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The effects of dose and duration of neuroleptic administration on dopamine receptor sensitivity Dewey, Kevin John 1981

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THE EFFECTS OF DOSE AND DURATION OF NEUROLEPTIC ADMINISTRATION ON DOPAMINE RECEPTOR SENSITIVITY by KEVIN JOHN DEWEY B . S c . (Hons) , Concordia U n i v e r s i t y , 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF MEDICINE DEPARTMENT OF PSYCHIATRY DIVISION OF NEUROLOGICAL SCIENCES We accept t h i s t h e s i s as conforming to the r e q u i r e d s tandard THE UNIVERSITY OF BRITISH COLUMBIA November, 1981 (c) Kevin John Dewey, 1981 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Co lumbia , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re fe rence and s tudy . I f u r t h e r agree t h a t permiss ion f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Department o f PSYCHIATRY The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date i i ABSTRACT I t i s w e l l e s t a b l i s h e d t h a t c h r o n i c t reatment w i t h n e u r o l e p t i c agents which s e l e c t i v e l y b lock dopamine (DA) recepto rs i n the b r a i n leads to the development o f DA r e c e p t o r s u p e r s e n s i t i v i t y . However comparing the degree and d u r a t i o n o f the changes i n r e c e p t o r s e n s i t i v i t y ob ta ined by d i f f e r e n t i n v e s t i g a t o r s has been extremely d i f f i c u l t , because of the numerous d i f f e r e n c e s t h a t e x i s t i n i n d i v i d u a l methods o f producing and examining DA recepto r s u p e r s e n s i t i v i t y . By examining the DA r e c e p t o r s u p e r s e n s i t i v i t y t h a t ensues f o l l o w i n g c h r o n i c t reatment w i t h d i f f e r e n t doses and d u r a t i o n s of p imoz ide , at va r ious i n t e r v a l s a f t e r withdrawal from t r e a t m e n t , the o v e r a l l paramet r i c changes can be more d i r e c t l y compared. To measure the changes i n DA recepto r s e n s i t i v i t y f o l l o w i n g c h r o n i c pimozide t r e a t m e n t , both behav io ra l (d.-amphetamine-induced locomotor a c t i v i t y ; apomorphine-induced s t e r e o t y p y ) and b iochemical (DA r e c e p t o r b i n d i n g assay) techniques were u t i l i z e d . With i n c r e a s i n g doses o f c h r o n i c pimozide t r e a t m e n t , the degree and d u r a t i o n o f the r e s u l t i n g DA r e c e p t o r s u p e r s e n s i t i v i t y i n c r e a s e d as measured both b e h a v i o r a l l y and b i o c h e m i c a l l y . S i m i l a r i l y , the longer d u r a t i o n s of c h r o n i c pimozide t reatment had a g r e a t e r e f f e c t on the degree and d u r a t i o n o f the i n c r e a s e d DA recepto r s e n s i t i v i t y than d i d the s h o r t e r d u r a t i o n s o f t reatment . C o r r e l a t i o n s were found between the b iochemical and behav io ra l r e s u l t s both between groups of animals t r e a t e d c h r o n i c a l l y w i t h d i f f e r e n t doses and d u r a t i o n s of pimozide and w i t h i n i n d i v i d u a l groups o f a n i m a l s . In a d d i t i o n , the changes i n r e c e p t o r s e n s i t i v i t y f o l l o w i n g c h r o n i c pimozide t reatment was due to an i n c r e a s e i n the number of DA recepto rs w i th no change i n the a f f i n i t y o f these recepto rs to DA. i i i These r e s u l t s f o l l o w i n g c h r o n i c t reatment w i t h n e u r o l e p t i c s demonstrate t h a t the behav io ra l s u p e r s e n s i t i v i t y observed i n animals i n response to e i t h e r the d i r e c t DA a g o n i s t apomorphine or the i n d i r e c t DA a g o n i s t d-amphetamine, may be a r e s u l t o f an i n c r e a s e d number of DA r e c e p t o r s . F i n a l l y , the s u p e r s e n s i t i v e DA recepto rs t h a t develop as a r e s u l t o f c h r o n i c t reatment w i t h n e u r o l e p t i c s are d i s c u s s e d w i t h regard to t h e i r p o s s i b l e re levance as an animal model o f the i a t r o g e n i c d i s e a s e , t a r d i v e d y s k i n e s i a , observed c l i n i c a l l y i n s c h i z o p h r e n i c p a t i e n t s withdrawn from n e u r o l e p t i c therapy . i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 Chemistry o f N e u r o l e p t i c s 2 Pharmacology of N e u r o l e p t i c s 3 Acute B iochemical A c t i o n s of N e u r o l e p t i c s 5 I The Dopamine Receptor 5 II E f f e c t s on P o s t s y n a p t i c Dopamine Receptors 7 I I I E f f e c t s on P r e s y n a p t i c Dopamine Receptors 9 Acute Behav io ra l A c t i o n s of N e u r o l e p t i c s 10 I E f f e c t s on Inherent Behaviors 10 II E f f e c t s on Drug-Induced Behaviors 11 I ' l l E f f e c t s on Cond i t ioned Behaviors 12 E f f e c t s o f Chronic N e u r o l e p t i c Treatment 13 I B e h a v i o r a l S tud ies 14 II B iochemical S tud ies ' 1 7 I I I N e u r o p h y s i o l o g i c a l S tud ies 18 IV P r e s y n a p t i c S tud ies ._ 18 C l i n i c a l E f f e c t s of N e u r o l e p t i c s 19 I Ex t rapyramida l S ide E f f e c t s o f Chron ic N e u r o l e p t i c Treatment 20 II B r a i n Mechanisms Under ly ing Ta rd i ve D y s k i n e s i a 22 I I I Animal Models of Tard ive D y s k i n e s i a 24 Summary 25 STATEMENT OF PURPOSE 27 GENERAL MATERIALS AND METHODS 28 . Sub jects .V 28 Drug Treatment 28 V Procedures ". 28 I Locomotor A c t i v i t y 28 II Stereotypy 29 I I I Dopamine Receptor B ind ing 29 i Drugs 32 S t a t i s t i c s 32 RESULTS 33 Experiment 1 : P r e l i m i n a r y S tud ies 33 Experiment 2 : Paramet r i c Time Course Study -E f f e c t of Dose of Pimozide 42 Experiment 3 : Paramet r i c Time Course Study -E f f e c t of Durat ion of Pimozide A d m i n i s t r a t i o n . . 52 DISCUSSION 69 P r e l i m i n a r y S t u d i e s 70 E f f e c t of Dose of Pimozide on Dopamine Receptor S e n s i t i v i t y 72 E f f e c t of Durat ion o f Pimozide A d m i n i s t r a t i o n on Dopamine Receptor S e n s i t i v i t y 75 E f f e c t of Chronic Pimozide Treatment on the Dopamine Receptor . . 77 C o r r e l a t i o n s Between Behav io ra l and B iochemical R e s u l t s 79 Animal Models of Tard ive D y s k i n e s i a 81 REFERENCES 85 v i LIST OF FIGURES F igure 1 : The e f f e c t o f c h r o n i c p imozide a d m i n i s t r a t i o n on the locomotor s t i m u l a n t e f f e c t of severa l doses of d-amphet-amine s u l f a t e 35 F igure 2: The e f f e c t of c h r o n i c pimozide a d m i n i s t r a t i o n on the t o t a l locomotor a c t i v i t y e l i c i t e d by severa l doses o f d-amphetamine s u l f a t e , examined a t va r ious i n t e r v a l s f o l l o w i n g withdrawal from c h r o n i c t reatment 37 F igure 3 : Dose response curve of apomorphine- induced s te reotypy . . . . 39 F igure 4 : The e f f e c t o f p r i o r (24 hours) a d m i n i s t r a t i o n of d-amphet-amine s u l f a t e on apomorphine- induced s tereotyped behav ior i n c h r o n i c pimozide or v e h i c l e t r e a t e d animals 41 F igure 5 : The e f f e c t of c h r o n i c t reatment w i t h v e h i c l e or severa l doses o f p imozide on the locomotor s t i m u l a n t e f f e c t s o f d-amphetamine s u l f a t e measured 4 , 1 0 , 20 and 40 days f o l l o w i n g withdrawal from c h r o n i c t reatment 44 F igure 6: The e f f e c t o f c h r o n i c t reatment w i t h v e h i c l e or severa l doses of pimozide on the t o t a l locomotor a c t i v i t y f o l l o w i n g the a d m i n i s t r a t i o n o f d-amphetamine s u l f a t e , examined a t v a r i o u s i n t e r v a l s f o l l o w i n g wi thdrawal 46 F igure 7: The e f f e c t of c h r o n i c t reatment w i t h v e h i c l e or severa l doses o f pimozide on apomorphine- induced s tereotyped b e h a v i o r , examined a t v a r i o u s i n t e r v a l s f o l l o w i n g withdrawal from c h r o n i c t reatment 48 F igure 8 : The e f f e c t o f c h r o n i c t reatment w i t h v e h i c l e or severa l doses o f pimozide on the amount of s p e c i f i c [ 3 H ] -s p i r o p e r i d o l b i n d i n g , examined a t v a r i o u s i n t e r v a l s f o l l o w i n g withdrawal 50 F igure 9: The e f f e c t of d i f f e r e n t d u r a t i o n s o f c h r o n i c pimozide or v e h i c l e t reatment on the locomotor s t i m u l a n t e f f e c t s of d-amphetamine s u l f a t e , examined a t va r ious i n t e r v a l s f o l l o w i n g withdrawal from c h r o n i c t reatment 54 F igure 10: The e f f e c t o f d i f f e r e n t d u r a t i o n s of c h r o n i c pimozide or v e h i c l e t reatment on the t o t a l locomotor a c t i v i t y 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 d-amphetamine s u l f a t e , examined a t va r ious i n t e r v a l s f o l l o w i n g withdrawal c h r o n i c t reatment 56 F i g u r e 11: The e f f e c t s of d i f f e r e n t d u r a t i o n s of c h r o n i c pimozide or v e h i c l e t reatment on apomorphine- induced s te reo typed b e h a v i o r , examined at v a r i o u s i n t e r v a l s f o l l o w i n g withdrawal from c h r o n i c t reatment F igure 12: The e f f e c t o f d i f f e r e n t d u r a t i o n s o f c h r o n i c pimozide or v e h i c l e t reatment on the amount of s p e c i f i c [ 3 H] -s p i r o p e r i d o l b i n d i n g , examined a t va r ious i n t e r v a l s fo11owi ng wi thdrawal F igure 13 : The s a t u r a t i o n curves of s p e c i f i c b i n d i n g of [ 3 H ] -s p i r o p e r i d o l to combined s t r i a t a l t i s s u e as a f u n c t i o n of i t s c o n c e n t r a t i o n i n groups of animals t r e a t e d c h r o n i c a l l y w i t h e i t h e r pimozide or v e h i c l e F igu re 14: Scatchard a n a l y s i s of s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g to combined s t r i a t a l t i s s u e from groups of an imals t r e a t e d c h r o n i c a l l y w i t h e i t h e r pimozide o r v e h i c l e F igu re 15 : The c o r r e l a t i o n between behav io ra l and b iochemica l data i n groups of animals t r e a t e d c h r o n i c a l l y w i t h v e h i c l e or s e v e r a l doses o f p imozide and examined 10-1.2 days f o l l o w i n g withdrawal from c h r o n i c t reatment F igure 16: The c o r r e l a t i o n between b e h a v i o r a l and b iochemica l data i n i n d i v i d u a l animals t r e a t e d c h r o n i c a l l y w i t h pimozide and examined 10-12 days f o l l o w i n g withdrawal from c h r o n i c t reatment v i i i ACKNOWLEDGEMENTS I would l i k e to s i n c e r e l y thank my s u p e r v i s o r Dr. H.C. F i b i g e r f o r h i s d i r e c t i o n and support throughout the course of t h i s p r o j e c t . I would a l s o l i k e to express my a p p r e c i a t i o n to Dr. C. S p y r a k i , Dr. K. A s i n and Wm. S t a i n e s f o r t h e i r f r i e n d l y a d v i c e and h e l p f u l d i s c u s s i o n s and to E. Lyson f o r her t ime spent i n the f i n a l p r e p a r a t i o n o f t h i s t h e s i s . F i n a l l y , I am g r a t e f u l to the many members o f the Kinsmen Laboratory who made my study here en joyab le and f u l f i l l i n g . 1 INTRODUCTION A n a t o m i c a l , b i o c h e m i c a l , p h a r m a c o l o g i c a l , p h y s i o l o g i c a l and b e h a v i o r a l s t u d i e s have c l e a r l y e s t a b l i s h e d the importance of dopamine (DA) as a n e u r o t r a n s m i t t e r i n the mammalian b r a i n and i n p a r t i c u l a r the n i g r o s t r i a t a l and mesol imbic systems. In a d d i t i o n c l i n i c a l research has i m p l i c a t e d DA as being of p o s s i b l e major importance to severa l p s y c h i a t r i c and n e u r o l o g i c a l d i s o r d e r s i n c l u d i n g s c h i z o p h r e n i a ( C a r l s s o n , 1977) , P a r k i n s o n ' s d i s e a s e (Hornyk iewicz , 1972) and Hunt ington 's chorea (Chase, 1973) . A n t i p s y c h o t i c agents i n c l u d e the c l a s s of compounds tha t have the a b i l i t y to c o n t r o l many p s y c h o t i c symptoms i n p a t i e n t s and to be p a r t i c u l a r l y usefu l in the t reatment of s c h i z o p h r e n i a . The use of a n t i p s y c h o t i c drugs f o r the t reatment of s c h i z o p h r e n i a was o r i g i n a l l y repor ted i n 1952 by Delay and Deniker who found the s e d a t i n g agent ch lorpromaz ine r e l i e v e d s c h i z o p h r e n i c symptoms i n p a t i e n t s a f f l i c t e d w i t h the d i s o r d e r . S h o r t l y t h e r e a f t e r , i t was reported tha t the Rauwol f ia a l k a l o i d r e s e r p i n e had t h e r a p e u t i c a c t i o n s i n s c h i z o p h r e n i a s i m i l a r t o ch lorpromazine (Den iker , 1970) . However, because ch lorpromaz ine and r e s e r p i n e produced motor s i d e e f f e c t s i n many p a t i e n t s i n a d d i t i o n t o b e n e f i c i a l a n t i s c h i z o p h r e n i c a c t i o n s , i t was specu lated t h a t the s i d e e f f e c t s of these agents may be necessary f o r t h e i r t h e r a p e u t i c a c t i o n s (Den ike r , 1970) . In l i g h t of t h i s o b s e r v a t i o n , Delay and Deniker c a l l e d these a n t i p s y c h o t i c agents " n e u r o l e p t i c s , " de f ined as agents which induce a syndrome of behav io ra l and psych ic symptoms t h a t i s observed i n p a t i e n t s 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 ch lorpromaz ine l i k e d rugs . Thus, n e u r o l e p t i c drugs are those a n t i p s y c h o t i c drugs which produce n e u r o l o g i c a l s i d e e f f e c t s i n a d d i t i o n to t h e i r a n t i p s y c h o t i c a c t i o n s . Below i s an 2 i n t r o d u c t o r y survey of the cur rent s t a t e of knowledge concern ing n e u r o l e p t i c drugs i n c l u d i n g t h e i r c h e m i s t r y , pharmacology, b iochemica l and behav io ra l a c t i o n s , t h e i r c l i n i c a l use and the ext rapyramidal s i d e e f f e c t s t h a t ensue f o l l o w i n g t h e i r use i n man. Chemistry of N e u r o l e p t i c s N e u r o l e p t i c s c o n s i s t of f i v e major c l a s s e s of drugs whose members are u s e f u l i n the t reatment of s c h i z o p h r e n i a and o ther psychoses : p h e n o t h i a z i n e s , butyrophenones, t h i o x a n t h e n e s , d ihyd ro indo lones and d ibenzoxazep ines . The f i r s t n e u r o l e p t i c agent syn thes i zed and used c l i n i c a l l y was the p h e n o t h i a z i n e , ch lorpromazine ( C h a r p e n t i e r , 1950) . S h o r t l y t h e r e a f t e r , the f i r s t butyrophenone, h a l o p e r i d o l was i d e n t i f i e d and f o l l o w i n g i t s pharmacological i n v e s t i g a t i o n (Janssen et a l . , 1960) , the s y n t h e s i s and examinat ion of the p r o p e r t i e s of many d e r i v a t i v e s of the butyrophenones r e s u l t e d . For example, a m o d i f i c a t i o n i n the s i d e cha in of the butyrophenones r e s u l t e d i n a new c l a s s of n e u r o l e p t i c a g e n t s , the d i p h e n y l b u t y l p i p e r i d i n e s . Pimozide was the f i r s t compound in t h i s new s e r i e s and i s i t s e l f a d e r i v a t i v e of the butyrophenone benper ido l (Janssen et a l . , 1968) . A l l n e u r o l e p t i c s are t e r t i a r y or secondary amines wi th a chemical s t r u c t u r e c o n s i s t i n g of at l e a s t one aromat ic r i n g (Ar) l i n k e d to an amine group (-NC) by an i n t e r m e d i a t e s t r a i g h t c h a i n , w i th the general formula Ar -X -CH2-N ( Janssen , 1973) . For example, the butyrophenones c o n s i s t of a 4 - f l u o r o p h e n y l group, a keto group and methylene groups wi th the f o r m u l a : and when the keto f u n c t i o n i s rep laced by a 4 - f luo ropheny lmeth ine m o i e t y , the r e s u l t i n g s t r u c t u r e i s r e p r e s e n t a t i v e of the d i p h e n y l b u t y l p i p e r i d i n e s . F-Aside from a s i m i l a r chemical s t r u c t u r e , n e u r o l e p t i c s have many s i m i l a r chemical p r o p e r t i e s i n c l u d i n g e l e c t r o n donor a b i l i t y , i n h i b i t i o n o f o x i d a t i v e phosphory la t ion and the c a p a b i l i t y to lower su r face t e n s i o n ( Janssen , 1965; M a t t h y s s e , 1973) . The s i m i l a r i t i e s among the v a r i o u s c l a s s e s of n e u r o l e p t i c drugs suggest that they a l l have a common mechanism of a c t i o n . P r e s e n t l y , a great deal of ev idence supports the concept tha t n e u r o l e p t i c s exert t h e i r a n t i p s y c h o t i c e f f e c t by b l o c k i n g DA receptors i n the b ra in (Van Rossum, 1966) , but the exact mechanisms remain unknown. It has been p o s t u l a t e d that n e u r o l e p t i c s are ab le to b lock DA receptors because of a comformational compl imentar i t y between c e r t a i n p o r t i o n s of these drugs and DA (Horn et a l . , 1975). Pharmacology of N e u r o l e p t i c s F o l l o w i n g t h e i r s y n t h e s i s and the e v a l u a t i o n of t h e i r chemical p r o p e r t i e s , the pharmacological c h a r a c t e r i s t i c s of n e u r o l e p t i c s were examined. As would be expected from t h e i r chemical s i m i l a r i t i e s , the pharmacological p r o p e r t i e s of n e u r o l e p t i c s are a l s o s i m i l a r , wi th obvious d i f f e r e n c e s i n t h e i r potency (Niemegeers and Janssen , 1979) . At low doses not a f f e c t i n g the normal behavior of the a n i m a l , n e u r o l e p t i c s s p e c i f i c a l l y i n h i b i t such behaviors as apomorphine or d-amphetamine induced s t e r e o t y p y , i n t r a c r a n i a l s e l f s t i m u l a t i o n (ICSS) and c o n d i t i o n e d operant behavior i n a n i m a l s . With i n c r e a s i n g doses , n e u r o l e p t i c s induce c a t a l e p t i c i m m o b i l i t y in the a n i m a l , reducing both spontaneous movement and e x p l o r a t o r y b e h a v i o r . N e u r o l e p t i c agents used c l i n i c a l l y are known to c o n t r o l such p s y c h o t i c symptoms as h a l l u c i n a t i o n s and mental confus ion and to reduce 4 psychomotor a g i t a t i o n . Even though a s p e c i f i c animal model of human psychos is has yet to be found , the i n h i b i t i o n of d-amphetamine or apomorphine induced s tereotyped b e h a v i o r , i n t r a c r a n i a l s e l f s t i m u l a t i o n and c o n d i t i o n e d operant behav ior i n r a t s a l l have been shown to c o r r e l a t e we l l w i th the a n t i p s y c h o t i c e f f e c t s of n e u r o l e p t i c s i n man (Janssen et a l . , 1965; 1967; Wauquier and Niemegeers, 1972). Therefore severa l c l i n i c a l e f f e c t s of n e u r o l e p t i c drugs can be p r e d i c t e d from t h e i r pharmacological c h a r a c t e r i s t i c s , i n c l u d i n g potency , d u r a t i o n of a c t i o n and t h e i r o ra l e f f e c t i v e n e s s . In a d d i t i o n to b l o c k i n g DA r e c e p t o r s , l e s s s p e c i f i c n e u r o l e p t i c s at a n t i p s y c h o t i c dose l e v e l s are ab le to b lock severa l o ther recepto r s i t e s i n c l u d i n g n o r a d r e n a l i n e (Peroutka et a l . , 1977) , s e r o t o n i n (Creese and Snyder , 1978) , a c e t y l c h o l i n e (Snyder et a l . , 1974) and h is tamine (Chang e t a l . , 1979) , r e s l t i n g i n an inc reased l i a b i l i t y to produce s e d a t i v e , autonomic and n e u r o l o g i c a l s i d e e f f e c t s . For example, the r e l a t i v e a b i l i t y of n e u r o l e p t i c s to antagonize NA a c t i v i t y i n r e l a t i o n to DA antagonism r e s u l t s i n the c l i n i c a l l y observed s i d e e f f e c t s of t a c h y c a r d i a , o r t h o s t a t i c hypotension and other symptoms of autonomic b lockade . The n e u r o l o g i c a l s i d e e f f e c t s such as muscular r i g i d i t y , a k a t h i s i a , d y s k i n e s i a and tremor are the most common s i d e e f f e c t s and the most d i f f i c u l t to a v o i d . Whereas the butyrophenones and the d i p h e n y l b u t y l p i p e r i d i n e s have s i m i l a r chemical p r o p e r t i e s , they have d i f f e r e n t pharmacolog ica l c h a r a c t e r i s t i c s s i n c e the l a t t e r c l a s s of n e u r o l e p t i c s are c h a r a c t e r i z e d by the absence of s e d a t i v e and autonomic s i d e e f f e c t s and a very low i n c i d e n c e of n e u r o l o g i c a l s i d e e f f e c t s (Janssen et a l . , 1968) . 