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Mechanisms of excitation and inhibition in the nigrostriatal system Richardson, Thomas L. 1979

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MECHANISMS OF EXCITATION AND INHIBITION IN THE NIGROSTRIATAL SYSTEM by THOMAS L. RICHARDSON B.Sc. U n i v e r s i t y of B r i t i s h Columbia, 1974 A THESIS SOBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n F a c u l t y of Graduate Stud i e s (Department of Physiology) we accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1979 (51) T h o m a s L . R i c h a r d s o n In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it 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. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department nf Physiology The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 HatP May 25, 1979 D E - 6 B P 7 5 - 5 1 1 E i i > ABSTRACT The e x t r a c e l l u l a r responses of neurons i n the corpus s t r i a t u m f o l l o w i n g s i n g l e pulse s t i m u l a t i o n of the s u b s t a n t i a n i g r a or d o r s a l raphe nucleus were i n v e s t i g a t e d i n urethane a n a e s t h e t i z e d r a t s , n i g r a l s t i m u l a t i o n a t low i n t e n s i t i e s {10 v) evoked s i n g l e l a r g e amplitude s p i k e s while higher i n t e n s i t i e s {10 to 20 v) evoked, i n a d d i t i o n , a high frequency bu r s t of smal l amplitude s p i k e s or waves. Spontaneous l a r g e s p i k e s , or those induced by the a d m i n i s t r a t i o n of glutamate, were i n h i b i t e d by n i g r a l s t i m u l a t i o n . The onset of i n h i b i t i o n c o i n c i d e d with the onset of the b u r s t . I f the b u r s t was prevented, i n h i b i t i o n no longer occurred. Neither the i n h i b i t o r y nor the b u r s t response evoked by n i g r a l s t i m u l a t i o n was i n f l u e n c e d by i o n t o p h o r e t i c a l l y or s y s t e m i c a l l y a dministered a n t a g o n i s t s of dopamine or by chemi c a l l e s i o n s of the dopaminergic neurons of the n i g r o s t r i a t a l pathway. However the e x c i t a t i o n of l a r g e u n i t s by n i g r a l s t i m u l a t i o n was r e v e r s i b l y blocked by dopamine a n t a g o n i s t s . S t i m u l a t i o n of the d o r s a l raphe nucleus produced i n h i b i t i o n of spontaneously a c t i v e s t r i a t a l neurons. No e x c i t a t o r y response was ever observed. HEP i n j e c t e d i n t o the s t r i a t u m was t r a n s p o r t e d to c e l l s i n the d o r s a l raphe nucleus and i n j e c t i o n of t r i t i a t e d l e u c i n e i n t o the d o r s a l raphe nucleus produced s i g n i f i c a n t t r a n s p o r t of r a d i o l a b e l l e d p r o t e i n t o the caudate nucleus. I t i s concluded t h a t the b u r s t response i s produced i±i by excitation of s t r i a t a l interneurons through c o l l a t e r a l s of the s t r i a t o n i g r a l pathway which are i n t r i n s i c to the nucleus. Nigral stimulation causes an antidromic activation of the axon and a subsequent orthodromic activation of i t s c o l l a t e r a l s . The interneurons activated by this "axon r e f l e x " are i n h i b i t o r y i n function. It i s further concluded that the dopaminergic neurons of the n i g r o s t r i a t a l tract make excitatory synaptic contact with s t r i a t a l neurons i n the central region of the nucleus. At le a s t some of these target neurons project, in turn, to the globus p a l l i d u s . i v TABLE OF CONTENTS Acknowledgements .- V i i Table Of Abbreva t i o n s V-Abstract ' i i I n t r o d u c t i o n - - • - 5 Methods ... . . ..... 36 S u r g i c a l P r e p a r a t i o n - 36 S t i m u l a t i o n Procedure . . ............. 37 M i c r o e l e c t r o d e Peparation 37 Recording Procedures And Data A n a l y s i s ............ 38 H i s t o l o g y ..... - 40 L e s i o n i n g And Assay Procedures 41 Re s u l t s . - 43 Burst Response 43 S i n g l e U n i t s 51 Antidromic P o t e n t i a l s ............................. 60 Lesioned P r e p a r a t i o n s 62 Ac t i o n s Of Pharmacological Agents 66 Anatomical S t u d i e s .... 69 D i s c u s s i o n - 74 Burst Response -- 74 Pathway Mediating The Burst Response 77 I n h i b i t o r y Response 82 Large Amplitude C e l l s 85 Conclusion . 86 References 89 TABLE OF ABBRSVATIONS Ach a c e t y l c h o l i n e AchE a c e t y l c h o l i n e s ! e r a s e CAT c h o l i n e a c e t y l t r a n s f e r a s e c a caudate nucleus DA dopamine DC d i r e c t c u r r e n t DFP d i i s o p r o p y l fluorophosphate DRN Dorsal Raphe Nucleus E.PSP e x c i t a t o r y p o s t - s y n a p t i c p o t e n t i a l G ABA gamma aminobutyric a c i d GAD glu t a m i c a c i d decarboxylase gm gram GP globus p a l l i d u s HEP h o r s e r a d i s h peroxidase HVA h o m o v a n i l l i c a c i d Hz h e r t z IC i n t e r n a l capsule IPS? i n h i b i t o r y p o s t - s y n a p t i c p o t e n t i a l IPT i n t r a l a m i n a r and p a r a f a s c i c u l a r n u c l e i of the thalamus kg kilogram M molar ml m i l l i l i t r e mm m i l l i m e t e r mH m i l l i m o l a r msec m i l l i s e c o n d WEN mesencephalic raphe nucleus NA n o r a d r e n a l i n PST post s t i m u l u s time histogram SN s u b s t a n t i a n i g r a SNC zona compacfa of the s u b s t a n t i a n i g r a SUB zona r e t i c u l a t a o f the s u b s t a n t i a n i g r a \ a microam peres um micrometers uv m i c r o v o l t s 5-HT 5-hydroxytryptamine 6-OHDA 6-hydroxydopamine TABLE OE EIGUBES F i g u r e 1 - ....... 44 Figu r e 2. 45 F i g u r e 3. 47 Fi g u r e 4 48 Fi g u r e 5 49 F i g u r e 6. 51 F i g u r e 7. 52 F i g u r e 8. 54 F i g u r e 9. 55 Figure 10 57 F i g u r e 11 .. - ........... 59 F i g u r e 12. 61 Fi g u r e 13 63 F i g u r e 14. 65 F i g u r e 15. .......................................... 67 Figu r e 16 - 68 F i g u r e 17 70 Figure 18 71 F i g u r e 19. ..... ....... ... 88 v i i ACKNOWLEDGEMENTS I t i s tny pleasure to thank Dr. H. McLennan and Dr. J . J . M i l l e r f o r t h e i r s u p e r v i s i o n , t e a c h i n g and continued i n t e r e s t i n t h i s p r o j e c t . I would a l s o l i k e to thank Yvonne Heap and Ron Walker t T f o r t h e i r t e c h n i c a l a s s i s t a n c e , Helen Brandis f o r a s s i s t a n c e i n p r e p a r a t i o n of the HRP h i s t o l o g y and Dr. C. F i b i g e r and a s s o c i a t e s f o r performing the catecholamine and t r i t i a t e d p r o t e i n assays. F i n a l l y I would l i k e t o thank Joanne, my wife, f o r continued p a t i e n c e and a s s i s t a n c e i n b r i n g i n g t h i s e f f o r t to completion. LEAVES 1 - 4 OMITTED IN PAGE NUMBERING 5 INTRODUCTION The e a r l y anatomists d e f i n e d the e x t r a p y r a m i d a l motor system as a l l c e n t r a l motor mechanisms not mediated through the pyramidal t r a c t s {Jung and H a s s l e r , 1960). However t h i s d e f i n i t i o n has l e d t o d i f f i c u l t i e s . Areas of the c e r e b r a l c o r t e x c l a s s i c a l l y d e f i n e d as e x t r a p y r a m i d a l i n f u n c t i o n have been shown t o c o n t r i b u t e a s i g n i f i c a n t number of f i b r e s to the pyramidal t r a c t s , while the pyramidal c o r t e x , area 4 gamma, has a major p r o j e c t i o n t e r m i n a t i n g i n s u b c o r t i c a l e x t r a p y r a m i d a l s t r u c t u r e s (Carman et a l , 1963; R u s s e l l and DeMyer, 1961). Thus every c o r t i c a l motor area has both pyramidal and e x t r a p y r a m i d a l f u n c t i o n s . Furthermore, s i n c e lower v e r t e b r a t e s do not have a pyramidal t r a c t t h e i r e n t i r e motor system i s , of n e c e s s i t y , e x t r a p y r a m i d a l . More r e c e n t l y a f u n c t i o n a l l y r e l e v a n t concept of e x t r a p y r a m i d a l motor mechanisms has developed based mainly on c l i n i c o - p a t h o l o g i c a l s t u d i e s i n man. A group of r e l a t e d syndromes, r e f e r r e d t o as e x t r a p y r a m i d a l motor d i s e a s e s , r e s u l t from l e s i o n s of the caudate- putamen (Cd), globus p a l l i d u s (GP), subthalamic nucleus and s u b s t a n t i a n i g r a (SN) (Vogt and Vogt, 1920; Wilson, 1912; T r e t i a k o f f , 1919). Each disease i n v o l v e s an abnormality of unconscious or stereotyped motor behavior. The h y p e r k i n e t i c syndromes are c h a r a c t e r i z e d by an excess of spontaneous, aimless and i n v o l u n t a r y movements. These syndromes i n c l u d e chorea, r e s u l t i n g from l o s s of small c e l l s i n the Cd and GP; 6 athetosis, r e s u l t i n g from lesions damaging the large c e l l s of these nuclei and ballismus, r e s u l t i n g from lesions of the subthalamic nucleus. The hypokinesis of Parkinson's disease i s characterized by an absence of spontaneous reactive and automatic movements as well as a persistent increase i n muscle tone without spastic paresis or es s e n t i a l changes in s p i n a l reflexes. This syndrome i s associated with c e l l l o s s in the pars compacta of the SN. On the basis of these syndromes the term extrapyramidal motor system i s now used to refer to the motor nuclei and c o r t i c a l regions involved i n the integration and regulation of unconscious and stereotyped motor behavior. The neuronal c i r c u i t r y of the extrapyramidal motor system i s complex and not yet f u l l y understood. However, by considering only the major and well established f i b r e systems, certain organizational patterns are evident. The extrapyramidal nuclei are under the influence of motor as well as sensory afferents mainly v i a c o r t i c a l and thalamic projections to the Cd. This nucleus receives a somatotopically organized projection from a l l regions of the i p s i l a t e r a l cerebral cortex (Carman et a l , 1963) as well as from the supplementary motor area and area 5 of the c o n t r a l a t e r a l cortex (Carman et a l , 196 5) . The heaviest projection occurs from the i p s i l a t e r a l somato-sensory and motor regions. The intralaminar nuclei of the thalamus (I.P.T.), including the parafascicular and centromedian nu c l e i , have a major projection terminating in the i p s i l a t e r a l Cd (Powell and Cowan, 1956) . These 7 n u c l e i r e c e i v e sensory a f f e r e n t s from s p i n a l and r e t i c u l a r o r i g i n s and are a l s o s t r o n g l y i n f l u e n c e d by a c t i v i t y i n the motor c o r t e x . The Cd r e c e i v e s a d d i t i o n a l a f f e r e n t s from other e x t r a p y r a m i d a l motor n u c l e i . The most e x t e n s i v e l y s t u d i e d of these o r i g i n a t e s i n the SN (Anden et a l , 196 6) . E f f e r e n t f i b r e s of the Cd are thought t o terminate almost e x c l u s i v e l y i n the SN and globus p a l l i d u s (Nauta and Mehler, 1966; Szabo, 1967). Both p r o j e c t i o n s are s o m a t o t o p i c a l l y w e l l organized. The s m a l l e r p r o j e c t i o n i s to the SN, the p a l l i d a l p r o j e c t i o n i s the major outflow from the Cd. The GP, u n l i k e the Cd, does not r e c e i v e a f f e r e n t s from e i t h e r c o r t i c a l or thalamic sources. The 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 s i t s primary i n p u t although the SN and subthalamic nucleus a l s o c o n t r i b u t e f i b r e s (Carpenter and Strominger, 1967). The e f f e r e n t p r o j e c t i o n s from the two segments of the GP d i f f e r . The e x t e r n a l segment p r o j e c t s to the subthalamic nucleus while the i n t e r n a l segment p r o j e c t s t o the SN, midbrain tegmentum and the thalamus (Nauta and Mehler, 1966; Carpenter and Strominger, 1967; Sanson and Hanson, 1942) . Thus both the SN and subthalamic nucleus have r e c i p r o c a l connections with the GP. The thalamic p r o j e c t i o n s terminate i n the v e n t r a l a n t e r i o r and ventro-l a t e r a l n u c l e i as w e l l as the centromedian nucleus of the i n t r a l a m i n a r group. The thalamic motor n u c l e i , v e n t r a l 8 anterior and ventro-lateral, project in turn to the motor cortex completing a neuronal loop from the cortex through the Cd, GP, thalamus and back to the cortex. The centromedian nucleus, since i t has both efferents to and afferents from extrapyramidal n u c l e i , i s part of a second loop involving a pathway from the centromedian through the Cd, GP and back to the centromedian. Modifications of motor behavior by the extrapyramidal system re s u l t s largely through thalamic relays to the motor cortex. However, before extrapyramidal influences reach the cortex they are integrated, in the thalamic n u c l e i , with influences from other c e n t r a l motor and sensory mechanisms. The ventral anterior and ventro-l a t e r a l nuclei, both thalamic motor structures, are s i t e s for convergence of cerebellar a c t i v i t y via dentato- and rubro-thalamic f i b r e s and extrapyramidal a c t i v i t y via the pallido-thalamic projection. The a c t i v i t y of the ventral anterior and ventral l a t e r a l nuclei can then influence the motor cortex through a dir e c t thalamo-cortical pathway. The centromedian nucleus receives extrapyramidal afferents from the GP and sensory afferents from spinal and r e t i c u l a r o r i g i n s . Descending influences from the motor cortex are also present. Although the major projection of the centromedian nucleus i s to the Cd providing a feed-back to the extrapyramidal system, i t s a c t i v i t y i s known to influence the cortex v i a i n t r i n s i c projections to the other thalamic nuclei (Purpura and Yahr, 1966) . 