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Acetylcholinesterase and the basal ganglia : from cytology to function Lehmann, John 1980

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ACETYLCHOLINESTERASE AND THE BASAL GANGLIA FROM CYTOLOGY TO FUNCTION  by  JOHN LEHMANN B.Sc,  California Institute  o f Technology,  1974  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in... THE FACULTY OF GRADUATE STUDIES (Interdisciplinary  We a c c e p t t h i s t h e s i s to the r e q u i r e d  Studies)  as  conforming  standard  THE UNIVERSITY OF BRITISH COLUMBIA May 1980 John Lehmann, 1980  In p r e s e n t i n g t h i s  thesis  an advanced degree at  further  fulfilment  of  the  requirements  the U n i v e r s i t y of B r i t i s h Columbia, I agree  the L i b r a r y s h a l l make it I  in p a r t i a l  freely  available  for  agree t h a t p e r m i s s i o n for e x t e n s i v e copying o f  of  representatives.  this  thesis for  It  financial  this  thesis  g a i n s h a l l not be allowed without my  of  The U n i v e r s i t y o f B r i t i s h Columbia 2075  Wesbrook  Vancouver, V6T  6  1W5  Place  Canada  or  i s understood that copying or p u b l i c a t i o n  written permission.  Department  that  reference and study.  f o r s c h o l a r l y purposes may be granted by the Head of my Department by h i s  for  ii  ABSTRACT  B i o c h e m i c a l , a n a t o m i c a l , and h i s t o c h e m i c a l s t u d i e s were performed i n t h e b a s a l g a n g l i a w i t h an emphasis on the l o c a l i z a t i o n o f t h e enzyme a c e t y l c h o l i n e s t e r a s e dopaminergic  (AChE).  The e x i s t e n c e of t h e enzyme i n  n i g r o - s t r i a t a l neurons was demonstrated.  Descending  s t r i a t o - n i g r a l and p a l l i d o - n i g r a l axons d i d not c o n t a i n d e t e c t a b l e amounts of AChE. cellularis,  A c e l l group  c a l l e d t h e n u c l e u s b a s a l i s magno-  i n t i m a t e l y a s s o c i a t e d w i t h the globus p a l l i d u s ,  found t o c o n t a i n h i g h l e v e l s of AChE; f u r t h e r m o r e , these  was  neurons  were shown t o be t h e source of a c h o l i n e r g i c p r o j e c t i o n t o t h e neocortex. In  the s t r i a t u m , l a r g e neurons  AChE were found t o be l i k e l y of  the striatum.  c o n t a i n i n g h i g h l e v e l s of  c a n d i d a t e s as..the c h o l i n e r g i c  neuron  C h o l i n e r g i c p e r i k a r y a were found t o be absent  i n t h e neocortex; nor were p e r i k a r y a s y n t h e s i z i n g l a r g e amounts of AChE found i n the n e o c o r t e x .  An e m p i r i c a l h y p o t h e s i s was f o r m u l a t e d  on the b a s i s of these and other f i n d i n g s r e g a r d i n g c h o l i n e r g i c neurons:  High l e v e l s of AChE a r e a n e c e s s a r y but not s u f f i c i e n t  criterion for identifying cholinergic  perikarya.  iii  TABLE OF CONTENTS Abstract  i i  T a b l e of Contents  i i i  L i s t of f i g u r e s  iv  Acknowledgements  v  GENERAL INTRODUCTION  1  a) H i s t o r i c a l overview of AChE  1  b) The b a s a l g a n g l i a and c h o l i n e r g i c f u n c t i o n  8  c) B r i e f d e s c r i p t i o n of r e s e a r c h rationale  and  GENERAL DISCUSSION  10  13  a) Review of new c o n t r i b u t i o n s r e p o r t e d in this thesis  13  b) The r o l e of the s t r i a t u m i n motor and psychological function  15  c) Psychopharmacology of t h e s t r i a t u m  17  1) .Psychopharmacology of dopamine i n the s t r i a t u m  17  2) Psychopharmacology of a c e t y l c h o l i n e i n the s t r i a t u m  18  d) I n t e r a c t i o n s between dopamine and a c e t y l choline  19  1) P s y c h o p h a r m a c o l o g i c a l s t u d i e s  19  2) Measurement of b i o c h e m i c a l parameters following pharmacological manipulations  20  e) The c y t o l o g i c a l r e l a t i o n s h i p between the dopaminergic and the c h o l i n e r g i c neuron  21  REFERENCES  26  APPENDIX  35  iv  LIST OF FIGURES F i g u r e 1. T r a d i t i o n a l c y t o l o g i c a l model e x p l a i n i n g dopamine:.- a c e t y l c h o l i n e i n t e r a c t i o n s  24  F i g u r e 2. A l t e r n a t i v e c y t o l o g i c a l model proposed to e x p l a i n t h e same dopamine - a c e t y l c h o l i n e interactions  25  V  ACKNOWLEDGEMENTS  My f i r s t  thanks go t o a l l t h e members, p a s t  Division of Neurological part.  As a c o o p e r a t i v e  Sciences,  and p r e s e n t , o f t h e  o f which i t was an honour t o be a  u n i t w i t h the goal of advancing  scientific  knowledge, a l l t h e members o f the D i v i s i o n s e t examples and gave me help  and encouragement.  S p e c i a l thanks go to my s u p e r v i s o r ,  F i b i g e r , f o r h i s uncanny a b i l i t y  Dr. C h r i s  to m o t i v a t e and d i r e c t my work.  The  a c t i n g Department head, Dr. E d i t h McGeer, was i n s p i r a t i o n a l through g i v i n g me a sense o f p e r s p e c t i v e ible  source o f new i d e a s .  A m e l i a Wong, and B e t t y  i n n e u r o s c i e n c e , and a s an i n e x h a u s t -  Our groups' t e c h n i c a l s t a f f , S t e l l a  Richter, not only  taught me a g r e a t  Atmadja,  deal of  p r a c t i c a l use, b u t we worked t o g e t h e r so t h a t a t times t h e s c i e n t i f i c g o a l was t h e secondary reward.  The o t h e r graduate s t u d e n t s o f t h e  D i v i s i o n , always ready f o r d i s c u s s i o n amount of academic s t i m u l a t i o n .  or debate, s u p p l i e d  the g r e a t e s t  F i n a l l y , my thanks go o u t to a l l t h e  members o f my committee and t e a c h e r s o f c o u r s e s . S p e c i f i c acknowledgements f o r each of t h e p u b l i c a t i o n s  listed  i n t h e appendix of t h i s t h e s i s a r e as f o l l o w s : P u b l i c a t i o n A ) : S. Atmadja f o r l e s i o n s , h i s t o l o g y , photomicroscopy; H.C. F i b i g e r f o r d i s s e c t i o n s , photomicroscopy.  P u b l i c a t i o n B ) : S. Atmadja f o r l e s i o n s ; H.C. F i b i g e r  f o r d i s s e c t i o n s ; L . L . Butcher f o r h i s t o l o g y and p h o t o m i c r o s c o p y . P u b l i c a t i o n C ) : J . I . Nagy f o r l e s i o n s and l e s i o n diagrams; S. Atmadja f o r photomicroscopy, HRP-AChE combined h i s t o l o g y , F i b i g e r f o r d i s s e c t i o n , photomicroscopy. for lesions, histology.  layout;  H.C.  P u b l i c a t i o n D) : S. Atmadja  1  GENERAL  The  general  INTRODUCTION  s t r a t e g y adopted i n t h i s t h e s i s was t o i n v e s t i g a t e  c h o l i n e r g i c systems of the e x t r a p y r a m i d a l studying  t h e enzyme a c e t y l c h o l i n e s t e r a s e  advantage t o s t u d y i n g  system, e s p e c i a l l y by (AChE).  There i s a s p e c i a l  AChE - i t may be assayed b i o c h e m i c a l l y  and a l s o  l o c a l i z e d h i s t o c h e m i c a l l y , w i t h e x c e l l e n t r e s o l u t i o n and c o n t r a s t . Moreover, by p r e t r e a t i n g animals w i t h an i r r e v e r s i b l e AChE i n h i b i t o r , commonly d i i s o p r o p y l p h o s p h o r o f l u o r i d a t e sacrifice  preceding  (Lynch e t a l . , 1972), f u r t h e r advantages may be r e a l i z e d :  Neurons which s y n t h e s i z e guished.  (DEP), some time  t h e enzyme a t d i f f e r e n t r a t e s may be d i s t i n -  By t h e use of DFP, the s u b c e l l u l a r d i s t r i b u t i o n o f t h e  enzyme may be r e s o l v e d w i t h the l i g h t microscope, w i t h i n l i m i t a t i o n s . The  transport  of AChE along  a x o n a l p r o j e c t i o n s may be determined.  For  t h e s e reasons and o t h e r s  has  been an i n t e g r a l p a r t of each o f t h e i n v e s t i g a t i o n s  t o be e l a b o r a t e d  below, the study of AChE described  in this thesis. a) H i s t o r i c a l overview of AChE S i n c e much of t h e work comprising  t h i s t h e s i s i s concerned w i t h  AChE, what i s known and what has been s p e c u l a t e d p a s t decades w i l l be reviewed i n some d e t a i l .  about AChE over t h e  The major e a r l y  advances i n u n d e r s t a n d i n g p o t e n t i a l f u n c t i o n a l r o l e s o f AChE were made by K o e l l e i n g AChE: presynaptic  (1962).  First,  He noted t h r e e major g e n e r a l i z a t i o n s  i t s highest  concentrations  concern-  appeared t o be on  c h o l i n e r g i c terminals, with the p o s s i b l e exception of  the neuromuscular j u n c t i o n .  Second, t h e enzyme was found i n many  neurons n o t thought t o be c h o l i n e r g i c , a l t h o u g h i n d r a m a t i c a l l y  lower  2  l e v e l s than i n neurons thought to ;be c h o l i n e r g i c .  Finally,  there d i d  appear t o be n i c o t i n i c r e c e p t o r s on t h e t e r m i n a l s of c h o l i n e r g i c neurons, s p e c i f i c a l l y motoneuron t e r m i n a l s , which c o u l d g i v e r i s e t o a n t i d r o m i c a c t i o n p o t e n t i a l s recorded  i n t h e alpha-motoneuron.  o b s e r v a t i o n would of course present  presynaptically.  two of these h y p o t h e s e s .  This  last  g i v e AChE a r a i s o n d ' e t r e f o r b e i n g  Subsequent r e s e a r c h has borne out t h e f i r s t With r e g a r d t o t h e t h i r d ,  today t h e e x i s t e n c e  of the n i c o t i n i c r e c e p t o r s on motoneuron t e r m i n a l s appears t o be v a l i d , although  i t i s dubious t h a t they a r e of 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  (Miyamoto,. 1978).  Thev, f i r s t h y p o t h e s i s ,  t h a t h i g h AChE l e v e l s  appear to be a u n i v e r s a l c h a r a c t e r i s t i c o f c h o l i n e r g i c neurons, appears to  be t r u e a t l e a s t as f a r as the p e r i k a r y o n i s concerned.  Unfortunately,  t h i s hypothesis  has been r e c e i v e d p o o r l y by the neuro-c.:.-  s c i e n c e community, s i n c e Shute and Lewis (1961, 1965) m o d i f i e d t h e hypothesis  t o s t a t e t h a t a l l neurons which c o n t a i n e d  AChE i n t h e i r axons were c h o l i n e r g i c . without  any l e v e l of  T h i s m o d i f i c a t i o n was  accepted  f u r t h e r e x a m i n a t i o n by a number o f o t h e r p r o l i f i c i n v e s t i g a t o r s  (e.g., K r n j e v i c and S i l v e r , 1965, 1966), d e s p i t e e a r l i e r p u b l i s h e d by K o e l l e  (1954)^which c l e a r l y i n v a l i d a t e d  data  such;a.hypothesis.  The p o s t u l a t e t h a t t h e p o s t - s y n a p t i c occurence o f AChE i m p l i e s c h o l i n o c e p t i o n remains unproven. p e r i p h e r a l nervious  I t i s c e t a i n l y p l a u s i b l e i n the  system ( K o e l l e , 1962), b u t t h e r e e x i s t s a t  l e a s t one major e x c e p t i o n  i n the c e n t r a l nervous system:  The  dentate  gyrus of the hippocampal f o r m a t i o n r e c e i v e s a massive c h o l i n e r g i c input  (Lewis,  Shute, and S i l v e r ,  the e n t i r e hippocampal f o r m a t i o n (Lewis,  Shute, and S i l v e r ,  1967), but none of t h e neurons o f c o n t a i n a p p r e c i a b l e amounts o f AChE  1967; Lehmann, u n p u b l i s h e d  observations).  3  Some attempts have been made to support the h y p o t h e s i s t h a t low intermediate_levels  of AChE i n ^ c e r t a i n neurons o f the c e n t r a l nervous  system a r e i n d i c a t i v e of c h o l i n o c e p t i o n but  the m a j o r i t y  (Lehmann and Two  (Butcher and  Talbot,  1978a,b),  of the e v i d e n c e f o r t h e s e s p e c i a l cases i s  negative  F i b i g e r , 1979).  hypotheses were proposed decades ago  i n an attempt  e x p l a i n c o m p l e t e l y AChE i n a l l i t s l o c a t i o n s . b e l i e v e d t h a t a c e t y l c h o l i n e was e n t i r e extent  r e l e a s e d and  of axons i n g e n e r a l ,  to nerve impulse c o n d u c t i o n . a c e t y l c h o l i n e was and  contained  and  Burn and  to  Nachmansohn (1959) hydrolyzed  along  t h a t t h i s p r o c e s s was  i n sympathetic  (noradrenergic)  the essential  Rand (1959) b e l i e v e d  that terminals  t h a t the r e l e a s e of a c e t y l c h o l i n e , e l i c i t e d by the a c t i o n p o t e n t i a l ,  mediated the subsequent r e l e a s e of n o r a d r e n a l i n e ,  by  pre-junctional, e x t r a c e l l u l a r cholinergic receptor. perhaps data to support these hypotheses a t one  stimulating a While t h e r e were  time, abundant  d a t a garnered over the decades have r e f u t e d both..hypotheses 1974).  Champions of the Nachmansohn (1959) and  hypotheses are s t i l l  Burn and  (Silver,  Rand  noradrenergic they may  system.  The  neurons i n the p e r i p h e r y  experimentally 1978a,b).  (Sharma and  e x i s t on catecholamine t e r m i n a l s latter  Thus exogenous a c e t y l c h o l i n e may  Recently, as a p r e c u r s o r  exist  on  Banerjee, 1978),  i n the c e n t r a l nervous  case i s of c o u r s e much more d i f f i c u l t  ( f o r review of l i t e r a t u r e ,  on catecholamine  (1959)  to be found, however (Butcher et a l . , 1975).  I t remains l i k e l y , however, t h a t c h o l i n e r g i c r e c e p t o r s  and  to  see Butcher and  to  resolve  Talbot,  have a d i r e c t a c t i o n  terminals.  i t has been proposed t h a t b u t y r y l c h o l i n e s t e r a s e f o r AChE, on the b a s i s of.some r a t h e r  equivocal  serves  4  pharmacological data laboratory  ( K o e l l e et a l . , 1976).  