5 Acute B iochemical A c t i o n s of N e u r o l e p t i c s E a r l y b iochemica l s t u d i e s w i th n e u r o l e p t i c drugs examined many o f t h e i r n o n s p e c i f i c a c t i o n s on membranes and enzyme systems throughout the body (Guth and S p i r t e s , 1964) . However most of these e f f e c t s of n e u r o l e p t i c s show on ly a very weak c o r r e l a t i o n wi th c l i n i c a l potency , u n l i k e the a c t i o n s of n e u r o l e p t i c s on n e u r o t r a n s m i t t e r mechanisms i n the b r a i n . I. The Dopamine Receptor The e x i s t e n c e o f m u l t i p l e DA pathways i n the mammalian c e n t r a l nervous system i s c l e a r l y e s t a b l i s h e d , but t h e r e i s much u n c e r t a i n t y concern ing what types of DA receptors are i n the b r a i n (Creese and S i b l e y , 1979; M e l t z e r , 1979; S o k o l o f f et a l . , 1980). Receptor l a b e l i n g techn iques have proven to be usefu l t o o l s i n n e u r o l o g i c a l r e s e a r c h . Because of t h e i r s p e c i f i c i t y , these recepto r b i n d i n g assays have been use fu l f o r d e f i n i n g the b iochemical and pharmacolog ica l c h a r a c t e r i s t i c s of n e u r o t r a n s m i t t e r recepto r s i t e s (Creese and S i b l e y , 1979; S o k o l o f f et a l . , 1980). For example, the d i r e c t b iochemical l a b e l i n g of DA receptors has r e s u l t e d i n a b e t t e r understanding of both the mechanisms o f a c t i o n o f n e u r o l e p t i c drugs and the receptor changes tha t occur i n c o n j u n c t i o n wi th the normal ag ing process and n e u r o p s y c h i a t r y d i s o r d e r s . P r e s e n t l y , [ 3 H] -dopamine , the DA a g o n i s t s [ 3 H] -apomorph ine , [ 3 H ] -a m i n o - 6 , 7 - d i h y d r o x y t e t r a h y d r o n a p h t h a l e n e and [ 3 H] -p ropy lapomorph ine , and the DA a n t a g o n i s t s [ 3 H ] - h a l o p e r i d o l , [ 3 H ] - s p i r o p e r i d o l , [ 3 H ] - a - f 1 upenth ixo l and [ 3 H ] - d i h y d r o e r g o c r y p t i n e are used as l i g a n d s f o r l a b e l i n g DA receptors i n the CNS (Burt et a l . , 1975; Creese and Snyder , 1977; Leysen and Gommeren, 1981; Leysen et a l . , 1978; Seeman et a l . , 1975; T i t e l e r et a l . , 6 1979). Recent ly [ 3 H ] - s p i r o p e r i d o l has been shown to be a v a l u a b l e l i g a n d f o r s t u d i e s i n v o l v i n g DA recepto rs because of i t s h igher a f f i n i t y f o r DA recepto rs and i t s lower a f f i n i t y f o r a-NA receptors than the w i d e l y used l i g a n d h a l o p e r i d o l (Laduron et a l . , 1978; Leysen et a l . , 1978) . However, s t u d i e s from severa l l a b o r a t o r i e s have concluded tha t a l though [ 3 H ] -s p i r o p e r i d o l b i n d i n g i n the s t r i a t u m l a b e l s on ly DA r e c e p t o r s , [ 3 H ] -s p i r o p e r i d o l b inds almost e x c l u s i v e l y to s e r o t o n i n recepto rs in the f r o n t a l co r tex where DA p r o j e c t i o n s are l e s s ev ident (Leysen et a l . , 1978; Quik et a l . , 1978). It i s thought tha t both t r i t i a t e d DA a g o n i s t s and a n t a g o n i s t s bind to the same r e c e p t o r s i n the b r a i n s i n c e s t u d i e s have shown that DA r e c e p t o r a g o n i s t s and a n t a g o n i s t s have s i m i l a r potenc ies i n competing f o r the t r i t i a t e d l i g a n d s (Burt et a l . , 1975; 1976) . In a d d i t i o n , the t r i t i a t e d DA a g o n i s t s and a n t a g o n i s t s d i s p l a y s i m i l a r l e v e l s of b i n d i n g i n p a r t i c u l a r b r a i n r e g i o n s , wi th the h ighest b i n d i n g o c c u r r i n g i n the s t r i a t u m , nucleus accumbens and o l f a c t o r y t u b e r c l e , and no b i n d i n g i n the tha lamus , or cerebe l lum (Creese et a l . , 1975) . Although t r i t i a t e d DA a g o n i s t s and a n t a g o n i s t s appear to l a b e l s i m i l a r recepto r s i t e s , d i f f e r e n c e s i n a f f i n i t y have been r e p o r t e d . For example, a g o n i s t s have a g rea te r a f f i n i t y f o r the [ 3 H] agon is t s i t e and a n t a g o n i s t s have a h igher a f f i n i t y f o r the [ 3 H] a n t a g o n i s t s i t e (Creese et a l . , 1975a). From these o b s e r v a t i o n s , two d i f f e r e n t views of the DA recepto r have been p o s t u l a t e d . One view i s tha t t h e r e i s on ly one DA receptor e x i s t i n g i n two s t a t e s (Leysen, 1979; Monod et a l . , 1965) , as has been proposed f o r m u s c a r i n i c c h o l i n e r g i c , and s e r o t o n e r g i c receptors i n the b r a i n (Snyder , 1975) . P o s s i b l y , the agon is t and antagon is t s t a t e s of the recepto r e x i s t s i n e q u i l i b r i u m . In t h i s way, DA a n t a g o n i s t s would 7 bind t o the an tagon is t s t a t e of the r e c e p t o r , dec reas ing the number of ag on is t s i t e s a v a i l a b l e to the n e u r o t r a n s m i t t e r . The second v iew of the DA recepto r i s tha t t h e r e are at l e a s t two separate receptors w i th d i f f e r e n t degrees of s p e c i f i c i t y , one of which has a h igher a f f i n i t y f o r a g o n i s t s and one of which has a h igher a f f i n i t y f o r a n t a g o n i s t s (Cools and Van Rossum, 1976; Creese and S i b l e y , 1979; S o k o l o f f et a l . , 1980) . P r e s e n t l y , n e i t h e r v iew of the DA receptor can be d i sca rded and only f u t u r e research w i l l e l u c i d a t e the exact nature of the DA recepto r or r e c e p t o r s . C o r r e l a t i o n s have been found between the pharmacological potency ( i n h i b i t i o n of amphetamine-induced behav ior ) of n e u r o l e p t i c drugs in animals and man and t h e i r a f f i n i t y f o r [ 3 H ] - h a l o p e r i d o l b i n d i n g (Creese et a l . , 1976a) . In a d d i t i o n , the c l i n i c a l potency of n e u r o l e p t i c s i n man a l s o c o r r e l a t e very c l o s e l y w i th compei t ion of these drugs f o r [ 3 H ] - h a l o p e r i d o l b i n d i n g (Creese et a l . , 1976b; Hy t te l , 1978; Seeman et a l . , 1976) . These c o r r e l a t i o n s i n d i c a t e t h a t n e u r o l e p t i c agents act at the l e v e l of the p o s t s y n a p t i c DA r e c e p t o r . I I . E f f e c t s on P o s t s y n a p t i c Dopamine Receptors I n i t i a l i n v e s t i g a t i o n s on the p o s s i b l e a c t i o n s of n e u r o l e p t i c drugs on DA recepto rs examined the e f f e c t s of these drugs on the DA s e n s i t i v e adeny late c y c l a s e . I t was thought t h a t DA recepto rs were l i n k e d to the enzyme adeny late c y c l a s e i n the CNS because s t i m u l a t i o n of the receptors by DA r e s u l t e d in an inc reased l e v e l of c y c l i c AMP and because the d i s t r i b u t i o n of t h i s enzyme in the b r a i n p a r a l l e l e d the d i s t r i b u t i o n of DA receptors i n the b r a i n (Kebabian et a l . , 1972) . When the e f f e c t of n e u r o l e p t i c s on the DA s e n s i t i v e adeny late c y c l a s e was examined i t was found t h a t the p h e n o t h i a z i n e s , but not the butyrophenones or the 8 d i p h e n y l b u t y l p i p e r i d i n e s , were e f f e c t i v e enzyme i n h i b i t o r s (Clement-Cormiere et a l . , 1974; M i l l e r et a l . , 1974) . I n j e c t i o n s of the neurotox in 6-hydroxydopamine (6-OHDA) i n t o the n i g r o s t r i a t a l pathway have been shown to produce no changes i n l e v e l s of s t r i a t a l DA s e n s i t i v e adeny late c y c l a s e a c t i v i t y (Mishra et a l . , 1974) . On the other hand, f o l l o w i n g the i n j e c t i o n of k a i n i c a c i d i n t o the s t r i a t u m , which des t roys l o c a l neuronal c e l l bodies but leaves nerve t e r m i n a l s and f i b e r s of passage i n t a c t , t h e r e was a marked decrease i n the DA s e n s i t i v e adeny late c y c l a s e a c t i v i t y (Schwarcz and C o y l e , 1977) . These r e s u l t s i m p l i c a t e an a s s o c i a t i o n of adeny late c y c l a s e wi th the p o s t s y n a p t i c r e c e p t o r s ; however, s i n c e the butyrophenones f a i l to a l t e r the l e v e l s of adeny late c y l a s e a c t i v i t y , f u r t h e r i n v e s t i g a t i o n s are r e q u i r e d . When the e f f e c t s of n e u r o l e p t i c s on CNS catecholamines were examined i t was observed t h a t , a l though 3-methoxytyramine l e v e l s were e leva ted f o l l o w i n g n e u r o l e p t i c a d m i n i s t r a t i o n , the l e v e l s o f DA and NA were not changed (Car l sson and L i n d q u i s t , 1963) . S ince n e u r o l e p t i c s were shown t o i n c r e a s e the c a t a b o l i s m of DA to 3 -methoxytyramine, C a r l s s o n and L i n d q u i s t (1963) suggested t h a t these agents produce an i n c r e a s e i n the s y n t h e s i s and r e l e a s e of DA. E l e c t r o p h y s i o l o g i c a l s t u d i e s have s i n c e shown t h a t the r a t e of DA s y n t h e s i s and r e l e a s e i s d i r e c t l y r e l a t e d t o the f i r i n g r a t e of DA neurons i n the s u b s t a n t i a n i g r a o f r a t s (Bunney and A g h a j a n i a n , 1973) . T h e r e f o r e , i t has been suggested tha t by b l o c k i n g p o s t s y n a p t i c DA recepto rs i n the s t r i a t u m , n e u r o l e p t i c s , would v i a a feedback l o o p , send i n f o r m a t i o n back t o DA neurons w i t h i n the s u b s t a n t i a n i g r a , i n c r e a s i n g t h e i r f i r i n g r a t e and thus a l s o i n c r e a s i n g the s y n t h e s i s and r e l e a s e of DA (Car lsson and L i n d q u i s t , 1963) . This p r e d i c t i o n has s i n c e been conf i rmed by severa l 9 methods, such a s : the accumulat ion of DA f o l l o w i n g monoamine ox idase i n h i b i t i o n ; the c o n c e n t r a t i o n of h o m o v a n i l l i c a c i d i n t i s s u e s ; the appearance of r a d i o a c t i v e DA f o l l o w i n g the in t ravenous i n j e c t i o n of r a d i o a c t i v e t y r o s i n e , and the d isappearance of r a d i o a c t i v e DA a f t e r p r e v i o u s l y l a b e l i n g b r a i n s torage s i t e s (Fuxe and Ungers ted t , 1976; Kendler et a l . , 1981; Sedval l et a l . , 1975; VonPraag and K o r f , 1975) . From these s t u d i e s i t has been p o s t u l a t e d tha t the pr imary a c t i o n of n e u r o l e p t i c s i s t o b lock DA t r a n s m i s s i o n w i th the i n c r e a s e d tu rnover of DA being a compensatory mechanism of t h i s a c t i o n . I I I . E f f e c t s on P r e s y n a p t i c Dopamine Receptors In a d d i t i o n to p o s t s y n a p t i c DA r e c e p t o r s , i t has been p o s t u l a t e d from b iochemica l and e l e c t r o p h y s i o l o g i c a l s t u d i e s t h a t DA recepto rs a l s o e x i s t on the p r e s y n a p t i c DA t e r m i n a l s i n the s t r i a t u m , as we l l as on DA c e l l bodies in the s u b s t a n t i a n i g r a ( C a r l s s o n , 1975) . The s t i m u l a t i o n of p r e s y n a p t i c or au to recepto rs by DA or DA a g o n i s t s produces an i n h i b i t o r y e f f e c t on the DA neuron , d e c r e a s i n g both the s y n t h e s i s and r e l e a s e of DA ( Iversen et a l . , 1976; Kehr et a l . , 1972; Nowycky and R o t h , 1978; Roth et a l . , 1976; Strombom, 1976; Wal ters et a l . , 1975; W e s t f a l l et a l . , 1976) . For example, m i c r o i o n t o p h o r e s i s of DA or apomorphine to DA c e l l bodies in the s u b s t a n t i a n i g r a decreases both the r e l e a s e of DA and the f i r i n g r a t e o f these neurons (Bunney and A g h a j a n i a n , 1975) . S i m i l a r l y , DA a n t a g o n i s t s have been proposed to i n c r e a s e the r e l e a s e of DA f o l l o w i n g t h e i r i n t e r a c t i o n w i th p r e s y n a p t i c recepto rs ( C h r i s t i a n s e n and S q u i r e s , 1974; Farnebo and Hamberger, 1971; G o l d s t e i n et a l . , 1973; Z i v k o v i c et a l . , 1974) . 10 Acute Behav io ra l A c t i o n s of N e u r o l e p t i c s 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 n e u r o l e p t i c drugs to animals OR man, a s i m i l a r set of symptoms i s observed ( F i e l d i n g and L a i , 1975) . In man, low doses of n e u r o l e p t i c s r e s u l t i n d r o w s i n e s s , the reduct ion of spontaneous movements and the suppress ion of p s y c h o t i c symptoms such as h a l l u c i n a t i o n s and mental c o n f u s i o n . With i n c r e a s i n g doses a v a r i e t y of n e u r o l o g i c a l , autonomic and e n d o c r i n o l o g i c a l e f f e c t s o c c u r . In a n i m a l s , low doses of n e u r o l e p t i c s decrease e x p l o r a t o r y and operant behav ior w h i l e h igher doses produce c a t a l e p s y and p o s s i b l e autonomic e f f e c t s . In a d d i t i o n , n e u r o l e p t i c s are a n t i e m e t i c agents in both man and a n i m a l s , hav ing the a b i l i t y to act on the chemoreceptor t r i g g e r zone i n the h i n d b r a i n . Along w i th b iochemica l and e l e c t r o p h y s i o l o g i c a l s t u d i e s , i n v e s t i g a t i o n s o f the behav io ra l a c t i o n s of n e u r o l e p t i c s c o n t r i b u t e to a b e t t e r understanding o f the mechanism of a c t i o n s of n e u r o l e p t i c d rugs . I. E f f e c t s on Inherent Behavior N e u r o l e p t i c s have been shown to e f f e c t such inherent behav iors as spontaneous m o t i l i t y , e a t i n g and d r i n k i n g . A l l n e u r o l e p t i c s depress spontaneous locomotor a c t i v i t y i n animals by producing c a t a l e p s y (Janssen et a l . , 1965) . However, i t i s unknown whether t h i s depress ion of m o t i l i t y i s a d i r e c t a c t i o n of n e u r o l e p t i c drugs or whether i t i s secondary t o the e f f e c t s of i n d u c i n g c a t a l e p s y . Dopamine mechanisms i n the l a t e r a l hypothalamus have been i m p l i c a t e d i n r e g u l a t i n g e a t i n g and d r i n k i n g behav iors (Hynes et a l . , 1975) . Although n e u r o l e p t i c s have been shown t o suppress feed ing and d r i n k i n g behav iors i n animals (Janssen et a l . , 1965; R o l l s . e t a l . , 1974) , h igh doses are u s u a l l y r e q u i r e d . Thus these a c t i o n s may be s i d e e f f e c t s of these drugs . With the i n c r e a s i n g knowledge of the 11 mechanisms u n d e r l y i n g n e u r o l e p t i c drug a c t i o n s i n the CNS, t h e i r p r e c i s e e f f e c t on spontaneous m o t i l i t y , e a t i n g and d r i n k i n g may soon be e l u c i d a t e d . I I . E f f e c t s on Drug Induced Behaviors N e u r o l e p t i c drugs are ab le to i n h i b i t many drug induced behav iors i n man and a n i m a l s . Amphetamines produce s t i m u l a n t a c t i o n s on locomotor a c t i v i t y and operant behav ior in a n i m a l s . At low doses there i s an i n c r e a s e i n spontaneous motor a c t i v i t y w h i l e , at i n c r e a s i n g d o s e s , t h i s s t i m u l a n t e f f e c t on motor a c t i v i t y i s g r a d u a l l y reduced a long w i th an i n c r e a s e i n the frequency of s tereotyped behav iors (Russel and P i h l , 1978) . Amphetamine induced s t e r e o t y p y , observed i n a l l mammalian spec ies i n c l u d i n g man (Randrup and Munkvad, 2967; R y l a n d e r , 1971) , can be de f ined as the r e p e t i t i v e performance of a constant sequence of movements i n v o l v i n g p r i m a r i l y the head, and f a c i a l musculature and mouth pa r t s (Randrup and Munkvad, 1967). Amphetamine i s thought to cause the r e l e a s e of DA from nerve t e r m i n a l s , making more DA a v a i l a b l e to act on the DA receptors (Hanson, 1966) . The two d i f f e r e n t behav io ra l e f f e c t s of amphetamine are thought to s p e c i f i c a l l y r e f l e c t a c t i v i t y i n d i f f e r e n t DA c o n t a i n i n g pathways. Amphetamine induced locomotor a c t i v i t y i s thought to be mediated by the mesol imbic dopaminergic sys tem, w h i l e the n i g r o s t r i a t a l dopaminergic system has been i m p l i c a t e d in the i n i t i a t i o n of s te reotypy (Ernst and S m e l i k , 1966; K e l l y et a l . , 1975) . N e u r o l e p t i c drugs i n h i b i t many of the known a c t i o n s of amphetamine. For example, i n a l l l a b o r a t o r y a n i m a l s , n e u r o l e p t i c s antagonize amphetamine induced s tereotyped behav ior (Janssen et a l . , 1965; 1967) . S i m i l a r l y i n man, n e u r o l e p t i c s reduce the s t i m u l a n t and euphor iant e f f e c t s of l a r g e doses of amphetamine in p r e v i o u s l y add ic ted sub jec ts ( Jonsson, 1972) . 12 These agents a l s o i n h i b i t s tereotyped behavior 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 apomorphine (Janssen et a l . , 1965; 1967) , which u n l i k e amphetamine, i s a DA analog t h a t d i r e c t l y s t i m u l a t e s DA recepto rs ( E r n s t , 1967) . Fo l l ow ing the u n i l a t e r a l i n j e c t i o n of 6-OHDA i n the s u b s t a n t i a n i g r a , which s e l e c t i v e l y dest roys the n i g r o s t r i a t a l DA pathway, t h e r e i s a l o s s of p r e s y n a p t i c DA receptors and the development of s u p e r s e n s i t i v e p o s t s y n a p t i c DA recepto rs on the l e s i o n e d s i d e (Ungers tedt , 1971a). Amphetamine admin is te red to the animal w i l l on l y r e l e a s e DA on the i n t a c t s i d e of the b r a i n , r e s u l t i n g in the animal r o t a t i n g i p s i l a t e r a l to the s i d e of the l e s i o n (Ungers tedt , 1971b). By b l o c k i n g DA r e c e p t o r s , n e u r o l e p t i c s are ab le to i n h i b i t the amphetamine induced i p s i l a t e r a l t u r n i n g i n animals p r e v i o u s l y t r e a t e d with 6-OHDA (Stawarz et a l . , 1975) . I I I . E f f e c t s on Cond i t ioned Behaviors Cond i t ioned or learned behav iors i n c l u d e such behav iors as avoidance l e a r n i n g , i n t r a c r a n i a l s e l f - s t i m u l a t i o n (ICSS) and the s e l f - a d m i n i s t r a t i o n of d rugs . Avoidance behav ior i n c l u d e s operant responses which the animal performs to avo id a v e r s i v e s t i m u l i , such as e l e c t r i c a l shock. DA mechanisms have been i m p l i c a t e d as p l a y i n g an important u n d e r l y i n g r o l e i n avoidance l e a r n i n g and n e u r o l e p t i c agents are known t o i n h i b i t on l y the c o n d i t i o n e d avoidance response i n a n i m a l s , not the escape response (Beninger et a l . , 1980; F i b i g e r et a l . , 1975; Fregnan and Chi e l i , 1980; Niemegeers et a l . , 1969). Fur thermore , a p o s i t i v e c o r r e l a t i o n e x i s t s between antiamphetamine potency and the a n t i a v o i d a n c e a c t i o n of n e u r o l e p t i c s (Niemegeers et a l . , 1969) . E l e c t r i c a l s t i m u l a t i o n of severa l b r a i n reg ions produces e f f e c t s which are p o s i t i v e l y r e i n f o r c i n g i n many s p e c i e s . In small doses n e u r o l e p t i c 13 drugs have been shown to i n h i b i t i n t r a c r a n i a l s e l f - s t i m u l a t i o n i n r a t s , dogs and monkeys (Broekkamp and Van Rossum, 1975; Mora et a l . , 1976; R o l l s et a l . , 1974) . The r e i n f o r c i n g mechanisms u n d e r l y i n g ICSS have been thought to be mediated by catecholamine mechanisms i n the b r a i n ; however, t h i s hypothes is i s s t i l l c o n t r o v e r s i a l and awai ts f u r t h e r i n v e s t i g a t i o n (Crow, 1972; F i b i g e r , 1978; German and Bowden, 1974) . N e u r o l e p t i c s have a l s o been demonstrated to i n h i b i t the s e l f -a d m i n i s t r a t i o n of such drugs as morphine , h e r o i n , apomorphine and amphetamine i n animals and to p r e v e n t , as wel l as r e v e r s e , many of the s igns of drug w i t h d r a w a l , i n c l u d i n g hypothermia , wet dog shakes , i r r i t a b i l i t y and a g r e s s i o n (Cox et a l . , 1975; Lai et a l . , 1971) . In man i t has been reported tha t h a l o p e r i d o l i s b e n e f i c i a l i n r e l i e v i n g many of the symptoms of hero in withdrawal (Karka las and L a i , 1973) . S ince i t i s specu la ted t h a t there e x i s t h y p e r a c t i v e DA systems i n p a t i e n t s who are drug dependent ( L a i , 1975) , n e u r o l e p t i c agents might p o s s i b l y i n h i b i t the u n d e r l y i n g neural b a s i s f o r the c o n t i n u a l d r i v e f o r drug a d m i n i s t r a t i o n i n animals and man. E f f e c t s of Chronic N e u r o l e p t i c Treatment The r e g u l a t i o n of recepto rs i n the b r a i n has probable c l i n i c a l importance s i n c e a l t e r a t i o n s i n receptor p r o p e r t i e s may occur i n p a t i e n t s a f f l i c t e d w i t h v a r i o u s p s y c h i a t r i c and n e u r o l o g i c a l d i s o r d e r s . Fur thermore , they may have a s i g n i f i c a n t r o l e i n the development and modulat ion of synapses (Heidmann and Changeux, 1978) . Receptor changes can be detected by e i t h e r b i o c h e m i c a l , e l e c t r o p h y s i o l o g i c a l , or behav io ra l methods and have been shown to be regu la ted in both d i r e c t i o n s -h y p e r s e n s i t i v i t y ( K a l i s k e r et a l . , 1973; Sporn et a l . , 1976) and 14 h y p o s e n s i t i v i t y ( S c h u l t z , 1976; Vetu lan i and S u l s e r , 1975) . The p r e c i s e mechanisms f o r such r e g u l a t o r y changes are unknown, but cou ld e i t h e r i n v o l v e the number of f u n c t i o n a l recepto rs or the r e l a t i v e a f f i n i t y of the r e c e p t o r s . Nerve i n n e r v a t i o n i n f l u e n c e s the long term p h y s i o l o g i c a l and pharmacolog ica l p r o p e r t i e s of a l l t i s s u e s , i n c l u d i n g o ther neurons. It has been observed repeated ly tha t when the i n n e r v a t i o n by a neuron i s d i s r u p t e d , the s e n s i t i v i t y of the p o s t s y n a p t i c c e l l i s i n c r e a s e d . The development of denervat ion s u p e r s e n s i t i v i t y i s a general b i o l o g i c a l phenomenon, which was i n i t i a l l y p o s t u l a t e d by Cannon i n 1939: "When i n a s e r i e s of e f f e r e n t neurons a u n i t i s d e s t r o y e d , an i n c r e a s e i n the i r r i t a b i l i t y to chemical agents develop i n the i s o l a t e d s t r u c t u r e or s t r u c t u r e s , the e f f e c t being maximal in the part d i r e c t l y d e n e r v a t e d . " In the case of the n i g r o s t r i a t a l DA sys tem, i t i s c l e a r from b i o c h e m i c a l , behav io ra l and p h y s i o l o g i c a l s t u d i e s t h a t l e s i o n i n g or the c h r o n i c a d m i n i s t r a t i o n of n e u r o l e p t i c agents a c t i n g at the DA nerve t e r m i n a l o r the p o s t s y n a p t i c DA recepto r leads to an inc reased responsiveness to DA agoni s t s . I. Behav iora l S tud ies F o l l o w i n g b i l a t e r a l 6-OHDA l e s i o n s of the n i g r o s t r i a t a l t r a c t , r a t s d i s p l a y an enhanced s tereotyped behav io ra l response to apomorphine and respond to subthresho ld doses t h a t p r e v i o u s l y had no e f f e c t (Creese and I v e r s e n , 1975b; P r i c e and F i b i g e r , 1974; Ungers tedt , 1971a). L i k e w i s e , a f t e r u n i l a t e r a l 6-OHDA l e s i o n s in the n i g r o s t r i a t a l system behav io ra l s u p e r s e n s i t i v i t y i s ev ident by the apomorphine- induced r o t a t i o n of the a n i m a l , i n a d i r e c t i o n c o n t r a l a t e r a l to the s i d e of the l e s i o n (Thornburg and Moore, 1975; Ungers tedt , 1971b; Von Vo ig t lander and Moore, 1973) . 