UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

GABA, substance P and the efferents of the striatum 1980

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
UBC_1980_A1 V56.pdf
UBC_1980_A1 V56.pdf [ 10.82MB ]
UBC_1980_A1 V56.pdf
Metadata
JSON: 1.0076987.json
JSON-LD: 1.0076987+ld.json
RDF/XML (Pretty): 1.0076987.xml
RDF/JSON: 1.0076987+rdf.json
Turtle: 1.0076987+rdf-turtle.txt
N-Triples: 1.0076987+rdf-ntriples.txt
Citation
1.0076987.ris

Full Text

GABA, SUBSTANCE P AND THE EFFERENTS OF THE STRIATUM by STEVEN ROBERT VINCENT , B.Sc. Carleton U n i v e r s i t y , 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY / THE FACULTY OF GRADUATE STUDIES ( I n t e r d i s c i p l i n a r y Studies) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1980 (c^ Steven Robert Vincent, 1980 In presenting this thesis in partial fulf i lment of the requirements for an advanced degree at the University of Bri t ish Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of Bri t ish Columbia 2075 Wesbropk Place Vancouver, Canada V6T 1W5 D E - 6 B P 75-51 1 E i i ABSTRACT The efferent pathways from the striatum to the other n u c l e i of the basal ganglia were examined biochemically and histochemically. Acetylcho- l i n e , GABA, enkephalin and substance P have a l l been suggested to occur i n s t r i a t a l neurons, and markers for these possible transmitters were therefore measured i n various n u c l e i of the basal ganglia following k n i f e cuts of the s t r i a t a l efferent f i b r e s . These studies confirmed the existence of GABA projections from the head of the striatum to the globus p a l l i d u s (GP), entopeduncular nucleus (EP) and substantia n i g r a (SN). In addi t i o n the presence of substance P i n the s t r i a t a l p r o j e c t i o n to the EP was demonst- rated and the substance P projection to the SN was confirmed using a radio- immunoassay. The f i r s t evidence suggesting the presence of both substance P and methionine-enkephalin i n the s t r i a t o p a l l i d a l f i b r e s was also obtained. Also the important observation that methionine-enkephalin i s not present i n the projections from the head of the striatum to the EP and SN was noted. In order to v i s u a l i z e substance P f i b r e s i n the br a i n a new method for immunohistochemical studies of the nervous system was developed based on the b i o t i n - a v i d i n system. Using t h i s powerful technique substance P f i b r e s and terminals were observed i n the striatum, GP, EP and SN, as we l l as i n various other areas including the amygdaloid complex, the habenula and the interpeduncular nucleus. This represents the f i r s t report of substance P f i b r e s i n the basal ganglia demonstrated using an immunoperoxidase procedure. The enzyme GABA-transaminase (GABA-T) was examined as a p o t e n t i a l marker for the GABA neurons of the basal ganglia. Using s e l e c t i v e l e s i o n s and a biochemical assay procedure the enzyme was found to be present i n the neurons of the striatum and i n the s t r i a t o n i g r a l pathway. GABA-T was apparently not present i n the g l i a l elements of the striatum nor was i t present i n the n i g r o s t r i a t a l dopamine neurons. Histochemical experiments i i i demonstrated GABA-T to be present i n the terminals of the s t r i a t a l and p a l l i d a l efferents which are thought to use GABA as a transmitter. These experiments e s t a b l i s h the usefulness of GABA-T histochemistry as a new method for the analysis of the topography of the GABA systems i n the basal ganglia. The response of the GABA and substance P c e l l s i n the basal ganglia to the s e l e c t i v e removal of the dopamine c e l l s of the SN was examined and compared with the pathological findings observed i n Parkinson's disease. In contrast with the decrease reported i n glutamate decarboxylase a c t i v i t y i n the basal ganglia i n Parkinson's disease, an increase i n the a c t i v i t y of t h i s enzyme was observed i n the animal model. Also, a s i g n i f i c a n t decrease i n n i g r a l and s t r i a t a l substance P l e v e l s occurred following t h i s l e s i o n . The implications of these findings f o r the etiology and pharmacological therapy of Parkinsonism are discussed. F i n a l l y , the n i g r o t e c t a l pathway was examined u l t r a s t r u c t u r a l l y and biochemically since i t represents a major output pathway of the basal ganglia. A s e l e c t i v e decrease was found i n the glutamate decarboxylase a c t i v i t y of the superior c o l l i c u l u s following l e s i o n s of the SN. This observation provides the f i r s t i n d i c a t i o n that the n i g r o t e c t a l p r o j e c t i o n may use GABA as a transmitter. Electron microscopic examination of axon terminals of the n i g r o t e c t a l pathway indicated the axons were probably myelinated and that the terminals form symmetric synapses with the major dendrites of neurons i n the deep layers of the superior c o l l i c u l u s . i v T A B L E OF CONTENTS ABSTRACT i i T A B L E OF CONTENTS i v L I S T OF T A B L E S v i L I S T OF F I G U R E S x i i i L I S T OF A B B R E V I A T I O N S x ACKNOWLEDGEMENTS x i INTRODUCTION 1 STATEMENT OF THE PROBLEMS TO BE EXAMINED 9 E X P E R I M E N T 1 : N e u r o t r a n s m i t t e r s c o n t a i n e d i n t h e e f f e r e n t s o f t h e s t r i a t u m 11 EXPER IMENT 2 : T h e i m m u n o h i s t o c h e m i c a l d e m o n s t r a t i o n o f s u b s t a n c e P i n t h e b a s a l g a n g l i a 27 EXPER IMENT 3 : T h e l o c a l i z a t i o n o f G A B A - t r a n s a m i n a s e i n t h e s t r i a t o n i g r a l s y s t e m 4 0 EXPER IMENT 4 : T h e h i s t o c h e m i c a l l o c a l i z a t i o n o f G A B A - t r a n s a m i n a s e i n t h e b a s a l g a n g l i a 53 EXPER IMENT 5 : B i o c h e m i c a l c h a n g e s f o l l o w i n g 6 - h y d r o x y d o p a m i n e l e s i o n s o f t h e n i g r o s t r i a t a l d o p a m i n e n e u r o n s : a n a n i m a l m o d e l o f P a r k i n s o n i s m ' EXPER IMENT 6 : T h e n i g r o t e c t a l p r o j e c t i o n : a b i o c h e m i c a l a n d u l t r a s t r u c t u r a l s t u d y 76 GENERAL D I S C U S S I O N 85 N i g r o s t r i a t a l r e g u l a t i o n 8 5 GABA r e g u l a t i o n o f t h e n i g r o s t r i a t a l d o p a m i n e s y s t e m 89 S u b s t a n c e P r e g u l a t i o n o f t h e n i g r o s t r i a t a l d o p a m i n e s y s t e m . . . 92 GABA , s u b s t a n c e P a n d t h e o u t p u t o f t h e s t r i a t u m 94 7 66 V Outputs of the substantia nigra 97 A model of the basal ganglia 104 REFERENCES : 107 APPENDIX 133 v i LIST OF TABLES Table 1. Glutamic acid decarboxylase and choline acetyltransferase a c t i v i t i e s i n various areas a f t e r lesions i s o l a t i n g the globus p a l l i d u s from the striatum (GP islands) 16 Table 2. Glutamic acid decarboxylase and choline acetyltransferase a c t i v i t i e s i n various areas a f t e r hemitransections anterior to the globus p a l l i d u s 18 Table 3. Substance P l e v e l s i n various areas a f t e r lesions i s o l a t i n g the globus p a l l i d u s from the striatum (GP islands) 20 Table 4. The l e v e l s of substance P and met-enkephalin i n various areas a f t e r hemitransections anterior to the globus p a l l i d u s 21 Table 5. The a c t i v i t i e s of neurotransmitter-related enzymes i n the striatum and substantia nigra one month a f t e r the i n j e c t i o n of 6-0HDA into the n i g r o s t r i a t a l bundle . . . 44 Table 6. The a c t i v i t i e s of neurotransmitter-related enzymes i n the striatum and substantia nigra two weeks a f t e r the i n j e c t i o n of f i v e or ten nmoles of kai n i c acid into the striatum 45 Table 7. The a c t i v i t i e s of neurotransmitter-related enzymes i n the striatum a f t e r the i n j e c t i o n of 20 nmoles of k a i n i c acid into the striatum of ten-day old rats 50 Table 8. Glutamic acid decarboxylase a c t i v i t y i n various brain areas a f t e r u n i l a t e r a l lesions of the n i g r o s t r i a t a l pathway with 6-OHDA 69 v i i Table 9. Enzyme a c t i v i t i e s and neuropeptide l e v e l s i n the striatum and substantia nigra three months a f t e r the i n j e c t i o n of s a l i n e or 6-OHDA into the l e f t n i g r o - s t r i a t a l pathway 71 Table 10. D i s t r i b u t i o n of s i l v e r grains i n the superior c o l l i c u l u s 24 hr a f t e r the i n j e c t i o n of [ 3H]leucine into the substantia nigra 80 Table 11. Biochemical changes i n the superior c o l l i c u l u s a f t e r l e s i o n s of the substantia nigra 83 V l l l LIST OF FIGURES Figure 1. The knife cuts separating the globus p a l l i d u s from the striatum Figure 2. A comparison of the b i o t i n - a v i d i n method with the peroxi- dase-antiperoxidase procedure for immunohistochemistry . Figure 3. Control section of susbtantia nigra incubated with pre-absorbed anti-substance P sera Figure 4. Substance P immunohistochemistry i n the striatum and i t s pr o j e c t i o n areas Figure 5. Substance P immunohistochemistry i n the l a t e r a l habenula and the amygdaloid complex Figure 6. Substance P immunohistochemistry i n the substantia nigra Figure 7. C o r r e l a t i o n between s t r i a t a l glutamate decarboxylase and GABA-transaminase following the i n j e c t i o n of k a i n i c acid into the striatum Figure 8. C o r r e l a t i o n between n i g r a l glutamate decarboxylase and GABA-transaminase following the i n j e c t i o n of k a i n i c a c i d into the striatum Figure 9. S a g i t a l section through the rat b r a i n stained f o r GABA-transaminase a c t i v i t y Figure 10. E f f e c t of k a i n i c acid on GABA-transaminase s t a i n i n g i n the striatum Figure 11. The e f f e c t of k a i n i c acid l e s i o n s of the striatum on GABA-transaminase s t a i n i n g i n the p r o j e c t i o n areas i x Figure 12. GABA-transaminase histochemistry i n the subthalamic nucleus a f t e r k a i n i c acid lesions of the globus p a l l i d u s 59 Figure 13. GABA-transaminase histochemistry i n the habenula following k a i n i c acid lesions of the entopeduncular nucleus 60 Figure 14. Increased s t r i a t a l glutamate decarboxylase following 6-0HDA lesions of the n i g r o s t r i a t a l dopamine neurons . . 68 Figure 15. Schematic representation of the s i t e and extent of the [ % ] l e u c i n e i n j e c t i o n i n the substantia nigra . . . 79 Figure 16. Labeled terminals i n the superior c o l l i c u l u s following the i n j e c t i o n of [ aH]leucine into the substantia nigra 82 Figure 17. Summary diagram of the outputs of the striatum 86 Figure 18. Hypothetical mechanisms of action for h a l o p e r i d o l and amphetamine 101 X LIST OF ABBREVIATIONS AOAA amino-oxyacetic acid BA b i o t i n - a v i d i n CAT choline acetyltransferase DOPAC dihydroxyphenylacetic acid EOS ethanolamine-o-sulphate EP entopeduncular nucleus GABA -y-aminobutyric acid GABA-T GABA-transaminase GAD glutamate decarboxylase GP globus p a l l i d u s HVA homovanillic acid KA k a i n i c acid leu-enkephalin leucine-enkephalin met-enkephalin methionine-enkephalin 6-OHDA 6-hydroxy dopamine PAP peroxidase anti-peroxidase PBS phosphate buffered s a l i n e SN substantia nigra SNC substantia nigra pars compacta SNR substantia nigra pars r e t i c u l a t a x i ACKNOWLEDGEMENTS I would l i k e to thank my supervisor Dr. Edie McGeer for allowing me a free r e i n to explore any of the crazy ideas that popped in t o my head during the course of t h i s work. The f r i e n d l y i n t e r e s t , advice and encouragement of Dr. Chris F i b i g e r i s also e s p e c i a l l y appreciated. I would l i k e to acknowledge my debt to my two sen sei Drs. T. H a t t o r i and H. Kimura, without whom none of the h i s t o l o g i c a l work would have been possible. F i n a l l y , i t i s a pleasure to remember the many members of the Kinsmen Laboratory and, i n p a r t i c u l a r , B i l l Staines and Jim Nagy, for making t h i s work a pleasure. INTRODUCTION The b a s a l g a n g l i a are a group of s u b c o r t i c a l n u c l e i which i n c l u d e s the s t r i a t u m , globus p a l l i d u s (CP), entopeduncular nucleus (EP), subthalamic nucleus and s u b s t a n t i a n i g r a (SN). Together these s t r u c t u r e s comprise a major p o r t i o n of what has been termed the extrapyramidal motor system and are involved i n the c o n t r o l of posture and locomotion. D y s f u n c t i o n of the basal g a n g l i a r e s u l t i n a v a r i e t y of c l i n i c a l c o n d i t i o n s i n v o l v i n g motor behaviour. Parkinson's disease i s the best understood of these d i s o r d e r s and i s c h a r a c t e r i z e d by a l o s s of the dopamine-containing neurons of the SN and a corresponding r e d u c t i o n i n s t r i a t a l dopamine l e v e l s (Hornykiewicz, 1973). Huntington's disease i s another d i s o r d e r i n v o l v i n g the b a s a l g a n g l i a . I t i s c h a r a c t e r i z e d by n e u r o p a t h o l o g i c a l changes i n the c o r t e x and the s t r i a - tum (Lange et a l . , 1976). Although choreiform movements are the hallmark of t h i s d i s e a s e , dementia i s a l s o a s t r i k i n g f e a t u r e of Huntington's chorea (Garron, 1973). The b a s a l g a n g l i a have a l s o been i m p l i c a t e d i n the e t i o l o g y of s c h i z o - phrenia. Motor disturbances i n c l u d i n g chorea, a k a t h i s i a , and o r a l d y s k i n e s i a s occur f r e q u e n t l y i n p s y c h o t i c s , and t h i s o b s e r v a t i o n l e d M e t t l e r (1955) to propose o r i g i n a l l y that schizophrenia i s a d i s o r d e r of the b a s a l g a n g l i a . With the i n t r o d u c t i o n of the n e u r o l e p t i c drugs f o r the symptomatic treatment of t h i s disease a t t e n t i o n has focused p a r t i c u l a r l y on the r o l e of the n i g r a l dopamine neurons i n schizophrenia. These c l i n i c a l c o n d i t i o n s i l l u s t r a t e that d i s o r d e r s of the b a s a l g a n g l i a may be c h a r a c t e r i z e d not only by impairments of motor behaviour, but a l s o by d i s r u p t i o n s of c o g n i t i v e f u n c t i o n . Thus, study of the b a s a l g a n g l i a may provide i n s i g h t i n t o the neural mechanisms i n v o l v e d i n both of these important processes. In p a r t i c u l a r , a study of the output pathways of the 2 b a s a l g a n g l i a may i n d i c a t e t h e s i t e s a t w h i c h t h i s s y s t e m c a n i n t e r a c t w i t h t h e r e s t o f t h e n e r v o u s s y s t e m t o i n f l u e n c e b e h a v i o u r . T h e s t r i a t u m i s t h e l a r g e s t s t r u c t u r e i n c l u d e d i n t h e b a s a l g a n g l i a a n d i n d e e d , i s t h e l a r g e s t s u b c o r t i c a l c e l l - m a s s i n t h e m a m m a l i a n b r a i n . A s s u c h i t p r o b a b l y r e p r e s e n t s t h e m a i n i n t e g r a t i v e c e n t e r o f t h e b a s a l g a n g l i a . T h e s t r i a t u m r e c e i v e s p r o j e c t i o n s f r o m a l l p a r t s o f t h e n e o c o r t e x , i n c l u d i n g m o t o r a r e a s a s w e l l a s a r e a s i m p l i c a t e d i n p e r c e p t i o n , a s s o c i a t i o n a n d memory ( C a r m e n e t a l . , 1 9 6 3 ; D e V i t o a n d S m i t h , 1 9 6 4 ; G a r c i a - R i l l e t a l . , 1 9 7 9 ; G l e e s , 1 9 4 4 ; G o l d m a n a n d N a u t a , 1 9 7 7 ; J o n e s e t a l . , 1 9 7 7 ; Kemp a n d P o w e l l , 1 9 7 0 ; K u n z l e , 1 9 7 5 ; 1 9 7 7 ; N i i m i e t a l . , 1 9 6 3 ; W e b s t e r , 1 9 6 1 ; 1 9 6 5 ; Y e t e r i a n a n d v a n H o e s e n , 1 9 7 8 ) . T h i s i s t h e m a j o r i n p u t t o t h e s t r i a t u m a n d a c c o u n t s f o r a p p r o x i m a t e l y o n e t h i r d o f t h e a f f e r e n t t e r m i n a l s t o t h i s n u c l e u s (Kemp a n d P o w e l l , 1 9 7 1 b ) . T h e c o r t i c o - s t r i a t a l p a t h w a y i s t h o u g h t t o b e e x c i t a t o r y ( K i t a i e t a l . , 1 9 7 6 a ; 1 9 7 6 b ; K o c s i s e t a l . , 1 9 7 7 ) a n d t o u s e g l u t a m a t e a s a t r a n s m i t t e r ( D i v a c e t a l . , 1 9 7 7 ; K i m e t a l . , 1 9 7 7 b ; M c G e e r e t a l . , 1 9 7 7 ; R e u b i e t a l . , 1 9 7 9 ; S p e n c e r , 1 9 7 6 ) . E l e c t r o p h y s i o l o g i c a l ( K i t a i e t a l . , 1 9 7 6 b ) a n d a n a t o m i c a l ( E n d o e t a l . , 1 9 7 3 ) e v i d e n c e i n d i c a t e s t h a t t h e c o r t i c o s t r i a t a l f i b e r s a r e i n d e p e n d e n t o f t h e c o r t i c o s p i n a l o r c o r t i c o b u l b a r s y s t e m s . A l t h o u g h t h e p a r a f a s i c u l a r a n d c e n t r o m e d i a n n u c l e i p r o v i d e t h e m a j o r t h a l a m i c i n p u t s ( K u r o d a e t a l . , 1 9 7 5 ; K a l i l , 1 9 7 8 ; R o y c e , 1 9 7 8 ) a l l o f t h e i n t r a l a m i n a r n u c l e i a p p e a r t o p r o j e c t t o t h e s t r i a t u m ( J o n e s a n d L e a v i t t , 1 9 7 4 ) . T o g e t h e r t h e s e i n p u t s make u p o n e q u a r t e r o f t h e a f f e r e n t t e r m i n a l s i n t h e s t r i a t u m (Kemp a n d P o w e l l , 1 9 7 1 b ) . A l t h o u g h c h a n g e s i n 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 n d a c e t y l c h o l i n e l e v e l s h a v e b e e n r e p o r t e d i n t h e h e a d o f t h e s t r i a t u m f o l l o w i n g p a r a f a s i c u l a r l e s i o n ( S a e l e n s e t a l . , 1 9 7 9 ; S i m k e a n d S a e l e n s , 1 9 7 7 ) t h e a n t e r i o r o n e - t h i r d o f t h e s t r i a t u m r e c e i v e s f e w i f a n y p a r a f a s i c u l a r a f f e r e n t s ( J o n e s a n d L e a v i t t , 1 9 7 4 ) . T h i s a n d t h e l a c k o f s t a i n i n g of p a r a f a s i c u l a r c e l l s f o r CAT (H. Kimura, personal communication) raises doubt about t h i s being a c h o l i n e r g i c t r a c t . Thus, at present the biochemical nature of t h i s input remains a matter of speculation, although e l e c t r o p h y s i o l o g i c a l r e s u l t s suggest that i t i s an e x c i t a t o r y pathway (Kocsis et a l . , 1977; M a l l i a n i and Purpura, 1967; Purpura and M a l l i a n i , 1967). The t h i r d major s t r i a t a l afferent a r i s e s i n the midbrain and supplies the striatum with approximately f i f t e e n percent of i t s a f f e r e n t terminals (Kemp and Powell, 1971b). This pathway contains dopamine and a r i s e s from both the SN and the adjacent v e n t r a l tegmental area (Anden et a l . , 1964; Beckstead et a l . , 1979; F a l l o n and Moore, 1978; L i n d v a l l and Bjorklund, 1974). The n i g r o - s t r i a t a l system was o r i g i n a l l y thought to be an i n h i b i t o r y projection; but there i s now considerable evidence that the dopamine t e r - minals depolarize s t r i a t a l neurons (Davies and Tongroach, 1978; K i t a i et a l . , 1975; Richardson et a l . , 1977). A second non-dopaminergic n i g r o s t r i a t a l system has also been suggested on both p h y s i o l o g i c a l and biochemical grounds (Feltz and deChamplain, 1972; Fibiger et a l . , 1972; Ljundahl et a l . , 1975; Guyenet and Aghajanian, 1978). In addition to these major s t r i a t a l a f f e r e n t s , two aminergic afferents from the brainstem have also been i d e n t i f i e d . Serotonin i s contained i n the s t r i a t a l afferents from the dorsal raphe nucleus (Azmitia and Segal, 1978; Jacobs et a l . , 1978; M i l l e r et a l . , 1975). Biochemical studies suggest that t h i s p r o j e c t i o n i s most concentrated i n the ventrocaudal region of the striatum (Ternaux et a l . , 1977). Also, the ubiquitous axons of the locus coeruleus have been shown to project to the striatum, providing a noradrener- gic innervation to t h i s area (Mason and F i b i g e r , 1979; Moore, 1978). In contrast to the d i v e r s i t y of the a f f e r e nts to the striatum, the out- put of t h i s nucleus i s quite r e s t r i c t e d . The striatum i s known to innervate d i r e c t l y only three structures, the GP, EP and SN. Thus, the e n t i r e output of the striatum i s confined to other nuclei within the basal ganglia. A l l parts of the striatum, including the head (Cowan and Powell, 1966; Graybiel et a l . , 1979, Nagy et a l . , 1978a, Tulloch et a l . , 1978) and t a i l (Tulloch et a l . , 1978) project to the pallidum. Although the EP receives fibers from the head of the striatum (Adinolfi, 1969; Nagy et a l . , 1978a) the extent to which i t receives afferents from the t a i l of the striatum is uncertain. The SN receives a rich innervation from diverse areas of the striatum (Bunney and Aghajanian, 1976; Grofova, 1975; Kanazawa et a l . , 1976; Nagy et a l . , 1978a; Richardson et a l . , 1977; Tulloch et a l . , 1978) both to the pars compacta (SNC) and to the pars reticulata (SNR). Which of the neuronal types of the striatum gives rise to these efferent pathways has been a matter of some debate. It has been estimated that over 95% of s t r i a t a l neurons are medium size spiny cells (Kemp and Powell, 1971a). The striatum also contains a few small neurons as well as a population of giant aspiny cells (Kemp and Powell, 1971a). These giant ce l l s , which account for only about one percent of the s t r i a t a l neurons (Kemp and Powell, 1971a) were originally suggested on the basis of Golgi material to be the projection neurons of the striatum (Fox et a l . , 1971). However, with the introduction of retrograde transport as an anatomical tool, this conclusion has been questioned. Thus, these giant cells have not been labeled following the injection of horseradish peroxidase into the GP, EP or SN. Recently, however, some large neurons have been labeled following injections into the retrorubral area (Grofova, 1979), a dorsal extension of the SN (Beckstead et a l . , 1979; Nauta et a l . , 1978). Although horseradish peroxidase injections into the GP, EP or SN proper have failed to label the giant aspiny neurons of the striatum, many medium size cells are labeled following these injections (Bunney and Aghajanian, 1976; Graybiel et a l . , 1979; Grofova, 1975; Kanazawa et a l . , 1976; Tulloch e t a l . , 1 9 7 8 ) . T h i s i n d i c a t e s t h a t t h e s e c e l l s p r o v i d e t h e m a j o r p a t h w a y s f o r t h e s t r i a t a l o u t f l o w . E v i d e n c e i n d i c a t e s t h a t t h e i n h i b i t o r y n e u r o t r a n s m i t t e r Y _ a m i n o b u t y r i c a c i d (GABA) i s c o n t a i n e d i n t h e s t r i a t a l a f f e r e n t s t o t h e GP ( F o n n u m e t a l . , 1 9 7 8 ; H a t t o r i e t a l . , 1 9 7 3 ; N a g y e t a l . , 1 9 7 8 a ) E P ( F o n n u m e t a l . , 1 9 7 8 a ; N a g y e t a l . , 1 9 7 8 a ) a n d SN ( B r o w n s t e i n e t a l . , 1 9 7 7 ; F o n n u m e t a l . , 1 9 7 8 a ; 1 9 7 4 ; G a l e e t a l . , 1 9 7 7 b ; H a t t o r i e t a l . , 1 9 7 3 b ; J e s s e l e t a l . , 1 9 7 8 ; K i m e t a l . , 1 9 7 7 a ; N a g y e t a l . , 1 9 7 8 a ) . T h i s s u g g e s t s t h a t GABA i s c o n t a i n e d i n a t l e a s t some o f t h e m e d i u m s p i n y c e l l s o f t h e s t r i a t u m , a n d i n f a c t some o f t h e s e c e l l s h a v e b e e n f o u n d t o s t a i n f o r t h e GABA s y n t h e t i c e n z y m e g l u t a m a t e d e c a r b o x y l a s e (GAD) i n i m m u n o h i s t o c h e m i c a l s t u d i e s ( R i b a k e t a l . , 1 9 7 9 ) . R e c e n t l y , e v i d e n c e h a s b e e n o b t a i n e d t h a t t h e n e u r o p e p t i d e s u b s t a n c e P i s a l s o c o n t a i n e d i n t h e s t r i a t a l p r o j e c t i o n s t o t h e n i g r a ( H o n g e t a l . , 1 9 7 7 c ; K a n a z a w a e t a l . , 1 9 7 7 a ; M r o z e t a l . , 1 9 7 7 ; P a l k o v i t s e t a l . , 1 9 7 8 ; P a x i n o s e t a l . , 1 9 7 8 a ) a n d EP ( P a x i n o s e t a l . , 1 9 7 8 b ) , s u g g e s t i n g t h a t some o f t h e m e d i u m s p i n y c e l l s s h o u l d c o n t a i n t h i s p e p t i d e . I n f a c t , i m m u n o - h i s t o c h e m i c a l s t u d i e s h a v e s h o w n some s m a l l a n d m e d i u m s i z e s t r i a t a l c e l l s d o c o n t a i n s u b s t a n c e P i m m u n o r e a c t i v e m a t e r i a l ( C u e l l o a n d K a n a z a w a , 1 9 7 8 ; L j t f n g d a h l e t a l . , 1 9 7 8 a ) . R e c e n t b i o c h e m i c a l e x p e r i m e n t s h a v e s h o w n t h a t t h e GABA a n d t h e s u b s t a n c e P p r o j e c t i o n s a r i s e f r o m d i f f e r e n t s i t e s w i t h i n t h e s t r i a t u m ( B r o w n s t e i n e t a l . , 1 9 7 7 ; G a l e e t a l . , 1 9 7 7 ; J e s s e l e t a l . , 1 9 7 8 ) i n d i c a t i n g t h a t t h e s e t r a n s m i t t e r s a r e p r o b a b l y c o n t a i n e d i n s e p a r a t e p o p u l a t i o n s o f s t r i a t a l n e u r o n s . T h e s t r i a t u m h a s a l s o b e e n f o u n d t o c o n t a i n o t h e r p e p t i d e s t h a t may f u n c t i o n a s n e u r o t r a n s m i t t e r s . I n p a r t i c u l a r s ome e v i d e n c e s u g g e s t s t h a t t h e e n d o g e n o u s o p i o i d p e p t i d e s m e t h i o n i n e - a n d l e u c i n e - e n k e p h a l i n may b e p r e s e n t i n s t r i a t o p a l l i d a l f i b e r s ( C u e l l o a n d P a x i n o s , 1 9 7 8 b ; H o n g e t a l . , 6 1977a). Although the d i r e c t output of the s t r i a t u m i s confined to the b a s a l g a n g l i a , pathways must e x i s t to transmit t h i s output beyond t h i s system, u l t i m a t e l y to the motor neurons. Both the EP (Carpenter, 1973; Kim et a l . , 1976; Kuo and Carpenter, 1973; Larsen and McBride, 1979; Nauta and Mehler, 1966) and SN (Beckstead et a l . , 1979) send p r o j e c t i o n s to the pars compacta of the nucleus tegmenti pedunculopontis, a mesencephalic r e t i c u l a r formation nucleus l y i n g w i t h i n the brachiumconjunctivum. The e f f e r e n t connections of t h i s nucleus are unknown, but p r e l i m i n a r y r e p o r t s suggest that i t s axons descend to the s p i n a l cord (Ross et a l . , 1979) and ascend towards the f o r e b r a i n , perhaps synapsing i n the pallidum (Gonya-Magee and Anderson, 1979). A p o p u l a t i o n of dopamine c e l l s i n the medial SN and the v e n t r a l tegmen- t a l area (Fuxe et a l . , 1974; L i n d v a l l et a l . , 1974; T h i e r r y et a l . , 1973) and a p o p u l a t i o n of c h o l i n e r g i c c e l l s i n and around the GP and EP ( K e l l e y and Moore, 1978a; Lehmann et a l . , 1980) p r o j e c t to the c e r e b r a l cortex. However, these pathways have only r e c e n t l y been discovered and t h e i r connections w i t h the s t r i a t u m and t h e i r r o l e i n mediating b a s a l g a n g l i a output have not been examined. The pathways most commonly thought to provide the main output f o r the b a s a l g a n g l i a a r i s e from the EP and from the SNR. As discussed above these two n u c l e i both r e c e i v e d i r e c t inputs from the s t r i a t u m . I n a d d i t i o n the GP p r o j e c t s d i r e c t l y to the SN (Bunney and Aghajanian, 1976; Grofova, 1975; H a t t o r i et a l . , 1975; Kanazawa et a l . , 1976; Kim et a l . , 1976; T u l l o c h et a l . , 1978) and to the subthalamic nucleus ( H a t t o r i et a l . , 1975; Kim et a l . , 1976; Nauta and Mehler, 1966) which i n t u r n p r o j e c t s to the EP (Carpenter and Strominger, 1967; Nauta and C o l e , 1978; W h i t t i e r and M e t t l e r , 1949) and the SN (Hammond et a l . , 1978; Kanazawa et a l . , 1976; Nauta and 7 Cole, 1978; Tulloch et a l . , 1978; Whittier and Mettler, 1949). A major p r o j e c t i o n from the EP to the l a t e r a l habenula (Carter and Fibi g e r , 1978; Herkenham and Nauta, 1977; Kim et a l . , 1976; Larsen and McBride, 1979; Nauta, 1974) has been demonstrated and biochemical studies have suggested that GABA i s a transmitter i n t h i s pathway (Gottesfeld et a l . , 1977; Nagy et a l . , 1978b). However, the l a t e r a l habenula i s not thought to be c r i t i c a l l y involved i n the control of motor behaviour. Rather, the projection from the EP to the thalamus has been suggested to serve such a function (Hassler, 1978). The p r i n c i p a l s i t e s of termination of p a l l i d a l f i b e r s i n the thalamus are the centromedian and p a r a f a s i c u l a r n u c l e i , and more importantly, the ventrolateral-ventroanterior complex and the ventro- medial nucleus (Carter and F i b i g e r , 1978; Kim et a l . , 1976; Kuo and Carpenter, 1973; Larsen and McBride, 1979; Nauta and Mehler, 1966). Since the nucleus v e n t r a l i s l a t e r a l i s projects to the motor cortex areas four and s i x , the p a l l i d a l p r o j e c t i o n to t h i s nucleus provides a pathway for the basal ganglia to a f f e c t d i r e c t l y the motor output of the cortex. A substantial p r o j e c t i o n to the thalamus from the SNR has also been demonstrated. This i s a non-dopaminergic p r o j e c t i o n and i s d i s t r i b u t e d to the medial part of the nucleus v e n t r a l i s l a t e r a l i s , the magnocellular part of the nucleus v e n t r a l i s anterior and the paralaminar part of the nucleus dorsomedialis (Beckstead et a l . , 1979; B e n t i v i g l i o et a l . , 1979; Carpenter et a l . , 1976; Carpenter and Peter, 1972; Cole et a l . , 1964; F a u l l and Mehler, 1978; F i b i g e r , et a l . , 1972; Mettler, 1970; Rinvik, 1975). A p r o j e c t i o n to the p a r a f a s i c u l a r nucleus has also been reported (Ahlenius, 1978; Beckstead et a l . , 1979; C l a v i e r et a l . , 1976). Again, v i a the connection with the nucleus v e n t r a l i s l a t e r a l i s , t h i s output pathway provides the basal ganglia with access to the motor cortex. F i n a l l y , f i b e r s from the SNR, a r i s i n g at l e a s t i n part as c o l l a t e r a l s of the nigrothalamic p r o j e c t i o n (Anderson and Yoshida, 1977; Bentivoglio et a l . , 1979), innervate the superior c o l l i c u l u s ( A f i f i and Kaelber, 1965; Beckstead et a l . , 1979; F a u l l and Mehler, 1978; Graybiel, 1978; Hopkins and Niessen, 1976; Jayaraman et a l . , 1977; Rinvik et a l . , 1976). The n i g r o t e c t a l f i b e r s terminate i n the deeper layers of the superior c o l l i c u l u s (Anderson and Yoshida, 1977; Beckstead et a l . , 1979; Graybiel, 1978, Jayaraman et a l . , 1977; Rinvik et a l . , 1976), which are known to give r i s e to the t e c t o r e t i c u l a r and te c t o s p i n a l pathways (Kuypers and Maisky, 1975). Indeed, e l e c t r o p h y s i o l o g i c a l experiments have i n d i c a t e d that the p r o j e c t i o n from the SNR to the superior c o l l i c u l u s i n t e r a c t s with the t e c t o s p i n a l system which regulates the neck muscles involved i n head o r i e n t a t i o n (York and Faber, 1977). Thus, t h i s system could well be involved i n the expression of the motor asymmetries observed a f t e r u n i l a t e r a l manipulations of the basal ganglia. 9 STATEMENT OF THE PROBLEMS TO BE EXAMINED In the present study a biochemical and h i s t o l o g i c a l a nalysis of the output pathways of the basal ganglia i s presented. We begin with an examination of the efferents of the caudate-putamen to the other n u c l e i of the basal ganglia. As mentioned i n the Introduction, GABA, ac e t y l c h o l i n e , enkephalin and substance P have a l l been suggested to be contained i n s t r i a t a l c e l l s . In Experiment 1 the existence of these transmitters i n the s t r i a t a l projections to the GP, EP and SN was biochemically examined following lesions of the s t r i a t a l output pathways. The biochemical approach used i n Experiment 1 can provide evidence that a chemical such as GABA or substance P i s contained i n a f i b r e t r a c t . This technique w i l l not however give information regarding the topography and synaptic r e l a t i o n s h i p s of the f i b r e systems. For t h i s h i s t o l o g i c a l methods are e s s e n t i a l . Therefore i n Experiment 2 the d i s t r i b u t i o n of the substance P f i b r e s was examined using an immunohistochemical approach. To study the GABA systems of the basal ganglia the l o c a l i z a t i o n of the enzyme GABA-transaminase (GABA-T), which catabolizes GABA, was examined. The biochemical studies i n Experiment 3 suggest that GABA-T i s l o c a l i z e d i n the GABA neurons of the striatum. Therefore, i n Experiment A, a histochemical method for t h i s enzyme was adapted to examine the s t r i a t a l and p a l l i d a l GABA ef f e r e n t s . The s t r i a t a l efferents are intimately associated with the n i g r o s t r i a t a l dopaminergic neurons. In order to examine the nature of t h i s i n t e r a c t i o n the responses of the s t r i a t a l GABA and substance P c e l l s to the s e l e c t i v e loss of the n i g r a l dopaminergic neurons were biochemically examined i n Experiment 5. As t h i s l e s i o n of the dopaminergic neurons has been used as a model of Parkinson's disease (Marsden, 1977), the r e s u l t s of t h i s study have been compared with the biochemical changes reported to occur i n 10 Parkinsonism. F i n a l l y i n Experiment 6 the biochemical nature of the n i g r o t e c t a l pathway, which probably represents a major output of the basal ganglia, was examined by measuring various neurotransmitter markers i n the superior c o l l i c u l u s following n i g r a l l e s i o n s . The morphology of the n i g r o t e c t a l p r o j e c t i o n was also examined using electron microscopy. The r e s u l t s provide the f i r s t i n d i c a t i o n that GABA may be a transmitter i n the n i g r a l output pathways. The implications of t h i s observation f o r basal ganglia function are therefore discussed. 1 1 EXPER IMENT 1 ; NEUROTRANSMITTERS CONTAINED I N THE E F F E R E N T S OF THE S T R I A T U M T h r e e n e u r o t r a n s m i t t e r c a n d i d a t e s h a v e b e e n f o u n d t o b e c o n t a i n e d i n t h e f i b e r s e m a n a t i n g f r o m t h e s t r i a t u m . T h e s e a r e GABA , s u b s t a n c e P a n d e n k e p h a l i n . GABA h a s b e e n s h o w n t o b e c o n t a i n e d i n s t r i a t o p a l l i d a l ( F o n n u m e t a l . , 1 9 7 8 a ; H a t t o r i e t a l . , 1 9 7 3 b ; N a g y e t a l . , 1 9 7 8 a ) , s t r i a t o e n t o p e d u n c u l a r ( F onnum e t a l . , 1 9 7 8 a ; N a g y e t a l . , 1 9 7 8 a ) a n d s t r i a t o n i g r a l ( B r o w n s t e i n e t a l . , 1 9 7 7 : F o n n u m e t a l . , 1 9 7 8 a ; 1 9 7 4 ; H a t t o r i e t a l . , 1 9 7 3 b ; J e s s e l e t a l . , 1 9 7 8 ; K i m e t a l . , 1 9 7 1 ; N a g y e t a l . , 1 9 7 8 a ) f i b e r s . T h e s t r i a t o - e n t o p e d u n c u l a r a n d s t r i a t o n i g r a l p r o j e c t i o n s h a v e a l s o b e e n s h o w n t o c o n t a i n s u b s t a n c e P b y i m m u n o f l u o r e s c e n t ( P a x i o n s e t a l . , 1 9 7 8 a ; 1 9 7 8 b ) a n d r a d i o i m m u n o c h e m i c a l ( B r o w n s t e i n e t a l . , 1 9 7 7 ; G a l e e t a l . , 1 9 7 7 ; H o n g e t a l . , 1 9 7 7 c ; J e s s e l e t a l . , 1 9 7 8 ; K a n a z a w a e t a l . , 1 9 7 7 a ) t e c h n i q u e s , r e s p e c t i v e l y . A s t r i a t o p a l l i d a l l e u - e n k e p h a l i n - c o n t a i n i n g p a t h w a y h a s b e e n d e m o n s t r a t e d b y i m m u n o h i s t o f l u o r e s c e n t m e t h o d s ( C u e l l o a n d P a x i n o s , 1 9 7 8 ) a n d p a l l i d a l m e t - e n k e p h a l i n l e v e l s h a v e b e e n o b s e r v e d t o d e c r e a s e 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 o f t h e s t r i a t u m ( H o n g e t a l . , 1 9 7 7 a ) . A c o m p r e h e n s i v e a n d s y s t e m a t i c e x a m i n a t i o n o f e a c h o f t h e s e s y s t e m s h a s n o t b e e n c o n d u c t e d . T h e p r e s e n t s t u d y p r o v i d e s f u r t h e r i n f o r m a t i o n o n t h e c o n t r i b u t i o n o f e a c h o f t h e s e s u b s t a n c e s t o t h e s t r i a t a l p r o j e c t i o n s t o t h e G P , E P a n d S N . METHODS M a l e W i s t a r r a t s w e i g h i n g a b o u t 3 0 0 g i n t h e h e m i t r a n s e c t i o n g r o u p a n d 4 5 0 g i n t h e GP i s l a n d g r o u p w e r e a n e s t h e t i z e d w i t h p e n t o b a r b i t a l a n d p l a c e d i n a K o p f s t e r e o t a x i c i n s t r u m e n t . F o r t h e s e m i c i r c u l a r c u t s s e p a r a t i n g t h e GP f r o m t h e s t r i a t u m (GP i s l a n d s ) a H a l a s z k n i f e f o u r mm i n r a d i u s a n d t h r e e mm i n h e i g h t w a s f a s h i o n e d f r o m t u n g s t e n w i r e a n d m o u n t e d i n a 26 g a u g e c a n n u l a . A f t e r r e m o v a l o f a b o n e f l a p a n d d e f l e c t i o n 12 of the dura the k n i f e was lowered at a parasaggital o r i e n t a t i o n to an empirically determined set of coordinates. The axis of r o t a t i o n of the k n i f e was 3.5 mm posterior to bregma and 2.3 mm l a t e r a l to the midline. The t i p of the k n i f e was 8.1 mm v e n t r a l to the c r a n i a l surface. Once positioned the k n i f e was rotated slowly 10-15 degrees towards the midline and then rotated 130 degrees laterocaudally. A f t e r returning to the entry o r i e n t a t i o n the k n i f e was withdrawn and the wound treated with 0.1% Zephiran. Hemitransections j u s t a n t e rior to the GP were produced with a st r a i g h t tungsten wire lowered at the midline to the v e n t r a l surface of the brain and drawn to the l a t e r a l extreme i n the plane of the anterior commissure. Animals were s a c r i f i c e d two to three weeks postoperatively by c e r v i c a l f r a c t u r e and the brains r a p i d l y removed and placed on i c e . A l l areas were dissected freehand from sections obtained on a f r e e z i n g micro- tome. The head of the striatum was dissected only from those sections not v i s i b l y damaged by the k n i f e . The GP was not assayed i n animals i n which the k n i f e cut encroached on t h i s area. The t a i l of the striatum was dissected from the same sections as the GP. For enzyme a c t i v i t y measurements tissues were homogenized i n 20 to 50 volumes of 50 mM T r i s - a c e t a t e buffer, pH 6.4, containing 0.2% v/v T r i t o n X-100. Pro t e i n was determined on these homogenates according to the method of Lowry et a l . (1951). A modification of the method of Albers and Brady (1959) was used for the assay of GAD a c t i v i t y (Chalmers et a l . , 1970). Tissue homogenate (20 yl) was added to 50 y l of incubation mixture containing ( f i n a l concentrations) 2.0 mM [l- l i fC]glutamate ( s p e c i f i c a c t i v i t y 0.1 to 0.2 mCi/ mmol, Amersham), 0.02% bovine serum albumin (Sigma) and 0.1 mM pyridoxal 13 p h o s p h a t e ( C a l b i o c h e m ) i n 2 0 mM p o t a s s i u m p h o s p h a t e b u f f e r , p H 7 . 