9 Extrapyramidal influences on the motor cortex are l i k e l y to re s u l t in modification of a c t i v i t y descending to spinal l e v e l s i n the pyramidal t r a c t s . Projections to the midbrain tegmentum from the SN and globus p a l l i d u s , and to the tectum from the SN may also have important influences on motor behavior since these midbrain regions are sources of major descending pathways modifying the output of sp i n a l motor neurons. The integration and modification of a c t i v i t y i n these various pathways i s a re s u l t of the anatomical and functional organization of the neurons within the nuclei. However our understanding of the i n t r i n s i c organization of the motor nuclei i s far from complete. The Cd, the largest subcortical structure in the mammalian nervous system, and i t s associated n u c l e i , the SN and GP, have received the most thorough investigation. Cajal and Ramon (1911) f i r s t investigated the Cd with Golgi stains and the l i g h t microscope. More recent workers have expanded on Cajal's c l a s s i c a l description of the neuronal organization and with the advent of the electron microscope have also investigated the synaptic organization of the nucleus. In view of t h i s detailed anatomical data the structure of the Cd can no longer be refered to as homogeneous, although i t i s not organized into discrete lamina of specialized c e l l s as i s seen i n c o r t i c a l structures, the neurons are grouped into c l u s t e r s of c e l l bodies surrounded by neuropil {Kemp and Powell, 10 1971 a; C h r o n i s t e r et a l , 1976). Furthermore the nucleus i s t r a v e r s e d by f a s c i c l e s of c o r t i c o - f u g a l f i b r e s t r a v e l l i n g towards the b a s i s p e d u n c u l i . The d i s t r i b u t i o n of these f i b r e s d i f f e r s i n the s t r i a t u m of va r i o u s s p e c i e s . Man, with a w e l l developed a n t e r i o r limb of the i n t e r n a l capsule (IC), has a s t r i a t u m almost devoid of l a r g e f a s c i c l e s of p a s s i n g f i b r e s . In the c a t the a n t e r i o r limb of the IC i s l e s s w e l l developed and many of the c o r t i c o - f u g a l f i b r e s pass through the adj a c e n t s t r i a t u m . However the r a t has no a n t e r i o r limb of the IC and the corresponding f i b r e s pass through the substance of the str i a t u m as l a r g e f a s c i c l e s o f axons 50 t o 200 um i n diameter. On c o r o n a l s e c t i o n s the f a s c i c l e s are cut i n c r o s s s e c t i o n and appear to be surrounded by c l u s t e r s of c e l l s . Each c l u s t e r has 10-14 c e l l bodies and i s about 60 um across. D e n d r i t e s stream out from these c l u s t e r s and form t i g h t bundles i n t e r c o n n e c t i n g the c e l l c l u s t e r s and surrounding the f a s c i c l e ( C h r o n i s t e r et a l , 1976). In s a g i t a l or f r o n t a l s e c t i o n s the f a s c i c l e s run with the plane of the s e c t i o n i n a r a d i a l f a s h i o n from the IC t o the c o r t e x . C l u s t e r s of c e l l bodies form columns of c e l l s p a r a l l e l to the f a s c i c l e s . T i g h t bundles of d e n d r i t e s are seen passing a c r o s s the f a s c i c l e s j o i n i n g c e l l groups on i t s two s i d e s . Kemp and Powell (1971 A, B, C) have found t h a t the neurons of the Cd can be d i v i d e d i n t o a t l e a s t s i x d i f f e r e n t v a r i e t i e s based on morphological c h a r a c t e r i s t i c s . These neurons form two f u n c t i o n a l groups, 11 interneurons and projecting neurons. The vast majority of Cd neurons, over 96%, are interneurons. At least 95% of these are medium sized spiny c e l l s . The somata are 12-14 u^m across with branched dendrites forming a spherical arborization extending 180-240^m away from the parent c e l l . Although the primary dendrite i s smooth i t s branches are studded with t i g h t l y packed spinous processes. The axons i n some examples may t r a v e l long distances however most have multiple c o l l a t e r a l s terminating within the dendritic tree of the parent c e l l . The remaining 5% of interneurons can be divided into three v a r i e t i e s . The f i r s t group also consists of neurons with c e l l bodies of medium s i z e . However, t h e i r dendrites are long and slender, often exceeding 300 urn i n length. Only occasional spinous processes are present. The axon often bifurcates and the c o l l a t e r a l s usually terminate within the dendritic tree of the parent c e l l . The second variety has medium sized c e l l bodies which give r i s e to spineless dendrites with multiple varicosed and twisted branches. The dendrites form a dense arborization surrounding the parent c e l l . The short axons have multiple bifurcations which usually terminate near the c e l l body. The l a s t variety consists of interneurons with small c e l l bodies 5-9 i^ rn across and very dense dendritic networks within 50-60 u^m of the soma. No axons have been i d e n t i f i e d . 12 No s p e c i f i c i t y between afferent systems and c e l l groups was detected by Kemp and Powell. Each variety of interneuron receives synaptic contacts en passent from afferent f i b r e s of the cortex, thalamus and SN. The majority are axospinous but axodendritic and axosomatic contacts are also present. The majority of afferent terminals are 1 um i n diameter but a few are 5 ujm across. The terminals contain many round vesicles and the synapses have asymmetrical s p e c i a l i z a t i o n of the pre and post synaptic membrane. They are described as Golgi type 1 synapses (Gray, 1959). The interneurons also receive mainly axodendritic and axosomatic synapses from other interneurons. Most of these terminals have flattened pleomorphic v e s i c l e s and symmetrical s p e c i a l i z a t i o n of the pre and post synaptic membrane. They are described as Golgi type 2 synapses. The majority of interneurons influence c e l l s within a radius of 450 i^ m. The second functional group of neurons project beyond the Cd to other structures. They form a small group of c e l l s amounting to only 3-4% of the t o t a l number of neurons. Two morphological v a r i e t i e s found i n approximately the same proportion are described by Kemp and Powell. The medium sized projecting neurons have thick dendrites studded with only a few spines. The axons are varicosed and occasionally give off c o l l a t e r a l s . However they do not form a profuse network. Similar c e l l s were said to project beyond the nucleus by C a j a l and Ramon 13 (1911). The second v a r i e t y c o n s i s t s of very l a r g e f u s i f o r m c e l l s 20-30 um i n l e n g t h . They have long s t r a i g h t d e n d r i t e s o f t e n extending as f a r as a m i l l i m e t e r beyond the parent c e l l . The d e n d r i t e s have a few spinous processes along t h e i r l e n g t h . The axons are very long and have few c o l l a t e r a l s . Kemp and Powell r e f e r to these neurons as " g i a n t c e l l s " . Axons from the c o r t e x , thalamus and SN form G o l g i type 1 synapses with the sp i n e s and d e n d r i t e s o f p r o j e c t i n g neurons. However the i n t e r n e u r o n s make G o l g i type 2 s y n a p t i c contact with t h e i r d e n d r i t e s , somata and i n t i a l segments. The p r o j e c t i n g neurons o f the Cd send axons to the GP and SN. The s t r i a t o - p a l l i d a l f i b r e s pass d i r e c t l y t o the adjacent GP and ter m i n a t e with a x o d e n d r i t i c synapses. Both G o l g i type 1 and 2 s y n a p t i c s p e c i a l i z a t i o n s are pr e s e n t . The s t r i a t o - n i g r a l f i b r e s must f i r s t pass through the GP and then t r a v e l i n the IC and b a s i s p e d u n c u l i . As they approach the SN the f i b r e s move l a t e r a l l y and pass d o r s a l l y i n t o the v e n t r a l aspect o f the pars r e t i c u l a t a of the SN. Here they synapse on the d e n d r i t e s of the r e t i c u l a t a c e l l s . Again, both G o l g i type 1 and 2 synapses are seen. In an attempt to e l u c i d a t e the f u n c t i o n s of the e x t r a pyramidal motor n u c l e i e a r l y i n v e s t i g a t o r s observed the behavior r e s u l t i n g from s t i m u l a t i o n and l e s i o n i n g of these s t r u c t u r e s . F e r r i e r (1873) found t h a t f a r a d i c s t i m u l a t i o n 14 of the Cd caused pronounced bending of the head and body to the c o n t r a l a t e r a l side. However these movements were thought to r e s u l t from unintentional stimulation of nearby capsular f i b r e s since movements were not observed i n animals with degeneration of the IC. Furthermore, Wilson (1914) was unable to demonstrate any effect of faradic stimulation of the putamen i n monkeys. On t h i s basis he considered the putamen an inexcitable structure. However l a t e r studies, using more discrete stimulation technigues report three general patterns of behavioral response each dependent on the freguency of Cd stimulation. Low frequency (0.2 to 10 HZ) b i l a t e r a l stimulation of the Cd for long periods of time may produce i n the unanaesthetized f r e e l y moving cat what Hess (1948) describes as p a r t i a l sleep (Parneggiani, 1962) . This state i s characterized by i n a c t i v i t y with l i t t l e spontaneous movement and a d e f i c i e n t motor responsiveness to external stimulation. Heath and Hodes (1952) have also reported sleep following stimulation of the Cd in monkey and man. However, these findings have not been confirmed by McLennan et a l (1964). The l a t t e r investigators detected neither sleep nor decreased alertness even i n an environment most conducive to sleep. In fact, an increased alertness was invariably observed. B i l a t e r a l stimulation of the Cd at higher frequencies (10 to 30 HZ) causes an arrest reaction (Jung and Hassler, 1960; McLennan et a l , 1964) s i m i l a r to that described by 1 5 Hunter and Jasper following stimulation of the intralaminar thalamic nuclei (Hunter and Jasper, 1949). Ongoing behavior of the cat, such as walking towards a dish of food, w i l l come to a sudden halt at the onset of stimulation, even though the animal remains a l e r t . Following cessation of the stimulation the cat w i l l resume i t s o r i g i n a l behavior. S i m i l a r l y intermediate frequencies of Cd stimulation increase reaction time by several hundred percent for performance of a well learned visual discrimination task. However, when the response i s i n i t i a t e d i t i s excecuted smoothly and r a p i d l y . Buchwald (Buchwald et a l , 1961 a) believes that the stimulation i n t e r f e r e s with the i n i t i a t i o n of the behavior rather than i t s subsequent performance. Unilateral stimulation of the Cd at intermediate frequencies produces an apparently purposeful turning of the head and body to the c o n t r a l a t e r a l side. The turning movement often develops into a well coordinated rotation of the animal in a d i r e c t i o n c o n t r a l a t e r a l to the s i t e of stimulation. The body regions affected are somatotopically related to the s i t e of stimulation within the Cd {Forman and Ward, 1957). Ventral s i t e s are associated with movements of the head, neck and forelimbs whereas dorsal s i t e s are associated with movements of the trunk and hindlimbs. Forman and Ward claim these contraversive movements are independent of c o r t i c o - s p i n a l systems. They found that motor responses to c o r t i c a l stimulation of anaesthetized cats are not influenced by simultaneous 16 stimulation of the Cd. On the other hand Hendley and Hodes (1953) demonstrated that turning movements are dependent on i n t a c t connections between the Cd and medial SN. Stimulation of the Cd at a high frequency (100 to 300 Hz) results i n behavioral arousal or an a l e r t i n g response i n drowsy animals (Buchwald and Wyers, 1961 b); a response s i m i l a r to that seen following stimulation of the r e t i c u l a r formation. I f the stimulus i s continued for a few seconds a tremor of the c o n t r a l a t e r a l fore or hindlimb w i l l be induced. More recently workers have studied the neurochemical properties of the extra pyramidal motor system. Analysis of the Cd, GP and SN has revealed s i g n i f i c a n t concentrations of several putative synaptic transmitters. The presence of acetylcholine (Ach), 5-hydroxytryptamine (5-HT) and dopamine (DA) suggests that they may function as transmitters either in pathways interconnecting extrapyramidal structures or in the i n t r i n s i c c i r c u i t r y of the nuclei. However to demonstrate that a substance functions as a transmitter is a d i f f i c u l t task. A number of c r i t e r i a Bust be f u l l f i l l e d . These were f i r s t formulated during investigation of the peripheral autonomic nervous system. It must be demonstrated that 1) the substance i s present in the terminals, 2) the neuron contains the appropriate precursors and enzymes necessary fo r synthesis of the substance, 3) the substance i s released upon stimulation of the neuron, 4) the substance 17 when applied a r t i f i c i a l l y to the synapse, mimics the response seen following stimulation of the neuron, and 5) that a mechanism f o r i n a c t i v a t i o n of the substance i s present at the synapse (Florey, 1960}. although Ach i s c l e a r l y established as a synaptic transmitter i n the periphery i t s r o l e i n the CNS i s less well defined. The highest concentration of Ach, choline acetyltransferase {CAT) and acetylcholine esterase (AchE) in the brain are found i n the Cd although considerable quantities are also found i n the GP and SN. CAT i s the synthetic enzyme required for conversion of choline to Ach. Therefore i t must be present i n cho l i n e r g i c neurons. However AchE, the catabolic enzyme required for i n a c t i v a t i o n of Ach at the synapse may be present i n either cholinergic neurons or neurons receiving a cholinergic input. Investigation of the role of Ach i n the basal ganglia i s based mainly on l o c a l i z a t i o n of CAT and AchE since Ach i s very l a b i l e i n brain t i s s u e . Subcellular fractionation of s t r i a t a l tissxie shows that most of the CAT i s concentrated i n nerve endings while a large portion of AchE i s membrane bound. Sternberger {1970) developed a very sensitive immunohistochemical technique for l o c a l i z i n g tissue enzymes. Using a complex sandwich of immunoglobulins, peroxidase i s bound to the enzyme and a brown reaction product results following addition of hydrogen peroxide and diaminobenzidine. Using t h i s marker, Hattori et a l 18 (1976 B) have i d e n t i f i e d a p o p u l a t i o n of medium s i z e d (7-14 nm) CAT c o n t a i n i n g neurons d i s t r i b u t e d i n l a r g e numbers throughout the Cd the e l e c t r o n microscope r e v e a l s CAT c o n t a i n i n g d e n d r i t i c processes r e c e i v i n g asymmetrical axospinous synapses mainly from CAT f r e e t e r m i n a l s . Terminals c o n t a i n i n g CAT make s i m i l a r synapses with CAT f r e e d e n d r i t e s . The axons of CAT c o n t a i n i n g c e l l s are not w e l l v i s u a l i z e d with t h i s technigue and l i t t l e d i r e c t evidence e x i s t s to determine i f the axons a l l remain w i t h i n the Cd or i f some form the e f f e r e n t p r o j e c t i o n s of the nucleus. However McGeer e t a l (1971) b e l i e v e that CAT c e l l s are c h o l i n e r g i c i n t e r n e u r o n s completely i n t r i n s i c t o the nucleus. They found t h a t e l e c t r o l y t i c l e s i o n s of the Cd d i d not i n f l u e n c e CAT or AchE l e v e l s i n r e g i o n s which r e c e i v e major s t r i a t a l e f f e r e n t s i n c l u d i n g the thalamus, GP and midbrain (mcGeer e t a l , 1969) . They reasoned that i f CAT i n the GP and SN i s present i n t e r m i n a l s of s t r i a t a l e f f e r e n t s , l e s i o n s to the Cd should have caused a s i g n i f i c a n t drop i n CAT c o n c e n t r a t i o n i n these s t r u c t u r e s . However, i n t e r p r e t a t i o n of t h e i r n e g a t i v e r e s u l t s must be made with c a u t i o n . I f the t a r g e t nucleus a l s o c o n t a i n s CAT i n c e l l bodies as w e l l as t e r m i n a l s from other n u c l e i , the change i n CAT l e v e l f o l l o w i n g Cd l e s i o n s may be undetectable. McGeer e t a l (1971) a l s o demonstrated t h a t l e s i o n s of the c o r t e x , thalamus, v e n t r a l tegmentum and GP do not i n f l u e n c e CAT or AchE l e v e l s i n the Cd. They reasoned t h a t , i f c h o l i n e r g i c neurons p r o j e c t beyond the s t r i a t u m , l e s i o n s of the t a r g e t n u c l e i should have caused 19 retrograde degeneration of efferent neurons and a subsequent decrease i n CAT levels in the striatum. However, i f the cholinergic neurons have multiple c o l l a t e r a l s with the majority synapsing within the nucleus and only a few projecting to other brain regions, retrograde degeneration of the somata and i n t r i n s i c c o l l a t e r a l s would not be expected. The c e l l u l a r l o c a l i z a t i o n of AchE i s e a s i l y examined in routinely fixed tissue since the enzyme retains a c t i v i t y even after exposure to formaldehyde. When brain