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The role of central noradrenergic systems in morphine tolerance development Klonoff, Pamela Susan 1979

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THE ROLE OF CENTRAL NORADRENERGIC SYSTEMS IN MORPHINE TOLERANCE DEVELOPMENT. by PAMELA SUSAN KLONOFF B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY; OF GRADUATE STUDIES DEPARTMENT OF PSYCHIATRY DIVISION OF NEUROLOGICAL SCIENCES We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1979 © Pamela Susan K l o n o f f , 1979 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 o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f P s y c h i a t r y , D i v i s i o n of N e u r o l o g i c a l Sciences The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e October 25. 1979. G i i ABSTRACT The r o l e of noradrenaline (NA) i n the behavioural and pharmacological e f f e c t s of morphine was evaluated i n r a t s . Animals r e c e i v e d s p e c i f i c i n j e c -t i o n s of 6-hydroxydopamine (6-OHDA) i n t o the d o r s a l noradrenergic bundle (DB) r e s u l t i n g i n s e l e c t i v e d e p l e t i o n of t e l e n c e p h a l i c NA l e v e l s and increased l e v e l s of noradrenaline i n the s p i n a l cord and cerebellum. Employing changes i n the hypoactive phase of morphine-induced locomotor a c t i v i t y as an index of tolerance development, i t was observed that i n j e c t i o n of 6-OHDA i n t o the dor-s a l noradrenergic bundle r e s u l t e d i n a slower r a t e and a l e s s e r degree of tolerance development to morphine. The e f f e c t of the DB-6-0HDA l e s i o n on p h y s i c a l dependence was assessed by measuring naltrexone-induced withdrawal i n l e s i o n e d and c o n t r o l animals who had recei v e d chronic morphine treatment. Results i n d i c a t e that although NA i s important i n tol e r a n c e development, i t does not mediate a dominant r o l e i n withdrawal, although behavioural evidence suggesting a secondary or modulatory r o l e i s presented. The i n t e r a c t i o n of amphetamine and morphine w i t h the dopamine (DA) system was a l s o assessed by studying the behavioural e f f e c t s of amphetamine i n animals f o l l o w i n g e i t h e r acute or chronic morphine treatment. I t was observed that amphetamine poten-t i a t e d the spontaneous locomotor h y p e r a c t i v i t y f o l l o w i n g both acute and chro-n i c morphine treatment. The DB-6-OHDA l e s i o n d i d not a f f e c t the locomotor p o t e n t i a t i o n of amphetamine i n morphine p r e - t r e a t e d animals, and the hypothe-s i s that another t r a n s m i t t e r system mediates t h i s e f f e c t , s p e c i f i c a l l y DA, i s discussed. i i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENT i x GENERAL INTRODUCTION 1 Acute Studies: I n t e r a c t i o n With the Dopamine System. 1 Acute Studies: I n t e r a c t i o n With the Noradrenergic System. 3 Chronic Studies. 4 STUDY I . EFFECT OF DB-6-OHDA LESIONS ON TOLERANCE DEVELOPMENT TO MORPHINE-INDUCED LOCOMOTOR ACTIVITY. 10 INTRODUCTION 10 Morphine Tolerance and Locomotor A c t i v i t y 10 METHODS 14 Subjects 14 Drugs 14 Apparatus 14 Procedure 15 Biochemistry 17 RESULTS 18 Ha b i t u a t i o n Data 18 P o s t - I n j e c t i o n Data 18 Weight Data 27 Biochemistry 36 DISCUSSION 38 Morphine Tolerance and Locomotor A c t i v i t y 38 STUDY I I . THE EFFECT OF A 6-OHDA LESION TO THE DORSAL NORADRENERGIC BUNDLE ON WITHDRAWAL IN MORPHINE-DEPENDENT.RATS. 41 i v Page INTRODUCTION 4 1 METHODS 4 5 STUDY H a 4 5 I r r i t a b i l i t y R.ating Scale 45 Withdrawal 4 5 STUDY l i b 4 6 Subjects 4 6 Drugs 4 6 Procedure 46 RESULTS 4 9 STUDY H a 4 9 I r r i t a b i l i t y Data 4 9 Withdrawal Weight Data 49 STUDY l i b 53 DISCUSSION 56 STUDY I I I . A STUDY OF THE PSYCHOPHARMACOLOGICAL INTERACTION OF MORPHINE AND AMPHETAMINE. 58 INTRODUCTION 58 STUDY I l i a . INTERACTION BETWEEN THE BEHAVIOURAL EFFECTS OF ACUTE MORPHINE AND D-AMPHETAMINE ADMINI-STRATION ON LOCOMOTOR ACTIVITY IN RATS. 60 METHODS 60 Subjects 6 0 Apparatus 60 Procedure 60 RESULTS 6 2 STUDY I l l b . THE EFFECT OF ACUTE D-AMPHETAMINE ADMINI-STRATION ON LOCOMOTOR ACTIVITY IN MORPHINE TOLERANT RATS. 6 6 METHODS 6 6 V Page Subjects and Procedures 66 Drugs 6 6 RESULTS 6 7 H a b i t u a t i o n Data 6 7 P o s t - I n j e c t i o n Data ^7 DISCUSSION 7 2 GENERAL DISCUSSION 7 4 REFERENCES 8 9 APPENDIX I 9 6 98 APPENDIX I I v i LIST OF TABLES Page Table I E f f e c t of b i l a t e r a l 6-OHDA i n j e c t i o n s i n t o the d o r s a l NA p r o j e c t i o n on NA l e v e l s i n the hippo-campus /cortex and caudate DA l e v e l s . 37 Table I I Summary of the morphine i n j e c t i o n procedure f o r i n d u c t i o n of p h y s i c a l dependence. 47 Table I I I I r r i t a b i l i t y data. 52 Table IV Withdrawal-counted signs. 54 Table V Withdrawal-present sign s . 55 v i i LIST OF FIGURES Page Figure 1 Model f o r the e f f e c t s of acute and chronic morphine treatment on noradrenergic tra n s m i s s i o n (Schwartz, 1979) 6 Figure 2 Mean locomotor a c t i v i t y during a 1 hour p r e - i n j e c t i o n h a b i t u a t i o n phase f o r days 1,4,7,10,13 and 16 of drug i n j e c t i o n . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r -c l e s = v e h i c l e - s a l i n e group, N=8. 20 Figure 3 Mean locomotor a c t i v i t y during the hypoactive phase f o r days 1,4,7,10,13. and 16 of drug i n j e c t i o n . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = v e h i c l e -morphine group, N=10; Open c i r c l e s = V e h i c l e - s a l i n e group, N=8. 22 Figure 4 Mean locomotor a c t i v i t y during the hyper a c t i v e phase f o r days 1,4,7,10,13 and 16 of drug i n j e c t i o n . Closed squares - DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = v e h i c l e -morphine group, N-10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 25 Figure 5 Mean locomotor a c t i v i t y during the 3 hour p o s t - i n j e c t i o n p e r i o d on day 1 of drug i n j e c t i o n . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-s a l i n e group, N=8; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 29 Figure 6 Mean locomotor a c t i v i t y during the 3 hour p o s t - i n j e c t i o n p e r i o d on day 7 of drug i n j e c t i o n . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-s a l i n e group, N=8; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 31 Figure 7 Mean locomotor a c t i v i t y during the 3 hour p o s t - i n j e c t i o n p e r i o d on day 16 of drug i n j e c t i o n . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-s a l i n e group, N=8; Closed c i r c l e s - vehicle-morphine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 33 Figure 8 Mean body weight data on days 1.6.12 and 18 of drug i n j e c t i o n . Closed squares - DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r c l e s -v e h i c l e - s a l i n e group, N=8. 35 Figure 9 Mean body weight data on the l a s t day of morphine i n j e c t i o n (day 24) and f o r 5 days f o l l o w i n g d i s c o n t i n u a t i o n of d a i l y morphine i n j e c t i o n s . Closed squares = DB-6-0HDA-morphine group, N=10; Closed c i r c l e s = vehicle-morphine group, N=10. 51 v i i i Page Figure I P Mean locomotor a c t i v i t y d u r i ng a 3 hour p e r i o d f o l l o w -i n g s a l i n e i n j e c t i o n . Closed squares - s a l i n e / amphetamine, N=10; Open squares = morphine/saline, N=8; Closed c i r c l e s = morphine/amphetamine, N=10; Open c i r c l e s = s a l i n e / s a l i n e , N=10 per group. 65 Figure 11. Mean locomotor a c t i v i t y during a 1 hour p r e - i n j e c t i o n h a b i t u a t i o n phase p r i o r to acute amphetamine i n j e c t i o n s . See Study I l l b . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 69 Figure 12 Mean locomotor a c t i v i t y during a 3 hour p e r i o d f o l l o w -ing acute amphetamine i n j e c t i o n . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-OHDA-saline group, N=8; Closed c i r c l e s = v e h i c l e -morphine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 71 i x ACKNOWLEDGEMENTS I would l i k e to express my a p p r e c i a t i o n to those people who co n t r i b u t e d to t h i s p r o j e c t . My s i n c e r e thanks are expressed to my a d v i s o r , Dr. C h r i s F i b i g e r , f o r h i s d i r e c t i o n and support. My s i n c e r e thanks are also ex-pressed to S t e l l a Atmadja and Amelia Wong f o r t h e i r t e c h n i c a l a s s i s t a n c e . I a l s o thank Dr. A.G. P h i l l i p s f o r s e r v i n g on my t h e s i s committee and John Lehmann f o r h e l p f u l d i s c u s s i o n s . 1 GENERAL INTRODUCTION The c h a r a c t e r i z a t i o n of op i a t e receptors by bi n d i n g and autoradiographic s t u d i e s and the i d e n t i f i c a t i o n of o p i o i d peptides as t h e i r endogenous l i g a n d s has aided i n the understanding of the acute and chronic behavioural a c t i o n s of morphine-like drugs. Lesi o n s t u d i e s i n d i c a t e that o p i a t e receptors are found on dopaminergic (DA) and noradrenergic (NA) nerve-endings and c e l l bodies (see review by Schwartz, 1979) and i t has been suggested that the biochemical mechanisms underlying the psychopharmacological a c t i o n s of morphine i n v o l v e a d i r e c t i n t e r a c t i o n w i t h the catecholamine systems. Acute Studies: I n t e r a c t i o n With the Dopamine System: The s t r i a t u m i s one of the r i c h e s t b r a i n areas f o r enkephalin and opi a t e b i n d i n g s i t e s ( P o l l a r d , L l o r e n s , Schwartz, Gros and Dray, 1978), and ther e f o r e much research has been aimed at determining the e f f e c t s of opiat e s on dopaminergic neurons. In r a t s , morphine a d m i n i s t r a t i o n i n i t i a l l y e l i c i t s acute symptoms of decreased DA neurotransmission, i . e . c a t a l e p s y , hypokinesia and muscular r i g i d i t y . F o llowing t h i s , a compensatory increase i n endogenous DA l e v e l s as a r e s u l t of enhanced DA synthesis and u t i l i z a t i o n , has been a t t r i b u t e d to a feedback a c t i v a t i o n of b i o s y n t h e s i s (Gauchy, Ag i d , G l o w i n s k i and Cheramy, 1973; F u k u i , Shiomi and Takagi, 1972; see review by Kuschinsky, 1976). Strong evidence f o r t h i s theory has been provided by Nowycky, Walters and Roth (1978), who p l o t t e d the time course of morphine's e f f e c t on DA metabolism. They reported that s t r i a t a l DOPA synthesis r a t e s were increased between 3Q-60 minutes a f t e r acute morphine a d m i n i s t r a t i o n and that s t r i a t a l DOPAC (3,4 dihydroxyphenylacetic acid) l e v e l s were not s i g n i f i c a n t l y d i f f e -rent from c o n t r o l l e v e l s 30 minutes f o l l o w i n g morphine i n j e c t i o n , but were 2 doubled by 60 minutes p o s t - i n j e c t i o n . Kuschinsky and Hornykiewicz (1972) found that acute i n j e c t i o n s of mor-phine induced catalepsy i n r a t s and r a i s e d the h o m o v a n i l l i c a c i d (HVA) con-c e n t r a t i o n i n the s t r i a t u m . The morphine-induced catalepsy could be abo-l i s h e d w i t h L-DOPA or apomorphine. These authors hypothesized that morphine i n f l u e n c e s DA metabolism p r e s y n a p t i c a l l y and that the increase i n HVA i s a consequence of a d i v e r s i o n of newly synthesized DA from storage s i t e s to s i t e s of catabolism. Increased breakdown of the newly formed DA r e s u l t s i n a dearth of the amine at the receptor s i t e . L a i , Gianutsos and P u r i (1975) compared the a c t i o n of morphine w i t h a n e u r o l e p t i c , h a l o p e r i d o l , known to bl o c k DA r e c e p t o r s , on a s e r i e s of beha-v i o u r a l measures ( i . e . stereotypy, c a t a l e p s y , e t c . ) . In these b e h a v i o u r a l t e s t s , both morphine and h a l o p e r i d o l resembled each other i n t h e i r acute a c t i o n s . However, the authors a l s o reported that the morphine-induced i n -crease i n the f i r i n g r a t e of DA c e l l s i n the zona compacta of the s u b s t a n t i a n i g r a can be f u r t h e r stimulated by h a l o p e r i d o l . In a d d i t i o n , a n t i c h o l i n e r -g i c drugs reverse many of h a l o p e r i d o l ' s a c t i o n s , but not the morphine e f f e c t s w i t h the same true of naloxone. The authors t h e r e f o r e concluded that mor-phine blocks p o s t s y n a p t i c DA r e c e p t o r s , but that the a c t i o n i s i n d i r e c t . Furthermore, behavioural evidence and biochemical evidence that endogenous o p i o i d peptides and morphine produce markedly d i f f e r e n t response p r o f i l e s than haloperidol-, has r e c e n t l y been presented (Weinberger, Arnsten and Segal, 1979). Diamond and Borison (1978) have reported that f o l l o w i n g u n i l a t e r a l 6-hy-droxydopamine (6-OHDA) l e s i o n s of the s u b s t a n t i a n i g r a , naloxone p o t e n t i a t e d agents w i t h p o s t s y n a p t i c dopaminergic a c t i o n s (apomorphine, L-DOPA) and anta-gonized agents w i t h p r e s y n a p t i c dopaminergic a c t i o n s (d-amphetamine, phenyl-ethylamine). The authors t h e r e f o r e proposed the f o l l o w i n g model f o r the ac-3 t i o n s of enkephalins i n the n i g r o s t r i a t a l system. A presynaptic enkephalin interneuron f a c i l i t a t e s n i g r o s t r i a t a l t r a n s m i s s i o n and a post s y n a p t i c enke-p h a l i n e r g i c interneuron i n h i b i t s a c t i v a t i o n of dopamine receptors. Drugs that p o t e n t i a t e enkephalin mechanisms i n the b r a i n , p o t e n t i a t e presynaptic dopaminergic ac t i o n s ( i . e . increase DA synthesis) w h i l e antagonizing post-s y n a p t i c e f f e c t s , w h i l e naloxone, p o t e n t i a t e s p o s t s y n a p t i c but not presynap-t i c mechanisms. P o l l a r d et a l (1978) explored the hypothesis f o r the presence of opiate receptors on DA neurons by assessing the e f f e c t s of extensive degeneration of DA neurons. E f f e c t s of i n t r a n i g r a l 6-OHDA l e s i o n s or hemitransections, i n t r a -s t r i a t a l a d m i n i s t r a t i o n of k a i n i c a c i d and 6-OHDA l e s i o n s of the s u b s t a n t i a n i g r a on opiate r e c e p t o r s , i n d i c a t e d that o n e - t h i r d of s t r i a t a l o p i a t e recep-t o r s are l o c a l i z e d on dopaminergic neurons, w h i l e two-thirds are l o c a l i z e d on neurons i n t r i n s i c to the s t r i a t u m . Furthermore, o p i a t e receptors might be present on DA c e l l s i n the s u b s t a n t i a n i g r a although contrary evidence i n d i -c a t i n g that opiate receptors are not on n i g r a l DA c e l l bodies, but may be on GABAergic or substance P a f f e r e n t s from the s t r i a t u m has r e c e n t l y been repor-ted ( R e i s i n e , Nagy, Beaumont, F i b i g e r and Yamamura, i n p r e s s ) . P o l l a r d et a l go on to describe the model e x p l a i n i n g the mechanisms un d e r l y i n g the acute a c t i o n of morphine: presynaptic o p i a t e receptors on DA neurons mediate pre-s y n a p t i c i n h i b i t i o n . This i n h i b i t i o n accounts f o r the symptoms of decreased dopaminergic tran s m i s s i o n observed a f t e r acute a d m i n i s t r a t i o n . This primary e f f e c t t r i g g e r s a second phase - a compensatory increase i n DA s y n t h e s i s , which i s t r a n s l a t e d i n t o symptoms of increased DA transm i s s i o n ( i . e . locomo-t o r a c t i v i t y , jumping, stereotyped and aggressive behaviours). Acute Studies: I n t e r a c t i o n With the Noradrenergic System: Opiate receptors have been l o c a t e d p r e s y n a p t i c a l l y on c o r t i c a l and c e r e b e l l a r noradrenergic nerve terminals o r i g i n a t i n g from the locus coeru-4 leus (LC) ( L l o r e n s , Martres, Baudry, Schwartz, 1978; P e r t and Snyder, 1973). Opiate a d m i n i n s t r a t i o n r e s u l t s i n i n h i b i t i o n of the spontaneous f i r i n g r a t e of NA c e l l s i n the locus coeruleus, and t h i s e f f e c t i s reversed by naloxone.