4 i n a s m a l l g l a s s t u b e . E a c h t u b e w a s p l a c e d i n a s c i n t i l l a t i o n v i a l w i t h h a l f a g e l a t i n c a p s u l e ( P a r k e - D a v i s ) c o n t a i n i n g a p i e c e o f f i l t e r p a p e r t o w h i c h 0 . 1 m l o f h y a m i n e h y d r o x i d e w a s a d d e d . T h e v i a l s w e r e c a p p e d w i t h r u b b e r s e p t u m s a n d i n c u b a t e d a t 3 7 ° C f o r 3 0 m i n . T h e r e a c t i o n w a s s t o p p e d b y t h e i n j e c t i o n o f 0 . 2 m l o f 2M H^SO^ i n t o t h e i n c u b a t i o n t u b e t h r o u g h t h e r u b b e r s e p t u m w i t h a 19 g a u g e n e e d l e . A f t e r t w o h r t h e r e a c t i o n t u b e s w e r e r e m o v e d f r o m t h e v i a l s a n d t h e t r a p p e d l t + C 0 2 c o u n t e d i n ACS ( A m e r s h a m ) w i t h a l i q u i d s c i n t i l l a t i o n c o u n t e r . CAT wa s a s s a y e d a c c o r d i n g t o t h e m e t h o d o f F o n n u m ( 1 9 7 5 ) . T h e r e a c t i o n wa s s t a r t e d b y a d d i n g 20 u l o f t i s s u e h o m o g e n a t e t o 5 0 y l o f a m i x t u r e w h i c h c o n t a i n e d ( f i n a l c o n c e n t r a t i o n s ) 3 0 0 mM N a C l , 8 . 0 mM c h o l i n e c h l o r i d e , 0 . 1 mi l e s e r i n e , 2 0 mM E D T A , a n d 0 . 2 mM [ 1 - 1 I + C ] a c e t y l c o e n z y m e A ( A m e r s h a m , s p e c i f i c a c t i v i t y 0 . 0 5 m C i / m m o l ) i n 5 0 mM s o d i u m p h o s p h a t e b u f f e r p H 7 . 4 . T h e m i x t u r e w a s i n c u b a t e d a t 3 7 ° C f o r 20 m i n i n a s c i n t i l l a t i o n v i a l . T h e r e a c t i o n w a s t h e n s t o p p e d b y a d d i n g s e v e n m l o f a s o l u t i o n c o n s i s t i n g o f two p a r t s a c e t o n i t r i l e a n d f i v e p a r t s t e n mM s o d i u m p h o s p h a t e b u f f e r pH 7 . 4 c o n t a i n i n g t w o m g / m l s o d i u m t e t r a p h e n y l b o r o n . T e n m l o f t o l u e n e c o n t a i n i n g 0.4% 2 , 5 - d i p h e n y l - o x a z o l e ( PPO ) a n d 0 . 0 1 5 % p _ - b i s - [ 2 - ( 5 - p h e n y l o x a z o l y l ) ] b e n - z e n e ( POPOP) w e r e t h e n a d d e d a n d t h e 1 ^ - a c e t y l c h o l i n e p r o d u c e d d u r i n g t h e i n c u b a t i o n w a s e x t r a c t e d i n t o t h e o r g a n i c p h a s e w i t h t h e t e t r a p h e n y l b o r o n a n d c o u n t e d i n a l i q u i d s c i n t i l l a t i o n c o u n t e r . S u b s t a n c e P a n d m e t - e n k e p h a l i n l e v e l s w e r e d e t e r m i n e d b y r a d i o - i m m u n o a s s a y o n l y o p h i l i z e d 1 . 0 N a c e t i c a c i d e x t r a c t s o f w e i g h e d t i s s u e ( s e e A p p e n d i x ) . R E S U L T S T h e e x t e n t t o w h i c h t h e k n i f e c u t s i s o l a t e d t h e GP f r o m t h e s t r i a t u m (GP i s l a n d s ) i s s h o w n i n F i g . 1 . R o s t r a l l y , t h e l e s i o n s s t a r t e d j u s t l a t e r a l 14 Figure 1. The k n i f e cuts separating the globus p a l l i d u s from the striatum. The diagram on the l e f t represents a h o r i z o n t a l section through the forebrain showing the s i t e s of both the c i r c u l a r cut forming the GP i s l a n d (arrow) and the hemitransection (double arrow). The photograph on the ri g h t i s a coronal section through the anterior globus p a l l i d u s showing the c i r c u l a r k n i f e cut. The c i r c u l a r cut i s seen l a t e r a l to the globus p a l l i d u s which i s out- l i n e d i n white dots. Shrinkage of s t r i a t a l t i s s u e i s evidenced by the d i l a t i o n of the l a t e r a l v e n t r i c l e (LV). AC nucleus accumbens, GP globus p a l l i d u s , CP caudate- putamen. Magnification: 25X. i ta 15 t o t h e s e p t u m a n d a n t e r i o r t o t h e d e c u s s a t i o n o f t h e a n t e r i o r c o m m i s s u r e . F r o m t h i s p o i n t , t h e l e s i o n s p r o c e e d e d l a t e r a l l y a n d t h e n p o s t e r i o r l y t o s u b s c r i b e a n a r c s u r r o u n d i n g t h e GP b o t h d o r s a l l y a n d l a t e r a l l y . I n o r d e r t o a v o i d d a m a g e t o t h e i n t e r n a l c a p s u l e t h e l e s i o n s w e r e p r o d u c e d s o a s t o i s o l a t e o n e - h a l f t o t w o - t h i r d s o f t h e GP f r o m t h e t a i l o f t h e s t r i a t u m . A l t h o u g h d i r e c t d a m a g e t o t h e p a l l i d u m w a s r a r e l y o b s e r v e d , t h e l e s i o n s o c c a s i o n a l l y a p p r o a c h e d t h e l a t e r a l a s p e c t o f t h i s s t r u c t u r e . Some g l i o s i s a n d s h r i n k a g e o f t h e p a l l i d u m w e r e e v i d e n t p r o b a b l y r e s u l t i n g f r o m t h e l o s s o f s t r i a t o - p a l l i d a l t e r m i n a l s a n d t h e r e t r o g r a d e l o s s o f some p a l l i d a l p r o j e c t i o n n e u r o n s . R e g i o n s o f t h e s t r i a t u m a n t e r i o r a n d l a t e r a l t o t h e l e s i o n s e x h i b i t e d some s h r i n k a g e a l t h o u g h no g l i o s i s w a s o b s e r v e d i n t h e s e a r e a s . T h e e f f e c t o f t h e s e k n i f e c u t s o n GAD a n d CAT a c t i v i t y i n t h e v a r i o u s b r a i n a r e a s e x a m i n e d a r e s h o w n i n T a b l e 1 . I n t h e GP a n d EP GAD w a s r e d u c e d s i g n i f i c a n t l y b y 44 a n d 22 p e r c e n t , r e s p e c t i v e l y . T h e a c t i v i t y o f t h i s e n z y m e was n o t s i g n i f i c a n t l y d e c r e a s e d i n t h e SN b y t h i s l e s i o n . T h e a c t i v i t y o f CAT wa s n o t a f f e c t e d i n t h e SN o r EP b u t w a s r e d u c e d b y 3 5 p e r c e n t i n t h e GP f o l l o w i n g t h i s l e s i o n . T h e p r o d u c t i o n o f t h e GP i s l a n d s r e s u l t e d i n a 49 p e r c e n t i n c r e a s e i n GAD a c t i v i t y i n t h e h e a d o f t h e s t r i a t u m a n t e r i o r t o t h e l e s i o n , w i t h o u t a f f e c t i n g CAT a c t i v i t y . T h e e f f e c t o f h e m i t r a n s e c t i o n s j u s t r o s t r a l t o t h e GP o n CAT a n d GAD i s s h o w n i n T a b l e 2 . T h e s e l e s i o n s w e r e m o r e p o s t e r i o r a n d e n c r o a c h e d u p o n t h e a n t e r i o r p o l e o f t h e G P . T h i s p r o b a b l y e x p l a i n s t h e g r e a t e r r e d u c t i o n s o f GAD o b s e r v e d i n t h e GP a n d EP f o l l o w i n g t h i s l e s i o n c o m p a r e d w i t h t h o s e o b s e r v e d a f t e r t h e c i r c u l a r c u t s a n d c o u l d a c c o u n t f o r t h e s i g n i f i c a n t r e d u c t i o n o b s e r v e d i n n i g r a l GAD . A s i n t h e c a s e o f t h e GP i s l a n d s , CAT was s i g n i f i c a n t l y r e d u c e d i n GP b u t u n a l t e r e d i n t h e E P o r S N . N e i t h e r CAT n o t GAD w a s s i g n i f i c a n t l y a l t e r e d i n t h e t a i l o f t h e s t r i a t u m l a t e r a l t o 16 T a b l e 1 . G l u t a m i c a c i d d e c a r b o x y l a s e a n d 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 v a r i o u s a r e a s a f t e r l e s i o n s i s o l a t i n g t h e g l o b u s p a l l i d u s f r o m t h e s t r i a t u m (GP i s l a n d s ) . G l u t a m i c a c i d d e c a r b o x y l a s e C h o l i n e a c e t y l t r a n s f e r a s e n m o l / m g p r o t e i n / h r n m o l / m g p r o t e i n / h r A r e a 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 H e a d o f s t r i a t u m 235±14 3 5 0 ± 1 6 * * 168±9 173±10 % o f c o n t r o l 1 4 9 1 0 3 G l o b u s p a l l i d u s 4 98±30 2 7 9 ± 2 6 * * 5 5 . 8 + 7 . 2 3 6 . 4 ± 3 . 3 * % o f c o n t r o l 56 65 E n t o p e d u n c u l a r n u c l e u s 290±27 2 2 5 ± 1 4 * 2 0 . 2 ± 1 . 5 1 6 . 9 ± 1 . 3 % o f c o n t r o l 78 84 S u b s t a n t i a n i g r a 683±78 545±50 % o f c o n t r o l 8 0 V a l u e s i n d i c a t e m e a n s ± S . E . M . o f e i g h t d e t e r m i n a t i o n s . * p < . 0 5 , * *p < . 0 1 , S t u d e n t ' s _t t e s t . 17 t h e GP . T h i s s u g g e s t s t h e r e d u c t i o n i n p a l l i d a l e n z y m e a c t i v i t i e s w a s n o t s i m p l y d u e t o a p o s t e r i o r s p r e a d o f t i s s u e d a m a g e a f t e r t h e h e m i t r a n s e c t i o n l e s i o n s . A s i n t h e c a s e o f t h e GP i s l a n d s , h e m i t r a n s e c t i o n s s i g n i f i c a n t l y i n c r e a s e d GAD a c t i v i t y b y 54% i n t h e h e a d o f t h e s t r i a t u m w h i l e CAT a c t i v i t y r e m a i n e d u n c h a n g e d . S u b s t a n c e P l e v e l s i n t h e h e a d a n d p r o j e c t i o n a r e a s o f t h e s t r i a t u m a f t e r l e s i o n s p r o d u c i n g t h e GP i s l a n d s a r e s h o w n i n T a b l e 3 . E x p r e s s e d a s p e r c e n t o f t h e u n l e s i o n e d c o n t r a l a t e r a l s i d e , t h e s e l e s i o n s s i g n i f i c a n t l y r e d u c e d s u b s t a n c e P l e v e l s b y 4 8 p e r c e n t i n t h e G P , 58 p e r c e n t i n E P a n d 47 p e r c e n t i n t h e S N . T h e l e v e l s i n t h e h e a d o f t h e s t r i a t u m w e r e u n a f f e c t - e d . T h e l e v e l s o f s u b s t a n c e P i n t h e s e same a r e a s a f t e r h e m i t r a n s e c t i o n s j u s t a n t e r i o r t o t h e GP a r e s h o w n i n T a b l e 4 . T h e r e d u c t i o n s o f t h i s p e p t i d e i n t h e t h r e e p r o j e c t i o n a r e a s o f t h e s t r i a t u m w e r e c o m p a r a b l e t o t h o s e o b s e r v e d i n t h e c a s e o f t h e GP i s l a n d s . S i m i l a r l y , s u b s t a n c e P l e v e l s i n t h e h e a d o f t h e s t r i a t u m w e r e u n a l t e r e d . T h e h e m i t r a n s e c t i o n s s i g n i f i c a n t l y r e d u c e d m e t - e n k e p h a l i n l e v e l s i n t h e GP b y 20 p e r c e n t b u t d i d n o t a l t e r l e v e l s i n t h e E P o r SN ( T a b l e 4 ) . I n a d d i t i o n , t h e s e l e s i o n s c a u s e d a 34 p e r c e n t i n c r e a s e i n m e t - e n k e p h a l i n l e v e l s i n t h e h e a d o f t h e s t r i a t u m . D I S C U S S I O N A l t h o u g h i t h a s b e e n k n o w n f o r some t i m e t h a t t h e m a j o r o u t p u t s o f t h e s t r i a t u m a r e t o t h e G P , EP a n d S N , t h e r e i s s t i l l c o n t r o v e r s y o v e r w h i c h n e u r o t r a n s m i t t e r s a r e i n v o l v e d i n e a c h o f t h e s e s y s t e m s . A t p r e s e n t , t h e t r a n s m i t t e r s t h o u g h t t o b e a s s o c i a t e d w i t h s t r i a t a l n e u r o n s i n c l u d e a c e t y l c h o l i n e , GABA , s u b s t a n c e P , a n d e n k e p h a l i n . We h a v e , t h e r e f o r e , e x a m i n e d t h e p o s s i b l e e x i s t e n c e o f e a c h o f t h e s e s u b s t a n c e s i n t h e v a r i o u s p r o j e c t i o n s o f t h e s t r i a t u m . 18 T a b l e 2 . G l u t a m i c a c i d d e c a r b o x y l a s e a n d 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 v a r i o u s a r e a s a f t e r h e m i t r a n s e c t i o n s a n t e r i o r t o t h e g l o b u s p a l l i d u s . A r e a G l u t a m i c a c i d d e c a r b o x y l a s e C h o l i n e a c e t y l t r a n s f e r a s e n m o l / m g p r o t e i n / h r n m o l / m g p r o t e i n / h r 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 H e a d o f s t r i a t u m % o f c o n t r o l 285±23 4 4 0 ± 3 3 * 154 2 2 4 ± 1 5 199±18 89 T a i l o f s t r i a t u m % o f c o n t r o l 3 26±19 417±44 128 1 6 7 ± 1 2 139±18 83 G l o b u s p a l l i d u s % o f c o n t r o l 5 06±22 2 1 5 + 2 4 * * 42 6 2 . 8 ± 6 . 5 4 1 . 4 + 2 . 9 * 66 E n t o p e d u n c u l a r n u c l e u s 3 38±25 1 9 6 ± 2 1 * * % o f c o n t r o l 58 2 8 . 5 ± 5 . 4 2 5 . 4 ± 5 . 5 89 S u b s t a n t i a n i g r a % o f c o n t r o l 632±39 3 6 9 ± 3 7 * 58 V a l u e s i n d i c a t e m e a n s ± S . E . M . o f s e v e n d e t e r m i n a t i o n s f o r SN a n d f i v e f o r t h e r e m a i n i n g t i s s u e s . *p < . 0 2 5 , * *p < . 0 0 5 , S t u d e n t ' s _t t e s t . 19 ( a ) T h e G l o b u s P a l l i d u s T h e d e c r e a s e i n s u b s t a n c e P l e v e l s i n t h e GP o b s e r v e d f o l l o w i n g i t s s e p a r a t i o n f r o m t h e s t r i a t u m p r o v i d e s t h e f i r s t e v i d e n c e t h a t s u b s t a n c e P may b e a t r a n s m i t t e r i n t h e s t r i a t o p a l l i d a l p a t h w a y . I t w o u l d a p p e a r t h a t h a l f t h e s u b s t a n c e P i n t h e p a l l i d u m comes f r o m t h e a n t e r i o r s t r i a t u m , w h i l e t h e r e m a i n i n g 5 0 p e r c e n t c o u l d e i t h e r a r i s e f r o m t h e t a i l o f t h e s t r i a t u m o r c o u l d r e p r e s e n t i n t r i n s i c s u b s t a n c e P n e u r o n s o f t h e G P . S u b s t a n c e P - l i k e i m m u n o r e a c t i v e c e l l s h a v e b e e n f o u n d i n t h e GP ( C u e l l o a n d K a n a z a w a , 1 9 7 8 ) . I n t h i s r e g a r d i t i s i n t e r e s t i n g t o n o t e t h a t i n H u n t i n g t o n ' s c h o r e a a 50 p e r c e n t d e c r e a s e i n s u b s t a n c e P l e v e l s h a s a l s o b e e n o b s e r v e d i n t h e GP ( G a l e e t a l . , 1 9 7 8 ) . S t r i a t o p a l l i d a l G A D - c o n t a i n i n g f i b e r s h a v e p r e v i o u s l y b e e n d e m o n s - t r a t e d ( F o n n u m e t a l . , 1 9 7 8 a ; H a t t o r i e t a l . , 1 9 7 3 b ; N a g y e t a l . , 1 9 7 8 a ) . I n t h e p r e s e n t s t u d y t h e r e d u c t i o n s i n p a l l i d a l GAD w e r e o n l y s l i g h t l y l e s s a f t e r GP i s l a n d l e s i o n s t h a n a f t e r h e m i t r a n s e c t i o n s . T h i s i n d i c a t e s t h a t t h e GP r e c e i v e s t h e m a j o r p o r t i o n o f i t s GABA i n p u t f r o m n e u r o n s o r i g i n a t i n g r o s t r a l t o b o t h l e s i o n s i n t h e s t r i a t u m . T h i s i s s u p p o r t e d b y t h e o b s e r v a t i o n t h a t e l e c t r o l y t i c l e s i o n s o f t h e t a i l o f t h e s t r i a t u m do n o t r e d u c e GAD a c t i v i t y i n t h e GP ( N a g y a n d F i b i g e r , 1 9 8 0 ) . T h e GP h a s b e e n r e p o r t e d t o c o n t a i n t h e h i g h e s t l e v e l s o f m e t - e n k e p h a l i n i n t h e b r a i n ( G r a m s c h e t a l . , 1 9 7 9 ; H o n g e t a l . , 1 9 7 7 b ) . I m m u n o h i s t o c h e m i c a l e v i d e n c e h a s b e e n p r e s e n t e d f o r a l e u - e n k e p h a l i n - c o n t a i n i n g 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 ( C u e l l o a n d P a x i n o s , 1 9 7 8 ) . T h e a n t i - s e r u m e m p l o y e d i n t h a t s t u d y ( C u e l l o a n d P a x i n o s , 1 9 7 8 ) h a d l i t t l e c r o s s - r e a c t i v i t y w i t h m e t - e n k e p h a l i n . On t h e o t h e r h a n d , m e t - e n k e p h a l i n l e v e l s h a v e b e e n o b s e r v e d t o d e c r e a s e i n t h e GP f o l l o w i n g k a i n i c a c i d ( K A ) i n j e c t i o n s i n t o t h e s t r i a t u m ( H o n g e t a l . , 1 9 7 7 a ) . I n t h a t s t u d y , h o w e v e r , t h e K A l e s i o n i n c l u d e d n o t o n l y t h e s t r i a t u m b u t a l s o t h e GP a n d p a r t o f 20 T a b l e 3 . S u b s t a n c e P l e v e l s i n v a r i o u s a r e a s a f t e r l e s i o n s i s o l a t i n g t h e g l o b u s p a l l i d u s f r o m t h e s t r i a t u m (GP i s l a n d s ) . A r e a S u b s t a n c e P p g / m g t i s s u e C o n t r o l L e s i o n % o f c o n t r o l H e a d o f s t r i a t u m 4 4 5 ± 1 1 0 448±82 6 1 0 1 G l o b u s p a l l i d u s 502± 90 2 0 6 ± 4 5 * 5 4 1 E n t o p e d u n c u l a r n u c l e u s 9 93+114 4 1 6 ± 9 0 * * 6 42 S u b s t a n t i a n i g r a 2 0 1 4 ± 1 7 5 1 0 6 7 ± 2 1 5 * * 6 5 3 V a l u e s a r e m e a n s ± S . E . M . o f t h e n u m b e r o f d e t e r m i n a t i o n s (N ) i n d i c a t e d . *p < . 0 2 5 , * * p < . 0 1 , S t u d e n t ' s _t t e s t . 21 T a b l e 4 . T h e l e v e l s o f s u b s t a n c e P a n d m e t - e n k e p h a l i n i n v a r i o u s b r a i n a r e a s a f t e r h e m i t r a n s e c t i o n s a n t e r i o r t o t h e g l o b u s p a l l i d u s S u b s t a n c e P M e t - e n k e p h a l i n p g / m g t i s s u e p g / m g t i s s u e A r e a C o n t r o l L e s i o n N C o n t r o l L e s i o n H e a d o f s t r i a t u m 322±16 301±24 15 1 1 2 6 ± 8 5 1 5 3 1 ± 1 1 0 * * * % o f c o n t r o l 94 134 G l o b u s p a l l i d u s 462±14 2 3 3 ± 5 0 * * * 16 3 3 5 1 ± 1 8 1 2 6 7 5 ± 2 4 9 * % o f c o n t r o l 50 8 0 E n t o p e d u n c u l a r n u c l e u s 614+99 3 0 0 ± 5 6 * * 9 7 28±80 749±96 % o f c o n t r o l 49 96 S u b s t a n t i a n i g r a 1 503±237 7 1 0 + 2 0 4 * * * 5 1 5 2 ± 2 8 187±28 % o f c o n t r o l 47 1 2 3 V a l u e s r e p r e s e n t t h e m e a n s ± S . E . M . o f t h e n u m b e r o f d e t e r m i n a t i o n i n d i c a t e d . *p < . 0 5 , * * p < . 0 2 5 , * * * p < . 0 1 , S t u d e n t ' s t t e s t . 22 the thalamus. Thus, i n view of the demonstration of d i s t i n c t populations of leucine- and methionine-enkephalin-containing neurons i n the caudate- putamen and GP (Larsson et a l . , 1979), the present r e s u l t s provide the f i r s t demonstration of the existence of a met-enkephalin containing 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 . The small but s i g n i f i c a n t decrease i n the l e v e l s of this peptide observed i n the GP a f t e r a l e s i o n which markedly depleted GAD and substance P, suggests an alternate source f o r the majority of the met-enkephalin i n the GP than the head of the striatum. Since KA lesions of the striatum which include the GP lead to a 50% decrease i n p a l l i d a l met-enkephalin l e v e l s (Hong et a l . , 1977a), i t i s probable that there are i n t r i n s i c p a l l i d a l met-enkephalin neurons and/or that the pallidum receives s u b s t a n t i a l met-enkephalin afferents from other sources. Although CAT a c t i v i t y i n the GP was decreased by both les i o n s i n t h i s study, these r e s u l t s are not s u f f i c i e n t to demonstrate the existence of a ch o l i n e r g i c s t r i a t o p a l l i d a l projection. I t has previously been observed that c o r t i c a l l e s i o n s r e s u l t i n the retrograde degeneration of the intense- l y s t a i n i n g acetylcholinesterase neurons located i n and around the GP which are thought to provide the c h o l i n e r g i c input to the cortex (Lehmann et a l . , 1980). It has also been observed (Wm. Staines and H.C. F i b i g e r , personal communication) that KA les i o n s confined wholly to the head of the striatum do not decrease p a l l i d a l CAT. The loss of p a l l i d a l CAT a c t i v i t y observed i n the present study may therefore be a t t r i b u t a b l e to the retrograde loss of the c h o l i n e r g i c p a l l i d a l neurons which project to areas r o s t r a l to the l e s i o n s i t e s such as the cerebral cortex (Johnston et a l . , 1979; K e l l e y and Moore, 1978a; Lehmann et a l . , 1980). (b) The Entopeduncular Nucleus The EP has previously been shown to contain substance P - l i k e immuno- r e a c t i v i t y by immunohistochemical methods (Cuello and Kanazawa, 1978; Ljiing- 23 d a h l e t a l . , 1 9 7 8 a ; P a x i n o s e t a l . , 1 9 7 8 b ) . H o w e v e r , l e v e l s o f s u b s t a n c e P i n t h e EP h a v e n o t p r e v i o u s l y b e e n q u a n t i f i e d b y b i o c h e m i c a l m e t h o d s . T h e l e v e l s m e a s u r e d i n t h e s t r i a t u m , GP a n d SN i n t h e p r e s e n t i n v e s t i - g a t i o n a r e s i m i l a r t o t h o s e r e p o r t e d i n s t u d i e s o f t h e r e g i o n a l d i s t r i b u - t i o n o f s u b s t a n c e P ( B r o w n s t e i n e t a l . , 1 9 7 6 ; K a n a z a w a a n d J e s s e l , 1 9 7 6 ) a n d f r o m t h i s i t w o u l d a p p e a r t h a t o n l y t h e S N , a n d t h e t r i g e m i n a l c o m p l e x c o n t a i n h i g h e r l e v e l s o f s u b s t a n c e P t h a n t h e E P . P a x i n o s e t a l . ( 1 9 7 8 b ) h a v e r e c e n t l y r e p o r t e d t h a t s u b s t a n c e P - l i k e i m m u n o f l u o r e s c e n c e i n t h e EP d i s a p p e a r e d f o l l o w i n g l e s i o n s s i m i l a r t o t h o s e u s e d i n t h e p r e s e n t s t u d y . T h e p r e s e n t d a t a c o n f i r m t h a t t h e m a j o r i t y o f t h e s u b s t a n c e P i n t h e EP i s p r o b a b l y p r e s e n t i n a f f e r e n t s f r o m t h e h e a d o f t h e s t r i a t u m . I n a d d i t i o n t o a G A B A - c o n t a i n i n g s t r i a t o - p a l l i d a l t r a c t , G A D - c o n t a i n i n g s t r i a t o e n t o p e d u n c u l a r f i b e r s h a v e a l s o b e e n r e c e n t l y d e m o n s t r a t e d ( F o n n u m e t a l . , 1 9 7 8 a ; N a g y e t a l . , 1 9 7 8 a ) . T h e p r e s e n t r e s u l t s s u p p o r t t h e p r e v i o u s e v i d e n c e f o r s u c h a p a t h w a y . T h e h e m i t r a n s e c t i o n s d e c r e a s e d EP GAD t o t h e same e x t e n t a s p r e v i o u s l y r e p o r t e d b y N a g y e t a l . ( 1 9 7 8 a ) . I n c o n t r a s t , t h e l e s i o n s e m p l o y e d t o p r o d u c e t h e GP i s l a n d s , w h i c h s t a r t e d m o r e a n t e r i o r t h a n t h e h e m i t r a n s e c t i o n s a n d s w e p t w i d e o f t h e GP l a t e r a l l y l e a v i n g some s t r i a t a l t i s s u e i n t a c t m e d i a l t o t h e l e s i o n , d i d n o t r e s u l t i n a s l a r g e a d e c r e a s e . T h i s i n d i c a t e s t h a t a m a j o r p o r t i o n o f t h e GABA i n p u t t o t h e EP a r i s e s f r o m s t r i a t a l c e l l s b o r d e r i n g t h e a n t e r i o r p o l e o f t h e G P . T h i s i s i n a g r e e m e n t w i t h t h e o b s e r v a t i o n s o f F o n n u m e t a l . ( 1 9 7 8 a ) who f o u n d t h a t s u c c e s s i v e l y m o r e p o s t e r i o r l e s i o n s i n v o l v i n g t h e h e a d o f t h e s t r i a t u m l e d t o p r o g r e s s i v e l y g r e a t e r r e d u c t i o n s i n e n t o - p e d u n c u l a r GAD a c t i v i t y . T h e l e v e l s o f m e t - e n k e p h a l i n i n t h e E P w e r e f o u n d t o b e o n l y s l i g h t l y l e s s t h a n t h o s e i n t h e s t r i a t u m ; h o w e v e r , t h e y w e r e n o t a f f e c t e d b y e i t h e r 24 type of l e s i o n . This suggests that, i n contrast to the s t r i a t o p a l l i d a l p r o jection, the afferents to the EP from the head of the striatum do not contain met-enkephalin. F i n a l l y , since CAT a c t i v i t y i n the EP was unaffected by any of the lesions i t would appear that acetylcholine i s not a transmitter i n the striato-entopeduncular projection. (c) The Substantia Nigra The SN was found to contain very low l e v e l s of met-enkephalin. The l e s i o n data i n d i c a t e that the SN, l i k e the EP, receives no c h o l i n e r g i c or met-enkephalin-containing input from the striatum. The absence of a c h o l i n e r g i c 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 n agreement with previous reports (Fonnum et a l . , 1978a; McGeer et a l . , 1971). Since the discovery of the existence of a GABA-containing p r o j e c t i o n from the s t r i a t o p a l l i d a l complex to the SN, numerous studies have been conducted to determine the exact l o c a t i o n of the neurons g i v i n g r i s e to this pathway (Brownstein et a l . , 1977; Fonnum et a l . , 1978a, Gale et a l . , 1977; J e s s e l et a l . , 1978). These recent studies and, i n p a r t i c u l a r , that by Brownstein et a l . (1977) have considerably c l a r i f i e d t h i s issue. These workers suggested that there i s a concentration of GAD-containing n i g r a l p r o j e c t i o n neurons i n the striatum j u s t anterior to the GP. The present r e s u l t s are i n agreement with t h i s proposal. Hemitransections i n the striatum near the r o s t r a l t i p of the GP, reduced n i g r a l GAD a c t i v i t y s u b s t a n t i a l l y . However, the GP i s l a n d l e s i o n s , which began at a more r o s t r a l l e v e l , did not s i g n i f i c a n t l y decrease n i g r a l GAD a c t i v i t y . These r e s u l t s together with recent observations (DiChiara et a l . , 1980; Nagy and F i b i g e r , 1980) that KA i n j e c t i o n s into the GP do not s i g n i f i c a n t l y a l t e r n i g r a l GAD are i n f u l l agreement with Brownstein et a l . (1977) who have suggested that the o r i g i n of the majority of GAD i n the SN i s derived 25 from neurons located outside the pallidum but cl o s e l y apposed to i t . I t i s noteworthy that while GP i s l a n d lesions did not decrease GAD i n the SN and only produced a s l i g h t decrease i n the EP GAD a c t i v i t y , the decrease i n the GP was only s l i g h t l y l e s s than a f t e r the hemitransection. This indicates that the GABA-containing neurons innervating the SN and EP can at least p a r t i a l l y be dissoc i a t e d from those p r o j e c t i n g to the GP. Of a l l the brain areas that have been examined, the SN contains the highest l e v e l s of substance P (Brownstein et a l . , 1976; Kanazawa and Je s s e l , 1976). In Huntington's chorea substance P l e v e l s and GAD a c t i v i t y have both been found to decrease i n the SN (Kanazawa et a l . , 1977b). A sim i l a r p a r a l l e l decrease has been observed following hemitransections at the l e v e l of the l a t e r a l hypothalamus (Mroz et a l . , 1977). These r e s u l t s led to the suggestion that both GABA and substance P are transmitters i n the s t r i a t o n i g r a l pathway (Hong et a l . , 1977c; Kanazawa et a l . , 1977b; Mroz et a l . , 1977; Paxinos et a l . , 1978b). More recent work has shown that the substance P and GAD-containing efferents appear to be d i s s o c i a b l e . The substance P f i b e r s appear to a r i s e from the an t e r i o r striatum and not from the caudal and l a t e r a l areas (Brownstein et a l . , 1977; Gale et a l . , 1977; Je s s e l et a l . , 1978; Palkovits et a l . , 1978). In contrast and as discussed above, the GABA afferents to the SN a r i s e from s t r i a t a l neurons c l o s e l y apposed to the GP (Brownstein et a l . , 1977). That the two lesio n s employed i n the present study produced a s i m i l a r depletion i n n i g r a l substance P, but had d i f f e r e n t e f f e c t s on n i g r a l GAD a c t i v i t y i s i n agree- ment with t h i s segregation of these two n i g r a l a f f e r e n t systems, (d) The Striatum The increases i n GAD a c t i v i t y and met-enkephalin l e v e l s i n the striatum anterior to the hemitransections and GP i s l a n d l e s i o n s could be due eit h e r to the shunting of enzyme and peptide to l o c a l c o l l a t e r a l branches or 26 terminals a f t e r severing long axon efferent c o l l a t e r a l s (Storm-Mathisen, 1975), or to an a l t e r a t i o n i n the a c t i v i t y of the neurons containing GAD and met-enkephalin. That t h i s change does not merely r e f l e c t t i s s u e shrinkage i s indicated by the unchanged l e v e l s of substance P and the normal CAT a c t i v i t y i n t h i s same tis s u e . At le a s t a portion of the GAD increase i n the head of the striatum can be att r i b u t e d to the severing of the dopaminer- gic input to this structure since we have found that 6-hydroxydopamine (6-OHDA) lesions of the n i g r o s t r i a t a l pathway increase GAD a c t i v i t y i n the striatum (Experiment 5). Hemitransections caudal to the GP do not appear to r e s u l t i n an increase i n the met-enkephalin l e v e l s i n the striatum (Pollard et a l . , 1978). This suggests that t h i s increase i s due to d i s - ruption of the s t r i a t o p a l l i d a l met-enkephalin pathway. Further work i s necessary to determine the s i g n i f i c a n c e of these changes i n the function- ing of the striatum and i n i t s recovery from i n j u r y . 27 EXPERIMENT 2: THE IMMUNOHISTOCHEMICAL DEMONSTRATION 0? SUBSTANCE P IN THE BASAL GANGLIA The undecapeptide substance P i s contained i n the efferents of the striatum, where i t may function as a neurotransmitter. In Experiment 1, the existence of substance P-containing projections from the head of the striatum to the SN (Brownstein et a l . , 1977; Gale et a l . , 1977; Hong et a l . , 1977c; J e s s e l et a l . , 1978; Kanazawa et a l . , 1977a; Paxinos et a l . , 1978a) and EP (Paxinos et a l . , 1978b) was confirmed using a radioimmuno- assay procedure. In addition, the presence of substance P i n the s t r i a t a l p r ojection to the GP was demonstrated. In the present study an immuno- histochemical approach was used to provide information on the d i s t r i b u t i o n of substance P f i b e r s i n these and other brain n u c l e i . Previous immunofluorescent studies have demonstrated dense p l e x i of substance P terminals i n the SN (Cuello and Kanazawa, 1978; Cuello et a l . , 1979; HSkfelt et a l . , 1977; J e s s e l et a l . , 1978; Ljungdahl et a l . , 1978a; 1978b; Paxinos et a l . , 1978a), EP (Cuello and Kanazawa, 1978; Ljungdahl et a l . , 1978a; Paxinos et a l . , 1978b; J e s s e l et a l . , 1978), and GP (LjUngdahl et a l . , 1978a). These fluorescent studies have been of great value i n the mapping of the substance P systems of the b r a i n . The f l u o r e s - cent method does not however provide the r e s o l u t i o n necessary to d i s c e r n the precise synaptic r e l a t i o n s h i p s of the substance P terminals and c e l l s . In p a r t i c u l a r the method i s not compatible with e l e c t r o n microscopic studies. For t h i s the immunoperoxidase method i s a v a i l a b l e . Immunoperoxidase stainin g f or substance P has been performed at both the l i g h t and electron microscopic l e v e l s i n the s p i n a l cord (Barber et a l . , 1979; HOkfelt et a l . , 1977; P i c k e l et a l . , 1977; Vacca et a l . , 1980), medulla (Chan-Palay, 1978; P i c k e l et a l . , 1979) and amygdala ( P e l l e t i e r et a l . , 1977) using the peroxidase-antiperoxidase (PAP) method developed by 28 Sternberger et a l . (1970). These studies have provided information on the l o c a l i z a t i o n of substance P i n nerve terminals i n synaptic r e l a t i o n - ships of various types. In the present study an immunoperoxidase pro- cedure was used to demonstrate substance P i n some terminal f i e l d s i n the basal ganglia. METHODS I n i t i a l l y the t r a d i t i o n a l PAP procedure of Sternberger et a l . (1970) was attempted using the guinea pi g antisera to substance P described i n the Appendix. This method was abandoned when a f t e r a year of unsuccessful work a l e t t e r was received from Cappel Laboratories s t a t i n g that t h e i r guinea pig PAP was being discontinued due to " t e c h n i c a l problems". As an a l t e r n a t i v e method, the b i o t i n - a v i d i n (BA) procedure has been u t i l i z e d (Heitzmann et a l . , 1974; Guesdon et a l . , 1979). As t h i s i s the f i r s t time that t h i s method has been used i n immunohistochemical studies of the nervous system, the t h e o r e t i c a l basis of t h i s technique w i l l be described i n some d e t a i l and contrasted with the more f a m i l i a r PAP method (Fig. 2). In both the BA technique and the PAP procedure, the i n i t i a l incubation of the t i s s u e i n guinea p i g anti-substance P sera i s the same (Step 1). In the PAP procedure the second step involves incubation i n rabbit anti-guinea p i g sera. This must be used at high concentration i n order to leave one binding s i t e of the rabbit a n t i s e r a free a f t e r reaction with the primary antibody. The use of high concentrations of t h i s sera increases the amount of nonspecific binding of t h i s sera to the tissue and t h i s can increase the a r t i f a c t u a l s t a i n i n g . In the BA procedure, the b i o t i n y l a t e d rabbit anti-guinea p i g sera i s used i n place of the rabbit antisera used i n the PAP procedure. I t has been found that extensive b i o t i n y l a t i o n of antibodies does not modify t h e i r antigen bind- ing capacity (Guesdon et a l . , 1979). In the BA procedure the second step 29 Figure 2: A comparison of the b i o t i n - a v i d i n (BA) method (b) with the peroxidase-antiperoxidase (PAP) procedure (a) for immuno- histochemistry. In Step 1 guinea p i g - a n t i substance P sera (GPA) binds to substance P i n the t i s s u e . In Step 2 of the PAP procedure r a b b i t - a n t i guinea p i g IgG (RAGP) binds the GPA and then the PAP i n Step 3. In the BA procedure the B i o t i n residues attached to the RAGP bound i n Step 2 are bound by a v i d i n (A) which has been coupled to horseradish peroxidase (HRP).  30 can be used at high d i l u t i o n since i t i s not necessary to have a free antibody binding s i t e a v a i l a b l e for Step 3. It i s i n the t h i r d step where the BA system i s most d i f f e r e n t from the PAP approach. In the PAP procedure, the free binding s i t e l e f t i n the second step binds the PAP complex which acts as an antigen. In the BA method the b i o t i n residues attached to the second antibody are bound by a v i d i n to which horseradish peroxidase has been chemically coupled. The i n t e r a c t i o n between a v i d i n and b i o t i n i s non-covalent, but extremely strong (Kd = 10 15M; Green, 1963). In f a c t the a f f i n i t y i s s i x to eight orders of magnitude higher than that of most antibodies for antigen. This means that the avidin-peroxidase complex can be used at very high d i l u t i o n . This again greatly reduces the chances for non-specific binding. Also, extensive washing can be performed a f t e r the incubation without d i s s o c i a - t i o n of the a v i d i n - b i o t i n complex since the half-time of d i s s o c i a t i o n requires months. In the present study the d i s t r i b u t i o n of substance P immunoreactivity was examined i n the cat brain using the BA procedure. Adult male cats were deeply anesthetized with pentobarbital and perfused through the heart with phosphate buffered (0.1 M, pH 7.4) s a l i n e (PBS) followed by cold 4% paraformaldehyde and 0.3% glutaraldehyde i n 0.1 M phosphate buffer pH 7.4. The brain was removed, cut into 5 mm coronal sections and kept i n 4% paraformaldehyde at 4°C for 6 hr. The t i s s u e was then stored overnight i n the buffer containing 15% sucrose and cut into 25 micron thick sections on a cryostat at -20°C. Sections f o r immunohistochemistry were washed i n PBS containing 0.01% T r i t o n X-100 for one hr at room temperature and then incubated overnight at 4°C i n guinea p i g - a n t i substance P sera (see Appendix) d i l u t e d 1:2,000 i n PBS containing 0.05% T r i t o n X-100. The sections were then washed i n PBS and incubated for two 31 Figure 3. Control section of substantia nigra incubated with pre- absorbed anti-substance P sera. Only a l i g h t brown background e x i s t s , and no s p e c i f i c substance P s t a i n i n g occurs. 