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Investigation into the chemical sensitivities of single nerve cells in the caudate nucleus in relation… York, Donald Harold 1966

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"AN INVESTIGATION INTO THE CHEMICAL SENSITIVITIES OF SINGLE NERVE CELLS IN THE CAUDATE NUCLEUS IN RELATION TO ASSOCIATED STRUCTURES OF THE BRAIN" by DONALD  HAROLD  YORK  BSc, U n i v e r s i t y o f B r i t i s h Columbia, 1965  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  i n the department of PHYSIOLOGY We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1966  In p r e s e n t i n g requirements Columbia, for  thesis  in p a r t i a l  f o r an a d v a n c e d  degree  a t the U n i v e r s i t y  1 agree that  the L i b r a r y  r e f e r e n c e and s t u d y .  tensive by  this  copying o f this  thesis  cial  gain  that shall  Department o f  *s></-e &Zs. /\ ;  Columbia  f o r ex-  p u r p o s e s may be g r a n t e d  o r by h i s r e p r e s e n t a t i v e s .  724  available  permission  of this  thesis  n o t be a l l o w e d w i t h o u t my w r i t t e n  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a Date  for scholarly  copying o r p u b l i c a t i o n  of British  make i t f r e e l y  I f u r t h e r agree that  t h e Head o f my D e p a r t m e n t  understood  shall  f u l f i l m e n t o f the  It  is  for finan-  permission.  ABSTRACT Considerable evidence has been accumulated suggesting the possible neurotransmitter functions of acetylcholine (Ach)  and dopamine i n the central nervous system. In part-  i c u l a r the caudate nucleus i s believed to contain cholinergi c and  dopaminergic c e l l s , as well as the specific enzymes  responsible for the synthesis and destruction of these chemical entities.  In t h i s study, the technique of microelectro-  phoresis has been applied to c e l l s of the caudate nucleus i n order to investigate  the p a r t i c u l a r location and pharmacolog-  i c a l 1 properties of caudate neurones i n r e l a t i o n to some other structures  of the b r a i n .  Neurones located between the surface and 4.5 mm deep i n the head of the caudate nucleus of cats have been tested for t h e i r acetylcholine and dopamine s e n s i t i v i t y . Of 427 c e l l s examined, about 10$ were excited by acetycholine, and a further 10$ depressed, while the remaining majority of the c e l l s were unaffected. The c e l l s which were excited l a y at an average depth of 860u below the surface (5$ confidence l i m i t s ± l 6 5 u ) , while those depressed were situated at 2170\x (5$ confidence l i m i t s ± 3 5 0 u ) . Both excitatory and depressant responses could also be e l i c i t e d by e l e c t r i c a l stimulation of the nucleus v e n t r a l i s anterior thalami (VA). The responses appeared to be "muscarinic" i n character,  since they were abolished by atropine  and were e l i c i t e d by acetyl-{i-methylcholine as effectively by acetylcholine i t s e l f .  as  Of 152 c e l l s examined, about 1% were e x c i t e d by dopamine, and 64% depressed, w h i l e the remaining m a j o r i t y of c e l l s were u n a f f e c t e d . The c h a r a c t e r i s t i c depth stratum f o r e x c i t a t o r y and depressant responses o f c h o l i n e r g i c c e l l s was  not found f o r dopaminergic c e l l s .  s t i m u l a t i o n o f the s u b s t a n t i a n i g r a  Electrical  (SN) e l i c i t e d  evoked response from caudate neurones which was by dopamine. I t was  an  depressed  found t h a t a dopamine induced d e p r e s s i o n  c o u l d be b l o c k e d by d i b e n z y l i n e , but not by d i c h l o r o i s o p r o p ylnoradrenaline  (DCI). T h i s would suggest t h a t the dopamine  s e n s i t i v e c e l l s have o f - a d r e n e r g i c r e c e p t o r s . s t i m u l a t i o n of the nucleus centromedianus  Electrical  thalami  (CM)  caused d e p r e s s i o n of s i n g l e c e l l a c t i v i t y i n the caudate, c o i n c i d i n g w i t h d e p r e s s i o n caused by  iontophoretically  a p p l i e d dopamine. Taken w i t h the e a r l i e r o b s e r v a t i o n t h a t the r e l e a s e o f Ach from the caudate can be enhanced by VA s t i m u l a t i o n , i t has been concluded t h a t the p r e s e n t r e s u l t s i n d i c a t e a f i n a l c h o l i n e r g i c l i n k i n the pathway from VA to the caudate l e u s . Furthermore,  nuc-  the demonstration of enhanced dopamine  output from the caudate upon CM s t i m u l a t i o n , w i t h r e f e r e n c e to p r e s e n t r e s u l t s ,  i n d i c a t e s an i n h i b i t o r y  l i n k i n t h i s thalamo-caudate  dopaminergic  pathway. A n i g r o s t r i a t a l  tract  c a u s i n g e x c i t a t o r y responses a t the caudate  termination  has been observed. However, the e f f e c t i v e blockage of the e x c i t a t o r y SN evoked response 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 dopamine would suggest a p o s s i b l e dominance of a t h a l a m i i n h i b i t o r y n e u r a l mechanism over the e x c i t a t o r y input from SN operated v i a a dopaminergic  link.  TABLE OF CONTENTS  INTRODUCTION I. The Caudate Nucleus A) Anatomy B) F u n c t i o n II..Chemical T r a n s m i t t e r s I I I . The M i c r o e l e c t r o p h o r e t i c Technique IV. C h o l i n e r g i c and Dopaminergic C e l l s  page 1-23 1 2 6 12 15 19  METHODS  24-2 7  RESULTS A) C h o l i n e r g i c C e l l s (i) E f f e c t of e l e c t r i c a l stimulation ( i i ) Pharmacology B) Dopaminergic C e l l s (i) E f f e c t of e l e c t r i c a l stimulation ( i i ) Pharmacology  28-48 28 31 36 40 43 46  DISCUSSION A) C h o l i n e r g i c C e l l s  49-5,8 49  B) Dopaminergic C e l l s  54  BIBLIOGRAPHY  59-70  APPENDIX  71-74  LIST OF TABLES  Table  I.  D i s t r i b u t i o n o f Ach s e n s i t i v e c e l l i n the head o f the caudate nucleus  Table I I . D i s t r i b u t i o n o f dopamine s e n s i t i v e c e l l s i n the head of the caudate nucleus.  vii  LIST OF FIGURES  F i g u r e 1.  E x c i t a t i o n o f a caudate neurone by DLH and by.; .Ach a p p l i e d iontophoretically.  page 29  F i g u r e 2. .Enhancement o f DLH e x c i t a t i o n produced by Ach.  page 30  F i g u r e 3.  Depressant a c t i o n s o f a p p l i e d Ach. A) E f f e c t upon two neurones f i r i n g spontaneously. B) E f f e c t o f Ach a p p l i e d w i t h two i n t e n s i t i e s of c u r r e n t upon the d i s c h a r g e o f a c e l l e x c i t e d by DLH.  page 32 page 33  F i g u r e 4.  Sketch o f the caudate nucleus i n transverse section, at f r o n t a l plane 17.  page 35  F i g u r e 5.  Enhancement o f DLH e x c i t a t i o n by s t i m u l a t i o n o f VA.  page 37  F i g u r e 6.  Depressant e f f e c t o f VA s t i m u l a t i o n upon a neurone e x c i t e d by DLH.  page 38  F i g u r e 7.  B l o c k i n g a c t i o n o f a t r o p i n e upon Ach e f f e c t s A) a t a neurone e x c i t e d by Ach B) a t a neurone depressed by Ach  page 39  F i g u r e 8.  The e f f e c t o f s y s t e m i c a l l y administe r e d a t r o p i n e upon Ach enhanced f i r ing and upon the evoked response t o VA s t i m u l a t i o n .  page 41  F i g u r e 9.  A) I n t e g r a t e d response o f a caudate c e l l showing the e f f e c t produced by s t i m u l a t i o n o f CM. B) I n t e g r a t e d response o f same c e l l induced t o f i r e w i t h DLH and depressed; by dopamine.  page 44  « ••  Figure  9.  C) O s c i l l o s c o p e t r a c e showing the depressant e f f e c t on c e l l f i r i n g of dopamine. D) An evoked response from the same c e l l produced by s t i m u l a t i o n o f SN which i s a l s o b l o c k e d by dopamine.  page 45  F i g u r e 10.  Depressant e f f e c t o f dopamine. A) Response o f caudate c e l l i n which DCI was unable t o b l o c k dopamine induced d e p r e s s i o n . B) Response o f same c e l l w i t h dopamine d e p r e s s i o n b l o c k e d by d i b e n z y l i n e .  page 47  F i g u r e 11.  A) A dopamine induced d e p r e s s i o n b l o c k e d by d i b e n z y l i n e . B) Evoked response from caudate c e l l .  page 48  i x  LIST OF PLATES  Plate  1.  U l t r a s t r u c t u r e of the caudate nucleus in rat brain.  page 3  P l a t e 2.  P a r t of a nerve c e l l w i t h d e n d r i t e i n caudate n u c l e u s .  page 4  Plate  E l e c t r o n m i c r o g r a p h of caudate nucleus showing v e s i c l e f i l l e d axon t e r m i n a l s .  page 5  A s e c t i o n of caudate nucleus showing a stratum of neurones w i t h complex dendritic structures.  page 7  P l a t e 5. .A l a y e r o f neurones w i t h more deeply p l a c e d compact c e l l s .  page 8  P l a t e 6.  page 9  3.  P l a t e 4.  A higher-power ial" cell.  view of one  "superfic-  ACKNOWLEDGMENT I am greatly indebted to Dr. H. McLennan for his helpful and constructive advice on the research of t h i s thesis.  I am especially grateful to Mr. P.  Graystone, Mr.R. Walker, and Mr. K. Henze for t h e i r excellent technical assistance.  