(Korf, Bunney and Aghajanian, 1974). Herz, Teschemacher, Albus and Zeiglgansberger (1972) i d e n t i f i e d the me-d u l l a r y and pontine areas of the lower b r a i n stem as primary s i t e s of mor-phine drug a c t i o n and have suggested involvement of such aminergic b r a i n s t r u c t u r e s as the locus coeruleus and raphe n u c l e i . In a d d i t i o n , Atweh and Kuhar (1977b)have demonstrated high concentrations of o p i a t e receptors i n the locus coeruleus using both b i n d i n g s t u d i e s and autoradiography tech-niques. Acute a d m i n i s t r a t i o n of morphine has been reported to increase the l e -v e l s of MHPG-SOIL (3 methoxy-4-hydroxyphenylglycol) , a major me t a b o l i t e of b r a i n norepinephrine i n the r a t b r a i n (Roffman, R e i g l e , Orsulak, Cassens and S c h i l d k r a u t , 1979), w h i l e Watanabe (1971) reported a s i g n i f i c a n t decrease i n b r a i n noradrenaline a f t e r an acute i n t r a v e n t r i c u l a r i n j e c t i o n of morphine. Chronic Studies; A major theory f i r s t proposed by C o l l i e r (1965) and J a f f e and Sharpless (1968), to e x p l a i n the mechanisms un d e r l y i n g t o l e r a n c e and p h y s i c a l depen-dence i s that of "disuse h y p e r s e n s i t i v i t y " . According to t h i s theory, o p i a t e a d m i n i s t r a t i o n r e s u l t s i n decreased catecholamine t r a n s m i s s i o n , and prolonged p r e s y n a p t i c i n h i b i t i o n during long-term morphine a d m i n i s t r a t i o n r e s u l t s i n a compensatory mechanism of increased responsiveness of the post-s y n a p t i c t a r g e t c e l l to catecholamines. With morphine withdrawal, catecho-lamine r e l e a s e i s r e s t o r e d and a rebound response of h y p e r s e n s i t i v e t a r g e t c e l l s r e s u l t s . D e t a i l s of the disuse h y p e r s e n s i t i v i t y theory are presented i n Figure A (Schwartz, 1979). 5 Figure 1: Model f o r the e f f e c t s of acute and • chronic morphine treatment on noradrenergic tran s m i s s i o n . (Schwartz, 1979). © ® N o r m a l s i tuat ion A c u t e morph ine Noradrenergic neurone O p i a t e receptors M o r p h i n e N A release O O O O O O O O O o Response N o r m a l -Noradrenergic receptors « - i Decreased © C h r o n i c morph ine M o r p h i n e o N o r m a l ® A b s t i n e n c e OOO o o o o o o Increased Fig. 1. Model for the effects of acute and chronic morphine treatment on noradrenergic transmission*. 7 Llor e n s et a l (1978) found that r a t s c h r o n i c a l l y t r e a t e d w i t h morphine developed a h y p e r s e n s i t i v i t y of po s t s y n a p t i c c e l l s to noradrenergic i n p u t , as i n d i c a t e d by an increased responsiveness to NA and i s o p r e n a l i n e . This f i n d i n g was p a r t l y a t t r i b u t e d to an increased number of g-adrenergic recep-t o r s . The authors suggested that t h i s h y p e r s e n s i t i v i t y to NA could account f o r the development of tol e r a n c e to the a c t i o n of morphine. P u r i , V o l i c e r and L a i (1977) t e s t e d the s u p e r s e n s i t i v i t y hypothesis b i o c h e m i c a l l y by measuring the changes i n s t r i a t a l dopamine turnover a f t e r a d m i n i s t r a t i o n of apomorphine i n morphine dependent r a t s . They found that chronic morphine treatment d i d not a l t e r the s t r i a t a l DA turnover, however, the a b i l i t y of apomorphine to decrease DA turnover was s i g n i f i c a n t l y en-hanced i n 72-hour withdrawn morphine dependent animals. In that i t has been suggested that apomorphine decreases dopamine turnover by s t i m u l a t i n g dopamine r e c e p t o r s , e l i c i t i n g a compensatory decrease i n presynaptic u t i l i -z a t i o n and r e l e a s e of dopamine, i t would appear that t h i s data suggests a s u p e r s e n s i t i z a t i o n of post s y n a p t i c DA rec e p t o r s . P u r i and L a i (1974) f u r t h e r hypothesized that i f morphine dependency were c h a r a c t e r i z e d by s u p e r s e n s i t i v i t y of DA r e c e p t o r s , there should be a r e -duction i n pharmacological responsiveness to drugs that block DA re c e p t o r s , such as h a l o p e r i d o l . A f t e r acute i n j e c t i o n of morphine or h a l o p e r i d o l , there was a marked increase i n the r a t e of DA d e p l e t i o n a f t e r i n h i b i t i o n of i t s synt h e s i s w i t h AMPT and hence a s i g n i f i c a n t increase i n turnover. However, animals made dependent on morphine showed t o l e r a n c e to the e f f e c t of morphine or h a l o p e r i d o l on DA turnover. Catalepsy was used as the behavioural c o r r e l a t e to the above neurochemical f i n d i n g s . Acute admini-s t r a t i o n of h a l o p e r i d o l or morphine r e s u l t e d i n catalepsy in.non-dependent animals, but the behavioural e f f e c t s to h a l o p e r i d o l or morphine were not present i n morphine dependent r a t s . The observation that the s u p e r s e n s i t i v e 8 DA receptors reduce the e f f e c t i v e n e s s of drugs that act by b l o c k i n g DA r e -ceptors ( i . e . h a l o p e r i d o l ) was i n t e r p r e t e d by the authors as support f o r the development of s u p e r s e n s i t i v e DA receptors during n a r c o t i c dependence. Smee and Overstreet (1976) reported a s i g n i f i c a n t i n c r e a s e i n o r a l cage or i e n t e d stereotyped behaviour i n chronic morphine t r e a t e d animals when ch a l lenged w i t h apomorphine, but no d i f f e r e n c e i n s a l i n e t r e a t e d animals. When pimozide, a DA antagonist was administered to chronic morphine t r e a t e d r a t s , the depressive e f f e c t s on a c t i v i t y were reduced. Further evidence i n support of t h i s theory has been provided by Baume, Patey, Marcais, P r o t a i s , Constentin and Schwartz (1979). They t e s t e d whether s e v e r a l features of the t y p i c a l dopaminergic h y p e r s e n s i t i v i t y syn-drome observed f o l l o w i n g blockade of DA receptors by h a l o p e r i d o l could be detected f o l l o w i n g chronic a d m i n i s t r a t i o n of morphine. A s i g n i f i c a n t i n -crease i n the behavioural responsiveness to apomorphine was observed on a cli m b i n g t e s t , as w e l l as a s i g n i f i c a n t decrease i n HVA l e v e l s . Therefore, both behavioural and biochemical data supported the theory of h y p e r s e n s i t i -v i t y to DA a f t e r sustained blockade of DA re c e p t o r s . A DA-sensitive adenylate c y c l a s e , found i n the s t r i a t u m has been sugges ted as the DA receptor (Clouet and Iwatsubo, 1975a). Inasmuch as the stimu-l a t i o n of DA-sensitive adenylate c y c l a s e can be i n h i b i t e d i n v i t r o by neuro-l e p t i c receptor b l o c k e r s , which a l s o increase DA turnover, the e f f e c t of n a r c o t i c a n a l g e s i c s on b a s a l and DA- s e n s i t i v e adenylate c y c l a s e i n s t r i a t a l nerve ending preparations were examined to determine whether opiates have a d i r e c t e f f e c t on the DA receptor. I t has found that morphine had no e f f e c t on DA-sensitive adenylate c y c l a s e i n crude ruptured nerve endings from the r a t s t r i a t u m , and i t was t h e r e f o r e suggested that morphine, u n l i k e h a l o p e r i -d o l , does not .react d i r e c t l y w i t h the "dopamine receptor". When, however, adenylate c y c l a s e was measured i n a smaller t i s s u e sample, a f r a c t i o n con-9 t a i n i n g ruptured synaptosomal contents and p o r t i o n s of the pre- and post-s y n a p t i c membrane, s t r i a t a l D A - s e n s i t i v e adenylate c y c l a s e was i n c r e a s e d , w h i l e b a s a l adenylate c y c l a s e remained constant, f o l l o w i n g chronic morphine treatment. I t i s t h e r e f o r e suggested that the increased DA s e n s i t i v i t y of adenylate c y c l a s e i n the s t r i a t u m of morphine t o l e r a n c e r a t s i s r e l a t e d to the s u p e r s e n s i t i v i t y to DA agonists ( i . e . apomorphine) found i n b e h a v i o u r a l experiments. Considering the evidence h e r e t o f o r e presented supporting the theory of p o s t s y n a p t i c receptor s u p e r s e n s i t i v i t y as an explanation f o r the mechanisms underlying morphine t o l e r a n c e , i t was decided to t e s t the theory by study-in g s e v e r a l b e h a v i o u r a l e f f e c t s of chronic morphine a d m i n i s t r a t i o n i n r a t s . 10 STUDY I . EFFECT OF DB-6-OHDA LESIONS ON TOLERANCE DEVELOPMENT TO MORPHINE-INDUCED LOCOMOTOR ACTIVITY. INTRODUCTION Morphine Tolerance and Locomotor A c t i v i t y : Morphine produces a n t i n o c i c e p t i v e a c t i o n s , as w e l l as e f f e c t s on l o c o -motor a c t i v i t y . F o llowing acute i n j e c t i o n , morphine produces a b i p h a s i c e f -f e c t on locomotor a c t i v i t y , c h a r a c t e r i z e d by an i n i t i a l depressant phase, fo l l o w e d by a s t i m u l a t o r y phase, approximately 2-3 hours p o s t - i n j e c t i o n (Babbini and Davis, 1972). Hosoya, Oguri and A k i t a (1963) observed tolerance development to the e f f e c t s of chronic morphine a d m i n i s t r a t i o n on spontaneous locomotor a c t i v i t y , s p e c i f i c a l l y to the i n i t i a l s e d a t i v e e f f e c t , by the t h i r d day of t e s t i n g . Although the authors claimed that t o l e r a n c e occurred only to the sed a t i v e e f f e c t s of morphine on locomotor a c t i v i t y , they noted that the e x c i t a t o r y e f f e c t s a l s o became enhanced by repeated morphine a d m i n i s t r a t i o n . This f i n d -i n g has sin c e been confirmed by Vasko and Domino (1978) and Smee and Over-s t r e e t (1976). Catecholamines have been i m p l i c a t e d i n morphine's a c t i o n on locomotor a c t i v i t y . E i d e l b e r g and Schwartz (1970) reported that ct-methylparatyrosine (AMPT), a catecholamine syn t h e s i s i n h i b i t o r , prevented the h y p e r a c t i v i t y evidenced by morphine t o l e r a n t r a t s , and that t h i s e f f e c t was reversed by p r e - i n j e c t i o n of L-dopa. These r e s u l t s i n morphine t o l e r a n t r a t s were con-firmed by Davis, Babbini and Khalsa (1972) and Buxbaum, Yarbrough and Carter (1973). Further support f o r these f i n d i n g s f o l l o w i n g acute morphine a d m i n i s t r a t i o n was p a r a l l e l e d by Oka and Hosoya (1976) and C a r r o l l and Sharp (1972). In a d d i t i o n , C a r r o l l and Sharp (1972) provided f u r t h e r e v i -dence that the morphine induced a c t i v a t i o n response i n mice i s modified by 11 drugs that a f f e c t catecholamines, w i t h the observations that i n h i b i t i o n of monoamine oxidase a c t i v i t y by p a r g y l i n e p o t e n t i a t e d acute morphine-induced h y p e r a c t i v i t y and blockade of a-adrenoreceptors w i t h phentolamine and phen-oxybenzamine reduced the response. Herman (1970) provided evidence that i n -t r a v e n t r i c u l a r NA increases locomotor a c t i v i t y i n r a t s , whereas Maj, Grabow-ska and M o g i l n i c k a (1971) suggested that motor s t i m u l a t i o n appears only i f DA l e v e l s are r a i s e d and i f NA l e v e l s are approximately normal. E s t l e r (1973) reported t h a t morphine caused marked e x c i t a t i o n i n mice and that s i -multaneous treatment w i t h the a-sympatholytic drug, phenoxybenzamine, abo-l i s h e d t h i s e f f e c t . Recent research, however, has i m p l i c a t e d a more dominant r o l e f o r DA than NA i n morphine induced locomotor h y p e r a c t i v i t y . Broekkamp, P h i l l i p s and Cools (1979) reported increased l e v e l s of spontaneous locomotor a c t i v i t y i n animals r e c e i v i n g i n t r a c e r e b r a l i n j e c t i o n s of the long a c t i n g s y n t h e t i c enkephalin analogue (D-ala 2) - Met 5enkephalinamide (AME) i n t o the dopaminer-g i c A10 region of the v e n t r a l tegmental area (VTA). C a r r o l l and Sharp (1972) reported t h a t h a l o p e r i d o l completely blocked the a c t i v a t i o n response of mice to acute morphine, w h i l e chlorpromazine had a s i g n i f i c a n t but l e s s potent e f f e c t . I t has been reported that these two drugs have equal a-adre-n e r g i c b l o c k i n g a c t i v i t y (Anden, Butcher, C o r r o d i , Fuxe and Ungerstedt, 1970), but that h a l o p e r i d o l i s ten times more a c t i v e than chlorpromazine against c e n t r a l DA mechanisms. I t was t h e r e f o r e suggested that c a t e c h o l a -mines are invo l v e d i n the normal a c t i v a t i o n response of mice to acute mor-phine i n j e c t i o n , but that the dopaminergic mechanism i s of primary impor-tance. Kuschinsky and Hornykiewicz (1974) and Kuschinsky (1976) reported that DA s t i m u l a t i o n i s r e s p o n s i b l e f o r morphine-induced locomotor s t i m u l a -t i o n , inasmuch as pre-treatment w i t h AMPT abolished the morphine e f f e c t , which could e a s i l y be res t e d by L-dopa, but not DOPS (a precursor of NA). 12 In a d d i t i o n , morphine produced an i n c r e a s e i n s t r i a t a l HVA and b i l a t e r a l e l e c t r o l y t i c l e s i o n s of the s t r i a t u m s i g n i f i c a n t l y decreased morphine's s t i -mulant e f f e c t on locomotor a c t i v i t y . Furthermore, these authors pointed out that drugs such as L-dopa, apomorphine and amphetamine, which are thought to act p r i m a r i l y v i a c e n t r a l DA mechanisms produce marked h y p e r a c t i v i t y , where-as NA agonists (DOPS, c l o n i d i n e ) do not produce motor s t i m u l a t i o n . The authors t h e r e f o r e i n f e r r e d that the b r a i n amine p r i m i a r l y i n v o l v e d i n mor-phine-induced locomotor a c t i v i t y i s DA and that the noradrenergic system may be i n v o l v e d i n r e g u l a t i n g the s e n s i t i v i t y of the e f f e c t o r system(s) through which DA exerts i t s e f f e c t . Smee and Overstreet (1976) hypothesized that the b i p h a s i c behavioural changes of morphine on locomotor a c t i v i t y are r e l a t e d to the i n i t i a l b l o c k -ade of dopamine transmission and that t h i s r e s u l t s i n an increase i n dopa-mine synth e s i s and turnover which o u t l a s t s the blockade. Chronic treatment r e s u l t s i n p o s t s y n a p t i c DA receptors becoming s u p e r s e n s i t i v e , as evidenced by a s u p e r s e n s i t i v e stereotypy response to amphetamine and apomorphine, and a s u b s e n s i t i v e response to pimozide a f t e r chronic morphine treatment. On the b a s i s of the above l i t e r a t u r e , i t i s p o s s i b l e to conclude that catecholamines are somehow i m p l i c a t e d i n the expression of morphine-induced locomotor a c t i v i t y , as w e l l as the mechanisms underlying the development of t o l e r a n c e , although c l e a r d i f f e r e n t i a t i o n of the s p e c i f i c r o l e s of the DA and NA systems i s as yet incomplete. P r e v i o u s l y , the s e l e c t i v e neurotoxin 6-hydroxydopamine has been used i n . d e f i n i n g the r o l e of catecholaminergic neurons i n morphine-mediated behaviour. 6-OHDA produces s e l e c t i v e destruc-t i o n of catecholaminergic nerve endings and c e l l bodies i n the c e n t r a l ner-vous system (see review by F i b i g e r , F i b i g e r and Z i s , 1973), and s p e c i f i c i n -j e c t i o n of the drug i n t o the v i c i n i t y of the d o r s a l noradrenergic p r o j e c t i o n 13 (DB) s u b s t a n t i a l l y depletes f o r e b r a i n NA and increases s i g n i f i c a n t l y the content of NA i n the cerebellum and s p i n a l cord (Mason, Roberts and F i b i g e r , 1975). This procedure r e s u l t s i n a s l i g h t , but n o n s i g n i f i c a n t e f f e c t on DA l e v e l s and there f o r e enables the d i s s o c i a t i o n of the NA and DA systems i n morphine-mediated behaviour. Roberts, Mason and F i b i g e r (1978) reported that 6-OHDA l e s i o n s to t h i s ascending NA p r o j e c t i o n p o t e n t i a t e d the l o c o -motor depressant e f f e c t s of morphine when i t i s ac u t e l y administered. In a d d i t i o n , i t has been reported that the same l e s i o n p o t e n t i a t e d morphine-in-duced catalepsy (Mason, Roberts and F i b i g e r , 1978). Considering t h i s e v i -dence, and p r i o r f i n d i n g s of tole r a n c e development to the suppressant e f -f e c t s of morphine on locomotor a c t i v i t y (Hosoya et a l , 1963), i t was hypothe-s i z e d that NA may mediate some aspects of to l e r a n c e development f o l l o w i n g c h r o n i c morphine a d m i n i s t r a t i o n . I t was the r e f o r e decided to explore the e f f e c t s of a 6-OHDA l e s i o n to the d o r s a l noradrenergic p r o j e c t i o n on morphine tol e r a n c e development, by studying changes i n spontaneous locomotor a c t i v i t y w i t h c h r o n i c morphine treatment. 