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The dorsal tegmental noradrenergic projection : an analysis of its role in learning Roberts, David Charles Stephen 1976

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THE DORSAL TEGMENTAL NORADRENERGIC PROJECTION: AN ANALYSIS OF ITS ROLE IN LEARNING by DAVID CHARLES STEPHEN ROBERTS B.Sc. (Hons.) University of V i c t o r i a , 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Division of Neurological Sciences) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1976 © David C. S. Roberts, 1976. In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree at 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 , I a g r ee t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree 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 pu rpo se s may be g r a n t e d by the Head o f my Department 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 not 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 . Department o f f a rfi, p JLe y ) The U n i v e r s i t y o f B r i t i s h Co l umb i a 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 ABSTRACT THE DORSAL TEGMENTAL NORADRENERGIC PROJECTION: AN ANALYSIS OF ITS ROLE IN LEARNING David C. S. Roberts The hypothesis that the noradrenergic projection from the locus coeruleus (LC) to the cerebral cortex and hippocampus i s an important neural substrate for learning was evaluated. Maze performance was studied in rats receiving either e l e c t r o l y t i c lesions of the LC, or 6-hydroxydopamine (6-0HDA) inj e c t i o n s i n -to the region of the dorsal tegmental noradrenergic projection. In contrast to the res u l t s of an e a r l i e r report (Anlezark, Crow, and Greenway, 1973), LC lesions did not disrupt the acq u i s i t i o n of a running response for food reinforcement i n an L-shaped run-way, even though hippocampal-cortical noradrenaline (NA) was re-duced to 29%. Greater telencephalic NA depletions (to 6 percent of control l e v e l s ) produced by 6-0HDA also f a i l e d to disrupt the acquisi t i o n of th i s behaviour or impair the acquisition of a food reinforced position habit i n a T-maze. Neither locomotor a c t i v i t y nor habituation to a novel environment was affected by the 6-0HDA lesions. Rats with such lesions were, however, „ found to be s i g n i f i c a n t l y more d i s t r a c t i b l e than controls during the performance of a previously trained response. In another group of rats with i d e n t i c a l 6-OHDA inj e c t i o n s , the esta b l i s h -ment of a lithium chloride-induced conditioned taste aversion was not affected by the lesions. The hypothesis that telencephalic NA i s of fundamental importance in learning was not supported. - i i i -TABLE OF CONTENTS LIST OF FIGURES iv INTRODUCTION Local i z a t i o n and Metabolism of CA in the Central Nervous System 1 Theoretical Consideration of Noradrenaline Involvement in Learning 4 GENERAL METHOD Subjects 10 Treatments 10 Noradrenaline Assay 11 S t a t i s t i c s 14 EXPERIMENT 1: THE EFFECT OF TELENCEPHALIC NA DEPLETION ON THE ACQUISITION OF AN L-MAZE RUNNING RESPONSE 1 4 EXPERIMENT 2: THE EFFECT OF TELENCEPHALIC NA DEPLETION ON THE ACQUI-SITION AND REVERSAL OF A T-MAZE POSITION HABIT 25 EXPERIMENT 3: THE EFFECT OF TELENCEPHALIC NA DEPLETION ON EXPLORATORY LOCOMOTOR ACTIVITY 28 EXPERIMENT 4: THE EFFECT OF TELENCEPHALIC NA DEPLETION ON THE D.IS-TRACTIBILITY OF RATS DURING THE PERFORMANCE OF A TRAINED RESPONSE 3 3 EXPERIMENT 5: THE EFFECT OF TELENCEPHALIC NA DEPLETION ON THE ACQUISITION OF A CONDITIONED TASTE AVERSION 3 8 GENERAL DISCUSSION 44 CONCLUSIONS 50 REFERENCES . 5 1 CURRICULUM VITAE 55 - iv -LIST OF FIGURES FIGURE 1 Photomicrograph of a b i l a t e r a l e l e c t r o l y t i c lesion of the nucleus locus coeruleus FIGURE 2 Effec t of telencephalic NA depletion on the acquisition of an L-maze running response 20 FIGURE 3 Eff e c t of telencephalic NA depletion on the acquisition of a T-maze position habit 26 FIGURE 4 Effect of 6-OHDA injections into the region of the dorsal NA bundle on locomotor a c t i v i t y 30 FIGURE 5 Effect of 6-OHDA injec t i o n s into the region of the dorsal NA bundle on the running times i n an L-maze in teh presence of noval st i m u l i 35 FIGURE 6 Eff e c t of 6-OHDA injec t i o n s into the region of the dorsal NA bundle on the acquisition of a conditioned taste aversion to saccharin 41 ACKNOWLEDGEMENTS I would l i k e to express my appreciation to those people who contributed enormously to t h i s project. I am deeply i n -debted to Dr. Marion Price for her assistance and support in c o l l e c t i n g a large part of the data reported here. I am also grateful to S t e l l a Atmadja for her help i n the preparation of experimental animals, and to Dr. Ron Clavier for photographic assistance. I would l i k e to thank Shiela Brooke for her major role i n the chemical analyses. Most of a l l , I would l i k e to thank my advisor, Dr. Chris Fibiger, for his u n f a i l i n g en-couragement and support. INTRODUCTION In 1953, Eejn d e s c r i b e d the " t r a n q u i l i z i n g " e f f e c t of ra u w o l f i a n a l k a l o i d s , and s p e c i f i c a l l y r e s e r p i n e . I t was noted that t h i s drug was capable of reducing m o t i l i t y of r a b b i t s without the b a r b i t u r a t e - l i k e s e d a t i o n and d i s e q u i -l i b r i u m . The b e h a v i o u r a l suppression was l a t e r l i n k e d to the a b i l i t y of r e s e r p i n e to deplete the neuronal s t o r e s of catecholamines (CAs) and s e r o t o n i n (5-HT) (Brodie et a l . , 1957). The f i n d i n g that small q u a n t i t i e s of DOPA, a metabolic p r e c u r s o r of dopamine (DA) and n o r a d r e n a l i n e (NA), could reverse the r e s e r p i n e - i n d u c e d h y p o m o t i l i t y suggested that CAswere important f o r t h i s a c t i o n of r e s e r p i n e . Other pharmacological manipulations of CA have s i n c e been shown to a f f e c t behaviour, and have sparked a widespread r e -search i n t e r e s t i n t o the f u n c t i o n a l importance of CAs i n the c e n t r a l nervous system (CNS) and t h e i r r e l a t i o n to v a r i o u s response p a t t e r n s . S p e c i f i c a l l y , a number of t h e o r i s t s have p o s t u l a t e d that CAs are necessary to the l e a r n i n g process and the formation of memory. The present r e s e a r c h r e p o r t i s an attempt to examine t h i s r e l a t i o n s h i p i n s e v e r a l l e a r n i n g s i t u a t i o n s . A short i n t r o d u c t o r y survey of the cu r r e n t s t a t e of knowledge concerning the l o c a l i z a t i o n and p h y s i o l o g y of CAs i n the CNS f o l l o w s . L o c a l i z a t i o n and Metabolism of CAs i n the C e n t r a l Nervous System. The presence of CAs i n the CNS was demonstrated b i o -- 2 -c h e m i c a l l y by Vogt (1954) f o r NA and C a r l s o n et a l . (1958) f o r DA. However, a c l e a r e r understanding of the d i s t r i b u t i o n and c e l l u l a r l o c a l i z a t i o n of these monoamines awaited the development of the formaldehyde h i s t o c h e m i c a l f l u o r e s c e n c e technique introduced by F a l c k and H i l l a r p (1962). T h i s procedure allowed d i r e c t v i s u a l i z a t i o n of catecholamine -c o n t a i n i n g c e l l bodies and h i g h l y f l u o r e s c e n t v a r i o c o s i t e s of the presume nerve t e r m i n a l s . Since the axons of CA c o n t a i n i n g c e l l bodies c o n t a i n very low c o n c e n t r a t i o n s of amines, pharmacological and l e s i o n . t e c h n i q u e s were necessary to map out the p r o j e c t i o n s and pathways of these f i b e r systems. I t was found.that f l u o r e s c e n t v a r i o c o s i t e s accumulated proximal to the c e l l body at the s i t e of a l e s i o n , presumably due to orthograde axonal t r a n s p o r t of the t r a n s m i t t e r or i t s s y n t h e s i z i n g enzymes. By making hemisections at v a r i o u s l e v e l s of the b r a i n , the course of s p e c i f i c pathways could be e s t a b l i s h e d (e.g. Dahlstrom and Fuxe, 1964, 1965; Anden, et a l . , 1966). In 1971, Ungerstedt using t h i s procedure provided a s t e r e o t a x i c map of the known monoamine pathways. L i n d v a l l and B j o r k l u n d (1974 a and b) have s i n c e been able to provide a more d e t a i l e d d e s c r i p t i o n with the a i d of the s e n s i t i v e g l y o x y l i c a c i d f l u o r e s c e n c e method. These authors have i d e n t i f i e d f i v e d i s t i n c t NE f i b e r bundles. The c e n t r a l t egmental t r a c t ascends (but a l s o has descending components) from the medulla oblongata to the caudal diencephalon; c o n t r i b u t i n g f i b e r s o r g i n a t e from - 3 -A l , A 2 , A5, A7 and t h e l o c u s c o e r u l e u s (A6) ( D a h l s t r o m and F u x e , 1964 , n o m e n c l a t u r e ) . The d o r s a l t e g m e n t a l b u n d l e ( f o r m e r l y ' d o r s a l b u n d l e ' o f U n g e r s t e d t , 1971) o r i g i n a t e s s o l e l y i n t h e l o c u s c o e r u l e u s and p r o j e c t s p r i n c i p a l l y t o t h e t h a l a m u s , h i p p o c a m p u s and c o r t e x . The p e r i v e n t r i -c u l a r s y s t em c o n t a i n s two c o m p o n e n t s ; t h e d o r s a l component c o n t a i n s c e l l b o d i e s d i f f u s e l y d i s t r i b u t e d a l o n g . i t s e x t e n t f r o m pons t h r o u g h p o s t e r i o r t h a l a m u s . The v e n t r a l component o r i g i n a t e s i n t h e r o s t r a l m e s e n c e p h a l o n and i n t e r m i n g l e s i n t h e d o r s o m e d i a l h y p o t h a l a m i c n u c l e u s w i t h t h e d o r s a l component t o f o r m an a s c e n d i n g h y p o t h a l m i c CA s y s t e m . The m e d i a l , f o r e b r a i n b u n d l e s y s t e m i s an e x t e n s i o n o f t h e d o r s a l t e g m e n t a l b u n d l e and c e n t r a l t e g m e n t a l t r a c t . F i b e r s a r e s e e n t o i n n e r v a t e t h e c i n g u l u m , h y p o t h a l a m i c n u c l e i , geni-^ c u l a t e b o d i e s , s e ptum and o l f a c t o r y b u l b s among o t h e r f o r e b r a i n s t r u c t u r e s . Two m a j o r DA a s c e n d i n g s y s t e m s have b e e n d e s c r i b e d . The n i g r o - s t r i a t a l bund1e (NSB) o r i g i n a t e s i n t h e p a r s c o m p a c t a o f t h e s u b s t a n t i a n i g r a and t e r m i n a t e s t o p o g r a p h i c a l l y i n t h e c a u d a t e - p u t a m e n . The m e s o - l i m b i c s y s t e m