23X.  32 hr at room temperature with b i o t i n y l a t e d rabbit anti-guinea p i g IgG (E-Y Labs.) d i l u t e d 1:200 i n PBS containing 0.01% T r i t o n X-100. Again the sections were washed i n PBS and f i n a l l y incubated for one hr at room temperature i n horseradish peroxidase-avidin (Vector Labs. Inc.) d i l u t e d 1:500 i n PBS. The sections were f i n a l l y washed i n PBS for one hr and the peroxidase then reacted for 20 min with 0.02% (w/v) 3,3'-diaminobenzidine (Sigma) and 0.006% H 20 z i n 50 mM T r i s - C l b u f f e r , pH 7.6. The. sections were then washed and mounted from PBS. The mounted sections were soaked i n d i s t i l l e d water 30 min to remove s a l t s and detergent and then treated with 0.01% (w/v) osmium tetroxide for three min to enhance contrast. Control sections were processed i d e n t i c a l l y except that the i n i t i a l a n t i s era was preabsorbed with 100 ug of synthetic substance P (Beckman) per ml at room temperature f or four hr p r i o r to use i n immunohistochemistry. Sections incubated with t h i s pre-absorbed sera did not ex h i b i t any s p e c i f i c s t a i n i n g (Fig. 3). RESULTS In sections from untreated adult cats which were stained f o r substance P immunoreactivity using the BA procedure immunoreactive c e l l bodies were not c o n s i s t e n t l y detected. This i s i n agreement with previous immuno- fluorescent studies i n the rat i n which c e l l bodies could only be repro- ducibly demonstrated following c o l c h i c i n e pretreatment (Cuello and Kanazawa, 1978; Ljiingdahl et a l . , 1978a). This presumably in d i c a t e s that the c e l l bodies normally contain very low l e v e l s of substance P. Although p o s i t i v e c e l l bodies were not apparent, widespread networks of substance P-positive terminals and f i b e r s were observed through the brain and s p i n a l cord. In the present report we s h a l l confine our discussion to substance P-positive structures i n areas associated with the basal ganglia. Both the putamen (Fig. 4a) and the caudate (Fig. 4b) contained a 33 d e l i c a t e network of f i n e f i b e r s which could be observed forming c l u s t e r s of axo-somatic contacts with medium siz e s t r i a t a l neurons. A s i m i l a r network of f i b e r s was also observed i n the claustrum, p a r t i c u l a r l y i n the ventro-medial portion of t h i s nucleus (Fig. 4c). Within the GP (Fig. 4d) and the EP (Fig. 4f) the neuropil was rather densely stained. In both areas varicose f i b e r s appeared to out l i n e the d e n d r i t i c processes of the p a l l i d a l neurons, presumably forming synapses along t h e i r length. The ansa l e n t i c u l a r i s , i n which the s t r i a t o n i g r a l substance P f i b e r s are thought to course (Palkovits et a l . , 1978), contained a dense network of varicose f i b e r s (Fig. 4e). Although not previously discussed i n t h i s report the amygdaloid complex i s another telencephalic structure often included i n the basal ganglia. Certain amygdaloid n u c l e i s t a i n very strongly for substance P. The c o r t i c a l nucleus contains an extremely dense network of p o s i t i v e l y stained punctate structures throughout i t s neuropil ( F i g . 5b). A somewhat less dense network i s present i n the medial amygdala where long varicose f i b e r s could often be seen (Fig. 5c). A dense punctate network of substance P-positive f i b e r s was also observed i n the l a t e r a l habenula, p a r t i c u l a r l y i n the medial portion of thi s nucleus (Fig. 5a). In the mesencephalon a d i f f u s e network of varicose axons appears to form a s h e l l over the dorsal and l a t e r a l borders of the interpeduncular nucleus (Fig. 6c). The s t a i n i n g i s strongest i n the v e n t r o - l a t e r a l portion of t h i s s h e l l where i t apparently i s i n continuum with the substance P immunoreactivity i n the SN. Within the SN the substance P-positive structures form a massive, d i f f u s e and punctiform pattern. The s t a i n i n g i s most intense i n the SNR (Fig. 6b). Bundles of intensely stained neuropil 34 Figure 4. Substance P immunoreactivity i n coronal sections of thereat basal ganglia demonstrated with the b i o t i n - a v i d i n method. In (a) a sparse network of p o s i t i v e l y stained puncta i s apparent. These can often be seen forming terminals about the medium size c e l l s (arrows). F i g i .(b) shows a s i m i l a r pattern of substance P s t a i n i n g i n the caudate nucleus. Again the punctate f i b e r s can often be seen covering the unstained c e l l bodies (arrows). Fig . (c) shows the same sparse pattern of punctate s t a i n i n g e x i s t s i n the claustrum. The c e l l bodies show a s l i g h t non- s p e c i f i c background s t a i n i n g . In (d) the neuropil of the globus p a l l i d u s can be seen to contain a dense network of substance P-positive f i b e r s . These appear to cover the c e l l bodies and major dendrites (arrow) of the pallidum with many v a r i c o s i t i e s . F i g . (e) demonstrates the substance P - l i k e immunoreactivity i n the f i b e r s of the ansa l e n t i c u l a r i s . These f i b e r s appear to have many v a r i c o s i t i e s along t h e i r length. The entopeduncular nucleus (f)-. the patternof s t a i n i n g i s very s i m i l a r to that seen i n the globus p a l l i d u s (d). Again the stained puncta cover the c e l l bodies (arrow) and appear to follow the major dendrites through the neuropil. C a l i b r a t i o n bars represent 20 microns in. a l l f i g u r e s .  35 Figure 5. Substance P immunoreactive f i b e r s demonstrated with the b i o t i n - a v i d i n immunoperoxidase procedure. In (a) the dense network of p o s i t i v e puncta found i n the medial portion of the l a t e r a l habenula nucleus i s shown i n a coronal section. A s i m i l a r dense network can also be seen i n the neuropil of the corticomedial amygdala (b). In t h i s coronal section the varicose f i b e r s appear to branch throughout the neuropil. Occasionaly, such as i n t h i s coronal section of the medial amygdala (c) substance P-containing varicose axons can be followed for some distance. The v a r i c o s i t i e s appear to be evenly spaced and about 2 microns apart. C a l i b r a t i o n bar represents 20 microns for a l l three photographs. 35 a 36 c a n be s e e n e x t e n d i n g f r o m t h e SNR i n t o t h e s u b a d j a c e n t c r u s c e r e b r i . I n t h e SNC t h e s u b s t a n c e P p o s i t i v e f i b e r s a p p e a r t o r u n i n a h o r i z o n t a l o r i e n t a t i o n . T h e u n s t a i n e d c e l l b o d i e s o f t h e SNC a r e q u i t e a p p a r e n t a n d d o n o t a p p e a r t o r e c e i v e many a x o - s o m a t i c c o n t a c t s . D I S C U S S I O N I n t h e p r e s e n t s t u d y we h a v e p r o v i d e d t h e f i r s t d e m o n s t r a t i o n o f s u b s t a n c e P - i m m u n o r e a c t i v i t y i n t h e b a s a l g a n g l i a w i t h a n i m m u n o p e r o x i d a s e t e c h n i q u e . T h e f i n d i n g s c o n f i r m a n d e x t e n d t h e p r e v i o u s r e p o r t s o f s u b s t a n c e P - i m m u n o r e a c t i v i t y i n t h e r a t b r a i n u s i n g i m m u n o f l u o r e s c e n t t e c h n i q u e s ( C u e l l o a n d K a n a z a w a , 1 9 7 8 ; L j u n g d a h l e t a l . , 1 9 7 8 a ) . I n t h o s e s t u d i e s a n e t w o r k o f f i n e , w e a k l y f l u o r e s c e n t s t r u c t u r e s w e r e o b s e r v e d i n t h e r a t s t r i a t u m . I n o u r m a t e r i a l a s i m i l a r f i n e n e t w o r k o f s u b s t a n c e P - i m m u n o r e a c t i v i t y wa s o b s e r v e d i n b o t h t h e c a u d a t e n u c l e u s a n d t h e p u t a m e n o f t h e c a t . No d i f f e r e n c e s i n t h e s t a i n i n g b e t w e e n t h e s e t w o n u c l e i w e r e a p p a r e n t . T h i s n e t w o r k p r o b a b l y a r i s e s f r o m l o c a l c o l l a t e r a l s o f t h e s u b s t a n c e P c e l l b o d i e s w h i c h h a v e b e e n o b s e r v e d i n t h e s t r i a t u m ( C u e l l o a n d K a n a z a w a , 1 9 7 8 ; J e s s e l e t a l . , 1 9 7 8 ; K a n a z a w a e t a l . , 1 9 7 7 ) . I n b o t h t h e GP a n d t h e EP t h e d e n s e p l e x u s o f f i b e r s p r e s e n t l i k e l y r e p r e s e n t s t h e t e r m i n a l s o f t h e s t r i a t o p a l l i d a l s u b s t a n c e P - c o n t a i n i n g p a t h w a y s ( s e e E x p e r i m e n t 1 ) . T h e s e f i b e r s o f t e n s e e m e d t o o u t l i n e t h e m a j o r d e n d r i t i c p r o c e s s e s p e r h a p s f o r m i n g s y n a p s e s e n p a s s a n t ( F o x e t a l . , 1 9 7 4 ) . I n p r e v i o u s i m m u n o f l u o r e s c e n t s t u d i e s C u e l l o a n d K a n a z a w a ( 1 9 7 8 ) r e p o r t e d a v e r y c o m p a c t n e t w o r k o f f i b e r s i n t h e m e d i a l a n d t h e c o r t i c a l a m y g d a l o i d n u c l e i . L j u n g d a h l e t a l . ( 1 9 7 8 a ) r e p o r t v e r y d e n s e s t a i n i n g i n t h e c a u d a l p o r t i o n o f t h e m e d i a l n u c l e u s , b u t o n l y s i n g l e f i b e r s i n t h e c o r t i c a l a m y g d a l a . T h e m e d i a l n u c l e u s i n t h e r a t h a s b e e n f o u n d t o c o n t a i n v e r y h i g h l e v e l s o f s u b s t a n c e P b y r a d i o i m m u n o a s s a y , w h i l e l e v e l s 37 Figure 6. Substance P - l i k e immunoreactivity i n coronal sections of the v e n t r a l mesencephalon demonstrated with the b i o t i n - a v i d i n immunoperoxidase technique. In (a) the dense f i b e r system occurs throughout the neuropil of the substantia nigra pars compacta. The c e l l s of the pars compacta are unstained (arrows) and do not appear to receive many axosomatic substance P synapses. Some substance P-positive f i b e r s extend d o r s a l l y from the nigra into the medial lemniscus (ml). In (b) the intense s t a i n i n g which e x i s t s i n the neuropil of the substantia nigra pars r e t i c u l a t a (snr) i s demon- strated. Bundles of f i b e r s can be seen extending v e n t r a l l y from the nigra into the crus c e r e b r i (cc). In (c) a d i s c r e t e network of substance P f i b e r s can be seen at the v e n t r o - l a t e r a l border of the interpeduncular nucleus (ip) adjacent to the v e n t r a l tegmental area (vta) C a l i b r a t i o n bar represents 50 microns f o r a l l three f i g u r e s .  38 i n the c o r t i c a l nucleus appear quite low (Ben-Ari et a l . , 1977). In the present study, the corticomedial nucleus of the cat was found to s t a i n most intensely for substance P, with the f i b e r s often forming basket-like structures about the c e l l bodies. Perhaps t h i s indicates a homology between the nucleus amygdaloideus medialis of the rat and the c o r t i c o - medial nucleus of the cat. In both species i t i s apparent that most of the substance P i s present i n the more p r i m i t i v e components of the amygda- l o i d complex. The intense s t a i n i n g observed i n the l a t e r a l habenula i s consistent with the previous immunofluorescent studies (Cuello and Kanazawa, 1978; Ljungdahl et a l . , 1978a; Cuello et a l . , 1978). These studies have shown that the substance P terminals l i e i n the medial portion of the l a t e r a l habenula. A s i m i l a r r e s u l t was observed i n the present study. In addition, a previously unknown band of dense terminals overlying the d o r s o - l a t e r a l surface of t h i s nucleus was observed. The l o c a t i o n of the substance P terminals i n the l a t e r a l habenula appears to be quite d i s t i n c t from that of the GABA terminals which appear to l i e i n the v e n t r o - l a t e r a l portion of the l a t e r a l habenula (see Experiment 4). Although the substance P terminals have been suggested to a r i s e from c e l l s i n the medial habenula (Cuello et a l . , 1978) anatomical studies have f a i l e d to demonstrate a connection between these n u c l e i (Herkenham and Nauta, 1979; Iwahori, 1977). Thus, the o r i g i n of the substance P i n the l a t e r a l habenula remains i n doubt. The medial habenula has also been suggested to be the source of the substance P i n the interpeduncular nucleus (Cuello et a l . , 1978; Emson et a l . , 1977; Hong et a l . , 1976; Mroz et a l . , 1976). In agreement with the immunofluorescent studies i n the rat 'the substance P immunoreactivity found i n the cat forms a s h e l l about the dorsal and l a t e r a l borders of the 39 interpeduncular nucleus adjacent to the area of the dopaminergic A-10 c e l l s (Dahlstrom and Fuxe, 1964). The ce n t r a l core of the interpedun- cular nucleus i s r e l a t i v e l y barren of substance P terminals. As the medial habenula i s known to provide the major innervation to th i s p a r t of the interpeduncular nucleus (Herkenham and Nauta, 1979), i t appears that the major transmitters i n t h i s system remain to be determined. Within the SN the substance P immunoreactivity i s most intense i n the SNR, i n agreement with radioimmunoassay data obtained i n the cat SN (Gauchy et a l . , 1979). The s t a i n i n g i n the SNC was less intense but many varicose f i b e r s were s t i l l apparent. This may.indicate that the dopamine c e l l s of the A9 group.and, i n p a r t i c u l a r , t h e i r d e n d r i t i c processes i n the SNR are d i r e c t l y innervated by substance P terminals (Ljungdahl et a l . , 1978b). This idea i s supported by the observation that iontopho- r e t i c a l l y applied substance P excites the dopaminergic c e l l s of the SN (Davies and Dray, 1976; Walker et a l . , 1976). Behavioural (James and Starr, 1977; 1979; Kelley and Iversen, 1978; 1979; Kelley et a l . , 1979; Olpe and Ko e l l a , 1977) and biochemical (Cheramy et a l . , 1977; 1978; Magnusson et a l . , 1976; Starr, 1978b) observations also support such an in t e r a c t i o n . Hopefully the extension of the immunoperoxidase procedure developed i n the present study to the electron microscopic l e v e l ( P i c k e l , 1979) w i l l allow the d i r e c t v i s u a l i z a t i o n of the synaptic r e l a t i o n s h i p s between the substance P terminals and the dopamine c e l l s of•the SN. 40 EXPERIMENT 3: THE LOCALIZATION OF GABA-TRANSAMINASE IN THE STRIATO-NIGRAL SYSTEM. In Experiment 1 the existence of GABA projections from the striatum to the GP, EP and SN was confirmed. Although these biochemical experi- ments provide good evidence for the existence of GABA pathways, they provide no information regarding the topographic d i s t r i b u t i o n of the GABA innervations. For t h i s a r e l i a b l e morphological technique i s required. A fluorescent method f or GABA has been devised, but the r e s u l t s obtained with t h i s technique have not been promising (Wolman, 1971). The immuno- histochemical method for GAD has made possible the v i s u a l i z a t i o n of GAD- containing nerve terminals and c e l l s (Roberts, 1979); however, t h i s method, has not been used to characterize the topography of GABA projections and the complexity of the technique precludes i t s general usage. Although a simple histochemical procedure for GABA-transaminase (GABA-T), the enzyme which catabolizea GABA, has long been known (Van Gelder, 1965) i t s use i n examining GABA systems has not been explored. High l e v e l s of GABA-T have been found i n the basal ganglia (Salvador and Albers,, 1959), and the striatum and SN have been found to exhibit intense histochemical s t a i n i n g for t h i s enzyme.(Robinson and Wells, 1973). However, the precise l o c a l i z a t i o n of GABA-T has not been c l e a r l y resolved. Histochemical studies have suggested that GABA-T a c t i v i t y could be present i n nerve c e l l s , terminals or g l i a l elements i n the basal ganglia, although which of these elements a c t u a l l y contains the enzyme has been d i f f i c u l t to discern (Robinson and Wells, 1973). High a f f i n i t y GABA uptake i s thought to be pr i m a r i l y responsible for the removal of s y n a p t i c a l l y released GABA (Martin, 1976), but recent studies using s p e c i f i c GABA-T i n h i b i t o r s have indicated that GABA-T may be d i r e c t l y involved i n regulating the transmitter pool of GABA. Thus, 41 i n t r a c e r e b r a l or systemic i n j e c t i o n s of GABA-T i n h i b i t o r s r e s u l t i n a marked elevation of bra i n GABA l e v e l s (Rando and Bangerter, 1977; Matsui and Kamioka, 1978). Also l o c a l i n j e c t i o n s of GABA-T i n h i b i t o r s into the basal ganglia r e s u l t i n marked behavioural e f f e c t s , i n v o l v i n g both GABA systems and the n i g r o s t r i a t a l dopaminergic system (Pycock et a l . , 1976; Matsui and Kamioka, 1978). The l o c a l i z a t i o n of GABA-T i n the basal ganglia has therefore been biochemically examined i n order to evaluate the s u i t a b i l i t y of t h i s enzyme for the histochemical demonstration of GABA pathways. METHODS To destroy the s t r i a t a l neurons, KA was inject e d into the caudate- putamen as previously described (McGeer and McGeer, 1976a). Nine rats received a u n i l a t e r a l i n j e c t i o n of f i v e nmoles of KA i n 0.5 y l 50 mM NaPOi+j pH 7, at a rate of 1 yl/5 min. An a d d i t i o n a l group of rats received an i n j e c t i o n of ten nmoles of KA i n 1 y l . Two weeks l a t e r , the rats were s a c r i f i c e d by c e r v i c a l f r a c t u r e , and the striatum was dissected on i c e . The SN was obtained from sections cut on a freezing microtome. Campochiara and Coyle (1978) and Lehmann and Fi b i g e r (1979) have found that i n j e c t i o n s of KA into the striatum of neonatal rats p r e f e r e n t i a l l y deplete CAT as compared to GAD. In the present report we have examined the e f f e c t of such les i o n s on GABA-T a c t i v i t y . Ten day old rats were inject e d with 20 nmoles of KA i n 1 y l of sodium phosphate buffered (pH 7.4) isosmolar Ringer s o l u t i o n . Fourteen days l a t e r the animals were s a c r i f i c e d and the striatum dissected for enzyme analyses. In an a d d i t i o n a l group of adult animals, 2.9 yg of 6-OHDA i n 1 y l of 0.9% s a l i n e , 0.1% ascorbate was inject e d into the l e f t n i g r o s t r i a t a l bundle to destroy s e l e c t i v e l y the n i g r o s t r i a t a l dopaminergic neurons (Clavier and Fi b i g e r , 1977). The i n j e c t i o n s were made at A + 4.4; L + 1.8; DV - 2.4 mm, 42 according to the a t l a s of Kb'nig and K l i p p e l (1963). These animals received 25 mg/kg desipramine 30 min p r i o r to the 6-OHDA to prevent damage to the noradrenergic neurons (Roberts et a l . , 1975). One month a f t e r the l e s i o n the striatum and SN were dissected as described above. For the biochemical analyses the tissues were homogenized i n 50 mM phosphate buffer, pH 7.4, containing 0.25% T r i t o n X-100. Tyrosine hydro- xylase was measured as previously described (McGeer et a l . , 1967). Forty y l of tissue homogenate were added to 80 y l of a mixture containing ( f i n a l concentrations) 1.0 mM 2-amino-5-hydroxy-6,7-dimethyl tetrahydropteridine (DMPH4, Sigma) , 0.1 mM 1- [ l t +C (U) ] tyrosine ( s p e c i f i c a c t i v i t y 3 to 5 mCi/ mmol, New England Nuclear), 0.3 mM f e r r i c sulphate, 50 mM 2-mercapto-ethanol (Eastman) i n 0.2 M sodium acetate buffer, pH 6.0. The re a c t i o n was incubated for 12 min at 37°C, then stopped by the addition of two ml of a s o l u t i o n containing 1.4% p e r c h l o r i c a c i d , 0.52% a c e t i c acid and 0.5 yg/ml of dihydro- xyphenylalanine. The reaction tubes were centrifuged at 1,000 g for f i v e min and the supernatants transferred to 25 ml beakers. The p e l l e t s were rinsed with two ml of 0.35 M potassium phosphate buffer, pH 6.0, centrifuged as before and the supernatants added to those i n the beakers. Twelve ml of 28 mM Na2EDTA were then added to each beaker and the samples were brought to pH 9 to 9.5 and then poured onto columns packed with about 0.3 g of alumina (Calbiochem, a c i d , AG^, 100-200 mesh). The columns were washed with 35 ml of d i s t i l l e d water, eluted into s c i n t i l l a t i o n v i a l s with 2.0 ml of 0.5 M a c e t i c acid and counted i n 14 ml of ACS (Amersham). The method of S t e r r i and Fonnum (1978) was used to assay GABA-T a c t i v i t y . Twenty y l of t i s s u e homogenate was placed d i r e c t l y into s c i n t i l l a - t i o n v i a l s i n an i c e bath. F i f t y y l of re a c t i o n mixture containing ( f i n a l concentrations) 5 mM y-amino [U- 1 1 +C] butyric acid ( s p e c i f i c a c t i v i t y 0.71 mCi/ mmol, Amersham), 2 mM a-ketoglutaric acid (Sigma), 10 mM d i t h i o t h r i e t o l 43 (Sigma), 3 mM ni c o t i n e adenine dinucleotide (NAD, Calbiochem), 1 mM succinate (Sigma), 0.3 mM pyridoxal phosphate (Calbiochem) i n 50 mM T r i s - C l buffer pH 8.2, containing 0.2% T r i t o n X-100 were then added and the v i a l s incubated at 37°C for 20 min. The reaction was stopped by the addition of one ml of 0.1 M sodium phosphate buffer, pH 7.4. The labeled succinate formed i n the reaction was extracted from the aqueous phase by the addition of one ml of isoamyl alcohol containing 0.2 M t r i - n - o c t y l - ammonium phosphate, f r e s h l y prepared from tri-n-octylamine (Sigma) according to the method of S t e r r i and Fonnum (1978). Ten ml of the toluene- based f l u o r used i n the CAT assay were then added and the r a d i o a c t i v i t y i n the organic phase counted. Before use i n the assay, the commercial sample of radioactive GABA was p u r i f i e d by extracting once with an equal volume of 20 mM tri-n-octylammonium phosphate i n chloroform. CAT and GAD were assayed according to the procedures outlined i n Experiment 1, and protein was determined by the method of Lowry et a l . (1951). RESULTS One month a f t e r the i n j e c t i o n of 6-OHDA, the a c t i v i t y of tyrosine hydroxylase i n the striatum was reduced by hal f i n d i c a t i n g a s i g n i f i c a n t destruction of the n i g r o s t r i a t a l dopamine neurons (Table 5). The a c t i v i t i e s of CAT and GAD i n the striatum were not s i g n i f i c a n t l y affected nor was GAD a c t i v i t y i n the SN alt e r e d . This l e s i o n had no e f f e c t on the GABA-T a c t i v i t y of either the striatum or the SN. This agrees with the report of Kim (1973) who found no change i n s t r i a t a l GABA-T a c t i v i t y four days a f t e r i n t r a v e n t r i c u l a r 6-OHDA. Following the i n j e c t i o n of KA into the striatum, the a c t i v i t i e s of s t r i a t a l CAT and GAD were reduced i n a dose-dependent manner. A s i m i l a r dose-dependent decrease was seen i n s t r i a t a l GABA-T a c t i v i t y (Table 6). 44 Table 5. THE ACTIVITIES OF NEUROTRANSMITTER-RELATED ENZYMES IN THE STRIATUM AND SUBSTANTIA NIGRA ONE MONTH AFTER INJECTION OF 6-OHDA INTO THE NIGROSTRIATAL BUNDLE . Control Lesion (nmol/mg protein/hr S.E.M.) % of cont r o l Striatum Tyrosine hydroxylase 82.0±3.0 39.0±7.8 48* Choline acetyltransferase 140.4±6.7 142.5±2.7 101 Glutamate decarboxylase 48.2±1.4 54.3±3.3 113 GABA-transaminase 109.3±5.0 110.5±5.0 101 Substantia nigra Glutamate decarboxylase 255.6±14.4 218.8±17.8 86 GABA-transaminase 101.0±1.6 99.2±6.7 98 n = 12 *p_ < .001, Student's two-tailed t_ t e s t . 45 Table 6. The a c t i v i t i e s of neurotransmitter-related enzymes i n the striatum and substantia nigra two weeks a f t e r the i n j e c t i o n of f i v e or ten nmoles of k a i n i c acid into the striatum. 5 nmol kainate (N=9) 10 nmol kainate (N=6) Control Lesion Control Lesion (nmol/mg protein/hr ± S.E.M.) Striatum CAT GAD GABA-T 72.6±5.2 46.5±5.0 (64% of c o n t r o l ) * * * 107.7±3.2 66.8±4.7 (62% of c o n t r o l ) * * * 105.9±3.2 18.9±1.7 (18% of c o n t r o l ) * * * 91.2±4.5 22.6±2.6 (25% of c o n t r o l ) * * * 107.3±3.1 35.9±2.4 (34% of c o n t r o l ) * * * Substantia nigra GAD GABA-T 254.6+6.1 188.6±8.9 (74% of c o n t r o l ) * * 109.7±4.7 110.0±4.8 (100% of control) 260.0±8.2 86.6±7.6 (33% of c o n t r o l ) * * * 117.6±2.8 104.6±3.4 (89% of c o n t r o l ) * *p_ < .01; **p_ < .005; ***p_ < .001; Student's two-tailed jt test \ 46 This decrease i n s t r i a t a l GABA-T a c t i v i t y correlated remarkably well with the decrease i n s t r i a t a l GAD a c t i v i t y ( F ig. 7). The i n j e c t i o n of f i v e nmoles of KA into the striatum which resulted i n a small but s i g n i f i c a n t decrease i n n i g r a l GAD, did not s i g n i f i c a n t l y reduce the a c t i v i t y of GABA-T i n the SN (Table 6). Following the i n j e c t i o n of 10 nmoles of KA, however, n i g r a l GAD was reduced by 75% and n i g r a l GABA-T a c t i v i t y also showed a s i g n i f i c a n t reduction (Table 6). As i n the striatum, the decrease observed i n GABA-T a c t i v i t y i n the SN correlated s i g n i f i c a n t l y with the decrease i n GAD (Fig. 8). Following the i n j e c t i o n of KA into the striatum of ten day old r a t s , the a c t i v i t y of CAT i n the in j e c t e d striatum was reduced by h a l f (Table 7). This i n j e c t i o n had no e f f e c t on the a c t i v i t i e s of GAD or GABA-T i n the striatum. DISCUSSION The dopamine neurons of the SN are thought to receive a major GABA innervation. Evidence suggests that a massive GABA proje c t i o n to the SN ar i s e s i n the striatum (Brownstein et a l . , 1977; Fonnum et a l . , 1978a; Nagy et a l . , 1978a). Immunocytochemical studies have reported that most of the boutons i n contact with n i g r a l dendrites s t a i n for GAD (Ribak et a l . , 1976). Stimulation of the striatum can i n h i b i t n i g r a l c e l l f i r i n g (Yoshida and Precht, 1971), and th i s e f f e c t i s blocked by p i c r o t o x i n (Precht and Yoshida, 1971) and mimicked by iontophoretic GABA ( F e l t z , 1971). A marked reduction i n n i g r a l 3H-GABA binding has been observed following 6-OHDA lesio n s of the n i g r o s t r i a t a l dopamine neurons (Guidotti et a l . , 1978). 3H-GABA binding has also been found to be reduced i n the SN of Parkinsonian patients, i n which the dopamine neurons have degene- rated (Lloyd et a l . , 1977b; Rinne et a l . , 1978). A wealth of biochemical, anatomical and p h y s i o l o g i c a l evidence therefore indicates that the dopamine 47 Figure 7. The c o r r e l a t i o n between glutamate decarboxylase and GABA- transaminase a c t i v i t i e s i n the striatum following k a i n i c acid i n j e c t i o n s of the striatum, squares = c o n t r o l ; t r i a n g l e = 5 nmoles k a i n i c acid; c i r c l e s = 10 nmoles k a i n i c a c i d . The l i n e was drawn from the l i n e a r regress- ion equation. *7 3 GAD (nmoles/mg protein/hr) 48 neurons receive a major GABA innervation. Thus, the lack of change i n GABA-T a c t i v i t y following 6-OHDA lesions of the dopamine neurons r a i s e s some i n t e r e s t i n g questions regarding GABA neurotransmission. It has previously been suggested that GABA released at the synapse i s taken up and catabolized i n the postsynaptic neuron and i n surrounding g l i a l elements since these were the structures thought to contain most of the GABA-T (Baxter, 1976). However, the present r e s u l t s i n d i c a t e that the dopamine neurons do not contain GABA-T, although they are thought to be postsynaptic to a major GABA system. This observation indicates that a l l neurons which receive GABA synapses do not nec e s s a r i l y contain GABA-T. This s i t u a t i o n i s i n sharp contrast with the s i t u a t i o n for a c e t y l - cholinesterase, the enzyme which catabolizes a c e t y l c h o l i n e . The dopa- minergic neurons of the SN have been shown to contain appreciable l e v e l s of t h i s enzyme (Lehmann and F i b i g e r , 1978). However, at present there i s l i t t l e evidence f o r the presence of c h o l i n e r g i c synapses onto the dopamine neurons. Thus, the function of the acetylcholinesterase i n these neurons remains a mystery. Schwarz et a l . , (1977) and Nicklas et a l . (1979) have recently reported a decrease i n GABA-T i n the striatum following i n t r a s t r i a t a l KA and have suggested a neuronal l o c a l i z a t i o n for t h i s enzyme. The r e s u l t s of the present experiments with KA suggest that v i r t u a l l y a l l of the GABA-T a c t i v i t y i n the striatum i s neuronal. Thus, the dose-dependent decrease i n GABA-T a c t i v i t y c o r r e l a t e s with that of the neuronal marker GAD (Fig. 7). In f a c t , the regression l i n e for th i s c o r r e l a t i o n approaches the o r i g i n , i n d i c a t i n g that i f GAD a c t i v i t y was completely abolished, the a c t i v i t y of GABA-T i n the striatum would also approach zero. Recent studies have shown that the i n j e c t i o n of KA into the striatum of neonatal rats p r e f e r e n t i a l l y depletes CAT compared with GAD (Campochiara 49 Figure 8. The c o r r e l a t i o n between glutamate decarboxylase and GABA- transaminase i n the substantia nigra following k a i n i c acid i n j e c t i o n s of the striatum, squares = cont r o l ; t r i a n g l e s = 5 nmoles k a i n i c a c i d ; c i r c l e s = 10 nmoles k a i n i c a c i d . The l i n e was drawn from the l i n e a r regression equation.  50 Table 7. The a c t i v i t i e s of neurotransmitter-related enzymes i n the striatum a f t e r the i n j e c t i o n of twenty nmoles of k a i n i c acid into the striatum of ten day r a t s . Control Lesion % of Control (nmol/mg protein/hr S.E.M.) Choline acetyltransferase 286.3±5.4 124.1±24.9 43* Glutamate decarboxylase 102.1±6.8 95.0+ 6.8 93 GABA-Transaminase 88.812.6 97.9± 3.3 110 n = 6; *p_ < .001; Student's two t a i l e d _t test 51 and Coyle, 1978; Lehmann and F i b i g e r , 1979). In f a c t , Lehmann and Fibig e r (1979) have found that these i n j e c t i o n s s e l e c t i v e l y destroy the large aspiny neurons of the striatum, which are thought to be the choli n e r g i c neurons (Lehmann and F i b i g e r , 1979; Kimura et a l . , 1980) and do not a f f e c t the density of the medium and small c e l l s . In the present experiment t h i s p r e f e r e n t i a l decrease i n CAT a c t i v i t y was again observed, while GAD and GABA-T a c t i v i t i e s were unaffected. This suggests that the large aspiny c h o l i n e r g i c neurons of the striatum do not contain GABA-T. Both GABA and substance P neurons are known to project to the SN from the striatum (see Experiment 1). Therefore destruction of eit h e r of these systems could account for the decrease i n n i g r a l GABA-T seen a f t e r s t r i a t a l KA i n j e c t i o n . However-, i f GABA transmission within the striatum i s s i m i l a r i n mechanism to that discussed above for the SN, then even i f the s t r i a t o n i g r a l substance P neurons receive a GABA.input i n the striatum, t h i s would not necessitate t h e i r containing GABA-T. On the other hand, j u s t as acetylcholinesterase i s found i n very high concentrations i n ch o l i n e r g i c neurons (Lehmann and F i b i g e r , 1979), perhaps i t i s the GABA neurons them- selves which contain the GABA-T a c t i v i t y i n t h i s system. This hypothesis i s supported by the s i g n i f i c a n t c o r r e l a t i o n between GAD and GABA-T found i n the striatum and SN a f t e r i n t r a s t r i a t a l KA. S t r i a t a l KA i n j e c t i o n s only reduced n i g r a l GABA-T by ten percent. Kataoka et a l . (1974) have reported a 25% decrease i n GABA-T a c t i v i t y i n the baboon SN a f t e r hemitransection between the striatum and SN which reduced n i g r a l GAD by 70%. Kim et a l . (1974) found a 30% reduction i n GABA-T a c t i v i t y i n the cat SN following removal of the caudate by suction, a procedure which reduced n i g r a l GABA'levels by 50%. The regression l i n e f or the c o r r e l a t i o n between n i g r a l GAD and GABA-T following s t r i a t a l KA 52 i n j e c t i o n s (Fig. 8) does not approach the o r i g i n . This contrasts with the s i t u a t i o n i n the striatum, where the regression l i n e does approach the o r i g i n (Fig. 7). This may in d i c a t e that GAD and GABA-T are i n d i f f e r e n t c e l l u l a r compartments. In the striatum, KA i n j e c t i o n s destroy both the i n t r i n s i c GABA c e l l bodies and the terminals of these c e l l s within the striatum. This r e s u l t s i n a p a r a l l e l decrease i n s t r i a t a l GAD and GABA-T. In the SN following the s t r i a t a l l e s i o n , the s t r i a t o n i g r a l terminals are destroyed and GAD i s p r e f e r e n t i a l l y decreased compared with GABA-T. This suggests that a high GAD to GABA-T r a t i o may e x i s t i n GABA terminals, but not i n GABA c e l l bodies. Most of the GABA-T i n the SN i s i n elements other than the s t r i a t o - n i g r a l terminals. As discussed above, the dopamine neurons do not appear to contain t h i s enzyme. GABA-T could be contained i n other n i g r a l a f f e r e n t s , i n other n i g r a l neurons, or i n g l i a l elements. It has been suggested that the SN contains a population of GABA neurons (Nagy et a l . , 1978d). Also, evidence i s presented i n Experiment 6 for a GABA pr o j e c t i o n from the SNR to the superior c o l l i c u l u s . In view of the arguments presented above, i t would not be s u r p r i s i n g i f most of the GABA-T a c t i v i t y i n the SN was contained i n these GABA neurons. 53 EXPERIMENT 4: THE HISTOCHEMICAL LOCALIZATION OF GABA-TRANSAMINASE IN THE BASAL GANGLIA. As discussed previously, biochemical studies have suggested the existence of many GABA pathways i n the basal ganglia. In Experiment 3 i t was demonstrated that GABA-T i s contained i n the s t r i a t a l p r o j e c t i o n to the SN and i t was suggested that t h i s enzyme may i n fac t be contained i n the s t r i a t o n i g r a l GABA neurons. As a simple histochemical procedure for GABA-T has long been known (Van Gelder, 1965) the use of t h i s technique for the histochemical demonstration of some GABA pathways i n the basal ganglia was therefore examined. METHODS Male Wistar rats weighing about 300 g were obtained from Woodlyn Laboratories, Guelph, Ontario and were used i n a l l the experiments. Histo- chemical s t a i n i n g for GABA-T was performed by a modification of the procedure of Van Gelder (1965). Rats were perfused i n t r a c a r d i a l l y with 50 ml of i c e - cold 0.1 M phosphate buffered s a l i n e , pH 7.4, followed by 200 ml of 2.0% paraformaldehyde and 2.0% glutaraldehyde i n 0.1 M phosphate buffer, pH 7.4. The f r e e - f l o a t i n g sections were preincubated for 20 min at 37°C i n a reaction mixture containing 5.0 mg/ml a-ketoglutarate 1.0 mg/ml malonate, and 0.05 mg/ml KCN i n 50 mM T r i s - C l , pH 8.6. A f t e r the preincubation the substrate and dye were added to give f i n a l concentrations of 5.0 mg/ml GABA, 1.0 mg/ml n i t r o blue tetrazolium and 0.01 mg/ml phenazine methosul- phate, and the incubation was continued for 30 min i n the dark. The reaction was stopped by d i l u t i n g i n phosphate buffer, and the sections were then mounted, dehydrated and cover-slipped. Control sections incubated without GABA, or with the GABA-T i n h i b i t o r amino-oxyacetic acid (AOAA) showed no rea c t i o n product. In order to destroy the s t r i a t a l neurons, KA was injected into the 54 striatum as previously described (McGeer and McGeer, 1976a). Rats received a u n i l a t e r a l i n j e c t i o n of 5 nmoles of KA i n 0.5 y l of 50 mM NaPO^, pH 7 at a rate of 1 yl/5 min. Two weeks l a t e r these animals were deeply anesthetized with pentobarbital and perfused and processed for GABA-T histochemistry at the i n j e c t i o n s i t e and i n the pr o j e c t i o n areas of the s t r i a t a l GABA efferents. In a second study the GABA-T s t a i n i n g of the efferents of the pallidum was examined. One group of animals was given a u n i l a t e r a l stereotaxic i n j e c t i o n of KA (2.0 nmol. i n 0.25 yl) into the GP (AP + 8.0; ML + 2.8; DV -J- 3.6; with respect to stereotaxic zero) while another group received the same i n j e c t i o n into the EP (AP + 6.3; ML + 3.0; DV + 2.2). One week l a t e r these animals were also processed for GABA-T histochemistry. RESULTS (a) GABA-T s t a i n i n g i n control animals A l l of the n u c l e i of the basal ganglia stain strongly for GABA-T. in control animals. This i s r e a d i l y apparent i n s a g i t a l sections through t h i s region (Fig. 9). In f a c t , i n many areas the s t a i n i n g i s so intense as to obscure which c e l l u l a r structures contain the enzyme. The striatum stains strongly for GABA-T. The r e a c t i o n product i s absent i n the white matter coursing through the striatum but i s d i f f u s e l y present throughout the neuropil (Fig. 10). Stained neurons can not be clearly.discerned and for the most part are hidden by the intense neuropil s t a i n i n g . The neuropil of the GP also stains strongly f o r GABA-T a c t i v i t y and i n addition some large, intensely stained neurons can be seen i n t h i s area (Fig. 11a). The white matter passing through the GP and the adjacent i n t e r - nal capsule are not stained. An even more intense s t a i n i n g pattern i s observed i n the neuropil of the EP, although stained neurons are not apparent here (Fig. 11c). 