subjects i n the amygdala and hippocampus groups. Figure 9 i l l u s t r a t e s the mean number of IIDs i n each of the four experimental groups recorded during the 2-min p r e s t i m u l a t i o n p e r i o d on each of the l a s t f o u r occasions before spontaneity when e l e c t r o g r a p h i c a c t i v i t y was monitored. Each sub-j e c t r e c e i v i n g amygdaloid or hippocampal s t i m u l a t i o n d i s p l a y e d IIDs before the development of spontaneous MSs. Subjects i n both of these groups r e -quired an average of about 200 s t i m u l a t i o n s before t h e i r f i r s t IIDs were observed; however, i t i s d i f f i c u l t to do more than estimate t h i s f i g u r e since e l e c t r o g r a p h i c a c t i v i t y was monitored so i n f r e q u e n t l y . On the other hand, no IIDs were ever recorded from those animals w i t h e l e c t r o d e s i n the caudate, and only three of the seven subjects w i t h e l e c t r o d e s i n the e n t o r h i n a l cortex d i s p l a y e d IIDs before they e x h i b i t e d spontaneous MSs. Figure 9 i l l u s t r a t e s the s i g n i f i c a n t d i f f e r e n c e s i n the incidence of IIDs between the four experimental groups i n the prespontaneity p e r i o d (F(3,23)=3.36, p=0.04), the l a c k of o v e r a l l d i f f e r e n c e between the incidence of IIDs recorded on Sessions -4 and -1 (F(3,64)=0.05, p=0.98), and the h i g h l y s i g n i f i c a n t i n t e r a c t i o n e f f e c t (F(3,9)=2.50, p=0.02) which seemed -56-F i g . 9; Incidence of i n t e r i c t a l discharges (Experiment 2). F i g . 9 i l l u s t r a t e s the mean incidence of i n t e r i c t a l discharges observed i n each of the four groups during the 2-min p r e s t i m u l a t i o n p e r i o d preceding each of the l a s t four s t i m u l a t i o n s before each animal d i s p l a y e d i t s f i r s t spontaneous MS. Thus, the p o i n t s above Recording Session -1 i n d i c a t e the mean incidence of i n t e r i c t a l discharges of each group observed during the l a s t r e c ording session before each animal d i s p l a y e d i t s f i r s t spontaneous MS. There were 2 or 3 weeks between each of these l a s t four recording sessions. -58-to r e s u l t from the d i f f e r e n t trends observed i n the amygdala and campus animals. The r e s u l t s of c o r r e l a t e d _ t - t e s t s i n d i c a t e d that the incidence of IIDs increased s i g n i f i c a n t l y i n subjects i n the amygdala group over t h i s p e r i o d (_t(6)=2.71, p<0.01), but that there was no s i g n i f i -cant change i n animals i n the hippocampus group (_t (3) = 1.11, p=0.05). The apparently s t r i k i n g change i n the hippocampus group was i n s i g n i f i c a n t because of the high v a r i a b i l i t y i n t h i s group. The idea that the development and p r o l i f e r a t i o n of IIDs i s a c r i t i c a l step i n the development of spontaneous MSs r e c e i v e s support from experiments on the k i n d l i n g of MSs. The most popular view of the k i n d l i n g of MSs i s that when ADs are f i r s t e l i c i t e d they become more ge n e r a l i z e d and then become a s s o c i a t e d w i t h MSs. A simple extension of t h i s l o g i c can be used to account f o r the development of spontaneous MSs. According to t h i s view, spontaneous IIDs at f i r s t remain r e l a t i v e l y l o c a l and i s o l a t e d , but each discharge f a c i l i t a t e s the production of other discharges and t h e i r g e n e r a l i z a t i o n to other s t r u c t u r e s u n t i l the point where they are s u f f i c i e n t l y frequent and general to be a s s o c i a t e d w i t h motor symptoms. In t h i s context one might view as somewhat s u r p r i s i n g the f i n d i n g i n Experiment 2 that many animals which d i s p l a y e d spontaneous MSs never d i s p l a y e d IIDs. However, i n a d d i t i o n to the r e s u l t s of E x p e r i -ment 1, s e v e r a l previous r e p o r t s i n d i c a t e that e p i l e p t o g e n i c discharges a s s o c i a t e d w i t h spontaneous MSs do not n e c e s s a r i l y o r i g i n a t e at the s i t e of s t i m u l a t i o n (Wada et a l . , 1974, 1975, 1976). Thus, i t i s most par-simonious at t h i s time to assume that IIDs preceded the development of spontaneous MSs i n a l l of the spontaneous animals i n Experiment 2, but that they were not n e c e s s a r i l y manifested at the s i t e of s t i m u l a t i o n . -59-Spontaneous MSs. As i