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UBC Theses and Dissertations

Anatomical and biochemical organization of the basal ganglia Nagy, James Imre 1979

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ANATOMICAL AND BIOCHEMICAL ORGANIZATION OF THE BASAL GANGLIA by JAMES IMRE NAGY B.Sc. U n i v e r s i t y of B r i t i s h Columbia, 1973 M.Sc. 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 FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY m THE FACULTY OF GRADUATE STUDIES I n t e r d i s c i p l i n a r y Studies, ( N e u r o l o g i c a l Sciences) We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1979 5a James -Imre Nagy, 1979 In present ing t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re ference and s tudy. I f u r t h e r agree that permiss ion f o r ex tens ive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted 'by the Head of my Department or by h i s r e p r e s en t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n pe rm iss i on . Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P lace Vancouver, Canada V6T 1W5 D E - 6 B P 75-51 1 E i i ABSTRACT The biochemistry and anatomy of various n u c l e i of the b a s a l g a n g l i a of the r a t were i n v e s t i g a t e d . The head of the s t r i a t u m was found to p r o j e c t to the a n t e r i o r globus p a l l i d u s (GP) and to the entopeduncular nucleus (EP). A p r o j e c t i o n from the a n t e r i o r s t r i a t u m to the s u b s t a n t i a n i g r a (SN) was con-firmed. The t a i l of the s t r i a t u m was found to p r o j e c t to the p o s t e r i o r part of the GP. No anatomical evidence was obtained f o r a p r o j e c t i o n from the t a i l of the s t r i a t u m to the EP. The p o s t e r i o r s t r i a t u m was found to p r o j e c t to the l a t e r a l SN. B i o c h e m i c a l l y , the presence of glutamic a c i d decarboxylase (GAD) i n the p r o j e c t i o n from the head of the s t r i a t u m to the GP has been confirmed. The head and t a i l of the s t r i a t u m were found to p r o j e c t GAD-containing f i b e r s to the EP. While the absence of n i g r a l GAD-containing a f f e r e n t s o r i g i n a t i n g i n the a n t e r i o r s t r i a t u m has been confirmed, i t was found that the SN does r e -ceive such a f f e r e n t s from more p o s t e r i o r regions of the s t r i a t u m . The SN was found to be devoid of a GAD-containing input from the GP. The EP e f f e r e n t s to the habenula were found to be GAD-containing. P r e l i m i n a r y evidence was ob-t a i n e d f o r the presence of c h o l i n e r g i c f i b e r s i n the s t r i a t a l p r o j e c t i o n s to the GP and EP. The c e l l u l a r l o c a l i z a t i o n of v a r i o u s enzyme systems i n the SN and of the dopamine (DA) receptor i n the SN and s t r i a t u m was i n v e s t i g a t e d using k a i n i c a c i d and 6-hydroxydopamine l e s i o n techniques. I t was concluded that c h o l i n e a c e t y l t r a n s f e r a s e and DA-sensitive adenylate cy c l a s e are contained i n n i g r a l a f f e r e n t s , a c e t y l c h o l i n e s t e r a s e i s contained i n both n i g r a l a f f e r e n t s and i n -t r i n s i c neuronal elements and t y r o s i n e hydroxylase i s contained i n n i g r a l p e r i k a r y a . The r e s u l t s concerning n i g r a l GAD were i n c o n c l u s i v e . Evidence was obtained f o r the existence of DA receptors on DA-containing neurons i n the SN and t h e i r t e rminals i n the s t r i a t u m . i i i TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v i i LIST OF FIGURES v i i i ABBREVIATIONS i x ACKNOWLEDGEMENTS x i PREFACE x i i i INTRODUCTION • 1 I General Review of the Basal Ganglia 1 a) The s t r i a t u m 1 1. i n t e r n a l o r g a n i z a t i o n 1 2. s t r i a t a l a f f e r e n t s 3 3. s t r i a t a l e f f e r e n t s 6 b) The globus p a l l i d u s 9 1. i n t e r n a l o r g a n i z a t i o n 9 2. p a l l i d a l a f f e r e n t s 11 3. e f f e r e n t s of the medial p a l l i d a l segment 15 4. e f f e r e n t s of the l a t e r a l p a l l i d a l segment 18 5. a c e t y l c h o l i n e s t e r a s e s t a i n i n g neurons of the globus p a l l i d u s 24 c) Subthalamic nucleus 24 d) Substantia n i g r a 25 1. i n t e r n a l o r g a n i z a t i o n 25 2. n i g r a l e f f e r e n t s 28 3. n i g r a l a f f e r e n t s 32 I I Adenylate Cyclase and Dopamine Receptors i n the Substantia N i g r a and Striatum 34 I I I The Present I n v e s t i g a t i o n 40 a) Methodological c o n s i d e r a t i o n s 40 b) Objectives 43 METHODS AND MATERIALS 45 I Animal Surgery 45 a) 6-Hydroxydopamine l e s i o n s of the n i g r o - s t r i a t a l pathway 45 b) K a i n i c a c i d l e s i o n s 46 c) Hemitransections 47 d) E l e c t r o l y t i c l e s i o n s 47 I I Anatomical Methods 48 a) Autoradiographic s t u d i e s 48 b) Retrograde t r a n s p o r t of hor s e r a d i s h peroxidase 48 c) D i s s e c t i o n s 49 d) H i s t o l o g y 50 I I I Biochemical Methods 50 a) Glutamic a c i d decarboxylase 50 b) Tyrosine hydroxylase 51 c) Choline a c e t y l t r a n s f e r a s e and a c e t y l c h o l i n e s t e r a s e 51 d) Adenylate c y c l a s e 52 e) P r o t e i n assay and s c i n t i l l a t i o n counting 53 f) Tissue packaging 53 RESULTS 53 I Neuroanatomy 53 a) A n t e r i o r s t r i a t a l e f f e r e n t s to the globus p a l l i d u s , entopeduncular nucleus and s u b s t a n t i a n i g r a 53 b) P o s t e r i o r s t r i a t a l e f f e r e n t s to the globus p a l l i d u s , entopeduncular nucleus and s u b s t a n t i a n i g r a 63 I I Biochemical Neuroanatomy 68 a) The c o n t r i b u t i o n of the head of the V s t r i a t u m to v a r i o u s neurotransmitter enzyme markers i n the globus p a l l i d u s , entopeduncular nucleus and s u b s t a n t i a n i g r a 68 b) The c o n t r i b u t i o n of the t a i l of the s t r i a t u m to v a r i o u s neurotransmitter enzyme markers i n the globus p a l l i d u s , entopeduncular nucleus and s u b s t a n t i a n i g r a 71 c) The c o n t r i b u t i o n of the globus p a l l i d u s to v a r i o u s neurotransmitter enzyme markers i n the entopeduncular nucleus and s u b s t a n t i a n i g r a 74 d) E f f e r e n t s of the entopeduncular nucleus 94 I I I Biochemical I n v e s t i g a t i o n s of Substantia N i g r a and Striatum 94 a) Neurotransmitter s y n t h e t i c enzyme l o c a l i z a t i o n i n . the s u b s t a n t i a n i g r a 94 b) Neurotransmitter receptor l o c a l i z a -t i o n i n the s u b s t a n t i a n i g r a and s t r i a t u m 104 DISCUSSION 111 I Anatomy of the S t r i a t o - P a l l i d a l and S t r i a t o - N i g r a l P r o j e c t i o n s 111 I I Biochemical Neuroanatomy of S t r i a t a l and P a l l i d a l E f f e r e n t P r o j e c t i o n s 114 a) S t r i a t o - p a l l i d a l p r o j e c t i o n s ; glutamic a c i d decarboxylase 114 b) S t r i a t o - p a l l i d a l p r o j e c t i o n s ; c h o l i n e a c e t y l t r a n s f e r a s e 116 c) S t r i a t o - n i g r a l p r o j e c t i o n s 121 d) P a l l i d o - n i g r a l p r o j e c t i o n s 123 I I I S t r i a t a l and P a l l i d a l E f f e r e n t s : Synthesis and Sp e c u l a t i o n 125 a) Topographic r e l a t i o n s 126 b) c) S t r i a t a l p r o j e c t i o n neurons Neurotransmitters i n s t r i a t a l 129 vx and p a l l i d a l e f f e r e n t s 130 d) Biochemical neuroanatomy of s t r i a t a l and p a l l i d a l e f f e r e n t s 133 IV P r e l i m i n a r y Observations of N i g r a l and Entopeduncular E f f e r e n t s 136 V The L o c a l i z a t i o n of Enzymes i n the Substantia N i g r a 138 VI The L o c a l i z a t i o n of Dopamine A c t i v a t e d Receptors and Adenylate Cyclase 140 a) Dopamine-sensitive adenylate c y c l a s e i n the s u b s t a n t i a n i g r a 140 b) Pre- and po s t - s y n a p t i c dopamine receptors i n the s u b s t a n t i a n i g r a and s t r i a t u m 142 c) Dopamine trans m i s s i o n i n the su b s t a n t i a n i g r a and s t r i a t u m : s y n t h e s i s and s p e c u l a t i o n 146 CONCLUSIONS 147 REFERENCES 150 V l l LIST OF TABLES Table 1 The a c t i v i t y of GAD i n v a r i o u s areas a f t e r l e s i o n s of the head of the s t r i a t u m . Table 2 The a c t i v i t y of GAD and CAT i n the GP, EP and SN a f t e r hemitran-s e c t i o n s a n t e r i o r to the GP. Table 3 . The a c t i v i t y of GAD and CAT i n the GP and t a i l of the s t r i a t u m a f t e r hemitransections a n t e r i o r to the GP. Table 4 The a c t i v i t y of GAD and CAT i n v a r i o u s areas a f t e r l e s i o n s of the t a i l of the s t r i a t u m . Table 5 The a c t i v i t y of GAD and CAT i n the EP and SN a f t e r e l e c t r o l y t i c l e s i o n s of the GP. Table 6 The a c t i v i t y of GAD, CAT and TH i n v a r i o u s areas a f t e r k a i n i c a c i d l e s i o n s of the GP. Table 7 The a c t i v i t y of GAD and CAT i n the habenula a f t e r e l e c t r o l y t i c l e s i o n s of the EP. Table 8 The a c t i v i t i e s of v a r i o u s enzymes a f t e r i n t r a n i g r a l i n j e c t i o n s of k a i n i c a c i d . Table 9 The e f f e c t of 6-0HDA l e s i o n s of the NSP and k a i n i c a c i d l e s i o n s of the s t r i a t u m on v a r i o u s enzymes i n the SN and s t r i a t u m . Table 10 3H-apomorphine b i n d i n g i n the SN and s t r i a t u m . Table 11 3 H - n e u r o l e p t i c b i n d i n g i n the SN and s t r i a t u m . y i x x LIST OF FIGURES Figure 1 I n j e c t i o n locus of 3 H - l e u c i n e i n the head of the s t r i a t u m . Figure 2 Anterograde t r a n s p o r t of 3 H - l e u c i n e from the head of the s t r i a t u m . Figure 3 Diagram of autoradiographic g r a i n d i s t r i b u t i o n a f t e r 3 H - l e u c i n e i n j e c t i o n s i n t o the head of the s t r i a t u m . Figure 4 Retrograde t r a n s p o r t of HRP from the GP and EP. Figure 5 Anterograde t r a n s p o r t of 3 H - l e u c i n e from the t a i l of the s t r i a t u m . Figure 6 Diagram of autoradiographic g r a i n d i s t r i b u t i o n a f t e r 3 H - l e u c i n e i n j e c t i o n i n t o the t a i l of the s t r i a t u m . Figure 7 Diagram of e l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m . Figure 8 Diagram of e l e c t r o l y t i c l e s i o n s of the GP. Figure 9 Diagram of k a i n i c a c i d l e s i o n s of the GP. Figure 10 Photomicrographs of e l e c t r o l y t i c and k a i n i c a c i d l e s i o n s . Figure 11 L o c a t i o n of a c e t y l c h o l i n e s t e r a s e - s t a i n i n g neurons i n the GP. Figure 12 Diagram of the l o c a t i o n of l a b e l e d neurons i n the GP a f t e r i n t r a -n i g r a l i n j e c t i o n s of HRP. Figure 13 Photomicrograph of HRP l a b e l e d neurons i n the GP. Figure 14 Photomicrograph of e l e c t r o l y t i c l e s i o n s of the EP. Figure 15 H i s t o l o g y of normal and k a i n i c a c i d - i n j e c t e d SN. Figure 16 Dopamine-sensitive adenylate c y c l a s e i n the SN. Figure 17 Scatchard p l o t of 3H-apomorphine bindi n g i n the s t r i a t u m . i x ABBREVIATIONS AC a n t e r i o r commissure ACh a c e t y l c h o l i n e AChE a c e t y l c h o l i n e s t e r a s e AMPH amphetamine cAMP adenosine c y c l i c 3',5'-monophosphate CAT c h o l i n e a c e t y l t r a n s f e r a s e CM centromedian nucleus CNS c e n t r a l nervous system DA dopamine DAC dopamine-sensitive adenylate c y c l a s e DBH dopamine-3-hydroxylase DRN d o r s a l raphe nucleus EM e l e c t r o n microscope EP entopeduncular nucleus EPSP e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l F f o r n i x FR f a s c i c u l u s r e t r o f l e x u s GABA gamma-aminobutyric a c i d GAD glutamic a c i d decarboxylase GP globus p a l l i d u s HRP h o r s e r a d i s h peroxidase IC i n t e r n a l capsule IP interpeduncular nucleus IPSP i n h i b i t o r y p o s t s y n a p t i c p o t e n t i a l LC locus coeruleus LGP globus p a l l i d u s , l a t e r a l segment (primate) MGP globus p a l l i d u s , medial segment (primate) ML medial lemniscus X ABBREVIATIONS (Cont'd) MMT mammillo-thalamic t r a c t MRN medial raphe nucleus NA noradrenaline NSP DA-containing n i g r o - s t r i a t a l pathway OT o p t i c t r a c t 6-OHDA 6-hydroxydopamine PF p a r a f a s c i c u l a r nucleus SC sup e r i o r c o l l i c u l u s SM s t r i a medularis St s t r i a t u m SN s u b s t a n t i a n i g r a SNC s u b s t a n t i a n i g r a , pars compacta SNR s u b s t a n t i a n i g r a , pars r e t i c u l a t a SUT subthalamic nucleus TH t y r o s i n e hydroxylase TPP nucleus tegmenti pedunculopontis VAp v e n t r a l a n t e r i o r thalamic nucleus, p r i n c i p a l part VM v e n t r a l medial thalamic nucleus (subprimate) VLo v e n t r a l l a t e r a l thalamic nucleus, o r a l part VTA ve n t r a l tegmental area x i ACKNOWLEDGEMENT S Someone once s a i d " I have only myself to blame f o r my f a i l u r e but many people to thank f o r my successes". This i s very t r u e i n my case. To begin, s e v e r a l years ago, the teaching a b i l i t y of Dr. Don C l a r k was second to none. He sparked i n t e r e s t and enthusiasm and brought a s c h o l a r l y a t t i t u d e to research which I w e l l remember. U n f o r t u n a t e l y , the chemistry department f a i l e d to grant Dr. C l a r k tenure. Even i f such i s the reward f o r a b i l i t y and s c h o l a r s h i p , I would be glad to f o l l o w i n Don Cl a r k ' s wake. While I was a Masters' student i n biochemistry, Dr. S.C. Sung taught me how to keep a l a b o r a t o r y . T h i s , though, was the l e a s t of h i s c o n t r i b u t i o n s to me. His continued, h e a r t f e l t concern f o r my personal w e l l - b e i n g and career was always appreciated. His p r a i s e , to the po i n t of my embarrassment, was both i n s p i r i n g and r e a s s u r i n g , e s p e c i a l l y to the insecure son of an immigrant Hungarian farmer. I consider myself f o r t u n a t e that my contemporaries and c o l l a b o r a t o r s , Steven Vincent and W i l l i a m S t a i n e s , have the i n g r e d i e n t s e s s e n t i a l i n any work-in g environment. T h e i r i n t e r e s t i n t h e i r work i s a u t h e n t i c . T h e i r keen eye f o r problem s o l v i n g i s unmistable. T h e i r d i s p o s i t i o n s are c h e e r f u l . By these q u a l i t i e s they have created an atmosphere i n which i t i s a d e l i g h t to work. I am glad to have known them. I would a l s o l i k e to express my g r a t i t u d e to my f e l l o w students f o r bearing w i t h me on those occassions when I could be a r e a l p a i n i n the ass ay. I am g r a t e f u l to Dr. Stephen Mason f o r b r i n g i n g to me from the other s i d e of the A t l a n t i c h i s measure of encouragement and support. A l s o f o r the i n t e -r e s t i n g and endless d i s c u s s i o n s and l i q u i d consumables; I can't remember which was more i n t e r e s t i n g and which was more endless. I f r e e l y admit t h a t the t e c h n i c a l e x p e r t i s e of S t e l l a Atmadja i s f a r more advanced than mine and because of t h i s her; a s s i s t a n c e i n the present endea-vours has proven to be i n v a l u a b l e . Thank you, S t e l l a , f o r your hard work and x i i and your patience w i t h me. I would l i k e to express my thanks to the members of my committee f o r sug-gestions that markedly enhanced the l i t e r a r y value of t h i s t h e s i s . F i n a l l y , I owe a great debt to my t h e s i s a d v i s o r , Dr. Hans C. F i b i g e r , without whom the path I might have taken would have been q u i t e d i f f e r e n t i n -deed. Dr. F i b i g e r i s exemplary of my conception of an e x c e l l e n t t h e s i s a d v i -so r . He has allowed me to make and l e a r n from my own mistakes w h i l e at the same time p r o v i d i n g guidance when my own r e s o u r c e f u l n e s s was exhausted. He has been r e c e p t i v e and w i l l i n g to d i s c u s s both good and bad i d e a s , always a b l e , of course, to choose the former. Whether the s i t u a t i o n looked bleak or other-wise he has c o n s t a n t l y been quick to encourage. The output of personal energy and time, the high q u a l i t y of work r e q u i r e d i n research and c e r t a i n i d e a l s have been taught to Dr. F i b i g e r ' s students by example. I have b e n e f i t e d from my a s s o c i a t i o n w i t h him enormously and am honored to have been one of h i s s t u -dents. I ' l l never be able to repay Dr. F i b i g e r f o r everything he has done f o r me but h o p e f u l l y I w i l l be given the opportunity to t r y . Perhaps the highest compliment a student can give h i s teacher i s to say w i t h c e r t a i n t y that i n the f u t u r e I w i l l be asking myself "Now, how would C h r i s have handled t h i s ? " x i i i PREFACE B r a i n research i s n e c e s s a r i l y an i n t e r d i s c i p l i n a r y f i e l d . I n s p i t e of the tremendous t e c h n i c a l advances i n each of the d i s c i p l i n e s c o n t r i b u t i n g to the growing body of knowledge of the machinery of the b r a i n , there remain precious few hard and f a s t r u l e s on which to hinge conceptual frameworks and which could provide g u i d e l i n e s f o r f u t u r e work of the ki n d that would provide major i n s i g h t s . For t h i s reason, i t i s of some advantage that a student o f , f o r example, the b a s a l g a n g l i a be able to draw upon i n f o r m a t i o n a v a i l a b l e i n many f i e l d s during any c o n s i d e r a t i o n of b a s a l g a n g l i a f u n c t i o n . While a formula-t i o n of a meaningful construct of the f u n c t i o n of t h i s part of the b r a i n may, at present, be u n s u c c e s s f u l , an attempt at the condensation and syn t h e s i s of a v a i l a b l e data may at l e a s t be worthwhile. Such an attempt has been made i n the I n t r o d u c t i o n of the present t h e s i s . I t has been learned by the present author ( r e l i a b l e sources, 1977) that works such as a d o c t o r a l t h e s i s provide a ra r e opportunity to extend the i n -t e r p r e t a t i o n of experimental data beyond the c o n s t r a i n t s of current knowledge. Some s p e c u l a t i v e l i c e n c e i s th e r e f o r e taken i n the d i s c u s s i o n of the present t h e s i s . 1 INTRODUCTION I General Review of the Bas a l Ganglia a) The s t r i a t u m 1. i n t e r n a l o r g a n i z a t i o n The apparent homogeneous i n t e r n a l o r g a n i z a t i o n of the s t r i a t u m (caudate-putamen) has made d i f f i c u l t the study of the d e t a i l e d anatomical r e l a t i o n s h i p s between t h i s s t r u c t u r e and as s o c i a t e d b r a i n areas. Nevertheless, the s t r i a t u m has y i e l d e d to some i n v e s t i g a t i o n s probing i t s s t r u c t u r a l framework. In general, the neurons i n the s t r i a t u m have been c l a s s i f i e d i n t o three types on the b a s i s of s i z e (Kemp and P o w e l l , 1971a; H a s s l e r , 1978). These are: 1) gi a n t aspiny neurons re p r e s e n t i n g l e s s than 1% of the t o t a l neuronal popu-l a t i o n , 2) sm a l l neurons w i t h few s p i n e s , a l s o present i n numbers l e s s than 1% of the t o t a l , and 3) medium s i z e d spiny neurons which c o n s t i t u t e greater than 95% of the t o t a l neuronal pool of the s t r i a t u m . The medium spiny c e l l s can be c l a s s i f i e d f u r t h e r on the b a s i s of spine d e n s i t y and den d r i t e and axon morphology (Kemp and P o w e l l , 1971a). Greater than 96% of these neurons have de n d r i t e s h e a v i l y laden w i t h spines and nume-rous axon c o l l a t e r a l s (Kemp and P o w e l l , 1971a). The aspiny neurons have al s o been f u r t h e r c l a s s i f i e d i n t o three types ( D i f i g l i a et a l , 1976); two of these are of the small v a r i e t y and the t h i r d i s the l a r g e aspiny neuron described by Kemp and Powell (1971a). With regard to i n v e s t i g a t i o n s of the arrangement of s t r i a t a l neurons, although some c e l l c l u s t e r i n g has been observed (Kemp and Po w e l l , 1971a; Mensah, 1977; Ch r o n i s t e r et a l , 1976), such st u d i e s have not revealed any s t r i k i n g f eatures of neuronal o r g a n i z a t i o n of .the s t r i a t u m ; never-t h e l e s s , they do suggest a degree of morphological d i v e r s i t y w i t h i n t h i s s t r u c -t u r e . The degree of morphological heterogeneity of the s t r i a t u m and of any b r a i n r e g i o n becomes an important c o n s i d e r a t i o n not only i n the i n t e r p r e t a t i o n of the o r g a n i z a t i o n and topography of a f f e r e n t s to the s t r i a t u m but a l s o i n neurochemical a n a l y s i s . For example, a f t e r production of any manner of l e s i o n , 2 p a r t i c u l a r l y an incomplete l e s i o n , the neurochemical consequences of the l e -s i o n may be :extrapolated to the whole i n the case of a homogeneous s t r u c t u r e . This c l e a r l y i s not the case f o r a heterogeneous s t r u c t u r e where each compo-nent must be t r e a t e d s e p a r a t e l y . This p o i n t has some bearing on the present r e s u l t s and w i l l be d e a l t w i t h f u r t h e r i n subsequent s e c t i o n s . Analogous w i t h the search f o r some r e g u l a r i t y i n the arrangement of neu-rons i n the s t r i a t u m , very l i t t l e i s known of the o r g a n i z a t i o n and extent of the i n t r i n s i c l o n g - or short-axon connections i n t h i s s t r u c t u r e . Degeneration of symmetrical and asymmetrical s y n a p t i c contacts have been observed only as f a r as 0.5 and 1.0 mm, r e s p e c t i v e l y , from the s i t e of a s m a l l l e s i o n i n the s t r i a t u m (Kemp and P o w e l l , 1971a). V i r t u a l complete s u r g i c a l i s o l a t i o n of the s t r i a t u m r e s u l t s i n the p e r s i s t e n c e of symmetrical and asymmetrical s y n a p t i c contacts i n t h i s s t r u c t u r e and i t has been concluded that a l l of the former s y n a p t i c type i s of i n t r i n s i c o r i g i n w h i l e the l a t t e r type i s of both i n t r i n -s i c and e x t r i n s i c o r i g i n (Kemp and P o w e l l , 1971a; Tennyson and Marco, 1973). I n t e r e s t i n g l y , r a t h e r than being s c a t t e r e d homogeneously throughout the s t r i a -t a l n e u r o p i l , i n t r i n s i c synapses have been observed to be arranged i n groups (Tennyson and Marco, 1973). I t should be mentioned that c u r r e n t l y the view i s h e l d by some that the nucleus accumbens i s a medial extension of the s t r i a t u m (Nauta et a l , 1978; Swanson and Cowan, 1975). T h i s i s based on the c y t o a r c h i t e c t o n i c appearance, connections and development of the s t r i a t u m and nucleus accumbens. Although these two areas have s i m i l a r anatomical connections, some d i f f e r e n c e s do e x i s t (Nauta et a l , 1978). The apparent grouping of these areas may simply depend on the c r i t e r i a of c l a s s i f i c a t i o n chosen. Although the nucleus accum-bens and s t r i a t u m may have s i m i l a r f u n c t i o n a l and processing c a p a b i l i t i e s , the i n f o r m a t i o n acted on by the two s t r u c t u r e s may d i f f e r . Since evidence bearing on t h i s p o i n t i s p r e s e n t l y not a v a i l a b l e , the nucleus accumbens and s t r i a t u m , f o r the sake of convenience i f not accuracy, w i l l be t r e a t e d as 3 d i s t i n c t e n t i t i e s . The s t r i a t u m contains numerous p u t a t i v e neurotransmitters and t h e i r meta-b o l i z i n g enzymes. These biochemical systems have been a t t r i b u t e d to the v a r i -ous a f f e r e n t or i n t r i n s i c neuronal elements of the s t r i a t u m . Although i n t e r -neurons c o n t a i n i n g other t r a n s m i t t e r s probably e x i s t i n the s t r i a t u m , the l e a s t e q u i v o c a l evidence f o r an i n t e r n e u r o n a l l o c a l i z a t i o n of s p e c i f i c t r a n s -m i t t e r systems i s that f o r the s y n t h e t i c enzymes of a c e t y l c h o l i n e (ACh) -c h o l i n e a c e t y l t r a n s f e r a s e (CAT) - and gamma-aminobutyric a c i d (GABA) - g l u t a -mic a c i d decarboxylase (GAD) (McGeer et a l , 1971a; H a t t o r i et a l , 1976; McGeer and McGeer, 1975). C o r r e l a t i o n s of the c e l l types c o n t a i n i n g CAT i n the s t r i a -tum have been attempted. Thus, i t appears that medium s i z e d neurons s t a i n immunohistochemically ( H a t t o r i et a l , 1976) and c y t o h i s t o c h e m i c a l l y f o r CAT (Kaiya et a l , 1979). 2. s t r i a t a l a f f e r e n t s The s t r i a t u m r e c e i v e s a f f e r e n t s from a number of d i v e r s e areas of the b r a i n . The l a r g e s t of these i s the a f f e r e n t system from a l l p a r t s of the c e r e b r a l cortex (Webster, 1961, 1965; Heimer and Wilson, 1975; Goldman and Nauta, 1977; Carmen et a l , 1963; Kemp and Po w e l l , 1970; Jones et a l , 1977). According to Kemp and Powell (1971b) the cortex s u p p l i e s 30 to 40% of the t e r -minals of e x t r i n s i c o r i g i n to the s t r i a t u m . 'These terminals form s y n a p t i c con-t a c t s that have membrane thickenings e x c l u s i v e l y on the post s y n a p t i c s t r u c t u r e . This type of sy n a p t i c contact i s r e f e r r e d to as asymmetrical. Degeneration s t u d i e s suggested (Kemp and Po w e l l , 1971b) and autoradiographic transport s t u -d i e s d r a m a t i c a l l y demonstrated (Goldman and Nauta, 1977; Jones et a l , 1977; Y e t e r i a n and Hoesen, 1978) a discontinuous nature of the te r m i n a t i o n of the c o r t i c o - s t r i a t a l f i b e r s . Thus, i n the s t r i a t u m the c o r t i c a l a f f e r e n t s d i s t r i -bute themselves i n c l u s t e r s , s t r i p s and bands. The t r a n s m i t t e r of the c o r t i c o -s t r i a t a l system i s thought to be glutamate. This has been suggested on the b a s i s of the f i n d i n g s t h a t c o r t i c a l l e s i o n s reduce the high a f f i n i t y uptake of 4 glutamate (McGeer et a l , 1977; Fonnum and Storm-Mathlsen, 1977) and glutamate l e v e l s (Kim et a l , 1977) i n the s t r i a t u m . E l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n s have shown the c o r t i c o - s t r i a t a l pathway to be e x c i t a t o r y ; c o r t i c a l s t i m u l a t i o n inducing monosynaptic e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l s (EPSP's) i n s t r i a t a l neurons ( K i t a i et a l , 1976a, 1976b; K o c s i s et a l , 1977). This i s i n accord w i t h the e x c i t a t o r y nature of glutamate on s t r i a t a l c e l l s (Spencer, 1976) and w i t h the hypothesis that glutamate may be the t r a n s m i t t e r of t h i s system. There i s a l s o e l e c t r o p h y s i o l o g i c a l evidence that the c o r t i c o - s t r i a t a l f i b e r s are independent of the c o r t i c o - s p i n a l or c o r t i c o - b u l b a r systems ( K i t a i et a l , 1976b). This i s i n agreement w i t h the d i f f e r e n t l o c a t i o n w i t h i n the c o r t i c a l l a y e r s of s t r i a t a l a f f e r e n t s ( l a y e r I I I , K i t a i et a l , 1976b; l a y e r V, Jones et a l , 1977) and s p i n a l and bulbar a f f e r e n t s ( l a y e r I V ) . The thalamus i s second only to the cortex w i t h regard to the d e n s i t y of asymmetrical s y n a p t i c terminals i t s u p p l i e s to the s t r i a t u m (20 to 25%,'Kemp and P o w e l l , 1971b). The neurons of o r i g i n of the t h a l a m o - s t r i a t a l system are lo c a t e d p r i m a r i l y i n the p a r a f a s c i c u l a r and centromedian i n t r a l a m i n a r thalamic n u c l e i (Jones et a l , 1977; Powell and Cowan, 1967; Johnson, 1961; Kuroda et a l , 1975) although a l l of the i n t r a l a m i n a r n u c l e i appear to p r o j e c t to the s t r i a -tum (Jones and L e a v i t t , 1974). The te r m i n a t i o n w i t h i n the s t r i a t u m of these thalamic n u c l e i (Kemp and P o w e l l , 1971b), p a r t i c u l a r l y the centromedian, ( K a l i l , 1978; Royce, 1978) not to be outdone by the c o r t i c a l a f f e r e n t s , a l s o e x h i b i t a s t r i k i n g array of c o n f i g u r a t i o n s from pat ches^arid bands to s e m i c i r c l e s . There are no candidates f o r the t r a n s m i t t e r of the t h a l a m o - s t r i a t a l system. A decrease i n CAT a c t i v i t y and ACh l e v e l s has been observed i n the head of the s t r i a t u m a f t e r p a r a f a s c i c u l a r nucleus l e s i o n s suggesting a c h o l i n e r g i c compo-nent i n t h i s p r o j e c t i o n (Simke and Saelens, 1977; Saelens et a l , 1979). T h i s , however, i s suspect s i n c e the a n t e r i o r o n e - t h i r d of the s t r i a t u m r e c e i v e s few, i f any, a f f e r e n t s from the p a r a f a s c i c u l a r nucleus (Jones and L e a v i t t , 1974). Thalamic s t i m u l a t i o n evokes d e p o l a r i z a t i o n responses i n the s t r i a t u m (Kocsis 5 et a l , 1977; Purpura and M a l l i a n i , 1967; M a l l i a n i and Purpura, 1967). In a d d i -t i o n , evidence has been presented (Kocsis et a l , 1977) that there i s consider-able convergence of c o r t i c a l and thalamic e f f e r e n t f i b e r s on medium spiny neu-rons i n the s t r i a t u m . The i m p l i c a t i o n s , i f not the means of proof, of the patchy d i s t r i b u t i o n of both c o r t i c a l and thalamic s t r i a t a l a f f e r e n t s and the convergence of these systems on the same neurons are obvious. The most i n -t r i g u i n g of these i s the p o s s i b i l i t y of a v a r i e t y of c l o s e d loop c i r c u i t s w i t h i n and/or between the b a s a l g a n g l i a , thalamus and cortex. Three monoamine-containing systems have been shown to innervate the s t r i a -tum. The r i c h e s t of these i s that o r i g i n a t i n g from the s u b s t a n t i a n i g r a (SN), pars compacta (SNC) and w i l l be discussed together w i t h SN e f f e r e n t s . A second monoamine system i n n e r v a t i n g the s t r i a t u m o r i g i n a t e s from the mesencephalic raphe nucleus (Pasquier et a l , 1977; Moore et a l , 1978) and, s p e c i f i c a l l y , the d o r s a l raphe nucleus (DRN) ( M i l l e r et a l , 1975; A z m i t i a and . Segal, 1978; Jacobs et a l , 1978). Consistent w i t h the f i n d i n g s that the raphe n u c l e i are r i c h i n s e r o t o n i n - c o n t a i n i n g neurons, l e s i o n s of raphe n u c l e i (Kuhar et a l , 1972) and, more p r e c i s e l y , the DRN reduce s e r o t o n i n l e v e l s and tryptophan hydroxylase - the s y n t h e s i z i n g enzyme f o r s e r o t o n i n - a c t i v i t y i n the s t r i a t u m (Geyer et a l , 1976; Dray et a l , 1978). Although anatomical s t u -d i e s i n d i c a t e a uniform i n n e r v a t i o n of the s t r i a t u m by the DRN (Moore et a l , 1978), i t appears from biochemical s t u d i e s that s e r o t o n i n t e r m i n a l s are mainly l o c a l i z e d i n the ventrocaudal area of t h i s s t r u c t u r e (Ternaux et a l , 1977). S t i m u l a t i o n of the DRN produces i n h i b i t i o n of neurons of the s t r i a t u m ( M i l l e r et a l , 1975; Davies and Tongroach, 1978) and t h i s i n h i b i t i o n i s presumably me-d i a t e d by s e r o t o n i n s i n c e i t was abolished by the s e r o t o n i n antagonist methy-sergide (Davies and Tongroach, 1978). A minor, but nevertheless a u t h e n t i c a t e d , noradrenaline (NA)-containing p r o j e c t i o n to the s t r i a t u m occurs from the locus coeruleus (LC) (Moore, 1978; 6 Mason and F i b i g e r , i n p r e s s ) . This p r o j e c t i o n must indeed be sparse as the l e v e l of NA i n the s t r i a t u m i s low ( F a r l e y and Hornykiewicz, 1977) or non-de-t e c t a b l e (Versteeg et a l , 1976) and l e s i o n s of the locus coeruleus f a i l e d to reduce dopamine-g-hydroxylase (DBH) - the s y n t h e s i z i n g enzyme of NA - i n the s t r i a t u m (Ross and R e i s , 1974). 3. s t r i a t a l e f f e r e n t s One of the major e f f e r e n t p r o j e c t i o n s of the s t r i a t u m i s that to the SN. This f i b e r system was recognized by e a r l y workers (Rundles and Papez, 1937; Riese, 1924; Ranson and Ranson, 1941). Subsequent s t u d i e s using the Nauta method or i t s Fink-Heimer m o d i f i c a t i o n supported those e a r l y observations and i n d i c a t e d that the p r o j e c t i o n was t o p o g r a p h i c a l l y organized ( N i i m i et a l , 1970; Voneida, 1960; Johnson, 1961; Johnson and Rosvold, 1971; Szabo, 1962, 1967, 1970; M i c k l e , 1976). These s t u d i e s a l l s t r e s s e d that the s i t e of t e r m i n a t i o n of s t r i a t a l e f f e r e n t s i n the s u b s t a n t i a n i g r a was p r i n c i p a l l y the p a r s . r e t i c u -l a t a (SNR) , although, Nauta and Mehler (1966) d i d observe some t e r m i n a t i o n i n the SNC. I n v e s t i g a t o r s applying e l e c t r o n microscopy and the more recent auto-r a d i o g r a p h i c and h o r s e r a d i s h peroxidase (HRP) t r a c i n g methods to the a n a l y s i s of t h i s p r o j e c t i o n have confirmed and extended these e a r l i e r s t u d i e s (Hajdu et a l , 1973; Kemp, 1970; Grofova and R i n v i k , 1970; Kanazawa et a l , 1976). I t has become evident that s t r i a t a l e f f e r e n t s to the SN a r i s e from a l l p a r t s of the s t r i a t u m except a c e n t r a l core r e g i o n (Richardson et a l , 1977; Bunney and Aghajanian, 1976a). In a d d i t i o n , such s t u d i e s provided c l e a r evidence of a p r o j e c t i o n to the SNC ( H a t t o r i et a l , 1975; Bunney and Aghajanian, 1976a; T u l l o c h et a l , 1978) and f u r t h e r i n d i c a t e d some topographical arrangement. The exact topographical arrangement, however, has been d i f f i c u l t to d i s c e r n . For example, i n the s t r i c t e s t sense, i t i s impossible to c o r r e l a t e topographi-c a l r e l a t i o n s h i p s from degeneration or autoradiographic p r o f i l e s i n the SN when l e s i o n s or i n j e c t i o n s of l a b e l , r e s p e c t i v e l y , are s h i f t e d i n more than one plane i n the s t r i a t u m . T h i s , notwithstanding, the d o r s a l - v e n t r a l dimension 7 i n the s t r i a t u m appears to be„ superimposed on the v e n t r a l - d o r s a l aspect of the SN; the a n t e r i o r - p o s t e r i o r s t r i a t a l plane may be represented i n the a n t e r i o r -p o s t e r i o r as w e l l as m e d i a l - l a t e r a l plane i n the SN. Although obviously not a l t o g e t h e r d e f i n i t i v e , such t o p o g r a p h i c a l r e l a t i o n s are of considerable i n t e -r e s t , p a r t i c u l a r l y i n view of recent f i n d i n g s by F a u l l and Mehler (1978). They suggest that the f u n c t i o n a l topography of the c o r t i c o - s t r i a t e system i s f a i t h f u l l y conserved i n the s t r i a t o - n i g r a l system, such that s t r i a t a l areas which r e c e i v e motor and v i s u a l input from the c o r t e x p r o j e c t to n i g r a l regions that have e f f e r e n t s to the thalamus and SC, r e s p e c t i v e l y . Of course, topo-g r a p h i c a l overlap does not n e c e s s a r i l y i n d i c a t e s y n a p t i c convergence. I f , however, these i n i t i a l f i n d i n g s are born out by demonstrations of d i r e c t synap-t i c r e l a t i o n s e i t h e r by e l e c t r o p h y s i o l o g i c a l or m u l t i l a b e l i n g anatomical tech-niques, then, i n the o p i n i o n of the present author, these advances would r e -present a s i g n i f i c a n t breakthrough i n understanding the o r g a n i z a t i o n of the b a s a l g a n g l i a . I t i s not c l e a r whether the s t r i a t o - n i g r a l pathway i s i n h i b i t o r y or e x c i -t a t o r y or whether i t contains f i b e r s mediating both types of these a c t i o n s . Constant l a t e n c y , i n h i b i t o r y as w e l l as e x c i t a t o r y , responses i n the SN have been reported to occur f o l l o w i n g s t r i a t a l s t i m u l a t i o n (Ferger and Ohye, 1975; Dray et a l , 1976a). I n t r a c e l l u l a r observations of short l a t e n c y EPSP's i n n i g r a l neurons l e d F r i g y e s i and Purpura (1967) to suggest a monosynaptic e x c i -t a t o r y component of the s t r i a t o - n i g r a l pathway. These s t r i a t a l evoked e x c i t a -t o r y events i n the SN are i n marked c o n t r a s t i n f i n d i n g s of the e x c l u s i v e l y i n h i b i t o r y monosynaptic response observed by Yoshida and Precht (1971) and Yoshida et a l (1972). In the study by Yoshida et a l (1972), evidence was presented that the i n -h i b i t o r y s t r i a t o - n i g r a l f i b e r s send c o l l a t e r a l s to the GP. K i t a i et a l (1975) have reported that s t r i a t a l neurons p r o j e c t i n g to the SN are not d i r e c t l y i n -nervated by the n i g r o ^ s t r i a t a l f i b e r s . In a d d i t i o n , Richardson et a l (1977) 8 have shown that s t r i a t a l neurons that are d i r e c t l y innervated by SNC neurons a l s o give r i s e to GP a f f e r e n t s which may be e x c i t a t o r y . These f i n d i n g s t o -gether can be taken to suggest, i n s o f a r as i t i s p o s s i b l e to g e n e r a l i z e , t h a t at l e a s t the s t r i a t a l e x c i t a t o r y f i b e r s which p r o j e c t to the SN do not give o f f c o l l a t e r a l s to the GP. The exact neuronal type(s) i n the s t r i a t u m that give r i s e to the e f f e r e n t f i b e r system of t h i s s t r u c t u r e has not been f i r m l y e s t a b l i s h e d . However, the l i t e r a t u r e on the subject makes c e r t a i n conclusions unavoidable. Fox et a l (1971-72) suggested,on the b a s i s of G o l g i s t u d i e s , that the l a r g e aspiny neu-rons were s o l e l y r e s p o n s i b l e f o r conveying s t r i a t a l output. They found that the only myelinated axons i n the s t r i a t u m were