to be r e p o r t e d  In s t u d i e s from  this  below, a s e l e c t i v e l e s i o n of dopaminergic  neurons i n the s u b s t a n t i a n i g r a r e s u l t e d i n a s u b s t a n t i a l d e p l e t i o n of AChE w i t h no d e p l e t i o n i n b u t y r y l c h o l i n e s t e r a s e , s u g g e s t i n g at  that  l e a s t i n these neurons b u t y r y l c h o l i n e s t e r a s e cannot s e r v e as  precursor  f o r AChE.  cholinesterase  I t has  s i n c e been shown t h a t AChE and  show l a r g e d i f f e r e n c e s i n b i n d i n g  immunological c r o s s - r e a c t i v i t y , and, gene l o c i  (Silman  the  butyryl-  properties, i n  i n human plasma, have s e p a r a t e  et a l . , 1979), making i t d i f f i c u l t  indeed to support  t h i s l a t e s t h y p o t h e s i s of K o e l l e et a l . (1976). A g r e a t d e a l of r e s e a r c h has  been done on the d i f f e r e n t m o l e c u l a r  forms of AChE, r e s o l v e d by a number of b i o c h e m i c a l s t u d i e s were a t disagreement w i t h r e s p e c t numbers of isoenzymes of AChE. degrees of a g g r e g a t i o n and  T h i s was  techniques.  to the m o l e c u l a r weights apparently  to  differing  s o l u b i l i z a t i o n o c c u r r i n g due  to  the  a l . (1976) succeeded i n s o l u b i l i z i n g  r e p r o d u c i b l y by u s i n g  Vigny  the enzyme t h o r o u g h l y  the v e r y h i g h sodium c h l o r i d e  and  concentration  of 1 M.  They found t h r e e c h a r a c t e r i s t i c forms of AChE i n muscle:  4s,  and  10s,  the 16s  16s,  form was  following ultracentrifugation.  I t appeared  s p e c i f i c a l l y a s s o c i a t e d w i t h muscle  10s  forms (Rieger and  V i g n y , 1976).  that  end-plate.  Subsequently, the same group found t h a t r a t b r a i n c o n t a i n e d 4s and  only  the  Furthermore, as r a t  b r a i n developed, a predominance of the 4s form gave way  to  an  almost e x c l u s i v e o c c u r r e n c e o f . t h e " 1 0 s ' f o r m . These data were v e r y suggestive AChE.  of a s y n a p t i c  In s u p e r i o r  and  due  d i f f e r e n t procedures used i n the d i f f e r e n t l a b o r a t o r i e s . et  Early  f u n c t i o n of an o l i g o m e r i c  c e r v i c a l ganglion,  4s,  6.5s,  form of  10s,  and  16s  forms  5  were found, a l t h o u g h a l l forms but and  the 16s were found i n p r e g a n g l i o n i c  p o s t g a n g l i o n i c nerves ( G i s i g e r et a l . , 1978).  disappeared  The  16s  form  from the s u p e r i o r c e r v i c a l g a n g l i o n f o l l o w i n g  denervation.  However, other workers found s m a l l amounts of 16s AChE i n p e r i p h e r a l nerves which a p p a r e n t l y (DiGiamberardino and  was  transported with unusually  Courard, 1978;  high v e l o c i t y  Fernandez e t a l . , 1979).  The^  f u n c t i o n a l i m p l i c a t i o n s of these f i n d i n g s f o r AChE i n s y n a p t i c await f u r t h e r study.  I t i s a l s o noteworthy t h a t AChE which i s bound to  the e x t e r n a l s u r f a c e of axons ( r e p r e s e n t i n g about 84% the nerve s e c t i o n ) a p p a r e n t l y  i s not  transported  at  v e l o c i t i e s i n e i t h e r the a n t e r o g r a d e nor r e t r o g r a d e Instead,  function  the m i n o r i t y  of AChE c o n t a i n e d  r a p i d axoplasmic t r a n s p o r t  of the t o t a l i n  appreciable direction.  w i t h i n the axon undergoes  ( B r i m i j o i n et a l . , 1978).  I n the nerve t e r m i n a l , AChE appears to have an e x c l u s i v e l y e x t e r n a l , membrane-bound l o c a l i z a t i o n ,  a l t h o u g h sometimes e l e c t r o n  s t u d i e s r e p o r t r e a c t i o n product w i t h i n m i t o c h o n d r i a apparatus, which was  not  e l i m i n a t e d by  c o n t r o l s , i . e . , i t does not r e p r e s e n t  microscopic  or the  the a p p r o p r i a t e  Golgi  histochemical  t r u e AChE (Er'anko et a l . ,  Bridges  e t a l . , 1973;  1974).  T h i s i s i n agreement w i t h s u b c e l l u l a r f r a c t i o n a t i o n s t u d i e s  ( T o s c h i , 1959; All  K o e l l e et a l . , 1974;  DeRobertis et a l . , 1963;  these o b s e r v a t i o n s  Kuhar and  1967;  Rommelspacher,  W h i t t a k e r et a l . , 1964).  emphasize the importance of p r e s y n a p t i c  p a r t i c u l a r l y t h a t which i s bound on the e x t e r n a l s u r f a c e of  AChE,  the  c h o l i n e r g i c t e r m i n a l , i n the c e n t r a l nervous system.  Obviously,  AChE has  transmission  a different role i n regulating acetylcholine  than monoamine oxidase ( G e f f e n and L i v e t t ,  has  i n r e g u l a t i n g catecholamine  1971), monoamine oxidase  having  transmission  an i n t r a t e r m i n a l ,  6  primarily mitochondrial A very  localization.  i n t r i g u i n g p o s s i b l e mechanism f o r r e g u l a t i n g c h o l i n e r g i c  t r a n s m i s s i o n v i a AChE has been r a i s e d i n r e c e n t y e a r s , namely t h e s e c r e t i o n of AChE e i t h e r by c h o l i n e r g i c neurons or by t h e neurons or organs which r e c e i v e c h o l i n e r g i c i n n e r v a t i o n . t h a t AChE was s e c r e t e d was f i r s t  The  hypothesis  suggested by e l e c t r o n m i c r o s c o p i c  h i s t o c h e m i c a l s t u d i e s ( F l u m e r f e l t e t a l . , 1973; K r e u t z b e r g 1974;  Kreutzberg  microscopic  and Schubert, 1975).  h i s t o c h e m i c a l study  In a rigorous electron  i n the motoneuron, the p o s s i b l e  c y t o l o g i c a l mechanism f o r s e c r e t i o n was proposed 1975):  Following  apparently  and T6th,  (Kreutzberg  eta l . ,  s y n t h e s i s i n the rough endoplasmic r e t i c u l u m , AChE  passes through^the G o l g i apparatus and becomes i n c o r p o r a t e d  i n t o the smooth endoplasmic r e t i c u l u m .  Since there i s d i r e c t c o n t i n u i t y  between the smooth endoplasmic r e t i c u l u m and the plasma membrane, t h i s suggested a g e n e r a l c y t o l o g i c a l mechanism f o r AChE s e c r e t i o n . The  o b s e r v a t i o n t h a t , r a r e l y , s y n a p t i c v e s i c l e s appear t o c o n t a i n  AChE (Bodian restricted  1970), suggested t h a t s e c r e t e d AChE may not o n l y be  t o e x t r a c e l l u l a r l o c a t i o n s , but may p l a y some r o l e i n  i n t e r c e l l u l a r communication as w e l l .  I t i s important  most i n d i c a t i o n s suggest t h a t i f AChE indeed it  i s by  been d e t e c t e d  (Chubb e t a l . , 1976; B a r e g g i  i n cerebrospinal  vesicles,  fluid  and G i a c o b i n i , 1978; S c a r s e l l a e t a l . ,  G r e e n f i e l d e t a l . , 1979).  Non-denaturing g e l e l e c t r o p h o r e s i s  s t u d i e s suggested t h a t one isoenzyme was s e c r e t e d 1976;  synaptic  endocytosis.  AChE has subsequently  1979;  enters  t o note t h a t  S c a r s e l l a e t a l . , 1979).  A d m i n i s t r a t i o n of  i n c r e a s e s the amount of AChE s e c r e t e d  (Chubb e t a l . , chlorpromazine  ( B a r e g g i and G i a c o b i n i , 1978;  7  G r e e n f i e l d e t a l . , 1979).  The r e l e a s e i s much more pronounced i n  c i s t e r n a l than i n v e n t r i c u l a r c e r e b r o s p i n a l  fluid  G i a c o b i n i , 1978; G r e e n f i e l d e t a l . , 1979).  The source o f the  r e l e a s e d AChE i s unknown; however, i t i s h i g h e r  in activity  plasma AChE, and t h e r e i s no concomitant i n c r e a s e ase,  a conventional  1979) .  (Bareggi and  than  i n l a c t a t e dehydroen-  marker f o r c e l l d i s r u p t i o n ( G r e e n f i e l d e t a l . ,  T h i s phenomenon i s o f obvious importance i n u n d e r s t a n d i n g  c o n t r o l mechanisms governing c h o l i n e r g i c tone i n the c e n t r a l nervous system.  I n the cases of the s u p e r i o r c e r v i c a l g a n g l i o n  V i g n y , 1977) and c a t g e n i o h y o i d  ( G i s i g e r and  muscle ( I n e s t r o s a e t a l . , 1977),  a f f e r e n t c h o l i n e r g i c neurons a r e not n e c e s s a r y t o support the r e l e a s e of AChE.  I n the i s o l a t e d r a t hemidiaphragm, however, the  r a t i o o f 10s and 4s AChE r e l e a s e d by e l e c t r i c a l d e p o l a r i z a t i o n match the_r.atio of these forms i n the p h r e n i c (Skau and B r i m i j o i n , 1978) .  nerve, and not muscle  The l a t t e r a u t h o r s suggested  synaptic,  e x o c y t o t i c r e l e a s e of AChE a s _ t h e u s o u r c e o f t e x t r a e e l l u l a r ^ A C h E i . a n d means of r e g u l a t i n g or perhaps s u p p l y i n g  postsynaptic  AChE.  as a  While  it  remains q u e s t i o n a b l e  whether c h o l i n e r g i c axons can s e c r e t e AChE,  it  c e r t a i n l y i s t r u e t h a t organs which n o r m a l l y r e c e i v e c h o l i n e r g i c  i n n e r v a t i o n can s e c r e t e AChE, f o l l o w i n g c h r o n i c d e n e r v a t i o n  and i n  non-depolarizing  et a l . ,  medium ( G i s i g e r and V i g n y , 1977; I n e s t r o s a  1977). AChE r e l e a s e from whole b r a i n synaptosomal f r a c t i o n i n nond e p o l a r i z i n g medium has been demonstrated What r o l e e x a c t l y AChE r e l e a s e p l a y s i s more d i f f i c u l t It  (Burgun e t a l . , 1977).  i n the c e n t r a l nervous system  t o answer than i t i s i n the p e r i p h e r a l nervous system.  i s not known, f o r i n s t a n c e , whether such r e l e a s e d AChE reaches  8  c h o l i n e r g i c synapses, but i t s p r e s e n c e has not been r e p o r t e d i n extracellular  spacelin^the./brain^except.Jforccerebrospinal:fluid.  b) The b a s a l g a n g l i a and c h o l i n e r g i c f u n c t i o n The  postulate  t h a t AChE i s concerned i n some way w i t h the f u n c t i o n  of a c e t y l c h o l i n e remains such an a t t r a c t i v e concept t h a t the study of t h i s enzyme was the major emphasis o f the p r o j e c t s comprising That t h e s t r i a t u m .is among the h i g h e s t for c h o l i n e r g i c transmission  i n biochemical  this thesis.  markers  i n the e n t i r e b r a i n (Hoover e t a l . , 1978;  Kobayashi e t a l . , 1978) makes t h i s an a t t r a c t i v e n u c l e u s i n which to study the c h o l i n e r g i c neuron.  Many o f the p s y c h o p h a r m a c o l o g i c a l  e f f e c t s produced by drugs a c t i n g on c h o l i n e r g i c r e c e p t o r s c e n t r a l nervous system, such as tremor and c a t a l e p s y  i n the  (Karczmar,11975)  may be mediated i n the s t r i a t u m . I n the e a r l y p a r t o f the 1970's i t became c l e a r t h a t t h e markers f o r _ c h o . l i n e r g i c _ r i e u r o n s , namely c h o l i n e a c e t y l t r a n s f e r a s e and a c e t y l c h o l i n e , were completely i n t r i n s i c t o the s t r i a t u m 1971;  Butcher and Butcher, 1974).  Lesions  of the b r a i n s u r r o u n d i n g  the s t r i a t u m had no e f f e c t on e i t h e r o f . t h e s e b i o c h e m i c a l f o r c h o l i n e r g i c neurons.  T h i s l e d to t h e s u g g e s t i o n  c h o l i n e r g i c neuron was an i n t e r n e u r o n  (McGeer e t a l . ,  markers  t h a t t h e . u ...  (McGeer e t a l . , 1971).  At about  the same time, s i x d i f f e r e n t m o r p h o l o g i c a l types o f neurons were i d e n t ified  i n the straitum  scientist  (Kemp and P o w e l l , 1971).  i s to construct  One g o a l of the neuro-  a w i r i n g diagram o f the b r a i n , as i f i t  were a computer, and then go on t o e x p l a i n i t s f u n c t i o n , and i n cases o f d i s e a s e first  dysfunction,  i n terms o f t h a t w i r i n g diagram.  The  s t e p , of course, i s t o i d e n t i f y the components. In the case o f the b a s a l g a n g l i a , the primary s p e c i f i c g o a l as  9  f a r as a c e t y l c h o l i n e i s concerned, was t o i d e n t i f y neuron m o r p h o l o g i c a l l y .  the c h o l i n e r g i c  T h i s was f i r s t accomplished by t h e  t e c h n i q u e o f immunohistochemistry, employing a n t i b o d i e s r a i s e d a g a i n s t purified  c h o l i n e a c e t y l t r a n s f e r a s e ( H a t t o r i e t a l . , 1976).  most common type o f neuron i n the s t r i a t u m ,  the medium s p i n y  were i d e n t i f i e d as t h e p u t a t i v e c h o l i n e r g i c neurons. pharmacological  data were used t o support  neurons,  Biochemico-  t h e i d e n t i f i c a t i o n of  t h i s as the c h o l i n e r g i c neuron, which r e c e i v e d a d i r e c t input  The  dopaminergic  ( H a t t o r i e t a l . , 1976). When the experiments r e p o r t e d  i n t h i s t h e s i s were begun, t h e  study o f AChE i n the s t r i a t u m appeared t o be as complex an approach as any other  f o r studying  cholinergic function.  As noted above,  AChE has complex forms and c e l l u l a r l o c a l i z a t i o n s , n o t o n l y  within  and  on t h e plasma membranes of neurons i n v a r y i n g l e v e l s of a c t i v i t y ,  but  a l s o q u i t e p o s s i b l y i n i n t e r c e l l u l a r space.  Histochemically,  AChE a c t i v i t y i n t h e s t r i a t u m appears dense and u n i f o r m 1954;  McGeer e t a l . , 1971).  which allowed  F o r t u n a t e l y , a t e c h n i q u e was developed  t h e r e s t r i c t i o n of h i s t o c h e m i c a l product formed by AChE  to the neurons which s y n t h e s i z e 1972;  (Koelle,  Butcher e t a l . , 1975):  t h e enzyme most r a p i d l y (Lynch  Administration  i t o r of AChE, d i i s o p r o p y l p h o s p h o r o f l u o r i d a t e AChE (as w e l l as o t h e r  et a l . ,  of t h e i r r e v e r s i b l e i n h i b (DFP), i n h i b i t s the  enzymes) .everywhere i n the a n i m a l .  