has i t s c e l l s of o r i g i n d o r s o - l a t e r a l t o t h e i n t e r p e d u n c u l a r n u c l e u s , and p r o j e c t s t o t h e n u c l e u s a c c u m b e n s , t u b e r c u l u m o l f a c -t o r i u m and n u c l e u s i n t e r s t i t i a l i s s t r i a t e r m i n a l i s . DA t e r m i n a l s have a l s o b e e n i d e n t i f i e d i n t h e f r o n t a l c o r t e x , s eptum and a n t e r i o r l i m b i c c o r t e x . I n 1 939, B i a s c h k o p r o p o s e d t h e e n z y m a t i c s e q u e n c e i n - 4 -the b i o s y n t h e s i s of t h e c a t e c h o l a m i n e s w h i c h i s g e n e r a l l y a g r e e d upon a t p r e s e n t . The amino a c i d , t y r o s i n e , i s c o n v e r t e d to d i h y d r o x y p h e n y l a l a n i n e (DOPA) v i a t h e enzyme t y r o s i n e h y d r o x y l a s e . T h i s i s c o n s i d e r e d to be a r a t e l i m i t i n g s t e p i n t h e s y n t h e s i s of CA. The d i s t r i b u t i o n of t h i s enzyme r o u g h l y p a r a l l e l s t h e known DA and NE pathways w i t h h i g h l e v e l s f o u n d i n t e r m i n a l r e g i o n s . The s e c o n d enzyme i n t h e s y n t h e s i s i s DOPA d e c a r b o x y l a s e w h i c h y i e l d s dopamine. T h i s enzyme i s not s p e c i f i c to n e u r a l t i s s u e b ut i s p r e s e n t i n many p e r i p h e r a l o r g a n s (e.' g. k i d n e y , l i v e r ) . T h i s enzyme i s n o t s p e c i f i c t o DOPA, and may more p r o p e r l y be r e f e r r e d to as L - a r o m a t i c amino a c i d d e c a r b o x y l a s e . A t h i r d enzyme, d o p a m i n e - B - h y d r o x y l a s e i s n o t f o u n d i n DA n e u r o n s , b u t i s s p e c i f i c to a d r e n e r g i c and NA n e u r o n s . T h i s enzyme, w h i c h c o n v e r t s DA to NA, may a l s o be r a t e c o n t r o l l i n g . Two i m p o r t a n t enzymes have been shown to m e t a b o l i z e NA and DA. C a t e c h o l - O - m e t h y l t r a n s f e r a s e i s t h o u g h t to f u n c t i o n e x t r a n e u r o n a l l y . The o t h e r , monoamine o x i d a s e (MAO), i s presumed to f u n c t i o n i n t r a n e u r o n a l l y to m e t a b o l i z e CA not c o n f i n e d to s t o r a g e g r a n u l e s . MAO i s f o u n d e x t r a n e u r o n a l l y as w e l l . I n a c t i v a t i o n of CA i n the s y n p a t i c c l e f t i s not p r i m a r i l y v i a t h e s e m e t a b o l i c enzymes, however, but i s c o n s i d e r e d to t a k e p l a c e m a i n l y by a r e - u p t a k e p r o c e s s i n t o t h e p r e s y n a p t i c c e l l . T h e o r e t i c a l C o n s i d e r a t i o n of NA I n v o l v e m e n t i n L e a r n i n g S e v e r a l t h e o r i e s of l e a r n i n g i n v o l v e t h e e s t a b l i s h m e n t o f new n e u r a l c o n n e c t i o n s o r a t l e a s t a change i n t h e c o n d u c t i v i t y of e x i s t i n g s y n a p s e s ( R o b e r t s & M a t t h y s s e , 1970) T h e o r i s t s have most o f t e n p r o p o s e d t h a t t h i s p l a s t i c c hange t a k e s p l a c e a t t h e c o r t i c a l l e v e l and c o n s i s t s of a two s t a g e p r o c e s s . The f i r s t p h a s e i s of s h o r t d u r a t i o n and l a b i l e t o many t y p e s o f e x t e r n a l i n t e r f e r e n c e . T h i s p h a s e c o u l d i n v o l v e an e l e c t r i c a l r e v e r b e r a t i n g p a t t e r n w h i c h i s l a t e r c o n s o l i d a t e d by some p h y s i c a l c h a n g e i n t h e n e u r a l n e t w o r k . T h e o r e t i c a l r e f i n e m e n t s h a v e drawn on v a r i o u s m e chanisms w h i c h a l l o w s e l e c t i v e s t o r a g e of a d a p t i v e r e s p o n s e p a t t e r n s , w i t h t h e o b v i o u s a d v a n t a g e t h a t m a l a d a p t i v e o r u n r e i n f o r c e d b e h a v i o r s w o u l d n o t be as l i k e l y t o r e o c c u r . One s u c h m e c h a n i s m p r o p o s e d by Crow (1968) i s t e r m e d a " d i s c r i m i n a t o r " . T h i s i s a s y s t e m of f i b e r s w h i c h have a w i d e s p r e a d t e r m i n a l d i s t r i b u t i o n , and a r e a b l e t o a f f e c t a change i n s y n a p t i c c o n n e c t i o n s of t h o s e c i r c u i t s c a r r y i n g a c t i v i t y a s s o c i a t e d w i t h a b e n e f i c i a l r e s p o n s e . T h i s p r o -p o s e d r e w a r d s y s t e m i s a n e u r o p h y s i o l o g i c a l a n a l o g u e o f S k i n n e r i a n r e i n f o r c e m e n t , s i n c e i t i n c r e a s e s t h e p r o b a b i l i t y o f r e o c c u r r e n c e o f t h a t p a r t i c u l a r n e u r a l r e s p o n s e w h i c h l e d to t h e a c t i v a t i o n o f t h e r e w a r d s y s t e m . Crow and A r b u t h n o t t ( 1 9 7 2) f u r t h e r s p e c u l a t e d on t h e i d e n t i f y o f s u c h a pa t h w a y . On t h e b a s i s of a n a t o m i c a l and b e h a v i o r a l e v i d e n c e , t h e s e a u t h o r s p r o p o s e d t h a t n o r a d r e -n e r g i c , n e u r o n s f r o m t h e n u c l e u s l o c u s c o e r u l e u s , w i t h i t s - 6 -a s s o c i a t e d c o r t i c a l d i s t r i b u t i o n , may f o r m t h e n e u r a l b a s i s o f r e i n f o r c e m e n t . The l o c u s c o e r u l e u s has t h e c h a r a c t e r i s t i c s o f a v i s c e r a l a f f e r e n t n u c l e u s ( R u s s e l l , 1955) and may r e c e i v e i n f o r m a t i o n f r o m g u s t a t o r y r e c e p -t o r s . The presumed a c t i v a t i o n o f t h i s n u c l e u s d u r i n g f e e d -i n g f i t s w e l l w i t h a t h e o r y o f e n v i r o n m e n t a l a c t i v a t i o n of a n e u r a l r e w a r d p a t h w a y . T h i s n u c l e u s a l s o p o s s e s s e s t h e r e q u i r e d c o r t i c a l t e r m i n a l d i s t r i b u t i o n w h i c h i s p r e d i c t e d by t h i s t h e o r y . A d d i t i o n a l s u p p o r t f o r t h e i m p l i c a t i o n o f t h i s n u c l e u s b e i n g i n v o l v e d i n a c o r t i c a l r e w a r d s y s t e m i s t h e d e m o n s t r a t i o n t h a t t h i s n u c l e u s w i l l s u p p o r t i n t r a -c r a n i a l s e l f - s t i m u l a t i o n ( R i t t e r and S t e i n , 1 9 7 3 ) . K e t y ( 1 9 7 0 , 1972) has a l s o p r o p o s e d a l e a r n i n g r o l e f o r t h e a d r e n e r g i c s y s t e m s o f t h e c o r t e x . T h i s t h e o r y i s much l i k e t h a t p u t f o r w a r d by Crow i n t h a t t h e r e l e a s e o f NA " c o n s o l i d a t e s " n e u r a l a s s o c i a t i o n s . I n d i r e c t s u p p o r t f o r t h e h y p o t h e s i z e d r o l e o f t h e c o r t i c a l NA p r o j e c t i o n i n l e a r n i n g t a k e s s e v e r a l f o r m s . P h a r m a c o l o g i c a l m a n i p u l a t i o n s o f CA s y n t h e s i s have l o n g been known t o d i s r u p t l e a r n e d r e s p o n s e s . An a g e n t w h i c h i n h i b i t s t h e enzyme t y r o s i n e h y d r o x y l a s e , a - m e t h y 1 - p a r a - t y r o s i n e , m a r k e d l y i n t e r f e r e s w i t h t h e p e r f o r m a n c e o f a c o n d i t i o n e d a v o i d a n c e r e s p o n s e (CAR) ( R e c h , B o r y s and M o o r e , 1 9 6 6 ) . S i n c e t h e p e r f o r m a n c e o f t h e l e a r n e d r e s p o n s e i s i m p a i r e d , i t i s n o t s u r p r i s i n g t o f i n d t h a t t h i s a g e n t a l s o d i s r u p t s t h e a c q u i s i t i o n o f t h e CAR ( E s s m a n , c i t e d by K e t y , 1 9 7 0 ) . - 7 -I n h i b i t i o n o f t h e enzyme dopamine — 3— h y d r o x y l a s e w i t h d i e t h y l d i t h i o c a r b a m a t e (DDC) has been shown t o d i s r u p t t h e f o r m a t i o n of a p a s s i v e a v o i d a n c e r e s p o n s e i n m i c e ( R a n d t , Q u a r t e r m a n , G o l d s t e i n and A n a g n o s t e , 1971) and r a t s ( S t e i n , B e l l u z z i and W i s e , 1975). The f a c t t h a t t h i s e f f e c t o f DDC c a n be r e v e r s e d w i t h i n t r a v e n t r i c u l a r a d m i n i s -t r a t i o n o f NA ( S t e i n e t a l . , 1975) f u r t h e r i n d i c a t e s t h a t NA s y n t h e s i s p l a y s an i m p o r t a n t r o l e i n memory f u n c t i o n . R e c o v e r y f r o m t h e a m n e s i a p r o d u c e d by DDC has been r e p o r t e d t o o c c u r by a d m i n i s t r a t i o n o f e i t h e r of t h e monoamine o x i d a s e i n h i b i t o r s ( M A O I ) , " C a t r o n " o r " P a r g y l i n e " ( B o t w i n i c k and Q u a r t e r m a i n , 1974) b e f o r e t h e r e t e s t . T h e s e d a t a s u g g e s t t h e p o s s i b i l i t y t h a t w h i l e memory may be s t o r e d u n d e r t h e DDC d r u g c o n d i t i o n , f u t u r e r e c a l l i s i m p a i r e d . T h i s f a c i l i t a t i o n o f r e c a l l by MAOI i s n o t s p e c i f i c t o a m n e s i a p r o d u c e d by DDC s i n c e an MAOI a l s o a t t e n u a t e s a m n e s i a i n a n i m a l s t r e a t e d w i t h a c e t o x y c y c l o h e x i m i d e (AXM), a p r o t e i n s y n t h e s i s i n h i b i t o r ( B o t w i n i c k and Q u a r t e r m a i n , 1974). W h i l e t h e p h a r m a c o l o g i c a l d a t a f r o m p e r i p h e r a l l y a d m i n i -s t e r e d d r u g s i m p l i c a t e NA i n t h e a c q u i s i t i o n o f l e a r n e d r e s p o n s e s , c l e a r l y c e n t r a l m a n i p u l a t i o n s a r e r e q u i r e d t o i d e n t i f y w h a t , i f a n y , s p e c i f i c NA p a t h w a y s a r e i n v o l v e d . S e v e r a l s t u d i e s h a v e e m p l o y e d t h e n e u r o t o x i c a g e n t 6 - h y d r o x y d o p a m i n e (6-OHDA) i n an a t t e m p t t o ex a m i n e CA i n v o l v e m e n t i n t h e a c q u i s i t i o n o f f o o d - r e w a r d e d b e h a v i o r s . T h i s a g e n t has b e e n shown t o p r o d u c e s e l e c t i v e d e s t r u c t i o n o f - 8 -CA n e r v e e n d i n g s and c e l l b o d i e s i n t h e CNS ( B l o o m e t a l . , 1969)'. C A - n e u r o n s a c c u m u l a t e CA ( I v e r s e n , 1 9 7 1 ) . One a n a l o g u e , 6-OHDA, i s a l s o a c t i v e l y t a k e n up by t h e s e c e l l s and i s t h o u g h t t o c a u s e n e u r o n a l damage v i a one of t h e o x i d a t i o n p r o d u c t s , h y d r o g e n p e r o x i d e ( H e i k k i l a and Cohen, 1 9 7 2 ) . C h o l i n e r g i c , s e r o t o n e r g i c and g a b a n e r g i c n e u r o n s a r e n o t damaged when 6-OHDA i s u s e d i n a p p r o p r i a t e d o s a g e s ( U r e t s k y and I v e r s e n , 1 9 7 0 ) ; McGeer e t a l . , 1 9 7 3 ) ; h o w e v e r , some n o n - s p e c i f i c damage does o c c u r a t t h e s i t e o f i n j e c t i o n s ( H o k f e l t and U n g e r s t e d t , 1 9 7 3 ) . Howard, G r a n t and B r e e s e (1974) a d m i n i s t e r e d 6-OHDA i n t r a c i s t e r n a l l y and s t u d i e d t h e e f f e c t s o f s u c h t r e a t m e n t i n r a t s on t h e a c q u i s i t i o n and p e r f o r m a n c e o f a d o u b l e T-maze. E a c h a n i m a l was r e q u i r e d t o c h o o s e b e t w e e n two l e v e r s a t ea c h end of a runway. D e p r e s s i o n o f one l e v e r w o u l d d e l i v e r a f o o d p e l l e t t w i c e , a f t e r w h i c h t h e a n i m a l was r e q u i r e d t o t r a v e r s e t h e runway and c h o o s e t h e c o r r e c t l e v e r a t t h e o t h e r end t o o b t a i n f u r t h e r f o o d . The number of r e w a r d e d r e s p o n s e s ( l e v e r p r e s s e s ) i n e a c h d a i l y t e s t s e s s i o n was t h e d e p e n d a n t v a r i a b l e . 