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The role of the drug-effect contingency in the development of cross tolerence to anticonvulsant drug… Kim, Chang Kwon 1989

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THE ROLE OF THE DRUG-EFFECT CONTINGENCY IN THE DEVELOPMENT OF CROSS TOLERANCE TO ANTICONVULSANT DRUG EFFECTS By CHANG KWON KIM B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1986. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n THE FACULTY OF GRADUATE STUDIES (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 October 1989 (c) Chang Kwon Kim In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of f%UC>holc>i The University of British Columbia Vancouver, Canada Date OcJ- /3 1181 DE-6 (2/88) i i A b s t r a c t I t was r e c e n t l y demonstrated t h a t t o l e r a n c e develops to the a n t i c o n v u l s a n t e f f e c t of ethanol on k i n d l e d c o n v u l s i o n s e l i c i t e d i n r a t s by e l e c t r i c a l s t i m u l a t i o n of the amygdala, f o l l o w i n g each of a s e r i e s of ethanol i n j e c t i o n s d e l i v e r e d on a b i d a i l y schedule (once every 48 h r ) . The t o l e r a n c e developed on l y when the c o n v u l s i v e s t i m u l a t i o n was administered d u r i n g the pe r i o d s o£ ethanol exposure: s u b j e c t s t h a t r e c e i v e d ethanol 1.5 hr before each c o n v u l s i v e s t i m u l a t i o n demonstrated t o l e r a n c e a f t e r j u s t f i v e e thanol i n j e c t i o n s ; whereas, no t o l e r a n c e was evident i n s u b j e c t s t h a t r e c e i v e d ethanol 1.5 hr a f t e r each s t i m u l a t i o n . Such t o l e r a n c e , which i s not the i n e v i t a b l e product of drug exposure but i s cont i n g e n t upon the e x p r e s s i o n of the drug e f f e c t - - t h e a n t i c o n v u l s a n t e f f e c t i n t h i s case--has been termed contingent t o l e r a n c e . In Experiment 1A, t o l e r a n c e developed to the a n t i c o n v u l s a n t e f f e c t s of b i d a i l y IP i n j e c t i o n s of p h e n o b a r b i t a l (30 mg/kg), trimethadione (270 mg/kg) and clonazepam (0.40 or 0.35 mg/kg) d e l i v e r e d 1 hr before each c o n v u l s i v e s t i m u l a t i o n . In Experiment IB, the r a t s t o l e r a n t to the a n t i c o n v u l s a n t e f f e c t s of p h e n o b a r b i t a l , trimethadione, or clonazepam r e c e i v e d b i d a i l y i n j e c t i o n s of carbamazepine (35 mg/kg, I P ) , administered 1 hr before each s t i m u l a t i o n . There was a s t a t i s t i c a l l y s i g n i f i c a n t t r a n s f e r of t o l e r a n c e from p h e n o b a r b i t a l to carbamazepine, but not from e i t h e r trimethadione or clonazepam to carbamazepine. Thus, c r o s s t o l e r a n c e appears to be g r e a t e s t between i i i a n t i c o n v u l s a n t drugs t h a t are e f f e c t i v e a g a i n s t a s i m i l a r p r o f i l e of c l i n i c a l and experimental s e i z u r e s and t h a t have s i m i l a r mechanisms of a c t i o n . In Experiment 2A, t o l e r a n c e developed to the a n t i c o n v u l s a n t e f f e c t of b i d a i l y p e n t o b a r b i t a l (15 mg/kg, IP) i n j e c t i o n s o n l y i n those r a t s t h a t r e c e i v e d the drug 1 hr before the c o n v u l s i v e s t i m u l a t i o n , but not i n those r a t s t h a t had r e c e i v e d the drug 1 hr a f t e r each s t i m u l a t i o n . Furthermore, those r a t s t h a t had r e c e i v e d the c o n v u l s i v e s t i m u l a t i o n s while under the i n f l u e n c e of p e n t o b a r b i t a l subsequently d i s p l a y e d a g r e a t e r degree of c r o s s t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol (1.5 g/kg, IP) than those t h a t had r e c e i v e d the drug a f t e r each s t i m u l a t i o n (contingent c r o s s t o l e r a n c e ) . Experiment 2B was the converse of Experiment 2A: co n t i n g e n t t o l e r a n c e was demonstrated to ethanol and c o n t i n g e n t c r o s s t o l e r a n c e to p e n t o b a r b i t a l . T h i s study provided the f i r s t unambiguous and b i d i r e c t i o n a l demonstration t h a t the d r u g - e f f e c t contingency pla y s an important r o l e i n the development of c r o s s t o l e r a n c e . In Experiment 3, t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol d i s s i p a t e d when b i d a i l y p e n t o b a r b i t a l (15 mg/kg, IP) i n j e c t i o n s were d e l i v e r e d 1 hr a f t e r each c o n v u l s i v e s t i m u l a t i o n (contingent c r o s s - d i s s i p a t i o n of t o l e r a n c e ) , but d i d not d i s s i p a t e when i t was d e l i v e r e d 1 hr before each s t i m u l a t i o n . Thus, the d r u g - e f f e c t contingency was shown to be important i n the d i s s i p a t i o n of t o l e r a n c e to one drug f o l l o w i n g the a d m i n i s t r a t i o n of another drug. i v I n E x p e r i m e n t 4, d i f f e r e n t g r o u p s o f r a t s r e c e i v e d d i f f e r e n t d o s e s o f p e n t o b a r b i t a l ( 10-50 mg/kg, I P ) on a b i d a i l y s c h e d u l e 1 h r b e f o r e t h e c o n v u l s i v e s t i m u l a t i o n . G r e a t e r t o l e r a n c e was f o u n d t o t h e a n t i c o n v u l s a n t e f f e c t o f p e n t o b a r b i t a l i n r a t s t h a t had r e c e i v e d s u c c e s s i v e l y l a r g e r d o s e s o f t h e d r u g , none o f w h i c h were l a r g e e nough t o s u p p r e s s t h e c o n v u l s i o n s , t h a n t h o s e r a t s t h a t were m a i n t a i n e d on a h i g h d o s e o f t h e d r u g t h a t c o m p l e t e l y s u p p r e s s e d t h e c o n v u l s i o n s . The g r e a t e r t o l e r a n c e i n t h e g r o u p t h a t r e c e i v e d s u c c e s s i v e l y g r e a t e r d o s e s was a t t r i b u t e d t o t h e f a c t t h a t t h e c o n v u l s i o n s were e x p e r i e n c e d i n t h e d r u g g e d s t a t e . T h i s s t u d y c h a l l e n g e d t h e g e n e r a l l y a c c e p t e d v i e w t h a t t o l e r a n c e d e v e l o p s more r a p i d l y and t o a g r e a t e r e x t e n t w i t h l a r g e r d r u g d o s e s . T h i s t h e s i s p r o v i d e s t h e f i r s t u n a m b i g u o u s a n d s y s t e m a t i c e v i d e n c e o f t h e r o l e o f t h e d r u g - e f f e c t c o n t i n g e n c y i n t h e t r a n s f e r o f t o l e r a n c e f r o m one d r u g t o a n o t h e r , and i n t h e d i s s i p a t i o n o f t o l e r a n c e t o one d r u g f o l l o w i n g t h e a d m i n i s t r a t i o n o f a n o t h e r . On t h e b a s i s o f t h e p r e s e n t e x p e r i m e n t s , s e v e r a l e l a b o r a t i o n s were p r o p o s e d t o t h e d r u g - e f f e c t t h e o r y o f d r u g t o l e r a n c e , w h i c h c l a i m s t h a t t o l e r a n c e d e v e l o p s t o d r u g e f f e c t s and n o t t o d r u g e x p o s u r e p e r s e . Table of Contents A b s t r a c t i i Table of Contents v L i s t of F i g u r e s v i i L i s t of Tables v i i i Acknowledgement i x I. General I n t r o d u c t i o n 1 1. Drug Tolerance 2 2. Cross T o l e r a n c e 4 3. T r a d i t i o n a l Drug-Exposure Theory of T o l e r a n c e . . . . 5 4. D r u g - E f f e c t Theory of Tolerance 6 5. G e n e r a l i t y of the D r u g - E f f e c t Theory 8 6. A n t i c o n v u l s a n t Drug E f f e c t 8 7. K i n d l e d c o n v u l s i o n s . 11 8 . General R a t i o n a l e 14 9 . General Purpose 15 I I . General Background f o r Experiments 1 and 2 17 1. Tolerance and Cross Tolerance to A n t i c o n v u l s a n t Drug E f f e c t 17 2. Contingent Cross Tolerance 17 I I I . General Methodology 27 Subjects 27 Surgery 27 K i n d l i n g Phase 28 No-Drug B a s e l i n e Phase 28 Drug B a s e l i n e Test 29 Treatment and Test Phase 30 H i s t o l o g y 30 S t a t i s t i c a l A n a l y s i s 31 IV. Experiment 1 32 Experiment 1A 34 Method 35 R e s u l t s 37 D i s c u s s i o n 42 Experiment IB 43 Method 43 R e s u l t s 44 D i s c u s s i o n 49 V. Experiment 2 51 General Method 52 Experiment 2A 54 Method 54 v i R e s u l t s 54 Experiment 2B 58 Method 58 R e s u l t s 58 D i s c u s s i o n 63 VI. Experiment 3 65 General Method 65 Experiment 3A 66 Method 66 R e s u l t s . 67 Experiment 3B 67 Method 67 R e s u l t s 70 Discuss ion 74 Experiment 3C 75 Method 75 R e s u l t s 76 D i s c u s s i o n 79 V I I . Experiment 4 81 Method 82 R e s u l t s 84 D i s c u s s i o n 87 V I I I . General D i s c u s s i o n 95 1. General D i s c u s s i o n of Experiments 1 and 2 95 2. General D i s c u s s i o n of Experiment 3 97 3. General D i s c u s s i o n of Experiment 4 98 4. T h e o r e t i c a l I m p l i c a t i o n s 98 5. C l i n i c a l I m p l i c a t i o n s 103 6. Conclu s i o n s and Future D i r e c t i o n s 107 References 109 v i i L i s t of F i g u r e s F i g u r e 1... . C l a s s i f i c a t i o n of C l i n i c a l S e i z u r e s 33 F i g u r e 2....Experiment 1A. Tolerance to P h e n o b a r b i t a l , Tr imethadione, and Clonazepam 38 F i g u r e 3....Experiment IB. Cross Tolerance to Carbamazepine 45 F i g u r e 4 .... H i s t o l o g i c a l R e s u l t s From Experiment 1 48 F i g u r e 5....Experiment 2A. Contingent Cross T o l e r a n c e : P e n t o b a r b i t a l to Ethanol 55 F i g u r e 6....Experiment 2B. Contingent Cross T o l e r a n c e : Ethanol to P e n t o b a r b i t a l 59 F i g u r e 7 .... H i s t o l o g i c a l R e s u l t s From Experiment 2 61 F i g u r e 8....Experiment 3A. Contingent Tolerance to P e n t o b a r b i t a l 68 F i g u r e 9....Experiment 3B. Contingent Cross T o l e r a n c e : P e n t o b a r b i t a l to Ethanol 72 F i g u r e 10...Experiment 3C. D i s s i p a t i o n of Ethanol Tolerance 77 F i g u r e 11... H i s t o l o g i c a l R e s u l t s From Experiment 3 80 F i g u r e 12...Experiment 4. Tolerance to P e n t o b a r b i t a l . . . . . . 85 F i g u r e 13 .. .Exper iment 4. R i g h t i n g Test 88 F i g u r e 14 .. .Exper iment 4. Tube Test 90 F i g u r e 15 ... H i s t o l o g i c a l R e s u l t s From Experiment 4 92 L i s t of Tables Table I Review of Contingent Drug Tolerance Table I I . . . . C l a s s i f i c a t i o n of K i n d l e d Convulsions Table III...Review of Tolerance to A n t i c o n v u l s a n t Drug E f f e c t s Table IV....Review of Cross Tolerance to A n t i c o n v u l s a n t Drug E f f e c t s . . . i x Acknowledgements I am indebted to Dr. John P.J. P i n e l f o r h i s support and h i s confidence i n me. I thank M. Mana f o r h i s a s s i s t a n c e and i n s i g h t s . I thank Dr. P. Graf and Dr. C. Rankin f o r t h e i r h e l p f u l c r i t i c i s m s and c o n t r i b u t i o n s to the f i n a l copy. I a l s o thank N. Roese, M. Hudda, D. Wong, A. Yung, D. P a u l , N. Lee, L. Hoover, J . Weinberg, And I f i n a l l y thank G i t t e f o r making t h i n g s e a s i e r and fo r being so t o l e r a n t . 1 I. General I n t r o d u c t i o n P i n e l , Colbourne, S i g a l e t , and Renfrey ( 1 9 8 3 ) demonstrated the development of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol on k i n d l e d c o n v u l s i o n s e l i c i t e d i n r a t s by e l e c t r i c a l s t i m u l a t i o n o£ the amygdala. The most i n t e r e s t i n g f e a t u r e of t h i s demonstration was t h a t the t o l e r a n c e developed o n l y i f c o n v u l s i v e s t i m u l a t i o n s was d e l i v e r e d d u r i n g the p e r i o d s of ethanol exposure. Two groups o£ r a t s r e c e i v e d an ethanol i n j e c t i o n every two days; the r a t s i n one group r e c e i v e d the ethanol 1.5 hr before a c o n v u l s i v e s t i m u l a t i o n , whereas those i n the other group r e c e i v e d the ethanol 1.5 hr a f t e r a c o n v u l s i v e s t i m u l a t i o n . A f t e r f i v e i n j e c t i o n s , the r a t s i n the e t h a n o l - b e f o r e - s t i m u l a t i o n group were almost t o t a l l y t o l e r a n t to the a n t i c o n v u l s a n t e f f e c t of e t h a n o l , whereas no t o l e r a n c e at a l l developed i n the e t h a n o l -a f t e r - s t i m u l a t i o n r a t s . The d i f f e r e n c e i n the degree of t o l e r a n c e d i s p l a y e d i n these two c o n d i t i o n s was a t t r i b u t e d to the f a c t t h a t the a n t i c o n v u l s a n t e f f e c t of ethanol was r e p e a t e d l y experienced only i n those r a t s t h a t r e c e i v e d ethanol before the s t i m u l a t i o n . Such t o l e r a n c e , which i s not the i n e v i t a b l e product of drug exposure but i s c o n t i n g e n t upon the repeated e x p r e s s i o n of the drug e f f e c t - - t h e a n t i c o n v u l s a n t e f f e c t i n t h i s case--has been termed con t i n g e n t t o l e r a n c e ( C a r l t o n & Wolgin, 1 9 7 1 ) . On the b a s i s of numerous demonstrations of c o n t i n g e n t t o l e r a n c e , P i n e l , Mana, and Kim (1989) proposed the d r u g - e f f e c t theory of drug t o l e r a n c e . According to t h i s theory, t o l e r a n c e develops to drug 2 e f f e c t s and not to drug exposure per se. The main purpose of the present experiments was to assess the r o l e of d r u g - e f f e c t s , as opposed to mere drug-exposure, on the development of c r o s s t o l e r a n c e between a n t i c o n v u l s a n t drugs. Does the a n t i c o n v u l s a n t drug e f f e c t have to be r e p e a t e d l y experienced f o r the t o l e r a n c e to t r a n s f e r to the a n t i c o n v u l s a n t e f f e c t of another drug, or i s drug exposure without the e x p r e s s i o n of the a n t i c o n v u l s a n t e f f e c t s u f f i c i e n t f o r the t r a n s f e r of t o l e r a n c e ? A c c o r d i n g l y , the General I n t r o d u c t i o n to t h i s t h e s i s i s d i v i d e d i n t o nine s e c t i o n s . S e c t i o n s 1 and 2 introduce the phenomena of drug t o l e r a n c e and c r o s s t o l e r a n c e , r e s p e c t i v e l y ; S e c t i o n 3 o u t l i n e s the t r a d i t i o n a l drug-exposure theory of drug t o l e r a n c e ; S e c t i o n 4 o u t l i n e s the d r u g - e f f e c t theory of drug t o l e r a n c e ; S e c t i o n 5 b r i e f l y reviews the l i t e r a t u r e on c o n t i n g e n t t o l e r a n c e ; and S e c t i o n s 5 and 6 d i s c u s s a n t i c o n v u l s a n t drug e f f e c t s , and the use of the k i n d l e d c o n v u l s i o n paradigm i n t h e i r measurement. The General I n t r o d u c t i o n concludes with the general r a t i o n a l e and the g e n e r a l purpose of the t h e s i s i n S e c t i o n s 8 and 9, r e s p e c t i v e l y . 1. Drug T o l e r a n c e : The B a s i c Phenomenon Drug t o l e r a n c e i s a decrease i n the e f f e c t of a drug t h a t occurs as the r e s u l t of exposure to the drug. I t can be manifested as a p r o g r e s s i v e decrease In the pharmacological e f f e c t of a p a r t i c u l a r dose of a drug with repeated a d m i n i s t r a t i o n s , or as the need f o r p r o g r e s s i v e l y g r e a t e r doses of a drug to maintain the same p h a r m a c o l o g i c a l . e f f e c t . In e f f e c t , drug t o l e r a n c e i s a rightward s h i f t i n the dose response curve of the drug f o l l o w i n g exposure to the drug. Although the v a r i o u s mechanisms of drug t o l e r a n c e are p o o r l y understood, they are c o n s i d e r e d to be of two g e n e r a l types: d i s p o s i t i o n a l or f u n c t i o n a l (e.g., Kalant, LeBlanc, & G i b b i n s , 1971). D i s p o s i t i o n a l t o l e r a n c e i s t o l e r a n c e t h a t r e s u l t s from a decrease i n the a v a i l a b i l i t y of the drug a t the s i t e of i t s a c t i o n due to changes i n the a b s o r p t i o n , d i s t r i b u t i o n , metabolism, or e x c r e t i o n of the drug. F u n c t i o n a l t o l e r a n c e Is t o l e r a n c e t h a t r e s u l t s from any change i n the s e n s i t i v i t y of the p h y s i o l o g i c a l systems a f f e c t e d by the drug; f o r example, a change i n the s e n s i t i v i t y or number of n e u r o t r a n s m i t t e r r e c e p t o r s (Rebec & Lee, 1983; Seeman, 1980), a change i n the l e v e l s of n e u r o t r a n s m i t t e r s (Mechior & Tabakoff, 1981), neuromodulators ( V o l l i c e r & Ullman, 1985) or hormones (Wood, 1977; Tabakoff & Yanai, 1979), a change i n c e l l membrane composition ( G o l d s t e i n , 1983), a change i n the a c t i v i t y of secondary messengers (e.g., S i g g i n s , 1979), or a change i n ion conductance (Ross, G a r r e t t , & Cardenas, 1979). A t t r i b u t i n g t o l e r a n c e to e i t h e r a d i s p o s i t i o n a l or a f u n c t i o n a l change or to a combination of them i s a reasonable f i r s t s tep i n the a n a l y s i s of a p a r t i c u l a r i n s t a n c e of t o l e r a n c e , but there are three problems with t h i s approach. F i r s t , t o l e r a n c e i s o f t e n i d e n t i f i e d as r e s u l t i n g from a f u n c t i o n a l change on l y by the f a i l u r e to d e t e c t an u n d e r l y i n g d i s p o s i t i o n a l change (e.g., Dews, 1978). Second, any d i s p o s i t i o n a l or f u n c t i o n a l change t h a t 4 i s i d e n t i f i e d i s not n e c e s s a r i l y c a u s a l l y r e l a t e d to the observed development of t o l e r a n c e (Demellweek & Goudie, 1983b). T h i r d , the d i s p o s i t i o n a l and f u n c t i o n a l changes to which s p e c i f i c i n s t ances of t o l e r a n c e are a t t r i b u t e d are o f t e n too s l i g h t to account f o r the hundred f o l d or g r e a t e r decreases i n responsiveness t h a t c h a r a c t e r i z e some t o l e r a n c e e f f e c t s (e.g., Seevers & Deneau, 1963) . Arguably, the key q u e s t i o n t h a t remains to be answered about drug t o l e r a n c e i s why t o l e r a n c e develops to some drug e f f e c t s but not o t h e r s . Tolerance may develop to some e f f e c t s of a drug while at the same time not develop to other e f f e c t s of the same drug (e.g., M a n s f i e l d , Benedict & Woods, 1983). Moreover, t o l e r a n c e may develop to a p a r t i c u l a r drug e f f e c t i n one study but not develop to the same drug e f f e c t i n another study i n which s i m i l a r doses of the drug were employed. Moreover, i n some cases, s e n s i t i z a t i o n — a n i n c r e a s e d s e n s i t i v i t y to a drug as the r e s u l t of p revious exposure to it--may develop to some e f f e c t s of a drug while t o l e r a n c e develops to other e f f e c t s of the same drug (Kczenski & L e i t h , 1981). 2. Cross Tolerance Cross t o l e r a n c e i s the t r a n s f e r of t o l e r a n c e from one drug to another drug. Cross t o l e r a n c e i s common between drugs t h a t have s i m i l a r mechanisms of a c t i o n , s i m i l a r p r o f i l e s of drug e f f e c t s , and s i m i l a r chemical s t r u c t u r e s . Because c r o s s t o l e r a n c e i s l e s s common between drugs of two d i f f e r e n t pharmacological c l a s s e s , demonstrations of c r o s s t o l e r a n c e between such drugs are 5 commonly con s i d e r e d to be evidence t h a t they have s i m i l a r mechanisms of a c t i o n (Khanna & Mayer, 1982). L i k e s p e c i f i c i n s t a n c e s of t o l e r a n c e , s p e c i f i c i n s t a n c e s of c r o s s t o l e r a n c e are c l a s s i f i e d as d i s p o s i t i o n a l or f u n c t i o n a l (e.g., Coper, 1978). 