n Experiment 1, there was considerable v a r i a b i l i t y i n the r a t e at which i n d i v i d u a l subjects developed spontaneous MSs. The number of s t i m u l a t i o n s r e q u i r e d to generate the e p i l e p t i c syndrome i n the 27 experimental animals ranged from 92 to 508, w i t h a mean of 348. An a n a l y s i s of variance i n d i c a t e d that there was no s i g n i -f i c a n t d i f f e r e n c e between the r a t e s at which subjects i n the four groups developed spontaneous MSs (F(3,23)=0.29, p>0.05). In most cases, the spontaneous MSs were the same as those e l i c i t e d by s t i m u l a t i o n , except f o r the f a c t that spontaneous MSs d i s p l a y e d by animals i n the caudate group lacked the i n i t i a l 5-sec l o s s of p o s t u r a l c o n t r o l . Thus, i t was impossible to d i f f e r e n t i a t e between the four groups on the b a s i s of the form of t h e i r spontaneous MSs. Figure 10 i l l u s t r a t e s the mean incidence of spontaneous MSs during the 35-day p o s t - s t i m u l a t i o n observation p e r i o d i n the subjects completing the experiment. Of the 27 subjects who d i s p l a y e d spontaneity, 16 d i d not su r v i v e the e n t i r e 35-day p e r i o d , and t h e i r data were not subjected to analy-s i s . Of those animals that d i d s u r v i v e , four were i n the amygdala group, three were i n the hippocampus group, and there were two each i n the caudate and e n t o r h i n a l groups. The e l e c t r o d e placements had no s i g n i f i c a n t o v e r a l l e f f e c t on the incidence of spontaneous MSs during t h i s 35-day p e r i o d (F(3,7)=0.95, p=0.47). Even though spontaneous s e i z u r e s were observed i n most of the subjects on Day 35, there was a s i g n i f i c a n t o v e r a l l decrease i n the in c i d e n c e of spontaneous MSs over the 35-day p e r i o d (F(34,238)=2.11, p=0.001). However s e v e r a l animals were observed f o r as long as 7 months f o l l o w i n g the c e s s a t i o n of s t i m u l a t i o n s and they continued to d i s p l a y spontaneous s e i z u r e s u n t i l the experiment was a r b i t r a r i l y terminated. -60-F i g . 10. Incidence of spontaneous MSs during p o s t s t i m u l a t i o n observations (Experiment 2). F i g . 10 i l l u s t r a t e s the mean incidence of spontaneous MSs f o r each of the four groups which were observed during each animal's 35 d a i l y p o s t s t i m u l a t i o n observation ses-sions. Each observation s e s s i o n was 1-hr long. NUMBER OF SEIZURES PER OBSERVATION -62-The r e s u l t s of Experiment 2 support the view that an e p i l e p t i c syndrome i s the i n e v i t a b l e c u l m i n a t i o n of the k i n d l i n g process. Regardless of the s i t e of s t i m u l a t i o n , the gradual i n t e n s i f i c a t i o n of e l i c i t e d s e i z u r e s e v e n t u a l l y l e d to the m a n i f e s t a t i o n of spontaneous MSs i n every subject who completed the experiment. This e p i l e p t i c syndrome, once induced, proved to be r e l a t i v e l y enduring, l a s t i n g i n some cases f o r up to 7 months a f t e r the c u r t a i l m e n t of s t i m u l a t i o n s . \ -63-GENERAL DISCUSSION Spontaneous Seizures The present experiments have e s t a b l i s h e d s e v e r a l important p o i n t s con-cerning the spontaneous MSs which are e v e n t u a l l y d i s p l a y e d by k i n d l e d r a t s . 1) Incidence. The present r e s u l t s confirm previous r e p o r t s that p e r i o d i c amygdaloid s t i m u l a t i o n may l e a d to the development of spontaneous MSs ( P i n e l et a l . , 1973, 1975; Wada et a l . , 1974, 1976). Although these previous s t u d i e s demonstrated that spontaneity could be induced by p e r i -o dic amygdaloid s t i m u l a t i o n , t h i s i s the f i r s t i n stance i n which spontaneity was demonstrated i n a reasonably l a r g e number of s u b j e c t s . Hence, i t pro-v i d e s the f i r s t i n f o r m a t i o n concerning the r e l i a b i l i t y w i t h which p e r i o d i c amygdaloid s t i m u l a t i o n can induce an. e p i l e p t i c syndrome. The r e s u l t s of both Experiments 1 and 2 suggest that an e p i l e p t i c syndrome may be the i n -e v i t a b l e r e s u l t of long-term, p e r i o d i c , amygdaloid s t i m u l a t i o n . In E x p e r i -ment 1, 16 of the 18 experimental subjects d i s p l a y e d spontaneous MSs before the experiment was a r b i t r a r i l y terminated. In Experiment 