6-OHDA t r e a t m e n t p r o d u c e d s i g n i f i c a n t d e c r e m e n t s i n t h e r e s p o n s e r a t e i n r a t s w h i c h had p r e v i o u s l y b een t r a i n e d , and t h i s r e d u c t i o n was c l e a r l y r e l a t e d t o t h e d e p l e t i o n of CAs. 6-OHDA t r e a t -ment p r i o r t o t r a i n i n g a l s o p r o d u c e d a p r o f o u n d i m p a i r m e n t i n t h e a c q u i s i t i o n o f t h e b a r p r e s s i n g r e s p o n s e . A n a l y s i s of t h e w h o l e b r a i n c o n t e n t o f NA and DA l e d t h e a u t h o r s - 9 -to c o n c l u d e t h a t t h e behav i o u r a l d e f i c i t s "were more r e l a t e d t o t h e r e d u c t i o n i n dopamine t h a n t h e y were t o t h e d e p l e t i o n o f b r a i n n o r e p i n e p h r i n e ( p . 9 9 5 ) . " Mason and I v e r s e n (1974) e m p l o y e d t h e i n t r a v e n t r i c u l a r r o u t e o f 6-OHDA a d m i n i s t r a t i o n t o r e d u c e w h o l e b r a i n t y r o -s i n e h y d r o x y l a s e a c t i v i t y t o 8, 16, and 1 5 % i n t h e s t r i a t u m , h y p o t h a l a m u s and c o r t e x r e s p e c t i v e l y . A n i m a l s w i t h s u c h l e s i o n s w e r e f o u n d t o be i m p a i r e d on a c o m p l i c a t e d p r o b l e m s o l v i n g t a s k . The r a t was r e q u i r e d t o e i t h e r p u s h o r p u l l a b a l l t h r o u g h a t u n n e l t o g a i n a c c e s s t o a f o o d r e w a r d . 6-OHDA t r e a t e d a n i m a l s r e q u i r e d more t r i a l s t o l e a r n t h e r e s p o n s e t h a n d i d c o n t r o l s . The a u t h o r s a r g u e d t h a t m o t i -v a t i o n a l , m o t o r o r c o - o r d i n a t i o n a l d e f i c i t s were n o t i n v o l v e d , b u t t h a t t h e d e f i c i t i n a c q u i s i t i o n o f t h e t a s k d e m o n s t r a t e d t h a t CAs were i m p o r t a n t t o t h e l e a r n i n g p r o c e s s . A t t e m p t s t o t e s t t h e i n v o l v e m e n t o f s p e c i f i c NA p a t h -ways i n t h e a c q u i s i t i o n o f a p p e t i t i v e l y m o t i v a t e d l e a r n i n g t a s k s have y i e l d e d c o n t r a d i c t o r y r e s u l t s . A n l e z a r k , Crow and Greenway (1973) r e p o r t e d t h a t e l e c t r o l y t i c l e s i o n s t o t h e l o c u s c o e r u l e u s ( L C ) , w h i c h d e p l e t e d c o r t i c a l NA, a l s o a b o l i s h e d t h e a b i l i t y to a c q u i r e a r u n n i n g r e s p o n s e i n an L - s h a p e d runway. A t t e m p t s t o r e p l i c a t e t h i s f i n d i n g h ave f a i l e d . A m a r a l and F o s s (1975) r e p o r t e d t h a t e l e c t r o l y t i c l e s i o n s t o t h e LC w h i c h p r o d u c e d e q u a l o r g r e a t e r d e p l e t i o n s of h i p p o c a m p a l - c o r t i c a l NA f a i l e d t o p r o d u c e d e f i c i t s i n - 10 -a c q u i s i t i o n i n e i t h e r " T " - o r " L " - s h a p e d mazes. The p r e s e n t i n v e s t i g a t i o n r e p r e s e n t s an a t t e m p t t o d e t e r m i n e t h e n a t u r e o f t h e i n v o l v e m e n t of t h e NA pathway f r o m t h e LC t o t h e c o r t e x . We ha v e shown ( R o b e r t s e t a l . , 1975) t h a t e x t e n s i v e d e p l e t i o n s o f c o r t i c a l NA c a n be a c h i e v e d t h r o u g h i n t r a c e r e b r a l i n j e c t i o n s o f 6-OHDA i n t h e v i c i n i t y of t h e d o r s a l t e g m e n t a l NA b u n d l e ( L i n d v a l l and B j o r k l u n d , 1 9 7 4 ) . S i n c e t h i s method i s s e l e c t i v e and more e f f e c t i v e t h a n e l e c t r o l y t i c l e s i o n s i n d e p l e t i n g f o r e b r a i n NA, 6-OHDA l e s i o n s w e r e u t i l i z e d t o examine f u r t h e r t h e i n v o l v e m e n t of f o r e b r a i n NA i n t h e a c q u i s i t i o n o f some l e a r n e d b e h a v i o r s . GENERAL METHOD S u b j e c t s M a l e W i s t a r r a t s ( W o o d l y n F a r m s , G u e l p h , O n t a r i o ) , w e i g h i n g 290-320 gm, were u s e d i n a l l e x p e r i m e n t s . A l l a n i m a l s were i n d i v i d u a l l y h o u s e d i n s t a i n l e s s s t e e l w i r e c a g e s and r e c e i v e d f o o d and w a t e r jad 1 i b i t u r n f o r t h r e e weeks p o s t o p e r a t i v e l y . T r e a t m e n t s A l l r a t s w e r e a n a e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l (50 mg/kg) and p r e p a r e d f o r s u r g e r y i n a K o p f s t e r e o t a x i c a p p a r a t u s . I n one g r o u p (LC) , b i l a t e r a l l e s i o n s of t h e n u c l e u s l o c u s c o e r u l e u s w e re a t t e m p t e d by p a s s i n g 1mA f o r - 11 -30 s e c t h r o u g h t h e b a r e d t i p o f an e l e c t r o d e , f a s h i o n e d f r o m two t w i s t e d s t r a n d s o f .277-mm-diameter n i c h r o m e w i r e . The c o - o r d i n a t e s a c c o r d i n g t o F i f k o v a and M a r s a l a (1967) were AP 8.5; ML + 1.2; DV - 6.5 mm. A n o t h e r g r o u p of r a t s (NA-6-0HDA) r e c e i v e d i n j e c t i o n s o f 6-OHDA i n t h e d o r s a l t e g m e n t a l NA b u n d l e . B i l a t e r a l i n j e c t i o n s o f 4 ug/2 y l / 1 0 min of 6-OHDA h y d r o b r o m i d e ( d o s e e x p r e s s e d as t h e f r e e b a s e ) i n 0.9% s a l i n e c o n t a i n i n g a s c o r b i c a c i d ( 0 .2 mg/ml) were i n f u s e d t h r o u g h a 34-gauge n e e d l e . C o - o r d i n a t e s , a c c o r d i n g t o K o n i g and K l i p p e l ( 1 9 6 3 ) , were AP + 2.6 mm: ML + 1.1 mm; DV + 3.7 mm. C o n t r o l a n i m a l s r e c e i v e d b u r r h o l e s t h r o u g h t h e s k u l l , w h i l e some a d d i t i o n a l a n i m a l s were u n o p e r a t e d . N o r a d r e n a l i n e A s s a y E x t r a c t i o n o f c a t e c h o l a m i n e s . F o l l o w i n g t h e c o m p l e t i o n of t h e b e h a v i o r a l m e a s u r e s , t h e a n i m a l s were k i l l e d by c e r v i c a l f r a c t u r e and t h e b r a i n s q u i c k l y removed. The h i p p o c a m p u s and c e r e b r a l c o r t e x were d i s s e c t e d o u t on i c e and c o m b i n e d . The t i s s u e was w e i g h e d , t h e n h o m o g e n i z e d i n 5 ml p e r c h l o r i c - a c e t i c a c i d . The h o m o g e n i z i n g t u b e was r i n s e d w i t h a f u r t h e r 2 ml of a c i d w h i c h was added t o t h e homogenate. T h i s was a l l o w e d t o s t a n d i n t h e c o l d f o r 1/2 h o u r and was t h e n c e n t r i f u g e d a t low s p e e d . The c l e a r s u p e r n a t a n t was d e c a n t e d . The r e s i d u e was r e s u s p e n d e d , c e n t r i f u g e d , and t h e s u p e r n a t a n t d e c a n t e d , t w i c e more. - 12 -To t h e c o m b i n e d s u p e r n a t a n t s was added 0.5 m l , 0.1 M e t h y l e n e d i a m i n e t e t r a c e t i c a c i d (EDTA). A t t h i s s t a g e t h e s a m p l e s w ere u s u a l l y f r o z e n o v e r n i g h t . An a l u m i n a c o l u m n was p r e p a r e d as f o l l o w s . A g l a s s t u b e 20 cm i n l e n g t h was f a s h i o n e d so t h a t t h e u p p e r p o r t i o n of t h e t u b e c o n s i s t e d of a 2 cm d i a m e t e r r e s e r v o i r of a b o u t 15 ml c a p a c i t y . The l o w e r p o r t i o n was a 4 mm d i a m e t e r s h a f t , d rawn o u t t o a f i n e t i p and p l u g g e d w i t h g l a s s w o o l . To t h e t i s s u e e x t r a c t was added 1 ml EDTA and 1.5 ml p o t a s s i u m p h o s p h a t e (.35 M), and t h e pH a d j u s t e d t o 9.2-9.4. A c o n s i s t e n t m e a s u r e of a l u m i n a ( a b o u t 0.4 gm) was added and s t i r r e d f o r 3 m i n u t e s . T h i s s l u r r y was p o u r e d t h r o u g h t h e g l a s s t u b e , t h e f l o w t h r o u g h w h i c h was a d j u s t e d by a p p l i c a t i o n of g e n t l e s u c t i o n . The a l u m i n a was washed w i t h 25-30 m i s of d i s t i l l e d w a t e r . The c a t e c h o l a m i n e s were e l u t e d w i t h 0.5 ml a c e t i c a c i d ( 0 . 5 N ) . F o r m a t i o n and e s t i m a t i o n of f l u o r e s c e n t d e r i v a t i v e s . To t h e 0.5 ml of e l u a n t was added 0.5 m l of 1 M s o d i u m a c e t a t e b u f f e r (pH6) and t h e pH a d j u s t e d t o 5.7-6.0 w i t h 0.5N NaOH. E a c h s a m p l e was t h e n b r o u g h t t o a v o l u m e o f 3.0 ml w i t h d i s t i l l e d H 2 0 . I n t o e a c h of two t e s t t u b e s was p l a c e d 0.5 ml o f t h e s a m p l e s o l u t i o n , and 0.5 m l , 0.5 M s o d i u m a c e t a t e b u f f e r , pH 6.4 was a d d e d , f o l l o w e d by 0.5 ml i o d i n e s o l u t i o n (0.254 gm I + 5. 0 gm K l / 2 2 7 Hil H 2 0 ) . The m i x t u r e was s h a k e n and a l l o w e d t o s t a n d . A f t e r 10 m i n 0.25 ml s o d i u m t h i o s u l p h a t e ( 0 . 