those belonging to aspiny neu-rons . Spiny neurons have unmyelinated axons. The s t r i a t a l e f f e r e n t s are myelinated as they enter the GP. Hence, l a r g e aspiny s t r i a t a l neurons form, at l e a s t i n p a r t , the e f f e r e n t s to the GP. There i s evidence, based on the assumption that these neurons t r a n s p o r t AChE down t h e i r axons, that they do not c o n t r i b u t e to the s t r i a t o - r i i g r a l system (Lehmann et a l , 1979). The s t r i a -t a l e f f e r e n t s i n the "comb system" are unmyelinated. This suggests t h e i r o r i -g i n to be s t r i a t a l spiny neurons. Such a c o n c l u s i o n i s supported and indeed warranted by recent f i n d i n g s that n i g r a l a f f e r e n t s from the s t r i a t u m o r i g i n a t e predominantly, i f not e x c l u s i v e l y , from medium s i z e d spiny neurons (Grofova, 1975; Bunney and Aghajanian, 1976a). Although the retrograde t r a n s p o r t of herpes simplex v i r u s has not yet been proven a r e l i a b l e anatomical research t o o l f o r the t r a c i n g of anatomical pathways, i t should be mentioned that l a r g e aspiny neurons as w e l l as many medium s i z e d neurons i n the s t r i a t u m were l a b e l e d a f t e r i n t r a n i g r a l herpes i n j e c t i o n s . ( B a k et a l , 1978). Thus, the pos-s i b i l i t y must be e n t e r t a i n e d that the f a i l u r e to l a b e l the l a r g e aspiny s t r i a -t a l neurons w i t h HRP a f t e r i n t r a n i g r a l i n j e c t i o n s may be due to t h e i r i n a b i l i -t y to accumulate t h i s p r o t e i n . From e l e c t r o p h y s i o l o g i c a l work the s t r i a t a l medium s i z e d spiny neurons 9 must n e c e s s a r i l y be d i v i d e d i n t o two p o p u l a t i o n s . K i t a i et a l (1975) have shown that n i g r o - s t r i a t a l f i b e r s do not converge on s t r i a t o - n i g r a l neurons, but t h i s group has a l s o shown that n i g r a l e f f e r e n t s to the s t r i a t u m synapse on medium spiny s t r i a t a l neurons (Kocsis et a l , 1977). Hence, there are presum-ably at l e a s t two populations of medium spiny neurons, one which p r o j e c t s to the SN and one which does not. Although t h i s i s the c o n c l u s i o n the present author has been l e d t o , i t should be pointed out that the reports by K i t a i and coworkers are sometimes d i f f i c u l t to comprehend, p a r t i c u l a r l y when they tend not only to c o n t r a d i c t themselves from paper to paper ( K i t a i et a l , 1975; Kocs i s and K i t a i , 1977) but from paragraph to paragraph (Kocis et a l , 1977). I f the above l i n e of reasoning i s c o r r e c t , t h i s would i n v a l i d a t e the argument of Fox et a l (1975) of extensive c o l l a t e r a l i z a t i o n of s t r i a t a l e f f e r e n t s i n the globus p a l l i d u s (see next s e c t i o n ) , before c o n t i n u i n g to the SN. b) The globus p a l l i d u s 1. i n t e r n a l o r g a n i z a t i o n O n t o g e n e t i c a l l y , the globus p a l l i d u s i s derived from the l a t e r a l part of the b a s a l w a l l of the prosencephalon and i s t h e r e f o r e r e l a t e d more to the d i -encephalon (Zeman and Innes, 1963) than the f o r e b r a i n w i t h which i t i s u s u a l l y a s s o c i a t e d . In f r e s h t i s s u e i t has a p a l e appearance due to a r i c h content of myelinated axons. The globus p a l l i d u s i n primates i s d i v i d e d i n t o medial and l a t e r a l (or i n t e r n a l and e x t e r n a l ) segments by the lamina p a l l i d a l l i s i n t e r n a . In non-primate mammals ( r a t , c a t , etc.) a d i s t i n c t lamina d i v i d i n g the globus p a l l i d u s i n t o two d i v i s i o n s cannot be found. In these species the nucleus commonly l a b e l e d the globus p a l l i d u s does not appear to be homologous to both the medial and l a t e r a l segments of the primate globus p a l l i d u s . Rather, there i s s u b s t a n t i a l anatomical and u l t r a s t r u c t u r a l evidence that the i n t e r n a l seg-ment i n non-primates i s represented by the entopeduncular nucleus (see below f o r r e f e r e n c e s ) . Henceforth, the i n t e r n a l and e x t e r n a l d i v i s i o n s of the g l o -bus p a l l i d u s i n primates w i l l be r e f e r r e d to as the MGP and LGP, r e s p e c t i v e l y , 10 and i n non-primates the EP and GP. For general purposes, these s t r u c t u r e s , whether i n the primate or non-primate, w i l l be r e f e r r e d to c o l l e c t i v e l y as the globus p a l l i d u s . In the human i t has been estimated t h a t there are 540,000 neurons i n the LGP and 170,000 i n the MGP (Fox and R a f o l s , 1976). I f the r a t i o of the number of P u r k i n j e c e l l s i n the human (Armstrong and S c h i l d , 1970) and r a t ( B r a i t e n -berg and Atwood, 1958) cerebellum p a r a l l e l s that of the r a t i o of neurons i n the globus p a l l i d u s , then i n the r a t these are about 13,000 and 4,000 neurons i n the GP and EP, r e s p e c t i v e l y . Neurons i n the GP are f a r apart and dispersed throughout the nucleus. The p e r i k a r y a are of the l a r g e f u s i f o r m type which range from 30 to 60 um i n l e n g t h . The dendrites of these c e l l s are smooth, r a d i a t i n g , s l i g h t l y branched s t r u c t u r e s and have been sene to be as long as 900 pm (Fox et a l , 1974). The dendrites and d e n d r i t i c branches are o c c a s i o n a l -l y v a r i c o s e , according to Fox et a l (1974), and commonly so according to Kemp (1970). This i s a l s o true of neurons i n the EP ( A d i n o l f i , 1969a). Spines on dendrites are sparse (Fox et a l , 1974'; Kemp, 1970). There are a few s h o r t -axon l o c a l c i r c u i t neurons i n the GP (Fox et a l , 1974). S i m i l a r l y , i n the EP there are i s l a n d s of c e l l bodies and n e u r o p i l which are i s o l a t e d by myelinated bundles of f i b e r s t r a v e r s i n g the r e g i o n ( A d i n o l f i , 1969a; 1969b). Surrounding the globus p a l l i d u s are what Das (1971) c a l l s i n t e r s t i t i a l nerve c e l l s , so named f o r the reason that they are l o c a t e d i n the white matter and d i s t i n c t l y separate from the neighbouring globus p a l l i d u s . On the b a s i s of morphological, d e n d r o a r c h i t e c t o n i c and h i s t o c h e m i c a l c r i t e r i a , these neurons are d i f f e r e n t from those of the globus p a l l i d u s (Das and Kreutzberg, 1968). With regard to the morphology of the a f f e r e n t s to the LGP and MGP, some of these form, long s l e e v e - l i k e plexuses of f i n e , l o n g i t u d i n a l l y running f i b e r s which ensheath the r a d i a t i n g d e ndrites of p a l l i d a l neurons (Fox et a l , 1974). At the e l e c t r o n microscope (EM) l e v e l these dendrites can be seen completely covered w i t h a continuous sheet of s y n a p t i c endings. Immediately p e r i p h e r a l 11 to these terminals are the c l o s e l y packed p r o f i l e s of f i n e a f f e r e n t f i b e r s which bear these endings and which run l o n g i t u d i n a l l y i n the d i r e c t i o n of the dendrites (Fox et a l , 1974; Fox and R a f o l s , 1976). This c o n f i g u r a t i o n , which has been termed the l o n g i t u d i n a l a x o - d e n d r i t i c synapse, i s c h a r a c t e r i s t i c of the synapses c l i m b i n g f i b e r s form w i t h P u r k i n j e c e l l s . Axo-somatic synapses are l e s s common (Kemp, 1970). The m a j o r i t y of the terminals i n the globus p a l l i d u s have symmetrical membrane thi c k e n i n g s although asymmetrical synapses are a l s o present and both types are found to be i n contact w i t h somata and dendrites (Fox et a l , 1974; Fox and R a f o l s , 1976; A d i h o l f i , 1969b). 2. p a l l i d a l a f f e r e n t s The globus p a l l i d u s r e c e i v e s a f f e r e n t s from the s t r i a t u m , the nucleus ac-cumbens and subthalamic nucleus. Of these the s t r i a t u m appears to provide the most dense i n n e r v a t i o n of the globus p a l l i d u s . I t has been demonstrated by degeneration t r a c i n g techniques that the s t r i a t u m p r o j e c t s to both the LGP and MGP (Cowan and P o w e l l , 1966; Fox and R a f o l s , 1975; Johnson and Rosvold, 1971; Kemp, 1970; Szabo, 1962, 1967, 1970; Nauta and Mehler, 1966; M i c k l e , 1976; Voneida, 1960). The p r o j e c t i o n i s t o p o g r a p h i c a l l y organized such that the head of the s t r i a t u m p r o j e c t s to the d o r s a l and r o s t r a l p a r t s of the pa l l i d u m , whereas the putamen p r o j e c t s to the v e n t r a l and caudal p a r t s (Cowan and Po w e l l , 1966; Szabo, 1962, 1967, 1970; Voneida, 1960). The e f f e r e n t s of the s t r i a t u m run i n r a d i a l l y arranged f i b e r bundles which converge on the GP " l i k e spokes i n a wheel" (Papez, 1941). Szabo (1962) l i k e n e d the whole s t r i a t u m to a dome, superimposed on the pallidum. These r a d i a l f i b e r s are thought to giv e r i s e to the bouton-en-passage f i b e r s and the l o n g i t u d i n a l a x o - d e n d r i t i c synapses i n the globus p a l l i d u s s i n c e both these and the r a d i a l f i b e r s dege-nerate a f t e r l e s i o n s of the s t r i a t u m (Fox and R a f o l s , 1975; Fox et a l , 1975). There i s good anatomical evidence that the r a d i a l f i b e r s , during t h e i r t r a n -s i t of the LGP and MGP, give o f f extensive c o l l a t e r a l s (Fox and R a f o l s , 1975, 1976; Fox et a l , 1975). I t i s u n c e r t a i n whether the same r a d i a l f i b e r s give 12 r i s e to c o l l a t e r a l s i n both p a l l i d a l segments before c o n t i n u i n g on to the SN. Fox et a l (1975) suggest that they do f o r the f o l l o w i n g reason: The r a d i a l f i b e r s a f t e r converging upon the globus p a l l i d u s are thought to be continuous w i t h the "comb" system. The "comb" system, so named because i t i s arranged i n the c e r e b r a l peduncle l i k e t e e t h i n a comb, i s the route of the s t r i a t o -n i g r a l f i b e r s (Szabo, 1962, 1967, 1970; Nauta and Mehler, 1966). Fox et a l (1975) found that the c a l i b e r of the r a d i a l f i b e r s , before e n t e r i n g the LGP i s l a r g e r than those e n t e r i n g the MGP which i n t u r n i s l a r g e r than those i n the "comb" system. They suggest that the successive decrease i n f i b e r diameter i s due to the s u b s t a n t i a l l o s s of axoplasm to extensive c o l l a t e r a l r a m i f i c a t i o n s i n both segments of the globus p a l l i d u s . In the few s t u d i e s that have been done employing the autoradiographic t r a c i n g technique, the s t r i a t u m has been shown to p r o j e c t to both the MGP and LGP i n the monkey (Kim et a l , 1976) and to the GP ( T u l l o c h et a l , 1978) and EP i n the r a t ( H a t t o r i et a l , 1975). With regard to the s t r i a t a l - E P pathway there remains a p a u c i t y of d e f i n i t i v e s t u d i e s . N i i m i et a l (1970) report only a few degenerating preterminals i n the EP a f t e r l e s i o n s i n the d o r s o l a t e r a l head of the s t r i a t u m i n the c a t . In the same spe c i e s , A d i n o l f i (1969) r e p o r t s numerous a x o d e n d r i t i c and r e l a t i v e l y sparse axosomatic t e r m i n a l degeneration i n the EP f o l l o w i n g undescribed s t r i a t a l l e s i o n s . M i c k l e (1976) a f t e r p l a c i n g l e s i o n s throughout the head of the caudate of the opossum, observed degenera-t i o n i n the intrapeduncular nucleus, a s t r u c t u r e which i s l o c a t e d i n a p o s i -t i o n s i m i l a r to that of the EP i n the r a t . T u l l o c h et a l (1978), i n an auto-r a d i o g r a p h i c study, made no mention of the involvement of the EP i n the s t r i a -t a l e f f e r e n t p r o j e c t i o n s . The question of a s t r i a t a l - E P pathway i n the r a t i s f u r t h e r complicated by the presence of c o r t i c o f u g a l f i b e r s of passage which are damaged by s t r i a t a l l e s i o n s . Knook (1965) reported s l i g h t l y more dense degeneration i n the EP of the r a t a f t e r s t r i a t a l l e s i o n s than a f t e r c o r t i c a l l e s i o n s suggestive of the existence of a s t r i a t a l - E P pathway. 13 B i o c h e m i c a l l y , the GP presents a h i g h l y complicated p i c t u r e as i t con-t a i n s many p u t a t i v e n e urotransmitters and t h e i r s y n t h e s i z i n g enzymes at f a i r l y s u b s t a n t i a l l e v e l s compared w i t h other b r a i n areas. These i n c l u d e GABA (Okatda et a l , 1971) and GAD (Tappaz et a l , 1976; L l o y d et a l , 1975), ACh (Jacobowitz and Goldberg, 1977) and CAT (Lloyd et a l , 1975; Brownstein et a l , 1975), s e r o t o n i n (Saavedra et a l , 1974), NA (Fa r l e y and Hornykiewicz, 1977; Versteeg et a l , 1976), dopamine (DA) (Versteeg et a l , 1976), substance P (Kanazawa and J e s s e l , 1976) and methionine-enkephalin (met-enkephalin) (Hong et a l , 1977b; Sar et a l , 1978). I t i s not known to what extent these substan-ces are represented i n the GP by v i r t u e of t h e i r presence i n neurons i n t r i n s i c to the GP. There i s , however, evidence f o r the l o c a l i z a t i o n of some of them i n a f f e r e n t s to the GP. That GAD and met-enkephalin i n GP are of s t r i a t a l o r i g i n i s i n d i c a t e d by the observation of a decrease i n GAD a c t i v i t y (McGeer et a l , 1974a; H a t t o r i et a l , 1973b; Fonnum et a l , 1978a; J e s s e l et a l , 1978), met-enkephalin l e v e l s (Hong et a l , 1977b) and met-enkephalin immunofluorescence (Cuello and Paxinos, 1978) f o l l o w i n g l e s i o n s of the s t r i a t u m . There i s pre-s e n t l y no datum bearing on the source of substance P, ACh and CAT i n the GP. S i m i l a r l y , there have been no comparative measurements of these various en-zymes and substances i n the EP or MGP. However, supportive of anatomical e v i -dence of a s t r i a t a l p r o j e c t i o n to MGP and EP, there i s biochemical data f o r GAD. (Fonnum et a l , 1978) and immunbhistochemical data f o r substance P (Paxinos et a l , 1978) that suggest these to be contained i n s t r i a t a l e f f e r e n t s to the EP. Studies of the e l e c t r o p h y s i o l o g i c a l i n f l u e n c e of s t r i a t a l e f f e r e n t s on neurons of the GP and EP have i n d i c a t e d p r i m a r i l y i n h i b i t o r y e f f e c t s ( M a l l i a n i and Purpura, 1967; Levine et a l , 1974; Yoshida et a l , 1972). I t has been f u r -t h e r suggested that i n h i b i t i o n i n the EP i s mediated by s t r i a t o - n i g r a l f i b e r s which send c o l l a t e r a l s to the EP (Yoshida et a l , 1972). This i s supported by the anatomical evidence obtained by Fox et a l (1975, see above). These f i n d -14 ings are c o n s i s t e n t w i t h biochemical observations demonstrating a GABA-contain-i n g p r o j e c t i o n from the s t r i a t u m to the p a l l i d u m . However, e x c i t a t o r y respon-ses i n the GP and EP have al s o been observed a f t e r s t r i a t a l s t i m u l a t i o n ( M a l l i a n i ^ and Purpura, 1967) and i t has been suggested by i n f e r e n c e that the s t r i a t o - p a l l i d a l system contains an e x c i t a t o r y component (Richardson et a l , 1977). The evidence f o r a substance P-containing pathway from the s t r i a t u m to at l e a s t the EP (Paxinos et a l , 1978) together w i t h the e x c i t a t o r y a c t i o n s of t h i s peptide on neurons i n the CNS ( K r n j e v i c and M o r r i s , 1974; P h i l l i s and Limacher, 1974) are c o n s i s t e n t w i t h the suggestion of a s t r i a t a l e x c i t a t o r y i n -put to the EP. There i s p r e s e n t l y no t r a n s m i t t e r candidate f o r the mediator of e x c i t a t i o n i n the s t r i a t o - G P pathway. The d o r s a l raphe nucleus has been shown to be the source of the high l e -v e l s of s e r o t o n i n i n the GP and EP (Moore et a l , 1978). Thus, t h i s s t r u c t u r e has been shown to p r o j e c t to both EP and GP and s e r o t o n i n - h i s t o f l u o r e s c e n c e i s decreased f o l l o w i n g l e s i o n s of the d o r s a l raphe (Kuhar et a l , 1972). The pre-sence of DA i n the GP (Versteeg et a l , 1976) i s c o n s i s t e n t w i t h the demonstra-t i o n of a p r o j e c t i o n to the GP from the DA-rich neurons of the SN ( F a l l o n and Moore, 1978). I t has been reported that l e s i o n s of the SN w i t h no apparent involvement of the subthalamic nucleus (SUT) r e s u l t s i n some degeneration of f i b e r s i n both MGP and LGP (Carpenter and Strominger, 1967; Moore et a l , 1971). Examination of the EP at the EM l e v e l a l s o revealed t e r m i n a l degeneration a f t e r l e s i o n s of the SN ( A d i n o l f i , 1968). With regard to NA, the GP contains l e v e l s comparable to most areas of the cortex. However, a p r o j e c t i o n from the NA-containing neurons of the locus coeruleus has not been observed (Jones and Moore, 1977). I t i s now w e l l e s t a b l i s h e d that the nucleus accumbens innervates the GP (Nauta et a l , 1978; Swanson and Cowan, 1975; W i l l i a m s et a l , 1977; Conrad and P f a f f , 1976), although there i s one autoradiographic study r e p o r t i n g the ab-15 sence of such a p r o j e c t i o n (Powell and Leman, 1976). These GP a f f e r e n t s are l o c a t e d mainly i n the ventromedial p o r t i o n of the GP. The accumbens appears not to p r o j e c t to the EP, as a l l the descending f i b e r s from t h i s s t r u c t u r e en route to the SN conspicuously avoid the EP (Nauta et a l , 1978). Both segments of the globus p a l l i d u s i n the primate and non-primate r e -c e i v e a f f e r e n t s from the subthalamic nucleus (SUT). I n the primate a topo-g r a p h i c a l i n n e r v a t i o n of the globus p a l l i d u s was observed and the p a t t e r n of t e r m i n a t i o n was i n d i s c r e t e bands o r i e n t e d p a r a l l e l to the medullary laminae. S t i m u l a t i o n of the SUT produces i n h i b i t i o n of p a l l i d a l neurons (Larsen and S u t i n , 1978; Yoshida, 1973). E l e c t r o p h y s i o l o g i c a l evidence suggests that SUT neurons simultaneously innervate the GP, EP and SN by way of axon c o l l a t e r a l s (Deniau et a l , 1978a). Although the t r a n s m i t t e r of t h i s system i s not known, i t has been suggested t h a t g l y c i n e may be i n v o l v e d (Yoshida, 1973). 3. e f f e r e n t s of the medial p a l l i d a l segment In c o n t r a s t to the l a t e r a l p a l l i d a l segment, the EP has a major p r o j e c t i o n to the habenula. Only w i t h the i n t r o d u c t i o n of autoradiographic t r a c i n g me-thods has f i r m evidence been provided f o r the existence of t h i s pathway (Nauta, 1974), and t h i s has r e c e n t l y been confirmed (Kim et a l , 1976; Herkenham and Nauta, 1977; Carter and F i b i g e r , 1978). The m e d i o - l a t e r a l dimension of the EP was suggested to be represented t o p o g r a p h i c a l l y i n a s i m i l a r f a s h i o n i n the l a t e r a l habenula (Herkenham and Nauta, 1977). In t h i s same study, a f t e r HRP i n j e c t i o n s i n t o the l a t e r a l habenula, i t was reported that v i r t u a l l y a l l neu-rons of the EP were l a b e l e d w i t h HRP. Since i t i s known that the EP has other p r o j e c t i o n areas, t h i s r a i s e s the p o s s i b i l i t y that some connections of the EP a r i s e by way of axon c o l l a t e r a l s of EP e f f e r e n t s . Although c o l l a t e r a l s of EP e f f e r e n t s have been demonstrated e l e c t r o p h y s i o l o g i c a l l y , l i t t l e evidence was obtained suggesting that EP neurons which p r o j e c t to the habenula a l s o pro-j e c t to other s i t e s (Larsen and S u t i n , 1978; F i l i o n and Harnois, 1978). There i s evidence that at l e a s t a component of the EP-habenula pathway contains GAD 16 ( G o t t e s f e l d et a l , 1977) and t h e r e f o r e u t i l i z e s GABA as a t r a n s m i t t e r . Depen-ding on the extent of c o l l a t e r a l i z a t i o n of the EP e f f e r e n t s , t h i s suggests th a t the other e f f e r e n t p r o j e c t i o n s of the EP may a l s o be GABA-containing. Although an extensive c h a r a c t e r i z a t i o n of the EP e f f e r e n t s to the t h a l a -mus was not undertaken here, t h i s p r o j e c t i o n of the p a l l i d u m represents per-haps one of the more i n t e r e s t i n g outputs of the b a s a l g a n g l i a . The globus p a l l i d u s s i t s at the apex of the convergence of f i b e r s from the cortex to the s t r i a t u m to the pallidum. I n t u i t i v e l y , then, the p a l l i d a l e f f e r e n t s may t r a n s -mit h i g h l y i n t e g r a t e d i n f o r m a t i o n , the nature of which may be a v a l u a b l e key to the understanding of b a s a l g a n g l i a f u n c t i o n . The MGP gives r i s e to two f i b e r t r a c t s , the ansa l e n t i c u l a r i s and the l e n t i c u l a r f a s c i c u l a r i s , that merge i n t o the thalamic f a s c i c u l u s which contains the p a l l i d o - t h a l a m i c p r o j e c -t i o n (Grofova, 1970; Kuo and Carpenter, 1973). In non-primates these f i b e r systems, a r i s i n g from the EP, are l e s s d i s t i n c t . I t seems f a i r l y c l e a r that the p a l l i d o - t h a l a m i c p r o j e c t i o n o r i g i n a t e s e x c l u s i v e l y from the MGP (Kuo and Carpenter, 1973; Kim et a l , 1976; Carpenter, 1976) and EP (Larsen and McBride, 1979; Carter and F i b i g e r , 1978; H a t t o r i et a l , 1975; Grofova and R i n v i k , 1974). Thus, t e c h n i c a l d i f f e r e n c e s (or d i f f i c u l t i e s ) may have c o n t r i b u t e d to the r e -s u l t s of the only recent study suggesting widespread p r o j e c t i o n s from the GP to the thalamus (Severin et a l , 1976). Degeneration (Carpenter, 1976; Kuo and Carpenter, 1973; Nauta and Mehler, 1966; Harding, 1973) and autoradiographic (Kim et a l , 1976) s t u d i e s i n the monkey have shown that the p r i n c i p a l termina-t i o n s i t e s w i t h i n the thalamus of p a l l i d a l f i b e r s i n c l u d e the v e n t r a l l a t e r a l nucleus o r a l part (VLo), v e n t r a l a n t e r i o r nucleus p r i n c i p a l p a r t (VAp) and centromedian nucleus (CM). In these s t u d i e s a topographical o r g a n i z a t i o n of p a l l i d o - t h a l a m i c f i b e r s has been observed such that r o s t r a l p a r ts of the MGP p r o j e c t predominantly to VAp, whereas caudal p a r t s of MGP p r o j e c t p r i m a r i l y to VLo. The greatest number of p a l l i d o - t h a l a m i c f i b e r s ending i r i CM o r i g i n a t e i n 17 the r o s t r a l and medial p o r t i o n of the MGP (Carpenter, 1976). In the r a t , EP e f f e r e n t s to the thalamus terminate i n the l a t e r a l ventromedial nucleus (VM) and immediately l a t e r a l to VM (Carter and F i b i g e r , 1978). I t i s s i g n i f i c a n t here that s t u d i e s of the n i g r a l ( R i n v i k , 1975; Carpenter et a l , 1976; C l a v i e r et a l , 1976; F a u l l and Carman, 1968) and c e r e b e l l a r ( F a u l l and Mehler, 1976) e f f e r e n t s to the thalamus i n the r a t , cat and monkey suggest that VM i n sub-primates i s homologous to VA and VL i n primates, and s p e c i f i c a l l y to VL pars m e d i a l i s and pars m a g n o c e l l u l a r i s of VA. In a d d i t i o n to these pathways, a f u r t h e r EP p r o j e c t i o n i n the r a t and cat has been observed to the p a r a f a s c i -c u l a r nucleus (PF) (Carter and F i b i g e r , 1978) and CM (Nauta, 1974) of the tha-lamus. A p r o j e c t i o n from the MGP to PF was not observed i n the monkey (Kim et a l , 1976) whereas a p r o j e c t i o n to CM was. Therefore, although there may be some correspondence between the PF i n the r a t and the CM i n the monkey, i t i s noteworthy, i n s o f a r as p a l l i d o - n i g r a l i n t e r a c t i o n s i n the thalamus are con-cerned, that whereas, a n i g r o - p a r a f a s c i c u l a r p r o j e c t i o n has been reported i n the r a t ( C l a v i e r et a l , 1976), i n at l e a s t one study no n i g r a l e f f e r e n t f i b e r s were found to p r o j e c t to any of the i n t r a l a m i n a r n u c l e i i n the monkey (Carpen-t e r et a l , 1976). The EP-thalamic p r o j e c t i o n i n the r a t appears to o r i g i n a t e throughout the nucleus and there seems to be no topographical o r g a n i z a t i o n of the p r o j e c t i o n (Larsen and McBride, 1979). Such observations seem to be c o n s i s t e n t w i t h e l e c t r o p h y s i o l o g i c a l demonstrations of some degree of c o l l a t e r a l i z a t i o n of EP e f f e r e n t f i b e r s (Larsen and S u t i n , 1978; F i l i o n and Harnois, 1978). For the most p a r t , e l e c t r o p h y s i o l o g i c a l s t u d i e s of EP-thalamic r e l a t i o n -ships have y i e l d e d l i t t l e i n f o r m a t i o n . Dormont and Ohye (1971) observed only weak e x c i t a t i o n of u n i t s i n the VL thalamus a f t e r EP s t i m u l a t i o n and were hard pressed to a t t r i b u t e t h i s s o l e l y to s t i m u l a t i o n of EP neurons. S t i m u l a t i o n of d i e n c e p h a l i c regions "which should i n c l u d e e f f e r e n t f i b e r s from the EP" (Yoshida et a l , 1971) produced monosynaptic IPSP's i n the EP. This suggests 18 an i n h i b i t o r y component of EP e f f e r e n t s and t h e i r r e c u r r e n t c o l l a t e r a l s . The o r i g i n or d e s t i n a t i o n of these f i b e r s , however, remained unknown. S t i m u l a t i n g i n the brachium conjunctivum and ansa l e n t i c u l a r i s D e s i r a j u and Purpura (1969) observed EPSP's i n the VL thalamus and concluded the existence of a d i r e c t monosynaptic e x c i t a t o r y convergence of f i b e r s from the cerebellum and "corpus s t r i a t u m " onto a common pool of VL neurons. In c o n t r a s t , Uno and Yoshida (1975) and Uno et a l (1978) observed only monosynaptic IPSP responses i n VL neurons upon EP s t i m u l a t i o n , found these neurons to form anatomically separate populations from those r e c e i v i n g c e r e b e l l a r input and concluded that very l i t t l e , i f any, d i r e c t convergence of c e r e b e l l a r or EP f i b e r s on s i n g l e VL neurons occurs. I t i s apparent that somewhat more a t t e n t i o n to d e t a i l i s r e -q u i r e d i n e l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n s i n order even to begin to make sense of the s y n a p t i c r e l a t i o n s h i p s of EP-thalamic f i b e r s and the i n t e r a c t i o n of these w i t h other thalamic a f f e r e n t s . To emphasize f u r t h e r the l a c k of knowledge i n t h i s area, v i r t u a l l y nothing i s known of the biochemistry of t h i s major f i b e r p r o j e c t i o n of the b a s a l g a n g l i a . Other than i t s e x i s t e n c e , very l i t t l e i s known about the pathway from the MGP (Kim et a l , 1976; Kuo and Carpenter, 1973; Nauta and Meh.ler:, 1966) and EP (Larsen and McBride, 1979; C a r t e r and F i b i g e r , 1978; F i l i o n and Harnois, 1978) to the nucleus tegmenti pedunculopontis (TPP). In a d d i t i o n , i t has been r e -ported that i n j e c t i o n of HRP i n t o areas of the b r a i n stem other than SN or TPP r e s u l t s i n l a b e l i n g of EP and GP i n the r a t and MGP i n the monkey (Hopkins, 1977). These pathways appear to be f a i r l y sparse and t h e r e f o r e f u r t h e r i n v e s -t i g a t i o n s are required before the suggestion can be taken s e r i o u s l y that they provide a means whereby the b a s a l g a n g l i a can d i r e c t l y and s i g n i f i c a n t l y exert an i n f l u e n c e on, f o r example, descending motor systems (Hopkins, 1977). 4. e f f e r e n t s of the l a t e r a l p a l l i d a l segment The LGP p r o j e c t i o n to the SUT has been known f o r some time ( W h i t t i e r and M e t t l e r , 1949; Nauta and Mehler, 1966) and has r e c e n t l y been demonstrated w i t h 19 autoradiographic t r a n s p o r t methods (Kim et a l , 1976; H a t t o r i et a l , 1975). This p r o j e c t i o n i s t o p o g r a p h i c a l l y organized such that the e f f e r e n t s of the LGP focus onto the SUT i n much the same manner as the s t r i a t u m p r o j e c t s to the LGP (Carpenter et a l , 1968). The MGP and EP appear not to p r o j e c t to SUT as no autoradiographic a c t i v i t y was observed i n the SUT a f t e r i n j e c t i o n s of r a d i o -t r a c e r i n t o these s t r u c t u r e s (Kim et a l , 1976; Carter and F i b i g e r , 1978). Le-si o n s of the GP have been shown to decrease GAD a c t i v i t y i n the SUT (Fonnum et a l , 1978b). Although the topo g r a p h i c a l changes observed i n GAD were reported to r e f l e c t the o r g a n i z a t i o n of the p a l l i d o p s u b t h a l a m i c p r o j e c t i o n , t h i s conclu-s i o n can be disputed s i n c e some of the GP l e s i o n s i n v o l v e d the i n t e r n a l cap-s u l e which contains a l l of the descending p a l l i d o - s u b t h a l a m i c f i b e r s (Nauta and Mehler, 1966). In the absence of e l e c t r o p h y s i o l o g i c a l data, the presence of GAD i n the e f f e r e n t f i b e r s of the GP to SUT suggests that t h i s pathway i s i n -h i b i t o r y . An e x c i t a t o r y component, however, cannot be r u l e d out i n view of immunohistochemical demonstrations of substance P - p o s i t i v e neurons i n the GP (Kanazawa et a l , 1977), an e x c i t a t o r y a c t i o n of ACh on SUT neurons (Ferger et a l , 1979), and that the m a j o r i t y of neurons i n the SUT respond by i n i t i a l ex-c i t a t i o n to s t i m u l a t i o n of the c o n t r a l a t e r a l area of the s k i n i n the v i c i n i t y of the v i b r a s s a e (Hammond et a l , 1978). I t has been v i r t u a l l y impossible to demonstrate a p r o j e c t i o n from the GP to the SN using degeneration techniques. Lesions of the GP i n v a r i a b l y i n v o l v e s t r i a t a l f i b e r s passing through the GP on t h e i r way to the SN. Indeed, Nauta and Mehler (1966) i n t h e i r review a r t i c l e concluded that the p a l l i d o - s u b t h a l a -mic p r o j e c t i o n " p o s s i b l y represents the only e x t r i n s i c e f f e r e n t connection of the e x t e r n a l segment". The f i r s t convincing anatomical demonstration of a f i b e r system between the GP and SN was an autoradiographic study by H a t t o r i e t a l (1975). They showed that the GP p r o j e c t s p r i m a r i l y to the zona compacta of the SN. Subsequent autoradiographic s t u d i e s confirmed the exi s t e n c e of n i g r a l a f f e r e n t s of., the LGP (Kim et a l , 1976). In n e i t h e r of these s t u d i e s 20 was l a b e l found i n the SN a f t e r the i n j e c t i o n of t r a c e r i n t o the EP or MGP. Employing the HRP retrograde t r a n s p o r t technique, l a b e l e d c e l l s i n the GP have been observed a f t e r i n j e c t i o n s of HRP i n t o the SN (Grofova, 1975; T u l l o c h et a l , 1978). In a d d i t i o n , HRP p o s i t i v e c e l l s were found i n the EP a f t e r i n j e c -t i o n s of HRP i n t o the medial caudal p a r t s of the SNC (Grofova, 1975). The d i f f e r e n c e s obtained between the HRP and autoradiographic techniques w i t h r e -gard to an EP p r o j e c t i o n to the SN have yet to be r e c o n c i l e d . The p r e f e r e n t i a l p r o j e c t i o n of the GP to the SNC was als o observed by Kanazawa et a l (1976) who found that pressure i n j e c t i o n s of HRP i n t o the SNC r e s u l t e d i n a greater number of l a b e l e d c e l l s i n the GP than i n j e c t i o n s i n t o the SNR. On the other hand, Bunney and Aghajanian (1976a) reported a l a r g e r number of l a b e l e d c e l l s i n the GP a f t e r HRP i n j e c t i o n s i n t o the SNR than SNC. The discrepancy between these r e s u l t s may be due to the f a c t that i n the l a t -t e r study HRP was d e l i v e r e d to the SN by m i c r o e l e c t r o p h o r e s i s and t h i s proce-dure has been shown to cause damage to and uptake of HRP by f i b e r s of passage (see s e c t i o n I l i a ) . P h y s i c a l and anatomical parameters might be such that the f i b e r of passage problem posed by e l e c t r o p h o r e t i c i n j e c t i o n s manifests i t s e l f to a greater degree a f t e r i n j e c t i o n s i n t o the SNR than SNC. The c o i n c i d e n t a l unequivocal demonstration of the existence of a p a l l i d o -n i g r a l pathway w i t h the onset of se r i o u s e f f o r t s to determine the biochemical nature of the s t r i a t o - n i g r a l system has l e d to an inseparable e v o l u t i o n of data on the o r i g i n of the neurons c o n t a i n i n g the neurotransmitters i n v o l v e d i n these n i g r a l a f f e r e n t s . For t h i s reason the biochemical neuroanatomy of the s t r i a t o - and p a l l i d o - n i g r a l systems are discussed simultaneously i n the f o l l o w i n g s e c t i o n . The o b s e r v a t i o n t h a t the SN contains r e l a t i v e l y high l e v e l s of GABA and GAD a c t i v i t y was made s h o r t l y a f t e r the discovery of GABA i n the CNS (Albers and Brady, 1959; M u l l e r and Langemann, 1962). The SNR, i n f a c t , contains the highest GAD a c t i v i t y of the 74 b r a i n areas measured by Tappaz et a l (1976). 21 In t h e i r o r i g i n a l observation McGeer et a l (1971b)showed that l e s i o n s of the GPL caused a r e d u c t i o n of n i g r a l GAD a c t i v i t y . Kim et a l (1971) showed that i n the r a t SN GABA content decreases a f t e r hemitransections at the l e v e l of the SUT or a f t e r s t r i a t a l l e s i o n s and suggested that some of the s t r i a t o - n i g r a l f i b e r s u t i l i z e GABA as a ne u r o t r a n s m i t t e r . From the h i s t o l o g y of the l e s i o n s i t e shown i n t h e i r paper the s t r i a t a l l e s i o n almost c e r t a i n l y i n v o l v e d the GP. In a d d i t i o n , although not d e t r a c t i n g from the q u a l i t a t i v e v a l i d i t y of t h e i r o b s e r v a t i o n , without t a k i n g s p e c i a l p r e c a u t i o n s , the measurement of GABA l e v e l s cannot be used to gain q u a n t i t a t i v e estimates of the t o t a l c o n t r i b u t i o n of n i g r a l GABA from a f f e r e n t sources s i n c e b r a i n GABA content has been shown to undergo post mortem changes (Baxter, 1969). Subsequent st u d i e s by the same group (Kataoka et a l , 1974) showed th a t n i g r a l GAD a c t i v i t y can be reduced 70% by hemitransections at the subthalamic l e v e l . One study has focused on d e f i -ning the exact source of GABA-containing a f f e r e n t s to the SN. H a t t o r i et a l (1973b) (see a l s o McGeer et a l , 1974a) demonstrated that hemitransections an-t e r i o r to the GP, severing descending p r o j e c t i o n s from about 60% of the s t r i a -tum, d i d not reduce n i g r a l GAD a c t i v i t y . On the other hand, hemitransections at the l e v e l of the hypothalamus reduced GAD a c t i v i t y i n the SN by 82%. These workers suggested that n i g r a l GAD-containing a f f e r e n t s o r i g i n a t e i n the t a i l of the s t r i a t u m and/or the GP. In c o n t r a s t to these r e s u l t s , Fonnum et a l (1974) observed GAD reductions (up to 90% i n some instances) i n the SN a f t e r l a r g e s t r i a t a l l e s i o n s , i n c l u d i n g those confined to the a n t e r i o r s t r i a t u m , which l e d these i n v e s t i g a t o r s to conclude "th a t only a small number of GABA-e r g i c f i b e r s to the s u b s t a n t i a n i g r a could be derived from other sources". Fonnum et a l (1974) a t t r i b u t e d the d i f f e r e n c e s between t h e i r work and that of H a t t o r i et a l (1973b) to be due to t h e i r observation that n i g r a l GAD l o s s i s r e g i o n a l and f o l l o w s the topography of the s t r i a t o - n i g r a l p r o j e c t i o n s , and such l o s s e s may have been overlooked by H a t t o r i et a l (1973b) who employed measurements of the whole SN. Such an explanation f o r the i n c o n g r u i t i e s be-22 tween the r e s u l t s of the two groups, although p o s s i b l e , i s d o u b t f u l . GAD a c t i -v i t y i s highest i n the medial h a l f of the SN (Fonnum et a l , 1974). From the l a r g e reductions i n GAD a c t i v i t y observed by Fonnum et a l (1974) only i n the medial h a l f of the SN a f t e r " r o s t r a l " s t r i a t a l l e s i o n s , i t i s h i g h l y u n l i k e l y that such l o s s e s of GAD would go unobserved i n whole SN assays. These d i s c r e -pancies are more l i k e l y to be due to species d i f f e r e n c e s between the cat (Fonnum et a l , 1974) and the r a t ( H a t t o r i et a l , 1973b) or to methodological d i f f e r e n c e s . For example, i t i s p r e s e n t l y not apparent what the equivalent of an a n t e r i o r hemitransection l e s i o n i n the r a t would be i n the c a t . F o l l o w i n g these i n i t i a l observations and probably due, i n p a r t , to the controversy surrounding them, there have been numerous reports d e a l i n g w i t h the source of n i g r a l GAD-containing a f f e r e n t s . The e a r l i e r work of H a t t o r i et a l (1973b) was confirmed (Brownstein et a l , 1977; J e s s e l et a l , 1978; Spano et a l , 1977). Brownstein et a l (1977) found that the r o s t r a l three-quarters of the s t r i a t u m does not send GABA-containing f i b e r s to the SN. In a d d i t i o n , they found that l e s i o n s i n v o l v i n g the l a t e r a l h a l f of the t a i l of the s t r i a t u m produce only modest decreases of GAD a c t i v i t y i n the SNR without a l t e r i n g i t i n the SNC. Brownstein and coworkers concluded that GAD c o n t a i n i n g neurons i n n e r v a t i n g the SN are l o c a t e d i n the GP and i n the s t r i a t u m immediately bor-dering the GP on i t s a n t e r i o r and l a t e r a l aspect. S i m i l a r l y , J e s s e l et a l (1978), a f t e r producing l e s i o n s which separated the e n t i r e s t r i a t u m from the GP, concluded that the GP contains s i g n i f i c a n t numbers of GABA neurons pro-j e c t i n g to the SN. As f o r Fonnum and coworkers (Fonnum et a l , 1978), they have r e f u t e d some of t h e i r e a r l i e r claims and have s t a t e d that "a s i g n i f i c a n t decrease i n n i g r a l GAD occurred only a f t e r hemitransections i n v o l v i n g the t a i l of the caudate-putamen". I t should be mentioned here that some workers (Kanazawa et a l , 1977; Gale et a l , 1977b) s t i l l m aintain that a n t e r i o r s t r i a -t a l regions c o n t r i b u t e to n i g r a l GAD-containing a f f e r e n t s . Although the pos-s i b i l i t i e s f o r the anatomical l o c a t i o n of the e l u s i v e GAD-containing s t r i a t o -p a l l i d o - n i g r a l neurons are narrowing, i t i s evident from ..the for g o i n g d i s c u s -s i o n that the exact d i s t r i b u t i o n of these neurons w i t h i n the s t r i a t o - G P com-p l e x remains unresolved. P a r a l l e l i n g the above s t u d i e s , the existence of s t r i a t o - and/or p a l l i d o -n i g r a l substance P-containing f i b e r s has a l s o been demonstrated ( J e s s e l et a l , 1978; Brownstein et a l , 1977; Mroz et a l , 1977; Gale et a l , 1977b; P a l k o v i t s et a l , 1978; Kanazawa et a l , 1977; Hong et a l , 1977c). Indeed, greater than 90% of n i g r a l substance P appears to be i n terminals of these descending f i -b e r s . For the greater part of the n i g r a l a f f e r e n t s of the s t r i a t u m , there appears to be a c l e a r s e p a r a t i o n of those c o n t a i n i n g GAD from those c o n t a i n i n g substance P. Thus, a s u b s t a n t i a l number of substance P-containing s t r i a t o -n i g r a l neurons are l o c a t e d i n the s t r i a t u m a n t e r i o r to the GP (Brownstein et a l , . 