3. T r a d i t i o n a l Drug-Exposure Theory of Drug Tolerance According to the t r a d i t i o n a l drug-exposure th e o r y of drug t o l e r a n c e , the exposure of the organism to the drug i s the c r i t i c a l c a u s a l f a c t o r i n the development of t o l e r a n c e to the drug's e f f e c t s . Within the context of t h i s theory, the f a c t o r s that are thought to i n f l u e n c e the development of drug t o l e r a n c e are the dose, the schedule, the d u r a t i o n , and the route of drug exposure. The major shortcoming of t h i s t r a d i t i o n a l theory i s t h a t i t cannot e x p l a i n why the very same regimen of drug a d m i n i s t r a t i o n produces t o l e r a n c e to a drug i n some ins t a n c e s but not i n o t h e r s . In the l a s t two decades, two s e r i o u s c h a l l e n g e s to the t r a d i t i o n a l theory of drug t o l e r a n c e have been i s s u e d . U n l i k e the t r a d i t i o n a l drug-exposure theory, these new t h e o r i e s are both based on the premise t h a t the experiences of the s u b j e c t s while they are drugged p l a y a major r o l e i n t o l e r a n c e development. The f i r s t of these two t h e o r i e s , the c o n d i t i o n e d t o l e r a n c e t h e o r y (e.g., S i e g e l , 1983), focuses on the r o l e i n the development of t o l e r a n c e of the environment i n which the s u b j e c t s experience the drug e f f e c t s . The second of these two t h e o r i e s , the d r u g - e f f e c t  theory focuses on the r o l e of the behavior of the s u b j e c t d u r i n g drug exposure. I t i s the d r u g - e f f e c t theory t h a t was the focus of 6 the experiments r e p o r t e d here. 4. D r u g - E f f e c t Theory of Drug Tolerance According to the d r u g - e f f e c t theory, drug t o l e r a n c e develops to the drug e f f e c t s , not to drug exposure; t o l e r a n c e i s seen as an a d a p t a t i o n to the repeated e x p r e s s i o n of a drug's d i s r u p t i v e e f f e c t on ongoing n e u r a l a c t i v i t y , not to the mere presence of the drug. A c c o r d i n g l y , drug exposure i s presumed necessary, but not s u f f i c i e n t f o r the development of f u n c t i o n a l t o l e r a n c e . That i s , exposure to the drug per se may not lead to t o l e r a n c e to a p a r t i c u l a r drug e f f e c t ; i n order f o r t o l e r a n c e to develop to a p a r t i c u l a r drug e f f e c t , t h a t e f f e c t must be r e p e a t e d l y manifested ( P i n e l et a l . , 1989) In order to t e s t the d r u g - e f f e c t theory of t o l e r a n c e , i t i s necessary to study drug e f f e c t s t h a t are not the i n e v i t a b l e consequence of drug exposure. In cases i n which drug exposure and the c r i t e r i o n drug e f f e c t are i n e x t r i c a b l y r e l a t e d , both the t r a d i t i o n a l drug-exposure t h e o r y and the d r u g - e f f e c t theory make the same p r e d i c t i o n s . However, there are drug e f f e c t s t h a t occur o n l y i f the s u b j e c t engages i n a p a r t i c u l a r response while drugged; f o r example, the a n t i c o n v u l s a n t drug e f f e c t s can be expressed o n l y when the organism i s e x p e r i e n c i n g a c o n v u l s i o n . In such cases, i t i s p o s s i b l e to p i t the p r e d i c t i o n s of the t r a d i t i o n a l drug-exposure t h e o r y of t o l e r a n c e a g a i n s t the p r e d i c t i o n s of the d r u g - e f f e c t theory. The d r u g - e f f e c t theory of t o l e r a n c e can be i l l u s t r a t e d with r e f e r e n c e to another more w e l l understood form of a d a p t a t i o n , the 7 a d a p t a t i o n t h a t occurs to the d i s r u p t i v e e f f e c t s of v i s u a l displacement on visu a l - m o t o r c o o r d i n a t i o n ( c f . Poulos & Hinson, 1984). When a s u b j e c t f i r s t wears d i s p l a c i n g prisms t h a t s h i f t her or h i s v i s u a l world a few degrees to one s i d e , v i s u a l - m o t o r c o o r d i n a t i o n i s s e v e r e l y d i s r u p t e d . But a f t e r some experience with the prisms, the s u b j e c t adapts ( i . e . , becomes t o l e r a n t ) to the v i s u a l displacement and visu a l - m o t o r c o o r d i n a t i o n r e t u r n s to normal. What i s the f a c t o r t h a t leads to t h i s a daptation? Is i t the exposure to d i s p l a c e d v i s i o n , which i s analogous to drug-exposure, or i s i t the experience of the d i s r u p t i o n of v i s u a l -motor c o o r d i n a t i o n , which i s analogous to the d r u g - e f f e c t ? The evidence overwhelmingly supports the l a t t e r view. L i t t l e v i s u a l -motor a d a p t a t i o n develops to the e f f e c t s of d i s p l a c i n g prisms i n s u b j e c t s t h a t do not perform v i s u a l - m o t o r tasks while wearing them (Held, 1972, Rock & H a r r i s , 1972). According to the drug-e f f e c t theory of t o l e r a n c e , the t o l e r a n c e to the prisms i s simply a type of sensory-motor a d a p t a t i o n , and l i k e other forms of sensory-motor a d a p t a t i o n i t r e s u l t s from the repeated experience of sensory-motor d i s r u p t i o n r a t h e r than from the a p p l i c a t i o n of the d i s r u p t i n g agent. Most demonstrations of the r o l e of d r u g - e f f e c t s i n the development of t o l e r a n c e have used the b e f o r e - a n d - a f t e r design (Kumer & Stolerman, 1977). In such experiments, there are two groups of s u b j e c t s : the s u b j e c t s i n the drug-before group r e c e i v e the drug before each t e s t t r i a l , whereas those i n the d r u g - a f t e r group r e c e i v e the drug a f t e r each t e s t t r i a l ; thus, the s u b j e c t s 8 i n the drug-before group experience the drug's e f f e c t on the t e s t behavior on each t r i a l , whereas those i n the d r u g - a f t e r group do not. On the t o l e r a n c e t e s t , the s u b j e c t s i n both groups r e c e i v e the drug before the t e s t t r i a l so t h a t the degree of t o l e r a n c e t h a t developed i n each group can be compared. Because the s u b j e c t s of both groups have had the same number of t e s t t r i a l s and drug a d m i n i s t r a t i o n s at the same dose, on the same schedule, and v i a the same route of a d m i n i s t r a t i o n , evidence of g r e a t e r t o l e r a n c e i n the drug-before group can be a t t r i b u t e d to the d i f f e r e n c e i n the contingency between the drug exposure and the t e s t response. The numerous demonstrations of s i g n i f i c a n t l y g r e a t e r t o l e r a n c e i n the drug-before c o n d i t i o n than i n the drug-a f t e r c o n d i t i o n of a b e f o r e - a n d - a f t e r experiment have been termed co n t i n g e n t t o l e r a n c e ( C a r l t o n & Wolgin, 1971)--or more ambiguously as b e h a v i o r a l t o l e r a n c e (e.g., Chen, 1972). 5. G e n e r a l i t y of the D r u g - E f f e c t Theory of Tolerance D r u g - e f f e c t c o n t i n g e n c i e s have been shown to be an important f a c t o r i n the development of t o l e r a n c e to a wide v a r i e t y of drug e f f e c t s . Reports of c o n t i n g e n t drug t o l e r a n c e are summarized i n Table I. 6. A n t i c o n v u l s a n t Drug E f f e c t In t h i s t h e s i s , the a n t i c o n v u l s a n t drug e f f e c t was chosen fo r study f o r two main reasons. F i r s t , with t h i s drug e f f e c t there i s a c l e a r d i s a s s o c i a t i o n between drug-exposure and drug-e f f e c t because the drug e f f e c t can occur only i f a c o n v u l s i v e s t i m u l a t i o n i s d e l i v e r e d and t h i s i s under complete c o n t r o l of Table I Review of Contingent Tolerance Drug E f f e c t S p e c i f i c Drug Reference A d i p s i a scopolamine Poulos & Hinson (1984) A n a l g e s i a ethanol Jorgenson & Hole (1984) Jorgenson et a l . , (1985) morphine Ferguson & M i t c h e l l (1969) Kayan & M i t c h e l l (1969) Anorexigenia amphetamine C a r l t o n & Wolgin (1971) Demellweek & Goudie (1983) S t r e a t h e r & Hinson (1985) cathinone F o l t i n & Schuster (1982) coca ine Woolverton et a l . , (1978) qu i p a z i n e Rowland & C a r l t o n (1983) Hypothermia ethanol Alkana et a l . , (1983) Hjeresen et a l . , (1986) Le et a l . , (1986) D i s r u p t i o n of motor c o o r d i n -a t i o n and balance ethanol Chen (1968) LeBlanc et a l . , (1976) M a n s f i e l d et a l . , (1983) Wenger et a l . , (1980) p h e n o b a r b i t a l Commissaris & Rech (1981) D i s r u p t i o n of operant tasks amphetamine Campbell & Seiden (1973) Emmett-Oglesby et a l . , (1984) delta-9-THC Carder & Olsson (1973) ethanol Chen (1979) Wi g g e l l & O v e r s t r e e t (1984) morphine Smith (1979) p e n t o b a r b i t a l Branch (1983) p h e n o b a r b i t a l Tang & Falk (1974) 10 A c c e l e r a t i o n ethanol i n the decay of p o s t t e t a n i c p o t e n t i a t i o n D i s r u p t i o n of ethanol male sexual behavior Ant i c o n v u l s a n t e f f e c t Traynor et a l . , (1981) P i n e l et a l . , (1989 ) carbamazepine Mana et a l . , ( i n press) diazepam Mana et a l . , ( i n press) ethanol P i n e l et a l . , (1983) P i n e l et a l . , (1985) v a l p r o a t e Mana et a l . , ( i n press) 11 the experimenter. With most drug e f f e c t s , there i s no such c l e a r -cut d i s a s s o c i a t i o n between the d r u g - e f f e c t and drug-exposure. For example, the s u b j e c t experiences many components of a drug's d i s r u p t i v e e f f e c t on maze running whether i t i s r u n n i n g the maze or walking around i n i t s home cage. The c l e a r - c u t d i s a s s o c i a t i o n between exposure to a n t i c o n v u l s a n t drugs and a n t i c o n v u l s a n t drug e f f e c t s i s a great advantage i n s t u d y i n g c o n t i n g e n t t o l e r a n c e . Second, s t u d i e s of t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s may have i m p l i c a t i o n s f o r the pharmacologic treatment of e p i l e p s y , an a f f l i c t i o n t h a t a f f e c t s n e a r l y 1% of the p o p u l a t i o n (Gilman, Goodman, & Gilman, 1980; Katzung, 1987). 7. K i n d l e d Convulsions In order to study a n t i c o n v u l s a n t drug e f f e c t s , one must have a way of induci n g c o n v u l s i o n s i n experimental s u b j e c t s . Convulsions can be induced e l e c t r i c a l l y or c h e m i c a l l y i n a l l v e r t e b r a t e s and i n some s p e c i e s , c o n v u l s i o n s can be induced by sound, l i g h t , or h a n d l i n g . In t h i s t h e s i s , c o n v u l s i o n s were 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 of the amygdala i n k i n d l e d r a t s . In the broad sense of the term, " k i n d l i n g " r e f e r s to a p r o g r e s s i v e increase i n the s e v e r i t y of motor s e i z u r e s e l i c i t e d by a s e r i e s of p e r i o d i c c o n v u l s i v e treatments, but i n the narrow sense, i t r e f e r s to Goddard's (e.g., Goddard, Mcl n t y r e , & Leach, 1969) o b s e r v a t i o n t h a t p e r i o d i c (e.g., one every 24 hr) low-i n t e n s i t y 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 through an implanted e l e c t r o d e leads to the development and 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 c o n v u l s i o n s . At f i r s t no c o n v u l s i v e 12 response i s e l i c i t e d , but repeated s t i m u l a t i o n s g r a d u a l l y come to e l i c i t m i l d c o n v u l s i v e responses t h a t p r o g r e s s i v e l y i n c r e a s e i n i n t e n s i t y with each s t i m u l a t i o n 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 g e n e r a l i z e d c l o n i c c o n v u l s i o n t h a t i s very s t a b l e and changes l i t t l e from s t i m u l a t i o n to s t i m u l a t i o n . The p r o g r e s s i v e i n c r e a s e s i n the g e n e r a l i t y of the c o n v u l s i o n s has been used as a b a s i s f o r the t o p o g r a p h i c a l c l a s s i f i c a t i o n system developed by Racine (1972) and extended by P i n e l and Rovner ( 1 9 7 8 ) — s e e Table I I . Although many s i t e s i n the b r a i n can be k i n d l e d , the amygdala k i n d l e s p a r t i c u l a r l y r a p i d l y and r e l i a b l y , and thus i t has been the p r e f e r r e d s t i m u l a t i o n s i t e . Although k i n d l i n g has been re p o r t e d i n many s p e c i e s , r a t s have been the most common s u b j e c t s i n k i n d l i n g experiments. K i n d l e d c o n v u l s i o n s have important advantages over other experimental c o n v u l s i o n s when i t comes to a s s e s s i n g a n t i c o n v u l s a n t drug e f f e c t s . For example, e l e c t r o c o n v u l s i v e -shock-induced and p e n t y l e n e t e t r a z o l - i n d u c e d c o n v u l s i o n s are o f t e n a s s o c i a t e d with s u b j e c t i n j u r y and f a t a l i t y , and they are extremely v a r i a b l e i n form and d u r a t i o n , which makes them d i f f i c u l t to measure. The problem of s u b j e c t a t t r i t i o n i s p a r t i c u l a r l y s e r i o u s i n s t u d i e s of t o l e r a n c e i n which a n t i c o n v u l s a n t e f f e c t s are r e p e a t e d l y assessed i n the same s u b j e c t s because any sy s t e m a t i c change i n the apparent a n t i c o n v u l s a n t a c t i o n of a drug are always confounded by the p r o g r e s s i v e d e b i l i t a t i o n and s e l e c t i v e a t t r i t i o n of those s u b j e c t s e x p e r i e n c i n g the most severe c o n v u l s i o n s . i n c o n t r a s t , 13 Table II C l a s s i f i c a t i o n of K i n d l e d Convulsions C l a s s Character i s t i c s 1 f a c i a l clonus 2 f a c i a l c l o n u s , head nodding 3 f a c i a l c l o n u s , head nodding, f o r e l i m b clonus 4 f a c i a l c l o n u s , head nodding, f o r e l i m b c l o n u s , r e a r ing 5 f a c i a l c l o n u s , head nodding, f o r e l i m b c l o n u s , rea r i n g , l o s s of e q u i l i b r i u m 6 f a c i a l c l o n u s , head nodding, f o r e l i m b c l o n u s , m u l t i p l e sequences of r e a r i n g and l o s s of e q u i l i b r i u m 7 a l l of the above plus a running f i t 8 any of the above plus tonus 14 k i n d l e d r a t s remain h e a l t h y and easy to handle f o r the d u r a t i o n of an experiment, and f a t a l i t i e s are extremely r a r e . Moreover, i n w e l l k i n d l e d r a t s , i t i s p o s s i b l e to e l i c i t c o n v u l s i o n s t h a t vary l i t t l e from s u b j e c t to s u b j e c t i n both form and d u r a t i o n , and b a s e l i n e s can be e s t a b l i s h e d i n i n d i v i d u a l s u b j e c t s that d i s p l a y almost no f l u c t u a t i o n from s t i m u l a t i o n to s t i m u l a t i o n ( P i n e l , P h i l l i p s , & MacNeil, 1973). The importance of such long-term s t a b i l i t y i n the study of the development of t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s i s obvious, and the s t e r e o t y p e d nature of k i n d l e d motor c o n v u l s i o n s makes the measurement of t h e i r i n t e n s i t y a r e l a t i v e l y simple matter. In most s t u d i e s of a n t i c o n v u l s a n t drug a c t i o n s on k i n d l e d c o n v u l s i o n s , the dependent measure has been the c l a s s of the c o n v u l s i o n (see Table 2), an o r d i n a l s c a l e of measure. In the present t h e s i s , although c o n v u l s i o n c l a s s was always recorded, the primary dependent measure was the d u r a t i o n of f o r e l i m b c l o n u s . The d u r a t i o n of f o r e l i m b clonus i s h i g h l y c o r r e l a t e d with c o n v u l s i o n c l a s s and v a r i o u s other measures of k i n d l e d s e i z u r e s e v e r i t y , but i t has the advantage of being a continuous r a t i o s c a l e . Experiments from t h i s l a b o r a t o r y have demonstrated the r e l i a b i l i t y of t h i s measure and i t s s e n s i t i v i t y to drug manipulations (e.g., P i n e l et a l . , 1989) 8. General R a t i o n a l e There were two general r a t i o n a l e s f o r s t u d y i n g the r o l e of the d r u g - e f f e c t contingency on the development of t o l e r a n c e and c r o s s t o l e r a n c e . The f i r s t was the apparent lack of a complete 15 understanding of the complex phenomena of drug t o l e r a n c e and cro s s t o l e r a n c e . Despite decades of r e s e a r c h and hundreds of re s e a r c h a r t i c l e s , many a r t i c l e s s t i l l end with perhaps the most overused c l i c h e 1 i n s c i e n c e , "more r e s e a r c h i s necessary." Perhaps the reason t h a t progress has been slow i s due to the narrowness of the t r a d i t i o n a l pharmacological drug-exposure theory t h a t has guided most r e s e a r c h . With the c u r r e n t understanding t h a t the s u b j e c t s ' d r u g - r e l a t e d experiences are important i n the development of t o l e r a n c e , the study of t o l e r a n c e and c r o s s t o l e r a n c e may y i e l d new and u s e f u l i n f o r m a t i o n of t h e o r e t i c a l and c l i n i c a l importance. I t may lead to a b e t t e r understanding of why t o l e r a n c e and/or s e n s i t i z a t i o n occurs or does not occur; increase our understanding of the u n d e r l y i n g mechanisms of drug a c t i o n ; provide a b e t t e r understanding of the r e l a t i o n s h i p between drug t o l e r a n c e and the phenomena of drug dependence, withdrawal, and abuse; and lead to more e f f e c t i v e p harmacological treatment regimens. The second reason was the apparent gap between the r e s e a r c h of mainstream pharmacologists and those t h a t study c o n t i n g e n t drug t o l e r a n c e . Despite the g e n e r a l i t y of the co n t i n g e n t t o l e r a n c e phenomenon, many mainstream pharmacologists have ignored t h i s work. By demonstrating t h a t the d r u g - e f f e c t contingency a l s o p l a y s a major r o l e i n c r o s s t o l e r a n c e , the robustness of t h i s phenomenon would be e s t a b l i s h e d ; and perhaps then i t would f i n d more widespread acceptance. 9. General Purpose 16 The g e n e r a l p u r p o s e o f t h e t h e s i s was t o a s s e s s t h e r o l e o f d r u g - e f f e c t s as opposed t o mere d r u g - e x p o s u r e on t h e d e v e l o p m e n t of c r o s s t o l e r a n c e t o a n t i c o n v u l s a n t d r u g e f f e c t s . A c c o r d i n g l y , E x p e r i m e n t 1 examined c r o s s t o l e r a n c e t o a n t i c o n v u l s a n t d r u g e f f e c t s w i t h o u t m a n i p u l a t i n g t h e d r u g - e f f e c t c o n t i n g e n c y ; w h i l e E x p e r i m e n t 2 examined c r o s s t o l e r a n c e t o a n t i c o n v u l s a n t d r u g e f f e c t s w h i l e m a n i p u l a t i n g t h e d r u g - e f f e c t c o n t i n g e n c y . E x p e r i m e n t 3 examined t h e r o l e o f t h e d r u g - e f f e c t c o n t i n g e n c y i n t h e c r o s s - d i s s i p a t i o n o f t o l e r a n c e t o a n t i c o n v u l s a n t d r u g e f f e c t s . E x p e r i m e n t 4 d e t e r m i n e d i f d i f f e r e n t d r u g a d m i n i s t r a t i o n r e g i m e n s would d i f f e r e n t i a l l y i n f l u e n c e t h e d e g r e e t o w h i c h t o l e r a n c e d e v e l o p s t o a n t i c o n v u l s a n t d r u g e f f e c t s . 17 I I . Background f o r Experiments 1 and 2 The main focus of the t h e s i s was c o n t i n g e n t cross tolerance to a n t i c o n v u l s a n t drug e f f e c t s . A c c o r d i n g l y , the l i t e r a t u r e on t o l e r a n c e and c r o s s t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s and of c o n t i n g e n t c r o s s t o l e r a n c e i s reviewed i n t h i s s e c t i o n . 1. Tolerance and Cross Tolerance to A n t i c o n v u l s a n t Drug E f f e c t s Tolerance has been demonstrated to the a n t i c o n v u l s a n t e f f e c t of v i r t u a l l y every a n t i e p i l e p t i c drug i n c u r r e n t u s e — s e e Table I I I . In c o n t r a s t , only a handful of s t u d i e s have examined c r o s s t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s - - s e e Table IV. 2. Contingent Cross Tolerance There have been on l y f i v e s t u d i e s t h a t have examined the r o l e of the d r u g - e f f e c t contingency on the development of c r o s s t o l e r a n c e between drugs, and i t s r o l e i s s t i l l somewhat u n c e r t a i n . These experiments e i t h e r s t u d i e d a drug e f f e c t i n which confounding may be a problem, the experimental design was flawed, the data were improperly analyzed, or the r e s u l t s were i n c o n s i s t e n t . Two s t u d i e s used the a n o r e x i g e n i c drug e f f e c t to study c o n t i n g e n t c r o s s t o l e r a n c e ; u n f o r t u n a t e l y , t h i s drug e f f e c t i s s u s c e p t i b l e to a s e r i o u s experimental confound. Tolerance to a n o r e x i a t h a t i s seen a f t e r c h r o n i c drug treatment may be a consequence of m o t i v a t i o n a l changes and/or body weight l o s s i n the r a t s t h a t r e c e i v e d the drug before the f e e d i n g s e s s i o n s ; and the amount of " t r u e " t o l e r a n c e t h a t develops may be n e g l i g i b l e . 