55 Figure 9. A s a g i t a l section through the r a t brain stained histochemi- c a l l y for GABA-transaminase a c t i v i t y (magnification = 9.2. x) Note the regional differences i n the i n t e n s i t y of the st a i n i n g . In p a r t i c u l a r areas associated with the basal ganglia show an intense reaction, ep entopeduncular nucleus; cp caudate-putamen; gp globus p a l l i d u s ; l h l a t e r a l habenula; sc superior c o l l i c u l u s ; sn substantia nigra; st subthalamic nucleus. £5 a 56 Figure 10. F i f t y micron vibratome section through the rat forebrain stained histochemically for GABA-transaminase a c t i v i t y . The intense reaction product, which i s present through- out the s t r i a t a l neuropil of the control side ( l e f t ) , i s almost completely absent on the c o n t r a l a t e r a l side following the destruction of the s t r i a t a l neurons by the i n j e c t i o n of f i v e nmoles of k a i n i c acid. Some loss of s t a i n i n g can also be seen i n the cortex on the lesioned side. This i s most apparent along the needle t r a c t (arrow). Magnification i s 10 x. SO a 57 Figure 11. GABA-transaminase histochemistry following the i n j e c t i o n of k a i n i c acid into the head of the striatum. (a) The con t r o l uninjected side of the brain can be seen i n t h i s coronal section through the t a i l of the caudate. Note the intense s t a i n i n g i n the t a i l of the caudate and i n the globus p a l l i d u s medial to i t . Magnification i s 16 x. (b) On the injected side the s t a i n i n g i n the globus p a l l i d u s i s markedly reduced (arrow) but the GABA-T a c t i v i t y i n the t a i l of the caudate l a t e r a l to the globus p a l l i d u s i s unaffected by the kainate l e s i o n of the head of the striatum. Magnification i s 16 x. (c) In t h i s coronal section s t a i n i n g i n the entopeduncular nucleus on the control side can be compared with that on the lesioned side. The GABA-T a c t i v i t y on the lesioned side (arrow) shows a marked reduction following the s t r i a t a l k a i n i c acid i n j e c t i o n . A s i m i l a r loss of s t a i n - ing can be seen i n the l a t e r a l hypothalamic area (*) as we l l . Magnification i s 15.1 x. (d) The GABA-transaminase a c t i v i t y i n the substantia nigra i s also d r a s t i c a l l y reduced (arrow) following the s t r i a t a l l e s i o n when compared with the c o n t r a l a t e r a l control nigra. This i s true both i n the pars compacta and the pars r e t i c u l a t a . Note the st a i n i n g i n the v e n t r a l tegmental area medial to the substantia nigra i s unaffect- ed by the s t r i a t a l l e s i o n s . Magnification i s 17 x.  58 Within the SN, the GABA-T st a i n i n g i s present as a dense band i n the r o s t r a l SNC (Fig. l i d ) , while i n the SNR the pattern of s t a i n i n g resembles that seen i n the GP (Fig. l i d ) . A few large, stained neurons s i m i l a r to.those i n the GP can also be found i n the SNR. The subthalamic nucleus shows the most intense GABA-T st a i n i n g of any area yet examined. The rea c t i o n product i s so strong i n t h i s nucleus that morphological features cannot be discerned (Fig. 9). Although the en t i r e nucleus shows high GABA-T a c t i v i t y the medial ha l f i s the most intensely stained ( Fig. 12). The crus c e r e b r i passing v e n t r a l to the subthalamus i s unstained except for some f i n e bands of rea c t i o n product extending from the subthalamic nucleus into the adjacent intrapeduncular area. F i n a l l y , the l a t e r a l habenula stains quite strongly f o r GABA-T while minimal a c t i v i t y i s present i n the medial habenula (Fig. 13). The stai n i n g i n the l a t e r a l habenula i s most intense i n the v e n t r o l a t e r a l d i v i s i o n and i s noticeably weaker i n the more medial portion of t h i s nucleus. The dorsal aspect of the l a t e r a l habenula stains as weakly as the medial habenula for GABA-T. (b) GABA-T st a i n i n g a f t e r l e s i o n s of GABA pathways Following the destruction of the neurons of the striatum by the i n j e c t i o n of KA, the GABA-T sta i n i n g i s almost abolished i n the s t r i a t a l neuropil (Fig. 10). There i s f a i n t s t a i n i n g of macrophages or monocytes along the needle t r a c t , and of astrocytes throughout the area of damage. I n f i l t r a t i o n by macrophages which s t a i n weakly for GABA-T was also observed i n the corpus callosum dorsal to the lesioned s t r i a t a . The l e s i o n was confined to the striatum r o s t r a l to the decussation of the anterior commissure but extended v e n t r a l l y to include the nucleus accumbens. The i n j e c t i o n of KA into the striatum also reduced the GABA-T sta i n i n g 59 Figure 12. GABA-transaminase a c t i v i t y i n the subthalamic nucleus of the r a t i s extremely strong on the co n t r o l side (arrow), but i s d r a s t i c a l l y reduced on the side i n which the globus p a l l i d u s was inje c t e d with k a i n i c acid (double arrow). Note that i n the c o n t r o l sub- thalamus the staini n g i s most intense i n the medial portion of the nucleus. Also, bands of r e a c t i o n pro- duct can be seen extending v e n t r a l l y from the subtha- lamus into the adjacent intrapeduncular area. Magnification i s 12.9 x.  60 Figure 13. GABA-transaminase histochemistry i n the habenula follow- ing k a i n i c acid lesions of the entopeduncular nucleus. In the lower f i g u r e , which i s a coronal section at the l e v e l of the i n j e c t i o n s i t e , the loss of s t a i n i n g i n the entopeduncular nucleus following the k a i n i c acid i n j e c t - ions i s r e a d i l y apparent (arrow). This l e s i o n dramatically reduced the GABA-T a c t i v i t y of the i p s i l a t e r a l l a t e r a l habenula (double arrow) compared with the c o n t r a l a t e r a l c o n t r o l . Magnification i s 15.7 x. In the upper figure the decrease i n habenular GABA-T a c t i v i t y following the entopeduncular l e s i o n can be seen i n greater d e t a i l . Note that i n the c o n t r o l habenula the GABA-T a c t i v i t y i s most concentrated i n the ventro- l a t e r a l portion of the l a t e r a l habenula. Magnification i s 36.6 x.  61 i n the GP, EP and SN i p s i l a t e r a l to the l e s i o n (Fig. 11). The s t a i n i n g i n the t a i l of the caudate was not affected by the l e s i o n (Fig. l i b ) . This suggests that the reduced p a l l i d a l s t a i n i n g was not due to the d i f f u s i o n of KA to t h i s structure. Large GABA-T p o s i t i v e neurons were s t i l l v i s i b l e i n the GP and SN and a few small GABA-T p o s i t i v e c e l l s could be seen i n the EP. In the control side these c e l l s are s t i l l concealed by the intense s t a i n i n g of the neuropil. Within the SN, both the intense s t a i n i n g of the SNC and the more d i f f u s e SNR s t a i n i n g were markedly reduced by the s t r i a t a l l e s i o n (Fig. l i d ) . Following the i n j e c t i o n of KA into the GP a marked reduction i n GABA- T a c t i v i t y was evident. In p a r t i c u l a r , the GABA-T p o s i t i v e neurons were no longer v i s i b l e . The KA l e s i o n of the GP also d r a s t i c a l l y reduced the GABA-T sta i n i n g i n the i p s i l a t e r a l subthalamic nucleus. This i s most evident i n the medial portion of the nucleus (Fig. 12). The s t a i n i n g i n the f i e l d s of F o r e l and the zona i n c e r t a around the subthalamic nucleus was not affected by the l e s i o n s . F i n a l l y , the i n j e c t i o n of KA into the EP re s u l t e d i n a marked reduction i n the GABA-T s t a i n i n g of t h i s nucleus, although some d i f f u s e r e a c t i o n product was s t i l l present i n the neuropil (Fig. 13). The GABA-T st a i n i n g i n the i p s i l a t e r a l l a t e r a l habenula was also dramatically decreased by t h i s l e s i o n , while the s t a i n i n g of the c o n t r a l a t e r a l habenula was not d i f f e r e n t from control (Fig. 13). DISCUSSION In Experiment 3, biochemical evidence was presented suggesting that GABA-T a c t i v i t y i n the striatum i s contained i n neurons. The present histochemical studies support such a conclusion. Thus, following the destruction of the s t r i a t a l neurons with KA the GABA-T s t a i n i n g i n t h i s nucleus was almost completely abolished. The lesioned s t r i a t a did 62 contain non-neuronal elements which stained weakly for GABA-T; however, invasion by these elements (monocytes, fibrous astrocytes) i s probably related to the pathology of the l e s i o n and they would not normally be present i n the striatum. Previous studies have found that lesions of the striatum r e s u l t i n a reduction i n GAD a c t i v i t y i n both the GP and the EP (see Experiment 1). The present experiments c l e a r l y i l l u s t r a t e that following the i n j e c t i o n of KA into the head of the striatum a marked reduction i n the GABA-T a c t i v i t y i n these areas also occurs. This suggests that GABA-T i s con- tained within the terminals of the s t r i a t o p a l l i d a l GABA neurons. Some neurons i n the GP and EP s t a i n for GABA-T a f t e r the l e s i o n , i n d i c a t i n g the presence of t h i s enzyme i n some p a l l i d a l c e l l bodies as well as i n afferent terminals. Both the GP (Fonnum et a l . , 1978b; H a t t o r i et a l . , 1973b) and the EP (Nagy et a l . , 1978b) are thought to contain GABA neurons, suggesting that i t i s these c e l l s which s t a i n for GABA-T. It i s i n t e r e s t i n g to note that the i n j e c t i o n of KA into the striatum reduces the a c t i v i t y of GABA-T i n the l a t e r a l hypothalamic area ventro- medial to the EP (Fig. 11c). This region stains intensely on the control side of the brain (Fig. 11c). The l a t e r a l hypothalamic area has been shown to possess the highest GABA l e v e l s (Kimura and Kuriyama, 1975a) and GAD a c t i v i t y (Kimura and Kuriyama, 1975b) i n the hypothalamus. Anatomical studies have shown that the nucleus accumbens projects massively to t h i s region (Nauta et a l . , 1978; Powell and Leman, 1976). Recently, biochemical studies have indicated that the accumbens projections to the substantia innominata, GP (Walaas and Fonnum, 1979), SN and v e n t r a l teg- mental area (Waddington and Cross, 1978b; Walaas and Fonnum, 1980) may contain GAD. Together, these r e s u l t s suggest that the decrease i n the GABA-T a c t i v i t y observed i n the l a t e r a l hypothalamic area following KA 63 i n j e c t i o n s o f t h e s t r i a t u m may b e d u e t o t h e d e s t r u c t i o n o f GABA n e u r o n s i n t h e n u c l e u s a c c u m b e n s p r o j e c t i n g t o t h i s a r e a . T h e e x i s t e n c e o f a s t r i a t o n i g r a l GABA p a t h w a y h a s b e e n r e p e a t e d l y d e m o n s t r a t e d , a l t h o u g h t h e p r e c i s e o r i g i n o f t h i s p a t h w a y h a s b e e n a m a t t e r o f s o m e d e b a t e ( s e e E x p e r i m e n t 1 ) . R e c e n t e v i d e n c e s u g g e s t s t h a t t h e r e i s a c o n c e n t r a t i o n o f s t r i a t o n i g r a l G A D - c o n t a i n i n g n e u r o n s i n t h e s t r i a t u m a p p o s e d t o t h e a n t e r i o r GP ( B r o w n s t e i n e t a l . , 1 9 7 7 ; E x p e r i m e n t 1 ) . A n a t o m i c a l s t u d i e s s u g g e s t t h a t t h e s e n e u r o n s p r o j e c t p r e d o m i n a n t l y t o t h e SNC , w h i l e t h e a n t e r i o r s t r i a t a l p r o j e c t i o n , w h i c h c o n t a i n s s u b s t a n c e P ( s e e E x p e r i m e n t 1 ) p r o j e c t s l a r g e l y t o t h e SNR ( H a t t o r i e t a l . , 1 9 7 5 ; T u l l o c h e t a l . , 1 9 7 8 ) . T h u s , t h e o b s e r v a t i o n t h a t t h e G A B A - T s t a i n i n g a l t h o u g h p r e s e n t t h r o u g h o u t t h e S N , i s m o s t i n t e n s e i n t h e SNC c o r r e l a t e s w e l l w i t h t h e p r o p o s e d d i s t r i b u t i o n o f t h e t e r m i n a l s o f t h e GABA p r o j e c t i o n f r o m t h e s t r i a t u m . I t h a s b e e n d e m o n s t r a t e d b i o c h e m i c a l l y t h a t t h e d o p a m i n e r g i c n e u r o n s o f t h e SNC d o n o t c o n t a i n G A B A - T ( E x p e r i m e n t 3 ) . T h e l o s s o f s t a i n i n g o b s e r v e d i n t h e p a r s c o m p a c t a f o l l o w i n g s t r i a t a l l e s i o n s i s c o n s i s t e n t w i t h t h i s f i n d i n g s i n c e t h e d o p a m i n e r g i c n e u r o n s a r e n o t d a m a g e d b y t h i s l e s i o n . A s i n t h e p a l l i d u m , some l a r g e c e l l s i n t h e SNR s t a i n f o r GABA -T b o t h i n c o n t r o l a n i m a l s a n d a f t e r s t r i a t a l l e s i o n s . T h e s e n e u r o n s may b e t h e n i g r a l c e l l s w h i c h p r o j e c t t o t h e t e c t u m a n d t h e v e n t r o - m e d i a l t h a l a m u s a n d a r e t h o u g h t t o c o n t a i n GABA ( s e e E x p e r i m e n t 6 ) . R e c e n t s t u d i e s h a v e s h o w n t h a t i n j e c t i o n s o f G A B A - T i n h i b i t o r s i n t o t h e GP o r SN r e s u l t i n m a r k e d e f f e c t s o n m o t o r b e h a v i o u r ( M a t s u i a n d K a m i o k a , 1 9 7 8 ; P y c o c k e t a l . , 1 9 7 6 ) . T h e p r e s e n t h i s t o c h e m i c a l o b s e r v a t i o n s s u g g e s t t h a t t h e G A B A - T p r e s e n t i n t h e t e r m i n a l s o f t h e s t r i a t a l GABA p r o j e c t i o n s c o u l d b e t h e s i t e o f a c t i o n f o r t h e s e e f f e c t s . T h i s w o u l d b e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t G A B A - T i n GABA N e r v e t e r m i n a l s i s d i r e c t l y i n v o l v e d i n t h e r e g u l a t i o n o f GABA t r a n s m i s s i o n . 64 Lesions of the EP have p r e v i o u s l y been found to decrease GABA l e v e l s and GAD a c t i v i t y i n the habenula i n d i c a t i n g that t h i s i s a GABA pathway (G o t t e s f e l d et a l . , 1977; Nagy et a l . , 1978b). In the present histochem- c a l study, the a c t i v i t y of the enzyme GABA-T i n the habenula was a l s o found to decrease f o l l o w i n g EP l e s i o n s . This suggests that GABA-T i n the habenula i s contained i n the terminals of the EP-habenula pathway. The GABA-T s t a i n i n g i n the l a t e r a l habenula i s most i n t e n s e i n the v e n t r o l a t e r a l p o r t i o n of t h i s nucleus, and i s weakest i n the dorso-medial r e g i o n . This may i n d i c a t e that the GABA terminals i n the l a t e r a l habenula are most concentrated i n the v e n t r o l a t e r a l area. The evidence from anatomical studies of the EP-habenula pathway i s i n f u l l agreement w i t h t h i s hypothesis. In autoradiographic s t u d i e s of the orthograde t r a n s p o r t of r a d i o l a b e l e d p r o t e i n from the EP the l a b e l has been found to d i s t r i b u t e predominantly i n the v e n t r o l a t e r a l p o r t i o n of the l a t e r a l habenula (Carter and F i b i g e r , 1978; Larsen and McBride, 1979; Nagy et a l . , 1978b) and not i n the dorso- medial p o r t i o n (Nauta, 1974). A s i m i l a r topography has been obtained from retrograde s t u d i e s w i t h horseradish peroxidase (Herkenham and Nauta, 1977; Larsen and McBride, 1979). The recent observation of Fonnum et a l . (1978b) that GP l e s i o n s i n the cat reduce subthalamic GAD a c t i v i t y i n d i c a t e s that t h i s may a l s o be a GABA pathway. Lesions of the GP i n the r a t reduce GABA-T a c t i v i t y i n the sub- thalamic nucleus, suggesting that t h i s enzyme i s present i n the ter m i n a l s of the p a l l i d a l a f f e r e n t s to t h i s nucleus. Fonnum et a l . (1978b) have found that the co n c e n t r a t i o n of GAD increases from the l a t e r a l to the medial part of the subthalamus. In the present experiments the i n t e n s i t y of GABA-T s t a i n i n g was found to f o l l o w a s i m i l a r p a t t e r n . Again t h i s agrees w i t h the topography of the p a l l i d o - s u b t h a l a m i c pathway found i n anatomical s t u d i e s . Autoradiographic s t u d i e s i n the r a t f o l l o w i n g GP i n j e c t i o n s of 65 t r i t i a t e d amino acids have found the medial portion of the subthalamic nucleus to be most heavily labeled (Carter and F i b i g e r , 1978). The present experiments indicate that GABA-T histochemistry may be a useful adjunct to biochemical studies for the analysis of GABA pathways. The l o c a l i z a t i o n of GABA-T staining i n the efferents of the striatum corresponds to the d i s t r i b u t i o n of the s t r i a t a l GABA effe r e n t s . Also the d i s t r i b u t i o n of GABA-T st a i n i n g i n the l a t e r a l habenula and subthalamic nucleus correlates well with the known topography of the p a l l i d a l GABA projections to these areas. Moreover, the combination of GABA-T h i s t o - chemistry with s e l e c t i v e lesions has provided a very graphic summary of what has been learned from several years of biochemical work. This approach provides a de t a i l e d p i c t u r e of the topography of these GABA pathways previously only hinted at from c o r r e l a t i o n s of lesion-biochemical work with anatomical studies. Thus, GABA-T histochemistry may provide a simple technique for the morphological analysis of known or suspected GABA pathways. 66 EXPERIMENT 5: BIOCHEMICAL CHANGES FOLLOWING 6-HYDROXYDOPAMINE LESIONS OF THE NIGROSTRIATAL DOPAMINE NEURONS: AN ANIMAL MODEL OF PARKINSONISM? Lesions of the n i g r o s t r i a t a l dopamine p r o j e c t i o n have been used as animal models of Parkinson's disease (see Marsden et a l . , 1975) since i t was discovered that t h i s system degenerates i n t h i s disorder (Ehringer and Hornykiewicz, 1960; Hornykiewicz, 1973). A d e t a i l e d study of the e f f e c t s of such lesions on the other transmitter systems of the basal ganglia has not been conducted. This i s of great importance f o r , although the most s t r i k i n g aspect of Parkinson's disease i s the marked decrease i n n i g r a l and s t r i a t a l dopamine, changes i n other transmitter systems have also been reported. A decrease i n GAD a c t i v i t y i n some n u c l e i of the basal ganglia has been c o n s i s t e n t l y observed i n Parkinson's disease (Lloyd and Hornykiewicz, 1973; McGeer et a l . , 1971; Rinne et a l . , 1974; 1979). This decrease has been reported to be reversed by chronic L-dopa therapy (Lloyd and Hornykiewicz, 1973; Rinne et a l . , 1979). Also, normal rats treated c h r o n i c a l l y with L-dopa have been reported to have an increased s t r i a t a l GAD a c t i v i t y (Lloyd and Hornykiewicz, 1973). These re s u l t s suggest an intimate 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 dopamine system and the GABA neurons of the striatum. Although the l e v e l s of substance P and met-enkephalin have not been measured i n Parkinson's disease, experimental data i n d i c a t e that the dopaminergic n i g r o s t r i a t a l system profoundly influences the neurons containing these peptides i n the striatum. Acute amphetamine treatment has been found to decrease the substance P l e v e l s i n the striatum (Pettibone et a l . , 1978a; 1978b). Chronic neuroleptic treatment decreases the l e v e l s of substance P i n the SN (Hong and Costa, 1978) and increases the l e v e l s (Hong and Costa, 1978) and accelerates the synthesis of met-enkephalin 67 (Hong et a l . , 1978b) i n the striatum, GP and nucleus accumbens. The present s e r i e s of experiments was designed to examine further the interactions between the dopaminergic neurons and the transmitters contained i n the efferents of the striatum. S e l e c t i v e l e s i o n s of the n i g r o s t r i a t a l dopamine neurons were performed with 6-OHDA and the effects of such l e s i o n s on the GABA, substance P and met-enkephalin systems of the basal ganglia examined. METHODS Male Wistar r a t s weighing about 300 g were placed under Nembutal anesthesia and given a u n i l a t e r a l stereotaxic i n j e c t i o n of four yg 6-OHDA into the n i g r o s t r i a t a l pathway as described i n Experiment 3. Control animals received an i n j e c t i o n of ve h i c l e only. A l l animals received desipramine (25 mg/kg) 30 min before the 6-OHDA i n j e c t i o n s . Following surgery the animals were sin g l y housed i n a 12 hr light - d a r k environment and given food and water ad l i b i t u m . The animals were s a c r i f i c e d by c e r v i c a l fracture at various times a f t e r the operation, the brains removed and dissected for biochemical analyses. Tissues for the enzyme assays were homogenized i n 20 to 30 volumes of 50 mM Tris-acetate buffer pH 6.4 containing 0.2% T r i t o n X-100. GAD was assayed as described i n Experiment 1, tyrosine hydroxylase as outl i n e d i n Experiment 3, and protein according to Lowry et a l . (1951). The l e v e l s of substance P and met-enkephalin were measured by radioimmunoassay following extraction i n one N ace t i c acid (Appendix). RESULTS In the f i r s t experiment, animals were s a c r i f i c e d 25 days a f t e r the i n j e c t i o n of 6-OHDA into the l e f t n i g r o s t r i a t a l pathway. The whole striatum, i n c l u d i n g the accumbens and GP was dissected freehand, while the SN was obtained from sections cut on a freezing microtome. This l e s i o n 67a Figure 1 4 . Enzyme a c t i v i t i e s i n the striatum and substantia nigra (SN) 25 days a f t e r u n i l a t e r a l lesions of the n i g r o s t r i a t a l pathway, expressed as percent of c o n t r o l . Each column represents the mean of the number of rats indicated i n brackets. V e r t i c a l bars represent the standard error of the mean. Absolute values f o r tyrosine hydroxylase (TH) and glutamate decarboxylase (GAD) i n control s t r i a t a were 4.2 - 0.2 and 98.9 - 3.7 nmoles/mg protein/h r e s p e c t i v e l y . For control SN, the TH and GAD values were 2.3 - 0.1 and 272 - 1 1 nmoles/mg protein/h. * JK.OOI, paired ^ - t e s t . ENZYME ACTIVITY (% OF CONTROL) 68 resulted i n an decrease i n s t r i a t a l and n i g r a l tyrosine hydroxylase a c t i v i t y of more than 90%, r e s u l t i n g from the orthograde and retrograde degeneration of the dopamine terminals and perikarya r e s p e c t i v e l y . This l e s i o n was associated with a highly s i g n i f i c a n t increase i n the a c t i v i t y of GAD i n the striatum on the i n j e c t e d side. In contrast, GAD a c t i v i t y i n the SN was not affected by the l e s i o n (Fig. 14). These r e s u l t s have recently been published (Vincent et a l . , 1978b). Since the s t r i a t a l sample i n which the GAD increase was observed i n the preliminary experiment included the caudate-putamen, the accumbens and the GP, the experiment was r e p l i c a t e d and GAD was assayed i n d i s c r e t e n u c l e i of the basal ganglia i n an attempt to l o c a l i z e the s i t e of t h i s change i n a c t i v i t y . One month af t e r the 6-OHDA^lesion animals were k i l l e d by c e r v i c a l fracture and the nucleus accumbens, the head and the t a i l of the striatum, the GP and the SN were dissected from coronal sections obtained on a freezing microtome and assayed for GAD a c t i v i t y . These lesions were found to reduce tyrosine hydroxylase more than 92% i n the head of the striatum i n d i c a t i n g that the l e s i o n of the dopamine neurons was e s s e n t i a l l y complete. As seen i n Table 8, these le s i o n s increased GAD a c t i v i t y i n the nucleus accumbens, the head and the t a i l of the striatum, and the GP by about the same extent. There was a tendency for GAD to increase i n the SN but t h i s did not reach s t a t i s t i c a l s i g n i - ficance (.05 < p < .1). To determine i f the observed increase i n s t r i a t a l GAD a c t i v i t y was a transient or a permanent change, the a c t i v i t y of GAD was measured i n the striatum and SN three months a f t e r the u n i l a t e r a l i n j e c t i o n of 6-OHDA into the n i g r o s t r i a t a l bundle. The l e v e l s of the neuropeptides substance P and met-enkephalin were also measured at t h i s s u r v i v a l time, and i n addition a group r e c e i v i n g a u n i l a t e r a l i n j e c t i o n of v e h i c l e only was 69 Table 8. Glutamic acid decarboxylase a c t i v i t y i n various b r a i n areas af t e r u n i l a t e r a l lesions of the n i g r o s t r i a t a l pathway with 6-OHDA. Enzyme (nmol/mg protein/hr) Area Control Lesioned % of con t r o l Accumbens 15815.3 19818.3 125** Body of caudate-putamen 10917.6 13517.9 124*** T a i l of caudate-putamen 99.4±6.7 12714.2 128** Globus p a l l i d u s 22117.8 26615.7 120*** Substantia nigra 29318.9 321113 110 Enzyme a c t i v i t i e s are the mean ± S.E.M. of the lesioned and contra- l a t e r a l side of 12 r a t s . S t a t i s t i c a l evaluation was conducted using the paired _t t e s t . **p < .01, ***p < .001. 70 included i n the an a l y s i s . As shown i n Table 9, the decrease i n s t r i a t a l tyrosine hydroxylase i n t h i s study was again v i r t u a l l y complete. This indicates that the l e s i o n was t o t a l and no regeneration of the dopamine neurons had occurred. The vehicl e injected control group showed a small reduction i n mean tyrosine hydroxylase a c t i v i t y i n the striatum, however, t h i s did not reach s t a t i s t i c a l s i g n i f i c a n c e at the f i v e percent l e v e l . I t i s worth noting i n t h i s regard that previous reports have found ascorbate- containing v e h i c l e to be s l i g h t l y t o x i c . t o the n i g r a l dopamine c e l l s (Wolfarth et a l . , 1977). The increase i n GAD a c t i v i t y observed at three months following 6-OHDA was i d e n t i c a l to that seen at 28 days. The GAD a c t i v i t y i n the denervated striatum was s i g n i f i c a n t l y higher than that i n eit h e r the co n t r a l a t e r a l uninjected s t r i a t a or i n the sa l i n e i n j e c t e d s t r i a t a of the control group. The l e s i o n of the n i g r o s t r i a t a l dopamine system with 6-OHDA dramati- c a l l y reduced the l e v e l s of substance P i n both the head of the striatum and i n the SN. Saline injected animals showed no change i n substance P l e v e l s . The l e v e l s of met-enkephalin were unaffected i n either the striatum or the SN following the 6-OHDA lesio n s (Table 9). DISCUSSION Although 6-OHDA has been found to be a s e l e c t i v e neurotoxin for catecholamine neurons (HCkfelt and Ungerstedt, 1973; Maler et a l . , 1973) the changes observed i n GAD a c t i v i t y and substance P l e v e l s i n the present study make i t evident that secondary and probably i n d i r e c t e f f e c t s on other neuronal systems can also occur following 6-OHDA i n j e c t i o n s . Thus, the s e l e c t i v e l e s i o n of the n i g r o s t r i a t a l dopamine system resulted i n an enhanced s t r i a t a l GAD a c t i v i t y . As GAD a c t i v i t y was measured at substrate Table 9. Enzyme a c t i v i t i e s and neuropeptide l e v e l s i n the striatum and substantia nigra three months a f t e r the i n j e c t i o n of s a l i n e or 6-hydroxydopamine into the l e f t n i g r o s t r i a t a l pathway. Striatum Substantia nigra R L R L Saline i n j e c t i o n s : Tyrosine hydroxylase (nmol/mg protein/hr) Glutamate decarboxylase (nmol/mg protein/hr) Substance P (pg/mg tissue) 6-OHDA i n j e c t i o n s : Tyrosine hydroxylase (nmol/mg protein/hr) Glutamate decarboxylase (nmol/mg protein/hr) Substance P (pg/mg tissue) Met-enkephalin (pg/mg tissue) *p < .001 compared to c o n t r a l a t e r a l side.or i p s i l a t e r a l s a l i n e i n j e c t i o n ( 2 - t a i l _t test) **p < .05 compared to c o n t r a l a t e r a l side (1 t a i l e d t test) or i p s i l a t e r a l s a l i n e i n j e c t i o n ( 2 - t a i l _t test) 1.62+ .33 0.98±.17 60.9+ 4.3 56.2±4.3 369+ 26 353±25 1918H55 1854+189 1.181.12 0.01±.01* (.85% of control) 56.1 +3.7 66.7±4.0** (119% of control) 393121 232±12* 1997+170 1013±67* (59% of control) (51% of control) 533±84 696±128 153±18 135±8 concentrations w e l l above saturation, i t appears that the increased a c t i v i t y measured indicates an increased enzyme v e l o c i t y , i n d i c a t i n g an actual increase i n the amount of enzyme rather than an a c t i v a t i o n of e x i s t i n g enzyme. This has i n fact been found i n a recent k i n e t i c analysis of the GAD increase following 6-OHDA lesions (Fibiger et a l . , 1980). It i s evident from the regional examination that these le s i o n s increase GAD a c t i v i t y i n the caudate-putamen as well as i n the nucleus accumbens and the GP. A non-significant tendency toward increased GAD a c t i v i t y was also observed i n the SN. Inasmuch as i t i s known that the d i s t r i b u t i o n of GAD within the SN i s not homogeneous (Fonnum et a l . , 1978a; Fonnum et a l . , 1974) i t i s possible that there occurred s i g n i f i - cant regional increases i n n i g r a l GAD which may have been obscured when the whole SN was assayed. Saavadra et a l . (1978) have recently provided evidence that such regional increases i n n i g r a l GAD do indeed occur a f t e r 6-OHDA le s i o n s of the dopamine neurons. These changes i n GAD a c t i v i t y i n the basal ganglia stand i n marked contrast to those observed i n Parkinson's disease which the 6-OHDA l e s i o n i s thought to mimic. In Parkinsonism, a consistent decrease i n basal ganglia GAD a c t i v i t y has been observed (Lloyd and Hornykiewicz, 1973; McGeer and McGeer, 1976b, McGeer, et a l . , 1971; Rinne et a l . , 1974). This decrease has led to the proposal that i n Parkinson's disease the decreases i n s t r i a t a l and n i g r a l GAD a c t i v i t y are compensatory changes and represent an attempt to maintain n i g r o s t r i a t a l dopamine transmission (Lloyd and Davidson, 1979). The present observations would seem to make t h i s hypothesis untenable. Thus, complete destruction of the nigro- s t r i a t a l dopamine system with 6-OHDA r e s u l t s not i n a compensatory decrease i n GAD a c t i v i t y , but rather, i n a s i g n i f i c a n t increase. The present findings thus have important implications for the etiology 73 of Parkinson's disease. They strongly suggest that i n addition to the demonstrated pathology of the dopaminergic systems i n t h i s disease a pathology of the s t r i a t a l GABA neurons may also be present. The decrease observed i n p a l l i d a l and n i g r a l GAD i n Parkinsonism (Lloyd and Hornykiewicz, 1973; McGeer and McGeer, 1976b; McGeer et a l . , 1971) i s e n t i r e l y consis- tent with t h i s hypothesis and suggests that 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 GABA t r a c t s atrophy i n t h i s disease. In t h i s regard, i t has been found that the cerebrospinal f l u i d l e v e l s of GABA are markedly reduced i n Parkinsonian patients (Lakke and Teelken, 1976) and i n fac t are even lower than those found i n patients with Huntington's disease where a degeneration of s t r i a t a l GABA neurons i s considered to be a main pathological f i n d i n g (Chase and Tammiga, 1979). Also, Rinne et a l . (1979) report a s i g n i f i c a n t reduction i n GABA l e v e l s i n the cerebral and cere b e l l a r c o r t i c e s i n Parkinsonism. Rinne et a l . (1979) have also observed s i g n i f i c a n t c o r r e l a t i o n s between GAD a c t i v i t y and the symptoms of Parkinson's disease. These d e f i c i t s i n the GABA system may have some importance i n designing a pharmacological therapy f o r t h i s disease. We have seen that i n the animal experiments s t r i a t a l GAD increases i n response to the loss of the dopamine neurons. In Parkinson's disease t h i s apparently i s not possible because of the concomitant atrophy of the GABA neurons. Thus, j u s t as L-dopa has been used to replace the l o s t dopamine innervation i n the striatum, perhaps a GABA agonist could be of use i n replac i n g the GABA d e f i c i t , p a r t i c u l a r l y i n the SN where both GAD and GABA receptors are reduced (Lloyd et a l . , 1977b; Rinne et a l . , 1978; 1979). In t h i s regard B a r t h o l i n i et a l . (1979) have found i n preliminary studies that the GABA agonist and prodrug SL 76003, when combined with L-dopa therapy, prevents L-dopa-induced involuntary movements and increases the dosage of L-dopa that can be given, r e s u l t i n g 74 i n greatly ameliorated Parkinsonian symptoms with the absence of involun- tary movements. Pharmacological manipulations of the n i g r o s t r i a t a l dopamine system have been found to a f f e c t the l e v e l s of substance P i n the striatum and SN (Hong and Costa, 1978; Hong et a l . , 1978a; Pettibone et a l . , 1978a; 1978b). The present experiments have shown that the destruction of the n i g r o s t r i a t a l dopamine system with i n t r a c e r e b r a l 6-OHDA i s associated with a marked reduction i n substance P l e v e l s i n the striatum and the SN. This could represent a change i n substance P turnover i n these areas, or i t could be due to the actual destruction of s t r i a t o n i g r a l substance P neurons by 6-OHDA. Several observations argue against the l a t t e r i n t e r p r e - t a t i o n . In Experiment 1, complete hemitransections at the anterior pole of the GP reduced substance P l e v e l s i n the EP, GP and SN, but did not a f f e c t the l e v e l s of substance P i n the head of the striatum. This indicates that destruction of substance P axons projecting to the pallidum and SN does not re s u l t i n the death of the s t r i a t a l , substance P neurons, presumably because they possess many i n t r i n s i c c o l l a t e r a l s (Experiment 2). Also, the 6-OHDA i n j e c t i o n was made into the n i g r o s t r i a t a l pathway at a l e v e l at which the descending s t r i a t o n i g r a l system i s separated from the ascending dopamine f i b e r s (Tulloch et a l . , 1978). In addition, i t has been reported that i n t r a c i s t e r n a l i n j e c t i o n s of 250 ug of 6-OHDA does not a f f e c t s p i n a l cord substance P l e v e l s (Singer et a l . , 1979) i n d i c a t i n g that 6-OHDA i s not i n general toxic to substance P neurons. I n c i d e n t a l l y , these l e s i o n s , had no ef f e c t on the met-enkephalin l e v e l s i n the striatum. The observations that pharmacological manipulations (Hong and Costa, 1978; Hong et a l . , 1978a; Pettibone et a l . , 1978a; 1978b) or lesions of the n i g r o s t r i a t a l dopamine neurons can reduce substance P l e v e l s i n the striatum and SN suggest that great caution should be exercised i n i n t e r p r e t i n g the 75 r e s u l t s of l e s i o n studies where a decrease i n peptide l e v e l s i s observed. Although such decreases could indicate the i n t e r r u p t i o n of a peptide pathway, i n the absence of other evidence, a secondary change i n peptide turnover due to d i s r u p t i o n of some unknown system would seem an equally l i k e l y explanation. The decreased l e v e l s of substance P i n the SN probably represent an increase i n the turnover of substance P. A s i m i l a r decrease has been observed a f t e r chronic haloperidol treatment (Hong and Costa, 1978; Hong et a l . , 1978a). I n t r a n i g r a l substance P increases the f i r i n g of the n i g r o s t r i a t a l dopamine neurons (Davies and Dray, 1976; Walker et a l . , 1976) thereby increasing the release of dopamine i n the striatum (Cheramy et a l . , 1977) . These observations suggest that substance P turnover increases i n the nigra i n response to a decrease i n dopamine transmission i n the striatum. The decrease i n s t r i a t a l substance P l e v e l s seen i n the present study may be a manifestation of the prolonged a c t i v a t i o n of these substance P neurons and may not have been apparent at the shorter s u r v i v a l times used i n the chronic haloperidol studies (Hong and Costa, 1978; Hong et a l . , 1978a). This hypothesis suggests that drugs which increase the effectiveness of substance P transmission, p a r t i c u l a r l y i n the SN, may be of value i n the therapy for Parkinson's disease. 