1977; J e s s e l et a l , 1977; Gale et a l , 1977b) and according to Brownstein et a l (1977) none o r i g i n a t e i n the t a i l of the s t r i a t u m or the GP. However, substance P has been demonstrated immunohistochemically i n t y p i c a l neurons of the GP (Kanazawa et a l , 1977) and by i n f e r e n c e the existence of p a l l i d o - n i g r a l substance P-containing f i b e r s i s i n d i c a t e d ( J e s s e l et a l , 1977). For the l o c a l i z a t i o n of both substance P- and GAD-containing neurons which p r o j e c t to the SN, i t appears that mechanical or e l e c t r o l y t i c l e s i o n s can provide only a l i m i t e d degree of r e s o l u t i o n which i s determined by l e s i o n s i z e , d i s s e c t i o n accuracy and assay s e n s i t i v i t y ; i n the case of p a l l i d o - n i g r a l p r o j e c t i o n s such s t u d i e s are impossible due to the fiber-of-passage problem. E l u c i d a t i o n of the a r c h i t e c t u r a l arrangement of the substance P and GAD-c o n t a i n i n g p r o j e c t i o n neurons i n the s t r i a t u m and GP w i l l r e q u i r e a combina-t i o n of h i s t o l o g i c a l , p o s s i b l y immunohistological techniques, w i t h retrograde t r a n s p o r t anatomical techniques, o r , i n the case of the GP l e s i o n s , techniques which do not destroy f i b e r s of passage. There i s c u r r e n t l y no e l e c t r o p h y s i o l o g i c a l datum a v a i l a b l e on the p a l l i d o -n i g r a l pathway. 24 5. a c e t y l c h o l i n e s t e r a s e s t a i n i n g neurons of the globus p a l l i d u s A source of confusion w i t h regard to the present experiments (see d i s c u s -sion) and i n d e f i n i n g the e f f e r e n t connections of the globus p a l l i d u s are the c e l l s , r e f e r r e d to by Das and Kreutzberg (1968) as i n t e r s t i t i a l neurons, which surround and i n t e r s p e r s e both segments of the globus p a l l i d u s . Compared to the smaller t r i a n g u l a r and f u s i f o r m neurons of the globus p a l l i d u s proper, these i n t e r s t i t i a l neurons are much l a r g e r , m u l t i p o l a r and s t a i n more int e n s e -l y f o r AChE. whether these c e l l s should be c l a s s i f i e d as an i n t e g r a l part of the globus p a l l i d u s i s d o u b t f u l . For example, u n l i k e neurons of the GP, they appear to p r o j e c t to c o r t i c a l areas ( K e l l y and Moore, 1978, Divac, 1975; Johnston et a l , 1979; Das, 1971). Grouping these i n t e r s t i t i a l c e l l s w i t h the s u b s t a n t i a innominata would not s o l v e matters but merely put the onus of c l a s -s i f i c a t i o n on a broader scope, v i s a v i s , the current controversy of whether the s u b s t a n t i a innominata i s part of the globus p a l l i d u s or a separate e n t i t y . The o r g a n i z a t i o n of these c e l l s i n the e x i s t i n g conceptual and anatomical framework of the b a s a l g a n g l i a must await a more d e t a i l e d account of t h e i r a f -f e r e n t s and e f f e r e n t s . c) Subthalamic nucleus In a G o l g i study of the SUT by R a f o l s and Fox (1976), three p r i n c i p a l neu-ron types were i d e n t i f i e d on the b a s i s of s i z e and morphology. These are r a -d a t i n g neurons, elongated f u s i f o r m neurons and l o c a l interneurons. I t may be of some s i g n i f i c a n c e that the l o c a l interneurons of the SUT bear some s i m i l a -r i t i e s to neurons i n other n u c l e i that have been shown to be presynaptic to axon terminals (see R a f o l s and Fox, 1976). The major connections of the SUT, those w i t h the globus p a l l i d u s and SN, are discussed i n previous and subsequent s e c t i o n s , r e s p e c t i v e l y . Some sparse f i b e r p r o j e c t i o n s of the SUT to the VA-VL thalamus, putamen and TPP pars compacta have al s o been observed (Nauta and Cole, 1978). A d e t a i l e d documentation of SUT a f f e r e n t s awaits examination w i t h the retrograde HRP technique. 25 d) Substantia n i g r a 1. i n t e r n a l o r g a n i z a t i o n A great deal of anatomical, e l e c t r o p h y s i o l o g i c a l and biochemical i n v e s t i -g a t i v e e f f o r t has been brought to bear on the SN. I n p a r t , t h i s may be due to the p o s s i b l e involvement of t h i s s t r u c t u r e , and i n p a r t i c u l a r , of the n i g r o -s t r i a t a l DA neurons i n a number of phenomena of p s y c h o l o g i c a l , pharmacological and c l i n i c a l importance. What f o l l o w s i s a b r i e f review of the data a v a i l a b l e on the SN w i t h emphasis on c e r t a i n features that p e r t a i n to the present work. The SN i s d i v i d e d i n t o two p o r t i o n s : the neuron dense pars compacta and the n e u r o p i l r i c h pars r e t i c u l a t a . Three types of neurons have been i d e n t i f i e d i n the SN by v a r i o u s techniques (Juroska et a l , 1977; R i n v i k and Grofova, 1970; Grofova and R i n v i k , 1970; G u l l e y and Wood, 1971; Schwyn and Fox, 1974). The l a r g e s t n i g r a l neurons, those i n the SNR, measure 45 to 74 um. Medium s i z e d (19-45 ym) heterogeneously shaped neurons, s i m i l a r i n morphological c h a r a c t e r i -s t i c s to the l a r g e neurons, are present i n both SNR and SNC. The smallest neu-rons, a l s o present i n both SN s u b s t r u c t u r e s , measure 11 to 26 ym. Neurons of a l l types have v a r y i n g numbers of spines and some degree of v a r i c o s i t i e s or beading of t h e i r d e n d r i t e s . Medium s i z e d neurons of the SNC p r o j e c t long v a r i -cose dendrites i n t o the SNR. These neurons a l s o have r e s t r i c t e d d e n d r i t i c f i e l d s i n the SNC. Dendrites a r i s i n g from neruons i n the SNR tend not to leave t h i s s t r u c t u r e . Axons of many l a r g e and medium s i z e d neurons i n the SNR have c o l l a t e r a l s , some of which are r e c u r r e n t w h i l e others f o l l o w the o r i e n t a -t i o n of the p r i n c i p a l axon. The medium s i z e d neruons of the SNC, which give" r i s e to the ascending DA-containing p r o j e c t i o n , s t a i n i n t e n s e l y f o r DA (Dahlstrom and Fuxe, 1964). The d e n d r i t e s of these neurons, where they invade the SNR, have a l s o been shown to c o n t a i n DA (Bjorklund and L i n d v a l l , 1975). I t i s of i n t e r e s t here that u l t r a s t r u c t u r a l s t u d i e s have d i s c l o s e d v e s i c l e -c o n t a i n i n g dendrites i n the SN and these appear to form dendro-dendritic synap-ses (Hajdu et a l , 1973). Moreover, Wilson et a l (1977a), using the osmophilic l a b e l 5-hydroxydopamine, have r e c e n t l y reported that l a b e l e d v e s i c l e - c o n t a i n -i n g dendrites and axons form en passage synapses i n the SNC and dendro-dendri-t i c synapses i n the SNR. These observations are s i g n i f i c a n t i n that they r e -l a t e to c e r t a i n e l e c t r o p h y s i o l o g i c a l and biochemical phenomena concerning DA-c o n t a i n i n g neurons which w i l l be discussed i n subsequent s e c t i o n s . E l e c t r o p h y s i o l o g i c a l ^ at l e a s t two types of neurons have been i d e n t i f i e d i n the SN (Guyenet and Aghajanian, 1978; Wilson et a l , 1977b). Type I neurons, l o c a t e d i n the SNC and probably g i v i n g r i s e to the dopaminergic n i g r o - s t r i a t a l system, have a slow b u r s t i n g p a t t e r n and a slow conduction v e l o c i t y . Type I I neurons have a higher f i r i n g r a t e and conduction v e l o c i t y and are l o c a t e d i n the SNR. Although most of the Type I I neurons could be a n t i d r o m i c a l l y a c t i v a -ted from the thalamus, some lo c a t e d subjacent to the SNC were a n t i d r o m i c a l l y a c t i v a t e d from the s t r i a t u m (Guyenet and Aghajanian, 1978). There i s only p a r t i a l agreement between e l e c t r o p h y s i o l o g i c a l and anatomical s t u d i e s as to the exact l o c a t i o n w i t h i n the SNR of neurons p r o j e c t i n g to the s t r i a t u m . B i o c h e m i c a l l y , the SN presents a p r o f i l e that i n some respects resembles the GP. The substances and enzymes, r e l e v a n t to neurotransmission, that have been observed i n the SN i n c l u d e NA, DA ( F a r l e y and Hornykiewicz, 1977; Ver-steeg et a l , 1976), .tyrosine hydroxylase (TH) (Reis et a l , 1975), DBH (Reis et a l , 1978; Ross and R e i s , 1974), adren a l i n e (van der Gugen et a l , 1976), s e r o t o n i n ( P a l k o v i t s et a l , 1974), GABA (Fahn and C6te, 1968; Okatda et a l , 1971), GAD (Tappaz et a l , 1976; L l o y d et a l , 1975) and substance P (Brownstein et a l , 1977; J e s s e l et a l , 1978). A major goal of numerous s t u d i e s has been to e s t a b l i s h the e f f e r e n t , a f f e r e n t and i n t r i n s i c anatomical systems of the SN that c o n t a i n these m a t e r i a l s and some success has been achieved i n these attempts. In the case of TH, i t i s probable that t h i s enzyme i s l o c a t e d w i t h i n dopamine c o n t a i n i n g p e r i k a r y a and d e n d r i t e s , and perhaps even terminals a l -though at present there i s no evidence of t h i s . The DA-containing neurons i n the SNC s t a i n immunohistochemically f o r TH (Hokfelt et a l , 1976) and a f t e r hemitransections a n t e r i o r to the SN or 6-hydroxydopamine (6-OHDA) i n j e c t i o n s i n t o the n i g r o - s t r i a t a l pathway (NSP), the retrograde degeneration of DA-con-t a i n i n g neurons c o i n c i d e s w i t h the r e d u c t i o n of TH i n the SN (Reis et a l , 1978; McGeer et a l , 1973). I t i s i n t e r e s t i n g to note that the 6-OHDA l e s i o n s r e s u l t i n severe d e p l e t i o n s of n i g r a l TH. However, a f t e r hemitransection about 20% of n i g r a l TH remains. This suggests that these neurons may have some c o l l a -t e r a l s which prevent t h e i r complete degeneration. A l t e r n a t i v e l y , the l i m i t e d c a p a c i t y to s u r v i v e t r a n s e c t i o n of t h e i r p r i n c i p a l axons may be r e l a t e d to the extensive r a m i f i c a t i o n s and s p e c i a l i z a t i o n of the dendrites of these neurons i n the SNR (Reis et a l , 1978). In a d d i t i o n , a p r o j e c t i o n to regions p o s t e r i o r to the hemitransections cannot be r u l e d out. With regard to GAD, Ribak et a l (1976) observed no immunohistochemical s t a i n i n g of neuronal p e r i k a r y a i n the SN although GAD-positive axon terminals were abundant. Apparently, t h i s absence of c e l l body s t a i n i n g was " a n t i c i p a -ted s i n c e there i s evidence that another neurotransmitter candidate, dopamine, i s concentrated w i t h i n neurons of the s u b s t a n t i a - n i g r a " ( R i b a k et a l , 1976). This e x p l a n a t i o n , however, i s not s a t i s f y i n g . Among other explanations i s the p o s s i b i l i t y that GAD l e v e l s i n neuronal soma are below the t h r e s h o l d of detec-t i o n by the immunohistochemical method. A c h o l i n e r g i c involvement i n n i g r a l f u n c t i o n i s suggested by the presence i n the SN of ACh (Jacobowitz and Goldberg, 1977), CAT (Brownstein et a l , 1975; Cheney et a l , 1975; L l o y d et a l , 1975) and AChE (Butcher and Hodge, 1976; Butcher et a l , 1975; P a l k o v i t s and Jacobowitz, 1974). In a d d i t i o n , ACh has been found to i n f l u e n c e the e l e c t r i c a l a c t i v i t y of n i g r a l neurons (Dray and Straughan, 1976; Aghajanian and Bunney, 1974a). The l o c a l i z a t i o n of c h o l i n e r -g i c markers i n the SN i s at present unclear. Although i t has been d i f f i c u l t to a t t r i b u t e a f u n c t i o n to the d i s p r o p o r t i o n a t e l y h i g h l e v e l s of n i g r a l AChE r e l a t i v e to CAT, i t has been shown, by h i s t o c h e m i c a l (Butcher and Hodge, 1976; 28 Butcher et a l , 1975) and biochemical c r i t e r i a (Lehmann and F i b i g e r , 1978) that dopaminergic neurons c o n t a i n some of the AChE of the SN. With regard to CAT, s i n c e hemitransections immediately a n t e r i o r to the SN f a i l to reduce the a c t i -v i t y of t h i s enzyme i n the SN (Kataoka et a l , 1974; McGeer et a l , 1971) a r o s -t r a l c h o l i n e r g i c p r o j e c t i o n to the SN i s u n l i k e l y . C u r i o u s l y , both the SN and GP possess ACh l e v e l s which are d i s p r o p o r t i o n a t e l y high compared to areas con-t a i n i n g greater amounts of CAT a c t i v i t y (Jacobowitz and Goldberg, 1977). The reason f o r t h i s remains unknown. The other substances mentioned above appear, f o r the most p a r t , to be pre-sent i n terminals of a f f e r e n t f i b e r s to the SN and are discussed i n that con-t e x t . 2. n i g r a l e f f e r e n t s The n i g r o - s t r i a t a l system has been much st u d i e d and w e l l documented (Anden et a l , 1964; H b k f e l t and Ungerstedt, 1969; H a t t o r i et a l , 1973a; F a l l o n and Moore, 1978). These s t r i a t a l a f f e r e n t s and t h e i r t e r minals are probably the source of the bulk of the remainder of terminals of e x t r i n s i c o r i g i n to the s t r i a t u m a f t e r the cortex and thalamus (Kemp and P o w e l l , 1971b). That these f i b e r s are dopamine-containing has been w e l l documented. In a d d i t i o n , l e s i o n s t u d i e s f o l lowed by DA measurments i n the s t r i a t u m (Moore et a l , 1971; Koob et a l , 1975) are i n good agreement w i t h anatomical s t u d i e s ( F a l l o n and Moore, 1978) i n demonstrating that both the SN and v e n t r a l tegmental area (VTA) pro-j e c t DA-containing f i b e r s to the s t r i a t u m , a l b e i t the VTA c o n t r i b u t i o n i s somewhat l e s s . The o r g a n i z a t i o n of the SN/VTA-striatal DA system has been shown to be h i g h l y topographic ( F a l l o n and Moore, 1978; Moore and Bloom, 1978). This s t r i c t t o p o g r a p h i c a l arrangement may provide some i n s i g h t i n t o the func-t i o n of the r e c i p r o c a l connections of the SN and s t r i a t u m and w i l l be discussed l a t e r . The SNC a l s o has widespread p r o j e c t i o n s to c o r t i c a l and other subcor-t i c a l areas. Thus, f o l l o w i n g the i n i t i a l demonstration of the presence of DA i n the cortex (T h i e r r y et a l , 1973), many papers q u i c k l y appeared demonstra-29 t i n g the SN-VTA to be the source of DA i n the c o r t e x , accumbens, o l f a c t o r y t u b e r c l e , s e p t a l nucleus, amygdala, habenula, cerebellum, thalamus, hypothala-mus and median eminence (Fuxe et a l , 1974; F a l l o n et a l , 1978a, 1978b; L i n d v a l l et a l , 1974; L i n d v a l l , 1975; K i z e r et a l , 1976; Berger et a l , 1976; Carter and F i b i g e r , 1977; Koob et a l , 1975; F a l l o n and Moore, 1978; Emson and Koob, 1978). Evidence has been provided that the po p u l a t i o n of DA-containing neurons i n the SNC p r o j e c t i n g to the s t r i a t u m are separate from those p r o j e c t i n g to other b r a i n areas ( F a l l o n et a l , 1978b; Moore, 1978). With regard to the e l e c t r o p h y s i o l o g i c a l nature of the DA input to the s t r i a t u m , the general concensus has s h i f t e d from the view that DA i s p r i m a r i l y i n h i b i t o r y on s t r i a t a l neurons to the c u r r e n t l y h e l d one of an e x c i t a t o r y ac-t i o n of DA (Richardson et a l , 1977; K i t a i et a l , 1975, 1976c; Davies and Tongroach, 1978). E x c i t a t o r y responses i n s e p t a l neurons have a l s o been ob-served by s t i m u l a t i o n of the VTA and t h i s was suggested to be mediated by DA (Assaf and M i l l e r , 1977). The d i s c r e p a n c i e s i n the determination of the e l e c -t r o p h y s i o l o g i c a l a c t i o n of the n i g r o - s t r i a t a l system have a l s o been suggested to be due to a dual i n n e r v a t i o n of the s t r i a t u m by the SN; a DA and non-DA pathway (Hedreen, 1971, Hedreen and Chalmers, 1972; Davies and Tongroach, 1978; F r i g y e s i and Purpura, 1967; F i b i g e r et a l , 1972). This p o s s i b i l i t y has not been f u l l y r e s o l v e d . A n i g r o t e c t a l p r o j e c t i o n has been demonstrated i n s e v e r a l species i n c l u -ding the r a t , cat and monkey (Hopkins and Niessen, 1976; Hopkins, 1977; G r a y b i e l , 1975; G r a y b i e l , 1978; R i n v i k et a l , 1976; F a u l l and Mehler, 1978). This p r o j e c t i o n a r i s e s e x c l u s i v e l y i n the SNR and terminates mainly i n the caudal two-thirds of the intermediate gray l a y e r of the i p s i l a t e r a l s u p e r i o r c o l l i c u l u s (SC) . The n i g r a l f i b e r s i n the SC array themselves i n remarkably r e g u l a r bands running the r o s t r a l - c a u d a l l e n g t h of the SC. From electrophy-s i o l o g i c a l and biochemical evidence the SNR-SC pathway appears to have both an e x c i t a t o r y and i n h i b i t o r y component. S t i m u l a t i o n of the SN has been shown to 30 produce e x c i t a t i o n of SC neurons (York and Farber, 1977). Support f o r an i n h i -b i t o r y n i g r o - c o l l i c u l a r pathway has been provided by the f i n d i n g that GAD i n the SC decreases f o l l o w i n g n i g r a l l e s i o n s (Vincent et a l , 1978). The SNR has a l s o been shown to p r o j e c t to the VA and VL thalamus i n the monkey (Carpenter and P e t e r s , 1972; Carpenter et a l , 1976) and the homologue of these n u c l e i i n the r a t ( R i n v i k , 1975; C l a v i e r et a l , 1976; F a u l l and Mehler, 1978). In a d d i t i o n , although i n s e v e r a l s t u d i e s no n i g r a l - i n t r a l a m i -nar p r o j e c t i o n s were observed ( R i n v i k , 1975; K u l t a s - I l i n s k y e t a l , 1978; Carpenter et a l , 1976), there i s one report of a p r o j e c t i o n to the PF from the SN ( C l a v i e r et a l , 1976). The nigro-rthalamic p r o j e c t i o n appears to be l a r g e l y i n h i b i t o r y as s t i m u l a t i o n of the SN has been shown to produce monosynaptic IPSP's i n thalamic neurons (Ueki et a l , 1977; Anderson and Yoshida, 1977; Deniau et a l , 1978b). There are no r e p o r t s of the biochemical nature of the ni g r o - t h a l a m i c pathway. Several of the above st u d i e s and others (Jayarman et a l , 1977; Hedreen and Chalmers, 1972) have reported on the r e g i o n a l d i s t r i b u t i o n w i t h i n the SN of neurons p r o j e c t i n g to the s t r i a t u m , SC and thalamus. However, the r e l a -t i o n s h i p to each other of neurons p r o j e c t i n g to these v a r i o u s t a r g e t s has been most c l e a r l y demonstrated i n a recent study by F a u l l and Mehler (1978). These i n v e s t i g a t o r s showed that n i g r a l neurons comprising the s t r i a t a l , tha-lamic and SC e f f e r e n t s f e l l i n t o d i s t i n c t s i z e groups and that w i t h i n the SNR and SNC there was very l i t t l e overlap of the l o c a t i o n of the neurons p r o j e c -t i n g to these separate n u c l e i . This suggests that the various SN p r o j e c t i o n s a r i s e from separate populations of neurons and i s t h e r e f o r e i n c o n s i s t e n t w i t h e l e c t r o p h y s i o l o g i c a l observations demostrating that a s i g n i f i c a n t p o r t i o n of neurons i n the SNR send axon branches to both the SC and thalamus (Anderson and Yoshida, 1977). Deniau et a l (1978c), r e f u t i n g t h e i r e a r l i e r work (Deniau et a l , 1977), have a l s o suggested on e l e c t r o p h y s i o l o g i c a l grounds the 31 existence of c o l l a t e r a l s of SNR e f f e r e n t s to m u l t i p l e s i t e s . These discrepan-c i e s are reminiscent of those already descirbed concerning the degree of c o l l a -t e r i z a t i o n of p r o j e c t i o n s from the EP to the habenula and thalamus. These i n -c o n g r u i t i e s between anatomical and e l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n s have yet to be r e c o n c i l e d . A f u r t h e r p o i n t regarding the study by F a u l l and Mehler (1978) i s t h e i r o b s e rvation of s u b s t a n t i a l numbers of n i g r o - s t r i a t a l neurons l o c a t e d i n the SNR. As mentioned e a r l i e r a pathway connecting the SN and s t r i a t u m i n a d d i -t i o n to the dopaminergic n i g r o - s t r i a t a l system has been proposed ( H a t t o r i et a l , 1973a; F i b i g e r et a l , 1972; F e l t z and DeChamplain, 1972). The non-SNC l o -c a t i o n of these c e l l s together w i t h the observation of the presence i n the SNR of non-catecholamine f l u o r e s c e n t HRP-positive neurons a f t e r s t r i a t a l HRP i n -j e c t i o n s (Berger et a l , 1978) suggest that these c e l l s may be the o r i g i n of the non-dopaminergic n i g r o - s t r i a t a l system. In the context of other p o s s i b l e sources (besides the SNR) of s t r i a t a l a f f e r e n t s o r i g i n a t i n g i n the v i c i n i t y of the SN, i t i s perhaps s i g n i f i c a n t that f o l l o w i n g HRP i n j e c t i o n s i n the s t r i a -tum of the cat l a b e l e d c e l l s have been observed i n the r e t r o r u b r a l nucleus and i n c e l l bridges between t h i s nucleus and the SN (Vandermaelen et a l , 1978). F a l l o n and Moore (1978) a l s o found l a b e l e d c e l l s i n t h i s area i n the cat a f t e r s t r i a t a l HRP i n j e c t i o n s . They, however, simply r e f e r r e d to t h i s zone as caudal SN. For a more d e t a i l e d d i s c u s s i o n of the r e l a t i o n s h i p of these out-l y i n g n i g r a l neurons to n i g r a l e f f e r e n t s and a f f e r e n t s and, i n p a r t i c u l a r , hypothalamo-nigral connections the reader i s r e f e r r e d to papers by Nauta and Domesick (1978) and Nauta et a l (1978). Further s t u d i e s are r e q u i r e d to de-termine the exact r e l a t i o n of these neurons to the SN proper. There are some a d d i t i o n a l descending p r o j e c t i o n s from the SNR to the c e n t r a l gray area, pons and medulla oblongata (Hopkins, 1977; Hopkins and Neissen, 1976). The t e r m i n a l f i e l d s of these f i b e r s and the c e l l s of o r i g i n 32 i n the SN r e l a t i v e to the thalamic, s t r i a t a l and t e c t a l p r o j e c t i o n neurons are as yet not w e l l defined. 3. n i g r a l a f f e r e n t s The SN r e c e i v e s numerous a f f e r e n t s from d i v e r s e b r a i n areas. In view of i m p l i c a t i o n s of the involvement of the SN and i t s e f f e r e n t s i n the mediation of c e r t a i n b e h a v i o r a l processes and i n the s i t e of a c t i o n of some pharmacolo-g i c a l agents, the n i g r a l a f f e r e n t s take on s p e c i a l s i g n i f i c a n c e . The SN r e c e i v e s monoamine inputs from the LC and raphe nucleus. Although not s p e c i f i c a l l y s t a t e d the LC appears to p r o j e c t to the SNE. as gleaned from the diagram i n a paper by Jones and Moore (1977). That t h i s p r o j e c t i o n i s NA-c o n t a i n i n g i s suggested by the presence i n t h i s area of NA (Versteeg et a l , 1976) and NA-containing f i b e r s (Ungerstedt, 1971a) and a decrease i n DBH f o l -lowing l e s i o n s of the p o s t e r i o r midbrain (Reis et a l , 1978). Swanson and Hartman (1975) d i d not observe immunohistochemical s t a i n i n g f o r DBH i n the SN. I t should be pointed out, however, that these workers c o n s i s t e n t l y observed l i t t l e or no s t a i n i n g i n areas known to r e c e i v e NA i n p u t . In t h i s context i t i s p e r p l e x i n g that they reported s t a i n i n g i n the f r o n t a l c ortex which contains lower DBH a c t i v i t y than the SN (Reis et a l , 1978). The SN r e c e i v e s a s u b s t a n t i a l p r o j e c t i o n from the midbrain raphe nucleus (MRN) (Moore et a l , 1978; F i b i g e r and M i l l e r , 1978). The SNC appears to r e -ceive a heavier p r o j e c t i o n than the SNR and indeed Azmitra and Segal (1978) r e p o r t the absence of a p r o j e c t i o n to the SNR. That t h i s p r o j e c t i o n i s sero-t o n e r g i c i s i n d i c a t e d by the f i n d i n g s that s e r o t o n i n l e v e l s decrease i n the SN f o l l o w i n g raphe l e s i o n s (Dray et a l , 1978; P a l k o v i t s et a l , 1977; F i b i g e r and M i l l e r , 1977). E l e c t r o p h y s i o l o g i c a l s t u d i e s have shown the raphe-SN p r o j e c -t i o n to be mainly i n h i b i t o r y ( F i b i g e r and M i l l e r , 1977; Dray et a l , 1978), and t h i s i n h i b i t i o n of SN neurons appears to be mediated by s e r o t o n i n ( F i b i g e r and M i l l e r , 1977). As i n the case of the extent of t e r m i n a t i o n of raphe e f f e r e n t s i n the SNR, there i s disagreement concerning the exact source of these f i b e r s 33 from w i t h i n the raphe n u c l e i . Some s t u d i e s suggest that the p r o j e c t i o n a r i s e s e x c l u s i v e l y from the DRN (Azmitia and Segal, 1978; F i b i g e r and M i l l e r , 1977; Pasquier et a l , 1977) w h i l e others suggest the ex i s t e n c e of ser o t o n e r g i c a f f e -r e n t s from both DRN and MEN (Dray et a l , 1978; Dray et a l , 1976b). The a f f e r e n t s to the SN from the nucleus accumbens have been adequately demonstrated (Nauta et a l , 1978; Swanson and Cowan, 1975; W i l l i a m s et a l , 1977; Conrad and P f a f f , 1976). The t e r m i n a l area w i t h i n the SN i s heavi e s t i n the medial h a l f of the SNC although there i s some ter m i n a t i o n i n the VTA and the SNR subjacent to the SNC. In an autoradiographic study Powell and Leman (1976) a l s o purported to demonstrate an accumbo-nigral p r o j e c t i o n . However, contrary to the above s t u d i e s , they f a i l e d to confirm accumbens connections w i t h the e x t e r n a l segment of the globus p a l l i d u s and found an accumbens p r o j e c t i o n to the caudate nucleus. In the report by Powell and Leman (1976), inasmuch as the l a b e l i n the caudate looks s u s p i c i o u s l y l i k e d i f f u s i o n of r a d i o a c t i v i t y up a needle t r a c t , t h i s l a b e l i n g i n the caudate confounds t h e i r a n a l y s i s of the e f f e r e n t p r o j e c t i o n s of the nucleus accumbens. E l e c t r o p h y s i o l o g i c a l experiments suggest that the n i g r a l and VTA a f f e -r e n t s of the accumbens are p r i m a r i l y i n h i b i t o r y although e x c i t a t i o n has a l s o been observed (Dray and Oakley,.1977; Wolf et a l , 1978). The i n h i b i t i o n was suggested to be mediated by GABA as i t was blocked by b i c u c u l l i n e (Wolf et a l , 1978). However, no decreases i n GABA l e v e l s i n the SN were observed f o l l o w i n g e l e c t r o l y t i c l e s i o n s of the accumbens (Dray and Oakley, 1977) or GAD i n the VTA f o l l o w i n g hemitransections at the l e v e l of the r o s t r a l GP (Fonnum et a l , 1977) . Lesions of the nucleus accumbens w i t h k a i n i c a c i d decreased GAD a c t i -v i t y i n the VTA but not i n the SN (Waddington and Cross, 1978). In view of the l a c k of e f f e c t of hemitransections, i t seems p o s s i b l e that the r e s u l t s of the study by Waddington and Cross (1978) may be a t t r i b u t e d to d i f f u s i o n of k a i n i c a c i d to the s t r i a t u m and/or GP. 34 From autoradiographic (Nauta and Cole, 1978) and HRP stu d i e s (Kanazawa et a l , 1976; T u l l o c h et a l , 1975) i t i s evident that both the SNR and SNC r e c e i v e a s u b s t a n t i a l p r o j e c t i o n from the SUT. No s t u d i e s have been conducted to c h a r a c t e r i z e the e l e c t r o p h y s i o l o g i c a l or biochemical nature of the n i g r a l a f f e -r ents from the SUT. Other a f f e r e n t s to the SN a r i s e from v a r i o u s p a r t s of the co r t e x , the habenula, hypothalamus and amygdala (Bunney and Aghajanian, 1976a). In a r e -cent paper by P h i l l i p s o n (1978) no l e s s than 37 d i v e r s e b r a i n areas were ob-served to co n t a i n l a b e l e d neurons f o l l o w i n g HRP i n j e c t i o n s i n t o the VTA. Further work, however, i s re q u i r e d to s u b s t a n t i a t e some of these afferents.and to determine t h e i r importance. I I Adenylate Cyclase and Dopamine Receptors i n the Substantia N i g r a and  Striatum The l i k e l i h o o d that Parkinson's disease and the p o s s i b i l i t y that s c h i z o -p hrenia and even some of the symptoms of Huntington's disease have t h e i r cause i n malfunctions of c e n t r a l DA systems has l e d to the expenditure of enormous e f f o r t s to determine the f a c t o r s that govern the a c t i v i t y of dopaminergic neu-rons i n the SN. One approach along these l i n e s i s the d e f i n i t i o n by anatomi-c a l methods of the n i g r a l a f f e r e n t s that might r e g u l a t e the a c t i v i t y of these neurons. Inputs to the:. SN from the s t r i a t u m have rece i v e d p a r t i c u l a r a t -t e n t i o n i n t h i s regard. Another approach i s d i r e c t e d toward the concept of the s e l f - r e g u l a t i o n of dopaminergic n i g r a l neurons: the importance of t h i s process, the extent to which i t occurs and the mechanisms by which i t i s achieved. What f o l l o w s i s a d e s c r i p t i o n of some of the key f i n d i n g s which have prompted the current view that there e x i s t both a s t r i a t o - n i g r a l feed-back system and a feedback system i n t r i n s i c to dopaminergic neurons. Both of these systems may a i d DA-containing n i g r a l neurons to gauge and adjust t h e i r output. An i n t r o d u c t i o n to the emergence of these concepts i s best done 35 w i t h i n a h i s t o r i c a l p e r s p e c t i v e . Consequently, both feedback systems are d i s -cussed simultaneously. More than f i f t e e n years ago Ca r l s s o n and L i n d q v i s t (1963) administered h a l o p e r i d o l and chlorpromazine (DA' receptor b l o c k e r s ; although even that was not c e r t a i n at the time) to r a t s and found that these agents augmented the accumulation of DA metabolites i n the b r a i n . They suggested that there i s "a compensatory a c t i v a t i o n of monoaminergic neurons a f t e r the blockade of mono-aminergic r e c e p t o r s " . A decade passed before the i n s i g h t of t h i s c o n c l u s i o n began to be appreciated. What t h i s c o n c l u s i o n d i d not e x p l a i n , however, was whether the feedback system was i n t r i n s i c to the DA neurons or mediated through a long axon system. The b a t t l e waged by the proponents of each of these mechanisms has been raging from the time i t became apparent that two such systems may e x i s t . The f i r s t suggestion that a neuronal feedback loop may account f o r the r e g u l a t i o n of dopaminergic tra n s m i s s i o n and b i o s y n t h e s i s i n the n i g r o - s t r i a t a l pathway was provided by Co r r o d i et a l (1967). These workers suggested that some of the e f f e c t s of d-amphetamine (AMPH) on DA metabolism might be secon-dary to a decrease i n the f i r i n g r a t e of dopaminergic neurons. They f u r t h e r suggested that t h i s decrease of DA neuronal a c t i v i t y might be due to the a b i -l i t y of AMPH to cause, i n d i r e c t l y , an increase i n the s t i m u l a t i o n of post-s y n a p t i c DA r e c e p t o r s , l e a d i n g to a compensatory decrease i n the f i r i n g r a t e of DA neurons mediated by an i n t e r n e u r o n a l feedback pathway. These sugges-t i o n s found support through the subsequent work of Bunney, Aghajanian and c o l -l a b o r a t o r s (Bunney et a l , 1973a, 1973b; Aghajanian and Bunney, 1973, 1974a, 1974b; Bunney and Aghajanian, 1975; Bunney and Aghajanian, 1973, 1976b). These workers found 1) that i n t r a v e n o u s l y administered AMPH causes a depres-s i o n of DA neuronal a c t i v i t y i n the SNC, 2) that p o s t s y n a p t i c c e l l s i n the s t r i a t u m are very s e n s i t i v e to m i c r o i o n t o p h o r e t i c a l l y a p p l i e d AMPH whereas n i g r a l neurons are not, and 3) that t r a n s e c t i o n of the b r a i n between the 36 s t r i a t u m and SN and more s p e c i f i c a l l y , l e s i o n s i n the crus c e r e b r i or i n the v i c i n i t y of the t a i l of the s t r i a t u m , g r e a t l y attenuate the depressant e f f e c t s of intravenous AMPH on n i g r a l neurons• At about the same time that the above work was begun, evidence f o r an in t r a n e u r o n a l dopaminergic feedback mechanism was provided by the specta c u l a r observations of Kehr et a l (1972).. I n i t i a l l y , Anden et a l (1972) found t h a t , w i t h i n the f i r s t day a f t e r hemitransections between the s t r i a t u m and SN, there occurs a marked increase i n DA l e v e l s i n DA neuron t a r g e t areas a n t e r i o r to the l e s i o n . Kehr et a l (1972) (see a l s o Carlsson et a l , 1972, 1976) demonstra-ted that t h i s was due to an increase i n DA synthesis a f t e r axotomy. They f u r -ther showed that the increased DA sy n t h e s i s was reversed by the DA ago n i s t , apomorphine, and that t h i s r e v e r s a l could be blocked by pretreatment w i t h h a l o -p e r i d o l . Since these drugs i n f l u e n c e d events i n DA-containing terminals even a f t e r the above hemitransections, these r e s u l t s l e d to the suggestion of the presence of DA receptors on dopaminergic terminals and the term "autoreceptors" was coined f o r these e n t i t i e s by Ca r l s s o n (1975). These experiments, however, could not exclude a postsynaptic e f f e c t of apomorphine and h a l o p e r i d o l f o l -lowed by an a c t i o n on dopaminergic terminals of a t r a n s s y n a p t i c messenger. Support f o r the existence of autoreceptors on DA-containing neurons i n the SN was provided by Groves et a l (1975). Contrary to the above c i t e d f i n d -ings of Bunney and Aghajanian, these workers found t h a t i n t r a n i g r a l i n f u s i o n s of AMPH depressed whereas h a l o p e r i d o l augmented neuronal a c t i v i t y i n the SNC. Moreover, i n t r a s t r i a t a l i n f u s i o n s of AMPH increased and h a l o p e r i d o l decreased SNC neuronal a c t i v i t y . Since these r e s u l t s were i n c o n s i s t e n t w i t h a s t r i a t o -n i g r a l negative feedback loop, Groves et a l (1975) suggested the existence of a p o s i t i v e s t r i a t o - n i g r a l feedback pathway and that the e f f i c a c y of t h i s i s masked by the e f f e c t of the above drugs on dopaminergic transmission w i t h i n the SN; s p e c i f i c a l l y , t h e i r a c t i o n on autoreceptors on DA-containing neurons i n the SNC. These r e s u l t s were c o n s i s t e n t w i t h the subsequent demonstrations 37 by Garcia-Munoz et a l (1977) that l e s i o n s of the s t r i a t o - n i g r a l t r a c t do not a l t e r the a b i l i t y of h a l o p e r i d o l to augment DA metabolism i n the s t r i a t u m . Apart from the conc l u s i o n by Groves et a l . (1975) of the s i t e of a c t i o n of h a l o p e r i d o l and AMPH, t h e i r suggestion of autoreceptors i n the SN was i n agree-ment w i t h e a r l i e r proposals of t h i s by Aghajanian and Bunney (1973, 1974a, 1974b) who found that i n t r a n i g r a l a d m i n i s t r a t i o n of DA and apomorphine caused a h a l o p e r i d o l r e v e r s i b l e i n h i b i t i o n of SNC neurons. In a d d i t i o n , the concept of n i g r a l autoreceptors was strengthened by demonstrations that 1) the den-d r i t i c processes of DA neurons c o n t a i n DA and v e s i c l e s (Haj.du et a l , 1973; Bj o r l u n d and L i n d v a l l , 1975; Paizek et a l , 1971), 2) c e l l s i n the SN e x h i b i t calcium-dependent DA rel e a s e (Geffen et a l , 1976; Korf et a l , 1976; N i e o u l l o n et a l , 1977), and 3) n i g r a l DA neurons engage i n dendro-dendritic s y n a p t i c contacts (Wilson et a l , 1977a). Another l i n e