15 S i m i l a r l y , i t i s w ide l y accepted t h a t c h r o n i c t reatment w i th agents t h a t i n t e r f e r e w i th catecholamine mechanisms in the CNS a l s o leads to changes i n the responsiveness of the receptors to ca techo lamines . Th is was o r i g i n a l l y observed by Schelkunov i n 1967; he noted tha t withdrawal of r a t s from long term h a l o p e r i d o l t reatment produced inc reased d-amphetamine- induced s t e r e o t y p y . S ince then numerous i n v e s t i g a t o r s have observed t h a t c h r o n i c t reatment w i t h n e u r o l e p t i c drugs produces an inc reased behav io ra l response to DA a g o n i s t s (Chr i s tensen et a l . , 1976; Clow et a l . , 1979; 1980; Dunstan and J a c k s o n , 1977; Gianutsos and Moore, 1977; Gianutsos et a l . , 1974; Jackson et a l . , 1975; Klawans and R u b o v i t z , 1972; M o l l e r - N i e l s o n et a l . , 1974; Sahakian et a l . , 1976; Sayers et a l . , 1975; S c a t t o n , 1977; Smith and D a v i s , 1976; S t o l k and Rech, 1968; Tarsy and B a l d e s s a r i n i , 1974; Thornburg and Moore, 1974; . V o i t h , 1977; Von V o i g t l a n d e r et a l . , 1975; Waddington and Gamble, 1980; Waldemeir and M a i t r e , 1976; Yen-Koo and B a l a z s , 1980) . These e f f e c t s appear to represent the development of s u p e r s e n s i t i v e DA receptors i n the b r a i n . It has been specu la ted t h a t the d u r a t i o n of s u p e r s e n s i t i v i t y may p a r a l l e l the d u r a t i o n of pretreatment w i th the n e u r o l e p t i c drug ( M u l l e r and Seeman, 1978). Recent s t u d i e s have demonstrated, both b e h a v i o r a l l y and b i o c h e m i c a l l y , an i n c r e a s e i n DA r e c e p t o r s e n s i t i v i t y a f t e r o n l y a s i n g l e dose of a n e u r o l e p t i c drug and t h i s i n c r e a s e p e r s i s t e d f o r days ( H y t t e l , 1977; Mar t res et a l . , 1977) . A d d i t i o n a l i n v e s t i g a t o n s on the t ime course e f f e c t s of n e u r o l e p t i c t reatment on DA recepto r s u p e r s e n s i t i v i t y are requ i red before any f i r m c o n c l u s i o n can be drawn. In a d d i t i o n to induc ing DA recepto r s u p e r s e n s i t i v i t y i n the s t r i a t u m , i t has been proposed t h a t c h r o n i c n e u r o l e p t i c t reatment may a l s o cause 16 s u p e r s e n s i t i v e DA receptors i n l i m b i c reg ions ( S c a t t o n , 1977). For example, f o l l o w i n g c h r o n i c n e u r o l e p t i c t reatment i n animals the l o c a l i n j e c t i o n of DA i n t o the nucleus accumbens or the systemic i n j e c t i o n of apomorphine both r e s u l t e d i n i n c r e a s e d locomotor a c t i v i t y (Jackson et a l . , 1975; Ungerstedt and L jungborg , 1977) . The p o s s i b l e development of s u p e r s e n s i t i v e DA recepto rs i n l i m b i c areas arose from these o b s e r v a t i o n s s i n c e locomotor a c t i v i t y i s thought to be e l i c i t e d from the nucleus accumbens (P i jnenburg and Von Rossum, 1973) . Based on the hypothes is t h a t p s y c h o t i c symptoms are connected wi th the l i m b i c system, these r e s u l t s suggest ing l i m b i c s u p e r s e n s i t i v e DA receptors are p u z z l i n g s i n c e , i f t h i s occur red c l i n i c a l l y , p s y c h o t i c symptoms in s c h i z o p h r e n i c p a t i e n t s would tend t o s l o w l y worsen. The development of s u p e r s e n s i t i v e DA receptors 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 n e u r o l e p t i c drugs i n man and animals appears t o v a r y depending on the agent used. For example, f o l l o w i n g the c h r o n i c a d m i n i s t r a t i o n of c l o z a p i n e , s u l p i r i d e or t h i o r i d a z i n e , some i n v e s t i g a t o r s have observed behav io ra l e f f e c t s which suggest s u p e r s e n s i t i v e DA recepto rs (Gianutsos and Moore, 1977; S c a t t o n , 1977; Smith and D a v i s , 1976) , w h i l e o thers have f a i l e d to observe these e f f e c t s c o n s i s t e n t l y (Sayers et a l . , 1975; Waldemeir and M a i t r e , 1976) . In an e f f o r t to e x p l a i n t h i s d i s c r e p a n c y , these a t y p i c a l n e u r o l e p t i c s have been suggested t o p r e f e r e n t i a l l y act on l i m b i c DA recepto rs (Costa l 1 and N a y l o r , 1976) . I f n e u r o l e p i c agents e x i s t t h a t are s p e c i f i c f o r l i m b i c DA r e c e p t o r s , t h e i r use c l i n i c a l l y might be b e n e f i c i a l s i n c e p s y c h o t i c symptoms might be r e l i e v e d i n p a t i e n t s wi thout the appearance of unwanted s i d e e f f e c t s . 17 I I . B iochemical S tud ies Severa l b iochemical approaches have been taken t o examine the e f f e c t s of c h r o n i c n e u r o l e p t i c t reatment on DA recepto r mechanisms. A g a i n , the re are c o n f l i c t i n g repor ts regard ing changes i n the a c t i v i t y of the s t r i a t a l DA s e n s i t i v e adeny late c y c l a s e f o l l o w i n g n i g r o s t r i a t a l l e s i o n s or prolonged t reatment w i th n e u r o l e p t i c s . Whereas some i n v e s t i g a t o r s have reported no changes i n the enzyme (Rotrosen et a l . , 1975; Von Vo ig t lander et a l . , 1973) , o thers have shown some enhanced a c t i v i t y (Mishra et a l . , 1974; Prement et a l . , 1975) . Thus, no f i r m c o n c l u s i o n s can be drawn concern ing whether t h e r e i s an inc reased s e n s i t i v i t y of DA s e n s i t i v e adeny late c y c l a s e i n v a r i o u s s t a t e s of s u p e r s e n s i t i v i t y . Other b iochemica l t e s t s have shown t h a t f o l l o w i n g c h r o n i c n e u r o l e p t i c t r e a t m e n t , the a b i l i t y of DA a g o n i s t s to i n h i b i t the ra te of s y n t h e s i s and tu rnover of DA in the s t r i a t u m i s changed. For example, c h r o n i c n e u r o l e p t i c t reatment i n c r e a s e s the i n h i b i t o r y e f f e c t of apomorphine on DA t u r n o v e r , i n d i c a t i n g s u p e r s e n s i t i v e s t r i a t a l DA receptors (Gianutsos and Moore, 1977; Smith et a l . , 1978) . B iochemical methods which have examined changes i n DA receptors f o l l o w i n g c h r o n i c n e u r o l e p t i c t reatment or n i g r o s t r i a t a l l e s i o n s a l s o u t i l i z e d DA recepto r l a b e l i n g techniques wi th t r i t i a t e d DA a g o n i s t s and a n t a g o n i s t s . Fo l l ow ing n i g r o s t r i a t a l l e s i o n s (Creese et a l . , 1977; M i s h r a et a l . , 1980) , or c h r o n i c t reatment w i th n e u r o l e p t i c s (Burt et a l . , 1976b; F r i e d h o f f et a l . , 1977; F r iend et a l . , 1978; Kamer et a l . , 1981; Kobayashi et a l . , 1978; M u l l e r and Seeman, 1977; Sayers et a l . , 1975; Staunton et a l . , 1981; Theodorou et a l . , 1981) , an i n c r e a s e i n the amount of b i n d i n g to DA recepto rs has been observed i n s t r i a t a l and l i m b i c a r e a s . This 18 i n c r e a s e i n b i n d i n g cou ld be due e i t h e r to a change i n the a f f i n i t y of the recepto r f o r DA or to a change i n the number o f b i n d i n g s i t e s . From more d e t a i l e d b iochemica l i n v e s t i g a t i o n s the major change seen f o l l o w i n g c h r o n i c n e u r o l e p t i c t reatment or n i g r o s t r i a t a l l e s i o n s i s a s s o c i a t e d w i t h an i n c r e a s e i n the number of DA recepto r s i t e s w i th smal l or no changes i n the a f f i n i t y of the DA receptor (Burt et a l . , 1977; Helmeste et a l . , 1981; Kobayashi et a l . , 1978; M u l l e r and Seeman, 1977; Theodorou et a l . , 1981) . I I I . N e u r o p h y s i o l o g i c a l Stud ies In e l e c t r o p h y s i o l o g i c a l s t u d i e s , d e s t r u c t i o n of the n i g r o s t r i a t a l pathway wi th 6-OHDA l e s i o n s ( F e l t z and DeChamplain, 1972; Schu l t z and Ungers ted t , 1978) , or c h r o n i c pretreatment w i th n e u r o l e p t i c s (Yarbrough, 1975) , produces a s i g n i f i c a n t i n c r e a s e i n the s e n s i t i v i t y of s t r i a t a l neurons to m i c r o i o n t o p h o r e t i c a l l y a p p l i e d DA. These n e u r o p h y s i o l o g i c a l s t u d i e s lend a d d i t i o n a l support to behav io ra l and b iochemica l s t u d i e s t h a t c h r o n i c n e u r o l e p t i c t reatment r e s u l t s i n the development of s u p e r s e n s i t i v e DA r e c e p t o r s . IV. P resynapt i c S tud ies Recent l y i t has been reported t h a t i n a d d i t i o n to an e f f e c t on p o s t s y n a p t i c DA r e c e p t o r s , t h e r e i s an a l t e r a t i o n i n the s e n s i t i v i t y of p r e s y n a p t i c DA receptors f o l l o w i n g c e s s a t i o n of c h r o n i c n e u r o l e p t i c t reatment (B igg io et a l . , 1980; G a l l a g e r et a l . , 1978; Gianutsos et a l . , 1975; Nowycky and R o t h , 1977) . However, the reported s u p e r s e n s i t i v e p r e s y n a p t i c receptors which ensue f o l l o w i n g c h r o n i c n e u r o l e p t i c t reatment have not been r e p l i c a t e d by other i n v e s t i g a t o r s ( M u l l e r et a l . , 1980; R a i t e r i et a l . , 1980) . In an e f f o r t to e x p l a i n t h i s d i s c r e p a n c y , e i t h e r the e x i s t e n c e of p r e s y n a p t i c recepto rs on s t r i a t a l nerve endings has been 19 quest ioned ( R a i t e r i et a l . , 1980) , or a p o s s i b l e blockade of the p r e s y n a p t i c s i t e s by r e s i d u a l drug remaining i n the s y n a p t i c c l e f t has been suggested ( M u l l e r et a l . , 1980) . Many of the a c t i o n s of c h r o n i c n e u r o l e p t i c t reatment cou ld be e x p l a i n e d by an e f f e c t on p r e s y n a p t i c recepto rs i n a d d i t i o n to p o s t s y n a p t i c r e c e p t o r s . However, because the e x i s t e n c e and the exact f u n c t i o n of DA p r e s y n a p t i c receptors i n the s t r i a t u m or s u b s t a n t i a n i g r a remains to be f i r m l y e s t a b l i s h e d , the hypothes ized e f f e c t of n e u r o l e p t i c drugs on p r e s y n a p t i c mechanisms must remain u n c e r t a i n . The f u n c t i o n a l s i g n i f i c a n c e of a l t e r a t i o n s i n receptor s e n s i t i v i t y i s unknown, but severa l hypotheses have been proposed. The most p l a u s i b l e hypothes is i s t h a t these recepto r changes represent neuronal homeostat ic mechanisms f o r m a i n t a i n i n g s y n a p t i c f u n c t i o n as c l o s e as p o s s i b l e to the " n a t u r a l s t a t e , " d e s p i t e l a r g e v a r i a t i o n s i n s y n a p t i c input (Schwartz et a l . , 1978) . U n t i l the p r e c i s e mechanisms by which recepto rs change s e n s i t i v i t y f o l l o w i n g l e s i o n s or drug t reatment are understood , on ly s p e c u l a t i o n s u n d e r l y i n g these a l t e r a t i o n s can be proposed. The C l i n i c a l E f f e c t s of N e u r o l e p t i c s The e a r l y c l i n i c a l success of the n e u r o l e p t i c agent ch lorpromazine to reduce the p s y c h o t i c behav ior of s c h i z o p h r e n i c s (Delay and D e n i k e r , 1952) r e s u l t e d i n the r a p i d development of c l i n i c a l psychopharmacology. In the f o l l o w i n g y e a r s , phenoth iaz ines were accepted as potent a n t i p s y c h o t i c drugs possess ing the a b i l i t y to reduce both the pr imary symptoms of s c h i z o p h r e n i a , (such as thought d i s o r d e r , b lunted a f f e c t , a u t i s t i c wi thdrawal and p s y c h o t i c behav ior ) and the accessary symptoms (such as h a l l u c i n a t i o n s , paranoid i d e n t i f i c a t i o n , h o s t i l i t y , b e l l i g e r e n c e and uncooperat iveness) ( S a r e s t k y , 1966; Shimkunas et a l . , 1966) . The search f o r b e t t e r 20 a n t i p s y c h o t i c drugs possess ing fewer s i d e e f f e c t s r e s u l t e d i n the d i s c o v e r y of the butyrophenones (Lafave et a l . , 1967) . Even though the phenoth iaz ines and butyrophenones have d i f f e r e n t chemical s t r u c t u r e s and s i d e e f f e c t p o t e n t i a l , they are both e f f e c t i v e as a n t i p s y c h o t i c a g e n t s , reduc ing s c h i z o p h r e n i c symptoms in a f f l i c t e d i n d i v i d u a l s . S ince n e u r o l e p t i c s are known to have a b l o c k i n g a c t i o n on the DA synapse and to b e n e f i t s c h i z o p h r e n i c s , the hypothes is of o v e r a c t i v e DA t r a n s m i s s i o n i n s c h i z o p h r e n i a has been suggested ( f o r rev iew , Crow and G i l l b e , 1974; Langer et a l . , 1981; M a t t h y s s e , 1974; Randrup and Munkvad, 1974; Snyder , 1976; 1981). Even though the hypothes is f o r the u n d e r l y i n g b a s i s of s c h i z o p h r e n i a i s o v e r s i m p l i f i e d , the combined knowledge about the mechanism of a c t i o n of n e u r o l e p t i c s on t h i s d i s e a s e , and the inc reased understanding of the e t i o l o g y and pathophys io logy of s c h i z o p h r e n i a , should lead to a more r a t i o n a l drug therapy f o r s c h i z o p h r e n i a . I. Ext rapyramidal S ide E f f e c t s of Chronic N e u r o l e p t i c Treatment F o l l o w i n g t h e i r i n i t i a l c l i n i c a l use i n the t reatment of s c h i z o p h r e n i a , i t was found tha t n e u r o l e p t i c drugs a l s o produce numerous ex t rapyramida l s i d e e f f e c t s (Schonecker , 1957; Sigwald et a l . , 1959) . The n e u r o l o g i c a l s i d e e f f e c t s , i n c l u d i n g the c l a s s i c a l b u c c a l - l i n g u a l -m a s t i c a t o r y syndrome, were examined by Faurbye (1960) and c o l l e a g u e s who i n i t i a l l y c a l l e d these o ra l d y s k i n e s i a s , " t a r d i v e d y s k i n e s i a " (TD), meaning an i a t r o g e n i c d i s e a s e caused by c h r o n i c n e u r o l e p t i c t reatment (Uhrbrand and Faurbye , 1960). By comparing groups of p a t i e n t s , t r e a t e d and unt reated wi th n e u r o l e p t i c a g e n t s , a s i g n i f i c a n t d i f f e r e n c e was found i n the i n c i d e n c e o f TD between the two groups and the s e v e r i t y and occur rence o f TD was r e l a t e d to the length of c h r o n i c n e u r o l e p t i c t reatment (Crane, 21 1968). The most f requent form of TD i s the o ra l syndrome, i n v o l v i n g a b n o r m a l i t i e s of movement i n the tongue , l i p s and f a c i a l muscles w i t h r e s p i r a t i o n and speech being i n v o l v e d i n more severe forms (Crane and Naranjo , 1971; Maxwell et a l . , 1970) . O c c a s i o n a l l y , the upper and lower e x t r e m i t i e s are i n v o l v e d , i n p a r t i c u l a r i r r e g u l a r , s low and rhythmic movements of the f i n g e r s , w r i s t s , toes and ankles (Crane and Naranjo , 1971) . Al though p r a c t i c a l l y a l l n e u r o l e p t i c s p r e s e n t l y being used cause s i d e e f f e c t s , some a g e n t s , such as the exper imental drugs c l o z a p i n e and t h i o r i d a z i n e a re p o s s i b l e e x c e p t i o n s , s i n c e no symptoms o f TD have been reported in severa l s t u d i e s of p a t i e n t s r e c e i v i n g these drugs (Sayers et a l . , 1975; Tarsy and B a l d e s s a r i n i , 1976) . Even though the onset of TD begins d u r i n g drug t r e a t m e n t , i n most p a t i e n t s the symptoms of TD become f u l l y mani fested f o l l o w i n g drug withdrawal (Degwitz et a l . , 1967). A f t e r the d i s c o n t i n u a t i o n of a l l drugs the symptoms of TD can p e r s i s t f o r prolonged and i n d e f i n i t e per iods of t ime i n a f f l i c t e d p a t i e n t s (Crane, 1971; Degwitz et a l . , 1967; Hunter et a l . , 1964; Schmidt and J a r c h o , 1966) . Tard ive d y s k i n e s i a i s the most s e r i o u s n e u r o l o g i c a l d i s o r d e r i n p a t i e n t s t r e a t e d wi th n e u r o l e p t i c drugs f o r long pe r iods of t i m e . However i n a d d i t i o n to TD, n e u r o l e p t i c s have been shown to produce a v a r i e t y of o ther ex t rapyramida l s i d e e f f e c t s i n p a t i e n t s t r e a t e d w i th t h e s e a g e n t s , many of which are r e v e r s i b l e . These i n c l u d e drug - induced Park insonism ( t remor , b r a d y k i n e s i a , r i g i d i t y ) , a k a t h i s i a , a k i n e s i a , acute d y s t o n i a , acute j u v e n i l e and t r a n s i e n t d y s k i n e s i a s , h y p o t o n i a , t remor and p o s s i b l y encephalopath ies ( chorea , d y s t o n i a and b a l l i s m u s ) ( B a l d e s s a r i n i 22 and T a r s y , 1980; Crane, 1975; Marsden and Jenner , 1980). The age and sex of the p a t i e n t are thought to i n f l u e n c e which n e u r o l o g i c a l s i d e e f f e c t s induced by c h r o n i c n e u r o l e p t i c t reatment are l i k e l y t o o c c u r . For example, o l d e r p a t i e n t s are more prone to P a r k i n s o n i s m , w h i l e acute dys ton ias are more common i n younger p a t i e n t s . Even though f u r t h e r i n v e s t i g a t i o n s are r e q u i r e d , i t appears t h a t TD, a l though present i n every age group, i s more common i n e l d e r l y female p a t i e n t s (Crane, 1975) . I I . B r a i n Mechanisms Under l y ing Ta rd i ve D y s k i n e s i a Permanent s t r u c t u r a l a l t e r a t i o n s i n the b r a i n have been specu la ted as p o s s i b l y u n d e r l y i n g the b a s i s of TD because of the prolonged and o f t e n i r r e v e r s i b l e nature of t h i s d i s o r d e r . Although a b n o r m a l i t i e s i n the basal g a n g l i a , i n c l u d i n g neuronal degenerat ion and g l i o s i s , have been reported i n e l d e r l y p a t i e n t s w i th TD (Gross and K a l t e n b a c h , 1968) , most n e u r o p a t h o l o g i c a l s t u d i e s have f a i l e d to i n d i c a t e s t r u c t u r a l changes ( C o l o n , 1975; Hunter et a l . , 1968) . S ince degenerat i ve changes have been repor ted i n e l d e r l y i n d i v i d u a l s w i thout ex t rapyramida l d i s o r d e r s , i n c l u d i n g TD f o l l o w i n g c h r o n i c n e u r o l e p t i c t reatment ( F o r r e s t et a l . , 1963) , i t has been suggested tha t the reported changes are p o s s i b l y due to i n c r e a s i n g age and u n r e l a t e d to the e f f e c t s of n e u r o l e p t i c d rugs . P r e s e n t l y t h e r e are very few e l e c t r o n m i c r o s c o p i c examinat ions of b r a i n t i s s u e f o l l o w i n g prolonged t reatment w i th n e u r o l e p t i c s ; p o s s i b l y t h e r e e x i s t s an important but so f a r undetected s t r u c t u r a l change i n e i t h e r the DA neuron or the p o s t s y n a p t i c neuron. An i n depth neurochemical examinat ion of b r a i n s from p a t i e n t s w i th TD has not yet been done, but a l t e r a t i o n s i n n e u r o t r a n s m i t t e r f u n c t i o n p o s s i b l y u n d e r l y i n g the b a s i s of t h i s d i s o r d e r are p o s t u l a t e d from f i n d i n g s 23 in o t h e r , s i m i l a r n e u r o l o g i c a l d i s e a s e s and from animal exper iments . Because DA has been shown to p lay an important r o l e i n many d i s o r d e r s o f the ex t rapyramida l system, i t has a l s o been i m p l i c a t e d in the pathophys io logy of TD. For example, i t has been reported ( B i r d et a l . , 1977) but not c o n f i r m e d , tha t the l e v e l s of DA and i t s m e t a b o l i t e , h o m o v a n i l l i c a c i d , may be inc reased i n s c h i z o p h r e n i c p a t i e n t s exposed t o c h r o n i c n e u r o l e p t i c t r e a t m e n t . With the recent development of more s o p h i s t i c a t e d b iochemica l t e c h n i q u e s , DA mechanisms i n the b r a i n have been shown to be i n v o l v e d i n the development and p o s s i b l e p e r s i s t e n t nature of TD. For example, s t u d i e s i n animals have shown that an i n t e r r u p t i o n of the n i g r o s t r i a t a l pathway w i th techn iques such as 6-OHDA treatment or n e u r o l e p t i c drug t r e a t m e n t , w i l l cause s u p e r s e n s i t i v e DA recepto rs due to denervat ion and d i s u s e (Burt et a l . , 1976b; Klawans and R u b o v i t s , 1972; Ungers tedt , 1971a; Yarbrough, 1975). S ince TD symptoms are accentuated both by L-DOPA and DA recepto r a g o n i s t s (Hershon et a l . , 1972; Jacobson et a l . , 1974; Klawans, 1973) and reduced by DA recepto r a n t a g o n i s t s , DA s y n t h e s i s b l o c k e r s and DA d e p l e t i n g drugs (Ger lach et a l . , 1974; Kazamatsuri et a l . , 1972) , DA recepto r s u p e r s e n s i t i v i t y may u n d e r l i e the pathophys io logy of t h i s d i s o r d e r , a s u p e r s e n s i t i v i t y presumably induced by the a b i l i t y of n e u r o l e p t i c s to b lock DA r e c e p t o r s . L i k e w i s e , the basal g a n g l i a o f s c h i z o p h r e n i c p a t i e n t s exposed to c h r o n i c n e u r o l e p t i c t reatment have been reported to c o n t a i n i n c r e a s e numbers of DA r e c e p t o r s from recepto r l a b e l i n g techn iques (Lee and Seeman, 1980; M u l l e r and Seeman, 1978; Owen et a l . , 1978; Snyder , 1981) . These r e s u l t s need to be r e p l i c a t e d and examined more i n t e n s i v e l y s i n c e i n a few p a t i e n t s not exposed to prolonged n e u r o l e p t i c t reatment t h e r e a l s o 24 appeared to be an i n c r e a s e i n the number of DA receptors (MacKay et a l . , 1980; Owen et a l . , 1978) . Although n e u r o l e p t i c - i n d u c e d s u p e r s e n s i t i v i t y may be an important mechanism i n the development of TD, i t s r e l a t i v e l y shor t l i v e d e f f e c t i n many s p e c i e s makes i t u n l i k e l y t h a t i t i s the major mechanism i n v o l v e d i n the prolonged and sometimes i r r e v e r s i b l e course of TD. The prolonged exposure t o n e u r o l e p t i c s probably r e s u l t s i n numerous compensatory adjustments i n the phys io logy and b i o c h e m i s t r y of DA neurons and o ther c e l l s which they i n t e r a c t w i t h i n the CNS. P o s s i b l y some of these compensatory changes become permanent r e s u l t i n g i n the i r r e v e r s i b l e form of TD i n some p a t i e n t s . A d d i t i o n a l research i s necessary to determine the p r e c i s e changes i n the b r a i n t h a t r e s u l t s in the prolonged and permanent course of TD s i n c e i t appears t h a t in a d d i t i o n to t h e i r b e n e f i c i a l t h e r a p e u t i c e f f e c t s i n s c h i z o p h r e n i a , the r o u t i n e l y used n e u r o l e p t i c agents may a l s o induce i r r e v e r s i b l e n e u r o t o x i c or degenerat ive changes i n the CNS. I I I . Animal Models of Tard ive D y s k i n e s i a