5 M) was m i x e d i n . To one - 13 -of t h e two t u b e s was added 0.5 ml of a c o m b i n a t i o n o f a s c o r b i c a c i d (5 mg/ml) and 5N NaOH i n a r a t i o o f 3:7. To t h e s e c o n d t u b e was added 0.35 ml of 5N NaOH o n l y . The s a m p l e s w ere a l l o w e d t o s t a n d a t room t e m p e r a t u r e , u n d e r f l u o r e s c e n t l i g h t i n g f o r 90 - 120 m i n . To t h e s e c o n d t u b e was added 0.15 ml a s c o r b i c a c i d (5 mg/ml); t h i s now c o n s t i t u t e d t h e " f a d e d b l a n k " . A l l s a m p l e s w ere r e a d i n a s p e c t r o p h o t o f l u o r o m e t e r . The a c t i v a t i o n peak was s e t a t 395 nm and t h e e x c i t a t i o n peak was 505 nm. B o t h c o l u m n and a s s a y s t a n d a r d s w e r e e m p l o y e d t o d e t e r m i n e t h e p e r c e n t r e c o v e r y o f am i n e s o f t h e c o l u m n , and r e l a t i o n s h i p o f t h e f l u o r o m e t e r r e a d i n g t o amount o f n o r a d r e n a l i n e p e r s a m p l e . P r i n c i p l e o f t h e n o r a d r e n a l i n e a s s a y . W h i l e c a t e -c h o l a m i n e s w i l l f l u o r e s c e ( e x c i t a t i o n p e a k , 285 nm; e m i s s i o n , 325 nm), t h i s i s no a i d t o t h e i r d e t e r m i n a t i o n i n s m a l l amounts. The r e a s o n l i e s i n t h e f a c t t h a t t h i s f l u o r e s c e n c e i s a n o n - s p e c i f i c p r o p e r t y due t o t h e p h e n o l r i n g p r e s e n t i n t h e compound. The t a s k t h e n i s t o c o n v e r t t h e NA, and o n l y t h e NA, i n t o s u b s t a n c e s w h i c h c a n be s p e c i f i c a l l y i d e n t i f i e d f l u o r o m e t r i c a l l y . The a s s a y method i s b a s e d on t h e d e t e r m i n a t i o n o f a d e r i v a t i v e of NA, and n o t NA i t s e l f . The d e r i v a t i v e e m p l o y e d i n t h e p r e s e n t l y d e s c r i b e d a s s a y t e c h n i q u e i s 3 , 5 , 6 , - t r i h y d r o x y i n d o 1 e . The r e a c t i o n s r e q u i r e d i n t h e f o r m a t i o n of t h i s d e r i v a t i v e a r e shown b e l o w ( f r o m N a g a t s u , 1 9 7 3 ) . 14 r. 3 ? 5 ? 6 ^ t r l h y d r o x y i n d o l e O H v ^ \ OH kKK OH**S, » I o x i d a t i o n H rearrangement H The o x i d a t i o n i s c a r r i e d out by the i o d i n e s o l u t i o n f o r 10 min a f t e r which time t h i s r e a c t i o n i s stopped by the sodium t h i o s u l p h a t e . Rearrangement occurs i n a l k a l i n e s o l u t i o n during exposure to l i g h t . S t a t i s t i c s S t a t i s t i c a l comparisons of b r a i n NA were done using Student's _ t - t e s t . B e h a v i o u r a l data were analysed using repeated measures a n a l y s i s of v a r i a n c e . EXPERIMENT 1 E f f e c t of T e l e n c e p h a l i c NA D e p l e t i o n on the A c q u i s i t i o n of an L-Maze Running Response The importance of t e l e n c e p h a l i c NA to the a c q u i s i t i o n of a food rewarded running response i n an L-shaped runway was i n v e s t i g a t e d . Both e l e c t r o l y t i c and 6-OHDA-induced l e s i o n s were used i n an attempt to r e p l i c a t e the f i n d i n g of Anlezark et a l . , (1973). These authors r e p o r t e d t h a t , u n l i k e c o n t r o l s , r a t s with l e s i o n s of the LC which decreased c o r t i c a l NA f a i l e d to decrease t h e i r running time over days. o Method Four weeks post o p e r a t i v e l y , a l l r a t s were handled f o r 5 min/day and food deprived 22 hr/day. T r a i n i n g commenced the f i f t h week. On the f i r s t experimental day, each r a t was allowed to explore the runway f o r 30 min without food present; f o r each of 10 subsequent days, each r a t r e c e i v e d f i v e food-rewarded t r i a l s . On each t r i a l , the r a t was placed i n the s t a r t box and a f t e r 3 sec a g u i l l o t i n e - s t y l e door was r a i s e d which allowed access to the runway. Another manually operated door prevented the animal from r e t r a c i n g a f t e r e n t e r i n g the goal box. The r a t was removed a f t e r i t had consumed the f i v e 45 mg food p e l l e t s (Noyes). P u r i n a Lab Chow was a v a i l a b l e f o r 2 hours a f t e r being returned to the home cage. The maze was c o n s t r u c t e d of wood, with dimensions as f o l l o w s : h e i g h t , 15 cm; width, 11 cm; runway, 140 cm. Two p h o t o - c e l l s , 120 cm apart, were u t i l i z e d to measure running time over the i n i t i a l long arm of the maze. The length of the runway conforms to the s p e c i f i c a t i o n s of Anlezark et a l . (1973). F i f t y r a t s were used, of which 22 were 6-OHDA t r e a t e d , 10 were e l e c t r o l y t i c a l l y l e s i o n e d , and 18 were c o n t r o l s . R e s u l t s Four r a t s were excluded from the a n a l y s i s (1 c o n t r o l , 1 NA-6-0HDA, and 2 LC). Each d i s p l a y e d a g g r e s s i v e and i r r i t a b l e behaviour c h a r a c t e r i z e d by f r e e z i n g i n the s t a r t - 16 -b o x , v o c a l i z i n g when h a n d l e d , and f a i l i n g t o consume f o o d when p l a c e d i n t h e g o a l box. The h i p p o c a m p a l - c o r t i c a l NA d e p l e t i o n s o f t h e two LC a n i m a l s w e re 18 and 87%, t h u s , t h e a b n o r m a l b e h a v i o r does n o t a p p e a r r e l a t e d t o d e s t r u c t i o n o f t h e l o c u s c o e r u l e u s p e r s e . T a b l e 1 shows t h e e f f e c t o f l e s i o n s t o t h e l o c u s c o e r u l e u s on NA c o n t e n t of t h e h i p p o c a m p u s p l u s c o r t e x . H i p p o c a m p a l - c o r t i c a l NA was r e d u c e d t o 29% o f c o n t r o l (Range = 1 6 - 4 7 % ) . I n t h e NA-6-0HDA g r o u p , h i p p o c a m p a l -c o r t i c a l NA was r e d u c e d t o 6.7% of c o n t r o l v a l u e s . Many of t h e r e a d i n g s f r o m t h i s g r o u p w e r e a t b l a n k l e v e l s . The l o w e r l i m i t of t h e s e n s i t i v i t y o f t h e a s s a y was 0.01 p g, H i s t o l o g i c a l e x a m i n a t i o n of t h e l o c u s c o e r u l e u s l e s i o n s r e v e a l e d t h a t i n most c a s e s t h e l e s i o n d e s t r o y e d t h e r o s t r a l p o r t i o n of t h e n u c l e u s . Some c e l l s i n t h e c a u d a l p o r t i o n o f t h e n u c l e u s a p p e a r e d t o be s p a r e d i n most of t h e a n i m a l s . F i g u r e 1 shows t h e 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 a t t h e r o s t r a l a s p e c t of t h e LC i n an a n i m a l w i t h NA d e p l e t e d t o 22% of c o n t r o l v a l u e s . No d i f f e r e n c e i n r u n n i n g s p e e d was f o u n d b e t w e e n t h e b u r r h o l e and n o n - o p e r a t e d c o n t r o l g r o u p s . T h e s e d a t a were p o o l e d i n s u b s e q u e n t a n a l y s e s . F i g u r e 2 shows t h e r e d u c t i o n of mean r u n n i n g t i m e o v e r d a y s . S t a t i s t i c a l a n a l y s i s f a i l e d t o show any s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e s e g r o u p s , F (2,44) = 1.44, p > .05. S i n g l e c o m p a r i s o n s b e t w e e n - 17 -TABLE 1 E f f e c t o f 6-OHDA L e s i o n s i n t h e D o r s a l Tegmentum and E l e c t r o l y t i c L e s i o n s t o t h e L o c u s C o e r u l e u s on Hi p p o c a m p u s p l u s C o r t e x N o r a d r e n a l i n e . NA (ng/g) Group C o r t e x p l u s H i p p o c a m p u s C o n t r o l s (n=28) 389 + 30 (1 0 0 % ) LC L e s i o n s (n=8) 113 + 20 * ( 2 9 . 0 % ) 6-OHDA D o r s a l B u n d l e L e s i o n (n=33) 2 6 + 7 * (6 . 7 % ) D a t a r e p r e s e n t means (+ S.E.M.). The d a t a f r o m a n i m a l s w i t h 6-OHDA d o r s a l b u n d l e l e s i o n s w e r e c o m b i n e d f r o m t h e d i f f e r e n t e x p e r i m e n t s . * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s p < 0.01 F i g u r e 1. P h o t o m i c r o g r a p h of 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 t h e n. l o c u s c o e r u l e u s w h i c h c a u s e d a 79% r e d u c t i o n o f h i p p o c a m p u s p l u s c o r t e x NA. A b b r e v i a t i o n s : Mes V = m e s e n c e p h a l i c t r a c t o f t h e t r i g e m i n a l n e r v e ; MLF = m e d i a l l o n g i t u d i n a l f a s c i c u l u s ; PVG = p e r i v e n t r i c u l a r g r e y ; SCP = s u p e r i o r c e r e b e l l a r p e d u n c l e ; IV = f o u r t h v e n t r i c l e . - 19 -F i g u r e 2. R u n n i n g t i m e s m e a s u r e d b e t w e e n two p h o t o c e l l s 120 cm a p a r t on t h e i n i t i a l arm of an L - s h a p e d maze. A n i m a l s w e re t e s t e d f o r 5 t r i a l s / d a y and e a c h p o i n t r e p r e s e n t s t h e mean of 5 t r i a l s . F o r c l a r i t y S.E.M.s a r e shown f o r one g r o u p o n l y ; v a r i a n c e s w e r e homogeneous on e a c h day. See methods f o r d e t a i l s . - 21 -20 1 2 3 4 5 6 7 8 9 10 DAYS - 22 -e x p e r i m e n t a l g r o u p s a g a i n s t c o n t r o l s a l s o f a i l e d t o r e a c h s t a t i s t i c a l s i g n i f i c a n c e a t p < .05 [NA-6-0HDA vs C o n t . F ( 1 ,37) = 2.71: LC vs C o n t . F ( 1 , 2 4 ) = 2 . 3 0 ] . A n a l y s i s r e s t r i c t e d t o t h e i n i t i a l t h r e e days was p e r f o r m e d t o t e s t w h e t h e r a p o s s i b l e d i f f e r e n c e a t t h i s s t a g e was o b s c u r e d by t h e s i m i l a r i t y o f t h e f o l l o w i n g d a y s . T h i s c o m p a r i s o n a l s o f a i l e d t o r e a c h s i g n i f i c a n c e , F ( 2 . 4 4 ) = 1.10, p > .05. Two a n i m a l s i n t h e LC g r o u p d i s p l a y e d a u r o g e n i t a l d i s o r d e r . N i n e o f f o u r t e e n r a t s u s e d t o c o n f i r m l e s i o n c o - o r d i n a t e s a l s o showed i d e n t i c a l symptoms. T h e s e w e r e : b l o o d i n t h e u r i n e , 1-3 days p o s t o p e r a t i v e l y ; p o l y d i p s i a and p o l y u r i a w h i c h s u b s i d e d i n ^ 5 - 1 4 d a y s . S e v e r a l a n i m a l s d i e d , and a u t o p s y r e v e a l e d t h a t t h e b l a d d e r had r u p t u r e d i n e a c h c a s e . T h i s d i s o r d e r d i d n o t a p p e a r r e l a t e d t o t h e d e g r e e of l o c u s c o e r u l e u s damage, as a s s e s s e d h i s t o l o g i c a l l y , n o r d e p l e t i o n of c o r t i c a l NA. I t was n e v e r o b s e r v e d i n a n i m a l s w i t h 6-OHDA l e s i o n s o f t h e d o r s a l t e g m e n t a l NA p r o j e c t i o n . D i s c u s s i o n A n l e z a r k e t a l . (1973) r e p o r t e d l e a r n i n g , as a s s e s s e d by i n c r e a s i n g r u n n i n g s p e e d i n an L - s h a p e d r u n w a y , was a b s e n t i n a n i m a l s w i t h c o m p l e t e e l e c t r o l y t i c l e s i o n s t o t h e l o c u s c o e r u l e u s . B e c a u s e t h e s e l e s i o n s a l s o s i g n i f i -c a n t l y r e d u c e d t e l e n c e p h a l i c NA, i t was c o n c l u d e d t h a t - 2 3 t h e s e n o r a d r e n e r g i c n e u r o n s w ere i m p o r t a n t s u b s t r a t e s f o r l e a r n i n g . The p r e s e n t r e s u l t s a r e n o t c o n s i s t e n t w i t h t h e c o n c l u s i o n s of t h i s e a r l i e r work. C o m p a r a b l e NA d e p l e t i o n s w e re a c h i e v e d w i t h e l e c t r o l y t i c l e s i o n s , w i t h o u t s i g n i f i c a n t i m p a i r m e n t on t h i s t a s k . I t s h o u l d be n o t e d , h o w e v e r , t h a t o u r LC l e s i o n s a p p e a r t o h a v e b e e n more r o s t r a l t h a n t h o s e u t i l i z e d by A n l e z a r k e t a l . ( 1 9 7 3 ) . To what e x t e n t t h i s v a r i a b l e may have c o n t r i b u t e d t o t h e d i s c r e p a n t r e s u l t s i s n o t known. 