tissue i s incubated with acetylthiocholine, an Ach analogue, AchE w i l l r e s u l t i n the production of an opaque pre c i p i t a t e . Examination of the tissue with the l i g h t microscope w i l l then reveal s i t e s of high enzyme concentration. When t h i s technique i s applied to the adult striatum a dense staining occurs throughout the nucleus and detailed c e l l u l a r l o c a l i z a t i o n i s impossible. Cl e a r l y , AchE i s present in high concentration i n the majority of c e l l bodies and processes. However in the newborn rat very l i t t l e AchE a c t i v i t y i s present i n the striatum. Butcher Hodge (1976) studied the subsequent development of AchE staining during maturation of the Cd and SN. During the f i r s t 3-10 days of l i f e , islands of AchE a c t i v i t y appear in the l a t e r a l regions of the Cd. Clusters of AchE containing c e l l bodies and th e i r processes are often observed within these islands. The neurons have multipolar somata and are usually triangular or fusiform in shape, with the majority from 13-20 um i n diameter. Although 20 s l i g h t l y larger than the medium sized c e l l s described by Kemp and Powell, Butcher believes that on the basis of their morphology and frequent occurrence these neurons should s t i l l be considered within the medium sized group. They may be either the medium projecting neurons or the medium smooth interneurons. Occasionally AchE neurons with very large fusiform somata and prominent c e l l u l a r processes are detected. They correspond to the 1% of Cd neurons c l a s s i f i e d as giant c e l l s by Kemp and Powell. The long thick axons of giant c e l l s are believed to project beyond the nucleus. After 10 days of l i f e an increasing number of c e l l u l a r processes become positive for AchE u n t i l at 15 days i t i s not possible to i d e n t i f y i n d i v i d u a l c e l l bodies. The loss of d i s t i n c t staining of neurons appears to be a function both of a diffuse staining of a multitude of c e l l u l a r processes as well as a lower intensity of staining in the somata. This suggests that AchE i s synthesized i n the somata at high rates u n t i l adequate concentrations are available i n the more d i s t a l regions of the c e l l . At that time the rate of synthesis decreases and as a result AchE concentration in the c e l l body also decreases. However Butcher Hodge (1976) found that treatment of the neostriatum of the adult rat with diisopropyl fluorophosphate (DFP), a potent i r r e v e r s i b l e AchE i n h i b i t o r w i l l induce synthesis of new enzyme. In this way the c e l l u l a r l o c a l i z a t i o n of AchE can be studied in the adult. Immediately after a l o c a l i n j e c t i o n of DFP into the Cd a region 2 mm in diameter at the in j e c t i o n 21 site i s devoid of AchE sta i n i n g . However by 10 hours a f t e r the inje c t i o n c e l l bodies and some processes are c l e a r l y stained. These c e l l s have the same c h a r a c t e r i s t i c s as those found i n the neonate. Systemic i n j e c t i o n of DFP causes a temporary loss of AchE a c t i v i t y throughout the striatum. Although the detailed c h a r a c t e r i s t i c s of AchE c e l l s are not seen with t h i s technique d i s t r i b u t i o n of the c e l l s can be investigated. Butcher et a l found the AchE c e l l dispersed throughout the nucleus with a pronounced tendency toward c l u s t e r i n g in the immediate v i c i n i t y of the f a s c i c l e s traversing the nucleus. In the SN of the rat AchE staining i s more intense i n the pars compacta than the pars r e t i c u l a t a at a l l stages from neonate to adult. In the neonate AchE staining i s d i f f u s e l y present throughout the compacta. However i n the pars r e t i c u l a t a discrete somata 8-40 u^m in diameter, as well as bundles of f i b r e s , are deeply stained from day 3-15. Later these c e l l s and fibres become less v i s i b l e against a background of AchE staining. However i n the adult rat treated with DFP no AchE c e l l s are observed i n the pars r e t i c u l a t a . Instead, intense staining of the pars compacta neurons i s observed (Butcher and B i l e z i k j i a n , 1975). Since the compacta neurons are believed to be dopaminergic i n function the AchE synthesized by these c e l l s may serve to inactivate Ach released from cholinergic afferents. This finding also serves to point out that neurons rich in AchE need not be cholinergic i n function. 2 2 A c h o l i n e s t e r a s i c s t r i a t o - n i g r a l pathway has been de s c r i b e d by O l i v i e r e t a l (1970). They found AchE s t a i n i n g of f i b r e s f o l l o w i n g the same anatomical r o u t e as the s t r i a t o - n i g r a l pathway demonstrated by s i l v e r impregnation (Voneida, 1960). E l e c t o l y t i c l e s i o n s of the Cd r e s u l t e d i n a l o s s of s t a i n i n g f o r AchE along the pathway although l e s i o n s of the SN were without e f f e c t -O l i v i e r suggested t h a t the s t r i a t o - n i g r a l pathway i s c h o l i n e r g i c s i n c e f i b r e s f o l l o w i n g the a p p r o p r i a t e r o u t e are r i c h i n AchE and both the Cd and the SN c o n t a i n high c o n c e n t r a t i o n s of Ach. However h i s c o n c l u s i o n must be reassessed i n l i g h t o f the r e c e n t demonstration of AchE s t a i n i n g i n dopaminergic neurons of the n i g r o s t r i a t a l path way. The p u t a t i v e t r a n s m i t t e r 5-HT i s < a l s o present i n r e l a t i v e l y high c o n c e n t r a t i o n i n the Cd (Anden e t a l , 1966; Bogdanski e t a l , 1957; Broch and Harsden 1972). The t e r m i n a l s r i c h i n 5-HT found i n f o r e b r a i n r e g i o n s are d e r i v e d e x c l u s i v e l y from c e l l bodies l o c a t e d i n the mesencephalic raphe n u c l e i (MRN) (Anden e t a l , 1966; Ungerstedt, 1971). L e s i o n s of these n u c l e i , p a r t i c u l a r l y the d o r s a l or median, or i n t e r r u p t i o n of the p r o j e c t i n g f i b r e s at the l e v e l of the v e n t r a l tegmentum or medial f o r e b r a i n bundle r e s u l t i n an e x t e n s i v e r e d u c t i o n of s t r i a t a l 5-HT and i t s s y n t h e t i c enzyme, tryptophan hydroxylase (Kostowski et a l , 1968; Kuhar et a l , 1972; P o i r i e r et a l , 1967; P o i r i e r et a l , 1969)- On the other 2 3 hand, an increased release of 5-HT from the striatum follows stimulation of the raphe n u c l e i (Holman and Vogt, 1972). Nauta et a l (1974) studied the d i s t r i b u t i o n of labelled c e l l bodies following the i n j e c t i o n of horse radish peroxidase (HBP) into the Cd. This protein i s taken up by nerve terminals and transported i n a retrograde direction to the c e l l body. He found l a b e l l e d c e l l s mainly in the cortex, thalamus, and the SN. However he also noted some c e l l s in the dorsal raphe nucleus (DBN). These data suggest the presence of a seritonergic pathway from the HEN to the striatum. The SN and GP contain the highest concentration of gamma-aminobutyric acid (GABA) and i t s synthetic enzyme, glutamic acid decarboxylase (GAD) , i n the brain (Lowe et a l , 1958; Baxter and Roberts, 1959; Fahn and Cote, 1968). The Cd also contains appreciable concentrations of both compounds but i n only 30 to 50% the concentrations found in the GP and SN (Enna et a l , 1975). GABA has been c l e a r l y established as an i n h i b i t o r y transmitter in Purkinje c e l l s of the cerebellum, however i t s role i n the basal ganglia i s less well defined. E l e c t r o l y t i c lesions of the GP or hemitransection of the brain at the l e v e l of the subthalamus r e s u l t i n an 80% decrease in GAD concentration in the i p s i l a t e r a l SN (HcGeer et a l , 1974) . No change i n GAD concentration i s seen i n the i p s i l a t e r a l Cd. Kim et a l (1971) have reported that e l e c t r o l y t i c lesions of the Cd cause a 20% drop i n l e v e l s of GABA i n the SN. However more recently McGeer et a l (1974) found that interruption of 24 the s t r i a t o - n i g r a l pathway, b y h e m i t r a n s e c t i o n of the b r a i n at the l e v e l o f the a n t e r i o r commissure, d i d not i n f l u e n c e l e v e l s of GAD i n the SN i n 11 of 14 animals- The remaining 4 animals had a r e d u c t i o n i n GAD of 30%. However these animals were noted to have a d d i t i o n a l damage to the GP. These f i n d i n g s suggest that the p a l l i d o n i g r a l pathway may be GABAnergic i n f u n c t i o n . However, GABA i s u n l i k e l y to f u n c t i o n as the t r a n s m i t t e r i n the s t r i a t o - n i g r a l t r a c t . Substance P, a p o l y p e p t i d e , i s a p u t a t i v e n e u r o t r a n s m i t t e r i n the somato-sensory system a t the s p i n a l l e v e l (Otsuka e t a l , 1975). However the SN c o n t a i n s the highest c o n c e n t r a t i o n of substance P of any r e g i o n of the s p i n a l cord or b r a i n (Kanazawa and J e s s e l 1976; Duffy et a l (1975) demonstrated that much of the substance P i s i n synaptosomes. L e s i o n s o f the s t r i a t o - n i g r a l pathway lea d t o a sharp drop i n l e v e l s of substance P found i n the SN {Kanazawa and J e s s e l 1976). Immunohistochemical s t u d i e s i n d i c a t e that axons c o n t a i n i n g substance P a r e found i n the SN, p e r i a q u e d u c t a l grey matter, amygdala and thalamus. However only the habenular nucleus has been found t o c o n t a i n l a b e l l e d c e l l bodies {Hockfelt e t a l , 1975) . DA i s found i n higher c o n c e n t r a t i o n i n the Cd and SN than any other CNS s t r u c t u r e . I t s s y n t h e t i c enzyme, 1-dopa decarboxylase, i s a l s o present i n high l e v e l s ( B e r t l e r and Bosengren, 1959 a & b; C a r l s s o n , 1959). In some b r a i n r e g i o n s , such as the hypothalamus, DA i s a p r e c u r s o r f o r s y n t h e s i s of n o r a d r e n a l i n e (NA) and accounts f o r only about 10% of the t o t a l catecholamine content of the t i s s u e . However i n the b a s a l g a n g l i a B e r t l e r and Rose.nger (1959 A and B) found DA i n 10 t o 100 times the c o n c e n t r a t i o n of the NA. They suggested that DA has a f u n c t i o n i n the b a s a l g a n g l i a unique from i t s r o l e as a s y n t h e t i c p r e c u r s o r to NA. P a r t i c u l a r i n t e r e s t i n the n i g r o s t r i a t a l p r o j e c t i o n was generated by the d i s c o v e r y that the DA c o n t e n t of the Cd was v a s t l y depleted i n b r a i n s s t u d i e d a t autopsy of p a t i e n t s s u f f e r i n g from i d i o p a t h i c or p o s t e n c e p h a l i t i c Parkinson's d i s e a s e (Ehringer and Hornykiewicz, 1960). I t had been known p r e v i o u s l y that the neurons cf the pars compacta, the pigmented p o r t i o n of the SN, undergoes an almost t o t a l degeneration i n the d i s e a s e ( T r e t i a k o f f , 1919). These f i n d i n g s suggested t h a t the neurons of the pars compacta are r i c h i n DA and t h a t axonal and t e r m i n a l degeneration of these c e l l s r e s u l t s i n a d e p l e t i o n of s t r i a t a l dopamine i n Parkinsonian p a t i e n t s . However i t was f i r s t necessary t o prove that the DA content of the striatum was s p e c i f i c a l l y i n t e r m i n a l s of SN neurons. Laverty et a l (1963) s t u d i e d the DA i n v a r i o u s s u b c e l l u l a r f r a c t i o n s of homogenates of the dog Cd. They found high c o n c e n t r a t i o n s of DA i n the synaptosomal f r a c t i o n and the s o l u b l e supernatant f r a c t i o n . They suggested t h a t DA i n the Cd i s present i n s y n a p t i c t e r m i n a l s i n a f r e e and e a s i l y r e l e a s a b l e form, although 26 the source of these t e r m i n a l s was unknown. P o i r i e r and Sourkes (1965) found t h a t a u n i l a t e r a l l e s i o n of the SN r e s u l t s i n a g r e a t e r than 50% d e p l e t i o n of DA i n the i p s i l a t e r a l Cd. T h i s f i n d i n g demonstrated a d i r e c t i n f l u e n c e of n i g r a l e f f e r e n t s on the DA content of the s t r i a t u m . Dahlstrom and Fuxe X19 64) , using the h i s t o f l u o r e s c e n c e technique of F a l c k et a l (1962), were able to demonstrate c l e a r l y the presence of dopamine i n neurons of the n i g r o s t r i a t a l pathway. Attempts to i d e n t i f y the axons of the pathway by c l a s s i c a l t e c h n i q u e s had been met with f r u s t r a t i o n , although i t was known that l e s i o n s of the Cd caused r e t r o g r a d e degenerative changes i n n i g r a l c e l l s . However l a t e r work by Shimizu and Ohnishi (1973) using Fink-Heimer's m o d i f i c a t i o n o f the Nauta method di d c l e a r l y demonstrate the e x i s t e n c e of t h i s pathway. The c e l l s of o r i g i n of the n i g r o s t r i a t a l pathway l i e i n the pars compacta of the SN. These l a r g e DA c o n t a i n i n g c e l l s have e x t e n s i v e a r b o r i z a t i o n of t h e i r d e n d r i t e s . They penetrate at r i g h t angles through the pars r e t i c u l a t a and r e c e i v e s y n a p t i c c o n t a c t from r e t i c u l a t a c e l l s . The pars r e t i c u l a t a i s populated by s m a l l e r c e l l s with d e n d r i t e s which a r b o r i z e w i t h i n the pars r e t i c u l a t a . T h e i r axons synapse with d e n d r i t e s of compacta neurons and a l s o g i v e r i s e to the n i g r o p a l i i d a l and nigrotegmental pathways. The axons of compacta c e l l s t r a v e l towards the Cd f i r s t i n the medial f o r e b r a i n bundle and then i n the medial aspect of the i n t e r n a l c a p s u l e . They are extremely f i n e unmyelinated axons with a r e l a t i v e l y low dopamine co n t e n t . They enter 27 the ventro-medial aspect of the Cd after passing, at least in part, through the GP. In the Cd the f i b r e s branch extensively and are then d i s t r i b u t e d throughout the nucleus. Each f i b r e has a multitude of v a r i c o s i t i e s , high i n dopamine content, each one making a synaptic contact within the dendritic system of a Cd neuron. In this way the axons from a r e l a t i v e l y small number of SN neurons are able to account for the very high content of dopamine in the striatum. Portig and Vogt (1969) f i r s t investigated the release of dopamine in the striatum following a c t i v a t i o n of the SN. They measured the concentration of various compounds in a solution perfusing the l a t e r a l cerebral v e n t r i c a l . E l e c t r i c a l stimulation of the SN resulted i n only an occasional r i s e i n dopamine concentration i n the solution. However since s t r i a t a l dopamine i s rapidly transformed enzymatically to homovanillic acid (HVft), dopamine released i n the Cd may be converted to HVA before reaching the surface of the nucleus. When HVA levels were measured, SN stimulation f o r 3 or 4 minutes resulted i n an increased concentration in the perfusate l a s t i n g over an hour. Krnjevic'and P h i l l i s (1963) examined the e f f e c t s of DA applied iontophoretically to c o r t i c a l neurons. By f i r s t applying glutamate they induced f i r i n g i n otherwise s i l e n t units. They found that concurrent application of DA depressed the f i r i n g of these c e l l s . In a second experiment, peripheral stimulation was used to evoke a 28 synaptic response. lontophoretic application