Animal models of v a r y i n g degrees of re levance have been u t i l i z e d t o study the mechanism of a c t i o n of n e u r o l e p t i c s in i nduc ing movement d i s o r d e r s s i m i l a r to TD. The a b i l i t y to reproduce the n e u r o l o g i c a l d i s o r d e r of TD i n l a b o r a t o r y animals f o l l o w i n g c h r o n i c n e u r o l e p t i c t reatment would f u r t h e r support the drug e t i o l o g y theory of TD, but so f a r t h e r e has only been l i m i t e d s u c c e s s . A f e a t u r e of the animal model t h a t i s s i m i l a r to TD i n man i s the demonstrat ion tha t the a d m i n i s t r a t i o n of a n e u r o l e p t i c drug p r i o r to apomorphine i n h i b i t s the appearance of s tereotyped behav ior (Tarsy and B a l d e s s a r i n i , 1974) . Even though animal 25 models of TD may e l u c i d a t e some of the chemical mechanisms of TD, these models d i s p l a y severa l d i f f e r e n c e s from t h i s n e u r o l o g i c a l d i s o r d e r i n man. For example, f o l l o w i n g prolonged n e u r o l e p t i c t reatment i n r a t s , n e u r o l o g i c a l symptoms are not seen except when the animals are c h a l l e n g e d w i th agents which d i r e c t l y or i n d i r e c t l y s t i m u l a t e DA a c t i o n (Klawans and R u b o v i t z , 1972; Tarsy and B a l d e s s a r i n i , 1974) . For example, apomorphine-induced "vacous chewing" was observed i n r a t s d u r i n g prolonged n e u r o l e p t i c t reatment (Waddington and Gamble, 1981) . Oral d y s k i n e s i a s have been produced spontaneously dur ing prolonged a d m i n i s t r a t i o n of n e u r o l e p t i c drugs i n monkeys and r a t s (Clow et a l . , 1979; Gunne and Barany , 1976; Sahakian et a l . , 1976; Weiss et a l . , 1977) . For example, o r a l - f a c i a l and l imb d y s k i n e s i a s appear i n rhesus monkeys d u r i n g long term ch lorpromazine t reatment and p e r s i s t e d f o r th ree months a f t e r drug withdrawal (McKinney et a l . , 1980) . S i m i l a r i l y , spontaneous mouth movements were observed in r a t s f o l l o w i n g withdrawal from c h r o n i c t r i f l u o p e r a z i n e or t h i o r i d a z i n e t reatment (Clow et a l . , 1979) . S ince the p e r s i s t e n c e of TD has only been observed i n man, f u r t h e r i n v e s t i g a t i o n s are requ i red t o e i t h e r produce t h i s phenomenon i n animal spec ies or t o understand why the sometimes i r r e v e r s i b l e nature o f TD i s unique t o man. Summary The b i o c h e m i c a l , p h a r m a c o l o g i c a l , b e h a v i o r a l , p h y s i o l o g i c a l and c l i n i c a l s t u d i e s of the n e u r o l o g i c a l e f f e c t s of the n e u r o l e p t i c agents i n the CNS have c o n t r i b u t e d to a g rea te r understanding of t h e i r mechanisms of a c t i o n . The major a c t i o n of the n e u r o l e p t i c drugs seems to be t o b l o c k s e l e c t i v e l y the a c t i o n of DA as a n e u r o t r a n s m i t t e r i n v a r i o u s reg ions of the b r a i n . The a n t i p s y c h o t i c e f f e c t s of n e u r o l e p t i c s have been 26 hypothesized to r e f l e c t t h e i r ant idopamine e f f e c t s i n the l i m b i c f o r e b r a i n , whereas the ex t rapyramida l and e n d o c r i n o l o g i c a l s i d e e f f e c t s of n e u r o l e p t i c s are probably due to t h e i r a c t i o n i n the basal g a n g l i a and hypothalamus r e s p e c t i v e l y . S ince the d i s c o v e r y of these compounds, p s y c h i a t r y has advanced tremendously and unto ld number of p a t i e n t s who r o u t i n e l y make use of these drugs have been permi t ted to f u n c t i o n more "normal l y " i n s o c i e t y . However d e s p i t e a l l t h e i r b e n e f i c i a l a c t i o n s , numerous s i d e e f f e c t s r e s u l t from the use of n e u r o l e p t i c agents . With the c o n t i n u i n g i n v e s t i g a t i o n s i n severa l s c i e n t i f i c d i s c i p l i n e s , i t i s to be hoped t h a t the n e u r o l o g i c a l s i d e e f f e c t s of these drugs i n p a t i e n t s w i l l one day be c o n t r o l l e d and tha t new and more s p e c i f i c a n t i p s y c h o t i c agents w i l l be developed which produce fewer unwanted s i d e e f f e c t s . 27 STATEMENT OF PURPOSE In the present i n v e s t i g a t i o n the behav io ra l and b iochemica l e f f e c t s o f dopamine b lockade by the long term n e u r o l e p t i c t reatment on p o s t s y n a p t i c dopamine r e c e p t o r s was a n a l y z e d . A l a r g e amount of i n v e s t i g a t i o n has been undertaken i n t h i s area r e s u l t i n g i n the g e n e r a l l y accepted view t h a t p o s t s y n a p t i c dopamine recepto r s u p e r s e n s i t i v i t y ensues f o l l o w i n g c h r o n i c n e u r o l e p t i c t reatment . However, each l a b o r a t o r y has examined the e f f e c t s of one or two doses w i t h one or two d u r a t i o n s of n e u r o l e p t i c t reatment and examined e i t h e r b e h a v i o r a l l y or b i o c h e m i c a l l y the e f f e c t s on dopamine r e c e p t o r s i n the CNS. Because o f v a r i a t i o n s t h a t e x i s t between the i n d i v i d u a l s t u d i e s , i n c l u d i n g d i f f e r e n c e s i n animal s p e c i e s and s t r a i n , the dosage and d u r a t i o n of drug t r e a t m e n t , the type o f n e u r o l e p t i c and the d i f f e r e n t behav io ra l and b iochemica l paradigms u t i l i z e d , i t i s d i f f i c u l t to compare r e s u l t s between the va r ious l a b o r a t o r i e s . In o ther words, the r e l a t i o n s h i p between the dosage and d u r a t i o n of c h r o n i c n e u r o l e p t i c a d m i n i s t r a t i o n and the ex tent and d u r a t i o n of DA recepto r s u p e r s e n s i t i v i t y remains p o o r l y understood. With a view to address ing t h i s problem, a study was conducted us ing severa l dosages and d u r a t i o n s o f drug t reatment and then examining the r e s u l t i n g e f f e c t s a t va r ious i n t e r v a l s f o l l o w i n g withdrawal from treatment u t i l i z i n g both behav io ra l and b iochemica l paradigms. 28 GENERAL MATERIALS AND METHODS Subjects Male W i s t a r r a t s (Canadian Breeding L a b o r a t o r i e s ; L a P r a i r i e , Quebec) weighing 225-275 grams were used i n a l l exper iments . The animals were housed i n groups of 5 in s t a i n l e s s s t e e l w i r e cages w i th f r e e access to food and water i n a temperature c o n t r o l l e d room (22°-25°C) and mainta ined on a 12 hour l i g h t / d a r k c y c l e (8 :30 a.m. to 8 :30 p . m . ) , beg inn ing one week before the exper iment . Drug Treatment Groups of r a t s (n = 8) were t r e a t e d c h r o n i c a l l y w i th v a r i o u s doses of p imoz ide . The c h r o n i c pimozide i n j e c t i o n s were admin i s te red to r a t s i n t r a p e r i t o n e a l l y tw ice a day at 8 :00 a.m. and 6:00 p.m. Contro l animals rece i ved the same number of v e h i c l e i n j e c t i o n s at the same t ime i n t e r v a l s and d u r a t i o n s as the exper imental r a t s . Pimozide i n a 1:6 r a t i o w i th t a r t a r i c a c i d was d i s s o l v e d i n hot (100°C) d i s t i l l e d water . Contro l animals rece i ved the v e h i c l e s o l u t i o n , t a r t a r i c a c i d . The body weights of the animals were monitored throughout the course of t reatment and were not found t o d i f f e r s i g n i f i c a n t l y from the c o n t r o l group i n any exper iment . At d i f f e r e n t withdrawal t imes f o l l o w i n g the l a s t t r e a t m e n t , behav io ra l and b iochemical t e s t s were c a r r i e d ou t . Procedures ( I ) Locomotor A c t i v i t y S i x photoactometer cages (BRS Fo r inger #PAC-001) measuring 61cm i n d iameter w i th 43 cm high w a l l s and t r a n s e c t e d by 6 i n f r a r e d p h o t o c e l l beams were used. I n t e r r u p t i o n of the l i g h t beams incremented e lec t romechan ica l 29 c o u n t e r s , l o c a t e d in an adjacent room. P h o t o c e l l beam i n t e r r u p t i o n s were cumulated over per iods of 10 minutes and then recorded by an automat ic p r i n t o u t counter (BRS Fo r inger #P0S-112). The p h y s i c a l environment in the room c o n s i s t e d of whi te no i se and temperature rang ing between 21°-24°C. ( I I ) . Stereotypy Stereotyped behavior was determined by p l a c i n g animals i n d i v i d u a l l y i n r e c t a n g u l a r cages (42 x 36 x 18 cm) wi th w i re mesh f l o o r s and w a l l s . S tereotypy was assessed by means of a r a t i n g s c a l e , p r e v i o u s l y developed from observa t ions of the behav ior of normal r a t s exposed to i n c r e a s i n g doses of amphetamine, (Creese and I v e r s e n , 1975a). 0 as leep or s t a t i o n a r y 1 a c t i v e 2 predominant ly a c t i v e but w i t h b u r s t s of s te reotyped s n i f f i n g or r e a r i n g 3 s tereotyped a c t i v i t y such as s n i f f i n g or r e a r i n g over a l a r g e area of the cage 4 s tereotyped s n i f f i n g or r e a r i n g mainta ined i n one l o c a t i o n 5 s te reotyped behav ior i n one l o c a t i o n w i th b u r s t s of l i c k i n g or gnawing 6 c o n t i n u a l l i c k i n g or gnawing of the cage bars Observat ions of each animal were made at 15 minute i n t e r v a l s f o r 30 seconds , s t a r t i n g 15 minutes a f t e r the drug i n j e c t i o n . Stereotypy was ra ted and any a d d i t i o n a l behav iors such as grooming, sway ing , yawning , f r e e z i n g and l e a p i n g were noted . ( I I I ) . Dopamine Receptor B ind ing The l i g a n d used f o r the dopamine receptor b i n d i n g assay was the 30 dopamine a n t a g o n i s t ( 3 H ) ~ s p i r o p e r i d o l . The b i n d i n g procedure u t i l i z e d was s i m i l a r t o t h a t used by Burt et a l . (1975; 1976) and Creese and Snyder (1977) w i th some m o d i f i c a t i o n s . T i ssue P r e p a r a t i o n Rats were s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n and the b r a i n s were immediately removed and p laced on i c e . Both the l e f t and r i g h t s t r i a t a were d i s s e c t e d from the b r a i n s , weighted and then f rozen at -80°C u n t i l assayed . F reez ing the t i s s u e f o r per iods of up to 4 months at -80°C d i d not a l t e r the amount of b i n d i n g when compared to f r e s h t i s s u e . The t i s s u e was i n i t i a l l y homogenized i n 50 volumes of i c e c o l d 50 mM T r i s b u f f e r , pH 7.7 at 25°C, w i th a Brinkmann Po ly t ron PT-10 ( s e t t i n g 7 , 10 seconds ) . A f t e r homogenizat ion , an a d d i t i o n a l 50 volumes of i c e c o l d b u f f e r was added to the homogenate, vor texed and then c e n t r i f u g e d at 27,000 x g (15,000 r .p .m. ) f o r 15 minutes at 4°C ( S o r v a l l RC-5B; S o r v a l l Rotor SS-3 4 ) . The supernatant was d i s c a r d e d and the p e l l e t was again homogenized i n 50 volumes of b u f f e r , d i l u t e d f u r t h e r by the a d d i t i o n of 50 more volumes of b u f f e r and c e n t r i f u g e d . The supernatant was again d i s c a r d e d and the f i n a l p e l l e t was homogenized i n 200 volumes of c o l d f r e s h l y prepared 50 mM T r i s b u f f e r c o n t a i n i n g 0 .1% a s c o r b i c a c i d , 10 yM p a r g y l i n e and the f o l l o w i n g i o n s : 120 mM N a C l , 5 mM KCI , 2 mM C a C l 2 , 1 mM MgS04 t o g i ve a f i n a l pH of 7.1 at 37°C. The f i n a l t i s s u e c o n c e n t r a t i o n was 5 mg/ml based on o r i g i n a l wet we ight . [ 3 H ] - S p i r o p e r i d o l Receptor B i n d i n g Assay [ 3 H ] - S p i r o p e r i d o l (21 Ci/mmol) was obta ined from Amersham Corpora t ion and s to red under n i t r o g e n at -20°C i n the d a r k . [ 3 H ] - S p i r o p e r i d o l was d i l u t e d on the day of use wi th 0.1% a s c o r b i c a c i d t o g ive a f i n a l 31 4 c o n c e n t r a t i o n of 2 .2 nM - 2 .4 nM f o r i n d i v i d u a l t i s s u e b i n d i n g de te rminat ion and ranging from 0.04 nM - 4 . 0 nM f o r Scatchard A n a l y s i s . G lass d i s p o s a b l e (10 x 75 mm) i n c u b a t i o n t u b e s , i n d u p l i c a t e r e c e i v e d i n o r d e r : 100 y l [ 3 H ] - s p i r o p e r i d o l , 100 y l dopamine ( I O - 3 M) f o r n o n s p e c i f i c b i n d i n g or the drug s o l v e n t 0 .1% a s c o r b i c a c i d , and 800 y l of the t i s s u e suspension to g i ve a t o t a l volume of 1 m l . B ind ing was a l lowed to reach e q u i l i b r i u m by i n c u b a t i n g the tubes a t 37°C f o r 15 minutes w i t h cont inuous shaking and then r a p i d l y f i l t e r e d under vacuum through Whatman GF/B f i l t e r s w i t h th ree 5 ml r i n s e s i f i c e c o l d T r i s b u f f e r , pH 7.7 a t 25°C. The e n t i r e s e p a r a t i o n procedure ( f i l t r a t i o n and three r i n s e s ) was es t imated to r e q u i r e 5 seconds. The [ 3 H ] - s p i r o p e r i d o l t rapped on the f i l t e r s was counted by l i q u i d s c i n f J i l l a t i o n spectrometry at an e f f i c i e n c y of 28% a f t e r remaining f o r s i x hours i n g l a s s s c i n t i l l a t i o n v i a l s , c o n t a i n i n g 10 mis of Amersham ACS s c i n t i l l a t i o n f l u i d . S a t u r a b l e or s p e c i f i c b i n d i n g o f [ H ] - s p i r o p e r i d o l was d e f i n e d as the t o t a l bound minus the n o n s p e c i f i c bound i n the presence of an excess of n o n r a d i o a c t i v e dopamine ( I O - 3 M). The t o t a l bound r a d i o a c t i v i t y was always l e s s than 10% o f the t o t a l [ 3 H ] - s p i r o p e r i d o l added to the assay . S p e c i f i c b i n d i n g of the [ 3 H ] - l i g a n d to s t r i a t a l membranes was about 75-80% of the t o t a l bound f o r [ 3 H ] - s p i r o p e r i d o l . In a l l an imals the l e f t s t r i a t u m was assayed i n d i v i d u a l l y a long w i t h c o n t r o l s t r i a t a w i t h [ 3 H ] - s p i r o p e r i d o l . The c o n t r o l group was randomized w i t h 8 s t r i a t a from d i f f e r e n t withdrawal pe r iods and assayed along w i t h each exper imenta l group. There was no s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t of wi thdrawal from c h r o n i c v e h i c l e t reatment on [ 3 H ] - s p i r o p e r i d o l b i n d i n g i n c o n t r o l a n i m a l s . For de te rminat ion o f the amount bound to the t i s s u e , 32 exper imental t i s s u e was compared to c o n t r o l samples , assayed at the same t ime and c a l c u l a t e d as a percentage of t h a t c o n t r o l . For some groups , the r i g h t s t r i a t a were pooled together from 2 or 3 animals and used f o r Scatchard A n a l y s i s (Rosentha l , 1967; S c a t c h a r d , 1949) , w i t h the d e t e r m i n a t i o n of r e c e p t o r number, Bmax ( in pmol/g wet weight t i s s u e ) and recepto r a f f i n i t y KD ( i n nM) f o r exper imental and c o n t r o l groups be ing determined by the method o f l e a s t squares l i n e a r r e g r e s s i o n . Drugs The f o l l o w i n g compounds were obta ined from the f o l l o w i n g companies: p imozide (Janssen Pharmaceut i ca ) , d-amphetamine s u l f a t e (Smi th , K l i n e and F r e n c h ) , apomorphine HC1 (Sigma C o r p o r a t i o n ) , dopamine (Ca lb iochem) , and [ 3 H ] - s p i r o p e r i d o l (Amersham C o r p o r a t i o n ) . A l l drug s o l u t i o n s were f r e s h l y prepared immediately before use. Pimozide i n a 1:6 r a t i o w i t h t a r t a r i c a c i d was d i s s o l v e d i n hot ( lOO'C) d i s t i l l e d water . d-Amphetamine was d i s s o l v e d i n d i s t i l l e d water . Apomorphine was d i s s o l v e d i n d i s t i l l e d water c o n t a i n i n g 0.3% a s c o r b i c a c i d w h i l e dopamine and [ 3 H ] - s p i r o p e r i d o l were d i s s o l v e d i n d i s t i l l e d water c o n t a i n i n g 0 .1% a s c o r b i c a c i d . S t a t i s t i c s The behav io ra l and b iochemical data were analyzed s t a t i s t i c a l l y us ing repeated measures a n a l y s i s o f v a r i a n c e . For pos t -hoc comparisons between i n d i v i d u a l groups the Newman-Keuls t e s t and the S t u d e n t ' s t - t e s t were used f o r the locomotor a c t i v i t y d a t a ; the Mann-Whitney U - t e s t was used f o r the s te reo typy data and the S t u d e n t ' s t - t e s t was used f o r the b iochemical d a t a . 33 RESULTS EXPERIMENT 1  P r e l i m i nary Experiments P r i o r to the paramet r i c t ime course s t u d i e s , t h r e e p r e l i m i n a r y experiments were performed. The dose response curves f o r d-amphetamine 0 (Experiment IA) and apomorphine (Experiment IB) were requ i red to determine the dose of these drugs to be used i n subsequent s t u d i e s t h a t would g i ve the g r e a t e s t d i f f e r e n c e i n behav io ra l responses between n e u r o l e p t i c t r e a t e d and c o n t r o l g roups . A lso s i n c e apomorphine induced s tereotyped behav ior was examined 24 hours a f t e r the same animals had rece ived a dose of d -amphetamine, the p o s s i b l e e f f e c t of p r i o r d-amphetamine a d m i n i s t r a t i o n on apomorphine induced s te reo typy was examined (Experiment IC ) . Experiment IC was c a r r i e d out to i n v e s t i g a t e whether p r i o r d-amphetamine a d m i n i s t r a t i o n had an e f f e c t on apomorphine induced s t e r e o t y p y . Methods Experiment IA Groups of r a t s (N = 8) were c h r o n i c a l l y t r e a t e d wi th e i t h e r pimozide (1 .5 mg/kg) or a v e h i c l e s o l u t i o n , t w i c e d a i l y f o r 10 days and then 4 , 1 0 , 20 and 40 days f o l l o w i n g c e s s a t i o n from t h i s t reatment locomotor a c t i v i t y in response to d i f f e r e n t doses of d-amphetamine (1 .0 mg/kg, 2 .0 mg/kg, 4 .0 mg/kg) was examined. I n i t i a l l y the animals were p laced i n d i v i d u a l l y i n the p h o t o c e l l a c t i v i t y cages at e i t h e r 9:00 a .m. or 1:00 p.m. f o r a 1 hour h a b i t u a t i o n p e r i o d . The r a t s were then i n j e c t e d i n t r a p e r i t o n e a l l y w i th d -amphetamine and immediate ly rep laced i n the a c t i v i t y cages f o r an a d d i t i o n a l 3 hour p e r i o d . The a c t i v i t y was recorded at 10 minute i n t e r v a l s dur ing both h a b i t u a t i o n and f o l l o w i n g the i n j e c t i o n of d-amphetamine. Each 34 animal rece i ved one i n j e c t i o n of d-amphetamine and was t e s t e d on ly once. Experiment IB Groups of r a t s (N = 8) were t e s t e d wi th an i n t r a p e r i t o n e a l dose of apomorphine (0 , .375 mg/kg, .75 mg/kg, 1.5 mg/kg, 3 .0 mg/kg, 6 .0 mg/kg) a f t e r the animals were hab i tuated t o the r e c t a n g u l a r cages f o r 1 hour . The r e s u l t i n g s tereotyped behav ior was then q u a n t i f i e d f o r 1 - 1.5 hours at 15 minute i n t e r v a l s . Experiment IC Groups of r a t s (N = 8) were c h r o n i c a l l y t r e a t e d wi th e i t h e r pimozide (1 .5 mg/kg) or a v e h i c l e s o l u t i o n , t w i c e d a i l y f o r 10 days and then 4 days a f t e r c e s s a t i o n from t r e a t m e n t , locomotor a c t i v i t y was examined f o l l o w i n g e i t h e r a dose of d-amphetamine (2 .0 mg/kg) or a v e h i c l e s o l u t i o n . The p r o t o c o l used f o r measuring locomotor a c t i v i t y was the same as f o r experiment IA. On the subsequent day (24 hours l a t e r ) , a l l an imals were t e s t e d f o r s te reotyped behav ior f o l l o w i n g a dose of 0 .75 mg/kg apomorphine. The p ro toco l used f o r measuring s te reo typy was the same as t h a t used f o r Experiment IB. R e s u l t s Experiment IA The t ime course of the locomotor a c t i v i t y induced by each dose of d -amphetamine i n j e c t e d 4 days a f t e r the l a s t pimozide or v e h i c l e t reatment i s d e p i c t e d i n F igure 1. Dur ing the 1 hour h a b i t u a t i o n p e r i o d , the locomotor a c t i v i t y of the pimozide p r e t r e a t e d and c o n t r o l groups d i d not d i f f e r from one another . Between the g roups , 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 d i f f e r e n c e was obta ined w i th the 2 .0 mg/kg dose of d-amphetamine (F = 8 . 9 2 , d f = 1 , 14, p < 0.01) but not w i th a lower dose of 1.0 mg/kg d-amphetamine (F = 1 . 8 8 , df = 1 , 14, p > 0.1) nor wi th a h igher dose of 4 . 0 mg/kg 35 F igure 1 The e f f e c t s of c h r o n i c pimozide a d m i n i s t r a t i o n ( 1 . 5 mg/kg twice/day f o r 10 days) on the locomotor s t i m u l a n t e f f e c t of severa l doses of d-amphetamine s u l f a t e . d-Amphetamine was a d m i n i s t e r e d f o l l o w i n g a 1 hour h a b i t u a t i o n pe r iod 4 days f o l l o w i n g withdrawal from c h r o n i c pimozide t reatment . The h a b i t u a t i o n curves are the mean response o f a l l p imozide o r v e h i c l e a n i m a l s . Each p o i n t represents the mean response of a group of animals (n=8). 35a 1Cf-8 CM O 6 0) r- 2 O Ol2 PIMOZIDE V E H I C L E HABITUATION • • • • i i I I I I I d-AMPHETAMINE 2.0mg/kg d-AMPHETAMINE 1.0 mg/kg d-AMPHETAMINE 4-0 mg/kg i ,i i i i i i 3 0 6 0 90 120 150 180 30 60 90 120 150 180 TIME (minutes) 36 d-amphetamine (F = 0 . 0 6 , df = 1 , 14, p > 0 . 1 ) . A s i g n i f i c a n t e f f e c t over the t ime per iods was observed f o l l o w i n g a l l doses of d-amphetamine ( 1 . 0 mg/kg - F = 3 3 . 1 2 , df = 17, 238, p < 0 . 0 0 5 ; 2 .0 mg/kg - F = 3 2 . 2 9 , df = 17 , 238, p < 0 . 0 0 5 ; 4 .0 mg/kg - F = 3 . 4 9 , df = 17 , 238, p < 0 . 0 0 5 ) , r e f l e c t i n g the p r o g r e s s i v e decrease of the r a t s ' a c t i v i t y i n both groups. In a d d i t i o n a s i g n i f i c a n t o v e r a l l group by t ime i n t e r a c t i o n was observed on ly f o r the two h igher doses of d-amphetamine (1 .0 mg/kg - F = 0 . 8 6 4 , df = 1 7 , 2 3 8 , p > 0 . 1 ; 2 .0 mg/kg - F = 2 . 1 3 , df = 17 , 238, p < 0 . 0 1 ; 4 .0 mg/kg - F = 6 . 5 5 , d f = 17, . 238, p < 0 . 