INTRODUCTION  I . The C a u d a t e The  Nucleus  c a u d a t e n u c l e u s i s a n e l o n g a t e d mass o f g r a y  bent on i t s e l f l i k e a horseshoe  and i s t h r o u g h o u t  matter  i t s entire  extent i n close proximity to the l a t e r a l v e n t r i c l e . I t s swoll e n r o s t r a l e x t r e m i t y o r head i s pear-shaped  and b u l g e s  into  t h e a n t e r i o r h o r n o f t h e l a t e r a l v e n t r i c l e . The r e m a i n d e r o f t h e n u c l e u s i s drawn o u t i n t o a l o n g , s l e n d e r , h i g h l y tail.  arched  Along the f l o o r of the c e n t r a l part of t h e v e n t r i c l e  the head g r a d u a l l y t a p e r s o f f i n t o t h e t a i l , curves around  which  finally  i n t o t h e r o o f o f t h e i n f e r i o r h o r n and e x t e n d s  r o s t r a l l y a s f a r . ' a s t h e a m y g d a l o i d n u c l e u s . The h e a d o f t h e caudate nucleus i s d i r e c t l y continuous w i t h t h e a n t e r i o r p e r f o r a t e d s u b s t a n c e , and v e n t r a l t o t h e a n t e r i o r l i m b o f t h e i n t e r n a l capsule i ti s fused with the l e n t i f o r m nucleus.(Rans o n & C l a r k , 1959). The  caudate n u c l e u s has been found t o c o n t a i n c h o l i n e r -  g i c and d o p a m i n e r g i c  c e l l s , as w e l l as t h e s p e c i f i c  enzymes  r e s p o n s i b l e f o r t h e s y n t h e s i s and d e s t r u c t i o n o f t h e s e chemi c a l - e n t i t i e s . I n t h i s study, the technique of microelectrop h o r e s i s has been a p p l i e d t o c e l l s o f t h e caudate n u c l e u s i n o r d e r t o i n v e s t i g a t e t h e p a r t i c u l a r l o c a t i o n and p h a r m a c o l o g i c a l p r o p e r t i e s o f c a u d a t e n e u r o n e s i n r e l a t i o n t o some other structures of the brain.  A. Anatomy Electron microscopic work by Fuxe, HOkfelt, and Nilsson (1964) demonstrated that the head of the caudate nucleus contained a highly  packed plexus b u i l t up of c e l l bodies, abund-  ant f i b e r s , and numerous c e l l processes the f i b e r s  (Plate 1-3). Most of  had the appearance of axon terminals (Gray,1959).  They were f i l l e d with synaptic v e s i c l e s and contained numerous mitochondria. Some of the terminals contained a few nucleated v e s i c l e s . The thickness of the axon terminals varied  between  0.1 and l.Ou- and many of them had a diameter below 0.4(J. (plate 3). They often l a y i n very close contact with each other. The axon terminals came into close contact also with nerve c e l l bodies and with processes that mainly seemed to belong to an extensive dendritic net (Plates 1, 2). These contact sites often showed the presence of synaptic membranes (Palay 1958;  1956,  Pellegrino de I r a l d i , P a r i n i Duggan, de Robertis, 1963).  The contact s i t e s , on the other hand, between f i b e r s containting  abundant synaptic v e s i c l e s never possessed  these s p e c i a l -  izations. A morphological  correlation f o r the d i s t r i b u t i o n of two  types of neurones within the caudate nucleus i s described by Cajal (1955). He stated that : "giant c e l l s with long axis cylinders are grouped p r i n c i p a l l y i n the infero-external region of the: nucleus and near the internal capsule", whereas "a very dense plexiform band composed of several series of large and medium sized c e l l s with short axis cylinders are found near  Plate 1  Ultrastrueture of the caudate nucleus i n the rat b r a i n . Many t h i n f i b e r s with interposed large l i g h t l y - s t a i n e d c e l l processes are seen. Most of the l i g h t l y - s t a i n e d processes are dendrites, containing neurofilaments and mitochondria, but some of them could be g l i a l processes. 12,000X. (Fuxe K.,H6'kfelt T . , N i l s s o n 0 . , 1964)  4  Plate Z  Part of a nerve c e l l with dendrite i n the caudate nucleus. Many v e s i c l e f i l l e d axon terminals are observed. 14,000X. (Fuxe K.,Hokfelt T . , N i l s s o n 0 . , 1964)  5  Plate 3  A higher power electronmicrograph of the caudate nucleus demonstrating • v e s i c l e f i l l e d axon terminals; several terminals forming synaptic membranes. 45»OOOX. (Puxe K.,H6kfelt T.,Nilsson 0., 1964)  the l a t e r a l v e n t r i c l e " . Sections of the caudate stained by the Golgi method (Moliner,1957) have been found to show , amongst others, two d i s t i n c t types of c e l l s . A layer of neurones having massive dendritic meshworks surrounding them, have frequentl y been seen l y i n g some 600fi i n from the ventricular border of the nucleus and extending to a distance of 1500p. from the surface (Plate 4,6). In the i n t e r i o r of the nucleus a second type of c e l l possessing a more compressed but s t i l l extensive dendritic structure and with apparently a longer axon are common (Plate: 5,). . B. Function On the basis of c l i n i c a l and neuropathological c o r r e l ations, Vogt and Vogt (1919) have postulated that the e x c i t ation of elements of medium-sized c e l l s i n the striatum f a c i l i t a t e bodily movements whereas the smaller c e l l s are inhibitory i n nature. Differences i n e x c i t a b i l i t y of two main types of neurones comprising the corpus striatum have been described by Stevens, Kim and Mac Lean (1961). The c i r c l i n g movements seen i n cats as an early effect of chemical excitation or the result of high frequency e l e c t r i c a l stimulation are due to excitation of medium large c e l l s having f a c i l i t a t o r y action; whereas the quietude and interference with conditioned avoidance testing observed during low frequency stimulation or as a l a t e chemical effect i s attributable to small c e l l s with inhibitory functions.  Plate 4>  A section showing a stratum of neurones with complex d e n d r i t i c structures l y i n g approximately between 650u and 14-OOu. below the v e n t r i c u l a r border, (golgi preparation of transverse section through the head of the caudate nucleus approximately at f r o n t a l plane 17).  Plate S  A section showing a layer of neurones with more deeply placed compact c e l l s . The v e n t r i c l e i s v i s i b l e i n the upper right-hand corner, (golgi preparation of transverse section* through the head of the caudate nucleus approximately at f r o n t a l plane 17).  00  Plate 6  A higher-power view of one " s u p e r f i c i a l " c e l l , ( g o l g i p r e p a r a t i o n of t r a n s v e r s e s e c t i o n through the head of the caudate nucleus approximately at f r o n t a l plane 17).  (0  10  Most of the clues to the functions of the caudate nucleus and the rest of the corpus striatum come from c l i n i c a l observations. On the basis of d e f i c i t s resulting from lesions, i t has been inferred that the corpus striatum i s part of a system controlling motor behavior and coordination. Experimental evidence, however, suggests that this interpretation represents too r e s t r i c t e d a view of i t s functions. For-example, i n an important early study, Rogers (1922) found that pigeons i n which the homologue of the striatum had been excised could neither r e t a i n nor aquire the a b i l i t y to place i n proper sequence the patterns of behavior making up the r i t u a l s of mating and nesting a c t i v i t y . More recently Gomez, Thompson & Mettler (1958) have found that b i l a t e r a l caudatal lesions interfere with the conditioned avoidance behavior of cats i n a manner quite comparable to the effects of stimulation reported by Stevens, Kim, & MacLean (1961). In experiments also involving lesions of the caudate i n cats Knott, Ingram & C o r r e l l (I960) have reported disturbances  of a transient nature i n conditioned  approach responses. F e r r i e r (1873) f i r s t reported the effects of e l e c t r i c a l stimulation of the head of the caudate nucleus, i n dogs. There i s a general tendency to regard the effects of such stimulation as f a l l i n g into three main d i v i s i o n s , delimited by the frequency of stimulation. Buchwald, Wyers, Lauprecht  11  and Heuser (1961) have defined them as (a) 0.2-10 impulses/ sec. " i n h i b i t o r y " effects including inactivation and  sleep;  (b) 20-30/sec, "arrest" similar to that described by Hunter & Jasper (1949) f o r stimulation of the intralaminar thalamus; and  (c) greater than 30/sec, arousal and such overt motor  phenomena as contraversive c i r c l i n g . McLennan,Emmons & Plummer  (1964) observed that u n i l a t e r a l stimulation of the caudate  produced the "arrest" reaction only at low frequencies, whereas the higher frequencies the "arrest" response may  be masked  through the appearance of turning and c i r c l i n g movemnets. Thus, the response of caudate neurones to d i f f e r e n t frequencies of stimulation may  involve two d i s t i n c t pathways.  In acute experiments, Delgado (1957) observed i n monkeys that food ingestion stopped on stimulation of the v i c i n i t y of the caudate nucleus. Also, i n acute s t r i a t a l stimulation experiments, Akert & Andersson (1951) noted that the attention of the exteroceptive sense organs remained preserved  i n cont-  rast to natural sleep. By i n j e c t i o n of alumina cream into the head of the caudate nucleus (Spiegel & Szekely, 1961), a prolonged stimulation could be produced i n cats, r e s u l t i n g i n catatonia, loss of spontaneous movements, and "active" catalepsy. Electrographic studies have shown that s t r i a t a l stimulation induces an increased amplitude of the e l e c t r i c a l discharges of the hypothalamus and the amygdala; slow waves and eventually seizure  discharges  12  appear i n the electroencephalogram. Thus s t r i a t o f u g a l impulses may act, on the one hand, upon the cerebral cortex, and, on the other hand, on the brain stem, at least to the l e v e l of the oculomotor n u c l e i . I I . Chemical Transmitters Dale (1914) f i r s t described the peripheral actions of acetylcholine as being of two d i s t i n c t types; "muscarinic" and  " n i c o t i n i c " . He defined the muscarinic action as "the  action which true muscarine exhibits i n i t s true form, uncomplicated by the n i c o t i n i c action". Similarly, n i c o t i n i c action was defined as that mimicked by the a l k a l o i d nicotine. Recent evidence has suggested that muscarine i t s e l f may have certain n i c o t i n i c action i n the superior c e r v i c a l ganglion and n i c t a t i n g membrane of the cat (Jones,1963)•  Muscarinic  action i s demonstrated by drugs, p r i n c i p a l l y synthetic modifications of acetylcholine (Ach), that appear to act only at the peripheral effector organs i n the parasympathetic system and at the "sympathetic" endings i n sweat glands. I t i s termed muscarinic because the a l k a l o i d muscarine acts only at these s i t e s (Wilson & G i s v o l d , 1 9 6 2 ) .  