of i n v e s t i g a t i o n that has added to the saga of the DA auto-receptor i n v o l v e s a species of adenylate c y c l a s e whose a c t i v i t y i s sti m u l a t e d by dopamine. When Kebabian et a l (1972) demonstrated a DA-sensitive adenylate c y c l a s e (DAC) i n the s t r i a t u m they suggested that the DA receptor i s an i n t e -g r a l p a r t of the c y c l i z i n g enzyme. The d e s i r e by many i n v e s t i g a t o r s to i n t e r -p r e t experimental r e s u l t s i n accordance w i t h t h i s suggestion marshalled the concept of a s i n g l e DA receptor-adenylate c y c l a s e e n t i t y i n t o dogma. This i n -v a r i a b l y i n f l u e n c e d views regarding the DA autoreceptor. F o l l o w i n g the i n i -t i a l demonstrations of DAC i n the SN ( T r a f i c o n t e et a l , 1976; P h i l l i p s o n and Horn, 1976; Spano et a l , 1976), i t was q u i c k l y determined that DAC i s not lo c a t e d on DA-containing n i g r a l neurons (Kebabian and Saavedra, 1976; Premont et a l , 1976) but p o s s i b l y on n i g r a l a f f e r e n t terminals from the s t r i a t u m (Premont et a l , 1976; Gale et a l , 1977a; P h i l l i p s o n et a l , 1977), and more s p e c i f i c a l l y , on n i g r a l a f f e r e n t terminals from s t r i a t a l neurons l o c a t e d pos-t e r i o r to the a n t e r i o r pole of the GP (Spano et a l , 1977). Although n i g r a l DA autoreceptors had gained support from other l i n e s of evidence, these r e -38 s u i t s cast some doubt as to t h e i r e x i s t e n c e . Further support f o r the l o c a l i z a t i o n of DAC on n i g r a l a f f e r e n t s was pro-vided by Reubi et a l (1977) who reported that both DA and AMPH induce a f l u -phenazine or h a l o p e r i d o l r e v e r s i b l e r e l e a s e of ^ H-GABA from n i g r a l s l i c e s . H-GABA r e l e a s e was a l s o e l i c i t e d by d i b u t y r y l - c y c l i c AMP. These f i n d i n g s d e a l t a p a r t i c u l a r l y damaging blow to the concept of n i g r a l DA autoreceptors s i n c e much of the e a r l i e r e l e c t r o p h y s i o l o g i c a l support f o r t h e i r existence could be r e i n t e r p r e t e d as being due to the a c t i o n of DA and apomorphine on t e r -minals causing GABA r e l e a s e which i n tu r n i n h i b i t s dopaminergic neurons. The a c t i o n of h a l o p e r i d o l and AMPH could a l s o be a p p r o p r i a t e l y r e i n t e r p r e t e d . At press time of t h i s t h e s i s , however, Bunney and Aghajanian (1978) have the l a t e s t word. These workers observed that GABA antagonists block and i n t r a - . s t r i a t a l i n j e c t i o n s of k a i n i c a c i d p a r t i a l l y b l ock the a b i l i t y of AMPH to i n -h i b i t SNC neuronal a c t i v i t y . Since k a i n i c a c i d l e s i o n s of the s t r i a t u m only p a r t i a l l y a b o l i s h the e f f e c t of AMPH and s i n c e t h i s p a r t i a l i n h i b i t i o n was not reversed by GABA b l o c k e r s , Bunney and Aghajanian r e f u t e d some of t h e i r e a r l i e r work and suggested that some of the e f f e c t s of AMPH may occur d i r e c t l y i n the SN as o r i g i n a l l y proposed by Groves et a l (1975). In a d d i t i o n , they argued s t r o n g l y against a GABA mediated i n h i b i t i o n of SNC c e l l s through the r e l e a s e of GABA by DA, s i n c e both DA and apomorphine s t i l l caused a potent, h a l o p e r i -d o l r e v e r s i b l e i n h i b i t i o n of n i g r a l neuronal a c t i v i t y which was not a f f e c t e d by s t r i a t a l l e s i o n s or GABA b l o c k e r s . Developments regarding DA autoreceptors on DA-containing terminals i n the s t r i a t u m have p a r a l l e l e d roughly those i n the SN. Fo l l o w i n g the i n i t i a l demon-s t r a t i o n by.Kehr et a l (1972) of the e f f e c t s of dopaminergic drugs on DA meta-bol i s m on.severed axons, evidence f o r the existence of these autoreceptors was provided by C h r i s t i a n s e n and Squires (1974a, b ) . They found that apomorphine causes the i n h i b i t i o n of DA synte h s i s i n synaptosomes and that t h i s i s r e -versed by a host of DA ant a g o n i s t s . S t r i a t a l DA autoreceptors gained a d d i -39 t i o n a l acceptance w i t h the i n v i t r o demonstrations that cAMP, through p o s s i b l y an adenosine c y c l i c 3', 5' monophospate (cAMP)-dependent p r o t e i n kinase, a l -t e r s the k i n e t i c s t a t e of TH (Lovenberg et a l , 1975a, b; Morgenroth et a l , 1975) . This was c o n s i s t e n t w i t h the presence of DAC i n the s t r i a t u m and sug-gested that DA exerted i t s presynaptic e f f e c t s through DAC. These views stood u n t i l i t was demonstrated that k a i n i c a c i d s t r i a t a l l e s i o n s v i r t u a l l y a b o l i s h DAC i n the s t r i a t u m suggesting an e x c l u s i v e l y p o s t s y n a p t i c l o c a l i z a t i o n of t h i s enzyme (McGeer et a l , 1976; Schwarcz and Coyle, 1977). The most recent data bearing on s t r i a t a l DA autoreceptors have been ob-t a i n e d by f u r t h e r u t i l i z a t i o n of i n t r a s t r i a t a l i n j e c t i o n s of k a i n i c a c i d . As mentioned e a r l i e r , dendro-axonic t r a n s m i s s i o n i n the s t r i a t u m could not be r u l e d out as a p o s s i b l e a l t e r n a t i v e to autoreceptors. In t h i s regard, i t i s noteworthy that ACh, f o r example, has been shown to i n f l u e n c e i n v i t r o the r e -l e a s e of DA from s t r i a t a l t i s s u e ( W e s t f a l l , 1974). What the work w i t h k a i n i c a c i d has demonstrated i s that a f t e r s t r i a t a l l e s i o n s w i t h t h i s compound, apo-morphine s t i l l decreases and h a l o p e r i d o l s t i l l i ncreases DA metabolism i n the s t r i a t u m (Fadda et a l , 1977; D i C h i a r a et a l , 1977; A r g i o l a s et a l , 1978). These l e s i o n s l a r g e l y e l i m i n a t e d s t r i a t a l p o s t s y n a p t i c elements and n i g r a l a f f e r e n t s from the s t r i a t u m and t h e r e f o r e would have l e f t the DA neurons i n the SNC i s o l a t e d both at t h e i r d e ndrites and terminals from p o s s i b l e t r a n s -s y n a p t i c i n f l u e n c e . Notwithstanding the p o s s i b i l i t y of incomplete l e s i o n s , these r e s u l t s supported the e x i s t e n c e of autoreceptors e i t h e r on DA terminals or dendrites or both. While i t became apparent that DAC was not present on n i g r a l DA neurons or t h e i r t e r minals i n the s t r i a t u m , such was the atmosphere surrounding DA autoreceptors that i n order to avoid d i s p e l l i n g the e x i s t e n c e of these a l t o -gether, i t was suggested by numerous workers (McGeer et a l , 1976; Gale et a l , 1977; P h i l l i p s o n et a l , 1977; Premont et a l , 1976) t h a t , i f DA autoreceptors e x i s t , they may not be coupled w i t h adenylate c y c l a s e . T h i s , of course, r e -40 quired a reworking of the ideas concerning the mechanisms of dopaminergic t r a n s m i s s i o n . In view of t h i s and s i n c e DA autoreceptors were " i n f e r r e d " to e x p l a i n data described above and i n some cases subsequently r e l i e d on to i n t e r -p r e t otherwise i n e x p l i c a b l e f i n d i n g s , c r i t i c a l experiments of a more d i r e c t nature were r e q u i r e d to t e s t the hypothesis of DA autoreceptors and these awaited the merger between the production of appropriate l e s i o n s and measure-ments u t i l i z i n g receptor b i n d i n g techniques. I l l The Present I n v e s t i g a t i o n a) Methodological c o n s i d e r a t i o n s The present s t u d i e s have u t i l i z e d some of the major neuroanatomical tech-niques c u r r e n t l y i n use to e l u c i d a t e b r a i n c i r c u i t r y . Some of these tech-niques together w i t h t h e i r advantages and disadvantages are discussed i n t h i s s e c t i o n . More than three decades ago Weiss and Hiscoe (1948) discovered that ma-t e r i a l s are transported from the c e l l body of a neuron along i t s axon to the axon t e r m i n a l . Some time l a t e r , T a y l o r and Weiss (1965) and Droz and Leblond (1963) demonstrated, using autoradiographic techniques, that neuronal p e r i -karya could accumulate 3 H - l e u c i n e i n j e c t e d i n t h e i r v i c i n i t y , i n c o r p o r a t e i t i n t o p r o t e i n and t r a n s p o r t the l a b e l e d 3 H - p r o t e i n along the axon processes of the c e l l to the nerve t e r m i n a l . Subsequent i n v e s t i g a t i o n s showed the poten-t i a l of t h i s technique i n the t r a c i n g of neuroanatomical pathways (Hendrickson, 1969;.Lasek et a l , 1968; Schonbach et a l , 1971). I t was shown that the s i l v e r g r a i n s seen i n autoradiographs enabled f i b e r systems to be traced from the c e l l body ( i n t h i s case, the d o r s a l root ganglion) to t e r m i n a l a r b o r i z a t i o n s (Lasek et a l , 1968). I t was not u n t i l 1972 that Cowan et a l (1972) developed and a p p l i e d the method to t r a c i n g pathways i n the CNS. This technique o f f e r s s e v e r a l advantages over other methods: 1) I t i s based on a p h y s i c a l proper-ty of nerve c e l l s and thus does not i n v o l v e p e r t u r b a t i o n of normal f u n c t i o n 41 which may l e a d to m i s i n t e r p r e t a t i o n s . 2) There i s no detectable retrograde t r a n s p o r t of l a b e l ( 3H-protein) f o l l o w i n g the i n j e c t i o n of t r i t i a t e d amino a c i d i n t o the r e g i o n of t e r m i n a t i o n of a neuronal pathway (Cowan et a l , 1972; Crossland et a l , 1973; Schonbach and Guenod, 1971). 3) Axons passing through an area i n t o which 3H-amino acid s have been i n j e c t e d do not synthesize the l a b e l i n t o p r o t e i n or transport i t . Thus, there i s no " f i b e r of passage" problem (Cowan et a l , 1972; G o t t l i e b and Cowan, 1973; Heuser and M i l e d i , 1970; Swanson et a l , 1974). 4) At the EM l e v e l grains can be a s s o c i a t e d w i t h e i t h e r f i b e r s of passage or t e r m i n a l s , and i n a t e r m i n a t i o n area the morpho-logy of the t e r m i n a l s of a p r o j e c t i o n can be e s t a b l i s h e d . 5) The technique i s very s e n s i t i v e , enabling the t r a c i n g of f i n e or sparse f i b e r systems (Hendrickson et a l , 1972; Meier, 1973). There are s e v e r a l l i m i t a t i o n s of autoradiographic t r a c i n g techniques. 1) At the LM l e v e l s i n c e only s i l v e r grains are v i s u a l i z e d , no i n f o r m a t i o n concerning the morphology of the axons or terminals can be gained. In a d d i -t i o n , although the d e n s i t y and p a t t e r n of d i s t r i b u t i o n of grains i s g e n e r a l l y a good i n d i c a t i o n e i t h e r of f i b e r s - o f - p a s s a g e or of a t e r m i n a l f i e l d , the u l -timate proof of t h i s must sometimes r e s t w i t h i n v e s t i g a t i o n s at the EM l e v e l . 2) An i n j e c t i o n of l a b e l i n t o neuronal s t r u c t u r e s r e s u l t s i n a gradient of l a b e l e d neurons away from the highest d e n s i t y which occurs at the focus of the i n j e c t i o n s i t e . An e v a l u a t i o n of the extent of spread of the l a b e l from the intended i n j e c t i o n s i t e to undesired adjacent areas i s t h e r e f o r e e s s e n t i a l i n order to determine whether contamination by these areas of the f i b e r pro-j e c t i o n under i n v e s t i g a t i o n has occurred. However, i t i s u n c e r t a i n how h e a v i l y l a b e l e d a c e l l must be i n order f o r l a b e l to be detected i n i t s axon or axon t e r m i n a l . As w e l l as the anterograde t r a n s p o r t of m a t e r i a l , i t i s now w e l l e s t a -b l i s h e d that retrograde t r a n s p o r t from the nerve t e r m i n a l to the neuronal c e l l body occurs ( J e f f r e y and A u s t i n , 1973; L a V a i l and L a V a i l , 1974). I t has been 42 found that c e r t a i n l a r g e exogenous macromolecules can be taken up by the t e r -minals of axons and transported to the neuronal p e r i k a r y a ( K r i s t e n s s o n and Olsson, 1971, 1974). Through the retrograde t r a n s p o r t of exogenously. a p p l i e d h o r s e r a d i s h peroxidase (HRP) and appropriate h i s t o c h e m i c a l techniques the c e l l s of o r i g i n of n e u r a l pathways can be determined ( L a V a i l and L a V a i l , 1972, 1974; L a V a i l et a l , 1973). As e f f e c t i v e as t h i s technique has become i n r e -p l a c i n g retrograde c e l l degeneration techniques, there are c e r t a i n l i m i t a t i o n s 1) In common w i t h anterograde t r a c i n g u s i n g 3H-amino a c i d s , there i s the pro-blem of d i f f u s i o n of HRP and of d e f i n i t i o n of the i n j e c t i o n s i t e . Thus, i t i s necessary to i n t e r p r e t r e s u l t s w i t h c o n s i d e r a t i o n of the extent to which d i s -t a n t c e l l s have been l a b e l e d due to d i f f u s i o n and subsequent t r a n s p o r t of HRP. 2) The amount of HRP found i n the soma i s p a r t i a l l y dependent on the s i z e , number and extent of a r b o r i z a t i o n of the axon terminals i n the i n j e c t e d area (Jones, 1975). The technique i n t h i s regard i s not standardized to a l l ana-t o m i c a l systems but may vary w i t h d i f f e r e n t types of systems. 3) I t appears that f o r the most pa r t f i b e r s - o f - p a s s a g e do not accumulate and t r a n s p o r t HRP. However, there i s evidence that axons severed or i n j u r e d by the i n j e c t i o n pro-cedure take up and r e t r o g r a d e l y t r a n s p o r t HRP ( K r i s t e n s s o n and Olsson, 1974; Kuypers and Maisky, 1975; Nauta et a l , 1974). 4) Although HRP i s transported p r e f e r e n t i a l l y i n the retrograde d i r e c t i o n from the i n j e c t i o n s i t e , t r a n s p o r t can a l s o occur anterogradely (Lynch et a l , 1973; Sherlock and Raisman, 1975). A f u r t h e r set of techniques which a i d i n the d e l i n e a t i o n of b r a i n s t r u c -ture can be subsumed under the heading of biochemical neuroanatomy. The sim-p l e s t of these i s the measurement i n d i s c r e t e b r a i n areas of l e v e l s of sub-stances t h a t are e s t a b l i s h e d or suspected neurotransmitters and a d d i t i o n a l l y the measurement of the a c t i v i t y of t h e i r r e l a t e d metabolic enzymes. Although t h i s method o f f e r s i n f o r m a t i o n concerning only the gross anatomical l o c a l i z a -t i o n of a substance or enzyme, i t i s an important f i r s t step s i n c e i t provides the impetus f o r the f u r t h e r l o c a l i z a t i o n of a substance. 43 This i s achieved i n p a r t by the production of l e s i o n s at s i t e s which are suspected to c o n t r i b u t e to the a f f e r e n t p r o j e c t i o n s of the s t r u c t u r e of i n t e -r e s t . Through the process of anterograde degeneration, metabolic systems as-s o c i a t e d w i t h nerve terminals whose axons have been i n t e r r u p t e d w i l l be r e -duced. This method simultaneously provides i n f o r m a t i o n as to the existence of a pathway as w e l l as the neurotransmitter i n v o l v e d . In a d d i t i o n , an estimate can be made of the r e l a t i v e c o n t r i b u t i o n of a neurotransmitter to an area from v a r i o u s a f f e r e n t sources. The most recent a d d i t i o n to biochemical neuroanatomical s t u d i e s i s the measurement of neurotransmitter receptor l e v e l s . This i s a v a l u a b l e a d d i t i o n to the a r s e n a l of techniques a v a i l a b l e to the i n v e s t i g a t o r of b r a i n f u n c t i o n s i n c e i t i s an e x c e l l e n t complement to neuroanatomical and neurochemical s t u -d i e s . Thus, by producing appropriate l e s i o n s , receptors of s p e c i f i c neuro-t r a n s m i t t e r s can be l o c a l i z e d to d i s t i n c t neuron types and to neuronal e l e -ments. These st u d i e s a i d i n the determination of 1) connections at the synap-t i c l e v e l , 2) the d i r e c t i o n of t r a n s m i t t e r r e l e a s e and, 3) the p o t e n t i a l s i t e of a c t i o n of drugs. Biochemical neuroanatomy s u f f e r s two major disadvantages. F i r s t , i n s t u -d i e s i n v o l v i n g l e s i o n s there i s the fiber-of-passage problem. T h i s , however, can be avoided by employing s e l e c t i v e neurotoxins. Second, i t i s becoming apparent that the CNS i s a h i g h l y p l a s t i c s t r u c t u r e and t h e r e f o r e c e r t a i n l e s i o n s may produce changes secondary to the i n i t i a l l e s i o n s such as d i a s c h i -s i s , transneuronal degeneration, s u p e r s e n s i t i v i t y or axon sprouting (Schoenfeld and Hamilton, 1977). These phenomena may l e a d to m i s i n t e r p r e t a t i o n of r e s u l t s , p a r t i c u l a r l y s i n c e they occur over w i d e l y v a r y i n g periods of time. b) Objectives The present experiments were undertaken to provide answers to s p e c i f i c questions regarding the neuroanatomy and neurochemistry of the b a s a l g a n g l i a . These questions vary somewhat i n t h e i r depth of focus on the assorted n u c l e i 44 of the b a s a l g a n g l i a . In anatomical and biochemical neuroanatomical s t u d i e s s e v e r a l n u c l e i were i n v o l v e d . I n other s t u d i e s , concerted e f f o r t was d i r e c -ted toward a s i n g l e nucleus, such as the SN. I n a t h i r d s e r i e s of i n v e s t i g a t i o n s , a t t e n t i o n was given to a s p e c i f i c neuronal element of the SN; the DA-containing neurons of the SNC. What f o l -lows i s a more d e t a i l e d account of the goals of these v a r i o u s experiments. Neuroanatomical s t u d i e s were aimed e x c l u s i v e l y at the e f f e r e n t p r o j e c -t i o n s of the s t r i a t u m . S p e c i f i c a l l y , a more l u c i d d e s c r i p t i o n , as afforded by the autoradiographic and HRP anatomical methods, was sought i n the r a t . The s t r i a t a l e f f e r e n t to the EP was of p a r t i c u l a r i n t e r e s t as t h i s had been only p o o r l y described i n the rodent. Biochemical neuroanatomical s t u d i e s concentrated on the s t r i a t a l e f f e -r e n t s to the GP, EP and SN and the GP e f f e r e n t s to the SN. These experiments were designed to determine the extent to which these pathways conta i n GAD and CAT and the o r i g i n of the neurons of these f i b e r systems w i t h i n the s t r i a t u m and GP. Although e a r l i e r work had been conducted on e x a c t l y t h i s t o p i c , the dearth of knowledge i n t h i s area and the ti m e l y nature of the present in v e s -t i g a t i o n s i s emphasized by the f a c t t h a t not l e s s than s i x reports on t h i s s ubject appeared i n the l i t e r a t u r e roughly concomitant w i t h the published r e -s u l t s of the present work. Some attempts were al s o made to define b i o c h e m i c a l l y the e f f e r e n t pro-j e c t i o n s of the EP and SN to the thalamus and habenula. Two key observations prompted a d e t a i l e d i n v e s t i g a t i o n of the SN. The f i r s t was the discovery t h a t k a i n i c a c i d appears to be t o x i c to neuronal p e r i k a r y a and not to axons or t h e i r t e r m i n a l s . The second was the suggestion that axon terminals i n the SN bear DAC. In experiments on the SN described here, the former was e x p l o i t e d to t e s t : t h e l a t t e r . 45 With regard to the DA-containing neurons of the SN, the advent of neuro-t r a n s m i t t e r receptor measurement techniques provided a means to t e s t the hypo-t h e s i s of the e x i s t e n c e of autoreceptors on DA-containing neurons and te r m i -n a l s . These experiments i n v o l v e d the s p e c i f i c d e s t r u c t i o n of DA-containing neurons w i t h 6-OHDA or the d e s t r u c t i o n of the s t r i a t a l p r o j e c t i o n area of these neurons w i t h k a i n i c a c i d . This was followed by the assessment of the e f f e c t of these l e s i o n s on neurotransmitter receptor l e v e l s i n the SN and st r i a t u m . METHODS AND MATERIALS I Animal Surgery Male Wistar r a t s obtained from Woodlyn l a b o r a t o r i e s (Ontario) were em-ployed i n a l l the experiments. Rats were kept on a 12 hr d a r k / l i g h t c y c l e , fed ad l i b i t u m and group housed except a f t e r surgery at which time they were s i n g l y housed. During surgery animals weighing about 300 g were placed under p e n t o b a r b i t a l anesthesia (50 mg/kg, i.p.) and immobilized i n a Kopf s t e r e o -t a x i c apparatus. A l l s t e r e o t a x i c co-ordinates are according to the a t l a s of Konig and K l i p p e l (1963). Following surgery wounds were cleaned w i t h Zepharin and c l o s e d by sewing or w i t h wound c l i p s . S u r v i v a l times were u s u a l l y from 9 to 14 days except where otherwise i n d i c a t e d . These procedures were common to a l l of the f o l l o w i n g s u r g i c a l o perations. a) 6-Hydroxydopamine l e s i o n s of the n i g r o - s t r i a t a l pathway The neurotoxin 6-OHDA when c o r r e c t l y administered i s known to be very e f -f e c t i v e at causing the degeneration of neurons which accumulate catecholamines. To achieve d e s t r u c t i o n of the DA-containing NSP 6-OHDA was i n j e c t e d i n t o the axons of t h i s system at about the l e v e l of the l a t e r a l hypothalamus. These l e s i o n s are known to cause near-complete anterograde and retrograde degenera-t i o n of the terminals and c e l l bodies of the NSP ( C l a v i e r and F i b i g e r , 1977). To increase f u r t h e r the s p e c i f i c i t y of 6-OHDA to only DA-containing neurons, 46 animals rece i v e d desipramine HC1 (25 mg/kg) 30 min before the 6-OHDA i n j e c t i o n . Desipramine has been shown to be e f f e c t i v e i n preventing the uptake of 6-OHDA i n t o noradrenergic axons and terminals (Evetts and Iversen, 1970). In the pre-sent experiments animals r e c e i v e d u n i l a t e r a l i n j e c t i o n s of 4.0 ]ig of 6-OHDA contained i n 2.0 u l . The i n j e c t i o n r a t e was 0.2 yl/min. The s t e r e o t a x i c co-ordin a t e s were AP + 4.4, ML + 1.8, DV + 2.5. The 6-OHDA was d i s s o l v e d i n 0.15 M NaCl which contained 0.2 mg/ml as c o r b i c a c i d as an a n t i o x i d a n t , b) K a i n i c a c i d l e s i o n s S t e r e o t a x i c i n j e c t i o n s of k a i n i c a c i d - a neurotoxic analogue of glutamic a c i d - i n t o d i s c r e t e b r a i n areas has been shown to produce l o c a l i z e d l e s i o n s (Coyle and Schwarcz, 1976; McGeer and McGeer, 1976). S u f f i c i e n t doses of k a i n i c a c i d cause complete degeneration of neuronal c e l l bodies and the p r o l i -f e r a t i o n of g l i a at the i n j e c t i o n s i t e . However, the extent of damage, i f any-, by t h i s compound to fibe r s - o f - p a s s a g e and to nerve terminals i s not c e r t a i n . The b u l k of the i n f o r m a t i o n a v a i l a b l e to date (Schwarcz and Coyle, 1977a, 1977b; Schwarcz et a l , 1977; McGeer et a l , 1976; Meibach et a l , 1978; Butcher and Rogers, 1978) suggests that damage to f i b e r s and axon terminals i s minimal and i n p a r t dependent on the dose and thus s i z e of the l e s i o n e d area and whether or not c a v i t a t i o n occurs. In the present experiments k a i n i c a c i d l e s i o n s were employed not only as an a l t e r n a t i v e and adjunct to other l e s i o n methods, but al s o to take advantage of i t s r e l a t i v e s p e c i f i c i t y f o r neuronal p e r i k a r y a as compared w i t h axon t e r m i -n a l s . Four areas of the r a t b r a i n were u n i l a t e r a l l y i n j e c t e d w i t h k a i n i c a c i d . These were the e n t i r e s t r i a t u m , the head of the s t r i a t u m , the GP and the SN. Numerous attempts were made i n every b r a i n r e g i o n to achieve the d e s i r e d c i r -cumscribed l e s i o n s i z e w h i l e maximizing the l e s i o n s p e c i f i c i t y . K a i n i c a c i d was d i s s o l v e d i n sodium phosphate b u f f e r e d s t e r i l e p h y s i o l o g i c a l s a l i n e , pH 6.9. For the l a r g e s t r i a t a l l e s i o n s 10 nmoles of k a i n i c a c i d was i n j e c t e d i n 1.0 u l over a per i o d of 5 min at the co-ordinates AP + 8.4, ML + 2.8, DV + 4.5. 47 For l e s i o n s of the head of the s t r i a t u m 5 nmoles of k a i n i c a c i d i n 1.0 n l was d e l i v e r e d over a p e r i o d of 5 min at the co-ordinates AP + 8.4,: ML + 2.4, DV + 0.8. For GP l e s i o n s 2.0 nmoles of k a i n i c a c i d i n 0.2 \il was i n j e c t e d over a pe r i o d of 7 min at the co-ordinates AP + 7.8, ML + 2.6, DV + 3.6. For the n i g r a l l e s i o n s 5 nmoles of k a i n i c a c i d i n 1.0 u l was i n j e c t e d over a pe r i o d of 5 min at the co-ordinates AP + 2.2, ML + 2.3, DV+ 2.3. c) Hemitransections Hemitransections, as the term i m p l i e s , i n v o l v e coronal t r a n s e c t i o n s of the b r a i n at some a n t e r i o r - p o s t e r i o r l e v e l . The l e s i o n s s t a r t a t m i d l i n e and sever as v e n t r a l and l a t e r a l as i s p o s s i b l e a l l c o r t i c a l and s u b c o r t i c a l s t r u c -t u r e s . Two types of u n i l a t e r a l hemitransections were produced. These were at the l e v e l of the a n t e r i o r commissure j u s t r o s t r a l to the GP (AP + 7.5) and p o s t e r i o r to the GP at about the l e v e l of the EP. These were achieved by lowering a t h i n blade a t the m i d l i n e to the v e n t r a l surface of the b r a i n and moving i t l a t e r a l l y about 4 mm. d) E l e c t r o l y t i c l e s i o n s E l e c t r o l y t i c l e s i o n s were achieved by lowering a metal e l e c t r o d e (28-30 gauge) i n t o the d e s i r e d area of the b r a i n . A thermal l e s i o n was produced by passing current through the e l e c t r o d e w i t h the current being completed by grounding the animal at some p o i n t . The s i z e of the l e s i o n was determined by the s i z e of the un i n s u l a t e d area of the t i p of the e l e c t r o d e , the amplitude of the current and the d u r a t i o n f o r which i t was a p p l i e d . I n i t i a l l y , nichrome r e s i n i n s u l a t e d w i r e elec t r o d e s were employed. Subsequently, much superi o r e l e c t r o d e s were f a b r i c a t e d by using s t a i n l e s s s t e e l w i r e . To i n s u l a t e these el e c t r o d e s the w i r e was placed i n s i d e f i n e hematocrit c a p i l l a r y tubes and the tubes were p u l l e d i n a flame. E l e c t r o l y t i c l e s i o n s were produced u n i l a t e r a l l y i n four b r a i n regions; the EP, the head of the s t r i a t u m , the GP and the t a i l of the s t r i a t u m . Lesions of the EP were achieved w i t h a current of 2 mA f o r 30 sec at the co-ordinates AP + 6.3, ML + 2.8, DV + 1.8. S i m i l a r current and 48 d u r a t i o n parameters were employed f o r l e s i o n s of the head of the s t r i a t u m at the co-ordinates AP + 8.5, ML + 2.4, DV + 0.8. For the GP a current of 2 mA was a p p l i e d f o r 20 sec at the co-ordinates AP + 8.1, ML + 2.6, DV + 3.6. For l e s i o n s of the t a i l of the s t r i a t u m a l a r g e r p o r t i o n . o f the e l e c t r o d e t i p was d e i n s u l a t e d to increase the d o r s a l - v e n t r a l dimension of the l e s i o n . In a d d i -t i o n two l e s i o n s were produced i n the t a i l of the s t r i a t u m , both w i t h a cur-r e n t of 3 mA f o r 20 sec. The f i r s t was at the co-ordinates AP + 8.6, ML + 4.5, DV + 3.5, the second at the co-ordinates AP + 7.6, ML + 5.0, DV + 3.0. I I Anatomical Methods a) . Autoradiographic s t u d i e s The autoradiographic anterograde t r a n s p o r t technique f o r the t r a c i n g of n e u r a l connections was u t i l i z e d to study two anatomical systems. These were the p r o j e c t i o n s from the head and the t a i l of the s t r i a t u m to the GP, entope-duncular nucleus and s u b s t a n t i a n i g r a . In each group 4 to 7 animals were i n -j e c t e d i n the appropriate areas w i t h 3 H - l e u c i n e (6.5 yC/yl) i n phosphate buf-f e r e d p h y s i o l o g i c a l s a l i n e . The head of the s t r i a t u m received 0.1 to 0.4 u l and the t a i l of the s t r i a t u m r e c e i v e d 0.1 u l of the 3 H - l e u c i n e s o l u t i o n . A f t e r 24 to 36 hrs the r a t s were given an overdose of p e n t o b a r b i t a l and per-fused w i t h normal s a l i n e followed by 10% f o r m a l i n - s a l i n e . The b r a i n s were removed and immersed i n 10% f o r m a l i n - s a l i n e f o r 2 to 3 days, then i n sucrose-f o r m a l i n f o r another two to three days. Frozen s e r i a l s e c t i o n s (40 urn) were taken i n the f r o n t a l or s a g g i t a l plane and immersed i n 5% f o r m a l i n - s a l i n e f o r 1 to 3 days. The s e c t i o n s were then mounted from a g e l a t i n - e t h a n o l s o l u t i o n onto g l a s s s l i d e s and allowed to dry. A f t e r 2 to 3 days the s l i d e s were dipped i n t o Kodak NTB. 3 emulsion, l e f t to dry, then placed i n t o l i g h t t i g h t boxes and stored at 4°C. A f t e r 3 weeks the s l i d e s were developed i n Kodak D-19 and counter s t a i n e d f o r N i s s l w i t h c r e s y l v i o l e t . b) Retrograde t r a n s p o r t of h o r s e r a d i s h peroxidase 49 For the i n v e s t i g a t i o n of anatomical pathways by the retrograde t r a n s p o r t of h o r s e r a d i s h peroxidase (HRP), the b r a i n n u c l e i were i n j e c t e d u n i l a t e r a l l y w i t h HRP. 0.1 u l of a 25% s a l i n e s o l u t i o n of HRP (Sigma, Type VI) was d e l i -vered i n t o the GP or EP by pressure through a 10 u l Hamilton microsyringe over a p e r i o d of about 20 min. In some experiments a 12% s o l u t i o n of HRP was d e l i -vered i n t o the EP and GP by m i c r o e l e c t r o p h o r e s i s u s i n g m i c r o p i p e t t e s w i t h t i p diameters of 30 to 60 urn and a current of 1.8 yA f o r 10 min. For retrograde t r a n s p o r t of HRP from the SN, 0.1 u l of a 30% s o l u t i o n of HRP was i n j e c t e d b i -l a t e r a l l y . A f t e r a 24 hr s u r v i v a l p e r i o d the animals were perfused w i t h normal s a l i n e followed by 400 ml of a mixture of 30 g paraformaldehyde, 25 g dextrose and 20 ml of 50% glutaraldehyde i n 1.0 l i t e r of 0.05 M phosphate b u f f e r pH 7.4. The b r a i n s were then removed and immersed overnight i n the same f i x a t i v e minus the glutaraldehyde. A few hrs before s e c t i o n i n g the b r a i n s were immersed i n a phosphate b u f f e r s o l u t i o n c o n t a i n i n g 2.5% dextrose. Frozen s e c t i o n s (60 um) were taken i n the f r o n t a l plane and t r e a t e d to r e v e a l HRP a c t i v i t y . Two sub-s t r a t e s f o r the HRP r e a c t i o n were employed; diaminobenzidine according to the method of Nauta et a l (1974) and d i c h l o r o b e n z i d i n e according t o the method of Mesalum and Rosene (1977). c) D i s s e c t i o n s D i s c r e t e b r a i n regions were d i s s e c t e d e i t h e r freehand from f r e s h b r a i n s as i n the case of the whole s t r i a t u m or from sec t i o n s obtained on a f r e e z i n g microtome. A l l d i s s e c t i o n s were conducted w h i l e maintaining the t i s s u e c o l d on i c e . Areas obtained from s e c t i o n s included the t a i l of the s t r i a t u m , GP, EP, SN and habenula. These areas were d i s s e c t e d freehand from the secti o n s except the GP which was obtained by using a punch 1.8 mm i n i n t e r n a l diameter. In one experiment i n v o l v i n g the a n t e r i o r hemitransections three s e c t i o n s were obtained; one c o n t a i n i n g the a n t e r i o r h a l f of the GP, another the adjacent p o s t e r i o r h a l f of the GP and the t h i r d p o s t e r i o r to the GP c o n t a i n i n g the caudal part of the t a i l of the s t r i a t u m . The GP was punched from each of the 50 / f i r s t two s e c t i o n s followed by d i s s e c t i o n of the t a i l of the s t r i a t u m (imme-d i a t e l y l a t e r a l to the GP) from w i t h i n the same s e c t i o n s , as w e l l as from the t h i r d s e c t i o n . d) H i s t o l o g y H i s t o l o g i c a l examination of b r a i n s was necessary i n order to evaluate the extent of damage produced by the v a r i o u s l e s i o n s . In p a r t i c u l a r , l a r g e numbers of b r a i n s w i t h GP k a i n i c a c i d l e s i o n s and e l e c t r o l y t i c s t r i a t a l t a i l l e s i o n s were examined i n order to gain some idea of the v a r i a b i l i t y of the l e s i o n s . Brains were removed and placed i n 10% f o r m a l i n - s a l i n e f o r 7 to 10 days. Sections (50 um) were placed i n 5% f o r m a l i n f o r 2 to 3 days, r i n s e d i n d i s t i l l e d water and mounted on s l i d e s . The s l i d e s were dipped i n 40% ethanol - 15% g e l a t i n , allowed to dry i n a i r and s t a i n e d w i t h c r e s y l v i o l e t . I l l Biochemical Methods Enzyme assays were employed e i t h e r to determine the e f f e c t of a l e s i o n on v a r i o u s enzyme a c t i v i t i e s or to confirm the e f f i c a c y of a l e s i o n . A l l en-zyme a c t i v i t i e s were shown to be l i n e a r w i t h time of i n c u b a t i o n and p r o t e i n c o n c e n t r a t i o n and these parameters were adjusted to a l l o w measurement of the a c t i v i t i e s i n each of the assays on the l i n e a r p o r t i o n of the curves. For CAT, GAD and TH b r a i n t i s s u e was homogenized i n 100 to 50 volumes of 50 mM T r i s - A c e t a t e b u f f e r pH 6.3 c o n t a i n i n g 0.2% T r i t o n X-100 ( v / v ) . a) Glutamic a c i d decarboxylase GAD was assayed by a m o d i f i c a t i o n of the method of A l b e r s and Brady (1959) as p r e v i o u s l y described (Chalmers et a l , 1970). The i n c u b a t i o n mixture con-t a i n e d 50 u l of a mixture c o n t a i n i n g ( f i n a l concentration) 2.0 mM L t l - ^ C ] glutamate ( s p e c i f i c a c t i v i t y 0.1 to 0.2 mCi/mmol, Radiochemical Centre, Amer-sham, England), 0.02% bovine serum albumin (Sigma), 0.1 mM p y r i d o x a l phosphate 51 (Calbiochem), 28 mM potassium phosphate b u f f e r pH 7.4 and 20 y l of t i s s u e homo-genate. This mixture was incubated f o r 30 min at 37°C i n small tubes placed i n t o s c i n t i l l a t i o n v i a l s c o n t a i n i n g a g e l a t i n capsule (Parke-Davis) loaded w i t h Whatman f i l t e r paper soaked w i t h 0.1 ml of hyamine hydroxide. The reac-t i o n was stopped by adding 0.2 ml of 2 M H 2 S O 4 to the mixture through a rubber washer i n the cap of the s c i n t i l l a t i o n v i a l w i t h a 5 ml sy r i n g e adapted w i t h a 19 gauge needle. A f t e r 2 hrs the r e a c t i o n v e s s e l was removed from the v i a l , s c i n t i l l a t i o n f l u i d added and the sample counted. b) Tyrosine hydroxylase TH was measured as p r e v i o u s l y described (McGeer et a l , 1967). The i n c u -b a t i o n mixture contained 80 y l of a mixture c o n t a i n i n g ( f i n a l c o n c e n t r a t i o n 1.0 mM 2-amino-5-hydroxy-6,7-dimethyl t e t r a h y d r o p t e r i d i n e (DMPH^, Sigma or Calbiochem), 0.1 mM L-[ l t +C (U) ] t y r o s i n e ( s p e c i f i c a c t i v i t y 3 to 5 mCi/mmol, New England N u c l e a r ) , 0.3 mM f e r r i c sulphate, 50 mM 2-mercapto-ethanol (East-man) 0.2 M sodium-acetate b u f f e r pH 6.0 and 40 y l of t i s s u e homogenate. The r e a c t i o n mixture was incubated at 37°C f o r 12 min a f t e r which time 2.0 ml of a s o l u t i o n c o n t a i n i n g 1.4% p e r c h l o r i c a c i d , 0.52% a c e t i c a c i d and 0.5 yg/ml of dihydroxyphenylalanine was added. The r e a c t i o n v e s s e l s were c e n t r i f u g e d at 2000 g f o r 5 min and the supernatants t r a n s f e r e d to 25 ml beakers. The ves-s e l s were r i n s e d w i t h 2.0 ml of 0.35 M KH 2P0i t pH 6.0, c e n t r i f u g e d as before and the supernatants pooled i n the beakers. A f t e r the a d d i t i o n of 2.0 ml of 0.2 M N a 2 E D T A and 12 ml, of d i s t i l l e d water to the beakers, the samples were brought to pH 9.0 to 9.5 and poured onto columns packed w i t h about 0.3 g of alumina (Calbiochem, a c i d , AG^, 100-200 mesh). The columns were washed w i t h 35 ml of d i s t i l l e d water, e l u t e d i n t o s c i n t i l l a t i o n v i a l s w i t h 2.0 ml of 0.5 M a c e t i c a c i d and counted. c) Choline a c e t y l t r a n s f e r a s e and a c e t y l c h o l i n e s t e r a s e Choline a c e t y l t r a n s f e r a s e was assayed according to the method of McGeer and McGeer (1971). The i n c u b a t i o n mixture c o n s i s t e d of 20 y l of a mixture 52 c o n t a i n i n g ( f i n a l c o ncentration 300 mM NaCl, 8.0 mM c h o l i n e c h l o r i d e , 0.2 mM e s e r i n e , 0.8 mM [ l - l l f C ] a c e t y l coenzyme A ( s p e c i f i c a c t i v i t y 3 to 4 mCi/mmol, Radiochemical Centre, Amersham, England), 100 mM sodium phosphate b u f f e r pH 7.4 and 20 u l of t i s s u e homogenate. The mixture was incubated at 37°C f o r 5 to 30 min depending on the area measured. The r e a c t i o n was terminated by adding 1.0 ml of a s o l u t i o n of 0.14% p e r c h l o r i c a c i d , 0.052% a c e t i c a c i d and 35 yg/ml a c e t y l c h o l i n e bromide. This was followed by the a d d i t i o n of 1.0 ml of 0.5 M t r i s - a c e t a t e b u f f e r pH 7.0. This mixture was then poured onto 4 cm columns packed w i t h Amberlite r e s i n CG-50H (Type 1 BDH) which had been e q u i l i -b rated w i t h 3.0 ml of 0.5 M t r i s - a c e t a t e b u f f e r pH 7.0. The columns were washed w i t h 35 ml of water, e l u t e d i n t o s c i n t i l l a t i o n v i a l s w i t h 3.0 ml of 4.1 M a c e t i c a c i d , s c i n t i l l a t i o n f l u i d was added to the v i a l s and the samples were counted. AChE was assayed as p r e v i o u s l y described (Fonnum, 1969). F i n a l con-c e n t r a t i o n s of 15 mM sodium phosphate b u f f e r pH 7.0 and 5.0 mM [ a c e t y l - l - l l t C ] a c e t y l c h o l i n e i o d i d e ( s p e c i f i c a c t i v i t y 0.218 mCi/mmol, New England Nuclear) were employed. Two e x t r a c t i o n s w i t h 1.5% tetraphenylboron i n heptanone were used to remove the l a b e l e d s u b s t r a t e . F o l l o w i n g c e n t r i f u g a t i o n a p o r t i o n of the aqueous phase was added to s c i n t i l l a t i o n v i a l s and counted, d) Adenylate c y c l a s e Dopamine-sensitive adenylate c y c l a s e i n the SN was measured according to the method of M i l l e r et a l (1974). The t i s s u e was homogenized i n 25 to 50 volumes (w/v) of 2.0 mM t r i s - m a l e a t e b u f f e r pH 7.4 c o n t a i n i n g 2.0 mM EGTA. F i f t y u l a l i q u o t s of t h i s homogenate were added to 250 u l of 80 mM t r i s -maleate b u f f e r c o n t a i n i n g 2.0 mM MgSO^, 0.2 mM EGTA and the d e s i r e d concentra-t i o n s of DA. The i n c u b a t i o n tubes were kept i n an i c e bath w h i l e ATP was ad-ded to a f i n a l c o n c e n t r a t i o n of 0.5 mM. The r e a c t i o n mixture was incubated at 3Q°C f o r 2.5 min and then t r a n s f e r r e d to a b o i l i n g water bath f o r an a d d i -t i o n a l 2.5 min. The tubes were c e n t r i f u g e d f o r 5 min at 2000 g and 40 y l a l i -quots of the supernatant were assayed f o r c y c l i c AMP content by the method of 53 Gilman (1970) using a l i n e a r standard curve produced w i t h a l i q u o t s of authen-t i c c y c l i c AMP between 0.2 and 8.0 pmol. e) P r o t e i n assay and s c i n t i l l a t i o n counting P r o t e i n assays were conducted by the method of Lowry et a l (1951). In g e n e r a l , r a d i o a c t i v i t y was counted i n .5 to 15 ml of ACS (Amersham) w i t h the ex-c e p t i o n of M i l l i p o r e f i l t e r s which were counted i n 10 ml of a toluene based s c i n t i l l a t i o n f l u i d . Samples were counted i n a Nuclear-Chicago s c i n t i l l a t i o n counter w i t h an e f f i c i e n c y of about 80% f o r carbon-14 samples and 30% f o r t r i t i u m samples. f) T issue packaging In c o l l a b o r a t i v e work i t was necessary to t r a n s p o r t t i s s u e samples. B r a i n t i s s u e was d i s s e c t e d as described above, placed w i t h i n 3 min i n t o auto-analyzer cups ( T r i l a b I n d u s t r i e s ) , sealed and stored at -70°C u n t i l shipment. Samples were packaged w i t h about 10 kg of dry i c e i n styrofoam boxes and shipped by . a i r f r e i g h t . The methods f o r the receptor b i n d i n g assays employed i n these experiments are described elsewhere (Seeman et a l , 1976; R e i s i n e et a l , 1979a, b ) . RESULTS I Neuroanatomy a) A n t e r i o r s t r i a t a l e f f e r e n t s to the globus p a l l i d u s , entopeduncular nu-cleus and s u b s t a n t i a n i g r a I n j e c t i o n s of [ 3H] l e u c i n e i n t o the head of the s t r i a t u m r e s u l t e d i n a l o c a l i z e d homogeneous d i s t r i b u t i o n of grains which were confined to t h i s nu-cleus ( F i g . 