of DA completely blocked the evoked potentials-Bloom et a l (1965) studied the response of s t r i a t a l neurons to iontophoretically applied DA. They found very large numbers of spontaneously active units i n the Cd of unanaesthetized cats. Often several units were recorded simultaneously from one position of the recording electrode. F i f t y percent of these c e l l s showed a decrease in discharge rate following iontophoretic application of DA. However 141 had an increase in f i r i n g rate. Anaesthesia caused a s i g n i f i c a n t change i n the a c t i v i t y of s t r i a t a l units. After administration of chloralose or barbiturate almost no spontaneous units could by detected. When the influence of iontophoretically applied DA was tested on glutamate induced a c t i v i t y , 60% of the units were depressed and only 2.5% showed a f a c i l i t a t i o n of t h e i r f i r i n g rate. Herz and Zieglgansberger ( 1968) also studied the e f f e c t s of iontophoretically applied DA on s t r i a t a l neurons. They confirmed that DA depresses the spontaneous or glutamate induced a c t i v i t y of the majority of neurons in the Cd. Dopamine also blocked unit responses evoked by thalamic stimulation. However i f a unit was depolarized by a high dose of glutamate, DA often caused the c e l l to return to a high frequency of f i r i n g . The authors suggested that DA may depress f i r i n g of most neurons by causing a hyperpolarization or r e p o l a r i z a t i o n of the 29 membrane. McLennan and York (1967) found that the a c t i v i t y of 60% of s t r i a t a l neurons was depressed and the a c t i v i t y of 9% was f a c i l i t a t e d by iontophoretic application of DA. The effects of DA could be prevented by the previous iontophoretic administration of phenoxybenzamine, an alpha adrenergic blocker, but not by dichloroisopropylnoradrenaline, a beta blocker. Stimulation of the SN was found to evoke e i t h e r a single unit or a burst of action potentials with an average latency of 15 - 30 msec. Both responses were depressed by iontophoretic application of DA. The depression was blocked by previous application of the alpha blocker. The alpha blocker alone did not influence the response. These findings suggested that DA may function as an i n h i b i t o r y transmitter within the Cd. However i f DA functions as a transmitter in the n i g r o s t r i a t a l pathway, stimulation of the SN and iontophoretic application of DA would be expected to influence s t r i a t a l neurons i n a s i m i l a r manner. McLennan suggested that two classes of DA receptor may exist. The neurons excited by iontophoretic application of DA or n i g r a l stimulation would have excitatory receptors and the 60% of neurons responding with a depression of spontaneous a c t i v i t y would have in h i b i t o r y receptors. However none of the neurons responding with an increased discharge rate to application of DA were responsive to n i g r a l stimulation. Furthermore units evoked by n i g r a l stimulation responded to 30 a p p l i c a t i o n of DA with a decreased f i r i n g r a t e . Connor (1970) s t u d i e d the i n f l u e n c e of n i g r a l s t i m u l a t i o n on spontaneous or glutamate induced a c t i v i t y of s t r i a t a l neurons. F o l l o w i n g the a p p l i c a t i o n of a t r a i n of 4 s t i m u l i at 100 p u l s e s per second t o the SN, 50% of s t r i a t a l neurons responded with a decreased frequency of discharge l a s t i n g about 50 msec. Eighty percent of these neurons were a l s o depressed by i o n t o p h o r e t i c a p p l i c a t i o n of DA. The f a l s e t r a n s m i t t e r , alpha-methyldopamine, blocked both the s t i m u l u s and DA induced depression of f i r i n g . On the other hand, f o r 20% of the neurons, n i g r a l s t i m u l a t i o n produced a f a c i l i t a t i o n of f i r i n g frequency l a s t i n g up to '40 msec. I f glutamate a p p l i c a t i o n was stopped n i g r a l s t i m u l a t i o n o f t e n continued to evoke a s i n g l e u n i t at a constant l a t e n c y . The average l a t e n c y f o r d i f f e r e n t u n i t s was 20 msec. Seven out of ten of these neurons were a l s o e x c i t e d by i o n t o p h o r e t i c a p p l i c a t i o n of DA. The i n f l u e n c e of alpha-methyldopamine was not reported. On the b a s i s of these data Connor suggested t h a t the depression of s t r i a t a l u n i t a c t i v i t y produced by s t i m u l a t i o n of the SN i s mediated by a d i r e c t dopaminergic n i g r o s t r i a t a l pathway. However the e x c i t a t o r y e f f e c t s of n i g r a l s t i m u l a t i o n remained unexplained. Ohye et a l (1970) s t u d i e d the i n f l u e n c e of n i g r a l l e s i o n s on the spontaneous a c t i v i t y of the s t r i a t u m . Ohye reasoned that i f the n i g r o s t r i a t a l pathway has a t o n i c i n h i b i t o r y i n f l u e n c e on neuronal a c t i v i t y i n the Cd, 3 1 l e s i o n s of the SN should r e l e a s e the i n h i b i t i o n and r e s u l t i n an i n c r e a s e d f i r i n g frequency of s t r i a t a l u n i t s . F o l l o w i n g c h r o n i c e l e c t r o l y t i c l e s i o n s of the SN the average r a t e of neuronal discharge was g r e a t e r on the i p s i l a t e r a l as compared to the c o n t r a l a t e r a l s i d e . The r e s u l t s from one c o n t r o l animal i n d i c a t e d t h a t the u n i t a c t i v i t y i n the c o n t r a l a t e r a l s i d e remained unchanged. Based on the these f i n d i n g s i t became g e n e r a l l y b e l i e v e d that DA f u n c t i o n e d as an i n h i b i t o r y t r a n s m i t t e r i n the n i g r o s t r i a t a l pathway. In summary, 1) DA and i t s s y n t h e t i c enzyme are present i n the Cd, and DA i s l o c a l i z e d t o t e r m i n a l s ; 2) DA i s r e l e a s e d i n the Cd f o l l o w i n g s t i m u l a t i o n of the SN; 3) n i g r a l s t i m u l a t i o n or i o n t o p h o r e t i c a p p l i c a t i o n of DA r e s u l t s i n a d e p r e s s i o n of spontaneous a c t i v i t y of the m a j o r i t y of s t r i a t a l neurons and alpha-methyldopamine blocks both e f f e c t s and f i n a l l y <4) an e l e c t r o l y t i c l e s i o n of the SN a p p a r e n t l y r e s u l t s i n a r e l e a s e of a t o n i c i n h i b i t i o n of s t r i a t a l u n i t s . However c e r t a i n f i n d i n g s c o n t r a r y to the h y p o t h e s i s remained unexplained. McLennan and York (196 7) r e p o r t e d e x c i t a t i o n of s t r i a t a l u n i t s f o l l o w i n g n i g r a l s t i m u l a t i o n . Although an a d r e n e r g i c b l o c k e r blocked the e f f e c t s of i o n t o p h o r e t i c a p p l i c a t i o n of DA, the e x c i t a t i o n observed f o l l o w i n g n i g r a l s t i m u l a t i o n was not i n f l u e n c e d . Connor a l s o reported the n i g r a l s t i m u l a t i o n evoked s i n g l e u n i t s with a constant l a t e n c y . Alpha-methyldopamine was only o c c a s i o n a l l y e f f e c t i v e i n b l o c k i n g the response. F r i g y e s i and Purpura (196 7) observed e x c i t a t i o n of s i n g l e u n i t s with an average l a t e n c y of 20 msec f o l l o w i n g s t i m u l a t i o n of the SN. The u n i t s f o l l o w e d well up t o a frequency of 40 Hz. They were detected most of t e n i n the c e n t r a l r e g i o n s of the s t r i a t u m . Previous l e s i o n s o f the c e r e b r a l c o r t e x and other s t r u c t u r e s with e f f e r e n t s t o the Cd d i d not i n f l u e n c e the p r o b a b i l i t y o f detec t tig evoked u n i t s . F e l t z and Albe-Fessard (1972) detected 419 c e l l s evoked by n i g r a l s t i m u l a t i o n . Again they were found most o f t e n i n the medial two t h i r d s of the nucleus, had a l a t e n c y of 10 to 25 msec, and had a s t a b l e l a t e n c y f o l l o w i n g s t i m u l a t i o n a p p l i e d at f r e q u e n c i e s up t o 50 Hz. They a l s o r e p o r t e d i n h i b i t i o n of 102 of a 166 spontaneously a c t i v e neurons. I n t r a c e l l u l a r s t u d i e s (Buchwald e t a l , 1973; H u l l e t a l , 1970, 1974; K i t a i et a l , 1975, 1976) have shown that an EPSP or an EPSP-IPSP sequence i s the predominant response recorded in s t r i a t a l neurons f o l l o w i n g s t i m u l a t i o n of the SN. Le s i o n s of dopaminergic neurons by i n j e c t i o n of 6-OHDA i n t o the medial f o r e b r a i n bundle do not i n f l u e n c e the e x c i t a t i o n or i n h i b i t i o n observed f o l l o w i n g n i g r a l s t i m u l a t i o n ( F e l t z and DeChamplain, 1972). F i n a l l y , H u l l e t a l (1973) performed a s e r i e s of el e g a n t and well c o n t r o l l e d experiments to re-examine the t o n i c i n f l u e n c e of t h e n i g r o s t r i a t a l pathway on s t r i a t a l neurons. In the f i r s t experiment e l e c t r o d e s were p o s i t i o n e d i n the Cd on both the l e f t and r i g h t s i d e . S i n g l e u n i t s were recorded simultaneously from both s i d e s t o c o n t r o l f o r changes i n the s t a t e of a r o u s a l of the animal. In seven c o n t r o l animals the mean f i r i n g r a t e was not s i g n i f i c a n t l y 33 d i f f e r e n t on the two s i d e s . F o l l o w i n g c h r o n i c l e s i o n s of the medial f o r e b r a i n bundle or SN the mean f i r i n g r a t e f o r neurons i n the i p s i l a t e r a l Cd was unchanged from t h a t o f c o n t r o l animals. However the mean f o r neurons i n the c o n t r a l a t e r a l Cd was reduced by about 75%. Although the ra t e of f i r i n g was unchanged i n the i p s i l a t e r a l Cd the pa t t e r n of a c t i v i t y was altered, from the u s u a l b u r s t i n g p a t t e r n to a more r e g u l a r f i r i n g p a t t e r n with fewer s h o r t or long i n t e r v a l s . F o l l o w i n g the acute experiments the dopamine content of each s t r i a t u m was determined. A 75 to 90% r e d u c t i o n i n DA was found i n t h e i p s i l a t e r a l s t r i a t u m as compared to the c o n t r a l a t e r a l s i d e or to c o n t r o l animals. L e s i o n s of the tegmentum above the SN a l s o r e s u l t e d i n a decrease i n the mean f i r i n g r a t e of spontaneous u n i t s i n the c o n t r a l a t e r a l Cd with no change on the i p s i l a t e r a l s i d e . However the DA l e v e l s i n the i p s i l a t e r a l Cd were unchanged from c o n t r o l s . In the second experiment t h r e e monkeys had c h r o n i c a l l y implanted l e s i o n i n g e l e c t r o d e s placed i n the region j u s t d o r s a l to the SN on one s i d e , and a s i n g l e u n i t r e c o r d i n g device was implanted over both caudate n u c l e i . Before l e s i o n i n g the animals, simultaneous c o n t r o l r e c o r d s of a number of s t r i a t a l u n i t s were obtained over a pe r i o d of s e v e r a l months. E l e c t r o l y t i c l e s i o n s were then placed i n the v e n t r a l tegmentum on one s i d e by using the alrea d y implanted l e s i o n i n g e l e c t r o d e s . A f t e r w a i t i n g two weeks f o r rec o v e r y , data from another sample of spontaneous u n i t s were obt a i n e d . The r e s u l t s were s i m i l a r 3 4 to the f i r s t experiment. Again there was no change i n the mean f i r i n g r a t e on the i p s i l a t e r a l s i d e compared to c o n t r o l values and there was a s i g n i f i c a n t i n c r e a s e i n the mean f i r i n g r a t e on the i n t a c t s i d e . There was no s i g n i f i c a n t d i f f e r e n c e i n DA con t e n t of the two caudate n u c l e i . These f i n d i n g s i n d i c a t e t h a t l e s i o n s of the n i g r o s t r i a t a l pathway do not r e l e a s e s t r i a t a l neurons from a t o n i c i n h i b i t o r y i n f l u e n c e as suggested by Ohye. In f a c t l e s i o n s i n and near the pathway have a major e f f e c t on the c o n t r a l a t e r a l s t r i a t u m . Furthermore these changes are independent of the DA content o f the Cd. C l e a r l y , the hy p o t h e s i s t h a t n i g r a l s t i m u l a t i o n e x e r t s an i n h i b i t o r y i n f l u e n c e on the s t r i a t u m by r e l e a s i n g DA as a s y n a p t i c t r a n s m i t t e r must be r e -evaluated. F r i g y e s i and Purpura (1967) p o s t u l a t e d two d i s t i n c t n i g r o s t r i a t a l pathways s u b s e r v i n g the i n h i b i t o r y and e x c i t a t o r y a c t i o n s on s t r i a t a l neurons. F e l t z and Albe-Fessard (1972) suggested t h a t there i s a s i n g l e e x c i t a t o r y input impinging on s t r i a t a l t a r g e t c e l l s and that the i n h i b i t o r y i n f l u e n c e i s the r e s u l t of an i n t r i n s i c mechanism w i t h i n the s t r i a t u m . However, these suggestions have remained s p e c u l a t i v e i n view of the lack of 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 va r i o u s p o p u l a t i o n s of neurons w i t h i n the s t r i a t u m r e s p o n s i v e t o n i g r a l s t i m u l a t i o n . The present experiments were designed t o c h a r a c t e r i z e the s y n a p t i c i n f l u e n c e of the n i g r o s t r i a t a l pathway. The 35 e x t r a c e l l u l a r responce of neurons in the striatum were recorded in urethane anaesthetized r a t s . P i r s t , stimulation of the SN, GP, IC, or DRN was used to determine the e l e c t r o p h y s i o l o g i c a l properties of s t r i a t a l neurons and to i d e n t i f y subpopulations of neurons within the nucleus. Second, pharmacological agents were systemically or iontophoretically administered to determine their influence on s t r i a t a l neurons already e l e c t r o p h y s i o l o g i c a l l y i d e n t i f i e d . Third, chemical or e l e c t r o l y t i c lesions were placed in the n i g r o s t r i a t a l and associated pathways to examine the dependence of the electrophysiological properties on known neuronal systems. The results indicate that a) dopamine functions as an excitatory transmitter in the n i g r o s t r i a t a l pathway, b)that the i n h i b i t i o n observed in the striatum following n i g r a l stimulation i s the r e s u l t of an i n h i b i t o r y c o l l a t e r a l system dependent on interneurons within the Cd, (Richardson et a l , 1977) and c) that a neuronal pathway exis t s between the dorsal raphe nucleus and the striatum. Stimulation of t h i s pathway produces a potent i n h i b i t i o n of s t r i a t a l neurons ( H i l l e r et a l , 1975). 3 6 METHODS Surgical Preparation Acute recording experiments were performed on a t o t a l of 8 3 male Wistar rats weighing between 250 and 400 g. A l l surgical preparation and subsequent experimentation were performed under urethane anaesthesia. Urethane was given I.P. in a dose of 1.5 g/Kg and a s a t i s f a c t o r y l e v e l of anaesthesia was maintained by supplemental I. P. i n j e c t i o n s during the experiment. Body temperature was monitored by a r e c t a l thermistor probe and maintained between 36 and 37 degrees centigrade by a thermostatically controlled heating pad. The animals were placed in a Kopf stereotaxic frame with the i n c i s o r bar at 4.0 to 5.0 mm below zero thereby positioning the s k u l l in a horizontal plane. A rectangular region of calvarium was removed to expose an area of cortex roughly corresponding to boundaries 2.5 mm anterior and 4 mm posterior to bregma and 4 mm on either side of the saggital suture. The exposed cortex was covered with warm saline throughout the experiment. 