6-OHDA l e s i o n s t o t h e d o r s a l t e g m e n t a l b u n d l e c a u s e d a g r e a t e r NA d e p l e t i o n , y e t a l s o f a i l e d t o a l t e r s i g n i f i c a n t l y t h e r e s p o n s e p a t t e r n as compared w i t h c o n t r o l s . The r u n n i n g t i m e s d i f f e r e d i n - a n o t h e r r e s p e c t f r o m t h o s e r e p o r t e d by A n l e z a r k e t a l . ( 1 9 7 3 ) . R u n n i n g t i m e s a t t h e b e g i n n i n g o f t r a i n i n g . i n t h e p r e s e n t e x p e r i m e n t were s h o r t e r t h a n t h e t i m e s r e p o r t e d by t h o s e i n v e s t i g a t o r s . P r o c e d u r a l d i f f e r e n c e s may e x p l a i n t h i s d i s c r e p a n c y . We h a n d l e d t h e r a t s e x t e n s i v e l y and a l l o w e d e x p l o r a t i o n of t h e runway b e f o r e b e g i n n i n g t r a i n i n g . I t i s n o t known w h e t h e r A n l e z a r k e t a l . f o l l o w e d a s i m i l a r p r o c e d u r e . Our s h o r t e r t i m e s may r e f l e c t a g r e a t e r d e g r e e o f a d a p t a t i o n t o h a n d l i n g and t o t h e runway. T h i s d i s c r e p a n c y does n o t d e t r a c t , h o w e v e r , f r o m t h e m a j o r c o n c l u s i o n , i . e . , t h a t c o n t r a r y t o p r e v i o u s o b s e r v a t i o n s , h i p p o c a m p a l - c o r t i c a l NA does n o t seem t o be c r i t i c a l f o r - 24 -t h e d e m o n s t r a t i o n ; o f l e a r n i n g i n an L-maze, as a s s e s s e d by r u n n i n g t i m e s . The f o u r a n i m a l s w h i c h w e re e x c l u d e d f r o m t h e a n a l y s i s d e s e r v e comment. They w o u l d t y p i c a l l y b e h a v e n o r m a l l y on t h e f i r s t t r i a l s i n a d a y ; h o w e v e r , w i t h i n c r e a s e d h a n d l i n g , f r e e z i n g , d e f e c a t i o n , u r i n a t i o n , and v o c a l i z a t i o n became e v i d e n t . T h e s e a n i m a l s w e re r u n t h r o u g h day 5, w i t h a c u t o f f v a l u e of 5 m i n p e r t r i a l . I f a v e r a g e r u n n i n g t i m e s were u s e d as t h e s o l e c r i t e r i o n f o r l e a r n i n g , t h e n i t m i g h t be c o n c l u d e d t h a t t h e s e a n i m a l s had a " l e a r n i n g d e f i c i t " . However, s i n c e some r u n n i n g t i m e s d e c r e a s e d t o as l i t t l e as 1.2 s e c , we c o n c l u d e d t h a t l e a r n i n g d i d o c c u r , b u t t h a t i t s d e m o n s t r a t i o n was o b s c u r e d by a c o m p e t i n g b e h a v i o u r , n a m e l y f r e e z i n g . I t i s n o t c l e a r f r o m t h e r e p o r t o f A n l e z a r k e t a l . (1973) w h e t h e r t h e LC l e s i o n a n i m a l s w h i c h d i d n o t show d e c r e a s e s i n r u n n i n g s p e e d b e h a v e d i n a n o r m a l manner o r d i s p l a y e d u n u s u a l b e h a v i o r s as were s o m e t i m e s o b s e r v e d i n t h e p r e s e n t e x p e r i m e n t s . The p r e s e n t r e s u l t s a r e i n c l o s e a g r e e m e n t w i t h t h o s e of A m a r a l and F o s s ( 1 9 7 5 ) . These a u t h o r s a l s o f a i l e d t o show a d e f i c i t i n runway l e a r n i n g a f t e r l e s i o n s t o t h e LC. U r o g e n i t a l d i s o r d e r s s i m i l a r t o t h o s e r e p o r t e d h e r e were a l s o o b s e r v e d . T h e s e d i s o r d e r s h a v e r e c e n t l y b e e n s t u d i e d i n d e t a i l by O sumi, O i s h i , F i m i w a r a and T a k a o r i ( 1 9 7 5 ) . - 25 -EXPERIMENT 2 The E f f e c t o f T e l e n c e p h a l i c NA D e p l e t i o n on t h e A c q u i s i t i o n and R e v e r s a l o f a T-Maze P o s t i o n H a b i t D e c r e a s e d r u n n i n g t i m e s i n an L - s h a p e d runway may i n d i -c a t e t h a t an a n i m a l has l e a r n e d t h a t f o o d i s p r e s e n t i n t h e g o a l box. Howe v e r , i n t h i s p r o c e d u r e f o o d i s a v a i l a b l e r e g a r d l e s s o f r u n n i n g s p e e d , a n d r e i n f o r c e m e n t i s n o t c o n -t i n g e n t on a s p e c i f i c b e h a v i o r , o t h e r t h a n e v e n t u a l l y f i n d i n g t h e f o o d . I n a d d i t i o n , c h a n g e s i n r u n n i n g s p e e d may r e f l e c t m o t i v a t i o n a l , m o t o r o r s e n s o r y f a c t o r s r a t h e r t h a n l e a r n i n g . F o r t h e s e r e a s o n s , t h e a c q u i s i t i o n o f a T-maze p o s i t i o n h a b i t was s e l e c t e d as a s i t u a t i o n more s u i t e d t o an a n a l y s i s o f l e a r n i n g . I n t h i s s i t u a t i o n , o n l y a p p r o p r i a t e r e s p o n s e s a r e r e i n f o r c e d , i . e . , c h o o s i n g t h e c o r r e c t g o a l box. I n t h e f o l l o w i n g e x p e r i m e n t , t h e h y p o t h e s i s t h a t c o r t i c a l NA i s c r i t i c a l f o r l e a r n i n g was t h e r e f o r e i n v e s t i g a t e d u s i n g 6-OHDA l e s i o n e d a n i m a l s i n a T-maze p o s i t i o n t a s k . 6-OHDA l e s i o n s w e r e p e r f o r m e d p r i o r t o t h e t r a i n i n g o f a p o s i t i o n h a b i t a f t e r w h i c h t h e a n i m a l s w e re t r a i n e d t o r u n t o t h e o p p o s i t e arm o f t h e T-maze. R e s u l t s D e p l e t i o n s o f h i p p o c a m p a l - c o r t i c a l NA c o m p a r a b l e t o Exp. 1 were o b t a i n e d and a r e shown i n T a b l e 1. F i g u r e 3 shows t h e a v e r a g e e r r o r p e r g r o u p i n b l o c k s of 5 t r i a l s . S t a t i s t i c a l a n a l y s i s r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e two g r o u p s on e r r o r s , F ( 1 . 2 0 ) < 1.0; F i g u r e 3. A v e r a g e e r r o r s of two g r o u p s of a n i m a l s i n b l o c k s of 5 t r i a l s d u r i n g t h e a c q u i s i t i o n o f a p o s i t i o n h a b i t i n a T-maze. E a c h a n i m a l was t e s t e d f o r 10 t r i a l s / day w i t h f o o d r e w a r d a v a i l a b l e i n one g o a l box o n l y . On days 8-10 f o o d r e w a r d was p r e s e n t e d i n t h e g o a l box on t h e o p p o s i t e s i d e f r o m o r i g i n a l t r a i n i n g . See methods f o r d e t a i l s . - 27 -- 23 -o r r u n n i n g s p e e d , F (1.20) = 1.32, p > .05. A n a l y s i s o f e r r o r s on t h e r e v e r s a l t a s k a l s o f a i l e d t o y i e l d a s i g n i f i -c a n t d i f f e r e n c e , F ( 1 , 2 0 ) < 1.0. D i s c u s s i o n The h y p o t h e s i s t h a t t e l e n c e p h a l i c NA i s i m p o r t a n t f o r t h e l e a r n i n g o f a f o o d - r e w a r d e d r e s p o n s e was n o t s u p p o r t e d . E x a m i n a t i o n o f F i g u r e 3 shows e s s e n t i a l l y i d e n t i c a l l e a r n i n g c u r v e s f o r t h e e x p e r i m e n t a l g r o u p and c o n t r o l s , d e s p i t e n e a r t o t a l d e p l e t i o n s o f h i p p o c a m p a l -c o r t i c a l NA. A p r e v i o u s r e p o r t ( A m a r a l and F o s s , 1975) i n d i c a t e d e l e c t r o l y t i c l e s i o n s t o t h e l o c u s c o e r u l e u s , w h i c h d e p l e t e d c o r t i c a l NA t o 18%, were a l s o i n e f f e c t i v e i n p r o d u c i n g a l e a r n i n g d e f i c i t . T h e s e a u t h o r s e m p l o y e d an o l f a c t o r y s t i m u l u s t o i d e n t i f y t h e c o r r e c t g o a l box of a T-maze. The p r e s e n t r e s u l t s e x t e n d t h e s e f i n d i n g s t o more c o m p l e t e d e p l e t i o n s o f h i p p o c a m p a l - c o r t i c a l NA. The r e v e r s a l d a t a i n d i c a t e t h a t e x t e n s i v e l e s i o n s of t h e d o r s a l NA b u n d l e do n o t s i g n i f i c a n t l y a f f e c t t h e a b i l i t y o f t h e a n i m a l t o a l t e r a p o s i t i o n h a b i t as t h e r e i n f o r c e m e n t c o n t i n g e n c i e s a r e m o d i f i e d . EXPERIMENT 3 The E f f e c t o f T e l e n c e p h a l i c NA D e p l e t i o n on E x p l o r a t o r y L o c o m o t o r A c t i v i t y E x p l o r a t o r y b e h a v i o r and h a b i t u a t i o n t o a n o v a l e n v i r o n -ment a r e two i m p o r t a n t p r o c e s s e s i n t h e e s t a b l i s h m e n t o f an - 29 -a p p e t i t i v e r e s p o n s e . S u f f i c i e n t i n v e s t i g a t i o n o f a maze i s r e q u i r e d f o r t h e a n i m a l t o f i n d t h e r e w a r d ; f u r t h e r , h a b i t u a t i o n t o n o n - r e i n f o r c i n g a s p e c t s o f t h e e n v i r o n m e n t must o c c u r f o r t h e a p p r o p r i a t e r e s p o n s e t o p r e d o m i n a t e e v e n t u a l l y o v e r t h e e x p l o r a t o r y b e h a v i o r . The f o l l o w i n g two e x p e r i m e n t s r e p r e s e n t an a t t e m p t t o exa m i n e t h e d e g r e e o f i n v o l v e m e n t of h i p p o c a m p a l - c o r t i c a l NA i n t h e s e p r o c e s s e s . I n t h e f i r s t e x p e r i m e n t t h e amount of l o c o m o t o r a c t i v i t y and t h e t i m e c o u r s e o f h a b i t u a t i o n t o a n o v a l e n v i r o n m e n t w e r e m e a s u r e d i n a c t i v i t y c a g e s . Method S u b j e c t s were t h o s e u s e d i n E x p e r i m e n t 2. A c t i v i t y was m e a s u r e d i n s i x c i r c u l a r 6 1 - c m - d i a m e t e r a c t i v i t y c a g e s ( L e h i g h V a l l e y E l e c t r o n i c s ) h o u s e d i n an i l l u m i n a t e d , sound a t t e n u a t e d room. E a c h c a g e was e q u i p p e d w i t h 12 p h o t o c e l l s e n s o r u n i t s , mounted on t h e o u t e r w a l l a t e q u a l d i s t a n c e s , 2 cm a b o v e t h e w i r e mesh f l o o r . I n t e r -r u p t i o n s of t h e l i g h t beams were r e c o r d e d on a 10-min t i m e d p r i n t o u t c o u n t e r f o r 1 h r . A c t i v i t y was m e a s u r e d b o t h b e f o r e and a f t e r 4 d a y s o f a 22 h r / d a y f o o d d e p r i v a t i o n s c h e d u l e a t t h e same t i m e o f day f o r e a c h a n i m a l . R e s u l t s S t a t i s t i c a l a n a l y s i s f a i l e d t o r e v e a l a s i g n i f i c a n t d i f f e r e n c e b e t w e e n c o n t r o l a n i m a l s and t h o s e w i t h 6-OHDA l e s i o n s to t h e d o r s a l NA b u n d l e , i n e i t h e r t h e d e p r i v e d F i g u r e 4 . L o c o m