18 Table I I I Review of Tolerance to A n t i c o n v u l s a n t Drug E f f e c t s S p e c i f i c  Drug Acetazol-amide Convulsant Model maximal e l e c t r o -shock maximal e l e c t r o -shock maximal e l e c t r o -shock Species Tolerance Reference mouse yes Anderson et a l . , (1989) mouse mouse yes Koch & Woodbury (1958) yes M i l l i c h a p et a l . , (1955) e l e c t r o -shock t h r e s h o l d mouse yes Anderson et a l . , (1989) A c e t y l e n i c GABA a i r b l a s t g e r b i 1 yes Loscher (1986) Aminooxy-acet i c a c i d a i r b l a s t g e rbi1 yes Loscher (1986) C a n n a b i d i o l ma x i ma 1 e l e c t r o -shock mouse yes K a r l e r et a l . , (1974) maximal e l e c t r o -shock mouse no K a r l e r & Turkanis (1980) e l e c t r o -shock t h r e s h o l d mouse no K a r l e r & Turkanis (1980) Carbama-zepine ma x i ma 1 e l e c t r o -shock mouse yes Masuda et a l . , (1979 ) k i n d l i n g r a t yes Mana et a l . , (under review) C h l o r d i a - metrazol mouse yes G a r r a t t et a l . , (1988) zepoxide 19 maximal e l e c t r o -shock mouse yes Goldberg et a l . , (1967) Clobazam me t r a z o l mouse yes Gent et a l . , (1984) metrazol mouse yes Gent et a l . , (1985) metrazol mouse yes Gent et a l . , (1986) metrazol mouse yes Haigh et a l . , (1987) k i n d l i n g r a t yes Rosenberg et a l . , (1989) k i n d l i n g r a t yes Vajda et a t . , (1987) k i n d l i n g r a t yes Young et a l . , (1988) Clo r a z e p a t e metrazol rhesus monkey no P l o t n i k o f f & (1974) 0'Br ien metrazol mouse yes S c h e r k l et a l ., (1988) metrazol dog yes S c h e r k l et a l ., (1989) Clonazepam metrazol mouse yes Gent et a l . , (1980) metrazol mouse yes Gent et a l . , (1986) metrazol dog yes S c h e r k l et a l ., (1985) metrazol mouse yes S c h e r k l et a l ., (1988) l i g h t baboon yes K i l l a m et a l . , (1973) k i n d l i n g r a t yes Young et a l . , (1987) k i n d l i n g r a t yes Young et a l . , (1988) k i n d l i n g r a t yes Rosenberg et a l . , (1989) De l t a - 9 - maximal mouse yes K a r l e r et a l ., (1974) THC e l e c t r o -shock maximal mouse no K a r l e r & Turkanis (1980) e l e c t r o -shock e l e c t r o - mouse no K a r l e r & Turkanis (1980) shock t h r e s h o l d 20 k i n d l i n g r a t yes F r i e d & Mclntyre (1973) Diazepam metrazol mouse yes F i l e (1983a) metrazol mouse yes F i l e (1983b) metrazol dog yes Frey e t a l . , (1984) metrazol mouse yes Gent et a l . , (1986) metrazol mouse no Juhasz & Dairman (1977) metrazol mouse no Li p p a & Regan (1977) metrazol rhesus monkey no P l o t n i k o f f & O'Brien (1974) b i c u c u l -1 ine r a t yes Gonsalves & G a l l a g e r (1988) b i c u c u l -1 ine mouse yes Jahasz & Dairman (1977) b i c u c u l -1 ine mouse yes Lip p a & Regan (1977) s t r y c h -nine mouse yes Lip p a & Regan (1977) 3-MP A mouse yes Turner et a l . , (1981) l i g h t baboon yes K i l l a m et a l . , (1973) a i r b l a s t g e r b i l Loscher (1986) k i n d l i n g r a t yes Loscher & Schwark (1985) k i n d l i n g r a t yes Mana et a l . , (under review) k i n d l i n g r a t yes Rosenberg et a l . , (1989) Desmethy-diazepam metrazol dog yes Frey et a l . , (1984) Ethanol m a x i m a l e l e c t r o -mouse no McQuarrie & F i n q l e (1958) s h o c k 21 e l e c t r o -shock t h r e s h o l d mouse no A l l a n & Swinyard (1949) e l e e t r o -shock t h r e s h o l d mouse- no Chen (1968) e l e c t r o -shock t h r e s h o l d mouse no Zarrow e t a l . , (1962) k i n d l i n g r a t yes Mana & P i n e l (1987) k i n d l i n g r a t yes P i n e l et a l . , (1983) k i n d l i n g r a t yes P i n e l et a l . , (1985) k i n d l i n g r a t yes P i n e l & Puttaswamaiah (1983) Ethosuc-c i n i m i d e metrazol maximal e l e c t r o -shock mouse mouse no no Fr e y & Kampmann (1965) Frey & Kampmann (1965) Gaboxadol a i r b l a s t g e r b i l yes Loscher (1986) L i t h i u m k i n d l i n g c a t yes Minabe et a l . , (1988) Lorazepam metrazol mouse yes Gent et a l . , (1986) Mepro-bamate metrazol mouse yes Chin & Swinyard (1959) Metha-qualone maximal e l e c t r o -shock mouse yes Boggon et a l . , (1977) Midazolam metrazol mouse yes G a r r a t t et a l . , (1988) Nitrazepam metrazol mouse yes G a r r a t t et a l . , (1988) Oxazepam metrazol mouse yes Gluckman & Baxter (1971) Phenacemide metrazol r a t yes DeSalva (1956) e l e c t r o -shock r a t yes DeSalva (1956) 22 Phenagly-codol metrazol mouse yes Chin & Swinyard (1959) e l e c t r o -shock t h r e s h o l d mouse yes Chin & Swinyard (1958) Phenobar b-i t a l me t r a z o 1 metrazol metrazol maximal e l e c t r o -shock maximal e l e c t r o -shock maximal e l e c t r o -shock maximal e l e c t r o -shock maximal e l e c t r o -shock maximal e l e c t r o -shock e l e c t r o -shock t h r e s h o l d e l e c t r o -shock t h r e s h o l d mouse mouse mouse mouse-mouse mouse mouse yes Frey & Kampmann (1965) yes K i l i a n & Fr e y (1973) no Schmidt et a l . , (1980) yes Frey & Kampmann (196 5) mouse no K a r l e r & Turkanis (1980) yes K a r l e r et a l . , (1965) mouse yes K i l i a n & Fr e y (1973) yes Masada et a l . , (1979) mouse yes Schmidt et a l . , (1980) mouse yes K a r l e r & Turkanis (1980) no Schmidt et a l . , (1980) Phenytoin metrazol mouse maximal mouse e l e c t r o -shock yes Frey & Kampmann (1965) yes F r e y & Kampmann (1965) maximal mouse no K a r l e r & Turkanis (1980) e l e c t r o -shock maximal e l e c t r o -shock mouse yes K a r l e r et a l . , (1974) maximal e l e c t r o -shock mouse yes Masuda et a l . , (1979) e l e c t r o -shock t h r e s h o l d mouse no K a r l e r & Turkanis (1980) Primidone maximal e l e c t r o -shock mouse yes Bourgeois (1986) RO 16-6028 metrazol mouse yes Haigh & F e e l y (1988) Sulfamoyl-methyl B e n z i -oxole maximal e l e c t r o -shock mouse no Masuda et a l . , (1979) Tr imetha-dione metrazol e l e c t r o -shock mouse r a t yes yes Frey & Kretschmer (1971) DeSalva (1956) V a l p r o a t e metrazol mouse no Gent et a l . , (1986) a i r b l a s t g e r b i l •? Loscher (1986) k i n d l i n g r a t yes Mana et a l . , (under review) k i n d l i n g r a t no Young et a l . , (1987) 24 Table IV Review of Cross Tolerance to A n t i c o n v u l s a n t Drug E f f e c t s Cross Tolerance Tolerance Convulsant Cross Drug Drug Model Tolerance Reference Clobazam Clonazepam k i n d l i n g Diazepam Flurazepam k i n d l i n g Pr imidone Clobazam Clonazepam Diazepam Clobazam Clonazepam Lorazepam Clobazam Diazepam Lorazepam Clonazepam Diazepam Lorazepam Clobazam Clonazepam Diazepam Lorazepam Clobazam Clonazepam Diazepam Lorazepam Pheno- maximal b a r b i t a l e l e c t r o -shock Lorazepam metrazol Diazepam metrazol Clonazepam metrazol Clobazam metrazol V a l p r o a t e metrazol Pheno- metrazol b a r b i t a l yes yes yes yes yes yes yes no Vajda et a l . , (1987) yes Rosenberg et a l . , (1989) Bourgeois (1986) Gent et a l . , (1986) Gent et a l . , (1986) Gent et a l . , (1986) Gent et a l . , (1986) Gent et a l . , (1986) Gent et a l . , (1986) S t r e a t h e r and Hinson (1985) c o n t r o l l e d f o r t h i s and demonstrated t h a t c r o s s t o l e r a n c e to the a n o r e x i g e n i c e f f e c t of apomorphine was g r e a t e r i n r a t s allowed to eat while under the i n f l u e n c e of amphetamine than those t h a t were not; however, co n t i n g e n t c r o s s t o l e r a n c e was e v i d e n t a t only one t e s t dose of apomorphine, and there was no evidence of c o n t i n g e n t c r o s s t o l e r a n c e from amphetamine to f e n f l u r a m i n e . Wooverton, Kandel, and Schuster (1978) demonstrated t h a t c r o s s t o l e r a n c e to the a n o r e x i g e n i c e f f e c t of amphetamine was g r e a t e r i n r a t s allowed to eat while under the i n f l u e n c e of cocaine; but t h i s study was confounded, making i n t e r p r e t a t i o n of the r e s u l t s d i f f i c u l t . Two other s t u d i e s used the motor-coordination-and-balance task, a poor c h o i c e of drug e f f e c t f o r the study of the d r u g - e f f e c t contingency because there i s no c l e a r d i s a s s o c i a t i o n between drug-exposure and drug-e f f e c t . The s u b j e c t may experience components of a drug's d i s r u p t i v e e f f e c t whether performing the c r i t e r i o n task or walking around i n i t s home cage. Commissar i s and Rech (1981) found t h a t under one c o n d i t i o n , there was no s i g n i f i c a n t d i f f e r e n c e i n the degree of t o l e r a n c e to p e n t o b a r b i t a l i n those r a t s t h a t were allowed to p r a c t i c e while drugged and those t h a t were not, but there was a g r e a t e r degree of c r o s s t o l e r a n c e to ethanol i n r a t s allowed to p r a c t i c e while under the i n f l u e n c e of p e n t o b a r b i t a l . Under another s e t of c o n d i t i o n s , there was a d i f f e r e n c e i n the degree of t o l e r a n c e to p e n t o b a r b i t a l , but no s i g n i f i c a n t d i f f e r e n c e i n the degree of c r o s s t o l e r a n c e to e t h a n o l . Le, El-Ghundl, Khanna, and Kalant (1986) found no 26 s i g n i f i c a n c e d i f f e r e n c e i n the degree of t o l e r a n c e to ethanol i n those r a t s allowed to p r a c t i c e while drugged and those t h a t were not, but found that c r o s s t o l e r a n c e to p e n t o b a r b i t a l was g r e a t e r i n r a t s allowed to p r a c t i c e while under the i n f l u e n c e of e t h a n o l . Jorgensen, Fasmer, and Hole (1986) r e p o r t e d t h a t c r o s s t o l e r a n c e to the i n h i b i t o r y e f f e c t of c l o n i d i n e on the t a i l - f l i c k r e f l e x of s p i n a l l y t r a n s a c t e d r a t s was g r e a t e r when t e s t e d while under the i n f l u e n c e of e t h a n o l ; but there was a problem with the way the data was analyzed, making the f i n d i n g s somewhat q u e s t i o n a b l e . Thus, the few s t u d i e s of co n t i n g e n t c r o s s t o l e r a n c e have not c l e a r l y i n d i c a t e d whether or not the d r u g - e f f e c t contingency i s important i n the development of c r o s s t o l e r a n c e . I I I . General Method This s e c t i o n d e s c r i b e s the methods common to most of the present experiments. S p e c i f i c a d d i t i o n s or changes to t h i s g e neral methodology are d e s c r i b e d i n the Methods s e c t i o n of each experiment. Subjects The s u b j e c t s i n a l l the experiments were a d u l t , male, hooded Long-Evans r a t s (from Charles R i v e r , Canada) weighing 300 to 500 g at the time of surgery. Each r a t was i n d i v i d u a l l y housed i n standard s t a i n l e s s s t e e l wire mesh hanging cages with continuous access to Purina r a t chow and water. A l l experimental manipulations occurred d u r i n g the l i g h t phase of the 12/12-hr l i g h t / d a r k c y c l e . Surgery F o l l o w i n g the a d m i n i s t r a t i o n of sodium p e n t o b a r b i t a l (65 mg/kg, IP) a n e s t h e s i a , and a t r o p i n e sulphate (0.04 mg, IP) to help prevent r e s p i r a t o r y f a i l u r e , a s i n g l e c h r o n i c b i p o l a r e l e c t r o d e ( P l a s t i c Products Company, MS 303/2) was d i r e c t e d a t the l e f t b a s o l a t e r a l amygdala of each r a t . I t was secured to the s k u l l with s t a i n l e s s s t e e l screws and d e n t a l a c r y l i c . The t i p of each e l e c t r o d e was p o s i t i o n e d using one of two c o o r d i n a t e systems: 2.8 mm p o s t e r i o r , 5.0 mm l a t e r a l , and 8.7 mm v e n t r a l to the bregma s k u l l s u r f a c e , with the i n c i s o r bar s e t a t +3.3 mm (co o r d i n a t e s from Paxinos & Watson, 1982) f o r Experiments 1 and 4; or 1.2 mm a n t e r i o r , 5.0 mm l a t e r a l , and 10 mm v e n t r a l to the 28 bregma s k u l l s u r f a c e , with the i n c i s o r bar s e t at +5.0 mm (co o r d i n a t e s from P e l l e g r i n o , P e l l e g r i n o , & Cushman, 1979) f o r Experiments 2 and 3. T e t r a c y c l i n e was s p r i n k l e d over the i n c i s i o n before s u t u r i n g to help prevent i n f e c t i o n s . K i n d l i n g Phase A f t e r at l e a s t 5 days of p o s t s u r g i c a l r ecovery, each of the r a t s was s t i m u l a t e d (400 uA, 60 Hz, 1 sec) three times per day, 5 days a week f o r 3 weeks, with at l e a s t 2 hr s e p a r a t i n g c o n s e c u t i v e s t i m u l a t i o n s . For each s t i m u l a t i o n , the r a t was removed from i t s home cage, the s t i m u l a t i o n l e a d was connected, and then the r a t was placed i n a 58 x 58 x 25 cm opaque p l a s t i c chamber c o n t a i n i n g a l a y e r of S a n - i - c e l bedding m a t e r i a l . The s t i m u l a t i o n was d e l i v e r e d immediately, and the r a t was retu r n e d to i t s home cage once c o n v u l s i v e a c t i v i t y ceased. By the end of t h i s regimen of 45 k i n d l i n g s t i m u l a t i o n s , each s t i m u l a t i o n produced g e n e r a l i z e d c l o n i c c o n v u l s i o n s . No-Drug B a s e l i n e Phase Th i s phase began 48 hr f o l l o w i n g the l a s t of the 45 k i n d l i n g s t i m u l a t i o n s , and i t i n v o l v e d four s t i m u l a t i o n s d e l i v e r e d one every 48 hr (+2 h r ) . Th i s b i d a i l y s t i m u l a t i o n schedule once i n i t i a t e d d u r i n g the no-drug b a s e l i n e phase was maintained f o r the d u r a t i o n of each experiment. For each c o n v u l s i o n , the l a t e n c y and d u r a t i o n of f o r e l i m b clonus and the c o n v u l s i o n c l a s s was recorded, but the dependent measure i n a l l except Experiment 4 was the d u r a t i o n of f o r e l i m b clonus e l i c i t e d by each s t i m u l a t i o n . The i s o t o n i c s a l i n e v e h i c l e (0.9% s a l i n e ) , matched i n volume f o r 29 the subsequent drug i n j e c t i o n s (the i n j e c t i o n volume v a r i e d f o r the experiments), was i n j e c t e d 1 hr p r i o r to the f o u r t h and l a s t no-drug b a s e l i n e s t i m u l a t i o n to determine the e f f e c t s of the i n j e c t i o n procedure on the d u r a t i o n of f o r e l i m b clonus e l i c i t e d by the s t i m u l a t i o n . A l l the i n j e c t i o n s , f o r the drugs and the s a l i n e v e h i c l e , were I n j e c t e d IP a t room temperature. Any r a t s that d i d not d i s p l a y at l e a s t 20 s of f o r e l i m b clonus on t h i s no-drug s a l i n e b a s e l i n e t r i a l or d i s p l a y e d running f i t s ( c l a s s 7 c o n v u l s i o n a c c o r d i n g to the c l a s s i f i c a t i o n scheme of P i n e l & Rovner, 1978; see Table 2) were not s t u d i e d f u r t h e r . The r e j e c t i o n c r i t e r i o n was enforced to e l i m i n a t e any s u b j e c t s t h a t d i d not c o n s i s t e n t l y d i s p l a y c o n v u l s i v e a c t i v i t y i n the absence of the drug or d i s p l a y e d v i o l e n t c o n v u l s i o n s t h a t are d i f f i c u l t to measure. T h i s c r i t e r i o n was not met by 6.1% of the r a t s from a l l the experiments. Drug B a s e l i n e Test Approximately 48 hr a f t e r the no-drug s a l i n e b a s e l i n e t r a i l , a l l r a t s r e c e i v e d the a p p r o p r i a t e drug i n an i s o t o n i c s a l i n e v e h i c l e 1 hr p r i o r to the c o n v u l s i v e s t i m u l a t i o n . Those r a t s t h a t d i d not show a s u f f i c i e n t a n t i c o n v u l s a n t response to the drug; t h a t i s , those t h a t d i s p l a y e d more than 20 s of f o r e l i m b c l o n u s , were not s t u d i e d f u r t h e r . T h i s r e j e c t i o n c r i t e r i o n was enf o r c e d because the development of t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s cannot be s t u d i e d i f s u b j e c t s do not d i s p l a y i n i t i a l s e n s i t i v i t y to the drug's a n t i c o n v u l s a n t e f f e c t . T h i s c r i t e r i o n l e d to the r e j e c t i o n of 2.8% of the r a t s . The p a r t i c u l a r doses 30 employed i n the present experiments were determined from previous p u b l i s h e d s t u d i e s (e.g., A l b e r t s o n , Peterson & Stark, 1980; Mana & P i n e l , 1987; Minabe, T a n i i & Kurache, 1987; P i n e l et a l . , i n press) and from p i l o t o b s e r v a t i o n s . In a l l but Experiment 4, an attempt was made to s e l e c t doses that were the minimum r e q u i r e d to completely suppress the f o r e l i m b clonus i n a l l r a t s . Treatment and Test Phase The treatment and t e s t phases d i f f e r e d f o r the experiments, but each had s e v e r a l t h i n g s i n common: the r a t s were assigned to the experimental groups i n such a way t h a t the mean d u r a t i o n of f o r e l i m b clonus e l i c i t e d on the no-drug s a l i n e b a s e l i n e t r i a l and the drug b a s e l i n e t e s t , and t h e i r body weights were approximately equal f o r the groups; i t began 48 hr a f t e r the drug b a s e l i n e t e s t ; the treatment and t e s t t r i a l s occurred on a b i d a i l y schedule (every 4 8 + 2 hr) f o r the d u r a t i o n of the experiment; and on each treatment and t e s t t r i a l , the s u b j e c t was removed from i t s home cage, weighed, and the drug or the s a l i n e v e h i c l e was administered e i t h e r 1 hr before or 1 hr a f t e r the c o n v u l s i v e s t i m u l a t i o n . H i s t o l o g y At the c o n c l u s i o n of each experiment, a l l of the r a t s were k i l l e d with CO a c c o r d i n g to Canada C o u n c i l on Animal Care G u i d e l i n e s . T h e i r b r a i n s were removed, preserved i n f o r m a l i n , f r o z e n , s l i c e d along the c o r o n a l plane (approximately 30 urn t h i c k n e s s ) , mounted on s l i d e s , and then s t a i n e d with c r e s y l v i o l e t to c o n f i r m the l o c a t i o n s of the e l e c t r o d e t i p s . 31 S t a t i s t i c a l A n a l y s i s In most cases, the s t a t i s t i c a l s i g n i f i c a n c e of the r e s u l t s was evaluated u s i n g nonparametric s t a t i s t i c a l methods ( S i e g e l & C a s t e l l a n , 1988) because of repeated gross d e v i a t i o n s from n o r m a l i t y and from homogeneity of v a r i a n c e — i t was not uncommon for the scores i n an experimental c o n d i t i o n to be a l l z e r o s . Only i n Experiment 4 were the data parametric, and thus parametric methods were used only i n t h i s experiment. The b a s i c s i g n i f i c a n c e l e v e l was s e t at p<.05, but i n each study b o n f e r r o n i a d j u s t e d alpha values were used to avoid the problem of e l e v a t e d alpha v a l u e s a s s o c i a t e d with m u l t i p l e comparisons. Only the data of those r a t s t h a t completed as experiment were s u b j e c t e d to s t a t i s t i c a l a n a l y s i s . IV. Experiment 1 32 Tolerance has been demonstrated e x p e r i m e n t a l l y to v i r t u a l l y every a n t i e p i l e p t i c drug i n c u r r e n t use (see Table 3; Frey, 1985, 1986, 1987). But there i s a notable lack of r e s e a r c h on c r o s s t o l e r a n c e between a n t i c o n v u l s a n t drug e f f e c t s (see Table 4). The few s t u d i e s t h a t have examined c r o s s t o l e r a n c e have u s u a l l y l i m i t e d the choice of drugs between those of the same or s i m i l a r pharmacological c l a s s e s (Bourgeois, 1986; Rosenberg, T e i t z & Chiu, 1985; Vajda, Lewis, H a r r i s , J a r r o t t , & Young, 1986). In t h i s study, c r o s s t o l e r a n c e was examined between s e v e r a l drugs that d i f f e r e d i n the degree of s i m i l a r i t y with each other; the t r a n s f e r of t o l e r a n c e was assessed from p h e n o b a r b i t a l , t r i m e t h a d i o n e , and clonazepam to carbamazepine. From among the approximately 20 a n t i e p i l e p t i c drugs i n c u r r e n t use, these four drugs were s e l e c t e d f o r study on the b a s i s of three c r i t e r i a . The f i r s t was t h a t they were e f f e c t i v e a g a i n s t d i f f e r e n t c l i n i c a l s e i z u r e s (see F i g u r e 1); p h e n o b a r b i t a l and carbamazepine are most commonly used i n the treatment of p a r t i a l s e i z u r e s and g e n e r a l i z e d t o n i c - c l o n i c s e i z u r e s , trimethadione i s used e x c l u s i v e l y i n the treatment of absence s e i z u r e s , and clonazepam i s used i n the treatment of the e n t i r e spectrum of s e i z u r e types, although mostly i n the treatment of absence s e i z u r e s (see Ead i e , 1985; R a i l & S c h l e i f e r , 1980). The second was the f a c t t h a t they have d i f f e r e n t p u t a t i v e mechanisms of a c t i o n ; c u r r e n t evidence suggests t h a t p h e n o b a r b i t a l and carbamazepine a c t by d e c r e a s i n g 33 F i g u r e 1. The c l a s s i f i c a t i o n of the types of e p i l e p t i c s e i z u r e s , and the r e l a t i v e e f f i c a c y of the a n t i e p i l e p t i c d r u g s — p h e n o b a r b i t a l , trimethadione, clonazepam and carbamazepine--at c o n t r o l l i n g them. E p i l e p t i c s e i z u r e s can be c l a s s i f i e d i n t o g e n e r a l i z e d and p a r t i a l s e i z u r e s , with f u r t h e r s u b d i v i s i o n s w i t h i n each category; and a n t i e p i l e p t i c drugs v a r y i n t h e i r e f f e c t i v e n e s s at c o n t r o l l i n g the d i f f e r e n t types of e p i l e p t i c s e i z u r e s . Trimethadione i s most e f f e c t i v e a g a i n s t absence s e i z u r e s ; p h e n o b a r b i t a l and carbamazepine i s