76 EXPERIMENT 6: THE NIGROTECTAL PROJECTION: A BIOCHEMICAL AND ULTRASTRUCTURAL STUDY As confirmed i n Experiment 1, the SN receives both a GABA and a substance P p r o j e c t i o n from the striatum. In return, the SN provides the striatum with a dense dopaminergic innervation a r i s i n g from the SNC. The non-dopaminergic SNR c e l l s have also been suggested to send a sparse projection to the striatum (Fibiger et a l . , 1972) and i n addition t h i s area projects "massively to the superior c o l l i c u l u s ( F a u l l and Mehler, 1978; Graybiel, 1978; Hopkins and Niessen, 1976; Rinvik et a l . , 1976) and the thalamus (Carpenter and Peters, 1972; Carpenter et a l . , 1976; C l a v i e r et a l . , 1976; F a u l l and Mehler, 1978; Rinvik, 1975). Lesion studies have shown that the s t r i a t o n i g r a l pathway i s e s s e n t i a l for the expression of some types of striatal-mediated behaviour (Lee et a l . , 1980; Marshall and Ungerstedt, 1971). For example, the rotatory behaviour induced by dopamine agonists i n r a t s with u n i l a t e r a l lesions of the n i g r o s t r i a t a l dopamine c e l l s i s dependent upon the i n t e g r i t y of t h i s pathway (Marshall and Ungerstedt, 1971). It has been suggested that the p r o j e c t i o n from the SNR to the superior c o l l i c u l u s i n t e r a c t s with the t e c t o s p i n a l system which regulates the neck muscles involved i n head o r i e n t a t i o n (York and Faber, 1977). Thus, t h i s pathway may be the 'output of the basal ganglia involved i n r o t a t i o n a l behaviour. In t h i s study, the e f f e c t s of l e s i o n s of the SN on neurochemical parameters i n the superior c o l l i c u l u s was examined i n an attempt to determine the transmitters i n t h i s system. The f i n e structure of the terminal boutons of t h i s p r o j e c t i o n was also examined to understand further the synaptic r e l a t i o n s h i p s of t h i s p r o j e c t i o n with the c o l l i c u l a r c e l l s . METHODS Male Wistar r a t s weighing about 300 g were used for a l l experiments. For electron microscopic analysis s i x r a t s received a stereotaxic i n j e c t i o n of 10 yCi of [ 3H]leucine (New England Nuclear, 80 Ci/mmol) i n 0.5 Vl s a l i n e into the l e f t SNR. The coordinates of the i n j e c t i o n were AP + 2.9; ML + 2.1; DV + 3.0, with respect to stereotaxic zero, with the i n c i s o r bar 5 mm above the h o r i z o n t a l plane. With these coordinates the needle t r a c t completely avoids the superior c o l l i c u l u s . The i n j e c t i o n was 20 min, and the cannula was l e f t i n place an a d d i t i o n a l f i v e min a f t e r the i n j e c t i o n . The r a t s were perfused through the heart 24 hr l a t e r with 500 ml of 4% paraformaldehyde, 0.5% glutaraldehyde, and 0.6% dextrose i n 0.1 M sodium A phosphate buffer, pH 7.4. One mm cubes were cut from the deeper layers of the i p s i l a t e r a l superior c o l l i c u l u s and processed for electron microscopic autoradiography according to the method of H a t t o r i et a l . (1973a). The blocks were postfixed i n the above so l u t i o n overnight and then i n 1% buffered osmium tetroxide (pH 7.4) for two hours. The tiss u e was then embeded in epon-araldite mixture and gold sections cut on an LKB microtome. Sections were picked up on formvar-coated copper grids , and I l f o r d L4 emulsion was applied by the standard loop technique. A f t e r one month exposure at 4°C the sections were developed i n Microdol X and fi x e d i n Kodak Rapid F i x . Sections were counterstained with uranyl acetate and lead c i t r a t e . Grain d i s t r i b u t i o n and area determinations were performed on ele c t r o n micrographs taken with a P h i l i p s 201 electron microscope, and the r e l a t i v e grain density over various c e l l structures was calculated according to Salpeter and McHenry (1973). T h i r t y micron sections through the i n j e c t i o n s i t e were cut on a freezing microtome and examined with a l i g h t microscopic autoradiographic technique. Slides were dipped i n emulsion (Kodak NTB 3), dried and then stored i n l i g h t - t i g h t boxes containing s i l i c a at 4°C for two weeks. The s l i d e s were developed i n D-19, fixed and counterstained with c r e s y l v i o l e t . In the biochemical studies, the l e f t SN was injected with f i v e nmoles 78 of KA i n one y l of buffered s a l i n e . Three weeks l a t e r , the animals were k i l l e d by c e r v i c a l f r a c t u r e . The superior c o l l i c u l i were dissected fresh from coronal s l i c e s and homogenized i n 30 volumes of 0.32 M sucrose. A 75 y l aliquot was removed and added to 75 y l of 50 mM Tris-acetate buffer (pH 7) containing 0.2% T r i t o n X-100. Aliquots of t h i s were used to assay GAD and CAT as described i n Experimental 1. The P 2 f r a c t i o n was obtained from the o r i g i n a l homogenate by the method of Simon et a l . (1976) and was resuspended i n 50 volumes of 0.32 M sucrose. Aspartate and glycine uptake were measured i n 5 min incubations of 60 y l of t h i s suspen- sion i n Krebs-Ringer phosphate buffer with 10 6M of [U-^C]aspartate (New England Nuclear 30 Ci/mol) , or [U- 1 L fC]glycine (New England Nuclear; 68 C i / moi). Af t e r incubation, the samples were rinsed onto M i l l i p o r e f i l t e r s and washed with 0.9% s a l i n e . The f i l t e r s were counted i n a mixture of one ml water and 9 ml of a s o l u t i o n of 10% naphthalene and 0.4% PPO i n dioxane. In control incubations sodium ion was replaced by choline or potassium i n the buffer. Protein was determined on both the sucrose homogenates used fo r uptake and on the samples used for the enzyme assays by the method of Lowry et a l . (1951). RESULTS The r e s u l t s of t h i s study have recently been published (Vincent et a l . , 1978a). In the autoradiographic experiments to be discussed the i n j e c t i o n s i t e was confined to SNR (Fig. 15). The d i s t r i b u t i o n of s i l v e r grains i n the superior c o l l i c u l u s one day a f t e r the i n j e c t i o n of [ 3H]leucine into the SNR i s summarized i n Table 10. Myelinated axons were p r e f e r e n t i a l l y labeled compared to unmyelinated axons. Boutons forming symmetrical synapses with major dendrites were labeled with a high r e l a t i v e grain density of 2.4, i n d i c a t i n g that l a b e l transported from the SN was l o c a l i z e d i n a preferen- t i a l manner i n these structures (Salpeter and McHenry, 1973). The labeled 79 Figure 15. Schematic representation of the s i t e and extent of the [ H ] l e u c i n e i n j e c t i o n s i n the substantia nigra pars r e t i c u l a t a (SNR). CC crus c e r e b r i ; IP interpeduncular nucleus; LM medial leminiscus; SNC substantia nigra pars compacta. '71a 80 Table 10. D i s t r i b u t i o n of s i l v e r grains i n the superior c o l l i c u l u s 24 hr af t e r the i n j e c t i o n of [ 3H]leucine into the substantia nigra. Number of Percent Percent Relative Structure grains grains area grain density Bouton Symmetric 51 15.9 6.5 2.4* Asymmetric 5 1.4 4.4 0.32 Preterminal 8 2.5 2.6 0.96 Axon Myelinated 98 30.5 21.5 1.4* Unmyelinated 40 12.4 15.3 0.81 Soma 23 7.2 10.8 0.67 Dendrite 66 20.7 24.3 0.85 G l i a 27 8.2 12.8 0.64 Blood vessel 4 1.2 1.8 0.67 Tota l 322 100 100 *RGD > 1. 81 boutons were one to two microns i n diameter and contained moderately packed s l i g h t l y pleomorphic v e s i c l e s (Fig. 16). The r e s u l t s of the biochemical studies of the superior c o l l i c u l u s a f t e r u n i l a t e r a l KA lesions of the SN are summarized i n Table 11. No s i g n i f i c a n t changes were observed i n aspartate or glycine uptake or i n the a c t i v i t y of CAT i n the superior c o l l i c u l u s a f t e r l e s i o n i n g the SN. However, GAD a c t i v i t y showed a s i g n i f i c a n t decreases i n the superior c o l l i c u l u s i p s i l a t e r a l to the l e s i o n when compared to the c o n t r a l a t e r a l control side. DISCUSSION Glutamic acid decarboxylase i s the rate l i m i t i n g enzyme i n the syn- thesis of GABA and i s a us e f u l marker f o r GABA nerve endings. The d i s t r i b u t i o n of GAD correlates well with that of GABA (Fahn, 1975) and GAD a c t i v i t y i s concentrated i n synaptosomes i n areas known to receive GABA afferents (Fonnum and Walberg, 1973). Thus, the s i g n i f i c a n t drop i n GAD a c t i v i t y observed i n the superior c o l l i c u l u s following the SN l e s i o n suggests that a GABA pr o j e c t i o n to the tectum was lesioned. It has been shown that i n t r a n i g r a l i n j e c t i o n s of KA s i m i l a r to those used i n the present study r e s u l t i n a sub s t a n t i a l reduction i n n i g r a l GAD a c t i v i t y (Nagy et a l . , 1978d). Injections of KA have been suggested to damage only neurons with c e l l bodies at the s i t e of i n j e c t i o n , sparing f i b e r s of passage (Coyle and Schwarcz, 1976; McGeer and McGeer, 1976a). Thus, the decrease i n GAD a c t i v i t y observed i n the c o l l i c u l u s appears to be due to the loss of n i g r a l GABA neurons which project to the tectum. The morphological data obtained i n the present study confirm e a r l i e r reports of a pr o j e c t i o n from the SNR to the superior c o l l i c u l u s . Further- more, u l t r a s t r u c t u r a l examination revealed that the terminals labeled a f t e r SN i n j e c t i o n s form symmetrical synapses with the major dendrites of t e c t a l neurons and contain pleomorphic v e s i c l e s . This morphology i s i d e n t i c a l 82 Figure 16. Examples of labeled boutons i n the superior c o l l i c u l u s following i n j e c t i o n of [ 3H]leucine into the substantia nigra pars r e t i c u l a t a . The labeled terminals form symmetrical synaptic contacts (arrow heads) with major dendrites (D) and contain s l i g h t l y pleomorphic v e s i c l e s Magnification A: X 46,309; B: X 62,842.  83 Table 11. Biochemical changes i n the superior c o l l i c u l u s a f t e r lesions of the substantia n i g r a . Uptake expressed as pmol/mg protein/ 5 min. Enzyme a c t i v i t y as nmol/mg protein/hr. n Control Lesion % of control Glycine uptake 6 209127 223+29 107 Aspartate uptake 6 352+40 302+30 86 Choline acetyltransferase 12 18.211.3 18.5+1.2 102 Glutamate decarboxylase 8 221+5.7 132+14^ 60* *p < 0.001; Student's _t test 84 to that of Purkinje c e l l terminals i n the deep cer e b e l l a r n u c l e i labeled by radioactive GABA transport (McGeer et a l . , 1975) and with presumed GABA boutons i n the SN labeled by uptake of rad i o a c t i v e GABA (Hattori et a l . , 1973b). They are also s i m i l a r to those synapses of the ce r e b e l l a r cortex or SN which s t a i n immunocytochemically for GAD (McLaughlin et a l . , 1974; Ribak et a l . , 1976). Anderson and Yoshida (1977) have recently suggested that some nigro- t e c t a l neurons send c o l l a t e r a l s to the ventromedial thalamus, and have shown that SN stimulation r e s u l t s i n monosynaptic i n h i b i t i o n i n the thalamus. Anatomical studies have also found that many n i g r a l neurons project to both the tectum and the ventromedial thalamus (Bentivoglio et a l . , 1979), and the morphology of the nigrothalamic boutons i s i d e n t i c a l to that of the n i g r o t e c t a l terminals (Kultas-Ilinsky et a l . , 1978). This suggests that the n i g r o t e c t a l p r o j e c t i o n may also be i n h i b i t o r y . Although York and Faber (1977) o r i g i n a l l y reported that a few t e c t a l units were activated by stimulation i n the SN, more recent studies have demonstrated that t e c t a l units are monosynaptically i n h i b i t e d by SN stimulation (Deniau et a l . , 1978). This i s consistent with the present evidence for a GABA-containing proj e c t i o n from the SNR to the superior c o l l i c u l u s and suggests that the nigrothalamic p r o j e c t i o n may u t i l i z e GABA as well. In f a c t , evidence has very recently been published supporting t h i s hypothesis (DiChiara et a l . , 1979; F e l t e r et a l . , 1979). 85 GENERAL DISCUSSION The striatum has been implicated i n both motor behaviour and i n more cognitive functions. In the present study the pathways by which the striatum can a f f e c t these processes have been examined using biochemical and h i s t o l o g i c a l techniques. Evidence has been provided i n d i c a t i n g that a p a r a l l e l p a i r of s t r i a t a l efferent systems exists projecting to the GP, EP and SN. One of these systems contains GABA, the other substance P. A met-enkephalin-containing s t r i a t o p a l l i d a l pathway has also been proposed (Experiment 1). Further experiments have suggested a r o l e for GABA i n the output pathways of the basal ganglia. The histochemical r e s u l t s obtained with GABA-T support the hypothesis of a pallidohabenular GABA pathway, while the r e s u l t s of Experiment 6 provided the f i r s t evidence for a n i g r o t e c t a l GABA projection. These pathways are summarized i n Fi g . 17. In the following discussion some speculations on the possible r o l e of these"pathways i n mediating the functions of the basal ganglia w i l l be offered. As l i t t l e i s known concerning the functions of the s t r i a t a l projections to the GP and EP, the discussion w i l l concentrate on the s t r i a t o n i g r a l system and the n i g r a l output pathways. NIGROSTRIATAL REGULATION The s t r i a t o n i g r a l pathway appears to serve two major functions:' i t provides an output pathway from the striatum and,;. i n addition, i t serves as a feedback system regulating the n i g r o s t r i a t a l dopamine system. The regulation of the n i g r o s t r i a t a l dopamine neurons has been a subject of intense debate for almost twenty years. In 1963 Carlsson and L i n q v i s t noted that neuroleptic drugs increased the turnover of dopamine i n the striatum and suggested that t h i s was due to a compensatory a c t i v a t i o n of the monoamine neurons subsequent to dopamine receptor blockade. The observations of Kehr et a l . (1972) that the dopamine agonist apomorphine 86 Figure 17: Summary diagram of the inputs and outputs of the basal ganglia. G = GABA pathway P = Substance P pathway CEREBRAL CORTEX [> STRIATUM [> ENTOPEDUNCULAR INTRALAMINAR THALAMUS SUBSTANTIA A NIGRA 1 / NUCLEUS G VENTROLATERAL THALAMUS ^ o SUPERIOR MOTOR COLLICULUS CORTEX & X^L SPINAL C O R D 87 and antagonist haloperidol could influence dopamine metabolism i n the striatum even a f t e r hemitransections separating the dopamine terminals from t h e i r c e l l bocies l e d Carlsson (1975) to postulate the existence of "autoreceptors" or dopamine receptors on the dopamine terminals. Autoreceptors for dopamine have also been postulated to e x i s t i n the SN (Aghajanian and Bunney, 1973; Groves et a l . , 1975). Iontophore- t i c a l l y applied dopamine or apomorphine i n h i b i t s the dopamine c e l l s i n the n i g r a i n a h a l o p e r i d o l - r e v e r s i b l e manner (Aghajanian and Bunney, 1973). In fact the dopamine c e l l s have been found to be much more s e n s i t i v e than s t r i a t a l neurons to the action of dopamine agonists ( S k i r b o l l et a l . , 1979). E l e c t r o p h y s i o l o g i c a l studies have found that systemic apomorphine i n h i b i t s dopamine c e l l f i r i n g even a f t e r destruction of the s t r i a t a l neurons with KA (Baring et a l . , 1980), suggesting that the SN i s the s i t e of action for t h i s drug. This i s supported by the obser- vation that i n t r a n i g r a l and systemic apomorphine have s i m i l a r e f f e c t s on s t r i a t a l dopamine turnover (Maggi et a l . , 1977). The concept of n i g r a l dopamine receptors has also received support from anatomical and biochemical demonstrations of the mechanisms necessary for dopamine transmission i n the nigra. Thus, n i g r a l dendrites have been shown to contain dopamine (Bjorklund and L i n d v a l l , 1975; Felton, 1977) as well as the synthetic enzyme tyrosine hydroxylase (Pickel et a l . , 1976). Also s p e c i a l i z e d organelles which accumulate the dopamine analog 5-hydroxy- dopamine have been observed (Hattori et a l . , 1979; Mercer et a l . , 1979; Wilson et a l . , 1977) and both dendro-dendritic (Hajdu et a l . , 1973; Wilson et a l . , 1977) and dendro-axonic (Hattori et a l . , 1979; Reubi and Sandri, 1979) connections have been reported. In addition, a calcium- dependent dopamine release ( H e f t i and L i c h t e n s t e i g e r , 1978; Geffen et a l . , 1976; Korf et a l . , 1976; Nieoullon et a l . , 1977; Paden et a l . , 1976), 88 dopamine binding s i t e s (Nagy et a l . , 1978c; Quick et a l . , 1979) and a dopamine-sensitive adenylate cyclase (Kebabian and Saavadra, 1976; P h i l l i p s o n and Horn, 1976; Spano et a l . , 1976; Traficante et a l . , 1976) have been shown to e x i s t i n the SN. Although the existence of dopamine autoreceptors i n the striatum and SN appears to explain the actions of systemic apomorphine on dopamine neuronal a c t i v i t y , some d i f f i c u l t i e s a r i s e when the actions of other dopaminergic drugs are considered. Amphetamine i s thought to release dopamine and to block the neuronal uptake of t h i s amine i n both terminal regions (Carlsson et a l . , 1965; Glowinski et a l . , 1966) and at the dopamine c e l l bodies (Groves et a l . , 1975; Nieoullon et a l . , 1977; Paden et a l . , 1976). This i s accompanied by a depression of neuronal f i r i n g i n both the striatum and SN (Bunney and Aghajanian, 1973; Rebec and Groves, 1975). The i n h i b i t i o n of dopamine c e l l f i r i n g i s markedly attenuated by l e s i o n s of the s t r i a t o n i g r a l pathway providing support for the hypothesis of Corrodi et a l . (1967) that amphetamine acts v i a a neuronal feedback loop to modulate dopamine c e l l a c t i v i t y . However, amphetamine also appears to act i n the SN to i n h i b i t dopamine c e l l s since at high doses i t s depressant e f f e c t on n i g r a l c e l l s s t i l l occurs i n the absence of the s t r i a t o n i g r a l system (Bunney and Aghajanian, 1978). The e f f e c t of amphetamine.on dopamine turnover i n the striatum i s also not abolished by the destruction of the s t r i a t o n i g r a l pathway (Argiolas et a l . , 1978). Systemic dopamine antagonists such as haloperidol increase the f i r i n g rate of the dopamine neurons of the SN. In contrast to the i n h i b i t i o n of dopamine c e l l f i r i n g by apomorphine and amphetamine which are respectively unaffected or p a r t i a l l y attenuated, the actions of haloperidol on dopamine c e l l f i r i n g are completely abolished by i n t e r r u p t i o n of the s t r i a t o n i g r a l 89 pathway (Kondo and Iwatsubo, 1980). This indicates that the s t r i a t o n i g r a l feedback system mediates the actions of neuroleptics on dopamine neuron a c t i v i t y . Strong support for t h i s hypothesis comes from observations on the k i n e t i c a c t i v a t i o n of s t r i a t a l tyrosine hydroxylase. This e f f e c t i s dependent upon an increase i n impulse flow i n the n i g r o s t r i a t a l neurons (Roth et a l . , 1975) and occurs following systemic neuroleptic treatment (Zivkovic et a l . , 1974). Neuroleptic-induced a c t i v a t i o n , l i k e the increase i n dopamine c e l l f i r i n g i s abolished by lesions of the s t r i a t o n i g r a l pathway (Gale et a l . , 1978). Also d i r e c t a p p l i c a t i o n of neuroleptics to the striatum r e s u l t s i n a c t i v a t i o n (Gale et a l . , 1978) while n i g r a l a p p l i c a t i o n does not (Gale and G u i d o t t i , 1976; Gale et a l . , 1978). Thus, i t appears that systemic dopamine antagonists a f f e c t the dopamine neurons predominantly v i a the s t r i a t o n i g r a l feedback loop. However, i t must be kept i n mind that haloperidol can block the actions of apomorphine and amphetamine which seem to be independent of t h i s feedback system. GABA REGULATION OF THE NIGROSTRIATAL DOPAMINE SYSTEM The presence of a s t r i a t o n i g r a l pathway has been known for quite some time, and much evidence indicates that GABA i s a transmitter i n t h i s system (see Experiment 1). Stimulation of the striatum r e s u l t s i n a p i c r o t o x i n - 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 neurons ( F e l t z , 1971; Precht and Yoshida, 1971). This suggests that GABA could function i n the s t r i a t o n i g r a l feedback system c o n t r o l l i n g dopamine c e l l a c t i v i t y . Support for t h i s comes from the observation that intravenous i n j e c t i o n s of p i c r o t o x i n reverse the amphetamine-induced i n h i b i t i o n of n i g r a l c e l l s , and t h i s e f f e c t i s dependent upon the s t r i a t o n i g r a l neurons (Bunney and Aghajanian, 1978). This suggests that amphetamine acts i n the striatum to increase the f i r i n g rate of s t r i a t o n i g r a l GABA neurons which i n turn i n h i b i t the 90 dopamine c e l l s of the SN. Intracerebroventricular (Biswas and Carlsson, 1977a) and i n t r a - p e ritoneal (Biswas and Carlsson, 1977b) i n j e c t i o n s of GABA have been reported to increase s t r i a t a l dopamine l e v e l s suggesting an i n h i b i t i o n of dopamine c e l l f i r i n g . This has also been observed with the GABA agonist and prodrug SL 76002 (Lloyd et a l . , 1979) or with muscimol, another GABA agonist (Anden et a l . , 1979; Lloyd et a l . , 1979). Elevation of endogenous GABA l e v e l s by systemic i n j e c t i o n of GABA-T i n h i b i t o r s such as AOAA also increases s t r i a t a l dopamine l e v e l s (Anden, 1974; Biswas and Carlsson, 1977b) and AOAA reverses the increase i n dopamine turnover induced by neuroleptics (Anden, 1974; L a h t i and Losey, 1974), while i s o n i a z i d or p i c r o t o x i n enhance haloperidol-induced a c t i v a t i o n of s t r i a t a l tyrosine hydroxylase (Gale et a l . , 1978). F i n a l l y , chronic elevation of brain GABA l e v e l s r e s u l t s i n dopamine receptor s u p e r s e n s i t i v i t y i n the striatum, suggesting that the n i g r o s t r i a t a l dopamine c e l l s have been c h r o n i c a l l y i n h i b i t e d (Ferkany et a l . , 1980). These r e s u l t s i n d i c a t e that measures which increase c e n t r a l GABA function i n h i b i t the f i r i n g of n i g r a l dopamine c e l l s . Although GABA may have some action on dopamine release d i r e c t l y i n the striatum (Doble et a l . , 1980; Giorgieff-Chesselet et a l . , 1979; Starr, 1978a; Stoof and Mulder, 1977; Stoof et a l . , 1979) i t s main influence c l e a r l y occurs i n the SN. 3H-GABA receptors appear to occur on the dopamine c e l l s of the' SN, since t h e i r density decreases follow- ing 6-OHDA l e s i o n (Guidotti et a l . , 1978). Similar decreases i n 3H-GABA binding have been observed i n the SN of Parkinsonian patients (Lloyd et al.', 1977b; Rinne et a l . , 1978; 1979) i n which the n i g r o s t r i a t a l dopamine c e l l s are known to degenerate. Lesions of the s t r i a t o n i g r a l pathway increase the density of 3H-GABA or 3H-muscimol binding s i t e s (Gale and Iadorola, 1980; Gu i d o t t i et a l . , 1979; Waddington and Cross, 1978a) i n 91 a way suggestive of denervation s u p e r s e n s i t i v i t y . The observation of Bunney and Aghajanian (.1978) that KA lesions of the s t r i a t o n i g r a l system greatly increase the responses of SNC c e l l s to iontophoretic GABA further supports the idea that these receptors can display supersensi- t i v i t y . Anden and Stock (1973) and K e l l y and Moore (1978b) have found that the l o c a l a p p l i c a t i o n of GABA onto n i g r a l dopamine neurons causes an increase i n s t r i a t a l dopamine l e v e l s s i m i l a r to that seen a f t e r systemic GABA agonists. This does not occur a f t e r i n t r a s t r i a t a l GABA a p p l i c a t i o n (Andeh and Stock, 1973). These r e s u l t s suggest that n i g r a l GABA receptors are involved i n the regulation of dopamine c e l l s . Gale and Guido t t i (1976) also point to the ro l e of GABA i n the SN with t h e i r observation that i n t r a n i g r a l muscimol blocks the neuroleptic-induced a c t i v a t i o n of s t r i a t a l tyrosine hydroxylase. B i c u c u l l i n e i n j e c t i o n s of the SN block t h i s e f f e c t i n d i c a t i n g that s p e c i f i c GABA receptors are involved. Walters et a l . (1979) have found that muscimol or GABA-T i n h i b i t o r s also i n h i b i t the haloperidol-induced increase observed when tyrosine hydroxylase a c t i v i t y i s measured i n vivo. Their observation that n-dipropylacetate (Valproate) i s more e f f e c t i v e than AOAA i s consistent with the observation of Iadoraola and Gale (1979) that Valproate s e l e c t i v e - l y increases GABA l e v e l s i n the s t r i a t o n i g r a l terminals while AOAA appears to increase p r i m a r i l y other n i g r a l GABA pools. These observations support the hypothesis put forward in. Experiment 3 that the GABA-T present i n the s t r i a t o n i g r a l terminals may be d i r e c t l y involved i n c o n t r o l l i n g n i g r a l GABA transmission. In summary, the r e s u l t s described above i n d i c a t e that GABA released from the s t r i a t o n i g r a l terminals acts to i n h i b i t the f i r i n g of the n i g r o s t r i a t a l dopamine neurons. In times of increased dopaminergic 92 stimulation i n the striatum ( i . e . a f t e r amphetamine or L-dopa) the s t r i a t o n i g r a l GABA neurons would show an increase i n t h e i r a c t i v i t y , thereby i n h i b i t i n g the n i g r a l dopamine c e l l s . Conversely, during a time of decreased dopaminergic a c t i v i t y i n the striatum ( i . e . a f t e r neurolep- t i c s ) the a c t i v i t y of the s t r i a t o n i g r a l GABA neurons decreases, allowing the dopamine c e l l s of the SN to increase t h e i r f i r i n g rate. SUBSTANCE P REGULATION OF THE NIGROSTRIATAL DOPAMINE SYSTEM Substance P i s also present i n the s t r i a t o n i g r a l system (Experiment 1) and thus could be invoked as a p a r t i c i p a n t i n the feedback co n t r o l of n i g r a l dopamine c e l l s . Systemic substance P has been reported to increase s t r i a t a l dopamine turnover (Starr et a l . , 1978) as have i n j e c t i o n s of substance P into the l a t e r a l v e n t r i c l e (Magnusson et a l . , 1976). Intra- n i g r a l a p p l i c a t i o n of substance P has been found to produce a s i m i l a r increase i n s t r i a t a l dopamine turnover, as evidenced by increased s t r i a t a l l e v e l s of the dopamine metabolitesDOPAC and HVA (Waldemier et a l . , 1978). James and Starr (1979) report that t h i s increase i n s t r i a t a l dopamine turnover i s associated with substance P i n j e c t i o n s of the SNC. When the i n j e c t i o n i s made into the SNR a decrease i n s t r i a t a l HVA r e s u l t s . In vivo studies have also shown that i n t r a n i g r a l a p p l i c a t i o n of substance P increases dopamine release i n the i p s i l a t e r a l caudate nucleus (Cheramy et a l . , 1977). In contrast, n i g r a l a p p l i c a t i o n of antibodies against substance P, which function as substance P antagonists, produces a decrease i n s t r i a t a l dopamine release (Cheramy et a l . , 1978). Behavioural experiments also suggest a r o l e f o r the s t r i a t o n i g r a l substance P system i n the regulation of dopamine neuronal a c t i v i t y . Oipe and Koella (1977) report that u n i l a t e r a l i n t r a n i g r a l substance P in j e c t i o n s induce c o n t r a l a t e r a l r o t a t i o n . James and Starr (1979) have found that t h i s r o t a t i o n following SNC i n j e c t i o n s i s associated with an 93 increased turnover of dopamine i n the i p s i l a t e r a l striatum, and were able to block t h i s response with haloperidol (James and Starr, 1977). These observations agree with the hypothesis that animals rotate away from the side with increased s t r i a t a l dopamine function (Ungerstedt et a l . , 1969). Injection of substance P eit h e r into the l a t e r a l v e n t r i c l e (Katz, 1979) or the SN (Kelley et a l . , 1979) produces a grooming response which can be blocked by lesio n s of the dopamine terminals i n the striatum with 6-OHDA (Kelley and Iversen, 1978; 1979). B i l a t e r a l n i g r a l a p p l i c a t i o n r e s u l t s i n a strong stereotyped rearing and s n i f f i n g with no concurrent enhancement of locomotion (Kelley and Iversen, 1979). This response i s also blocked by s t r i a t a l 6-OHDA le s i o n s (Kelly and Iversen, 1979). These r e s u l t s are consistent with the hypothesis that stereotypy r e s u l t s from an a c t i v a t i o n of the n i g r o s t r i a t a l dopamine neurons (Kelly et a l . , 1975) and that such an a c t i v a t i o n can be produced by substance P. In t r a n i g r a l substance P has also been found to produce a d r a s t i c retrograde amnesia f o r a passive avoidance task (Huston and Sta u b l i , 1978) This e f f e c t too can be explained by a stimulation of the n i g r o s t r i a t a l dopamine neurons (Fibiger and P h i l l i p s , 1976). F i n a l l y , i n j e c t i o n s of substance P into the mesolimbic A-10 dopamine c e l l region produce an increased locomotor response which can be blocked by the i n f u s i o n of neuroleptics i n t o the nucleus accumbens or by 6-OHDA lesions of these neurons (Kelley et a l . , 1979). Also the locomotor response to systemic amphetamine i s potentiated by these substance P in j e c t i o n s (Stinus et a l . , 1978). These r e s u l t s are consistent with the hypothesis that enhanced locomotor a c t i v i t y r e s u l t s from a c t i v a t i o n of the mesolimbic dopamine neurons (Kelly et a l . , 1975) and indicate that substance P could play a r o l e i n t h i s response. In summary i t appears that the efferents of the striatum can exert 94 opposite e f f e c t s on the n i g r a l dopamine neurons. The GABA system provides a negative feedback system i n h i b i t i n g dopaminergic a c t i v i t y , while the substance P system acts to increase the a c t i v i t y of these neurons. Perhaps through these two systems the f i r i n g rate of the dopamine neurons can be controlled with great p r e c i s i o n . GABA, SUBSTANCE P AND THE OUTPUT OF THE STRIATUM In addition to t h e i r r o l e i n the regulation of the n i g r a l dopamine c e l l s , the efferents of the striatum must provide output pathways for the expression of the many functions i n which dopamine and the striatum have been implicated. Dyskinetic disorders have been a t t r i b u t e d to a hyper- dopaminergic a c t i v i t y or to a cholinergic-dopaminergic imbalance i n the striatum. GABA involvement i n dyskinesic syndromes has also been suggested due t o j t h e s i g n i f i c a n t reduction i n s t r i a t o n i g r a l GABA and GAD observed i n Huntington's disease (Bird and Iversen, 1974; Enna et a l . , 1976; McGeer and McGeer, 1976; Perry et a l . , 1973; Urquhart et a l . , 1975). A reduction i n cerebrospinal f l u i d l e v e l s of GABA has also been found i n tardive dyskine- s i a patients (Neophytides et a l . , 1978). In animal studies i t has been found that decreasing s t r i a t a l GABA function by the i n t r a s t r i a t a l i n j e c t i o n of GABA antagonists r e s u l t s i n a GABA-reversible dyskinesia (Robin et a l . , 1979). These r e s u l t s suggest that treatment with drugs which increase GABA function in the striatum could be of benefit i n t r e a t i n g dyskinesias i n man. Both muscimol (Chase and Tamminga, 1979) and Valproate ( L i n n o i l a e l a l . , 1976) have been found to be of some benefit i n tardive dyskinesia. Also, the d i r e c t GABA agonist SL 76002 has been found to be of benefit i n tr e a t i n g the dyskinesias r e s u l t i n g from L-dopa therapy i n Parkinson's disease ( B a r t h o l i n i et a l . , 1979). It has also been found to be of some ' value i n the early stages of Huntington's disease. However, Valproate has not been found b e n e f i c i a l i n Huntington's disease (Lenman et a l . , 1976; 95 Shoulson et a l . , 1976). The poor r e s u l t s obtained with GABA drugs i n Huntington's disease, e s p e c i a l l y i n i t s l a t e r stages, could be due to the loss of s t r i a t a l GABA receptors i n t h i s disease (Iversen et a l . , 1979; Lloyd et a l . , 1977a). Also, as the usual treatment for Huntington's disease involves neuroleptic drugs, the decrease i n GABA receptors produced by these drugs (Trabbuchi et a l . , 1978) could also tend to render GABA agonists i n e f f e c t i v e i n t h i s disease. Akinesia and catalepsy are thought to r e s u l t from an i n h i b i t i o n of the action of dopamine i n the striatum. Thus, the motor d e f i c i t s produced by haloperidol or reserpine are reversed by L-dopa or apomorphine. It has been reported that AOAA, muscimol and SL 76002 injected systemically potentiate neuroleptic-induced catalepsy (Biggio et a l . , 1977; Kaariainen, 1976; K e l l e r et a l . , 1976; Lloyd and Davidson, 1979; Worms and Lloyd, 1978). It was suggested that t h i s was due to a f a c i l i t a t i o n of the GABA i n h i b i t i o n of the dopamine neurons i n the SN (Kell e r et a l . , 1976; Lloyd and Davidson, 1979; Matsui and Deguchi, 1977). However, the observation ( C o s t a l l and Olley, 1971) that l e s i o n s of the GP antagonize haloperidol catalepsy indicates that the s t r i a t o p a l l i d a l pathway may be involved i n t h i s response. Indeed, i t has been found that d i r e c t elevation of p a l l i d a l GABA l e v e l s or the i n j e c t i o n of muscimol into the GP potentiates the c a t a l e p t i c action of haloperidol (Matsui and Kamioka, 1978). Also ethanolamine-o-sulphate (EOS, a GABA-T i n h i b i t o r ) i n j e c t i o n s of the GP r e s u l t i n an akinesic state which i s not blocked by amphetamine (Pycock et a l . , 1976). The EOS i n j e c t i o n of the GP also blocks the hyperactive response induced-in the rat by d i r e c t stimulation of the dopamine receptors i n the accumbens (Pycock and Horton, 1976). Perhaps t h i s indicates that i n h i b i t i o n of the recently discovered GABA pathway from the accumbens to the GP (Walaas and Fonnum, 1979) mediates the increased locomotor response to amphetamine. 96 Neuroleptic drugs have been found to increase the turnover of GABA i n the GP and the nucleus accumbens (Marco et a l . , 1976). The increased turnover i n the accumbens p e r s i s t s a f t e r chronic haloperidol (Marco et a l . , 1976) suggesting that t h i s change could be a biochemical marker fo r the mechanisms involved i n the symptomatic r e l i e f of schizophrenia e l i c i t e d by these drugs (Costa et a l . , 1978). In contrast, tolerance develops to the increase i n the turnover of GABA i n the GP (Marco et a l . , 1976) . This may be a biochemical i n d i c a t o r of the tolerance that occurs to the cataleptogenic properties of neuroleptics (Ezrin-Waters and Seeman, 1977) . Also, Moroni et a l . (1979) have observed an increased GABA turnover i n the GP following either systemic or i n t r a s t r i a t a l morphine and suggest that t h i s may also be i n d i c a t i v e of opiate catalepsy. Evidence also e x i s t s implying that the elevation of n i g r a l GABA l e v e l s does not potentiate neuroleptic catalepsy. Elevation of n i g r a l GABA l e v e l s by the l o c a l i n j e c t i o n of EOS (Dray et a l . , 1975) or gabaculline, another GABA-T i n h i b i t o r induces h y p e r a c t i v i t y which i s blocked by i n t r a n i g r a l p i c r o t o x i n (Matsui and Kamioka, 1978). Furthermore, i n t r a n i g r a l i n j e c t i o n of muscimol produces behavioural stimulation and antagonizes h a l o p e r i d o l - induced catalepsy (Scheel-Kruger et a l . , 1977). Elevation of n i g r a l GABA l e v e l s by l o c a l i n j e c t i o n s of gabaculline also antagonizes haloperidol catalepsy (Matsui and Kamioka, 1978). As discussed below, these behavioural e f f e c t s probably depend upon the i n h i b i t i o n of efferents from the SNR. In summary, these r e s u l t s i n d i c a t e that the systemic elevation of bra i n GABA function potentiates neuroleptic-induced catalepsy and antagonizes the hyperactive response to dopamine agonists v i a an action i n the GP and not the SN. This suggest that some of the behavioural functions of the n i g r o s t r i a t a l dopamine system might be mediated through an action on the s t r i a t o p a l l i d a l GABA pathway. 