2, a l l of the amygdaloid subjects e v e n t u a l l y d i s p l a y e d spontaneous MSs. The main d i f -ference between these experiments and others i n which the development of spontaneity has not been observed i n r a t s k i n d l e d w i t h amygdaloid stimu-l a t i o n i s that i n the present case considerably more s t i m u l a t i o n s were administered over a longer p e r i o d of time. 2) Placements. The present f i n d i n g s provide the f i r s t systematic evidence that an e p i l e p t i c syndrome can be induced by the p e r i o d i c s t i m u l a t i o n of s t r u c t u r e s other than the amygdala. The only previous report of spontaneity r e s u l t i n g from the s t i m u l a t i o n of a s t r u c t u r e other -64-than the amygdala was published by Wada et a l . (1975), but i t was the report of only a s i n g l e subject. In Experiment 2 i t was found that l o n g -term p e r i o d i c s t i m u l a t i o n of the hippocampus, caudate, or e n t o r h i n a l cor-t e x , as w e l l as the amygdala, r e l i a b l y leads to the development of spontaneous MSs. The number of s t i m u l a t i o n s which preceded spontaneity d i d not vary s i g n i f i c a n t l y between these s i t e s , and the behavioural form of the spontaneous MSs was i n d i s t i n g u i s h a b l e i n a l l of the animals. 3) Permanence. The r e s u l t s of Experiment 2 provide the f i r s t systematic i n f o r m a t i o n regarding the permanence of the e p i l e p t i c syndrome induced by k i n d l i n g . Although other i n v e s t i g a t o r s have i n d i c a t e d that the syndrome p e r s i s t e d f o r some time a f t e r s t i m u l a t i o n s were c u r t a i l e d ( c f . P i n e l et a l . , 1975; Wada et a l . , 1974) they d i d not observe i t s permanence i n a systematic f a s h i o n . A l l of the subjects i n Experiment 2 continued to d i s p l a y spontaneous MSs during the 35-day ob s e r v a t i o n p e r i o d f o l l o w i n g the t e r m i n a t i o n of s t i m u l a t i o n s , and those animals which were observed i r r e g u l a r l y f o r an a d d i t i o n a l 6 months continued to d i s p l a y spontaneous MSs throughout t h i s p e r i o d . 4) Antecedents. The r e s u l t s of the present experiments confirm, and i n some cases q u a l i f y , the impressions of previous i n v e s t i g a t o r s that the development of IIDs seem to be an important event i n the development of spontaneity. In animals k i n d l e d w i t h amygdaloid s t i m u l a t i o n there was a development of IIDs before spontaneity, and i n Experiment 1 the pro-l i f e r a t i o n of these IIDs was found to be p a r t i c u l a r l y s t r i k i n g i n the c o n t r a l a t e r a l amygdala. IIDs were al s o observed i n a l l of the hippocampus subjects who e v e n t u a l l y d i s p l a y e d spontaneous MSs. However, although a l l of the e n t o r h i n a l and caudate animals e v e n t u a l l y became spontaneous, -65-only some of the e n t o r h i n a l subjects and none of the caudate animals d i s p l a y e d IIDs at the s i t e of s t i m u l a t i o n . Since the development of spontaneous MSs i n the amygdala animals seems to be a s s o c i a t e d w i t h a p r o l i f e r a t i o n of IIDs, and s i n c e the development of e l i c i t e d MSs i n the caudate and e n t o r h i n a l animals appeared to progress i n approximately the same manner, i t i s most reasonable at t h i s time to assume that these animals were experiencing IIDs, but they were not being generated from the s i t e of s t i m u l a t i o n . I f t h i s were true i t i n d i c a t e s t h a t , i n c o n t r a s t to the assumption that seems to be i m p l i c i t i n some i n v e s t i g a t i o n s ( c f . A r n o l d et a l . , 1973),the e p i l e p t i c focus i s not n e c e s s a r i l y at or near the s i t e of s t i m u l a t i o n . Supporting t h i s view i s the f a c t that i n Experiment 1 IIDs were f r e q u e n t l y recorded from the c o n t r a l a t e r a l amyg-dala even when they d i d not occur at the s i t e of s t i m u l a t i o n . Moreover, i t was p o s s i b l e f o r a spontaneous MS to begin without any a b n o r m a l i t i e s from e i t h e r of the e l e c t r o d e s i t e s (Figure 5). I t was s t r i k i n g that there were no systematic changes i n the e l i c i t e d s e i z u r e s that seemed to i n d i c a t e the imminent m a n i f e s t a t i o n of spontaneous MSs. This absence of a r e l a t i