I f the  animal i s then s a c r i f i c e d f o u r to twelve hours a f t e r a d m i n i s t r a t i o n of DFP, only t h e enzyme which has been newly s y n t h e s i z e d time i s v i s u a l i z e d h i s t o c h e m i c a l l y .  during  Thus, i t became p o s s i b l e t o  study t h e i n t r i n s i c neurons of t h e s t r i a t u m which s y n t h e s i z e a study which i s s t i l l b e i n g  pursued.  that  Preliminary  studies  AChE, (Butcher  10  et  a l . , 1975)  synthesized  showed t h a t o n l y a m i n o r i t y the enzyme at a p p r e c i a b l e  fewer s y n t h e s i z e d The  l e v e l s , and  of these  nature.  speculations  The  concerning  still  and  f u n c t i o n a l i m p l i c a t i o n s , whereby some the r o l e of the s t r i a t a l c h o l i n e r g i c neurons  psychomotor f u n c t i o n may  the d i s c u s s i o n .  5%)  l a r g e amounts of the enzyme.  papers which f o l l o w are e n t i r e l y of an a n a t o m i c a l  biochemical  in  very  of neurons ( l e s s than  be e n t e r t a i n e d , w i l l be  dealt with i n  In the p u b l i c a t i o n s which f o l l o w , as the main body  of t h i s t h e s i s , a broad d a t a base was  sought t o  substantiate  hypotheses which were^and perhaps s t i l l  are,considered  The  as i n a l l "endeavors, today's  c a n d i d a t e i s aware t h a t i n s c i e n c e ,  conclusions  have a way  of becoming tomorrow's  c) B r i e f d e s c r i p t i o n of r e s e a r c h  and  Butcher et a l . (1975) r e p o r t e d of the s u b s t a n t i a n i g r a contained m a i n l y on  t h a t the dopaminergic neurons  AChE.  T h i s c o n c l u s i o n was  catecholamine h i s t o f l u o r e s c e n c e  Fuxe, 1964;  the f i r s t  rationale  U n g e r s t e d t , 1971).  studies  T h i s was  not, however, a  argument_to support the c o n c l u s i o n . i n v e s t i g a t i o n of t h i s t h e s i s was  (Dahlstr'om  For  this  reason,  to t e s t r i g o r o u s l y  h y p o t h e s i s t h a t dopamine-containing neurons of the s u b s t a n t i a contained  based  dopamine-containing neurons, the l a t t e r h a v i n g been  l o c a l i z e d by  compelling  fallacies.  the s i m i l a r t o p o g r a p h i c d i s t r i b u t i o n of A C h E - c o n t a i n i n g  neurons and  and  unorthodox.  the nigra  AChE.  S e l e c t i v e , i f not neurons can be a c h i e v e d 6-hydroxydopamine by  s p e c i f i c , d e s t r u c t i o n of dopamine-containing by  the a d m i n i s t r a t i o n  e i t h e r of two  routes.  of the  The  neurotoxin  t o x i n mayche i n j e c t e d  d i r e c t l y i n t o the a s c e n d i n g axons of the dopaminergic  nigrostriatal  11  tract.n.'.Retrograde d e g e n e r a t i o n of the dopaminergic ensues.  A l t e r n a t i v e l y , the t o x i n may  ventricle.  neurons  be i n j e c t e d i n t o the  then lateral  Desmethylimipramine, which b l o c k s uptake of the t o x i n  i n t o n o r a d r e n e r g i c neurons, must be a d m i n i s t e r e d a l s o i f n o r a d r e n e r g i c f i b r e s and p e r i k a r y a a r e to be  spared.  Bothsof :: t h e s e c r o u t e s of a d m i n i s t r a t i o n were employed i n the r  first  investigation  (A) to o b t a i n a complete and s p e c i f i c  of the n i g r o s t r i a t a l dopaminergic l e s i o n s was  determined  which i s thought  projection.  The  lesion  e x t e n t of the  by measuring the enzyme t y r o s i n e h y d r o x y l a s e ,  to be a s p e c i f i c marker f o r catecholamine  neurons.  C h o l i n e a c e t y l t r a n s f e r a s e was  measured t o a s s e s s the s p e c i f i c i t y  the l e s i o n .  both .measured by r a d i o e n z y m a t i c  and  F i n a l l y , AChE was  examined h i s t o c h e m i c a l l y .  AChE, an;  of  assay  The b i o c h e m i c a l c h a r a c t e r i s t i c s of  enzyme which does not f o l l o w M i c h a e l i s - M e n t e n  kinetics,  were a l s o s t u d i e d . The h i s t o c h e m i c a l demonstration n i g r a l p r o j e c t i o n had been proposed The  second  of an AChE-containing earlier  ( O l i v i e r e t a l . , 1970).  i n v e s t i g a t i o n of t h i s t h e s i s s e t out to support  h y p o t h e s i s a l s o . The ..original i n v e s t i g a t i o n had employed e l e c t r o l y t i c  striato-  this  ( O l i v i e r e t a l . , 1970)  l e s i o n s , which d e s t r o y axons-passing  the l e s i o n e d a r e a , i n a d d i t i o n to neurons.  through  Since that i n v e s t i g a t i o n ,  a n e u r o t o x i n , k a i n i c a c i d , has been found which spares axons of passage but d e s t r o y s i n t r i n s i c neurons of the s t r i a t u m . second  investigation  (B) u t i l i z e d k a i n i c a c i d i n order to o b t a i n  the more s e l e c t i v e s t r i a t a l l e s i o n .  I n f o r m a t i o n on the  and sources of AChE i n the s t r i a t u m were a l s o o b t a i n e d . experiments  This  were performed  to determine  localization Histochemical  i f blockade of t r a n s p o r t  12  along the s t r i a t o n i g r a l axons caused an a c c u m u l a t i o n  of AChE w i t h i n  them. A group of neurons, the nucleus may  belong  to the e x t r a p y r a m i d a l  b a s a l i s m a g n o c e l l u l a r i s , which  system  (Divac, 1975)  r e t i c u l a r group of neurons (Das  and K r e u t z b e r g ,  focus of the next  (C).  investigation  h i g h l e v e l s of AChE, and  the  1968), became the  These neurons c o n t a i n v e r y  i t had been s p e c u l a t e d t h a t they were the  source of a c h o l i n e r g i c p r o j e c t i o n t o the neocortex All  or to  (Divac, 1975).  the most r i g o r o u s means of t e s t i n g t h i s h y p o t h e s i s , by  d i f f e r e n t s o r t s of l e s i o n s , neuroanatomical  utilizing  t r a c i n g techniques,  AChE h i s t o c h e m i s t r y , were employed to t e s t t h i s  and  hypothesis.  There has been a g r e a t d e a l of c o n f u s i o n and numerous hypotheses concerning  the i n t e r p r e t a t i o n s which may  of AChE a c t i v i t y  i n d i f f e r e n t neurons.  be drawn from the presence The  i n v e s t i g a t i o n s reported i n  t h i s t h e s i s have some b e a r i n g on these i n t e r p r e t a t i o n s . review  on the t o p i c was  some o r i g i n a l data, morphological  t h e r e f o r e w r i t t e n (D).  the most s t r i k i n g of which r e l a t e to  Coyle  c h o l i n e r g i c neuron.  (1978) had  developed  contains  the  T h i s turned out to be  a s e l e c t i v e l e s i o n of  the b a s i s of a n a t o m i c a l  the  the same m o r p h o l o g i c a l l y  d e s c r i b e d neuron p r e v i o u s l y s p e c u l a t e d t o be  B.  T h i s review  i d e n t i f i c a t i o n of the s t r i a t a l c h o l i n e r g i c neuron.  Campochiara and  publication  A short  the c h o l i n e r g i c neuron on 'j.'..  and h i s t o c h e m i c a l arguments s e t f o r t h i n  13  GENERAL DISCUSSION  a) Preview of new c o n t r i b u t i o n s r e p o r t e d From the p r e c e d i n g 1)  A hypothesis  i n this thesis  papers, t h r e e major f i n d i n g s have emerged:  f o r i d e n t i f y i n g p o t e n t i a l c h o l i n e r g i c neurons on  the b a s i s of t h e i r h i g h AChE a c t i v i t y has.been proposed. c h o l i n e r g i c neuron of the s t r i a t u m has been identified.  3)  2)  The  morphologically  The o r i g i n of a major c h o l i n e r g i c p r o j e c t i o n t o  the c o r t e x has been In the f i r s t  identified.  i n v e s t i g a t i o n (A), t h e h y p o t h e s i s  Butcher et a l . (1975) was supported:  proposed by  S e l e t i v e l e s i o n of the  dopaminergic neurons r e s u l t e d i n .a 30-40% d e p l e t i o n o f AChE i n the s u b s t a n t i a n i g r a , and about a 12% d e p l e t i o n of AChE i n t h e striatum.  These r e s u l t s suggest t h a t both t h e p e r i k a r y a and axons  of dopaminergic n i g r o s t r i a t a l neurons c o n t a i n AChE. vestigation,  In t h i s i n -  t h e source of a c h o l i n e r g i c i n p u t t o t h e s u b s t a n t i a  n i g r a c o u l d not be found. In the second i n v e s t i g a t i o n ( B ) , the h y p o t h e s i s et a l . (1970) was r e f u t e d . neurons d i d not r e s u l t substantia nigra.  of O l i v i e r  A massive k a i n i c a c i d l e s i o n of s t r i a t a l  i n a d e t e c t a b l e d e p l e t i o n of AChE i n t h e  Furthermore, c o l c h i c i n e i n j e c t i o n s i n t o the  axons of the s t r i a t o n i g r a l pathway, which i n g e n e r a l w i l l proximal  accumulatiori._of AChE i n axons which t r a n s p o r t t h e enzyme,  d i d not cause such an a c c u m u l a t i o n . all,  cause  While a major p o r t i o n , i f not  of t h e c h o l i n e a c e t y l t r a n s f e r a s e a c t i v i t y o f t h e s t r i a t u m  o r i g i n a t e s from neurons whose p e r i k a r y a r e s i d e w i t h i n the s t r i a t u m , about 50% o f the AChE a c t i v i t y i s d e r i v e d from an e x t e r n a l The  t h i r d i n v e s t i g a t i o n provided  compelling  source.  e v i d e n c e t h a t the  14  nucleus b a s a l i s m a g n o c e l l u l a r i s p r o j e c t i o n to t h e c o r t e x (C, F i g s . 1 and 2 ) .  i s the source of a c h o l i n e r g i c  ( C ) . The e f f e r e n t neurons were mapped  I t was n o t p o s s i b l e t o a s s i g n t h i s group of neurons  to e i t h e r the r e t i c u l a r f o r m a t i o n  or the e x t r a p y r a m i d a l  on t h e b a s i s of c u r r e n t l y a v a i l a b l e The  system,  evidence.  review (D), which e v a l u a t e s  t h e i n t e r p r e t a t i o n s which can be  made from the l o c a l i z a t i o n of AChE i n v a r i o u s neurons, a r r i v e d a t two b a s i c g e n e r a l and e m p i r i c a l c o n c l u s i o n s :  1) Very h i g h l e v e l s of  AChE f o u r t o twelve hours f o l l o w i n g DFP a d m i n i s t r a t i o n can be taken as a n e c e s s a r y but not s u f f i c i e n t c r i t e r i o n f o r a neuron to be c h o l i n e r gic.  This i s a valuable r u l e ,  s i n c e i t would e l i m i n a t e 99% o f the  neurons i n t h e b r a i n as c a n d i d a t e s  f o r c h o l i n e r g i c neurons,  they c o n t a i n but low t o i n t e r m e d i a t e the c e r e b r a l c o r t e x i s devoid and  thus,  l e v e l s of AChE.  since  For instance,  of i n t e n s e l y A C h E - r e a c t i v e neurons,  one would p r e d i c t , does n o t c o n t a i n c h o l i n e r g i c p e r i k a r y a .  T h i s example was e x p e r i m e n t a l l y time, AChE a c t i v i t y c o n t a i n e d  verified  (D).  2) A t t h i s p o i n t i n  i n a neuron cannot be taken as e v i d e n c e  t h a t such a neuron i s c h o l i n o c e p t i v e . In t h i s i n v e s t i g a t i o n , evidence was a l s o gathered t o support t h e hypothesis interneuron  t h a t the i n t e n s e l y A C h E - r e a c t i v e neuron of the s t r i a t u m .  was the c h o l i n e r g i c  When r a t pups t e n days of age r e c e i v e d  k a i n i c a c i d i n j e c t i o n s , c h o l i n e a c e t y l t r a n s f e r a s e a c t i v i t y and AChE a c t i v t y were s e l e c t i v e l y d e p l e t e d . neuron l o s s was d e t e c t a b l e , destroyed.  While h i s t o l o g i c a l l y no other  the i n t e n s e l y A C h E - r e a c t i v e neuron was  The p u t a t i v e c h o l i n e r g i c neuron makes up o n l y about  1% of t h e t o t a l s t r i a t a l neuron  population.  These fundamental s t u d i e s of t h e p u t a t i v e c h o l i n e r g i c neuron  15  of the s t r i a t u m are e s s e n t i a l as a b a s i s f o r a more comprehensive u n d e r s t a n d i n g of t h a t n e u r o n l s more complex f u n c t i o n s .  I t would  appear to p l a y an important p a r t i n the f u n c t i o n of the  striatum.  For  instance,  of a l l s t r i a t a l neurons, the l a r g e neurons ( i n c l u d i n g  the p u t a t i v e c h o l i n e r g i c neuron) undergo t h e i r f i n a l m i t o s i s than the r e s t of the s t r i a t a l neurons (Brand and  Rakic,  earlier  1979).  They appear to develop s e n s i t i v i t y to k a i n i c a c i d l s n e u r o t o x i c earlier  than the r e s t of the s t r i a t a l neurons.  These r e s u l t s r a i s e  the p o s s i b i l i t y t h a t they form the primary n e u r a l f o u n d a t i o n subsequent s t r i a t a l n e u r a l networks are b u i l t .  effects  upon which  A postnatal  develop-,  mental study of the p u t a t i v e c h o l i n e r g i c n e u r o n - i s c u r r e n t l y i n progress.  b) The  r o l e of the s t r i a t u m i n motor and  The  psychological  function  t i t l e of the t h e s i s promises some c o n s i d e r a t i o n of  the  f u n c t i o n of the s t r i a t a l c h o l i n e r g i c neuron.  This i s , a f t e r a l l ,  the u l t i m a t e  current  concerning w i l l now  i n t r i g u e of b r a i n r e s e a r c h .  the g e n e r a l  i t must be  concepts  p s y c h o l o g i c a l f u n c t i o n of the; s t r i a t u m  be reviewed v e r y  First,  The  briefly.  considered  t h a t the s t r i a t u m does not  an e s s e n t i a l r o l e i n the c o n t r o l of b a s i c m e t a b o l i c  play  function,  elementary s e n s o r i m o t o r f u n c t i o n s , or elementary c o g n i t i v e  functions  ( V i l l a b l a n c a et a l . , 1976).  initiation  to a c o n d i t i o n e d  However, d e f i c i t s  i n response  avoidance response occur f o l l o w i n g l e s i o n of  the  dopaminergic n i g r o s t r i a t a l p r o j e c t i o n ( F i b i g e r et a l . , 1974). d e f i c i t s can be r e v e r s e d amine and  atropine  by a n t i c h o l i n e r g i c drugs, such as  ( F i b i g e r et a l . , 1975).  