0 0 5 ) . From F i g u r e 1 , i t i s c l e a r t h a t w i th a dose of 2 .0 mg/kg d-amphetamine, the i n t e r a c t i o n i s a . r e s u l t of the pimozide p r e t r e a t e d animals being more a c t i v e than c o n t r o l s d u r i n g the f i r s t 90 minutes f o l l o w i n g the i n j e c t i o n o f d-amphetamine and not s i g n i f i c a n t l y d i f f e r e n t i n the l a t t e r h a l f of the 3 hour s e s s i o n . For the h ighest dose of d-amphetamine (4 .0 mg/kg), t h i s i n t e r a c t i o n i s due to the o b s e r v a t i o n tha t the pimozide p r e t r e a t e d animals locomotor a c t i v i t y was v e r y d i f f e r e n t from the c o n t r o l s m o t i l i t y , e s p e c i a l l y d u r i n g the peak of the d-amphetamine e f f e c t . F igu re 2 shows the dose response curve of the e f f e c t t h a t d i f f e r e n t doses of d-amphetamine have on t o t a l (3 hours) locomotor a c t i v i t y i n animals t r e a t e d c h r o n i c a l l y w i th e i t h e r pimozide or a v e h i c l e s o l u t i o n f o r 10 days and withdrawn from treatment f o r 4 , ' 1 0 , 20 and 40 days . On a l l f o u r withdrawal d a y s , t h e r e was an o v e r a l l s i g n i f i c a n t e f f e c t o f the dose of d-amphetamine used (day 4 - F = 1 4 . 0 4 , df = 2 , 4 2 , p < 0 . 0 0 5 ; day 10 - F = 1 9 . 8 0 , df = 2 , 4 2 , p < 0 . 0 0 5 ; day 20 - F = 8 . 9 6 , df = 2 , 42 , p < 0 . 0 0 5 ; day 40 - F = 7 . 1 1 , df = 2 , 4 2 , p < 0 . 0 0 5 ) . The p o t e n t i a t i n g e f f e c t o f c h r o n i c pimozide pretreatment on h y p e r m o t i 1 i t y induced by d-amphetamine at 4 days f o l l o w i n g t reatment (F = 4 . 5 5 , df = 1 , 4 2 , p < 0.05) i s s t i l l 37 F igure 2 The e f f e c t o f c h r o n i c pimozide a d m i n i s t r a t i o n ( 1 . 5 mg/kg twice/day f o r 10 days) on the t o t a l (3 hours) locomotor a c t i v i t y e l i c i t e d by s e v e r a l doses o f d-amphetamine s u l f a t e examined 4 , 1 0 , 20 and 40 days f o l l o w i n g withdrawal from c h r o n i c t reatment . Each exper imental and c o n t r o l group was examined at a l l f o u r wi thdrawal p e r i o d s . Each p o i n t represents the mean t o t a l locomotor a c t i v i t y (± S . E . M . ) i n groups o f animals (n=8). * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l group us ing t w o - t a i l e d S t u d e n t ' s t - t e s t p<0.025 37a C/>10 CC CO 8 co 6 o X 4| (/) 2 o UJ o o o x 0. 4 P I M O Z I D E V E H I C L E o — o Or -8 W I T H D R A W A L DAY 4 W I T H D R A W A L D A Y 20 W I T H D R A W A L D A Y 10 J L W I T H D R A W A L DAY 40 d-AMPHETAMINE DOSE (mg/kg) 38 ev ident 10 days a f t e r withdrawal (F = 6 . 5 8 , df = 1 , 42 , p < 0 . 0 2 5 ) . By 20 days (F = 0 . 0 1 , df = 1 , 4 2 , p > 0.1) and 40 days (F = 0 . 0 5 , d f = 1 , 42 , p > 0.1) of withdrawal t h e r e was no d i f f e r e n c e between the pimozide p r e t r e a t e d and c o n t r o l qroups locomotor a c t i v i t y . Post hoc a n a l y s i s w i t h the 2 - t a i l e d S t u d e n t ' s t - t e s t , shows t h a t the p o t e n t i a t i n g e f f e c t of c h r o n i c pimozide pretreatment on h y p e r m o t i l i t y over c o n t r o l animals at 4 and 10 days withdrawal was most s i g n i f i c a n t at a dose of 2.0 mg/kg (4 days - t = 2 . 9 7 , df = 14, p < 0 . 0 2 5 ; 10 days - t = 2 . 9 4 , df = 14, p < 0 . 0 2 5 ) . Experiment IB F i g u r e 3 shows the e f f e c t o f i n c r e a s i n g doses of apomorphine on s te reotyped behavior . - There was a s i g n i f i c a n t o v e r a l l e f f e c t of apomorphine dose on s te reotypy (F = 2 9 . 9 6 , df = 5 , 4 2 , p < 0 . 0 0 5 ) . The lower dose of apomorphine ( .75 mg/kg) r e s u l t e d i n an i n c r e a s i n g amount o f locomotor , s n i f f i n g and r e a r i n g b e h a v i o r s , w h i l e the h igher doses (1 .5 mg/kg, 3 . 0 mg/kg, 6 . 0 mg/kg) showed these behav iors a long wi th a g r e a t e r i n c i d e n c e of l i c k i n g , gnawing and jumping b e h a v i o r s . F o l l o w i n g the i n j e c t i o n of apomorphine, the behav ior of a l l an imals returned t o c o n t r o l l e v e l s w i t h i n 60 minutes w i th the except ion of the h ighest doses (3 .0 mg/kg, 6 .0 mg/kg), where the e f f e c t d imin i shed by 90 minutes . The s t e r e o t y p y e l i c i t e d by the lowest dose of apomorphine ( .375 mg/kg) was not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l s (U = 3 2 . 5 , df = 8 . 8 , p > 0 . 1 ) , but at a l l h igher doses t h e r e was a s i g n i f i c a n t d i f f e r e n c e (eg. .75 mg/kg -U = 4 . 5 , d f = 8 , 8 , p < 0 . 0 1 ) . Experiment IC F i g u r e 4 shows the r e s u l t s of c h r o n i c n e u r o l e p t i c pretreatment and/or a s i n g l e i n j e c t i o n of d-amphetamine 24 hours p r i o r to determin ing the 39 F igures 3A-B Dose response curve o f apomorphine- induced s t e r e o t y p y . A. Each p o i n t represents the mean s te reo typy score o f a group of animals (n=8). S tereotypy was ra ted accord ing to the Creese and Iversen (1975) s c a l e . B. Each bar represents the mean t o t a l s te reotypy score (± S . E . M . ) , which i s the sum of the f o u r i n d i v i d u a l scores obta ined dur ing the 60 minute p e r i o d i n groups of animals (n=8). * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l group us ing t w o - t a i l e d Mann-Whitney U - t e s t p<0.02 ** p<0.01 MEAN STEREOTYPY SCORE ro 0*1 ~r > I I I I f f > I I I I I .ri • I I I f I I • I I t I I I 33 3 3 3 3 I D ( D (D ID ID CD ( D I D (DID CO ID • > I I I I • > I • • > T3 O o ID TJ I Z m D O oo m TOTAL STEREOTYPY SCORE/l HOUR 00 ro 00 *6E 40 e f f e c t of apomorphine on s te reotyped b e h a v i o r . There was a s i g n i f i c a n t o v e r a l l e f f e c t of n e u r o l e p t i c pretreatment on s te reotyped behav ior when compared to c o n t r o l an imals (F = 3 8 . 9 7 , df = 1 , 28 , p < 0 . 0 0 5 ) . However i n both the pimozide and v e h i c l e p r e t r e a t e d g roups , p r i o r (24 hours) a d m i n i s t r a t i o n o f d-amphetamine had no e f f e c t on apomorphine- induced s te reo typy (F = 0 . 1 5 ; df = 1 , 2 8 , p > 0 . 1 ) . Post hoc a n a l y s i s w i th the Mann-Whitney U - t e s t conf i rms t h i s r e s u l t showing no d i f f e r e n c e between the pimozide p r e t r e a t e d groups (U = 2 8 . 5 , df = 8 , 8 , p > 0.1) and the v e h i c l e p r e t r e a t e d groups (U = 3 7 . 5 , df = 8 , 8 , p > 0 . 1 ) . 41 F igures 4A-B The e f f e c t of p r i o r (24 hour) a d m i n i s t r a t i o n o f d-amphetamine ( 2 . 0 mg/kg) on apomorphine (0 .75 mg/kg) induced s te reotyped behavior i n c h r o n i c pimozide ( 1 . 5 mg/kg twice/day f o r 10 days) o r v e h i c l e t r e a t e d a n i m a l s . A. Each p o i n t represents the mean s te reo typy score of a group o f animals (n=8). B. Each bar represents the mean t o t a l s te reotypy score obta ined i n 1 hour i n groups of animals (n=8). MEAN STEREOTYPY SCORE a o c/> m x m 2 • ro —r-Ul TOTAL STEREOTYPY SCORE/l HOUR _• __ ' ro ^ oo to o o 05 ~T~ < < T J T I mm — I I I I < ><> o 1 ^ mgmg 2 O N a m 3 CD 3 Ii < m I O r -m 00 42 EXPERIMENT 2 Paramet r i c Time Course Study - E f f e c t of Dose of Pimozide In t h i s experiment the e f f e c t of d i f f e r e n t doses of p i m o z i d e , admin i s te red c h r o n i c a l l y to animals f o r a f i x e d pe r iod (10 days) was examined both b e h a v i o r a l l y and b i o c h e m i c a l l y f o l l o w i n g d i f f e r e n t withdrawal per iods from the drug t r e a t m e n t . Methods Groups of r a t s (N = 8) were c h r o n i c a l l y t r e a t e d w i th e i t h e r pimozide (0 .75 mg/kg, 1.5 mg/kg, 3 .0 mg/kg) or v e h i c l e , t w i c e d a i l y f o r 10 days . Four , 10, 2 0 , and 40 days f o l l o w i n g c e s s a t i o n from treatment d-amphetamine (2 .0 mg/kg) induced locomotor a c t i v i t y was examined. The p r o t o c o l used f o r .measuring locomotor a c t i v i t y was the same'as t h a t used f o r Experiment IA. On the subsequent day , a l l an imals were t e s t e d f o r s te reotyped behavior f o l l o w i n g a dose of 0.75 mg/kg apomorphine. The p ro toco l used f o r measuring s te reo typy was d e s c r i b e d in Experiment IB. Twenty - four hours 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 apomorphine the animals were s a c r i f i c e d and the l e f t and r i g h t s t r i a t a were d i s s e c t e d from the b r a i n , weighed and then f rozen at -80°C u n t i l b i o c h e m i c a l l y assayed . Resu l t s Locomotor A c t i v i t y F i g u r e 5 shows the t ime course of the locomotor a c t i v i t y i n groups of animals withdrawn 4 , 10, 20 and 40 days a f t e r c h r o n i c t reatment w i th doses of p imoz ide . During the 1 hour h a b i t u a t i o n p e r i o d , the locomotor a c t i v i t y of the d i f f e r e n t pimozide t r e a t e d groups d i d not d i f f e r from c o n t r o l groups on any day a f t e r withdrawal (data not shown). Between the groups t h e r e was an o v e r a l l s i g n i f i c a n t e f f e c t of dose (F = 3 . 1 3 , df = 3 , 112, p < 0.05) and 43 of days of withdrawal (F = 1 4 3 . 2 2 , df = 17, 1904, p < 0.001) on locomotor a c t i v i t y . - There was a l s o an o v e r a l l s i g n i f i c a n t e f f e c t over the 3 hour t e s t per iod (F = 6 . 0 4 , df = 3 , 112, p < 0.005) r e f l e c t i n g the p r o g r e s s i v e decrease i n the r a t s ' m o t i l i t y . A s i g n i f i c a n t o v e r a l l i n t e r a c t i o n between the dose of pimozide and withdrawal was observed (F = 3 . 0 7 , df = 3 , 1904, p < 0 . 0 0 5 ) . From F igure 5 i t can be seen tha t t h i s i s due to the observa t ion t h a t c h r o n i c pretreatment w i th the h igher doses of pimozide enhanced locomotor a c t i v i t y f o r longer per iods f o l l o w i n g withdrawal from p r e t r e a t m e n t . For example, c h r o n i c t reatment w i t h 1.5 mg/kg pimozide s t i l l had an e f f e c t on locomotor a c t i v i t y a f t e r 10 days whereas c h r o n i c t reatment w i th 3 .0 mg/kg pimozide s t i l l enhanced m o t i l i t y 20 days a f t e r w i t h d r a w a l . By 40 days the a c t i v i t y of a l l groups were not d i f f e r e n t from c o n t r o l a n i m a l s . F igu re 6 shows the e f f e c t of d i f f e r e n t doses of c h r o n i c pimozide on the t o t a l locomotor a c t i v i t y accumulated d u r i n g the f i r s t 90 minutes (peak d-amphetamine e f f e c t ) of the 3 hour t e s t p e r i o d 4 , 10 , 2 0 , and 40 days of w i t h d r a w a l . During the 1 hour h a b i t u a t i o n p e r i o d , t h e r e was no 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 between exper imental and c o n t r o l groups (F = 0 . 7 4 , df = 3 , 112, p > 0 . 1 ) . S i m i l a r to the t ime course locomotor a c t i v i t y d a t a , t h e r e was an o v e r a l l s i g n i f i c a n t e f f e c t of dose (F = 4 . 4 3 , df = 3 , 112, p < 0.01) as we l l as an e f f e c t of the p e r i o d of withdrawal (F = 4 . 7 4 , df = 3 , 112, p < 0 . 0 0 5 ) . However there was no s i g n i f i c a n t o v e r a l l i n t e r a c t i o n between dose and withdrawal pe r iod (F = 2 . 1 9 , df = 9 , 112, p > 0 . 0 5 ) . Post hoc comparisons w i th the Newman-Keuls t e s t revea led t h a t the h igher doses o f p imozide ( 1 . 5 mg/kg, 3 . 0 mg/kg) had g r e a t e r e f f e c t s than the lower dose of pimozide (0 .75 mg/kg) i n 44 F igures 5A-D The e f f e c t o f c h r o n i c t reatment (10. days) w i t h v e h i c l e or severa l doses o f pimozide on the locomotor s t i m u l a n t e f f e c t s o f d-amphetamine s u l f a t e ( 2 . 0 mg/kg) measured 4 , 1 0 , 20 and 40 days f o l l o w i n g wi thdrawal from c h r o n i c t reatment . d-Amphetamine was admin is te red to animals f o l l o w i n g a 1 hour h a b i t u a t i o n pe r iod to the a c t i v i t y cages. D i f f e r e n t groups of animals were used a t each dose and withdrawal p e r i o d . Each p o i n t represents the mean response o f a group o f animals (n=8). 44a 10 8 PIMOZIDE PRETREATMENT .75 mg/kg A.....A 1.5 mg/kg • • 3.0 mg/kg • • V E H I C L E O O o WITHDRAWAL DAY 4 o o J L i i i i i • l I I 1 1 1 1 L LUiOr O O ? 8[ B WITHDRAWAL DAY 10 j i_ J L i I I L 30 60 90 120 TIME (minutes) 150 180 44b 45 p o t e n t i a t i n g the d-amphetamine e f f e c t . D e t a i l e d post hoc examinat ion betweeen i n d i v i d u a l groups u s i n g the t w o - t a i l e d S tudent ' s t - t e s t , showed t h a t each dose of pimozide used dur ing c h r o n i c pretreatment had a s i g n i f i c a n t e f f e c t on m o t i l i t y 4 days f o l l o w i n g pretreatment (0 .75 mg/kg -t = 3 . 4 3 , df = 14, p < 0 . 0 0 5 ; 1.5 mg/kg - t = 2 . 7 3 , df = 1 4 , p < 0 . 0 2 5 ; 3 .0 mg/kg - t = 2 . 2 3 , d f = 14, p < 0 . 0 5 ) . The m o t i l i t y o f groups p r e t r e a t e d w i th the two h ighest doses of pimozide were s t i l l enhanced 10 days f o l l o w i n g withdrawal ( 1 . 5 mg/kg - t = 2 . 2 1 , d f = 14, p < 0 . 0 5 ; 3 .0 mg/kg -t = 2 . 7 8 , df = 14, p < 0 . 0 2 5 ) , but by 20 days on ly the h ighest dose of pimozide used remained s i g n i f i c a n t (3 .0 mg/kg - t = 2 . 1 5 , d f = 14 , p < 0 . 0 5 ) . For ty days f o l l o w i n g drug withdrawal the e f f e c t of a l l doses of pimozide pretreatment on d-amphetamine induced m o t i l i t y in animals was no d i f f e r e n t from c o n t r o l a n i m a l s . Stereotypy The t o t a l s te reo typy score obta ined from groups of animals d u r i n g the one hour o b s e r v a t i o n p e r i o d a f t e r the a d m i n i s t r a t i o n of apomorphine i s d e p i c t e d i n F igure 7A. There was a ^ s i g n i f i c a n t o v e r a l l e f f e c t of dose of pimozide on the t o t a l s t e r e o t y p y score (F = 6 . 1 4 , df = 3 , 112, p < 0 . 0 0 5 ) . There was no o v e r a l l s i g n i f i c a n t e f f e c t of wi thdrawal p e r i o d (F = 1 . 7 2 , df = 3 , 112, p > 0 . 1 ) ; n e v e r t h e l e s s , by 41 days f o l l o w i n g withdrawal from c h r o n i c n e u r o l e p t i c t r e a t m e n t , t h e r e was no d i f f e r e n c e i n t o t a l s t e r e o t y p y by any of the exper imental groups when compared to c o n t r o l a n i m a l s . F igure 7B shows the peak s te reo typy score a t t a i n e d by groups of animals f o l l o w i n g d i f f e r e n t per iods of withdrawal from c h r o n i c n e u r o l e p t i c t r e a t m e n t . Again t h e r e was an o v e r a l l s i g n i f i c a n t e f f e c t of pimozide dose on the apomorphine- induced s t e r e o t y p y score (F = 6 . 2 1 , df = 3 , 112, p < 0.005) but 46 F igures 6A-B The e f f e c t o f c h r o n i c t reatment (10 days) w i t h v e h i c l e or severa l doses of pimozide on the t o t a l (90 minutes) locomotor a c t i v i t y f o l l o w i n g the a d m i n i s t r a t i o n o f d-amphetamine s u l f a t e ( 2 . 0 mg/kg), examined a t va r ious i n t e r v a l s a f t e r w i t h d r a w a l . A. Each bar represents the mean t o t a l response (± S . E . M . ) i n groups o f animals (n=8). The dot ted l i n e s represent the a c t i v i t i e s o f the c o n t r o l groups t r e a t e d c h r o n i c a l l y w i th a v e h i c l e s o l u t i o n . B. The percent i n c r e a s e of the t o t a l (90 minutes) d-amphetamine-induced locomotor a c t i v i t y i n pimozide t r e a t e d groups compared to c o n t r o l groups. * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l group "using t w o - t a i l e d S tudent ' s t - t e s t p<0.05 ** p<0.025 PHOTOCELL COUNTS (x103) / 90 min. NJ Xk 0) 00 o o o N N N — — — m o D o o m m m mco Vi b oi 0 1 3 3 3 CO (O CD 3T "T (S (O <Q 47 y not of withdrawal from pretreatment (F = 2 . 8 0 , df - 3 , 112, p > 0 . 1 ) . No o v e r a l l s i g n i f i c a n t i n t e r a c t i o n between dose and wi thdrawal p e r i o d occur red w i th e i t h e r the t o t a l s te reo typy (F = 0 . 6 3 ; df = 9 , 112, p > 0.1) or the peak s t e r e o t y p y score (F = 0 . 4 1 , df = 9 , 112, p > 0 . 1 ) . The l a r g e s t dose of pimozide used d u r i n g c h r o n i c pretreatment (3 .0 mg/kg) always tended to have the g r e a t e s t e f f e c t on e i t h e r the t o t a l s te reo typy (F igure 7A) or the peak s te reo typy score (F igure 7B) induced by apomorphine 5 , 1 1 , 2 1 , and 41 days withdrawn from pre t reatment . However t h i s e f f e c t by 3 .0 mg/kg was never 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 induced by apomorphine i n an imals p r e t r e a t e d wi th the lower doses of pimozide (0 .75 mg/kg, 1.5 mg/kg). F o l l o w i n g the i n j e c t i o n of the t e s t dose of apomorphine (0 .75 mg/kg) c o n t r o l an imals p r i m a r i l y showed such s tereotyped behav iors as s n i f f i n g , locomot ion and r e a r i n g w i th o c c a s i o n a l grooming and yawning whereas c h r o n i c pimozide t reatment (0 .75 mg/kg, 1.5 mg/kg, 3 .0 mg/kg), i n c r e a s e d the i n c i d e n c e of l i c k i n g , gnawing and jumping behav iors i n a n i m a l s . The o ra l behav iors of l i c k i n g and gnawing p e r s i s t e d f o r longer per iods of t ime f o l l o w i n g withdrawal from pretreatment with' the h ighest dose of p imozide ( 3 . 0 mg/kg) when compared t o lower doses ( .75 mg/kg, 1.5 mg/kg) but t h i s f a i l e d to reach s t a t i s t i c a l s i g n i f i c a n c e . In a d d i t i o n , many animals (65%) p r e t r e a t e d c h r o n i c a l l y w i th p i m o z i d e , i n p a r t i c u l a r w i th the h ighes t dose of 3 .0 mg/kg, d i s p l a y e d burs ts of chewing jaw movements not on the cage bars but a p p a r e n t l y purpose less d u r i n g the t e s t p e r i o d . This behav ior u s u a l l y ev ident w h i l e the animal was s t a t i o n a r y or l y i n g down, j u s t f a i l e d to reach s t a t i s t i c a l s i g n i f i c a n t l e v e l s (U = 16, df = 8 , 8 , p > 0 . 0 5 ) . Dopamine Receptor B i n d i n g F i g u r e 8 shows the e f f e c t of c h r o n i c pimozide a d m i n i s t r a t i o n on the 48 F igures 7A-B The e f f e c t of c h r o n i c t reatment (10 days) w i th v e h i c l e or severa l doses o f p imozide on apomorphine- induced (0 .75 mg/kg) s t e r e o t y p e d b e h a v i o r , examined at va r ious i n t e r v a l s f o l l o w i n g withdrawal from c h r o n i c t reatment . S tereotypy was ra ted accord ing to the Creese and Iversen (1975) s c a l e . A. Each bar represents the mean t o t a l (1 hour) s t e r e o t y p y score (± S . E . M . ) obta ined by groups o f animals (n=8). Dotted l i n e s represent the t o t a l s te reotypy score obta ined i n c o n t r o l a n i m a l s . B. Each p o i n t represents the mean of the peak s te reotypy score ob ta ined by groups o f animals (n=8). The peak score was determined as the h ighes t s te reotypy r a t i n g g iven to an animal dur ing the 1 hour t e s t p e r i o d . * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l group us ing o n e - t a i l e d Mann-Whitney U - t e s t p<0.05 ** p<0.025 *** p<0.01 **** p<0.005 PIMOZIDE -75 mg/kg PIMOZIDE 15 mg/kg PIMOZIDE 3.0 mg/kg B o I , LU u > ° -3 o LU £ 2 C/) 0. PIMOZIDE PRETREATMENT .75 mg/kg 1.5 mg/kg •- -• 3.0 mg/kg • — • VEHICLE O—O 00 21 41 5 WITHDRAWAL PERIOD (days) ll 21 41 49 changes i n the amount of s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g i n s t r i a t u m r e l a t i v e to c o n t r o l animals 6 , 12 , 22 and 42 days f o l l o w i n g w i t h d r a w a l . There was an o v e r a l l s i g n i f i c a n t e f f e c t of pimozide dose (F = 1 9 . 2 9 , df = 2 , 8 4 , p < 0.001) and of withdrawal (F = 2 7 . 1 0 , df = 3 , 8 4 , p < 0.001) on the a l t e r a t i o n s in recepto r b i n d i n g . No o v e r a l l s i g n i f i c a n t i n t e r a c t i o n occur red between dose of p imozide t reatment and pe r iod of withdrawal on recepto r b i n d i n g (F = 1 . 3 5 , df = 6 , 8 4 , p > 0 . 1 ) . Post hoc comparisons wi th the two t a i l e d S t u d e n t ' s t - t e s t , between groups revea led t h a t a l l doses of pimozide s i g n i f i c a n t l y inc reased recepto r b i n d i n g over c o n t r o l s 6 days f o l l o w i n g p re t reatment . In a d d i t i o n the i n c r e a s e i n recepto r b i n d i n g i n animals t r e a t e d w i th the h ighest dose of pimozide (3 .0 mg/kg) 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 produced by 0.75 mg/kg pimozide (t = 2 . 3 8 , df = 14, p < 0.005) but not w i th 1.5 mg/kg pimozide (t = 0 . 4 5 , df = 14, p > 0 . 1 ) . The enhancement of recepto r b i n d i n g appeared to reach a maximum l i m i t (40-45%) f o l l o w i n g c h r o n i c pimozide t r e a t m e n t , s i n c e both h igher doses of pimozide produced s i m i l a r i n c r e a s e s . Twelve days f o l l o w i n g w i t h d r a w a l , t h e r e was s t i l l a s i g n i f i c a n t i n c r e a s e i n recepto r b i n d i n g over c o n t r o l s r e g a r d l e s s of the dose of p imozide used. Again the i n c r e a s e i n b i n d i n g i n animals t r e a t e d w i th 3 .0 mg/kg pimozide 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 produced by 0 .75 mg/kg pimozide (t = 2 . 9 2 , d f = 1 4 , p < 0.025) but not w i th 1.5 mg/kg pimozide (t = 1 . 8 4 , d f = 14, p > 0 . 0 5 ) . By 22 days the inc reased r e c e p t o r b i n d i n g had returned to c o n t r o l l e v e l s i n animals p r e t r e a t e d wi th the lowest dose of pimozide (0 .75 mg/kg). In animals p r e t r e a t e d wi th 3 .0 mg/kg, the i n c r e a s e i n recepto r b i n d i n g was s i g n i f i c a n t l y g r e a t e r than i n the group p r e t r e a t e d w i t h 1.5 mg/kg (t = 2 . 5 1 , df = 14, p < 0 . 0 2 5 ) . For ty - two days f o l l o w i n g withdrawal w i t h 50 F igures 8A-B The e f f e c t o f c h r o n i c t reatment (10 days) w i t h v e h i c l e or severa l doses of p imozide on the amount o f s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g , examined a t v a r i o u s i n t e r v a l s a f t e r w i t h d r a w a l . In a l l animals the l e f t s t r i a t u m was i n d i v i d u a l l y assayed f o r DA r e c e p t o r b i n d i n g w i t h 2 . 3 nM [ 3 H ] -s p i r o p e r i d o l . A. Each bar represents the mean s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g (± S . E . M . ) i n groups o f animals (n=8). The dot ted l i n e s rep resent the s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g of the c o n t r o l groups t r e a t e d c h r o n i c a l l y w i t h a v e h i c l e s o l u t i o n . B. The percent i n c r e a s e o f the s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g i n pimozide t r e a t e d groups compared to c o n t r o l groups. * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l group us ing t w o - t a i l e d S t u d e n t ' s t - t e s t p<0.05 ** p<0.01 *** p<0.005 **** p<0.001 + s i g n i f i c a n t l y d i f f e r e n t from other t r e a t e d groups us ing t w o - t a i l e d S t u d e n t ' s t - t e s t p<0.05 ++ p<0.025 +++ p<0.01 ++++ p<0.005 VEHICLE PIMOZIDE .75 mg/kg PIMOZIDE 1.5 mg|kg g||ggg| PIMOZIDE 3.0mg|kg 6 12 22 42 6 WITHDRAWAL PERIOD (days) 1 2 2 2 42 o Pi 51 doses of 0 .75 mg/kq and 1.5 mg/kg the b i n d i n g i n animals was down t o c o n t r o l l e v e l s , w h i l e the group p r e t r e a t e d wi th the h ighest dose of p imozide ( 3 . 