o n s h i p between changes i n e l i c i t e d s e i z u r e s and the development of spontaneous s e i z u r e s has two important i m p l i c a t i o n s , one t h e o r e t i c a l and one p r a c t i c a l . The t h e o r e t i c a l i m p l i c a t i o n of t h i s l a c k of r e l a t i o n s h i p i s that the development of spontaneity does not simply r e s u l t from an extension of the same processes which are r e s p o n s i b l e f o r the i n t e n s i f i c a t i o n of e l i c i t e d s e i z u r e s . The p r o g r e s s i v e development of e l i c i t e d ADs and MSs appears to be complete long before the f i r s t m a n i f e s t a t i o n s of spontaneous motor symptoms. Moreover, there was no obvious r e l a t i o n between the development of e l i c i t e d MSs -66-and the r a t e at which spontaneous MSs developed. For example, there was no s i g n i f i c a n t c o r r e l a t i o n between the number of s t i m u l a t i o n s neces-sary to e l i c i t a s u b ject's f i r s t C lass 5 MS and the number before i t s f i r s t spontaneous MS (r(41)=0.118, p>0.05). Thus, whatever mechanisms u n d e r l i e the i n t e n s i f i c a t i o n of e l i c i t e d MSs they do not seem to be the same as those r e s p o n s i b l e f o r the l a t e r development of spontaneous MSs. I f a theory i s going to be developed to e x p l a i n the e n t i r e course of k i n d l i n g and i t s c u l m i n a t i o n i n spontaneity, i t appears that i t w i l l have to i n v o l v e more than a s i n g l e mechanism. On the p r a c t i c a l s i d e , t h i s l a c k of r e l a t i o n between the course of development of e l i c i t e d s e i z u r e s and the development of spontaneity has i m p l i c a t i o n s f o r the use of the k i n d l i n g paradigm as a model of e p i l e p t o g e n e s i s , e s p e c i a l l y as an assay device f o r convulsant and a n t i -convulsant drugs. I n v e s t i g a t o r s conducting s t u d i e s of t h i s type ( c f . Racine, L i v i n g s t o n , & Joaquin, 1975) u s u a l l y i n j e c t subjects w i t h a given drug p r i o r to each s t i m u l a t i o n , and then observe the drug's e f f e c t on e l i c i t e d s e i z u r e s . However, these s t u d i e s have u s u a l l y been terminated when animals have d i s p l a y e d t h e i r f i r s t g e n e r a l i z e d e l i c i t e d MSs, or s h o r t l y t h e r e a f t e r . Thus, i f drugs do not block the development of e l i c i t e d MSs to t h i s p o i n t , i t i s assumed that they have no e f f e c t on e p i l e p t o g e n e s i s . In l i g h t of the present f i n d i n g s that k i n d l i n g i s f a r from complete when g e n e r a l i z e d MSs are f i r s t e l i c i t e d , subsequent attempts to screen drugs f o r t h e i r a n t i e p i l e p t o g e n i c e f f e c t s should look at the e n t i r e course of e p i l e p t o g e n e s i s . I t may be that a drug which i s completely i n e f f e c t i v e i n b l o c k i n g e l i c i t e d s e i z u r e s may block the development of spontaneous s e i z u r e s . -67-E l i c i t e d Seizures Although the primary purpose of the present experiments was to study the spontaneous MSs produced by s t i m u l a t i o n of the .amygdala or other s i t e s , the p r o g r e s s i v e development of e l i c i t e d s e i z u r e s was a l s o documented during the course of the experiment. 1) AD Duration. Two d i f f e r e n t patterns of the development of ADs were noted i n the amygdaloid su b j e c t s . In Experiment 1 the durations of ADs increased d r a m a t i c a l l y over the f i r s t 18 s t i m u l a t i o n s , and then p a r t i a l l y d e c l i n e d . However, i n Experiment 2 the .durations of ADs increased monotonically. Although on the b a s i s of the present i n v e s t i g a -t i o n i t i s impossible to determine which f a c t o r s produced these two pat-t e r n s , both the monotonic increase and the b i p h a s i c p a t t e r n of develop-ment have been p r e v i o u s l y reported (cf. P i n e l et a l . , 1973; Racine, Burnham, Gartner, & L e v i t a n , 1973). 2) MS. Duration. The monotonic increases of the durations of the e l i c i t e d MSs i n the amygdaloid subjects i n both Experiments 1 and 2 were s i m i l a r to numerous previous reports ( c f . Mclntyre & Goddard, 1973). In p a r t i c u l a r , the s t r i k i n g increase i n the d u r a t i o n of e l i c i t e d MSs during the e a r l y phase of k i n d l i n g has been f r e q u e n t l y observed ( c f . P i n e l et a l . , 1974). 