of an antagonism which appears v e r y  T h i s i s one  Such  scopol-  example  f r e q u e n t l y between d o p a m i n e r g i c  16  and c h o l i n e r g i c t r a n s m i s s i o n i n the s t r i a t u m . The s t r i a t u m appears to p l a y i t s major r o l e as one of the h i g h e r feedback systems c o n t r o l l i n g movement. been c o n s i d e r e d a more p r i m i t i v e motor dependently motor  A l t h o u g h t r a d i t i o n a l l y i t has output system which o p e r a t e s i n -  of the p y r a m i d a l ( c o r t i c o - s p i n a l ) motor system,  direct  outputs have been found to be a t most a minor p o r t i o n of  e f f e r e n t s from the b a s a l g a n g l i a .  On the c o n t r a r y ,  circuitry  which l i n k s the s t r i a t u m w i t h t h a l a m i c pathways which f e e d back onto the c e r e b r a l c o r t e x i s the predominant n e u r o a n a t o m i c a l c h a r a c t e r i s t i c of the system (Carpenter, 1975).  There appear t o be r a t h e r  direct  r o u t e s f o r v i s u a l i n p u t to the s t r i a t u m v i a the c o r t e x , demonstrable both anatomically  (K'unzle and A k e r t , 1977) and by r e c o r d i n g  responses to v i s u a l s t i m u l i  (Pouderoux and F r e t o n , 1979).  a f f e r e n t s , not only from motor  Cortical  areas (Kunzle, 1975) but a l s o from  a l l s e n s o r y and a s s o c i a t i o n a l a r e a s , i n n e r v a t e the s t r i a t u m and Nauta, 1977;  Jones e t . . a l . , 1977).  (Goldman  Somatic and v i s u a l s e n s o r y  n e g l e c t can be induced by l e s i o n s of the e x t r a p y r a m i d a l motor (Feeney and Wier, 1979).  I n normal animals, s i n g l e - c e l l  system  recording  i n the s t r i a t u m d u r i n g v i s u a l l y and s o m a t i c a l l y guided hand movements i n d i c a t e t h a t the the t y p i c a l l y " q u i e t " s t r i a t u m f i r e s c o r r e c t i o n of t h e s e movements (Anderson.et a l . ,  1979).  during A direct  role  of the c h o l i n e r g i c system of the s t r i a t u m i n m o d u l a t i n g c o r t i c a l p r o c e s s i n g of sensory i n f o r m a t i o n has been demonstrated by Roemer et a l . (1978): I n t r a c a u d a t e i n j e c t i o n of c a r b a c h o l has d i r e c t on somatosensory-evoked c o r t i c a l p o t e n t i a l s .  effects  Cryogenic l e s i o n s  of the s t r i a t u m produce p u r s u i t - t r a c k i n g d e f i c i t s  (Bowen, 1969).  17  c) Psychopharmacology o f the s t r i a t u m Anthropomorphism i s c o n s i d e r e d to be a p i t f a l l a l l y and i n n e u r o s c i e n c e i n p a r t i c u l a r .  i n s c i e n c e gener-  However, s i n c e some drugs  used  i n the s t u d i e s t o be d i s c u s s e d can induce a syndrome t h a t c l o s e l y resemb l e s p a r a n o i d s c h i z o p h r e n i a (e.g., amphetamine), and o t h e r s a r e used t h e r a p e u t i c a l l y i n s c h i z o p h r e n i c s (Snyder, 1974), some o f the animal b e h a v i o r s e l i c i t e d by these drugs may be c o n s i d e r e d analogous p s y c h o l o g i c a l d i s o r d e r s (Mattyse, 1974).  to human  Thus the r e a d e r i s encouraged  to c o n s i d e r the f o l l o w i n g p s y c h o p h a r m a c o l o g i c a l  d i s c u s s i o n i n terms o f  what r o l e s i n thought p r o c e s s e s the t r a n s m i t t e r s dopamine and a c e t y l c h o l i n e may p l a y .  I t should be noted t h a t w h i l e t h e r e i s c o n s i d e r a b l e  evidence to i n d i c a t e t h a t s i t e o f a c t i o n s of drugs d e s c r i b e d below i s the s t r i a t u m , t h i s p o i n t i s ' - a c t u a l l y r a r e l y a d e q u a t e l y  demonstrated.  1) Psychopharmacology of dopamine i n the s t r i a t u m 6-Hydroxydopamine l e s i o n s o f the n i g r o s t r i a t a l p r o j e c t i o n r e s u l t i n decreased spontaneous locomotor Roberts e t a l . ,  (Creese and I v e r s e n , 1973;  1975), and an a t t e n u a t i o n i n the normal locomotor  l a t i o n induced by amphetamine. i n v o l v e d i n the locomotor response  activity  to h i g h e r doses  stimu-  A p p a r e n t l y the n u c l e u s accumbens i s more  response  to amphetamine, w h i l e the s t e r e o t y p y  of amphetamine i s l o c a l i z e d  putamen (Creese and Iversen,1975) .  to the caudate-  N e u r o l e p t i c s , which a r e thought to  b l o c k dopamine r e c e p t o r s , cause c a t a l e p s y i n h i g h doses  (Asper e t a l . ,  (1973) . E l e c t r o p h y s i o l o g i c a l ^ , i t i s thought by some t h a t dopamine i s monos y n a p t i c a l l y e x c i t a t o r y , although a t longer l a t e n c i e s i t s o v e r a l l i s i n h i b i t o r y , an e f f e c t p r o b a b l y mediated (Kitai et a l . ,  1976; R i c h a r d s o n e t a l . ,  effect  by a t l e a s t one i n t e r n e u r o n  1977).  Dopamine s t i m u l a t e s  a d e n y l a t e c y c l a s e i n the s t r i a t u m (McGeer e t a l . ,  1976; Kebabian,  1978),  18  making i t a c a n d i d a t e  as a slower  a c t i n g , humoral agent which.may n o t  function e x a c t l y l i k e a c l a s s i c a l synaptic transmitter. 2) Psychopharmacology of a c e t y l c h o l i n e i n the s t r i a t u m Because c h o l i n e r g i c systems a r e found  i n so many p a r t s of the  c e n t r a l and p e r i p h e r a l nervous systems, t h e o n l y d i r e c t  assessment  of the b e h a v i o r a l r o l e of a c e t y l c h o l i n e i n the s t r i a t u m i s o b t a i n e d by  i i n j e c t i o n of a c e t y l c h o l i n e a g o n i s t s and a n t a g o n i s t s  i n t o the s t r i a t u m .  I n j e c t i o n of muscarinic  directly  a g o n i s t s or AChE  i n h i b i t o r s i n t o the j s t r i a t u m r e s u l t s i n tremor, limb  dystonia,  r i g i d i t y , and a k i n e s i a - a l l c h a r a c t e r i s t i c s of P a r k i n s o n ' s (Connor e t a l . , 1966; 1978,  1979).  Goldman and Lehr,  disease  1976; Matthews and Chiou,  A l l these e f f e c t s a r e b l o c k e d by m u s c a r i n i c  antagonists.  I n t r a s t r i a t a l i n j e c t i o n o f a t r o p i n e , on the other hand, e l i c i t s typy -  stereo-  an e f f e c t which may a l s o be e l i c i t e d by i n c r e a s i n g dopaminergic  tone (Zambo e t a l . , 1979). E l e c t r o p h y s i o l o g i c a l ^ , t h e r e i s l e s s agreement about the e f f e c t s of a c e t y l c h o l i n e i n t h e s t r i a t u m . predominantly 1965;  i n h i b i t i o n of i o n t o p h o r e s e d  Some i n v e s t i g a t o r s found a c e t y l c h o l i n e (Bloom e t a l . ,  McLennan and York, 1966) which was b l o c k e d by m u s c a r i n i c  gonists  (McLennan and York, 1966).  Systemically  anta-  administered  c h o l i n e r g i c a g o n i s t s and a n t a g o n i s t s l e d o t h e r s to b e l i e v e t h a t c h o l i n e r g i c r e c e p t o r s i n the caudate n u c l e u s were i n h i b i t o r y and muscarinic  ( R o l l e r and B e r r y , 1976).  However, u s i n g s t r i a t a l  slices,  l o c a l s t i m u l a t i o n evokes e x c i t a t i o n which can be b l o c k e d by h i g h c o n c e n t r a t i o n s of c u r a r e or enhanced by physostigmine, muscarinic  a n t a g o n i s t s were without  I n c o n t r a s t , another  effect  while  ( M i s g e l d and Bak, 1979).  study r e p o r t s both s h o r t and l o n g l a t e n c y  19  e x c i t a t i o n by l o c a l s t i m u l a t i o n o f s t r i a t a l s l i c e s , b e i n g b l o c k e d by n i c o t i n i c a n t a g o n i s t s and  the  first  the l a t t e r b e i n g  blocked  by m u s c a r i n i c a n t a g o n i s t s ( W e i l e r e t a l . , 1979).  d) I n t e r a c t i o n s between dopamine and a c e t y l c h o l i n e On  the b a s i s of p h a r m a c o l o g i c a l  s t u d i e s a l o n e , i t i s not  s u r p r i s i n g to f i n d t h a t a l a r g e number of a n t a g o n i s t i c e f f e c t s between dopaminergic and  and  c h o l i n e r g i c a g o n i s t s , and between  cholinergic antagonists.  dopaminergic  While g e n e r a l l y Barbeau (1962) i s  c r e d i t e d w i t h c r e a t i n g the concept  of a  dopamine-acetylcholine  b a l a n c e i n the s t r i a t u m , f o r h i s t o r i c a l a c c u r a c y he c r e d i t s concept's  exist  i n c e p t i o n t t o McGeer e t a l . (1961).  the  Both groups of workers  drew t h e i r c o n c l u s i o n s from the e f f e c t s of drugs on v i c t i m s of Parki n s o n ' s d i s e a s e , i n whom dopamineragonists  or c h o l i n e r g i c  a m e l i o r a t e d , but dopamine a n t a g o n i s t s or c h o l i n e r g i c exacerbated  the symptoms.  Parkinson's  agonists  The c h o l i n o l y t i c drugs which a r e  f u l i n a m e l i o r a t i n g the symptoms of P a r k i n s o n ' s ( D u v o i s i n , 1967).  antagonists  Huntington's  success-  disease are muscarinic  d i s e a s e i s e s s e n t i a l l y the i n v e r s e of  d i s e a s e - i n these p a t i e n t s dopaminergic  a f f e r e n t s to the  s t r i a t u m a r e i n t a c t , but the neurons of the s t r i a t u m , i n c l u d i n g c h o l i n e r g i c neurons, have undergone massive a t r o p h y . a c e t y l c h o l i n e b a l a n c e model gained pharmacological  The  dopamine-  f u r t h e r support when the -inverse  r e l a t i o n s h i p h e l d t r u e once more - dopamine a n t a g o n i s t s  a n d ^ c h o l i n e r g i c a g o n i s t s a m e l i o r a t e the symptoms of the d i s e a s e ( A q u i l o n i u s and  the  Sjostrom,  1971).  1) P s y c h o p h a r m a c o l o g i c a l In p s y c h o p h a r m a c o l o g i c a l  studies  s t u d i e s i n experimental  examples of the i n v e r s e a c t i o n s of c h o l i n e r g i c and  animals,  dopaminergic  20  drugs are innumerable. here.  Methylphenidate  J u s t a few p e r t i n e n t examples w i l l be induces s t e r e o t y p y by i n c r e a s i n g  cited  dopaminergic  t r a n s m i s s i o n ; i t s e f f e c t s are r e v e r s e d by a d m i n i s t r a t i o n of the m u s c a r i n i c a g o n i s t oxotermorine agonist, pilocarpine, locomotor  (Davis et a l . , 1978).  muscarinic  decreases amphetamine-induced i n c r e a s e s i n  a c t i v i t y , w h i l e scopolamine,  p o t e n t i a t e s the locomotor et.-_al., 1970).  The  a muscarinic antagonist,  e x c i t a t i o n produced  by amphetamine  (Fibiger  In mice w i t h u n i l a t e r a l 6-hydroxydopamine l e s i o n s  of the n i g r o s t r i a t a l p r o j e c t i o n , scopolamine  produces  circling  towards the s i d e of the l e s i o n , i n d i c a t i n g p o t e n t i a t i o n of tone on the i n t a c t s i d e (Pycock  et a l . , 1978).  dopaminergic  This e f f e c t i s  b l o c k e d by a d m i n i s t r a t i o n of t y r o s i n e h y d r o x y l a s e i n h i b i t o r s prevent the s y n t h e s i s of dopamine).  Significantly, direct  i n d i r e c t c h o l i n e r g i c a g o n i s t s depressed  t r a n s m i s s i o n on the i n t a c t  or  the r a t e s of c i r c l i n g  by amphetamine or apomorphine, s u g g e s t i n g antagonism of  (which  caused  dopaminergic  side.  2) Measurement of b i o c h e m i c a l parameters f o l l o w i n g p h a r m a c o l o g i c a l manipulation When i n s t e a d of b e h a v i o r a l measurements, b i o c h e m i c a l parameters i n d i c a t i n g r a t e of t r a n s m i t t e r t u r n o v e r are measured i n response pharmacological  to  m a n i p u l a t i o n , d a t a c o n s i s t e n t w i t h the dopamine-  a c e t y l c h o l i n e b a l a n c e are o b t a i n e d .  Cholinergic agonists increase  dopamine t u r n o v e r i n the s t r i a t u m , w h i l e m u s c a r i n i c a n t a g o n i s t s decrease dopamine turnover i n the s t r i a t u m , presumably as some s o r t of compensatory mechanism, as reviewed Systemic  by B a r t h o l i n ! e t a l . (1975).  a d m i n i s t r a t i o n of n e u r o l e p t i c s (dopamine a n t a g o n i s t s )  causes a marked i n c r e a s e i n the amount of a c e t y l c h o l i n e which  can  21  b e ^ c o l l e c t d from c a t caudate n u c l e u s by p u s h - p u l l e t a l . , 1973; B a r t h o l i n i e t a l . , 1975). a transmitter  i s measured f o l l o w i n g  cannula  (Stadler  More commonly, the l e v e l of  the p h a r m a c o l o g i c a l m a n i p u l a t i o n ;  the l e v e l i s c o n s i d e r e d to be i n v e r s e l y r e l a t e d to the t u r n o v e r r a t e o f the  transmitter.  Thus, e l e c t r o l y t i c l e s i o n o f the s u b s t a n t i a  nigra  causes a t r a n s i e n t decrease i n s t r i a t a l l e v e l s o f a c e t y l c h o l i n e , amphetamine i n c r e a s e s  s t r i a t a l a c e t y l c h o l i n e l e v e l s , b u t r e q u i r e s an  intact n i g r o s t r i a t a l projection Neuroleptics  i n order t o do so (Agid  e t . a l . , 1975).  such as h a l o p e r i d o l , . s p i r o p e r i d o l , c h l o r p r o m a z i n e , and  pimozide, and the d e p l e t e r  of biogenic  creases i n s t r i a t a l a c e t y l c h o l i n e  amines, r e s e r p i n e ,  cause de^  l e v e l s (Cohsolo e t a l . , 1975; G l i c k  et a l . , 1976; Marco e t a l . , 1976; Consolo e t a l . , 1977;. B i a n c h i 1979) .  et a l . ,  I t may be noted t h a t such r e s u l t s have n o t c o n s i s t e n t l y been  obtained i n " o l f a c t o - s t r i a t a l " regions, and  while  olfactory tubercle  namely the n u c l e u s accumbens  (Consolo e t a l . , 1977).  