0 mg/kg), s t i l l showed some r e c e p t o r b i n d i n g , a l though t h i s j u s t f a i l e d t o reach s t a t i s t i c a l s i g n i f i c a n c e . The l e v e l of recepto r b i n d i n g i n the group p r e t r e a t e d wi th 3 .0 mg/kg 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 i n animals p r e t r e a t e d w i th e i t h e r 0 .75 mg/kg (t = 3 . 4 2 , d f = 1 4 , p < 0.005) or 1.5 mg/kg (t = 3 . 2 5 , df = 14, p < 0.01) 42 days withdrawal from pre t reatment . There was no o v e r a l l s i g n i f i c a n t e f f e c t of wi thdrawal from c h r o n i c v e h i c l e t reatment (F = 0 . 6 6 , df = 3 , 3 1 , p > 0 .1) on [ 3 H ] -s p i r o p e r i d o l b i n d i n g i n c o n t r o l a n i m a l s . Scatchard a n a l y s i s was used to determine i f the i n c r e a s e i n receptor b i n d i n g was due to e i t h e r a change i n the number of recepto r b i n d i n g s i t e s ( B m a x ) or in the a f f i n i t y of the recepto rs (Kn) . For the d e t e r m i n a t i o n of B m a x and Kn v a l u e s , the group of animals p r e t r e a t e d f o r 10 days wi th 1.5 mg/kg of pimozide and withdrawn from t h i s pretreatment f o r 12 days was used a long w i th i t s r e s p e c t i v e c o n t r o l group. With regard to the number of r e c e p t o r s , a s i g n i f i c a n t i n c r e a s e (t = 6 . 1 8 , df = 5 , p < 0.005) was observed between exper imental an imals (39.74 ± 1.20 pmol/g t i s s u e ) and c o n t r o l animals (26.57 ± 1.76 pmol/g t i s s u e ) , the i n c r e a s e be ing about 50%. However t h e r e was no s i g n i f i c a n t change i n recepto r a f f i n i t y (t = 0 . 2 8 , df = 5 , p > 0.1) between exper imental an imals (0 .556 ± 0.096 nM) and c o n t r o l animals, (0 .613 ± 0.182 nM). D e t a i l e d a n l a y s i s of the b i n d i n g k i n e t i c s u s i n g the [ 3 H ] - s p i r o p e r i d o l r e c e p t o r b i n d i n g a s s a y , a long wi th s a t u r a t i o n and Scatchard p l o t s of r e s u l t s from exper imental and c o n t r o l s t r i a t a are d e p i c t e d i n Experiment 3 . 52 EXPERIMENT 3 Parametr ic Time Course Study - E f f e c t of Durat ion of Pimozide A d m i n i s t r a t i o n The e f f e c t of the d u r a t i o n of p imozide a d m i n i s t r a t i o n on s t r i a t a l dopamine recepto rs was examined b e h a v i o r a l l y and b i o c h e m i c a l l y f o l l o w i n g d i f f e r e n t withdrawal pe r iods from drug t r e a t m e n t . In c o n t r a s t t o the p rev ious s t u d y , in t h i s experiment the dose of pimozide remained constant w h i l e the d u r a t i o n of pimozide a d m i n i s t r a t i o n was v a r i e d . Methods Groups of r a t s ( N = 8) were c h r o n i c a l l y t r e a t e d w i th e i t h e r 1.5 mg/kg of p imozide or the v e h i c l e s o l u t i o n , t w i c e d a i l y f o r e i t h e r 5 , 1 0 , 20 or 40 days and then 4 , 10 , 20 and 40 days f o l l o w i n g c e s s a t i o n from treatment behav io ra l and b iochemica l procedures were conducted to examine the e f f e c t s of these t reatments on dopamine r e c e p t o r s . For a l l groups of animals the p ro toco l was i d e n t i c a l to tha t used i n Experiment 2: B r i e f l y , locomotor a c t i v i t y was examined i n animals f o l l o w i n g the i n j e c t i o n of 2.0 mg/kg of d -amphetamine and then 24 hours l a t e r the animals were t e s t e d f o r s te reotyped behav ior e l i c i t e d by a dose of 0 .75 mg/kg apomorphine. Twenty - four hours 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 apomorphine, the animals were s a c r i f i c e d and the l e f t and r i g h t s t r i a t a d i s s e c t e d and f rozen at -80°C u n t i l assayed us ing the [ 3 H ] - s p i r o p e r i d o l recepto r b i n d i n g procedure . Resu l t s  Locomotor A c t i v i t y The t ime course of the locomotor a c t i v i t y induced by d-amphetamine i n groups of animals, withdrawn 4 , 10 , 20 and 40 days f o l l o w i n g c h r o n i c pimozide i s shown in F igure 9. The o v e r a l l p a t t e r n of m o t i l i t y d u r i n g the 53 1 hour h a b i t u a t i o n pe r iod tha t was e x h i b i t e d by animals t r e a t e d f o r d i f f e r e n t d u r a t i o n s wi th pimozide d i d not d i f f e r from one another on any withdrawal day (data not shown). S i m i l a r l y , t h e r e was no s i g n i f i c a n t . e f f e c t of d i f f e r e n t d u r a t i o n s of n e u r o l e p t i c t reatment on the t o t a l locomotor a c t i v i t y accumulated d u r i n g the 1 hour h a b i t u a t i o n pe r iod (F = 2 . 0 5 , df = 3 , 112, p > 0 . 1 ) . There was an o v e r a l l s i g n i f i c a n t e f f e c t of the d u r a t i o n of n e u r o l e p t i c t reatment (F = 4 . 1 1 , df = 3 , 112, p < 0.01) and of withdrawal from c h r o n i c t reatment (F = 1 1 8 . 2 6 , d f = 17, 1904, p < 0.001) on locomotor a c t i v i t y induced by d-amphetamine (F igure 9 ) . The longer d u r a t i o n s of pimozide t reatment (20 and 40 days) had a g reate r e f f e c t on m o t i l i t y than d i d the s h o r t e r d u r a t i o n s of p imozide t reatment (5 and 10 d a y s ) . In a d d i t i o n locomotor a c t i v i t y g r a d u a l l y returned to c o n t r o l l e v e l s from 4 to 40 days of w i t h d r a w a l . A s i g n i f i c a n t e f f e c t over the 3 hour t e s t pe r iod i s a l s o ev ident (F = 8 ; 9 5 , df = 3 , 112, p < 0.005) which i s the r e s u l t of the p r o g r e s s i v e decrease i n the r a t s m o t i l i t y over the d u r a t i o n of the t e s t p e r i o d i n a l l groups. A s i g n i f i c a n t o v e r a l l i n t e r a c t i o n between the d u r a t i o n of pimozide a d m i n i s t r a t i o n and withdrawal was observed (F = 1 . 8 5 , df = 51 , 112, p < 0 . 0 1 ) . From F igure 9 i t can be seen tha t t h i s i s due to the obse rva t ion t h a t c h r o n i c t reatment w i th longer d u r a t i o n s of p imozide enhanced locomotor a c t i v i t y f o r longer per iods f o l l o w i n g withdrawal from t r e a t m e n t . For example, c h r o n i c t reatment w i th pimozide f o r 20 days s t i l l had an e f f e c t on locomotor a c t i v i t y a f t e r 10 days whereas c h r o n i c pimozide treatment f o r 40 days s t i l l enhanced m o t i l i t y 20 days a f t e r w i t h d r a w a l . F i g u r e 10 shows the e f f e c t of d i f f e r e n t d u r a t i o n s of pimozide a d m i n i s t r a t i o n on the t o t a l d-amphetamine induced locomotor a c t i v i t y 54 F igures 9A-D The e f f e c t of d i f f e r e n t d u r a t i o n s o f c h r o n i c pimozide (1 .5 mg/kg twice/day) or v e h i c l e t reatment on the locomotor s t i m u l a n t e f f e c t o f d-amphetamine s u l f a t e ( 2 . 0 mg/kg), examined at v a r i o u s i n t e r v a l s f o l l o w i n g withdrawal from c h r o n i c t reatment . d-Amphetamine was admin is te red f o l l o w i n g a 1 hour h a b i t u a t i o n pe r iod to the a c t i v i t y cages . D i f f e r e n t groups o f animals were used a t each t reatment d u r a t i o n and withdrawal p e r i o d . The v e h i c l e curves represents the mean response f o r a l l c o n t r o l a n i m a l s . Each p o i n t represents the mean response of a group of animals (n=8). 54a LU O ' ' ' i i i i 1 i i i i i 1 1 1 1 i TIME (minutes) 54b I I I 1 1 1 1 1 1 — 30 60 90 120 150 180 TIME (minutes) 55 accumulated d u r i n g the f i r s t 90 minutes (peak d-amphetamine e f f e c t ) of the 3 hour t e s t pe r iod 4 , 10 , 2 0 , and 40 days a f t e r w i t h d r a w a l . The r e s u l t s from the t o t a l locomotor a c t i v i t y d a t a , l i k e the t ime course r e s u l t s show t h a t t h e r e i s an o v e r a l l s i g n i f i c a n t e f f e c t o f the d u r a t i o n o f n e u r o l e p t i c t reatment (F = 2 . 8 1 , df = 3 , 112, p < 0.05) and of withdrawal from t h i s t reatment (F = 1 0 . 6 0 , df = 3 , 112, p < 0.001) on t o t a l m o t i l i t y f o l l o w i n g d-amphetamine. However t h e r e was no s i g n i f i c a n t o v e r a l l i n t e r a c t i o n between d u r a t i o n of t reatment and withdrawal p e r i o d (F = 0 . 8 3 , d f = 9 , 112, p > 0 . 1 ) . Post hoc comparisons wi th the Newman-Keuls t e s t showed tha t pimozide f o r e i t h e r 5 or 10 days produced s i m i l a r e f f e c t s on d-amphetamine induced m o t i l i t y in a n i m a l s . L ikewise 20 or 40 day t reatment w i th pimozide d i d not r e s u l t i n d i f f e r e n t e f f e c t s on locomotor a c t i v i t y . However the longer per iods of pimozide a d m i n i s t r a t i o n (20 and 40 days) a f f e c t e d the animals m o t i l i t y d i f f e r e n t l y than the s h o r t e r d u r a t i o n s (5 and 10 d a y s ) . Post hoc a n a l y s i s between i n d i v i d u a l groups us ing the t w o - t a i l e d S t u d e n t ' s t - t e s t s u b s t a n t i a t e d the r e s u l t s o f the Newman-Keuls t e s t i n t h a t the e f f e c t s of c h r o n i c a d m i n i s t r a t i o n f o r 20 or 40 days had a g r e a t e r e f f e c t on m o t i l i t y than d i d 5 or 10 day t r e a t m e n t . The longer d u r a t i o n s s t i l l had a s t rong s i g n i f i c a n t e f f e c t on animals locomotor a c t i v i t y 10 days f o l l o w i n g the l a s t i n j e c t i o n o f p imozide (20 days - t = 4 . 1 4 , d f = 14 , p < 0 . 0 0 1 ; 40 days t = 3 . 4 8 , df = 14, p < 0 . 0 0 5 ) , whereas the e f f e c t on m o t i l i t y of the s h o r t e r d u r a t i o n s of p imozide d i d not s i g n i f i c a n t l y d i f f e r from c o n t r o l animals (5 days - t = 1 . 1 2 , df = 14, p > 0.1) or j u s t reached s i g n i f i c a n t l e v e l s (10 days - t = 2 . 2 1 , d f = 14, p < 0 . 0 5 ) . Twenty days f o l l o w i n g w i t h d r a w a l , on l y the group of animals t r e a t e d f o r 40 days w i th pimozide s t i l l d i s p l a y e d enhanced m o t i l i t y (t = 2 . 3 3 , df = 14 , p < 0.05) and t h i s had returned to c o n t r o l l e v e l s by 40 days . 56 F igures 10A-B The e f f e c t of d i f f e r e n t d u r a t i o n s of c h r o n i c p imozide ( 1 . 5 mg/kg twice/day) or v e h i c l e t reatment on the t o t a l (90 minutes) locomotor a c t i v i t y f o l l o w i n g the a d m i n i s t r a t i o n o f d-amphetamine s u l f a t e ( 2 . 0 mg/kg), examined a t va r ious i n t e r v a l s a f t e r wi thdrawal from c h r o n i c t reatment . A. Each bar represents the mean t o t a l response (± S . E . M . ) i n groups of animals (n=8). The dot ted l i n e s represent the r e s p e c t i v e c o n t r o l groups t r e a t e d c h r o n i c a l l y f o r d i f f e r e n t d u r a t i o n s w i t h a v e h i c l e s o l u t i o n . B. The percent i n c r e a s e of the t o t a l (90 minutes) d-amphetamine-induced locomotor a c t i v i t y i n pimozide t r e a t e d groups compared to c o n t r o l groups. * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l groups us ing t w o - t a i l e d S t u d e n t ' s t - t e s t p<0.05 ** p<0.025 *** p<0.005 **** p<0.001 B VEHICLE l l i PIMOZIDE 5 days 4 10 20 40 4 10 2 0 4 0 WITHDRAWAL PERIOD (days) 57 An o v e r a l l s i g n i f i c a n t e f f e c t of the d u r a t i o n of v e h i c l e t reatment in c o n t r o l animals (F = 3 . 1 3 , df = 3 , 56 , p < 0.05) but not of withdrawal from treatment (F = 0 . 8 8 , d f = 1 , 56, p > 0.1) was observed on t o t a l m o t i l i t y f o l l o w i n g d-amphetamine. From F igure 10 i t can be seen t h a t t h i s e f f e c t i s due to a decrease i n the t o t a l a c t i v i t y of c o n t r o l groups w i th i n c r e a s i n g d u r a t i o n of t r e a t m e n t . Increased age and hand l ing of the animals w i th i n c r e a s i n g d u r a t i o n s of t reatment as we l l as yet o ther undetermined f a c t o r s cou ld c o n t r i b u t e to t h i s e f f e c t . Stereotypy The t o t a l s te reotypy score obta ined d u r i n g the one hour t e s t p e r i o d f o l l o w i n g the a d m i n i s t r a t i o n o f apomorphine (0 .75 mg/kg) i s shown i n F i g u r e 11A i n groups of animals 5 , 11 , 21 and 41 days a f t e r withdrawal from p imoz ide . There was an o v e r a l l s i g n i f i c a n t e f f e c t of the d u r a t i o n o f p imozide t reatment (F = 2 1 . 5 1 , df = 3 , 112, p < 0.005) and of withdrawal pe r iod from t h i s t reatment (F = 6 . 4 6 , df = 3 , 112, p < 0.005) on the t o t a l s te reotypy score obta ined by groups of a n i m a l s . The peak s te reo typy score a t t a i n e d by groups of animals f o l l o w i n g d i f f e r e n t per iods of withdrawal i s d e p i c t e d i n F igure 11B. Again the d u r a t i o n of pimozide t reatment (F = 5 . 5 5 , d f = 3 , 1 1 2 , p < 0.005) and the withdrawal p e r i o d (F = 4 . 2 9 , df = 3 , 112, p < 0.01) both had an o v e r a l l s i g n i f i c a n t e f f e c t on the peak s te reo typy response. No i n t e r a c t i o n between the d u r a t i o n of p imozide a d m i n i s t r a t i o n and withdrawal pe r iod occur red w i t h e i t h e r the t o t a l s te reotypy (F = 0 . 3 8 , df = 9 , 112, p > 0.1) or the peak s t e r e o t y p y score (F = 0 . 5 1 , df = 9 , 112, p > 0 . 1 ) . Post hoc examinat ion w i th the Mann-Whitney U - tes t revea led tha t the enhancement of e i t h e r the t o t a l s te reo typy score (F igure 11A) or the peak s te reotypy response (F igure 11B) induced by 58 apomorphine 5 , 1 1 , 21 and 41 days a f t e r withdrawal from e i t h e r 5 or 10 days of p imozide pretreatment was s i m i l a r (eg. 5 days withdrawal - U = 3 2 . 5 , d f = 8 , 8 , p > 0 . 0 5 ) . L ikewise 20 or 40 days of pimozide pretreatment a l s o produced s t a t i s t i c a l l y i n d i s t i n g u i s h a b l e e f f e c t s on these two measures o f s t e r e o t y p y (eg. 5 days withdrawal - U = 4 5 . 5 , d f = 8 , 8 , p > 0 . 0 5 ) ; however these e f f e c t s were always s i g n i f i c a n t l y g r e a t e r than those induced by 5 or 10 days of pimozide t reatment (F igure 11A). It was a l s o found t h a t whereas the t o t a l s te reo typy score i n animals t r e a t e d w i th pimozide f o r 5 days was s i g n i f i c a n t l y g rea te r than t h a t obta ined by c o n t r o l an imals 5 days a f t e r w i t h d r a w a l , animals t r e a t e d f o r 10 days and longer (20 or 40 d a y s ) , d i s p l a y e d enhanced s te reotypy compared t o c o n t r o l s 21 and 41 days r e s p e c t i v e l y f o l l o w i n g w i t h d r a w a l . Regardless of the d u r a t i o n of p imozide t r e a t m e n t , the t o t a l s te reotypy score (F igure 11A) or peak s te reo typy response (F igure 1IB) d i m i n i s h e d over the 41 days o f w i t h d r a w a l , e s p e c i a l l y w i t h the longer per iods of c h r o n i c a d m i n i s t r a t i o n . No o v e r a l l s i g n i f i c a n t e f f e c t of the d u r a t i o n of v e h i c l e t reatment i n c o n t r o l animals or of wi thdrawal from treatment was observed on e i t h e r the t o t a l or peak s te reotypy score (data not shown). The s te reo typy d i s p l a y e d by c o n t r o l an imals 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 apomorphine i n c l u d e d s n i f f i n g , l ocomoto r , r e a r i n g and grooming b e h a v i o r s . Pimozide p r e t r e a t e d animals a l l d i s p l a y e d l i c k i n g , gnawing and jumping b e h a v i o r s , being more s e v e r e , r e p e t i t i v e and l a s t i n g f o r longer per iods of t ime i n groups t r e a t e d c h r o n i c a l l y f o r 20 and 40 days . As i n Experiment 2 , many animals (75%) t r e a t e d c h r o n i c a l l y w i th p i m o z i d e , e s p e c i a l l y f o r the long d u r a t i o n s (20 and 40 d a y s ) , d i s p l a y e d burs ts of chewing jaw movement t h a t were not d i r e c t e d towards any s o l i d ob jec t or par t of the cage dur ing the t e s t p e r i o d . This o ra l behav ior 59 F igures 11A-B The e f f e c t s o f d i f f e r e n t d u r a t i o n s of c h r o n i c pimozide ( 1 . 5 mg/kg twice/day) or v e h i c l e t reatment on apomorphine- induced (0 .75 mg/kg) s te reotyped b e h a v i o r , examined a t va r ious i n t e r v a l s f o l l o w i n g withdrawal from c h r o n i c t reatment . S tereotypy was ra ted accord ing to the Creese and Iversen (1975) s c a l e . A. Each bar represents the mean t o t a l (1 hour) s te reotypy score (± S . E . M . ) i n groups of animals (n=8). Dotted l i n e s represent the t o t a l s te reotypy score obta ined i n the r e s p e c t i v e c o n t r o l a n i m a l s . B. Each p o i n t represents the mean of the peak s te reo typy score ob ta ined by groups o f animals (n=8). • s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l groups us ing o n e - t a i l e d Mann-Whitney U - t e s t p<0.05 p<0.025 p<0.01 p<0.005 + s i g n i f i c a n t l y d i f f e r e n t from other t r e a t e d groups us ing t w o - t a i l e d Mann-Whitney U - t e s t p<0.05 ++ p<0.001 WITHDRAWAL PERIOD (days) 60 p r i m a r i l y observed i n animals f o l l o w i n g long d u r a t i o n s of pimozide t reatment 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 observed i n c o n t r o l an imals (20 or 40 days - U = 12, df = 8 , 8 , p < 0 . 0 5 ) . In a d d i t i o n i n 4 an imals (12%) t r e a t e d c h r o n i c a l l y f o r 40 d a y s , purpose less chewing jaw movements were o c c a s i o n a l l y observed d u r i n g the course o f the t r e a t m e n t . Dopamine Receptor B ind ing The e f f e c t s of d i f f e r e n t d u r a t i o n s of pimozide a d m i n i s t r a t i o n on the amount of s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g to the s t r i a t u m wi th respect to c o n t r o l an imals 6 , 12, 22 , and 42 days f o l l o w i n g withdrawal are shown i n F igure 12. The d u r a t i o n of p imozide pretreatment d i d not produce an o v e r a l l s i g n i f i c a n t e f f e c t on s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g (F = 0 . 7 0 , df = 3 , 112, p > 0 . 1 ) . Post hoc a n a l y s i s w i th the t w o - t a i l e d S t u d e n t ' s t -t e s t shows tha t r e g a r d l e s s of the d u r a t i o n of pimozide t r e a t m e n t , 6 and 12 days f o l l o w i n g w i t h d r a w a l , a l l groups of animals d i s p l a y e d a s i m i l a r i n c r e a s e i n receptor b i n d i n g (35-40%) compared to c o n t r o l l e v e l s . For example, in groups p r e t r e a t e d f o r 5 or 40 days w i th pimozide and withdrawn f o r 6 d a y s , t h e r e was no s i g n i f i c a n t d i f f e r e n c e between t h e i r i n c r e a s e i n receptor b i n d i n g (t = 2 . 0 9 , d f = 14 , p > 0 . 0 5 ) . A s i g n i f i c a n t e f f e c t of withdrawal pe r iod on changes i n the amount of s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g was obta ined (F = 3 1 . 4 2 , df = 3 , 112, p < 0 . 0 0 1 ) . This was ev ident s i n c e whereas t h e r e was no d i f f e r e n c e i n the change i n recepto r b i n d i n g from 6 to 12 days f o l l o w i n g w i t h d r a w a l , by 22 days the amount o f r e c e p t o r b i n d i n g decreased s i m i l a r l y i n a l l groups. By 42 days t h i s decrease in recepto r b i n d i n g l e v e l s had cont inued so t h a t i n a l l groups the amount o f s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g had near l y returned to c o n t r o l l e v e l s . No o v e r a l l s i g n i f i c a n t i n t e r a c t i o n occur red between the d u r a t i o n of 61 F igures 12A-B The e f f e c t of d i f f e r e n t d u r a t i o n s o f c h r o n i c pimozide (1 .5 mg/kg twice/day) or v e h i c l e t reatment on the amount of s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g , examined a t va r ious i n t e r v a l s a f t e r w i t h d r a w a l . In a l l animals the l e f t s t r i a t u m was i n d i v i d u a l l y assayed f o r DA r e c e p t o r b i n d i n g w i t h 2 . 3 nM [ 3 H ] - s p i r o p e r i d o l . A. Each bar represents the mean s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g (± S . E . M . ) i n groups of animals (n=8). The dot ted l i n e s represent the s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g to the c o n t r o l groups t r e a t e d c h r o n i c a l l y w i t h a v e h i c l e s o l u t i o n . B. The percent i n c r e a s e o f the s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g i n pimozide t r e a t e d groups compared to c o n t r o l groups. * s i g n i f i c a n t l y d i f f e r e n t from conto l group us ing t w o - t a i l e d S t u d e n t ' s t - t e s t p<0.05 ** p<0.01 *** p<0.005 **** p<0.001 T .TJMT 180h o OC160 o O Li. o 140 ft 120| O CC LU CL 100! 8* B I E VEHICLE PIMOZIDE 5 days PIMOZIDE 10 days PIMOZIDE 20 days PIMOZIDE 40 days 42 6 12 WITHDRAWAL PERIOD (days) 62 pimozide a d m i n i s t r a t i o n and the per iod of withdrawal on recepto r b i n d i n g (F = 1 . 9 1 , df = 9 , 112, p > 0 . 0 5 ) . There was no o v e r a l l s i g n i f i c a n t e f f e c t o f e i t h e r the d u r a t i o n of v e h i c l e t reatment (F = 1 . 1 2 , df = 3 , 56 , p > ,0.1) or of withdrawal from treatment (F = 0 . 0 1 , df = 1 , 56 , p > 0.1) on [ 3 H ] -s p i r o p e r i d o l b i n d i n g i n c o n t r o l a n i m a l s . For both exper imental and c o n t r o l groups the b i n d i n g of [ 3 H ] -s p i r o p e r i d o l to s t r i a t a l t i s s u e in r a t s i s s a t u r a b l e . The s a t u r a t i o n curves and Scatchard p l o t s of the s a t u r a t i o n data f o r both exper imental and c o n t r o l groups from a s i n g l e experiment are d e p i c t e d i n F igures 13 and 14, r e s p e c t i v e l y . S t r i a t a l t i s s u e from animals t r e a t e d w i t h e i t h e r 1.5 mg/kg of pimozide f o r 40 days or v e h i c l e and withdrawn from t h i s pretreatment f o r 12 days were examined by Scatchard a n a l y s i s to determine whether the observed i n c r e a s e i n recepto r b i n d i n g was due to e i t h e r a change i n the number of recepto r b i n d i n g s i t e s or i n the a f f i n i t y of the r e c e p t o r s . A s i g n i f i c a n t i n c r e a s e (t = 3 . 