37 S t i m u l a t i o n Procedure C o n c e n t r i c b i p o l a r metal e l e c t r o d e s were used > f o r e l e c t r i c a l s t i m u l a t i o n . These had t i p s e p a r a t i o n s of 0.3 to 0.5 mm and a DC r e s i s t a n c e o f 75 t o 100 K ohms i n normal s a l i n e . Using c o o r d i n a t e s from the a t l a s of Konig and K l i p p e l (1963) the e l e c t r o d e s were p o s i t i o n e d i n one or more of the f o l l o w i n g r e g i o n s : SN, GP, TC, i n t r a l a m i n a r and p a r a f a s c i c u l a r n u c l e i of the thalamus, IPT or n i g r o s t r i a t a l bundle. S i n g l e sguare wave pulses of 0.1 msec d u r a t i o n and 5 to 20 v o l t s (v) i n t e n s i t y were d e l i v e r e d through a Grass i s o l a t i o n u n i t . An Ortec c r y s t a l c l o c k c o n t r o l l e d the r a t e and t i m i n g of s t i m u l a t i o n . i l i c r o e l e c t r o d e P e p a r a t i o n E x t r a c e l l u l a r u n i t a c t i v i t y was recorded using e i t h e r s i n g l e m i c r o p i p e t t e s prepared from Corning c a p i l l a r y t u b i n g , or m u l t i p i p e t t e assemblies prepared from custom made 7 b a r r e l e l e c t r o d e blanks. The p i p e t t e s were heated and drawn to f i n e t i p s i n a v e r t i c a l m i c r o e l e c t r o d e p u l l e r - The t i p s were then broken under l i c r o s c o p i c o b s e r v a t i o n t o diameters of 1-2 g^m f o r s i n g l e p i p e t t e s and 4-8 um f o r m u l t i p i p e t t e s . The s i n g l e p i p e t t e s and c e n t r a l r e c o r d i n g b a r r e l of p i p e t t e assemblies were f i l l e d with e i t h e r 4 M NaCl or Pontamine sky blue i n 4 M sodium ac e t a t e . The remaining 38 b a r r e l s of the p i p e t t e assemblies were f i l l e d with the f o l l o w i n g s o l u t i o n s : sodium 1-glutamate <1 M, pH 4.0, Regis Chemical), dopamine h y d r o c h l o r i d e (1 fl, pH 4.0, Regis Chemical) and alpha-.flupenthi.xol (0.5 M, pH 4.0, fl. Limbeck and Co.). The drugs were e j e c t e d i o n t o p h o r e t i c a l l y using a p p r o p r i a t e a n i o n i c and c a t i o n i c c u r r e n t s . H a l o p e r i d o l (0.5 to 2.5 mg/kg, McNeil L a b o r a t o r i e s ) and a l p h a - f l u p e n t h i x o l (0.5 to 2.5 mg/kg) were a l s o given i n t r a v e n o u s l y . Recordi ng Procedures And Data A n a l y s i s M i c r o p i p e t t e s were p o s i t i o n e d a c c o r d i n g to s t e r e o t a x i c c o o r d i n a t e s from the a t l a s of Konig and K l i p p e l (1963). An AB Transvertex Microstep u n i t was used to lower the e l e c t r o d e i n steps of 1 u^ m. A l l recorded p o t e n t i a l s were f i r s t passed through a custom made v o l t a g e f o l l o w e r f o r impedence matching. The s i g n a l s were then l e d through a bandpass f i l t e r (1 t o 10 K Hz), a m p l i f i e d by a T e k t r o n i x 3A9 d i f f e r e n t i a l a m p l i f e r and d i s p l a y e d on a Te k t r o n i x 564 st o r a g e o s c i l l o s c o p e or a RM 565 dual beam o s c i l l o s c o p e . A P o l a r o i d camera was used to photograph the sweeps. The a m p l i f i e d s i g n a l was a l s o passed through a volta g e d i s c r i m i n a t o r . The d i s c r i m i n a t o r produced an output pulse i f an a c t i o n p o t e n t i a l occurred with an amplitude above a manually set t h r e s h o l d . The pulses were i n t e g r a t e d and d i s p l a y e d on a paper c h a r t t o gi v e a continuous record of f i r i n g frequency. The pulses also f i r e d a Schmidt trigger of a PDP-3L computer for real time generation and display of post stimulus time histograms (PST). Permanent records were obtained from an analogue X Y plotter. The PST was used to assess a change in neuronal f i r i n g following stimulation of a p a r t i c u l a r brain region. The X, or latency axis, of the histogram was divided into a predetermined number of equal time periods referred to as bins. The latency of each discharge was measured re l a t i v e to a timing pulse from the c r y s t a l clock, and the appropriate bin was incremented. Data were collected during 25 to 100 cycles of the clock. The r e l a t i v e probability of discharge at a p a r t i c u l a r latency was represented on the Y axis by the height of the histogram bar for that bin. The clock also i n i t i a t e d a stimulus presentation at a constant latency from the timing pulse. Therefor the p r o f i l e of the histogram represented the influence of the stimulus on neuronal discharge. A peak indicated an increased, and a trough a decreased probability of discharge, whereas a f l a t p r o f i l e indicated a lack of influence by the stimulus (Gerstein and Kiany, 1960). 40 Histology Sites of stimulation were marked by passing 10 to 15 ma of DC anodal current for 10 seconds through the center core of the stimulating electrode. Perfusion of the brain with potassium ferrocyanide allowed subsequent i d e n t i f i c a t i o n of a Prussian blue spot in h i s t o l o g i c a l sections. Recording s i t e s were marked by leaving the t i p in place during f i x a t i o n so that the electrode t r a c t could be visualized h i s t o l o g i c a l l y . In some preparations the recording electrode contained Pontamine sky blue for precise i d e n t i f i c a t i o n of the recording s i t e . After dye was ejected e l e c t r o p h o r e t i c a l l y a blue spot could be located on sections. Following each experiment the animal was perfused i n t r a c a r d i a l l y with 200 ml of 0.9 % sodium chloride followed by 100 ml of a mixture of potassium ferrocyanide and 10% buffered formalin. Frozen sections were then cut at int e r v a l s of 50 u^ m. These sections were mounted on glass s l i d e s , dehydrated and stained with c r e s y l v i o l e t or saf f r a n i n . Electrode s i t e s could then be located under a microscope. Retrograde transport of horseradish peroxidase (HEP, type 4 Sigma Chemicals) was examined i n 11 preparations. The animals were anaesthetized with pentobarbital (50 mg/kg, I.P.). In 6 animals 0.1 ul of a 10% solution of HRP in saline was injected u n i l a t e r a l l y into the SN by a ste r e o t a x i c a l l y guided microsyringe. Similar i n j e c t i o n s 41 were made u n i l a t e r a l l y or b i l a t e r a l l y i n t o the Cd of 5 animals. Following a sur v i v a l period of 24 hours the rats were k i l l e d and perfused at room temperature with a solution of .335 paraformaldehyde and 2% glutaraldehyde in 0.05 M phosphate buffer (pH 7-5,). The brains were removed and allowed to stand for 24 hours i n phosphate buffer containing 5% sucrose. Frozen sections were then cut at 50 um i n t e r v a l s , treated to reveal peroxidase a c t i v i t y according to the method of Graham and Karnovsky (1966) and examined microscopically under dark f i e l d i l l u m i n a t i o n . Lesioning And Assay Procedures Lesions interrupting afferent pathways to the striatum were car r i e d out in preliminary operations 3 weeks to 2 months prior to acute electrophysiological experiments. E l e c t r o l y t i c lesions were performed under pentobarbital anaesthesia (50 mg/kg, I.P.) by passing up to 2 ma of DC current for 15 to 30 seconds through ste r e o t a x i c a l l y placed metal electrodes. Lesions were made in the intralaminar and parafascicular nuclei of the thalamus of 3 animals, i n the SN of 5 animals, and i n the ventral tegmentum at the l e v e l of the SN of 3 animals. In another 4 animals aspiration of the cerebral cortex dorsal and r o s t r a l to the striatum was carried out. In six animals s e l e c t i v e depletion of s t r i a t a l DA was achieved by using 6-hydroxydopamine hydrobromide (6-OHDA, Regis Chemical). Animals were pretreated with 42 desmethylimipramine (25 mg/kg I.P.) 1 hour prior to uni l a t e r a l i n j e c t i o n of 6-OHDA (12 ug dissolved i n 4 u l of 0.15 H NaCl containing 1 mg/ml of ascorbic acid) i n t o the n i g r o s t r i a t a l bundle at the l e v e l of the hypothalamus. The effectiveness of the chemical lesions was confirmed by measuring the dopamine content of the i p s i l a t e r a l striatum compared with the contra l a t e r a l control according to the method of NcGeer and McGeer (1962). Orthograde axonal transport of t r i t i a t e d leucine from the dorsal raphe nucleus to the striatum was examined i n 5 ra t s . U n i l a t e r a l e l e c t r o l y t i c lesions (2 ma for 30 seconds) were f i r s t made in the medial forebrain bundle at the l e v e l of the hypothalamus. Twenty four hours l a t e r 0.6 ul of a solution containing 5.76 Ci/mmole of t r i t i a t e d leucine ( s p e c i f i c a c t i v i t y 50.0 Ci/mmole) was injected by microsyringe into the dorsal raphe nucleus. Animals were s a c r i f i c e d 24 hours l a t e r and the brains were removed and dissected r a p i d l y into samples from both Cd and overlying c o r t i c e s . Each sample was weighed and the content of t r i t i a t e d protein determined by the method of Fibiger et a l (1972). '4 3 RESULTS Burst Response A burst of excitation was the most frequent response recorded in the Cd following e l e c t i c a l stimulation of the i p s i l a t e r a l SN. The burst response was usually in the form of 2 to 8 " r i p p l e s " of the recording baseline with superimposed low amplitude spikes (50 to 300 uv), although occassionally spikes were not present. The frequency of spikes within the burst was from 200 to 900 Hz. The response began at a latency of 3 to 10 msec and had a duration of 3 to 7 msec. In figure 1, 4 r i p p l e s with superimposed spikes are seen in the response to n i g r a l stimulation at i n t e n s i t y of 20 v o l t s . Threshold stimulation evoked a response consisting of a few low amplitude spikes. As the stimulus i n t e n s i t y was increased the amplitude of the i n d i v i d u a l components of the burst became larger, and r i p p l e s or spikes occurred with longer, as well as shorter, l a t e n c i e s . At low frequencies of stimulation (1 Hz) the burst response had a very consistent latency and configuration. However, with increased rates of SN stimulation (above 10 Hz) the amplitude of the bursts decreased and the configuration became variable. They f a i l e d to occur at stimulus frequencies above 40 Hz (figure 1 and 2). The response occurred throughout a l l regions of the Cd explored. Several regions responding with a burst of 4'4 A-FIGURE 2i fln example of the influence of stimulus i n t e n s i t y of n i g r a l stimulation on the burst response recorded i n the Cd (7, 10, 15, 20 v o l t s ) . Note that a stimulus intensity of 7 V evokes a single spike. This response becomes incorporated into the burst as the stimulus i n t e n s i t y i s increased. Note: these and each subsequent photograph show 5 superimposed oscilloscope sweeps unless otherwise stated. The arrow r e f e r s to the stimulus a r t i f a c t . 44 B 7 V 10 V 15 V 20 v ^JOI mV 2 msec 45 A FIGURE 2. Cha r a c t e r i s t i c s of di f f e r e n t frequencies of 20,40 Hz). The response 40 Hz. the burst response at n i g r a l stimulation (1, f a i l e d at a frequency of 45 B I HZ 20 HZ 40 Hz Recovery (30 sec) t ijoimv 2 msec 46 e x c i t a t i o n would be encountered on one e l e c t r o d e t r a c t , each with a d i f f e r e n t l a t e n c y and d u r a t i o n . Within a r e g i o n responding to n i g r a l s t i m u l a t i o n the response was c o n t i n u o u s l y present during l a r g e v e r t i c a l rfoveraents of the e l e c t r o d e ( f i g u r e 3). Each a c t i v e r e g i o n spanned about 200 to 300 urn separated by a 200 t o 400 urn r e g i o n of " s i l e n c e " . Small movements of the e l e c t r o d e w i t h i n an a c t i v e r e g i o n r e s u l t e d i n q u a l i t a t i v e changes i n the recorded response ( f i g u r e 4). R e l a t i v e amplitude of s p i k e s i n the response would change duri n g the movement and s p i k e s sometimes appeared or disappeared. When the p o s i t i o n of an e l e c t r o d e r e c o r d i n g a burst, was marked with Pontaraine sky blue, i t was always l o c a t e d w i t h i n the neuronal t i s s u e between the f a s c i c l e s of c o r t i c o - s p i n a l f i b e r s t r a v e r s i n g the s t r i a t u m . Conversly the b u r s t responses were not recorded when the e l e c t r o d e t i p was l o c a t e d within r e g i o n s of high f i b e r d e n s i t y . Bursts of e x c i t a t i o n could be recorded i n the Cd only when the s t i m u l a t i o n s i t e was a c c u r a t e l y l o c a t e d w i t h i n the SN. Placement of the s t i m u l a t i n g e l e c t r o d e d o r s a l or caudal to the SN a b o l i s h e d the response ( f i g u r e 5). S i m i l a r burst responses were recorded i n the Cd f o l l o w i n g s t i m u l a t i o n of the GP, IC, or IPT. Although the responses were of a s l i g h t l y s h o r t e r l a t e n c y t h e i r c h a r a c t e r i s t i c s were i d e n t i c a l to those seen f o l l o w i n g SN s t i m u l a t i o n . Often a r e c o r d i n g l o c a t i o n i n the Cd was r e s p o n s i v e to more than one s t i m u l a t i o n s i t e . For example 47 A FIGURE 3_. V a r i a b i l i t y in the burst response evoked by n i g r a l stimulation. The electrode was moved along a v e r t i c a l t r a c t in the striatum extending from 4.50 to 4.82 mm below the surface of the cortex. The photograph on the right i s of a coronal section through the striatum. The arrows point to Pontamine sky blue marks l e f t by the recording electrode. The l e f t spot is i n the middle of a region responding with a burst. The two spots on the r i g h t mark the begining and end of a region of the electrode t r a c t continuously responsive to n i g r a l stimulation. PIGOB'E 4. Changes in the burst response recorded i n the striatum following n i g r a l stimulation as a r e s u l t of small changes in the p o s i t i o n of the recording electrode (4.00, 4.04, 4.06 mm). Note the change in sharpness and amplitude of the peaks. 48 B V = 4.00 1 1 | t V=4.04 Is • 'Si J 0. ! . ( i i t V = 4.06 M 1 mi 1 ! llii ^JO.I mV 2 msec. 49 A F IGURE 5^ A b u r s t r e s p o n s e i s r e c o r d e d i n t h e Cd o n l y i f t h e s t i m u l a t i n g e l e c t r o d e i s a c c u r a t e l y p l a c e d i n t h e SN ( t o p ) . S t i m u l a t i o n a t a s i t e p o s t e r i o r o r d o r s a l t o t h e SN f a i l s t o e v o k e a r e s p o n s e ( bo t t om) . 