3) MS P a t t e r n s . The p r o g r e s s i v e increase i n the s e v e r i t y of MSs e l i c i t e d w i t h amygdaloid s t i m u l a t i o n was the same (up to Class 5) as that observed by other i n v e s t i g a t o r s ( c f . Racine , 1972b). However, w i t h continued s t i m u l a t i o n s MSs continued to grow more severe, although the r a t e of change was slower than that observed i n the e a r l i e r phases of k i n d l i n g . The m a n i f e s t a t i o n s of these more severe e l i c i t e d MSs included-the -68-development of m u l t i p l e sequences of r e a r i n g and f a l l i n g , running f i t s , and running f i t s which culminated i n a r i g i d body tonus. On the b a s i s of these observations, three c l a s s e s of MSs were added to Racine's o r i g i n a l f i v e - c l a s s s c a l e . This h i g h l i g h t s the f a c t that most previous k i n d l i n g s t u d i e s have been a r b i t r a r i l y c u r t a i l e d not only long before spontaneous MSs developed, but even long before the development of e l i c i t e d MSs was complete. 4) Placement. The same general course of the development of ADs and e l i c i t e d MSs that was observed i n the amygdala . animals was a l s o seen i n subjects r e c e i v i n g hippocampal, e n t o r h i n a l , or caudate s t i m u l a t i o n . There were, however, two exceptions. F i r s t , the durations of ADs i n the hippocampal animals were c o n s i s t e n t l y longer than those of the other subjects. Secondly, the c l a s s e s of e l i c i t e d MSs of the caudate animals ranged only from 3 to 6, and the MSs began w i t h an immediate l o s s of p o s t u r a l c o n t r o l before the d i s p l a y of the usual l i m b i c - s e i z u r e p a t t e r n . In form, these MSs c l o s e l y resembled those e l i c i t e d by s t i m u l a t i o n of the c i n g u l a t e cortex (Racine, 1973). One of the most s u r p r i s i n g observations i n Experiment 2 was that there d i d not seem to be any s i g n i f i c a n t d i f f e r e n c e s i n the rat e s of k i n d l i n g of animals i n the four experimental groups, even though s t r i k i n g d i f f e r e n c e s has been reported by other i n v e s t i g a t o r s , namely Goddard et a l . (1969). As suggested e a r l i e r , these l a r g e d i f f e r e n c e s observed by Goddard et a l . may have been due to e i t h e r the d i f f e r e n t i a l s e n s i t i v i t y to k i n d l i n g of these l i m b i c s t r u c t u r e s , or to the f a c t that the AD thresholds i n some s t r u c t u r e s may have been higher than the s t i m u l a t i o n l e v e l s (50 uA) employed, or a combination of both. However, the r e l a t i v e l y -69-high current i n t e n s i t y employed i n the present study r e l i a b l y e l i c i t e d ADs at a l l four s i t e s , and there was l i t t l e evidence that these s i t e s were d i f f e r e n t i a l l y s e n s i t i v e to k i n d l i n g . Aggressive Behaviour and K i n d l i n g In both Experiments 1 and 2 many of the experimental subjects but none of the c o n t r o l s became extremely aggressive and h y p e r r e a c t i v e . They were extremely r e s i s t a n t to handling, and some became q u i t e adept at drawing the experimenter's blood. Although these behaviours were not o b j e c t i v e l y scored or s y s t e m a t i c a l l y observed, they were o b v i o u s l y d i f f e r e n t from those of the c o n t r o l animals. This aggressiveness u s u a l l y developed i n the l a t t e r stages of k i n d l i n g , and seemed to i n t e n s i f y as IIDs p r o l i f e r a t e d . I t was f i r s t assumed that t h i s behaviour was a r e s u l t of the many s t i m u l a t i o n s and MSs these animals had experienced, but i n Experiment 2 the caudate animals d i d not e x h i b i t t h i s h y p e r r e a c t i v i t y , even though they had as many s t i m u l a t i o n s and more e l i c i t e d MSs than the other r a t s . Therefore, i t would appear that the aggressive behaviour a s s o c i a t e d w i t h k i n d l i n g i s a f u n c t i o n of neural' changes s p e c i f i c to the s i t e of s t i m u l a t i o n r a t h e r than to the s t i m u l a t i o n s or s e i z u r e s per se. These observations are p a r t i c u l a r l y i n t e r e s t i n g when viewed from the p e r s p e c t i v e of reports of a r e l a t i o n between e p i l e p s y and aggression ( c f . Kligman & Goldberg, 1975; Stevens, 1966), and re p o r t s of the s u c c e s s f u l treatment of aggressive d i s o r d e r s by e x c i s i o n of suspected e p i l e p t i c f o c i ( c f . Falconer, 1973; James, 1960; Heimberger, Whitlock, & Kalsbeck, 1966) and by anticonv u l s a n t drugs (cf• Munroe, 1975). Although there has yet to be a convincing demonstration that e p i l e p t i c discharges are c a u s a l l y r e l a t e d to -70-aggressive behaviour, the p o s s i b i l i t y cannot be r u l e d out. Because of the l a c k of a s u i t a b l e animal model of the r e l a t i o n between e p i l e p s y and aggression, research i n t h i s area has been l i m i t e d to c o r r e l a t i o n a l s t u d i e s ( n a t u r a l experiments). However, i t i s f e a s i b l e that f u r t h e r study of the r e l a t i o n s h i p between k i n d l i n g and h y p e r r e a c t i v i t y might e s t a b l i s h the k i n d l i n g paradigm as a u s e f u l t o o l i n studying e p i l e p s y - r e l a t e d aggression. ECS K i n d l i n g and Spontaneous MSs The development of spontaneous MSs i n animals k i n d l e d w i t h l o c a l b r a i n s t i m u l a t i o n may be r e l a t e d i n some way to the development of spontaneous s e i z u r e s a f t e r repeated e l e c t r o c o n v u l s i v e shocks. The occur-rence of spontaneous s e i z u r e s f o l l o w i n g a s e r i e s of e l e c t r o c o n v u l s i v e shocks has been widely reported i n both the experimental and c l i n i c a l l i t e r a t u r e ( c f . E s s i g , Groce, & Williamson, 1961; Blumenthal, 1955; P a c e l l a & Ba r r e r a , 1945). This i s extremely i n t e r e s t i n g i n view of a recent report that e l e c t r o c o n v u l s i v e shocks, when administered to r a t s at 3-day i n t e r v a l s , produce a pro g r e s s i v e increase i n the s e v e r i t y of the e l i c i t e d MS p a t t e r n — e s s e n t i a l l y , ECS k i n d l i n g (Ramer & P i n e l , 1976). Other Methods of Producing Experimental E p i l e p s y In a d d i t i o n to the obvious s i m i l a r i t i e s between the k i n d l i n g of e l i c i t e d MSs and c l i n i c a l e p i l e p t o g e n e s i s , i t i s now c l e a r that the k i n -d l i n g procedure can r e l i a b l y produce a l o n g - l a s t i n g and r e l a t i v e l y s t a b l e e p i l e p t i c syndrome i n r a t s . Thus, i t appears that k i n d l i n g may be a valua b l e a d d i t i o n to the methods a v a i l a b l e f o r producing experimental e p i l e p s y . Although experimental e p i l e p s y had been produced i n animals -71-w i t h s t r y c h n i n e (Ralston, 1958), p e n i c i l l i n (Faeth, Walker, & Warder, 1956), mescaline ( C r i g h e l & S t o i c a , c i t e d i n K r e i n d l e r , 1965), alumina cream (Kopeloff, B u r rera, & K o p e l o f f , 1942), l o c a l f r e e z i n g of the cortex (Speransky, 1943), and e t h y l c h l o r i d e ( M o r r e l l & F l o r e n z , 1958), i n most cases more c o n t r o l i s a f f o r d e d by the k i n d l i n g procedure. For example, there i s more c o n t r o l over the s i t e of a p p l i c a t i o n , and i t would appear that gross c e l l u l a r damage i s minimal. However, the main advantage of k i n d l i n g procedure over the other methods i s that i t allows one to study the process of e p i l e p t o g e n e s i s i t -s e l f i n a step-by-step f a s h i o n . Other methods have s e v e r a l shortcomings i n t h i s regard; the development of e p i l e p s y i s l a r g e l y out of the exper-menter's c o n t r o l , and he has no way of c h a r a c t e r i z i n g t h i s development. In a d d i t i o n , the r e l i a b i l i t y of these methods i n producing the e p i l e p t i c syn-drome appears to be somewhat l e s s than .that of the k i n d l i n g procedure. In f a c t , these other procedures are so u n s u i t a b l e f o r studying e p i l e p -togenesis that such a p p l i c a t i o n s are extremely r a r e . However, w i t h the k i n d l i n g procedure the experimenter can c o n t r o l the frequency and i n -t e n s i t y of s t i m u l a t i o n s , and can f o l l o w the development and i n t e n s i f i -c a t i o n of e l i c i t e d s e i z u r e s as. w e l l as the development and p r o l i f e r a t i o n of IIDs u n t i l the point at which spontaneous MSs are f i r s t d i s p l a y e d . Neuroanatomical C o r r e l a t e s of E p i l e p t o g e n e s i s That very l i t