The n i c o t i n i c action of a c e t y l -  choline involves stimulation of a l l the peripheral autonomic ganglia and s k e l e t a l muscles, s i m i l a r to the stimulating action of the a l k a l o i d drug nicotine. Bradley, Dhawan & Wolstencroft (1966) found a clear d i s t i n c t i o n could be made between the excitatory and inhibitory effects observed at neurones of the pons and medulla by the  13  use of muscarinic and n i c o t i n i c agents. The excitatory  effects  were mimicked by both muscarinic and n i c o t i n i c agents i n contrast to the i n h i b i t o r y effects which were mimicked only by musca r i n i c agents. S i m i l a r l y , the n i c o t i n i c blocking; agent dihydro)3-erythroidine (DHBE) did not antagonize the i n h i b i t o r y responses but often blocked the excitatory effects, whereas the muscarinic blocking agent atropine antagonized both effects. Muscarinic excitation of neurones has been found i n the cerebral cortex by Krnjevid: & P h i l l i s (1963c), and mixed muscarinic-nicotinic type of excitation has been found i n other parts of the brain stem, i n the l a t e r a l geniculate nucleus (Curtis & Davis,1963) i n certain ventrobasal thalamic nuclei (Andersen & Curtis,1964 a,b),  and i n the cerebellum (Crawford,Curtis,Voorhoeve & Wilson  1963; McCance & P h i l l i s , 1964). In contrast,  the cholinergic excitation of Renshaw c e l l s  i s mainly n i c o t i n i c (Curtis & Eccles,1958a,b) and the i n h i b i t i o n by acetylcholine of c e l l s i n the gracile and cuneate nuclei has n i c o t i n i c properties  (J.H.Wolstencroft, unpublished). Curtis  & Ryall (1964) have demonstrated that acetylcholine excites Renshaw c e l l s by acting on both n i c o t i n i c and muscarinic recept o r s , the former being responsible only for the early response to motor axon stimulation. Excitatory and i n h i b i t o r y responses have also been described i n the hypothalamus (Bloom,Oliver & Salmoiraghi,1963), caudate nucleus (BLoom,Costa,Oliver & Salmoiraghi,1964), olfactory bulb (Bloom,Costa & Salmoiraghi,1964), and cerebral cortex ( R a n d i £ , S i m i n o f f & Straughan,1964).  The interpretation of  the actions of various chemical  agents on the caudate nucleus  has particular  difficulties.  Substances injected into the blood c i r c u l a t i o n may never reach the caudate neurones on account of the blood-brain barrier,, and they may produce indirect effects by a l t e r i n g the blood supply to the brain or by exciting peripheral structures with central connections. S i m i l a r l y , direct application to the surface of the caudate, as with direct application to the surface-of  the cortex, does not give a more definite answer  because e x c i t a b i l i t y i s abolished by i n h i b i t i o n or an excess of  excitation and, i n general, changes i n e l e c t r i c a l records  from the surface can be quite ambiguous (compare the analyses of  the action of $-aminobutyric acid (GABA) by Purpura, Girado,  Smith,Gallen & Grundfest, 1959 on the one hand, and by Jasper I960 and Goldring & O'Leary, I960 on the other hand). The caudate nucleus i s not a homogeneous structure, and thus different neurones may not respond to' drugs i n the same: way. This may not be revealed by recording the a c t i v i t y of single units, since the numerous excitatory and inhibitory interconnections between neurones may cause the reaction of the single unit to be altered r a d i c a l l y by the massive d i s charge of other c e l l s . For ing  a l l these reasons the iontophoretic method of apply-  substances to single units i s the method of choice whenever  the substance i s ionized i n solution. The amounts released are minute and therefore the concentration i n the tissue negligible  15  except i n the immediate v i c i n i t y of the neurone under observat i o n . Many neurones can thus be studied i n succesion, i n the same animal with l i t t l e interference being caused to brain function. The microelectrophoretic technique was f i r s t  applied to  studies i n v i t r o on the neuro-muscular junction by Fatt & Katz (1951). Many studies followed, on spinal neurones (Curtis & Eccles,1958;  C u r t i s , P h i l l i s & Watkins 1959,1960); i n studies  of neurones i n the brain stem (Curtis & Koizumi,1961; Curtis & Davis,1962; Bradely,Dhawan & Wolstencroft, 1966); i n the l a t e r a l geniculate  nucleus (Curtis & Davis,1962);  i n the o l -  factory bulb (von Baumgarten,Bloom,Oliver & Salmoiraghi,1963); at the c o r t i c a l terminations of the v i s u a l pathway (Spehlmann, 1963); i n sensory and motor areas of the cortex (Krnjevi<5 & Phillis,1961,1962). III.  The Microelectrophoretic Technique The iontophoretic method i s based on the well-known p r i n -  ciple of migration of ions under the effects of an e l e c t r i c a l f i e l d (electrophoresis).  From a micropipette containing an  aqueous solution of an ionizable drug, the b i o l o g i c a l l y active ion (cation or anion, depending upon the chemical  structure)  can be ejected from the pipette, when desired, by current of the appropiate p o l a r i t y . To a f i r s t  approximation, when the elect-  r i c a l conductivity of the solution i n the pipette i s high compared to that of the tissue f l u i d , the number of ions released i s expressed by Faraday's law (Pauling,1955;  Falkenham & Kelbg,  16  1 9 5 9 ) , according to which each nanoampere of current flow would eject approximately 1 x 1 0  - 1  ^ gram equivalent of ion  per second. More exact quantification of the electrophoretic dose can be determined by the calculation of  "efficiency"  constants, which express the flux of each ionizable chemical achieved under actual experimental conditions (Overbeek & Lijklema,1959; C u r t i s , Perrin & Watkins,1960; Krnjevid, Mitchell... & Szerb,1963). At present, dose i s estimated i n terms of current f l u x , and devices which w i l l deliver r e l a t i v e l y constant amounts of current are available to minimize the effect of varations i n the e l e c t r i c a l resistance of the micropipette i n the course of the experiment. Spontaneous diffusion of drug can be checked by the application of a retaining current (of p o l a r i t y opposite to that of the ejecting current), which holds the active ion within the micropipette. Careful controls are necessary to rule out the effects of pH, e l e c t r i c a l current, and actions of the complementary drug i o n . Microelectrophoresis has p a r t i a l l y overcome the l i m i t a t i o n s of c l a s s i c a l  neurophar-  macological techniques, but additional modifications have been required"in i t s p r a c t i c a l application (Salmoiraghi & Bloom, 1964). The microelectrophoretic technique was fireit applied during studies i n v i t r o on chemical transmission at the neuromuscular junction (Fatt & K a t z , 1 9 5 l ) , where i t was possible to position several micropipettes independently under v i s u a l c o n t r o l . Record-  17  ing micro-electrodes could be inserted into muscle fibers  close  to an end plate (the neuromuscular synapse) or at some distance away along the muscle f i b e r . Micropipettes containing drugs were used to eject ions, d i r e c t l y at the end plate or at other points along the nerve and muscle f i b e r s .  