33 S.N. Poster ior to S.N. 0.2 mV 50 e i t h e r SN or thalamic s t i m u l a t i o n was f o l l o w e d by a burst response i n c e r t a i n r e g i o n s of the Cd. Most s t r i k i n g , however, was c l o s e c o r r e l a t i o n between r e g i o n s responding to s t i m u l a t i o n of the IC and SN. Once the IC e l e c t r o d e was p o s i t i o n e d t o produce a b u r s t i n a r e g i o n r e s p o n s i v e to SN s t i m u l a t i o n , a l l other r e g i o n s of the Cd e x p l o r e d responded i n a s i m i l a r f a s h i o n to s t i m u l a t i o n of e i t h e r s i t e { f i g u r e 6) . siaais Hulls S i n g l e u n i t a c t i v i t y has been recorded from more than 300 neurons i n the Cd. Some were spontaneously a c t i v e with a mean freguency of 5.2 spikes/sec and a range of 1 to 20 s p i k e s / s e c . However the m a j o r i t y only f i r e d when glutamate was e j e c t e d i o n t o p h o r e t i c a l l y from one b a r r e l of an e l e c t r o d e assembly. Spontaneous or glutamate induced a c t i v i t y of 65% of these was depressed by i o n t o p h o r e t i c a l l y a p p l i e d D a { f i g u r e 7). The remaining c e l l s were non-responsive o r , i n a m i n o r i t y of cases, responded with an i n c r e a s e d f i r i n g r a t e . The s i n g l e u n i t s c o u l d be d i f f e r e n t i a t e d i n t o two groups based on s p i k e amplitude and t h e i r response to s t i m u l a t i o n of the SN. The m a j o r i t y of u n i t s had low amplitudes r a n g i n g from 150-300 uv. They were detected with equal p r o b a b i l i t y i n a l l r e g i o n s of the Cd explored. A spontaneous u n i t and a b u r s t response f o l l o w i n g SN s t i m u l a t i o n were o f t e n recorded simultaneously from the same e l e c t r o d e s i t e . The 51 A FIGURE 6._ Effect of an e l e c t r o l y t i c lesion of the IC on the burst response evoked by n i g r a l stimulation. Note the s i m i l a r i t y of the response to stimulation of the SN (top l e f t ) , IC (middle), and GP (bottom) . a l l responses were recorded from the same animal without adjusting the position of the recording electrode. An acute lesion was performed by passing current through the already positioned capsular electrode. Following the les i o n , n i g r a l stimulation (top right) no longer evoked a response. 51 B f S N ( C o n t r o l ) f S N ( I C L e s i o n ) IC GP ^ J O . I m V 2 m s e c 52 A F I G D B E 7 . A) the response of spontaneously discharging s t r i a t a l neurons to single pulse stimulation of the SN. The photographs show spontaneous units responding with a period of i n h i b i t i o n . In both examples a burst occurred within the f i r s t 10 msec of the response (masked by the stimulus a r t i f a c t ) . The PST below each photograph shows the summation of the responses to 50 s t i m u l i . B) rate records of the same neurons in d i c a t i n g the inhibi t o r y action of iontophoreticaly applied DA (DA 60 and 50 nA). The periods of application are shown by the s o l i d horizontal bars. Note: these and each subsequent PST show the summation of 50 responses. The stimulus a r t i f a c t s are indicated by the f i r s t large deflection of the histogram. The binwidth is 10msec i n each case. r 52 B 5 3 spontaneous unit, i n some cases, had an amplitude similar to one of the spikes within the burst. If the v e r t i c a l position of the recording electrode was moved the spontaneous unit and the spike within the burst underwent the same a l t e r a t i o n i n amplitude. Following a burst response the spontaneous a c t i v i t y was completely inhibited for 60 to 300 msec (mean 175 msec) (figure 8). A s l i g h t rebound of excitation l a s t i n g 50 to 150 msec frequently followed the period of i n h i b i t i o n . The minimum stimulation in t e n s i t y required to produce i n h i b i t i o n of spontaneous a c t i v i t y was, i n most cases, equal to or greater than the intensity required to produce a burst response. Small amounts of glutamate ejected iontophoretically often prolonged and i n t e n s i f i e d the burst response. However, higher ejection currents of glutamate caused a decomposition of the response and a coincidental loss of the i n h i b i t o r y e f f e c t normally observed following n i g r a l stimulation (figure 9). Low amplitude units responded i n a sim i l a r manner following stimulation of the IPT or GP. The unit often appeared within the burst response and i t s spontaneous a c t i v i t y was i n h i b i t e d for up to 300 msec. The second group of single units had action potentials with amplitudes greater than 300 uV. Spontaneous or glutamate induced large amplitude units were also encountered most often in regions responding with a burst of excitation following nigral stimulation and were always strongly i n h i b i t e d for 60 to 300 msec 54 A FIGURE 8_. The top photograph i s of a burst response recorded i n the Cd following n i g r a l stimulation. The PST shows the inh i b i t o r y influence of n i g r a l stimulation on the spontaneous a c t i v i t y of a neuron i n the same region. S p i k e s o o I I 55 A F IG USE 9 T h e i n f l u e n c e o f g l u t a m a t e on t h e r e s p o n s e e v o k e d i n t h e s t r i a t u m by n i g r a l s t i m u l a t i o n . A) t h e b u r s t r e s p o n s e h a s b e e n p r o l o n g e d by a p p l i c a t i o n o f 10 nA o f g l u t a m a t e . The PST shows i n h i b i t i o n o f a s p o n t a n e o u s n e u r o n f o l l o w i n g e a c h s t i m u l u s . B) g l u t a m a t e a p p l i e d a t 75 nA a t t e n u a t e d t h e b u r s t r e s p o n s e . T h e r e was a c o i n c i d e n t a l l o s s o f i n h i b i t i o n o f t h e s p o n t a n e o u s n e u r o n a s d e m o n s t r a t e d by t h e PST . 50-CO Glu (25 nA) JfO.lmV 2 msec. 56 following the burst response. However, unlike low amplitude units, they were more l i k e l y to be detected i n the central core of the Cd and were only r a r e l y detected i n the l a t e r a l region of the nucleus. These units often responded with single action potential during the burst. The high amplitude of the unit discharge made them c l e a r l y distinguishable from the low amplitude burst response (figure 10). The latency of an evoked discharge was in the range of 4 to 18 msec (mean 10.8 msec). At a stimulus frequency of 1 - 5 Hz the latency was constant although at higher frequencies there was considerable v a r i a b l i l i t y . Units were unable to follow at stimulus frequencies above 50 Hz. High amplitude units could often be evoked with stimulus i n t e n s i t i e s below threshold f o r the burst response. If the stimulus i n t e n s i t y was then increased the burst appeared and the unit discharge would occur during the time course of the burst. However i f the stimulus was increased to an i n t e n s i t y s u f f i c i e n t to evoke a maximal burst response the high amplitude unit often f a i l e d to discharge (figure 10). In some instances a spontaneous or glutamate induced high amplitude unit was encountered in a region that did not respond with a burst following n i g r a l stimulation. These units were never activated by n i g r a l stimulation although t h e i r spontaneous a c t i v i t y was i n h i b i t e d . Thalamic or GP stimulation also i n h i b i t e d the high 57 A FIGDHE _1Q-. Activation of a large amplitude neuron i n the striatum following stimulation of the SN. A) ef f e c t s of increasing the stimulus i n t e n s i t y from 5 to 20 v o l t s . Note that the large amplitude spike i s blocked during the burst response at the highest i n t e n s i t y of n i g r a l stimulation. Activation of the large spike following n i g r a l stimulation at 1 and 10 Hz. The response f a i l e d to follow at stimulus frequencies above 50 Hz. C) the same c e l l evoked antidromically by p a l l i d a l stimulation. The neuron could follow a stimulus frequency of 100 Hz-57 B 58 amplitude u n i t s f o r up to 300 msec but d i d not evoke a u n i t d i s c h a r g e . S t i m u l a t i o n of the tegmentum d o r s a l t o the SN caused a potent i n h i b i t i o n o f both high and low amplitude u n i t d i s c h a r g e i n the Cd. However an i n i t i a l evoked d i s c h a r g e or b u r s t of e x c i t a t i o n was never observed. S i n c e ascending f i b e r s from the raphe n u c l e i are known to course through t h i s r e g i o n the e f f e c t of raphe s t i m u l a t i o n was determined. In 15 r a t s s t i m u l a t i n g e l e c t r o d e s were p o s i t i o n e d i n the d o r s a l raphe nucleus (DRN)- S t i m u l a t i o n of t h i s r e g i o n never produced e x c i t a t i o n i n the Cd. However raphe s t i m u l a t i o n d i d r e s u l t i n a potent i n h i b i t i o n of 33 of 45 glutamate induced or spontaneously a c t i v e Cd u n i t s { f i g u r e 11). The i n h i b i t i o n l a s t e d 50 -380 msec and was o f t e n f o l l o w e d by a 50 - 100 msec p e r i o d of rebound e x c i t a t i o n . S t i m u l a t i o n of the median raphe or a t s i t e s d o r s a l or v e n t r a l to the DBN d i d not i n f l u e n c e Cd u n i t s . 59 A FIGURE Influence of raphe stimulation on spontaneous a c t i v i t y of s t r i a t a l c e l l s . A) t h i s c e l l was inhibited for 160 msec. Note that no early activation occurred. B) the absence of i n h i b i t i o n following raphe stimulation in an animal with an e l e c t r o l y t i c lesion of the ventral tegmentum. The lesion was performed 4 weeks previous to the acute experiment. S p i k e s o o o o 3 60 Antidromic Potentials Antidromic activation of s t r i a t a l units by n i g r a l stimulation was only rarely encountered (12 c e l l s ) during the course of these experiments. A response was considered antidromic i f i t had a r e l a t i v e l y short latency of f i r i n g (1.9 -4.5 msec), a constant latency with threshold s t i m u l i and the a b i l i t y to follow a stimulus frequency of 100 Hz. In several neurons responding with these c h a r a c t e r i s t i c s , IS-SD breaks i n the action potentials were observed and stimulation at frequencies of 150 to 200 Hz resulted in f a i l u r e of the SD component. None of these antidromically activated units were spontaneously active. H i s t o l o g i c a l v e r i f i c a t i o n of the recording placements indicated that neurons evoked antidromically following n i g r a l stimulation were r e s t r i c t e d to the ventral aspect and peripheral s h e l l of the Cd (figure 12). Stimuli applied to the GP were also observed to evoke large amplitude antidromic spikes i n the Cd with latencies of 1-1 to 2.6 msec. The responses occurred with a constant latency following threshold s t i m u l i and they were able to follow frequencies of 100 to 200 Hz (figure 10). Of 52 c e l l s with these c h a r a c t e r i s t i c s 27 were also orthodromically activated by n i g r a l stimulation. These c e l l s were the high amplitude units described i n the section e n t i t l e d "single units". Their refractory period was estimated as 1.0 to 2.0 msec since double pulse stimulation with an interstimulus i n t e r v a l of less than 6 1 A F I G U 1 E 12^ A) di a g r a m a t i c r e p r e s e n t a t i o n of the d i s t r i b u t i o n of s t r i a t a l c e l l s l a b e l l e d with HBP. Each dot marks the p o s i t i o n of one c e l l . Hatched area i n d i c a t e s the r e g i o n where l a r g e amplitude neurons were synapt.ical.ly e x c i t e d by n i g r a l s t i m u l a t i o n . AC, a n t e r i o r commissure; CC, corpus callosum; S, septum. B) an t i d r o m i c s p i k e s evoked by n i g r a l s t i m u l a t i o n at a frequency of 100 Hz. These responses were recorded from p o s i t i o n s i n the v e n t r a l and " p e r i p h e r a l s h e l l " o f the nucleus corresponding to the dotted r e g i o n shown i n (A). Large d e f l e c t i o n i s the s t i m u l u s a r t i f a c t . C) photomicrograph of neurons l a b e l l e d with HBP i n the s t r i a t u m corresponding to area o u t l i n e d i n (A) . D) the i n j e c t i o n s i t e i n the SN. 62 thi s period f a i l e d to i l l i c i t a double response- I f the n i g r a l orthodromic ac t i v a t i o n preceeded GP stimulation by less than 5 msec the GP response was blocked. C o l l i s i o n extinction with a c r i t i c a l latent period of 5 msec i s reasonable for the neurons since i t i s approximately equal to the conduction time for the antidromic response (1.1 to 2-6 msec) plus the refractory period (1.0 to 2.0 msec). Hi s t o l o g i c a l v e r i f i c a t i o n of the location of 15 neurons responsive to stimulation of either the GP or SN indicated that they were r e s t r i c t e d to the centro-medial "core" of the striatum. Lesioned Preparations Large e l e c t r o l y t i c lesions were placed in the IPT of 3 animals and the DRN of another 3 . In 4 animals the cerebral cortex was aspirated from the dorsal and r o s t r a l aspects of the Cd. Three weeks to two months l a t e r acnte electrophysiological experiments were performed. These lesions did not influence the properties of the burst response, the high or low amplitude evoked unit discharge, or the potent i n h i b i t i o n of spontaneous a c t i v i t y observed in the Cd following stimulation of the SN (figure 13). However si m i l a r e l e c t r o l y t i c lesions of the IC i n 5 animals completely blocked the e f f e c t s of n i g r a l stimulation. In two experiments, after the SN and IC electrodes were c a r e f u l l y positioned to produce a s i m i l a r response at the same recording s i t e i n the Cd, a discrete 63 A FIGURE 13. Persistence of the burst response following lesions of the IPT (left) or suction of the cerebral cortex overlying the Cd (right). In both examples the response was recorded i n the Cd following stimulation of the i p s i l a t e r a l SN- The lesions were performed one month prior to the acute experiments. The PST demonstrates the persistence of the i n h i b i t o r y influence of n i g r a l stimulation on single unit a c t i v i t y a f t e r lesioning the IPT. A s i m i l a r i n h i b i t i o n was observed in animals with previous ablation of the cortex. 63 B 64 e l e c t r o l y t i c lesion was placed in the IC using the already positioned stimulating electrode. Following the l e s i o n , SN stimulation no longer e l i c i t e d a burst response even after allowing one and one-half hours for recovery of tissue surrounding the l e s i o n . Furthermore no responsive regions were detected during a subsequent careful search of the Cd with the recording electrode (figure 6) . In three animals lesions of the ventro-medial tegmentum were made at the l e v e l of the SN and acute experiments were performed 3 - 4 weeks l a t e r . Stimulation of the DSN i n these animals did not influence Cd a c t i v i t y (figure 11). A group of 6 animals had large e l e c t r o l y t i c lesions placed in the region of the SN. Four weeks la t e r acute experiments were performed. Stimulation of the IC of these animals produced a burst response in the i p s i l a t e r a l Cd (figure 14). Each tr a c t of the recording electrode passed through several regions responding with a burst of excitation continuously present for 100 to 300 um. Spontaneous or glutamate induced a c t i v i t y of both high and low amplitude units was i n h i b i t e d for up to 300 msec. The cha r a c t e r i s t i c s of both the burst response and the i n h i b i t i o n were i d e n t i c a l to those observed in inta c t animals following stimulation of the IC or SN. However, i n lesioned animals, high amplitude units were never evoked during the burst response. After each experiment the lesions were examined h i s t o l o g i c a l l y and the dopamine 6 5 A FIGURE 14.. Comparison of the burst response and i n h i b i t o r y a c t i v i t y observed a f t e r s t i m u l a t i o n of the SN (top) and IC (middle) - Both s e t s of data were recorded from the same animal without a d j u s t i n g the p o s i t i o n of the r e c o r d i n g e l e c t r o d e . On the r i g h t i s a diagramatic c r o s s " s e c t i o n of a r a t b r a i n at the l e v e l o f the hypothalamus. The dots i n d i c a t e the r e g i o n where c a p s u l a r s t i m u l a t i o n was e f f e c t i v e i n evoking a bu r s t response. The response was blocked by e l e c t r o l y t i c l e s i o n s of the IC d e s t r o y i n g the region o u t l i n e d by the dashed l i n e . Bottom) c a p s u l a r s t i m u l a t i o n s t i l l produces a b u r s t and an i n h i b i t o r y response i n an animal with an e l e c t r o l y t i c l e s i o n o f the SN. The l e s i o n was performed 4 weeks p r e v i o u s to the acute experiment. IC Stimuiition (SN Lt'Sion) . 