3 8 , df = 3 , p < 0.025) i n the number of recepto r b i n d i n g s i t e s was observed between exper imental (40.90 ± 3 .15 pmol/g t i s s u e ) , and c o n t r o l animals (27.10 ± 2 .60 pmol/g t i s s u e ) , the i n c r e a s e being around 50%. There was no s i g n i f i c a n t change i n the a f f i n i t y of the recepto r (t = 0 . 0 6 5 , df = 3 , p > 0.1) between exper imental (0.426 ± .005 nM) and c o n t r o l animals (0.417 ± .138 nM). C o r r e l a t i o n s Between Behav io ra l and Biochemical Data Behav io ra l and b iochemica l i n d i c e s o f dopamine r e c e p t o r changes f o l l o w i n g c h r o n i c n e u r o l e p t i c pretreatment were analyzed both between groups and w i t h i n groups to determine i f t h e r e were s i g n i f i c a n t c o r r e l a t i o n s between these measures. When locomotor a c t i v i t y and s te reotypy were compared wi th the amount of s p e c i f i c [ 3 H ] - s p i r o p e r i d o l 63 F igure 13 The s a t u r a t i o n curves of s p e c i f i c b i n d i n g of [ 3 H ] - s p i r o p e r i d o l to combined s t r i a t a l t i s s u e as a f u n c t i o n of i t s c o n c e n t r a t i o n i n groups of animals (n=8) t r e a t e d c h r o n i c a l l y (40 days) w i t h e i t h e r pimozide (1 .5 mg/kg twice/day) or v e h i c l e . A l l animals were s a c r i f i c e d 12 days f o l l o w i n g withdrawal from t reatment . S p e c i f i c b i n d i n g was determined as the d i f f e r e n c e between the t o t a l b i n d i n g and the n o n s p e c i f i c b i n d i n g (presence of 10~3M DA). Each p o i n t represents the r e s u l t s of a s i n g l e experiment performed i n q u a d r u p l i c a t e . 63a 64 F igure 14 Scatchard a n a l y s i s o f s p e c i f i c [ 3H ] - sp i roper ido l b i n d i n g to combined s t r i a t a l t i s s u e from groups of animals (n=8) t r e a t e d c h r o n i c a l l y (40 days) w i th e i t h e r pimozide (1 .5 mg/kg twice/day) or v e h i c l e . A l l an imals were s a c r i f i c e d 12 days f o l l o w i n g withdrawal from t reatment . The f i n a l t i s s u e c o n c e n t r a t i o n remained constant a t 5 mg/ml based on the o r i g i n a l wet we ight . Each p o i n t represents the r e s u l t s of a s i n g l e experiment performed i n q u a d r u p l i c a t e . * s i g n i f i c a n t c o r r e l a t i o n a t p<0.001 64a • PIMOZIDE o VEHICLE Bmax=148.44pM K o - 0 . 5 5 4 n M r= -Q992*" J _1 40 5(5 120 160 200 (3H)-SPlROPERIDOL BOUND (pM) 65 bound t o s t r i a t a l t i s s u e between groups t r e a t e d e i t h e r f o r 10 days w i th d i f f e r e n t doses of pimozide (0 , . 7 5 mg/kg, 1.5 mg/kg, 3 .0 mg/kg) or w i t h 1.5 mg/kg of pimozide f o r d i f f e r e n t d u r a t i o n s of t ime (5 d a y s , 10 d a y s , 20 days , 40 d a y s ) , t h e r e was always a g r e a t e r tendency f o r s i g n i f i c a n t c o r r e l a t i o n s to e x i s t between t o t a l s te reotypy and s p e c i f i c [ 3 H ] -s p i r o p e r i d o l b i n d i n g . For example, F igure 15 shows the c o r r e l a t i o n between s te reo typy and b i n d i n g (F igure 15A) and between locomotor a c t i v i t y and b i n d i n g (F igure 15B) f o r groups of animals t r e a t e d f o r 10 days w i th d i f f e r e n t doses of pimozide ( 0 , .75 mg/kg, 1.5 mg/kg, 3 .0 mg/kg) and withdrawn from t h i s t reatment f o r 10 days . Whereas a s i g n i f i c a n t c o r r e l a t i o n was obta ined between the t o t a l s t e r e o t y p y score and the amount of s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g (r = 0 . 9 8 7 , df = 2 , p < 0 . 0 2 ) , no s i g n i f i c a n t c o r r e l a t i o n e x i s t e d between the t o t a l locomotor a c t i v i t y and [ 3 H ] - s p i r o p e r i d o l b i n d i n g (r = 0 . 7 6 2 , df = 2 , p > 0 . 1 ) . S i m i l a r l y i n one group o f a n i m a l s , when the t o t a l locomotor a c t i v i t y or s te reo typy were examined a long w i th the amount of [ 3 H ] - s p i r o p e r i d o l b i n d i n g t h e r e was again a g rea te r tendency f o r s i g n i f i c a n t c o r r e l a t i o n s t o occur between s te reo typy and the b iochemica l measure of dopamine recepto r b i n d i n g . An example of the c o r r e l a t i o n s between s te reo typy and b i n d i n g (F igure 16A) and between locomotor a c t i v i t y and b i n d i n g (F igure 16B) i n an i n d i v i d u a l group of animals t r e a t e d f o r 20 days w i th 1.5 mg/kg of pimozide and withdrawn f o r 10 days i s shown i n F igure 16. The c o r r e l a t i o n s between the t o t a l locomotor a c t i v i t y score and the l e v e l of [ 3 H ] - s p i r o p e r i d o l b i n d i n g f a i l e d to reach s i g n i f i c a n c e (r = 0 . 2 0 , df = 6 , p > 0 . 1 ) , whereas t h e r e was a s i g n i f i c a n t c o r r e l a t i o n between t o t a l s te reo typy and recepto r b i n d i n g (r = 0 . 7 3 5 , df = 6 , p < 0 . 0 5 ) . The c o r r e l a t i o n between t o t a l locomotor a c t i v i t y and t o t a l s te reotypy e i t h e r between groups of animals or 66 w i t h i n an i n d i v i d u a l group of animals never reached s i g n i f i c a n t l e v e l s (data not shown). 67 F igures 15A-B The c o r r e l a t i o n between behav io ra l and b iochemica l data i n groups o f animals (n=8) t r e a t e d c h r o n i c a l l y (10 days) w i t h v e h i c l e or severa l doses o f p imozide and examined 10-12 days f o l l o w i n g withdrawal from c h r o n i c t rea tment . A . C o r r e l a t i o n between the t o t a l (1 hour) s t e r e o t y p y score e l i c i t e d by apomorphine (0 .75 mg/kg) and the s p e c i f i c [ 3 H ] - s p i r o p e r i d o l bound to s t r i a t a l t i s s u e . B. C o r r e l a t i o n between the t o t a l (90 minutes) locomotor a c t i v i t y e l i c i t e d by d-amphetamine s u l f a t e ( 2 . 0 mg/kg) and the s p e c i f i c [ 3 H ] - s p i r o p e r i d o l bound to s t r i a t a l t i s s u e . * s i g n i f i c a n t c o r r e l a t i o n a t p<0.02 67a Ol8[ UJ o t >;iOr O UJ 8\ CC UJ CO _J f* Q 2\ PIMOZIDE PRETREATMENT] •75 mg/kg A I.5 mg/kg • 3.0 mg/kg • VEHICLE O r=0.987 -/A J I I I I I I ' 1 B 10h 8 m T L O 7 6 CO o o UJ o 2h b 1 1 0. r = 0.762 U L J 1 L I J I L 20" 120 140 160 180 200 (3H)-SPIROPERIDOL BOUND ( pM) 68 F igures 16A-B The c o r r e l a t i o n between behav io ra l and b iochemical data i n i n d i v i d u a l animals t r e a t e d c h r o n i c a l l y (20 days) w i t h p imozide ( 1 . 5 mg/kg twice/day) and examined 10-12 days f o l l o w i n g withdrawal from c h r o n i c t reatment . A. C o r r e l a t i o n between the t o t a l (1 hour) s te reo typy score e l i c i t e d by apomorphine (0 .75 mg/kg) and the s p e c i f i c [ 3 H ] - s p i r o p e r i d o l bound to s t r i a t a l t i s s u e . B. C o r r e l a t i o n between the t o t a l (90 minutes) locomotor a c t i v i t y e l i c i t e d by d-amphetamine s u l f a t e ( 2 . 0 mg/kg) and the s p e c i f i c [ 3 H ] - s p i r o p e r i d o l bound to s t r i a t a l t i s s u e * s i g n i f i c a n t c o r r e l a t i o n a t p<0.05 69 DISCUSSION Prolonged t reatment w i th drugs t h a t i n t e r f e r e w i th normal c a t e c h o l a m i n e r g i c mechanisms i n the b r a i n leads to changes i n the s e n s i t i v i t y of catecholamine r e c e p t o r s . Using b e h a v i o r a l , b iochemica l and e l e c t r o p h y s i o l o g i c a l t e c h n i q u e s , n e u r o l e p t i c drugs have been shown to act as a n t a g o n i s t s at p o s t s y n a p t i c DA r e c e p t o r s i t e s . The present study conf i rms the r e s u l t s obta ined by numerous other l a b o r a t o r i e s by demonstrat ing tha t long term, n e u r o l e p t i c a d m i n i s t r a t i o n produces an i n c r e a s e i n the behav io ra l e f f e c t s of dopamine a g o n i s t s (Klawans and R u b o v i t z , 1972; Tarsy and B a l d e s s a r i n i , 1974; Thornburg and Moore, 1974) as w e l l as an i n c r e a s e i n the number of DA recepto rs i n the s t r i a t u m (Burt et a l . , 1977; Kobayashi et a l . , 1978; M u l l e r and Seeman, 1977) . The purpose o f the present paramet r i c study was to compare the degree and d u r a t i o n of DA recepto r s u p e r s e n s i t i v i t y through the use of behav io ra l and b iochemica l methods f o l l o w i n g c h r o n i c a d m i n i s t r a t i o n of d i f f e r e n t doses of pimozide f o r v a r i o u s d u r a t i o n s of t i m e . These r e s u l t s i n d i c a t e t h a t f o l l o w i n g c h r o n i c t reatment w i th pimozide ( w i t h i n the dose and d u r a t i o n per iods u s e d ) , enhanced DA recepto r s e n s i t i v i t y occurs f o r at l e a s t 4 d a y s . These r e s u l t s demonstrat ing t h a t pimozide i s ab le t o induce DA recepto r s u p e r s e n s i t i v i t y are i n agreement w i th data reported by other i n v e s t i g a t o r s us ing pimozide (Thornburg and Moore, 1974) as we l l as other n e u r o l e p t i c d r u g s , i n c l u d i n g c h l o r p r o m a z i n e , a l p h a - f l u p e n t h i x o l , f l u p e n a z i n e , h a l o p e r i d o l , l o x a p i n e , p e n f l u r i d o l , t h i o r i d a z i n e and t r i f 1 u p e r a z i n e (Burt et a l . , 1977; Clow et a l . , 1979; Jackson et a l . , 1975; Kamer et a l . , 1981; Klawans and R u b o v i t z , 1972; M u l l e r and Seeman, 1977; S a h a k i a n , 1976; Sayers 70 et a l . , 1975; Smith and D a v i s , 1976; Tarsy and B a l d e s s a r i n i , 1974; V o i t h , 1977) . P r e l i m i n a r y S tud ies In experiment IA , the dosage of d-amphetamine which produced the most s i g n i f i c a n t d i f f e r e n c e i n locomotor a c t i v i t y i n animals p r e t r e a t e d c h r o n i c a l l y w i th pimozide or v e h i c l e was determined. For d-amphetamine, a s t i m u l a n t drug which i n c r e a s e s the s y n a p t i c r e l e a s e of DA and i n h i b i t s i t s reuptake (Farnebo, 1971; Hanson, 1966) a dose of 2 mg/kg was necessary t o s i g n i f i c a n t l y i n c r e a s e locomotor a c t i v i t y i n p imozide p r e t r e a t e d an imals compared to c o n t r o l s (F igure 1 ) . F o l l o w i n g c h r o n i c pimozide t r e a t m e n t , doses of 1 mg/kg and 4 mg/kg d-amphetamine produced no d i f f e r e n c e i n t o t a l locomotor a c t i v i t y between exper imental and c o n t r o l a n i m a l s . S i m i l a r r e s u l t s demonstrat ing t h a t the e f f e c t s of d-amphetamine are p o t e n t i a t e d a t 1.5 - 2 .0 mg/kg, but not at lower dosages , have been r e c e n t l y observed i n animals t r e a t e d c h r o n i c a l l y w i th t r i c y l i c a n t i d e p r e s s a n t d r u g s , a t reatment t h a t i s proposed to produce s u p e r s e n s i t i v e p o s t s y n a p t i c DA recepto rs i n the mesol imbic dopaminergic system (Spyraki and F i b i g e r , i n p r e s s ) . No e x p l a n a t i o n i s p r e s e n t l y apparent w i th regard to the u n d e r l y i n g b a s i s of the narrow dose range at which the locomotor s t i m u l a n t e f f e c t s of d -amphetamine are p o t e n t i a t e d by c h r o n i c p imoz ide . From the present r e s u l t s i t i s c l e a r t h a t whereas the h igher doses of d-amphetamine (4 mg/kg) cont inued to i n c r e a s e locomotor a c t i v i t y i n c o n t r o l animals compared to t h a t e l i c i t e d by the lower doses of d-amphetamine ( 1 - 2 mg/kg), t h i s dose of d-amphetamine decreased the locomotor a c t i v i t y i n animals t r e a t e d c h r o n i c a l l y w i th p imoz ide . With the development of s u p e r s e n s i t i v e DA 71 recepto rs i n pimozide p r e t r e a t e d a n i m a l s , t h i s dose of d-amphetamine (4 mg/kg) r e s u l t e d i n the appearance o f i n t e n s e r e p e t i t i v e s te reotyped behav iors such as s n i f f i n g , r e a r i n g , l i c k i n g and gnawing, and t h i s probably caused the s imultaneous reduc t ion of the locomotor s t i m u l a n t e f f e c t s . From experiment IB , the dose response curve f o r apomorphine (F igure 3 ) , a d i r e c t DA recepto r agon is t ( E r n s t , 1 9 6 7 ) , i n d i c a t e s t h a t a dose of 0 .75 mg/kg i s the minimum dose necessary t o induce s i g n i f i c a n t s te reotyped / behav iors i n c o n t r o l a n i m a l s . T h e r e f o r e , t h i s dose was chosen f o r the subsequent s t u d i e s on the e f f e c t s of pimozide on apomorphine- induced s t e r e o t y p y . As observed i n experiments 2 and 3 , i n c o n t r a s t to the locomoto r , s n i f f i n g and r e a r i n g behav iors observed i n c o n t r o l a n i m a l s , f o l l o w i n g apomorphine, c h r o n i c pimozide t r e a t e d animals showed the more i n t e n s e forms of s t e r e o t y p y , such as l i c k i n g and gnawing. In Experiment IC i t was observed t h a t a dose of 2 mg/kg d-amphetamine 24 hours p r i o r to the i n j e c t i o n of apomorphine had no e f f e c t on s tereotyped behav ior i n e i t h e r v e h i c l e or p imozide p r e t r e a t e d a n i m a l s . This experiment was conducted s i n c e i n subsequent s t u d i e s , a l l an imals were t e s t e d f o r apomorphine- induced s t e r e o t y p y 24 hours f o l l o w i n g d-amphetamine induced locomotor a c t i v i t y . Contrary to severa l r e p o r t s (Hitzemann et a l . , 1980; Howlett and N a h o r s k i , 1979) , the c u r r e n t r e s u l t s i n d i c a t e t h a t a s i n g l e dose of d-amphetamine d i d not change or a l t e r the DA recepto r i n any way when examined e i t h e r i n d i r e c t l y (apomorphine- induced s te reo typy -Experiment IC) or d i r e c t l y ( [ 3 H ] - s p i r o p e r i d o l recepto r b i n d i n g - data not shown). These s t u d i e s r e p o r t i n g an enhanced s t e r e o t y p y induced by 1.0 mg/kg apomorphine and a decreased [ 3 H ] - s p i r o p e r i d o l receptor b i n d i n g i n the s t r i a t u m 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 d-amphetamine, used much h igher doses of t h i s drug (6 mg/kg twice/day or 15 mg/kg once/day) and 72 s t u d i e d the e f f e c t s on ly 16 - 20 hours and 90 minutes l a t e r (Hitzemann et a l . , 1980; Howlett and N a h o r s k i , 1979) , r e s p e c t i v e l y . E f f e c t of Dose of Pimozide on Dopamine Receptor S e n s i t i v i t y It has been repor ted t h a t a s i n g l e i n j e c t i o n of a n e u r o l e p t i c can l e a d t o the development of DA recepto r s u p e r s e n s i t i v i t y ( C h r i s t i a n s e n et a l . , 1976; Mart res et a l . , 1977) . The inc reased number of DA recepto rs has been specu la ted t o be a compensatory mechanism of the p o s t s y n a p t i c neuron i n response to decreased s t i m u l a t i o n by DA d u r i n g the n e u r o l e p t i c chemical b l o c k a d e . This o b s e r v a t i o n suggests tha t d u r i n g c h r o n i c n e u r o l e p t i c t r e a t m e n t , dopaminergic s u p e r s e n s i t i v i t y would i n i t i a l l y develop a f t e r the f i r s t drug i n j e c t i o n , w i th an i n c r e a s e i n the degree of s e n s i t i v i t y c o n t i n u i n g to a maximum l e v e l w i th long term n e u r o l e p t i c a d m i n i s t r a t i o n . In the present s t u d y , changes i n the s e n s i t i v i t y of DA recepto rs induced by c h r o n i c n e u r o l e p t i c t reatment were f i r s t examined 4 days f o l l o w i n g pimozide w i t h d r a w a l , at which t ime dopaminergic s u p e r s e n s i t i v i t y measured both b e h a v i o r a l l y and b i o c h e m i c a l l y was apparent . The h a l f l i f e of p imozide i n the b r a i n has been reported to be 5.6 hours (Janssen and A l l e w i j n , 1968). T h e r e f o r e , and from the present r e s u l t s i t may be assumed t h a t any r e s i d u a l drug remaining i n the b r a i n 4 days f o l l o w i n g the l a s t pimozide i n j e c t i o n would probably be i n s u f f i c i e n t to i n t e r f e r e w i th the examinat ion of DA recepto rs us ing behav io ra l and b iochemical methods. The per iod of withdrawal from c h r o n i c n e u r o l e p t i c t reatment t h a t was necessary t o reduce the dopaminergic recepto r s e n s i t i v i t y to c o n t r o l l e v e l s as measured by behav io ra l and b iochemica l methods was shown to be dose dependent. Treatment w i th the h igher doses augmented the locomotor a c t i v i t y induced by d-amphetamine to a g r e a t e r magnitude and f o r a longer 73 withdrawal p e r i o d than d i d t reatment w i th the lower doses of pimozide (F igures 5 and 6 ) . However, whereas the degree o f apomorphine- induced s te reotyped behavior i n animals t r e a t e d w i th the h igher doses of pimozide was g r e a t e r than t h a t i n an imals t r e a t e d w i th the lower doses o f p i m o z i d e , the d u r a t i o n of t h i s i n c r e a s e f o l l o w i n g withdrawal remained the same (F igure 7 ) . When s tereotyped behav iors were examined i n d i v i d u a l l y , i t was found tha t the l i c k i n g and gnawing behav iors tended to p e r s i s t f o r longer per iods of t ime f o l l o w i n g t reatment withdrawal w i th the h ighes t dose of p imozide when compared to the lower dose ; however t h i s j u s t f a i l e d to reach s t a t i s t i c a l s i g n i f i c a n c e . I f d i f f e r e n t s te reo typy responses ( s n i f f i n g , r e a r i n g , l o c o m o t i o n , l i c k i n g , gnawing) were i n d i v i d a l l y scored on separate r a t i n g s c a l e s i n s t e a d of being lumped t o g e t h e r , more d e t a i l e d i n f o r m a t i o n might be e x t r a c t e d from the examinat ion of apomorphine- induced s t e r e o t y p y . When changes i n the s e n s i t i v i t y of DA recepto rs f o l l o w i n g d i f f e r e n t doses o f n e u r o l e p t i c t reatment were examined by recepto r l a b e l i n g t e c h n i q u e s , h igher doses of pimozide inc reased recepto r s e n s i t i v i t y to both a g r e a t e r degree and f o r a longer pe r iod of t ime than d i d the lower doses (F igure 8 ) . These behav io ra l and b iochemica l r e s u l t s i n d i c a t e tha t a dose response r e l a t i o n e x i s t s between the dose of pimozide admin is te red c h r o n i c a l l y and the degree and d u r a t i o n of the change i n the s e n s i t i v i t y of DA r e c e p t o r s . The r e s u l t s demonstrat ing t h a t the dose of pimozide admin is te red c h r o n i c a l l y to animals has an e f f e c t on the degree and d u r a t i o n of the r e s u l t i n g changes i n the number of DA recepto rs are i n disagreement w i th those observed by o ther i n v e s t i g a t o r s (Burt et a l . , 1977) . Burt et a l . , (1977) admin i s te red 0 . 5 mg/kg and 5 . 0 mg/kg h a l o p e r i d o l c h r o n i c a l l y t o animals f o r 1 or 3 weeks and observed no changes i n the degree of the 74 inc reased DA recepto r b i n d i n g . In the present study th ree d i f f e r e n t doses of p imozide were admin i s te red c h r o n i c a l l y to animals and changes i n DA recepto r s e n s i t i v i t y were examined w i th behav io ra l and b iochemica l measures 4 , 10, 20 and 40 days f o l l o w i n g withdrawal from t r e a t m e n t , e n a b l i n g a d e t a i l e d examinat ion of the degree and t ime course of the r e s u l t i n g DA recepto r s u p e r s e n s i t i v i t y . Burt et a l . , (1977) examined b i o c h e m i c a l l y the e f f e c t s of 2 doses of h a l o p e r i d o l at a s i n g l e t ime (5 days) f o l l o w i n g withdrawal from c h r o n i c t r e a t m e n t . The dose/response e f f e c t observed i n the present study would not have been so ev ident i f changes i n DA recepto r s e n s i t i v i t y were examined at on ly a s i n g l e withdrawal p e r i o d i n s t e a d of at four d i f f e r e n t per iods f o l l o w i n g withdrawal from c h r o n i c t r e a t m e n t . In a d d i t i o n , Burt et a l . , (1977) use h a l o p e r i d o l as the [ 3 H ] - l i g a n d f o r l a b e l i n g DA receptors i n s t e a d of [ 3 H ] - s p i r o p e r i d o l , which has been shown to have a h igher a f f i n i t y f o r DA r e c e p t o r s and a lower a f f i n i t y f o r oc-NA recepto rs than h a l o p e r i d o l (Laduron et a l . , 1978; Leysen et a l . , 1978) . Therefore u t i l i z i n g [ 3 H ] - s p i r o p e r i d o l i n the b i n d i n g assay r e s u l t s i n a more p r e c i s e measure of DA receptor s u p e r s e n s i t i v i t y . Fur thermore, the more i n depth present study w i th p a r a l l e l b e h a v i o r a l and b iochemica l r e s u l t s shows more c o n c l u s i v e l y tha t a r e l a t i o n does e x i s t s between pimozide dose and the magnitude and d u r a t i o n of DA recepto r s u p e r s e n s i t i v i t y . In a review of the l i t e r a t u r e , M u l l e r and Seeman (1978) examined the r e s u l t s obta ined by d i f f e r e n t i n v e s t i g a t o r s and concluded t h a t the e f f e c t s of c h r o n i c n e u r o l e p t i c t reatment on the maximal i n c r e a s e in n e u r o l e p t i c b i n d i n g were not dose dependent. With the s u b s t a n t i a l v a r i a t i o n s t h a t e x i s t between the i n d i v i d u a l s t u d i e s , the v a l i d i t y i n a t tempt ing to e x t r a p o l a t e from the i n f o r m a t i o n i n these d i f f e r e n t s t u d i e s i s 75 q u e s t i o n a b l e . The present study shows t h a t t h e r e i s d e f i n i t e l y a dose-dependent e f f e c t of c h r o n i c n e u r o l e p t i c a d m i n i s t r a t i o n on the degree and d u r a t i o n of DA recepto r s u p e r s e n s i t i v i t y . M u l l e r and Seeman (1978) d i d s p e c u l a t e t h a t , at lower n e u r o l e p t i c c o n c e n t r a t i o n s (as used i n t h i s s tudy) t h e r e i s p o s s i b l y a dose-dependent e f f e c t on the i n c r e a s e i n recepto r s e n s i t i v i t y . A maximum i n c r e a s e i n recepto r s e n s i t i v i t y does appear t o occur w i t h the h igher doses of pimozide admin is te red c h r o n i c a l l y when examined b i o c h e m i c a l l y , but the d u r a t i o n of the observed i n c r e a s e was g r e a t e r f o l l o w i n g t reatment w i th the h igher doses o f p imozide than w i t h the lower doses . E f f e c t of Durat ion of Pimozide A d m i n i s t r a t i o n on Dopamine Receptor  S e n s i t i v i t y . Just as the degree and d u r a t i o n of DA recepto r s u p e r s e n s i t i v i t y was shown to be dependent on the dose of p i m o z i d e , t h i s a l s o appears to be dependent on the d u r a t i o n of c h r o n i c n e u r o l e p t i c t r e a t m e n t . Thus, i n terms of d-amphetamine- induced locomotor a c t i v i t y , the longer d u r a t i o n s o f c h r o n i c pimozide t reatment (20 and 40 days) appeared to have had a g rea te r e f f e c t on the extent and d u r a t i o n of the inc reased DA r e c e p t o r s e n s i t i v i t y than d i d the s h o r t e r d u r a t i o n s of ch ron ic t reatment (5 and 10 days) (F igures 9 and 1 0 ) ; the same was t r u e f o r apomorphine- induced s t e r e o t y p y (F igure 11) . When the DA receptors were examined d i r e c t l y w i th recepto r b i n d i n g techn iques (F igure 1 2 ) , the degree of the inc reased DA recepto r number appeared to p la teau at a maximum l e v e l r e g a r d l e s s of the d u r a t i o n of c h r o n i c pimozide t r e a t m e n t . In a d d i t i o n , the inc reased number detected b i o c h e m i c a l l y decreased s i m i l a r i l y from 12 to 42 days i n animals withdrawn from d i f f e r e n t d u r a t i o n s of c h r o n i c pimozide t r e a t m e n t . 