1). I n the region caudal to the i n j e c t i o n s i t e and j u s t r o s t r a l to the GP the g r a i n s were p r e f e r e n t i a l l y d i s t r i b u t e d over f i b e r bundles ( F i g . 2A). Further c a u d a l l y , w i t h i n the GP, the grains were more h e a v i l y concentra-ted over the n e u r o p i l of the GP and to a l e s s e r extent i n the f i b e r bundles of t h i s region ( F i g . 2B). The a n t e r i o r r egion of the GP appeared to cont a i n the 54 greatest c o n c e n t r a t i o n of l a b e l . In a s i m i l a r f a s h i o n , l a b e l r o s t r a l to the EP was l o c a t e d over the i n t e r -n a l capsule ( F i g . 2C), w h i l e i n the EP gra i n s were observed to be pre f e r e n -t i a l l y d i s t r i b u t e d over the n e u r o p i l ( F i g . 2D). Caudal to the EP the d e n s i t y of grains decreased markedly, as the l a b e l e d f i b e r s passed w i t h i n the i n t e r n a l capsule to terminate i n the SNR ( F i g . 2E-F). Of the t e r m i n a l areas examined, the highest d e n s i t y of grains was observed over GP. S u b s t a n t i a l l y l e s s l a b e -l i n g was found over EP, w h i l e the a c t i v i t y over SNR was intermediate. I n b r a i n s sectioned s a g i t a l l y , these r e l a t i o n s were e s p e c i a l l y c l e a r . F i g . 3 shows the d i s t r i b u t i o n of s i l v e r g r a i n s i n a s a g i t t a l s e c t i o n a f t e r an i n j e c -t i o n of [ H] l e u c i n e i n t o the head of the s t r i a t u m . D i f f u s e accumulation of grai n s could be observed over the n e u r o p i l of GP, EP and SNR. In between these n u c l e i the s i l v e r grains were arranged i n l i n e a r arrays w i t h i n the i n -t e r n a l capsule, t y p i c a l of that observed i n l a b e l e d axons. A f t e r m i c r o e l e c t r o p h o r e t i c i n j e c t i o n s of HRP i n t o the GP, which r e s u l t e d i n HRP r e a c t i o n product w e l l l o c a l i z e d w i t h i n t h i s s t r u c t u r e ( F i g . 4A), l a -beled c e l l s were observed i n the s t r i a t u m ( F i g . 4C). These c e l l s , which were medium s i z e d , were confined to the d o r s a l - c e n t r a l , a n t e r i o r 'core' region of the CP and the cytoplasm of these c e l l s contained the type of f i n e granules described by previous authors (Herkenham and Nauta, 1977; Jones and L e a v i t t , 1974). Whether s t r i a t a l c e l l s o u t s ide t h i s core region of the s t r i a t u m a l s o pro-j e c t to GP could not be determined w i t h c e r t a i n t y from t h i s m a t e r i a l . Micro-e l e c t r o p h o r e t i c i n j e c t i o n s of HRP i n t o EP ( F i g . 4C) a l s o r e s u l t e d i n the ap-pearance of r e a c t i v e p e r i k a r y a i n the s t r i a t u m ( F i g . 4D). Although c l e a r topographic r e l a t i o n s could not be di s c e r n e d , i t was apparent from examination of a l a r g e number of animals t h a t , w i t h the exception of the caudal t a i l , the e n t i r e s t r i a t u m appears to p r o j e c t to EP. Examination of b r a i n s i n which HRP had been d e l i v e r e d to the EP v i a pressure i n j e c t i o n s through a Hamilton micro-55 L i g h t f i e l d photomicrograph of a N i s s l - s t a i n e d s a g i t t a l s e c t i o n showing i n j e c t i o n s i t e of 3 H - l e u c i n e i n the head of the s t r i a t u m . M a g n i f i c a t i o n x 31. 57 F i g ure 2 A: d a r k - f i e l d photomicrograph of a s a g i t t a l s e c t i o n showing. autoradiographic l a b e l i n g of f i b e r bundles of the i n t e r n a l cap-s u l e caudal to the s t r i a t a l i n j e c t i o n s i t e ( F i g . 1) and j u s t r o s t r a l to the globus p a l l i d u s (x 190). B: accumulation of s i l v e r g rains i n the a n t e r i o r edge of globus p a l l i d u s . Note r e -l a t i v e l y sparse l a b e l i n g j u s t a n t e r i o r to GP on the r i g h t s i d e of photograph. S a g i t t a l s e c t i o n (x 190). C: d i s t r i b u t i o n of s i l v e r g rains i n the i n t e r n a l capsule j u s t a n t e r i o r to the ento-peduncular nucleus. Note l i n e a r arrays of g r a i n s , suggesting l a b e l i n g of axons. S a g i t t a l s e c t i o n (x 190). D: accumulation of autoradiographic g r a i n s i n the entopeduncular nucleus. S a g i t -t a l s e c t i o n (x 190). E: l i g h t f i e l d photomicrograph of a s a g i t -t a l s e c t i o n of the s u b s t a n t i a n i g r a . Note zona compacta i n the d o r s a l - r i g h t quadrant (x 90). F: d a r k - f i e l d photomicrograph of f i e l d i d e n t i c a l to t h a t shown i n (E). Large accumulations of s i l v e r g r a i n s can be observed i n the zona r e t i c u l a t a , p a r t i c u l a r -l y i n the v e n t r a l h a l f (x 90). 59 Figure 3 Camera l u c i d a drawing of a s a g i t t a l s e c t i o n showing d i s t r i b u t i o n of autoradiographic g r a i n s a f t e r an i n j e c t i o n of [ 3H] l e u c i n e i n t o head of s t r i a t u m . GP, globus p a l l i d u s ; AC, a n t e r i o r com-missure; EP, entopeduncular nucleus; OT, o p t i c t r a c t ; SNR, sub-s t a n t i a n i g r a , pars r e t i c u l a t a . 61 Figure 4 A: i n j e c t i o n s i t e of m i c r o e l e c t r o p h o r e t i c a p p l i c a t i o n of HRP i n t o the globus p a l l i d u s (x 19.5). B: d a r k - f i e l d photomicro-graph showing HRP r e a c t i v e neurons i n the c e n t r a l core region of the s t r i a t u m a f t e r HRP i n j e c t i o n i n t o globus p a l l i d u s (x 195). C: ' i n j e c t i o n s i t e of m i c r o e l e c t r o p h o r e t i c a p p l i c a t i o n of HRP i n t o the entopeduncular nucleus (x 19.5). D and E: l a b e l e d neurons i n s t r i a t u m (D) and c e r e b r a l cortex (E) a f t e r HRP i n -j e c t i o n i n t o entopeduncular nucleus (x 195). 63. syringe revealed a s i m i l a r p a t t e r n of l a b e l i n g i n the s t r i a t u m . Both e l e c t r o -p h o r e t i c and pressure i n j e c t i o n s of HRP i n t o EP r e s u l t e d i n the o c c a s i o n a l ap-pearance of l a b e l e d c e l l s i n the SN, pars compacta, and i n the c e r e b r a l cortex ( F i g . 4E). The appearance of these c e l l s d i f f e r e d from those i n the s t r i a t u m i n that they contained l a r g e r , more i n t e n s e l y s t a i n i n g granules and the neuro-n a l cytoplasm had a brownish appearance. These l a t t e r r e s u l t s r a i s e the pos-s i b i l i t y t hat some of the HRP l a b e l i n g i n the s t r i a t u m a f t e r i n j e c t i o n s i n t o EP may have been due to accumulation of HRP by damaged axons i n the crus cere-b r i . The extent to which t h i s f a c t o r may have i n f l u e n c e d s t r i a t a l l a b e l i n g cannot be determined w i t h c e r t a i n t y at present. However, i t i s r e l e v a n t that i n c o n t r o l experiments m i c r o e l e c t r o p h o r e t i c i n j e c t i o n s of HRP i n t o the crus c e r e b r i r e s u l t e d i n a p a t t e r n of l a b e l i n g i n the s t r i a t u m , SN and cortex s i m i -l a r to that observed a f t e r i n j e c t i o n s i n t o EP. No l a b e l e d c e l l s were found i n any of these areas a f t e r pressure i n j e c t i o n s of HRP i n t o the crus c e r e b r i . b) P o s t e r i o r s t r i a t a l e f f e r e n t s to the globus p a l l i d u s , entopeduncular nu-cleus and s u b s t a n t i a n i g r a The r e s u l t s of the [ 3H] l e u c i n e i n j e c t i o n i n t o the t a i l of the s t r i a t u m i s shown i n the dark f i e l d photomicrographs i n F i g . 5 and d i a g r a m a t i c a l l y i n F i g . 6. The i n j e c t i o n s of [ 3H] l e u c i n e were l o c a l i z e d d o r s a l and l a t e r a l to the GP midway through t h i s nucleus i n the AP dimension ( F i g . 5A). The l a b e l d i f f u s e d from the i n j e c t i o n s i t e to the l e v e l of the a n t e r i o r extreme of the GP ( F i g . 6A). There was no spread of l a b e l from the i n j e c t i o n s i t e i n the t a i l of the s t r i a t u m to the c o r t e x or the GP. Further c a u d a l l y ( F i g s . 5B and 6C).there was dense l a b e l i n g of the v e n t r a l l a t e r a l p o r t i o n of the p o s t e r i o r GP. Although some gra i n s were present i n l a r g e f i b e r bundles t r a v e r s i n g t h i s r e g i o n the m a j o r i t y of the grains were concentrated and p r e f e r e n t i a l l y l a b e l e d the n e u r o p i l of the GP. This was so pronounced that the r e l a t i v e l y sparse l a -b e l i n g of the f i b e r bundles i n t h i s r e g i o n r e s u l t e d i n t h e i r d e l i n e a t i o n by the surrounding h e a v i l y l a b e l e d areas. Further c a u d a l l y , at the l e v e l of the 64 Figure 5 A: d a r k - f i e l d photomicrograph showing the i n j e c t i o n s i t e of 3 H - l e u c i n e i n t o the t a i l of the s t r i a t u m l a t e r a l to the GP (x 12.7). B: accumulation of s i l v e r g rains i n the p o s t e r i o r GP (x 11.3). Note that the f i b e r bundles passing through the : area of the GP c o n t a i n i n g s i l v e r grains are devoid of l a b e l . C: accumulation of autoradiographic grains i n the SN (x 25.6). Diagramatic r e p r e s e n t a t i o n of the d i s t r i b u t i o n of autoradio graphic grains a f t e r i n j e c t i o n of 3 H - l e u c i n e i n t o the t a i l the s t r i a t u m . 68 EP ( F i g . 6D), the d e n s i t y of grains i n the i n t e r n a l capusle decreased dramati-c a l l y and was l o c a l i z e d mainly over f i b e r bundles. Although r a r e , some l a b e l from the i n t e r n a l capsule t r i c k l e d v e n tromedially w i t h i n the n e u r o p i l towards the EP. At the l e v e l of the subthalamic nucleus (SUT), p o s t e r i o r to the EP ( F i g . 6E), the l a b e l w i t h i n the i n t e r n a l capsule (IC) appeared to be more c l u s -t e r e d , r e l a t i v e to that observed at the l e v e l of the EP, and occupied a p o s i -t i o n i n the center of the long a x i s of the IC and immediately l a t e r a l to the d o r s a l l a t e r a l t i p of the SUT. No l a b e l i n g of the SUT occurred. I t could not be discerned whether there was a decrease i n g r a i n d e n s i t y at a l e v e l j u s t pos-t e r i o r to the EP. At the a n t e r i o r p o r t i o n of the SN ( F i g . 6F) the l a b e l was observed invading the extreme l a t e r a l regions of the SN pars r e t i c u l a t a (SNR) from the IC. Midway through the SN the accumulation of l a b e l increased com-pared to that observed i n the IC and occupied a p o s i t i o n i n the l a t e r a l one-t h i r d of the SNR ( F i g s . 5C and 6B). This l o c a t i o n of l a b e l w i t h i n the SNR d i d not change i n the most p o s t e r i o r extreme of the SN ( F i g . 6H). No l a b e l was observed i n any s t r u c t u r e s caudal to the SN. I I Biochemical Neuroanatomy a) The c o n t r i b u t i o n of the head of the s t r i a t u m to v a r i o u s neurotransmit-t e r enzyme markers i n the globus p a l l i d u s , entopeduncular nucleus and s u b s t a n t i a n i g r a Hemitransections at a l e v e l s l i g h t l y caudal to the EP were produced i n order to determine the maximal c o n t r i b u t i o n to n i g r a l glutamic a c i d decarboxy-l a s e (GAD) from s t r u c t u r e s r o s t r a l to these l e s i o n s . A d d i t i o n a l l y , i n order to evaluate the biochemical nature of the p r o j e c t i o n s from the head of the s t r i a t u m to the GP, EP and SN, hemitransections, e l e c t r o l y t i c and k a i n i c a c i d l e s i o n s of the head of the s t r i a t u m were produced. The hemitransections were j u s t a n t e r i o r to the GP. The e f f e c t s of these l e s i o n s on GAD a c t i v i t y are shown i n Table 1. The a n t e r i o r hemitransections r e s u l t e d i n a s i g n i f i c a n t de-Table 1 The a c t i v i t y of GAD i n v a r i o u s areas a f t e r l e s i o n s of the head of the s t r i a t u m Area L e s i o n technique Hemitransection (11) E l e c t r o l y t i c (8) K a i n i c a c i d (9) Head of s t r i a t u m - - 23.6±2.9*** GP 79.9±5.4* 74.9±13.4** 37.L+5.0*** EP 63.6±8.5*** 76.7±11.4** 80.0±9.6* SN 92.4±3.9 101.0±7.4 74.5±2.08*** Values are expressed as per cent of c o n t r o l ± S.E.M. The number of animals used i s shown i n pa r e n t h e s i s a f t e r the l e s i o n technique employed. Expressed i n terms of nmoles/mg p r o t e i n / h r , the GAD a c t i v i t y on the unlesioned s i d e was: s t r i a t u m , 90.1±5.1 GP, 220±16; EP, 122±9.9; SN, 310±8.4. S t a t i s t i c a l d i f f e r e n c e s compared to c o n t r a l a t e r a l c o n t r o l s i d e . * p < 0.05. ** p < 0.02. *** p < 0.01. 70 crease i n GAD a c t i v i t y of about 20 and 35% i n the GP and EP r e s p e c t i v e l y . E l e c t r o l y t i c l e s i o n s of the a n t e r i o r aspect of the s t r i a t u m , which d i d not en-croach upon the GP, decreased GAD a c t i v i t y i n the GP and EP by about 25 and 23% r e s p e c t i v e l y . N e i t h e r of these l e s i o n s s i g n i f i c a n t l y a l t e r e d GAD a c t i v i t y i n the whole SN. 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 the head of the s t r i a t u m r e -duced GAD a c t i v i t y i n the i n j e c t e d area by 76%. These l e s i o n s reduced GAD i n the GP and EP by 63 and 20% r e s p e c t i v e l y . In a d d i t i o n , there was a s i g n i f i -cant 25% r e d u c t i o n i n n i g r a l GAD a c t i v i t y . I t i s noteworthy that the k a i n i c a c i d t r e a t e d animals showed s i g n i f i c a n t l y greater reductions i n GAD i n the GP than d i d e i t h e r the hemitransection or e l e c t r o l y t i c groups (p < 0.01). This may be explained by d i f f u s i o n of k a i n i c a c i d to regions i n the s t r i a t u m and GP caudal to the a n t e r i o r pole of the GP. Indeed, h i s t o l o g i c a l examination of the extent of the k a i n i c a c i d l e s i o n revealed d e s t r u c t i o n of neurons i n both the GP and the t a i l of the s t r i a t u m . Hemitransections j u s t caudal to the EP reduced GAD i n the SN by about 84% (data not shown). In order to evaluate the p o s s i b l e c h o l i n e r g i c c o n t r i b u t i o n of the head of the s t r i a t u m to the GP, EP and SN, a second s e r i e s of experiments was conduc-ted i n v o l v i n g hemitransections a n t e r i o r to the GP. These were placed s l i g h t l y more p o s t e r i o r than the previous hemitransections. The r e s u l t s of these ex-periments are shown i n Table 2. The decrease i n p a l l i d a l GAD was greater (58%) compared w i t h the more r o s t r a l t r a n s e c t i o n (20%). A l s o there was a s i g -n i f i c a n t decrease of 16% i n n i g r a l GAD. I n t e r e s t i n g l y , however, the decrease i n GAD i n the EP was about the same a f t e r both hemitransections. These l e -sions had no e f f e c t on c h o l i n e a c e t y l t r a n s f e r a s e (CAT) a c t i v i t y i n the EP or SN. P a l l i d a l CAT a c t i v i t y , however, was s i g n i f i c a n t l y reduced (50%). With hemitransections c l o s e to the GP, changes i n enzyme a c t i v i t i e s i n the GP may be i n t e r p r e t e d as being due to spread of the l e s i o n to the GP rather than the se v e r i n g of a p r o j e c t i o n emanating to the GP from regions an-t e r i o r to the l e s i o n . To t e s t t h i s the l a t t e r hemitransections were repeated 71 and the a n t e r i o r and p o s t e r i o r GP, as w e l l as the t a i l of the s t r i a t u m imme-d i a t e l y l a t e r a l to the GP, were each assayed s e p a r a t e l y f o r GAD and CAT a c t i -v i t i e s . In a d d i t i o n , the t a i l of the s t r i a t u m p o s t e r i o r to the GP was exa-mined. The r e s u l t s are given i n Table 3. These l e s i o n s had no e f f e c t on GAD or CAT a c t i v i t i e s i n the t a i l of the s t r i a t u m p o s t e r i o r to the GP. In the a n t e r i o r GP GAD was decreased s i g n i f i c a n t l y by 57%. CAT was decreased by 38%, although t h i s d i d not reach s i g n i f i c a n c e due to an excessive v a r i a t i o n i n the standard e r r o r . I n the s t r i a t a l t i s s u e immediately l a t e r a l to the a n t e r i o r GP, GAD and CAT a c t i v i t i e s were unalt e r e d . This was t r u e of the GAD and CAT a c t i v i t i e s i n the s t r i a t a l t i s s u e l a t e r a l to the p o s t e r i o r GP. b) The c o n t r i b u t i o n of the t a i l of the s t r i a t u m to v a r i o u s neurotransmit-t e r enzyme markers i n the globus p a l l i d u s , entopeduncular nucleus and s u b s t a n t i a n i g r a The extent of the e l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m i s shown d i a g r a m a t i c a l l y i n F i g . 7 and i n the photomicrograph i n F i g . 10A. This example of the l e s i o n i s f a i r l y r e p r e s e n t a t i v e of the eleven b r a i n s t h a t were examined h i s t o l o g i c a l l y . A n t e r i o r l y , the l e s i o n s s t a r t e d at the l e v e l of the a n t e r i o r commissure and were l o c a t e d i n the l a t e r a l o n e - t h i r d of the s t r i a t u m ( F i g . 7A). Midway through the GP, the l e s i o n s occupied the main body of the t a i l of the s t r i a t u m and never encroached on the GP ( F i g . 7B). The most me-d i a l p o r t i o n of the d o r s a l s t r i a t u m and the most v e n t r a l p o r t i o n s were u s u a l l y spared but to va r i o u s degrees. At the caudal extreme of the GP, the l e s i o n was u s u a l l y r e s t r i c t e d to the d o r s a l h a l f of the t a i l of the s t r i a t u m w h i l e the v e n t r a l h a l f was spared ( F i g . 7C). Again there was no damage to the GP. At the r o s t r a l l e v e l of the EP ( F i g . 7D), the l e s i o n s were u s u a l l y q u i t e small and r e s t r i c t e d to the d o r s a l h a l f of the s t r i a t u m . O c c a s i o n a l l y (two b r a i n s ) , however, some g l i o s i s was observed i n the d o r s a l h a l f of the i n t e r n a l capsule, although no l e s i o n could be detected. The a c t i v i t i e s of GAD i n the GP, EP and SN and of CAT i n the GP and EP 72 Table 2 A c t i v i t y of GAD and CAT i n the globus p a l l i d u s , entopeduncular nucleus and s u b s t a n t i a n i g r a a f t e r hemitransections a n t e r i o r to the globus p a l l i d u s . Area Enzyme A c t i v i t y (nmoles/mg p r o t e i n / hr) GAD % c o n t r o l CAT % c o n t r o l GP l e s i o n c o n t r o l 243±46* 576±45 42 7.25±.68** 14.4±.94 50 EP l e s i o n 240±36* c o n t r o l 390±40 62 8.31±1.0 9.26±1.5 89 SN l e s i o n 884±47* c o n t r o l 1047±38 84 5.04±.49 4.46±.22 113 Values represent the mean ± S.E.M.. of seven determinations. * p < .025 ** p < .001 Table 3 The a c t i v i t y of GAD and CAT i n various areas a f t e r hemitransections a n t e r i o r to the globus p a l l i d u s Enzyme A c t i v i t y (nmoles/mg P r o t e i n / h r ) Area R e l a t i v e to GP A n t e r i o r GP GP Striatum P o s t e r i o r GP GP St r i a t u m P o s t e r i o r to GP Striatum GAD Les i o n C o n t r o l % of c o n t r o l 295±44** 698±85 43 389±37 349128 111 419±63 551191 76 394+40 337120 117 474143 466145 102 CAT Les i o n C o n t r o l % of c o n t r o l 2.961.25 4.811.89 62 46.712.9 50.112.9 93 4.181.49* 6.361.57 66 33.812.5 35.612.4 95 16.61.93 17.811.0 93 Values represent means 1 S.E.M. of eleven determinations, p < .01 p < .001 74 a f t e r e l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m are shown i n Table 4. In a separate experiment the SN was assayed f o r GAD w h i l e the remainder of the b r a i n was examined h i s t o l o g i c a l l y . Table 4 a l s o contains enzyme values from the l e f t and r i g h t s i d e of unoperated animals i n order to assure that any ob-served d i f f e r e n c e s i n l e s i o n e d animals were not due to d i s s e c t i o n e r r o r . No d i f f e r e n c e s were observed between t h e r i g h t and l e f t s i d e of unoperated a n i -mals. E l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m d i d not a l t e r GAD or CAT a c t i v i t i e s i n the GP. These l e s i o n s d i d , however, cause a s i g n i f i c a n t de-crease of 22 and 24% i n GAD and CAT, r e s p e c t i v e l y , i n the EP. Small:, but s i g -n i f i c a n t decreases of GAD were al s o observed i n both the SN (12%) obtained from b r a i n s used s o l e l y f o r enzyme a n a l y s i s as w e l l as i n the SN (17%) taken from b r a i n s which were u t i l i z e d f o r h i s t o l o g i c a l a n a l y s i s . c) The c o n t r i b u t i o n of the globus p a l l i d u s to v a r i o u s neurotransmitter en-zyme markers i n the entopeduncular nucleus and s u b s t a n t i a n i g r a In order to c h a r a c t e r i z e the biochemical nature of the e f f e r e n t s from the GP, t h i s s t r u c t u r e was l e s i o n e d e l e c t r o l y t i c a l l y and w i t h k a i n i c a c i d . The l o -c a t i o n of the e l e c t r o l y t i c l e s i o n of the GP i s shown i n F i g s . 8 and 10B. A n t e r i o r l y , the l e s i o n s destroyed the m a j o r i t y of the GP ( F i g . 8A). Midway through the GP the l e s i o n destroyed a l l but the l a t e r a l one-quarter of t h i s s t r u c t u r e ( F i g . 8B). Further c a u d a l l y ( F i g . 8C) the l e s i o n spared the poste-r i o r one-quarter to o n e - t h i r d of the GP. The e f f e c t s of these e l e c t r o l y t i c l e -s i o n s of the p a l l i d u m on GAD a c t i v i t y i n the EP and SN and c h o l i n e a c e t y l t r a n s -f e r a s e a c t i v i t y i n the EP are shown i n Table 5. CAT a c t i v i t y i n the EP was un-a l t e r e d by the l e s i o n . Glutamic a c i d decarboxylase (GAD) a c t i v i t y i n the EP and SN was s i g n i f i c a n t l y reduced by 35 and 14% r e s p e c t i v e l y . In order to circumvent the drawbacks of e l e c t r o l y t i c l e s i o n s , p a r t i c u l a r l y w i t h respect to f i b e r s - o f - p a s s a g e , k a i n i c a c i d l e s i o n s of the GP were employed. The l e s i o n e d area, shown i n F i g s . 9 and IOC, i s r e p r e s e n t a t i v e of the s i z e and l o c a t i o n of the l e s i o n e d area i n the ei g h t b r a i n s examined. The l e s i o n ante-75 Figure 7 Diagramatic r e p r e s e n t a t i o n of e l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m . The extent of t i s s u e d e s t r u c t i o n i s shown by the hatched area. 7 V Table 4 A c t i v i t y of GAD and CAT i n various areas a f t e r e l e c t r o l y t i c l e s i o n s of the t a i l of the st r i a t u m . Enzyme GP EP SN SN + H i s t o l o g y GAD les i o n e d 310±25 (17) 202116* (16) 728135** (15) 648147* (5) c o n t r o l % of c o n t r o l 340±25 (17) 91 260122 (16) 78 825119 (15) 88 785119 (5) 83 CAT lesioned 11.710.8 (18) 5.32i.44***(18) c o n t r o l % of c o n t r o l 13.511.0 (18) 87 7.Oil.40 (18) 75.9 GAD c o n t r o l 366127 (16) 276+21 (15) 773121 (13) c o n t r o l % of c o n t r o l 314123 (16) 117 281119 (15) 98 764130 (13) 101 CAT c o n t r o l 12.511.1 (16) 8.501.67 (12) c o n t r o l % of c o n t r o l 13.21.96 (16) 95 8.341.46 (12) 102 Enzyme a c t i v i t e s are i n terms of nmoles/mg p r o t e i n / h r Values represent means 1 S.E.M., number of determinations are enclosed i n parentheses. * p < .05 ** p < .025 *** p < .0.1 78 Figure 8 Diagramatic r e p r e s e n t a t i o n of e l e c t r o l y t i c l e s i o n s of the GP. Hatched area shows regions of t i s s u e d e s t r u c t i o n . 7<? Table 5 A c t i v i t y of GAD and CAT i n the entopeduncular nucleus and s u b s t a n t i a n i g r a a f t e r e l e c t r o l y t i c l e s i o n s of the globus p a l l i d u s . Enzyme Enzyme EP A c t i v i t y (nmoles/mg P r o t e i n / h r ) % of C o n t r o l SN % of C o n t r o l L e s i o n 171 ± 36** (6) 65 619 ± 19* (9) 86 GAD Co n t r o l 265 ± 15 (6) 724 ± 18 (9) Le s i o n 6.33 ± .41 (6) 101 CAT Contr o l 6.30 ± .23 (6) Values represent means ± S.E.M., number of determinations are enclosed i n parentheses. "p < .05 p < .001 81 Figure 9 Diagramatic r e p r e s e n t a t i o n of k a i n i c a c i d l e s i o n s of the GP. Hatched areas show regions where neuron d e s t r u c t i o n and intense g l i o s i s were observed. 83 r i o r to the a n t e r i o r commissure was small and incomplete i n that t o t a l neuron d e s t r u c t i o n w i t h i n t h i s area was seldom observed ( F i g . 9A). At the l e v e l of the a n t e r i o r commissure ( F i g . 9B) and midway through the GP i n the AP dimen-s i o n ( F i g . 9C) the l e s i o n enveloped the e n t i r e GP. Neuron l o s s i n t h i s area was extensive. At these l e v e l s neuron d e s t r u c t i o n i n the t a i l of the s t r i a t u m l a t e r a l to the GP was v a r i a b l e . In some cases there was l i t t l e Tdamagef be-yond the l a t e r a l border of the GP. In other b r a i n s neuron l o s s was observed i n an area c i r c u m s c r i b i n g one-half of the t a i l of the s t r i a t u m immediately sur-rounding the GP. The l e s i o n was never observed beyond t h i s r e g i o n ; thus the p e r i p h e r a l h a l f of the p o s t e r i o r s t r i a t u m was always spared. Further c a u d a l l y ( F i g . 9D) d e s t r u c t i o n was u s u a l l y observed i n the medial and d o r s a l h a l f of the GP but, p o s t e r i o r to t h i s l e v e l where numerous p a l l i d a l neurons could s t i l l be i d e n t i f i e d , damage was never observed. At t h i s l e v e l ( F i g . 9E) the r e t i c u -l a r nucleus of the thalamus seemed p a r t i c u l a r l y s e n s i t i v e to k a i n i c a c i d as d e s t r u c t i o n of t h i s nucleus o f t e n occurred. Of i n t e r e s t was the w e l l l o c a -l i z e d and c o n s i s t e n t d e s t r u c t i o n of the claustrum l o c a t e d l a t e r a l to the e x t e r -n a l capsule ( F i g . 9). No g l i o s i s or neuron l o s s was ever detected between t h i s s t r u c t u r e and the main body of the l e s i o n suggesting that the claustrum i s e i t h e r e x q u i s i t e l y s e n s i t i v e to k a i n i c a c i d or that i t undergoes degenera-t i o n f o r some as yet unknown reason ( i . e . , retrograde or transneuronal dege-n e r a t i o n or p e r t u r b a t i o n of a f f e r e n t : i n p u t ) . The a c t i v i t i e s of GAD, CAT and TH i n v a r i o u s areas f o l l o w i n g k a i n i c a c i d GP l e s i o n s are shown i n Table 6. TH was s i g n i f i c a n t l y increased by 25% i n the t a i l of the s t r i a t u m on the s i d e of the l e s i o n . CAT a c t i v i t y i n the t a i l of the s t r i a t u m was unaltered whereas i t was s i g n i f i c a n t l y decreased by 42% i n the GP. GAD i n the t a i l of the s t r i a t u m , GP and EP were s i g n i f i c a n t l y de-creased by 25, 42 and 34% r e s p e c t i v e l y . GAD i n the SN taken from b r a i n s which were employed f o r enzyme a n a l y s i s of GP, EP and t a i l of the s t r i a t u m showed a 10% n o n - s i g n i f i c a n t decrease as d i d GAD i n the SN taken from b r a i n s Table 6 A c t i v i t y of GAD, CAT and globus p a l l i d u s . TH i n various areas a f t e r k a i n i c a c i d l e s i o n s of the Enzyme T a i l of Striatum GP EP SN SN + Hi s t o l o g y GAD l e s i o n c o n t r o l 213±19* 283±23 183H7** 315±27 195122** 295±32 695±36 774123 701133 783127 % of c o n t r o l 75 58 66 90 90 CAT l e s i o n c o n t r o l % of c o n t r o l 58.3±3.5 58.913.5 99 5.551.86** 9.51±.56 58 TH l e s i o n c o n t r o l % of c o n t r o l 1.85±.ll* 1.481.09 125 Enzyme a c t i v i t i e s are expressed i n terms of nmols/mg p r o t e i n / h r Values represent means 1 S.E.M. of eight determinations. * p < .05 ** p < .025 85 Figure 10 Photomicrographs of e l e c t r o l y t i c and k a i n i c a c i d l e s i o n s . A: e l e c t r o l y t i c l e s i o n of the t a i l of the s t r i a t u m (x 15.4). B: e l e c t r o l y t i c l e s i o n of the GP (x 15.4). C: k a i n i c a c i d l e s i o n of the GP (x 13.4). Note the needle t r a c t and s i t e of k a i n i c a c i d i s i n the medial part of the GP. 87 which were analyzed h i s t o l o g i c a l l y f o r the assessment of the k a i n i c a c i d l e -s i o n . I t was mentioned e a r l i e r t h a t , at the m i d - l e v e l of the GP, k a i n i c a c i d de-stroyed v i r t u a l l y a l l neurons. However, i n c r e s y l v i o l e t s t a i n e d s e c t i o n s , i n the midst of the l e s i o n a few l a r g e neurons were seen to be l e f t i n t a c t . These were l o c a t e d i n the medial aspect of the GP, j u s t l a t e r a l to the i n t e r n a l cap-s u l e (IC) and at the v e n t r a l extreme of the GP. A c e t y l c h o l i n e s t e r a s e (AChE) s t a i n i n g of the k a i n i c a c i d lesioned-GP b r a i n s was conducted to determine i f the known l a r g e , i n t e n s e l y AChE-staining neurons were those which were l e f t i n t a c t . Shown i n F i g . 11 are the p o s i t i o n s of the AChE s t a i n i n g neurons l e f t i n t a c t by the l e s i o n . These areas correspond c l o s e l y to the p o s i t i o n s of the neurons seen to be remaining i n c r e s y l v i o l e t s t a i n e d s e c t i o n s . The area of absence of the small to medium s i z e d , moderate AChE s t a i n i n g neurons i n the GP and t a i l of the s t r i a t u m i n l e s i o n e d b r a i n s e x a c t l y over-lapped the area of the l e s i o n described i n F i g . 9. However, l a r g e i n t e n s e l y AChE s t a i n i n g neurons were present i n the medial and v e n t r a l l e s i o n e d GP and more p o s t e r i o r l y , they were i n t e r s p e r s e d i n the i n t e r n a l capsule ( F i g . 11C). Although i t was d i f f i c u l t to q u a n t i t a t e the degree of l o s s , i f any, of the AChE s t a i n e d neurons, they d i d appear to have undergone some atrophy as i s evident from t h e i r smaller p e r i k a r y a and dendrites compared to the neurons on the c o n t r o l s i d e . The HRP retrograde transport method was employed to determine whether the l a r g e AChE s t a i n i n g neurons p r o j e c t to the SN. A f t e r the i n j e c t i o n of HPR b i -l a t e r a l l y i n t o the SN, the GP was examined f o r HRP s t a i n e d neurons. The r e -s u l t s are shown i n F i g s . 12 and 13. In F i g . 12 the l e f t i s the k a i n i c a c i d l e s i o n e d and the r i g h t i s the c o n t r o l s i d e . The numerous HRP s t a i n e d neurons i n the s t r i a t u m are not depicted. In the a n t e r i o r h a l f of the GP on the con-t r o l s i d e , HRP s t a i n e d p e r i k a r y a could be seen throughout the GP ( F i g . 12A). Further c a u d a l l y , l a b e l e d neurons were most obvious i n the l a t e r a l v e n t r a l 88 Figure 11 L o c a t i o n of intense a c e t y l c h o l i n e s t e r a s e s t a i n i n g neurons i n and surrounding the GP. These neurons are represented by the open c i r c l e s i n the diagram. SI 90 Figure 12 Diagram of the l o c a t i o n of l a b e l e d neurons i n the GP a f t e r i n t r a n i g r a l i n j e c t i o n s of HRP. The l e f t GP received k a i n i c a c i d l e s i o n s as depicted i n Figure 9. The i n j e c t i o n of HRP i n t o the SN was b i l a t e r a l . The b l a c k dots represent HRP-l a b e l e d neurons i n the GP. Note t h a t l a b e l e d neurons on the l e s i o n s s i d e are sparse. 92 Figure 13 Photomicrograph of l a b e l e d neurons i n the GP a f t e r i n t r a n i g r a l HRP i n j e c t i o n s . A: l a b e l e d neurons i n the GP on the r i g h t s i d e of the b r a i n . B: l a b e l e d neurons i n the same area of the GP on the l e f t s i d e which had r e c e i v e d k a i n i c a c i d l e s i o n s . These few neurons are present i n that p a r t of the GP unlesioned by k a i n i c a c i d (x 262.5). 94 h a l f of the GP ( F i g . 12C). They were scarse i n the p o s t e r i o r h a l f but when present were l o c a t e d i n the v e n t r a l p o r t i o n of the p o s t e r i o r GP. Noteworthy i s the f a c t that there was v i r t u a l l y no overlap i n the GP of the p o s i t i o n s of the HRP-containing neurons a f t e r n i g r a l HRP i n j e c t i o n s and the AChE s t a i n e d neurons (compare F i g s . 11 and 12). d) E f f e r e n t s of the entopeduncular nucleus In order to c h a r a c t e r i z e the biochemical nature of the e f f e r e n t s of the EP t h i s s t r u c t u r e was l e s i o n e d e l e c t r o l y t i c a l l y . A h i s t o l o g y of a representa-t i v e l e s i o n of the EP i s given i n F i g . 14. I t i s apparent that the l e s i o n de-s t r o y e d most of the EP at t h i s l e v e l and extended beyond i t s l i m i t s both dor-s a l l y and v e n t r a l l y . The l e s i o n d i d not encroach upon the GP. The e f f e c t s of s i m i l a r l e s i o n s on habenular CAT and GAD a c t i v i t i e s are given i n Table 7. EP l e s i o n s produced a s i g n i f i c a n t decrease i n GAD of 63% without s i g n i f i c a n t l y a f f e c t i n g CAT a c t i v i t y . Another s e r i e s of experiments were undertaken i n order to c h a r a c t e r i z e b i o c h e m i c a l l y the EP and SN p r o j e c t i o n s to the v e n t r a l a n t e r i o r and p a r a f a s c i -c u l a r n u c l e i of the thalamus. Numerous attempts met w i t h f a i l u r e . The reason i s not d e f i n i t e l y known although l a c k of consistency i n the production of l e -sions and d i s s e c t i o n s may have been a f a c t o r . In any case the p r o j e c t was abandoned. I t might be mentioned t h a t c o n s i s t e n t decreases i n CAT were ob-served i n the v e n t a l a n t e r i o r nucleus of the thalamus a f t e r e l e c t r o l y t i c l e -s i o n s of the SN. This f i n d i n g should be i n v e s t i g a t e d f u r t h e r . I l l Biochemical I n v e s t i g a t i o n s of Substantia N i g r a and Striatum a) Neurotransmitter s y n t h e t i c enzyme l o c a l i z a t i o n i n the s u b s t a n t i a n i g r a I n order to 'characterize- the , n i g r a . f u r t h e r from a biochemical stand-p o i n t , t h i s s t r u c t u r e was i n j e c t e d w i t h k a i n i c a c i d . The s t e r e o t a x i c i n j e c t i o n of 5 nmoles of k a i n i c a c i d i n t o the SN r e s u l t e d i n a v i r t u a l l y complete des-95 Figure 14 Photomicrograph of e l e c t r o l y t i c l e s i o n s of the EP (x 81.5). Table 7. The a c t i v i t y of GAD and CAT i n the habenula on the l e s i o n e d and c o n t r o l s i d e a f t e r u n i l a t e r a l l e s i o n s of the entopeduncular nucleus. Side GAD nmoles/mg protein/hour CAT Per cent c o n t r o l nmoles/mg protein/hour Per cent c o n t r o l L e s i o n C o n t r o l 6 6 13.6±2.40* 21.4±2.34 63.6-100.0 122±18.1 124±10.8 98.4 100.0 * S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l P < 0.05. 98 t r u c t i o n of neuronal c e l l bodies i n both the pars compacta and the pars r e t i -c u l a t a ( F i g . 