14 METHODS Subjects: A t o t a l of f o r t y male Wistar a l b i n o r a t s from Woodlyn Farms, Guelph, Ontario were used i n the f o l l o w i n g s e r i e s of s t u d i e s . Using p e n t o b a r b i t a l (Nembutal) anesthesia, one group (n=20), weighing 290 - 330 grams, rece i v e d b i l a t e r a l i n j e c t i o n s of 6-hydroxydopamine (4 yg/2 y l ) expressed as the base (6-OHDA-HBr, Regis) i n 0.9% s a l i n e c o n t a i n i n g 0.3 mg/ml a s c o r b i c a c i d i n the d o r s a l noradrenergic p r o j e c t i o n . An i n j e c t i o n r a t e of 2 y l / 5 min through a 32 gauge 10 y l Hamilton s y r i n g e was maintained. F o l l o w i n g the i n j e c t i o n , the s yringe was l e f t i n place f o r 2 min to al l o w f o r any d i f f u s i o n . The s t e r e o t a x i c coordinates were A.P. + 2.6, from the i n t e r a u r a l l i n e ; M.L. ± 1.1 mm from the m i d l i n e and D.V. + 3.7 mm from the i n t e r a u r a l l i n e , w i t h the animal's head p o s i t i o n e d i n a Kopf s t e r e o t a x i c and the i n c i s o r bar adjusted 4.2 mm below the i n t e r a u r a l l i n e . C o n t r o l animals (n=20) were i d e n t i c a l l y l e s i o n e d , w i t h the exception that only a s c o r b i c s a l i n e was i n j e c t e d . Drugs: Doses of morphine sulphate were expressed i n terms of s a l t s . The s o l u -t i o n s were made w i t h p h y s i o l o g i c a l s a l i n e and i n j e c t e d IP at room tempera-t u r e . Apparatus: Locomotor a c t i v i t y : Spontaneous locomotor a c t i v i t y was recorded i n s i x c i r c u l a r photoactometer cages (BRS Foringer #PAC-ool), measuring 61 cm i n diameter, and 43 cm i n height. Each cage contained 12 p h o t o c e l l sensor u n i t s placed e q u i d i s t a n t around the w a l l of the cage. I n t e r r u p t i o n of the p h o t o c e l l beams was a u t o m a t i c a l l y recorded on electromechanical counters (BRS Foringer //POS-112) , which p r i n t e d cumulated scores and then r e s e t to zero every 10 minutes. Room temperature and l i g h t i n g c o n d i t i o n s were main-15 tained i n a constant s t a t e throughout.the d u r a t i o n of the study. Procedure: F o l l o w i n g surgery, a l l animals r e c e i v e d ad l i b i t u m food and water, and were housed i n d i v i d u a l l y . A 12 hour d a r k - l i g h t c y c l e was maintained through-out the experiment. Behavioural t e s t i n g commenced two weeks a f t e r the l e -s i o n s , to permit completion of anterograde degeneration (Ross and Reis,1974). Animals from each of the two l e s i o n e d groups were randomly assigned to two drug c o n d i t i o n s , 1) morphine, or 2) s a l i n e , thereby d e f i n i n g four groups: 1. d o r s a l bundle-6-hydroxydopamine (DB-6-0HDA) l e s i o n e d , morphine i n j e c t e d ; 2. d o r s a l bundle-6-hydroxydopamine (DB-6-0HDA) l e s i o n e d , s a l i n e i n j e c t e d ; 3. v e h i c l e , morphine i n j e c t e d and 4. v e h i c l e , s a l i n e - i n j e c t e d . Before behavioural t e s t i n g began, a l l animals r e c e i v e d three days of pre-handling to reduce s t r e s s a s s o c i a t e d w i t h the i n j e c t i o n procedure. A l l animals r e c e i v e d the appropriate d a i l y IP drug i n j e c t i o n ( e i t h e r s a l i n e or morphine) throughout the study. Each day, morphine sulphate was d i s s o l v e d i n p h y s i o l o g i c a l s a l i n e (0.9%) and a dose of 25 mg/kg of body weight was i n j e c t e d to the appropriate groups of animals. A l l i n j e c t i o n s were given i n a volume of 1 mg/kg. The drug s o l u t i o n s were prepared each morning and between the morning and afternoon i n j e c t i o n s , were wrapped i n l i g h t i n s e n s i t i v e p l a s t i c and stored i n a r e f r i g e r a t o r . Body weight data were recorded each day, and organized according to mean group weights. The data were analyzed using a 3 f a c t o r repeated measures ANOVA ( s u b j e c t s ) , using a simple main e f f e c t s model (groups). S i g n i f i c a n t d i f f e r e n c e s between groups were t e s t e d using the Duncan M u l t i p l e Range Test, p < .05. To f a c i l i t a t e b ehavioural t e s t i n g , two i n j e c t i o n schedules were i n t r o -duced. One group, c o n s i s t i n g of one-half of the subjects, from each of the 16 four groups p r e v i o u s l y d e scribed, was i n j e c t e d between 10:30 and 11:30 A.M., and the remaining animals were i n j e c t e d between 1:30 and 2:30 P.M. Each animal was maintained on i t s own s t r i c t i n j e c t i o n schedule, r e c e i v i n g the same drug i n j e c t i o n once d a i l y at the same time each day f o r an 18 day per i o d . Only 12 animals could be te s t e d d a i l y on the locomotor apparatus (6 i n the A.M., and 6 i n the P.M.), and the i n i t i a t i o n of the i n j e c t i o n schedules was staggered over a three day p e r i o d so as to accommodate a c t i v i t y t e s t i n g on the f i r s t day of drug treatment f o r a l l animals. Therefore, although a l l animals were i n j e c t e d once d a i l y , spontaneous locomotor a c t i v i t y was r e -corded every three days f o r each animal, i . e . day 1, day 4, day 7, day 10, etc. u n t i l each animal had undergone t e s t i n g f o r s i x s e s s i o n s , each three days apart. At l e a s t one animal from each of the four l e s i o n / d r u g groups p a r t i c i p a t e d i n every t e s t i n g s i t u a t i o n . Animals were i n j e c t e d i n t h e i r home cages on the days they were not tes t e d i n the a c t i v i t y cages. Locomotor a c t i v i t y : Animals were placed i n d i v i d u a l l y i n the a c t i v i t y cages at e i t h e r 9:00 A.M., or 1:30 P.M. and t h e i r spontaneous locomotor a c t i -v i t y was recorded f o r one hour. This c o n s t i t u t e d the h a b i t u a t i o n phase. Following t h i s , animals were removed from t h e i r cages, i n j e c t e d IP w i t h e i -t h e r s a l i n e or morphine (25 mg/kg), according to t h e i r group d e s i g n a t i o n and then replaced i n t h e i r o r i g i n a l cages. A c t i v i t y l e v e l s were then recorded f o r a three hour p e r i o d , at which time animals were removed and immediately returned to t h e i r home cages. Data from each of the four groups was organized according to s e s s i o n s , where s e s s i o n 1 corresponded to data from a l l animals c o l l e c t e d on the f i r s t day of drug i n j e c t i o n ; s e s s i o n 2 corresponded.to day 4 of drug i n j e c t i o n ; 17 s e s s i o n 3 corresponded to day 7 of drug i n j e c t i o n , e t c . , u n t i l data f o r 6 sessions (corresponding to day 16) had been c o l l e c t e d . Data from the one hour h a b i t u a t i o n p e r i o d was summed across the 6 ten minute periods f o r each group and analyzed across days using a Three Factor Repeated Measures Analy-s i s of Variance (Subjects) w i t h the simple main e f f e c t s model (groups). S i g n i f i c a n t d i f f e r e n c e s between groups were t e s t e d using the Duncan Post Hoc M u l t i p l e Range Test, p < .05. P o s t - i n j e c t i o n a c t i v i t y data were organized according to 20 minute periods (=9 v a r i a b l e s ) and analyzed u s i n g the above technique. The same data were a l s o subdivided i n t o two phases: the i n i t i a l hypoactive phase corresponded to data from the f i r s t 4 ten minute periods summed f o r each group and the subsequent h y p e r a c t i v e phase corresponded to the l a s t 140 minutes of t e s t i n g (ten minute periods 5-18 summed f o r each group). Data were analyzed according to the methods o u t l i n e d above. Biochemistry: Upon completion of the behav i o u r a l t e s t i n g , s i x animals from each of the d o r s a l bundle-6-0HDA l e s i o n e d groups and four animals from each of the v e h i c l e groups were s a c r i f i c e d by c e r v i c a l f r a c t u r e . The b r a i n s were imme-d i a t e l y d i s s e c t e d on i c e , as p r e v i o u s l y described (Roberts, Z i s and F i b i g e r , 1975). Noradrenaline l e v e l s were then measured i n the hippocampus and cere-b r a l c o r t e x , and dopamine was measured i n the s t r i a t u m by the method of McGeer and McGeer (1962). Student's _t Test was used to analyze the data. 18 RESULTS Ha b i t u a t i o n Data: Figure 1 shows the h a b i t u a t i o n data of the four groups across days. S t a t i s t i c a l a n a l y s i s revealed a s i g n i f i c a n t group e f f e c t F=7.15, df=5, 145, p < .001; and a s i g n i f i c a n t group x days i n t e r a c t i o n F=2.05, df=15, 145, p < .02. The Duncan M u l t i p l e Range Test i n d i c a t e d that no s i g n i f i c a n t group d i f -ferences were observed on Day 1 (Fig-. 1 ). By day 4 of drug treatment, however, the vehicle-morphine group had demonstrated s i g n i f i c a n t l y l e s s l o -comotor a c t i v i t y than the other three groups ( F i g . 1). On days 7, 10, 13 and 16 of drug treatment, the DB-6-0HDA-morphine and the vehicle-morphine groups demonstrated s i g n i f i c a n t l y l e s s locomotor a c t i v i t y during h a b i t u a t i o n than d i d the DB-6-0HDA-saline and v e h i c l e s a l i n e groups ( F i g . 1). P o s t - I n j e c t i o n Data; Figures 2 and 3 show the p o s t - i n j e c t i o n locomotor a c t i v i t y of the four groups over days, organized according to the hypoactive and hy p e r a c t i v e pha-ses . Hypoactive Phase: A s i g n i f i c a n t days e f f e c t was observed, F=23.32, df=5, 145, p < .001, as w e l l as a s i g n i f i c a n t group x days i n t e r a c t i o n , F=8.26, df=15, 145, p < .001. The Duncan M u l t i p l e Range Test i n d i c a t e d that the 6-OHDA-saline and v e h i c l e - s a l i n e groups demonstrated no s i g n i f i c a n t change i n t h e i r l o c o -motor a c t i v i t y over the days t e s t e d , whereas the DB-6-0HDA-morphine and vehicle-morphine groups demonstrated a s i g n i f i c a n t increase i n a c t i v i t y over days t e s t e d . On day 1 of drug i n j e c t i o n ( F i g . 2 ) , the Duncan M u l t i p l e Range Test i n d i c a t e d that both the DB-6-0HDA-morphine and the vehicle-morphine groups 19 Figure 2 Mean locomotor a c t i v i t y during a 1 hour p r e - i n j e c t i o n h a b i -t u a t i o n phase f o r days 1,4,7,10,13 and 16 of drug i n j e c t i o n . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = vehicle-mor-phine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 20 21 Figure ,3 Mean locomotor a c t i v i t y during the hypoactive phase f o r days 1,4,7,10,13 and 16 of drug i n j e c t i o n . Closed squares = DB-6-OHDA-morphine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 22 23 demonstrated s i g n i f i c a n t h y p o a c t i v i t y when compared to the DB-6-0HDA-saline and v e h i c l e - s a l i n e groups, c h a r a c t e r i s t i c of morphine's i n i t i a l a c t i o n on locomotor a c t i v i t y i n a c u t e l y i n j e c t e d animals. Repeated morphine a d m i n i s t r a t i o n r e s u l t e d i n the development of t o l e r -ance to the hypoactive phase, as i n d i c a t e d by the gradual i n c r e a s e i n a c t i -v i t y l e v e l s f o r both the DB-6-0HDA-morphine and the vehicle-morphine groups when t e s t e d on day 4 of drug treatment ( F i g . 2). More r a p i d t o l e r a n c e deve-lopment to the hypoactive phase i s evidence by the vehicle-morphine group than the DB-6-0HDA-morphine group, as evidence by t h e i r a c t i v i t y l e v e l s on day 7 and day 10 of chr o n i c drug treatment and by the s i g n i f i c a n t l y higher l e v e l s of locomotor a c t i v i t y d i s p l a y e d by the vehicle-morphine animals when compared to the s a l i n e i n j e c t e d groups on day 10 ( F i g . 2). Complete tol e r a n c e to the depressant e f f e c t s of morphine on locomotor a c t i v i t y was dis p l a y e d by both the DB-6-0HDA-morphine and vehicle-morphine groups on days 13 and 16 of chronic morphine treatment, i n that both groups showed s i g n i f i c a n t l y higher l e v e l s of locomotor a c t i v i t y than the two s a l i n e i n j e c t e d groups ( F i g . 2). In a d d i t i o n , the a c t i v i t y l e v e l s of the v e h i c l e -morphine group were s i g n i f i c a n t l y higher than those of the s a l i n e i n j e c t e d groups as w e l l as the DB-6-0HDA-morphine gr e a t e r , i n d i c a t i n g greater t o l e r -ance development i n the vehicle-morphine group ( F i g . 2). Hyperactive Phase: The Duncan M u l t i p l e Range Test i n d i c a t e d that on day 1, both the vehicle-morphine and the DB-6-0HDA-morphine groups demonstrated s i g n i f i -c a n t l y higher a c t i v i t y l e v e l s than the v e h i c l e - s a l i n e and DB-6-0HDA-s a l i n e groups, c h a r a c t e r i s t i c of the 2nd phase of morphine b i p h a s i c a c t i o n on l o c o m o t o r . a c t i v i t y . This f i n d i n g was a l s o observed on day 4 of drug 24 Figure 4 Mean locomotor a c t i v i t y during the hyper a c t i v e phase f o r days 1,4,7,10,13 and 16 of drug i n j e c t i o n . Closed squares = DB-6-OHDA-morphine group, N=10; Open squares = DB-6-OHDA-saline group; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r c l e s = v e h i c l e s a l i n e group, N=8. 25 H Y P E R A C T I V E P H A S E 51001 <2 3900|-z 3 o u U J 2700J-I -O X 0. 1500h 300r 26 treatment w i t h the added observation that animals from the DB-6-0HDA-mor-phine and vehicle-morphine groups showed an enhanced l e v e l of h y p e r a c t i v i t y that increased s t e a d i l y as morphine a d m i n i s t r a t i o n continued, and i s there-f o r e r e f e r r e d to as "reverse t o l e r a n c e " ( F i g . 3). I t was a l s o observed that although the enhanced h y p e r a c t i v i t y was evident f o r both morphine-injected groups, t h i s e f f e c t was s i g n i f i c a n t l y l e s s marked i n the DB-6-0HDA-morphine than the vehicle-morphine group on days 7, 10, 13 and 16 of t e s t i n g ( F i g . 3). Fi g u r e s , 4, 5 and 6 show the time course of the p o s t - i n j e c t i o n l o c o -motor a c t i v i t y of the 4 groups f o r days 1, 7 and 16 of drug i n j e c t i o n . Sta-t i s t i c a l a n a l y s i s summarized i n Appendix I , revealed an o v e r a l l s i g n i f i c a n t group e f f e c t except f o r the f i r s t 2 v a r i a b l e s (40 minutes), and a s i g n i f i -cant days e f f e c t on a l l 9.variables (180 minutes), w i t h a s i g n i f i c a n t i n -crease i n a c t i v i t y over days, r e g a r d l e s s of group. A s i g n i f i c a n t group x days i n t e r a c t i o n was observed f o r a l l but the l a s t 40 minutes ( v a r i a b l e s 8 and 9) of t e s t i n g . The Duncan M u l t i p l e Range Test revealed that on day 1, both the v e h i c l e -morphine and DB-6-0HDA-morphine groups d i s p l a y e d suppressed l e v e l s of a c t i -v i t y f o r the f i r s t 20 minute p e r i o d , c h a r a c t e r i s t i c of the b i p h a s i c a c t i o n of morphine on locomotor a c t i v i t y ( F i g . 4). No s i g n i f i c a n t d i f f e r e n c e s i n a c t i v i t y were evident f o r the next 2 hours. However, at 120-140 minutes p o s t - i n j e c t i o n , both the v e h i c l e morphine and the DB-6-0HDA-morphine groups showed s i g n i f i c a n t l y more a c t i v i t y than the v e h i c l e - s a l i n e and DB-6-0HDA-s a l i n e groups ( F i g . 4). By day 7 ( F i g . 5 ) , both the vehicle-morphine and the DB-6-0HDA-morphine groups d i s p l a y e d some t o l e r a n c e to the suppressant e f f e c t of morphine. Sta -t i s t i c a l a n a l y s i s revealed that although the morphine i n j e c t e d groups d i d not demonstrate s i g n i f i c a n t l y higher l e v e l s of a c t i v i t y than the s a l i n e i n -j e c t e d groups during the hypoactive phase (0-40 minutes), the vehicle-mor-27 phine group demonstrated s i g n i f i c a n t l y higher o v e r a l l locomotor a c t i v i t y than the DB-6-0HDA-morphine group. During the hyper a c t i v e phase (50-180 minutes p o s t - i n j e c t i o n ) , the ve-hicle-morphine and the. DB-6-0HDA-morphine groups showed s i g n i f i c a n t l y higher l e v e l s of locomotor a c t i v i t y than the v e h i c l e - s a l i n e and DB-6-0HDA-saline groups. I n a d d i t i o n , a d i s s o c i a t i o n i n a c t i v i t y occurred between the v e h i -cle-morphine and DB-6-0HDA-morphine groups, whereby the vehicle-morphine group d i s p l a y e d a s i g n i f i c a n t l y higher a c t i v i t y o v e r a l l than the DB-6-0HDA-morphine group throughout the hyperactive phase. By day 16 ( F i g . 6 ) , both the vehicle-morphine and the DB-6-0HDA-mor-phine groups demonstrated s i g n i f i c a n t l y higher l e v e l s of locomotor a c t i v i t y than the v e h i c l e - s a l i n e and DB-6-0HDA-saline groups i n d i c a t i n g that both groups were completely t o l e r a n t to the suppressant e f f e c t s of morphine. In a d d i t i o n , a d i s s o c i a t i o n i n a c t i v i t y again occurred between the v e h i c l e -morphine and the DB-6-0HDA-morphine groups, whereby the vehicle-morphine group d i s p l a y e d s i g n i f i c a n t l y higher a c t i v i t y than the DB-6-0HDA-morphine group during the hypoactive phase. In reviewing the h y p e r a c t i v e phase on day 16 ( F i g . 6 ) , s t a t i s t i c a l a n a l y s i s revealed t h a t , as w i t h day 7, the vehicle-morphine and DB-6-0HDA-morphine groups demonstrated s i g n i f i c a n t l y higher l e v e l s of locomotor a c t i -v i t y than the s a l i n e i n j e c t e d groups, w i t h a d i s s o c i a t i o n i n a c t i v i t y be-tween the vehicle-morphine and DB-6-0HDA-morphine groups, whereby the v e h i -cle-morphine group d i s p l a y e d s i g n i f i c a n t l y higher l e v e l s of a c t i v i t y than the DB-6-0HDA-morphine group during the hyper a c t i v e phase. Weight Data: Figure 7 shows the mean body weight data of the 4 groups f o r days 1, 6, 12 and 18 of drug i n j e c t i o n . S t a t i s t i c a l a n a l y s i s r evealed a s i g n i f i c a n t days e f f e c t F=16.45, df=17, 510, p < .001, and a s i g n i f i c a n t group x days 28 Figure 5 Mean locomotor a c t i v i t y during the 3 hour p o s t - i n j e c t i o n p e r i o d on day 1 of drug i n j e c t i o n . Closed squares = DB-6-0HDA-mor-phine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 29 30 Fig u r e 6 Mean locomotor a c t i v i t y during the 3 hour p o s t - i n j e c t i o n p e r i o d on day 7 of drug i n j e c t i o n . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r c l e s = v e h i c l e -s a l i n e group, N=8. 31 32 Figure 7 Mean locomotor a c t i v i t y during the 3 hour p o s t - i n j e c t i o n p e r i o d on day 16 f o r drug i n j e c t i o n . Closed squares = DB-6-0HDA-mor-phine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 33 34 Figure 8 Mean body weight data on days 1,6,12 and 18 of drug i n j e c t i o n . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = vehicle-mor-phine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 35 A 36 i n t e r a c t i o n F=7.20, df=51, 510, p < .001. The Duncan M u l t i p l e Range Test i n -d i c a t e d that on day 1 and day 6, there were no s i g n i f i c a n t group d i f f e r e n c e s ; however, on days 12 and 18, the DB-6-0HDA-morphine and vehicle-morphine groups weighed s i g n i f i c a n t l y l e s s than the DB-6-OHDA-saline and v e h i c l e - s a -l i n e groups. Biochemistry: The e f f e c t of the 6-OHDA l e s i o n on b r a i n noradrenaline and dopamine l e -v e l s i s summarized i n Table 1 . Animals i n the s a l i n e i n j e c t e d group that r e c e i v e d b i l a t e r a l i n j e c t i o n s i n the d o r s a l noradrenergic bundle showed s i g -n i f i c a n t d e p l e t i o n of hippocampus/cortex noradrenaline l e v e l s to 6.34% of v e h i c l e - s a l i n e group, _t = 12.55, p < .002. Animals i n the morphine i n j e c t e d group that r e c e i v e d b i l a t e r a l i n j e c -t i o n s i n the d o r s a l noradrenergic bundle, showed s i g n i f i c a n t d e p l e t i o n of the hippocampus/cortex noradrenaline l e v e l s to 3.00% of the vehicle-morphine group, _t = 24.2, p < .002. A s m a l l , but n o n s i g n i f i c a n t , e f f e c t on s t r i a t a l dopamine l e v e l s was measured i n both 6-OHDA l e s i o n e d groups. 37 TABLE I E f f e c t of b i l a t e r a l 6-OHDA i n j e c t i o n s i n t o the d o r s a l NA p r o j e c t i o n on NA l e v e l s i n the hippocampus/cortex and caudate DA l e v e l s . NA yg/g DA yg/g Cortex/hippocampus Caudate Vehicle-morphine N=4 .450±.030 9.46±2.50 DB-6-0HDA-morphine N=6 0.0135± .003N 12.92±1.36 % of c o n t r o l 3.00% 136.58% ,._ V e h i c l e - s a l i n e N=4 .410±.050 12.49±l-83 DB-6-OHDA-saline N=6 .026±.006 11.84±0.86 % of c o n t r o l 6.34% 94.80% 38 DISCUSSION Morphine Tolerance and Locomotor A c t i v i t y : The e f f e c t of i n j e c t i o n s of 6-OHDA i n t o the d o r s a l noradrenergic pro-j e c t i o n was evaluated w i t h respect to to l e r a n c e development i n chronic mor-phine t r e a t e d animals. Inasmuch as animals d i s p l a y a b i p h a s i c response to acute morphine i n j e c t i o n s , c h a r a c t e r i z e d by an i n i t i a l suppressant or hypo-a c t i v e and a subsequent hyperactive phase, and that t o l e r a n c e can be shown to develop to the suppressant e f f e c t s (Hosoya et a l , 1963; Vasko and Domino, 1978; and Smee and Overstreet, 1976), i t was decided to employ the suppres-sant phase of morphine induced locomotor a c t i v i t y as an index of t o l e r a n c e development. On day 1 of drug a d m i n i s t r a t i o n , animals from the DB-6-0HDA-morphine and the vehicle-morphine groups demonstrated the c h a r a c t e r i s t i c hypoactive phase. With repeated i n j e c t i o n s and f u r t h e r b e h a v i o u r a l t e s t i n g , these animals d i s p l a y e d r a p i d t o l e r a n c e development to the hypoactive phase, as i n d i c a t e d by the gradual increase i n a c t i v i t y l e v e l s f o r both morphine i n -j e c t e d groups. With continued drug a d m i n i s t r a t i o n , both the DB-6-0HDA-mor-phine and the vehicle-morphine groups demonstrated i n c r e a s i n g l e v e l s of locomotor a c t i v i t y when compared to the two s a l i n e - i n j e c t e d groups, i n d i c a -t i n g i n c r e a s i n g t o l e r a n c e to the depressant e f f e c t s of morphine on locomotor a c t i v i t y . In a d d i t i o n , the r a t e and degree of tolerance development to the depressant e f f e c t s of morphine on locomotor a c t i v i t y was d i f f e r e n t i n the two groups i n that the vehicle-morphine group d i s p l a y e d s i g n i f i c a n t l y higher l e v e l s of h y p e r a c t i v i t y than the DB-6-0HDA-morphine group on days 13 and 16 of chronic morphine a d m i n i s t r a t i o n . When changes i n the i n i t i a l depressant or hypoactive phase of l o c o -motor a c t i v i t y are used as an index of t o l e r a n c e development, i t can be 39 concluded from these data that w i t h chronic morphine treatment, the DB-6-OHDA l e s i o n r e s u l t e d i n p o t e n t i a t i o n of the hypoactive phase of locomotor a c t i v i t y i n d i c a t i n g t h a t the r a t e of development and degree of to l e r a n c e were impeded. I t should be noted that i t was reported p r e v i o u s l y that DB-6-0RDA l e -sions p o t e n t i a t e d catalepsy and locomotor h y p o a c t i v i t y f o l l o w i n g acute mor-phine i n j e c t i o n s (Roberts et a l , 1978; Mason et a l , 1978) and these beha-v i o u r a l f i n d i n g s were not r e p l i c a t e d i n t h i s study (see Figure 4 = day 1 of drug i n j e c t i o n ) . One p o s s i b l e e x p l a n a t i o n f o r t h i s negative f i n d i n g i s that the higher dose of morphine used i n t h i s study may have r e s u l t e d i n a "bottoming-out e f f e c t " . A l t e r n a t i v e l y , c l o s e r c o n s i d e r a t i o n of the study of Roberts et a l (1978) r e v e a l s that the same groups of animals underwent beha-v i o u r a l t e s t i n g f o l l o w i n g 3 drug doses of morphine, separated by 6 drug f r e e days. There i s evidence that animals p r e - t r e a t e d w i t h morphine and subjec-ted to a delay p e r i o d w i l l s t i l l e x h i b i t t o l e r a n c e when i n j e c t i o n s are r e -e s t a b l i s h e d ( S i e g e l , 1975; Cochin and Kornetsky, 1964). I t t h e r e f o r e ap-pears that the design employed by these authors may have introduced a t o l e r -ance e f f e c t which was overlooked i n the i n t e r p r e t a t i o n of the data. This c r i t i c i s m , however, does not apply to data obtained using a dose of 10 mg/kg morphine, i n t h a t t h i s represented the f i r s t drug i n j e c t i o n , however, t h i s dose i s s u b s t a n t i a l l y lower than the dose employed i n the present study (25 mg/kg), thereby making comparison of these data d i f f i c u l t . Nonetheless, i n the present study, the i n i t i a l i n j e c t i o n of a dose of 25 mg/kg morphine r e -s u l t e d i n the c h a r a c t e r i s t i c hypoactive phase w i t h no p o t e n t i a t e d depressant phase f o r the DB-6-0HDA-morphine group and t h e r e f o r e i t i s f e l t that group d i f f e r e n c e s between the vehicle-morphine and the DB-6-0HDA-morphine groups and t h e i r r e l a t i o n to t o l e r a n c e development can be made assuming equal base-l i n e behaviour f o r the two groups. 40 S i m i l a r f i n d i n g s were observed f o r the h y p e r a c t i v e phase. With chronic morphine treatment, both groups d i s p l a y e d i n c r e a s i n g l e v e l s of h y p e r a c t i v i t y , or "reverse t o l e r a n c e " . By day 7, the vehicle-morphine group demonstrated s i g n i f i c a n t l y higher l e v e l s of a c t i v i t y than the DB-6-0HDA group and again t h i s e f f e c t was r e p l i c a t e d on days 10, 13:and 16. Weight changes during chronic morphine treatment have been used as an index of t o l e r a n c e development (Mucha, Kalant and Linseman, 1979). In accor-dance w i t h previous f i n d i n g s , i t was observed that morphine caused an i n i t i a l suppression of weight gain, followed by a gradual, but l e s s marked i n c r e a s e , when compared to s a l i n e t r e a t e d groups. No s i g n i f i c a n t d i f f e r e n c e s were ob-served when comparing the DB-6-0HDA-morphine and vehicle-morphine groups, i n -d i c a t i n g that perhaps t h i s b e h a v i o u r a l measure i s a l e s s s e n s i t i v e i n d i c a t o r of t o l e r a n c e development. In c o n c l u s i o n , the f i n d i n g s that DB-6-0HDA l e s i o n s r e s u l t e d i n the en-hancement of the hypoactive phase of locomotor a c t i v i t y , thereby r e s u l t i n g i n a slower r a t e and a l e s s e r degree of t o l e r a n c e development, i m p l i c a t e s a r o l e f o r noradrenaline i n the mediation of t o l e r a n c e development to chronic morphine treatment. S i m i l a r l y , the s t i m u l a t o r y e f f e c t s of morphine become enhanced w i t h repeated morphine a d m i n i s t r a t i o n , but t h i s e f f e c t i s s i g n i f i -c a n t l y l e s s pronounced i n the DB-6-0HDA l e s i o n e d animals. I n t e r p r e t a t i o n of these f i n d i n g s i s d e t a i l e d i n the General D i s c u s s i o n . 41 STUDY I I . THE EFFECT OF A 6-OHDA LESION TO THE DORSAL NORADRENERGIC BUNDLE ON WITHDRAWAL IN MORPHINE-DEPENDENT RATS. INTRODUCTION The r o l e of catecholamines i n the abstinence syndrome i n morphine-depen-dent animals has been examined p r e v i o u s l y (Huidobro, Contreras and Croxatto, 1963; Schwartz and E i d e l b e r g , 1970; and Maruyama and Takemori, 1973). Maruyama and Takemori (1973) i m p l i c a t e d noradrenaline and dopamine i n the abstinence syndrome i n mice w i t h the observation that d i s u l f i r a m and AMPT caused a s i g n i f i c a n t i n h i b i t i o n of naloxone-induced jumping. I t was there-f o r e concluded that the f u l l e xpression of the abstinence syndrome i n mor-phine-dependent mice r e q u i r e d the i n t e g r i t y of the c e n t r a l s t o r e of catecho-lamines . S i m i l a r f i n d i n g s were reported by Schwartz and E i d e l b e r g (1970). They reported that AMPT reduced wet dog shakes and hypothermia induced by n a l o r -phine a d m i n i s t r a t i o n to morphine-dependent r a t s . In a d d i t i o n , Watanabe (1971) reported that i n t r a v e n t r i c u l a r pretreatment w i t h noradrenaline or dopamine ameliorated l e v a l l o r p h a n induced withdrawal. However, c o n t r a d i t o r y evidence to the above r e s u l t s a l s o e x i s t s . Gunne, Jonsson and Fuxe (1969) reported that withdrawal signs of tremor, p i l o e r e c -t i o n , i r r i t a b i l i t y and di a r r h e a induced by nalorphine were not modified by pretreatment w i t h AMPT. Segal, Deneau and Seevers (1972) reported that methyldopa d i d not a l t e r the morphine abstinence syndrome i n monkeys, where-as Pozeulo and Kerr (1972) reported t h a t AMPT i n h i b i t e d the withdrawal syn-drome i n morphine-dependent monkeys. F r i e d l e r , Bhargahava, Quock and Way (1972) reported that p r e c i p i a t e d abstinence, as measured by nalozone-induced withdrawal, jumping was enhanced by 6-OHDA pretreatment i n t r a c e r e b r a l l y , and that weight l o s s a f t e r abrupt 42 withdrawal was a l s o increased by 6-OHDA pretreatment. The enhanced jumping response was explained according to the theory of denervation s u p e r s e n s i t i -v i t y : i f p h y s i c a l dependency i s a m a n i f e s t a t i o n of c e n t r a l denervation s u p e r s e n s i t i v i t y , the withdrawal phenomena would r e f l e c t a s t a t e of rebound h y p e r e x c i t a b i l i t y . There i s some evidence i n the l i t e r a t u r e that noradrenaline may p l a y a more important r o l e than dopamine i n the expression of withdrawal. Herz, B l a s i g and Papeschi (1974) reported that s e l e c t i v e i n h i b i t i o n of NA synthe-s i s by FLA-63 r e s u l t e d i n a r e d u c t i o n i n withdrawal i n t e n s i t y , whereas d e s i -pramine, a drug that s p e c i f i c a l l y i n h i b i t s NA reuptake mechanisms aggravated the withdrawal syndrome. In a d d i t i o n , i t was observed that the antagonism of withdrawal w i t h AMPT was reversed w i t h L-dopa only when the s y n t h e s i s of NA was not prevented by i n h i b i t i o n of DA-beta-hydroxylase. I t was t h e r e f o r e concluded that NA i s more h i g h l y i n v o l v e d i n the m a n i f e s t a t i o n of the mor-phine-withdrawal syndrome. C i c e r o , Meyer and B e l l (1974) demonstrated that noradrenergic b l o c k i n g agents ( i . e . phenoxybenzamine) caused a dose-depen-dent suppression of wet dog shakes and d i a r r h e a - two b e h a v i o u r a l charac-t e r i s t i c s of naloxone-induced withdrawal. Recently, c l o n i d i n e , which i s the most powerful of the 0:2 agonists known to i n h i b i t the f i r i n g of locus coeruleus (LC) neurons has been repor-ted to suppress the symptoms of o p i a t e withdrawal i n humans (Gold, Redmond and K l e b e r , 1978). Aghajanian (1978) reported that t o l e r a n c e developed to the i n h i b i t o r y e f f e c t of morphine on the f i r i n g of LC neurons and that d i -r e c t a p p l i c a t i o n of naloxone to LC neurons by microiontophoresis induced a withdrawal response of > 100% a c t i v a t i o n of f i r i n g . During periods of n a l o -xone-induced o p i a t e - r e c e p t o r blockade and withdrawal a c t i v a t i o n , the micro-i o n t o p h o r e s i s of c l o n i d i n e was able to depress the LC c e l l f i r i n g to below b a s e l i n e r a t e s . This study a l s o i n d i c a t e d that morphine and c l o n i d i n e act 43 at independent LC c e l l - r e c e p t o r s , i n that naloxone antagonized morphine, but not the a-blocker piperoxane antagonized c l o n i d i n e , but not morphine. There-f o r e , i t was suggested that s i n c e morphine and c l o n i d i n e act on independent receptors w i t h the LC, but have s i m i l a r depressant e f f e c t on o v e r a l l LC c e l l a c t i v i t y , c l o n i d i n e might suppress c e r t a i n symptoms of o p i a t e withdrawal by means of a p a r a l l e l , but independent a c t i o n on c e l l a c t i v i t y . Laverty and Roth (1979) and Crawley, Laverty and Roth (1979) p a r a l l e l e d the e l e c t r o p h y s i o l o g i c a l r e s u l t s of Aghajanian (1978) w i t h biochemical f i n d -i n g s . Laverty and Roth (1979) reported that NA turnover, as measured by AMPT d e p l e t i o n of NA, increases during naloxone p r e c i p i t a t e d withdrawal and that t h i s i n c r e a s e i s attenuated by c l o n i d i n e . In a d d i t i o n , Crawley et a l (1979) reported that 3-methoxy-4-hydroxy-phenethyleneglycol (MHPG), a NA m e t a b o l i t e , increased during naloxone-induced withdrawal and c l o n i d i n e r e -versed t h i s i n c r e a s e . These r e s u l t s taken together serve to i m p l i c a t e a primary r o l e f o r NA i n withdrawal. In s t u d i e s where withdrawal i s p r e c i p i t a t e d by s a l i n e s u b s t i t u t i o n , or by n a r c o t i c a n t a g o n i s t s , weight l o s s has been shown to be a r e l i a b l e index ( T i l s o n , Rech, Stolman, 1973; Akera and Brody, 1967; Hosoya et a l , 1963; and F r i e d l e r et a l , 1972). I t was t h e r e f o r e of i n t e r e s t to examine the e f f e c t of t h i s l e s i o n on the withdrawal response, using weight l o s s as an i n d i c a t o r (Study H a ) . In addition., a growing tendency f o r some animals to d i s p l a y i r r i t a b i -l i t y on handling and i n j e c t i o n was observed during chronic morphine admini-s t r a t i o n , and t h e r e f o r e a r a t i n g s c a l e was developed and a p p l i e d to evaluate the e f f e c t s of the DB-6-0HDA l e s i o n on chronic morphine-induced i r r i t a b i l i t y (Study H a ) . A l s o , the f a c t that there i s considerable l i t e r a t u r e that i m p l i c a t e s 44 catecholamine i n the morphine abstinence syndrome, prompted an examination of the e f f e c t of 6-OHDA l e s i o n s of the d o r s a l noradrenergic bundle on narco-t i c antagonist-induced withdrawal i n morphine dependent r a t s , using the measurement technique of B l a s i g , Herz, Reinhold and Zieglansberger (1973) and Linseman (1975), (Study l i b ) . 45 STUDY I l a METHODS I r r i t a b i l i t y Rating Scale: I t was observed that animals from Study I began to show i r r i t a b i l i t y on handling and i n j e c t i o n during the course of the study. The animals' be-haviour during i n j e c t i o n was t h e r e f o r e rated s t a r t i n g day 14 of the study according to the f o l l o w i n g s c a l e : 0 = Calm, passi v e behaviour during handling and i n j e c t i o n . 1 = Crying when removed by the t a i l from the home cage. 2 = Crying when removed by the t a i l from the home cage and c r y i n g or m i l d w r i g g l i n g during the IP i n j e c t i o n . 