most e f f e c t i v e a g a i n s t p a r t i a l and g e n e r a l i z e d t o n i c - c l o n i c s e i z u r e s ; and clonazepam i s e f f e c t i v e a g a i n s t the whole spectrum of s e i z u r e s although i t i s used mostly a g a i n s t absence s e i z u r e s . E p i l e p t i c S e i z u r e s I G e n e r a l i z e d P a r t i a l I I I Absences Myoclonic j e r k s T o n i c - c l o n i c I I Trimethadione I I Carbamazepine I P h e n o b a r b i t a l i Clonazepam I 34 s o d i u m and c a l c i u m i o n movement a c r o s s membranes, and t h a t t r i m e t h a d i o n e and c l o n a z e p a m a c t by o t h e r m e c h a n i s m s ( s e e F r e y & J a n z , 1985; K a t z u n g , 1 9 8 7 ) . And t h e t h i r d was t h e i r d i f f e r e n t p r o f i l e o f e f f e c t i v e n e s s a g a i n s t m a x i m a l e l e c t r o s h o c k and p e n t y l e n e t e t r a z o l ( m e t r a z o l ) i n d u c e d c o n v u l s i o n s ; p h e n o b a r b i t a l a n d c a r b a m a z e p i n e a r e more e f f e c t i v e a g a i n s t m a x i m a l e l e c t r o s h o c k c o n v u l s i o n s t h a n a g a i n s t p e n t y l e n e t e t r a z o l c o n v u l s i o n s , w h e r e a s t h e r e v e r s e i s t r u e f o r t r i m e t h a d i o n e and c l o n a z e p a m ( s e e F r e y & J a n z , 1985) . B a s e d on t h e s i m i l a r i t y among t h e f o u r d r u g s i n t h e t y p e o f c l i n i c a l s e i z u r e s t h a t t h e y most e f f e c t i v e l y c o n t r o l , t h e i r p u t a t i v e m e c h a n i s m o f a c t i o n , and t h e i r r e l a t i v e e f f e c t i v e n e s s i n s u p p r e s s i n g e l e c t r o s h o c k and p e n t y l e n e t e t r a z o l i n d u c e d c o n v u l s i o n s , i t was h y p o t h e s i z e d t h a t c r o s s t o l e r a n c e w o u l d be g r e a t e s t f r o m p h e n o b a r b i t a l t o c a r b a m a z e p i n e , l e s s f r o m c l o n a z e p a m t o c a r b a m a z e p i n e , and l e a s t f r o m t r i m e t h a d i o n e t o c a r b a m a z e p i n e . The p u r p o s e o f E x p e r i m e n t s 1A and I B was t o t e s t t h i s h y p o t h e s i s . The t o l e r a n c e and c r o s s t o l e r a n c e were e x a m i n e d w i t h o u t m a n i p u l a t i n g t h e d r u g - e f f e c t c o n t i n g e n c y : t h e c o n v u l s i v e s t i m u l a t i o n a l w a y s f o l l o w e d t h e d r u g i n j e c t i o n s s o t h a t t h e a n t i c o n v u l s a n t d r u g e f f e c t s were e x p e r i e n c e d by t h e s u b j e c t s on e a c h t r i a l . E x p e r i m e n t 1A The p u r p o s e o f E x p e r i m e n t 1A was t o d e m o n s t r a t e t h a t t o l e r a n c e d e v e l o p s t o t h e a n t i c o n v u l s a n t e f f e c t s o£ p h e n o b a r b i t a l , t r i m e t h a d i o n e , and c l o n a z e p a m , and i n d o i n g s o t o 35 prepare the r a t s f o r the c r o s s t o l e r a n c e t e s t to carbamazepine i n Experiment IB. Previous s t u d i e s had a l r e a d y demonstrated t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t s of these three drugs (e.g., Frey & Kampmann, 1965; Frey & Kretschmer, 1971; Gent, F e e l y , & Haigh, 1985), but t h i s was the f i r s t demonstration of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t p h e n o b a r b i t a l and trimethadione on k i n d l e d c o n v u l s i o n s . Method Su b j e c t s . Of the 57 r a t s completing the k i n d l i n g phase of Experiment 1, two r a t s were r e j e c t e d because they d i d not meet the c r i t e r i o n on the no-drug s a l i n e b a s e l i n e t r i a l , four r a t s were r e j e c t e d because they d i d not meet c r i t e r i o n on the drug b a s e l i n e t e s t , and another three were l o s t due to i l l n e s s or r e j e c t i o n of t h e i r e l e c t r o d e assembly. Thus, 48 r a t s completed Experiment 1A. Drugs. The drugs used i n Experiment 1A were p h e n o b a r b i t a l (30 mg/kg; i n a sodium s a l t form; BDH Chemicals), trimethadione (270 mg/kg; Abbott P h a r m a c e u t i c a l s ) , .and clonazepam (0.40 or 0.35 mg/kg; Hoffmann-La Roche). The v e h i c l e was i s o t o n i c s a l i n e with 2% Tween 80 (J.T. Baker Chemical) to a i d i n suspension of the drugs. A l l drug and s a l i n e v e h i c l e i n j e c t i o n s were d e l i v e r e d i n a volume of 5 ml/kg. S o n i f i c a t i o n was r e q u i r e d to get trimethadione and clonazepam i n t o suspension. Drug B a s e l i n e T e s t . On the drug b a s e l i n e t e s t , a l l r a t s r e c e i v e d e i t h e r p h e n o b a r b i t a l , t r i m e t h a d i o n e , or clonazepam (0.40 mg/kg) 1 hr p r i o r to the c o n v u l s i v e s t i m u l a t i o n . 36 Tolerance-Development Phase. During the t o l e r a n c e -development phase, each group of r a t s t h a t had been screened on e i t h e r one of the three drugs on the drug b a s e l i n e t e s t were d i v i d e d i n t o two e q u i v a l e n t groups: the drug group (n=9 f o r p h e n o b a r b i t a l ; n=8 f o r both trimethadione and clonazepam) and the corresponding s a l i n e group (n=8 f o r p h e n o b a r b i t a l and trim e t h a d i o n e ; n=7 f o r clonazepam). On each t o l e r a n c e development t r i a l , the a p p r o p r i a t e d r u g — t h e same drug t h a t was d e l i v e r e d on the drug b a s e l i n e test--was d e l i v e r e d to the drug group and the s a l i n e v e h i c l e to the s a l i n e group 1 hr before the c o n v u l s i v e s t i m u l a t i o n . The were 10 t o l e r a n c e development t r i a l s f o r the ph e n o b a r b i t a l and trimethadione t r e a t e d r a t s , and 21 f o r the clonazepam t r e a t e d r a t s . For clonazepam, the dose administered on the drug b a s e l i n e t e s t and the f i r s t 15 t o l e r a n c e development t r i a l s was 0.40 mg/kg, but the dose was lowered to 0.35 mg/kg f o r the l a s t s i x t r i a l s . T olerance T e s t . On the t o l e r a n c e t e s t , which occurred 48 hr a f t e r the l a s t t o l e r a n c e development t r i a l , each r a t of the drug group and the corresponding s a l i n e group r e c e i v e d the a p p r o p r i a t e drug 1 hr before the c o n v u l s i v e s t i m u l a t i o n so t h a t the degree of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of the drug c o u l d be compared between the drug groups and t h e i r r e s p e c t i v e s a l i n e c o n t r o l groups. The t e s t dose of clonazepam was 0.35 mg/kg. S t a t i s t i c a l A n a l y s i s . The s t a t i s t i c a l s i g n i f i c a n c e of between-subject d i f f e r e n c e s was evaluated with the Wilcoxon-Mann-Whitney U t e s t , and of w i t h i n - s u b j e c t d i f f e r e n c e s with the Sign 37 t e s t . For the a n a l y s i s of t o l e r a n c e to p h e n o b a r b i t a l and tri m e t h a d i o n e , the s i g n i f i c a n c e l e v e l was s e t a t p<.017, one-t a i l e d , because three comparisons were made; f o r clonazepam, i t was s e t a t p<.025, o n e - t a i l e d , because two comparisons were made. Re s u l t s As i l l u s t r a t e d i n the three panels of F i g u r e s 2, t o l e r a n c e developed to the a n t i c o n v u l s a n t e f f e c t s of p h e n o b a r b i t a l (Panel A), trimethadione (Panel B), and clonazepam (Panel C), r e s p e c t i v e l y . At the s t a r t of the experiment, the drug and the corresp o n d i n g s a l i n e groups d i d not d i f f e r i n e i t h e r t h e i r responsiveness to the c o n v u l s i v e s t i m u l a t i o n on the no-drug s a l i n e b a s e l i n e t r i a l or to t h e i r responsiveness to the a n t i c o n v u l s a n t e f f e c t of each drug on the drug b a s e l i n e t e s t , but t o l e r a n c e developed to each drug i n the drug group over the course of the t o l e r a n c e development t r i a l s . For p h e n o b a r b i t a l and tri m e t h a d i o n e , on the drug b a s e l i n e t e s t the drugs completely suppressed f o r e l i m b clonus i n almost every r a t i n the drug groups; whereas, on the t o l e r a n c e t e s t , a f t e r the 10 drug i n j e c t i o n s , i t had markedly l e s s of an a n t i c o n v u l s a n t e f f e c t (p<.001 f o r p h e n o b a r b i t a l ; p<.004 f o r t r i m e t h a d i o n e ) . In c o n t r a s t , the r a t s of the corresponding s a l i n e groups d i s p l a y e d no evidence of t o l e r a n c e whatsoever: each drug suppressed f o r e l i m b clonus on both the drug b a s e l i n e t e s t and the t o l e r a n c e t e s t (p>.017 f o r both p h e n o b a r b i t a l and t r i m e t h a d i o n e ) . A c c o r d i n g l y , the d u r a t i o n s of f o r e l i m b clonus d i s p l a y e d was much gre a t e r f o r the drug groups then the corresponding s a l i n e groups 38 F i g u r e 2. Tolerance to the a n t i c o n v u l s a n t e f f e c t s of ph e n o b a r b i t a l (30 mg/kg; Panel A ) , trimethadione (270 mg/kg; Panel B) and clonazepam (0.40 mg/kg on the f i r s t 15 t o l e r a n c e development t r i a l s and 0.35 mg/kg on the l a s t s i x ; Panel C) on amygdala-kindled c o n v u l s i o n s i n r a t s . For each of the three drugs, there were two groups of r a t s t h a t d i d not d i f f e r at the s t a r t of the experiment on the no-drug s a l i n e b a s e l i n e t r i a l (SB) or the drug b a s e l i n e t e s t (DB). On t o l e r a n c e development t r i a l , the drug group r e c e i v e d the drug and the s a l i n e group r e c e i v e d the s a l i n e v e h i c l e 1 hr before the c o n v u l s i v e s t i m u l a t i o n . On the t o l e r a n c e t e s t (T) when both groups r e c e i v e d the a p p r o p r i a t e drug 1 hr before the s t i m u l a t i o n , o n l y the drug group d i s p l a y e d t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of the drug. P A N E L A : T O L E R A N C E T O P H E N O B A R B I T A L 601 IT S B D B 1 2 3 4 5 6 7 8 9 10 T TOLERANCE DEVELOPMENT TRIALS P A N E L B : T O L E R A N C E T O T R I M E T H A D I O N E 60-j z 5<H ' 1 r ' 1 1 r r- 1 -, , • , SB DB 1 2 3 4 5 6 7 1 g 1 0 T TOLERANCE DEVELOPMENT TRIALS o Q> (0. 6O-1 50-P A N E L C : T O L E R A N C E T O C L O N A Z E P A M Saline group o r 3 40-Q (/) 3 . 3 O CD ~ 20 LU o 30 H < L J TOLERANCE DEVELOPMENT TRIALS 42 on the t o l e r a n c e t e s t (p<.0039 f o r p h e n o b a r b i t a l ; p<.0003 f o r tr i m e t h a d i o n e ) . For clonazepam, t o l e r a n c e developed to i t s a n t i c o n v u l s a n t e f f e c t i n the drug group over the 21 t r i a l s , but at a slow r a t e . On the t o l e r a n c e t e s t , the r a t s of the drug group d i s p l a y e d markedly more clonus than the r a t s of the s a l i n e group but i t j u s t missed s t a t i s t i c a l s i g n i f i c a n c e s e t at p<.025 (p=.027) . However, when the drug and s a l i n e groups were compared on the change i n d u r a t i o n of f o r e l i m b clonus t h a t occurred f o r each s u b j e c t between the drug b a s e l i n e t e s t and the t o l e r a n c e t e s t , there was a s i g n i f i c a n t l y g r e a t e r i n c r e a s e i n the clonus d u r a t i o n s of the drug group (p<.015) . T h i s type of comparison was used, i n s t e a d of a d i r e c t comparison between the drug b a s e l i n e t e s t and the t o l e r a n c e t e s t , because the dose of clonazepam used on these two t e s t s were d i f f e r e n t . D i s c u s s i o n Tolerance developed to the a n t i c o n v u l s a n t e f f e c t s of ph e n o b a r b i t a l , trimethadione, and clonazepam when the drug i n j e c t i o n s occurred before the c o n v u l s i v e s t i m u l a t i o n . T h i s demonstration of t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s was i n agreement with other s t u d i e s t h a t have demonstrated t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t s of p h e n o b a r b i t a l (e.g., F r e y & Kampmann, 1965; Schmidt, Kuperberg, Yonekawa, & Penry, 1980) trimethadione (e.g., Desalva 1956; Frey & Kretschmer, 1971), and clonazepam (e.g., Gent, F e e l y , & Haigh, 1985; s c h e r k l , Kurudi, & Frey, 1988; S c h e r k l , Scheuler, & Frey, 1985); but t h i s was the 43 f i r s t such demonstration with p h e n o b a r b i t a l and trimethadione on k i n d l e d c o n v u l s i o n s . Experiment IB The purpose of Experiment IB was to demonstrate t h a t the degree of c r o s s t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of carbamazepine was g r e a t e s t i n r a t s t o l e r a n t to the a n t i c o n v u l s a n t e f f e c t of p h e n o b a r b i t a l , o n l y s l i g h t i n those t o l e r a n t to clonazepam, and the l e a s t i n those t o l e r a n t to t r i m e t h a d i o n e . Only a s m a l l number of other s t u d i e s have examined c r o s s t o l e r a n c e between a n t i c o n v u l s a n t drug e f f e c t s (see Table 4), and t h i s was the f i r s t examination between these p a r t i c u l a r drugs Method Su b j e c t s . The s u b j e c t s i n Experiment IB were the same r a t s t h a t had completed Experiment 1A: the three drug groups remained i n t a c t (n=9 f o r p h e n o b a r b i t a l ; n=8 f o r both trimethadione and clonazepam), but the three s a l i n e groups were c o l l a p s e d i n t o one (n=23) because 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 among them. Cross-Tolerance Phase. The c r o s s - t o l e r a n c e phase began 48 hr a f t e r the t o l e r a n c e t e s t i n Experiment 1A. The s u b j e c t s of the three drug groups and the s a l i n e group r e c e i v e d carbamazepine (35 mg/kg; Gelgy Pharmaceuticals) 1 hr before each of 10 b i d a i l y c o n v u l s i v e s t i m u l a t i o n s . Carbamazepine r e q u i r e d s o n i f i c a t i o n to get i n t o suspension; and the i n j e c t i o n s were d e l i v e r e d i n the same volume and the same v e h i c l e as the i n j e c t i o n s of Experiment 1A. Cross t o l e r a n c e to carbamazepine was assessed i n two ways: 1) by the i n a b i l i t y of the f i r s t i n j e c t i o n of carbamazepine to 44 suppress f o r e l i m b clonus and 2) by the r a t e at which each r a t achieved a c r i t e r i o n of t o l e r a n c e to carbamazepine, which was s e t at f o r e l i m b clonus d u r a t i o n s of at l e a s t 50% of t h a t d i s p l a y e d on the no-drug s a l i n e b a s e l i n e t r i a l on two c o n s e c u t i v e c r o s s t o l e r a n c e t r i a l s . S t a t i s t i c a l A n a l y s i s . The K r u s k a l - W a l l i s one-way a n a l y s i s of va r i a n c e was used to compare the four groups, with the s i g n i f i c a n c e l e v e l was s e t a t p<.017, t w o - t a i l e d , because three comparisons were made. If the r e s u l t s were s t a t i s t i c a l l y s i g n i f i c a n t , post hoc m u l t i p l e comparisons were made between the i n d i v i d u a l groups; the s i g n i f i c a n c e l e v e l was s e t at p<.0083 because s i x comparisons were made among the four groups. The w i t h i n - s u b j e c t comparisons were performed u s i n g the Sign t e s t , with the s i g n i f i c a n c e l e v e l s e t a t p<.013, o n e - t a i l e d , because four comparisons were made. Re s u l t s F i g u r e 3 i l l u s t r a t e s t h a t the degree of c r o s s t o l e r a n c e to carbamazepine was g r e a t e s t with p h e n o b a r b i t a l , o n l y s l i g h t with clonazepam, and the l e a s t with t r i m e t h a d i o n e . S t a t i s t i c a l l y s i g n i f i c a n t t r a n s f e r of t o l e r a n c e was observed on l y p h e n o b a r b i t a l to carbamazepine. The r e s u l t s were s i m i l a r f o r both measures of cro s s t o l e r a n c e : the K r u s k a l - W a l l i s t e s t showed t h a t there were s i g n i f i c a n t d i f f e r e n c e s among the groups on the f i r s t c r o s s t o l e r a n c e t r i a l (L3<.001) and on the r a t e at which the c r i t e r i o n of c r o s s t o l e r a n c e was achieved (p<.001). M u l t i p l e comparisons re v e a l e d t h a t the r a t s of the p h e n o b a r b i t a l group d i s p l a y e d 45 F i g u r e 3. Cross t o l e r a n c e from the a n t i c o n v u l s a n t e f f e c t s of p h e n o b a r b i t a l , trimethadione or clonazepam to the a n t i c o n v u l s a n t e f f e c t of carbamazepine on amygdala-kindled c o n v u l s i o n s i n r a t s . Carbamazepine (35 mg/kg) was d e l i v e r e d 1 hr before the c o n v u l s i v e s t i m u l a t i o n i n r a t s t o l e r a n t to p h e n o b a r b i t a l , trimethadione or clonazepam and the s a l i n e c o n t r o l group on each of 10 c r o s s t o l e r a n c e t r i a l s . There was s i g n i f i c a n t t r a n s f e r from p h e n o b a r b i t a l to carbamazepine, but not from e i t h e r trImethadione or clonazepam to carbamazepine. C R O S S T O L E R A N C E T O C A R B A M A Z E P I N E ~1 " 1— 1— , —, , __ 2 3 4 5 6 7 8 9 To CROSS TOLERANCE TRIALS 47 s i g n i f i c a n t l y g r e a t e r t o l e r a n c e to carbamazepine than the r a t s of the s a l i n e group (p<.0083 on both measures), and i n f a c t were completely t o l e r a n t on the very f i r s t t r i a l . The clonazepam group d i s p l a y e d s l i g h t l y more cr o s s t o l e r a n c e than the s a l i n e group, although not to a s i g n i f i c a n t extent (p>.0083 on both measures). And i n t e r e s t i n g l y , the trimethadione group d i s p l a y e d l e s s f o r e l i m b clonus on the f i r s t t r i a l and took longer to reach the c r i t e r i o n of t o l e r a n c e than the s a l i n e group, although not to a s i g n i f i c a n t extent (p>.0083 on both measures). Moreover, the ph e n o b a r b i t a l group was s i g n i f i c a n t l y more t o l e r a n t to carbamazepine than the trimethadione group (p<.0083 on both measures). The mean number of t r i a l s to reach the c r i t e r i o n of cr o s s t o l e r a n c e f o r each group was M=2.0 for p h e n o b a r b i t a l , M=3.5 for clonazepam, M=7.5 f o r trimethadione and M=4.5 f o r s a l i n e . On the 10th and f i n a l c r o s s t o l e r a n c e t r i a l , the four groups 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 t h e i r degree of t o l e r a n c e to carbamazepine (p>.0167). The w i t h i n - s u b j e c t i n c r e a s e i n the degree of t o l e r a n c e to carbamazepine from the f i r s t to the l a s t (10th) carbamazepine a d m i n i s t r a t i o n was found to be s i g n i f i c a n t f o r the trimetha d i o n e (p<.008) and the s a l i n e (p<.002) groups, but not f o r the phe n o b a r b i t a l (p>.0125) and the clonazepam (p>.0125) groups. H i s t o l o g i c a l a n a l y s i s of the e l e c t r o d e placements r e v e a l e d t h a t a l l e l e c t r o d e t i p s were l o c a t e d i n the amygdala complex or near i t s boundaries (see F i g u r e 4 ) . 49 Discuss Ion This was the f i r s t examination of the t r a n s f e r of t o l e r a n c e from p h e n o b a r b i t a l , t r i m e t h a d i o n e , or clonazepam to carbamazepine. And the r e s u l t s c o n f i r m the hypothesis t h a t the t r a n s f e r of t o l e r a n c e would be g r e a t e s t from p h e n o b a r b i t a l to carbamazepine and the l e a s t from trimethadione to carbamazepine. This hypothesis was based on the s i m i l a r i t y between the drugs on the type of c l i n i c a l s e i z u r e s the drugs most e f f e c t i v e l y c o n t r o l l e d , the p u t a t i v e mechanism of a c t i o n , and the r e l a t i v e e f f i c a c y with which c o n v u l s i o n s induced by p e n t y l e n e t e t r a z o l and e l e c t r o s h o c k were c o n t r o l l e d . The present r e s u l t s were i n agreement with the s u g g e s t i o n t h a t a n t i c o n v u l s a n t drugs f a l l Into two g e n e r a l c a t e g o r i e s (see Vanden Bussche, De Beukelaar, & Wauquier, 1985; P o r t e r & P i t l i c k , 1987; R a i l & S c h l e i f e r , 1980). Type 1 drugs c o n t r o l p a r t i a l s e i z u r e s and g e n e r a l i z e d t o n i c - c l o n i c s e i z u r e s ; work by d e c r e a s i n g the movement of sodium and c a l c i u m ions a c r o s s membranes; are more e f f e c t i v e a g a i n s t maximal e l e c t r o s h o c k than p e n t y l e n e t e t r a z o l induced c o n v u l s i o n s ; and work by p r e v e n t i n g the spread of the s e i z u r e . The drugs of t h i s c l a s s i n c l u d e p h e n o b a r b i t a l and carbamazepine. Type 2 drugs c o n t r o l g e n e r a l i z e d s e i z u r e s , e s p e c i a l l y absence s e i z u r e s ; are more e f f e c t i v e a g a i n s t p e n t y l e n e t e t r a z o l then maximal e l e c t r o s h o c k induced c o n v u l s i o n s ; and work by i n c r e a s i n g the s e i z u r e t h r e s h o l d . The drugs of t h i s group i n c l u d e trimethadione and clonazepam. Although t h i s experiment was not designed to study the 50 development of t o l e r a n c e to carbamazepine, i t was examined, a l b e i t without a c o n t r o l group, by measuring the i n c r e a s e i n t o l e r a n c e from the f i r s t to the l a s t (10th) a d m i n i s t r a t i o n of carbamazepine i n the s a l i n e group. S i g n i f i c a n t i n c r e a s e s i n the degree of t o l e r a n c e to carbamazepine was seen; t h i s i s i n agreement with Mana et a l . (under review) which u n e q u i v o c a l l y demonstrated the development of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of carbamazepine on amygdala-kindled c o n v u l s i o n s of r a t s . 