15), and t h i s was accompanied by intense g l i o s i s . The e f f e c t of these l e s i o n s on GAD, CAT and AChE a c t i v i t i e s i n the SN and on t y r o s i n e hyroxy-l a s e (TH) i n the s t r i a t u m i s shown i n Table 8. S u b s t a n t i a t i n g the h i s t o l o g i -c a l assessment of dopamine-containing neuron l o s s i n the SN i s the greater than 94% r e d u c t i o n i n s t r i a t a l TH a c t i v i t y i p s i l a t e r a l to the l e s i o n e d SN. Fol l o w i n g k a i n i c a c i d i n j e c t i o n s , i t was necessary to express the enzyme a c t i v i t i e s of the l e s i o n e d n i g r a on both a per p r o t e i n and a per SN b a s i s to c o n t r o l f o r the s i g n i f i c a n t t i s s u e shrinkage which was found to occur i n the l e s i o n e d t i s s u e . As shown i n Table 8, n i g r a l GAD a c t i v i t y was decreased on the l e s i o n e d s i d e by 51% when expressed per mg p r o t e i n , and 41% when expressed on a per t i s s u e b a s i s . In marked c o n t r a s t to the decrease i n CAT a c t i v i t y which occurs i n the s t r i a t u m a f t e r s t r i a t a l k a i n i c a c i d i n j e c t i o n s , CAT a c t i -v i t y i n the SN was unaffected by n i g r a l k a i n i c a c i d i n j e c t i o n s . There was no d i f f e r e n c e i n CAT a c t i v i t y between c o n t r o l and l e s i o n e d SN when a c t i v i t y was expressed i n terms of e i t h e r p r o t e i n or t i s s u e . The AChE a c t i v i t y i n the SN on the l e s i o n e d compared to the c o n t r a l a t e r a l c o n t r o l s i d e was reduced by 44% when c a l c u l a t e d on a per p r o t e i n b a s i s , and by 64% on a per nucleus b a s i s . Again t h i s d i f f e r e n c e between the extent of the r e d u c t i o n of AChE a c t i v i t y by k a i n i c a c i d when expressed i n the two ways may r e f l e c t t i s s u e shrinkage. Data on the b a s a l and dopamine st i m u l a t e d a c t i v i t y of adenylate c y c l a s e are shown i n F i g . 16. A s i g n i f i c a n t dopamine s t i m u l a t i o n was found i n both the l e s i o n e d and the c o n t r o l SN. This was true whether the data were c a l c u -l a t e d per mg p r o t e i n , F(3,30) = 12.57, P < 0.001; ( F i g . 16A), or per nucleus, F(3,24) = 30.13, P < 0.001; ( F i g . 16B). The l e s i o n d i d not r e s u l t i n a d i f f e -r e n t i a l e f f e c t of the v a r i o u s dopamine concentrations on the adenylate c y l c a s e a c t i v i t y as compared to c o n t r o l [per mg p r o t e i n , F(3,30) = 1.5, P < 0.05; or per nucleus, F(3,24) = 0.68, P < 0.05]. However, i n terms of a c t i v i t y per mg p r o t e i n , the dopamine sti m u l a t e d adenylate cyc l a s e a c t i v i t y was s i g n i f i c a n t l y 99 F i g ure 15 H i s t o l o g y of normal and k a i n i c a c i d - i n j e c t e d s u b s t a n t i a n i g r a . A: c o n t r a l a t e r a l u n i n j e c t e d SN. B: k a i n i c a c i d i n j e c t e d SN. SNC, pars compacta; SNR, pars r e t i c u l a t a . The i n j e c t e d SN shows complete l a c k of neuronal c e l l bodies, and an increased d e n s i t y of g l i a . M a g n i f i c a t i o n x 250. I DO Table 8 Enzyme a c t i v i t i e s f o l l o w i n g i n t r a n i g r a l k a i n i c a c i d . Enzyme (n) Per mg p r o t e i n Per SN TH (Striatum) GAD (SN) CAT (SN) AChE (SN) Con t r o l Lesion C o n t r o l L e s i o n C o n t r o l L e s i o n C o n t r o l L e s i o n 3.01±0.25 0.18±0.12** 278±10.1 142±12.9** 24.5±2.6 30.511.7 6.9310.41 3.8810.27** 203132.8 84.4126.7** 19.512.9 15.4+1.2 5.4810.51 1.9810.19** Values represent the mean 1 S.E.M. of the number of determinations i n d i -cated. Enzyme a c t i v i t i e s are expressed i n terms of nmoles/h, except AChE which i s i n umoles/h. * p < 0.05. ** p < 0.001, P a i r e d t t e s t . 102 Figure 16 A: The e f f e c t of i n t r a n i g r a l k a i n i c a c i d (5nmoles) on the dopa-m i n e - s e n s i t i v e adenylate c y c l a s e a c t i v i t y of the s u b s t a n t i a n i g r a expressed as pmoles of cAMP formed per mg p r o t e i n per min. Dopamine concentrations are p l o t t e d on a l o g s c a l e . Each p o i n t represents the mean ± S.E.M. f o r 6 animals. A c t i v i t y i n the presence of 10 mM NaF i s shown to the r i g h t of the curve. B: The e f f e c t of i n t r a n i g r a l k a i n i c a c i d (5 nmoles) on the dopamine-sensitive adenylate c y c l a s e a c t i v i t y of the s u b s t a n t i a n i g r a expressed as pmoles of cAMP formed per SN per min. Dopa-mine concentrations are p l o t t e d on a l o g s c a l e . Each p o i n t r e -presents the mean ± S.E.M. f o r 5 animals. A c t i v i t y i n the pre-sence of 1 mM NaF i s shown to the r i g h t of the curve. 105 104 greater i n the l e s i o n e d n i g r a compared to the c o n t r a l a t e r a l c o n t r o l , F(l,10) = 6.79, P < 0.05. This trend towards higher dopamine s t i m u l a t e d a c t i v i t y i n the l e s i o n e d SN was als o seen when the a c t i v i t i e s were expressed on a per t i s -sue b a s i s ; however, i n t h i s case the increase f a i l e d to reach s i g n i f i c a n c e . The s t i m u l a t i o n of adenylate c y c l a s e by 10 mM sodium f l u o r i d e was not s i g -n i f i c a n t l y d i f f e r e n t on the l e s i o n e d s i d e compared to c o n t r o l per p r o t e i n ( F i g . 16A) or per nucleus ( F i g . 16B). b) Neurotransmitter receptor l o c a l i z a t i o n i n the s u b s t a n t i a n i g r a and st r i a t u m Experiments were undertaken to determine the c e l l u l a r l o c a l i z a t i o n of the receptors f o r the neurotransmitter DA i n the SN and st r i a t u m . To t h i s end l e -sio n s of the NSP w i t h 6-OHDA and of the s t r i a t u m w i t h k a i n i c a c i d were em-ployed. The e f f e c t of these l e s i o n s on var i o u s enzyme a c t i v i t i e s i n the SN and s t r i a t u m i s shown i n Table 9. The e f f e c t s of 6-OHDA l e s i o n s of the NSP and k a i n i c a c i d l e s i o n s of the st r i a t u m on t y r o s i n e hydroxylase (TH), glutamic a c i d decarboxylase (GAD), and ch o l i n e a c e t y l t r a n s f e r a s e (CAT) i n the s t r i a t u m and SN are shown i n Table 9. 6-OHDA l e s i o n s of the NSP r e s u l t e d i n a 95% and 91% decrease i n s t r i a t a l and n i g r a l TH a c t i v i t y , a s i g n i f i c a n t i n c r e a s e of 19% i n s t r i a t a l GAD a c t i v i t y and no change i n n i g r a l GAD or s t r i a t a l CAT a c t i v i t y . S t r i a t a l .kainic a c i d l e -sions r e s u l t e d i n a s i g n i f i c a n t decrease of 19% i n s t r i a t a l TH a c t i v i t y w h i l e n i g r a l TH a c t i v i t y remained u n a l t e r e d . These l e s i o n s decreased s t r i a t a l and n i g r a l GAD a c t i v i t y by 56% and 42% r e s p e c t i v e l y . S t r i a t a l CAT a c t i v i t y was reduced by 70% on the l e s i o n e d compared to the c o n t r a l a t e r a l s i d e . I n t e r e s -t i n g l y , however, CAT a c t i v i t y c o n t r a l a t e r a l to the k a i n i c a c i d l e s i o n e d s t r i a -tum increased s i g n i f i c a n t l y by about 18% when compared to the a c t i v i t y i n sa-l i n e i n j e c t e d s t r i a t u m (data not shown). There was no a l t e r a t i o n i n e i t h e r GAD or TH on the s i d e c o n t r a l a t e r a l to the l e s i o n s r e l a t i v e to s a l i n e i n j e c t e d c o n t r o l s . 105 Table 9 The e f f e c t of 6-OHDA l e s i o n s of the NSP and k a i n i c a c i d l e s i o n s of the st r i a t u m on TH, GAD and CAT a c t i v i t i e s i n the s t r i a t u m and SN. Le s i o n Area measured Enzyme A c t i v i t i e s nmoles/mg pr o t / h r TH GAD CAT 6-OHDA l e s i o n of the NSP Striatum c o n t r o l 5.03±.19 le s i o n e d ND c o n t r o l 3.05±.25 242±12 65.6±3.0 288±14*** 69.5±2.5 930±43 SN le s i o n e d 0.26±.09*** 912±62 K a i n i c a c i d l e s i o n of the s t r i a t u m Striatum SN c o n t r o l 5.38±.54 242±9.1 76.1±4.4 l e s i o n e d 4.37±.30** 107±9.9* 23.0±2.9* c o n t r o l 2.86±.50 862±58 l e s i o n e d 3.4L+.93 501±23* Values represent the mean ± S.E.M. of 7 or 8 determinations of the l e -sioned and c o n t r a l a t e r a l c o n t r o l s i d e . ND - not dete c t a b l e . * p < .001 ** p < .05 *** p < .01 106 The r e s u l t s of i n v i t r o b i n d i n g of 3H-apomorphine f o r DA-receptors i n s t r i a t a l and SN t i s s u e a f t e r 6-OHDA l e s i o n s of the NSP i s shown i n Table 10. The s p e c i f i c b i n d i n g of 3H-apomorphine i n the SN was reduced on the l e s i o n e d s i d e by 76%. S i m i l a r l y , compared w i t h the c o n t r o l s i d e , NSP l e s i o n s reduce 3H-apomorphine b i n d i n g i n the s t r i a t u m by 56%. The l i n e a r r e g r e s s i o n l i n e s i n the Scatchard p l o t shown i n F i g . 17 f u r t h e r i n d i c a t e that both the a f f i n i t y of 3H-apomorphine f o r the DA receptor and the maximum number of bin d i n g s i t e s (B max) i n the NSP-lesioned s t r i a t u m are s i g n i f i c a n t l y reduced compared to c o n t r o l s t r i a t u m . Table 11 contains the r e s u l t s of the b i n d i n g of var i o u s l i g a n d s to DA r e -ceptors i n the s t r i a t u m and SN a f t e r 6-OHDA l e s i o n s of the NSP and k a i n i c a c i d l e s i o n s of the s t r i a t u m . In marked c o n t r a s t w i t h the re d u c t i o n i n s t r i a t a l 3H-apomorphine b i n d i n g , 3 H - h a l o p e r i d o l b i n d i n g was s i g n i f i c a n t l y increased by 22% a f t e r NSP 6-OHDA l e s i o n s . This was a l s o t r u e of 3 H - s p i r o p e r i d o l b i n d i n g at both the concentrations t e s t e d . Thus 3 H - s p i r o p e r i d o l b i n d i n g was increased by 18% at 80 pM ( f i n a l c o n c e n t r a t i o n i n the b i n d i n g assay) and 19% at 500 pM. In the SN a f t e r 6-OHDA l e s i o n s of the NSP there was no s i g n i f i c a n t decrease i n 3 H - s p i r o p e r i d o l b i n d i n g a t 500 pM. However, there was a s i g n i f i c a n t decrease of 42% at 80 pM 3 H - s p i r o p e r i d o l . K a i n i c a c i d l e s i o n s of the s t r i a t u m d i d not s i g n i f i c a n t l y a f f e c t 3 H - s p i r o p e r i d o l b i n d i n g i n the SN. 107 Table 10 Binding of 3H-Apomorphine i n the s t r i a t u m and SN a f t e r 6-OHDA induced l e s i o n s of the n i g r o s t r i a t a l pathway. H-Apomorphine bindi n g % of n fmol/mg p r o t e i n c o n t r o l C o n t r o l 11 11.2 + 1.2 Striatum L e s i o n 8 4.9 + 1.3* 44 Co n t r o l 6, 8.0 + 0.8 SN Le s i o n 5 1.9 + 0.8** 24 Values represent means ± S.E.M. of the number of determinations i n d i c a t e d . p < .01 p < .001 108 Figure 17 Scatchard a n a l y s i s of s p e c i f i c 3H-apomorphine bi n d i n g to r a t s t r i a t a l homogenates from c o n t r o l unoperated s i d e (open c i r c l e s ) and from c o n t r a l a t e r a l 6-OHDA-lesioned s i d e (closed c i r c l e s ) . Table 11 Neurotransmitter receptor b i n d i n g i n the s t r i a t u m and SN a f t e r 6-OHDA l e s i o n s of the n i g r o s t r i a t a l pathway and k a i n i c a c i d l e s i o n s of the str i a t u m . 6-OHDA - NSP l e s i o n s K a i n i c a c i d s t r i a t a l l e s i o n s Striatum SN SN (fmoles/mg p r o t e i n ) (fmoles/mg p r o t e i n ) L e s i o n 181 ± 5.9 (5) 11 ± 2.3* (5) 18 ± 3.9 (5) 3 H - S p i r o p e r i d o l (80pM) Con t r o l 154 ± 7.1 (5) 19 ± 2.8 (5) 20 ± 3.9 (5) % of c o n t r o l 118 58 90 Lesi o n 173 ± 4.9* (5) 31.7±3.7 (8) 3 H - S p i r o p e r i d o l (500pM) Con t r o l 147 ± 8.0 (5) 34.9±3.3 (8) % of c o n t r o l 119 91 Lesion 128 ± 7.8* (4) 3 H - H a l o p e r i d o l C o n t r o l 105 ± 8.0 (6) % of c o n t r o l 122 Values represent the means ± S.E.M. of the number of determinations shown i n parentheses. *p < .05 I l l DISCUSSION I Anatomy of the S t r i a t e — P a l l i d a l and S t r i a t o - N i g r a l P r o j e c t i o n s As elaborated i n the i n t r o d u c t i o n , the s t r i a t a l p r o j e c t i o n s to both seg-ments of the globus p a l l i d u s i n the primate and to the SN i n s e v e r a l species have been w e l l e s t a b l i s h e d by degenerative anatomical techniques. However, the advantages of the HRP and autoradiographic t r a c i n g techniques have l e d , i n many i n s t a n c e s , to a r e i n v e s t i g a t i o n of anatomical pathways i n the CNS. The present i n v e s t i g a t i o n s employing these techniques confirm and extend previous s t u d i e s of the s t r i a t a l e f f e r e n t p r o j e c t i o n s . In accordance w i t h these e a r l i e r anatomical s t u d i e s , i n j e c t i o n s of r a d i o l a b e l e d amino a c i d i n t o the d o r s o - l a t e r a l quadrant of the s t r i a t u m r e s u l t e d i n the t r a n s p o r t of 3 H - p r o t e i n to the GP and hence a heavy accumulation of autoradiographic grains i n t h i s s t r u c t u r e . S i m i l a r l y , w e l l l o c a l i z e d i n j e c t i o n s of r a d i o t r a c e r i n t o the t a i l of the s t r i a t u m , l a t e r a l to the GP, r e s u l t e d i n dense l a b e l i n g of the GP. From e i t h e r i n j e c t i o n s i t e there was no d i f f u s i o n of l a b e l to the cortex or GP as i n d i c a t e d by the l a c k of g r a i n accumulations i n the thalamus or SUT, r e -s p e c t i v e l y . That the GP represents a t e r m i n a l f i e l d of s t r i a t a l e f f e r e n t f i b e r s i s d i c t a t e d by a l l the c r i t e r i a g e n e r a l l y employed to draw such conclu-s i o n s by the autoradiographic t r a c i n g method (see w r i t t e n comprehensive exami-n a t i o n s , J . I . Nagy, 1978). The present r e s u l t s are a l s o i n complete agreement w i t h the p r e v i o u s l y described topography of the s t r i a t a l e f f e r e n t s to the GP (see I n t r o d u c t i o n ) . A n t e r i o r s t r i a t a l 3 H - l e u c i n e i n j e c t i o n s r e s u l t e d i n the highest g r a i n d e n s i t i e s i n the a n t e r i o r GP. In f a c t , the l a b e l i n g i n t h i s r e -gion of the GP was so intense that the s i l v e r grains c l e a r l y demarcated the a n t e r i o r border of the GP from the s t r i a t u m immediately r o s t r a l to i t . In the r a t , the GP i s l o c a t e d l a t e r a l to the i n t e r n a l capsule and f o l l o w s the arc circumscribed by t h i s f i b e r system to a considerable d i s t a n c e c a u d a l l y . I n -j e c t i o n s of . H-leucine i n t o the t a i l of the s t r i a t u m l e d to an area of unmis-112 takable t e r m i n a t i o n i n p r e c i s e l y these caudal regions of the GP. Further c o n f i r m a t i o n of the s t r i a t o - G P p r o j e c t i o n was obtained i n e x p e r i -ments u t i l i z i n g HRP which demonstrated that i n j e c t i o n s i n t o the GP r e s u l t e d i n the appearance of HRP r e a c t i v e neurons i n t h e ' s t r i a t u m . These l a b e l e d neurons appeared to be confined to the c e n t r a l core of t h i s s t r u c t u r e . One i n t e r p r e -t a t i o n of t h i s HRP l a b e l i n g p a t t e r n i s that only the c e n t r a l core r e g i o n of the s t r i a t u m surrounding the GP pr.bj.ect.es to the p a l l i d u m . This i s c l e a r l y not the case s i n c e the autoradiographic t r a n s p o r t data show that i n j e c t i o n s of l a b e l i n t o the a n t e r i o r and p o s t e r i o r extremes of the s t r i a t u m l a b e l s the an-t e r i o r and p o s t e r i o r aspects of the GP, r e s p e c t i v e l y . I t i s noteworthy that i n an attempt to keep the HRP r e s t r i c t e d to the GP, the HRP i n j e c t i o n s were aimed at the center of t h i s nucleus. A more p l a u s i b l e a l t e r n a t i v e , then, i s that the HRP p a t t e r n obtained i n the s t r i a t u m i s a f u r t h e r i n d i c a t i o n of the topography of the s t r i a t o - G P p r o j e c t i o n . From the present m a t e r i a l , p a r t i c u -l a r l y s i n c e the l e s s s e n s i t i v e diaminobenzidine HRP method was employed, i t was d i f f i c u l t to a s c e r t a i n the types of s t r i a t a l neurons which p r o j e c t to the GP. I t d i d appear, however, that most were of medium s i z e . As described i n the I n t r o d u c t i o n there i s a p a u c i t y of d e f i n i t i v e s t u d i e s on the s t r i a t o - E P pathway. The present autoradiographic experiments i n v o l v i n g °H-leucine i n j e c t i o n s i n t o the a n t e r i o r s t r i a t u m provide strong support f o r the e x i s t e n c e of t h i s pathway i n the r a t and f u r t h e r strengthen the concept th a t the EP i n subprimates represents the homologue of the i n t e r n a l segment of the globus p a l l i d u s i n the primate. I t was somewhat more d i f f i c u l t to d i s c e r n a p o s t e r i o r s t r i a t a l p r o j e c t i o n to the EP. F o l l o w i n g i n j e c t i o n s of 3 H - l e u c i n e i n t o the t a i l of the s t r i a t u m autoradiographic grains could be traced descen-ding in..the i n t e r n a l capsule caudal to the GP. At the l e v e l of the EP l a b e l was l o c a t e d j u s t d o r s o l a t e r a l to the EP w i t h i n the i n t e r n a l capsule. Due to the poor d e f i n i t i o n of the d o r s o l a t e r a l border of the EP i n t h e . i n t e r n a l cap-113 s u l e i t could not be determined whether there was sparse l a b e l i n g i n t h i s r e -mote p o r t i o n of the nucleus. I t was apparent, however, that where the EP was d i s t i n c t no l a b e l was observed and t h a t the bulk of the descending f i b e r s o r i -g i n a t i n g i n the t a i l of the s t r i a t u m avoid the EP as they forge toward the SN. The present attempts to study the s t r i a t o - E P pathway employing the HRP technique a l s o met w i t h some d i f f i c u l t i e s . As a l l u d e d to i n the I n t r o d u c t i o n , damaged axons at an i n j e c t i o n s i t e can accumulate HRP and t r a n s p o r t i t i n a retrograde f a s h i o n to the c e l l bodies. In the present study, HRP i n j e c t i o n aimed at the EP i n v a r i a b l y i n v o l v e d the adjacent i n t e r n a l capsule through which axons course en route to d i s t a n t t e r m i n a l f i e l d s . Labeled c e l l s were o c c a s i o n a l l y observed i n the c e r e b r a l cortex and SNR a f t e r microelectrophore-t i c i n j e c t i o n s i n t o the EP. The f a c t that m i c r o e l e c t r o p h o r e t i c i n j e c t i o n s i n t o the crus c e r e b r i a l s o l a b e l e d c e l l s i n the s t r i a t u m , SN and c e r e b r a l cor-tex confirms that axons d i d accumulate and transport HRP. These r e s u l t s echo the ca u t i o n which has been s t r e s s e d i n anatomical s t u d i e s u t i l i z i n g HRP. How-ever, pressure i n j e c t i o n s of HRP i n t o the crus c e r e b r i d i d not r e s u l t i n the appearance of r e a c t i v e c e l l s i n any of the above re g i o n s , suggesting that t h i s method of d e l i v e r y may be l e s s l i k e l y to l a b e l c e l l s as a r e s u l t of accumula-t i o n and t r a n s p o r t by axons i n the v i c i n i t y of the i n j e c t i o n . From e x p e r i -ments i n v o l v i n g pressure i n j e c t i o n s i n t o the EP i t i s apparent t h a t , w i t h the exception of the caudal t a i l , the e n t i r e s t r i a t u m p r o j e c t s to the EP. I t should be noted that the area of the s t r i a t u m which appeared to be devoid of a p r o j e c t i o n to the EP, as i n d i c a t e d by the HRP method, i s l o c a t e d f u r t h e r c a u d a l l y to the placements of the 3 H - l e u c i n e i n j e c t i o n s . As expected from the w e l l e s t a b l i s h e d s t r i a t o - n i g r a l p r o j e c t i o n , i n j e c -t i o n of l a b e l i n t o the s t r i a t u m r e s u l t e d i n g r a i n accumulations i n the SNR. No l a b e l was observed i n the SNC. A f t e r d o r s o a n t e r i o r s t r i a t a l i n j e c t i o n s , l a b e l was d i s t r i b u t e d p r i m a r i l y i n the medial SNR. I n j e c t i o n s i n t o the dorso-114 l a t e r a l t a i l of the s t r i a t u m l a b e l e d the extreme v e n t r a l p o r t i o n of the l a t e -r a l SNR whereas more v e n t r a l t a i l i n j e c t i o n l a b e l e d the d o r s o l a t e r a l SNR. I I Biochemical Neuroanatomy of S t r i a t a l and P a l l i d a l E f f e r e n t P r o j e c t i o n s a) S t r i a t o - p a l l i d a l p r o j e c t i o n s ; glutamic a c i d decarboxylase The present i n v e s t i g a t i o n s i n v o l v i n g l e s i o n s of the a n t e r i o r and poste-r i o r s t r i a t u m have d i s c l o s e d some in f o r m a t i o n as to the biochemical organiza -t i o n of the s t r i a t a l e f f e r e n t f i b e r s . I t must be s t r e s s e d , however, that these s t u d i e s are by no means d e f i n i t i v e and i n c e r t a i n instances conclusions drawn by the present author may r e q u i r e f u t u r e m o d i f i c a t i o n i n l i g h t of new data. E l e c t r o l y t i c l e s i o n s i n the head of the s t r i a t u m and hemitransections \ w e l l a n t e r i o r or j u s t a n t e r i o r to the GP r e s u l t e d i n s i g n i f i c a n t r eductions i n GAD i n the GP. This suggests that GABA i s u t i l i z e d as a t r a n s m i t t e r by at l e a s t some of the s t r i a t o - G P f i b e r s . A f u r t h e r observation i s that hemitran-s e c t i o n s which more c l o s e l y approached the GP, although s t i l l a v oiding i t , r e -s u l t e d i n greater GAD reductions (compare r e s u l t s i n Table 2 and 3). This might be expected i f the s t r i a t o - G P GAD-containing f i b e r s were organized topo-g r a p h i c a l l y . Support f o r t h i s i s derived from experiments where the a n t e r i o r and p o s t e r i o r GP was analyzed s e p a r a t e l y a f t e r a n t e r i o r hemitransections. These l e s i o n s d i d not cause a s i g n i f i c a n t decrease i n the p o s t e r i o r h a l f of the GP, but t h i s may have been due to the unusually l a r g e standard e r r o r s of these GAD values. The r e s u l t s , however, i n d i c a t e c l e a r l y that the hemitran-s e c t i o n s caused a greater s i g n i f i c a n t GAD decrease i n the a n t e r i o r than the p o s t e r i o r GP. Thus, i t may be concluded that the s t r i a t a l f i b e r s which have been repeatedly demonstrated to p r o j e c t t o p o g r a p h i c a l l y onto the GP (see i n t r o d u c t i o n ) a l s o may be, at l e a s t i n p a r t , the f i b e r s which cont a i n GAD. I n c i d e n t a l l y , because of the u n c e r t a i n t y of the GAD values i n the p o s t e r i o r GP the author i s not prepared to suggest that t h i s p a r t of the GP does not r e c e i v e gaba-ergic input from the a n t e r i o r s t r i a t u m , but r a t h e r that t h i s i s 115 l e s s dense than a n t e r i o r GP regions. K a i n i c a c i d l e s i o n s of the a n t e r i o r s t r i a t u m a l s o l e d to a decrease i n GP GAD. The r e s u l t s w i t h t h i s l e s i o n technique, however, were equivocal i n that h i s t o l o g i c a l examination of the extent of the l e s i o n revealed d e s t r u c t i o n of p a r t s of the GP and s t r i a t a l areas l a t e r a l to the GP. Consistent w i t h the anatomical observations of a s t r i a t o - E P p r o j e c t i o n , l e s i o n s of the a n t e r i o r s t r i a t u m caused a s i g n i f i c a n t GAD r e d u c t i o n i n the EP. This i s suggestive of a GAD-containing p r o j e c t i o n from the a n t e r i o r s t r i a t u m to the EP. I n t e r e s t i n g l y , k a i n i c a c i d l e s i o n s which destroyed the greater part of the body of the s t r i a t u m f a i l e d to cause a greater r e d u c t i o n i n EP GAD a c t i v i t y than d i d the hemitransections. Moreover, hemitransections which caused a greater r e d u c t i o n i n GP GAD d i d not produce a simultaneous propor-t i o n a l decrement i n EP GAD. This suggests two a l t e r n a t i v e s f o r the l o c a l i z a -t i o n of the neurons w i t h i n the s t r i a t u m that give r i s e to the s t r i a t o - E P GAD-c o n t a i n i n g pathway. I t may be that most of these neurons are l o c a t e d i n the a n t e r i o r reaches of the s t r i a t u m or that there i s a d i s c o n t i n u i t y of these neurons i n the r o s t r o c a u d a l dimension of the s t r i a t u m . The l a t t e r a l t e r n a t i v e i s favored i n view of the present HRP anatomical f i n d i n g s together w i t h the f a c t that e l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m r e s u l t e d i n s i g n i -f i c a n t reductions of GAD i n the EP. E l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m d i d not a l t e r GAD or CAT a c t i v e l y i n the GP. This r e s u l t together w i t h the present demonstration of a p r o j e c t i o n to the GP from the t a i l of the s t r i a t u m suggests that the f i b e r s comprising t h i s p r o j e c t i o n are devoid of GAD and CAT. However, cauti o n must be emphasized i n drawing such a c o n c l u s i o n . As noted e a r l i e r , the t a i l of the s t r i a t u m p r o j e c t s to q u i t e p o s t e r i o r p a r t s of the GP. This r e g i o n of the GP i s surrounded by the i n t e r n a l capsule m e d i a l l y , the amygdala v e n t r a l l y and the c a u d a l , t a i l of the s t r i a t u m l a t e r a l l y . In an e f f o r t to increase the accuracy of the GP d i s s e c t i o n and to exclude s t r i a t a l t i s s u e from these samples, only 116 the main body ,of the GP was d i s s e c t e d . I t i s e n t i r e l y p o s s i b l e that these "backwoods" regions of the GP were not included i n the present a n a l y s i s . I f d i s s e c t i o n d i f f i c u l t i e s could be overcome the experiments i n v o l v i n g the pro-j e c t i o n of the t a i l of the s t r i a t u m to these p o s t e r i o r areas of the GP cer-t a i n l y warrant r e i n v e s t i g a t i o n . The r e d u c t i o n of GAD i n the EP a f t e r e l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m appears to c o n t r a d i c t the autoradiographic anatomical f i n d i n g where one was hard pressed to observe a p r o j e c t i o n to the EP from s t r i a t a l areas l a t e r a l to the GP. On the other hand, HRP l a b e l e d c e l l s were present i n t h i s s t r i a t a l area a f t e r EP HRP i n j e c t i o n s . I t i s noteworthy i n t h i s regard that the e l e c t r o l y t i c s t r i a t a l l e s i o n s were d e c i s i v e l y l a r g e r than the °H-leu-ci n e i n j e c t i o n s i t e . Thus, a sparse p r o j e c t i o n to the EP from s t r i a t a l r e -gions l a t e r a l to the GP together w i t h a r e l a t i v e l y r e s t r i c t e d 3 H - l e u c i n e i n -j e c t i o n i n t h i s area may have r e s u l t e d i n the i n a b i l i t y to detect t h i s p r o j e c -t i o n anatomically. b) S t r i a t o - p a l l i d a l p r o j e c t i o n s ; c h o l i n e a c e t y l t r a n s f e r a s e A h i t h e r t o unexpected r e s u l t i s that s t r i a t a l l e s i o n s cause reductions i n CAT a c t i v i t y i n the GP and EP. These decreases were s i g n i f i c a n t i n the pos-t e r i o r GP a f t e r hemitransection a n t e r i o r to the GP and i n the EP a f t e r e l e c -t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m . Although a r e l a t i v e l y l a r g e de-crease was observed i n the a n t e r i o r GP a f t e r hemitransections, t h i s r e s u l t was s e r i o u s l y compromised by the l a r g e standard e r r o r of the CAT a c t i v i t i e s i n these samples. However, i n cases where s i g n i f i c a n c e was obtained, there are s e v e r a l p o s s i b l e i n t e r p r e t a t i o n s as to the cause of the CAT d e p l e t i o n s . Some of the p o s s i b l e c r i t i c i s m s that may be l e v i e d against the present biochemical work i n general are discussed i n r e l a t i o n to the present observations concer-ni n g CAT. The reason i s that these observations, i n p a r t i c u l a r , have c e r t a i n i m p l i c a t i o n s . r e g a r d i n g the c o n f i g u r a t i o n of s t r i a t a l e f f e r e n t s to va r i o u s n u c l e i and the d e f i n i t i o n of the neurotransmitters i n v o l v e d i n these s t r i a t a l 117 e f f e r e n t s . The f i r s t of the p o s s i b l e reasons f o r the lower CAT a c t i v i t y i n the GP of animals that had r e c e i v e d s t r i a t a l l e s i o n s i s that the CAT l o s s e s are only ap-parent and merely r e f l e c t a c o n s i s t e n t d i s s e c t i o n e r r o r between l e s i o n e d and c o n t r o l samples. This i s r u l e d out s i n c e , as shown i n the r e s u l t s , the l e f t and r i g h t sides of the b r a i n regions examined can be d i s s e c t e d a c c u r a t e l y from unoperated animals to y i e l d r e l i a b l e agreement of CAT and GAD between the two s i d e s . The second p o s s i b i l i t y i s a c r i t i c i s m of data obtained on the GP by hemi-t r a n s e c t i o n s c l o s e l y approaching the GP. This argument suggests that enzyme changes i n the GP may be due to spread of damage from the l e s i o n s i t e poste-r i o r to the GP thereby d e s t r o y i n g terminals and/or interneurons i n the GP con-t a i n i n g the enzymes under i n v e s t i g a t i o n . There i s no reason to b e l i e v e that a f t e r hemitransections such a process would not be o p e r a t i v e i n the t a i l of the s t r i a t u m immediately l a t e r a l to the GP and thus, a f f e c t enzymes contained i n n e u r a l elements of t h i s s t r u c t u r e . This c r i t i c i s m i s countered by the pre-sent observation that i n s e c t i o n s c o n t a i n i n g the t a i l of the s t r i a t u m imme-d i a t e l y l a t e r a l to the GP and from which the a n t e r i o r and p o s t e r i o r GP was ob-t a i n e d , n e i t h e r CAT nor GAD a c t i v i t y was a l t e r e d by hemitransections. The t h i r d p o s s i b i l i t y i n v o l v e s the h e a v i l y AChE-staining neurons in-and-around the GP and EP. As described i n the I n t r o d u c t i o n these c e l l s may be c h o l i n e r g i c , CAT-containing, p r o j e c t i o n neurons to the cortex. Indeed, some of these neurons undergo retrograde degeneration a f t e r c o r t i c a l l e s i o n s ( J . I . Nagy, unpublished o b s e r v a t i o n s ) . I t must be emphasized that t h i s retrograde l o s s i s only p a r t i a l . The extent to which these c e l l s undergo retrograde de-generation a f t e r a n t e r i o r hemitransection i s not known. The CAT decrease i n the GP, then, may have been due to a l o s s of t h i s enzyme i n neurons whose c o r t i c o p e t a l axons have been severed by hemitransection. This e x p l a n a t i o n , although i t cannot be r u l e d out e n t i r e l y at the present time, i s u n l i k e l y f o r 118 two reasons. F i r s t , retrograde degeneration r e q u i r e s more time to occur than the 9 to 14 days post o p e r a t i v e s u r v i v a l p e r i o d employed i n the present study (Das, 1971). Moreover, at s h o r t e r s u r v i v a l times, having.severed the axons of these c e l l s , i t might be expected that the d e p r i v a t i o n of t h e i r a b i l i t y to t r a n s p o r t CAT to d i s t a l regions of t h e i r axon would lea d to an accumulation of CAT i n the p e r i k a r y a r a t h e r than a d e p l e t i o n . Second, by f r a c t i o n a t i o n tech-niques neurotransmitter s y n t h e t i c enzymes have been shown to be concentrated i n nerve terminals to a greater degree than i n the c e l l soma. A s i m i l a r argu-ment holds f o r fibers-of-passage that may c o n t a i n CAT and which might undergo degeneration a f t e r having been severed. Thus, the CAT l o s s i n the GP i s more l i k e l y to represent a d e p l e t i o n from t e r m i n a l s than nerve c e l l bodies. I t may be argued that the d e p l e t i o n i s from r e c u r r e n t axon c o l l a t e r a l s of these c e l l s . However, i f CAT i n r e c u r r e n t c o l l a t e r a l s of these neurons c o n t r i b u t e d a s i g n i -f i c a n t p r o p o r t i o n of t h i s enzyme to the GP, then these c o l l a t e r a l s might a l s o be s u f f i c i e n t to prevent t h e i r retrograde degeneration. F o u r t h l y , the CAT decreases may have been due to a compensatory r e a c t i o n to the l e s i o n s of i n t r i n s i c n e u r a l systems of the GP and EP. The l a r g e a l t e -r a t i o n s i n CAT a c t i v i t y a f t e r a r e l a t i v e l y short s u r v i v a l p e r i o d , together w i t h the f a c t that these changes occurred i n both the EP and GP a f t e r two q u i t e d i f f e r e n t l e s i o n s , makes the occurrence of a compensatory r e a c t i o n to the l e s i o n s remote. This admittedly weak argument makes t h i s p o s s i b i l i t y worthy of f u r t h e r i n v e s t i g a t i o n as indeed are a l l the other p o s s i b i l i t i e s . The f i n a l p o s s i b i l i t y and the one favored here i s the existence of a cho-l i n e r g i c p r o j e c t i o n to the GP and EP from the l e s i o n e d s t r u c t u r e s or from other areas whose axons-of-passage were severed at the l e s i o n s i t e . On ana-tom i c a l grounds the only axons p r o j e c t i n g to the GP that would have been se-vered, other than those from the s t r i a t u m , are the accumbens-GP f i b e r s . At present an accumbens-GP c h o l i n e r g i c p r o j e c t i o n cannot be r u l e d out. In t h i s regard, i t i s perhaps s i g n i f i c a n t that the accumbens appears not to p r o j e c t 119 to the EP (Nauta et a l , 1978) and a n t e r i o r hemitransections d i d not a l t e r EP CAT a c t i v i t y . On the other hand, CAT was reduced i n the EP a f t e r l e s i o n s of the t a i l of the s t r i a t u m which probably would not have i n t e r r u p t e d e f f e r e n t accumbens f i b e r s . Further support f o r t h i s l a t t e r suggestion i s derived from the present experiments i n v o l v i n g k a i n i c a c i d l e s i o n s of the GP. H i s t o l o g i c a l examination of the k a i n i c a c i d l e s i o n s revealed that neuronal degeneration had occurred i n most of the GP. The only domain of the GP spared by these l e s i o n s was the most p o s t e r i o r extreme which, i n c i d e n t a l l y , overlaps that area spared by the e l e c t r o l y t i c l e s i o n s of the GP. Varying degrees of k a i n i c acid-induced neuron d e s t r u c t i o n was al s o observed i n the medial p o r t i o n of the t a i l of the s t r i a -tum immediately l a t e r a l to the GP and i n the s t r i a t u m j u s t a n t e r i o r and ante-r o l a t e r a l to the GP. The extent to which k a i n i c a c i d i s t o x i c to axons i s s t i l l c o n t r o v e r s i a l (see methods s e c t i o n ) . However, c o n s i s t e n t w i t h some views that f i b e r s - o f - p a s s a g e are not d i s t u r b e d by k a i n i c a c i d , i n j e c t i o n s of t h i s compound i n t o the GP which contains ascending DA-containing f i b e r s to the s t r i a t u m d i d not reduce but, i n f a c t , s i g n i f i c a n t l y increased TH i n the t a i l of the s t r i a t u m . The GAD and CAT reductions i n the GP, which were of equal magnitude a f t e r GP i n j e c t i o n s , probably represent d e s t r u c t i o n by k a i n i c a c i d of p e r i k a r y a i n the GP c o n t a i n i n g those enzymes. Q u a n t i t a t i v e l y , these reductions are comple-mentary to those caused by hemitransections a n t e r i o r to the GP. The destruc-t i o n of axon terminals i n the GP c o n t a i n i n g these enzymes cannot at present be r u l e d out, although the weight of evidence i s against a neurotoxic e f f e c t of k a i n i c a c i d on these neuronal elements. That d i f f u s i o n of k a i n i c a c i d and thus neuronal, d e s t r u c t i o n i n the t a i l of the s t r i a t u m occurred i s i n d i c a t e d by the s i g n i f i c a n t r e d u c t i o n of GAD i n samples of t h i s s t r u c t u r e . I t i s both s u r p r i s i n g and perhaps meaningful that i n these same samples no r e d u c t i o n of CAT was observed. T h r e e i p o s s i b i l i t i e s are given f o r t h i s r e s u l t . F i r s t , cho-120 l i n e r g i c neurons may be l e s s s e n s i t i v e to the t o x i c e f f e c t s of k a i n i c a c i d . T h i s , however, i s u n l i k e l y c o n s i d e r i n g e a r l i e r observations i n v o l v i n g k a i n i c a c i d i n j e c t i o n s i n t o the s t r i a t u m . These i n j e c t i o n s produced equal r e d u c t i o n of CAT and GAD at v a r i o u s doses of k a i n i c a c i d (McGeer and McGeer, 1976; Schwarcz and Coyle, 1977) (see, however, s e c t i o n on i n t r a n i g r a l k a i n i c a c i d ) . Second, c h o l i n e r g i c neurons may be l o c a t e d p r i m a r i l y i n the l a t e r a l r e gion of the t a i l of the s t r i a t u m which, according to the h i s t o l o g i c a l a n a l y s i s , was not a f f e c t e d by k a i n i c a c i d . T h i r d , c h o l i n e r g i c neurons i n the t a i l of the s t r i a t u m may, i n f a c t , have been destroyed. However, i f these were e f f e r e n t p r o j e c t i o n neurons, t h e i r l o s s , by CAT a n a l y s i s , may have gone undetected f o r arguments s i m i l a r to those proposed f o r d i s c o u n t i n g the retrograde l o s s of AChE-staining neurons i n the GP as being the cause of CAT decrease i n the GP a f t e r a n t e r i o r hemitransections. At present, i t i s d i f f i c u l t to decide between these a l t e r n a t i v e s . The l a t t e r p o s s i b i l i t y , however, i s c o n s i s t e n t w i t h the present proposal of the e x i s t e n c e of c h o l i n e r g i c s t r i a t a l e f f e r e n t f i b e r s . Further support i s derived f o r CAT-containing s t r i a t a l e f f e r e n t s to both GP and EP from observations made during the developmental stages of the k a i n i c a c i d GP l e s i o n . I n i t i a l l y , these l e s i o n s were l a r g e but were stepwise reduced i n s i z e as c o r r e c t i n j e c -t i o n volumes and k a i n i c a c i d concentrations were developed. Enough animals were in c l u d e d i n these experiments to make the f o l l o w i n g observations f a i r l y r e l i a b l e . When CAT a c t i v i t y i n the t a i l of the s t r i a t u m was reduced by 50%, GP CAT was reduced profoundly by 85%. A 21% CAT r e d u c t i o n i n the t a i l of the s t r i a t u m was accompanied by a 53% r e d u c t i o n i n the GP, and f i n a l l y when CAT was u n a l t e r e d i n the s t r i a t u m , GP CAT was reduced by 42%. This p a t t e r n was a l s o true of GAD i n the GP, EP and notably the SN. Since the l e s i o n s of the GP i n these successive experiments could not have been more complete than the one f i n a l l y chosen, the p a r a l l e l diminutions of CAT i n the GP and s t r i a t u m s t r o n g l y suggest the CAT l o s s e s i n the GP i n the I n i t i a l experiments to be 121 due to the d e s t r u c t i o n of CAT-containing s t r i a t a l e f f e r e n t p r o j e c t i o n neurons to the GP by k a i n i c a c i d . Yet another observation that bears on a l l the l e s i o n s that caused CAT r e -ductions i n the GP i s the e f f e c t of k a i n i c a c i d on the l a r g e AChE-staining c e l l s of the GP. The l o c a t i o n of these c e l l s i n the GP has been shown ( F i g . 