3 = Crying when removed by the t a i l from the home cage and extreme s t r u g g l i n g during the IP i n j e c t i o n . These data obtained were analyzed using the Mann Whitney U Test. Withdrawal: A f t e r 24 days of once d a i l y IP i n j e c t i o n s of 25 mg/kg to both the DB-6-OHDA-morphine and the vehicle-morphine groups and completion of the beha-v i o u r a l t e s t i n g f o r experiments I , I I and I l l b , i n j e c t i o n s were discontinued and body weight of a l l animals was recorded once d a i l y at 12:00 noon f o r a 5 day p e r i o d . The mean group body weight data were used as an index of w i t h -drawal and were analyzed using a Three Factor Repeated Measures A n a l y s i s of Variance (subjects) w i t h a simple main e f f e c t s model (groups). S i g n i f i c a n t d i f f e r e n c e s between groups were t e s t e d using the Duncan M u l t i p l e Range Test, p < .05. 46 STUDY l i b METHODS Subjects: A separate group of f o r t y animals was used i n the f o l l o w i n g experiment. The same procedure f o r l e s i o n i n g and group determination was used, as pre-v i o u s l y described i n Experiment I . Drugs: Doses of morphine sulphate and naltrexone h y d r o c h l o r i d e were expressed i n terms of the s a l t s . The s o l u t i o n s were made w i t h p h y s i o l o g i c a l s a l i n e and i n j e c t e d IP at room temperature. Procedure: A l l animals r e c e i v e d the appr o p r i a t e d a i l y IP drug i n j e c t i o n ( e i t h e r s a l i n e or morphine) throughout the study. Each day, morphine sulphate was d i s s o l v e d i n p h y s i o l o g i c a l s a l i n e (0.9%). A l l i n j e c t i o r s were given IP i n a volume of 1 ml/kg. Drug s o l u t i o n s were prepared each day, wrapped i n l i g h t i n s e n s i t i v e p l a s t i c , and stored i n a r e f r i g e r a t o r . Animals from the DB-6-0HDA-morphine and vehicle-morphine groups were put on the IP i n j e c t i o n schedule as presented i n Table I I . Once animals were r e c e i v i n g 100 mg/kg d a i l y , or hig h e r , i n j e c t i o n s were made twice d a i l y to prevent animals from e n t e r i n g withdrawal between i n j e c -t i o n s . Animals from the DB-6-0HDA-saline and v e h i c l e - s a l i n e groups were i n j e c -ted according to the above schedule, except that animals from these groups were i n j e c t e d w i t h only p h y s i o l o g i c a l s a l i n e . On day 25, f i v e animals from each of the DB-6-0HDA-morphine and the vehicle-morphine groups underwent naltrexone p r e c i p i t a t e d withdrawal. 47 TABLE I I Summary of the morphine i n j e c t i o n procedure f o r i n d u c t i o n of p h y s i c a l dependence Day IP I n j e c t i o n Dose (mg/kg) 1 2.5 3 5 5 . 10 7-14 i n c l u s i v e 20. 15 40 16 40 17 60 18 60 19 80 20 80 21 100 22 100 23 100 24 160 25 160 26 200 48 Withdrawal T e s t i n g : Animals from each of the DB-6-0HDA-morphine and the vehicle-morphine groups were a l t e r n a t e l y i n j e c t e d IP w i t h morphine (100 mg/kg) every 30 minutes. One h a l f hour a f t e r t h i s i n j e c t i o n , animals r e c e i v e d an i n j e c t i o n of naltrexone (2 mg/kg), and t h e i r behaviour during withdrawal was observed f o r 3 ten minute periods immediately f o l l o w i n g t h e i r naltrexone i n j e c t i o n . Animals were placed i n a cardboard box (40x40x65 cm) f o r observation. The order of t e s t i n g was balanced across both groups, w i t h an equal propor-t i o n of animals i n each group t e s t e d a f t e r s i m i l a r morphine-naltrexone i n t e r v a l s and a f t e r the same amount of morphine experience. A s c o r i n g procedure s i m i l a r to that of B l a s i g , Herz, Reinhold and Zieglansberger (1973) and Linseman (1975) was used. The incidence of the f o l l o w i n g behaviours was counted: c i r c l i n g (complete c i r c l e s w i t h i n the box, an index of locomotor a c t i v i t y ) , r e a r i n g (an index of e x p l o r a t o r y be-haviour, jumping ( l e a p i n g onto the edge of the box, four f e e t o f f the ground at the same time), wet dog shakes, t e e t h c h a t t e r i n g (episodes), and w r i t h i n g (abdominal s t r e t c h i n g ) . The presence of the f o l l o w i n g signs was checked every 10 minutes: scream-on-touch, h o s t i l i t y on h a n d l i n g , p t o s i s , eye t w i t c h i n g , r h i n o r r h e a , l a c r i m a t i o n , d i a r r h e a and p e n i l e e r e c t i o n . A score of 1 was awarded when the s i g n was present, and 0 i f the s i g n was not. Data from the counted signs were analyzed f o r each 10 minute p e r i o d using an a n a l y s i s of v a r i a n c e and data from the signs present were summed f o r the 3 ten minute period^, w i t h scores ranging from 0-3, depending on whether or not the signs were c o n s i s t e n t l y present during the 30 minute p e r i o d , and the data were then analyzed using Student's jt t e s t . 49 STUDY H a RESULTS I r r i t a b i l i t y Data: Results of the I r r i t a b i l i t y Rating Scale were analyzed u s i n g the Mann Whitney U Test and are presented i n Table I I I . S t a t i s t i c a l a n a l y s i s r e -vealed that the DB-6-0HDA-morphine group was s i g n i f i c a n t l y more i r r i t a b l e on a l l r a t i n g days than the vehicle-morphine group, p < .01. I n comparing the DB-6-OHDA-saline and the v e h i c l e - s a l i n e groups, no s i g n i f i c a n t group d i f f e r e n c e was observed, nor was a s i g n i f i c a n t group d i f f e r e n c e observed when comparing the vehicle-morphine and v e h i c l e - s a l i n e groups, i n d i c a t i n g t hat i r r i t a b i l i t y could not be a t t r i b u t e d s o l e l y to a l e s i o n , or drug e f f e c t . Withdrawal Weight Data: Figure 8 shows the r e s u l t s of the mean body weight measurements taken f o r the l a s t day of morphine i n j e c t i o n (corresponding to day 24) and f o r 5 days f o l l o w i n g abrupt d i s c o n t i n u a t i o n of d a i l y morphine i n j e c t i o n s . S t a t i s -t i c a l a n a l y s i s revealed a s i g n i f i c a n t day e f f e c t , F=101.31, df=4, 56, p < .001, but no s i g n i f i c a n t group x day i n t e r a c t i o n , F < 1.57, i n d i c a t e d s i g n i -f i c a n t weight changes over time, but no group d i f f e r e n c e s between the v e h i -c l e and DB-6-0HDA l e s i o n e d animals. Although both the morphine i n j e c t e d groups had l o s t weight by 48 hours f o l l o w i n g the l a s t i n j e c t i o n , the Stu-dent's _t t e s t revealed no s i g n i f i c a n t d i f f e r e n c e s between groups (t < 1.31). 50 Figure .9 Mean body weight data on the l a s t day of morphine i n j e c t i o n (day 24) and f o r 5 days f o l l o w i n g d i s c o n t i n u a t i o n of d a i l y morphine i n j e c t i o n s . Closed squares = DB-6-0HDA-morphine group, N=10; Closed c i r c l e s = vehicle-morphine group, N=10. 51 W E I G H T ( g r a m ) 4^  O O 6 to O 4^  GO o 4^  o ro i s / \ \ \ \ CO \ \ \ \ \ \ \ N \ N \ \ \ \ • • \ \ \ \ \ \ 52 TABLE I I I I r r i t a b i l i t y Data V e h i c l e - s a l i n e DB-6-OHDA-saline Vehicle-morphine DB-6-0HDA-morphine Days 1 .25 1.0 .33 1.33* 5 0 .25 0 1.11* 8 0 0 0 .78* 10 0 0 .22 1.63* I n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e between the DB-6-0HDA-morphine and vehicle-morphine groups, p < .01. Table III."Mean group scores f o r i r r i t a b i l i t y on days 1, 5, 8 and 10 of r a t i n g using the i r r i t a b i l i t y r a t i n g s c a l e . 53 STUDY l i b RESULTS Data from the naltrexone p r e c i p i t a t e d withdrawal are presented i n Tables IV&V & Appendix II.The a n a l y s i s of var i a n c e revealed no s i g n i f i c a n t group d i f -ferences (F < 2.23) and no s i g n i f i c a n t group x time i n t e r a c t i o n term, (F < .28). However, a s i g n i f i c a n t time d i f f e r e n c e was observed F=24.38, df=2, 24, p < .001, i n d i c a t i n g that counted signs decreased s i g n i f i c a n t l y over time, regardless of group. Of the counted s i g n s , only c i r c l i n g and r e a r i n g , jumping and teeth c h a t t e r i n g were observed i n the two morphine groups. No incidence of wet dog shakes or w r i t h i n g were ever noted i n any animals during the observation p e r i o d . The a n a l y s i s of the checked signs revealed no s i g n i -f i c a n t group d i f f e r e n c e s on any parameter. 54 TABLE IV Withdrawal-Counted Signs DB-6-OHDA-Morphine Vehicle-Morphine C i r c l i n g & t e a r i n g 0-10 min 16.22 10.0 10-20 min 3.89 .40 20-30 min 3.78 .20 Jumping 0-10 min 25.67 17.6 10-20 min 15.56 6.4 20-30 min 5.78 4.0 Teeth c h a t t e r i n g 0-10 min 8.56 9.0 10-20 min .44 1.60 20-30 min 0 ' .20 Table IV: Mean group scores of counted signs present during three 10 minute periods immediately f o l l o w i n g IP i n j e c t i o n of naltrexone (2 mg/kg). 55 TABLE V Withdrawal-Present Signs DB-6-0HDA-Morphine Vehicle-Morphine Screaming on touch 2.20 2.33 H o s t i l i t y on handling 2.60 2.78 P t o s i s 3.00 2.56 Eye t w i t c h i n g 2.60 2.56 Rhinorrhea 2.80 2.33 Lac r i m a t i o n 0 0 Diarrhea 1.40 1.22 P e n i l e e r e c t i o n 2.20 .78 Table V: Mean group scores of present signs summed f o r a 30 minute p e r i o d immediately f o l l o w i n g IP i n j e c t i o n of naltrexone (2 mg/kg). 56 DISCUSSION A growing tendency f o r some animals to d i s p l a y i r r i t a b i l i t y on handling and i n j e c t i o n was observed, and t h e r e f o r e a r a t i n g s c a l e was developed and ap p l i e d during the i n j e c t i o n procedure. Results i n d i c a t e that the DB-6-0HDA le s i o n e d animals d i s p l a y e d the most i r r i t a b i l i t y and i n that these animals were s i g n i f i c a n t l y more i r r i t a b l e than the vehicle-morphine group and the l e s i o n d i d not produce s i g n i f i c a n t i r r i t a b i l i t y i n s a l i n e i n j e c t e d animals, i t can be concluded that the l e s i o n , i n i n t e r a c t i o n w i t h chronic morphine treatment was r e s p o n s i b l e f o r the observed i r r i t a b i l i t y . These r e s u l t s are i n accordance w i t h those presented by F r i e d l e r et a l (1972) who reported d i f f i c u l t y i n handling morphine dependent animals i n j e c t e d w i t h 6-OHDA i n -t r a c e r e b r a l l y . Naltrexone p r e c i p i t a t e d withdrawal and weight l o s s a f t e r abrupt w i t h -drawal were not a f f e c t e d by 6-OHDA i n j e c t i o n s i n t o the d o r s a l noradrenergic bundle. These i n j e c t i o n s r e s u l t e d i n a very s u b s t a n t i a l d e p l e t i o n of cortex/ hippocampus NA, and using t h i s t e s t of withdrawal, i t appears that f o r e b r a i n noradrenaline may not be i n v o l v e d i n the expression of withdrawal. I t has been suggested p r e v i o u s l y that dopamine may be more important than NA i n the expression of withdrawal. L a i and P u r i (1972) reported that morphine withdrawal aggression was blocked by drugs which block dopamine r e -ceptors ( h a l o p e r i d o l ) and enhanced by drugs which s t i m u l a t e DA a c t i v i t y (apomorphine, amphetamine, levo-dopa). Gianutsos, Hynes, P u r i , Drawbaugh and L a i (1974) reported that withdrawal aggression measured 30 days a f t e r the l a s t morphine i n j e c t i o n was blocked by morphine or l e s i o n s of the n i g r o -s t r i a t a l bundle. Aggression was r e i n s t a t e d when the l e s i o n e d animals were tr e a t e d w i t h a small dose of apomorphine. Apomorphine a l s o reduced the t u r n -over of dopamine i n 30-day withdrawn animals at doses which were i n e f f e c t i v e 57 i n non-dependent r a t s . These r e s u l t s are i n t e r p r e t e d according to the theory of dopamine s u p e r s e n s i t i v i t y . Maruyama and Takemori (1973) reported that r e p l e t i o n . o f dopamine l e v e l s (AMPT + DOPA treatment) p a r t i a l l y r e s t o r e s withdrawal jumping i n morphine-dependent animals, w h i l e r e p l e t i o n of NA l e v e l s (AMPT + DOPS treatment) does not. L a i , P u r i and Karkelas (1971) reported that h a l o p e r i d o l , a DA receptor b l o c k e r , markedly reduces withdrawal i n r a t s and humans. Several explanations have been proposed f o r the di s c r e p a n c i e s between the r o l e of DA and NA i n withdrawal (Herz et a l , 1974; C o l l i e r , F r a n c i s and Schneider, 1972), included the f o l l o w i n g : a) o f t e n only one s i g n of mor-phine withdrawal i s considered, i . e . naloxone-induced jumping i n mice; b) withdrawal i s p r e c i p i t a t e d i n animals that have developed q u i t e d i f f e r e n t degrees of dependence; and c) the a c t i o n of drugs that modify withdrawal de-pends on the time at which the drug i s administered i n the course of depen-dency development and withdrawal. In c o n c l u s i o n , i t appears that 6-OHDA d e p l e t i o n of f o r e b r a i n noradrena-l i n e i s inv o l v e d i n some :tests of withdrawal but not ot h e r s , and these f i n d -ings i n d i c a t e a nondominant r o l e f o r NA and may lend i n d i r e c t support to the l i t e r a t u r e supporting a dopaminergic i n f l u e n c e . Further d i s c u s s i o n df these data are made i n the General D i s c u s s i o n . 58 STUDY I I I . A STUDY OF THE PSYCHOPHARMACOLOGICAL INTERACTION OF MORPHINE AND AMPHETAMINE INTRODUCTION Amphetamine i s a w e l l known stimulant drug that r e l e a s e s catecholamines. There i s a l s o evidence that v a r i o u s b e h a v i o u r a l e f f e c t s of morphine ( i . e . locomotor a c t i v i t y ) are mediated by catecholamine systems, i n that c a t e c h o l a -mine synt h e s i s i n h i b i t o r s (AMPT and ( b i s - ( l - m e t h y l - 4 - h o m o p i p e r a z i n y l - t h i o c a r -banyl) d i s u l p h i d e ) (FLA-63)), DA receptor b l o c k i n g drugs (spiramide and pimo-zide) and noradrenaline receptor b l o c k i n g drugs (aceperone and phenoxybenza-mine) have been reported to antagonize morphine induced e x c i t a t i o n (Ayhan and Randrup, 1973). Ayhan and Randrup (1973) reported that the b e h a v i o u r a l p r o f i l e s of mor-phine and amphetamine induced e x c i t a t i o n were d i f f e r e n t , inasmuch as morphine sti m u l a t e d some items of behaviour, i n c l u d i n g motor a c t i v i t y , grooming, eat-i n g and d r i n k i n g , whereas amphetamine s t i m u l a t e d motor a c t i v i t y and l e a r n i n g , but had no e f f e c t , or i n h i b i t i o n on grooming, e a t i n g and d r i n k i n g . A l s o mor-phine caused an increase i n b r a i n l e v e l s of HVA and DOPAC, whereas ampheta-mine caused a decrease i n DOPAC and an i n c r e a s e i n HVA (Fukui et a l , 1972; R o f f l e r - T a r l o v , Sharman and Tegerdine, 1971). Further i n t e r a c t i o n s t u d i e s between the behavioural a c t i o n s of morphine and amphetamine i n d i c a t e a mutual antagonism w i t h respect to stereotyped be-haviour (Fog, 1970) and enhancement of the a n a l g e s i c potency of morphine by amphetamine (Sprague and Takemori, 1978). F i n a l l y , Broekkamp (unpublished d a t a ) , has reported increased l e v e l s of spontaneous locomotor a c t i v i t y i n animals r e c e i v i n g e i t h e r an IP i n j e c t i o n of amphetamine, or i n t r a c e r e b r a l (IC) i n j e c t i o n of enkephalin i n t o the dopa-minergic A10 r e g i o n , w i t h a f u r t h e r p o t e n t i a t i o n of the locomotor h y p e r a c t i -59 v i t y i n animals r e c e i v i n g i n j e c t i o n s of enkephalin (IC) and amphetamine (IP) together. Since some behavioural e f f e c t s of both amphetamine and morphine are mediated by the catecholamine system, i t was decided to examine the e f f e c t s of acute i n j e c t i o n of amphetamine and morphine together and sep a r a t e l y on spontaneous locomotor a c t i v i t y i n r a t s . Smee and Overstreet (1976) have presented evidence u s i n g acute i n j e c -t i o n s of amphetamine f o l l o w i n g c h r o n i c morphine i n j e c t i o n s i n support of the "postsynaptic DA s u p e r s e n s i t i v i t y " hypothesis. They reported an in c r e a s e i n o r a l cage-directed stereotyped behaviour i n chronic morphine t r e a t e d animals f o l l o w i n g a d m i n i s t r a t i o n of d-amphetamine. I t was there f o r e concluded that morphine t r e a t e d animals demonstrated a s u p e r s e n s i t i v i t y to d-amphetamine. Considering the l i t e r a t u r e h e r e t o f o r e presented supporting the theory of p o s t s y n a p t i c DA receptor s u p e r s e n s i t i v i t y as explanation f o r the mecha-nisms underlying morphine t o l e r a n c e , i t was decided to f u r t h e r t e s t the theory by studying the e f f e c t of an acute i n j e c t i o n of d-amphetamine on the spontaneous locomotor a c t i v i t y of c h r o n i c a l l y t r e a t e d morphine r a t s . In a d d i t i o n , the e f f e c t of DB-6-0HDA lesions on the behavioural i n t e r a c t i o n of amphetamine and morphine was a l s o evaluated. 