51 V. Experiment 2 The importance of the d r u g - e f f e c t contingency i n the development of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t s of ethanol ( P i n e l et a l . , 1983, 1985) and the a n t i e p i l e p t i c drugs, carbamazepine, diazepam, and v a l p r o a t e (Mana, Kim, P i n e l & Jones, under review) has r e c e n t l y been demonstrated. In each case, t o l e r a n c e developed r a p i d l y In r a t s t h a t r e c e i v e d the drug I n j e c t i o n before each b i d a i l y c o n v u l s i v e s t i m u l a t i o n but not at a l l i n those r a t s i n j e c t e d a f t e r the s t i m u l a t i o n — t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s o n l y developed when the c o n v u l s i v e s t i m u l a t i o n occurred i n the drugged s t a t e so t h a t the a n t i c o n v u l s a n t drug e f f e c t s were experienced by the s u b j e c t s . The importance of the d r u g - e f f e c t contingency i n the development of t o l e r a n c e has been demonstrated to numerous other drug e f f e c t s . I t s r o l e i n the development of c r o s s t o l e r a n c e i s s t i l l u n c e r t a i n (see Background f o r Experiments 1 and 2). A c c o r d i n g l y , the main purpose of Experiment 2 was to e s t a b l i s h without ambiguity t h a t the d r u g - e f f e c t contingency does p l a y a r o l e i n the development of c r o s s t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s . More s p e c i f i c a l l y , the purpose of Experiment 2A was to demonstrate t h a t the e x p r e s s i o n of the a n t i c o n v u l s a n t drug e f f e c t was important i n the t r a n s f e r of t o l e r a n c e from p e n t o b a r b i t a l to e t h a n o l ; and the purpose of Experiment 2B was to demonstrate t h i s t r a n s f e r of t o l e r a n c e from ethanol to p e n t o b a r b i t a l Experiment 2B. These experiments focussed on p e n t o b a r b i t a l and ethanol 52 because c r o s s t o l e r a n c e has been r e p o r t e d to many e f f e c t s of these drugs (Khanna & Mayer, 1982), although never to t h e i r a n t i c o n v u l s a n t e f f e c t , and because they had been used i n two previous s t u d i e s of co n t i n g e n t c r o s s t o l e r a n c e (Commissaris & Rech, 1981; Le, El-Ghundi, Khanna, & Kalant, 1986). General Method The experimental methodology of the two s t u d i e s of Experiment 2 was i d e n t i c a l except f o r the order i n which the drugs were presented. Drugs The drugs used were p e n t o b a r b i t a l (15 mg/kg; i n a sodium s a l t form; BDH Chemicals) and ethanol (1.5 g/kg i n a 25% v/v s o l u t i o n i n i s o t o n i c s a l i n e ) . The i n j e c t i o n v e h i c l e was i s o t o n i c s a l i n e and a l l drug and s a l i n e i n j e c t i o n s were d e l i v e r e d i n a volume of 7.5 ml/kg. T h i s high volume was r e q u i r e d to d i l u t e the c o n c e n t r a t i o n of ethanol because high ethanol c o n c e n t r a t i o n s can i r r i t a t e the p e r i t o n e a l c a v i t y l i n i n g . Drug B a s e l i n e Test The s u b j e c t s r e c e i v e d e i t h e r p e n t o b a r b i t a l i n Experiment 2A or ethanol i n Experiment 2B 1 hr before the c o n v u l s i v e s t i m u l a t i o n . Tolerance-Development Phase The tolerance-development phase comprised 10 b i d a i l y s t i m u l a t i o n s and 10 b i d a i l y i n j e c t i o n s of the same drug as on the drug b a s e l i n e t e s t . The r a t s were d i v i d e d i n t o two e q u i v a l e n t groups and on each t o l e r a n c e development t r i a l , the r a t s i n one 53 group (the drug-before group) r e c e i v e d the drug 1 hr before the c o n v u l s i v e s t i m u l a t i o n , and the r a t s i n the other group (the d r u g - a f t e r group) r e c e i v e d the drug 1 hr a f t e r the s t i m u l a t i o n . Tolerance Test The t o l e r a n c e t e s t occurred 48 hr a f t e r the l a s t (10th) t o l e r a n c e development t r i a l . Each r a t r e c e i v e d the appropriate, drug ( p e n t o b a r b i t a l i n Experiment 2A; ethanol i n Experiment 2B) 1 hr before the c o n v u l s i v e s t i m u l a t i o n so t h a t the development of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of the drug could be compared between the drug-before and d r u g - a f t e r groups. Cross-Tolerance Phase The c r o s s - t o l e r a n c e phase began 48 hr a f t e r the t o l e r a n c e t e s t . The s u b j e c t s r e c e i v e d ethanol i f they had p r e v i o u s l y r e c e i v e d p e n t o b a r b i t a l i n the tolerance-development phase (Experiment 2A) and p e n t o b a r b i t a l i f they had p r e v i o u s l y r e c e i v e d ethanol (Experiment 2B) 1 hr before each of 10 b i d a i l y c o n v u l s i v e s t i m u l a t i o n s . Contingent c r o s s t o l e r a n c e was assessed i n two ways: 1) by the i n a b i l i t y of the f i r s t i n j e c t i o n of the second drug to suppress f o r e l i m b clonus and 2) by the r a t e a t which the s u b j e c t s i n the two groups achieved a c r i t e r i o n of t o l e r a n c e to the second drug s e t at f o r e l i m b clonus d u r a t i o n s of a t l e a s t 50% of the no-drug s a l i n e b a s e l i n e t r i a l on two c o n s e c u t i v e t r i a l s . S t a t i s t i c a l A n a l y s i s The s t a t i s t i c a l s i g n i f i c a n c e of the r e s u l t s were evaluated with the Wilcoxon-Mann-Whitney U t e s t f o r between s u b j e c t comparisons and the Sign t e s t f o r w i t h i n s u b j e c t comparisons. 54 Both the t o l e r a n c e and c r o s s t o l e r a n c e data were analyzed a t p<.017, o n e - t a i l e d , because three comparisons were made. Experiment 2A The purposes of Experiment 2A were to demonstrate 1) t h a t t o l e r a n c e develops to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l ; 2) t h a t t h i s t o l e r a n c e occurs o n l y when the c o n v u l s i v e s t i m u l a t i o n was d e l i v e r e d i n the presence of the drug, and 3) that the t r a n s f e r of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol i s a l s o dependent on the s t i m u l a t i o n o c c u r r i n g i n the drugged s t a t e . Method E l e c t r o d e s were implanted i n 30 r a t s , but the s c r e e n i n g c r i t e r i a were not met by three r a t s on the no-drug s a l i n e b a s e l i n e t r i a l and one on the drug b a s e l i n e t e s t , and one other was dropped due to i l l n e s s . Thus, 25 r a t s completed the experiment: n=13 i n the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n group and n=12 i n the pentobarbital-a£ter-stimulation group. R e s u l t s The r e s u l t s of Experiment 2A are i l l u s t r a t e d i n F i g u r e 5, which summarize the r e s u l t s of the two phases of the experiment: the tolerance-development phase and the c r o s s - t o l e r a n c e phase. I t i s r e a d i l y apparent that the two groups d i d not d i f f e r at the s t a r t of the experiment In e i t h e r t h e i r responsiveness to the c o n v u l s i v e s t i m u l a t i o n on the no-drug s a l i n e b a s e l i n e t r i a l or to t h e i r responsiveness to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l on the drug b a s e l i n e t e s t . 55 F i g u r e 5. The e f f e c t of p e n t o b a r b i t a l (15 mg/kg) a d m i n i s t r a t i o n e i t h e r 1 hr before or 1 hr a f t e r b i d a i l y c o n v u l s i v e s t i m u l a t i o n s , and the subsequent ethanol (1.5 g/kg) a d m i n i s t r a t i o n 1 hr before the s t i m u l a t i o n s . There were no d i f f e r e n c e s a t the s t a r t of the experiment between the two groups i n e i t h e r t h e i r r esponsiveness to the c o n v u l s i v e s t i m u l a t i o n on the no-drug s a l i n e b a s e l i n e t r i a l (SB) or t h e i r responsiveness to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l on the drug b a s e l i n e t e s t (DB). During the tolerance-development phase, t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l developed, but o n l y In the p e n t o b a r b i t a l -b e f o r e - s t i m u l a t i o n c o n d i t i o n . During the c r o s s - t o l e r a n c e phase, a g r e a t e r degree of c r o s s t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol was apparent i n the r a t s t hat had p r e v i o u s l y been on a regimen of p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n treatments than those t h a t had been on p e n t o b a r b l t a l - a f t e r - s t i m u l a t i o n treatments. CONTINGENT CROSS TOLERANCE: PENTOBARBITAL TO ETHANOL < I 1 1 I I I I I I 1 1 I I T I I | | | | ) | S8 0 B 1 2 3 4 . 5 6 7 8 9 10 T 1 2 3 4 5 6 7 8 9 10 TOLERANCE DEVELOPMENT TRIALS CROSS TOLERANCE TRIALS 57 During the tolerance-development phase, t o l e r a n c e developed to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l i n the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n group. On the drug b a s e l i n e t e s t , p e n t o b a r b i t a l completely suppressed f o r e l i m b clonus i n every r a t i n t h i s group; whereas, on the t o l e r a n c e t e s t , a f t e r the 10 i n j e c t i o n s , i t had no a n t i c o n v u l s a n t e f f e c t whatsoever (p<.001). In c o n t r a s t , the r a t s of the p e n t o b a r b i t a l - a f t e r -s t i m u l a t l o n group d i s p l a y e d no evidence whatsoever of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l : p e n t o b a r b i t a l completely suppressed f o r e l i m b clonus on both the drug b a s e l i n e t e s t and the t o l e r a n c e t e s t (p>.017). A c c o r d i n g l y , although the r a t s of both groups r e c e i v e d the same number of p e n t o b a r b i t a l i n j e c t i o n s and c o n v u l s i v e s t i m u l a t i o n s , there was a marked d i f f e r e n c e between the d u r a t i o n s of f o r e l i m b clonus d i s p l a y e d by the two groups on the t o l e r a n c e t e s t (p_<.001). During the c r o s s - t o l e r a n c e phase, the r a t s t h a t had r e c e i v e d the p e n t o b a r b i t a l i n j e c t i o n before each s t i m u l a t i o n d u r i n g the tolerance-development phase were s i g n i f i c a n t l y more t o l e r a n t to the a n t i c o n v u l s a n t e f f e c t of ethanol than were those t h a t had r e c e i v e d the p e n t o b a r b i t a l i n j e c t i o n a f t e r each s t i m u l a t i o n . The f i r s t ethanol i n j e c t i o n was s i g n i f i c a n t l y l e s s e f f e c t i v e a t suppres s i n g f o r e l i m b clonus of the r a t s i n the p e n t o b a r b l t a l -b e f o r e - s t i m u l a t i o n group than of the p e n t o b a r b i t a l - a f t e r -s t i m u l a t i o n group (p<.01). Moreover, the p e n t o b a r b i t a l - b e f o r e -s t i m u l a t i o n s u b j e c t s reached the c r i t e r i o n of t o l e r a n c e a f t e r a mean of on l y M=2.7 ethanol i n j e c t i o n s compared to a mean of M=6.2 58 r e q u i r e d by the p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n s u b j e c t s (p<.001). Two of the r a t s i n the p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n group d i d not reach the c r i t e r i o n of t o l e r a n c e and were thus assigned a score of 10 (the t o t a l number of ethanol i n j e c t i o n s ) for the purposes of c a l c u l a t i n g the group means. On the 10th and f i n a l e thanol i n j e c t i o n t r i a l , 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 degree of t o l e r a n c e between the groups (p>.017). H i s t o l o g i c a l a n a l y s i s of the e l e c t r o d e placements r e v e a l e d t h a t a l l e l e c t r o d e t i p s were l o c a t e d i n the amygdaloid complex or near i t s boundaries (see F i g u r e 7). Experiment 2B The purpose of Experiment 2B was the converse of the purpose of Experiment 2A: to demonstrate the t r a n s f e r of co n t i n g e n t t o l e r a n c e o£ the a n t i c o n v u l s a n t e f f e c t of ethanol to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l . Methods E l e c t r o d e s were implanted i n 30 r a t s , but one r a t f a i l e d to meet the s c r e e n i n g c r i t e r i o n on the no-drug s a l i n e b a s e l i n e t r i a l , and another was excluded because of i l l n e s s . Thus, 28 r a t s completed the experiment, n=14 i n each of the e t h a n o l - b e f o r e -s t i m u l a t i o n and the e t h a n o l - a f t e r - s t i m u l a t i o n groups. R e s u l t s The r e s u l t s of Experiment 2B are I l l u s t r a t e d i n F i g u r e 6, which summarize the r e s u l t s of the tolerance-development phase and the c r o s s - t o l e r a n c e phase. I t can be seen t h a t the two groups 59 F i g u r e 6. The e f f e c t of ethanol (1.5 g/kg) a d m i n i s t r a t i o n e i t h e r before or a f t e r b i d a i l y c o n v u l s i v e s t i m u l a t i o n s , and the subsequent p e n t o b a r b i t a l (15 mg/kg) a d m i n i s t r a t i o n before the s t i m u l a t i o n s . There were no d i f f e r e n c e s at the s t a r t of the experiment between the two groups i n e i t h e r t h e i r responsiveness to the c o n v u l s i v e s t i m u l a t i o n on the no-drug s a l i n e b a s e l i n e t r i a l (SB) or t h e i r responsiveness to the a n t i c o n v u l s a n t e f f e c t of ethanol on the drug b a s e l i n e t e s t (DB), During the t o l e r a n c e -development phase, t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol developed, but o n l y i n the p e n t o b a r b i t a l - b e f o r e -s t i m u l a t i o n c o n d i t i o n . During the c r o s s - t o l e r a n c e phase, a g r e a t e r degree of c r o s s t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l was apparent i n the r a t s t h a t had p r e v i o u s l y been on a regimen of e t h a n o l - b e f o r e - s t i m u l a t i o n treatments than those t h a t had been on e t h a n o l - a f t e r - s t i m u l a t i o n treatments. CONTINGENT CROSS TOLERANCE: ETHANOL TO PENTOBARBITAL « ' 1 ' « 1 " 1 1 1 1 1 1 1 1 F 1 1 1 1 1 1 1 SB OB 1 2 3 4 5 6 7 8 9 10 T 1 2 3 4 5 6 7 8 9 10 TOLERANCE DEVELOPMENT TRIALS CROSS TOLERANCE TRIALS <r» o 61 F i g u r e 7 . E l e c t r o d e placements i n the 53 r a t s completing Experiment 2 . 62 d i d not d i f f e r a t the s t a r t of the experiment i n e i t h e r t h e i r responsiveness to the c o n v u l s i v e s t i m u l a t i o n on the no-drug b a s e l i n e t r i a l or t h e i r responsiveness to the a n t i c o n v u l s a n t e f f e c t of ethanol on the drug b a s e l i n e t e s t . During the tolerance-development phase, complete t o l e r a n c e developed to the a n t i c o n v u l s a n t e f f e c t of ethanol i n the e t h a n o l -b e f o r e - s t i m u l a t i o n group over the course of the 10 i n j e c t i o n s : on the drug b a s e l i n e t e s t , e thanol suppressed f o r e l i m b clonus i n every r a t , but on the t o l e r a n c e t e s t , i t had no a n t i c o n v u l s a n t e f f e c t (p<.001). In c o n t r a s t , the r a t s i n the e t h a n o l - a f t e r -s t i m u l a t i o n group d i s p l a y e d no evidence of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol on e i t h e r the drug b a s e l i n e t e s t or the t o l e r a n c e t e s t (p>.017). A c c o r d i n g l y , the e t h a n o l - b e f o r e -s t i m u l a t i o n group d i s p l a y e d s i g n i f i c a n t l y longer f o r e l i m b clonus d u r a t i o n s on the t o l e r a n c e t e s t than d i d the e t h a n o l - a f t e r -s t i m u l a t i o n group (p<.001). During the c r o s s - t o l e r a n c e phase, the r a t s t h a t had been exposed to ethanol before the s t i m u l a t i o n s on the t o l e r a n c e development phase were more t o l e r a n t to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l than those t h a t r e c e i v e d ethanol a f t e r the s t i m u l a t i o n s . Although on the f i r s t i n j e c t i o n of p e n t o b a r b i t a l , both groups d i s p l a y e d l i t t l e or no t o l e r a n c e to i t s a n t i c o n v u l s a n t e f f e c t (p>.017), the c r i t e r i o n of t o l e r a n c e was achieved by the r a t s of the e t h a n o l - b e f o r e - s t i m u l a t i o n group much f a s t e r (M=4.6 p e n t o b a r b i t a l I n j e c t i o n s ) than d i d the r a t s i n the e t h a n o l - a f t e r - s t i m u l a t i o n group (M=7.7 i n j e c t i o n s ) (p<.005). One 63 r a t i n the e t h a n o l - b e f o r e - s t i m u l a t i o n group and three i n the e t h a n o l - a f t e r - s t i m u l a t i o n group d i d not reach the c r i t e r i o n of p e n t o b a r b i t a l t o l e r a n c e , and thus they were assig n e d a score of 10, the t o t a l number of drug i n j e c t i o n s , f o r the purposes of c a l c u l a t i n g the group means. On the 10th and f i n a l t r i a l of p e n t o b a r b i t a l i n j e c t i o n s , the groups 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 t h e i r degree of t o l e r a n c e (p>.017). H i s t o l o g i c a l a n a l y s i s of the e l e c t r o d e placements r e v e a l e d t h a t a l l e l e c t r o d e t i p s were l o c a t e d i n the amygdaloid complex or near i t s boundaries (see F i g u r e 7). Discuss ion The two s t u d i e s of Experiment 2 accomplish s e v e r a l t h i n g s . F i r s t , i t confirms previous r e p o r t s t h a t t o l e r a n c e does develop to the a n t i c o n v u l s a n t e f f e c t of ethanol (e.g., P i n e l et a l . , 1983, 1985), and a l s o confirms previous r e p o r t s t h a t t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol developed o n l y when the c o n v u l s i v e s t i m u l a t i o n s was d e l i v e r e d d u r i n g the p e r i o d s of drug exposure ( P i n e l et a l . , 1983, 1985). Second, t h i s was the f i r s t demonstration of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l , and t h i s t o l e r a n c e was shown to develop o n l y when the c o n v u l s i v e s t i m u l a t i o n occurred d u r i n g the p e r i o d s of drug exposure. T h i r d , t h i s was the f i r s t experimental demonstration of c r o s s t o l e r a n c e between p e n t o b a r b i t a l and ethanol on t h e i r a n t i c o n v u l s a n t e f f e c t . And f o u r t h , the present r e s u l t s p rovide the f i r s t unambiguous and s y s t e m a t i c evidence of the r o l e of the 64 d r u g - e f f e c t contingency i n the t r a n s f e r of t o l e r a n c e from one drug to another. U n l i k e previous examinations of c o n t i n g e n t c r o s s t o l e r a n c e , the t r a n s f e r of c o n t i n g e n t t o l e r a n c e was shown to be b i d i r e c t i o n a l and to be c o n s i s t e n t with the r o l e of the drug-e f f e c t contingency i n the development of t o l e r a n c e to the f i r s t drug. Furthermore, the r e s u l t s of Experiment 2 r a i s e an important methodological p o i n t . In most s t u d i e s , c r o s s t o l e r a n c e has been assessed by only a s i n g l e t e s t t r i a l , and the f a i l u r e to observe s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s on t h i s t r i a l has l e d to the c o n c l u s i o n t h a t c r o s s t o l e r a n c e d i d not develop. In Experiment 2B, the groups 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 response to t h e i r f i r s t i n j e c t i o n of p e n t o b a r b i t a l , but there was c l e a r evidence of a subsequent d i f f e r e n t i a l r a t e of t o l e r a n c e development. A c c o r d i n g l y the r a t e of t o l e r a n c e development may be a more s e n s i t i v e measure of t o l e r a n c e than the response to a s i n g l e t e s t i n j e c t i o n ; and thus f a i l u r e to observe s i g n i f i c a n t t o l e r a n c e e f f e c t s on a s i n g l e t r i a l i s not unequivocal evidence t h a t t o l e r a n c e d i d not develop. VI. Experiment 3 The o b s e r v a t i o n of co n t i n g e n t c r o s s - t o l e r a n c e i n Experiment 2 con s i d e r e d i n combination with the rec e n t r e p o r t