e) The c y t o l o g i c a l r e l a t i o n s h i p between the dopaminergic and c h o l i n e r g i c neuron The  v e r y o r d e r l y way i n which the p h a r m a c o l o g i c a l d a t a have sug-  gested a c o n s t a n t and c o n s i s t e n t inhibitory influence histochemical  antagonistic  e f f e c t o f dopaminergic  on the c h o l i n e r g i c neuron, t o g e t h e r w i t h immuno-  e v i d e n c e , make the model o f a d i r e c t s y n a p t i c  contact  from the dopamine neuron onto the c h o l i n e r g i c neuron v e r y a p p e a l i n g ( H a t t o r i e t a l . , 1976), and t h i s model has been p o p u l a r f o r a number of years  (see F i g . 1 ) . However, some o f :.the p o i n t s  r a i s e d by the  research  c o n t a i n e d i n t h i s t h e s i s and to be found i n the l i t e r a t u r e  make t h i s model appear l e s s a t t r a c t i v e . F i r s t , e l e c t r o n m i c r o s c o p i c data suggest t h a t dopaminergic neurons  22  account f o r approximately et and to  15% of the t e r m i n a l s of the s t r i a t u m ( H a t t o r i  a l . , 1973; H o k f e l t and Ungerstedt, these  1973; A r l u i s o n e t a l . , 1978a,b),  t e r m i n a l s synapse almost e x c l u s i v e l y on s p i n e s , o r a c c o r d i n g  o t h e r s , the d e n d r i t i c s h a f t s of s p i n y neurons ( f o r review, see  P a s i k e t a l . , 1979).  I t i s q u i t e d i f f i c u l t to argue f o r the e x i s t e n c e  of d e n d r i t i c s p i n e s on the p u t a t i v e c h o l i n e r g i c neuron. apparent i n DFP-pretreated  Spines  are not  m a t e r i a l s t a i n e d f o r AChE; i f they do e x i s t  one must p o s t u l a t e t h a t they f o r some reason do n o t c o n t a i n AChE, as does the r e s t of the d e n d r i t e a t the same post-DFP s u r v i v a l  time.  Furthermore, s i n c e the p u t a t i v e c h o l i n e r g i c neuron o n l y c o n s t i t u t e s about 1% of the t o t a l neuron p o p u l a t i o n , i t must have v e r y d e n d r i t i c a r b o r i z a t i o n i n order of the t o t a l s t r i a t a l  to r e c e i v e a l a r g e p r o p o r t i o n o f 15%  terminals.  psychopharmacologically,  The p o s s i b i l i t y  a neuron other  i s thus r a i s e d t h a t ,  than the c h o l i n e r g i c neuron  r e c e i v e s the major p o r t i o n of dopaminergic  input.  In the f i n a l a n a l y s i s , i t i s the psychopharmacological which w i l l make o r break a h y p o t h e s i s e a r l y study  extensive  of n e u r o n a l  experiment  connections.  I n an  ( F i b i g e r e t a l . , 1970), i t was shown t h a t r a t s o l d e r than  25 days modulated t h e i r locomotor s t i m u l a t i o n by amphetamine i n r e sponse to p i l o c a r p i n e ( d e c r e a s i n g the s t i m u l a t i o n ) and scopolamine ( i n c r e a s i n g the s t i m u l a t i o n ) .  However, a t 15 days of age, when the  amphetamine s t i m u l a t i o n c o u l d be demonstrated q u i t e e a s i l y , the c h o l i n e r g i c agents were without to  effect.  I f the dopamine neuron were  t r a n s m i t i t s i n f o r m a t i o n e x c l u s i v e l y through the c h o l i n e r g i c neuron,  as diagrammed i n F i g u r e 1, then these r e s u l t s would be i m p o s s i b l e to obtain:  With no c h o l i n e r g i c r e c e p t o r  to a f f e c t the amphetamine r e -  sponse, the c h o l i n e r g i c neuron i s , a t day 15, mute.  The dopaminergic  neuron must t r a n s m i t i t s i n f o r m a t i o n through some o t h e r neuron, whose  23  p o s t - s y n a p t i c r e c e p t o r has matured by t h i s  age.  The model of the dopamine neuron synapsing  d i r e c t l y onto  the  c h o l i n e r g i c neuron was  the a p p r o p r i a t e model f o r the data a v a i l a b l e a t  the time,- s i n c e i t was  the s i m p l e s t model.  I n s t e a d of a " s e r i e s "  cir-  c u i t , i n which the c h o l i n e r g i c neuron a c t s as the e s s e n t i a l l i n k  through  which dopamine must a c t , we must now  next  s i m p l e s t model may  i n v o l v e a dopaminergic and  a t i n g i n p a r a l l e l on  a t l e a s t f o u r types  The  c h o l i n e r g i c neuron  the same neuron, where they may  e f f e c t s , as diagrammed i n F i g u r e 2. and  c o n s i d e r o t h e r models.  exert t h e i r  terminopposite  With s i x types of neurons ( a t l e a s t )  of a f f e r e n t s to the s t r i a t u m , the model i s bound  to become more complex as s t u d i e s  progress.  Experiments a r e c u r r e n t l y i n p r o g r e s s  to t e s t the h y p o t h e s i s  proposed  above, namely t h a t the c h o l i n e r g i c neuron does not r e c e i v e a l a r g e p r o p o r t i o n of the dopaminergic i n p u t to the s t r i a t u m .  More r i g o r o u s b i o r  chemical  and  h i s t o c h e m i c a l s t u d i e s are b e i n g performed to see i f "the  s e l e c t i v e c h o l i n e r g i c neuron l e s i o n produced by a c i d i n neonatal  striatal  r a t s i s as s e l e c t i v e as i t appears.  i n j e c t s of k a i n i c  Binding  studies,  employing n e u r o l e p t i c s as l i g a n d s as w e l l as dopamine a g o n i s t s , i n d i c a t e no  decrease i n dopamine r e c e p t o r s f o l l o w i n g l e s i o n of  c h o l i n e r g i c neurons i f the h y p o t h e s i s dopamine-stimulated a d e n y l a t e  proposed here i s c o r r e c t .  c y c l a s e should not be a l t e r e d by  s e l e c t i v e c h o l i n e r g i c neuron l e s i o n .  Rotation  (i.e.,  should the Likewise, the  psychopharmaco-  l o g i c a l ) s t u d i e s are planned to see i f t h e r e i s a f u n c t i o n a l assymmetry i n r e c e p t o r s i n the a n i m a l . the n e c e s s a r y time, we may nothing  S t u d i e s such as these w i l l e v e n t u a l l y  i n f o r m a t i o n to c o n s t r u c t a "road map"  yield  of the s t r i a t u m .  In  be a b l e to comprehend the " t r a f f i c p a t t e r n s " t h a t c o n s t i t u t e  l e s s than thoughts.  24  s u b s t a n t i a n i g r a  F i g u r e 1. P h a r m a c o l o g i c a l and immunohistochemical d a t a were a l l n e a t l y e x p l a i n e d by t h i s s i m p l e model, i n w h i c h t h e c h o l i n e r g i c neuron i s p a r t of a " s e r i e s " c i r c u i t . The dopamine neuron from the s u b s t a n t i a n i g r a synapses d i r e c t l y onto the s t r i a t a l c h o l i n e r g i c neuron, which then c o n t a c t s o t h e r neurons of the s t r i a t u m . I n t h i s model, t h e c h o l i n e r g i c neuron i s an e s s e n t i a l l i n k i n dopaminergic neurotransmission. The e x p r e s s i o n of d o p a m i n e r g i c f u n c t i o n thus r e q u i r e s : 1) A dopamine r e c e p t o r on the c h o l i n e r g i c neuron; 2) F u n c t i o n a l a c e t y l c h o l i n e r e l e a s e ; 3) A f u n c t i o n a l c h o l i n e r g i c r e c e p t o r on other s t r i a t a l neurons.  25  substantia nigra  F i g u r e 2. Some p s y c h o p h a r m a c o l o g i c a l and a n a t o m i c a l c o n s i d e r a t i o n s suggest t h a t the c h o l i n e r g i c neuron does not r e c e i v e d i r e c t dopaminergic i n p u t . N o n e t h e l e s s , t h e " d o p a m i n e - a c e t y l c h o l i n e " h y p o t h e s i s has an i n s c r u t a b l e and l a r g e d a t a base to s u p p o r t it. I f t h i s dopamine-acetylcholine i n t e r a c t i o n occurs i n t h e s t r i a t u m , a " p a r a l l e l " c i r c u i t model may e x p l a i n the interaction. A v e r y s i m p l e model t o e x p l a i n the i n t e r a c t i o n i s diagrammed above. The dopamine neuron and c h o l i n e r g i c neuron b o t h synapse onto another s t r i a t a l , each e x e r t i n g o p p o s i t e e f f e c t s on t h a t neuron.  26  REFERENCES  A g i d Y., Guyenet P., G l o w i n s k i J . , Beaujouan J.C., and Javoy F. (1975) I n h i b i t o r y i n f l u e n c e of the n i g r o s t r i a t a l dopamine system on the s t r i a t a l c h o l i n e r g i c neurons i n the r a t . B r a i n Res. 86:488-492. 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Neuropharm. 18:727-730.  35  APPENDIX L i s t of P u b l i c a t i o n s A) Lehmann J . and F i b i g e r H.C. (1978) A c e t y l c h o l i n e s t e r a s e 36 i n t h e s u b s t a n t i a n i g r a and caudate-putamen of t h e r a t : p r o p e r t i e s and l o c a l i z a t i o n i n dopaminergic neurons. J . Neurochem. 30:615-624. B) Lehmann J . , F i b i g e r H.C, and Butcher L . L . (1979) The ^ l o c a l i z a t i o n o f a c e t y l c h o l i n e s t e r a s e i n t h e corpus s t r i a t u m f o l l o w i n g k a i n i c a c i d l e s i o n o f t h e corpus s t r i a t u m : A b i o c h e m i c a l and h i s t o c h e m i c a l study. N e u r o s c i . 4_:217-225.  45  C) Lehmann J . , Nagy J . I . , Atmadja S., and F i b i g e r H.C. (1980) The n u c l e u s b a s a l i s m a g n o c e l l u l a r i s : The u... o r i g i n of a c h o l i n e r g i c p r o j e c t i o n t o the n e o c o r t e x of t h e r a t . N e u r o s c i , i n press.!.  53  D) Lehmann J . and F i b i g e r H.C. (1979) A c e t y l c h o l i n e s t e r a s e and t h e c h o l i n e r g i c neuron. L i f e S c i . 25:1939-1947.  87  36  Journal  (>/ \<'l/^l^('/n'(lN^F^^•.  |«»78. Vol. 30. pp. 615-624. Pergamon Press. Primed in Great Britain.  ACETYLCHOLINESTERASE IN THE SUBSTANTIA NIGRA AND CAUDATE-PUTAMEN OF THE RAT: PROPERTIES AND LOCALIZATION IN DOPAMINERGIC NEURONS JOHN LEHMANN and H . C . FIBIGER  Division of Neurological Sciences, Department or Psychiatry, University of British Columbia, Vancouver, B.C., V6T 1W5, Canada  (Received 20 June 1977. Accepted 11 August 1977) Abstract—In order to examine the hypothesis that acetylcholinesterase (AChE) is contained within dopaminergic neurons of the nigro-striatal projection, the effects of selective destruction of these neurons by 6-hydroxydopamine (6-OHDA) on cholinesterase, tyrosine hydroxylase, and choline acetyltransferase in substantia nigra (SN) and caudate-putamen (CP) were studied in the rat. Four to five weeks after intraventricular or intracerebral 6-OHDA injections tyrosine hydroxylase in these structures was reduced by 90% or more. Choline acetyltransferase was not affected in the SN or CP, but cholinesterase was reduced by about 40% in the SN and by 12% in the CP. To determine that the observed decreases in cholinesterase activity reflected true AChE and not butyrylcholinesterase (BChE). further experiments were conducted on tissues from animals with intracerebral 6-OHDA lesions. (1) Acetylcholine (ACh) was replaced by either acetyl-£-methyl-choline (Ac/?MeCh) or butyrylcholine (BCh) in the cholinesterase assay. SN and CP from 6-OHDA lesioned rats showed 54% and 92% of control tissue cholinesterase activity respectively with Ac/?MeCh as substrate, in good agreement with values found using ACh. No decrease in activity toward BCh was observed. (2) The decrease in cholinesterase activities at different concentrations of ACh was determined. Analysis of the data revealed that cholinesterase in dopaminergic neurons was inhibited by high ACh concentrations, a characteristic property of AChE but not BChE. (3) In the SN, cholinesterase in dopaminergic neurons was inhibited by the selective AChE inhibitors BW284C51 and ambenonium with a dose-response curve similar to erythrocyte AChE but different from serum BChE. The selective BChE inhibitor, tetraisopropylpyrophosphoramide. inhibited the enzyme in dopaminergic neurons only at concentrations which inhibited erythrocyte AChE, concentrations somewhat higher than those which inhibited serum BChE. These results support recent histochemical observations indicating that AChE is contained in dopaminergic neurons of the SN. Moreover, these experiments represent the first characterization of AChE from a homogeneous population of non-cholinergic neurons in mammalian CNS.  Substrate specificity.  Kinetics.  Selective inhibitors.  neurotoxin for catecholaminergic neurons (JAVOY et ai. 1976). F o r example, intraventricular injection of 6 - O H D A can produce widespread destruction of catecholaminergic neurons without having significant effects upon neurons which contain other neurotransmitters (URETSKY & IVERSEN, 1970; M C G E E R et ai, 1973). Furthermore, it has recently been demonstrated that stereotaxic injection of 6 - O H D A into the axons of the nigrostriatal bundle results in both anterograde degeneration of D A terminals in the C P and retrograde degeneration of D A perikarya in the pars compacta of the S N (CLAVIER & FIBIGER. 1977). Thus, by comparing the properties of A C h E in the S N and C P of control and 6 - O H D A lesioned rats, it is possible to deduce some of the characteristics of this 6-OHDA. 6-hydroxydopamine; enzyme in Ac/?the D A neurons. MeCh, acetyl-/?-methylcholine; BCh, butyrylcholine; Cholinesterase .was characterized by three criteria BChE. butyrylcholinesterase (EC 3.1.1.8); CAT. choline used to distinguish true A C h E from BChE acetyltransferase (EC2.3.1.6); CP, caudate-putamen; DA. (EC3.1.1.8). These criteria were: (1) Kinetics. Under dopamine; D M P H . 6.7-dimethyl-5,6.7,8-tetrahydropteridine; isoOMPA. tetraisopropylpyrophosphoramide; NSB. certain conditions. A C h E demonstrates inhibition by nigrostriatal bundle; SN. substantia nigra; T H . tyrosine high concentrations of substrate, while B C h E shows hydroxylase (EC 1.14.16.2). a monotonically increasing velocity with increasing  ALTHOUGH it has been known for some years that the zona compacta of the substantia nigra contains high levels of A C h E (EC 3.1.1.7), only recently has evidence been provided for the existence of this enzyme within the dopaminergic neurons of this nucleus (BUTCHER et ai, 1975). A t present, however, there is no information regarding the biochemical properties of the cholinesterase in these neurons, and it is to this question that the present experiments were addressed. T w o techniques were viewed as providing the means for studying A C h E in D A cells of the S N as well as their terminals in the caudate-putamen. When used appropriately. 