o t o r a c t i v i t y o f two g r o u p s o f a n i m a l s m e a s u r e d d u r i n g 1 h r i n p h o t o c e l l a c t i v i t y c a g e s . E a c h p o i n t r e p r e s e n t s t h e mean (+S.E.M.) p h o t o c e l l i n t e r r u p -t i o n s o v e r 1 0 m i n . See methods f o r d e t a i l s . - 31 -M I N U T E S - 32 -F (1, 20) < 1.0 or non-deprived c o n d i t i o n , F (1, 20) < 1.0. The time course of the d e c l i n e of motor a c t i v i t y over the 1 hour t e s t p e r i o d a l s o d i d not d i f f e r between the groups in the dep r i v e d , F (5,100) < 1.0, or non-deprived c o n d i t i o n , F (5,100) < 1.0. Fig u r e 4 shows the a c t i v i t y measures of the two groups i n the non-food deprived s t a t e over 1 hour i n 10 minute segments. The scores f o l l o w i n g 4 days of food d e p r i v a t i o n y i e l d e d e s s e n t i a l l y the same h a b i t u a t i o n curve except that a s i g n i f i c a n t l y lower response p a t t e r n was seen, F (1, 20) = 7.63, p < .05. T e s t i n g order was not counterbalanced and t h e r e f o r e t h i s e f f e c t might have been due to having been i n the a c t i v i t y cages p r e v i o u s l y . D i s c u s s i o n In as much as a c t i v i t y cages can measure h a b i t u a t i o n to a noval environment, c o r t i c a l - h i p p o c a m p a l NA does not appear to play a s i g n i f i c a n t r o l e i n t h i s b ehavior. These r e s u l t s are i n agreement with e a r l i e r r e s u l t s from t h i s l a b o r a t o r y (Roberts, Z i s and F i b i g e r , 1975). Anlezark et a l . (1973) a l s o found no d i f f e r e n c e i n e x p l o r -atory behavior between L C - l e s i o n e d animals and c o n t r o l s - 33 -in an open f i e l d t e s t . In c o n t r a s t , Amaral and Foss (1975) re p o r t e d that a c t i v i t y , measured over a 90-minute t e s t p e r i o d , c o r r e l a t e d p o s i t i v e l y with NA content f o l l o w i n g e l e c t r o l y t i c LC l e s i o n s . Motor d i s t u r b a n c e s were a l s o noted, however, and may have accounted f o r the decreased a c t i v i t y i n l e s i o n e d animals which was observed by these workers. D i f f e r e n c e s i n t e s t apparatus may a l s o account f o r t h i s apparent d i s c r e p a n c y . Amaral and Foss used animex a c t i v i t y cages which are more s e n s i t i v e to small body movements than are p h o t o c e l l cages. EXPERIMENT 4 The E f f e c t of T e l e n c e p h a l i c NA D e p l e t i o n on the D i s t r a c t i b i i i t y of Rats during the Performance of Tra i n e d Response In Experiment 3, i t was shown that the e x p l o r a t o r y a c t i v i t y of animals with d e p l e t i o n s of h i p p o c a m p a l - c o r t i c a l NA d e c l i n e d at the same rate as that of c o n t r o l s , and thus - 34 -m i g h t i n d i c a t e t h e same r a t e of h a b i t u a t i o n t o t h e n o v e l t e s t e n v i r o n m e n t . H o w e v e r , i n a c t i v i t y c a g e s , no r e s p o n s e i s s p e c i f i c a l l y r e i n f o r c e d . The f o l l o w i n g e x p e r i m e n t was d e s i g n e d t o m e a s u r e t h e e f f e c t o f n o v e l t a c t i l e and v i s u a l s t i m u l i on a t r a i n e d f o o d - r e w a r d e d r e s p o n s e . M e t h o d s D o r s a l t e g m e n t a l b u n d l e 6-OHDA a n i m a l s and c o n t r o l s f r o m E x p e r i m e n t 1 were u s e d . The L - s h a p e d runway as d e s -c r i b e d a b o v e , was f i t t e d w i t h h e a v y g r a d e s a n d p a p e r on t h e runway f l o o r b e t w e e n t h e two p h o t o c e l l s . Two 1 0 - w a t t l i g h t b u l b s were f i x e d , 30 cm a p a r t , t o t h e w i r e mesh c e i l i n g o f t h e r u n w a y , d i r e c t l y a bove t h e s a n d p a p e r . T h e s e were programmed t o f l a s h a t 10 Hz commencing when t h e f i r s t p h o t o c e l l was a c t i v a t e d and t e r m i n a t i n g when t h e r a t r e a c h e d t h e s e c o n d p h o t o c e l l ; t h a t i s , t h e l i g h t s f l a s h e d d u r i n g t h e t i m i n g i n t e r v a l . The a n i m a l s were t r a i n e d o v e r a 10-day p e r i o d . On e x p e r i m e n t a l day 1 1 , t h e t a c t i l e and v i s u a l s t i m u l i w e r e a d d e d , and r u n n i n g s p e e d s were r e c o r d e d o v e r f i v e t r i a l s . R e s u l t s F i g u r e 5 r e p r e s e n t s t h e a v e r a g e g r o u p r u n n i n g s p e e d s on e a c h o f t h e f i v e t r i a l s . S t a t i s t i c a l a n a l y s i s r e v e a l e d a s i g n i f i c a n t d i f f e r e n c e b e t w e e n g r o u p s . F ( 1 , 3 7 ) = 4.68, p < .05. I n d i v i d u a l c o m p a r i s o n s y i e l d e d a s i g n i f i c a n t d i f f e r e n c e on t h e f i r s t t r i a l o n l y , t (37) = 2.21, p < .05. No q u a l i t a t i v e d i f f e r e n c e was s e e n i n t h e r e s p o n s e t o t h e F i g u r e 5. R u n n i n g t i m e s i n t h e p r e s e n c e of n o v e l t a c t i l e and v i s u a l s t i m u l i m e a s u r e d b e t w e e n two p h o t o c e l l s 120 cm a p a r t on t h e i n i t i a l arm of an L - s h a p e d maze. A n i m a l s were p r e t r a i n e d f o r 10 days b e f o r e e x p o s u r e t o t h e n o v e l s t i m u l i on t h e 1 1 t h day. E a c h p o i n t r e p r e s e n t s t h e g r o u p mean (+ S.E.M.) on e a c h t r i a l . See methods f o r d e t a i l s . - 3.7' -n o v e l s t i m u l i . On t h e f i r s t t r i a l , t h e s u b j e c t s t y p i c a l l y w o u l d m o m e n t a r i l y f r e e z e a t t h e o n s e t on t h e f l a s h i n g l i g h t s , f o l l o w e d by e x p l o r a t o r y s n i f f i n g b e h a v i o r D f t h e f l o o r and c e i l i n g . By t h e f i f t h t r i a l , r u n n i n g s p e e d s were c o m p a r a b l e t o t h o s e p r i o r t o t h e d i s t r a c t i n g s t i m u l i , i . e . , day 10 on F i g u r e 2. D i s c u s s i o n R a t s w i t h l e s i o n s t o t h e d o r s a l t e g m e n t a l NA b u n d l e were more d i s t r a c t i b l e t h a n c o n t r o l s i n a s i t u a t i o n w h i c h r e q u i r e d t h e p e r f o r m a n c e o f a p r e v i o u s l y t r a i n e d r e s p o n s e . T h i s e f f e c t , t h o u g h s t a t i s t i c a l l y s i g n i f i c a n t , was t r a n s i e n t ; a f t e r f i v e t r i a l s b o t h g r o u p s had c o m p l e t e l y h a b i t u a t e d t o t h e n o v e l s t i m u l i . The s i m i l a r i t y b e t w e e n t h e d e c r e a s e i n r u n n i n g t i m e i n th e p r e s e n t e x p e r i m e n t and t h e d e c r e a s e i n r u n n i n g t i m e o v e r d a y s i n E x p e r i m e n t 1 i s n o t e w o r t h y . W h i l e t h e 6-OHDA l e s i o n g r o u p was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s i n t h e o r i g i n a l L-maze l e a r n i n g t a s k , t h e g r o u p means f o r t h i s g r o u p t e n d e d t o be above t h o s e o f c o n t r o l s on t h e f i r s t s i x da y s ( F i g u r e 1 ) . T h i s may have b e e n due t o i n c r e a s e d d i s t r a c t i -b i l i t y d u r i n g t h e a c q u i s i t i o n p h a s e o f t h i s e x p e r i m e n t . H o wever, t h i s t e n d e n c y was n o t s t a t i s t i c a l l y s i g n i f i c a n t . Due t o t h e i n h e r e n t v a r i a b i l i t y i n t h e r u n n i n g s p e e d s on t h e f i r s t few d a y s o f t r a i n i n g , l a r g e g r o u p s o f a n i m a l s w o u l d be r e q u i r e d t o t e s t s u c h d i f f e r e n c e s . I n any e v e n t , l e s i o n s t o t h e d o r s a l NA b u n d l e a p p e a r t o a f f e c t t h e r a t e o f h a b i t u a t i o n t o n o v e l s t i m u l i d u r i n g t h e p e r f o r m a n c e o f a t r a i n e d r e s p o n s e . - 38 -EXPERIMENT 5 The E f f e c t o f T e l e n c e p h a l i c NA D e p l e t i o n on t h e A c q u i s i t i o n o f a C o n d i t i o n e d T a s t e A v e r s i o n Many e x p e r i m e n t a l l e a r n i n g p a r a d i g m s have been d e v e l o p -ed as an a i d t o t h e i n v e s t i g a t i o n o f p r o c e s s e s i n v o l v e d i n t h e f o r m a t i o n o f l e a r n e d a s s o c i a t i o n s . M o st o f t h e s e h a v e em p l o y e d s t i m u l i o r s i t u a t i o n s w h i c h a r e u n n a t u r a l t o t h e a n i m a l . T h a t i s , t h e l e a r n i n g s i t u a t i o n w o u l d n o t be e n c o u n t e r e d i n t h e n o r m a l e n v i r o n m e n t o f a n o n - e x p e r i m e n t a l r a t . One e x c e p t i o n i s t h e c o n d i t i o n e d t a s t e a v e r s i o n p r o c e d u r e . I n 1 9 5 5 , G a r c i a e t a l . d e m o n s t r a t e d t h a t r a t s w i l l a v o i d i n g e s t i n g a s o l u t i o n w h i c h has f o r m e r l y b e e n a s s o c i a t -ed w i t h i l l n e s s . They d i d t h i s by s u b j e c t i n g t h e a n i m a l s t o gamma r a d i a t i o n d u r i n g t h e i r f i r s t e x p o s u r e t o s a c c h a r i n f l a v o r e d w a t e r . Two d a y s l a t e r , b o t h t h e s a c c h a r i n s o l u t i o n and t a p w a t e r w ere made a v a i l a b l e s i m u l t a n e o u s l y and t h e amount of e a c h f l u i d consumed was m e a s u r e d . C o n t r o l ( n o n -i r r a d i a t e d ) r a t s d r a n k 85% of t h e i r d a i l y f l u i d i n t a k e f r o m t h e s a c c h a r i n c o n t a i n e r , w h i l e t h e x - i r r a d i a t e d a n i m a l s c o m p l e t e l y a v o i d e d t h e f l a v o u r e d s o l u t i o n f o r t h e f i r s t week and s t i l l showed an a v e r s i o n a t t h e end o f t h e two month e x p e r i m e n t . N o t i c e t h a t t h e i l l n e s s was n o t b r o u g h t a b o u t by t h e n o v e l t a s t i n g s o l u t i o n , b u t was u n d e r t h e d i r e c t c o n t r o l of t h e e x p e r i m e n t e r . The s u p p r e s s i o n o f i n t a k e ( h e n c e f o r t h - 39 -r e f e r r e d t o as c o n d i t i o n e d t a s t e a v e r s i o n , CTA) has s i n c e b e en a c c o m p l i s h e d u s i n g a w i d e v a r i e t y of s t i m u l i w h i c h c a u s e i l l n e s s . L i C l ( 2 % BW, .15 M) c a u s e s s i c k n e s s i n t h e r a t w i t h i n 15 m i n (Nachman, 1970) of i . p . a d m i n i s -t r a t i o n as a d d u c e d by u n r e s p o n s i v e n e s s and r e d u c e d a c t i v i t y . Such i n j e c t i o n s , f o l l o w i n g i n g e s t i o n o f n o v e l t a s t i n g s o l u t i o n s w i l l c a u s e CTAs much l i k e t h a t s e e n i n t h e i r r a d i a t i o n p a r