of Mana and P i n e l (1987) t h a t the d r u g - e f f e c t contingency a l s o p l a y s a major r o l e In the d i s s i p a t i o n of t o l e r a n c e suggested an i n t e r e s t i n g p o s s i b i l i t y . Remarkably, Mana and P i n e l found t h a t the d i s s i p a t i o n of t o l e r a n c e to ethanol was not found to be dependent on the withdrawal of e t h a n o l , but r a t h e r on the occurrence of the c o n v u l s i v e s t i m u l a t i o n i n the absence of e t h a n o l . Thus, the t o l e r a n c e d i s s i p a t e d i n r a t s given e t h a n o l i f i t was admin i s t e r e d a f t e r each of s i x c o n v u l s i v e s t i m u l a t i o n s , but maintained when etha n o l was given before each s t i m u l a t i o n . The main purpose of Experiment 3C was to demonstrate t h a t m a n i p u l a t i n g the d r u g - e f f e c t contingency d u r i n g a s e r i e s of p e n t o b a r b i t a l i n j e c t i o n s can Influence the d i s s i p a t i o n of a p r e v i o u s l y developed t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of e t h a n o l . But before t h i s was examined, the importance of the d r u g - e f f e c t contingency i n the development of t o l e r a n c e t o p e n t o b a r b i t a l and the t r a n s f e r of t h i s t o l e r a n c e to eth a n o l demonstrated i n Experiment 2 was confirmed. General Method Drugs The drugs used were p e n t o b a r b i t a l (20 mg/kg In Experiment 3A or 15 mg/kg i n Experiments 3B and 3C; BDH Chemicals) and ethanol (1.5 g/kg). A l l drug and s a l i n e i n j e c t i o n s were i n a 7.5 ml/kg 66 volume of i s o t o n i c s a l i n e . S t a t i s t i c a l A n a l y s i s i n each experiment, the s t a t i s t i c a l s i g n i f i c a n c e of the r e s u l t s were eva l u a t e d with the Wllcoxon-Mann-Whitney U t e s t f o r between s u b j e c t comparisons and the Sign t e s t f o r w i t h i n s u b j e c t comparisons: p<.017, o n e - t a i l e d , because three comparisons were made. Experiment 3A The purpose of Experiment 3A was to demonstrate c o n t i n g e n t t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l ; the dose of p e n t o b a r b i t a l chosen In t h i s experiment was higher than i n the p r e v i o u s demonstration of c o n t i n g e n t t o l e r a n c e i n Experiment 2A. Method S u b j e c t s . Of the 30 s u b j e c t s completing the k i n d l i n g phase of Experiment 3, one r a t was r e j e c t e d because I t d i d not meet the c r i t e r i o n on the no-drug s a l i n e b a s e l i n e t r i a l , t h ree because they d i s p l a y e d running f i t s , and two because of i l l n e s s or l o s s of e l e c t r o d e assembly. Thus, 24 r a t s completed Experiment 3A. Drug B a s e l i n e Phase. A l l the s u b j e c t s r e c e i v e d p e n t o b a r b i t a l (20 rag/kg) 1 hr before the c o n v u l s i v e s t i m u l a t i o n . Tolerance-Development Phase. The tolerance-development phase comprised of 10 b i d a i l y p e n t o b a r b i t a l i n j e c t i o n s and 10 b i d a i l y c o n v u l s i v e s t i m u l a t i o n s . The r a t s were d i v i d e d i n t o two e q u i v a l e n t groups, and on each t o l e r a n c e development t r i a l , the r a t s i n the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n group (n=12) r e c e i v e d the drug 1 hr before the s t i m u l a t i o n and the r a t s of the p e n t o b a r b l t a l - a f t e r - s t i m u l a t i o n group (n=12) r e c e i v e d the drug 1 hr a f t e r the s t i m u l a t i o n . Tolerance T e s t . The t o l e r a n c e t e s t occurred 48 hr a f t e r the l a s t t o l e r a n c e development t r i a l . Each r a t r e c e i v e d p e n t o b a r b i t a l 1 hr before the s t i m u l a t i o n to assess the degree of t o l e r a n c e i n the two groups. R e s u l t s I t Is c l e a r from F i g u r e 8 t h a t there was no evidence of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of 20 mg/kg of p e n t o b a r b i t a l i n e i t h e r experimental group. The a b i l i t y of 20 mg/kg of p e n t o b a r b i t a l to block k i n d l e d c o n v u l s i o n s was not s i g n i f i c a n t l y reduced by the 10 treatment I n j e c t i o n s i n e i t h e r the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n group (p>.017) or the p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n group (p>.017). A c c o r d i n g l y , the d i f f e r e n c e between the two groups on the t o l e r a n c e t e s t was not s i g n i f i c a n t (p>.017). Experiment 3B The purpose of Experiment 3A was to demonstrate c o n t i n g e n t t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l u s i n g a lower dose than that used i n Experiment 3A; and a l s o to demonstrate c o n t i n g e n t c r o s s t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of e t h a n o l . Method Su b j e c t s . The s u b j e c t s were the same r a t s t h a t completed Experiment 2A, but one was e l i m i n a t e d because i t d i s p l a y e d running f i t s . Thus, 23 r a t s completed Experiment 3B. 68 F i g u r e 8. The e f f e c t of p e n t o b a r b i t a l (20 mg/kg) a d m i n i s t r a t i o n e i t h e r 1 hr b e f o r e or 1 hr a f t e r the c o n v u l s i v e s t i m u l a t i o n . T o l e r a n c e was not a p p a r e n t a f t e r 10 b i d a i l y t r e a t m e n t i n j e c t i o n s i n e i t h e r the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n or the p e n t o b a r b i t a l - a f t e r - s t I m u l a t i o n group. CONTINGENT TOLERANCE TO PENTOBARBITAL 60i 1 ' ' —i 1 1 1— 1 1 , , , SB DB 1 2 3 4 5 6 7 8 9 i o T TOLERANCE DEVELOPMENT TRIALS 70 No-Drug B a s e l i n e Phase. Experiment 3B commenced 48 hr a f t e r the t o l e r a n c e t e s t i n Experiment 3A. I t began, as had Experiment 3A, with the four no-drug b a s e l i n e s t i m u l a t i o n s , and agai n the f o u r t h of these c o n s t i t u t e d the no-drug s a l i n e b a s e l i n e t r i a l . Drug B a s e l i n e T e s t . On the drug b a s e l i n e t e s t , a l l r a t s r e c e i v e d p e n t o b a r b i t a l (15 mg/kg) 1 hr before the c o n v u l s i v e s t i m u l a t i o n . Tolerance-Development Phase. T h i s was s i m i l a r to Experiment 3A except t h a t there were 15 t o l e r a n c e development t r i a l r a t h e r than 10 and a lower dose of p e n t o b a r b i t a l was used. The 23 r a t s were e q u a l l y (+1) d i s t r i b u t e d from the two groups of Experiment 3A to the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n (n=12) and the p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n (n=ll) groups. Tolerance T e s t . T h i s was s i m i l a r to Experiment 3A except t h a t the lower dose of p e n t o b a r b i t a l was used. Cross-Tolerance Phase. The c r o s s - t o l e r a n c e phase began 48 hr a f t e r the t o l e r a n c e t e s t . A l l s u b j e c t s r e c e i v e d ethanol 1 hr before each of 16 b i d a i l y c o n v u l s i v e s t i m u l a t i o n s . Contingent c r o s s t o l e r a n c e was assessed i n two ways: 1) by the i n a b i l i t y of the f i r s t i n j e c t i o n of ethanol to suppress f o r e l i m b clonus and 2) by the r a t e at which the s u b j e c t s i n the two groups achieved a c r i t e r i o n of t o l e r a n c e to ethanol s e t at f o r e l i m b clonus d u r a t i o n s of a t l e a s t 50% of the no-drug s a l i n e b a s e l i n e t r i a l on two c o n s e c u t i v e t r i a l s . R e s u l t s The r e s u l t s of Experiment 3A are I l l u s t r a t e d i n F i g u r e 9, 71 which summarize the r e s u l t s of the two phases of the experiment: the tolerance-development phase and the c r o s s - t o l e r a n c e phase. I t i s r e a d i l y apparent t h a t the two groups d i d not d i f f e r a t the s t a r t of the experiment i n e i t h e r t h e i r responsiveness to the c o n v u l s i v e s t i m u l a t i o n on the no-drug s a l i n e b a s e l i n e t r i a l or to t h e i r responsiveness to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l on the drug b a s e l i n e t e s t . During the tolerance-development phase, t o l e r a n c e developed to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l i n the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n group. On the drug b a s e l i n e t e s t , p e n t o b a r b i t a l completely suppressed f o r e l i m b clonus i n every r a t i n t h i s group; whereas, on the t o l e r a n c e t e s t i t had no a n t i c o n v u l s a n t e f f e c t whatsoever (p<.002). In c o n t r a s t , there was l i t t l e evidence of t o l e r a n c e i n the p e n t o b a r b i t a l - a f t e r -s t i m u l a t i o n c o n d i t i o n ; p e n t o b a r b i t a l completely suppressed f o r e l i m b clonus on the drug b a s e l i n e t e s t and i t suppressed i t again on the t o l e r a n c e t e s t , a l b e i t to a s l i g h t l y l e s s e r degree (p>.017). A c c o r d i n g l y , although the r a t s of both groups r e c e i v e d the same number of p e n t o b a r b i t a l i n j e c t i o n s and c o n v u l s i v e s t i m u l a t i o n s , there was a marked d i f f e r e n c e between the d u r a t i o n s of f o r e l i m b clonus d i s p l a y e d by the two groups on the t o l e r a n c e t e s t (p<.001). During the c r o s s - t o l e r a n c e phase, the r a t s t h a t had r e c e i v e d the p e n t o b a r b i t a l I n j e c t i o n before each s t i m u l a t i o n d u r i n g the tolerance-development phase were s i g n i f i c a n t l y more t o l e r a n t to the a n t i c o n v u l s a n t e f f e c t of ethanol than were those t h a t had 72 F i g u r e 9. The e f f e c t of p e n t o b a r b i t a l (15 mg/kg) a d m i n i s t r a t i o n e i t h e r 1 hr before or 1 hr a f t e r b i d a i l y c o n v u l s i v e s t i m u l a t i o n s , and the subsequent ethanol (1.5 g/kg) a d m i n i s t r a t i o n 1 hr before the s t i m u l a t i o n s . There were no d i f f e r e n c e s at the s t a r t of the experiment between the two groups In e i t h e r t h e i r r esponsiveness to the c o n v u l s i v e s t i m u l a t i o n on the no-drug s a l i n e b a s e l i n e t r i a l (SB) or t h e i r r esponsiveness to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l on the drug b a s e l i n e t e s t (DB). During the tolerance-development phase, t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l developed, but o n l y i n the p e n t o b a r b i t a l -b e f o r e - s t i m u l a t i o n c o n d i t i o n . During the c r o s s - t o l e r a n c e phase, a g r e a t e r degree of c r o s s t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol was apparent i n the r a t s t h a t had p r e v i o u s l y been on a regimen of p e n t o b a r b i t a l - b e f o r e - s t l m u l a t I o n treatments than those t h a t had been on p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n treatments. CONTINGENT CROSS TOLERANCE: PENTOBARBITAL TO ETHANOL 74 r e c e i v e d the p e n t o b a r b i t a l i n j e c t i o n a f t e r each s t i m u l a t i o n . The f i r s t ethanol i n j e c t i o n was s i g n i f i c a n t l y l e s s e f f e c t i v e a t supp r e s s i n g f o r e l i m b clonus of the r a t s i n the p e n t o b a r b i t a l -b e f o r e - s t i m u l a t i o n group than of the p e n t o b a r b i t a l - a f t e r -s t i m u l a t i o n group (p<.001). Moreover, the p e n t o b a r b i t a l - b e f o r e -s t i m u l a t i o n s u b j e c t s reached the c r i t e r i o n of t o l e r a n c e a f t e r a mean of onl y M=3.1 ethanol i n j e c t i o n s compared to a mean of M=8.5 r e q u i r e d by the p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n s u b j e c t s (p<.001). One r a t i n the p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n group d i d not reach the c r i t e r i o n of t o l e r a n c e and was thus assigned a score of 16 (the t o t a l number of ethanol i n j e c t i o n s ) f o r the purposes of c a l c u l a t i n g the group means. On the 16th and f i n a l e t hanol i n j e c t i o n t r i a l , there was 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 degree of t o l e r a n c e between the groups (p>.017) . D i s c u s s i o n The d r u g - e f f e c t contingency was shown to p l a y an important r o l e i n the development of t o l e r a n c e to p e n t o b a r b i t a l and c r o s s t o l e r a n c e to e t h a n o l ; t h i s confirms the r e s u l t s of Experiment 2A of t h i s t h e s i s . In view of the robust c o n t i n g e n t t o l e r a n c e e f f e c t s i n Experiment 3B as w e l l as i n Experiment 2A, i t s absence in Experiment 3A r e q u i r e s comment. I t i s p o s s i b l e t h a t p e n t o b a r b i t a l i n j e c t e d a t 15 mg/kg produces l a r g e t o l e r a n c e e f f e c t s t h a t are not produced at a l l by 20 mg/kg i n j e c t i o n s , but I favor the view t h a t t o l e r a n c e was de v e l o p i n g i n Experiment 3A but was not r e f l e c t e d i n a s i g n i f i c a n t i n c r e a s e i n clonus 75 d u r a t i o n because 20 mg/kg was w e l l above the t h r e s h o l d dose f o r complete clonus s u p p r e s s i o n . T h i s view i s supported by the o b s e r v a t i o n of P i n e l et a l . (1983) t h a t t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of l a r g e doses of eth a n o l was not apparent u n t i l both the ethanol and v e h i c l e c o n t r o l r a t s were c h a l l e n g e d by a sm a l l e r t e s t dose. Perhaps t o l e r a n c e would have e v e n t u a l l y been observed i n Experiment 3A, even a t the 20 mg/kg dose, i f more i n j e c t i o n s had been ad m i n i s t e r e d or i f a sm a l l e r t e s t dose was ad m i n i s t e r e d . The r e s u l t s emphasize the dangers of co n c l u d i n g , on the b a s i s of experiments i n v o l v i n g a l i m i t e d range of doses or a l i m i t e d number of i n j e c t i o n t r i a l s , t h a t t o l e r a n c e to a p a r t i c u l a r drug e f f e c t does not develop. Experiment 3C The purpose of Experiment 3C was to examine the r o l e of the d r u g - e f f e c t contingency i n the d i s s i p a t i o n of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol f o l l o w i n g p e n t o b a r b i t a l a d m i n i s t r a t i o n s . I t was hypothesized t h a t t o l e r a n c e to ethanol would d i s s i p a t e when p e n t o b a r b i t a l i s ad m i n i s t e r e d a f t e r the s t i m u l a t i o n , but not d i s s i p a t e when p e n t o b a r b i t a l i s given before the s t i m u l a t i o n . Method Su b j e c t s . The 22 s u b j e c t s completing Experiment 3B t h a t had achieved the c r i t e r i o n of t o l e r a n c e — t w o c o n s e c u t i v e c o n v u l s i o n s of f o r e l i m b clonus d u r a t i o n s of a t l e a s t S0% of t h a t achieved on the no-drug s a l i n e b a s e l i n e t r i a l - - w e r e r e a s s i g n e d to two new groups so as to d i s t r i b u t e s u b j e c t s with d i f f e r i n g experimental 76 h i s t o r i e s e q u a l l y (±1) and to equate the two new groups f o r t o l e r a n c e to ethanol on the l a s t c r o s s t o l e r a n c e t r i a l i n Experiment 3B. One of these 22 r a t s became i l l d u r i n g the course of t h i s experiment; i t s data were not a n a l y z e d . D i s s i p a t i o n Phase. T h i s phase began 48 hr a f t e r the l a s t c r o s s t o l e r a n c e t r i a l of Experiment 3B; i t comprised s i x b i d a i l y p e n t o b a r b i t a l i n j e c t i o n s and s i x b i d a i l y c o n v u l s i v e s t i m u l a t i o n s . The s u b j e c t s i n the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n group (n=10) r e c e i v e d each i n j e c t i o n 1 hr before the s t i m u l a t i o n and the r a t s of the p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n group r e c e i v e d the drug 1 hr a f t e r each s t i m u l a t i o n . Tolerance T e s t . On the t o l e r a n c e t e s t , 48 hr a f t e r the s i x t h p e n t o b a r b i t a l treatment i n j e c t i o n , a l l s u b j e c t s r e c e i v e d e t h a n o l 1 hr before the c o n v u l s i v e s t i m u l a t i o n . R e s u l t s The r e s u l t s of Experiment 3C, summarized i n F i g u r e 10, were as p r e d i c t e d . The p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n regimen was a s s o c i a t e d with a s i g n i f i c a n t d e c l i n e i n the d u r a t i o n of f o r e l i m b clonus between the l a s t t r i a l of the ethanol i n j e c t i o n i n Experiment 3B and the ethanol t e s t i n j e c t i o n f o l l o w i n g the s i x p e n t o b a r b i t a l i n j e c t i o n s i n Experiment 3C (p>.001), whereas the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n regimen was not (p>.017). A c c o r d i n g l y , the d u r a t i o n of the f o r e l i m b clonus e l i c i t e d i n the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t l o n group on the e t h a n o l t o l e r a n c e t e s t was s i g n i f i c a n t l y g r e a t e r than t h a t e l i c i t e d i n the p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n group (p<.005). 77 F i g u r e 10. The d i s s i p a t i o n of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of ethanol a f t e r p e n t o b a r b i t a l i n j e c t i o n s e i t h e r 1 hr before or 1 hr a f t e r the c o n v u l s i v e s t i m u l a t i o n s . The f i r s t p o i n t s (XT) on the graph r e p r e s e n t the i n i t i a l l y equal degree of t o l e r a n c e to ethanol t h a t the two groups d i s p l a y e d on the l a s t s e s s i o n of Experiment 3B; the l a s t p o i n t s (T) r e p r e s e n t the responses of the two groups on the f i n a l e t h a n o l t o l e r a n c e t e s t a f t e r the i n t e r v e n i n g p e n t o b a r b i t a l treatments. The p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n treatments produced a s i g n i f i c a n t d e c l i n e i n ethanol t o l e r a n c e , but the p e n t o b a r b i t a l - b e f o r e -s t i mu 1 a t i o n t r e a t me n t s d i d n o t . DISSIPATION OF ETHANOL TOLERANCE O or Q CO Z Q < Ld 60 n S O - L 40 H 30 H O CQ ^ 20-_ J LJ or Q Penfobarbital-after-stimulation group Pentobarbital-before-stimulation group • XT 2 3 4 5 PENTOBARBITAL TRIALS —i T H i s t o l o g i c a l a n a l y s i s of the e l e c t r o d e placements r e v e a l e d t h a t a l l e l e c t r o d e t i p s were l o c a t e d i n the amygdaloid complex or near i t s boundaries (see F i g u r e 11). Discuss ion The r e s u l t s of Experiment 3C demonstrate t h a t t o l e r a n c e t h a t had been e s t a b l i s h e d to ethanol d i s s i p a t e d f o l l o w i n g a s e r i e s of p e n t o b a r b i t a l i n j e c t i o n s when they occurred a f t e r c o n v u l s i v e s t i m u l a t i o n s (contingent c r o s s - d i s s i p a t i o n of t o l e r a n c e ) ; however, t o l e r a n c e d i d not d i s s i p a t e when the p e n t o b a r b i t a l i n j e c t i o n s occurred before the c o n v u l s i v e s t i m u l a t i o n . However, Experiment 3 does not d i f f e r e n t i a t e between the two p o s s i b l e i n t e r p r e t a t i o n s of the e f f e c t . One p o s s i b i l i t y i s t h a t the r a t s i n the p e n t o b a r b i t a l - b e f o r e - s t i m u l a t i o n group r e t a i n e d the t o l e r a n c e to ethanol t h a t they had developed i n Experiment 2, whereas the r a t s i n the p e n t o b a r b i t a l - a f t e r - s t i m u l a t i o n group l o s t t h e i r s because they were s t i m u l a t e d i n a dr u g - f r e e s t a t e d u r i n g the schedule of p e n t o b a r b i t a l i n j e c t i o n s . A second p o s s i b i l i t y i s that both groups of r a t s l o s t t h e i r t o l e r a n c e to ethanol's a n t i c o n v u l s a n t e f f e c t because they were a l l s t i m u l a t e d i n the absence of e t h a n o l , while the r a t s i n the p e n t o b a r b l t a l -b e f o r e - s t i m u l a t i o n group developed c o n t i n g e n t t o l e r a n c e to p e n t o b a r b i t a l that was manifested i n the development of conti n g e n t c r o s s - t o l e r a n c e to e t h a n o l . V I I . Experiment 4 81 A l a r g e body of evidence suggests t h a t t o l e r a n c e t o a drug's e f f e c t develops to a gr e a t e r extent and at a f a s t e r r a t e when l a r g e r treatment doses are administered (Aston, 1965; Jorgenson, Fasmer, & Hole, 1986; K a l a n t , LeBlanc, & Gibbons, 1971; LeBlanc, Kalant, Gibbons, & Berman, 1969) . The purpose of Experiment 4 was to demonstrate t h a t under c e r t a i n c o n d i t i o n s , g r e a t e r t o l e r a n c e would develop i n s u b j e c t s exposed to a lower dose a d m i n i s t r a t i o n regimen. A c c o r d i n g l y , d i f f e r e n t dose regimens were compared i n the extent and r a t e of t o l e r a n c e development to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l . The main comparisons were made between two dose regimens: one i n which the s u b j e c t s were maintained on a r e l a t i v e l y high dose of the drug t h a t completely suppressed f o r e l i m b clonus on every t r i a l f o r the d u r a t i o n of the experiment; and one i n which the s u b j e c t s were i n i t i a l l y g i v e n a r e l a t i v e l y low dose, t h a t d i d not completely suppress the c o n v u l s i o n s , but had the dose r e p e a t e d l y i n c r e a s e d by s m a l l amounts, none of which suppressed f o r e l i m b c l o n u s , as t o l e r a n c e developed to the dose. I t was hypothesized t h a t there would be a gre a t e r degree of t o l e r a n c e , as measured by whether f o r e l i m b clonus was suppressed or not, i n the l a t t e r group, d e s p i t e the f a c t t h a t t h i s group r e c e i v e d a lower dose regimen, because f o r e l i m b clonus was experienced i n the drugged s t a t e , compared to the former group i n which f o r e l i m b clonus was not experienced i n the drugged s t a t e . 