6 - O H D A is a selective  Abbreviations used: 4  615  37 JOHN LEHMANN and H. C. FIBIGER  616  acetylcholine concentrations. (2) Substrate selectivity. AChE and BChE hydrolyze different substrates with different velocities. For AChE, the hydrolysis rates for different substrates are ACh > Ac/?MeCh > B C h while for BChE, B C h > ACh > Ac/3MeCh (ADAMS, 1949). (3) Selective inhibitors. Several selective inhibitors of AChE and BChE exist (AUSTIN & BERRY, 1953; DUBOIS et ai, 1950; ALDRIDGE, 1953; LANDS et al., 1955), but these inhibitors have not been tested in the brain using radioenzymatic assay techniques or on specific nuclei within the extrapyramidal system. METHODS Male Wistar rats were obtained from Woodlyn Laboratories, Guelph, Ontario. 6-OHDA (250 ng, dissolved in 25 y\ of 0.9% saline, 0.1% ascorbic acid) was injected into the left lateral ventricle under light ether anesthesia, 1 h following pretreatment with pargyline (50 mg/kg). This procedure has been shown to produce extensive damage to both dopaminergic and noradrenergic neurons (BREESE & TRAYLOR, 1971). Controls received vehicle injections. Experimental animals were aphagic for an average of 3 days following injection, and were fed iniragastrically to maintain body weight. Control and experimental animals were killed 4-5 weeks following injection. Another group of rats received 4 /ig of 6-OHDA dissolved in 2 ftl of the same vehicle, stereotaxically injected into the left NSB at a rate of 0.2 /d/min under pentobarbital anesthesia. These animals received intraperitoneal injections of desipramine H Q (25 mg/kg) 30 min before the 6-OHDA injection to prevent concomitant damage to noradrenergic neurons (ROBERTS et ai, 1975). These animals were also allowed to survive 4-5 weeks following surgery. Animals were killed by cervical fracture. Brains were removed rapidly and the C P was dissected on ice. These tissues included globus pallidus and nucleus accumbens, and averaged 45 mg wet tissue weight. The mesencephalon was sectioned on a freezing microtome and the SN carefully dissected from these sections on ice. Tissues included A9 and A10 areas and pars reticulata, and averaged 8 mg wet tissue weight The right (i.e. contralateral) SN and CP served as control for all enzyme assays in the unilateral NSB lesioned group. Homogenization was in 10 vol of lOmM-sodium phosphate buffer (pH7.4) containing 0.25% Triton X-100. T H (EC 1.14.16.2) was assayed immediately by a modification of the method of COVLE (1972). Final incubation volume was 30/A with final concentrations: D M P H , 1.11 m M ; 2-mercaptoethanol, 111 mM; catalase, 13.9 units; F e S 0 , 6mM; NaAc, l l l m M (pH 5.80); tyrosine, 50^M. Incubations were for 20 min at 30°C. Enzyme activity was corrected for recovery from alumina (60%) and expressed as K^,. Homogenates were frozen and aliquots were taken as needed for subsequent assays. CAT was assayed in the incubation mixture described by MCCAMAN & DEWHURST (1970) in a final volume of 50 ft\. Incubations were for 30 min at 37°C. The product was extracted into 200 /il of 1.5% sodium tetraphenylboron in 3-heptanone. as described by FONNUM (1969). Since most batches of 3-heptanone acidify the aqueous phase, the solvent was previously washed in a separatory funnel with 0.5 M-NaOH. followed by six washes with distilled water. This procedure reduces the blank and results in consislenl 100% efficiency for ACh extraction. Following vigorous 4  4  agitation and centrifugation, 100 /il of the supernatant was added to scintillation vials. Omission of choline or physostigmine virtually eliminated measured CAT activity. Cholinesterase activity, using ACh, BCh, or Ac/?MeCh, was assayed in a final incubation volume of 50 jil, with final concentrations of 15mM-sodium phosphate buffer (pH 7.00) and 5 mM-substrate, except where noted. Tissues were diluted in water by at least a factor of 500, so that endogenous substrates and ions known to affect AChE (e.g. K , C a * , ACh) had insignificant final concentrations. Incubations were at i l " C for 30 min, and in no case was more than 10% of the substrate consumed. The reaction was linear with lime. Extraction with tetraphenylboron (FONNUM, 1969) was used in this assay to remove the labeled substrate: 200 jil of 1.5% tetraphenylboron in washed 3-heptanone was added to stop the incubation, and agitated vigorously. Following centrifugation, the supernatant was aspirated, and the extraction procedure repeated. The pH of the aqueous phase remained at 7.0. Following the second aspiration, 25 /il of the aqueous phase was added to scintillation- vials containing 0.5 ml of 0.1 M-NaOH. +  2  Ten millilitres of Bray's solution was added to scintillation vials for the T H assay, and 6 ml of Aquasol was added to scintillation vials for C A T arid cholinesterase assays. Internal standards were used to determine specific activity. Histochemical staining was performed by the method of KARNOVSKY & ROOTS (1954). Butyrylthiocholine was used as substrate in control sections. The following chemicals were obtained from the sources listed: Catalase, Boehringer-Mannheim; ACh bromide, B D H chemicals; tyrosine, BCh, Ac/JMeCh, sodium tetraphenylboron, bovine erthyrocyte AChE (Type I), and horse serum BChE (Type X), Sigma; acetylcoenzyme A, D M P H , Calbiochem; 3-heptanone, Eastman: [acetyl-l- H]choline. [acetyl-l-^CJcholine, [butyry]-!-" C]choline. [acetyll- C]/?MeCh, [U- C]tyrosine. New England Nuclear; tetraisopropyl pyrophosphoramide (isoOMPA) K & K Laboratories; 1.5-bis-(4 allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51). Burroughs Wellcome Co., North Carolina; and ambenonium chloride. Sterling-Winthrop. Rensselaer, New York. 4  3  ,  M  M  RESULTS Four to five weeks following intraventricular injection of 6-OHDA. T H in the CP and SN were reduced by 90%, while CAT activity in these areas showed no significant change (Table 1). Cholinesterase activity decreased by 31% in the SN and by 12% in the CP, both of which results were statistically significant (P < 0.001, Student's two-tailed t test). Four to five weeks following unilateral 6-OHDA injection into the NSB, similar decreases in T H were obtained in the ipsilateral C P and SN without altering CAT activity in these structures (Table 2). Cholinesterase activity was decreased in the SN by 43% (P < 0.001) and by 12% in the CP (P < 0.01). AChE histochemistry by the method of KARNOVSKY & ROOTS (1954) in a NSB-6-OHDA lesioned animal is shown in Fig. 1. The lesioned side (left) shows reduction in staining in the pars compacta and the ventral tegmental area [the A10 group of dopaminergic cells according to UNGERSTEDT (1971)]. This  38  617  FIG. 1. Section of mesencephalon has been stained for AChE. The pars compacta of the substantia nigra (SNC) and the A10 area stain quite densely for AChE. This staining is decreased on the left half of the section, where dopaminergic cells have been selectively lesioned with an injection of 6-OHDA placed in their ascending axons (the NSB).  39 AChE in dopaminergic neurons  619  TABLE 1. THE ACTIVITIES OF THREE ENZYMES FROM SN AND C P OF RATS INJECTED INTRAVENTR1CULARLY WITH  SN CP  6-OHT \ EXPRESSED AS PER CENT OF CONTROL ± S.E.M.  TH  CAT  AChE  10.3% ±1.7%* 9.3% ± 1.7%*  88.0% ± 7 . 1 % 93.6% ± 2.6%  69.0% ± 1.4%* 87.6% ± 1.8%*  Control values for SN were: T H . 1.14nmol/mg tissue/h; CAT. 2.54nmol/mg tissue/h; AChE. 659nmol/mg tissue/h. Control values for CP were: T H . 1.36nmol/mg tissue/h; CAT. 18.8nmol/mg tissue/h: AChE. 2.12/jmol/mg tissue/h. n = 6. * P < 0.001. TABLE 2. THE ACTIVITIES OF ENZYMES FROM SN AND CP OF RATS INJECTED WITH 6-OHDA IN THE NSB EXPRESSED AS PER CENT OF CONTROL + S.E.M.  Cholinesterase with different substrates  SN CP  TH  CAT  ACh  Ac/3MeCh  BCh  9.9% + 1.4%*** 3.4% ± 0.9%***  99.6% + 6.0% 106.0% ± 4.3%  56.7% + 1.6%*** 87.9% ± 1.7%**  53.7% ± 3.1%*** 92.0% ± 3.5%**-  96.4% ± 6.0% 110.7% ± 6.5%*  Control values for SN were: T H . 1.21 nmol/mg tissue/h: CAT. 2.37 nmol/mg tissue/h. Control values for C P were: T H . 1.33 nmol/mg tissue/h; CAT. 18.8 nmol/mg tissue/h. Cholinesterase values for control tissues are listed in Table 3. n = 6. * P < 0.05. ** P < 0.01. *** P < 0.001. observation confirms the extensive cell loss in the pars compacta of the S N and ventral tegmental area caused by retrograde degeneration after N S B lesions (CLAVIER et al, 1977). In discussing the results of this and subsequent experiments, S N A C h E will be defined as the cholinesterase activity of control substantia nigra tissue; C P A C h E refers to the cholinesterase activity of control caudate-putamen tissue; and A A C h E refers to the difference between S N A C h E and cholinesterase from the S N of 6 - O H D A treated animals (i.e. the A C h E which is probably contained within the dopaminergic neurons). These three categories of cholinesterase were compared with commercially available purified bovine erythrocyte A C h E and horse serum B C h E , which served as standard enzymes. The first criterion employed to characterize these enzymes was the use of selective substrates (Table 2 and 3). Erythrocyte A C h E hydrolyzcd B C h at 1.6% of the rate that it hydrolyzed A C h , while B C h E hydrolyzed B C h at 254% of the rate that it hydrolyzed A C h . Erythrocyte A C h E hydrolyzed Ac/?MeCh at  11% of the rate that it hydrolyzed A C h , while B C h E hydrolyzed Ac/?MeCh at only 0.4% of the rate that it hydrolyzed A C h . Activities reported here were not corrected for the racemic nature of Ac/JMeCh, only one enantiomer of which is reportedly hydrolyzable (HOSKIN. 1963). As shown in Table 3, the 6 - O H D A lesions did not affect the already low rate of B C h hydrolysis in the S N . while there was a slight increase in B C h hydrolysis in the C P , which proved statistically significant (P < 0.05). In contrast, there was a marked decrease in the rate of Ac/?MeCh hydrolysis in the S N (46.3%, P < 0.001) and an 8% decrease in the C P (P < 0.001). This decrease in Ac/?MeCh hydrolysis correlates quite well with the decrease in A C h hydrolysis in both S N and C P after 6 - O H D A lesions. Also, the relative rates of Ac/JMeCh hydrolysis to A C h hydrolysis in S N and C P compared closely to the ratio observed for erythrocyte A C h E (12-14%). For the comparison of the kinetic properties of the enzymes each point was graphed as per cent of activity at 1 m M - A C h in order to superimpose the  TABLE 3. SPECIFIC ACTIVITIES OF CHOLINESTERASES FROM FOUR DIFFERENT SOURCES USING THE THREE SUBSTRATES LISTED  ACh SN CP Erythrocyte AChE Serum BChE  684.3 1992 44.300 583.000  Specific activities of cholinesterases with different substrates (nmol/mg/h) Ac/?MeCh 82.8 278 4940 2500  BCh  70.1 70.5 725 1.480.000  All substrates are 5 mM. n = 6 for brain tissues, n = 2 for commercially supplied erythrocyte AChE and serum BChE.  40 JOHN LEHMANN and H. C. FIBIGER  620  I  i 3.00  i  i  2.60  2.30  I  J  2.00  1.60  1  1  1.30  -log [ACh]  FIG. 2. Kinetics of cholinesterase from various sources expressed as per cent of velocity at 1 mM-ACh. O S N : • C P : O Erythrocyte AChE; • Serum BChE (insei).  curves (Fig. 2). SN AChE and CP AChE apparently differ slightly in the substrate concentration giving highest activity, and erythrocyte AChE demonstrated a higher and sharper peak activity; all three enzymes, however, are inhibited by high concentrations of ACh, a property characteristic of AChE, but not BChE (inset, Fig. 2). In order to quantitate the cholinesterase which is found in dopaminergic neurons, the difference between control and 6-OHDA animals at a single concentration of ACh was measured. That difference (AAChE) can be taken as a measure of the AChE activity found in the dopaminergic neuron, assuming that there was negligible plasticity in the other cholinesterase-containing neurons in the region. In order to characterize the kinetics of the dopaminergic cholinesterase, velocities at different concentrations of ACh were measured in SN from control and unilateral NSB 6-OHDA lesioned animals, and AAChE values were calculated and plotted in Fig. 3. AAChE showed the same typical kinetics as the control SN AChE, indicating that it is true AChE. Unfortunately, the low magnitude of AChE decrease in the CP (as well "as the slight increase in BChE) prevented the same analysis from being performed reliably in that region. Figure 4 shows the effect of increasing concentrations of isoOMPA on cholinesterase activity from the two brain areas, the two standard enzymes, and AAChE, calculated as described. isoOMPA selectively inhibited BChE only at concentrations around 10" M. Figures 5 and 6 show that BW284C51 and ambenonium were potent inhibitors of AChE, although there was some inhibition of BChE at higher concentrations. In Figs. 4-6 it is evident that SN AChE and AAChE resemble erythrocyte AChE more closely than they resemble BChE on these graphs, but C P AChE resembles erythrocyte AChE more 5  faithfully than the enzymes from SN. The only case where AAChE differs markedly from SN AChE is in its dose-response to ambenonium: the Kj for AAChE is an order of magnitude lower than for SN AChE, and AAChE is more completely inhibited by 10 Mambenonium as well (Fig. 6). _4  DISCUSSION The decreases in AChE activity in SN and C P that were observed following 6-OHDA lesions of the dopaminergic nigrostriatal projection by two different methods confirm recent histochemical studies which have suggested that AChE is synthesized by the dopa-  3.00  2.60 2.30 2.00  1.60 1.30  -log[ACh]  FIG. 3. Kinetics of A C h E from SN of animals lesioned unilaterally by 6-OHDA injections into the nigrostriatal bundle. O S N . control side; • S N , lesioned side; A difference calculated between SN control and SN lesioned.  