97 Rats with u n i l a t e r a l 6-OHDA lesio n s of the n i g r o s t r i a t a l dopamine system rotate away from the side with the l e s i o n when challenged with apomorphine (Ungerstedt et a l . , 1969). This has been a t t r i b u t e d to denervation super- s e n s i t i v i t y of the dopamine receptors following the l e s i o n . This response has been found to be reduced by electrocoagulation of the caudate nucleus i p s i l a t e r a l to the 6-OHDA l e s i o n (Marshall and Ungerstedt, 1977) i n d i c a t i n g that s t r i a t a l neurons are involved. As crus c e r e b r i lesions or hemitransec- tions j u s t r o s t r a l to the SN also reduce t h i s r o t a t i o n a l response, i t has been suggested that the s t r i a t o n i g r a l f i b e r s mediate t h i s behaviour (Marshall and Ungerstedt, 1977). Garcia-Munoz et a l . (1977) have found that l e s i o n s of the s t r i a t o n i g r a l pathway r e s u l t s i n a marked turning response towards the lesioned side with both apomorphine and amphetamine, i n s pite of i n t a c t dopamine neurons. Similar e f f e c t s have been found a f t e r extensive u n i l a t e r a l ablations of the striatum (Anden et a l . , 1966) or more recently following KA induced degeneration of s t r i a t a l c e l l bodies (Schwarcz et a l . , 1979). Also, e l e c t r i c a l stimulation of the striatum has been found to r e s u l t i n c o n t r a l a t e r a l head turning, and t h i s behaviour i s abolished by lesions of the GP or SN on the stimulated side (Lee et a l . , 1980). These r e s u l t s i n d i c a t e that some of the motor asymmetries observed a f t e r manipulations of s t r i a t a l dopaminergic function are mediated through the s t r i a t o n i g r a l pathway. OUTPUTS OF THE SUBSTANTIA NIGRA It i s known that the efferents which a r i s e i n the head of the striatum synapse p r e f e r e n t i a l l y i n the SNR on non-dopaminergic neurons (Hattori et a l . , 1973b; Tulloch et a l . , 1978). The SNR contains neurons projecting predominantly to the VM-VL thalamus (Beckstead et a l . , 1979; Carpenter et a l . , 1976; Carpenter and Peter, 1972; Cole et a l . , 1964; F a u l l and Mehler, 1978; Fi b i g e r et a l . , 1972; Mettler, 1970; Rinvik, 1975) the 98 the superior c o l l i c u l u s ( A f i f i and Kaelber, 1965; Beckstead et a l . , 1979; Graybiel, 1978; Hopkins and Niessen, 1976; Jayaraman et a l . , 1977; Rinvik et a l . , 1976) and the nucleus tegmentus pedunculopontis, par compacta (Beckstead et a l . , 1979). E l e c t r o p h y s i o l o g i c a l and anatomical evidence suggests that many SNR neurons project to both the v e n t r o l a t e r a l thalamus and the superior c o l l i c u l u s (Anderson and Yoshida, 1977; Bentivoglio et a l . , 1979). It i s therefore of i n t e r e s t that Deniau et a l . (1976) have shown that those neurons i n the SNR which are i n h i b i t e d a f t e r stimulation of the striatum are also antidromically driven from the ventro- l a t e r a l thalamus. This indicates that the n i g r a l efferents to the thalamus and tectum may represent major output systems f o r the striatum. The behavioural e f f e c t s seen following the i n j e c t i o n of KA i n t o the SN strongly support t h i s suggestion. Thus, u n i l a t e r a l , i n t r a n i g r a l administration of KA r e s u l t s i n chronic turning away from the lesioned side (DiChiara et a l . , 1977; Olianas et a l . , 1978a), while b i l a t e r a l n i g r a l i n j e c t i o n s produce chronic stereotyped behaviours (Olianas et a l . , 1978a). These e f f e c t s mimic a s t r i a t a l dopamine receptor stimulation, although they are independent of the n i g r o s t r i a t a l dopamine pathway (Olianas et a l . , 1978a). Thus, the stereotypy i s not antagonized by haloperidol, which also f a i l s to produce catalepsy i n these animals (Olianas et a l . , 1978a). These r e s u l t s suggest that dopamine receptor stimulation i n the striatum r e s u l t s v i a the s t r i a t o n i g r a l pathway i n the i n h i b i t i o n of the SNR output c e l l s . This could be accomplished by eit h e r an increase i n the f i r i n g of the s t r i a t o n i g r a l GABA c e l l s or by a decrease i n the a c t i v i t y of the s t r i a t o n i g r a l ' substance P c e l l s . As discussed above, the behavioural responses to i n t r a n i g r a l substance P appear to be dependent upon the ascending dopamine systems. In contrast, i n t r a n i g r a l administration of GABA agonists produces an acute syndrome- 99 s i m i l a r to that seen a f t e r KA lesi o n s of the SN. That i s , u n i l a t e r a l n i g r a l i n j e c t i o n s of GABA, muscimol or GABA-T i n h i b i t o r s r e s u l t s i n c o n t r a l a t e r a l turning (Dray et a l . , 1975; Olianas et a l . , 1978b; Oberlander et a l . , 1977; Scheel-KrOger et a l . , 1977), while that of GABA antagonists produces i p s i l a t e r a l r o t a t i o n (Olianas et a l . , 1978b; Scheel-Kruger et a l . , t 1977). B i l a t e r a l i n j e c t i o n s of p i c r o t o x i n r e s u l t s i n catalepsy which i s r e s i s t a n t to apomorphine administration ((linanas et a l . , 1978b), while b i l a t e r a l i n j e c t i o n s . o f muscimol produce intense stereotypy that i s un- affected by haloperidol (Olianas et a l . , 1978b; Scheel-Krttger et a l . , 1977). It can, therefore, be suggested that s t r i a t a l dopamine receptor stimulation r e s u l t s i n increased a c t i v i t y i n the s t r i a t o n i g r a l GABA neurons increasing n i g r a l GABA release and thereby i n h i b i t i n g the non-dopaminergic SNR output c e l l s . This leads to stereotypy. Conversely, catalepsy i n response to s t r i a t a l dopamine receptor blockade depends upon a reduction i n GABA release within the SN, with the consequent a c t i v a t i o n of the n i g r a l non- dopaminergic e f f e r e n t s . This hypothesis suggests thatneuroleptics should decrease n i g r a l GABA release during catalepsy. However, we have already seen that neuro- l e p t i c s increase GABA action i n the GP (Marco et a l . , 1976). This implies that 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 GABA systems are fu n c t i o n a l l y d i s s o c i a b l e . This i s i n agreement with the r e s u l t s found i n Experiment 1 that these two systems are anatomically d i s t i n c t . What neurotransmitters are contained i n the n i g r a l neurons which provide these output pathways from the ;basal ganglia? Recently, a decrease i n n i g r a l GAD has been observed following the destruction of n i g r a l neurons with KA (Nagy et a l . , 1978d). This has led to the suggestion that the nigra , i n addition to rec e i v i n g a GABA innervation from the striatum, contains a population of GABA neurons (Nagy et a l . , 1978d). Could GABA be 100 contained i n the n i g r a l output pathways? This p o s s i b i l i t y was examined i n Experiment 6 where a s i g n i f i c a n t decrease i n GAD a c t i v i t y was observed i n the superior c o l l i c u l u s following KA lesi o n s of the SN. This suggests that GABA i s a transmitter i n the n i g r a l efferents to the tectum. E l e c t r o p h y s i o l o g i c a l (Anderson and Yoshida, 1977) and anatomical (Bentivoglio et a l . , 1979) evidence indicates that the n i g r o t e c t a l neurons send c o l l a t e r a l s to the VM-VL thalamus. This implies that the nigrothalamic neurons may also u t i l i z e GABA as a transmitter. In f a c t , Yoshida and Omata (1978) have reported that the monosynaptic i n h i b i t i o n of the ventromedial thalamic neurons i n response to n i g r a l stimulation i s blocked by p i c r o t o x i n . Also, a decrease i n GAD a c t i v i t y i n the thalamus has recently been found following n i g r a l KA or e l e c t r o l y t i c lesions (DiChiara et a l . , 1979a; F e l t e r et a l . , 1979). The GABA n i g r o t e c t a l and nigrothalamic projections are thus l i k e l y candidates for the mediation of s t r i a t a l efferent information. In t h i s scheme, dopamine agonists, by increasing the a c t i v i t y of the s t r i a t o n i g r a l GABA system and thus i n h i b i t i n g the n i g r a l GABA efferents i n the SNR, would act to release c e r t a i n thalamic and t e c t a l units from i n h i b i t i o n . Conversely, neuroleptics, by decreasing the a c t i v i t y of the s t r i a t o n i g r a l GABA c e l l s would i n h i b i t c e r t a i n t e c t a l and thalamic units v i a these pathways. These two conditions are summarized i n Fig. 18. DiChiara et a l . (1979b) have examined the p o s s i b i l i t y that the GABA pathway to the ventromedial thalamus may mediate basal ganglia output. These workers have found that the i n j e c t i o n of muscimol into the ventro- medial thalamus produces a catalepsy which i s not blocked by apomorphine. Also, the intrathalamic i n j e c t i o n of muscimol had no e f f e c t on the stereotypy induced by apomorphine. Thus, these animals display both catalepsy and.stereotypy simultaneously. P i c r o t o x i n i n j e c t i o n s of the 101 Figure 18. Hypothetical mechanisms of action for haloperidol and amphetamine. In condition A amphetamine causes an increased release of dopamine (DA) within the striatum. This r e s u l t s i n a decrease i n the f i r i n g of the s t r i a t o n i g r a l substance P c e l l s (P) and an increase i n the a c t i v i t y of the s t r i a t o - n i g r a l GABA f i b e r s (GABA^. Together these systems act to decrease the e f f e c t of amphetamine on s t r i a t a l dopamine release by i n h i b i t i n g the n i g r a l dopamine neurons. In addition, the increased a c t i v i t y of the s t r i a t o n i g r a l GABA f i b e r s i n h i b i t s the GABA c e l l s of the SNR which project to the thalamus and tectum (GABA 2). This i n h i b i t i o n i s behaviourally expressed i n the form of stereotypy. In condition B haloperidol acts to i n h i b i t dopamine a c t i v i t y i n the striatum. This r e s u l t s i n an increase i n s t r i a t o n i g r a l substance P a c t i v i t y and a decrease i n s t r i a t o n i g r a l GABA a c t i v i t y , e f f e c t s which act to increase the a c t i v i t y of the n i g r o s t r i a t a l dopamine neurons. The decrease i n a c t i v i t y of the s t r i a t o n i g r a l GABA neurons also r e s u l t s i n increased a c t i v i t y i n the GABA pr o j e c t i o n neurons of the SNR. This leads to the development of catalepsy. io! a A Thalamus Tectum stereotypy B / Striatum ^ 9 9 / Thalamus Tectum O c a t a l e p s y 102 ventromedial thalamus produce hyperactivity but no stereotypy, and in addition reverse haloperidol catalepsy. Thus, the catalepsy produced by the blockade of s t r i a t a l dopamine receptors appears to result from the activation of the nigrothalamic neurons. It is noteworthy that manipulation of GABA in the ventromedial thalamus affects cataleptic behaviour but not stereotypy. As discussed above, increasing GABA function in the SN results in profound stereotyped behaviours that are independent of the dopamine system (Olianas et a l . , 1978b; Scheel-Kruger et a l . , 1977). Thus, we would expect that some nigral efferents would be involved in this process. A clue to which efferents are involved may come from the work of Cools (1979) who has found that the blockade of GABA action in the parafasicular-centromedian complex produces stereotypy which is not blocked by haloperidol. As SNR neurons are known to project to this region (Ahlenius, 1978; Beckstead et a l . , 1979; Clavier et a l . , 1976) this observation raises the possibility that inhibition of a GABA projection from SNR to the parafasicular-centromedian complex could be the basis for the stereotypic response to dopamine agonists. The electrophysiological evidence that the projection from the SNR to the superior colliculus interacts with the tectospinal system which regulates the neck muscles involved in head orientation (York and Faber, 1977) suggests that this pathway may mediate the rotational behaviours associated with unilateral manipulations of the nigrostriatal dopamine system. Support for this concept comes from the observation that lesions of the tectospinal pathway or the superior colliculus markedly attenuate apomorphine-induced rotation in rats with unilateral 6-OHDA lesions (Wirtshafter et a l . , 1978). However, other workers have questioned this result (Crossman and Sanbrook, 1978; Lee et a l . , 1980; Reavill et a l . , 103 1 9 7 9 ) l e a v i n g t h e r o l e o f t h e n i g r o t e c t a l s y s t e m i n t h e s e r e s p o n s e s i n d o u b t . T h e d e e p l a y e r s o f t h e s u p e r i o r c o l l i c u l u s a p p e a r t o b e i n v o l v e d i n o p t o k i n e t i c n y s t a g m u s a n d i n v i s u a l t r a c k i n g ( S p r a g u e e t a l . , 1 9 7 3 ) . T h u s , t h e t e r m i n a t i o n o f t h e n i g r o t e c t a l p a t h w a y w i t h i n t h i s z o n e ( i . e . t h e u p p e r p a r t o f t h e i n t e r m e d i a t e g r e y l a y e r ) c o u l d p r o v i d e t h e b a s a l g a n g l i a w i t h a c c e s s t o t h e o c c u l o m o t o r m e c h a n i s m s . I n t h i s r e g a r d , l e s i o n s o f t h e s t r i a t u m h a v e b e e n f o u n d t o r e s u l t i n e y e d e v i a t i o n s ( C h a n d l e r a n d C r o s b y , 1 9 7 5 ) a n d t o i m p a i r v i s u a l t r a c k i n g ( B o w e n , 1 9 6 9 ) . M o h l e r a n d W u r t z ( 1 9 7 6 ) h a v e f o u n d t h a t u n i t s i n t h e u p p e r p a r t o f t h e i n t e r m e d i a t e g r e y l a y e r o f t h e s u p e r i o r c o l l i c u l u s d i s c h a r g e w i t h t h e s h o r t e s t t i m e - l e a d b e f o r e s a c c a d e s . T h e s e w o r k e r s h a v e a l s o r e c o r d e d n e u r a l r e s p o n s e s i n t h i s l a y e r i n t e r p r e t e d a s s i g n a l l i n g " r e a d i n e s s " o f t h e v i s u o m o t o r s y s t e m ( M o h l e r a n d W u r t z , 1 9 7 6 ) . T h u s , i n v i e w o f t h e t h e o r y t h a t t h e b a s a l g a n g l i a a c t i n t h e i n i t i a t i o n o f movemen t ( D e n n y - B r o w n a n d Y a n a g i s a w a , 1 9 7 6 ) t h e r o l e o f t h e n i g r o t e c t a l p a t h w a y i n t h e g e n e r a t i o n o f t h e s e " r e a d i n e s s " p o t e n t i a l s may b e c o n s i d e r e d . F i n a l l y , t h e b a s a l g a n g l i a h a v e b e e n s u g g e s t e d t o f u n c t i o n a s a n i n t e r n a l f e e d b a c k s y s t e m f o r v i s u a l l y a n d s o m a t i c a l l y g u i d e d m o v e m e n t s ( A n d e r s o n e t a l . , 1 9 7 9 ) . T h e s p i n a l p r o j e c t i o n s a r i s i n g i n t h e t e g m e n t u m a n d s u p e r i o r c o l l i c u l u s a r e i n v o l v e d i n c o n t r o l l i n g t h e i n t e g r a t e d m o v e m e n t s o f t h e h e a d a n d b o d y ( K u y p e r s , 1 9 7 3 ) . T h u s , t h e n i g r o t e c t a l p a t h w a y may p r o v i d e a r o u t e b y w h i c h t h e b a s a l g a n g l i a c a n a c t i n s u c h a f e e d b a c k r e g u l a t i o n o f m o t o r b e h a v i o u r . 104 A MODEL OF THE BASAL GANGLIA It i s often u s e f u l to formulate models to explain the behaviour of complex systems, even i f those models appear premature and o v e r s i m p l i f i e d . Roberts (1976) has employed the concept of d i s i n h i b i t i o n i n discussing the functions of the basal ganglia. In view of the many recently discovered GABA pathways i n t h i s system such a view may be appropriate, and i t may be us e f u l to expand upon t h i s model. As shown i n F i g . 17, the striatum receives input from the ent i r e cerebral cortex and from the i n t r a - laminar thalamus, which receives afferents from the r e t i c u l a r formation. Thus, the striatum i s i n a p o s i t i o n to receive information of a general nature regarding the emotional and physical state, the l e v e l of conscious- ness and the degree of alertness of the animal. The striatum also receives an input from the dopamine c e l l s of the SNC. In a highly s i m p l i f i e d scheme t h i s input might be thought of as providing "requests" to the striatum for c e r t a i n movements or behaviours to occur. The striatum could then compare these requests with the general state of the animal and make a "decision" as to whether to allow the response or not. This response would then be sent out v i a the s t r i a t a l e f f e r e n t s . The s t r i a t o - n i g r a l pathways could provide a feedback v i a the GABA and substance P neurons to the dopamine c e l l s informing them that the response e i t h e r has or has not been made. If the response has been made the s t r i a t o n i g r a l GABA f i b e r s would increase t h e i r f i r i n g r a te, while the a c t i v i t y of the substance P c e l l s would be decreased. This would prevent further a c t i v a t i o n of the appropriate dopamine c e l l s u n t i l the need f o r the response again arose. Certain SNR c e l l s would also be i n h i b i t e d as w e l l . These GABA p r o j e c t i o n neurons, which may be t o n i c a l l y a c t i v e , would then decrease t h e i r f i r i n g rate and thereby d i s i n h i b i t c e r t a i n t e c t a l and thalamic units allowing the requested response to occur. This may be thought of as the 105 d i s i n h i b i t i o n of a pre-programmed neuronal c i r c u i t f o r patterned postural co n t r o l , normally held i n a tonic i n h i b i t i o n by these GABA neurons. If other s t r i a t a l a c t i v i t i e s are incompatible with the response requested and i t i s not complied with a d i f f e r e n t r e s u l t would ensue. In t h i s case the s t r i a t o n i g r a l substance P c e l l s would increase t h e i r rate and the GABA c e l l s decrease t h e i r rate of f i r i n g , thereby increasing the a c t i v i t y of the n i g r a l dopamine c e l l s . This would also r e s u l t i n the d i s i n h i b i t i o n of the n i g r a l projection neurons. They would therefore f i r e more and act to i n h i b i t the appropriate t e c t a l and thalamic c e l l s thus blocking the response. Can t h i s scheme be related to what i s known regarding the a c t i v i t y of the basal ganglia? In Parkinson's disease, or i n an animal following the i n j e c t i o n of neuroleptics, the dopaminergic input to the striatum i s reduced. In t h i s condition there i s d i f f i c u l t y i n i n i t i a t i n g movements. In the present model the dopaminergic signals necessary f o r such i n i t i a - t i o n would be very weak and t h i s would be associated with a decrease i n the a c t i v i t y of the s t r i a t o n i g r a l GABA f i b e r s and an increase i n the a c t i v i t y of the substance P system. As a feedback system t h i s response would act to increase the a c t i v i t y of the dopamine neurons. I t would also increase the a c t i v i t y of the n i g r o t e c t a l and nigrothalamic GABA neurons and thereby prevent the appropriate response patterns from being i n i t i a t e d . In contrast, during times of dopaminergic h y p e r a c t i v i t y , i . e. i n Huntington's disease or following amphetamine administration, an inappropriate release of action patterns r e s u l t s . The model would suggest that t h i s i s due to a r e l a t i v e l y greater a c t i v i t y of the s t r i a t o - n i g r a l GABA neurons as compared to the substance P c e l l s . This response would tend to decrease the a c t i v i t y of the n i g r a l dopamine c e l l s i n an 106 attempt to return the s t r i a t a l dopaminergic a c t i v i t y to normal. It would also r e s u l t i n the i n h i b i t i o n of the n i g r o t e c t a l and nigro- thalamic GABA neurons. This would r e s u l t i n the release of the thalamic and t e c t a l units c o n t r o l l i n g posture, r e s u l t i n g i n inappropriate movements. Although models such as t h i s may appear overly s i m p l i s t i c i n view of the complexity of the basal ganglia, i t i s u s e f u l to attempt to gain a perspective on the function of the basal ganglia as a whole. Such an overview often y i e l d s new predictions and i n s i g h t s which may r e s u l t i n further testable hypotheses. Hopefully, as further knowledge of the basic anatomy, physiology and chemistry of the basal ganglia becomes available a better understanding of the functions of t h i s system w i l l be possible. 107 REFERENCES A d i n o l f i , A.M. (1969) Degenerative changes i n the entopeduncular nucleus following l e s i o n s of the caudate nucleus: an el e c t r o n microscopic study. Exp. Neurol. 25, 246-254. A f i f i , A.K. and Kaclber, W.W. (1965) Efferent connections of the substan- t i a nigra i n the cat. Exp. Neurol. 11, 474-482. Aghajanian, G.K. and Bunney, B.S. (1973) E l e c t r o p h y s i o l o g i c a l e f f e c t s of amphetamine on dopaminergic neurons. In: "F r o n t i e r s i n Catecholamic Research". S.H. Snyder and E. Usdin, eds. Pergamon Press, New York, pp. 643-651. Ahlenius, S. (1978) Po t e n t i a t i o n by haloperidol of the catalepsy produced by lesi o n s of the parafasicular nucleus of the r a t . Brain Res. 150, 648-652. Alberts, R.W. and Brady, R.O. (1959) The d i s t r i b u t i o n og glutamate decar- boxylase i n the nervous system of the rhesus monkey. J . B i o l . Chem. 234, 926-928. Anden, N.-E. (1974) I n h i b i t i o n of the turnover of the brain dopamine a f t e r treatment with gamma-aminobutyrate 2-oxyglutarate transaminase i n h i b i t o r aminooxyacetic a c i d . Naunyn-Schmiedeberg's Arch. Pharmacol. 283, 419-424. Anden, N.-E. and Stock, G. (1973) Inhibitory e f f e c t of gammahydroxybuty- r i c acid and gammaaminobutyric acid on the dopamine c e l l s i n the substantia nigra. Naunyn-Schmiedeberg's Arch. Pharmacol. 270, 89-92. Anden, N.-E., Carlsson, A., Dahstrom, A., Fuxe, K., H i l l a r p , N.A. and Larsson, K. (1964) Demonstration and mapping out of n i g r o - n e o s t r i a t a l dopamine neurons. L i f e S c i . 3, 523-530. Anden, N-E., Dahlstrom, A., Fuxe, K. and Larsson, K. (1966) Functional r o l e of the n i g r o - n e o s t r i a t a l dopamine neurons. Acta pharmac. 24, 263-274. Anden, N.-E., Grabowski-Anden, M. and Wachtel, H. (1980) E f f e c t s of GABA and GABA-like drugs on the brain dopamine and on the motor a c t i v i t y of rat s . In: "GABA Neurotransmitters". P. Krogsgaard-Larsen, J. Scheel- Krtiger and H. Kofod, eds. Munksgaard, Copenhagen, pp. 135-146. Anderson, M. and Yoshida, M. (1977) E l e c t r o p h y s i o l o g i c a l evidence for branching n i g r a l p r o j e c t i o n to the thalamus and the superior c o l l i c u l u s . Brain Res. 137, 361-364. Anderson, R.J., Aldridge, J.W. and Murphy, J.T. (1979) Function of caudate neurons during limb movements i n awake primates. Brain Res. 173, 489-501. Argiolas, A., Fadda, F., Stef a n i n i , E. and Gessa, G.L. (1978) Evidence for a d i r e c t a c t i o n of amphetamine on dopamine metabolism i n the r a t substantia nigra i n vivo. Naunyn-Schmiedeberg's Arch. Pharmacol. 301, 171-174. 108 Azmitia, E.C. and Segal, M. (1978) An autoradiographic analysis of the d i f f e r e n t i a l ascending projections of the dorsal and median raphe n u c l e i i n the r a t . J. Comp. Neurol. 179, 641-668. Barber, R.P., Vaughn, J.E., Slemmon, J.R., Salvaterra, P.M., Roberts, E. and Leeman, S.E. (1979) The o r i g i n , d i s t r i b u t i o n and synaptic r e l a t i o n - ships of substance P axons i n rat s p i n a l cord. J. Comp. Neurol. 184, 331-352. Baring, M.C., Walters, J. R. and Eng, N. (1980) Act i o n of systemic apomorphine on dopamine c e l l f i r i n g a f t e r n e o s t r i a t a l k a i n i c acid lesions. Brain Res. 181, 214-218. B a r t h o l i n i , G., Lloyd, K.G., Worms, P., Constantinidis, J. and T i s s o t , R. (1979) GABA and GABA-ergic medication: r e l a t i o n to s t r i a t a l dopamine function and Parkinsonism. Adv. Neurol. 24, 253-257. Baxter, C L . (1976) Some recent advances i n studies of GABA metabolism and compartmentation. In: "GABA i n Nervous System Fun-tion". E. Roberts, T.N. Chase and D.B. Tower, eds. Raven Press, New York, pp. 61-87. Beckstead, R.M., Domesick, V.B. and Nauta, W.J.H. (1979) Eff e r e n t connections of the substantia nigra and v e n t r a l tegmental area i n the rat . Brain Res. 175, 191-217. Ben-Ari, U., Le Gal La S a l l e , G. and Kanazawa, I. (1977) Regional d i s t r i b u t i o n of substance P within the amygdaloid complex and bed nucleus of the s t r i a t e rminalis. Neurosci. L e t t . 4, 299-302. Bentivoglio, M., Van der Kooy, D. and Kuypers, H.G.J.M. (1979) The organization of the efferent projections of the substantia nigra i n the rat. A retrograde fluorescent double l a b e l l i n g study. Brain Res. 174, 1-17. Biggio, G., Casu, M., Corda, M.G., Vernaleone, F. and Gessa, G.L. (1977) E f f e c t s of muscimol, a GABA mimetic agent, on dopamine metabolism i n the mouse brain. L i f e S c i . 21, 525-532. Bir d , E.D. and Iversen, L.L. (1974) Huntington's chorea: post-mortem measurement of glutamic acid decarboxylase, choline acetyltransferase and dopamine i n basal ganglia. Brain 97, 457-472. Biswas, B. and Carlsson, A. (1977a) The e f f e c t of i n t r a c e r e b r o v e n t r i c u l a r l y administered GABA on br a i n monoamine metabolism. Naunya-Schmiedeberg's Arch. Pharmacol. 299, 41-46. Biswas, B. and Carlsson, A. (1977b) The e f f e c t of i n t r a p e r i t o n e a l l y administered GABA on brain monoamine metabolism. Naunyn-Schmeideberg's Arch. Pharmacol. 299, 47-51. Bjorklund, A. and L i n d v a l l , 0. (1975) Dopamine i n dendrites of substantia nigra neurons: Suggestions for a r o l e i n d e n d r i t i c terminals. Brain Res. 83, 531-537. 109 Bowen, F.P. (1969) Visuomotor d e f i c i t s produced by cryogenic l e s i o n s of the caudate. Neuropsychologia 7, 59-65. Brownstein, M.J.,Mroz, E.A. Kizer, J.S., Palk o v i t s , M. and Leeman, S.E. (1976) Regional d i s t r i b u t i o n of substance P i n the br a i n of the r a t . Brain Res. 116, 299-305. Brownstein, M.J., Mroz, E.A., Toppaz, M.L. and Leeman, S.E. (1977) On the o r i g i n of substance P and glutamic a c i d decarboxylase (GAD) i n the substantia nigra. Brain Res. 135, 315-323. Bunney, B.S. and Aghajanian, G. K. (1976) The precise l o c a l i z a t i o n of n i g r a l afferents i n the rat as determined by a retrograde t r a c i n g technique. Brain Res. 117, 423-435. Bunney, B.S. and Aghajanian, G.E. (1978) a-Amphetamine-induced depression of c e n t r a l dopamine neurons: evidence f o r mediation by both autoreceptors and a s t r i a t o - n i g r a l feedback pathway. Naunyn-Schmiedeberg's Arch. Pharmacol. 304, 255-261. Campochiaro, P. and Coyle, J.T. (1978) Ontogenetic development of kainate neurotoxicity: correlates with glutamatergic innervation. Proc. N a t l . Acad. S c i . USA, 75, 2025-2029. Carlsson, A. (1975) Receptor-mediated control of dopamine metabolism. In: "Pre- and Postsynaptic Receptors". E. Usdin and W.E. Bunney, J r . eds. Mercel Dekker, New York, pp. 45-65. Carlsson, A. and Lin d q v i s t , M. (1963) E f f e c t of chlorpromazine and halo- p e r i d o l on formation of 3-methoxytyramine and normetanephrine i n mouse brain. Acta Pharmacol. Tox i c o l . 20, 140-144. Carlsson, A., Li n d q v i s t , M., Dahlstrom, A., Fuxe, K., Masuoka, D. (1965) E f f e c t s of the amphetamine group on intraneuronal b r a i n amines i n vivo and i n v i t r o . J. Pharm. Pharmacol. 18, 521-523. Carmen, J.B., Cowan, W.M. and Powell, T.P.S. (1963) The organization of the c o r t i c o s t r i a t e connections i n the rabbit. Brain 86, 525-562. Carpenter, M.B. and Peter, P. (1972) N i g r o s t r i a t a l and nigrothalamic f i b e r s i n the rhesus monkey. J. Comp. Neurol. 144, 93-116. Carpenter, M.B. and Strominger, N.L. (1967) Efferent f i b e r s of the subthalamic nucleus i n the monkey. A comparison of the effe r e n t pro- j e c t i o n s of the subthalamic nucleus, substantia nigra and globus p a l l i d u s . Am. J. Anat. 121, 41-72. Carpenter, M.B., Nakano, K. and Kim, R. (1976) Nigrothalamic projections i n the monkey demonstrated by autoradiographic techniques. J . Comp. Neurol. 165, 401-416. Carter, D.A. and F i b i g e r , H.C. (1978) The projections of the endopedun- cular nucleus and globus p a l l i d u s i n rat as demonstrated by autoradio- graphy and horseradish peroxidase histochemistry. J . Comp. Neurol. 177, 113-124. 110 Chalmers, A., McGeer, E.G., Wickson, V. and McGeer, P.L. (1970) D i s t r i b u t i o n of glutamic acid decarboxylase a c t i v i t y i n the brain of various mammalian species. Comp. gen. Pharmacol. 1, 385-390. Chandler, W.F. and Crosby, E.G. (1975) Motor e f f e c t s of stimulation and ablation of the caudate nucleus of the monkey. Neurol. (Minneap.) 25, 1160-1163. Chan-Palay, V. (1978) The paratrigeminal nucleus I I . I d e n t i f i c a t i o n and i n t e r r e l a t i o n s of catecholamine axons, indolamine axons, and substance P immunoreactive c e l l s i n the neuropil. J . Neurocytol. 7, 419-442. Chase, T.N. and Tamminga, C.A. (1979) GABA system p a r t i c i p a t i o n i n human motor, cognitive and endocrine function. In "GABA Neurotransmitters". P. Krogsgaard-larsen, J. Scheel-Kruger and H. Kofod, eds. Munksgaard, Copenhagen, pp. 283-294. Cheramy, A., Michelot, R., L e v i e l , V., Nieoullon, A., Glowinski, J . and Kerdelhue, B. (1978) E f f e c t of immunoneutralization of substance P i n the cat substantia nigra on the release of dopamine from dendrites and terminals of dopaminergic neurons. Brain Res. 155, 404-408. Cheramy, A., Nieoullon, A., Michelot, R. and Glowinski, J. (1977) E f f e c t s of i n t r a n i g r a l a p p l i c a t i o n of dopamine and substance P on the i n vivo release of newly synthesized [3H]-dopamine i n the i p s i l a t e r a l caudate nucleus of the cat. Neurosci. L e t t . 4, 105-109. C l a v i e r , R.M. and F i b i g e r , H.C. (1977) Evidence that common f i b e r s contribute to both dorsal pontine and substantia nigra s e l f - s t i m u l a t i o n : combined biochemical assay and s i l v e r impregnation study. Brain Res. 131, 271-281. Cl a v i e r , R.M., Atmadja, S. and F i b i g e r , H.C. (1976) Nigrothalamic projections i n the rat as demonstrated by orthograde and retrograde trac i n g techniques. Brain Res. B u l l . 1, 379-384. Cole, M., Nauta, W.J.H. and Mehler, W.R. (1964) The ascending efferent projections of the substantia nigra. Trans. Amer. Neurol. Ass. 89, 74-78. Cools, A.R. (1979) GABA and thalamo-striatal c o n t r o l of apomorphine- l i k e e f f e c t s . In: "GABA Neurotransmitters". P. Krogsgaard-larsen, J. Scheel-Kruger and H. Kofod, eds. Munksgaard, Copenhagen, pp. 500- 517. Corrodi, H., Fuxe, K. and Hokfelt, T. (1967) The e f f e c t of some psycho- active drugs on c e n t r a l monoamine neurons. Eur. J . Pharmacol. 1, 363-368. Costa, E., Cheney, D.L. , Mao, C C . and Moroni, F. (1978) Action of antischizophrenic drugs on the metabolism of Y - a m i n O D U t y : r i c a c i d and acetylcholine i n globus p a l l i d u s , striatum and nucleus accumbens. Fed. Proc. 37, 2408-2414. I l l C o s t a l l , B. and Olley, J.E. (1971) Cholinergic and neuroleptic induced catalepsy: modification by lesions i n the globus p a l l i d u s and substantia nigra. Neuropharmacology 10, 581-594. Cowan, W.M. and Powell, T.P.S. (1966) 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 i n the monkey. J. Neurol. Neurosurg. Psychiat. 29, 426.439. Coyle, J.T. and Schwarcz, R. (1976) Lesions of s t r i a t a l neurones with k a i n i c acid provides a model for Huntington's chorea. Nature (Lond.) 263, 244-246. Crossman, A.R. and Sambrook, M.A. (1978) The neur o l o g i c a l basis of motor asymmetry following u n i l a t e r a l n i g r o s t r i a t a l l e s i o n s i n the r a t : the e f f e c t of secondary superior c o l l i c u l u s l e s i o n s . Brain Res. 159, 211-213. Cuello, A.C. and Kanazawa, I. (1978) The d i s t r i b u t i o n of substance P immunoreactive f i b e r s i n the rat central nervous system. J . Comp. Neurol. 178, 129-156. Cuello, A.C. and Paxinos, G. (1978) Evidence for a long leu-enkephalin s t r i a t o - p a l l i d a l pathway i n rat brain. Nature (Lond.) 271, 178-180. Cuello, A.C, Emson, P.C., Paxinos, G. and J e s s e l , T. (1978) Substance P containing and c h o l i n e r g i c projections from the habenula. Brain Res. 149, 413-429. Cuello, A.C. Ga l f r e , G. and M i l s t e i n , C. (1979) Detection of substance P i n the c e n t r a l nervous system by a monoclonal antibody, Proc. Natl. Acad. S c i . USA, 76, 3532-3536. Dahlstrom, A. and Fuxe, K. (1964) Evidence for the existence of monoamine neurons i n the c e n t r a l nervous system. Acta P h y s i o l . Scand. 64, 1-55. Davies, J. and Dray, A. (1976) Substance P i n the substantia nigra. Brain Res. 107, 623-627. Davies, J. and Tongroach, P. (1978) Neuropharmacological studies on the n i g r o - s t r i a t a l and r a p h e - s t r i a t a l system i n the r a t . Eur. J. Pharmacol. 51, 91-100. Deniau, J. M. Chevalier, G. and Feger, J. (1978) E l e c t r o p h y s i o l o g i c a l study of the n i g r o - t e c t a l pathway i n the r a t . Neurosci. L e t t . 10, 215-220. Deniau, J.H. Feger, J. and Le Guyader, C. (1976) S t r i a t a l evoked i n h i b i t i o n of i d e n t i f i e d nigrothalamic neurons. Brain Res. 104, 152- 156. Denny-Brown, D. and Yanaagisawa, N. (1976) The r o l e of the basal ganglia i n the i n i t i a t i o n of movement. In: "The Basal Ganglia, Research Pu b l i c a t i o n s : A s s o c i a t i o n for Research i n Nervous and Mental Disease", v o l . 55. M. D. Yahr, ed. Raven Press, New York, pp. 115-149. 1 1 2 De V i t o , J . L . a n d S m i t h , O . A . J r . 1964) S u b c o r t i c a l p r o j e c t i o n s o f t h e p r e f r o n t a l l o b e o f t h e m o n k e y . J . Comp. N e u r o l . 1 2 3 , 4 1 3 - 4 2 4 . D i C h i a r a , G . , O l i a n a s , M. , D e l F i a c c o , M . , S p a n o , P . F . a n d T a g l i a m o n t e , A . ( 1 9 7 7 ) I n t r a n i g r a l k a i n i c a c i d i s e v i d e n c e t h a t n i g r a l n o n - d o p a m i n e r - g i c n e u r o n s c o n t r o l p o s t u r e . N a t u r e ( L o n d . ) 2 6 8 , 7 4 3 - 7 4 5 . D i C h i a r a , G . , P o r c e d d u , M . L . , M o r e l l i , M . , M u l a s , M . L . a n d G e s s a , G . L . ( 1 9 7 9 a ) E v i d e n c e f o r a G A B A e r g i c p r o j e c t i o n f r o m t h e s u b s t a n t i a n i g r a t o t h e v e n t r o m e d i a l t h a l a m u s a n d t o t h e s u p e r i o r c o l l i c u l u s o f t h e r a t . B r a i n R e s . 1 7 6 , 2 7 3 - 2 8 4 . D i C h i a r a , G . , P o r c e d d u e , M . L . , M o r e l l i , M . , M u l a s , M . L . a n d G e s s a , G . L . ( 1 9 7 9 b ) S t r i o - n i g r a l a n d n i g r o - t h a l a m i c G A B A - e r g i c n e u r o n s a s o u t p u t p a t h w a y s f o r s t r i a t a l r e s p o n s e s . I n : " G A B A - n e u r o t r a n s m i t t e r s " . P . K r o g s g a a r d - l a r s e n , J . S c h e e l - K r u g e r a n d H . K o f o d , e t a . M u n s g a a r d , C o p e n h a g e n , p p . 4 6 5 - 4 8 1 . D i v a c , I . , F o n n u m , F . a n d S t o r m - M a t h i s e n , J . ( 1 9 7 7 ) H i g h a f f i n i t y u p t a k e o f g l u t a m a t e i n t h e t e r m i n a l s o f c o r t i c o - s t r i a t a l a x o n s . N a t u r e ( L o n d . ) 2 6 6 , 3 7 7 - 3 7 8 . D o b l e , A . , M i d d l e m i s s , D n . n . , S h a w , J . a n d T u m b u l l , M. ( 1 9 8 0 ) ( 1 ) B a c l o f e n d e c r e a s e s n e u r o t r a n s m i t t e r r e l e a s e i n t h e m a m m a l i a n CNS b y a n a c t i o n a t a n o v e l GABA r e c e p t o r . N a t u r e ( L o n d ) , 2 8 3 , 9 2 - 9 4 . D r a y , A . , O a k l e y , N . R . a n d S i m m o n d s , N . A . ( 1 9 7 5 ) R o t a t i o n a l b e h a v i o r f o l l o w i n g i n h i b i t i o n o f GABA m e t a b o l i s m u n i l a t e r a l l y i n t h e r a t s u b s t a n t i a n i g r a . J . P h a r m . P h a r m a c o l . 2 7 , 6 2 7 = 6 2 9 . E h r i n g e r , H . , H o r n r y k i e w i c z , 0 . a n d V e r t e i l u n g , H . v o n . ( 1 9 6 0 ) N o r a d r e n a l i n e a n d d o p a m i n e ( 3 - h y d r o x y t y r a m i n ) i n G e h i r n d e s M e n s c h e n u n d i h r v e r h a l t e n b e : E r k r a n k u n g e n d e s e x t r a p y r a m i d d e n s y s t e m s . K l i n . W o c h e n - s c h r . 3 8 , 1 2 3 6 - 1 2 3 9 . E m s o n , P . C . , C u e l l o , A . C , P a x i n o s , G . , J e s s e l l , T . a n d I v e r s e n , L . L . ( 1 9 7 7 ) T h e o r i g i n o f s u b s t a n c e P a n d a c e t y l c h o l i n e p r o j e c t i o n s t o t h e v e n t r a l t e g m e n t a l a r e a a n d i n t e r p e d u n c u l a r n u c l e u s i n t h e r a t . A c t a P h y s i o l . S c a n d . S u p p l . 4 5 2 , 4 3 - 4 6 . E n d o , K . , A r a k i , T . a n d Y a g i , N . ( ( 1 9 7 3 ) T h e d i s t r i b u t i o n a n d p a t t e r n o f a x o n b r a n c h i n g o f p y r a m i d a l t r a c t c e l l s . B r a i n R e s . 5 7 , 4 8 4 - 4 9 1 . E n n a , S . J . , B i r d , E . D . , B e n n e t t , J . P . , B y l u n d , D . B . , Y a m a m u r a , H . I . , I v e n s e n , L . L . a n d S n y d e r , S . H . ( 1 9 7 6 ) H u n t i n g t o n ' s c h o r e a : c h a n g e s i n n e u r o t r a n s m i t t e r r e c e p t o r s i n t h e b r a i n . N . E n g l . J . M e d . 