1 ^^m^mmmi. ?msec 0 nisi-. 9 0 0 66 content of the Cd was measured. In each animal the l e s i o n had r e s u l t e d i n a complete d e s t r u c t i o n of the SN as w e l l as a d e p l e t i o n of OA below d e t e c t a b l e l e v e l s i n the i p s i l a t e r a l Cd. Actions Of Pharmacological Agents The i n t r a v e n o u s i n j e c t i o n o f h a l o p e r i d o l {0.5 to 2- 5mg/kg) or a l p h a - f l u p e n t h i x o l {0.5 to 2.5mg/kg) i n 7 animals, and the i o n t o p h o r e t i c a p p l i c a t i o n of alpha-f l u p e n t h i x o l i n 3 animals f a i l e d t o i n f l u e n c e e i t h e r the b u r s t response or the a s s o c i a t e d i n h i b i t i o n of spontaneous a c t i v i t y {figure 15). However the same dose of intravenous h a l o p e r i d o l c o n s i s t e n t l y blocked t h e high amplitude u n i t s evoked by n i g r a l s t i m u l a t i o n { f i g u r e 16}. During the blockade even a maximal sti m u l u s f a i l e d t o evoke the u n i t although the u n i t would continue to respond a n t i d r o m i c a i l y to p a l l i d a l s t i m u l a t i o n and the t h r e s h o l d f o r the burs t and i n h i b i t o r y response were unchanged. Recovery of the evoked response o c c u r r e d 20 to 40 min a f t e r the intra v e n o u s i n j e c t i o n . S i x animals r e c e i v e d u n i l a t e r a l i n j e c t i o n s of 6-OHDA i n t o the n i g r o s t r i a t a l bundle. In l a t e r e l e c t r o p h y s i o l o g i c a l experiments there was no change i n e i t h e r the b u r s t or i n h i b i t o r y response to n i g r a l s t i m u l a t i o n . However high amplitude s i n g l e u n i t s were never evoked. F o l l o w i n g the acute experiment each Cd was assayed f o r DA content. The i n j e c t i o n s were e f f e c t i v e i n d e p l e t i n g DA l e v e l s i n the i p s i l a t e r a l Cd by 90 to 35% when compared to the c o n t r a l a t e r a l s i d e . 67 A FIGURE 1J5. Influence of dopaminergic antagonism, or depletion, on the response recorded i n the Cd following stimulation of the SN. Intraveneous haloperidol (1 mg/kg) (A) , iontophoretic application of alpha-flupenthixol (B) , or previous chemical lesions of the medial forebrain bundle with 6-OH.DA fC), did not influence either the i n h i b i t o r y or excitatory response to n i g r a l stimulation. 67 B A 60 r n J1 i ^ v . - ^ J < Flupenthixol 6 - O H D A Les ion , _ J O i r T W 50 msec msec 6 0 0 msec 6 0 0 68 A FIG ORE 16-_ E f f e c t of i n t r a v e n e o u s a d m i n i s t r a t i o n of h a l o p e r i d o l (0.5 mg/kg) on a l a r g e a m p l i t u d e u n i t evoked by n i g r a l s t i m u l a t i o n . On the l e f t s t i m u l i were e q u a l to t h r e s h o l d f o r t h i s u n i t . On the r i g h t a s t i m u l u s i n t e n s i t y o f t w i c e t h r e s h o l d was , used. Each photograph i s o f 5 super imposed sweeps of the o s c i l l o s c o p e . Note t h a t h a l o p e r i d o l b l o c k e d the l a r g e a m p l i t u d e u n i t even when the s t i m u l u s was a p p l i e d a t the h igher i n t e n s i t y , a l though the b u r s t response was not i n f l u e n c e d . 68 B Control Haloperidol (3 rnin ) Recovery ( 2 0 m i n ) 0 . 2 m v 1 0 m s e c 69 Anatomical S t u d i e s When i n j e c t e d i n t o neuronal t i s s u e , HRP i s taken up by t e r m i n a l s and t r a n s p o r t e d i n a r e t r o g r a d e d i r e c t i o n to c e l l bodies. Subseguent treatment f o r peroxidase a c t i v i t y w i l l cause the formation of opaque granules i n the soma and proximal d e n d r i t e s of l a b e l l e d c e l l s . These can then be i d e n t i f i e d h i s t o l o g i c a l l y by t h e i r s t i p p l e d appearance under dark f i e l d i l l u m i n a t i o n . Five animals r e c e i v e d a u n i l a t e r a l i n j e c t i o n of HEP i n the SN. Some d i f f u s i o n o f HEP occurred i n t o the o v e r l y i n g tegmentum but the m a j o r i t y remained w i t h i n the nucleus. L a b e l l e d c e l l s were found mainly i n the v e n t r a l aspect and " p e r i p h e r a l s h e l l " of the Cd. Very few were detected i n the c e n t r a l core o f the nucleus ( f i g u r e 12). There were no d i f f e r e n c e s i n the d i s t r i b u t i o n of l a b e l l e d neurons i n the Cd f o l l o w i n g i n j e c t i o n i n t o the a n t e r i o r or p o s t e r i o r extent of the SN. Six animals r e c e i v e d u n i l a t e r a l or b i l a t e r a l i n j e c t i o n s of HEP i n t o the Cd. The d i f f u s i o n of HRP from the i n j e c t i o n s i t e was l i m i t e d to the boundaries of the Cd and none was detected i n the o v e r l y i n g c o r t e x . L a b e l l e d c e l l s were found i n the c o r t e x , pars compacta of the SN and i n the IPT ( f i g u r e 17). However t h e r e was a l s o extensive l a b e l l i n g of c e l l bodies i n the DRN along i t s e n t i r e a n t e r i o r p o s t e r i o r extent ( f i g u r e 18). The l a b e l l e d c e l l s were concentrated mainly i n the r e g i o n dorsomedial 70 A FIGURE 12 a d i a g r a m a t i c r e p r e s e n t a t i o n of l a b e l l e d neurons i n the IPT, zona compacta of the SN, and DSN, f o l l o w i n g i n j e c t i o n of HRP i n t o the Cd. HIPP, hippocampus; IL, i n t r a l a m i n a r nucei of the thalamus; PG, p e r i a q u e d u c t a l grey matter; SNC, zona compacta of the s u b s t a n t i a n i g r a ; DRN, d o r s a l raphe nucleus; MEN, median raphe nucleus. 70 33 71 A FIGURE 18_._ Photomicrographs showing lab e l l e d neurons i n the dorsal raphe nucleus following i n j e c t i o n of HBP into the Cd. A) low power micrograph (x4 0) following u n i l a t e r a l i n j e c t i o n of 0.1 ul of HRP. B) high magnification (x250) following u n i l a t e r a l i n j e c t i o n of 0.3 u l of HRP. Note the s t i p p l e d appearance of the reaction product i n somata and dendrites. Some c e l l s are out of focus because of the thickness of the sections. CA, cerebral aqueduct; DRN, dorsal raphe nucleus; MLF, medial longidutinal f a s c i c l e s . 72 to the medial l o n g i t u d i n a l f a s c i c l e s . Some neurons were observed both d o r s a l l y i n the p e r i a g u a d u c t a l grey matter and v e n t r o m e d i a l l y i n the nucleus l i n e a r i s raphe. No l a b e l l e d c e l l s were observed i n the median raphe-Orthograde axonal t r a n s p o r t o f p r o t e i n from the raphe to the Cd was a l s o measured. Amino a c i d s are a c t i v e l y taken up by the soma, i n c o r p o r a t e d i n t o p r o t e i n , and transported to the t e r m i n a l of the neuron. In 5 animals u n i l a t e r a l e l e c t r o l y t i c l e s i o n s were made i n the medial f o r e b r a i n bundle followed i n 24 hours, by i n j e c t i o n of t r i t i a t e d l e u c i n e i n t o the DRN. A f t e r an a d d i t i o n a l 24 hours s i g n i f i c a n t l e v e l s of t r i t i a t e d p r o t e i n were found i n both the Cd and cortex o f the i n t a c t s i d e {table 1) . The l e v e l s of r a d i o a c t i v i t y i n samples from the l e s i o n e d s i d e were 33.5% and 39.8% of the corres p o n d i n g c o n t r o l s f o r the Cd and co r t e x r e s p e c t i v e l y . 7 3 TABLE 1 the e f f e c t s of u n i l a t e r a l lesions of the medial forebrain bundle on accumulation of t r i t i a t e d protein i n the caudate nncleus and cerebral cortex a f t e r i n j e c t i o n of t r i t i a t e d leucine into the dorsal rapha nucleus. Data represent mean +^S.E.M. from 5 rats Control side (disint. /min/mg) lesioned side % (disint./min/mg) contr Caudate nucleus 53.0 +- 9.2 17.8 +- 3.5 33.5 Cortex 36.4 +- 4.1 14.5 +- 0.9 39.8 7 a DISCUSSION Previous i n v e s t i g a t o r s have emphasized the i n h i b i t i o n of s t r i a t a l neurons observed f o l l o w i n g n i g r a l s t i m u l a t i o n . Since the f i r i n g of most s t r i a t a l neurons i s depressed by i o n t o p h o r e t i c a p p l i c a t i o n of DA and DA i s r e l e a s e d i n the Cd f o l l o w i n g n i g r a l s t i m u l a t i o n , i t has become g e n e r a l l y accepted that DA i s an i n h i b i t o r y t r a n s m i t t e r i n the s t r i a t u m . However the present f i n d i n g s provide evidence fo r a d i f f e r e n t mechanism of i n h i b i t i o n and suggest t h a t DA may f u n c t i o n as an e x c i t a t o r y t r a n s m i t t e r i n the n i g r o s t r i a t a l pathway. Burst Response A b u r s t of e x c i t a t i o n was the predominant response evoked i n the Cd by n i g r a l s t i m u l a t i o n . The response was c o n t i n u o u s l y observed over l a r g e displacements of the r e c o r d i n g e l e c t r o d e . In many cases a movement of 300 i^ m was r e q u i r e d to pass through an a c t i v e r e g i o n of s t r i a t u m . However s t r i a t a l neurons are u s u a l l y only about 15 u^m i n diameter. C l e a r l y , a s i n g l e soma i s not the neuronal s t r u c t u r e r e s p o n s i b l e f o r g e n e r a t i o n of the b u r s t response. On the other hand the f a s c i c l e s of c o r t i c o f u g a l f i b e r s t r a v e r s i n g the Cd have a diameter of 50 to 200 ujn. I f n i g r a l s t i m u l a t i o n a l s o a c t i v a t e d axons i n the pyramidal t r a c t u n d e r l y i n g the SN the c o r t i c o f u g a l f i b e r s may have been a n t i d r o m i c a l l y a c t i v a t e d . A b u r s t of s m a l l p o t e n t i a l s would then be recorded with an e l e c t r o d e 75 located within a f a s c i c l e of f i b e r s i n the striatum. However, i n the present experiments th i s explanation i s unacceptable for the following reasons. A) Pontamine sky blue spots l e f t by the recording electrode indicate that a burst response was recorded only i n c e l l u l a r regions of the striatum and never i n a bundle of c o r t i c o f u g a l f i b e r s . B) Previous lesions of the cortex overlying the Cd did not a l t e r the burst response recorded during acute experiments. C) Stimulation of the pyramidal t r a c t s s l i g h t l y posterior to the SN did not evoke a burst response. D) The burst response did not follow n i g r a l stimulation at freguencies above 50 Hz. Fibers activated antidromically would follow much higher freguencies. E) Unlike axons, the neuronal structures underlying the response were sensitive to i o n t o p h o r e t i c a l l y applied glutamate. Therefore, i t i s concluded that the burst response was recorded from either somata or dendrites of neurons synaptically excited by n i g r a l stimulation. The properties of the response suggest that i t was generated by a c l u s t e r of neurons responding in a s i m i l a r manner. Neurons near the electrode t i p would contribute sharp spikes and the potentials from a large number of neurons at a greater distance from the t i p would summate to produce the " r i p p l e s " or waves of the baseline. As the electrode i s moved short distances i t would approach new c e l l s and move away from others. The evoked potential would r e f l e c t the changing orientation of the electrode to the surrounding structures. New peaks would appear in the 76 burst and others would decrease i n size exposing an underlying f i e l d p otential. Regions responding with a burst of exci t a t i o n were present throughout the Cd. On each electrode tract several active regions were detected. The neurons producing the response therefore must be very common. Several l i n e s of reasoning indicate that the most l i k e l y candidate i s the medium sized spiny c e l l described by Kemp and Powell (1971 A). F i r s t l y , they are the most numerous c e l l , making up 95% of the neurons in the striatum. Secondly, they have numerous dendritic branches extending 180-240 urn away from the parent c e l l . T hirdly, t h e i r axons usually terminate within the dendrit i c tree and therefore could influence nearby c e l l s with a common or overlapping dendritic f i e l d . F i n a l l y , these neurons are often observed in cl u s t e r s (Chronister et a l , 1976) . Interaction between neurons within the cl u s t e r would account for the complex form of the burst response. 77 P a t h way M e d i a t i n g The B u r s t R e s p o n s e A r e s p o n s e e v o k e d b y s t i m u l a t i o n o f a n u c l e u s i s o f t e n a s s u m e d t o r e s u l t f r o m a c t i v a t i o n o f s o m a t a n e a r t h e t i p o f t h e e l e c t r o d e and s u b s e q u e n t a c t i v i t y i n a p a t h w a y p r o j e c t i n g f r o m t h e n u c l e u s t o t h e r e c o r d i n g s i t e . H o w e v e r e l e c t r i c a l s t i m u l a t i o n o f a r e g i o n o f b r a i n c a n a c t i v a t e a n y n e u r o n a l s t r u c t u r e s i n t h e v i c i n i t y o f t h e s t i m u l a t i n g e l e c t r o d e . F o r e x a m p l e , a x o n s f r o m a s e c o n d n u c l e n s p r o j e c t i n g t o t h e r e c o r d i n g s i t e may be a c t i v a t e d i f t h e y p a s s n e a r t h e s t i m u l a t i n g e l e c t r o d e . H o w e v e r i n t h e p r e s e n t e x p e r i m e n t s s t i m u l a t i o n a t s i t e s s l i g h t l y v e n t r a l , p o s t e r i o r o r d o r s a l t o t h e SN n e v e r p r o d u c e d e x c i t a t i o n i n t h e s t r i a t u m . A b u r s t r e s p o n s e was o n l y e v o k e d when t h e s t i m u l a t i n g e l e c t r o d e was a c c u r a t e l y p o s i t i o n e d i n t h e S N . T h e r e f o r e t h e r e s p o n s e i n u n l i k e l y t o r e s u l t f r o m s t i m u l a t i o n o f " f i b e r s o f p a s s a g e " . N i g r a l s t i m u l a t i o n w i l l a l s o a c t i v a t e s o m a t a o r a x o n s p r o j e c t i n g t o r e g i o n s o f t h e b r a i n o t h e r t h a n t h e C d . S i n c e s t i m u l a t i o n o f t h e I P T a l s o p r o d u c e d a b u r s t r e s p o n s e i t i s p o s s i b l e t h a t n i g r a l s t i m u l a t i o n f i r s t a c t i v a t e d t h a l a m i c n e u r o n s . T h e y i n t u r n may p r o j e c t t o t h e Cd a n d a c c o u n t f o r t h e b u r s t o f e x c i t a t i o n . S i m i l a r l y a p o l y s y n a p t i c p a t h w a y v i a t h e c o r t e x may m e d i a t e t h e b u r s t r e s p o n s e . H o w e v e r n e i t h e r o f t h e s e m e c h a n i s m s c o u l d a c c o u n t f o r t h e p r e s e n t f i n d i n g s s i n c e p r e v i o u s l e s i o n s o f t h e I P T o r a b l a t i o n o f t h e c o r t e x o v e r l y i n g t h e C d d i d n o t i n f l u e n c e t h e r e s p o n s e t o n i g r a l s t i m u l a t i o n . 