76 R e s u l t s from the two behav io ra l measures i n d i c a t e tha t a c o r r e l a t i o n e x i s t s between the d u r a t i o n of p imozide t reatment and the degree and d u r a t i o n of the change i n the s e n s i t i v i t y of DA r e c e p t o r s . However, when the i n c r e a s e i n DA recepto r s e n s i t i v i t y was examined b i o c h e m i c a l l y , no d i f f e r e n c e i n the extent and d u r a t i o n of the inc reased [ 3 H ] - s p i r o p e r i d o l b i n d i n g was e v i d e n t , across t reatment d u r a t i o n s . In an e f f o r t to e x p l a i n t h i s d i sc repancy between the behav io ra l and b iochemical r e s u l t s i t may be specu la ted t h a t dur ing the c o n t i n u a l b lockade of DA recepto rs f o r 10 to 20 d a y s , an a d d i t i o n a l a l t e r a t i o n i n the DA system o c c u r s . For example, i n a d d i t i o n to changes in the number of DA receptors as measured b i o c h e m i c a l l y , o ther f a c t o r s t h a t are not detected by recepto r b i n d i n g t e c h n i q u e s , such as a l t e r a t i o n s i n the f u n c t i o n of the DA neuron i t s e l f or i n nondopaminergic neurons t h a t are i n t r i c a t e l y a s s o c i a t e d wi th the DA sys tem, might o c c u r . In support of t h i s s p e c u l a t i o n , c h r o n i c n e u r o l e p t i c t reatment has been shown t o produce e f f e c t s on n o r a d r e n e r g i c , s e r o t o n e r g i c and c h o l i n e r g i c systems and these cou ld d i r e c t l y or i n d i r e c t l y i n f l u e n c e b e h a v i o r a l l y dependent dopaminergic mechanisms (Dunstan and J a c k s o n , 1977; Kobayashi et a l . , 1978; M u l l e r and Seeman, 1977) . That i s , whereas the b iochemica l measure on ly examines changes i n the DA recepto r per s e , the behav io ra l r e s u l t s cou ld be i n f l u e n c e d by i n t e r a c t i o n s between DA and nondopaminergic systems. Whereas these r e s u l t s are in agreement w i th o ther o b s e r v a t i o n s where the length of c h r o n i c n e u r o l e p t i c t reatment was shown not to a f f e c t the degree and d u r a t i o n of a l t e r e d recepto r number when examined d i r e c t l y w i th r e c e p t o r b i n d i n g techn iques (Burt et a l . , 1977) , when measured i n d i r e c t l y by apomorphinme-induced s t e r e o t y p y , they are c o n t r a d i c t o r y ( V o i t h , 1977) . 77 Vo i th (1977) admin is te red 3 mg/kq f l u p h e n a z i n e d i h y d r o c h l o r i d e o r a l l y f o r 6 and 21 days to r a t s and observed t h a t the d u r a t i o n of the inc reased s e n s i t i v i t y seen f o l l o w i n g an i n j e c t i o n of 0 .2 mg/kg apomorphine was s i g n i f i c a n t l y enhanced over c o n t r o l s f o r 4 weeks f o l l o w i n g withdrawal from drug t reatment i n both groups . However t h e r e was a tendency f o r the t o t a l s te reo typy score to be g r e a t e r i n the 21 day t r e a t e d animals than i n animals t r e a t e d wi th the s h o r t e r d u r a t i o n a l though not s i g n i f i c a n t l y g r e a t e r as observed i n the c u r r e n t s tudy . No e x p l a n a t i o n i s p r e s e n t l y apparent w i th regard to the d i sc repancy between the two s t u d i e s . However the present r e s u l t s showing a r e l a t i o n between the d u r a t i o n of c h r o n i c pimozide t reatment and the degree and d u r a t i o n of DA recepto r s u p e r s e n s i t i v i t y are i n d i c a t e d not on ly by measuring apomorphine- induced s t e r e o t y p y w i th the more d e t a i l e d s te reo typy r a t i n g s c a l e developed by Creese and Iversen (1975a) i n s t e a d o f t h a t developed by C o s t a l l and Nay lor (1973) and used by Vo i th (1977) , but i s f u r t h e r s u b s t a n t i a t e d by comparable r e s u l t s obta ined wi th d-amphetamine- induced locomotor a c t i v i t y . E f f e c t of Chronic Pimozide Treatment on the Dopamine Receptor When examining the DA recepto r d i r e c t l y by recepto r b i n d i n g assay t e c h n i q u e s , the t r i t i a t e d l a b e l e d l i g a n d [ 3 H ] - s p i r o p e r i d o l , i s p r e s e n t l y cons idered t o be the l i g a n d of cho ice (Laduron et a l . , 1978; Leysen et a l . , 1978) . I n v e s t i g a t o r s have determined t h a t whereas [ 3 H ] - s p i r o p e r i d o l p r i m a r i l y l a b e l s s e r o t o n i n recepto rs in the f r o n t a l c o r t e x , t h i s l i g a n d b inds almost e x c l u s i v e l y to DA receptors i n the s t r i a t u m (Creese and Snyder , 1978; Howard et a l . , 1978; Leysen et a l . , 1978) . To d e f i n e the DA recepto rs l a b e l e d by [ 3 H ] - s p i r o p e r i d o l i n the s t r i a t u m as being s p e c i f i c f o r the n e u r o t r a n s m i t t e r , dopamine ( 1 0 - 3 M) was u t i l i z e d i n the assay f o r determin ing the degree of s p e c i f i c a l l y bound [ 3 H ] - s p i r o p e r i d o l . 78 In agreement w i th other i n v e s t i g a t o r s ( F i e l d s et a l . , 1977; Howlett et a l . , 1978; Laduron et a l . , 1978; Sundermann and Wooten, 1980) i t was found tha t [ 3 H ] - s p i r o p e r i d o l b i n d i n g to s t r i a t a l t i s s u e i s s a t u r a b l e ( F igu re 13) and Scatchard a n a l y s i s revea led t h a t t h i s l i g a n d l a b e l s a s i n g l e s p e c i f i c recepto r b i n d i n g s i t e (F igure 14) . Wi th in the l i m i t s used h e r e , p imozide s e l e c t i v e l y inc reased the amount of s p e c i f i c a l l y bound [ 3 H ] - s p i r o p e r i d o l i n the s t r i a t u m . When s p e c i f i c b i n d i n g of [ 3 H ] - s p i r o p e r i d o l to DA receptors was examined 6 and 12 days f o l l o w i n g the l a s t day of t reatment i n animals admin i s te red 1.5 mg/kg or 3 .0 mg/kg f o r 10 days (F igure 8) and 1.5 mg/kg f o r v a r i o u s d u r a t i o n s (5 d a y s , 10 d a y s , 20 days , 40 days) (F igure 1 2 ) , the percentage i n c r e a s e in b i n d i n g over c o n t r o l l e v e l s appeared to p la teau around 130-140%. With the lowest dose of p imozide admin is te red ( .75 mg/kg) the i n c r e a s e i n [ 3 H ] - s p i r o p e r i d o l was s i g n i f i c a n t l y l e s s and i t s d u r a t i o n was c o r r e s p o n d i n g l y l e s s than w i th the h ighest dose of p imozide admin is te red c h r o n i c a l l y (F igure 8 ) . Other i n v e s t i g a t o r s have reported s i m i l a r i n c r e a s e s i n s p e c i f i c b i n d i n g in the s t r i a t u m f o l l o w i n g the c h r o n i c a d m i n i s t r a t i o n of n e u r o l e p t i c agents (Helmeste et a l . , 1981; Kobayashi et a l . , 1978; M u l l e r and Seeman, 1977) and f o l l o w i n g 6-OHDA n i g r o s t r i a t a l l e s i o n s (Creese et a l . , 1977; Staunton et a l . , 1981). These r e s u l t s support the s p e c u l a t i o n (Mu l le r and Seeman, 1978) , t h a t f o l l o w i n g chemical b lockade or denervat ion of DA receptors i n the ra t s t r i a t u m the i n c r e a s e i n recepto r b i n d i n g p la teaus at a maximum l e v e l . The i n c r e a s e i n receptor b i n d i n g cou ld r e f l e c t e i t h e r an i n c r e a s e i n the number of DA recepto rs or a change i n the a f f i n i t y of these b i n d i n g s i t e s . D e t a i l e d examinat ion of the inc reased b i n d i n g r e s u l t i n g from 79 c h r o n i c n e u r o l e p t i c t reatment was conducted us ing Scatchard a n a l y s i s ( R o s e n t h a l , 1967; S c a t c h a r d , 1949) . The obta ined v a l u e s f o r r e c e p t o r number ( B m a x = 27 pmol/g t i s s u e ) and recepto r a f f i n i t y (Kn = 0 .5 nM) i n c o n t r o l an imals are i n agreement w i th p rev ious r e s u l t s (Leysen, 1979; Sundermann and Wooten, 1980) . In animals t r e a t e d c h r o n i c a l l y w i t h 1.5 mg/kg p i m o z i d e , t h e r e was an i n c r e a s e i n the number o f r e c e p t o r b i n d i n g s i t e s , s i m i l a r t o the i n c r e a s e obta ined i n the amount of s p e c i f i c a l l y bound [ 3 H ] - s p i r o p e r i d o l , whereas no s t a t i s t i c a l l y s i g n i f i c a n t change was detected i n the a f f i n i t y of the r e c e p t o r s . This observed i n c r e a s e i n the l e v e l of [ 3 H ] - s p i r o p e r i d o l b i n d i n g to DA recepto rs r e s u l t i n g from an i n c r e a s e i n the d e n s i t y of DA recepto r s i t e s w i th no s i g n i f i c a n t changes i n recepto r a f f i n i t y i s s i m i l a r to t h a t repor ted by other i n v e s t i g a t o r s f o l l o w i n g both c h r o n i c n e u r o l e p t i c (Burt et a l . , 1977; Helmeste et a l . , 1981; Kobayahsi et a l . , 1978; M u l l e r and Seeman, 1977; Theodorou et a l . , 1981) and 6-OHDA t reatments (Creese et a l . , 1977; Staunton et a l . , 1981). C o r r e l a t i o n s Between Behav io ra l and Biochemical Resu l t s S u p e r s e n s i t i v i t y of dopamine recepto rs was observed both b e h a v i o r a l l y and b i o c h e m i c a l l y f o l l o w i n g the c h r o n i c a d m i n i s t r a t i o n of p imoz ide . The i n c r e a s e d number of DA recepto rs as determined by receptor b i n d i n g assay t e c h n i q u e s , appears to u n d e r l i e the r e s u l t i n g b e h a v i o r a l s u p e r s e n s i t i v i t y to d-amphetamine and apomorphine i n r a t s f o l l o w i n g long term treatment w i t h p imoz ide . When the b iochemica l changes were compared w i th the behav io ra l o b s e r v a t i o n s , both between groups of animals t r e a t e d w i t h d i f f e r e n t doses or d u r a t i o n s of pimozide (F igure 15) and w i t h i n i n d i v i d u a l groups of animals (F igure 1 6 ) , s i g n i f i c a n t c o r r e l a t i o n s were obta ined In groups of animals t r e a t e d c h r o n i c a l l y w i th d i f f e r e n t doses o f 80 pimozide t h e r e was a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between the b iochemica l r e s u l t s and the t o t a l s t e r e o t y p y s c o r e . There was always a g r e a t e r tendency f o r the recepto r b i n d i n g data to c o r r e l a t e w i th s t e r e o t y p y scores than wi th the d-amphetamine- induced locomotor a c t i v i t y . There may be severa l reasons f o r t h i s . Both apomorphine- induced s te reo typy and the DA recepto r b i n d i n g assay are ab le to examine changes i n the DA recepto rs more d i r e c t l y than does d-amphetamine- induced locomotor a c t i v i t y , d -Amphetamine, u n l i k e the d i r e c t DA agon is t apomorphine, has many a c t i o n s i n the CNS, on ly one of which i s to r e l e a s e DA from nerve t e r m i n a l s (Co lpaer t et a l . , 1976; Hanson, 1966) . Second ly , w h i l e changes i n DA r e c e p t o r b i n d i n g were examined i n the ra t s t r i a t u m and the l i c k i n g and gnawing s te reotyped behav iors are thought to be mediated p r i m a r i l y i n the s t r i a t a l dopaminergic system, the s t r i a t u m i s thought to p lay on ly a minor r o l e i n e l i c i t i n g locomotor a c t i v i t y ( C o s t a l l et a l . , 1977; Ernst and S m e l i k , 1966; K e l l y et a l . , 1975; P i jnenburg and VanRossum, 1973). When comparisons were made between locomotor a c t i v i t y and s t e r e o t y p y , no s i g n i f i c a n t c o r r e l a t i o n s were o b t a i n e d . In groups o f animals t r e a t e d c h r o n i c a l l y f o r d i f f e r e n t d u r a t i o n s w i th p i m o z i d e , the re were s i m i l a r but weaker c o r r e l a t i o n s between the b iochemica l r e s u l t s and the apomorphine s te reotypy scores than i n the dose/response exper iment . This r e s u l t i s of s i g n i f i c a n c e s i n c e a l l d u r a t i o n s of c h r o n i c pimozide t reatment (5 -40 days) appeared to i n c r e a s e recepto r b i n d i n g to a s i m i l a r e x t e n t . No s i g n i f i c a n t c o r r e l a t i o n s were found between the b iochemical r e s u l t s and locomotor a c t i v i t y i n animals t r e a t e d c h r o n i c a l l y w i th d i f f e r e n t d u r a t i o n s of p imoz ide . Of g rea te r importance was the f i n d i n g t h a t when the b iochemica l changes were compared to the behav io ra l r e s u l t s w i t h i n a group, t h e r e was 81 again always a g r e a t e r tendency f o r s i g n i f i c a n t c o r r e l a t i o n s to e x i s t between the b i n d i n g data and apomorphine- induced s t e r e o t y p y than w i t h t h e d-amphetamine locomotor a c t i v i t y d a t a . Th is i s i n agreement w i th Creese et a l . (1977) who found a c o r r e l a t i o n between b iochemica l a l t e r a t i o n s and behav io ra l s u p e r s e n s i t i v i t y to apomorphine w i t h i n a group of animals f o l l o w i n g 6-OHDA induced n i g r o s t r i a t a l l e s i o n s . These c o r r e l a t i o n s i n i n d i v i d u a l groups of animals s t r o n g l y suggest t h a t c h r o n i c a d m i n i s t r a t i o n of n e u r o l e p t i c s or l e s i o n s of the n i g r o s t r i a t a l DA pathways (Creese et a l . , 1977) leads to an inc r ease i n the number of DA recepto rs w h i c h , i n t u r n , u n d e r l i e s the enhanced b e h a v i o r a l response observed i n animals f o l l o w i n g apomorphine. However, o ther yet undetermined f a c t o r s r e s u l t i n g from long term n e u r o l e p t i c t reatment cou ld a l s o i n f l u e n c e the enhanced behav io ra l response to DA a g o n i s t s . Animal Models of Tard ive D y s k i n e s i a The DA hypothes is i m p l i c a t i n g o v e r a c t i v e DA systems i n the pathophys io logy of s c h i z o p h r e n i a i s p r e s e n t l y thought to be on ly par t o f the b r a i n mechanisms u n d e r l y i n g t h i s n e u r o l o g i c a l d i s o r d e r (Crow and G i l l b e , 1974; Langer et a l . , 1981; M a t t h y s s e , 1974; Randrup and Munkvad, 1974; Snyder , 1976; 1981) . Th is hypothes is r e s t s l a r g e l y on the observed b e n e f i c i a l t h e r a p e u t i c e f f e c t s of n e u r o l e p t i c drugs i n s c h i z o p h r e n i a . S h o r t l y f o l l o w i n g the i n t r o d u c t i o n of n e u r o l e p t i c drugs f o r the t reatment of s c h i z o p h r e n i a by Delay and Deniker (1952) , severa l ex t rapyramida l s i d e e f f e c t s were observed (Schonecker , 1957; Sigwald et a l . , 1959) . The most common s i d e e f f e c t t h a t develops d u r i n g c h r o n i c n e u r o l e p t i c t reatment ( t a r d i v e d y s k i n e s i a ) has been hypothes ized to be due to the development of s u p e r s e n s i t i v e DA recepto rs f o l l o w i n g long term r e c e p t o r b lockade by n e u r o l e p t i c agents (Crane, 1968; Klawans, 1973) . 82 Animal models have been u t i l i z e d to study the u n d e r l y i n g chemical a b n o r m a l i t i e s of TD. F o l l o w i n g c h r o n i c n e u r o l e p t i c t reatment i n a n i m a l s , s u p e r s e n s i t i v e DA recepto rs have been d e s c r i b e d b e h a v i o r a l l y as an i n c r e a s e d response to DA a g o n i s t s (Klawans and R u b o v i t z , 1972; Tarsy and B a l d e s s a r i n i , 1974; Thornburg and Moore, 1974) , b i o c h e m i c a l l y as an i n c r e a s e i n DA recepto r b i n d i n g (Burt et a l . , 1977; M u l l e r and Seeman, 1977; Sayers et a l . , 1975) and e l e c t r o p h y s i o l o g i c a l l y as an i n c r e a s e i n the a c t i v i t y of DA neurons to m i c r o i o n t o p h o r e t i c a l l y a p p l i e d DA (Yarbrough, 1975) . In both c l i n i c a l TD and the p h a r m a c o l o g i c a l l y induced s u p e r s e n s i t i v e s t a t e i n a n i m a l s , the r e s u l t i n g motor a b n o r m a l i t i e s are aggravated or worsened by DA receptor a g o n i s t s (Jacobson et a l . , 1974; Klawans, 1973) and are t e m p o r a r i l y reduced by DA r e c e p t o r a n t a g o n i s t s (Ger lach et a l . , 1974; Kazamatsuri et a l . , 1972) . Because severa l s i m i l a r i t i e s e x i s t between the animal model of TD and the n a t u r a l d i s o r d e r i n man, DA recepto r s u p e r s e n s i t i v i t y has been p o s t u l a t e d to u n d e r l i e the pathophys io logy of TD. However, the re levance of t h i s animal model has been quest ioned by many i n v e s t i g a t o r s because severa l major d i f f e r e n c e s e x i s t between TD and i t s animal model . For example, the development of s u p e r s e n s i t i v e DA receptors i n animals i s more r a p i d and p e r s i s t s f o r s h o r t e r d u r a t i o n s of t ime (Chr i s tensen et a l . , 1976) when compared to the s low ly deve lop ing and sometimes i r r e v e r s i b l e TD symptoms i n p a t i e n t s (Crane, 1971; Degwitz et a l . , 1967; Hunter et a l . , 1964; Schmidt and J a r c h o , 1966). In a d d i t i o n , even though enhanced s te reo typy has been observed i n animals d u r i n g c h r o n i c n e u r o l e p t i c t reatment (Clow et a l . , 1979) , s i m i l a r t o the development of TD d u r i n g n e u r o l e p t i c therapy i n man (Degwitz et a l . , 1967) , u n l i k e TD where symptoms occur " sp o ntaneo u s l y , " i t s demonstrat ion u s u a l l y r e q u i r e s the 83 a d m i n i s t r a t i o n of a DA agon is t (Klawans and R u b o v i t z , 1972; Tarsy and B a l d e s s a r i n i , 1974) . Regardless of the dose or d u r a t i o n of c h r o n i c pimozide t r e a t m e n t , apomorphine- induced s te reo typy was always s i g n i f i c a n t l y g rea te r i n t r e a t e d animals than i n c o n t r o l a n i m a l s . However, the d u r a t i o n of c h r o n i c pimozide t reatment i n f l u e n c e d s t e r e o t y p y more than the dose of p imoz ide . Thus, d e s p i t e the d i f f e r e n t doses of p imoz ide , a l l groups d i s p l a y e d s i m i l a r degrees and d u r a t i o n s of enhanced s te reo typy f o l l o w i n g withdrawal from c h r o n i c pimozide p re t reatment . It was a l s o t r u e t h a t , i n many animals t r e a t e d c h r o n i c a l l y w i th the h ighes t dose of p i m o z i d e , spontaneous b u r s t s of r e p e t i t i v e chewing jaw movements were observed , but t h i s never reached s t a t i s t i c a l s i g n i f i c a n c e . On the other hand, the extent and d u r a t i o n o f the enhanced s te reo typy observed in groups f o l l o w i n g treatment w i t h longer d u r a t i o n s of pimozide (20 and 40 days) were s i g n i f i c a n t l y g r e a t e r than i n groups t r e a t e d w i th s h o r t e r d u r a t i o n s of pimozide (5 and 10 d a y s ) . A l so the seemingly " p u r p o s e l e s s " b u r s t s of r e p e t i t i v e jaw movements observed i n an imals f o l l o w i n g the longer d u r a t i o n s of pimozide t reatment (20 and 40 days) were s i g n i f i c a n t l y g r e a t e r than those observed i n c o n t r o l a n i m a l s . These observa t ions showing tha t the d u r a t i o n of pimozide t reatment i n animals has a g r e a t e r e f f e c t on apomorphine- induced s te reo typy than does the dosage of p i m o z i d e , i s comparable to the development of TD i n man f o l l o w i n g long term n e u r o l e p t i c therapy (Crane, 1968) . Spontaneous b u r s t s of r e p e t i t i v e jaw movements were a l s o observed i n 4 animals (12%) d u r i n g c h r o n i c t reatment w i th pimozide f o r 40 days . These mouth movements were not d i r e c t e d towards any s o l i d ob jec t i n c l u d i n g the cage b a r s . S i m i l a r i l . y , b u r s t s of spontaneous mouth movements have been 84 repor ted i n monkeys and r a t s exposed t o c h r o n i c n e u r o l e p t i c t reatment (Clow et a l . , 1979; Gunne and Baramy, 1976; McKinney et a l . , 1980; Sahakian et a l . , 1976; Weise et a l . , 1977) . Clow et a l . (1979) repor ted t h a t f o l l o w i n g c h r o n i c t r i f l u o p e r a z i n e or t h i o r i d a z i n e t reatment f o r 12 months, r a t s showed an inc reased i n c i d e n c e of spontaneous mouth movements and abnormal f a c i a l movements when compared to c o n t r o l a n i m a l s . Even though these r e p e t i t i v e movements appear to be r e l a t e d to the symptoms of TD i n man, the exact o r i g i n and neural s u b s t r a t e u n d e r l y i n g these abnormal movements w i l l r e q u i r e f u r t h e r i n v e s t i g a t i o n s . S u p e r s e n s i t i v e DA recepto rs per se .cannot be the s o l e u n d e r l y i n g b a s i s f o r t h e i r development, s i n c e such s u p e r s e n s i t i v i t y , u n l i k e t h i s behav ior appears i n a l l animals t r e a t e d f o r much s h o r t e r per iods of t ime (5 -20 d a y s ) . The c o n t i n u i n g i n v e s t i g a t i o n s i n t o the u n d e r l y i n g mechanisms of TD, p o s s i b l y through the use of b e t t e r animal models , w i l l h o p e f u l l y lead to a b e t t e r understanding of and the eventual e l i m i n a t i o n of t h i s d e v a s t a t i n g n e u r o l o g i c a l d i s e a s e . 85 REFERENCES B a l d e s s a r i n i , R . J . , and Tarsy , D. 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