2 9 4 , 1 3 0 5 - 1 3 0 9 . E z r i n - W a t e r s , C . a n d S e e m a n , P . ( 1 9 7 7 ) T o l e r a n c e t o h a l o p e r i d o l c a t a l e p s y . E u r . J . P h a r m . 4 1 , 3 2 1 - 3 2 7 . F a h n , S . ( 1 9 7 5 ) R e g i o n a l d i s t r i b u t i o n s t u d i e s o f GABA a n d o t h e r p u t a t i v e n e u r o t r a n s m i t t e r s a n d t h e i r e n z y m e s . I n : " G A B A i n N e r v o u s S y s t e m F u n c t i o n " . E . R o b e r t s , T . N . C h a s e , a n d D . B . T o w e r , e d s . R a v e n P r e s s , New Y o r k , p p . 1 6 9 - 1 8 6 . 113 Fall o n , J.H. and Moore, R.Y. (1978) Catecholamine innervation of the basal forebrain, IV. Topography of the dopamine p r o j e c t i o n to the basal forebrain and neostriatum. J. Comp. Neurol. 180, 545-579. F a u l l , R.L.M. and Mehler, W.R. (1978) The c e l l s of o r i g i n of n i g r o t e c t a l , nigrothalamic and n i g r o s t r i a t a l projections i n the ra t . Neuroscience 3, 989-1002. F e l t e r , A., James, T.A., K i l p a t r i c k , I.C., MaCleod, N.K. and St a r t , M.S. (1979) Neurochemical and e l e c t r o p h y s i o l o g i c a l evidence f o r gabaergic and glutamatergic nigro-thalamic neurones. Neursoci. L e t t , Suppl. 3, 5222. Felton, D.L. (1977) Dendritic monoamines i n the s q u i r r e l monkey brai n . Brain Res. 120, 553-558. F e l t z , P. (1971) Y _Aminobutyric acid and a caudate-nigral i n h i b i t i o n . Can. J . Phy s i o l . Pharmacol. 49, 1113-1115. F e l t z , P. and de Champlain, J. (1972) Persistence of caudate unitary responses to n i g r a l stimulation a f t e r destruction and fu n c t i o n a l impairment of the s t r i a t a l dopaminergic terminals. Brain Res. 42, 595-600. Ferkany, J.W., Strong, R. and Enna, S.J. (1980) Dopamine receptor super- s e n s i t i v i t y i n the corpus striatum following chronic e l e v a t i o n of brain y-aminobutyric a c i d . J. Neurochem. 34, 247-249. Fibig e r , H.C. and P h i l l i p s , A.G. (1976) Retrograde amnesia a f t e r e l e c t r i c a l stimulation of the substantia nigra: mediation by the dopaminergic n i g r o - n e o s t r i a t a l bundle. Brain Res. 116, 23-33. Fib i g e r , H.C, Nagy, J . I . , Staines, W.A. and Vincent, S.R. (1980) Organization and p l a s t i c i t y of GABAergic neurons i n some extrapyramidal n u c l e i of the ra t . Brain Res. B u l l . ( i n press). Fi b i g e r , H.C, Pudritz, R.E. McGeer, P.L. and McGeer, E.G. (1972) Axonal transport i n n i g r o - s t r i a t a l and nigro-thalamic neurons: e f f e c t s of medial forebrain buncle lesions and 6-hydroxydopamine. J. Neurochem. 19, 1697-1708. Fonnum, F. (1975) A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24, 407-409. Fonnum, F. and Walberg, F. (1973) An estimation of concentration of y-aminobutyric acid and glutamate decarboxylase i n the i n h i b i t o r y Purkinje axon terminals i n the cat. Brain Res. 54, 115-117. Fonnum, F., Gottesfeld, Z and Grofova, I. (1978a) D i s t r i b u t i o n of glutamate decarboxylase, choline acetyltransferase and aromatic amino acid decarboxylase i n the basal ganglia of normal and operated r a t s . Evidence for s t r i a t o - p a l l i d a l , striato-entopeduncular and s t r i a t o - n i g r a l GABAergic f i b e r s . Brain Res. 143, 125-138. 114 Fonnum, F., Grofova, I. and Rinvlk, E. (1980) O r i g i n and d i s t r i b u t i o n of glutamate decarboxylase i n the nucleus substhalamicus of the ra t . Brain Res. 153, 370-374. Fonnum, F., Grofova, I., Rinvik, E., Storm-Mathisen, J . and Walberg, F. (1974) Or i g i n and d i s t r i b u t i o n of glutamate decarboxylase i n substantia nigra of the cat. Brain Res. 71, 77-92. Fox, C.A., Andrade, A.N., Lu Qui, I.I. and Rafols, J.A. (1974) The primate globus p a l l i d u s : a Golgi and electron microscope study. J . Hirnforsch. 15, 75-93. Fox, C.A., Andrade, A.N., Schwyn, R.C. and Rafols, J.A. (1971) The aspiny neurons and the g l i a i n the primate striatum: a Golgi and e l e c - tron microscope study. J. Hirnforsch. 13, 341-362. Fuxe, K., Hokfelt, T., Johansson, 0., Lidbrink, P. and Ljungdahl, A. (1974) The o r i g i n of the dopamine nerve terminals i n limbic and f r o n t a l cortex, evidence for meso-corticl dopamine neurons. Brain Res. 82, 349-355. Gale, J.S., Bi r d , E.D., Spokes, E.G., Iversen, L.L. and J e s s e l , T. (1978) Human brain substance P: d i s t r i b u t i o n i n controls and Huntington's chorea. J. Neurochem. 30, 633-634. Gale, K. and Guido t t i , A. (1976) GABA-mediated con t r o l of rat n e o s t r i a t a l tyrosine hydroxylase revealed by i n t r a n i g r a l muscimol. Nature (Lond.) 263, 691-693. Gale, K. and Iadorola, M.J. (1980) GABAergic denervation of rat substantia nigra: functional and pharmacological properties. Brain Res. 183, 217-223. Gale, K. , Cost, E. , Toffano, G., Hong, J.-S. and G u i d o t t i , A. (1978) Evidence for a r o l e of n i g r a l Y - a m i n 0 D U t . y r i c a c i d and substance P i n the haloperidol-induced a c t i v a t i o n of s t r i a t a l tyrosine hydroxylase. J. Pharmacol. Exp. Ther. 206, 29-37. Gale, K., Hong, J.-S. and Gu i d o t t i , A. (1977) Presence of substance P and GABA i n separate s t r i a t o n i g r a l neurons. Brain Res. 136, 371-375. Garcia-Munoz, M., Nicolaou, N.M., Tullock, I.F., Wright, A.K. and Arbuthnott, G.W. (1977) Feedback loop or output pathway i n s t r i a t o - n i g r a l f i b r e s ? Nature (Lond.) 265, 363-366. G a r c i a - R i l l , E., Nieto, A., A d i n o l f i , A., H u l l , C.D. and Buchwald, N.A. (1979) Projections to the neostriatum from the cat precruciate cortex. Anatomy and physiology. Brain Res. 170, 393-407. Garron, D.C. (1973) Huntington's chorea and schizophrenia. In: "Advances i n Neurology", vol.1. A. Barbeau, T.N. Chase, and G.W. Paulson, eds. Raven Press, New York, pp. 729-734. 1 1 5 G a u c h y , C , B e a u j o u a n , J . C . B e s s o n , M . J . , K e r d e l h u e , B . , G l o w i n s k i , J . a n d M i c h e l o t , R. ( 1 9 7 9 ) T o p o g r a p h i c a l d i s t r i b u t i o n o f s u b s t a n c e P i n t h e c a t s u b s t a n t i a n i g r a . N e u r o s c i . L e t t . 1 2 , 1 2 7 - 1 3 1 . G e f f e n , L . B . , J e s s e l , T . M . , C u e l l o , A . C . a n d I v e r s e n , L . L . ( 1 9 7 6 ) R e l e a s e o f d o p a m i n e f r o m d e n d r i t e s i n r a t s u b s t a n t i a n i g r a l . N a t u r e ( L o n d . ) 2 6 0 , 2 5 4 - 2 6 0 . G i o r g u i e f f - C h e s s e l e t , M . L . , K e r n e l , M . L . , W a n d s c h e e r , D. a n d G l o w i n s k i , J . ( 1 9 7 9 ) A t t e m p t s t o l o c a l i z e t h e GABA r e c e p t o r s i n v o l v e d i n t h e G A B A - i n d u c e d r e l e a s e o f n e w l y - s y n t h e s i z e d [ 3 H ] d o p a m i n e i n r a t s t r i a t a l s l i c e s . B r a i n R e s . 1 7 5 , 3 8 3 - 3 8 6 . G l e e s , P . ( 1 9 4 4 ) T h e a n a t o m i c a l b a s i s o f c o r t i c o s t r i a t e c o n n e c t i o n s . J . A n a t . ( L o n d . ) 7 8 , 4 7 - 5 1 . G l o w i n s k i , J . , A x e l r o d , J . a n d I v e r s e n , L . L . ( 1 9 6 6 ) R e g i o n a l s t u d i e s o f c a t e c h o l a m i n e s i n t h e r a t b r a i n . I V . E f f e c t s o f d r u g s o n d i s p o s i t i o n a n d m e t a b o l i s m o f 3 H - n o r e p i n e p h r i n e a n d 3 H - d o p a m i n e . J . P h a r m a c o l . E x p . T h e r . 1 5 3 , 3 0 - 4 1 . G o l d m a n , P . S . a n d N a u t a , W . J . H . ( 1 9 7 7 ) A n i n t r i c a t e l y p a t t e r n e d p r e f r o n t o - c a u d a t e p r o j e c t i o n i n t h e r h e s u s m o n k e y . J . Comp . N e u r o l . 1 7 1 , 3 6 9 - 3 8 6 . G o n y a - M a g e e , T . a n d A n d e r s o n , M. ( 1 9 7 9 ) E l e c t r o p h y s i o l o g i c a l c h a r a c t e r i - x a t i o n o f a p r o j e c t i o n f r o m p e d u n c u l o p o n t i n e n u c l e u s t o e n t o p e d u n c u l a r n u c l e u s i n t h e c a t . N e u r o s c i . A b s t r . 5 , 2 3 3 . G o t t e s f e l d , Z . , M a s s a r i , V . J . , M u t h , E . A . a n d J a c o b o w i t z , D . M . ( 1 9 7 7 ) S t r i a m e d u l l a r i s : a p o s s i b l e p a t h w a y c o n t a i n i n g G A B A e r g i c a f f e r e n t s t o t h e l a t e r a l h a b e n u l a . B r a i n R e s . 1 3 0 , 1 8 4 - 1 8 9 . G r a m s c h , C . , H o l l t , V . , M e h r a e i n , P . , P a s i , A . a n d H e r z , A . ( 1 9 7 9 ) R e g i o n a l d i s t r i b u t i o n o f m e t h i o n i n e - e n k e p h a l i n a n d b e t a - e n d o r p h i n - l i k e i m m u n o r e a c t i v i t y i n human b r a i n a n d p i t u i t a r y . B r a i n R e s . 1 7 1 , 2 6 1 - 2 7 0 . G r a y b i e l , A . M . ( 1 9 7 8 ) O r g a n i z a t i o n o f t h e n i g r o t e c t a l c o n n e c t i o n : a n e x p e r i m e n t a l t r a c e r s t u d y i n t h e c a t . B r a i n R e s . 1 4 3 , 3 3 9 - 3 4 8 . G r a y b i e l , A . M . R a g s d a l e , C W . J r . a n d E d l e y , S . M . ( 1 9 7 9 ) C o m p a r t m e n t s i n t h e s t r i a t u m o f t h e c a t o b s e r v e d b y r e t r o g r a d e c e l l l a b e l i n g . E x p . B r a i n R e s . 3 4 , 1 8 9 - 1 9 5 . G r e e n , N . M . , A v i d i n ( 1 9 6 3 ) T h e u s e o f l l + C b i o t i n f o r k i n e t i c s t u d i e s a n d f o r a s s a y . B i o c h e m . J . 8 9 , 5 8 5 - 5 9 1 . G r o f o v a , I . ( 1 9 7 5 ) T h e i d e n t i f i c a t i o n o f s t r i a t a l a n d p a l l i d a l n e u r o n s p r o j e c t i n g t o s u b s t a n t i a n i g r a . A n e x p e r i m e n t a l s t u d y b y m e a n s o f r e t r o g r a d e a x o n a l t r a n s p o r t o f h o r s e r a d i s h p e r o x i d a s e . B r a i n R e s . 9 1 , 2 8 6 - 2 9 1 . G r o f o v a , I . ( 1 9 7 9 ) T y p e s o f s t r i a t o n i g r a l n e u r o n s l a b e l e d b y r e t r o g r a d e t r a n s p o r t o f h o r s e r a d i s h p e r o x i d a s e . A p p l . N e u r o p h y s i o l . 4 2 , 2 5 - 2 8 . 116 Groves, P.M., Wilson, C.J., Young, S.J. and Rebec, G.V. (1975) S e l f - i n h i b i t i o n by dopaminergic neurons. Science 190, 522-529. Guesdon, J.-L. Ternynck, T. and Aurameas, S. (1979) The use of a v i d i n - b i o t i n i n t e r a c t i o n i s immunoenzymatic techniques. J . Histochem. Cytochem. 27, 1131-1139. Guido t t i , A., Gale, K. and Suria, A. (1978) S p e c i f i c GABA binding i n nigra: enhancement a f t e r s t r i a t o - n i g r a l l e s i o n s . Trans. Am. Soc. Neurochem. 9, 125. Guidot t i , A., Gale, K. , Suria, A. and Toffano, G. (1979) Biochemical evidence f o r two classes of GABA receptors i n rat brai n . Brain Res. 172, 566-571. Guyenet, P.G. and Aghajanian, G.K. (1978) Antidromic i d e n t i f i c a t i o n of dopaminergic and other output neurons of the r a t substantia nigra. Brain Res. 150, 69-84. Hajdu, F., Hassler, R. and Bak, I.J. (1973) El e c t r o n microscopic study of the substantia nigra and the s t r i o - n i g r a l p r o j e c t i o n i n the r a t . Z. A e l l f o r s c h . 146, 207-221. Hammond, C., Deniau, J.M. , Rizk, A. and Feger, J . (1978) Electrophysio- l o g i c a l demonstration of an excitatory subthalamonigral pathway i n the rat. Brain Res. 151, 235-244. Hassler, R. (1978) S t r i a t a l control of locomotion, i n t e n t i o n a l actions and of int e g r a t i n g and perceptive a c t i v i t y . J. Neurol. S c i . 36, 187-224. H a t t o r i , T. , F i b i g e r , H. C. and McGeer, P.L. (1975) Demonstration of a p a l l i d o - n i g r a l p r o j e c t i o n innervating dopaminergic neurons. J. Comp. Neurol. 162, 487-504. H a t t o r i , T. , F i b i g e r , H.C, McGeer, P.L. and Maler, L. (1973a) Analysis of the f i n e structure of the dopaminergic n i g r o s t r i a t a l p r o j e c t i o n by electron microscopic autoradiography. Exp. Neurol. 41, 599-611 Hat t o r i , T. , McGeer, P.L., F i b i g e r , H.C. and McGeer, E.G. (1973b) On the source of GABA-containing terminals i n the substantia nigra. Electron microscopic, atuoradiographic and biochemical studies. Brain Res. 54, 103-114. H a t t o r i , T., McGeer, P.L. and McGeer, E.G. (1979) Dendro axonic neurotransmission. II Morphological s i t e s for the synthesis, binding and release of neurotransmitters i n dopaminergic dendrites i n the substantia nigra and cholinergic dendrites i n the neostriatum. Brain Res. 170, 71-83. H e f t i , F. and Licht e n s t e i g e r , W. (1978) Dendritic dopamine: studies on on the release of endogenous dopamine from s u b c e l l u l a r p a r t i c l e s derived from dendrites of rat n i g r o - s - r i a t a l neurons. Neurosci. L e t t . 10, 65- 70. 117 Heitzman, H. and Richards, F.M. (1974) Use of the a v i d i n - b i o t i n complex for s p e c i f i c s t a i n i n g of b i o l o g i c a l membranes i n electron microscopy. Proc. Natl. Acad. S c i . USA 71, 3537-3541. Herkenham, M. and Nauta, W.J.H. (1977) Afferent connections of the habenula n u c l e i i n the rat. A horseradish peroxidase study, with a note on the fiber-of-passage problem. J. Comp. Neurol. 173, 123-146. Herkenham, M. and Nauta, W.J.H. (1979) Efferent connections of the habenula n u c l e i i n the r a t . J. Comp. Neurol. 187, 19-48. Hokfelt, T. and Ungerstedt, U. (1973) S p e c i f i c i t y of 6-hydroxydopamine induced degeneration of centrol monoamine neurons; an electron and fluorescence microscope study with s p e c i a l reference to i n t r a c e r e b r a l i n j e c t i o n on the n i g r o s t r i a t a l dopamine system. Brain Res. 60, 269-298. o Hokfelt, T., Johannson, 0., K e l l e r t h , J.-0. Ljungdahl, A., Nil s s o n , G. Nygords, A. and Pernow, B. (1977) Immunohistochemical d i s t r i b u t i o n of substance P. In: "Substance P". U.S. von Euler and B. Pernow, eds. Raven Press, New York, pp. 117-145. Hong, J.S. and Costa, E. (1978) P a r t i c i p a t i o n of substance P i n the action of halo p e r i d o l . Psychopharm. B u l l . 14, 23-24. Hong, J.S., Costa, E. and Yang, H.-Y. T. (1976) E f f e c t s of habenula lesions on the substance P content of various brain regions. Brain Res. 118, 523-525. Hong, J.S., Yang, H.Y. and Costa, E. (1977a) On the l o c a l i z a t i o n of methio- nine enkephalin neurons i n rat striatum. Neuropharmacology 16, 451-453. Hong, J.S., Yang, H.-Y. T. and Costa, E. (1978a) Substance P convent of substantia nigra a f t e r chronic treatment with antischizophrenic drugs. Neuropharmacology 17, 83-85. Hong, J.S., Yang, H.-Y. T., Fratt a , W. and Costa, E. (1978b) Rat s t r i a t a l nine-enkephalin i n d i s c r e t e regions of rat brain. Brain Res. 134, 383-386. Hong, J.S., Yang, H.-Y.T., F r a t t a , W. and Costa, E. (1978b) Rat s t r i a t a l methionine-enkephalin content a f t e r chornic treatment with cataleptogenic and noncataleptogenic antischizophrenic drugs. J. Pharm. Exp. Ther. 205, 141-147. Hong, J.S., Yang, H-Y.T. , G i l l i n , J . C , D i G i u l i o , A.M., F r a t t a , W. and Costa, E. (1979) Chronic treatment with h a l o p e r i d o l accelerates the biosynthesis of enkephalins i n the striatum. Brain Res. 160, 192-195. Hong, J.S., Yang, H.-Y.T., Racagni, G. and Costa, E. (1977c) Projections of substance P containing neurons from neostriatum to substantia nigra. Brain Res. 122, 541-544. Hopkins, D.A. and Niessen, L.W. (1976) Substantia n i g r a projections to the r e t i c u l a r formation, superior c o l l i c u l u s and c e n t r a l gray i n the r a t , cat and monkey. Neurosci. L e t t . 2, 253-259. 118 Hornykiewicz, 0. (1973) Parkinson's disease: from b r a i n homogenate to treatment. Fed. Proc. 32, 183-190. Huston, P.P. and St a u b l i , U. (1978) Retrograde amnesia produced by p o s t - t r i a l i n j e c t i o n of substance P into substantia n i g r a . Brain Res. 159, 468-472. Iadarola, M.J. and Gale, K. (1979) D i s s o c i a t i o n between drug-induced increases i n nerve terminal and non-nerve terminal pools of GABA i n vivo. Eur. J . Pharmacol. 59, 125-129. Ivensen, L.L., Bi r d , E., Spokes, E., Nicholson, S.H. and Suckling, C.J. (1979) Agonist s p e c i f i c i t y of GABA binding s i t e s i n human brain and GABA i n Huntington's disease and schizophrenia. In: "GABA-Neurotrans- mitters". P. Krogsgaard-Larsen, J. Scheel-Kruger and H. Kofod, eds. Munksgaard, Copenhagen, pp. 179-190. Iwahori, N. (1977) A Golgi study on the habenula nucleus of the cat. J. Comp. Neurol. 171, 319-344. Jacobs, B.L., Foote, S.L. and Bloom, F.E. (1978) D i f f e r e n t i a l projections of neurons within the dorsal raphe nucleus of the r a t : A horseradish peroxidase (HRP) study. Brain Res. 147, 149-153. James, T.A. and Starr, M.S. (1977) Biochemical and biochemical e f f e c t s of substance P injec t e d into the substantia nigra of the r a t . J. Pharm. Pharmacol. 29, 181-182. James, T.A. and Sta r r , M.S. (1979) E f f e c t s of substance P in j e c t e d i n t o the substantia nigra. Br. J. Pharmacol. 65, 423-432. Jayaraman, A., Batton, R.R. I l l and Carpenter, M.B. (1977) N i g r o t e c t a l projections i n the monkey: an autoradiographic study. Brain Res. 135, 147-152. J e s s e l , T.M., Emson, P.C., Paxinos, G. and Cuello, A.C. (1978) Topographic proj e c t i o n of substance P and GABA pathways i n the s t r i a t o - and p a l l i d o - n i g r a l system: a biochemical and immunohistochemical study. Brain Res. 152, 487-498. Johnston, M.V., McKinney, M. and Coyle, J.T. (1979) Evidence for a choli n e r g i c p r o j e c t i o n to neocortex from neurons i n basal forebrain. Proc. Natl. Acad. S c i . USA 76, 5392-5396. Jones, E.G. and L e a v i t t , R.Y., (1974) Retrograde axonal transport and the demonstration of non-specific projections to the cerebral cortex and striatum from thalamic intralaminar n u c l e i i n the r a t , cat and monkey. J. Comp. Neurol. 154, 349-378. Jones, E.G., Coulter, J.D. , Burton, H. and Porter, R. (1977) C e l l s o r i g i n and terminal d i s t r i b u t i o n of c o r t i c o s t r i a t a l f i b e r s a r i s i n g i n the sensory-motor cortex of monkeys. J. Comp. Neurol. 173, 53-80. 119 Kaariainen, I. (1976) E f f e c t of AOAA and baclofen on the catalepsy and on the increase of mesolimbic and s t r i a t a l DA turnover induced by haloperidol i n r a t s . Acta Pharmacol. T o x i c o l . 39, 393-400. K a l i l , K. (1978) Patch-like termination of thalamic f i b e r s i n the putamen of the rhesus monkey, an autoradiographic study. Brain Res. 140, 333-339. Kanazawa, I. and J e s s e l , T. (1976) Postmortem changes and regional d i s t r i b u t i o n of substance P i n the rat and mouse nervous system. Brain Res. 117, 362-367. Kanazawa, I., Emson, P.C. and Cuello, A.C. (1977a) Evidence f or the existence of substance P-containing f i b e r s i n s t r i a t o - n i g r a l and p a l l i d o - n i g r a l pathways i n rat brain. Brain Res. 119, 447-453. Kanazawa, I., Marshall, G.R. and K e l l y , J.S. (1976) Afferents to the rat substantia nigra studied with horseradish peroxidase, with s p e c i a l r e f e r - ence to f i b e r s from the subthalamic ncuelus. Brain Res. 115, 485-491. Kanazawa, I., O'Connell, R. and Powell, D. (1977) Evidence f or a decrease i n substance P content of substantia nigra i n Huntington's chorea. Brain Res. 120, 387-392. Kataoka, K., Bak, I.J., Hassler, R., Kim, J.S. and Wagner, A. (1974) L-Glutamate decarboxylase and choline acetyltransferase i n the substantia nigra and the striatum a f t e r s u r g i c a l i n t e r r u p t i o n of the s t r i o - n i g r a l f i b e r s of the baboon. Exp. Brain Res. 19, 217-227. Katz, R.J., (1979) Central i n j e c t i o n of substance P e l i c i t s grooming behaviour and motor i n h i b i t i o n i n mice. Neurosci. L e t t . 12, 133-136. Kebabian, J.W. and Saavedra, J.M. (1976) Dopamine-sensitive adenylate cyclase occurs i n a region of substantia nigra containing dopamine dendrites. Science 193, 683-685. Kehr, W., Carlsson, A., L i n q v i s t , M., Magnusson, T. and Atack, C. (1972) Evidence for a receptor-mediated control of s t r i a t a l tyrosine hydroxylase a c t i v i t y . J. Pharm. Pharmacol. 24, 744-746. K e l l e r , H.H., Schnaffner, R. and Haefely, W. (1976) Int e r a c t i o n of benzo- diazepines with neuroleptics at cen t r a l dopamine neurons. Naunyn-Schmie- deberg' s Arch. Pharmacol. 294, 1-7. Kelley, A.E. and Iversen, S.D. (1978) Behavioural response to b i l a t e r a l i n j e c t i o n s of substance P into the substantia nigra of the r a t . Brain Res. 158, 474-478. Kelley, A.E. and Iversen, S.D. (1979) Substance P i n f u s i o n into substantia nigra of the r a t : behavioural analysis and involvement of s t r i a t a l dopamine. Eur. J . Pharmacol. 60, 171-179. Kelley, A.E., Stinus, L. and Iversen, S.D. (1979) Behavioural a c t i v a t i o n induced i n the rat by substance P in f u s i o n into v e n t r a l tegmental area: implications of dopaminergic A10 neurons. Neurosci. L e t t . 11, 335-339. 120 K e l l y , P.H. and Moore, K.E. (1978a) Decrease of n e o c o r t i c a l choline acetyltransferase a f t e r l e s i o n of the globus p a l l i d u s i n the r a t . Exp. Neurol. 61, 479-484. K e l l y , P.H. and Moore, K.E. (1978b) Dopamine concentrations i n the rat brain following i n j e c t i o n s into the substantia n i g r a of baclofen y-aminobutyric a c i d , y-hydroxybutyric acid, apomorphine and amphetamine. Neuropharmacology 17, 169-174. K e l l y , P.H., Seviour, P.W. and Iversen, S.D. (1975) Amphetamine responses i n the rat following 6-OHDA lesions of the nucleus accumbens s e p t i and corpus striatum. Brain Res. 94, 507-515. Kemp, J.M. and Powell, T.P.S. (1970) The c o r t i c o s t r i a t e projections i n the monkey. Brain 93, 526-546. Kemp, J.M. and Powell, T.P.S. (1971a) The synaptic organization of the caudate nucleus. P h i l . Trans. R. Soc. Lond. B. 262, 403-412. Kemp, J.M. and Powell, T.P.S. (1971b) The s i t e of termination of afferent f i b e r s i n the caudate nucleus. P h i l . Trans. R. Soc. Lond. B., 262, 413- 427. Kim, J.-S. (1973) E f f e c t s of 6-hydroxydopamine on a c e t y l c h o l i n e and GABA metabolism i n rat striatum. Brain Res. 55, 472-475. Kim, J.S. Bak, I.J. Hassler, R. and Okada, Y. (1977a) Role of y-amino- butyric acid (GABA) i n the extrapyramidal motor system. 2. Some evidence f o r the existence of a type of GABA-rich s t r i a t o - n i g r a l neuron. Exp. Brain Res. 132, 370-374. Kim, J.S., Wagner, A. and Hassler, R. (1974) E f f e c t of caudate l e s i o n on GABA, and the a c t i v i t i e s of GAD, GABA-T, ChAc and AChE i n the substantia nigra and the pallidum of the cat. J. Pharmacol. 5, 51. Kim,R.K., Nakano, A., Jayaraman, A. and Carpenter, M.B. (1976) Projections of the globus p a l l i d u s and adjacent structures: an autoradiographic study i n the monkey. J. Comp. Neurol. 169, 263-289. Kimura, H. and Kuriyama, K. (1975a) D i s t r i b u t i o n of gamma-aminobutyric acid (GABA) i n the rat hypothalamus: f u n c t i o n a l c o r r e l a t e s of GABA with a c t i v i t i e s of appetite c o n t r o l l i n g mechanisms. J . Neurochem. 24, 903-907. Kimura, H. and Kuriyama, K. (1975b) A new microassay method for 1-glutamic acid decarboxylase (GAD) a c t i v i t y . Jap. J. Pharmacol. 25, 189-195. Kimura, H., McGeer, P.L., Peng, F. and McGeer, E.G. (1980) Choline acetyltransferase containing neurons i n rodent b r a i n by immunohisto- chemistry. Science ( i n press). K i t a i , S.T. , Kocsis, J.D., Preston, R.J. and Sugimori, M. (1976a) Mono- synaptic inputs to caudate neurons i d e n t i f i e d by i n t r a c e l l u l a r i n j e c t i o n of horseradish peroxidase. Brain Res. 109, 601-606. 121 K i t a i , S.T., Kocsis, J.D. and Wood, J. (1976b) O r i g i n and c h a r a c t e r i s t i c s of the cortico-caudate a f f e r e n t s : an anatomical and e l e c t r o p h y s i o l o g i c a l study. Brain Res. 118, 137-141. K i t a i , S.T., Wagner, A., Precht, W. and Ohno, T. (1975) Nigro-caudate and caudato-nigral r e l a t i o n s h i p : an e l e c t r o p h y s i o l o g i c a l study. Brain Res. 85, 44-48. Kocsis, J.A., Sugimori, M. and K i t a i , S.T. (1977) Convergence of excitatory synaptic inputs to caudate spiny neurons. Brain Res. 124, 403-413. Kondo, Y. and Iwatsubo, K. (1980) Diminished responses of n i g r a l dopaminer- gic neurons of haloperidol and morphine following l e s i o n s i n the striatum. Brain Res. 181, 237-240. Kbnig, J.F.R. and K l i p p e l , R. A. (1963) "The Rat Brain. A Stereotaxic A t l a s of the Forebrain and Lower Parts of the Brain Stem". Williams and Wilkins, Baltimore, Md. Korf, J . , Zieleman, M. and Westerink, B.H.C. (1976) Dopamine release i n substantia nigra? Nature (Lond.) 260, 257-258. Kul t a s - I l i n s k y , K., I l i n s k y , I.A., Massopust, L.C. , Young, P.A. and Smith, K.R. (1978) Nigrothalamic pathway i n the cat demonstrated by autoradio- graphy and electron microscopy. Exp. Brain Res. 33, 481-492. Kunzle, H. (1977) Projections from the primary somatosensory cortex to the basal ganglia and thalamus i n the monkey. Exp. Brain Res. 30, 481-492. Kuo, J.S. and Carpenter, M.B. (1973) Organization of pallidothalamic projections i n the rhesus monkey. J. Comp. Neurol. 151, 201-236. Kuroda, R., Murai, H., Akagi, K., Kamikawa, K. and Mogami, H. (1975) Efferent connections of the centromedian nucleus and the magnocellular part of the medial geniculate body i n cates. Conf. Neurol. 37, 120, 127. Kuypers, H.G.J.M. (1973) The anatomical organization of the descending pathways and t h e i r contributions to motor c o n t r o l , e s p e c i a l l y i n primates. In: "New Developments i n Electromyography and C l i n i c a l Neurophysiology, v o l . 3, J.E. Desmelt, ed. S. Karger, Basel, pp. 38-68. Kuypers, H.G.J.M. and Maisky, U.A. (1975) Retrograde axonal transport of horseradish peroxidase from s p i n a l cord to brain stem c e l l groups i n the cat. Neurosci. L e t t . 1, 9-14. La h t i , R.A. and Losey, E.G. (1974) Antagonism of the e f f e c t s of chlor- promazine and morphine on dopamine metabolism by GABA. Res. Commun. Chem. Path. Pharmacol. 7, 31-39. Lakke, J.P.W.F. and Teelken, A.W. (1976) Amino acid abnormalities i n cerebrospinal f l u i d of patients with parkinsonism and extrapyramidal disorders. Neurology 26, 489-493. 122 Lange H. , Thorner, G. , Hopf, A. and Schroder, K.F. (1976) Morphometric studxes of the neuropathological changes i n choreatic diseases J. Neurol. S c i . 28, 401-425. Larsen, K.D. and McBride, R.L. (1979) The organization of f e l i n e ento- peduncular nucleus projections: anatomical studies. J. Comp. Neurol 184, 293-308. F Larsson, L.-L. Childers, S. and Snyder, S.H. (1979) Met-and-leu- enkephalin immunoreactivity i n separate neurons. Nature (Lond ) 282, 407-410. Lee, L.A. Crossman, A.R. and Slater, P. (1980) The neuro l o g i c a l basis of s t r i a t a l l y induced head-turning i n the r a t : the e f f e c t s of lesi o n s i n putative output pathways, Neuroscience 5, 73-79. Johnmann, J. and F i b i g e r , H.C. (1978) Acetylcholinesterase i n the substantia nigra and caudate-putamen of the r a t : properties and l o c a l i z a t i o n i n dopaminergic neurons. J. Neurochem. 30, 615-624. Lehmann, J. and F i b i g e r , H.C. (1979) Acetylcholinesterase and c h o l i n e r g i c neuron. L i f e S c i . 25, 1939-1947. Lehman, J.A.R., Ferguson, I.T., Fleming, A.M. Herzberg, L., Robb, J.E. and Turnbull, M.J. (1976) Sodium valproate i n chorea. Br. Med. J . Nov. 6, 1107-1108. L i n d v a l l , 0. and Bjbrklund, A. (1974) The organization of the ascending catecholamine neuron system i n the rat b r a i n as revealed by the g l y o x y l i c acid fluorescence method. Acta Physiol. Scand. 412, 1-48. L i n d v a l l , 0., Bjbrklund, A., Moore, R. Y. and Steneui, U. (1974) Mesenphalic dopamine neurons p r o j e c t i n g to neocortex. Brain Res. 81, 325-331. L i n n o i l a , M., V i u k a r i , M. and H i e t a l a , 0. (1976) E f f e c t of sodium v a l - proate on Tardive dyskinesia. Br. J. Psychiatry 129, 114-119. Ljlingdahl, A., Hbkfelt, T., Goldstein, M. and Park, D. (1975) Retrograde peroxidase t r a c i n g of neurons combined with transmitter histochemistry. Brain Res. 84, 313-319. Ljungdahl, A., Hbkfelt, T. and Nilsson, G. (1978a) D i s t r i b u t i o n of subs- tance P - l i k e immunoreactivity i n the cen t r a l nervous system of the rat - I. c e l l bodies and nerve terminals. Neuroscience 3, 861-943. Ljungdahl, A., Hbkfelt, T., Nilsson, G. and Goldstein, M. (1978b) D i s t r i b u t i o n of substance P - l i k e immunoreactivity i n the c e n t r a l nervous system of the ra t - I I . l i g h t microscope l o c a l i z a t i o n i n r e l a t i o n to catecholamine-containing neurons. Neurscience 3, 945-976. Lloyd, K.G. and Davidson, L. (1979) Involvement of GABA neurons and receptors i n Parkinson's disease and Huntington's chorea: a compensatory mechanism? Adv. Neurol. 24, 293-301. 123 Lloyd, K.G. and Hornykiewicz, 0. (1973) L-Glutamic acid decarboxylase i n Parkinson's disease: e f f e c t of L-DOPA therapy. Nature (Lond.) 243, 521-523. Lloyd, K.G., Dreksler, S. and Bi r d , E.D. (1977a) A l t e r a t i o n s i n 3H-GABA binding i n Huntington's chorea. L i f e S c i . 21, 747-754. Lloyd, K.G., Shemen, L. and Hornykiewicz, 0. (1977b) D i s t r i b u t i o n of high- a f f i n i t y sodium-independent [3H]gamma-aminobutyric acid ([ 3H]GABA)bind- ing i n the human brain: a l t e r a t i o n s i n Parkinson's disease. Brain Res. 127, 269-278. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, E.J. (1951) Protein measurement with the F o l i n phenol reagent. J. b i o l . Chem. 193, 265-275. Maggi, A., Cattabeni, F., Bruno, F. and Racagni, G. (1977) Haloperidol and clozapine: s p e c i f i c i t y of action on GABA i n the n i g r o s t r i a t a l system. Brain Res. 133, 382-385. Magnusson, T., Carlsson, A., Fisher, G.H., Chang, D. and Folkers, K. (1976) E f f e c t of synthetic substance P on monoaminergic mechanisms i n brain. J. Neural Transm. 38, 89-93. Maier, L. , F i b i g e r , H.C. and McGeer, P.L. (1973) Demonstration of the n i g r o s t r i a t a l p r o j e c t i o n by s i l v e r s t a i n i n g a f t e r n i g r a l i n j e c t i o n s of 6-hydroxydopamine, Exp. Neurol. 40, 505-515. M a l l i a n i , A. and Purpura, D.P. (1967) I n t r a c e l l u l a r studies of the corpus striatum. I I . Patterns of synaptic a c t i v i t i e s i n l e n t i c u l a r and ento- peduncular neurons. Brain Res. 6, 341-354. Marco, E. , Mar, C C . , Cheney, D.L. , Revuelta, A. and Costa, E. (1976) The e f f e c t s of antipsychotics on the turnover rate of GABA and a c e t y l - choline i n rat brain n u c l e i . Nature (Lond.), 264, 363-365. Marsden, CD., Duvoisin, R.C, Jenner, P., Parkes, J.D. , Pycock, C. and Tarsy, D. (1975) Relationship between animal models and c l i n i c a l parkinsonism. In: "Advances i n Neurology, v o l . 9: Dopaminergic Mechanisms". D.B. Calne, T.N. Chase and A. Barbeau, eds. Raven Press, New York, pp. 165-175. Marshall, J.F. and Ungerstedt, U. (1977) S t r i a t a l efferent f i b r e s play a r o l e i n maintaining r o t a t i o n a l behavior i n the r a t . Science 198, 62-64. Martin, D.L., (1976) Carrier-mediated transport and removal of GABA from synaptic regions. In: "GABA i n nervous System Function". E. Roberts, T.N. Chase and D.B. Tower, eds. Raven Press, New York, pp. 348-386. Mason, S.T. and F i b i g e r , H. C. (1979) Regional topography w i t h i n noradrener- gic locus coeruleus as revealed by retrograde transport of horseradish peroxidase. J . Comp. Neurol. 187, 703-724. 124 Matsui, Y. and Deguchi, T. (1977) Cataleptic a c t i o n of nitrazepam and brain dopamine function i n mice. Neuropharmacology 16, 253-258. Matsui, Y. and Kamioka, T. (1978) Cataleptic and a n t i c a t a l e p t i c e f f e c t s of muscimol and gabaculine injected into globus p a l l i d u s and substantia nigra and in t e r a c t i o n s with haloperidol or benzodiazepines. Arch. Pharmacol. 305, 219-225. McGeer, E.G. and McGeer, P. L. (1976a) Duplication of biochemical changes of Huntington's chorea by i n t r a s t r i a t a l i n j e c t i o n s of glutamic and kai n i c acids. Nature (Lrmd. •> Oh"* ^ i 7 - " " Nature ond.) 263, 517-519. McGeer, E.G. and McGeer, P.L. (1976b) Enzymes associated with the metabolism of catecholamines, acetylcholine and GABA i n human controls and patients with Parkinson's disease and Huntington's chorea. J. Neurochem. 26 65-76. McGeer, E.G., Gibson, S. and McGeer, P.L. (1967) Some c h a r a c t e r i s t i c s of brain tyrosine hydroxylase. Can. J . Biochem. 45, 1557-1563. McGeer, P.L. and McGeer, C.G. (1971) Cholinergic enzyme systems i n Parkin- son's disease. Arch. Neurol. 25, 265-268. McGeer, P.L., H a t t o r i , T. and McGeer, U.G. (1975) Chemical and autoradio- graphic analysis of y-aminobutyric acid transport i n Purkinje c e l l s of the cerebellum. Exp. Neurol. 47, 26-41. McGeer, P.L. McGeer, E.G., Scherer, U. and Singh, K. (1977) A glutamater- gic c o r t i c o s t r i a t a l path? Brain Res. 128, 369-373. McGeer, P.L. McGeer, E.G., Wada, J.A. and Jung, E. (1971) E f f e c t s of globus p a l l i d u s l e s i o n s and Parkinson's disease on brain glutamic acid decarboxylase. Brain Res. 32, 425-431. McLaughlin, B.J., Wood, J.G., Saito, K., Barber, R., Vaughn, J.E., Roberts, E. and Wu, J.-Y. (1974) The f i n e s t r u c t u r a l l o c a l i z a t i o n of glutamate decarboxylase i n synaptic terminals of rodent cerebellum. Brain Res. 76, 377-391. Mercer, L., d e l Fiacco, M. and Cuello, A.C. (1979) The smooth endoplasmic reticulum as a possible storage s i t e for d e n d r i t i c dopamine i n subs- t a n t i a nigra neurons. Experientia 35, 101-103. Mettler, F.A. (1970) Nigrofugal connections i n the primate brain. J . Comp. Neurol. 128, 291-320. Mettler, F.A. (1955) Perceptual capacity, functions of the corpus striatum and schizophrenia. Psychiat. Quart. 29, 89-121. M i l l e r , J . J . , Richardson, T.L., F i b i g e r , H.C. and McLennan, H. (1975) Anatomical and 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 f i c a t i o n of a p r o j e c t i o n from the mesencephalic raphe to the caudate-putamen i n the r a t . Brain Res. 97, 133-138. 125 Mohler, C.W. and Wurtz, R.H. (1976) Organization of monkey superior c o l l i c u l u s : intermediate layer c e l l s discharging before eye movements. J. Neurophysiol. 39, 722-744. Moore, R.Y..(1978) Catecholamine innervation of the basal forebrain. I. The septal area. J. Comp. Neurol. 177, 665-684. Moroni, F., Pe r a l t a , E., Cheney, D.L. and Costa, E. (1979) On the regulation of y-aminobutyric acid neurons i n caudates, p a l l i d u s and ni g r a : e f f e c t s of opioids and dopamine agonists. J. Pharm. Exp. Ther. 208, 190-194. Mroz, E.A., Brownstein, M.J. and Leeman, S.E. (1976) Evidence for substance P i n the habenulo-interpeduncular t r a c t . Brain Res. 113, 597-599. Mroz, E.A., Brownstein, M.J. and Leeman, S.E. (1977) Evidence f or substance P i n the s t r i a t o - n i g r a l t r a c t . Brain Res. 125, 305-311. Nagy, J . I . and F i b i g e r , H.C. (1980) A s t r i a t a l source of glutamic acid decarboxylase a c t i v i t y i n the substantia nigra. Brain Res. 187, 237-242. Nagy, J . I . , Carter, D.A. and Fi b i g e r , H.C. (1978a) Anterior s t r i a t a l projections to the globus p a l l i d u s , entopeduncular nucleus and substantia nigra i n the r a t : the GABA connection. Brain Res. 158, 15-29. Nagy, J . I . , Carter, D.A., Lhemann, J. and F i b i g e r , H.C. (1978b) Evidence for a GABA-containing projection from the entopeduncular nucleus to the l a t e r a l habenula i n the ra t . Brain Res. 145, 360-364. Nagy, J . I . , Lee, T., Seeman, P. and F i b i g e r , H.C. (1978c) D i r e c t evidence for presynaptic and postsynaptic dopamine receptors i n brai n . Nature (Lond.) 274, 278-281. Nagy, J . I . , Vincent, S.R., Lehmann, J . , F i b i g e r , H.C. and McGeer, E.G. (1978d) The sue of kai n i c acid i n the l o c a l i z a t i o n of enzymes i n the substantia nigra. Brain Res. 149, 431-441. Nauta, H.J.W. (1974) Evidence of pallidohabenular pathway i n the cat. J. Comp. Neurol. 156, 19-28. Nauta, H.J.W. and Cole, M. (1978) Efferent projections of the subthalamic nucleus: an autoradiographic study i n monkey and cat. J . Comp. Neurol. 180, 1-16. Nauta, W.J.H. and Mehler, W.J. (1966) Projections of the le n t i f o r m nucleus i n the monkey. Brain Res. 1, 3-42. Nauta, W.J.H., Smith, G.P., F a u l l , R.L.M. and Domesick, V.B. (1978) Efferent connections and n i g r a l afferents of the nucleus accumbens s e p t i i n the rat . Neuroscience 3, 385-401. Neophytides, A.N., Suria, A., Waniewski, R.A. and Chase, T.N. (1978) Cerebrospinal f l u i d GABA i n neurological disease. Neurology 32, 37-45. 126 Nieollon, A., Cheramy, A. and Glowinski, J. (1977) Release of dopamine l n V 1 V 0 f r o m cat substantia nigra. Nature (Lond.) 266, 375-377. Nilmi, K., Kishi, S., Miki, M. and Fujita, S. (1963) An experimental study of the course and termination of the projection fibers from cortical areas 4 and 6 in the cat. Folia Psychiatr. Neurol. Jpn. 17, 167-216. Nicklas, W.J., Nunez, R., Berl, S. and Duvoisin, R. (1979) Neuronal g l i a l contributions to transmitter amino acid metabolism: studies with kainic acid-induced lesions of rat striatum. J. Neurochem. 