a d i g m ( B a r k e r and S m i t h , 1 9 7 4 ) . O t h e r d i s t r e s s - i n d u e i n g a g e n t s w h i c h have b e e n shown t o p r o d u c e CTAs a r e a p o m o r p h i n e and e m e t i n e ( R e v u s k y and G o r r y , 1973) h y p e r t o n i c s a l i n e ( B r a v e m a n and C a p r e t t a , 1965) and i n s u l i n ( L o v e t t , G o o d c h i l d and B o o t h , 1 9 6 8 ) . T h e s e s t i m u l i a r e presumed t o have d i f f e r e n t l o c i , of a c t i o n s i n t h e b o d y , h o w e v e r , a l l p r o d u c e a g e n e r a l m a l a i s e w h i c h has b e e n p o s t u l a t e d as e s s e n t i a l t o t h e c o n d i t i o n i n g o f a v e r s i o n s t o n o v e l t a s t e s ( G a r c i a e t a l . , 1 9 6 7 ) . The p r e s e n t e x p e r i m e n t was u n d e r t a k e n t o e x a m i n e t h e a b i l i t y of r a t s w i t h d e c r e a s e d t e l e n c e p h a l i c NA t o l e a r n an a s s o c i a t i o n a n a l o g o u s t o t h a t r e q u i r e d o f an a n i m a l i n t h e n a t u r a l s e t t i n g . M ethod 6-OHDA i n j e c t i o n s (n=12) were made i n t o t h e a r e a o f d o r s a l t e g m e n t a l NA b u n d l e as p r e v i o u s l y d e s c r i b e d ( E x p e r i m e n t 1 ) . C o n t r o l a n i m a l s (n=12) r e c e i v e d sham - 40 " o p e r a t i o n s . A l l a n i m a l s w e re h o u s e d i n d i v i d u a l l y a n d , t h r e e weeks p o s t o p e r a t i v e l y , were p l a c e d on a 23.5 h r / d a y w a t e r d e p r i v a t i o n s c h e d u l e . Food was a v a i l a b l e ad l i b i t u m a t a l l t i m e s . F l u i d i n t a k e was m e a s u r e d by t h e w e i g h t d i f f e r e n c e o f t h e w a t e r b o t t l e b e f o r e and a f t e r p r e s e n t a t i o n . T h i s m e a s u r e was u n c o r r e c t e d f o r s p i l l a g e , w h i c h was assumed t o be c o n s t a n t a c r o s s g r o u p s . A f t e r w a t e r i n t a k e had s t a b i l i z e d , a 0.1% s a c c h a r i n s o l u t i o n was made a v a i l a b l e t o e a c h r a t i n l i e u o f t h e i r d a i l y w a t e r p r e s e n t a t i o n . F i v e m i n f o l l o w i n g t h e r e m o v a l o f .the s a c c h a r i n s o l u t i o n , e a c h r a t was i n j e c t e d i . p . w i t h L i C l ( 2 % Body W e i g h t , 0.15 M) F o r t h e n e x t t h r e e d a y s w a t e r was a g a i n made a v a i l a b l e f o r 0.5 h r / d a y . On t h e f o u r t h day f o l l o w i n g t h e L i C l i n j e c t i o n , s a c c h a r i n was a g a i n o f f e r e d t o t h e r a t s , and t h e amount consumed m e a s u r e d . R e s u l t s I n j e c t i o n s of 6-OHDA i n t o t h e d o r s a l t e g m e n t a l NA b u n d l e p r o d u c e d d e p l e t i o n s o f t e l e n c e p h a l i c NA s i m i l a r t o t h o s e p r o d u c e d i n t h e p r e v i o u s e x p e r i m e n t s . C o n t r o l v a l u e s f o r NA c o n t e n t o f t h e h i p p o c a m p u s p l u s c o r t e x were c a l c u l a t e d t o be 487 + 40 ng/gm. R a t s w h i c h r e c e i v e d i n j e c t i o n s o f 6-OHDA were f o u n d t o have a mean NA c o n t e n t o f 16 + 1 ng/gm. T h i s r e p r e s e n t s a mean g r o u p r e d u c t i o n o f 97% f r o m c o n t r o l v a l u e s . F i g u r e 6 shows t h e e f f e c t s o f t h e 6-OHDA t r e a t m e n t on t h e f l u i d c o n s u m p t i o n d u r i n g t h i s e x p e r i m e n t . The b a r g r a p h F i g u r e 6 E f f e c t o f 6-OHDA l e s i o n s on t h e a c q u i s i t i o n o f a c o n d i t i o n e d t a s t e a v e r s i o n i n d u c e d by an i . p . i n j e c t i o n o f L i C l . The two b a r s on t h e l e f t o f t h e f i g u r e show mean d a i l y w a t e r i n t a k e d u r i n g t h e 1/2 h o u r d r i n k i n g p e r i o d . The p o i n t s on t h e g r a p h show mean (± S.E.M.) gr o u p s a c c h a r i n i n t a k e b e f o r e and t h r e e d a y s a f t e r t h e L i C l i n j e c t i o n . - 42 -W A T E R S A C C H A R I N - 43 -on t h e l e f t o f t h e f i g u r e shows no d i f f e r e n c e i n t h e w a t e r i n t a k e b e t w e e n t h e s e two g r o u p s . T h i s m e a s u r e was t a k e n on t h r e e s e p a r a t e d a y s ; on no day was a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e o b s e r v e d . The m i d d l e p o i n t on t h e g r a p h shows t h e i n t a k e of s a c c h a r i n of e a c h g r o u p on t h e i r i n i t i a l e x p o s u r e t o t h i s s o l u t i o n . A s i g n i f i c a n t d i f f e r e n c e was o b s e r v e d b e t w e e n t h e two g r o u p s on t h i s day (t^ = 2.93, d f = 2 1 , p < 0 . 0 1 ) . No s t a t i s t i c a l d i f f e r e n c e was f o u n d b e t w e e n c o n t r o l a n i m a l s and e x p e r i m e n t a l s on s a c c h a r i n i n t a k e on t h e r e t e s t day. S i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d , h o w e v e r , w i t h i n e a c h g r o u p f r o m i n i t i a l e x p o s u r e t o r e t e s t day. T h a t i s , b o t h , g r o u p s d i s p l a y e d a s i g n i f i c a n t a v e r s i o n t o t h e n o v e l t a s t e f o l l o w i n g L i C l i n j e c t i o n . D i s c u s s i o n T h e s e d a t a a r e c o n s i s t e n t w i t h t h o s e r e p o r t e d by R o b e r t s and F i b i g er (1975) who showed t h a t 6—OHDA a d m i n i s -t e r e d i n t r a v e n t r i c u l a r l y had no e f f e c t on a t a s t e a v e r s i o n t o s a c c h a r i n i n d u c e d by r e p e a t e d i n j e c t i o n s o f L i C l . I n t h e p r e s e n t e x p e r i m e n t t h e o n l y d i f f e r e n c e o b s e r v e d b e t w e e n t h e two g r o u p s was i n t h e r e l u c t a n c e of t h e NA d e p l e t e d a n i m a l s t o consume t h e s a c c h a r i n on t h e i r f i r s t e x p o s u r e . T h i s n e o p h o b i a has b een o b s e r v e d p r e v i o u s l y i n t h i s l a b o r a t o r y i n o t h e r g r o u p s o f a n i m a l s w i t h i d e n t i c a l l e s i o n s . W h i l e - 44 -t h i s e f f e c t a p p e a r s r e l i a b l e , t h e d i f f e r e n c e i s t r a n s i e n t and i s n o t f o u n d i n s u b s e q u e n t n o n - p u n i s h e d p r e s e n t a t i o n s . The p r e s e n t e x p e r i m e n t o f f e r s no s u p p o r t t o t h e h y p o t h e s i s t h a t c o r t i c a l NA i s n e c e s s a r y t o t h e l e a r n i n g of a c o n d i t i o n e d t a s t e a v e r s i o n . GENERAL DISCUSSION D e p l e t i o n s of h i p p o c a m p a l - c o r t i c a l NA w e re f o u n d t o have l i t t l e e f f e c t on t h e a c q u i s i t i o n o f f o o d - r e w a r d e d m a z e - r u n n i n g r e s p o n s e s . N e i t h e r e l e c t r o l y t i c l e s i o n s t o t h e n u c l e u s l o c u s c o e r u l e u s , n o r 6-OHDA l e s i o n s o f t h e d o r s a l t e g m e n t a l b u n c l e a f f e c t e d t h e a c q u i s i t i o n o r p e r f o r m a n c e compared t o c o n t r o l a n i m a l s i n an L - s h a p e d runway T h e s e d a t a do n o t s u p p o r t t h e h y p o t h e s i s p r o p o s e d by o t h e r s ( C r o w , 1968; K e t y , 1 9 7 0 ) , w h i c h c o n t e n d s t h a t n o r a d r e n e r g i c t e r m i n a l s i n t h e c e r e b r a l c o r t e x m e d i a t e s y n a p t i c c h a n g e s d u r i n g l e a r n i n g . The f i r s t e x p e r i m e n t was u n d e r t a k e n i n an a t t e m p t t o r e p l i c a t e p r e v i o u s l y r e p o r t e d r e s u l t s ( A n l e z a r k e t a l . , 1973) i n d i c a t i n g t h a t a n i m a l s w i t h l e s i o n s t o t h e NA c e l l b o d i e s of t h e LC were i m p a i r e d i n t h e l e a r n i n g o f a f o o d - m o t i v a t e d r u n n i n g t a s k . F u r t h e r , i f t h i s i m p a i r m e n t was due t o t h e d e c r e a s e i n t e l e n c e p h a l i c NA, t h e n l e s i o n s o f t h e a s c e n d i n g pathway s h o u l d a l s o r e s u l t i n s u c h an i m p a i r m e n t . E l e c t r o -l y t i c l e s i o n s t o t h e LC, w h i c h c a u s e d d e p l e t i o n s of c o r t i c a l - 45 " NA c o m p a r a b l e t o t h o s e o f A n l e z a r k e t a l . ( 1 9 7 3 ) , w e re f o u n d t o have no e f f e c t on L-maze r u n n i n g when compared t o c o n t r o l s . Near t o t a l f o r e b r a i n NA d e p l e t i o n , c a u s e d by i n t r a c e r e b r a l i n j e c t i o n s o f 6-OHDA a l s o f a i l e d t o a f f e c t t h i s r u n n i n g r e s p o n s e f o r f o o d . Changes i n r u n n i n g s p e e d may p o s s i b l y s u p p l y an i n d i r e c t m e a s ure o f l e a r n i n g i n t h e runway s i t u a t i o n , b u t a l s o r e f l e c t m o t o r a b i l i t y , m o t i v a t i o n , h a b i t u a t i o n , and d i s t r a c -t i o n . A more d i r e c t a s s e s s e m e n t o f l e a r n i n g i s a v a i l a b l e , h o w e v e r , i n t h e T-shaped maze by m e a s u r i n g t h e d e v e l o p m e n t of a p o s i t i o n h a b i t t o t h e f o o d ~ r e i n f o r c e d s i d e o f t h e maze. A n i m a l s w i t h n e a r t o t a l d e p l e t i o n s of f o r e b r a i n NA l e a r n e d a p o s i t i o n d i s c r i m i n a t i o n i n t h i s p a r a d i g m , and were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s . T h e s e r e s u l t s a g r e e w i t h a p r e v i o u s r e p o r t ( A m a r a l and F o s s , 1975) i n w h i c h LC e l e c t r o l y t i c l e s i o n s had no e f f e c t on a T-maze d i s -c r i m i n a t i o n t a s k . Mason and I v e r s e n (1974) f o u n d t h a t i n t r a v e n t r i c u l a r a d m i n i s t r a t i o n o f 6-OHDA d i s r u p t e d a c q u i s i t i o n o f a c o m p l i c a t e d p r o b l e m s o l v i n g t a s k . The a n i m a l was r e q u i r e d t o e i t h e r p u s h o r p u l l a b a l l t h r o u g h a t u n n e l t o g a i n a c c e s s t o a f o o d r e w a r d . 