It was possible,  therefore, under these experimental conditions, to compare the effects of  microelectrophoretically administered acetylcholine  with the effects of motor-nerve stimulation on the precise conductance (that i s , ion permeability) changes occuring i n the junctional area. In addition, the effects of known a c e t y l choline antagonists, cholinesterase  i n h i b i t o r s , and neuromuscu-  l a r blocking agents could be compared on the same parameters of transmission. These experiments were instrumental i n confirming the long-held thesis that the transmitter at the neuromuscular junction was acetylcholine. Recently this approach has been extended to the study of another set of peripheral cholinergic synapses, the sympathetic ganglia of the frog (Blackman & Ginsborg,1963)• The exploration of the brain with microelectrodes i s  es-  s e n t i a l l y aablimd procedure. Although a general area of the nervous tissue can be preselected by means of topographical landmarks, estimation, of the position of ithe electrode t i p r e l a t i v e to a given neurone must rest soley on observations of the nature of the recorded potentials  (Frank & Fuortes,  1955). Detection of neural units depends either upon spontaneous a c t i v i t y or a c t i v i t y induced by stimulation of a  18  specific neural pathway. The ease with which c e l l s can be encountered with the electrode t i p i s related i n part to the size and type of the microelectrode used and to the surgical or chemical means by which the animal has been made insensitive to the manipulations required for exposure and immobilization (Salmoiraghi & B l o o m , 1 9 6 4 ) . As Curtis ( 1 9 6 1 ) has observed, the mere demonstration of the effect  of an electrophoretically administered substance  on either spontaneous or induced a c t i v i t y does not immediately indicate which of the many possible c e l l u l a r s i t e s of drug action i s involved. Presynaptic and postsynaptic sites of drug action probably could be distinguished by simultaneous i n t r a c e l l u l a r recording and extracellular drug administration, i f only the method were of easier a p p l i c a b i l i t y . With current microelectrophoretic techniques there i s often a delay of several seconds between the onset of application and the recording of an effect,- a delay which makes i t d i f f i c u l t to distinguish between synaptic or  non-synaptic sites of action on the record-  ed nerve c e l l s and similar sites on a functionally related neighbor. Although this latency may be partly b i o l o g i c a l , i t may also be attributable i n part to accumulation of tissue debris around the t i p of the microelectrode. The use of pharmacological agents with synergistic and antagonistic effects has proved helpful i n characterizing suspected chemical transmitters.  However, the specific  effects  19  of the modifying drug upon the transmitter substance at a given central s i t e cannot be generalized to other central s i t e s without further proof, since i t i s well known that certain peripheral synapses operated by the same autonomic transmitter show a wide range of s p e c i f i c i t i e s and responsiveness  (Nickerson,1949).  To date, the best characterizations  of transmitters at central s i t e s has been obtained  i n studies  of the Renshaw c e l l , of t h e l a t e r a l geniculate nucleus, of neurones i n the cortex, and of the olfactory bulb, because i n each of these cases the state of physiological understanding  of l o c a l synaptic relations was s u f f i c i e n t l y f a r advanced  to allow comparative pharmacological studies. IV. Cholinergic and Dopaminergic C e l l s The caudate nucleus i s one region of the brain i n which the abundant existence of cholinergic synapses may reasonably be expected to occur. It contains high concentrations  of a c e t y l -  choline i t s e l f  (Peldberg  & Vogt,1948;  (Macintosh,1941) of choline acetylase  Hebb & S i l v e r , 1 9 5 6 ) and of acetylcholinesterase  (Nachmansohn,1940; Burgen & Chipman,195l). The l a t t e r enzyme i s p r i n c i p a l l y associated with the dense neuropil of the nucleus while the neurones remain unstained aration (Shute & Lewis,1963;  i n histochemical prep-  McLennan H .,unpublished observa-  t i o n s ) . Since the existence of acetylcholinesterase indicates that the neurone containing i t i s cholinergic, i t may be assumed that the c e l l s of the caudate receive cholinergic synapses, but are themselves non-cholinergic  i n action.  20  Studies i n which the release of acetylcholine from the nucleus has been measured (Mitchell & Szerb,1962; McLennan, 1964)  also suggest that cholinergic synaptic processes  occur  there. Dopamine (3-hydroxytyramine) was f i r s t demonstrated i n the brain by Montagu (1957) and shown to be a normal brain constituent (Carlsson,Rosengren,Bertler  & Nilsson,1957). It  was also found that 3>4 dihydroxyphenylalanine  decarboxylase  (DOPA decarboxylase) occurred i n brain (Dietrich,1953; Holtz & Westermann,1956) and i s generally believed responsible f o r the formation of dopamine from DOPA i n the body. Dopamine can then be converted to noradrenaline by hydroxylation i n the side chain, and i s considered to be the normal precursor of noradrenaline  (Blaschko,1957).  Puxe, Hflkfelt & Nilsson to yellow-green  (1964)  demonstrated a strong green  fluorescence appearing i n a l l parts of the  caudate nucleus, i n which the fibrae capsulae internae stood out as dark areas. This s p e c i f i c fluorescence completely  dis-  appeared some hours after the administration of reserpine (5mg/ kg) to the animals, and has been shown to be due to the presence of dopamine (Oarlsson,Falck & Hillarp,1962). When the histochemical reaction was perfect i t could be seen that f l u o r escent material was concentrated to very fine nerve fibers that had abundant v a r i c o s i t i e s with an intense fluorescence. The f i b e r s had the same general appearance as the nerve terminals found to contain very high concentrations of noradrenaline i n  21  other parts of the central nervous system  (Carlsson,Palck,  Fuxe,Hillarp,1964; Dahlstrflm & Fuxe,1964). From i t s l o c a l i z a t i o n i n the corpus striatum i t may  be  suggested that dopamine plays a part i n the function of these nuclei which constitute an important unit of the  extrapyramidal  system. It i s of interest to note that reserpine, a drug known to release both noradrenaline  and dopamine from the cat and  rabbit brains (Holzbauer & Vogt,1956; Carlsson et al..1957: Carlsson et al.,1958), has a marked influence on motor a c t i v i t y and may  cause a motor disturbance  similar to Parkinson's syn-  drone. On the other hand, excess of dopamine induced by admini s t r a t i o n of DOPA to animals, causes motor hyperactivity (Carlsson, et al.,1958). There i s thus evidence to suggest that brain dopamine i s involved i n the control of motor functions. In view of the close functional relationship which appears to exist between the corpus striatum and the cerebral cortex, i t does not seem impossible that the brain dopamine may  influence other  c o r t i c a l functions as well. McLennan (1964) demonstrated an increased output of dopamine from the caudate nucleus i n response to stimulation of nucleus centralis c e n t r a l i s (centromedianus) of the thalamus. It has also been shown that stimulation of the substantia nigra brings about a marked increase i n dopamine output from the putamen, although stimulation of nucleus c e n t r a l i s c e n t r a l i s had effect upon the output from the putaman (McLennan,1965)•  little  22  The observation that the substantia nigra appears to send f i b e r s to the corpus striatum (Carpenter,1961; Brodal,1963) i s i n agreement with the finding that the nigro-neostriatal dopamine neurones form one of the dominant ascending systems. They are  thought to arise from catecholamine c e l l groups i n the lower  brain stem as seen from the fact that 80% or more of the brain's entire dopamine content occurs i n the caudate nucleus and putamen (Carlsson,196l; Bertler,196l; Hornykiewicz,1964). S p e c i f i c a l l y , the presence of a very high dopamine content i n the caudate nucleus has been demonstrated both biochemically (Bertler & Rosengren,1959; Carlsson,1959; Bertler,1961; P o i r i e r & Sourkes, 1965) and histochemically (Carlsson,Falck & Hillarp,1962). Holtz,Osswald & Stock (i960) have suggested that dopamine i t s e l f may be of importance to the functions of the nuclei concerned, rather than i t s metabolic product noradrenaline which i s present only i n much smaller amounts. Evidence f o r dopaminecontaining neurones whose c e l l bodies l i e i n the pars compacta of the substantia nigra has been presented by And^n,Carlsson, Dahlstram,Puxe  et al.(l964) and Bertler (1961). The axons of  these neurones form bundles i n the crus cerebri and run i n the internal capsule to reach the neostriatum. The significance of these findings has been further strengthened by the observation that i n a l l mammals examined (mice, rats, guinea-pigs, cats, and monkeys) the substantia nigra contains a large nutaber of special nerve c e l l s storing a primary catechol-  23  amine, probably dopamine (Dahlstrflm & Fuxe,1964). Since i t has been shown that dopamine neurones exist i n mammals - namely i n the retina (Haggendal & Malmfors,1963) - i t seems possible that the., catecholamine containing nerve c e l l s i n the substantia nigra might be those which terminate i n the neostriatum. These observations would indicate the l i k l i h o o d that dopamine may  act as a chemical transmitter of synaptic function  within the caudate nucleus.  METHODS Experiments were performed on adult cats of both sexes (weighing about 3 k g . ) . In most cases the animals were anaesthetized with intravenous pentothal sodium and then decerebrated e l e c t r o l y t i c a l l y ; thereafter no pharmacological agents were administered systemically. The animal was mounted i n a stereotaxic apparatus (Horsley & Clark, 1908) and a cerveau isole preparation was performed by an inclined section of the brain stem at 6 0 ° along the plane of the bony tentorium. A constant body temperature device with a heating pad and anal probe was used to maintain a normal body temperature during the experiment (Appendix I ) . The head of the caudate nucleus was exposed on the dorsal aspect by removal of the overl y i n g cerebral tissue, approximately between frontal planes 10 and 20 (Jasper & Ajmone-Marsan, 1954). The exposed tissue was covered with a layer of dark polythene to prevent drying and to reduce cooling. The area l e f t free for the insertion of the micropipette was i r r i gated by a continuous drip of oxygenated Locke's solution modified by the omission of bicarbonate and warmed to 3 8 ° C (Appendix I I ) . Five barrelled microelectrodes were made from lengths of hard glass tubing, 6.5 mm external diameter, fused together at one end and separated at the other. The electrode was heated and drawn to a fine t i p i n an electrode p u l l e r .  