33, 839-844. Oberlander, C., Dumont, C. and Boissier, J.R. (1977) Rotational Behaviour after unilateral nigral injection of muscimol in rats. Eur. J. Pharmacol. 43, 389-390. Olianis, M.C, DeMontis, CM., Concu, A., Tagliomonte, A. and DiChiara, G. (1978a) Intranigral kainic acid: evidence for nigral non-dopaminergic neurons controlling posture and behavior in a manner opposite to the dopaminergic ones. Eur. J. Pharmacol. 49, 223-232. Olianas, M.C. DeMontis, CM. , Mulas, G. and Tagliamonte, A. (1978b) The s t r i a t a l dopaminergic function i s mediated by the inhibition of a nigral, non-dopaminergic neuronal system via a strio-nigral gabaergic pathway. Eur. J. Pharmacol. 49, 233-241. Olpe, H.-R. and Koella, W.P. (1977) Rotatory behavior i n rats by intranigral application of substance P and an eledoisin fragment. Brain Res. 126, 576-579. Paden, C , Wilson, C.J. and Groves, P.M. (1976) Amphetamine-induced release of dopamine from the substantia nigra in vitro. L i f e Sci. 19, 1499-1506. Palkovits, M., Mroz, E.A., Brownstein, M.J. and Leeman, S.E. (1978) Descending substance P containing pathway: a component of the ansa lenticularis. Brain Res. 156, 124-128. Paxinos, G., Emson, P.C and Cuello, A.C. (1978a) The substance P projections to the frontal cortex and the substantia nigra. Neurosci. Lett. 7, 127-131. Paxinos, G., Emson, P.C. and Cuello, A.C. (1978b) Substantia P projections to the entopeduncular nucleus, the medial preoptic area and the lateral septum. Neurosci. Lett. 7, 133-136. Pelletier, G. Leclerc, R. and Dupont, A. (1977) Elec-ron microscope immuno- histochemical localization of substance P in the central nervous system of the rat. J. Histochem. Cytochem. 25, 1373-1380. Perry, T.L. , Hansen, S. and Kloster, M. (1973) Huntington's chorea, defi- ciency of gamma-aminobutyric acid in brain. N. Engl. J. Med. 288, 337-342. 127 Pettibone, D.J., Wurtman, R.J. and Leeman, S.E. (1978a) D-amphetamine administration reduces substance P concentration i n the rat striatum Biochem. Pharmacol. 27, 839-842. Pettibone, D.J., Wurtman, R.J. and Leeman, S.E. (1978b) D-amphetamine administration reduces substance P concentration i n the r a t striatum. Psychopharmacol. B u l l . 14, 24-26. P h i l l i p s o n , O.T. and Horn, A.S. (1976) Substantia nigra of the rat contains a dopamine s e n s i t i v e adenylate cyclase. Nature (Lond.) 261, 418-420. P i c k e l , V.M. (1979) Immunocytochemical l o c a l i z a t i o n of neuronal antigens: tyrosine hydroxylase, substance P, and [Met 5]-enkephalin, Fed. Proc. 38, 2374-2380. P i c k e l , V.M. Reis, D.J. and Leeman, S.E. (1977) U l t r a s t r u c t u r a l l o c a l i z a - t i o n of substance P i n neurons of rat s p i n a l cord. Brain Res. 122, 534-540. P i c k e l , V.M., Tong, H.J. and Reis, D.J. (1976) Monoamine synthesizing enzymes i n ce n t r a l dopaminergic, noradrenergic and serotonergic neurons. J. Histochem. Cytochem. 24, 792-806. P o l l a r d , H., Lorens, G., Schwartz, J.C., Gros, C. and Dray, F. (1978) L o c a l i z a t i o n of opiate receptors and enkephalins i n the ra t striatum i n re l a t i o n s h i p with the n i g r o - s t r i a t a l dopaminergic system: l e s i o n studies. Brain Res. 151, 392-398. Powell, E.W. and Leman, R.B. (1976) Connections of the nucleus accumbens. Brain Res. 105, 389-403. Precht, W. and Yoshida, M. (1971) Blockage of caudate evoked i n h i b i t i o n of neurons i n the substantia nigra by pi c r o t o x i n . Brain Res. 32, 229- 233. Purpura, D.P. and M a l l i a n i , A. (1967) I n t r a c e l l u l a r studies of the corpus striatum. I. Synaptic potentials and discharge c h a r a c t e r i s t i c s of cau- date neurons activated by thalamic stimulation. Brain Res. 6, 325-340. Pycock, C. and Horton, R. (1976) Evidence for an accumbens-pallidal pathway i n the ra t and i t s possible gabaminergic c o n t r o l . Brain Res. 110, 629-634. Pycock, C., Horton, R.W. and Marsden, C.D. (1976) The behavioral e f f e c t s of manipulating GABA function i n the globus p a l l i d u s . Brain Res. 116, 353-359. Quik, M., Emson, P.C. and Joyce, E. (1979) D i s s o c i a t i o n between the pre- synaptic dopamine-sensitive adenylate cyclase and [ 3H]spiperone binding s i t e s i n r a t substantia nigra. Brain Res. 167, 355-365. Rando, R.R. and Bangerter, F.W. (1977) The i n vivo i n h i b i t i o n of GABA- transaminase by gabaculine. Biochem. Biophys. Res. Comm. 76, 1276- 1281. 1 2 8 R e a v i l l , C , L e i g h , N . , J e n n e r , P . a n d M a r s d e n , C D . ( 1 9 7 9 ) D o p a m i n e - m e d x a t e d c i r c l i n g b e h a v i o u r d o e s n o t i n v o l v e t h e n i g r o - t e c t a l p a t h w a y . E x p . B r a i n R e s . 3 7 , 3 0 9 - 3 1 6 . R e b e c , C V . a n d G r o v e s , P . M . ( 1 9 7 5 ) D i f f e r e n t i a l e f f e c t s o f t h e o p t i c a l i s o m e r s o f a m p h e t a m i n e o n n e u r o n a l a c t i v i t y i n t h e r e t i c u l a r f o r m a t i o n a n d c a u d a t e n u c l e u s o f t h e r a t . B r a i n R e s . 8 3 , 3 0 1 - 3 1 8 . R e u b i , J . C . a n d C u e n o d , M. ( 1 9 7 9 ) G l u t a m a t e r e l e a s e i n v i t r o f r o m c o r t i c o - s t r i a t a l t e r m i n a l s . B r a i n R e s . 1 7 6 , 1 8 5 - 1 8 8 . R e u b i , J . C . a n d S a n d r i , C . ( 1 9 7 9 ) U l t r a s t r u c t u r a l o b s e r v a t i o n s o n i n t e r - c e l l u l a r c o n t a c t s o f n i g r a l d e n d r i t e s . N e u r o s c i . L e t t . 1 3 , 1 8 3 - 1 8 8 . R i b a k , C . E . , V a u g h n , J . E . a n d R o b e r t s , E . ( 1 9 7 9 ) T h e GABA n e u r o n s a n d t h e i r a x o n t e r m i n a l s i n r a t c o r p u s s t r i a t u m a s d e m o n s t r a t e d b y GAD i m m u n o c y t o c h e m i s t r y . J . Comp. N e u r o l . 1 8 7 , 2 6 1 - 2 8 4 . R i b a k , C . E . , V a u g h n , J . E . , S a i t o , K. B a r b e r , R. a n d R o b e r t s , E . ( 1 9 7 6 ) I m m u n o c y t o c h e m i c a l l o c a l i z a t i o n o f g l u t a m a t e d e c a r b o x y l a s e i n r a t s u b s t a n t i a n i g r a . B r a i n R e s . 1 1 6 , 2 8 7 - 2 9 3 . R i c h a r d s o n , T . L . , M i l l e r , J . J . a n d M c L e n n a n , H . ( 1 9 7 7 ) M e c h a n i s m s o f e x c i t a t i o n a n d i n h i b i t i o n i n t h e n i g r o s t r i a t a l s y s t e m . B r a i n R e s . 1 2 7 , 2 1 9 - 2 3 4 . R i n n e , U . K . , K o s k i n e n , V . , L e a k s o n e n , H . , L b n n b e r g , P . a n d S o n n i n e n , V . ( 1 9 7 8 ) GABA r e c e p t o r b i n d i n g i n t h e P a r k i n s o n i a n b r a i n . L i f e S c i . 2 2 , 2 2 2 5 - 2 2 2 8 . R i n n e , U . K . , L a a k s o n e n , H . , R i e k k i n e n , P . a n d S o n n i n e n , V . ( 1 9 7 4 ) B r a i n g l u t a m i c a c i d d e c a r b o x y l a s e a c t i v i t y i n P a r k i n s o n ' s d i s e a s e . E u r . N e u r o l . 1 2 , 1 3 - 1 9 . R i n n e , U . K . , S o n n i n e n , V . a n d L a a k s o n e n , H . ( 1 9 7 9 ) R e s p o n s e s o f b r a i n n e u r o c h e m i s t r y t o l e v o d o p a t r e a t m e n t i n P a r k i n s o n ' s d i s e a s e . A d v . N e u r o l . 2 4 , 2 5 9 - 2 7 4 . R i n v i k , E . ( 1 9 7 5 ) D e m o n s t r a t i o n o f n i g r o t h a l a m i c c o n n e c t i o n s i n t h e c a t b y r e t r o g r a d e a x o n a l t r a n s p o r t o f h o r s e r a d i s h p e r o x i d a s e . B r a i n R e s . 9 0 , 3 1 3 - 3 1 8 . R i n v i k , E . ( 1 9 7 6 ) G r o f o v a , I . a n d O t t e r s e n , 0 . 0 . ( 1 9 7 6 ) D e m o n s t r a t i o n o f n i g r o t e c t a l a n d n i g r o r e t i c u l a r p r o j e c t i o n s i n t h e c a t b y a x o n a l t r a n s p o r t o f p r o t e i n s . B r a i n R e s . 1 1 2 , 3 8 8 - 3 9 4 . R o b e r t s , D . C . S . , Z i s , A . P . a n d F i b i g e r , H . C . ( 1 9 7 5 ) A s c e n d i n g c a t e c h o l a m i n e p a t h w a y s a n d a m p h e t a m i n e - i n d u c e d l o c o m o t o r a c t i v i t y : i m p o r t a n c e o f d o p a m i n e a n d a p p a r e n t n o n - i n v o l v e m e n t o f n o r e p i n e p h r i n e . B r a i n R e s . 9 3 , 4 4 1 - 4 5 4 . R o b e r t s , E . ( 1 9 7 9 ) New d i r e c t i o n s i n GABA r e s e a r c h I : I m m u n o c y t o c h e m i c a l s t u d i e s o f GABA n e u r o n s . I n : " G A B A - N e u r o t r a n s m i t t e r s : P h a r m a c o c h e m i c a l , B i o c h e m i c a l a n d P h a r m a c o l o g i c a l A s p e c t s " . P . K r o s g s g a a r d - l a r s e n , J . S c h e e l - K r u g e r a n d H . K o f o d , e d s . M u n k s g a a r d , C o p e n h a g e n , p p . 2 8 - 4 5 . 129 Robin M.M. Palfreyman, M.G. and Schechter, P.J. (1979) Dyskinetic e f f 25V110T1II0" 7 l n J e C t e d G A M - t r a n s a m i n a s e i n h i b i t o r s . L i f e S c i ects Robinson, N. and Wells, P. (1973) D i s t r i b u t i o n and l o c a l i z a t i o n of s i t e s of gamma aminobutyric acid metabolism i n the adult rat brai n . J. Anat. 114, 365-378. Ross, C , Ruggiero, D.A. and Reis, D.J. (1979) Descending connections from the brain to the s p i n a l cord i n the r a t , as studied with horse- radish peroxidase. Neurosci. Abst. 5, 167. Roth, R.H., Walters, J.R., Murrin, L.C. and Morgenroth, V.H. (1975) Dopamine neurons: r o l e of impulse flow and presynaptic receptors i n the regulation of tyrosine hydroxylase. In: "Pre- and Postsynaptic receptors. E. Usdin, W.E. Bunney, J r . , eds. Marcel Dekker, New York, pp. 5-46. Royce, G.J. (1978) Autoradiographic evidence for a discontinuous pro- j e c t i o n to the caudate nucleus from the centromedian nucleus i n the cat. Brain Res. 146, 145-150. Saavedra, J.M. , S e t l e r , P.E. and Kebabian, J.W. (1978) Biochemical changes accompanying u n i l a t e r a l 6-hydroxydopamine l e s i o n s i n the r a t substantia nigra. Brain Res. 151, 339-352. Saelens, J.K. Edwards-Neale, S. and Simke, J.P. (1979) Further evidence for c h o l i n e r g i c thalamo-striatal neurons. J. Neurochem. 32, 1093-1094. Salpeter, M.M. and McHenry, F.A. (1973) Electron microscope autoradiography. In: "Advanced Techniques i n B i o l o g i c a l E lectron Microscopy". J . K. Koehler, Ed. Springer-Verlag, New York, pp. 113-152. Salvador, R.A. and Al b e r t s , R.W. (1959) The d i s t r i b u t i o n of glutamic-y- aminobutyric transaminase i n the nervous system of the rhesus monkey. J. b i o l . Chem. 234, 922-925. Scheel-Kruger, J . , Arnt, J. and Magelund, G. (1977) Behavioural stimula- t i o n induced by muscimol and other GABA agonists i n j e c t e d into the substantia nigra. Neurosci. L e t t . 4, 351-356. Schwarcz, R., Bennett, J.P. J r . and Coyle, J.T. (1977) I n h i b i t o r s of GABA metabolism: implications for Huntington's disease. Ann. Neurol. 2, 299-303. Schwarcz, R. Fuxe, K., Agnati, L.F., Hokfelt, T. and Coyle, J.T. (1979) Rotational behaviour i n rats with u n i l a t e r a l s t r i a t a l k a i n i c acid l e s i o n s : a behavioural model for studies on i n t a c t dopamine receptors Brain Res. 170, 485-495. Shoulson, I., K a r t z i n e l , R. and Chase, T.N. (1976) Huntington's disease treatment with d i p r o p l a c e t i c acid and gamma-aminobutyric a c i d . Neurology 26, 61-63. 130 Simke, J.P. and Saelens, J.K. (1977) E v i d e n c e f o r a c h o I i n e r g i c f i b e r t r a c t connecting the thalamus w i t h the head of (.he stri;iUnn of t h e r a t . Brain Res. 126, 487-495. Simon, J.R. , Contrera, J.F. and Kuhar, M. J . (1976) B i n d i n g of | 'ii | k.i i n i e acid, as analogue of L-glutamate, t o b r a i n membrances. ,1. Neurocbem. 2 6 , 141-147. Singer, E., Sperk, G., Placheta, P. and Leeman, S.E. ( 1 9 7 9 ) R e d u c t i o n of substance P l e v e l s i n the ventral c e r v i c a l s p i n a l c o r d of the rat: a f t e i n t r a c i s t e r n a l 5,7-dihydroxytryptamine I n j e c t i o n . B r a i n Res 174 362-365. S k i r b o l l , L.R. Grace, A.A. Bunney, B.S. (.1.979) Dopamine a u t o - and p o s t - synaptic receptors: e l e c t r o p h y s i o l o g i c a l e v i d e n c e f o r d i f f e r e n t Lai s e n s i t i v i t y to dopamine agonists. S c i e n c e 206, 8 0 - 8 2 . Spano, P.F., DiChiara, G., Tonon, G. C. and T r a b u c c h i , M. (1976) A dopa- mine-stimulated adenylate cyclase i n r a t s u b s t a n t i a n i g r a . J. Neurochem. 27, 1565-1568. Spencer, H.J. (1976) Antagonism of c o r t i c a l e x c i t a t i o n of s t r i a t a l neurons by glutamic acid d i e t h y l ester: e v i d e n c e f o r g l u t a m i c a c i d as an excitatory transmitter i n the rat s t r i a t u m . B r a i n Res. 1 0 2 , 9 1 - 1 0 1 . Sprague, J.M., Berlucchi, G. and R i z z o l a t t i , G. ( 1 9 7 3 ) The r o l e o f the superior c o l l i c u l u s and pretectum i n v i s i o n and v i s u a l l y g u i d e d b e h a v i o u r . In: "Handbook of Sensory Physiology". H. Antrum, R. J u n g , W.R. Lower- s t e i n , D. M. McKay and H.L. Teuber, eds. S p r i n g e r - V e r l a g , New Y o r k , pp. 517-528. Starr, M.S., (197.8a) Gaba potentiates p o t a s s i u m - s t i m u l a t e d 'll-dopamine release from s l i c e s of rat substantia n i g r a and c o r p u s s t r i a t u m . Eur. J. Pharmacol. 48, 325-328. Starr, M.S. (1978b) Investigation of p o s s i b l e i n t e r a c t i o n s between s u b s t a n c e P and transmitter mechanisms i n the s u b s t a n t i a n i g r a and c o r p u s s t r i a t u m of the r a t . J. Pharm. Pharmacol. 30, 359-363. Starr, N. J . , James, T. A. and Gaytten, D. (1978) B e h a v i o u r a l d e p r e s s a n t and antinociceptive properties of s u b s t a n c e P i n the mouse: possible? implication of brain monoamines. Eur. J . P h a r m a c o l . 4 8 , 2 0 3 - 2 1 2 . Sternberger, L.A., Hardy, P.H. J r . , C o c u l i s , J . J . and Meyer, H.G. ( 1 9 7 0 ) The unlabelled antibody-enzyme method of immuiioh i s t o e h e m i s t r y : Preparation of antigen-antibody complex ( p e r o x i d a s e - a n t i p e r o x i d a s e ) i n i d e n t i f i c a t i o n of spirochetes. J. Histochem. Cytochem. 18, 3 1 5 - 3 4 0 . S t e r r i , S.H. and Fonnum, F. (1978) I s o l a t i o n o f o r g a n i x a n i o n s by e x t r a c - t i o n with l i q u i d aninion exchangers and i t s a p p l i c a t i o n t o microniothods for acetylcholinesterase and 4-aminobutyrate a m i n o t r a n s f e r a s e . Eur. J . Biochem. 91, 215-222. 131 Stinus, L., Kelley, A.E. and Iversen, S.D. (1978) Increased spontaneous a c t i v i t y following substance P in f u s i o n into A10 dopaminergic area. Nature (Lond.) 276, 616-618. Stoof, J.C. and Mulder, A.H. (1977) Increased dopamine release from rat s t r i a t a l s l i c e s by i n h i b i t o r s of GABA-aminotransferase. Eur. J . Pharmacol. 46, 177-184. Stoof, J . C , Den Breejen, E.J.S. and Mulder, A.H. (1979) GABA modulates the release of dopamine and acetylcholine from r a t caudate nucleus s l i c e s . Eur. J . Pharmacol. 57, 35-42. Storm-Mathisen, J. (1975) Accumulation of glutamic acid decarboxyalse i n the proximal parts of presumed GABAergic neurons a f t e r axotomy. Brain Res. 87, 107-109. Ternaux, J.P., Hery, F., Bourgoin, S., Adrien, J . Glowinski, J . and Hamon, M. (1977) The topographical d i s t r i b u t i o n of serotonergic terminals i n the neostriatum of the rat and the caudate nucleus of the cat. Brain Res. 121, 311-326. Thierry, A.M., Stinus, L., Blanc, G. and Glowinski, J . (1973) Some evidence f o r the existence of dopamine neurons i n the rat cortex. Brain Res. 50, 230-234. Trabucchi, M., Govoni, S., Tonon, G.C and Spano, P.F. (1978) Functional i n t e r a c t i o n between receptors of dopamine antagonists and GABA c e n t r a l receptors. L i f e S c i . 23, 1751-1756. Tra f i c a n t e , L.J., Friedman, E., Oleshansky, M.A. and Gershon, S. (1976) Dopamine-sensitive adenylate cylcase and cAMP phosphodiesterase i n substantia nigra and corpus striatum of rat brain. L i f e S c i . 19, 1061-1066. Tulloch, I.F., Arbuthnott, G.W. and Wright, A.K. (1978) Topographical organization of the s t r i a t o - n i g r a l pathway revealed by anterograde and retrograde neuroanatomical tracing techniques. J . Anat. 127, 425-441. Ungerstedt, U., Butcher, L.L. Butcher, S.G., Anden, N.-E. and Fuxe, K. (1969) Direct chemical stimulation of dopaminergic mechanisms i n the neostriatum of the r a t . Brain Res. 14, 461-471. Urquhart, N., Perry, T.L., Hansen, S. and Kennedy, J. (1975) GABA content and glutamic acid decarboxylase a c t i v i t y i n brain of Huntington's chorea patients and cont r o l subjects. J . Neurochem. 24, 1071-1075. Vacca, L.L., Abrahams, S.J. and Naftchi, N.E. (1980) A modified peroxidase- antiperoxidase procedure for improved l o c a l i z a t i o n of t i s s u e antigens: L o c a l i z a t i o n of substance P i n rat sp i n a l cord. J . Histochem. Cytochem. 28, 297-307. Van Gelder, N.M. (1965) The histochemical demonstration of y-aminobutyric acid and metabolism by reduction of a tetrazolium s a l t . J . Neurochem. 12, 231-237. 13 2 Vincent, S.R. H a t t o r i , T. and McGeer, E.G. (1978) The n i g r o t e c t a l p r o j e c t i o n : a biochemical and u l t r a s t r u c t u r a l c h a r a c t e r i z a t i o n . Brain Res. 151, 159-164. Vincent, S.R., Nagy, J.I. and F i b i g e r , H.C. (1978) Increased s t r i a t a l glutamate decarboxylase a f t e r lesions of the n i g r o s t r i a t a l pathway. Brain Res. 143, 168-173. Waddington, J.L. and Cross, A.J. (1978a) Denervation s u p e r s e n s i t i v i t y i n the s t r i a t o n i g r a l GABA pathway. Nature (Lond.) 276, 618-620. Waddington, J.L. and Corss, A.J. (1978b) Neurochemical changes following k a i n i c acid lesions of the nucleus accumbens: implications for a GABAergic accumbal-ventral tegmental pathway. L i f e S c i . 22, 1011-1014. Walaas, I. and Fonnum, F. (1979) The d i s t r i b u t i o n and o r i g i n of glutamate cecarboxylase and choline acetyltransferase i n v e n t r a l pallidum and other basal forebrain regions. Brain Res. 177, 325-336. Walaas, I. and Fonnum, F. (1980) Biochemical evidence f o r y-aminobutyrate containing f i b r e s from the nucleus accumbens to the substantia nigra and v e n t r a l tegmental area i n the ra t . Neuroscience 5, 63-72. Waldmeier, P.C., Kam, R. and Sto c k l i n , K. (1978) Increased dopamine metabolism i n rat striatum a f t e r infusion of substance P into the substantia nigra. Brain Res. 159, 223-227. Walker, R.J., Kemp, J.A. Yajima, H., Kitagawa, K. and Woodruff, G.N. (1976) The action of substance P on mesencephalic r e t i c u l a r and substantia nigra neurones i n the rat. Experientia 32, 214-215. Walters, J.R., Lakoski, J.M., Eng, N. and Waszczak, B. L. (179) E f f e c t of muscimol, AOAA and Na Valproate on the a c t i v i t y of dopamine neurons and dopamine synthesis. In: "GABA Neurotransmitters". P. Krogsgaard- Larsen, J.Scheel-Kruger, H. Kofods, eds. Munksgaard, Copenhagen, pp. 118- 134. Webster, K.E. (1961) C o r t i c o - s t r i a t e i n t e r r e l a t i o n s i n the albino r a t . J. Anat. (Lond.) 95, 532-544. Webster, K.E. (1965) 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 i n the cat. J. Anat. (Lond.) 99, 329-337. Whi t t i e r , J.R. and Mettler, F.A. (1949) Studies on the subthalamus of the rhesus monkey. J . Comp. Neurol. 90, 281-317. Wilson, C.J., Groves, P.M. and Fifkova, E. (1977) Monoaminergic synapses, including dendro-dendritic synapses i n the rat substantia nigra. Exp. Brain Res. 30, 161-174. Wolfarth, S., Coelle, E.-F., Osborne, N.N. and Sontag, K.-H. (1977) Evidence f o r a neurotoxic e f f e c t of ascorbic a c i d a f t e r an i n t r a n i g r a l i n j e c t i o n i n the cat. Neurosci. L e t t . 6, 183-186. 133 Wolman, M. (1971) A fluorescent histochemical procedure for gamma-aminobu- t y r i c a cid. Histochemie 28, 118-130. Worms, P. and Lloyd, K.G. (1978) Influence of GABA-agonists and antagonists on neuroleptic-induced catalepsy i n r a t s . L i f e S i c . 23, 475-478. Yeterian, E.H. and van Hoesen, G.W. (1978) C o r t i c o - s t r i a t e projections i n the rhesus monkey: the organization of c e r t a i n cortico-caudate connections. Brain Res. 139, 43-63. York, D.H. and Faber, J.E. (1977) An e l e c t r o p h y s i o l o g i c a l study of nigro- t e c t a l r e l a t i o n s h i p s : a possible role i n turning behavior. Brain Res. 130, 383-386. Yoshida, M. and Omata, S. (1978) Blocking by p i c r o t o x i n of nigra-evoked i n h i b i t i o n of neurons of ventromedial nucleus of the thalamus. Experientia 35, 794. Yoshida, M. and Precht, W. (1971) Monosynaptic i n h i b i t i o n of neurons of the substantia n i g r a by caudato-nigral f i b r e s . Brain Res. 32, 225-228. Zivkovic, B., G u i d o t t i , A. and Costa, E. (1974) E f f e c t s of neuroleptics on s t r i a t a l tyrosine hydroxylase: changes i n a f f i n i t y for the p t e r i d i n e cofactor. Moi. Pharmacol. 10, 727-735. 134 APPENDIX THE SUBSTANCE P RADIOIMMUNOASSAY (a) Preparation of the Antigen Substance P i s a r e l a t i v e l y small molecule and must therefore be couple to a larger c a r r i e r molecule to give an antigen large enough for immunization. In the f i r s t report of a radioimmunoassay for substance P, Powell et a l . (1973) coupled synthetic substance P to bovine y- g l o b u l i n with a carbodiimide. In the present study synthetic substance P (Beckman) was coupled to bovine serum albumin following the method developed by Goodfriend et a l . (1964). Albumin (3.72 g., bovine RIA grade, Sigma), 1.90 mg synthetic substance P (Beckman) and 100 mg l-cyclohexyl-3(2-mor- pholinoethyl)-carbodiimide metho-p-toluene-sulphonate (Aldrich) were dissolved i n t h i s order i n 0.5 ml d i s t i l l e d water and agitated gently at room temperature for one hr. The gelatinous mixture was then dialysed against d i s t i l l e d water for 24 hr at 4°C and l y o p h i l i z e d . (b) Immunization The immunization procedure of V a i t u k a i t i s et a l . (1971) was used to produce s p e c i f i c a n t i s e r a to substance P. Our i n i t i a l attempts to r a i s e antisera to substance P i n rabbits met with l i m i t e d success and so guinea pigs were used i n the present studies. For the i n i t i a l immunization one mg of antigen was dissolved i n three ml water and emulsified with three ml Freund's complete adjuvant (Cappel Labs). One ml of t h i s emulsion was injected per guinea pig intradermally i n 20-30 wheals on the back. Animals received a booster i n j e c t i o n of 100 yg antigen i n 200 y l water and 200 y l Freund's incomplete adjuvant (Cappel Labs) every four weeks. (c) Screening of Antisera Sera were examined for t h e i r a b i l i t y to bind substance P two weeks afte r each booster. The guinea pigs were bled by cardiac puncture while 135 under halothane anesthesia. The blood was allowed to stand at room tempera- ture for one hr, then overnight at 4°C to f a c i l i t a t e c l o t t i n g . Following centrifugation at 3,000 g for 15 min to remove c e l l s , the sera were kept frozen at -20°C. Each a n t i s e r a was checked for i t s a b i l i t y to bind t r a c e r , and for the a b i l i t y of a small amount of unlabeled substance P to displace the tracer. A range of antisera d i l u t i o n s (1:100 to 1:312,500) was i n - cubated with about 5,000 cpm of tracer both with and without an a d d i t i o n a l one ng unlabeled substance P. With the antisera used i n a l l the experiments reported here, about 30% of the tracer was bound at a sera d i l u t i o n of 1:62,500 and 65% of t h i s could be displaced by one ng of unlabeled substance P (Fig. I ) . The c r o s s - r e a c t i v i t y of t h i s a n t i s e r a with physalamen and e l e d o i s i n , two nonmammalian peptides s t r u c t u r a l l y s i m i l a r to substance P and with the peptides somatostatin, b a c i t r a c i n , and l e u - or met-enkephalin was les s than one percent. Although only one immunoreactive f r a c t i o n was obtain- ed from tissue extracts chromatographed on Sephadex G-25, peptides other than substance P would contribute to t h i s immunoreactivity. Therefore the sub- stance P l e v e l s reported i n these experiments and the immunohistochemical st a i n i n g should properly be termed substance P - l i k e immunoreactivity. (d) Preparation of 1^ 5I-substance P The chloramine T procedure of Greenwood et a l . (1963) can be used to iodinate peptides or proteins possessing a tyrosine residue. Substance P does not contain a tyrosine group, however, one may be substituted f o r the phenylalanine residue i n p o s i t i o n eight without s e r i o u s l y a f f e c t i n g the b i o l o g i c a l or radioimmunological a c t i v i t y of the peptide. Ten yg of ( T y r 8 ) - substance P (Beckman) i n 100 y l of 500 mM sodium phosphate buffer pH 7.4 was added to one mCi of Iodine 125 (Amersham IMS-30) i n a pyrex tube (#9820). The reaction was started by the addition of 52 yg chloramine T 136 Figure I.: The e f f e c t of sera d i l u t i o n on the amount of d i s p l a c e - able 1 2 5 I - s u b s t a n c e P bound, closed c i r c l e s = t o t a l bound; open c i r c l e s = binding i n the presence of one ng unlabeled substance P.  137 (Sigma) i n 20 y l H 20. Af t e r 12 sec the reaction was stopped by the addition of 185 yg sodium metabisulphite i n 50 y l H 20. The iodinated substance P was then p u r i f i e d according to the method of Yalow and Berson (1966). One ml of water was added to the reaction mixture followed by 10 mg of microfine s i l i c a (QUSO G32, P h i l a d e l p h i a Quartz Co.). Aft e r standing for 10 min at room temperature the suspension was centrifuged at 1,000 g for f i v e min and the supernatant discarded. The p e l l e t was washed f i v e times with one ml of d i s t i l l e d water and the labeled substance P then extracted into one ml of 20% acetone-1% a c e t i c a c i d , or one ml of the buffer. Both extraction procedures yielded about 0.2 mCi of t r a c e r , and both showed equal apparent binding i n the absence of antibody (damaged label) of 5%. Upon storage at 4°C the "damage l a b e l " of the tracer stored i n buffer increased more than that i n acid-acetone but ei t h e r tracer was usable for at l e a s t 10 weeks. The tracer used i n the present experiments was always prepared within one month of use. (e) Sample Preparation Tissue samples were boiled i n about 20 volumes of 1.0 N a c e t i c acid for f i v e min, placed on i c e and homogenized, b o i l e d again f o r f i v e min and centrifuged at 1,000 g for 10 min. The supernatant was then l y o - p h i l i z e d . The sample was resuspended i n i n an appropriate volume of assay buffer immediately before assay and, i f necessary, spun to remove sediment When 1 2 5 I - s u b s t a n c e P was added to the o r i g i n a l homogenate i n t h i s proce- dure the recovery was greater than 90%. (f) The Assay The assay buffer was 50 mM sodium b a r b i t a l t i t r a t e d with a c e t i c a c i d to pH 8.6. It contained an a n t i b a c t e r i a l agent (0.001% merthiolate), a protease i n h i b i t o r (500 KlU/ml apro t i n i n , Sigma) and 0.2% bovine serum albumin (RIA grade, Sigma) to minimize the loss of substance P onto the 133 surface of the incubation tubes. The assay was set up on i c e i n disposable b o r o s i l i c a t e tubes (10 x 75 mm). A standard curve was set up i n t r i p l i c a t e with concentrations of unlabeled substance P ranging from 31.25 pg to eight ng. Determination of the concentration of substance P i n preliminary extractions allowed the samples to be assayed to be taken up i n an appro- p r i a t e volume of buffer such that the concentration of substance P i n each lay approximately at the midpoint of the standard curve. Assay of tissues d i l u t e d over a t h r e e - f o l d range gave a dose-response curve p a r a l l e l to the standard curve. The f i n a l volume of each incubation was 0.5 ml. Antisera ( d i l u t e d i n buffer to give a f i n a l concentration of 1:60,000) was mixed with buffer to give 300 yl si and sample or standard i n 100 y l was then added. A f t e r a f i v e hr preincubation at 4°C the labeled 1 2 5 I - s u b s t a n c e P was added (about 5,000 cpm/tube) i n 100 ul and the tubes l e f t at 4°C for 48 hr. To separate the tracer bound to antisera from free t r a c e r , the coated charcoal technique was used (Herbert et a l . , 1965). A suspension of 10 g charcoal (Norit A neutral d e c o l o r i s i n g carbon) and 0.1 g Dextran T-40 (Pharmacia) was prepared i n 10 ml of the assay buffer without the a p r o t i n i n . After the incubation of the assay, 200 y l of the charcoal-dextran suspension was added to each incubation tube, and, a f t e r standing at 4°C for f i v e min the tubes were spun for 10 min at 1,000 g. A 500 y l a l i q u o t of each supernatant was sampled and counted for four min i n 10 ml of ACS (Amersham) i n a l i q u i d s c i n t i l l a t i o n counter at a r e l a t i v e e f f i c i e n c y of 80% as determined by the channels r a t i o method using chloroform quenched standards of 1 2 5 I - s u b s t a n c e P. The concentration of substance P i n each sample was the determined from the standard curve (Fig. I I ) . To p r a c t i c a l s e n s i t i v i t y of the assay was defined as 10% displacement of tracer (equivalent to 50 fmoles of substance P per sample). A substance 139 Figure I I . I n h i b i t i o n of 1 2 5I-substance P binding by unlabeled substance P; the substance P standard curve. ma 140 P concentration of 186 fmol/assay gave a 50% displacement of bound trac e r . In the absence of antisera about f i v e percent of the tracer remained i n the supernatant and t h i s blank (damaged label) was subtracted from a l l samples. It i s i n t e r e s t i n g to compare the present assay with the o r i g i n a l sub- stance P radioimmunoassay reported by Powell et a l . (1973). The sera d i l u t i o n i n that report was 1:8,000 compared with 1:60,000 i n the present assay. The "damage l a b e l " or blank i n both assays was f i v e percent, however, the s e n s i t i v i t y of the present assay i s an order of magnitude greater than that reported by Powell et a l . (1973). THE METHIONINE-ENKEPHALIN RADIOIMMUNOASSAY Rabbit a n t i s e r a to methionine-enkephalin was obtained from immuno Nuclear Corporation, S t i l l w a t e r , Minnesota. The a n t i s e r a could be used at a f i n a l d i l u t i o n of 1:1,000 i n the assay (Fig. I I I ) . Methionine- enkephalin labeled with t r i t i u m on the tyrosine residue to a s p e c i f i c a c t i v i t y of 18.77 Ci/mmol (New England Nuclear) was used as tracer at about 5,000 cpm per assay. Unlabeled methionine-enkephalin (Sigma) was used at concentrations from 78 pg to 10 ng f o r a standard ( F i g . IV). In t h i s assay two pmoles of synthetic met-enkephalin caused displacement of 50% of the bound tr a c e r , while the p r a c t i c a l l i m i t of s e n s i t i v i t y was 0.5 pmoles, which gave a 10% displacement. This i s s i m i l a r to the s e n s i t i v i t y reported by others ( S u l l i v a n et a l . , 1977; Wesche et a l . , 1977; Yang et a l . , 1977). The t i s s u e preparation and assay conditions were i d e n t i c a l to those des- scribed above for the substance P assay, except that the incubation was i n polypropylene tubes as recommended by Yang et a l . (1977). The bound tracer was counted i n a l i q u i d s c i n t i l l a t i o n counter at an e f f i c i e n c y of 35% f o r t r i t i u m . The c r o s s - r e a c t i v i t y of the assay with leucine-enkephalin 1 4 1 Figure I I I . The e f f e c t of sera d i l u t i o n on the amount of displaceable 3H-methionine enkephalin bound, closed c i r c l e s = t o t a l bound; open c i r c l e s = binding i n the presence of one ng unlabeled methionine enkephalin. Serum Dilution 142 Figure IV. I n h i b i t i o n of 3H-meLhionine enkephalin binding by unlabeled methionine enkephalin; the methionine- enkephalin standard curve.  143 (Sigma) was found to be less than two percent. REFERENCES Goodfriend, T.L., Levine, L. and Fasman, G.D. Antibodies to bradykinin and angiotensin: a use of carbodiimides i n immunology. Science, 144 (1964) 1344-1346. Greenwood, F.C., Hunter, W.M. and Glover, J.S. The preparation of 1 2 5 I - labeled human growth hormone of high s p e c i f i c r a d i o a c t i v i t y . Biochem. J . , 89 (1963) 114-123. Herbert, V., Lau, K.-S., Got t l i e b , CW. and Bleicher, S.J. Coated charcoal immunoassay of i n s u l i n . J . C l i n . Endocrinol., 25 (1965) 1375-1384. Mroz, E.A. and Leeman, S.E. Substance P, In: "Methods of Hormone Radio- immunoassay", B.M. J a f f e and H.R. Behram, eds., Academic Press, New York, (1979), pp. 121-137. Powell, D., Leeman, S.E., Tregear, CW., N i a l l , H.D. and Potts, J.T. J r . Radioimmunoassay for substance P. Nature (Lond.), New B i o l . 241 (1973) 252-254. Su l l i v a n , S., A k i l , H., Watson, S.J. and Barchas, J.D. Antibodies to enkephalins: coupling of antigens and a s p e c i f i c methionine-enkephalin radioimmunoassay. Commun. Psychopharmacol. 1 (1977) 605-610. V a i t u k a i t i s , J . , Robbins, J.B., Nieschlag, E. and Ross, C T . A method for producing s p e c i f i c a n t isera with small doses of immunogen. J . C l i n . Endocrinol. 33 (1971) 988-991. Wesch, D., H o l l t , V. and Herz, A. Radioimmunoassay of enkephalins. Regional d i s t r i b u t i o n i n rat brain a f t e r morphine treatment and hypo- physectomy. Arch. Pharmacol. 301 (1977) 79-82. Yalow, R.S. and Berson, S.A. P u r i f i c a t i o n of 1 3 ^-I-parathyroid hormone with microfine granules of pr e c i p i t a t e d s i l i c a . Nature (Lond.) 212 (1966) 357-358. Yang, H.-Y., Hong, J.S. and Costa, E. Regional d i s t r i b u t i o n of l e u and met enkephalin i n r a t brain. Neuropharmacology 16 (1977) 303-307. PUBLICATIONS Gardner, D.R. and Vincent, S.R., Insecticidal and neuroexcitant actions of DDT analogs on the cockroach, Periplaneta americana, Bull. Environ. Contam. Toxicol. 20 (1978) 294-302. Vincent, S.R., Nagy, J.I. and Fibiger, H.C, Increased s t r i a t a l glutamate decarboxylase after lesions of the nigrostriatal pathway, Brain Research, 143 (1978) 168-173. Nagy, J.I., Vincent, S.R., Lehmann, J., Fibiger, H.C. and McGeer, E.G., The use of kainic acid in the localization of enzymes in the substantia nigra, Brain Research, 149 (1978) 431-441. Vincent, S.R., Hattori, T. and McGeer, E.G., The nigrotectal projection: a biochemical and ultrastructural characterization, Brain Research, 151 (1978) 159-164. Nagy, J.I., Vincent, S.R. and Fibiger, H.C, Altered neurotransmitter synthetic enzyme a c t i v i t y in some extrapyramidal nucei after lesions of the nigro-striatal dopamine projection, L i f e Sciences, 22 (1978) 1777-1782. Vincent, S.R. and McGeer, E.G., Kainic acid binding to membranes of s t r i a t a l neurons, L i f e Sciences, 24 (1979) 265-270. McGeer, E.G., McGeer, P.L., Hattori, T. and Vincent, S.R., Kainic acid neurotoxicity and Huntington's Disease. In: Advances in Neurology, Vol. 23: Huntington's Disease. T.N. Chase, N.S. Wexler and A. Barbeau, eds., Raven Press, New York, (1979) pp. 577-591. McGeer, E.G., McGeer, P.L. and Vincent, S.R., Morphine, naloxone and kainic acid neurotoxicity, Res. Comm. Chem. Path. Pharmacol. 25 (1979) 411-414. Vincent, S.R. and McGeer, E.G., Structural s p e c i f i c i t y of high a f f i n i t y H-glutamate uptake in rat striatum, J. Pharm. Pharmacol., 31 (1979) 703- 704. Vincent, S.R. and McGeer, E.G., A comparison of sodium-dependent glutamate binding with high-affinity glutamate uptake in rat striatum, Brain Research, 184 (1980) 99-108. Nagy, J.I., Vincent, S.R., Staines, W.A., Fibiger, H.C, Reisine, T.D. and Yamamura, H.I., Neurotoxic action of capsaicin on spinal substance P neurons, Brain Research, 186 (1980) 435-444. Vincent, S.R., Kimura, H. and McGeer, E.G., The pharmacohistochemical demonstration of GABA-transaminase, Neurosci. Lett., 16 (1980) 345-348. Fibiger, H.C, Nagy, J.I., Staines, W.A. and Vincent, S.R., Organization and p l a s t i c i t y of GABAergic neurons in some extrapyramidal nuclei of the rat, Brain Research Bull., in press (1980). Staines, W.A., Nagy, J.I., Vincent, S.R. and Fibiger, H.C, Neurotrans- mitters contained i n the efferents of the striatum, Brain Research, in press (1980). -2- Vincent, S.R., Lehmann, J. and McGeer, E.G., The localization of GABA- transaminase in the s t r i a t r n i g r a l system, L i f e Sciences, i n press (1980), Vincent, S.R., Staines, W.A., McGeer, E.G. and Fibiger, H.C., Neuro- transmitters contained in the efferents of the habenula, Brain Research, in press (1980).

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
China 10 37
United States 9 0
Japan 2 0
Switzerland 1 0
Italy 1 0
Ghana 1 0
City Views Downloads
Beijing 9 0
Unknown 5 1
Seattle 3 0
Tokyo 2 0
Redmond 1 0
Lausanne 1 0
Hangzhou 1 0
Sunnyvale 1 0
Ashburn 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}

Share

Share to:

Comment

Related Items