41 A C h E in dopaminergic neurons  O-i  o  Jo _c 6?  50 H  100 Con. 9  8 -log fisoOMPA  FIG.  T 7 6  -log fisoOMPA]  4. Per cent inhibition of cholinesterase by isoOMPA, 1 x 10~ M to 1 x 1 0 " * M . O S N (control); A ASN (SN control-SN lesioned); • C P (control); O Erythrocyte A C h E ; + Serum BChE. 9  100  Con. 9 8  7 6  -log  [BW 284C51J  F I G . 5. Per cent inhibition of cholinesterases by BW 284C51. 1 x 1 0 ~ M t o l x 10"* M . O SN (control); 9  A ASN (SN control-SN lesioned); •  C P (control: O Erythrocyte A C h E : • S e r u m BChE.  -log jambenonium j  - log Jotnbenonium]  F I G . 6. Per cent inhibition of cholinesterases by ambenonium, 1 x 10  9  M to 1 x 10  trol); A ASN (SN control-SN lesioned) Cl C P (control); O Erythrocyte A C h E ; •  4  M.  O S N (con-  Serum BChE.  4Z 622  JOHN LEHMANN ;  minergic neurons of the SN (BUTCHER et ai, 1975;  d H. C. FIBIGER  constant, in tissue obtained from different regions of  BUTCHER & BILEZIKJIAN, 1975; BUTCHER & HODGE,  the nervous system (TUNNICLIFF et ai, 1976). The  1976). Both AChE levels measured by radioenzymatic assay and AChE histochemical staining were observed to decrease following treatment with 6-OHDA (Fig. 1 and Tables 1 and 2 ) Some transsynaptic plasticity of AChE in nondopaminergic cells in the SN and CP following lesion of DA neurons cannot be ruled out However, in a histochemical study BUTCHER et ai (1975) examined the synthesis of AChE in the SN following irreversible inhibition of cholinesterase with diisopropylfluorophosphate and traced the de novo synthesis of AChE within the DA cell bodies. Thus, transsynaptic plasticity can be ruled out as the sole factor mediating the decrease in AChE in the SN following 6-OHDA. This is not a surprising result, since it has been established that all known catecholamine neurons stain for AChE  qualitative differences of AChE with respect to kinetic properties and response to inhibitors may be related to the heterogeneous molecular properties of this enzyme (CHAN et ai, 1972; V U A Y A N & BOWNSON, 1974; MCINTOSH & PLUMMER, 1976; RIEGER & VIGNY, 1976; SOMOGYI & CHUBB, 1976; G U R D , 1976). There  are hazards, of course, with imputing a different molecular structure of AChEs from different sources on the basis of assays performed on a crude homogenate. Although the tissues were diluted at least 500 fold, some very potent component in the tissue may have exerted a modulatory effect on the enzyme's properties. Likewise, the properties of the purified erythrocyte AChE may be due to an artifact of purification, e.g. a partial proteolysis at some stage of the procedure. (JACOBOWITZ & PALKOVITS, 1974; PALKOVITS & JACOThe presence of AChE on noncholinergjc neurons BOWITZ, 1974) and that total brain AChE decreases has been suggested by some to indicate that those by 13% following intraventricular 6-OHDA injection neurons receive a cholinergic input (PARENT & in mice (BENTON et ai, 1975). BUTCHER, 1976). The present data and other considerThe histochemical regimen may not be as reliable ations suggest however that AChE cannot be conas biochemical methods with respect to differentiating sidered a reliable marker for cholinoception. Primarbetween BChE and AChE (CONTESTABILE, 1976; ily, this is because there is a dramatic lack of correlaBRIDGES et ai, 1973). In the case of the dopaminergic tion between CAT and AChE in some regions of the neurons of the nigrostriatal system, however, the use nervous system. For example, the cerebellum of of several substrates, selective inhibitors, and different various species contains high levels of AChE, but low concentrations of ACh have confirmed the suggestion levels of CAT, and there is no correlation between that dopaminergic neurons contain true AChE the two enzyme levels in the different strata of cere(BUTCHER et ai, 1975). AAChE, the AChE which is bellar cortex (GOLDBERG & M C C A M A N , 1967). In depleted from the SN by 6-OHDA lesions, was char- addition, rabbit dorsal root ganglion contains high acterized by kinetic properties, substrate specificity, levels of AChE, but insignificant levels of CAT and response to selective inhibitors as true AChE. ( M C C A M A N & H U N T , 1965). AChE appears therefore It is likely that AAChE is AChE contained in dopa- to be playing an unknown role in these _reas where minergic neurons of the SN. The failure to observe ACh is not being synthesized in significant amounts. a decrease in BCh hydrolysis after 6-OHDA treatIt is generally agreed that compared to the striatum ments indicates that BChE is not contained in these the SN also contains a relatively low CAT to AChE neurons. ratio (MCGEER et ai, 1973; FONNUM et ai, 1974) and The difference between AChE and BChE, using the this raises the question as to the function, if any, of three biochemical criteria, were confirmed with com- AChE in the dopaminergic neurons of the nigro-striamercial preparations from erythrocytes and serum, re- tal projection. At present a cholinergic afferent to the spectively. The cholinesterases from the CP and SN SN has not been demonstrated. Hemitransections fulfill all the criteria for true AChE: (1) SN AChE anterior or posterior to this nucleus have no effects and CP AChE are inhibited by high concentrations upon nigral CAT activities (MCGEER et ai, 1973; unof ACh. (2) The relative rates of ACh to Ac^MeCh published observations). Furthermore, the failure of hydrolysis by SN AChE and CP AChE compared kainic acid injections into the SN to affect CAT acquite closely to the rates observed in this study for tivity in this nucleus suggests that CAT is not conerythrocyte AChE. (3) The responses of AChE from tained within perikarya in the SN which might these three sources to three selective inhibitors were synapse with DA neurons (Nagy, Vincent, Lehmann, essentially similar and were distinct from the response Fibiger & McGeer, in preparation). At present thereof BChE. Some qualitative differences between eryth- fore the localization of CAT within the SN is unrocyte, SN and CP AChE were observed Specifically, known and .there is no firm evidence to indicate that SN AChE showed some differences from CP AChE the dopaminergic perikarya or dendrites receive chowith respect to kinetics (Fig. 2) and effects of selective linergic innervation. If AChE in the DA perikarya inhibitors (Figs. 4-6). Second, the effects of selective does not serve to hydrolyze ACh released upon them, inhibitors on CP AChE paralleled the effects of these this raises the possibility that the enzyme is syntheinhibitors on erythrocyte AChE more closely than did sized in the SN but that its function with respect to SN AChE. Previous workers have also found differ- cholinergic transmission occurs in the axon terminals ences in a kinetic parameter of AChE, the Michaelis of the NSB in the striatum. Specifically in view of  43 AChE in dopaminergic neurons the growing evidence supporting dendro-axonic transmission (LLINAS& HESS, 1976), it is possible that dopaminergic neurons synthesize AChE to inactivate ACh released by cholinergic dendrites upon which the dopaminergic terminals are thought to synapse (HATTORI et ai, 1976; GIORGUIEFF et ai, 1976). Alternatively, cholinergic axons in the striatum may synapse • on dopaminergic axons or boutons. According to conventional criteria, however, there is no ultrastructural evidence for the presence of axo-axonic synapses in the striatum (KEMP & POWELL, 1971; HATTORI, personal communication). As a rule, cholinoceptive cells may be incapable of localizing AChE to the area of cholinoception. In addition to the above considerations, further support for this hypothesis is found in the peripheral sympathetic system, where AChE has been identified in noradrenergic terminals in the pineal gland (ERANKO et ai, 1970; RODRIGUEZ DE LORES ARNAIZ & PELLIGRINO DE IRALDI, 1972), and where C A T is virtually negligible (Lehmann, unpublished observations). In view of the well-established cholinergic input to the noradrenergic cell bodies in the superior cervical ganglion which give rise to the noradrenergic terminals in the pineal gland, the presence of AChE in those terminals may be due to a transport process incapable of specifically localizing AChE to the area of cholinoception. Therefore, although AChE may prove useful as a marker for cells which are cholinoceptive at some locus of the cell, it clearly cannot be utilized to identify the point of cholinergic contact. To the extent that the decrease in AChE in the CP can be attributed entirely to AChE present in dopaminergic axons and terminals, the cell bodies in the SN appear to transport a greater amount of the enzyme to their terminals than resides in the cell bodies: in terms of absolute enzyme activities, a decrease in the CP of 10,000 nmol/h/CP (i.e. 240nmol/mg tissue/h x 45 mg tissue/CP) correlates with a decrease in the SN of 2400 nmol/h/SN (i.e. 300nmol/mg tissue/h x 8mg tissue/SN). Approximately four times as much AChE is exported to axons and terminals as is retained in the dopaminergic cell bodies and dentrites of the SN—roughly the same ratio as is observed for T H in this system. The presence of AChE in dopaminergic neurons of the SN has been confirmed biochemically, and the transport of AChE by nigrostriatal axons is suggested The results presented here shed no light on the question of cholinoception by these dopaminergic neurons. Qualitative differences between AChE from CP, SN and bovine erythrocytes has been shown, with respect to kinetics and response to selective inhibitors. The nigrostriatal system may offer a good system for studying molecular isoenzymes of AChE from a homogeneous population of neurons in the CNS. The specific role of AChE on dopaminergic neurons in the SN is not understood When the origin of C A T in this nucleus is elucidated, and when the question of cholinoception by dopaminergic terminals is  623  resolved, the function of the enzyme may be better understood.  Acknowledgements—Supported by the Medical Research Council. The excellent technical assistance of S. ATMADJA is gratefully acknowledged. We thank B. D. 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Neurochem. 27, 23.121-129. 47-53. MCGEER P. L... FIBIGER H. C. HATTORI T., SINGH V. K.. . ROBERTS D. C. S.. ZIS A. P. & FIBIGER H. C. (1975) MCGEER E. G. & MALER L. (1974) Res. 93, 441-454. (MYERS R. D. & DRUCKER-COLIN R. R. DE LORES ARNAIZ G. & PELLEGRINO DE IRALDI RODRIGUEZ  45  NWii.w-i.w-i-. Vol. 4. pp. 217-225. Pcrgamon Press L i d , 1974. Primed in Great Britain. O  0306-4522/79)0201 -0217S02.0O/O  IBRO  THE LOCALIZATION OF ACETYLCHOLINESTERASE IN THE CORPUS STRIATUM AND SUBSTANTIA NIGRA OF THE RAT FOLLOWING KAINIC ACID LESION OF THE CORPUS STRIATUM: A BIOCHEMICAL AND HISTOCHEMICAL STUDY J. L E H M A N N and H . C . FIBIGER Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver. B.C. V6T 1W5, Canada and L. L. BUTCHER Department of Psychology and Brain Research Institute. University of California. Los Angeles. California. U.S.A. Abstract—The distribution ofacetylcholinestera.se in the corpus striatum and substantia nigra was examined with the use of kainic acid lesions of the corpus striatum and pharmacohistochemical experiments. Histochemically identified acetylcholinesterase-comaining neurons in the striatum were among those which were destroyed by kainic acid. Complementary biochemical studies demonstrated that approximately 50";, of the total acetylcholinesterase activity in the striatum was localized in these acetylcholineslerase-containing neurons. Intrastriatal injections of kainic acid produced a substantial decrease in the activity of the glutamic acid decarboxylase in the substantia nigra, thus demonstrating that neurons contributing to the striato- and/or pallidonigral pathways had been lesioned. However, nigral acetylcholinesterase activity was not significantly reduced by the striatal kainic acid injections. Furthermore, stereotaxic injections of colchicine along the course of the striatonigral projection failed to produce an accumulation of acetylcholinesterase in these fibers proximal to the injection. In contrast, injections of colchicine into the nigrostriatal projection led to a proximal accumulation of acetylcholinesterase in the fibers of this system, thus confirming the presence of acetylcholinesterase in the ascending dopaminergic neurons. It is concluded that the striato- and pallidonigral projections in the rat do not contain significant levels of acetylcholinesterase. Furthermore, acetylcholinesterase-containing neurons in the striatum appear to be interneurons rather than the source of striatal efferents. It is suggested that some of these acetylcholinesterase-containing neurons may be striatal cholinergic interneurons.  CERTAIN  nuclei  within  the  extrapyramidal  system  in pharmacohistochemical experiments to stain inten-  pars compacta. contain among the highest levels of  sely for A C h E  (BUTCHER,  acetylcholinesterase  1975a). In the  present experiments, we sought  (acetylcholine  hydrolase,  EC  3.1.1.7; A C h E ) in the central nervous system (SILVER, '  1974). Recent studies from  this laboratory suggest  that approximately 40% of the A C h E activity in the substantia nigra of the rat is contained within axons '  CHAND & BUTCHER. 1977). which have been shown  such as the caudate, putamen and substantia nigra,  or terminals afferent to this nucleus ( N A G Y , VINCENT, LEHMANN. FIBIGER & M C G E E R , 1978). Consistent with this finding are the results of earlier  lesion studies  which led POIRER & co-workers (OLIVIER.  