78 Nigral stimulation w i l l also activate somata of n i g r o s t r i a t a l neurons. The dopamine released from the terminals of t h i s pathway may excite s t r i a t a l c e l l s . However on the basis of the present experiments DA does not mediate the burst response. Neither haloperidol nor alpha-flupenthixol given systernically influenced the burst response. Iontophoretic application of alpha-flupenthixol was also without eff e c t . Furthermore a chemical lesion of the dopaminergic neurons of the n i g r o s t r i a t a l pathway with 6-OHDA did not a l t e r the burst response although s t r i a t a l l e v e l s of DA were depleted to very low l e v e l s . There remain two other possible explanations for the burst response. The response may be mediated by non-dopaminergic n i g r o s t r i a t a l f i b r e s . Alternately, n i g r a l stimulation may activate terminals of the s t r i a t o n i g r a l and cause an antidromic activation of the c e l l bodies i n the Cd. Subsequent orthodromic act i v a t i o n of c o l l a t e r a l s within the striatum could then mediate the burst response-One method of d i f f e r e n t i a t i n g between these two mechanisms i s to lesion the SN, wait for orthograde degeneration of a l l f i b r e s systems projecting from the SN to the Cd and then stimulate along the pathway mediating the burst response. I f the response i s preserved the somata of the f i b r e system mediating the burst cannot l i e i n the SN. The f i r s t step was to i d e n t i f y a s i t e f o r stimulation along the pathway mediating the response. The s t r i a t o n i g r a l and n i g r o s t r i a t a l pathways both l i e i n the 79 IC between the Cd and SH. S t i m u l a t i o n of the IC a n t e r i o r to the SN produced a burst response i n the Cd. The s i m i l a r p r o p e r t i e s of the response f o l l o w i n g n i g r a l and c a p s u l a r s t i m u l a t i o n suggests t h a t they may a c t i v a t e the same pathway. T h i s was confirmed by f i n d i n g t h a t a d i s c r e t e l e s i o n placed i n the IC by the a l r e a d y p o s i t i o n e d s t i m u l a t i n g e l e c t r o d e completely a b o l i s h e d the b u r s t response f o l l o w i n g n i g r a l s t i m u l a t i o n . The response to c a p s u l a r s t i m u l a t i o n was then t e s t e d i n animals with previous e l e c t r o l y t i c l e s i o n s of the SN. The n i g r a l l e s i o n s d i d not i n f l u e n c e the burst response recorded i n the Cd although the DA content of the i p s i l a t e r a l Cd of each animal was depleted to l e v e l s below those d e t e c t a b l e by the assay procedure. C l e a r l y , the l e s i o n s had r e s u l t e d i n degeneration of the dopaminergic n i g r o s t r i a t a l pathway. Any other p r o j e c t i o n with c e l l bodies l y i n g i n the SN would a l s o have degenerated. Therefore the b u r s t response evoked by n i g r a l or c a p s u l a r s t i m u l a t i o n must be a r e s u l t of an a n t idromic a c t i v a t i o n of the s t r i a t o n i g r a l pathway with subsequent orthodromic a c t i v a t i o n of c o l l a t e r a l s w i t h i n the Cd. Antidromic p o t e n t i a l s evoked by n i g r a l s t i m u l a t i o n were i n f r e q u e n t l y detected i n the s t r i a t u m and were only recorded from neurons i n the " p e r i p h e r a l s h e l l " of the nucleus. Betrograde t r a n s p o r t of HHP from the SN a l s o produced l a b e l l e d c e l l s e x c l u s i v e l y i n the p e r i p h e r a l s h e l l of the Cd. However the burst response was recorded throughout the s t r u c t u r e . Therefore the neurons of the 80 s t r i a t o n i g r a l pathway must give off large numbers of c o l l a t e r a l s and many of these must terminate at long distances from the parent c e l l . P a l l i d a l stimulation also evokes both antidromic and burst responses i n the striatum. The antidromic potentials were more frequently detected than those evoked by n i g r a l stimulation and were recorded almost exclusively in the c e n t r a l cere of the striatum. The present investigation does not eliminate the p o s s i b i l i t y that, the burst response was mediated through f i b r e s of passage or through a polysynaptic pathway. However the striatum has a large projection to the GP and antidromic activation of these neurons may produce a burst response via c o l l a t e r a l s of the s t r i a t o p a l l i d a l pathway. Kemp and Powell (1971A) described a medium sized and a "giant" projecting neuron. However, both c e l l s are dispersed throughout the nucleus and both have only a small number of c o l l a t e r a l s near the soma. Based on the present experiments i t i s not cle a r which, i f either, of these neurons may be the c e l l s of o r i g i n for the s t r i a t o n i g r a l and s t r i a t o p a l l i d a l pathways. Other workers found that n i g r a l stimulation evoked single units in the Cd. (Feltz and Albe-Fessard 1972;Frigyesi and Purpura 1973). The responses were not blocked by systemic haloperidol or lesions of the n i g r o s t r i a t a l pathway. I n t r a c e l l u l a r studies also indicate that an EPSP i s the f i r s t event evoked i n s t r i a t a l c e l l s by n i g r a l stimulation. In the present experiments n i g r a l stimulation at low i n t e n s i t i e s evoked a burst response 81 with only one or two spikes (see figure 1, 7v) . Perhaps some of the excitatory respones studied by other workers also r e s u l t from an antidromic axon r e f l e x mediated by the s t r i a t o n i g r a l pathway. There are some inconsistencies i n the l i t e r a t u r e regarding the antidromic responses of s t r i a t a l neurons evoked by n i g r a l stimulation. The latencies observed i n the present study agree with those reported by F r i g y e s i and Purpura (1967) and York{1970). However other workers have reported antidromic responses with latencies i n the range of 8 to 20 msec (Kitai et a l , 1975; i i l e s , 1974) . In the present study responses with these longer latencies invariably demonstrated a l l of the c h a r a c t e r i s t i c s of an orthodromic response. They had variable l a t e n c i e s at threshold stimulation and f a i l e d at stimulus frequencies above 40 Hz. 82 i s h i h i t o r v Response Spontaneously a c t i v e neurons were only o c c a s i o n a l l y detected i n the s t r i a t u m . The a c t i v i t y of these neurons was completely i n h i b i t e d f o r up to 300 msec f o l l o w i n g n i g r a l s t i m u l a t i o n . Neurons a c t i v a t e d by i o n t o p h o r e t i c a p p l i c a t i o n of glutamate responded i n a s i m i l a r manner. The i n h i b i t o r y response a l s o occurred i f the s t i m u l a t i n g e l e c t r o d e was p o s i t i o n e d s l i g h t l y d o r s a l t o the SN. Neurons o f the mesencephalic raphe n u c l e i p r o j e c t through the v e n t r a l tegmentum near the SN and are thought t o terminate, i n the s t r i a t u m (Nauta e t a l , 1974). A c t i v i t i o n of these " f i b r e s of passage" may have produced i n h i b i t i o n of s t r i a t a l neurons. In the present study i n j e c t i o n of HRP i n t o the s t r i a t u m produced a dense l a b e l l i n g of neurons s p e c i f i c a l l y i n the DRN and i n j e c t i o n of t r i t i a t e d l e u c i n e i n t o the DRN r e s u l t e d i n a s i g n i f i c a n t t r a n s p o r t of t r i t i a t e d p r o t e i n t o the Cd- S t i m u l a t i o n of the DRN produced i n h i b i t i o n of spontaneous and glutamate induced a c t i v i t y of s t r i a t a l neurons f o r p e r i o d s l a s t i n g up t o 400 msec. E l e c t r o l y t i c l e s i o n s of the v e n t r a l tegmentum at the l e v e l of the SN completely a b o l i s h e d the i n h i b i t o r y i n f l u e n c e of n i g r a l s t i m u l a t i o n and blocked the t r a n s p o r t of t r i t i a t e d p r o t e i n . These f i n d i n g s p rovide evidence f o r a r a p h e - s t r i a t a l pathway- Axons of t h i s pathway t r a v e l through or near the SN and could account f o r the i n h i b i t i o n of s t r i a t a l u n i t s produced by n i g r a l s t i m u l a t i o n . However fou r weeks 8 3 f o l l o w i n g e l e c t r o l y t i c l e s i o n s o f the DP.N, n i g r a l s t i m u l a t i o n continued to produce i n h i b i t i o n of s t r i a t a l u n i t s . Therefore a second mechanism o f i n h i b i t i o n must be op e r a t i n g i n the s t r i a t u m . The m a j o r i t y of s t r i a t a l neurons are i n h i b i t e d by the i o n t o p h o r e t i c a p p l i c a t i o n of DA. N i g r a l s t i m u l a t i o n i s known to cause the r e l e a s e of DA from t e r m i n a l s of the n i g r o s t r i a t a l pathway and subsequently the DA c o u l d cause i n h i b i t i o n of s t r i a t a l neurons. However dopaminergic blockade by systemic h a l o p e r i d o l or a l p h a - f 1 u p e n t h i x o l d i d not i n f l u e n c e the i n h i b i t o r y response. Chemical l e s i o n s of the n i g r o s t r i a t a l pathway with 6-OHDA were a l s o without i n f l u e n c e although DA l e v e l s i n the i p s i l a t e r a l Cd were depleted t o very low l e v e l s . F i n a l l y e l e c t r o l y t i c l e s i o n s of the SN and subsequent degeneration of the s t r i a t o n i g r a l pathway d i d not a l t e r the i n h i b i t i o n of s t r i a t a l u n i t s produced by c a p s u l a r s t i m u l a t i o n . C l e a r l y , DA does not mediate the i n h i b i t o r y i n f l u e n c e of n i g r a l s t i m u l a t i o n . Furthermore l e s i o n s o f the IPT or cortex d i d not i n f l u e n c e the i n h i b i t o r y response. These f i n d i n g s suggest t h a t the c e l l s e x c i t e d by c o l l a t e r a l s of the s t r i a t o n i g r a l pathway are i n f a c t i n h i b i t o r y i n t e r n e u r o n s . S e v e r a l f i n d i n g s support t h i s h y p o t h e s i s . A) A spontaneous u n i t i n h i b i t e d by n i g r a l s t i m u l a t i o n and a burst response were u s u a l l y recorded s i m u l t a n e o u s l y from the same e l e c t r o d e s i t e . B) As the stimulus i n t e n s i t y was s l o w l y i n c r e a s e d from zero the onset of i n h i b i t i o n c o i n c i d e d with the development of the b u r s t . C) A s t i m u l u s i n t e n s i t y s u f f i c i e n t to produce a 84 maximal burst also produced a maximum i n h i b i t i o n . D) I f additional application of glutamate caused degeneration of the burst the i n h i b i t i o n was abolished. Spontaneously active or glutamate induced units i n the striatum could be d i f f e r e n t i a t e d into two populations on the basis of amplitute. The majority had a low amplitude. Often threshold stimulation of the SN evoked a low amplitude spike at a constant latency. As the stimulus intensity was increased the unit became incorporated into the burst response. The spontaneous a c t i v i t y of the same c e l l would then be i n h i b i t e d following each burst. This finding suggests that the i n h i b i t o r y interneurons have recip r o c a l synaptic connections. Therefore i f the medium sized spiny c e l l s are the inhibitory interneurons, they should receive both excitatory and i n h i b i t o r y synaptic contacts. Kemp and Powell (1971B) found that the spiny c e l l s receive synaptic input from other neurons within the Cd. These synapses have membrane s p e c i a l i z a t i o n s associated with both excitation (Golgi type 1, Gray, 1959) and i n h i b i t i o n (Golgi type 2) . 85 Larae Amplitude C e l l s The second population of spontaneous units had amplitudes d i s t i n c t l y greater than the burst response. They responded to n i g r a l stimulation with a single orthodromic action p o t e n t i a l . Usually a burst response was evoked at the same recording s i t e . However the high amplitude units could be d i f f e r e n t i a t e d from the underlying burst by their greater amplitude, lower threshold and single action potential. Spontaneous a c t i v i t y of these units was also i n h i b i t e d following the burst response. In f a c t , as the stimulus i n t e n s i t y was increased to produce a maximal burst the high amplitude unit often f a i l e d to respond(fig 10, a). This suggests that the influence of the in h i b i t o r y interneuron was s u f f i c i e n t to overcome the synaptic e x c i t a t i o n produced by n i g r a l stimulation. Unlike other responses to n i g r a l stimulation, large amplitude units were detected only i n the central core of the striatum. The same units were antidromically activated by p a l l i d a l stimulation. Therefore they may be s t r i a t o p a l l i d a l neurones. The present experiments indicate that the large amplitude units were activated by the dopaminergic n i g r o s t i a t a l pathway. Systemic haloperidol r e v e r s i b l y blocked the response, the response was not detected i n animals with previous chemical lesions of the dopaminergic n i g r o s t r i a t a l pathway, and capsular stimulation did not 86 evoke the response i n animals with lesions of the SN. Conclusion In contrast to e a r l i e r reports, the present findings do not support the hypothesis that DA mediates the i n h i b i t i o n of s t r i a t a l neurons following n i g r a l stimulation. In fact the n i g r o s t r i a t a l pathway may excite s p e c i f i c neurons in the striatum. However, an important question remains. If DA i s an excitatory transmitter by what mechanism does iontophoretic application of DA produce i n h i b i t i o n i n the majority of neurons. Two possible explainations can be considered. F i r s t l y , York (1970) has suggested that s t r i a t a l neurons may possess two types of DA receptors, one excitatory and one i n h i b i t o r y in function. The excitatory receptor may be s p e c i f i c a l l y located i n dopaminergic synapses whereas the i n h i b i t o r y receptors may be located on other regions of the c e l l . Secondly, the i n i t i a l response to iontophoretically applied DA may be e x c i t a t i o n . However, continued application over a period of longer than a few hundred msec may produce a pharmacological "overload" of the receptors r e s u l t i n g i n a non-physiological depression of the neuron. K i t a i (1976) recorded the i n t r a c e l l u l a r response to e x t r a c e l l u l a r application of DA. He found that very short pulses l a s t i n g only a f r a c t i o n of a second consistently produced an FPSP. In conclusion the present experiments have 87 demonstrated that n i g r a l stimulation activates at le a s t two pathways (figure 19)- Stimulation of the n i g r o s t r i a t a l pathway excites neurons of the s t r i a t o p a l l i d a l pathway. Nigral stimulation also antidromically activates the s t r i a t o n i g r a l pathway and i t s c o l l a t e r a l s within the Cd. Inhibitory interneurons excited by these c o l l a t e r a l s i n h i b i t the a c t i v i t y of both s t r i a t o p a l l i d a l neurons and low amplitude units. At least some of the low amplitude units may also be i n h i b i t o r y interneurons. F i n a l l y a raphe s t r i a t a l pathway ex i s t s and stimulation of t h i s pathway causes a direct i n h i b i t i o n of s t r i a t a l units 88 A FIGURE 19. A schematic i l l u s t r a t i o n of the proposed s y n a p t i c arrangements of s t r i a t a l neurons (Cd) with those of the s u b s t a n t i a n i g r a (SN) and globus p a l l i d u s (GP). S t i m u l a t i o n of the zona compacta of the SN (SNC) produces an a c t i v a t i o n - i n h i b i t i o n sequence of s t r i a t a l t a r g e t c e l l s . The i n h i b i t o r y component i s mediated by r e c u r r e n t axon c o l l a t e r a l s impinging on i n h i b i t o r y i n t e r n e u r o n s (shown i n b l a c k ) . S t i m u l a t i o n of the GP e l i c i t s a n t i d r o m i c s p i k e s i n the same axon c o l l a t e r a l system. 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