t l e i s known about the neuroanatomical c o r r e l a t e s of e p i l e p t o g e n e s i s has perhaps been due to the l a c k of s u i t a b l e methods f o r i t s study. The usual method of studying these c o r r e l a t e s i s to examine the b r a i n s of e p i l e p t i c animals or p a t i e n t s and then to t r y to i n f e r the changes that -72-have l e d up to that pathological condition. Madge and Arnold Scheibel, for example, have examined the brains of human e p i l e p t i c s and found a number of changes i n the hippocampal pyramidal c e l l s and dentate granular c e l l s , ranging from minor pathology along si n g l e dendrites to massive degenerative changes culminating i n the death of many neurons (Scheibel, Crandall, & Scheibel, 1974). However, they pointed out that although t h e i r r e s u l t s seemed to indic a t e a systematic develop-ment of t h i s pathology, the precise r e l a t i o n between those changes and epileptogenesis could not be determined u n t i l the pathology could be studied as i t was developing. As a r e s u l t of the int e r e s t of the Scheibels i n epileptogenesis the brains of some of the animals i n the present experiment have been sent to them f or ana l y s i s , and t h e i r i n v e s t i g a t i o n i s s t i l l i n progress. There have been two notable attempts to study the neuroanatomical correlates of k i n d l i n g . Both an electron-microscope study (Goddard & Douglas, 1975) and a Golgi-Cox study (Racine, Tuff, & Zaide, 1975) f a i l e d to reveal s i g n i f i c a n t differences i n the brains of kindled rats and un-stimulated controls. However, i n both of these cases the stimulations had been c u r t a i l e d r e l a t i v e l y e arly i n k i n d l i n g , at a point at which one would expect that the neuroanatomical changes would not be so obvious as they would i n the l a t e r stages of k i n d l i n g . In view of the s t r i k i n g pathology i n human e p i l e p t i c t i s s u e i t i s l i k e l y that there are some obvious changes i n the brains of rats which display spontaneous MSs. Once these changes are observed, the Scheibels, i n c o l l a b o r a t i o n with John P i n e l of the University of B r i t i s h Columbia Department of Psychology, w i l l conduct a study i n which the genesis of -73-these f i n a l changes i s traced i n r a t s at v a r i o u s stages of e p i l e p t o g e n e s i s . Thus, the k i n d l i n g paradigm should be a v a l u a b l e a d d i t i o n to the methods of studying e p i l e p t o g e n e s i s and i t s c o r r e l a t e s , and the data provided by the present experiments should g r e a t l y f a c i l i t a t e i t s sub-sequent use i n t h i s regard. -74-REFERENCES Arno l d , P.S., Racine, R.J. & Wise, R.A. E f f e c t s of a t r o p i n e , r e s e r p i n e , 6 hydroxydopamine, and handling on s e i z u r e development i n the r a t . Experimental Neurology, 1973, 4X2, 457-470. Babington, R.G., & Wedeking, P.W. The pharmacology of s e i z u r e s induced by s e n s i t i z a t i o n w i t h low i n t e n s i t y of b r a i n s t i m u l a t i o n . Pharma- cology, Biochemistry and Behavior, 1973, J^, 461-467. C o l l i n s , R.L. Audiogenic s e i z u r e s . In D.P. Purpura, J.K. Penry, D.B. Tower, D.M. Woodbury & R.D. Walter (Eds.), Experimental models of  e p i l e p s y . New York: Raven P r e s s , 1972. 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Temporal lobe e p i l e p s y and aggression. J o u r n a l of Nervous and Mental Disease, 1975, 160, 324-331. Ko p e l o f f , L.M., Burrera, E., & Ko p e l o f f , N. Recurrent co n v u l s i v e s e i z u r e s i n animals produced by immunologic and chemical means. American  J o u r n a l of Psychiatry,; 1942, 98, 881-902. K r e i n d l e r , A. Experimental e p i l e p s y . New York: E l s e v i e r , 1965. Leech, C...K. Comparison of audiogenic s e i z u r e development and k i n d l e d convulsions i n seven s t r a i n s of mouse. Unpublished d o c t o r a l d i s - 1 s e r t a t i o n , Dalhousie U n i v e r s i t y , 1972. Mclntyre, D.C, & Goddard, G.V. Tra n s f e r , i n t e r f e r e n c e and spontaneous recovery of convulsions k i n d l e d from the r a t amygdala. E l e c t r o - encephalography and C l i n i c a l Neurophysiology, 1973, _35, 533-543. Monroe, R. Anticonvulsants i n the treatment of aggression. 