60 STUDY I l i a . INTERACTION BETWEEN THE BEHAVIOURAL EFFECTS OF ACUTE MORPHINE AND D-AMPHETAMINE ADMINISTRATION ON LOCOMOTOR ACTIVITY IN RATS. METHODS Subjects: A separate group of 40 naive Wistar a l b i n o r a t s from Woodlyn Farms, Guelph, Ontario were used i n t h i s study. In that i t was of i n t e r e s t to study the hyperactive phase of morphine induced locomotor a c t i v i t y i n i n t e r a c t i o n w i t h amphetamine induced h y p e r a c t i v i t y , a p i l o t study was conducted, and the time course of a dose of 2.5 mg/kg morphine was determined. The r e s u l t s i n d i c a -ted that the hypoactive phase was of d u r a t i o n of .5 hour and was followed by a hyperactive phase of 1.5 hours. Therefore, i t was decided to injectd-amphe-tamine .5 hour f o l l o w i n g the i n i t i a l morphine i n j e c t i o n , i n order to ade-quately assess the combined e f f e c t s of the drugs on locomotor h y p e r a c t i v i t y . Animals were the r e f o r e assigned randomly to one of the four f o l l o w i n g drug groups: 1. s a l i n e , amphetamine (.5 mg/kg); 2. morphine (2.5 mg/kg), s a l i n e ; 3. morphine (2.5 mg/kg), amphetamine (.5 mg/kg); and 4. s a l i n e , s a l i n e . A l l animals r e c e i v e d two IP drug i n j e c t i o n s 30 minutes apart and a l l t e s t i n g occurred between 10:00 A.M. and 2:30 P.M., d a i l y . Apparatus: Locomotor A c t i v i t y : Spontaneous locomotor a c t i v i t y was recorded i n s i x c i r c u l a r photoactometer cages, as described i n Experiment 1. Procedure: A l l animals were housed i n d i v i d u a l l y and given f r e e access to food and water. A 12 hour d a r k - l i g h t c y c l e was maintained throughout the d u r a t i o n of the experiment. Locomotor A c t i v i t y : S i x animals representing two drug treatment groups were i n d i v i d u a l l y 61 t e s t e d i n the locomotor a c t i v i t y apparatus s t a r t i n g at 10:00 A.M. The f o l -lowing day, another 6 animals r e p r e s e n t i n g the other two drug groups were te s t e d . This procedure of t e s t i n g a l t e r n a t e drug groups continued u n t i l an n=10 was reached f o r each group. Animals were placed i n the locomotor a c t i v i t y cages f o r a one hour ha-b i t u a t i o n p e r i o d , whereupon each animal was removed, given i t s f i r s t IP i n -j e c t i o n and replaced i n i t s wooden c a r r y i n g cage f o r 0.5 hour. At t h i s time, the second IP i n j e c t i o n was d e l i v e r e d and the animal was returned to the photoactometer apparatus where spontaneous locomotor a c t i v i t y was recorded f o r a three hour period. At the completion of t h i s phase of t e s t i n g , a l l animals were returned to t h e i r home cages. P o s t - i n j e c t i o n data were organized according to 18 minute t r i a l s and analyzed using a Three Factor A n a l y s i s of Variance ( s u b j e c t s ) , w i t h the simple main e f f e c t s model (groups). S i g n i f i c a n t d i f f e r e n c e s between groups were t e s t e d using the Duncan Post Hoc M u l t i p l e Range Test, p < .05. 62 STUDY I l i a RESULTS F i g u r e . 9 shows the time course of the locomotor responses of the 4 drug groups. S t a t i s t i c a l a n a l y s i s i n d i c a t e d : a s i g n i f i c a n t group e f f e c t , F= 21.60, df=3, 36, p < .001; a s i g n i f i c a n t time e f f e c t , F=11.89, df=9, 324, p < .001; and a s i g n i f i c a n t groups x time i n t e r a c t i o n , F=2.79, df=27, 324, p < .001. The Duncan M u l t i p l e Range Test i n d i c a t e d that the saline/ampheta-mine and the morphine/amphetamine groups demonstrated a gradual decrease i n a c t i v i t y over time, whereas the morphine/saline groups showed no change. The s a l i n e / s a l i n e group showed a s i g n i f i c a n t l y higher a c t i v i t y i n the f i r s t 36 minutes (2 t r i a l s ) , but t h e r e a f t e r showed no s i g n i f i c a n t d i f f e r e n c e i n a c t i v i t y over time. In accordance w i t h the b i p h a s i c a c t i o n of morphine on locomotor a c t i -v i t y , animals i n the morphine/saline groups i n i t i a l l y demonstrated s i g n i f i -c a n t l y l e s s a c t i v i t y than the other three groups. I t i s perhaps noteworthy that the p i l o t study i n d i c a t e d the hypoactive response was of a d u r a t i o n of 30 minutes, but when locomotor t e s t i n g commenced 30 minutes a f t e r the mor-phine i n j e c t i o n , animals s t i l l demonstrated the i n i t i a l hypoactive phase. This perhaps i n d i c a t e s an environmental i n f l u e n c e on the b e h a v i o u r a l a c t i o n s of morphine on locomotor a c t i v i t y . F o l l o w i n g t h i s , animals of the morphine/ s a l i n e group demonstrated the c h a r a c t e r i s t i c h y p e r a c t i v e phase, m a i n t a i n i n g s i g n i f i c a n t l y higher l e v e l s of locomotor a c t i v i t y than the s a l i n e / s a l i n e group f o r the next 2 hours. C h a r a c t e r i s t i c of the stimulant a c t i o n of amphetamine, animals of the amphetamine/saline group demonstrated s i g n i f i c a n t l y higher l e v e l s of l o c o -motor a c t i v i t y than the s a l i n e / s a l i n e group f o r approximately 2 hours pos-i n j e c t i o n . 63 Of p a r t i c u l a r i n t e r e s t was the behaviour of those animals who received a f i r s t i n j e c t i o n of morphine and a second i n j e c t i o n 30 minutes l a t e r of amphetamine. The Duncan M u l t i p l e Range Test revealed that o v e r a l l , the mor-phine/amphetamine group demonstrated s i g n i f i c a n t l y higher l e v e l s of l o c o -motor a c t i v i t y than the other three groups. These animals i n i t i a l l y demon-s t r a t e d s i g n i f i c a n t l y higher l e v e l s of locomotor a c t i v i t y than the s a l i n e / s a l i n e and the morphine/saline groups, but the same l e v e l of h y p e r a c t i v i t y as the amphetamine/saline group. However, by 72-108 and 144-162 minutes p o s t - i n j e c t i o n , animals of the morphine/amphetamine group demonstrated s i g -n i f i c a n t l y higher l e v e l s of locomotor a c t i v i t y than the other three drug groups, i n c l u d i n g the amphetamine/saline group. By the completion of the 3 hour t e s t i n g s e s s i o n , the morphine/amphetamine group s t i l l demonstrated s i g -n i f i c a n t l y higher l e v e l s of locomotor a c t i v i t y than the saline/amphetamine and s a l i n e / s a l i n e groups. 64 Figure 10 Mean locomotor a c t i v i t y during the 3 hour p o s t - i n j e c t i o n p e r i o d . Closed squares = saline/amphetamine, N=10; Open squares = morphine/saline, N=10; Closed c i r c l e s = morphine/ amphetamine, N=10; Open c i r c l e s = s a l i n e / s a l i n e , N=10 per group. 66 STUDY I l l b . THE EFFECT OF DB-6-OHDA LESIONS IN MORPHINE TOLERANT RATS ON LOCOMOTOR ACTIVITY FOLLOWING ACUTE AMPHETAMINE ADMINISTRATION METHODS Subjects and Procedure: At the completion of the previous study, a l l animals had been i n j e c t e d w i t h e i t h e r morphine or s a l i n e f o r a 21 day p e r i o d . According to the l o c o -motor t e s t i n g schedule p r e v i o u s l y d e s c r i b e d , animals from a l l four groups were r e t e s t e d i n the p h o t o c e l l cages. F o l l o w i n g the one hour standard h a b i -t u a t i o n p e r i o d , animals from a l l four groups r e c e i v e d an IP i n j e c t i o n of d-am-phetamine (1 mg/kg) according to body weight and were then replaced i n the p h o t o c e l l cages. Their spontaneous locomotor a c t i v i t y was then recorded f o r a three hour p e r i o d (as p r e v i o u s l y d e s c r i b e d ) . Animals were then removed from the p h o t o c e l l cages, given t h e i r appropriate d a i l y IP i n j e c t i o n s of sa-l i n e or morphine, and returned to t h e i r home cages. Data from the one hour h a b i t u a t i o n p e r i o d was summed across the 6 ten minute t r i a l s f o r each group and analyzed using a One Way A n a l y s i s of V a r i -ance. S i g n i f i c a n t between group d i f f e r e n c e s were t e s t e d using the Duncan Post Hoc M u l t i p l e Range Test, p < .05. P o s t - i n j e c t i o n data were organized according to 20 minute periods (=9 v a r i a b l e s ) and analyzed using a Three Factor Repeated Measures A n a l y s i s of Variance (subjects) w i t h the simple main e f f e c t s model (groups). S i g n i f i -cant d i f f e r e n c e s between groups were again t e s t e d using the Duncan Post Hoc M u l t i p l e Range Test, p < .05. Drugs: Doses of morphine sulphate and d-amphetamine sulphate were expressed i n terms of the s a l t s . The s o l u t i o n s were prepared d a i l y w i t h p h y s i o l o g i c a l s a l i n e and i n j e c t e d IP at room temperature, i n a volume of 1 kg/ml. 67 STUDY I I l b RESULTS Ha b i t u a t i o n Data: F i g u r e 10 shows the time course of the locomotor a c t i v i t y during the h a b i t u a t i o n p e r i o d f o r the four groups. S t a t i s t i c a l a n a l y s i s revealed a s i g -n i f i c a n t between groups d i f f e r e n c e , F=17.007, df=3, p < .001. The Duncan M u l t i p l e Range Test i n d i c a t e d that the DB-6-0HDA-morphine group showed over-a l l s i g n i f i c a n t l y l e s s locomotor a c t i v i t y than the vehicle-morphine group, which i n t u r n showed s i g n i f i c a n t l y l e s s locomotor a c t i v i t y than the DB-6-OHDA-saline and v e h i c l e - s a l i n e groups. P o s t - I n j e c t i o n Data: Figure 11 shows the time course of the locomotor a c t i v i t y of the four groups, p o s t - i n j e c t i o n . S t a t i s t i c a l a n a l y s i s i n d i c a t e s a s i g n i f i c a n t group e f f e c t , F=21.60, df=3, 36, p < .001, a s i g n i f i c a n t time e f f e c t , F=11.89, df= 9, 324, p < .001 and a s i g n i f i c a n t groups x time i n t e r a c t i o n , F=2.79, df= 27, 324, p < .001. The Duncan M u l t i p l e Range Test i n d i c a t e d that a l l four groups showed a decrease i n a c t i v i t y over time. No s i g n i f i c a n t group d i f f e r e n c e was revealed f o r the f i r s t 20 minutes p o s t - i n j e c t i o n . However, f o r the f o l l o w i n g one hour p e r i o d , animals that had undergone chronic morphine pretreatment, regardless of l e s i o n , demonstra-ted s i g n i f i c a n t l y higher l e v e l s of locomotor a c t i v i t y than those animals from both groups w i t h s a l i n e pretreatment. 68 Figure 11 Mean locomotor a c t i v i t y during a 1 hour p r e - i n j e c t i o n h a b i t u a -t i o n phase p r i o r to acute amphetamine I n j e c t i o n s . See Study I H b . Closed squares = DB-6-0HDA-morphine group, N=10; Open squares = DB-6-0HDA-saline group, N=8; Closed c i r c l e s = v e h i -cle-morphine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 69 T I M E ( m i n ) 70 Figure ,.12 Mean locomotor a c t i v i t y during a 3 hour period f o l l o w i n g : acute amphetamine i n j e c t i o n . Closed squares = DB-60HDA-morphine group, N=10; Open squares = DB-60HDA-saline group, N=8; Closed c i r c l e s = vehicle-morphine group, N=10; Open c i r c l e s = v e h i c l e - s a l i n e group, N=8. 71 72 DISCUSSION Amphetamine/Morphine I n t e r a c t i o n : Acute IP i n j e c t i o n of a low dose of amphetamine (.5 mg/kg) followed 30 minutes l a t e r by acute i n j e c t i o n of a low dose of morphine (2.5 mg/kg) r e s u l -ted i n s i g n i f i c a n t l y higher l e v e l s of locomotor a c t i v i t y than observed i n animals r e c e i v i n g e i t h e r ampehtamine (.5 mg/kg) or morphine (2.5 mg/kg) i n -j e c t i o n . As p r e v i o u s l y noted, i t was r e c e n t l y reported that o p i a t e s produce beha-v i o u r a l e x c i t a t i o n by a c t i v a t i n g DA neurons ( C a r r o l l and Sharp, 1972) and that i n j e c t i o n of the long a c t i n g s y n t h e t i c enkephalin analogue (D-ala 2) -Met 5enkephalinamide (AME) i n t o the nucleus accumbens and the dopaminergic A-10 area of the v e n t r a l tegmental area (VTA) , produced an in c r e a s e i n l o c o -motor a c t i v i t y (Pert and S i v i t , 1977; Broekkamp et a l , 1979). The nucleus accumbens i s one of the p r o j e c t i o n areas of DA c e l l s i n the VTA, and there i s a l s o evidence of involvement of t h i s mesolimbic DA pathway i n the stimu-l a n t e f f e c t s of amphetamine ( K e l l e y , Seviour and Iversen, 1975). One may the r e f o r e hypothesize that the enhancement of locomotor a c t i v i t y w i t h acute amphetamine + morphine i n j e c t i o n s i s r e l a t e d to increased a c t i v a t i o n of dopaminergic systems i n the mesolimbic pathway. The p o t e n t i a t i o n of spontaneous locomotor h y p e r a c t i v i t y f o l l o w i n g an acute amphetamine i n j e c t i o n (1 mg/kg) a l s o was observed i n chronic morphine p r e t r e a t e d animals when compared to s a l i n e p r e t r e a t e d animals i n accordance w i t h r e s u l t s p r e v i o u s l y i m p l i c a t i n g a s y n e r g i s t i c r o l e of amphetamine and morphine (Smee and Overstreet, 1976-; Ahyan and Randrup, 1973). Inasmuch as t h i s b e h a v i o u r a l e f f e c t was unaffe c t e d by a DB-6-0HDA l e s i o n , i t seems l i k e l y that i t i s mediated by another t r a n s m i t t e r system, p o s s i b l y DA. I t should be noted that these r e s u l t s cannot be a t t r i b u t e d to a c e i l i n g e f f e c t , 73 i n that i t was demonstrated p r e v i o u s l y t h a t animals who recei v e d b i l a t e r a l i n j e c t i o n s of k a i n i c a c i d i n t o t h e . s t r i a t u m d i s p l a y e d a c t i v i t y count l e v e l s as h i g h as 900 counts/10 min f o l l o w i n g an i n j e c t i o n of 1 mg/kg amphetamine ( P i s a , Sanberg and F i b i g e r , 1979), whereas count l e v e l s i n the present study reached a maximum of approximately 1200 counts/20 min. Furthermore, i n j e c t i o n s of 2 mg/kg i n t o animals w i t h the same l e s i o n r e s u l t e d i n a c t i v i t y l e v e l s of 1100/10 min (Mason and F i b i g e r , 1978). In a d d i t i o n , our f i n d i n g s that a DB-6-0HDA l e s i o n did not a f f e c t the amphetamine-induced p o t e n t i a t i o n of locomotor h y p e r a c t i v i t y f o l l o w i n g acute and chronic morphine do not lend support to the hypothesis that NA mediates a modulatory, or t r a n s y n a p t i c r o l e i n DA-mediated locomotor s t i m u l a t i o n (Maj et a l , 1971; Kuschinsky and Hornykiewicz, 1974). I t i s p o s s i b l e that morphine and amphetamine may be a c t i n g on two com-p l e t e l y d i f f e r e n t b r a i n systems that i n t e r a c t , or a l t e r n a t i v e l y , i t has been suggested that both act on the same DA neuronal system. Smee and Overstreet (1976) observed enhanced amphetamine induced stereotypy i n animals that had receiv e d chronic morphine pretreatment, when compared to s a l i n e p r e t r e a t e d animals and they a s c r i b e d these f i n d i n g s to 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 rec e p t o r s , r e s u l t i n g from chronic morphine treatment. Based on t h i s evidence and other previous f i n d i n g s i m p l i c a t i n g p o s t s y n a p t i c DA receptor supersensi-t i v i t y i n the e f f e c t s of. chronic morphine treatment ( P u r i et a l , 1977; P u r i and L a i , 1974; Baume et a l , 1979; and, Clouet and Iwatsubo, 1975a), i t seems reasonable to a s c r i b e t h i s mechanism to our observation of enhanced ampheta-mine-induced locomotor a c t i v i t y i n animals c h r o n i c a l l y p r e t r e a t e d w i t h mor-phine . 74 GENERAL DISCUSSION Several behavioural e f f e c t s of morphine were stud i e d f o l l o w i n g s p e c i f i c i n j e c t i o n s of 6-OHDA i n t o the d o r s a l noradrenergic bundle, i n an attempt to evaluate the i n t e r a c t i o n of morphine w i t h the catecholamine systems. Studies I and I I explored the e f f e c t s of chronic morphine on the development of t o -leranc e and p h y s i c a l dependence, r e s p e c t i v e l y . A s t a t e of to l e r a n c e i s s a i d to be reached when a f t e r repeated use, a given dose of drug produces a de-creased e f f e c t , or when increased doses must be taken to o b t a i n the e f f e c t s of the o r i g i n a l dose. P h y s i c a l dependence r e s u l t s when repeated a d m i n i s t r a -t i o n of a drug a l t e r s the p h y s i o l o g i c a l s t a t e and n e c e s s i t a t e s the continued use of the drug to prevent withdrawal ( J a f f e and M a r t i n , 1975). F i r s t l y , t h e . e f f e c t of DB-6-0HDA l e s i o n s on tole r a n c e was assessed em-p l o y i n g changes i n the hypoactive phase of morphine-induced locomotor a c t i -v i t y as an index of tole r a n c e development. I t was observed that a r o l e f o r the catecholamine systems, s p e c i f i c a l l y f o r NA can be i m p l i c a t e d i n t o l e r -ance development to chronic morphine treatment, i n that i n j e c t i o n of 6-OHDA i n t o the d o r s a l noradrenergic bundle r e s u l t e d i n a slower r a t e and a l e s s e r degree of to l e r a n c e development to the hypoactive phase of morphine-induced locomotor a c t i v i t y . Several models can be proposed i n r e l a t i o n to the data obtained, a l l of which maintain the b a s i c premise that the b i p h a s i c a c t i o n of morphine on locomotor a c t i v i t y can be a t t r i b u t e d to the i n t e r a c t i o n of the NA and DA systems. 