25  The t i p was broken back to a diameter of 3.5-15.0 u.. This was done to ensure that the barrels did not have an excess i v e l y high e l e c t r i c a l resistance. The micropipettes were then f i l l e d by b o i l i n g i n d i s t i l l e d water. The central barrel was f i l l e d with 4M NaCl after removal of the water except at the t i p , by means of a syringe and fine polythene tube. The remaining four barrels were s i m i l a r l y f i l l e d with drugs. The micropipettes were then stored 24-36 hours i n a refrigerator at 2-5°C before use to allow diffusion of the dissolved substances into the t i p s . The resistance of each electrode was measured i n a 0.9$ NaCl splution, by employing a 6V battery with a 10 M ohm resistance i n series to a galvanometer from which the measurement was compared to a previously calculated graphi c a l plot of galvanometer reading against resistance. The central barrel was carried on an electrode holder (Appendix III)  similar to that described by Debroske, Anderson &  Crisp (i960), and was connected v i a a chlorided s i l v e r wire to an a . c .  coupled amplifier of short time constant  (2 msec).  One barrel was usually f i l l e d with a solution of NaDL-homocysteate (DLH)(0.2M,pH 8) or Na-L-glutamate (1.5M, pH 6) for the activation and l o c a l i z a t i o n of quiescent neu* rones. Another barrel contained acetylcholine chloride (Ach) (lM). The two remaining barrels were used to apply a number of other substances to the c e l l s , including atropine (10 mM), strychnine (10 mM), acetyl-|3-methylcholine (lM), tetramethylammonium (1.5M), hexamethonium (50 mM), 3-hydroxytyramine  26  (dopamine)(1M), dibenzyline (10 mM), dichloroisopropylnoradrenaline (10 mM) and noradrenaline (lM). One barrel of the five-barrelled electrode contained saline (lM NaCl) or a c i d i f i e d saline (0.1M HAc,pH 3.2) which was used as a control for distinguishing the interference with true drug effects from possible electronic effects of current. When amino acids were used, strong acid or a l k a l i was added to bring the pH well away from the i s o - e l e c t r i c point, as i n the experiments of Curtis & Watkins (i960). S i l v e r wires were inserted into the drug containing barrels and connected to a device for passing a constant current through a 1000 M ohm series resistance to each barr e l . The current was routinely measured, being monitored on a series galvanometer with an accuracy of ±5nA, and could be varied or reversed. The constant application of a suitable backing current to neutralize the efflux by diffusion of substances from the t i p of the pipettes (cf. del C a s t i l l o & Zatz, 1955) was also required. Bradley et al.(l966) reported that a current of 100 nA, the electrode being p o s i t i v e , sometime caused a reduction i n the rate of f i r i n g of a neurone unless the overall t i p diameter was < 4u-, when the opposite effect,  i.e.  excitation,  similar to that described by Strumwasser & Rosenthal (i960) was often obtained. When currents of 75nA or less were used with five-barrelled electrodes of 6-8p, overall t i p diameter, i t was usually a simple matter to make a clear d i s t i n c t i o n between the drug and current effects. The s e n s i t i v i t y of  27  brain stem neurones to applied current has been discussed by Curtis & Koizumi (1961). Control experiments i n which four barrels were f i l l e d with saline showed that similar effects were obtained by passing current through each barr e l (Bradley et al..1966). The actual amount of Ach or other materials  released  from the pipettes was not determined, but i f i t i s assumed that the transport number for Ach i n the pipettes used was the same as the mean value (0.4) found by Krnjevi 6, Mitchell & Szerb (1965) then the amount released would have been approximately 4 x 10~^[i moles/nA/sec. The position of the micropipette was controlled by a micromanipulator (Debroske et al.,1960) which allowed changes i n position i n three dimensional coordinates with v e r t i c a l movements i n steps of l.Ou. The microelectrode was i n serted into the caudate nucleus under v i s u a l c o n t r o l . Square wave stimuli of 0.1 msec duration were delivered to the nucleus v e n t r a l i s anterior thalami, nucleus centromedianus thalami, and substantia nigra through a concentric bipolar electrode introduced stereotaxically.  The amplified neuronal potentials  were displayed upon an oscilloscope and were also used to t r i g ger a pulse generator whose output could be integrated and d i s played upon a paper chart (Appendix IV). In some cases the t o t a l number of action potentials produced by b r i e f pulses of DLH were recorded using an electronic counter. Spikes on a slow time base were photographed v i a a beam i n t e n s i f i e r described by Pokrovsky  (i960).  28  • —  RESULTS  In these experiments, i n c o n t r a s t to those by Bloom,Costa & Salmoiraghi  described  (1965), o n l y a s m a l l  proportion  of neurones of the caudate e x h i b i t e d spontaneous a c t i v i t y . order  to l o c a t e c e l l s t h e r e f o r e , e i t h e r p u l s e s  10-30  nA)  or continuous c u r r e n t s  the b a r r e l c o n t a i n i n g DLH  (ca. 5 nA)  (0.5-1.0 sec,  were a p p l i e d to  as the e l e c t r o d e penetrated  t i s s u e . By t h i s means otherwise s i l e n t neurones were to f i r e ,  and  could e a s i l y be  were v e r y s e n s i t i v e to DLH,  l o c a t e d and and  In  the induced  i s o l a t e d . The  cells  f r e q u e n t l y were e x c i t e d by  the m a t e r i a l d i f f u s i n g from the t i p when the  "backing  current"  a p p l i e d to the e l e c t r o d e b a r r e l to prevent such d i f f u s i o n  was  turned o f f . A)  Cholinergic Cells Ach  s e n s i t i v e e e l l s were o n l y r a r e l y encountered  before  the e l e c t r o d e had been i n s e r t e d 200yu below the s u r f a c e of nucleus.  Thereafter  c e l l s were encountered which were e x c i t e d  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 Ach considerable  the  (15-100 nA)  latency. A correspondingly  but o n l y a f t e r a  lengthy p e r i o d of enhan-  ced f i r i n g succeeded the c e s s a t i o n of Ach  application (Fig.1).  A number of c e l l s were encountered at which Ach able to induce f i r i n g but the e x c i t a t i o n due  i t s e l f was  to DLH  was  not  enhanced.  An example o f a response of t h i s type i s i l l u s t r a t e d by Fig.2  .  29  on off on off 30 sec. P i g . 1 E x c i t a t i o n . o f a caudate neurone by DLH and by Ach: applied, i o n t o p h o r e t i c a l l y . Integrated record. Depth: 217ji.  800  -t  O  6 0 0 O  CD W "=n  400 -  CL \ CO  <D 2 0 0  CO CL  I  0  Pulse No. F i g . 2. Enhancement of DLH excitation produced by Ach. The ordinate shows the number of impulses e l i c i t e d from a neurone by current pulses (25 nA,;, 0.6 see) applied;, every 4 sec to the DLH b a r r e l . Depth 1205H.  O  31  As the e l e c t r o d e was a zone o f c e l l s was  f u r t h e r lowered i n t o the caudate  t y p i c a l l y encountered which again  neurones i n s e n s i t i v e to a p p l i e d Ach. one  i n which Ach  Beyond t h i s r e g i o n  a p p l i c a t i o n depressed  neurones ( F i g . 3 A ) . Since the c e l l s , r a r e l y spontaneously  as noted  induced by DLH  was  the responses of the above were o n l y  a c t i v e , t h i s d e p r e s s i o n was  m a n i f e s t when f i r i n g was  had  usually only  (Fig.3B).  The p e n e t r a t i o n s upon which these r e s u l t s are based were made r o u g h l y i n t o the center of the head of the nucleus, i . e . between f r o n t a l planes to depths 4 mm The  and  below the s u r f a c e  numbers of.Ach  shown i n Table  l a t e r a l planes  3.5-5.5 and  (Jasper & Ajmone-Marsan,1954).  s e n s i t i v e c e l l s and t h e i r d i s t r i b u t i o n  are  1. From t h i s r e s u l t i t seemed p o s s i b l e t h a t the  c e l l s responding depressed  16-19  by e x c i t a t i o n formed a lamina  surrounding  neurones, and t h a t a t g r e a t e r depths near the  ventro-  l a t e r a l s u r f a c e , and near the medial edge of the nucleus groups of c e l l s e x c i t e d by Ach might be  other  located. This expectation  i s s u b s t a n t i a t e d by the r e s u l t s presented  i n Fig.4  . Each p o i n t  i n the f i g u r e r e p r e s e n t s a group of c e l l s responding e x c i t a t i o n or by d e p r e s s i o n ;  the  either  by  i n d i v i d u a l i s o l a t e d c e l l s have not  been i n d i c a t e d . ( i ) E f f e c t of e l e c t r i c a l s t i m u l a t i o n McLennan (1964) r e p o r t e d t h a t s t i m u l a t i o n of nucleus  ventr-  ACh off  I  1 5 sec.  3A  Depressant actions of applied Ach. The effect upon, two neurones (whose action potentials appear of different size) f i r i n g spontaneously. Depth 155H.  33  O (D  40  co  CD CO  1 o DLH ACh ACh 15 70 off 3 0 I  DLH off 1  30 sec. l ? i g . 2BJ Depressant, actions of applied, Ach.