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Electroencephalography and C l i n i c a l Neurophysiology, 1973, 35, 1-12. Racine, R.J., Burnham, W.M., & Gartner, J.G. F i r s t t r i a l motor s e i z u r e s t r i g g e r e d by amygdaloid s t i m u l a t i o n i n the r a t . Electroencephalography  and C l i n i c a l Neurophysiology, 1973, _3_5, 487-494. Racine, R.J., Burnham, W.M., Gartner, J.G., & L e v i t a n , D. Rates of motor s e i z u r e development i n r a t s subjected to e l e c t r i c a l b r a i n s t i m u l a t i o n : s t r a i n and i n t e r - s t i m u l a t i o n i n t e r v a l e f f e c t s . Electroencephalo- graphy and C l i n i c a l Neurophysiology, 1973, _3_5, 553-556. Racine, R.J., Gartner, J.G., & Burnham, W.M. E p i l e p t i f o r m a c t i v i t y and ne u r a l p l a s t i c i t y i n l i m b i c s t r u c t u r e s . B r a i n Research, 1972, 47, 262-268. Racine, R.J., L i v i n g s t o n , K., & Joaquin, A. E f f e c t s of procaine hydro-c h l o r i d e , diazepam, and diphenylhydantoin on s e i z u r e development i n c o r t i c a l and s u b c o r t i c a l s t r u c t u r e s i n r a t s . Electroencephalography  and C l i n i c a l Neurophysiology, 1975, _3JJ, 355-365. Racine, R., Newberry, F., & Burnham, W.M. Post a c t i v a t i o n p o t e n t i a t i o n and the k i n d l i n g phenomenon. Electroencephalography and C l i n i c a l  Neurophysiology, 1975, 3_9, 261-271. Racine, R., Okujava, V., & C h i p a s h v i l i , S. M o d i f i c a t i o n of s e i z u r e a c t i v i t y by e l e c t r i c a l s t i m u l a t i o n : I I I . mechanisms. E l e c t r o e n - cephalography and C l i n i c a l Neurophysiology, 1972, _32_, 295-299. Racine, R. , T u f f , L., & Zaide, J . K i n d l i n g , u n i t discharge patterns and ne u r a l p l a s t i c i t y . Canadian J o u r n a l of N e u r o l o g i c a l Sciences, 1975, 2, 395-406. Ral s t o n , B.L. 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Boston: L i t t l e , Brown and Company, 1969. -78-Appendix A A n a l y s i s of Variance Tables f o r Experiment 2 A n a l y s i s of Variance Table f o r AD Duration (Panel A) Source SS df MS Between Subj ects Groups Subj/groups 15894.8 52869.6 3 23 5298.3 2298.7 2.30 With i n Subjects Sessions 43541.1 Sessions x Group 7722.6 Sessions x Subj/groups 71640.2 3 9 69 14513.7 858.1 1038.3 13.98* 0.83 p<.05 A n a l y s i s of Variance Table f o r AD Duration (Panel B) Source SS df MS Between Subjects Groups: 34797.5 Subj/groups 39652.9 Wit h i n Subjects Session 341.0 Sessions x Groups 4677.4 Sessions x Subj/group 70403.1 3 23 3 9 69 11599.2 1724.0 113.7 519.7 1020.3 6.73* 0.95 0.86 p<.05 -79-A n a l y s i s of Variance Table f o r MS Class (Panel A) Source SS df MS Between Subjects Groups Subj/groups Wi t h i n Subjects Sessions Sessions x Groups Sessions x Subj/groups 10.2 146.6 242.8 47.4 103.5 3 23 3 9 69 3.4 6.4 80.9 5.3 1.5 0.53 53.9* 3.5 p<.05 A n a l y s i s of Variance Table f o r MS Class (Panel B) Source SS df MS Between Subjects Groups Subj/groups Wi t h i n Subjects Sessions Sessions x Groups Sessions x Subj/groups 51.4 115.8 5.41 12.51 211.99 3 23 3 9 69 17.1 3.41* 5.0 1.8 0.59 1.4 0.45 3.1 P<.05 -80-A n a l y s i s of Variance Table f o r MS Duration (Panel A) Source SS df MS Between Subjects Groups 1375.4 Subj/groups 7338.7 Wit h i n Subjects Sessions 13169.2 Sessions x Groups 1817.3 Sessions x Subj/groups 10541.5 3 23 3 9 69 458.5 319.1 4388.7 201.9 152.8 1.44 28.73* 1.32 p<.05 A n a l y s i s of Variance Table f o r MS Duration (Panel B) Source SS df MS Between Subjects Groups 102.7 Subj/groups 5096.6 Within Subjects Sessions 375.9 Session x Groups 845.7 Sessions x Subj/groups 9835.0 3 23 3 9 69 34.2 221.6 125.3 93,'9 142.5 0.15 0.88 0.66 -81-A n a l y s i s of Variance Table f o r IID Incidence Source SS df MS Between Subjects Groups Sub/groups 650.1 1481.9 3 23 216.7 64.4 3.36* Wit h i n Subjects Session Session x Groups Sessions x Subj/groups 1.6 234.6 718.4 3 9 69 0.5 26.1 10.4 0.05 2.50* p<.05 -82-A n a l y s i s of Variance Table f o r Spontaneous MS Incidence (35 days) Source SS df MS Between Subjects Groups 398.9 3 Subj/groups 981.6. 7 Wi t h i n Groups Sessions 251.9 34 Session x Groups 515.8 102 Sessions x Subj/groups 834.9 238 132.9 140.2 7.4 5.1 3.5 0.95 2.11* 1.44* p<.05 A n a l y s i s of Variance Table f o r Spontaneous MS Incidence (Day 1 & 35) Source SS df MS Between Subjects Groups Subj/groups Wi t h i n Groups Sessions Sessions x Groups Sessions x Subj/groups 4.7 39.7 25.1 1.1 13.7 1 3 7 1.6 5. 7 25.1 0.4 1.9 0.28 12.82* 0.19 p<.05 

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