11). Although t h e i r numbers were not q u a n t i t a t e d , i t appeared t h a t , apart from some atrophy, they were l e f t i n t a c t by k a i n i c a c i d . This i n d i c a t e s at l e a s t that CAT i s present i n neuronal elements i n the GP other than these pu-t a t i v e c h o l i n e r g i c neurons and that i t i s p o s s i b l e to o b t a i n CAT decreases i n the GP without t h e i r degeneration. From the foregoing d i s c u s s i o n i t i s proposed that ACh be added to the l i s t of substances contained i n the s t r i a t a l e f f e r e n t s to the GP and EP. T h i s , of course, does not preclude the e x i s t e n c e of s t r i a t a l c h o l i n e r g i c i n t e r n e u -rons . c) S t r i a t o - n i g r a l p r o j e c t i o n s I t has been p r e v i o u s l y shown that hemitransections at the l e v e l of the hypothalamus do not a l t e r n i g r a l CAT a c t i v i t y (McGeer et a l , 1973; Kataoka et a l , 1974). I t i s t h e r e f o r e not s u r p r i s i n g that i n the present experiments n i g r a l CAT was not a f f e c t e d by hemitransections a n t e r i o r to the GP. This nega-t i v e f i n d i n g takes on p a r t i c u l a r s i g n i f i c a n c e s i n c e these same hemitransec-t i o n s reduced CAT.in the GP. Furthermore, e l e c t r o l y t i c GP l e s i o n s d i d not l e a d to a CAT decrease i n the EP and t h i s l e s i o n would c e r t a i n l y have des-troyed s t r i a t o - E P f i b e r s o r i g i n a t i n g i n the a n t e r i o r s t r i a t u m . This r e s u l t then i s i n agreement w i t h the observation that hemitransections a n t e r i o r to the GP f a i l e d to a l t e r EP CAT a c t i v i t y . The s i g n i f i c a n c e of these f i n d i n g s i s discussed more f u l l y i n the next s e c t i o n . The present attempts to determine the l o c a t i o n w i t h i n the s t r i a t u m and/or GP of the neurons g i v i n g r i s e to the GAD-containing a f f e r e n t s to the SN were r e l a t i v e l y u n successful. A s t a r t i n g p o i n t i n such an i n v e s t i g a t i o n i s to 122 determine the maximal c o n t r i b u t i o n to n i g r a l GAD of a f f e r e n t f i b e r s from the s t r i a t u m and GP. In agreement w i t h previous observations (see I n t r o d u c t i o n ) hemitransections at the l e v e l of the EP r e s u l t e d i n a r e d u c t i o n of GAD i n the SN of 84%. This sets the b a s e l i n e to which the e f f e c t s of various l e s i o n s i n s t r u c t u r e s a n t e r i o r to t h i s hemitransection should be compared. The remaining GAD i n the SN may r e s i d e i n n i g r a l interneurons or i n a f f e r e n t s to the SN from s t r u c t u r e s caudal to the l e v e l of the EP. Of the v a r i e t y of l e s i o n s placed i n the s t r i a t u m or GP, none r e s u l t e d i n a r e d u c t i o n of GAD a c t i v i t y that even approached that caused by hemitransec-t i o n s at the EP l e v e l , although s m a l l s i g n i f i c a n t decreases were observed. Hemitransections s l i g h t l y a n t e r i o r to the GP or e l e c t r o l y t i c l e s i o n s placed i n the head of the s t r i a t u m d i d not cause a s i g n i f i c a n t r e d u c t i o n i n n i g r a l GAD whereas hemitransections more c l o s e l y approaching the GP produced a small but s i g n i f i c a n t decrease. K a i n i c a c i d l e s i o n s of the head of the s t r i a t u m a l s o reduced n i g r a l GAD but, as mentioned e a r l i e r , these l e s i o n s i n v o l v e d p a r t s of the GP and the s t r i a t u m l a t e r a l to the GP. I n s o f a r as n i g r a l GAD i s concerned, these r e s u l t s i n d i c a t e that there i s a s e n s i t i v e area i n the s t r i a t u m at the l e v e l of the a n t e r i o r border of the GP. E l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m a l s o caused small s i g n i f i c a n t decreases i n n i g r a l GAD. These l e s i o n s d i d not extend as f a r a n t e r i o r as the hemitransections and t h e r e f o r e a separate popu-l a t i o n of GAD-containing s t r i a t o - n i g r a l f i b e r s may have been a f f e c t e d than those damaged by the hemitransections. This being the case, about 47% of n i g r a l GAD can be accounted f o r . The c o n t r i b u t i o n from the s t r i a t u m j u s t ante-r i o r to the GP i s about,: 16%, the t a i l of the s t r i a t u m c o n t r i b u t e s about 15%, and 16% i s i n t r i n s i c or from sources caudal to the EP. The remaining 50% may o r i g i n a t e from regions not enveloped by the hemitransections or the e l e c t r o -l y t i c s t r i a t a l l e s i o n s . The gross anatomy of the t a i l of the s t r i a t u m causes consi d e r a b l e d i f f i c u l t i e s i n producing l e s i o n s which destroy the f u l l extent 123 of t h i s s t r u c t u r e . In the present attempts to l e s i o n t h i s s t r u c t u r e , the dor-somedial and v e n t r o p o s t e r i o r regions were spared. The c o n t r i b u t i o n to n i g r a l GAD from these areas i s t h e r e f o r e unknown, d) P a l l i d o - n i g r a l p r o j e c t i o n s To determine whether the GP e f f e r e n t f i b e r s to the SN c o n t a i n GAD, e l e c -t r o l y t i c l e s i o n s of the GP were produced. H i s t o l o g i c a l examinations showed that these l e s i o n s destroyed a l l but the p o s t e r i o r o n e - t h i r d of the GP. On the b a s i s of the present autoradiographic s t u d i e s these l e s i o n s would not have impinged on the descending s t r i a t a l f i b e r s o r i g i n a t i n g i n the t a i l of the s t r i a t u m but would have severed some of those o r i g i n a t i n g i n the a n t e r i o r s t r i a t u m . Damage by these l e s i o n s of f i b e r s passing through the GP compromi-ses the i n t e r p r e t a t i o n of the s i g n i f i c a n t reductions of GAD i n the EP and SN. I t i s t h e r e f o r e impossible to conclude whether these reductions r e s u l t e d from d e s t r u c t i o n of neurons i n the GP or from damage to fibers-of-passage o r i g i n a -t i n g from regions a n t e r i o r to the GP. Furthermore, because the l e s i o n s missed the l a t e r a l and p o s t e r i o r GP, the GAD c o n t r i b u t i o n to the SN from these areas remains unknown. What i s s i g n i f i c a n t , however, i s that the GP e l e c t r o l y t i c l e s i o n s d i d not produce a r e d u c t i o n of GAD i n e i t h e r the EP or SN greater than that produced by hemitransections a n t e r i o r to the GP. This suggests that the areas of the GP destroyed by the e l e c t r o l y t i c l e s i o n s do not p r o j e c t GAD-con-t a i n i n g f i b e r s to the EP or SN. Hemitransections a n t e r i o r to the GP causing only minor reductions of n i g r a l GAD together w i t h the demonstration by numerous i n v e s t i g a t o r s of the p a l l i d o - n i g r a l p r o j e c t i o n made t h i s pathway a major candidate f o r the o r i g i n of n i g r a l GAD. I t i s t h e r e f o r e of considerable i n t e r e s t that the k a i n i c a c i d l e s i o n s of the GP d i d not s i g n i f i c a n t l y a l t e r GAD a c t i v i t y i n the SN. Because of the s i g n i f i c a n c e of t h i s negative r e s u l t i t may be appropriate here to d i g r e s s to answer three c r i t i c i s m s of the present experiments. F i r s t , i n drawing on h i s t o l o g i c a l data from a group.of animals separate from those i n 124 which biochemical data were obtained, the assumption i s made that the l e s i o n s i n b r a i n s taken f o r biochemical a n a l y s i s were the same as i n those analyzed h i s t o l o g i c a l l y . F o l l o w i n g both e l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a -tum and k a i n i c a c i d GP l e s i o n s , i n some b r a i n s only biochemical s t u d i e s were conducted w h i l e i n others SN GAD was measured and the remainder of the b r a i n was taken f o r h i s t o l o g y . A f t e r both types of l e s i o n s the c l o s e agreement i n SN GAD values between b r a i n s taken f o r enzyme a n a l y s i s and b r a i n s taken f o r h i s t o l o g y i n d i c a t e s the v a l i d i t y of e x t r a p o l a t i n g the area covered by the l e -s i o n as assessed h i s t o l o g i c a l l y to a l l animals r e c e i v i n g a given l e s i o n . Furthermore, from the h i s t o l o g i c a l a n a l y s i s of a l a r g e number of l e s i o n s , t h e i r u n i f o r m i t y was evident. Moreover, l e s i o n s were inspected during d i s s e c -t i o n s and only those i n which the d e s i r e d placement was achieved were u t i l i z e d f o r biochemical s t u d i e s . Second, i t may be argued that l a r g e l o c a l i z e d l o s s e s of GAD w i t h i n subregions of the SN may have occurred a f t e r any of the above l e s i o n s . Therefore, i f n i g r a l GAD o r i g i n a t e d from neurons dispersed through-out the GP and s t r i a t u m , any i n d i v i d u a l l e s i o n would cause only small or unde-t e c t a b l e decreases of GAD i n the SN as a whole. T h i s , however, can be taken to r e d u c t i o ad absurdum i n that i f areas i n the SN the s i z e of axon: terminals could be assayed f o r GAD a f t e r given l e s i o n i n the s t r i a t o - p a l l i d a l complex, many samples would y i e l d reductions of 100%! To a f i r s t approximation, f o r biochemical s t u d i e s the extent to which the SN should be subdivided i n t o v a r i o u s components should correspond to the extent of the l e s i o n s produced i n the s t r u c t u r e s g i v i n g r i s e to i t s a f f e r e n t s . In the present study any i n d i v i -dual l e s i o n encompassed a l a r g e area of t i s s u e , thus, the SN was taken i n i t s e n t i r e t y f o r a n a l y s i s . T h i r d l y , given that hemitransections immediately ante-r i o r to the GP cause s i g n i f i c a n t , a l b e i t s m a ll decreases, i n n i g r a l GAD; that some GAD-containing n i g r a l p r o j e c t i o n neurons are l o c a t e d a n t e r i o r to the GP; and, that the f i b e r s of those neurons pass through the GP en route to the SN, the f a i l u r e of k a i n i c a c i d i n j e c t i o n s i n t o the GP to reduce n i g r a l GAD i s e v i -125 dence that under the present i n j e c t i o n parameters k a i n i c a c i d d i d not destroy f i b e r s - o f - p a s s a g e . There are two p o s s i b l e explanations f o r the f a i l u r e of k a i n i c a c i d GP l e -sions to e f f e c t n i g r a l GAD. F i r s t , that p a l l i d o - n i g r a l GAD-containing neurons e x i s t but that k a i n i c a c i d does not destroy them. To t e s t t h i s e v e n t u a l i t y and determine i f any p a l l i d o - n i g r a l neurons survived k a i n i c a c i d , the SN of animals which had r e c e i v e d u n i l a t e r a l k a i n i c a c i d GP l e s i o n s was i n j e c t e d b i -l a t e r a l l y w i t h HRP. Numerous l a r g e neurons i n the GP were l a b e l e d on the un-l e s i o n e d s i d e . Some l a b e l e d neurons were present i n the p o s t e r i o r GP of the l e s i o n e d s i d e and t h i s i s i n agreement w i t h the h i s t o l o g i c a l examinations which showed t h i s area to be spared by the k a i n i c a c i d l e s i o n s . The r e s t of the GP was devoid of c e l l s c o n t a i n i n g HRP. The h e a v i l y AChE-staining c e l l s , which appeared to be r e s i s t a n t to the neurotoxic e f f e c t s of k a i n i c a c i d , were not l a b e l e d w i t h HRP i n d i c a t i n g that these neurons have p r o j e c t i o n f i e l d s to areas other than the SN. In view of these r e s u l t s a second more l i k e l y a l t e r -n a t i v e i s t h a t no p a l l i d o - n i g r a l GAD-containing neurons e x i s t w i t h i n the con-f i n e s of the k a i n i c a c i d l e s i o n . I l l S t r i a t a l and P a l l i d a l E f f e r e n t s : Synthesis and S p e c u l a t i o n S e v e r a l important aspects of the s t r i a t a l and p a l l i d a l systems warrant d e t a i l e d c o n s i d e r a t i o n i n s o f a r as they p e r t a i n to the present f i n d i n g s and the l i t e r a t u r e a v a i l a b l e on the s t r i a t u m and p a l l i d u m . Four is s u e s which are c u r r e n t l y r e c e i v i n g considerable a t t e n t i o n are: 1) The topography of the s t r i a t o - p a l l i d a l and s t r i a t o - n i g r a l p r o j e c t i o n s i n r e l a t i o n to n i g r a l effe^-r e n t s and s t r i a t a l a f f e r e n t p r o j e c t i o n s , 2) The c h a r a c t e r i z a t i o n of the types of neurons which form the s t r i a t a l e f f e r e n t p r o j e c t i o n s , 3) The i d e n -t i t y of the neurotransmitters contained i n the s t r i a t a l and p a l l i d a l e f f e r e n t f i b e r s , and 4) The l o c a t i o n of the neurons w i t h i n the s t r i a t o - p a l l i d a l com-pl e x which are r e s p o n s i b l e f o r each of the neurotransmitters that are con-126 t a i n e d i n f i b e r s emanating from these s t r u c t u r e s , a) Topographic r e l a t i o n s Detailed.topographic r e l a t i o n s of the s t r i a t o - G P , s t r i a t o - E P and s t r i a t o -n i g r a l p r o j e c t i o n s could not be determined from the l i m i t e d number of i n j e c -t i o n s i t e s f o r e i t h e r HRP or 3 H - l e u c i n e employed i n the present study. Cer-t a i n features of the s t r i a t a l e f f e r e n t s , however, are worthy of comment. The present observations i n the r a t are i n agreement w i t h previous demonstrations of the o r g a n i z a t i o n of the s t r i a t a l e f f e r e n t s to the globus p a l l i d u s i n other species whereby a l l p a r t s of the s t r i a t u m , at l e a s t i n the a n t e r i o r - p o s t e r i o r and d o r s a l - v e n t r a l plane, p r o j e c t to the globus p a l l i d u s i n such a way that the former i s superimposed on the l a t t e r (Szabo, 1962, 1967, 1969; N i m i i , 1971; Johnson and Rosvold, 1971). Whether some s t r i a t a l areas p r o j e c t d i f f e r e n t i a l -l y to the GP and EP i s unresolved. There are suggestions i n the l i t e r a t u r e of a m e d i a l - l a t e r a l segregation of s t r i a t a l e f f e r e n t s to the LGP and MGP (Cowan and P o w e l l , 1966; Voneida, 1960; Szabo, 1962, 1967, 1969) such that progres-s i v e l y more medial s t r i a t a l areas p r o j e c t p r e f e r e n t i a l l y to MGP. E l e c t r o -p h y s i o l o g i c a l work suggesting the comparmentalization of s t r i a t a l e f f e r e n t s i s derived from the observation that antidromic responses i n the s t r i a t u m a f t e r GP s t i m u l a t i o n are l o c a t e d mainly i n the c e n t r a l core region (Richardson et a l , 1977). Some divergence of s t r i a t a l p r o j e c t i o n s to the two p a l l i d a l segments may occur i n the t a i l of the s t r i a t u m . As observed here, there appears to be a pa u c i t y of f i b e r s p r o j e c t i n g to the EP from t h i s area. T u l l o c h et a l (1978) a l s o show very l i t t l e l a b e l i n g i n . t h e v i c i n i t y of EP a f t e r p o s t e r i o r s t r i a t a l °H-leucine i n j e c t i o n s . In c o n t r a s t , the t a i l of the s t r i a t u m has a p r o j e c t i o n to the GP. The concept of d i f f e r e n t i a l s t r i a t o - p a l l i d a l p r o j e c t i o n s takes on added dimensions i n view of c e r t a i n f i n d i n g s regarding the s t r i a t o - n i g r a l pro-j e c t i o n . I t appears that a l l p a r t s of the s t r i a t u m i n the r a t p r o j e c t to the SN except a c e n t r a l core r e g i o n (Bunney and Aghajanian, 1976a). In a d d i t i o n , 127 the most dense p r o j e c t i o n s to the SN from the s t r i a t u m appear to a r i s e from the t a i l of the s t r i a t u m (Bunney and Aghajanian, 1976) and p e r i p h e r a l s t r i a t a l areas (Richardson et a l , 1977; T u l l o c h et a l , 1978). The o r i g i n w i t h i n the s t r i a t u m of e f f e r e n t p r o j e c t i o n s to the GP, EP and SN and the extent to which these overlap.has important anatomical and func-t i o n a l i m p l i c a t i o n s . Each of the GP, EP and SN have some mutually e x c l u s i v e p r o j e c t i o n s . This i s a l s o true on a more l o c a l s c a l e w i t h i n the SN ( F a u l l and Mehler, 1978). Thus, whether the s t r i a t u m i s a f u n c t i o n a l l y heterogeneous s t r u c t u r e may be determined by the degree to which s t r i a t a l a f f e r e n t p r o j e c -t i o n s are congregated and s t r i a t a l e f f e r e n t p r o j e c t i o n s segregated. D e t a i l e d i n f o r m a t i o n on these p o i n t s i s not a v a i l a b l e s i n c e , to date, i n v e s t i g a t i o n s along these l i n e s have been l a r g e l y , s u p e r f i c i a l . New, more s e n s i t i v e , HRP methods (Mesulam, 1978) w i l l undoubtedly prove u s e f u l i n r e s o l v i n g some of these problems. A c o r o l l a r y of the view of d i f f e r e n t i a l s t r i a t a l p r o j e c t i o n s to i t s t a r -get areas i n v o l v e s the c o l l a t e r a l i z a t i o n of s t r i a t a l e f f e r e n t f i b e r s . The oc-currence of c o l l a t e r a l i z a t i o n of these f i b e r s has been s t r e s s e d on both ana-t o m i c a l (Fox and R a f o l s , 1975; Fox et a l , 1975) and e l e c t r o p h y s i o l o g i c a l (Yoshida et a l , 1971, 1972) grounds. I t i s apparent from the present e x p e r i -ments, however, that a n t e r i o r s t r i a t a l areas which p r o j e c t GAD-containing f i -bers to the GP do not have c o l l a t e r a l p r o j e c t i o n s to the SN. I f f u r t h e r , more d e t a i l e d s t u d i e s demonstrate a considerable l a c k of overlap w i t h i n the s t r i a -tum of p r o j e c t i o n neurons to the GP, EP and SN, then t h i s would r a i s e serious questions as to the r o l e of axon c o l l a t e r a l s of the s t r i a t a l e f f e r e n t s as a major o r g a n i z a t i o n a l p r i n c i p l e . A f u r t h e r p o i n t regarding the s t r i a t o - n i g r a l p r o j e c t i o n i s i t s r e l a t i o n -s h i p to the c o r t i c o - s t r i a t a l p r o j e c t i o n . F a u l l and Mehler (1978) have c i t e d unpublished observations that the v i s u a l c o r t e x p r o j e c t s to s t r i a t a l areas that have e f f e r e n t p r o j e c t i o n s to that r e g i o n of the SN which p r o j e c t s to the 128 SC. S i m i l a r l y , the motor co r t e x p r o j e c t s to s t r i a t a l areas that have e f f e r e n t p r o j e c t i o n s to n i g r a l regions p r o j e c t i n g to the thalamus. The present obser-v a t i o n s suggest that the d o r s a l - a n t e r i o r s t r i a t u m p r o j e c t s to n i g r a l areas which according to F a u l l and Mehler (1978) and F a l l o n and Moore (1978), give r i s e to thalamic p r o j e c t i o n s , whereas the t a i l of the s t r i a t u m p r o j e c t s to SN areas which innervate the SC. As a d e t a i l e d map of the s t r i a t a l i n n e r v a t i o n from the cortex has not been p u b l i s h e d , i t i s not p o s s i b l e to determine which c o r t i c a l areas innervate the s t r i a t a l p r o j e c t i o n areas s t u d i e d here. In any case, i t should be pointed out t h a t the conservation of c o r t i c o - n i g r a l func-t i o n a l r e l a t i o n s h i p s through the s t r i a t u m i s d i f f i c u l t to e n v i s i o n i n view of recent autoradiographic demonstrations of the widespread d i s t r i b u t i o n of s t r i a -t a l a f f e r e n t s from the cortex (Goldman and Nauta, 1977; Y e t e r i a n and Hoeson, 1975; Kunzle, 1977; Jones et a l , 1977). In the ..context of the topography of the s t r i a t a l i n n e r v a t i o n of the SN i t may be appropriate to mention the r e c i p r o c a l i n n e r v a t i o n of the s t r i a t u m by the DA-containing neurons of the SNC. A d i r e c t i n f l u e n c e of these systems on each other would n a t u r a l l y r e q u i r e a r e c i p r o c a l t o p o g r a p h i c a l arrangement. However, a c o m p l i c a t i o n i n e s t a b l i s h i n g such r e c i p r o c o c i t y i s that the DA neu-rons of the SNC have dendrites which r a d i a t e i n t o the SNR. The determination of the p a r t i c u l a r DA neurons whose den d r i t e s i n the SNR r e c e i v e p a r t i c u l a r s t r i a t a l input r e q u i r e s more elaborate techniques than simply observing the topography of these i n p u t s . In the absence of hard data the present author i s given to the f o l l o w i n g s p e c u l a t i o n s . In the d o r s a l - v e n t r a l dimension the SN appears to be arranged i n t o three t i e r s . These t i e r s c o n t a i n neurons of e i t h e r the n i g r o - s t r i a t a l , n i g r o - t h a l a m i c or n i g r o - t e c t a l systems ( F a u l l and Mehler, 1978; F a l l o n and Moore, 1978) . The dendrites of the DA n i g r a l neurons may permeate a l l three of these t i e r s or seek an appropriate one. At the same time as n i g r a l a f f e r e n t input i s r e c e i v e d to any i n d i v i d u a l t i e r to be subse-quently r e l a y e d on to the s t r i a t u m , thalamus or SC, the DA dendrites i n t h i s 129 t i e r may a l s o r e c e i v e t h i s same input and transmit perhaps t o p o g r a p h i c a l l y to the s t r i a t a l areas from which the o r i g i n a l a f f e r e n t input arose. That i s , the DA neuron may monitor a f f e r e n t input to a n i g r a l t i e r through i t s d e n d r i t e and re g u l a t e t h i s through i t s axon. A l t e r n a t i v e l y , the DA neurons may monitor s t r i a t a l and p a l l i d a l input to a l l three t i e r s simultaneously, i n t e g r a t e t h i s and r e l a y appropriate feedback to the s t r i a t u m . These two a l t e r n a t i v e s have d i f f e r e n t i m p l i c a t i o n s w i t h regard to n i g r o - s t r i a t a l topography. The l a t t e r a l t e r n a t i v e would a l l o w t h i s to be l e s s s t r i n g e n t , i n t h a t , i f the DA neurons do indeed serve as a feedback mechanism, sampling and r e g u l a t i n g b a s a l g a n g l i a output, then such feedback i n t h i s case would i n v o l v e d i v e r s e areas of the s t r i a t u m , perhaps a f f e c t i n g simultaneously areas that r e c e i v e , f o r example, v i s u a l and motor c o r t i c a l i n p u t . b) S t r i a t a l p r o j e c t i o n neurons The i s s u e as to which of the v a r i o u s c l a s s e s of neurons i n the s t r i a t u m form the e f f e r e n t s of t h i s s t r u c t u r e i s not s e t t l e d . Nor f o r that matter, i s the problem of the types of neurons that e x i s t i n the s t r i a t u m . To be b r i e f , i t appears that there are three s i z e s of s t r i a t a l neurons; s m a l l , medium and l a r g e , or a s i z e to s u i t everybody. Spiny neurons seem to be e x c l u s i v e l y of the medium s i z e . Aspiny neurons come i n a l l three s i z e s . The reader i s d i r e c -ted to s e c t i o n Ia-3 of the I n t r o d u c t i o n f o r arguments as to why only medium spiny s t r i a t a l neurons p r o j e c t to the SN and why Fox and coworkers (1971-1972) o r i g i n a l l y assumed that only l a r g e aspiny neurons i s s u e s t r i a t a l e f f e r e n t pro-j e c t i o n s . With regard to the co n c l u s i o n that medium spiny neurons give r i s e to the s t r i a t o - n i g r a l f i b e r s , there i s no evidence to the contrary. That these neurons a l s o p r o j e c t to the globus p a l l i d u s i s suggested by the observa-t i o n that medium s i z e d spiny s t r i a t o - n i g r a l neurons i s s u e c o l l a t e r a l s to the globus p a l l i d u s (Yoshida.et a l , 1971, 1972) and by the demonstration that these neurons are l a b e l e d a f t e r p a l l i d a l HRP i n j e c t i o n s ( G r a y b i e l , 1979). Whether other types of s t r i a t a l neurons p r o j e c t to the globus p a l l i d u s i s de-130 batable. In view of evidence presented by Fox et a l (1971, 1972, 1975) i t would appear that l a r g e aspiny neurons have e f f e r e n t s to t h i s s t r u c t u r e . G r a y b i e l et a l (1979) observed t h a t some l a r g e s t r i a t a l c e l l s contained l a b e l a f t e r HRP i n j e c t i o n s i n t o the GP and EP. However, they hedged as to whether these were neurons or g l i a l c e l l s which had incorporated HRP r e a c t i o n product. I f these were indeed neurons, and i n p a r t i c u l a r l a r g e aspiny neurons, t h i s would i n p a r t v i n d i c a t e Fox et a l (1971, 1972, 1975) and have c e r t a i n i m p l i c a -t i o n s w i t h regard to the present suggestion of a s t r i a t a l c h o l i n e r g i c e f f e r e n t p r o j e c t i o n . As mentioned e a r l i e r , the l a r g e aspiny neurons of the s t r i a t u m s t a i n h e a v i l y f o r AChE (Butcher et a l , 1975; P o i r i e r et a l , 1977). T h i s im-p l i e s , although not n e c e s s a r i l y (see Lehmann et a l , 1979 and Lehmann and F i b i g e r , 1978), that these may be c h o l i n e r g i c neurons. Thus, although h i g h l y t e n t a t i v e , the l a r g e aspiny AChE-staining s t r i a t a l neurons may give r i s e to the c h o l i n e r g i c i n n e r v a t i o n of the GP and EP. T h i s , of course, does not ex-clude the p o s s i b i l i t y that the medium s i z e d neurons are r e s p o n s i b l e f o r con-t a i n i n g ACh i n t h i s p r o j e c t i o n , p a r t i c u l a r l y s i n c e there appears to be at l e a s t three subclasses of neurons of t h i s s i z e . c) Neurotransmitters i n s t r i a t a l and p a l l i d a l e f f e r e n t s As d e t a i l e d i n the I n t r o d u c t i o n , the neurotransmitter candidates con-t a i n e d i n s t r i a t a l e f f e r e n t p r o j e c t i o n s are GABA and enkephalin to the GP and GABA and substance P to both the EP and SN. Neither a substance P-containing s t r i a t o - G P pathway nor a s t r i a t o - E P or -SN enkephalin pathway has been demon-s t r a t e d . The present r e s u l t s of a s t r i a t o - n i g r a l GABA-containing pathway i s In accord w i t h previous demonstrations of t h i s p r o j e c t i o n (Kim et a l , 1971; H a t t o r i et a l , 1973b; Fonnum et a l , 1974). In a d d i t i o n , the presence of GABA-co n t a i n i n g f i b e r s i n the s t r i a t o - G P pathway as shown here s u b s t a n t i a t e s ear-l i e r suggestions of t h i s 6 ( H a t t o r i et a l , 1973b) and i s i n agreement w i t h work conducted simultaneously by others ( J e s s e l et a l , 1977; Fonnum et a l , 1978a). F i n a l l y , the present f i n d i n g of a s t r i a t o - E P GABA-containing pathway i s i n 131 e s s e n t i a l agreement w i t h the independent f i n d i n g s of Fonnum et a l (1978a). The l a c k of GABA-containing G P - n i g r a l pathways as i n t e r p r e t e d from the r e s u l t s obtained here i s contrary to the suggestions of Brownstein et a l (1977) and J e s s e l et a l (1978). This i s more f u l l y discussed i n the next s e c t i o n . The present r e s u l t s a l s o i n d i c a t e the p o s s i b l e existence of ACh i n s t r i a -to-GP and -EP f i b e r s . In t h i s context i t i s perhaps noteworthy that biochemi-c a l neuroanatomy enjoys a healthy r e p u t a t i o n s i n c e i n numerous instances the use of t h i s technique has r e s u l t e d i n the f i r s t suggestions of p r e v i o u s l y un-known pathways. Nevertheless, as pointed out e a r l i e r , the demonstration by combined l e s i o n and biochemical methods of anatomical pathways c o n t a i n i n g spe-c i f i c n e u rotransmitters s u f f e r s from c e r t a i n l i m i t a t i o n s and much a d d i t i o n a l support i s r e q u i r e d before the present suggestions of an ACh-containing s t r i a -t o - p a l l i d a l pathway can be taken as proven. Such st u d i e s could i n c l u d e : 1) the c o r r e l a t i o n of the time course of CAT d e p l e t i o n i n the GP w i t h t e r m i n a l degeneration i n t h i s area f o l l o w i n g s t r i a t a l l e s i o n s , 2) the uptake and r e -l e a s e of ACh i n the GP and the e f f e c t of s t r i a t a l l e s i o n s on these, 3) the e f -f e c t of i n j e c t i o n s of axoplasmic t r a n s p o r t i n h i b i t o r s such as c o l c h i c i n e on CAT i n the GP, 4) immunoprecipitation of 3H-CAT obtained from the GP a f t e r 3 H - l e u c i n e i n j e c t i o n s i n the s t r i a t u m , 5) immunohistochemical s t a i n i n g of CAT i n the GP and the e f f e c t on t h i s of s t r i a t a l l e s i o n s , 6) retrograde degenera-t i o n of unequivocally i d e n t i f i e d c h o l i n e r g i c neurons i n the s t r i a t u m f o l l o w i n g l e s i o n s of the GP, 7) the e l e c t r o p h y s i o l o g i c a l i d e n t i t y of a c t i o n of ACh and s t r i a t a l s t i m u l a t i o n on GP. neurons, 8) the e f f e c t of c h o l i n e r g i c drugs on s t r i a t a l evoked GP responses. The p o s s i b i l i t y of a c h o l i n e r g i c s t r i a t o - p a l l i d a l pathway may add yet another s i t e of a c t i o n i n the b r a i n of c h o l i n e r g i c drugs. The f o l l o w i n g i s the r a t i o n a l e f o r such a s i t e of a c t i o n . DA antagonists are known to i n c r e a s e ACh metabolism i n the s t r i a t u m (Marco et a l , 1976; S t r a d l e r et a l , 1973; McGeer et a l , 1974b) but decrease i t i n the globus p a l l i d u s (Marco et a l , 132 1976). C h o l i n e r g i c a ntagonists are known to reverse the increased output of DA caused by DA antagonists (Anden and Bedard, 1971). P a l l i d a l neurons are known to d i r e c t l y i n n ervate DA-containing neurons i n the SN ( H a t t o r i et a l , 1975). I t may be assumed that the decreased ACh output i n the GP may, i n p a r t , give r i s e to the increased DA output i n the s t r i a t u m . In a d d i t i o n , the increased DA output i n the s t r i a t u m may p a r t i a l l y overcome the DA blockade by DA-receptor antagonists. Thus, i n p a t i e n t s s u f f e r i n g from schizophrenia or Huntington's d i s e a s e , where DA antagonists are the treatment of choice, one p o s s i b l e s i t e of a c t i o n of the b e n e f i c i a l e f f e c t s of cholinomimetics (Davis and Berger, 1978) i s the GP where decreased ACh a c t i v i t y would be reversed r e -s u l t i n g i n reduced DA output i n the s t r i a t u m by way of the SN which would add to the DA b l o c k i n g e f f e c t s of a n t i p s y c h o t i c drugs. By s i m i l a r reasoning, a n t i c h o l i n e r g i c s may have a b e n e f i c i a l e f f e c t i n Parkinson's disease (Klawans, 1968) by decreasing c h o l i n e r g i c a c t i v i t y i n the GP thereby augmenting DA out-put i n the s t r i a t u m . As the above suggests there i s considerable pharmacological and some ana-t o m i c a l ( H a t t o r i et a l , 1976) evidence f o r an i n t e r a c t i o n between the n i g r o -s t r i a t a l DA system and c h o l i n e r g i c neurons i n the s t r i a t u m . I t i s suggested here that some of the interdependence of these systems i s manifested at the c h o l i n e r g i c end by the s t r i a t o - p a l l i d a l c h o l i n e r g i c neurons. The "some" i n t h i s l a s t statement should be emphasized s i n c e , as i n d i c a t e d above s t r i a t a l and p a l l i d a l ACh metabolism can simultaneously undergo changes i n opposite d i r e c t i o n (Marco et a l , 1976). This suggests that at l e a s t two f u n c t i o n a l l y d i s t i n c t c h o l i n e r g i c neuron populations e x i s t i n the s t r i a t u m ; those p r o j e c -t i n g to the GP and perhaps a separate p o p u l a t i o n of interneurons. A f u r t h e r c o n s i d e r a t i o n i s the e l e c t r o p h y s i o l o g i c a l nature of the s t r i a -t o - p a l l i d a l pathway w i t h regard to c h o l i n e r g i c mechanisms. E x c i t a t o r y r e -sponses to s t i m u l a t i o n of the s t r i a t u m have been observed i n the GP and EP, although whether these were monosynaptic was not d e a l t w i t h i n d e t a i l 133 ( M a l l i a n i and Purpura, 1967; Levine et a l , 1974). Richardson et a l (1977) have a l s o suggested, by i n f e r e n c e , an e x c i t a t o r y input to the GP from the s t r i -atum. There are p r e s e n t l y no neurotransmitter candidates which may mediate the e x c i t a t o r y responses i n t h i s system. I f the a c t i o n of ACh on GP neurons i s e x c i t a t o r y as i t i s i n many other CNS neurons, t h i s at l e a s t would be con-s i s t e n t w i t h the proposal of a c h o l i n e r g i c s t r i a t o - p a l l i d a l pathway and would suggest the e x c i t a t i o n i n the p a l l i d u m to be mediated by ACh. d) Biochemical neuroanatomy of s t r i a t a l and p a l l i d a l e f f e r e n t s The determination of the o r i g i n and d i s t r i b u t i o n w i t h i n the s t r i a t u m and GP of the e f f e r e n t p r o j e c t i o n neurons c o n t a i n i n g each of the neurotransmit-t e r s that have been i d e n t i f i e d as o r i g i n a t i n g from these n u c l e i would be of considerable value to both p s y c h o l o g i c a l and pharmacological s t u d i e s i n attemp-t i n g to define s t r i a t a l f u n c t i o n and to the eventual e l u c i d a t i o n of s t r i a t a l a r c h i t e c t u r e . Although, to date, such s t u d i e s have given only gross l o c a l i z a -t i o n s , i t should be remembered that some of the pathways concerned and the n e u r o t r a n s m i t t e r substances contained t h e r e i n have only r e c e n t l y been demons-t r a t e d . From the r e s u l t s of the present study i t i s u n l i k e l y that the SN r e c e i v e s a s i g n i f i c a n t GABA-containing p r o j e c t i o n from the GP. This i s i n c o n t r a s t to the c o n c l u s i o n drawn by J e s s e l et a l (1978) which was based on the f a i l u r e of l e s i o n s completely s e p a r a t i n g the GP from the s t r i a t u m to reduce n i g r a l GAD to those l e v e l s achieved by hemitransections p o s t e r i o r to the GP. Brownstein et a l (1977), by a s i m i l a r l i n e of reasoning, assumed that the n i g r a l GAD that they could not account f o r , o r i g i n a t e d not from the GP but the nucleus accum-bens. This assumption, however, i s probably i n c o r r e c t s i n c e Dray and Oakely (1977) observed no r e d u c t i o n i n n i g r a l GAD f o l l o w i n g accumbens l e s i o n s . From the present autoradiographic observations of the t r a j e c t o r y of the p o s t e r i o r s t r i a t o - n i g r a l f i b e r s together w i t h the f a c t that some of these are GAD-con-t a i n i n g , i t i s p o s s i b l e that both Brownstein et a l (1977) and J e s s e l et a l 134 (1978) d i d not sever these f i b e r s w i t h t h e i r s e m i c i r c u l a r l e s i o n s between the GP and s t r i a t u m . The n i g r a l r e d u c t i o n obtained a f t e r t h e i r l e s i o n s together w i t h the decrease obtained i n the present study a f t e r l e s i o n s i n the p o s t e r i o r s t r i a t u m accounts f o r a l l but about 10 to 15% of the n i g r a l GAD o r i g i n a t i n g from outside the SN. Thus, the r e s u l t s of the work of these groups, as i n t e r -preted here, and the present r e s u l t s place the neurons c o n t r i b u t i n g GAD-con-t a i n i n g e f f e r e n t s to the SN outside the GP. The m a j o r i t y of the work conducted to date shows that the c o n t r i b u t i o n to n i g r a l GAD from the e n t i r e s t r i a t u m a n t e r i o r to the GP does not amount to more than 10 to 15% (Brownstein et a l , 1977; H a t t o r i et a l , 1973b; Fonnum et a l , 1978a; Spano et a l , 1977; J e s s e l et a l , 1978). This l i m i t s the l o c a t i o n of the m a j o r i t y of s t r i a t o - n i g r a l GAD-containing neurons to s t r i a t a l areas poste-r i o r to the l e v e l of the r o s t r a l pole of the GP. P o s t e r i o r l y , s i n c e l e s i o n s s e p a r a t i n g the GP from the s t r i a t u m r e s u l t i n only small n i g r a l GAD r e d u c t i o n s , (Brownstein et a l , 1977) any s i g n i f i c a n t p r o p o r t i o n of n i g r a l GAD-containing a f f e r e n t s o r i g i n a t i n g from t h i s r e g i o n , must do so from areas immediately l a -t e r a l to the GP. That t h i s may indeed by the case i s suggested by the f i n d i n g that l e s i o n s i n v o l v i n g the l a t e r a l border between the GP and s t r i a t u m cause l a r g e reductions i n n i g r a l GAD (Brownstein et a l , unpublished data). From the above r e s u l t s and the present work the l o c a t i o n of the GABA-e r g i c p r o j e c t i o n neurons to the SN i s n e c e s s a r i l y decided by the process of e l i m i n a t i o n . The e l e c t r o l y t i c l e s i o n s of the t a i l of the s t r i a t u m together w i t h the a n t e r i o r hemitransections were to some extent complementary w i t h the k a i n i c a c i d GP l e s i o n s . K a i n i c a c i d GP l e s i o n s only reduced n i g r a l GAD when the l e s i o n s extended beyond the GP. Hemitransections only reduced n i g r a l GAD when these approached the GP. 