-.. Integrated record showing the effect of Ach'applied with two intensities^ of current upon the discharge of a c e l l excited by DLH; Depth. 2205a-. :  34 1  TABLE I . D i s t r i b u t i o n , of Ach sensitive c e l l s : in. the centre of the head of the caudate nucleus.,: depths to 4 mm*  Total c e l l s studied.  :  427  Excited by Ach  :  4.7  Mean depth, of cells., excited  :  Depressed by Ach Mean.depth of. c e l l s  :  depressed  :  860M. (5% confidence limits-j : ± I65u) 40 2170^ (5J6 confidence limits', ± 350u.)  35  Caudate  Nucleus. —  i  i  1 I mm.  F i g . 4 Sketch of the caudate nucleus i n transverse section, at f r o n t a l plane 17. + indicates groups: of cells:; excited by, Ach and - groups, depressed by the a p p l i c a t i o n . The r e s u l t s of a number of experiments, have been combined,; andi i n d i v i d u a l c e l l s l y i n g outside the groups have note; been included.  36  alis  anterior thalami  (VA) enhanced the output o f Ach from  the caudate n u c l e u s . In the p r e s e n t s e r i e s o f experiments caudate neurones have been d e t e c t e d which responded to Ach and to VA s t i m u l a t i o n . The e l e c t r i c a l response o f one such c e l l to a s i n g l e shock a p p l i e d to VA i s i l l u s t r a t e d  in Fig.  5; together w i t h the enhancement o f f i r i n g which f o l l o w e d a b r i e f r e p e t i t i v e stimulus to the thalamus. T h i s c e l l  was  e x c i t e d by Ach. Depressant e f f e c t s due to VA e x c i t a t i o n have a l s o been found  ( F i g . 6 ) . In g e n e r a l i t has been found t h a t o n l y  cells  r e s p o n s i v e to Ach, whether by e x c i t a t i o n or d e p r e s s i o n , have responded to s t i m u l a t i o n o f VA,  and the e f f e c t s produced by  e l e c t r i c a l and by chemical s t i m u l a t i o n have always been i n the same d i r e c t i o n , ( i i ) Pharmacology Ach responses, whether e x c i t a t o r y or depressant, were b l o c k e d by the p r i o r i o n t o p h o r e t i c a p p l i c a t i o n o f a t r o p i n e to the neurones ( F i g . 7 ) , and both types o f response c o u l d be e l i c i t e d by a c e t y l - / 3 - m e t h y l c h o l i n e as e f f e c t i v e l y as by Ach i t s e l f . Although comparatively few t e s t s have been c a r r i e d olit, tetramethylammonium and n i c o t i n e d i d not s i m i l a r l y mimic *  the a c t i o n o f Ach, nor was hexamethonium I t may  able to prevent  t h e r e f o r e be concluded t h a t the r e c e p t o r s upon the  it.  A  V Stim.  100/sec. 200 CO  1  CL \ co  0  CO  0  E  T"  10  5  Pulse No  P i g . 5 Enhancement of DLH e x c i t a t i o n by stimulation of VA. The numbers of impulses e l i c i t e d by DLH pulses (30 nA, 0.8 sec, 1/4 sec) are plotted as ordinate. VA stimulation (10V, 0.1 msec, 100/sec) applied for 1 sec at the arrow. Inset shows the response of the c e l l to a single shock applied to VA ( t o t a l duration of sweep, 200 msec). This c e l l was excited by Ach applied with a current of 80 nA. Depth 226M-.  38  o  30 sec. Fig 1 . 6- Depressant effect of VA stimulation upon a neurone excited by DLH. At the second arrow, s t i m u l i (12 V,, 0.1 msec,- 100/sec) were applied to VA for 2 sec. Depth 1592u.  Depth: 5 8 4 yu A.  Depth: I 8 6 0 / J  Control After Atropine, 90 sec. at 70 nA.  °—o x—x  B.  i  ACh 45 nA.  °—o x—x  Control After Atropine, 120 sec.at 5 5 nA.  x-x  o  20 -i  CD CO  r 300  'VV  ACh 50nA  - 250 g •o cr CO  \ CO CD W  - 200 <J> \ "0 1-150 E  15 -  r? Q.  CO CD  io - 100  O  cr CD  P o xz o  5-  - 50 X  CO  b  I  20  40  X  1  60  o r x  *  r~ 80  - 1 —  100  120 I  5  10  -I  0  15  Pulse No. Seconds P i g . 7 Blocking action,of atropine upon: Ach e f f e c t s . A) at. a neurone excited by, Ach and B) at, a neurone depressed by Ach. In each case the c i r c l e s represent the effects of Ach before and the crosses a f t e r iontophoretic. application of atropine. A) Atropine a p p l i c a t i o n 70 nA, 9 0 sec. Depth 584iu B| Atropine application 55 nA,, 120 sec. Depth 1860|i.  40  caudate  neurones f a l l  i n t o t h e " m u s c a r i n i c " r a t h e r than the  " n i c o t i n i c " category. The e l e c t r i c a l responses  to stimulation  of VA c o u l d s i m i l a r l y be prevented by a t r o p i n e , although i n g e n e r a l the drug must be administered s y s t e m i c a l l y r a t h e r than i o n t o p h o r e t i c a l l y f o r t h i s e f f e c t t o be seen. An exam^ p i e o f the p a r a l l e l d e p r e s s i o n o f evoked and Ach induced f i r i n g i s shown i n F i g . 8 . As noted a l s o by Bloom, jet al. (1965) a n a e s t h e t i c agents g r e a t l y reduced the e x c i t a t o r y e f f e c t s o f Ach upon neurones. In animals  caudate  l i g h t l y anaesthetized with pentobarbital  (35 mg/kg) o r w i t h D i a l  (65 mg/kg) depressant a c t i o n s o f Ach  could s t i l l be o b t a i n e d b u t o n l y a very o c c a s i o n a l e x c i t a t o r y response was d e t e c t e d . The depressant e f f e c t s were as r e a d i l y elicited  i n a n a e s t h e t i z e d as i n unanaesthetized animals, and  the d i s t r i b u t i o n o f such c e l l s w i t h i n the nucleus was u n a l t e r e d . B) Dopaminergic C e l l s The d i s t r i b u t i o n o f dopamine s e n s i t i v e c e l l s d i d not f o l l o w the laminar s t r u c t u r e o f the Ach s e n s i t i v e c e l l s . The caudate number  c e l l s which were e x c i t e d by dopamine were few i n (7%) compared t o those which were depressed  (64%).  Table I I shows the numbers o f dopamine s e n s i t i v e c e l l s and those u n a f f e c t e d . The dopamine s e n s i t i v e c e l l s were q u i t e randomly l o c a t e d and were r a r e l y encountered  b e f o r e the e l e c t -  41 »  Control  After  i  Atropine  1  I sec.  50 msec. Fig.  8 The effect of systemically administered atropine upon Ach enhanced f i r i n g and upon the evoked response to VA stimulation. A , C and E.before; K,, D and F 10 min after 0.1 mg/kg atropine sulphate I . V . A and C controls; B and D during iontophoretic a p p l i c a t i o n of Ach (75 nA). The large v e r t i c a l deflexions represent the beginning and end of the DLH pulses (20 nA, 0.8 sec) i n each record. In E and F single s t i m u l i (15 V,. 0.1 msec) were delivered to VA. Depth 1475|i.  TABLE II  Distributions of dopamine sensitive c e l l s i m the centre the head of the caudate nucleus,depths; to 4 mm.  Total cells... studied-  152  Excited by dopamine  11  Depressed by, dopamine  9L7.  Not affected by, dopamine  44  43  rode had The  p e n e t r a t e d 300yj. below the s u r f a c e of the  s t e r e o t a x i c coordinates  the head o f the  DLH;  f o r these p e n e t r a t i o n s  s e n s i t i v e c e l l s . The  dopamine dep-  c e l l s were i d e n t i f i e d i n i t i a l l y by a p p l i c a t i o n of  t h e r e a f t e r dopamine (50-100 nA)  applied  was  i o n t o p h o r e t i c a l l y :\  (Fig.9B,C).  ( i ) E f f e c t of e l e c t r i c a l An  into  caudate nucleus were s i m i l a r to those men-  t i o n e d above f o r the Ach ressed  nucleus.  increased  stimulation  output of dopamine from the  caudate nucleus  i n response to s t i m u l a t i o n of nucleus centromedianus (CM) the thalamus has  been r e p o r t e d  by McLennan (1964). In  p r e s e n t study, caudate neurones which were induced to by DLH  responded to CM  i c a l l y a p p l i e d dopamine (50-90 nA)  Carpenter  CM  fire  iontophoret-  (Fig.9B). E x c i t a t o r y  resp-  s t i m u l a t i o n were r a r e l y observed.  (1961) and  a r i s i n g i n the s u b s t a n t i a striatum.  the  s t i m u l a t i o n by a depressant response  (Fig.9A). The ,same c e l l s were a l s o depressed by  onses evoked by  of  Brodal  (1963) have d e s c r i b e d  n i g r a and  terminating  i n the  fibers corpus  Present experiments have shown t h a t upon e l e c t r i c a l  s t i m u l a t i o n of the response was  substantia nigra a c h a r a c t e r i s t i c excitatory  produced  (Fig.9D,llB). This e l e c t r i c a l  response could be b l o c k e d by (50-100 nA)  excitatory  i o n t o p h o r e t i c a l l y a p p l i e d dopamine  (Fig.9D) i n about one-half of the c e l l s examined,  44  DLH 35 on  STIM STIM DLH 5/sec off off on  40-i  DLH 35 orr Fig. 9  Dopo Dopo DLH 90 off off on  A) Integrated response of a caudate c e l l showing the effect produced by stimul ation of CM ( 8 V , 0.1 msec). B) Integrated response of the same c e l l induced to f i r e with DLH (35 nA) and depressed by dopamine (90 nA). Depth 300u.  lOO-i V  o-J  I  1 20msec  lOO-i  O-  Pig.  3  1  C) Oscilloscope trace showing the depressant effect on; c e l l f i r i n g (induced by DLH, 7 nA) of dopamine 75 nA. D) An evoked response from the same c e l l produced by stimulation. (8V, 0.1 msec) of the substantia nigra which i s also blocked by dopamine 75 nA. Depth 557u..  46  but was u n a f f e c t e d i n the remainder. ( i i ) Pharmacology The c h a r a c t e r i s t i c dopamine depressant response e x h i b i t e d by caudate  (Fig.9C)  c e l l s was q u i t e s p e c i f i c i n i t s a b i l i t y  to be b l o c k e d by p h a r m a c o l o g i c a l agents. T y p i c a l r e s u l t s obt a i n e d upon a neurone depressed by dopamine are shown i n F i g . 