82 Method Subjects Subjects In t h i s experiment had been p r e v i o u s l y exposed to a n t i c o n v u l s a n t drugs i n p i l o t s t u d i e s . In order to erase any t o l e r a n c e t h a t may have developed to the drug e f f e c t s , each r a t r e c e i v e d 17 b i d a i l y c o n v u l s i v e s t i m u l a t i o n s i n the absence of any drug. The a d m i n i s t r a t i o n of c o n v u l s i v e s t i m u l a t i o n s i n the absence of an a n t i c o n v u l s a n t drug causes the d i s s i p a t i o n of t o l e r a n c e to t h a t drug e f f e c t (Experiment 3C of t h i s t h e s i s ; Mana & P i n e l , 1987) . No-drug S a l i n e B a s e l i n e T r i a l On the 17th and f i n a l s t i m u l a t i o n t r i a l , a l l r a t s r e c e i v e d the s a l i n e v e h i c l e (5 ml/kg) 1 hr before the c o n v u l s i v e s t i m u l a t i o n to assess the e f f e c t of the s t i m u l a t i o n i n the absence of any drug. Drug-Baseline Test A l l the r a t s r e c e i v e d p e n t o b a r b i t a l (30 mg/kg i n a 5 ml/kg volume) 1 hr before the c o n v u l s i v e s t i m u l a t i o n to assess the a n t i c o n v u l s a n t potency of the drug i n each r a t . In a d d i t i o n to measuring the a n t i c o n v u l s a n t e f f e c t of the drug, the a t a x i a demonstrated by the r a t s In response to the drug was a l s o measured i n two ways: 1) the r i g h t i n g t e s t i n which the r a t was placed on i t s back on a S a n - i - c e l bedding covered s u r f a c e , and the l a t e n c y to r i g h t I t s e l f onto a l l fours was measured; 2) the tube t e s t In which the r a t was placed head f i r s t i n t o a 15 x 6 cm p l a s t i c tube placed at a 50 degree angle from the v e r t i c a l , and 83 the l a t e n c y to crawl out was measured. The r i g h t i n g and tube t e s t s were conducted 50 and 55 min, a f t e r the p e n t o b a r b i t a l i n j e c t i o n , r e s p e c t i v e l y . On each t e s t , the r a t was allowed 5 min to perform the task; r a t s t h a t f a i l e d to perform the task w i t h i n the time l i m i t were given a l a t e n c y score of 5 min. Tolerance-Development Phase The r a t s were d i v i d e d i n t o three e q u i v a l e n t groups based on the mean d u r a t i o n of f o r e l i m b clonus expressed on the no-drug s a l i n e b a s e l i n e t r i a l and the drug b a s e l i n e t e s t , the r i g h t i n g and tube t e s t scores on the drug b a s e l i n e t e s t , drug h i s t o r i e s , and body weights. A l l r a t s r e c e i v e d p e n t o b a r b i t a l 1 hr before the c o n v u l s i v e s t i m u l a t i o n on each of 20 b i d a i l y t r i a l s . One group r e c e i v e d a high dose of 50 mg/kg (50 group) throughout; t h i s dose completely suppressed f o r e l i m b clonus i n every s u b j e c t on each t r i a l . Another group r e c e i v e d a low dose of 10 mg/kg (10 group) throughout; t h i s dose d i d not completely suppress f o r e l i m b clonus i n most s u b j e c t s on most t r i a l s The t h i r d group ( I n c r e a s i n g group) r e c e i v e d 10 mg/kg, th a t d i d not suppress f o r e l i m b c l o n u s , and then the dose was i n c r e a s e d by 1 mg/kg f o r 13 t r i a l s , at which p o i n t f o r e l i m b clonus became suppressed i n over 50% of the s u b j e c t s . The r a t s of t h i s group were then maintained a t t h i s dose (23 mg/kg) f o r the remaining s i x t o l e r a n c e development t r i a l s . Tolerance Test The t o l e r a n c e t e s t occurred 48 hr a f t e r the l a s t (20th) t o l e r a n c e development t r i a l . I t i n v o l v e d 10 b i d a i l y t r i a l s of 84 p e n t o b a r b i t a l (20 mg/kg) a d m i n i s t r a t i o n s , each followed 1 hr l a t e r by the c o n v u l s i v e s t i m u l a t i o n . Tolerance was assessed i n two ways: 1) by the i n a b i l i t y of the drug to suppress f o r e l i m b clonus on the f i r s t t e s t t r i a l and 2) by the r a t e a t which a c r i t e r i o n of t o l e r a n c e of two co n s e c u t i v e t r i a l s of f o r e l i m b clonus e x p r e s s i o n was achi e v e d . In a d d i t i o n , both a t a x i a t e s t s were admi n i s t e r e d on the f i r s t t o l e r a n c e t r i a l i n the same manner as on the drug b a s e l i n e t e s t : a t 50 and 55 min f o l l o w i n g the t e s t dose . S t a t i s t i c a l A n a l y s i s Comparisons of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l among the three experimental groups were made with the Chi Square t e s t f o r nominal data (whether f o r e l i m b clonus occurred or n o t ) , and the K r u s k a l - W a l l i s one-way a n a l y s i s of va r i a n c e f o r non-nominal data; the l e v e l of s i g n i f i c a n c e was s e t at p<.017, because three comparisons were made. The post hoc comparisons of the nominal data were made us i n g two group Chi Square t e s t s with p<.017. For the e f f e c t of p e n t o b a r b i t a l on the a t a x i a measures, the parametric analyses of v a r i a n c e was used because the data were parametric; the l e v e l of s i g n i f i c a n c e was set at p<.025 because two comparisons were made. Re s u l t s F i g u r e 12 i l l u s t r a t e s t h a t the development of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l was s i g n i f i c a n t l y g r e a t e r i n the group that r e c e i v e d the i n c r e a s i n g l y higher doses of the drug ( I n c r e a s i n g group) as compared to the group t h a t 85 F i g u r e 12. The e f f e c t of d i f f e r e n t dose a d m i n i s t r a t i o n regimens of p e n t o b a r b i t a l on the development of t o l e r a n c e t o i t s a n t i c o n v u l s a n t e f f e c t . The group t h a t r e c e i v e d s u c c e s s i v e l y l a r g e r doses of the drug ( I n c r e a s i n g - g r o u p ) d i s p l a y e d s i g n i f i c a n t l y g r e a t e r t o l e r a n c e t h a n the group t h a t was m a i n t a i n e d on the h i g h dose t h r o u g h o u t ( 5 0 - g r o u p ) , but d i d not d i f f e r s i g n i f i c a n t l y from the group t h a t r e c e i v e d the low dose t h r o u g h o u t ( 1 0 - g r o u p ) ; and the 10- and 50-groups d i d not s i g n i f i c a n t l y d i f f e r . TOLERANCE TO PENTOBARBITAL 87 r e c e i v e d the high dose (50 mg/kg) of p e n t o b a r b i t a l throughout the tolerance-development phase. On the f i r s t t o l e r a n c e t e s t t r i a l , there were s i g n i f i c a n t d i f f e r e n c e s among the groups (p<.01); and post hoc comparisons r e v e a l e d t h a t the I n c r e a s i n g group expressed g r e a t e r t o l e r a n c e than the 50 group; however there was no s i g n i f i c a n t d i f f e r e n c e i n the degree of t o l e r a n c e between the I n c r e a s i n g group and the 10 group, or the 10 group and the 50 group. On the r a t e a t which the c r i t e r i o n of t o l e r a n c e was reached, there was a trend f o r the Increasing-group to become t o l e r a n t more q u i c k l y than the other groups but i t was not s t a t i s t i c a l l y s i g n i f i c a n t (p>.017): the Increasing-group reached i t In M=2.3 t r i a l s , the 10-group i n 4.9 t r i a l s , and the 50-group i n 5.4 t r i a l s . On the l a s t (10th) t o l e r a n c e t e s t t r i a l , there were no s i g n i f i c a n c e d i f f e r e n c e s among the groups i n the degree of t o l e r a n c e to p e n t o b a r b i t a l ' s a n t i c o n v u l s a n t e f f e c t (p>.017). F i g u r e s 13 and 14 i l l u s t r a t e t h a t the groups 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 the degree of a t a x i a produced by p e n t o b a r b i t a l on the f i r s t t o l e r a n c e t e s t t r i a l , as measured by both the r i g h t i n g t e s t (p>.025) and the tube t e s t (p>.025), r e s p e c t i v e l y . H i s t o l o g i c a l a n a l y s i s of the e l e c t r o d e placements r e v e a l e d t h a t a l l e l e c t r o d e t i p s were l o c a t e d i n the amygdaloid complex or near i t s boundaries (see F i g u r e 15). Discuss ion The r e s u l t s of t h i s study confirmed the hypothesis that t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l would be g r e a t e r i n those r a t s t h a t r e c e i v e d s u c c e s s i v e l y l a r g e r doses of 88 F i g u r e 13. The e f f e c t of the d i f f e r e n t dose a d m i n i s t r a t i o n regimens of p e n t o b a r b i t a l on i t s e f f e c t on t h e r i g h t i n g t e s t . The groups t h a t r e c e i v e d e i t h e r the p r o g r e s s i v e l y i n c r e a s i n g dose ( i n c r e a s i n g - g r o u p ) , the low (10-group) or the h i g h (50-group) dose of p e n t o b a r b i t a l d i d not d i f f e r s i g n i f i c a n t l y amongst themselves on e i t h e r the drug b a s e l i n e t e s t or t h e t o l e r a n c e t e s t . R I G H T I N G T E S T DRUG BASELINE TOLERANCE TEST oo 90 F i g u r e 14. The e f f e c t of the d i f f e r e n t dose a d m i n i s t r a t i o n regimens of p e n t o b a r b i t a l on i t s e f f e c t on the tube t e s t . The groups t h a t r e c e i v e d e i t h e r the p r o g r e s s i v e l y i n c r e a s i n g dose ( I n c r e a s i n g - g r o u p ) , the low (10-group) or the high (50-group) dose of p e n t o b a r b i t a l d i d not d i f f e r s i g n i f i c a n t l y amongst themselves on e i t h e r the drug b a s e l i n e t e s t or the t o l e r a n c e t e s t . T U B E T E S T 93 the drug, none of which were l a r g e enough to suppress f o r e l i m b c l o n u s , than those r a t s t h a t were maintained on a high dose of the drug that suppressed f o r e l i m b clonus throughout. The gr e a t e r t o l e r a n c e i n the I n c r e a s i n g group could be a t t r i b u t e d to the f a c t t h a t c o n v u l s i o n s were experienced i n the drugged s t a t e . The group t h a t r e c e i v e d the high dose of the drug, never experienced f o r e l i m b clonus i n the drugged s t a t e . T h i s r e s u l t was i n c o n t r a s t to the g e n e r a l l y accepted view of drug t o l e r a n c e . For example, Aston (1965) found t h a t the degree of t o l e r a n c e to s l e e p i n g time of a 40 mg/kg t e s t dose of p e n t o b a r b i t a l was d i r e c t l y p r o p o r t i o n a l to the s i z e of the treatment dose of p e n t o b a r b i t a l t h a t ranged from 25 to 45 mg/kg. A problem with s t u d i e s t h a t examine the r a t e and extent of t o l e r a n c e development between v a r i o u s doses of a drug i s t h a t the d e t e c t i o n of t o l e r a n c e i n the s u b j e c t s t h a t r e c e i v e the high treatment dose may be masked by the in c r e a s e d drug accumulation i n the s u b j e c t ' s body or because the high dose r e s u l t e d i n some n o n - s p e c i f i c change i n the s u b j e c t t h a t i n t e r f e r e s with, the assessment of t o l e r a n c e (Kalant et a l . , 1971). However, t h i s was not a problem i n t h i s study because, although t o l e r a n c e was gre a t e r i n the I n c r e a s i n g group to the a n t i c o n v u l s a n t e f f e c t , the same tr e n d was not seen with the a t a x i a measure. I f drug accumulation were to account f o r the l e s s observed t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l i n the higher dose c o n d i t i o n , one would a l s o have expected the drug accumulation to produce the most a t a x i a i n the higher dose c o n d i t i o n , but t h i s was no found. 95 V I I I . General D i s c u s s i o n The g e n e r a l purpose o£ the t h e s i s was to determine the r o l e of d r u g - e f f e c t s as opposed to mere drug-exposure i n the development of c r o s s t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s . The experiments of t h i s t h e s i s provide unequivocal evidence of the importance of the d r u g - e f f e c t contingency i n c r o s s t o l e r a n c e . A c c o r d i n g l y , the General D i s c u s s i o n of t h i s t h e s i s i s d i v i d e d i n t o s i x s e c t i o n s . S e c t i o n 1 d e a l s with the f i r s t two experiments, which examined c r o s s t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s with and without the d r u g - e f f e c t contingency present. S e c t i o n 2 d e a l s with the t h i r d experiment which examined the r o l e of the d r u g - e f f e c t contingency i n the d i s s i p a t i o n of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of one drug f o l l o w i n g exposure to another drug. S e c t i o n 3 d e a l s with the f o u r t h experiment, which demonstrated t h a t the degree of t o l e r a n c e to the a n t i c o n v u l s a n t drug e f f e c t was i n f l u e n c e d by the drug a d m i n i s t r a t i o n regimen. S e c t i o n s 4 and 5 present t h e o r e t i c a l and c l i n i c a l I m p l i c a t i o n s of the present r e s e a r c h , r e s p e c t i v e l y . And the l a s t s e c t i o n c o n s i d e r s f u t u r e d i r e c t i o n s of t h i s r e s e a r c h . 1. General D i s c u s s i o n of Experiments 1 and 2 The purpose of Experiments 1 and 2 was to determine the r o l e of the d r u g - e f f e c t contingency i n the development of c r o s s t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s . The present r e s u l t s provide the f i r s t unambiguous and s y s t e m a t i c evidence of the r o l e of the d r u g - e f f e c t contingency i n the t r a n s f e r of t o l e r a n c e from 96 one drug to another. In Experiment 1, t o l e r a n c e and c r o s s t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s were examined with the d r u g - e f f e c t contingency present; the drug i n j e c t i o n s always preceded the c o n v u l s i v e s t i m u l a t i o n s . Tolerance developed to the a n t i c o n v u l s a n t e f f e c t s of p e n t o b a r b i t a l , t r i m e t h a d i o n e , and clonazepam; s i g n i f i c a n t t r a n s f e r of t o l e r a n c e occurred from p e n t o b a r b i t a l to clonazepam but not from e i t h e r trimethadione or clonazepam to carbamazepine. The r e s u l t s confirmed the hypothesis t h a t degree of c r o s s t o l e r a n c e depended on the s i m i l a r i t y between the drugs on three f a c t o r s : the type of c l i n i c a l s e i z u r e s they were most e f f e c t i v e at c o n t r o l l i n g , t h e i r p u t a t i v e mechanism of a c t i o n , and t h e i r r e l a t i v e e f f e c t i v e n e s s a t s u p p r e s s i n g experimental c o n v u l s i o n s induced by e l e c t r o s h o c k and p e n t y l e n e t e t r a z o l . In Experiment 2, t o l e r a n c e and c r o s s t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s were examined with and without the d r u g - e f f e c t contingency present; i n one c o n d i t i o n a drug i n j e c t i o n preceded each c o n v u l s i v e s t i m u l a t i o n , whereas i n another c o n d i t i o n a drug i n j e c t i o n f o l l o wed each s t i m u l a t i o n . When the contingency was present, t o l e r a n c e developed q u i c k l y to p e n t o b a r b i t a l and e t h a n o l , and b i d i r e c t i o n a l c r o s s t o l e r a n c e a l s o developed between these drugs. In c o n t r a s t , i n the absence of the d r u g - e f f e c t contingency, there was l i t t l e evidence of t o l e r a n c e or c r o s s t o l e r a n c e . These r e s u l t s were confirmed i n Experiment 3B, which demonstrated the t r a n s f e r of t o l e r a n c e to 97 a n t i c o n v u l s a n t drug e f f e c t s from p e n t o b a r b i t a l to e t h a n o l . A c c o r d i n g l y , u n l i k e the r e s u l t s of previous examinations of conti n g e n t c r o s s t o l e r a n c e , the t r a n s f e r of c o n t i n g e n t t o l e r a n c e was shown to be b i d i r e c t i o n a l and to be c o n s i s t e n t with the r o l e of the d r u g - e f f e c t contingency i n the development of c r o s s t o l e r a n c e to the f i r s t drug. 2. General D i s c u s s i o n of Experiment 3 The main purpose of Experiment 3 was to examine the r o l e of the d r u g - e f f e c t contingency i n the d i s s i p a t i o n of t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of a drug f o l l o w i n g the a d m i n i s t r a t i o n of another drug. The present r e s u l t s provide the f i r s t evidence t h a t the d r u g - e f f e c t contingency was important i n the d i s s i p a t i o n of t o l e r a n c e to one drug f o l l o w i n g the a d m i n i s t r a t i o n of another. In Experiment 3C, i t was found t h a t t o l e r a n c e t h a t had been e s t a b l i s h e d to ethanol d i s s i p a t e d f o l l o w i n g a s e r i e s of p e n t o b a r b i t a l i n j e c t i o n s when they occurred a f t e r the c o n v u l s i v e s t i m u l a t i o n (contingent c r o s s - d i s s i p a t i o n of t o l e r a n c e ) ; however, t o l e r a n c e d i d not d i s s i p a t e when the p e n t o b a r b i t a l i n j e c t i o n s occurred before the c o n v u l s i v e s t i m u l a t i o n . The t o l e r a n c e to ethanol d i s s i p a t e d when the c o n v u l s i v e s t i m u l a t i o n o c c u r r e d i n the absence of p e n t o b a r b i t a l , d e s p i t e the a d m i n i s t r a t i o n s of the drug; but t o l e r a n c e d i d not d i s s i p a t e when the s t i m u l a t i o n occurred i n the drugged s t a t e . The key f a c t o r i n the d i s s i p a t i o n of ethanol t o l e r a n c e was the a d m i n i s t r a t i o n of the c o n v u l s i v e s t i m u l a t i o n i n the absence of p e n t o b a r b i t a l . These r e s u l t s c o n f i r m and extend the f i n d i n g of Mana and P i n e l (1986) t h a t 98 t o l e r a n c e to one drug (ethanol) d i s s i p a t e d when the c o n v u l s i v e s t i m u l a t i o n s were d e l i v e r e d i n the absence of the same drug. 3. General D i s c u s s i o n of Experiment 4 The purpose of Experiment 4 was to determine i f d i f f e r e n t dose a d m i n i s t r a t i o n regimens of a drug d i f f e r e n t i a l l y a f f e c t the extent of t o l e r a n c e development to i t s a n t i c o n v u l s a n t e f f e c t . Greater t o l e r a n c e was found to the a n t i c o n v u l s a n t e f f e c t of p e n t o b a r b i t a l i n r a t s t h a t r e c e i v e d s u c c e s s i v e l y l a r g e r doses of the drug, which were not l a r g e enough to suppress f o r e l i m b c l o n u s , than i n those r a t s t h a t were maintained on a high dose t h a t completely suppressed the f o r e l i m b c l o n u s . The gr e a t e r t o l e r a n c e i n the former group was a t t r i b u t e d to the f a c t t h a t the co n v u l s i o n s were experienced i n the drugged s t a t e , compared to the l a t t e r group t h a t d i d not experience f o r e l i m b clonus i n the drugged s t a t e . These r e s u l t s c h a l l e n g e the g e n e r a l l y accepted view t h a t t o l e r a n c e develops to a gr e a t e r extent with l a r g e r doses (e.g., Aston, 1965; Jorgenson et a l . , 1986; Kalant et a l , , 1971; LeBlanc et a l . , 1969) . 