75 T h e o r e t i c a l Considerations: There i s much evidence that motor s t i m u l a t i o n i s mediated by increased catecholamine t r a n s m i s s i o n , s p e c i f i c a l l y of DA (Kuschinsky, 1976; Creese and Iversen, 1973; Moore, 1977; Roberts et a l , 1978), i . e . the c e n t r a l s t i m u l a n t a c t i o n of amphetamine i s blocked p r i m a r i l y by drugs which have DA-receptor b l o c k i n g p r o p e r t i e s ( i . e . pimozide), but not drugs which b l o c k a- and g-adrenergic receptors ( i . e . phentolamine or p r o p a n o l o l ) . In a d d i t i o n , the c e n t r a l stimulant a c t i o n s of amphetamine were blocked i n animals w i t h s e l e c -t i v e l o s s of DA neurons, but not a l t e r e d i n animals w i t h l o s s of NA neurons. A l s o , the hypothesis that the s t i m u l a t o r y e f f e c t s of morphine are mediated by the DA system has been reviewed e a r l i e r (see I n t r o d u c t i o n ) . The present f i n d i n g that acute amphetamine a d m i n i s t r a t i o n f o l l o w i n g acute or chronic morphine pre-treatment r e s u l t e d i n p o t e n t i a t e d h y p e r a c t i v e responses, regardless of the DB-6-0HDA l e s i o n , f u r t h e r i m p l i c a t e s a r o l e f o r DA i n the mediation of the s t i m u l a t o r y e f f e c t s of morphine. In a d d i t i o n , i t has been suggested that the b i p h a s i c a c t i o n s of mor-phine on locomotor a c t i v i t y may i n v o l v e more than one neurotransmitter sub-stance (Vasko and Domino, 1978; Mason et a l , 1978). Roberts et a l (1978) reported that 6-OHDA-induced d e p l e t i o n of f o r e b r a i n NA caused p o t e n t i a t i o n of the depressant a c t i o n s of acute morphine a d m i n i s t r a t i o n , whereas the stimulant a c t i o n s appeared independent of noradrenergic mechanisms. Buxbaum et a l (1973) observed that h y p e r a c t i v i t y observed a f t e r acute a d m i n i s t r a t i o n of morphine was antagonized by treatments that deplete b r a i n catecholamines and that h y p o a c t i v i t y produced by high doses of morphine was antagonized by treatment that deplete s e r o t o n i n l e v e l s . The authors suggested that the e f -f e c t of morphine on locomotor a c t i v i t y i s dependent upon a balance between the two systems. Of p a r t i c u l a r i n t e r e s t , i s the evidence of Broekkamp, Van de Bogaard, Heijnen, Rops, Cools, and Van Rossum (1975) that i n t r a c e r e b r a l 76 i n j e c t i o n s of morphine separated the e x c i t a t o r y and i n h i b i t o r y e f f e c t s of mor-phine. They reported that pure i n h i b i t o r y e f f e c t s were obtained from the cen-t r a l grey substance surrounding the aqueduct and locus coeruleus, whereas pure e x c i t a t o r y e f f e c t s were measured a f t e r i n j e c t i o n s i n t o the p o s t e r i o r hypotha-lamus and v e n t r a l tegmental area. Based on the above f i n d i n g s , i t seems r e a -sonable to a s c r i b e the b i p h a s i c e f f e c t of morphine on locomotor a c t i v i t y to an i n t e r a c t i o n between the hypoactive response mediated by the NA system,and the h y p e r a c t i v e response mediated by the DA system. The b e h a v i o u r a l e f f e c t s on l o -comotor a c t i v i t y f o l l o w i n g chronic morphine a d m i n i s t r a t i o n can then be ex-p l a i n e d as f o l l o w s : i n i t i a l l y the hypoactive phase.characterized by a decrease i n NA t r a n s m i s s i o n , i s dominant and masks the DA i n f l u e n c e . The secondary r e -sponse of h y p e r a c t i v i t y represents the i n c r e a s i n g dominance of the DA system and i s c h a r a c t e r i z e d by increased r e l e a s e of DA. According to t h i s f o r m u l a t i o n , the DB-6-0HDA l e s i o n e d animals d i s p l a y a lower l e v e l of h y p e r a c t i v i t y f o l l o w -ing chronic morphine treatment because they show a p o t e n t i a t e d NA-mediated hy-p o a c t i v e response,which when added to the secondary DA-mediated hy p e r a c t i v e response,results i n the observed response of a lower l e v e l of h y p e r a c t i v i t y . With t h i s b a s i c premise i n mind, s e v e r a l models can be proposed f o r the e f f e c t s of chronic and acute morphine or locomotor a c t i v i t y . The f i r s t i s perhaps the most parsimonious and i n t e r p r e t s the data according to the theory of "disuse s u p e r s e n s i t i v i t y " , i n i t i a l l y proposed by C o l l i e r (1965) and J a f f e and Sharpless (1968), and more r e c e n t l y summarized by L l o r e n s et a l (1978). Acute morphine i n j e c t i o n s r e s u l t i n p r e s y n a p t i c noradrenergic i n h i b i t i o n , which r e s u l t s i n decreased NA t r a n s m i s s i o n . With chronic mor-phine a d m i n i s t r a t i o n , long-term p r e s y n a p t i c i n h i b i t i o n r e s u l t s i n a compen-sato r y i n c r e a s e i n the responsiveness of the p o s t s y n a p t i c c e l l to NA. I t should be noted that 6-OHDA l e s i o n s i n t o the f i b e r s of the d o r s a l noradrener-g i c bundle r e s u l t i n widespread d e p l e t i o n of f o r e b r a i n noradrenaline and t h a t 77 d e s t r u c t i o n of nerve f i b e r s may be p a r t i a l l y compensated f o r by supersensi-t i v e p o s t s y n a p t i c NA r e c e p t o r s , due to denervation s u p e r s e n s i t i v i t y . There-f o r e , according to t h i s model, because the DB-6-0HDA animals would already have pos t s y n a p t i c NA s u p e r s e n s i t i v i t y , one would hypothesize that an acute i n j e c t i o n would r e s u l t i n decreased or no observed h y p o a c t i v i t y , i n d i c a t i n g t o l e r a n c e development to the hypoactive phase w i t h ah acute morphine i n j e c -t i o n . This p r e d i c t i o n i s , of course, contrary to the observed f i n d i n g s of h y p o a c t i v i t y i n l e s i o n e d animals f o l l o w i n g an acute i n j e c t i o n of morphine. Secondly, when co n s i d e r i n g the response to chronic morphine, i f t o l e -rance development i s a t t r i b u t e d to increased p o s t s y n a p t i c NA receptor super-s e n s i t i v i t y , as L l o r e n s et a l (1979) suggest, and DB-6-0HDA animals have de-veloped p o s t s y n a p t i c NA s u p e r s e n s i t i v i t y p r i o r to the beginning of chronic morphine a d m i n i s t r a t i o n , i t i s not p o s s i b l e to make p r e d i c t i o n s w i t h respect to tolerance development to the hypoactive phase when the response to acute morphine i n j e c t i o n i s considered. I t t h e r e f o r e appears that the mere c o n s i -d e r a t i o n of postsynaptic s u p e r s e n s i t i v i t y i s inadequate to e x p l a i n the de-velopment of to l e r a n c e f o l l o w i n g repeated morphine a d m i n i s t r a t i o n . The second model i s s i m i l a r to the f i r s t presented, i n that i t postu-l a t e s a r o l e f o r increased p o s t s y n a p t i c NA s u p e r s e n s i t i v i t y f o l l o w i n g chro-n i c morphine a d m i n i s t r a t i o n . In a d d i t i o n , i t makes two assumptions: 1) there i s a c e i l i n g e f f e c t r e g u l a t i n g the amount of po s t s y n a p t i c receptor p r o l i f e r a t i o n ( s u p e r s e n s i t i v i t y ) t h a t can occur i n the l e s i o n e d and non-le-sioned animals, and 2) i n the DB-6-0HDA animals, increased p o s t s y n a p t i c NA receptor s u p e r s e n s i t i v i t y i s outweighed by the presence of s u b s t a n t i a l l y fewer NA nerve terminals (as a r e s u l t of the 6-OHDA l e s i o n ) , r e s u l t i n g i n an o v e r a l l r e d u c t i o n i n NA tone. I n c l u s i o n of the presence of decreased NA tone , a f f o r d s an a l t e r n a t e explanation when i n t e r p r e t i n g the behav i o u r a l e f -f e c t s of the DB-6-0HDA l e s i o n , e s p e c i a l l y w i t h regard to tol e r a n c e develop-78 merit f o l l o w i n g chronic morphine treatment. In t h i s case, the model would p r e d i c t that decreased NA tone would r e s u l t i n l e s s t o l e r a n c e development i n the DB-6-OHDA group or increased l e v e l s of h y p o a c t i v i t y i n r e l a t i o n to the vehicle-morphine group w i t h repeated.morphine a d m i n i s t r a t i o n . This p r e d i c t i o n i s supported by the present f i n d i n g s . The model i s perhaps l e s s e f f e c t i v e i n p r e d i c t i n g the acute a c t i o n s of morphine. Acute morphine i n j e c t i o n i n non-lesioned animals r e s u l t s i n the i n i t i a l hypoactive locomotor response, as a r e s u l t of decreased NA r e l e a s e . The model p o s t u l a t e s that the DB-6-OHDA l e s i o n r e s u l t s i n decreased NA tone. I t i s d i f f i c u l t on the b a s i s of the model to q u a l i t a t i v e l y compare the d i f -ferences i n tone between the non-lesioned and DB l e s i o n e d animals upon r e -c e i p t of acute morphine i n j e c t i o n . One p o s s i b l e recourse i s to consider the observed f i n d i n g s that both groups d i s p l a y the same degree of h y p o a c t i v i t y f o l l o w i n g acute i n j e c t i o n of a dose of 25 mg/kg morphine and assume that the two groups t h e r e f o r e have approximately the same degree of r e d u c t i o n i n NA tone. I t appears, however, that f u r t h e r biochemical i n v e s t i g a t i o n i s neces-sary to f u l l y evaluate the e f f e c t s of a DB-6-0HDA l e s i o n on NA r e l e a s e and turnover i n the f o r e b r a i n , so as to avoid making inferences about the b i o -chemical changes f o l l o w i n g acute morphine treatment s o l e l y from behavioural evidence. I n summary, i t appears that the second model, which i n c l u d e s considera-t i o n of NA tone as w e l l as NA p o s t s y n a p t i c s u p e r s e n s i t i v i t y , i s somewhat i n e f f e c t i v e i n p r e d i c t i n g the e f f e c t s of acute morphine i n j e c t i o n , but i s the model b e t t e r s u i t e d as an explanation .for the behavioural e f f e c t s of chronic morphine observed i n c o n t r o l and DB-6-0HDA l e s i o n e d animals. 79 An a l t e r n a t i v e and perhaps e q u a l l y important c o n s i d e r a t i o n i s the e f f e c t of DB-6-OHDA l e s i o n s on c e r e b e l l a r and s p i n a l cord l e v e l s of NA. Atweh and Kuhar (1977b) have demonstrated o p i a t e receptors i n the s p i n a l cord using autoradiographic i d e n t i f i c a t i o n ,of s t e r e o s p e c i f i c (%) d i s p r e -norphine (a potent o p i a t e antagonist) b i n d i n g s i t e s and Garcin and Coyle (1977) reported t h a t p e r i p h e r a l treatment of newborn r a t s w i t h 6-OHDA s i g -n i f i c a n t l y increased the l e v e l s of NA and o p i a t e receptor b i n d i n g i n the cerebellum, suggesting that o p i a t e receptors i n the cerebellum may be l o -c a l i z e d on NA f i b e r s i n n e r v a t i n g t h i s r e g i o n . I t has been reported that DB-6-0HDA l e s i o n s s i g n i f i c a n t l y increase the l e v e l s of NA i n the cerebellum and s p i n a l cord (Mason et a l , 1978; U'Prichard, R e i s i n e , Mason, F i b i g e r and Yamamura, i n p r e s s ) . Recent i n -v e s t i g a t i o n has revealed that the increased l e v e l s of NA r e s u l t i n a com-pensatory decrease i n the number of 3-adrenergic receptors a v a i l a b l e f o r NA to act upon (U'Prichard et a l , i n p r e s s ) . I t i s suggested that cere-b e l l a r receptor s i t e s p o s t s y n a p t i c to NA t e r m i n a l s can become desensi-t i z e d or " s u b s e n s i t i v e " as a r e s u l t of increased NA l e v e l s (U'Prichard et a l , i n p r e s s ) . A t h i r d model f o r the acute and chronic e f f e c t s of morphine can t h e r e f o r e be proposed, i n c o r p o r a t i n g these neuronal changes i n the s p i n a l cord f o l l o w i n g a DB-6-0HDA l e s i o n . In c o n s i d e r i n g the non-lesioned animals, acute morphine r e s u l t s i n decreased catecholamine r e l e a s e , and a concomitant increase i n turnover. 80 With chronic morphine a d m i n i s t r a t i o n , non-lesioned animals demonstrate t o l e -rance to the e f f e c t of morphine on turnover ( P u r i and L a i , 1974'J Gauchy et a l , 1973; see review by Clouet and Iwatsubo, 1975b) as w e l l as po s t s y n a p t i c s u p e r s e n s i t i v i t y ( C o l l i e r , 1965; J a f f e and Sharpless, 1968; L l o r e n s et a l , 1978). Therefore, chronic morphine a d m i n i s t r a t i o n r e s u l t s i n increased NA tone f o l l o w i n g morphine i n j e c t i o n , manifested b e h a v i o u r a l l y by decreased l e -v e l s of locomotor h y p o a c t i v i t y . In c o n s i d e r i n g the response of the DB-6-OHDA group to chronic morphine treatment, i t i s assumed that the DB-6-0HDA group shows a s i m i l a r t o l e r a n c e response to turnover however, DB-6-0HDA l e -sions a l s o r e s u l t i n increased l e v e l s of NA i n the cerebellum and s p i n a l cord w i t h the concomitant r e d u c t i o n i n the number of g-adrenergic receptors a v a i l a b l e f o r NA to act upon. I t can th e r e f o r e be hypothesized that chronic morphine treatment would r e s u l t i n the development of l e s s s u p e r s e n s i t i v i t y i n the DB-6-0HDA group due to the presence i n i t i a l l y of a l e s s e r number of posts y n a p t i c receptors as a r e s u l t of the l e s i o n . This would r e s u l t i n lower NA tone, manifested b e h a v i o u r a l l y by pr o l o n g a t i o n of the locomotor hy-poactive response. These p r e d i c t i o n s were again i n accordance w i t h the ob-served behavioural responses f o l l o w i n g chronic morphine treatment, and pro-v i d e support f o r the hypothesis that the i n i t i a l hypoactive phase of mor-phine-induced locomotor a c t i v i t y i s mediated by the NA system. As w i t h the models presented e a r l i e r , t h i s model i s l e s s adequate i n e x p l a i n i n g the e f f e c t s of acute morphine a d m i n i s t r a t i o n i n the naive animal. The neurochemical e f f e c t s of the DB-6-0HDA l e s i o n i n the s p i n a l cord and cerebellum have already been described, namely an increase i n NA l e v e l s and a re d u c t i o n i n the number of po s t s y n a p t i c g-adrenergic receptors. However, i t i s not p o s s i b l e to p r e d i c t the e f f e c t s of acute morphine treatment on NA tone s o l e l y from these observations. For t h i s reason, p r e d i c t i o n s are made according to the behavioural data observed. Locomotor h y p o a c t i v i t y has been 81 c i t e d as a behavioural c o r r e l a t e of the r e d u c t i o n i n r e l e a s e of NA, and the same l e v e l s of hypoactivity.were observed i n both the l e s i o n s and non-le-sioned animals f o l l o w i n g acute morphine treatment. I t i s t h e r e f o r e assumed based on the present b e h a v i o u r a l observations, that the DB-6-0HDA and con-t r o l groups showed the same re d u c t i o n i n NA r e l e a s e f o l l o w i n g acute morphine a d m i n i s t r a t i o n . I t i s p o s s i b l e to apply one f i n a l i n t e r p r e t a t i o n to the data based on the observed neurochemical changes i n the s p i n a l cord and cerebellum f o l l o w -in g a DB-6-0HDA l e s i o n described e a r l i e r . I t has been reported that acute morphine a d m i n i s t r a t i o n r e s u l t s i n increased l e v e l s of MHPG, a major metabo-l i t e of b r a i n NA (Roffman et a l , 1979) as w e l l as decreases i n b r a i n NA f o l -lowing i n t r a v e n t r i c u l a r i n j e c t i o n of morphine (Watanabe, 1971). In a d d i t i o n , i t has been reported that acute a d m i n i s t r a t i o n of an a n a l g e s i c dose of mor-phine increases the concentration of another NA m e t a b o l i t e , normetanephrine (NM) i n the d o r s a l h a l f of the s p i n a l cord (Shiomi and Takagi, 1974; Takagi, Shiomi, K u r a i s h i , Fukui and Ueda, 1979) and that animals that underwent chro-n i c morphine treatment showed to l e r a n c e to the NM i n c r e a s e . I t has t h e r e f o r e been suggested that morphine a c c e l e r a t e s the r e l e a s e of NA from descending noradrenergic f i b e r s (Shiomi and Takagi, 1974). On t h i s b a s i s , i t seems pos-s i b l e that the behavioural e f f e c t s of acute morphine, s p e c i f i c a l l y the sup-pressant phase of locomotor a c t i v i t y , may be mediated by an increase i n NA r e l e a s e i n the s p