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 l a t e r a l to the GP reduced GAD only s l i g h t l y . ' Therefore, the GAD-containing s t r i a t o -p a l l i d a l n i g r a l neurons must be sandwiched between the GP and s t r i a t u m , along t h e i r mutual borders, both a n t e r i o r l y and l a t e r a l l y and, although no informa-135 t i o n has been obtained i n the present s t u d i e s , perhaps d o r s a l l y . Such an a r -rangement would i n c l u d e the p o s t e r i o r GP which was not l e s i o n e d by any of the above procedures. Since n i g r a l i n j e c t i o n s of HRP r e s u l t i n a heavy de n s i t y of l a b e l e d neu-rons i n the caudal and p e r i p h e r a l s h e l l of the s t r i a t u m (Bunney and Aghajanian, 1976a; T u l l o c h et a l , 1978; Richardson et a l , 1977) and sin c e the m a j o r i t y of the substance P i n the SN o r i g i n a t e s from neurons i n the a n t e r i o r s t r i a t u m , these, together w i t h the above conclusions concerning GAD, suggest a t h i r d n e u r o t r a n s m i t t e r i n the p o s t e r i o r s t r i a t o - n i g r a l f i b e r s . Since the GP-nigral pathway appears not to co n t a i n GAD, t h i s b r ings i n t o the question the nature of the t r a n s m i t t e r of t h i s f i b e r system. However, substance P may be a can d i -date s i n c e neurons i n the GP s t a i n immunohistochemically f o r t h i s peptide. J e s s e l et a l (1978) suggest that a s i g n i f i c a n t p r o p o r t i o n of the n i g r a l sub-stance P o r i g i n a t e s from the GP whereas Brownstein et a l (1977) have concluded that no GP - n i g r a l substance P pathway e x i s t s . GP k a i n i c a c i d l e s i o n s of the k i n d employed i n the present study may r e s o l v e t h i s p o i n t . There i s l i t t l e i n f o r m a t i o n i n the l i t e r a t u r e regarding the p r e c i s e l o c a -t i o n of the GAD-containing s t r i a t o - p a l l i d a l neurons. J e s s e l et a l (1978) found t h a t hemitransections considerably more r o s t r a l to the GP than those employed here d i d not cause a s i g n i f i c a n t r e d u c t i o n i n GP GAD. Since t h e i r separations of the GP from the s t r i a t u m r e s u l t e d i n l a r g e d e p l e t i o n s , J e s s e l e t a l (1978) concluded t h a t the m a j o r i t y of GP GAD-containing a f f e r e n t s o r i g i -nate from neurons i n the caudal s t r i a t u m . This r e s u l t i s i n agreement w i t h the observation of Fonnum et a l (1978a) who found that p r o g r e s s i v e l y more cau-d a l l e s i o n s i n v o l v i n g the s t r i a t u m a n t e r i o r to the GP r e s u l t e d i n p r o g r e s s i v e -l y l a r g e r reductions i n GP GAD. The present r e s u l t s suggest that about 50% of the GAD i n the GP o r i g i n a t e s i n s t r i a t a l neurons l o c a t e d a n t e r i o r to the GP. This estimate and the observation that the a n t e r i o r GP regions s u f f e r greater l o s s e s of GAD than p o s t e r i o r regions f o l l o w i n g a n t e r i o r s t r i a t a l l e s i o n s are 136 i n c l o s e agreement w i t h the f i n d i n g s of Fonnum et a l (1978a). No conclusions . can be drawn from the present work regarding the c o n t r i b u t i o n of the t a i l of the s t r i a t u m to GP GAD. With regard to the source of GAD i n the EP, the a n t e r i o r s t r i a t u m c o n t r i -butes about 40% and the t a i l of the s t r i a t u m about 20%. This i s i n rough agreement w i t h the report of Fonnum et a l (1978a) who presented reductions of EP GAD i n i n d i v i d u a l animals f o l l o w i n g o b l i q u e hemitransections at the mid-GP l e v e l ranging from 46 to 88%. A l l the r e s u l t s to date i n d i c a t e that the neu-rons from which the GAD-containing GP and EP a f f e r e n t a r i s e are d i s t r i b u t e d uniformly and d i s c o n t i n u o u s l y , r e s p e c t i v e l y , i n the s t r i a t u m at l e a s t i n the a n t e r i o r - p o s t e r i o r dimension. E a r l i e r d i s c u s s i o n of the topography of these p r o j e c t i o n s suggests that there may be a m e d i a l - l a t e r a l d i f f e r e n t i a l p r o j e c -t i o n of these neurons to EP and GP. The l o c a t i o n of the neurons i n the s t r i a t u m that may provide a c h o l i n e r -g i c p r o j e c t i o n to the GP and EP i s u n c e r t a i n as are the existence of these pathways. Assuming t e n t a t i v e l y that c h o l i n e r g i c s t r i a t a l e f f e r e n t f i b e r s e x i s t , the a n t e r i o r . s t r i a t u m appears to send such f i b e r s to the GP but not to the EP. Although no c o n c l u s i o n can be drawn as to whether the t a i l of the s t r i a t u m has c h o l i n e r g i c e f f e r e n t s to the GP, such e f f e r e n t s may e x i s t to the EP. I f the l a r g e aspiny AChE-staining neurons are the source of s t r i a t a l cho-l i n e r g i c e f f e r e n t f i b e r s , then i t may be concluded that these e f f e r e n t s a r i s e from widespread areas of the s t r i a t u m s i n c e these AChE-staining neurons are ra t h e r uniformly d i s t r i b u t e d throughout t h i s s t r u c t u r e . IV P r e l i m i n a r y Observations of N i g r a l and Entopeduncular E f f e r e n t s I t was the i n t e n t i o n of the present set of experiments to c h a r a c t e r i z e b i o c h e m i c a l l y the e f f e r e n t s of the EP to the habenula and the e f f e r e n t s of both the EP and SN to the thalamus. This was undertaken s i n c e these pathways have r e c e n t l y been unequivocally demonstrated (Nauta, 1974; Kim et a l , 1976; 137 Herkenham and Nauta, 1977; Cart e r and F i b i g e r , 1978; Carpenter et. a l , 1976; C l a v i e r et a l , 1976; F a u l l and Mehler, 1978; R i n v i k , 1975).and s i n c e i t was i n keeping w i t h the present scheme of the i d e n t i f i c a t i o n of neurotransmitters i n the connections of the b a s a l g a n g l i a . These i n v e s t i g a t i o n s , however, are i n -complete and only b r i e f l y described here i n order to a i d p o s s i b l e f u t u r e work along these l i n e s . The f a c t that l e s i o n s of the EP r e s u l t e d i n a re d u c t i o n i n the habenula of GAD but not CAT i n d i c a t e s the existence of GABA-containing f i b e r s i n the EP-habenular pathway. These r e s u l t s are i n p a r t i a l agreement w i t h those of G o t t e s f e l d et a l (1977). These authors observed that 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 medularis, GP or GP and EP s i g n i f i c a n t l y decreased GAD i n the l a t e -r a l habenula. They suggested that some GABA-containing neurons i n the GP pro-j e c t to the l a t e r a l habenula, w i t h a f u r t h e r c o n t r i b u t i o n from the EP. How-ever, that damage to the EP and not the GP i s the cause of reduced habenular GAD i s suggested by 1) the l a c k of a p r o j e c t i o n to the habenula from the GP (Carter and F i b i g e r , 1978) and 2) the observation that the present EP l e s i o n s decrease GAD a c t i v i t y i n the habenula as much as the combined EP and GP l e -sions employed by G o t t e s f e l d et a l (1977) . The r e s u l t s obtained by the l a t t e r workers may have been due to damage of EP-habenular f i b e r s passing i n the v i -c i n i t y of the GP. These workers have more r e c e n t l y demonstrated a red u c t i o n of GABA uptake i n the l a t e r a l habenula a f t e r l e s i o n s of the s t r i a m e d u l l a r i s but make no mention of the o r i g i n of the neurons which have axons t r a v e l l i n g i n t h i s f i b e r bundle to the habenula ( G o t t e s f e l d and Jacobowitz, 1978). The experiments i n v o l v i n g the EP and SN e f f e r e n t p r o j e c t i o n s to the tha-lamus met w i t h l i t t l e success. Among the reasons f o r t h i s are the geometri-c a l l y compounding d i f f i c u l t i e s of the production of s u c c e s s f u l l e s i o n s , the d i s s e c t i o n of appropriate n u c l e i , and the i n t e r p r e t a t i o n of negative data. One f a i r l y c o n s i s t e n t observation which should be noted i s that a f t e r SN l e s i o n s there occurred a r e d u c t i o n i n CAT i n the v e n t r a l - l a t e r a l aspect of the 138 v e n t r a l - l a t e r a l , v e n t r a l - a n t e r i o r thalamic complex (the p r o j e c t i o n area of n i g r o - t h a l a m i c f i b e r s in' the r a t (Carter and F i b i g e r , 1978). The s c a r c i t y of knowledge of the neurotransmitters i n v o l v e d i n the p a l l i -d a l and n i g r a l e f f e r e n t s to the thalamus warrants a major t h r u s t of i n v e s t i g a -t i o n i n t h i s d i r e c t i o n . V The L o c a l i z a t i o n of Enzymes i n the S u b s t a n t i a N i g r a I t was the object of the present experiments to use k a i n i c a c i d as a r e -search t o o l r a t h e r than to t e s t i t s s e l e c t i v i t y of a c t i o n . However, some com-ments on i t s s e l e c t i v i t y are warranted as there are s t i l l doubts concerning the extent to which c e r t a i n neuronal elements s u f f e r the l e t h a l e f f e c t s of k a i n i c a c i d . The observed d e s t r u c t i o n of neurons i n the SN f o l l o w i n g the pre-sent i n t r a n i g 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 s i n accord w i t h e a r l i e r work which showed t h i s compound to be a neurotoxic agent f o r neuronal p e r i k a r y a and dendrites (Olney et a l , 1974; Coyle and Schwarcz, 1976; Schwarcz and Coyle, 1977a, 1977b; Schwarcz et a l , 1977; McGeer and McGeer, 1977). To date, h i s t o -chemical and biochemical a n a l y s i s of neuronal t i s s u e which has been exposed to k a i n i c a c i d suggests that minimal damage to t erminals and axons of passage occurs. K a i n i c a c i d i n j e c t i o n s i n t o the s t r i a t u m have been found to a f f e c t only those neurochemical parameters which are thought to be l o c a t e d i n neuro-n a l elements having c e l l bodies w i t h i n t h i s nucleus. The f u n c t i o n a l i n t e g r i t y of t e r m i n a l s at the s i t e of a k a i n i c a c i d i n j e c t i o n has not yet been f u l l y as-sessed, and i t i s p o s s i b l e that removal of the t a r g e t neurons of an axon t e r m i -n a l w i l l produce f u n c t i o n a l changes i n that t e r m i n a l , as i n the case of the decreased K m of TH f o r i t s c o f a c t o r observed i n dopaminergic terminals i n the s t r i a t u m f o l l o w i n g k a i n i c a c i d l e s i o n s of t h i s nucleus (Schwarcz and Coyle, 1977a). With regard to the degree of neuron l o s s i n the SN a f t e r k a i n i c a c i d i n -j e c t i o n s i n t o t h i s s t r u c t u r e , both h i s t o l o g i c a l a n a l y s i s and TH a c t i v i t y mea-139 surements i n d i c a t e d that t h i s was v i r t u a l l y complete. I t i s noteworthy that the present decrease of 94% of TH a c t i v i t y i n the s t r i a t u m was s u b s t a n t i a l l y greater than that obtained i n previous i n v e s t i g a t i o n s (McGeer and McGeer, 1976; Schwarcz and Coyle, 1977b). This may be due to d i f f e r e n c e s i n the exact l o c a t i o n of the k a i n i c a c i d i n j e c t i o n . In p r e l i m i n a r y i n v e s t i g a t i o n s w h i l e conducting simultaneous h i s t o l o g i c a l and biochemical assessment of v a r i o u s s t e r e o t a x i c coordinates, l a r g e v a r i a t i o n s were found i n the extent of the k a i n i c a c i d l e s i o n . The most extensive decreases i n s t r i a t a l TH were ob-served after, i n j e c t i o n s d i r e c t l y i n t o the SNC. These were the coordinates u t i l i z e d i n the present experiments. Keeping the above c o n s i d e r a t i o n s i n mind, some conclusions can be drawn as to the c e l l u l a r l o c a l i z a t i o n of the n e u r o t r a n s m i t t e r - r e l a t e d enzymes i n v e s -t i g a t e d here. From the present experiment i n v o l v i n g hemitransections between the SN and s t r i a t u m , i t appears that 84% of n i g r a l GAD o r i g i n a t e s i n the s t r i a -tum. I f the remaining 16% i s contained i n n i g r a l interneurons, then the 40 to 50% r e d u c t i o n i n GAD seen i n the SN a f t e r i n t r a n i g r a l k a i n i c a c i d i n j e c t i o n s suggests a maximum damage to GAD-containing terminals i n the SN of 25 to 35%. A l t e r n a t i v e l y , long term compensatory changes i n a c t i v i t y due to removal of p o s t s y n a p t i c elements may be a c o n t r i b u t o r y f a c t o r i n the decrease i n n i g r a l GAD. This i s supported by the observations that at 2-3 days (McGeer and McGeer, 1976) and at 5 days (Schwarcz and Coyle, 1977b) a f t e r i n t r a n i g r a l k a i n i c a c i d i n j e c t i o n s there was no change i n n i g r a l GAD, a l b e i t these s t u d i e s showed a smaller decrease i n TH a c t i v i t y i n the SN and s t r i a t u m than that ob-served here. A s t i l l more s p e c u l a t i v e a l t e r n a t i v e f o r the l a r g e k a i n i c a c i d -induced reductions i n GAD i s as f o l l o w s . The hemitransections between the SN and s t r i a t u m and the k a i n i c a c i d n i g r a l l e s i o n s were both p r e c a r i o u s l y c l o s e to the SUT which i s known to p r o j e c t to the SN. I f t h i s p r o j e c t i o n i s GAD-c o n t a i n i n g and both l e s i o n s d i d encroach on the SUT, then the s t r i a t a l c o n t r i -b u t i o n to n i g r a l GAD would be lower than suspected and the l a r g e r k a i n i c a c i d -140 induced n i g r a l GAD reductions would be p a r t l y explained. This p o s s i b i l i t y i s c u r r e n t l y under i n v e s t i g a t i o n . I n c i d e n t a l l y , i f t h i s s p e c u l a t i o n were to be c o r r e c t , t h i s would a l t e r the previous d i s c u s s i o n s of the n i g r a l GAD-contain-i n g a f f e r e n t s from the s t r i a t u m only q u a n t i t a t i v e l y and not q u a l i t a t i v e l y . The CAT a c t i v i t y i n the SN was unaffected by k a i n i c a c i d . This suggests that CAT i s not contained i n neuronal c e l l bodies which send e f f e r e n t p r o j e c -t i o n s from the SN or i n interneurons of the SN s i n c e i n e i t h e r case k a i n i c a c i d would have reduced t h i s a c t i v i t y . Rather, i t appears that CAT i s con-t a i n e d i n t e rminals which have t h e i r o r i g i n o u t side the SN. At present the sources of CAT-containing a f f e r e n t s to the SN are not known. 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 the SN caused a r e d u c t i o n i n AChE a c t i v i t y of 64% i n t h i s nucleus. This suggests that a l i t t l e over h a l f the AChE a c t i -v i t y i n the SN i s l o c a t e d i n neurons whose c e l l bodies l i e w i t h i n the SN. D e s t r u c t i o n of the dopaminergic neurons of the SN by i n j e c t i o n s of 6-hydroxy-dopamine i n t o the NSP has been shown to reduce AChE a c t i v i t y i n the n i g r a by 40%: (Lehmann and F i b i g e r , 1978). Taken together, these data suggest that about 40% of the AChE i n the SN i s present i n dopaminergic neurons, about 20% i n other types of neurons o r i g i n a t i n g i n the SN, and about 40% i n t e rminals a f f e r e n t to the SN which were spared by k a i n i c a c i d . F i n a l l y , a note of c a u t i o n regarding the i n t r a c e r e b r a l i n j e c t i o n s of k a i n i c a c i d . The r e s u l t s of the present study i n d i c a t e that t i s s u e shrinkage may be an important f a c t o r at the i n j e c t i o n locus as the SN showed v i s i b l e shrinkage f o l l o w i n g i n t r a n i g r a l i n j e c t i o n s of k a i n i c a c i d . This could a l t e r the s p e c i f i c a c t i v i t y of enzymes l o c a t e d on terminals not a f f e c t e d by t h i s com-pound . VI The L o c a l i z a t i o n of Dopamine A c t i v a t e d Receptors and Adenylate Cyclase a) Dopamine-sensitive adenylate c y c l a s e i n the s u b s t a n t i a n i g r a The absence of DAC on DA-containing n i g r a l neurons (Kebabian and Saavedra, 141 1976; Premont et a l , 1976), the r e s e r v a t i o n s t h i s caused regarding the e x i s -tence of DA autoreceptors together w i t h the novel suggestions of the presence of DAC on n i g r a l axon terminals (Gale et a l , 1977a; Spano et a l , 1977; P h i l -l i p s o n et a l , 1977), and the dendro-axonic DA t r a n s m i s s i o n t h i s i m p l i e d war-ranted f u r t h e r i n v e s t i g a t i o n s of the c e l l u l a r l o c a l i z a t i o n of DAC i n the SN. For example, a decrease i n n i g r a l DAC r e s u l t i n g from retrograde or t r a n s -neuronal changes i n n i g r a l neurons a f t e r l e s i o n s a n t e r i o r to the SN could not be r u l e d out. K a i n i c a c i d l e s i o n s of the SN circumvent p o s s i b l e i n t e r p r e t a -t i o n a l problems caused by these processes. The e f f e c t s of these l e s i o n s on v a r i o u s enzyme systems i n the SN was de-s c r i b e d i n the previous s e c t i o n . The present data obtained on DAC i n k a i n i c a c i d l e s i o n e d SN are c o n s i s t e n t w i t h previous f i n d i n g s that t h i s enzyme a c t i -v i t y i s not l o c a l i z e d on dopaminergic c e l l bodies (Kebabian and Saavedra, 1976; Premont et a l , 1976), but r a t h e r on t e r m i n a l a f f e r e n t s to the SN (Gale et a l , 1977a; Spano et a l , 1977). With the n e a r l y complete l o s s of c e l l bodies i n the SN obtained i n the present study i t can be f u r t h e r concluded t h a t no neu-r o n a l p e r i k a r y a or dendrites i n the SN possess a dopamine-sensitive adenylate c y c l a s e . DA was able to s t i m u l a t e adenylate cyclase i n the l e s i o n e d SN to the same extent as i n c o n t r o l SN. I n f a c t , when expressed on the per p r o t e i n b a s i s , the s t i m u l a t i o n of adenylate c y c l a s e by dopamine was s i g n i f i c a n t l y greater i n the l e s i o n e d SN compared to the c o n t r a l a t e r a l c o n t r o l . Although i t d i d not reach s i g n i f i c a n c e i n the present study, t h i s trend toward greater dopamine s t i m u l a t i o n i n the l e s i o n e d s i d e i s a l s o evident i f the data are pre-sented on a per SN b a s i s , i n d i c a t i n g that t i s s u e shrinkage may not f u l l y ex-p l a i n t h i s i n c r e a s e . This may i n d i c a t e a s u p e r s e n s i t i v e response of adenylate c y l c a s e to dopamine tr a n s m i s s i o n as has p r e v i o u s l y been observed i n the s t r i a -tum (Mishra et a l , 1976; Krueger et a l , 1976). I t i s u n l i k e l y that DAC i s present on g l i a l elements w i t h i n the n i g r a s i n c e the DAC i n the s t r i a t u m i s d r a m a t i c a l l y reduced (McGeer et a l , 1976; Schwarcz and Coyle, 1977a) a f t e r 142 k a i n i c a c i d s t r i a t a l l e s i o n s d e s p i t e massive g l i o s i s i n t h i s s t r u c t u r e . Thus, the observation that DAC d i d not decrease f o l l o w i n g d e s t r u c t i o n of n i g r a l p e r i k a r y a i s c o n s i s t e n t w i t h previous suggestions that d e n d r i t i c a l l y - l o c a t e d dopamine may c o n t r i b u t e to the r e g u l a t i o n of c y c l i c AMP synth e s i s i n a f f e r e n t terminals i n the SN (Reubi et a l , 1977). The i m p l i c a t i o n s these f i n d i n g s have on DA autoreceptors i n the SN are discussed i n s e c t i o n VI-c. of the D i s c u s s i o n , b) Pre- and pos t - s y n a p t i c dopamine receptors i n the s u b s t a n t i a n i g r a and s t r i a t u m The two types of l e s i o n s employed i n the present study to determine the l o c a l i z a t i o n of neurotransmitter receptors were 6-OHDA l e s i o n s of the NSP and k a i n i c a c i d l e s i o n s of the s t r i a t u m . The e f f e c t of these l e s i o n s on various enzyme systems are as f o l l o w s . In the s t r i a t u m , 6-OHDA l e s i o n s of the NSP caused a greater than 90% l o s s of TH, no change i n CAT a c t i v i t y and a s i g n i f i -cant i n c r e a s e i n s t r i a t a l GAD. The inc r e a s e i n s t r i a t a l GAD a f t e r these l e -sions confirms previous r e p r o t s of t h i s (Vincent et a l , 1978; Saavedra et a l , 1978). The a c t i v i t i e s of GAD and CAT i n the SN and s t r i a t u m f o l l o w i n g s t r i a -t a l k a i n i c a c i d i n j e c t i o n s are f o r the most part s i m i l a r to previous reports (Schwarcz and Coyle, 1977a, 1977b; McGeer and McGeer, 1976; Coyle and Schwarcz, 1976). The s i g n i f i c a n t 18% increase i n CAT i n the s t r i a t u m c o n t r a l a t e r a l to the k a i n i c a c i d l e s i o n e d s t r i a t u m compared to s a l i n e i n j e c t e d c o n t r o l s i n d i c a -ted that compensatory mechanisms may be ope r a t i v e i n t h i s s t r u c t u r e . While these a l t e r a t i o n s i n CAT a c t i v i t y may be r e l a t e d to the changes i n muscarinic c h o l i n e r g i c receptors which have been demonstrated i n the s t r i a t u m c o n t r a l a t e -r a l to 6-OHDA l e s i o n s of the SN (Kato et a l , 1978), the mechanisms underlying these changes remain undefined. With regard to TH, there have been previous observations of s i g n i f i c a n t l y increased a c t i v i t i e s of t h i s enzyme 2. days a f t e r s t r i a t a l k a i n i c a c i d l e s i o n s (Coyle and Schwarcz, 1976; McGeer and McGeer, 1976) and normal a c t i v i t i e s a f t e r 21 days (Schwarcz and Coyle, 1977a). In the present study, however, a f t e r a s u r v i v a l time of 30 days, there was a 19% de-143 crease i n s t r i a t a l TH a c t i v i t y . This observation i s c o n s i s t e n t w i t h the de-crease i n DA l e v e l s ( F r i e d l e et a l , 1978) and r e d u c t i o n of DA h i s t o f l u o r e -scence (Meiback et a l , 1978) which occurs at about 2 weeks a f t e r these l e s i o n s . A f t e r k a i n i c a c i d l e s i o n s of the s t r i a t u m i t has been shown that TH i s a c t i v a -ted having decreased IL^ f o r i t s c o f a c t o r (Schwarcz and Coyle, 1977a). The present TH assays were conducted at s a t u r a t i n g c o f a c t o r and s u b s t r a t e concen-t r a t i o n s . The concentrations of c o f a c t o r and s u b s t r a t e employed i n the TH assays of the above r e p o r t s could not be a s c e r t a i n e d . In any case, the l o s s i n DA-containing s t r u c t u r e s i n the k a i n i c a c i d l e s i o n e d s t r i a t u m represented by TH i s q u i t e modest r e l a t i v e to the l o s s of neurons represented by GAD and CAT. I n summary, the 6-OHDA l e s i o n s r e s u l t e d i n a profound l o s s of dopaminer-g i c elements i n the SN and s t r i a t u m w h i l e the k a i n i c a c i d s t r i a t a l l e s i o n s e l i m i n a t e d p o s t s y n a p t i c neurons i n the s t r i a t u m and some of the terminals of these p r o j e c t i o n s i n the SN. Apomorphine has been w e l l c h a r a c t e r i z e d as a DA agonist and h a l o p e r i d o l and s p i r o p e r i d o l as DA a n t a g o n i s t s . The v a l i d i t y of employing these compounds i n the assay of the DA receptor has been p r e v i o u s l y demonstrated (Seeman et a l , 1976; Leysen et a l , 1978; Thai et a l , 1978). The observation that s e l e c -t i v e and extensive retrograde l o s s of DA neurons i n the SN reduced ^-apomor-phine b i n d i n g i n t h i s s t r u c t u r e by 76% i s a d i r e c t demonstration of the e x i s -tence of DA receptors on these p e r i k a r y a . The f a c t that n i g r a l TH a c t i v i t y was reduced by greater than 90% whereas n i g r a l 3H-apomorphine b i n d i n g was r e -duced by only 76% suggests that there are a l s o DA receptors on other than dopaminergic neurons. The r e d u c t i o n by 56% of 3H-apomorphine b i n d i n g a f t e r v i r t u a l l y complete anterograde degeneration of dopaminergic elements i n the s t r i a t u m provides d i r e c t support f o r the presence of DA receptors on DA-con-t a i n i n g t e r m i n a l s . The Scat chard a n a l y s i s of s t r i a t a l 3H-apom'orphine b i n d i n g f u r t h e r i n d i c a t e s that both the a f f i n i t y and maximal number of receptors i s reduced a f t e r NSP l e s i o n s . The r e d u c t i o n i n the a f f i n i t y of 3H-apomorphine 144 f o r the DA receptor i s c o n s i s t e n t w i t h b e h a v i o r a l evidence f o r a presynaptic a c t i o n of low doses of apomorphine ( C a r l s s o n , 1975; Strombom, 1975). For example, i t i s apparent from the present data that both pre- and p o s t - s y n a p t i c b i n d i n g of apomorphine occurs i n the s t r i a t u m . I t i s a l s o probable that s t r i a t a l apomorphine receptor a f f i n i t y i s a composite of b i n d i n g to the two types of receptor. Thus, e l i m i n a t i o n of a higher a f f i n i t y p resynaptic recep-t o r would r e s u l t s i n a greater r e l a t i v e r e p r e s e n t a t i o n of the lower a f f i n i t y p o s t s y n a p t i c receptor and hence a higher Kd f o r apomorphine a f t e r the NSP l e -sions . In marked contrast w i t h the r e d u c t i o n i n 3H-apomorphine b i n d i n g i n the s t r i a t u m , NSP l e s i o n s caused a s i g n i f i c a n t i n c r e a s e i n 3 H - h a l o p e r i d o l and 3H-s p i r o p e r i d o l b i n d i n g . This suggests that the binding of these l i g a n d s occurs p r i m a r i l y to p o s t s y n a p t i c elements i n the s t r i a t u m . This observation may have some relevance to the r e p o r t s of enhanced b e h a v i o r a l e f f e c t s of d i r e c t l y - a c t -i n g DA agonists such as apomorphine a f t e r l e s i o n s of the NSP or a f t e r chronic treatment w i t h n e u r o l e p t i c s (Ungerstedt, 1971b; P r i c e and F i b i g e r , 1974; Christensen et a l , 1976). I t i s known (Creese et a l , 1977; Burt et a l , 1977; M u l l e r and Seeman, 1977) that these treatments produce an increase i n the num-ber of s t r i a t a l DA receptors assayed w i t h 3 H - h a l o p e r i d o l . The present r e s u l t s c o n f i r m the increase i n s t r i a t a l 3 H - h a l o p e r i d o l and 3 H - s p i r o p e r i d o l b i n d i n g a f t e r NSP l e s i o n s , and support the p o s s i b i l i t y that :this increase may be r e l a -t e d to the enhanced b e h a v i o r a l e f f e c t s of DA agonists a f t e r NSP l e s i o n s . The present observations regarding 3 H - s p i r o p e r i d o l b i n d i n g i n the SN pose some i n t e r p r e t a t i o n a l d i f f i c u l t i e s . At the higher c o n c e n t r a t i o n of ^ - s p i r o -p e r i d o l examined, NSP l e s i o n s had no e f f e c t on the b i n d i n g of t h i s l i g a n d . However, at the lower c o n c e n t r a t i o n 3 H - s p i r o p e r i d o l b i n d i n g was s i g n i f i c a n t l y reduced by 42%. The simplest e x p l a n a t i o n of these r e s u l t s i s that ^ - s p i r o -p e r i d o l binds p r e f e r e n t i a l l y to DA receptors l o c a t e d on non-dopaminergic e l e -ments i n the SN and that the NSP l e s i o n s caused an a f f i n i t y change i n these 145 receptors. Rather than invoke some of the other p o s s i b l e explanations at t h i s time, i t would be more s u i t a b l e to pursue t h i s f i r s t p o s s i b i l i t y by conducting Scatchard a n a l y s i s of 3 H - s p i r o p e r I d o l b i n d i n g i n the SN a f t e r NSP l e s i o n s . I n view of these r e s u l t s i n the SN and the f a c t that the b i n d i n g of DA antago-n i s t s increased i n the s t r i a t u m a f t e r NSP l e s i o n s , the present study provides no i n f o r m a t i o n as to the extent DA antagonists bind to dopaminergic neurons i n e i t h e r of these s t r u c t u r e s . However, that such receptors may be demonstrated w i t h more rigorous i n v e s t i g a t i o n s i s suggested by the observation that k a i n i c a c i d l e s i o n s of the s t r i a t u m do not d i m i n i s h the a b i l i t y of h a l o p e r i d o l to i n f l u e n c e DA metabolism i n the s t r i a t u m (Di Chiara et a l , 1977). A f u r t h e r p o t e n t i a l s i t e of 3 H - s p i r o p e r i d o l b i n d i n g i s to DA receptors thought to be l o c a t e d on terminals of s t r i a t a l a f f e r e n t s to the SN. However, i n the present study, k a i n i c a c i d s t r i a t a l l e s i o n s had no e f f e c t on t h i s b i n d i n g . This r e s u l t i s i n c o n s i s t e n t w i t h the observation that DA antagonists b l o c k the DA evoked r e l e a s e of GABA from n i g r a l s l i c e s (Reubi et a l , 1977). Thus, i t i s p o s s i b l e that s t r i a t a l k a i n i c a c i d l e s i o n s induced a f f i n i t y changes of DA-receptors on n i g r a l t e r m i n a l s . A l t e r n a t i v e l y , the k a i n i c a c i d l e s i o n s may not have destroyed those neurons i n the s t r i a t u m which send DA-bearing receptors to the SN. I t i s noteworthy i n t h i s regard that these l e -sions tend to be l o c a t e d a n t e r i o r l y i n the s t r i a t u m and reduce n i g r a l GAD by 42%, whereas i t i s l e s i o n s i n v o l v i n g the p o s t e r i o r s t r i a t u m that decrease DAC a c t i v i t y i n the SN (Spano et a l , 1977). Further s t u d i e s are r e q u i r e d to de-termine whether n i g r a l a f f e r e n t s o r i g i n a t i n g from the p o s t e r i o r regions of the s t r i a t u m c o n t a i n 3 H - s p i r o p e r i d o l b i n d i n g s i t e s . I t i s i n t e r e s t i n g that the r e s u l t s of the attempts to l o c a l i z e the source w i t h i n the s t r i a t u m of n i g r a l a f f e r e n t s c o n t a i n i n g DA-receptors and DAC p a r a l l e l remarkably the s t u d i e s of the o r i g i n of n i g r a l GAD. I t i s tempting to speculate that the s t r i a t a l -n i g r a l neurons whose terminals i n the SN c o n t a i n GAD, DAC and DA receptors are one and the same. 146 c) Dopamine tr a n s m i s s i o n i n the . s u b s t a n t i a n i g r a and.striatum: s y n t h e s i s and s p e c u l a t i o n The present observations of the d i f f e r e n t i a l e f f e c t s of NSP l e s i o n s on the b i n d i n g to s t r i a t a l membranes of 3H-apomorphine, on the one hand, and 3H-h a l o p e r i d o l and 3 H - s p i r o p e r i d o l , on the other, have c e r t a i n i m p l i c a t i o n s r e -garding the nature of DA r e c e p t o r s . Creese et a l (1975) have shown that i n s t u d i e s w i t h receptors that have bound DA a n t a g o n i s t s , DA agonists compete l e s s e f f e c t i v e l y f o r b i n d i n g than DA a n t a g o n i s t s . Conversely, DA antagonists compete l e s s e f f e c t i v e l y than DA agonists f o r receptors that have bound DA a g o n i s t s . Rather than p o s t u l a t e the e x i s t e n c e of two types of DA receptor, Creese et a l (1975) suggested that a s i n g l e receptor could e x i s t i n two forms, an agonist and an antagonist s t a t e . The present experiments cast doubt on t h i s i n t e r p r e t a t i o n , as 3H-apomorphine and 3 H - n e u r o l e p t i c b i n d i n g i n the s t r i a -tum would have been expected to show the same r e l a t i v e changes a f t e r the NSP l e s i o n s . However, t h i s was not the case; i n f a c t , s t r i a t a l b i n d i n g of the two l i g a n d s changed i n opposite d i r e c t i o n s . I t remains p o s s i b l e , of course, that the l e s i o n s locked the receptors i n t o an a n t a g o n i s t i c s t a t e . However, t h i s a l t e r n a t i v e would be d i f f i c u l t to r e c o n c i l e w i t h the known p o t e n t i a t i o n of the b e h a v i o r a l e f f e c t s of apomorphine a f t e r NSP l e s i o n s (Ungerstedt, 1971b; P r i c e and F i b i g e r , 1974). Another p o s s i b i l i t y i s that there are two types of DA r e c e p t o r , both l o c a t e d p o s t s y n a p t i c a l l y . In t h i s case, NSP l e s i o n s might change the r e l a t i v e numbers of each type of receptor. Again, however, an i n -crease i n 3 H - h a l o p e r i d o l and 3 H - s p i r o p e r i d o l receptors accompanied by a de-crease i n 3H-apomorphine r e c e p t o r s , together w i t h the lower a f f i n i t y of apo-morphine f o r the antagonist r e c e p t o r , would be d i f f i c u l t t o . r e c o n c i l e w i t h the above b e h a v i o r a l observations. An a l t e r n a t i v e i n t e r p r e t a t i o n of the f i n d i n g s of Creese et a l (1975) can be proposed i n l i g h t of the present data. I t appears that there may be two types of DA r e c e p t o r s ; those which i n t e r a c t p r e f e r e n t i a l l y w i t h apomorphine 147 and those which i n t e r a c t p r e f e r e n t i a l l y w i t h n e u r o l e p t i c s . I t may als o be t r u e that each of these l i g a n d s can bind to the unfavored form of the recep t o r . In the SN and s t r i a t u m apomorphine-type receptors e x i s t on dopaminergic neu-rons and t e r m i n a l s , r e s p e c t i v e l y . In a d d i t i o n , these receptors may a l s o be present on post s y n a p t i c neurons i n the s t r i a t u m and axon terminals i n the SN. Since n e i t h e r dopaminergic neurons or terminals possess DAC (Kebabian and Saavedra, 1976; Premont et a l , 1976), the apomorphine-type DA receptors on these s t r u c t u r e s appear to f u n c t i o n independently of t h i s enzyme and thus, cAMP. In c o n t r a s t , n e u r o l e p t i c r e c e p t o r s , at l e a s t i n the s t r i a t u m and p o s s i -b l y i n the SN, are l o c a t e d on nondopaminergic elements, as i s true of DAC i n these b r a i n regions (McGeer et a l , 1976; Schwarcz and Coyle, 1977a; the pre-sent st u d y ) . Thus, n e u r o l e p t i c - t y p e DA receptors i n the SN and s t r i a t u m may be coupled to DAC. These conclusions do not exclude the p o s s i b i l i t y that there may be adenylate cyclases on dopaminergic s t r u c t u r e s that f u n c t i o n i n -dependently of dopamine and that some DA receptors on nondopaminergic elements may f u n c t i o n independently of adenylate c y c l a s e . I t i s probably a safe estimate that the f u n c t i o n ( s ) served by the SN-VTA DA system has received generous c o n s i d e r a t i o n i n armchair s p e c u l a t i o n s . Unfor-t u n a t e l y , a u n i f i e d theory of the f u n c t i o n of these systems i n the b r a i n has not emerged. On the b a s i s of a v a i l a b l e i n f o r m a t i o n any such t h e o r i e s would be j u s t short of sheer f a b r i c a t i o n . S u f f i c e i t to say that an hypothesis of DA f u n c t i o n would have as a major c o n s i d e r a t i o n the s t r i n g e n t and m u l t i l e v e l con-t r o l to which DA neurons are su b j e c t . CONCLUSIONS The theme of the present t h e s i s was the study of the anatomical and b i o -chemical i n t e r a c t i o n s w i t h i n and between va r i o u s n u c l e i of the b a s a l g a n g l i a . The g o a l , to determine, at va r i o u s l e v e l s , some of the c i r c u i t r y of t h i s p a r t of the b r a i n , was met w i t h v a r y i n g degrees of success. The r e s u l t s of some of 148 of the experiments conducted were f a i r l y c o n c l u s i v e w h i l e others leave open s e v e r a l i n t e r p r e t a t i o n s and t h e r e f o r e r e q u i r e f u r t h e r i n v e s t i g a t i o n s . I t should be added that the r e s u l t s of numerous experiments, some of which were r a t h e r novel but a l l of which were miserable f a i l u r e s , were not i n c l u d e d . In s h o r t , the b a s a l g a n g l i a has been a h i g h l y complex and c h a l l e n g i n g proving ground. Since i t s i n c e p t i o n about a decade ago, biochemical neuroanatomy has served w e l l i n adding to the understanding of the neurotransmitters i n and con-nections of the b a s a l g a n g l i a . I t i s c l e a r , however, that i n order to c o n t i -nue to be powerful i n p r y i n g loose i n f o r m a t i o n about the b r a i n , the f a m i l y of techniques under t h i s heading must undergo major refinements, s i n c e , r e l a t i v e -l y speaking, only a glimpse of the t o t a l p i c t u r e has been gained to date. The present i n v e s t i g a t i o n s , then, together w i t h s i m i l a r ones conducted s i m u l t a -neously by o t h e r s , marks the end of an era and the l i m i t to which the current s t a t e of the methodology i n v o l v e d can be taken to a f f o r d answers concerning the biochemical c o n n e c t i v i t y of the b a s a l g a n g l i a . I t w i l l be i n t e r e s t i n g to observe what new innovations n e c e s s i t y w i l l demand. I t i s somewhat d i s s a t i s f y i n g t h a t , a f t e r more than four B i c pens, no con-c l u s i o n s were and can be drawn regarding f u n c t i o n s of the b a s a l g a n g l i a n u c l e i and the systems i n v e s t i g a t e d here; at l e a s t none that t h i s author would want to be c i t e d f o r , which may or may not be a reasonable c r i t e r i o n f o r d e c i d i n g such matters. In any case, t h i s outcome suggests a d i r e c t i o n f o r f u t u r e r e -search. On the one hand, no amount of knowledge regarding b r a i n c i r c u i t r y at the biochemical or anatomical l e v e l i s too much. On the other, the question becomes how much inf o r m a t i o n and what degree of d e t a i l i s needed before pre-c i s e computer-like f u n c t i o n s can be a s c r i b e d to s p e c i f i c c e l l groups of the brain? The n i g r o - s t r i a t a l DA system may be used as an example here. 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