10A,B. The dopamine d e p r e s s i o n was not b l o c k e d by d i c h l o r o i s o propyl-noradrenaline dibenzyline  (DCI) (Fig.10A) but was prevented by  (Fig.10B). Another  i l l u s t r a t i o n o f the d i b e n z y l i n e  blockade o f dopamine d e p r e s s i o n i s shown i n F i g . l l A . In t h i s figure,  a f t e r the drug has been a p p l i e d the DLH response i s  measured and the change i n amplitude demonstrates  the e f f e c t -  i v e n e s s o f the b l o c k o b t a i n e d . These r e s u l t s would thus ate t h a t some r e c e p t o r s upon caudate neurones conform "CX-adrenergic"  indic-  t o the  category, s i n c e the d i b e n z y l i n e b l o c k i s s p e c i f i c  to these r e c e p t o r s (Goodman & Gilman,  1965).  47 50-|  u  t t 1t  Q) </>  3 a.  £  DCI on 85  DLH on DCI Dopa Dopa 3 0 off on off 55  DLH off  10 s e c 50-|  u a) </> </>  <n 3  a. E  t t  Dibenzyline on 90  DLH on 30  Dopa on 55  Dopg off  Dibenzyline off F i g . 1 0 Depressant effects; of dopamine. A) Integrated response of. a caudate c e l l i n which DCI (85 nA) waa; unable to block the dopamine induced; depression. B;) Integrated response of the same c e l l with the dopamine depression blocked by dibenzyline (90 nA). Depth.4311*.  O-H30 © 3 |  DLH  25 Dopa 20 on  Dibenzyline 45 on  DLH 25 Dopa off  Dibenzyline off  10 sec  i  i  10msec  Pig.  11  A) A dopamine induced depression blocked by dibenzyline ( 4 5 nA). Depth 1718jx. B) Evoked response from caudate c e l l (stim 8V 0.1 msec, applied to substantia n i g r a ) . Depth 1841|X.  DISCUSSION A)  Cholinergic Cells The  to the  types of response e x h i b i t e d by caudate neurones  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 Ach  which have been d e s c r i b e d  resemble those  f o r c e r t a i n other  cells  the c e n t r a l nervous system - neurones of the (Krnjevic & P h i l l i s ,  1965). The  neocortex  1963a,b), v e n t r a l thalamus  & C u r t i s , 1964a,b) and b r a i n stem (Bradley &  within  (Andersen  Wolstencroft,  s i m i l a r i t y to c o r t i c a l c e l l s i s p a r t i c u l a r l y  c l o s e , i n t h a t the a c t i o n of Ach "muscarinic"  i n character,  a c t i o n of a t r o p i n e  appeared to be  purely  as e x e m p l i f i e d by the  blocking  (Dale,1914) and by the f a c t t h a t i t s  e f f e c t s were reproduced as p o w e r f u l l y choline. " N i c o t i n i c "  stimulants  by  acetyl-yB-methyl-  ( n i c o t i n e and  tetramethyl-  ammonium) or b l o c k i n g agents (hexamethonium) have been found  ineffective. C h a r a c t e r i s t i c of t h i s "muscarinic"  response are  slow onset o f a c t i o n and p e r s i s t e n t e f f e c t of Ach c e s s a t i o n o f the  the  after  i o n t o p h o r e t i c a p p l i c a t i o n , which i s i n  c o n t r a s t to the r a p i d e f f e c t observed at the  nicotinic  c h o l i n e r g i c synapses upon the Renshaw c e l l s of the s p i n a l cord  ( C u r t i s & E c c l e s , 1958a,b; Andersen & C u r t i s , 1964a).  Krnjevic & P h i l l i s  (1963) have noted t h a t the prolonged  50  a f t e r e f f e c t c o u l d suggest a lengthy attachment of Ach the c h o l i n o c e p t i v e elements of the c e l l onset  ; however the  to slow  c o u l d a l s o imply the e x i s t e n c e of e x t e n s i v e b a r r i e r s  to d i f f u s i o n and the a f t e r e f f e c t c o u l d then be due c o n t i n u i n g c o n c e n t r a t i o n o f Ach maintained  i n the  o f the r e c e p t o r s and need not mean a continued  to a  vicinity  combination  w i t h them. Although o n l y a few o b s e r v a t i o n s have been made, a n t i c h o l i n e s t e r a s e agents ( e s e r i n e and neostigmine) have been found to have l i t t l e Ach,  i n f l u e n c e upon the response to  agreeing with K r n j e v i c & P h i l l i s '  c o n c l u s i o n s t h a t the  a p p l i e d m a t e r i a l i s i n some manner p r o t e c t e d from enzymatic destruction. The  questions whether or not such "muscarinic"  as these r e p r e s e n t an a c t i o n of Ach upon subsynaptic and  i f so whether Ach  responses receptors,  i s the p h y s i o l o g i c a l l y a c t i v e t r a n s m i t t e r  are not easy to determine, w i t h i n the c e r e b r a l c o r t e x , where the deep pyramidal  c e l l s appear to be those most commonly  a f f e c t e d by:Ach ( K r n j e v i ^ & P h i l l i s , 1 9 6 3 ) , there i s histochemi c a l evidence  to suggest t h a t these r e c e i v e a c h o l i n e r g i c  i n e r v a t i o n by f i n e f i b e r s  ( K r n j e v i c & Silver,1965)  which  Bishop (1961) d e s c r i b e d as a p r i m i t i v e system, and which be the f i n a l l i n k i n the ascending  may  r e t i c u l a r a c t i v a t i n g system  (Shute & Lewis,1963). I t seems l i k e l y t h e r e f o r e t h a t i n c e r t a i n  51  c e l l s where the a c t i o n o f a p p l i e d Ach i s m u s c a r i n i c i t can indeed mimic a s y n a p t i c event. T h i s may  not be t r u e  i n circumstances/ p o s s i b l y r a r e , where both  nicotinic  and m u s c a r i n i c r e c e p t o r s occur upon the same c e l l , at  the Renshaw c e l l  as  ( C u r t i s & R y a l l , 1964) . In t h i s case  •  there i s evidence t h a t the slower m u s c a r i n i c type o f response may  not be o f p h y s i o l o g i c a l s i g n i f i c a n c e  (Curtis  & Ryall,1966). The  f i n d i n g t h a t Ach may  e x e r t both e x c i t a t o r y  and  depressant a c t i o n s upon d i f f e r e n t groups of c e l l s i n the caudate, to  and t h a t there are many other neurones which  fail  respond to Ach at a l l , strengthens the s u g g e s t i o n t h a t  the a c t i o n s of t h i s substance  are not u n s p e c i f i c a l l y e x e r t e d  but r a t h e r depend upon the presence o f s p e c i a l i z e d r e c e p t o r s (Curtis,1963). Anatomical evidence f o r a c o n n e c t i o n from VA t o the corpus s t r i a t u m (caudate nucleus and putamen) has been p r e s e n t e d by Von Monakow (1895), who  a p p a r e n t l y regarded  the f i b e r s as s t r i a t o f u g a l . However, l a t e r workers  felt  t h a t t h a l a m o s t r i a t e f i b e r s a r i s i n g from t h i s nucleus  may  be p r e s e n t as w e l l . The evidence a l s o suggests a c o n s i d e r a b l e c o r t i c a l p r o j e c t i o n from VA, regions  p a r t i c u l a r l y t o the  frontal  (Freeman & Watts,1947) and i t i s t h e r e f o r e p o s s i b l e  52  t h a t the e f f e c t s observed are a t t r i b u t a b l e to an i n d i r e c t a c t i o n through the c o r t e x r a t h e r than t o a d i r e c t one from the nucleus i t s e l f p r o j e c t i n g t o the caudate. A c l e a r r e l a t ion  between c o r t i c a l and caudate a c t i v i t y f o l l o w i n g  stimul-  a t i o n o f VA has been demonstrated by McLennan (1964).  Further-  more, evidence f o r a c o r t i c a l p r o j e c t i o n t o the caudate n u c l eus, again e s p e c i a l l y from the f r o n t a l r e g i o n s , accepted  (Walker,Andy  now seems  & Poggio,1955).  The r e s u l t s o f the p r e s e n t i n v e s t i g a t i o n imply t h a t the p r o j e c t i o n from VA t o the caudate nucleus may have a f i n a l c h o l i n e r g i c step,  and f u r t h e r t h a t e x c i t a t i o n o f VA  may lead t o e x c i t a t i o n o f some neurones o f the caudate and to i n h i b i t i o n o f o t h e r s .  Thus neurones have been  located  which respond t o s t i m u l a t i o n o f VA e i t h e r by e x c i t a t i o n o r depression  and the responses are q u a l i t a t i v e l y s i m i l a r f o l -  lowing a p p l i c a t i o n o f Ach. In a d d i t i o n , e a r l i e r evidence demonstrated t h a t VA s t i m u l a t i o n enhanced  the output o f Ach  from an i r r i g a t e d p o r t i o n o f the nucleus (McLennan,1964). The s i m i l a r i t y i n b l o c k i n g o f Ach e f f e c t s and those o f e l e c t r i c a l s t i m u l a t i o n o f VA f o l l o w i n g the a d m i n i s t r a t i o n  of atro-  pine supports the same view. Thus f i v e o f the c r i t e r i a r e q u i r e d ment o f t r a n s m i t t e r  function  f o r the e s t a b l i s h -  (McLennan,1963) appear t o have  53  been s a t i s f i e d . T h i s i s demonstrated by the f i n d i n g of high concentrations of a c e t y l c h o l i n e of c h o l i n e a c e t y l a s e  (Macintosh,1941),  (Feldberg & Vogt,1948; Hebb & S i l v e r ,  1956), and o f a c e t y l c h o l i n e s t e r a s e  (Nachmansohn,1940; Bur-  gen & Chipman,1951) taken w i t h the enhanced output o f Ach from the caudate upon s t i m u l a t i o n o f VA  (McLennan,1964),  i o n t o p h o r e t i c a l l y a p p l i e d Ach mimicking the response o f s t i m u l a t i o n o f the same neurone,  and f i n a l l y the a c t i o n o f  p h a r m a c o l o g i c a l agents which i n t e r f e r r e d both w i t h the opera t i o n o f the neurone and the a c t i o n o f a r t i f i c a l l y  applied  Ach. A m o r p h o l o g i c a l c o r r e l a t i o n f o r the d i s t r i b u t i o n o f the two types o f neurone w i t h i n the caudate, which  may  correspond to the two Ach responses d e s c r i b e d here may e x i s t . As s t a t e d e a r l i e r , C a j a l  (1955) d e s c r i b e d two  c e l l formations which correspond to the caudate ( p l a t e s 4-6)  also  distinct  sections  s t a i n e d by the G o l g i method (Moliner,1957). .,  The two d i s t i n c t types o f c e l l s demonstrate  a distribution  which i s i n f a i r agreement w i t h the e l e c t r o p h o r e t i c data presented. The f i n d i n g t h a t o n