4. T h e o r e t i c a l I m p l i c a t i o n s According to the d r u g - e f f e c t theory of drug t o l e r a n c e as proposed i n i t s o r i g i n a l form by P i n e l et a l . , (1989) , t o l e r a n c e develops to the drug e f f e c t s , not to drug exposure; t o l e r a n c e i s seen as an a d a p t a t i o n to the repeated e x p r e s s i o n of a drug's d i s r u p t i v e e f f e c t on ongoing neur a l a c t i v i t y , not to the mere presence of the drug. A c c o r d i n g l y , drug exposure i s presumed necessary, but not s u f f i c i e n t f o r the development of f u n c t i o n a l 99 t o l e r a n c e . The f o l l o w i n g a r e s e v e r a l e l a b o r a t i o n s t o t h e d r u g - e f f e c t t h e o r y o f t o l e r a n c e , w h i c h were d e r i v e d f r o m t h e r e s u l t s o f t h e p r e s e n t t h e s i s a nd f r o m t h e r e s u l t s o f o t h e r s t u d i e s o f t o l e r a n c e ( s e e T a b l e I I I f o r a c o m p l e t e r e v i e w ) 1) T o l e r a n c e d o e s d e v e l o p t o d r u g e f f e c t s w h e t h e r t h e c r i t e r i o n d r u g e f f e c t i s e x p l i c i t l y e x p r e s s e d , a s i s t h e c a s e when t h e d r u g a d m i n i s t r a t i o n p r e c e d e s t h e c r i t e r i o n r e s p o n s e , o r when t h e d r u g i s r e p e a t e d l y a d m i n i s t e r e d w i t h o u t e x p l i c i t l y e l i c i t i n g t h e c r i t e r i o n d r u g e f f e c t ; h o w e v e r , t o l e r a n c e d e v e l o p s more r a p i d l y a n d t o a g r e a t e r e x t e n t when t h e c r i t e r i o n d r u g e f f e c t i s e x p l i c i t l y e x p e r i e n c e d b y t h e s u b j e c t s . The q u e s t i o n o f w h e t h e r t o l e r a n c e d e v e l o p s i n t h e a b s e n c e o f t h e d r u g - e f f e c t c o n t i n g e n c y h a s b e e n t h e most h e a t e d d e b a t e i n t h e c o n t i n g e n t t o l e r a n c e l i t e r a t u r e . S e v e r a l l a b o r a t o r i e s h a v e a t t e m p t e d t o r e s o l v e t h i s i s s u e w i t h o u t s u c c e s s ( C h e n , 1968; Chen, 1972; L e B l a n c , G i b b i n s , & K a l a n t , 1 9 7 3 ; L e B l a n c , G i b b i n s , & K a l a n t , 1 9 7 5 ; Wenger, B e r l i n , & Woods, 1980; Wenger, T i f f a n y , B o m b a r d i e r , N i c h o l l s , & Woods, 1 9 8 1 ) . The p r o b l e m w i t h t h e s e s t u d i e s was t h e i n a p p r o p r i a t e c h o i c e o f d r u g e f f e c t ; t h e y e x a m i n e d t h e m o t o r - c o o r d i n a t l o n - a n d - b a l a n c e (maze r u n n i n g and t r e a d m i l l t a s k s ) d i s r u p t i n g e f f e c t o f e t h a n o l . W i t h t h i s d r u g e f f e c t , t h e r e i s no c l e a r - c u t d l s a s s o c i a t i o n b e t w e e n d r u g - e f f e c t a nd d r u g - e x p o s u r e . The s u b j e c t may e x p e r i e n c e many c o m p o n e n t s o f t h e d r u g e f f e c t w h e t h e r p e r f o r m i n g t h e maze o r t r e a d m i l l t a s k o r w a l k i n g a r o u n d I n I t s home c a g e . C o n s e q u e n t l y , t h e most 100 c o m p e l l i n g evidence that t o l e r a n c e can develop i n the absence o£ the d r u g - e f f e c t contingency i s from s t u d i e s t h a t have examined the a n t i c o n v u l s a n t drug e f f e c t , because with t h i s drug e f f e c t there i s a c l e a r - c u t d i s a s s o c i a t i o n between the d r u g - e f f e c t and drug-exposure. In a yet unpublished study of t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s r e v e a l e d t h a t t o l e r a n c e can develop i n the absence of the d r u g - e f f e c t contingency. For example, t o l e r a n c e was demonstrated to the a n t i c o n v u l s a n t e f f e c t of etha n o l when i t was admin i s t e r e d d a i l y f o r 20 days without the c o n v u l s i v e s t i m u l a t i o n ever o c c u r r i n g except on the t o l e r a n c e t e s t t r i a l f o l l o w i n g the treatment s e s s i o n s . S i m i l a r l y , Lippa and Regan (1977) demonstrated t o l e r a n c e to the a n t i c o n v u l s a n t e f f e c t of diazepam f o l l o w i n g seven days of drug treatment without the c o n v u l s i v e s t i m u l a t i o n o c c u r r i n g . And r e c e n t l y , i n two yet unpublished s t u d i e s from t h i s l a b o r a t o r y , t o l e r a n c e was demonstrated to the a n t i c o n v u l s a n t e f f e c t of ethanol when c o n v u l s i v e s t i m u l a t i o n s were d e l i v e r e d before the drug i n j e c t i o n s so t h a t the drug e f f e c t was not expressed. Subjects that r e c e i v e d ethanol a f t e r each c o n v u l s i v e s t i m u l a t i o n d i d not d i f f e r from the s a l i n e c o n t r o l group i n t h e i r response to ethanol on a s i n g l e t o l e r a n c e t e s t , and they d i s p l a y e d markedly l e s s t o l e r a n c e than the s u b j e c t s t h a t r e c e i v e d ethanol before the s t i m u l a t i o n s . The t o l e r a n c e was d e t e c t a b l e o n l y as an a c c e l e r a t i o n In the subsequent development of t o l e r a n c e as compared to the s a l i n e c o n t r o l group. 2) Tolerance development to a drug e f f e c t i s g r e a t e r i n the 101 absence of the d r u g - e f f e c t contingency with l a r g e r drug doses, with s h o r t e r i n t e r v a l s between drug a d m i n i s t r a t i o n s , and with longer d u r a t i o n s of drug treatment. The f a c t o r s Involved i n the development of t o l e r a n c e and cr o s s t o l e r a n c e are a complex i n t e r a c t i o n of the dose of the drug, the schedule of i t s a d m i n i s t r a t i o n , the d u r a t i o n of i t s treatment, and p o s s i b l y the route of i t s a d m i n i s t r a t i o n (see Kalant et a l . , 1971). In a d d i t i o n , the experiences of the s u b j e c t s a l s o p l a y a major r o l e --whether the c r i t e r i o n response occurs d u r i n g the p e r i o d s of drug exposure so t h a t the c r i t e r i o n drug e f f e c t i s experienced by the s u b j e c t . At lower doses, longer time i n t e r v a l s between drug a d m i n i s t r a t i o n s , and s h o r t e r drug treatment d u r a t i o n s , the impact of the drug on the organism i s r e l a t i v e l y more s u b t l e , and thus the experiences of the s u b j e c t s p l a y a l a r g e r r o l e ; and thus, a great e r d i f f e r e n c e i n the degree of t o l e r a n c e may be observed between those s u b j e c t s t h a t experienced the c r i t e r i o n drug e f f e c t i n the drugged s t a t e and those t h a t d i d not. At higher doses, s h o r t e r drug a d m i n i s t r a t i o n I n t e r v a l s , and longer treatment d u r a t i o n s , the impact of the drug on the s u b j e c t i s l e s s s u b t l e and the d r u g - e f f e c t contingency f a c t o r s are overwhelmed by the presence of the drug i n the organism; and t o l e r a n c e i s more l i k e l y to be observed whether the s u b j e c t s experience the drug e f f e c t In the drugged s t a t e or not. There i s on l y i n d i r e c t evidence f o r t h i s e l a b o r a t i o n . In a comparison of two s t u d i e s of t o l e r a n c e to ethanol's a n t i c o n v u l s a n t e f f e c t , P i n e l et a l . (1983) f a i l e d to observe 102 t o l e r a n c e when a r e l a t i v e l y low dose (1.5 g/kg) was a d m i n i s t e r e d t w i c e d a i l y f o r o n l y 5 days; and y e t i n an u n p u b l i s h e d s t u d y from t h i s l a b o r a t o r y , complete t o l e r a n c e d e v e l o p e d when a h i g h dose (5 g/kg) was d e l i v e r e d d a i l y f o r an extended p e r i o d of 20 days. There i s e x p e r i m e n t a l e v i d e n c e d e m o n s t r a t i n g t h a t i n the development of c o n d i t i o n e d t o l e r a n c e , a n o t h e r type of t o l e r a n c e i n which the e x p e r i e n c e s of the s u b j e c t a r e i m p o r t a n t — t h e environment i n which the s u b j e c t s e x p e r i e n c e d the drug e x p o s u r e - -the e n v i r o n m e n t a l cues p l a y e d a r e l a t i v e l y l e s s e r r o l e i n the t o l e r a n c e when h i g h d r ug doses and s h o r t e r drug a d m i n i s t r a t i o n i n t e r v a l s were used (Baker & T i f f a n y , 1985). And t h e r e i s the o b s e r v a t i o n t h a t i n g e n e r a l , t o l e r a n c e t o a drug's e f f e c t i s f a c i l i t a t e d by h i g h e r d o s e s , s h o r t e r i n t e r v a l s between drug a d m i n i s t r a t i o n s , and l o n g e r t r e a t m e n t d u r a t i o n s (see K a l a n t e t a l . , 1971). 3) The d i s s i p a t i o n of t o l e r a n c e t o a drug e f f e c t o c c u r s more r a p i d l y when the c r i t e r i o n d r u g e f f e c t i s a l l o w e d t o be e x p e r i e n c e d by the s u b j e c t s i n the non-drugged s t a t e , t h a n when the drug a d m i n i s t r a t i o n i s s i m p l y t e r m i n a t e d w i t h o u t the s u b j e c t s e x p e r i e n c i n g the c r i t e r i o n drug e f f e c t . Mana and P i n e l (1986) r e c e n t l y demonstrated t h a t t o l e r a n c e t o the a n t i c o n v u l s a n t e f f e c t of e t h a n o l d i s s i p a t e d , d e s p i t e the a d m i n i s t r a t i o n s of e t h a n o l on the same regimen t h a t produced the t o l e r a n c e , i f the drug I n j e c t i o n s f o l l o w e d each c o n v u l s i v e s t i m u l a t i o n ; and t o l e r a n c e a l s o d i s s i p a t e d when the c o n v u l s i v e s t i m u l a t i o n s were d e l i v e r e d a l o n e w i t h o u t any dr u g . And y e t t o l e r a n c e was m a i n t a i n e d when 103 ethanol a d m i n i s t r a t i o n was terminated without a p p l y i n g the c o n v u l s i v e s t i m u l a t i o n over the same r e t e n t i o n p e r i o d . The key f a c t o r i n the d i s s i p a t i o n of ethanol t o l e r a n c e , was not the withdrawal of e t h a n o l , but the a d m i n i s t r a t i o n of the c o n v u l s i v e s t i m u l a t i o n i n the absence of e t h a n o l ; e l i c l t a t i o n of the c r i t e r i o n response i n the non-drugged s t a t e causes the d i s s i p a t i o n of t o l e r a n c e . In Experiment 3C of t h i s t h e s i s , i t was shown t h a t t h i s may g e n e r a l i s e to the d i s s i p a t i o n of t o l e r a n c e to one drug f o l l o w i n g the a d m i n i s t r a t i o n of another drug. Tolerance to the a n t i c o n v u l s a n t e f f e c t of ethanol d i s s i p a t e d when p e n t o b a r b i t a l i n j e c t i o n s were d e l i v e r e d a f t e r each c o n v u l s i v e s t i m u l a t i o n , but not when i t was d e l i v e r e d before each s t i m u l a t i o n . 4) The p r i n c i p l e s of the d r u g - e f f e c t theory of t o l e r a n c e can be a p p l i e d to the development of c r o s s t o l e r a n c e to drug e f f e c t s . In t h i s t h e s i s , the c r o s s - d i s s i p a t i o n of t o l e r a n c e to one drug f o l l o w i n g the a d m i n i s t r a t i o n of another drug was a l s o c o n s i s t e n t with the d r u g - e f f e c t contingency. 5. C l i n i c a l I m p l i c a t i o n s Before the p o t e n t i a l l y important c l i n i c a l i m p l i c a t i o n s of t h i s r e s e a r c h are d i s c u s s e d , there i s a major d i s c r e p a n c y between the s t u d i e s conducted In experimental and the c l i n i c a l s i t u a t i o n t h a t needs to be addressed. The experimental data overwhelmingly and without any ambiguity whatsoever demonstrate t h a t t o l e r a n c e and c r o s s t o l e r a n c e do develop to a n t i c o n v u l s a n t e f f e c t s of a n t l e p i l e p t l c drugs (see Table 3; Frey 1985, 1986, 1987), yet 104 t h e r e have been few r e p o r t s t h a t t o l e r a n c e i s a major problem i n the t r e a t m e n t i f e p i l e p t i c p a t i e n t s ( e . g . , F r e y , 1985). There a r e two p o s s i b l e e x p l a n a t i o n s f o r t h i s d i s c r e p a n c y . F i r s t , t h e r e were d i f f e r e n c e s between the e x p e r i m e n t a l and c l i n i c a l s t u d i e s i n t h e i r methodology: the s u b j e c t s were a n i m a l s and not humans; the c o n v u l s i o n s were e l i c i t e d , u s u a l l y e l e c t r i c a l l y or c h e m i c a l l y , and not s p o n t a n e o u s l y o c c u r r i n g e p i l e p t i c s e i z u r e s ; the drug a d m i n i s t r a t i o n s were o f t e n i n j e c t e d a t spaced time i n t e r v a l s and not c o n t i n u a l o r a l i n g e s t i o n aimed a t m a i n t a i n i n g a c e r t a i n b l o o d plasma l e v e l of the dr u g ; and the doses used i n e x p e r i m e n t a l s t u d i e s tended t o be r e l a t i v e l y h i g h e r t h a n t h o s e used i n the c l i n i c a l s i t u a t i o n . I t may be p o s s i b l e t h a t the m e t h o d o l o g i c a l d i f f e r e n c e s c o u l d account f o r the v a s t l y d i f f e r e n t r e s u l t s . The more l i k e l y e x p l a n a t i o n i s t h a t i n the c l i n i c a l s e t t i n g , any observed d e c r e a s e i n the drug's e f f i c a c y d u r i n g a n t i e p i l e p t i c d r u g t r e a t m e n t has t y p i c a l l y been a t t r i b u t e d t o a number of f a c t o r s o t h e r t h a n t o l e r a n c e (Brown, 1976): 1) v a r i a b i l i t y of s e i z u r e i n c i d e n c e i n the e p i l e p t i c p a t i e n t , 2) p a t i e n t noncompliance--the p a t i e n t does not ta k e the drug as p r e s c r i b e d , 3) growth of the p a t i e n t t h a t w i l l reduce the r e l a t i v e dosage, and 4) drug m e t a b o l i t e s t h a t might I n t e r f e r e w i t h the e f f e c t of the p a r e n t d r u g . I t i s my f e e l i n g t h a t i n s p i t e of the m e t h o d o l o g i c a l d i f f e r e n c e s and i n l i g h t of such overwhelming e x p e r i m e n t a l e v i d e n c e , t o l e r a n c e c o u l d be demonstrated w i t h human s u b j e c t s In p r o p e r l y c o n t r o l l e d e x p e r i m e n t s . But t h i s i s a s i t u a t i o n t h a t i s o b v i o u s l y p r e c l u d e d 105 by e t h i c a l c o n s t r a i n t s , t h e r e f o r e u n t e s t a b l e . The f i r s t c l i n i c a l i m p l i c a t i o n i s t h a t the knowledge of t o l e r a n c e and cr o s s t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s c o u l d be of b e n e f i t i n the pharmacological treatment of e p i l e p t i c p a t i e n t s . T h i s i s an a f f l i c t i o n t h a t a f f e c t s n e a r l y 1% of the p o p u l a t i o n (Oilman, Goddman, & Oilman, 1980; Katzung, 1987). Quite o f t e n i n the c l i n i c a l s e t t i n g , the e p i l e p t i c p a t i e n t i s exposed not to one a n t l e p i l e p t l c drug, but r a t h e r to a v a r i e t y of them (polypharmacology), sometimes c o n c u r r e n t l y and sometimes s e q u e n t i a l l y (e.g., Eadie, 1985). Awareness of the drugs to which c r o s s t o l e r a n c e does develop would be v a l u a b l e i n making the a p p r o p r i a t e c h o i c e . For example, p h e n o b a r b i t a l , clonazepam, and carbamazepine a l l are e f f e c t i v e a g a i n s t p a r t i a l s e i z u r e s and g e n e r a l i z e d t o n i c - c l o n i c s e i z u r e s ; however, Experiment 1 has shown t h a t s i g n i f i c a n t c r o s s t o l e r a n c e was found between p h e n o b a r b i t a l and carbamazepine, and not between clonazepam and carbamazepine. T h e r e f o r e , i f a p a t i e n t d i s p l a y s evidence of t o l e r a n c e to carbamazepine, a b e t t e r a l t e r n a t i v e drug would be clonazepam and not p h e n o b a r b i t a l . The second p o t e n t i a l c l i n i c a l i m p l i c a t i o n of the t h e s i s was the o b s e r v a t i o n i n Experiment 4 t h a t t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s may be f a c i l i t a t e d by the e x p r e s s i o n of the co n v u l s i o n s i n the drugged s t a t e . In the treatment of e p i l e p s y , the p a t i e n t i s t y p i c a l l y given a r e l a t i v e l y low dose of the a n t i e p i l e p t i c drug, to avoid p o s s i b l e t o x i c s i d e e f f e c t s , and when an e p i l e p t i c s e i z u r e occurs, the dose i s Increased. T h i s may 106 be clone r e p e a t e d l y u n t i l an e f f e c t i v e t h e r a p e u t i c dose i s obtained. By a l l o w i n g the p a t i e n t to experience the c o n v u l s i o n s i n the drugged s t a t e , perhaps the development of t o l e r a n c e to the a n t i e p i l e p t i c e f f e c t i s f a c i l i t a t e d . An a l t e r n a t e t h e r a p e u t i c s t r a t e g y , which must be weighed a g a i n s t the adverse s i d e e f f e c t s of high drug doses, would be to give the p a t i e n t a r e l a t i v e l y high i n i t i a l dose to ensure t h a t the c o n v u l s i o n s are l e s s l i k e l y to be experienced, and thus en s u r i n g the development of slower and l e s s t o l e r a n c e to a n t i e p i l e p t i c drugs. The s t r a t e g y of a d m i n i s t e r i n g r e l a t i v e l y l a r g e r drug doses i n i t i a l l y i s b e t t e r because the f i n a l e f f e c t i v e t h e r a p e u t i c dose t h a t i s achieved may be lower s i n c e l e s s t o l e r a n c e has developed; and with lower doses, the p o s s i b i l i t y of adverse s i d e e f f e c t s are a l s o decreased. T h i s s t r a t e g y would a l s o b r i n g the s e i z u r e s under c o n t r o l more q u i c k l y , which i s Important f o r two reasons. F i r s t , there i s some evidence s u g g e s t i n g t h a t the occurrence of s e i z u r e s may r e s u l t i n b r a i n damage (Dam, 1982); t h i s was demonstrated i n human e p i l e p t i c s and i n animal models. Second, whether a c t u a l b r a i n damage occurs or not, there may be some permanent a l t e r a t i o n o c c u r r i n g i n the b r a i n as the r e s u l t of the s e i z u r e . I t has been demonstrated i n a v a r i e t y of experimental c o n v u l s a n t models i n animals t h a t repeated e l i c i t a t i o n of s e i z u r e s can lead to 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 them, and thus make s u c c e s s i v e s e i z u r e s more l i k e l y to occur; and these changes were permanent ( P i n e l & Van Dot, 1975). I f t h i s can g e n e r a l i z e to humans who r e p e a t e d l y experience s e i z u r e s , quick e f f e c t i v e c o n t r o l of the 107 s e i z u r e would be of prime importance. 6. Conclusions and Future D i r e c t i o n s According to the t r a d i t i o n a l pharmacological drug-exposure  theory of drug t o l e r a n c e , the exposure of the organism to the drug i s the c r i t i c a l f a c t o r i n the development of t o l e r a n c e to the drug's e f f e c t . T h i s i s very simple and s u c c i n c t , and could account f o r much of the ob s e r v a t i o n s of t o l e r a n c e . However, t h i s theory cannot e x p l a i n why the very same regimen of drug a d m i n i s t r a t i o n produces t o l e r a n c e to a drug e f f e c t i n some ins t a n c e s but not i n o t h e r s . In the past two decades, two c h a l l e n g e s were proposed to the t r a d i t i o n a l theory. U n l i k e the t r a d i t i o n a l theory, these new t h e o r i e s do not view the s u b j e c t as a p a s s i v e r e c i p i e n t of the drug, but are based on the premise t h a t the experiences of the s u b j e c t s while they are drugged p l a y a major r o l e i n the t o l e r a n c e development. The f i r s t of these two t h e o r i e s , the c o n d i t i o n e d t o l e r a n c e theory, focused on the r o l e i n the development of t o l e r a n c e of the environment i n which the s u b j e c t s have p r e v i o u s l y experienced the drug e f f e c t s . The second of these, the d r u g - e f f e c t theory, focused on the behavior of the s u b j e c t d u r i n g drug exposure. The d r u g - e f f e c t theory was the focus of the present t h e s i s . The t h e s i s has made s e v e r a l c o n t r i b u t i o n s : 1) i t has e s t a b l i s h e d the g e n e r a l i t y of the d r u g - e f f e c t contingency to the development of c r o s s t o l e r a n c e to a n t i c o n v u l s a n t drug e f f e c t s and to the c r o s s - d i s s i p a t i o n of t o l e r a n c e ; 2) i t has determined t h a t d i f f e r e n t dose a d m i n i s t r a t i o n regimens can Inf l u e n c e the extent 108 o f t o l e r a n c e d e v e l o p m e n t t o t h e a n t i c o n v u l s a n t d r u g e f f e c t s ; 3) I t h a s p r o p o s e d s e v e r a l e l a b o r a t i o n s t o t h e d r u g - e f f e c t t h e o r y t o p r o v i d e a more c o m p l e t e u n d e r s t a n d i n g o f t h i s phenomenon; 4) i t has p r o v i d e d some p o t e n t i a l l y i m p o r t a n t c l i n i c a l i m p l i c a t i o n s o f t h i s r e s e a r c h ; a n d 5) i t h a s d e m o n s t r a t e d t h e u t i l i t y o f t h e k i n d l e d - c o n v u l s i o n m o d e l i n t h e s t u d y o f t o l e r a n c e and c r o s s t o l e r a n c e t o a n t i c o n v u l s a n t d r u g e f f e c t s . 109 Re ferences A l b e r t s o n , T.E., Peterson, S.L., & Stark, L.G. 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