6-OHDA t r e a t e d a n i m a l s r e q u i r e d more t r i a l s t o l e a r n t h e r e s p o n s e . I t was a r g u e d t h a t t h i s d e f i c i t was n o t a r e s u l t o f mo t o r i m p a i r m e n t . I t i s i n t e r e s t i n g t o n o t e t h a t no d i f f e r e n c e s w e r e o b s e r v e d - 46 -b e t w e e n t h e s e a n i m a l s and c o n t r o l s when t h e t a s k was r e v e r s e d ; i . e . p u s h i n g t h e b a l l , when f o r m e r l y t r a i n e d t o p u l l . The p o s s i b i l i t y e x i s t s t h e r e f o r e t h a t a l e a r n i n g d e f i c i t a f t e r 6-OHDA l e s i o n s becomes e v i d e n t o n l y i n v e r y c o m p l i c a t e d t a s k s . The r e v e r s a l o f t h e p r e s e n t T-maze p r o b l e m may n o t h a v e b e e n s u f f i c i e n t l y d i f f i c u l t t o d e m o n s t r a t e t h e d i s r u p t i o n i n a c q u i s i t i o n o f l e a r n e d r e s p o n s e s . The p r e s e n t f i n d i n g t h a t a n i m a l s w i t h t e l e n c e p h a l i c NA d e p l e t i o n s w e re more d i s t r a c t i b l e s u p p o r t s t h i s n o t i o n . T h i s d i s t r a c t i b i l i t y may become d e b i l i t a t i n g o n l y when t h e a p p r o p r i a t e r e s p o n s e i s d i f f i c u l t t o l e a r n . A l t e r n a t i v e l y , t h e d e f i c i t o b s e r v e d by Mason and I v e r s e n ( 1974) f o l l o w i n g i n t r a v e n t r i c u l a r 6-OHDA a d m i n i s t r a t i o n d i s r u p t e d l e a r n i n g t h r o u g h a CA pat h w a y o t h e r t h a n t h e LC p r o j e c t i o n . D e f i c i t s i n a c q u i s i t i o n and p e r f o r m a n c e o f a d o u b l e T-maze f o o d - r e w a r d e d r e s p o n s e have b e e n r e p o r t e d a f t e r i n t r a c i s t e r n a l a d m i n i s t r a t i o n o f 6-OHDA (Howard e t a l . , 1 9 7 4 ) . A c l e a r r e l a t i o n s h i p was n o t e d b e t w e e n t h e s e i m p a i r -ments and w h o l e b r a i n d e p l e t i o n s o f CAs; h o w e v e r , t h e s e c o r r e l a t e d more w i t h DA t h a n NE d e p l e t i o n s . T h i s i s c o n s i s t e n t w i t h o b s e r v a t i o n s f r o m t h i s l a b o r a t o r y t h a t 6-OHDA l e s i o n s o f t h e n i g r o - n e o s t r i a t a l d o p a m i n e r g i c pathway p r o d u c e a p r o f o u n d i m p a i r m e n t i n t h e a c q u i s i t i o n o f a c o n -d i t i o n e d a v o i d a n c e r e p o n s e o r a s i m p l e a p p r o a c h r e s p o n s e f o r f o o d ( F i b i g e r , P h i l l i p s and Z i s , 1 9 7 4 ) . S u b s e q u e n t e x p e r i -ments have i n d i c a t e d however t h a t t h e s e i m p a i r m e n t s a r e due - 47 -t o a s p e c i f i c m o t o r d i s a b i l i t y r a t h e r t h a n a l e a r n i n g d e f i c i t ( P r i c e and F i b i g e r , 1975 ; Z i s , F i b i g e r and P h i l l i p s , 1974; F i b i g e r , Z i s and P h i l l i p s , 1 9 7 5 ) . W h i l e t h e p r e s e n t r e s e a r c h r e p o r t o f f e r s no s u p p o r t f o r t h e n e c e s s i t y o f NA t o ..the l e a r n i n g p r o c e s s , a c o n s i d e r a b l e amount of d a t a e x i s t w h i c h a r e . c o n s i s t e n t w i t h t h i s h y p o t h e s i s . T h e s e d a t a come i n t h e f o r m o f pharma-c o l o g i c a l m a n i p u l a t i o n s of NA s y n t h e s i s , u s i n g t h e d o p a m i n e -s - h y d r o x y l a s e i n h i b i t o r , d i e t h y l - d i t h i o c a r b a m a t e (DDC). I n t r a p e r i t o n e a l i n j e c t i o n s o f t h i s e i t h e r i m m e d i a t e l y b e f o r e o r a f t e r t r a i n i n g on a p a s s i v e a v o i d a n c e t a s k w i l l p r e v e n t t h e f o r m a t i o n o f a " l o n g - t e r m " b u t n o t a " s h o r t -t e r m " memory. T h i s i s d e m o n s t r a t e d by t h e r e t e s t l a t e n c i e s t o s t e p down f r o m a s a f e p l a t f o r m e i t h e r i m m e d i a t e l y a f t e r o r s e v e r a l h o u r s o r d a y s f o l l o w i n g t r a i n i n g . W h i l e t h i s d r u g may h a v e many p h a r m a c o l o g i c a l , e f f e c t s , S t e i n e t a l . (1975) h a v e p r e s e n t e d d a t a w h i c h i m p l i c a t e c e n t r a l NA i n t h e a c t i o n o f DDC. T h e s e w o r k e r s showed t h a t 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 s o f NA a r e c a p a b l e o f r e s t o r i n g t h e c a p a c i t y of a DDC t r e a t e d a n i m a l t o l e a r n a p a s s i v e a v o i d a n c e t a s k i f t h e s e i n j e c t i o n s f o l l o w i m m e d i a t e l y a f t e r t r a i n i n g . T h i s f i n d i n g has s i n c e b e e n c o n f i r m e d by M e l i g e n i e t a l . ( 1 9 7 5 ) . P e r i p h e r a l m echanisms have a l s o b e e n i m p l i c a t e d i n t h e a m n e s i c e f f e c t o f DDC. P o s t t r a i n i n g i n t r a -p e r i t o n e a l i n j e c t i o n s o f e i t h e r NA ( M e l i g i n i e t a l . , 1975) o r a d r e n a l i n e (Van B u s k i r k and G o l d , 1975) have b e e n - 48 -r e p o r t e d t o r e v e r s e t h e D D C - i n d u c e d d e f i c i t . T h i s e f f e c t may be due t o t h e NA g a i n i n g a c c e s s t o c e n t r a l NA r e c e p t o r s , a l t h o u g h t h i s p o s s i b i l i t y a p p e a r s u n l i k e l y . P e r i p h e r a l a d m i n i s t r a t i o n o f e p i n e p h r i n e has b e e n shown t o f a c i l i t a t e t i m e - d e p e n d e n t memory s t o r a g e p r o c e s s e s ( G o l d and Van B u s k i r k , 1 9 7 5 ) , t h u s d e m o n s t r a t i n g t h a t p e r i p h e r a l a d r e n e r g i c s y s t e m s may w e l l i n t e r a c t w i t h t h e CNS i n t h e s t o r a g e o f memory. The i n h i b i t i o n o f s y n t h e s i s i n p e r i p h e r a l a d r e n e r g i c s y s t e m s may t h u s be one e x p l a n a t i o n f o r t h e a m n e s i c e f f e c t o f DDC. Some a u t h o r s have q u e s t i o n e d w h e t h e r DDC e i t h e r i m p a i r s t h e c o n s o l i d a t i o n o f memories o r d i s r u p t s t h e s u b -s e q u e n t a b i l i t y o f t h e a n i m a l t o " r e t r i e v e " t h e memory w h i c h was s t o r e d . B o t w i n i c k and Q u a r t e r m a i n ( 1 9 7 4) have r e p o r t e d t h a t p r e - t e s t i n j e c t i o n s o f monoamine o x i d a s e i n h i b i t o r s ( C a t r o n o r P a r g y l i n e ) r e v e r s e t h e DDC a m n e s i a . T h e s e d a t a d e m o n s t r a t e t h a t b e c a u s e r e c a l l i s p o s s i b l e u n d e r some c o n d i t i o n s , some l e a r n i n g must h a v e b e e n c o n s o l i d a t e d d u r i n g t h e o r i g i n a l t r a i n i n g . Cohen e t a l . (1975) have r e p o r t e d t h a t DDC i m p a i r s p a s s i v e a v o i d a n c e r e t e n t i o n when g i v e n p r i o r t o t h e r e - t e s t , a l t h o u g h s u b -s e q u e n t t e s t i n g w h i l e n o t u n d e r t h e d r u g c o n d i t i o n showed t h a t l e a r n i n g had o c c u r r e d . T hese a u t h o r s i n t e r p r e t e d t h e d a t a as i n d i c a t i n g t h a t n o r a d r e n e r g i c s y s t e m s a r e n e c e s s a r y f o r " m e m o r y a c c e s s " . - 49 -I f d i s r u p t i o n o f NA s y n t h e s i s by DDG i s t h e c r i t i c a l p h a r m a c o l o g i c a l f a c t o r i n v o l v e d i n t h e d i s r u p t i o n o f memory, t h e n o t h e r d r u g s w h i c h e f f e c t NA s y n t h e s i s s h o u l d a l s o a f f e c t memory. D i s m u k e s and Rake (1972) showed t h a t r e s e r p i n e , an a g e n t w h i c h d e p l e t e s CA i m p a i r e d t h e c o n -s o l i d a t i o n o f a p a s s i v e a v o i d a n c e t a s k memory, and t h a t t h i s was r e v e r s e d by DOPA. W h i l e t h e s e d a t a c e r t a i n l y s u p p o r t t h e NA h y p o t h e s i s , o t h e r p h a r m a c o l o g i c a l a g e n t s do n o t y i e l d s u c h e v i d e n c e . A l p h a - m e t h y l - p a r a - t y r o s i n e (AMT), an e f f e c t i v e i n h i b i t o r o f CA s y n t h e s i s does n o t p r o d u c e memory f a i l u r e . T h i s d r u g however does p r o d u c e s t a t e d e p e n d e n t l e a r n i n g ( O v e r t o n , 1 9 6 8 ) . T h i s r e f e r s t o an a p p a r e n t a m n e s i a w h i c h i s s e e n i f t h e r e t e s t i s p e r f o r m e d u n d e r a d r u g c o n d i t i o n d i f f e r e n t t o t h a t o f t h e o r i g i n a l l e a r n i n g . A d m i n i s t r a t i o n o f AMT does n o t p r o d u c e a d e c r e m e n t i n . memory as l o n g as t h e r e t e s t i s p e r f o r m e d a f t e r an a d d i t i o n a l i n j e c t i o n o f AMT. A s i m i l a r e f f e c t has b e e n r e p o r t e d f o r F L A - 6 3 , a DBH i n h i b i t o r , ( A l h e n i u s , 1 9 7 3 ) . T h i s d r u g had no e f f e c t on t h e a c q u i s i -t i o n o r p e r f o r m a n c e o f a c o n d i t i o n e d a v o i d a n c e r e s p o n s e . I t a l s o , h o w e v e r , was shown t o p o s s e s s t h e p o t e n t i a l o f p r o d u c i n g s t a t e d e p e n d e n c y i n l e a r n i n g . T h i s s t a t e d e p e n d e n c y may be s i m i l a r t o t h e r e t r i e v a l o r memory a c c e s s f a i l u r e s p r o p o s e d as t h e d e f i c i t p r o d u c e d by DDC. From t h e p h a r m a c o l o g i c a l d a t a , one t h i n g i s c l e a r . M a n i p u l a t i o n s o f CA t h r o u g h p e r i p h e r a l a d m i n i s t r a t i o n o f - 50 -d r u g s , a l m o s t a l w a y s i n d u c e s a l e a r n i n g , c o n s o l i d a t i o n , o r r e t r i e v a l d e f i c i t . The q u e s t i o n as t o w h e t h e r t h e s e a r e s p e c i f i c and e x c l u s i v e t o CA s y s t e m s r e m a i n s u n a n s w e r e d . A s s u m i n g t h e y a r e , t h e r e i s a c o n s i d e r a b l e l i k e l i h o o d t h a t p e r i p h e r a l a d r e n e r g i c m echanisms p l a y a l a r g e r o l e i n t h e l e a r n i n g p r o c e s s , p a r t i c u l a r l y i n c o n d i t i o n i n g p a r a d i g m s u s i n g a v e r s i v e s t i m u l i . The p r e s e n t r e s e a r c h r e p o r t has d e a l t o n l y w i t h c e n t r a l NA m a n i p u l a t i o n s . C o n c o m m i t a n t p e r i p h e r a l m a n i p u l a t i o n s may a l l o w a b e t t e r u n d e r s t a n d i n g of t h e n a t u r e of NA i n v o l v e m e n t i n t h e l e a r n i n g p r o c e s s . CONCLUSIONS The p r e s e n t e x p e r i m e n t s o f f e r no s u p p o r t t o t h e h y p o t h e s i s t h a t t h e d o r s a l t e g m e n t a l n o r a d r e n e r g i c p r o j e c t i o n s i s of f u n d a m e n t a l i m p o r t a n c e t o t h e l e a r n i n g p r o c e s s , and p h a r m a c o l o g i c a l d a t a w h i c h s u p p o r t s u c h a t h e o r y m i g h t be e x p l a i n e d by d r u g a c t i o n on o t h e r , p o s s i b l y p e r i p h e r a l , a d r e n e r g i c m e c h a n i s m s . - 51 -REFERENCES A h l e n i u s , S. I n h i b i t i o n of catecholamine s y n t h e s i s and c o n d i -t i o n e d avoidance a c q u i s i t i o n . Pharmacology Bio c h e m i s t r y and Be-h a v i o r , 1973, 1:347-350. Amaral, D. G., and Foss, J . 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