l y a s m a l l p r o p o r t i o n of the t o t a l c e l l s t e s t e d have been found to respond to.Ach i s a r e s u l t a t v a r i a n c e w i t h t h a t r e p o r t e d by Bloom e t al.. (1965). These  authors a l s o d e s c r i b e d t h a t the m a j o r i t y o f the c e l l s encountered i n u n a n e s t h e t i z e d animals were spontaneously a c t i v e . The o n l y major d i f f e r e n c e between the experiments of Bloom e t a l . and those d e s c r i b e d here appears to have been t h a t the c o r t e x o v e r l y i n g the caudate was  left  intact  by the former a u t h o r s . Depths o f p e n e t r a t i o n i n t o the n u c l eus are not i n d i c a t e d by Bloom est a l . ,  so t h a t a more p r e c i s ,  comparison o f the two s e t s o f d a t a i s not p o s s i b l e ; however, it  i s c o n c e i v a b l e t h a t t h e i r p e n e t r a t i o n s extended o n l y t o  the zone o f c e l l s found i n the p r e s e n t experiments t o be e x c i t e d by Ach. B) Dopaminergic  Cells  The demonstration by f l u o r e s c e n t microscopy t h a t the catecholamines of the b r a i n are l o c a l i s e d w i t h i n nerve  fiber  ( C a r l s s o n e t al.,1962; Falck,1962) has strengthened e a r l i e r p r o p o s a l s t h a t dopamine i s a neurohumoral mediator o f the c e n t r a l nervous system  ( B r o d i e , S p e c t o r & Shore,1959;  b a l l e r , 1 9 5 9 ; Vogt,1959).  I t i s also of considerable  Rothinterest  t h a t i n the caudate and l e n t i f o r m n u c l e i o f the dog, r a t , monkey, and human the c o n c e n t r a t i o n s o f dopamine are 20 to 50 times those o f n o r e p i n e p h r i n e , whereas i n most o t h e r c e r e b r a l c e n t e r s and p e r i p h e r a l a d r e n e r g i c nerves dopamine accounts f o r o n l y one t h i r d t o one h a l f of the t o t a l  catechola-  55  mines. Thus dopamine may  have f u n c t i o n s o t h e r than as  a source of n o r e p i n e p h r i n e a t such s i t e s  (Holtz e t a l . ,  1960). Bloom,Costa & S a l m o i r a g h i (1965) d e s c r i b e d c e l l s i n the caudate nucleus which e x h i b i t e d a predominant s i o n o f spontaneous  depres-  u n i t d i s c h a r g e upon 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 dopamine. A s i m i l a r d i s t i n c t i v e d e p r e s s i o n was observed i n the p r e s e n t experiments. The f a c t t h a t dopamine depressed spontaneously a c t i v e c e l l s as w e l l as DLH  induced  responses strengthens the v a l i d i t y o f t h i s o b s e r v a t i o n . Chara c t e r i s t i c of the dopamine response i s the r a p i d onset o f a c t i o n and quick r e c o v e r y o f the c e l l a f t e r c e s s a t i o n of 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 , although spontaneously  firing  c e l l s recovered much more s l o w l y . The v e r y s m a l l number of dopamine induced e x c i t a t o r y responses may  i n d i c a t e t h a t the  i n i t i a l e f f e c t of dopamine upon s u b s y n a p t i c r e c e p t o r s may i n v o l v e a b r i e f p e r i o d of e x c i t a t i o n f o l l o w e d by d e p r e s s i o n . I t i s p o s s i b l e t h a t the e x c i t a t o r y response i n many cases has been masked by the DLH Ahlquist pathomimetic  e x c i t a t i o n used to l o c a t e  cells.  (1948) c l a s s i f i e d the r e c e p t o r s i t e s of sym-  drug a c t i o n as of and  fi,  on the b a s i s of t h e i r  responses to a v a r i e t y of sympathomimetic amines and t o a d r e n e r g i c b l o c k i n g agents. The a d r e n e r g i c b l o c k i n g  action  56  o f the compounds s t u d i e d , d i b e n z y l i n e and DCI,  are  rea-  sonably s p e c i f i c f o r of and y3 r e c e p t o r blockade. So s e l e c t i v e are these b l o c k i n g agents t h a t i t has become customary to i d e n t i f y the type of a d r e n a l i n e  receptors  i n a t i s s u e on  the b a s i s o f the e f f e c t of the a d r e n e r g i c b l o c k i n g agents on the responses to sympathomimetic amines. However, the above c l a s s i f i c a t i o n of a d r e n e r g i c  receptors  applies only  to the p e r i p h e r a l and c a r d i a c a c t i o n s of the drug.. Receptors f o r the c e n t r a l nervous system (CNS) e r g i c drugs cannot be to the  large overlap  simply  a c t i o n of v a r i o u s  adren-  c l a s s i f i e d intoo( andj& types  i n a c t i o n o f the two  groups i n the  due CNS  (Goodman & Gilman,1965). However, as the dopamine d e p r e s s i o n by d i b e n z y l i n e , an Of-adrenergic by DCI  b l o c k e r , but was  ( ^ - a d r e n e r g i c b l o c k i n g agent),  i s i m p l i e d . The p r e s e n t  c o u l d be  not  an<j(type of  blocked  receptor  r e s u l t s appear to c o n t r a d i c t the  g e n e r a l r u l e t h a t di' r e c e p t o r s  are e x c i t a t o r y and  i n h i b i t o r y , although there are other exceptions man  blocked  receptors known^ Good-  & Gilman,1965). The  anatomical evidence f o r a major e f f e r e n t pathway  of the nucleus centromedianus (CM) has been presented  to the corpus s t r i a t u m  by Vogt & Vogt{1941). However, there i s  u n c e r t a i n t y about whether both the caudate nucleus and putamen  57  r e c e i v e the p r o j e c t i o n f i b e r s . Some authors (eg. Powell & Cowan,1956) have f a i l e d t o f i n d evidence f o r connections from CM t o caudate; w h i l e o t h e r s (Hassler,1948) have r e p o r t e d t h a t the l a r g e - c e l l e d p o r t i o n o f the nucleus p r o j e c t s o n l y to  the caudate. Evidence p r e s e n t e d by l a t e r workers  would  f a v o r the l a t t e r view, e s p e c i a l l y the f i n d i n g t h a t a s p e c i f i c l i b e r a t i o n o f dopamine was o b t a i n e d from the caudate nucleus when CM was s t i m u l a t e d  (McLennan,1964). P r e s e n t r e s u l t s dem-  o n s t r a t e d a d e p r e s s i o n o f caudate s i n g l e c e l l a c t i v i t y upon s t i m u l a t i o n o f CM, thus i n d i c a t i n g t h a t t h i s pathway may have a f i n a l  i n h i b i t o r y s t e p . The p a r a l l e l i s m i n the depres-  s i o n response by dopamine i n the DLH induced c e l l , and the d e p r e s s i o n by CM s t i m u l a t i o n i n the same c e l l would a l s o tend to  support the i d e a o f a f i n a l  in this fiber  i n h i b i t o r y dopaminergic step  tract.  The f a c t t h a t the s u b s t a n t i a n i g r a appears t o send f i b e r s t o the corpus s t r i a t u m  (Carpenter,1961;  Brodal,1963)  strengthens the e a r l i e r view t h a t the n i g r o - n e o s t r i a t a l dopamine neurones form one o f the dominant  ascending systems. The  p r e s e n t experiments would suggest t h a t t h i s n i g r o - n e o s t r i a t a l t r a c t causes e x c i t a t o r y responses a t the caudate t e r m i n a t i o n . However, the e f f e c t i v e blockage o f the e x c i t a t o r y SN evoked response 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 o f dopamine would  indie-  58  ate a p o s s i b l e dominance o f a thalamic  i n h i b i t o r y neural  mechanism over the e x c i t a t o r y i n p u t operated v i a a dopaminergic l i n k , as i l l u s t r a t e d by the i n p u t of the s u b s t a n t i a n i g r a . T h i s i s supported by the f i n d i n g t h a t of the e x c i t a t ory c e l l s responding t o SN s t i m u l a t i o n , h a l f are a l s o depressed by dopamine. Furthermore, the n o n - s p e c i f i c l o c a t i o n of the dopamine s e n s i t i v e c e l l s would i n d i c a t e a wide d i s t r i b u t i o n c o i n c i d i n g w i t h t h e i r p o s s i b l e dominant i n h i b i t o r y r o l e i n the caudate nucleus.  However, the q u e s t i o n  o f why  30% o f  the c e l l s were t o t a l l y u n a f f e c t e d by dopamine i s not easy t o e x p l a i n i f a widespread dopaminergic i n h i b i t o r y mechanism i s operative. The i d e n t i f i c a t i o n o f the s p e c i f i c f l u o r e s c e n c e i n very f i n e nerve f i b e r s of the caudate due t o dopamine sson,Falck in brain  & Hillarp,1962); (Dietrich,1953;  (Carl-  the f i n d i n g o f dopa decarboxylase  H o l t z & Westermann,1956); the demon-  s t r a t e d i n c r e a s e d output o f dopamine from the caudate nucleus i n response t o CM s t i m u l a t i o n  (McLennan,1964); and the p r e s e n t -  l y observed i o n t o p h o r e t i c a p p l i c a t i o n o f dopamine mimicking the e l e c t r i c a l response would i n d i c a t e t h a t four o f the c r i t e r i a r e q u i r e d f o r the establishment  of t r a n s m i t t e r  (McLennan,1963) appear t o have been s a t i s f i e d .  function  59  BIBLIOGRAPHY  A h l q u i s t R.P. (1948) A study o f a d r e n o t r o p i c r e c e p t o r s . Amer.J.Physiol. 153,586-600. A k e r t K. & Andersson B. 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NaHCO, and a r e d u c t i o n ! i m the c o n c e n t r a t i o n : o f KC1 ( t o equal t h a t i n i tha c e r e b r o s p i n a l f l u i d ) .  APPENDIX III  Stereotaxic, apparatus and electrode  holder.  Galvanometer Preamp DC Supply  "  Iontophoresis Unit  Notch Filter  122  Band Pass Filter  Camera Scope  Stirr u US Isol at ion U  O  JL Saw Tooth Generator  Grass Stim. ( A )  Pulse Generator Grass Stim. (B)  Stimulus Isolation Unit  Integrator  > TJ  Paper Chart  m z o X  

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