i n a l cord and cerebellum. The observation of t o l e r a n c e to the increase i n turnover f o l l o w i n g chronic morphine treatment (Shiomi and Takagi, 1974; P u r i and L a i , 1974; Gauchy et a l , 1973; see review by Clouet and Iwatsubo, 1975) c o r r e l a t e s w i t h the behavioural observation of t o l e r a n c e to the hypoactive phase of morphine-induced locomotor a c t i v i t y . I t should be noted that these biochemical r e s u l t s are i n c o n s i s t e n t w i t h p h y s i o l o g i c a l evidence presented by Aghajanian (1978) that morphine causes i n h i b i t i o n of 82 the spontaneous f i r i n g r a t e of NA c e l l s i n the locus coeruleus. However, comparison of the a c t i o n s of amphetamine usi n g the same biochemical and phy-s i o l o g i c a l techniques has y i e l d e d the same i n c o n s i s t e n c i e s . There i s much evidence that the behavioural stimulant e f f e c t s of amphetamine are mediated by increased r e l e a s e of DA (Kuschinsky, 1976; Creese and Iversen, 1973; Moore, 1977; and Roberts et a l , 1978); however, p h y s i o l o g i c a l s t u d i e s i n d i -cate that d-amphetamine i n h i b i t s the f i r i n g of DA neurons i n the zona com-pacta and v e n t r a l tegmental area (Bunney, Aghajanian and Roth, 1973). Therefore, perhaps the a c t i o n s of morphine on NA neurons can be viewed as s i m i l a r to those of amphetamine on DA neurons. On t h i s b a s i s i t can be hypo-t h e s i z e d that morphine a d m i n i s t r a t i o n i n h i b i t s the spontaneous f i r i n g r a t e of LC c e l l s , as w e l l as mediates an increase i n the r e l e a s e of NA by an ac-t i o n on NA t e r m i n a l s . With these assumptions i n mind the beha v i o u r a l e f -f e c t s of morphine can be explained as f o l l o w s : as e a r l i e r s t a t e d , the DB-6-OHDA l e s i o n r e s u l t s i n increased l e v e l s of NA i n the b r a i n and s p i n a l cord (U'Prichard et a l , i n press) when compared to c o n t r o l s . I f increased l e v e l s of NA i n the s p i n a l cord of the DB-6-0HDA l e s i o n e d animals r e s u l t i n an en-hanced re l e a s e of NA, i t can be hypothesized that the DB-6-0HDA animals should demonstrate more pronounced h y p o a c t i v i t y and a l e s s e r degree of t o l e r -ance development f o l l o w i n g chronic morphine a d m i n i s t r a t i o n . However, i t should be noted that t h i s model i s s p e c u l a t i v e , inasmuch as l i t t l e i f any evidence i s c u r r e n t l y a v a i l a b l e on the e f f e c t s of acute and chronic morphine on NA r e l e a s e and turnover i n the s p i n a l cord and cerebellum. In summary, s e v e r a l models have been presented i n an attempt to e x p l a i n the observed behavioural f i n d i n g s . No one model adequately p r e d i c t s or ex-p l a i n s the behavioural and biochemical e f f e c t s of acute and chronic morphine a d m i n i s t r a t i o n . However, each one provides a framework i n which to d i s c u s s the data obtained. I t i s obvious that f u r t h e r research i s necessary before 83 a t r u l y s a t i s f a c t o r y model can be proposed, and that a l l assumptions regar-ding the biochemical e f f e c t s of the DB-6-0HDA l e s i o n on NA r e l e a s e and t u r n -over i n the s p i n a l cord, cerebellum and f o r e b r a i n f o l l o w i n g acute and chro-n i c morphine should be e m p i r i c a l l y t e s t e d . One e s s e n t i a l experiment c u r r e n t -l y underway i s the e v a l u a t i o n of the e f f e c t s of d e p l e t i o n of s p i n a l and cere-b e l l a r NA, so as to more c l e a r l y d e l i n e a t e the i n d i v i d u a l r o l e s of a l t e r a -t i o n s i n NA l e v e l s i n the s p i n a l cord/cerebellum and the f o r e b r a i n i n media-t i n g the e f f e c t s of acute and chronic morphine a d m i n i s t r a t i o n . 84 The 2nd study t e s t e d the e f f e c t of a DB-6-0HDA l e s i o n on the develop-ment of p h y s i c a l dependence. The c l a s s i c a l index of p h y s i c a l dependence i s withdrawal, and the degree of p h y s i c a l dependence i s measured by the s e v e r i t y of withdrawal ( J a f f e and M a r t i n , 1975). There are s e v e r a l tech-niques a v a i l a b l e f o r measuring withdrawal: f i r s t l y , chronic morphine pre-t r e a t e d animals can be i n j e c t e d w i t h an op i a t e antagonist ( i . e . naloxone) and the incidence of a s e r i e s of withdrawal behaviours measured. A second common m a n i f e s t a t i o n of withdrawal i s weight l o s s f o l l o w i n g d i s c o n t i n u a t i o n of morphine treatment. In a d d i t i o n , i n the st u d i e s o u t l i n e d above, i r r i -t a b i l i t y a s s o c i a t e d w i t h handling and i n j e c t i o n and locomotor a c t i v i t y l e -v e l s during the h a b i t u a t i o n p e r i o d were a l s o a t t r i b u t e d to a withdrawal response and th e r e f o r e used as i n d i c e s of p h y s i c a l dependence. The e f f e c t of the DB-6-0HDA l e s i o n on p h y s i c a l dependence was assessed by measuring naltrexone-induce withdrawal i n l e s i o n e d and v e h i c l e animals that had re c e i v e d chronic morphine treatment. R e s u l t s i n d i c a t e that although NA i s important i n tol e r a n c e development i t does not mediate a dominant r o l e i n p h y s i c a l dependence, as manifested i n n a l t r e x o n e - p r e c i p i t a t e d withdrawal. Some e f f e c t s of the l e s i o n on other measurements of dependence, however, were observed, s p e c i f i c a l l y on i r r i t a b i l i t y a s s o c i a t e d w i t h the handling and i n j e c t i o n procedure. Co n s i d e r a t i o n of the h a b i t u a t i o n data a l s o r e -vealed i n f o r m a t i o n about the e f f e c t of DB-6-0HDA l e s i o n s on p h y s i c a l depen-dence. 85 H a b i t u a t i o n Data: The Morphine Tolerance and the Amphetamine A f t e r Chronic Morphine s t u -d i e s (Study I l l b ) occurred i n the c h r o n o l o g i c a l order l i s t e d and t h e r e f o r e enable observation of the behaviour of the animals during the p r e - i n j e c t i o n h a b i t u a t i o n periods over an extended p e r i o d of morphine treatment. I n i t i a l l y i n s e s s i o n 1 of the Morphine Tolerance Study, no s i g n i f i c a n t d i f f e r e n c e s were observed. However, by s e s s i o n 2, the vehicle-morphine group demonstrated s i g n i f i c a n t l y l e s s locomotor a c t i v i t y than the other three groups, e s p e c i a l l y during the l a s t 30 minute p e r i o d . In sessions 3-6 i n c l u s i v e , the DB-6-0HDA-morphine and the vehicle-morphine groups demonstra-ted s i g n i f i c a n t l y l e s s a c t i v i t y than the DB-6-0HDA-saline and v e h i c l e - s a l i n e groups. This group d i f f e r e n c e i n a c t i v i t y was again e s p e c i a l l y pronounced during the l a s t 30 minute p e r i o d . This trend of morphine i n j e c t e d animals demonstrating l e s s a c t i v i t y during the h a b i t u a t i o n p e r i o d was a l s o evident i n subsequent s t u d i e s . During the f i n a l experiment of the s e r i e s , r e p r e s e n t i n g prolonged mor-phine treatment (see Study I l l b - Amphetamine A f t e r Chronic Morphine) ano-ther group d i s s o c i a t i o n became evident, whereby the DB-6-0HDA-morphine group demonstrated s i g n i f i c a n t l y l e s s locomotor a c t i v i t y than the v e h i c l e -morphine group, which i n t u r n demonstrated s i g n i f i c a n t l y l e s s a c t i v i t y than the DB-6-OHDA-saline and v e h i c l e - s a l i n e groups. In reviewing these f i n d i n g s , i t appears that chronic morphine t r e a t e d animals d i s p l a y e d s i g n i f i c a n t l y lower l e v e l s of locomotor a c t i v i t y during the h a b i t u a t i o n phase than the s a l i n e t r e a t e d animals. In a d d i t i o n , the DB-6-0HDA l e s i o n p o t e n t i a t e d t h i s trend towards suppression of locomotor a c t i v i t y of the morphine t r e a t e d groups during the h a b i t u a t i o n p e r i o d . Perhaps t h i s behaviour can be a t t r i b u t e d to a withdrawal e f f e c t , inasmuch as the 1 hour p r e - i n j e c t i o n h a b i t u a t i o n p e r i o d represents the f i n a l stages 86 of the 24 hour i n t e r v a l between i n j e c t i o n s . This i s c o n s i s t e n t w i t h the r e s u l t s of the i r r i t a b i l i t y data, wherein animals of the vehicle-morphine and DB-6-0HDA-morphine groups demonstrated s i g n i f i c a n t i r r i t a b i l i t y on handling and i n j e c t i o n during the course of the experiment, w i t h an en-hanced e f f e c t i n the DB-6-0HDA-morphine group (see Study H a ; F r i e d l e r et a l , 1972). Taken together, these data suggest that both groups were undergoing withdrawal, w i t h the DB-6-0HDA-morphine group showing a somewhat more se-vere r e a c t i o n . I t should be noted, however, that the f i n d i n g of increased withdrawal, w i t h the DB-6-0HDA-morphine group was hot reproduced when c o n s i -d e r a t i o n was made of other i n d i c e s , namely, naltrexone p r e c i p i t a t e d w i t h -drawal. Some explanation f o r these discrepant f i n d i n g s can be sought from c o n s i d e r a t i o n of the schedules employed f o r the i n d u c t i o n of dependence. The i r r i t a b i l i t y and h a b i t u a t i o n data were used as i n d i c e s of dependence i n animals that received once d a i l y i n j e c t i o n s of a dose of 25 mg/kg morphine, whereas animals who underwent n a l t r e x o n e - p r e c i p i t a t e d withdrawal, r e c e i v e d steady dose-increments, u n t i l the d a i l y dose reached 200 mg/kg. Perhaps i n -c o n s i s t e n c i e s i n behavioural f i n d i n g s can be explained by these d i f f e r e n c e s i n the morphine i n j e c t i o n schedule p r i o r to withdrawal t e s t i n g , and there-f o r e r e f l e c t d i f f e r e n t degrees of p h y s i c a l dependence. In e f f e c t , a schedule of once d a i l y i n j e c t i o n s of a s t a b l e dose of 25 mg/kg f o r a 24 day p e r i o d , may be s u f f i c i e n t f o r t o l e r a n c e development, but r e f l e c t only a s l i g h t degree of p h y s i c a l dependence. Furthermore, although the data are not presented, animals that underwent t h i s schedule of i n j e c t i o n , e x h i b i t e d no signs of withdrawal when challenged w i t h naloxone, again i n d i c a t i n g that the animals responded to only some t e s t s of withdrawal. Conversely, the second i n j e c t i o n schedule which c o n s i s t e d of a r a p i d i n d u c t i o n procedure may have r e s u l t e d i n h i g h l y dependent animals. The n o n s i g n i f i c a n t group- d i f f e r e n c e s may t h e r e f o r e be a t t r i b u t e d to a ' c e i l i n g e f f e c t ' . 87 I t seems p o s s i b l e that the b e h a v i o u r a l i n d i c e s of p h y s i c a l dependence may be a f f e c t e d by the drug a d m i n i s t r a t i o n procedure, and that c e r t a i n beha-v i o u r a l measures ( i . e . h a b i t u a t i o n and i r r i t a b i l i t y data) may c o r r e l a t e w i t h the e a r l y phases i n the development of p h y s i c a l dependence, whereas, a n t a g o n i s t - p r e c i p i t a t e d withdrawal- may be a b e t t e r index of a h i g h l y depen-dent s t a t e . P h y s i c a l dependence should perhaps be viewed as a n o n - s t a t i c process and c o n s i d e r a t i o n should be given to i t s dynamic p r o p e r t i e s when u t i l i z i n g i t s behavioural i n d i c e s . I t i s t h e r e f o r e suggested, that f u r t h e r research i s necessary to d e f i n e the p r o p e r t i e s of p h y s i c a l dependence as w e l l as design behavioural measures that adequately c h a r a c t e r i z e i t s develop-ment, before the r o l e of the catecholamine systems can be adequately assessed. I t i s a l s o noteworthy that d e p l e t i o n of f o r e b r a i n NA by a 6-OHDA l e s i o n a f f e c t e d tolerance development i n r e l a t i o n to locomotor a c t i v i t y , but was without e f f e c t when co n s i d e r i n g c l a s s i c a l symptoms of withdrawal. Several explanations can be o f f e r e d f o r a l e s i o n e f f e c t on t o l e r a n c e , but not p h y s i -c a l dependence. F i r s t l y , withdrawal t e s t i n g i s used as an index of p h y s i c a l dependence and the r e l a t i o n of t o l e r a n c e to withdrawal i s not c l e a r l y under-stood, nor are there b e h a v i o u r a l t e s t s a v a i l a b l e that c l e a r l y d e l i n e a t e t h e i r r e s p e c t i v e p r o p e r t i e s . Recently, Mucha et a l (1979) attempted to evaluate the r e l a t i o n of t o l e r a n c e to p h y s i c a l dependence by measuring withdrawal responses and r e l a -t i n g them to t o l e r a n c e development i n r e l a t i o n to t a i l f l i c k a n a l g e s i a . I t was reported that only some withdrawal signs c o r r e l a t e w e l l w i t h t o l e r a n c e measurement and t h e r e f o r e i t was concluded that the choice of responses to measure tolerance and dependence determines whether the phenomena are r e l a -ted. I t . i s p o s s i b l e t h e r e f o r e , that our choice of b e h a v i o u r a l t e s t s were res p o n s i b l e f o r our i n c o n s i s t e n t f i n d i n g s . 88 Secondly, i t i s p o s s i b l e that p h y s i c a l dependence and t o l e r a n c e are, i n f a c t , somewhat independent yet r e l a t e d phenomena and that a noradrenergic system may be i n v o l v e d i n one process but not the other. At t h i s time, t h e r e f o r e , i t appears that f u r t h e r i n v e s t i g a t i o n i s necessary to evaluate the r e l a t i o n of t o l e r a n c e and p h y s i c a l dependence and t h e i r r e s p e c t i v e u n d e r l y i n g mechanisms of a c t i o n . L a s t l y , the i n t e r a c t i o n of amphetamine and morphine w i t h the DA system was assessed by studying the behavioural e f f e c t s of amphetamine i n animals f o l l o w i n g e i t h e r acute or chronic morphine treatment. 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L i f e Sciences 24, 1637-1644. 96 APPENDIX I Summary of A n a l y s i s of Variance f o r P o s t - I n j e c t i o n Data f o r Days 1,7 and 16 of Drug I n j e c t i o n df SS F P v a r i a b l e 1 (0-20 minutes) Groups 3 620707.8 2.32 P< .01 Subjects 30 2673663.62 T r i a l s 2 2591391.82 25.20 P< .001 T r i a l s x Groups 6 2834717.05 9.19 P< .001 T r i a l s x Subjects 55 2828282.74 v a r i a b l e 2 (20-40 minutes) Groups 3 311951.40 1.49 a ns Subjects 30 2093091.67 T r i a l s 2 1356526.61 16.04 P< .001 T r i a l s x Groups 6 1042320.75 4.11 P< .001 T r i a l s x Subjects 55 2324462.47 v a r i a b l e 3 (40-60 minutes) Groups 3 1677314.76 7.08 a ns Subj ects 30 2369349.80 T r i a l s 2 2003864.43 20.56 P< .001 T r i a l s x Groups 6 1896825.99 6.49 P< .001 T r i a l s x Subjects 55 2680505.07 v a r i a b l e 4 (60-80 minutes) Groups 3 3241853.34 21.26 P< .001 Subjects 30 1524948.51 T r i a l s 2 2296370.11 18.93 P< .001 T r i a l s x Groups 6 2736636.83 7.52 P< .001 T r i a l s x Subj ects 55 3335353.39 v a r i a b l e 5 (80-100 minutes) Groups 3 5341340.59 44.64 P< .001 Subjects 30 1196556.56 T r i a l s 2 3095094.91 58.94 P< .001 T r i a l s x Groups 6 3280828.94 20.82 P< .001 T r i a l s x Subjects 55 1444208.82 v a r i a b l e 6 (100-120 minutes) Groups 3 5310304.06 43.18 P< .001 Subjects 30 1229677.85 T r i a l s 2 2228487.28 34.69 V< .001 T r i a l s x Groups 6 2209702.56 11.47 P< .001 T r i a l s x Subjects 55 1766420.99 Cont'd... 97 Appendix I - cont'd df SS I v a r i a b l e 7 (120-140 minutes) Groups 3 7023907.77 56. 78 Subjects 30 1237025.37 T r i a l s 2 .1433510.26 35. 43 T r i a l s x Groups 6 1276187.81 10. 51 T r i a l s x Subjects 55 1112581,43 v a r i a b l e 8 (140-160 minutes) Groups 3 7626961.19 99. 80 Subjects 30 764246.26 T r i a l s 2 844191.14 12. 86 T r i a l s x Groups 6 407070.30 2. 07 T r i a l s x Subjects 55 1804817.57 v a r i a b l e 9 (160-180 minutes) Groups 3 6960158.30 79. 62 Subjects 30 874121.82 T r i a l s 2 444568.84 11. 34 T r i a l s x Groups 6 215171.31 1. 83 T r i a l s x Subj ects 55 1078048.19 p< .001 p< .001 p< .001 .001 p< .001 a ns .001 p< .001 ns a ns = not s i g n i f i c a n t 98 APPENDIX I I Summary of A n a l y s i s of Variance f o r Withdrawal - Counted Signs df SS v a r i a b l e 1 (0-10 minutes) Groups Subj ects T r i a l s T r i a l s x Groups T r i a l s x Subjects Groups Subj ects T r i a l s T r i a l s x Groups T r i a l s x Subjects Groups Subjects T r i a l s T r i a l s x Groups T r i a l s x Subjects 1 12 2 2 24 245.38 1318.27 3813.90 44.05 1877.38 2.23 24.38 .28 v a r i a b l e 2 (10-20 minutes) 1 12 2 2 24 386.79 2840.67 2205.19 101.97 1769.51 1.63 14.95 .69 v a r i a b l e 3 (30-30 minutes) 1 12 2 2 24 3.47 479.60 639.57 1.58 722.84 0.09 10.62 .03 ns p< .001 a ns ns p< .001 a ns ns p< .001 ns ns = not s i g n i f i c a n t 

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