PARENT.  SIMARD & POIRIER. 1970) to propose that in the cat the projection  from  the striatum to the  substantia  nigra contains A C h E . Furthermore, it has been suggested that this projection neurons in the  striatum  may  arise from  aspiny  (POIRIER. PARENT, M A R -  TALBOT &  BILEZIKJIAN, to  evaluate further the existence and source of A C h E in the striatonigral and pallidonigral projections. In order to destroy these systems selectively without producing  concomitant  damage  to  the  dopaminergic  nigrostriatal projection, which is also known to contain A C h E (BUTCHER ci ai. 1975a; BUTCHER, TALBOT &  BILEZIKJIAN, 1975/?; BUTCHER, 1977; L E H M A N N &  FIBIGER,  1978). kainic acid, a neurotoxin  which is  thought to destroy perikarya selectively, leaving afferent axons and terminals intact (COYLE & SCHWARCZ, 1976: M C G E E R & M C G E E R , 1976), was injected  intra-  striatally and the effects on A C h E and other neuronal enzyme markers in the corpus striatum were determined. Furthermore, the brains of rats lesioned with kainic acid were examined  by A C h E histochemistry Abhreviaiions: AChE, acetylcholinesterase: ChAT. choto determine if cells which stained for A C h E were line acetyltransferase: D F P . diisopropylphosphorofluoridestroyed by kainic acid. date: G A D . glutamic acid decarboxylase. 17  46 J. LEHMANN, H. C. FIBIGER and L. L. BUTCHER  218 EXPERIMENTAL  1 9 7 1 6 ; M C G E E R & M C G E E R , 1 9 7 5 ; HATTORI, SINGH,  PROCEDURES  M C G E E R & M C G E E R , 1 9 7 6 ; RIBAK, 1978), i n d i c a t e d M a l e Wistar rats (300-325 g) were obtained from that the k a i n i c a c i d injections destroyed m o r e t h a n W o o d l y n Laboratories, Guelph, Ontario. Ten nmol kainic 7 0 % o f the - / - a m i n o b u t y r a t e - c o n t a i n i n g and c h o l i n e r acid in 1 //I 10 m,M sodium phosphate p H 6.5, 0.9% in N a C l gic cell b o d i e s . T h e a c t i v i t y o f A C h E was r e d u c e d was injected over 5 min unilaterally into the caudateputamen of rats anesthetized with pentobarbital. F r o m by 4 0 % i n the same tissues whereas there was s o m e stereotaxic zeros, coordinates were: anterior 9 . 6 m m : increase i n s t r i a t a l t y r o s i n e h y d r o x y l a s e . I n c o n t r a s t , lateral 2.8 m m : dorsal 4.5 m m . T w o weeks later the rats nigral t y r o s i n e h y d r o x y l a s e a n d C h A T activities were were killed by cervical fracture and the corpora striata not significantly affected. M o s t i m p o r t a n t i n terms o f dissected on ice. These tissues included the caudatethe possible presence o f A C h E i n the s t r i a t o n i g r a l putamen, the globus pallidus, and the nucleus accumbens, p r o j e c t i o n was the l a c k o f a significant decrease i n and averaged 45 mg wet tissue weight. The mesencephalon the a c t i v i t y of n i g r a l A C h E despite the extensive was sectioned on a freezing microtome and the substantia d a m a g e to the c o r p u s s t r i a t u m ( T a b l e 1 a n d F i g . 1) nigra was carefully dissected from these sections on ice. Tissues included A 9 and A 1 0 areas and pars reticulata, and the extensive d a m a g e t o the s t r i a t o n i g r a l a n d / o r averaging 8 mg wet tissue weight. Homogenization and p a l l i d o n i g r a l p r o j e c t i o n s as s h o w n by the decrease i n assay of tyrosine hydroxylase (L-tyrosine, tetrahydropterin i g r a l G A D a c t i v i t y . It s h o u l d be noted that h i s t o l o dine: oxygen oxidoreductase (3-hydroxylating) E C g i c a l e x a m i n a t i o n o f the extent o f the k a i n i c a c i d 1.14.6.2). choline acetyltransferase ( A c e t y l - C o A : choline-Olesion i n d i c a t e d that it i n c l u d e d the g l o b u s p a l l i d u s acetyltransferase E C 2.3.1.6; C h A T ) and A C h E were as as w e l l as the s t r i a t u m . N o significant cell d e s t r u c t i o n previously described (LEHMANN & FIBIGER, 1978). G l u t a was o b s e r v e d outside the c o r p u s s t r i a t u m . mic acid decarboxylase (L-glutamate 1-carboxylase E C 4.1.1.15: G A D ) was assayed by the method of MCGEER, MCGEER & WADA (1971a). Protein was assayed by the method of LOWRY, ROSEBROUGH, FARR & RANDALL (1951). Histochemical staining for A C h E was performed by the method of KARNOVSKY & ROOTS (1964) at 24-48 h followD F P pretreatment suppresses " b a c k g r o u n d ' A C h E ing pretreatment with diisopropylphosphorofluoridate s t a i n i n g ( w h i c h is p r e s u m a b l y c o n t a i n e d i n axons, ter( D F P ) . The irreversible inhibitfon of A C h E by the non-spem i n a l s a n d d e n d r i t i c processes o f neurons) a n d a l l o w s cific phosphorylating agent D F P allows A C h E to be visualized at various stages of regeneration, i.e. following de the v i s u a l i z a t i o n o f discrete p e r i k a r y a . A x o n a l l y t r a n s synthesis and subsequent transport to distal cell prop o r t e d A C h E is also seen m o r e clearly at l o n g e r s u r cesses. C o m b i n e d with other experimental manipulations v i v a l times. F o r these reasons, D F P pretreatment was as outlined in the figure captions, this technique permits e m p l o y e d for a l l A C h E h i s t o c h e m i c a l experiments. the identification of A C h E - p o s i t i v e neurons simultaneously Intrastriatal injections o f k a i n i c acid greatly with anatomical characterization. reduced the a m o u n t o f s t r i a t a l A C h E revealed by h i s -  Histochemistry  novo  Biochemistry  pare  neuronal  enzymes  w h i c h are  (MCGEER, MCGEER,  FIBIGER &  Fig. IB  and  C). In  the  uninjected  p u t a m e n , stained p e r i k a r y a appeared  contained  processes,  in neurons whose p e r i k a r y a are l o c a t e d i n the c o r p u s striatum  AChE-positive  seen i n the u n l e s i o n e d s t r i a t u m of the same rat ( c o m -  A s seen i n T a b l e 1, the m a r k e d fall i n the activities ChAT,  injections perikarya  the  v i s u a l i z e d by means o f D F P pretreatment. w h i c h were  RESULTS  o f G A D and  t o c h e m i s t r y ( F i g . 1A). F u r t h e r m o r e , these eliminated  at  least  at  the  light  caudate-  to have a s p i n y  microscopic  WICKSON,  d a l p o i n t s between the s t r i a t u m a n d substantia n i g r a  TABLE I. NEUROTRANSMITTER-RELATED ENZYMES IN CORPUS STRIATUM AND SUBSTANTIA NIGRA 2 WEEKS AFTER INJECTION OF KAINIC ACID (10 nmol) IN THE CAUDATE-PUTAMEN C o n t r o l value + S.E.M.  % Control Corpus striatum acetylcholinesterase choline acetyltransferase glutamic acid decarboxylase tyrosine hydroxylase  62.5% 27.5% 23.3% 126.8%  + ± ± ±  6.0%* 7.3%* 3.4%* 5.5%*  43.4 109.9 103.7 7.88  + 2.07 /imol/mg protein/h ± 4.01 n m o l / m g protein/h + 3.71 nmol/mg protein/h ± 0 . 3 1 9 nmol/mg protein/h  Substantia nigra acetylcholinesterase choline acetyltransferase glutamic acid decarboxylase tyrosine hydroxylase  92.0% 104.0% 51.1% 93.9%  ± ± ± ±  4.8%, 9.8% 3.8%* 4.9%  10.9 16.7 265.0 5.59  + + ± +  M = 12. *P < 0.001, Student's two-tailed test.  level  ( F i g . I B ) . Injection of c o l c h i c i n e at v a r i o u s r o s t r o - c a u -  0.132 /imol/mg protein/h 0.96 nmol/mg protein/h 8.75 nmol/mg protein/h 0.404 n m o l / m g protein/h  47 219  A  CX  FIG. 1. Loss of acetylcholinesterase-containing neuronal somata after infusion of I0nmol/1 / i l kainic acid into the right striatum (A). Non-infused side is shown on left side of A and in B: arrows point to individual cell bodies. Dashed lines in A delimit the area displaying loss of acetylcholinesterase activity, shown in detail in C. Acetylcholinesterase method as described previously (BUTCHER el al.. 1975a). 1.5 mg/kg D F P was injected intramuscularly 24 h prior to death, cx, cerebral cortex; fb, fiber bundle perforating the striatum. Scale in A is 4 m m ; scale in C is 400//m and this magnification applies also to B.  FIG. 2. Partial trajcclories of ihe nigrostriatal pathways (arrows, frames A and B) visualized by a c e u l cholinesterasc histochemistry following unilateral intracerebral infusion of colchicine (0.5 /<g in I ; i l 0.9"„ saline: rate = 0.25 /il/min) into the left globus pallidus and adjacent regions (frame A ) or into the left medial forebrain bundle regions and contiguous areas (frame B l . Rats were killed 48 h after treatment with 1.5 m g / k g D F P and 72 h after infusion of colchicine. In frame C is depicled the striatonigral pathway visualized according to I h e horseradish peroxidase procedure of D E OLMOS (1977): ( s e e also BUTCHER & GIKSLER. 1977): 0.5 /<] of a 40",, horseradish peroxidase solution was unilaterally infused into the caudate-putamen over a 5-min period: rats were killed 48 h after the injection. Anterograde transport of the enzyme reveals I h e partial trajectory of the striatonigral projection (frame C). which bears considerable resemblance l o the striatonigral palhwa> as demonstrated by protein-incorporation autoradiography with [ H ] p r o l i n c (cf. BUTCHER. 19781. H o r i z o n l a l sections are shown. Dashed lines in frame A bracket the area in which portions of I h e striatonigral pathway are contained. Scale = 4 mm. P C . substantia nigra, pars c o m p a c l a : P R . substantia nigra, pars reticulata: IC. internal capsule: ct. cannula tract. The PLLLIGRINO & CUSHMAN (I967| coordinates were: striatum: A P = 2.0. Lai = 3.0. vertical from c o n i c a l surface = 5.0: globus pallidus: A P — 0.8. Lat. 3.5. vertical = 6.5. Medial forebrain bundle and adjacent regions: A P = 1.8. Lat = 1.5. vertical = 8.2. 3  221  49  ••  FIG. 3. Accumulation of acetylcholinesterase in fibers of ascending dopaminergic pathways from the substantia nigra and probably also the ventromedial mesencephalic tegmentum (bracketed by arrows in F and H) following unilateral infusion of 0.5 ug 1 /J1 colchicine into the ventral thalamus (coordinates according to PELLEGRINO & CuSHMAN {1967): AP = 0.0. Lat = 1.5. vertical from cortical surface = 8.0). The non-infused side of the brain is shown at various levels in A, C, E and G corresponding to the same levels from the same brain sections on the infused side (B. D, F and H). Observe the absence of accumulation of acetylcholinesterase in processes of neuronal somata in the caudate-putamen complex on the infused side of the brain (B. D: compare with A and C. respectively). C and D are higher power depictions of A and B. respectively. E and F show the ventral thalamus and adjacent dorsolateral hypothalamus. G and H depict the substantia nigra and adjacent ventromedial mesencephalic tegmentum. Rats were killed 24 h after intramuscular injection of 1.5 mg/kg D F P and 72 h after intracerebral colchicine infusion. Scale in B is 500nm and applies to A-B and E-H; scale in D is 3 0 0 / i m and applies also to C. fb.fiberbundle perforating CP: PC. substantia nigra, pars compacta; PR. substantia nigra, pars reticulata.  50 Acetylcholinesterase in corpus striatum and substantia nigra reliably resulted in accumulation of A C h E in axons of the nigrostriatal projection proximal to the injection site (Fig. 2A and B, Fig. 3E-H). In contrast, accumulation of A C h E was never observed in the proximal segments of axons of the striatonigral or pallidonigral systems after colchicine injections in the vicinity of these projections (Fig. 2A and B, Fig. 3 A - D ) . Finally, unlike the accumulation and increased staining for A C h E in the processes of the cells of the zona compacta of the substantia nigra after these colchicine injections, such a phenomenon was never seen in the striatum (compare Fig. 3E-H with 3A-D).  DISCUSSION The present results failed to provide evidence for the presence of A C h E in the striatonigral or pallidonigral projections in the rat. Thus, although the kainic acid lesions extensively destroyed perikarya in the striatum and globus pallidus, including those which stain heavily for A C h E , no statistically significant loss of this enzyme could be detected biochemically in the substantia nigra ipsilateral to the lesion. Furthermore, in agreement with conclusions drawn by SHUTE & LEWIS (1967), colchicine injections in the vicinity of nigrostriatal and striatonigral fibers produced A C h E accumulation caudal but not rostral to the injection. The factors underlying the apparent discrepancy between these results and those of previous investigators (OLIVIER et ai, 1970) are presently not clear but could conceivably be related to species differences. However, inasmuch as OLIVIER et al. (1970) utilized electrolytic lesions and long survival times it is also possible that retrograde degeneration of the A C h E containing nigrostriatal projection may have contributed to their findings. This possibility has also recently been put forward by POIRIER et al. (1977). Nigral G A D activity was significantly decreased by these lesions, thus confirming previous suggestions that some of the striatonigral and/or pallidonigral fibers contain G A D (HATTORI, MCGEER, FIBIGER & M C G E E R , 1973; FONNUM, GROFOVA, RINVIK, STORMMATHISEN & WALBERG, 1974; GALE, H O N G & G u i -  DOTTI, 1977). The decrease in nigral G A D activity following striatal kainic acid lesions is complementary to the decrease in nigral G A D activity following intranigral kainic acid lesions (NAGY et ai, 1978), suggesting that nigral G A D is partially derived from intrinsic nigral neurons and partially derived from striatonigral and/or pallidonigral afferents. The modest decrease in striatal A C h E observed after kainic acid lesions indicates that approximately 50% of the A C h E activity in this structure is contained within neurons which are intrinsic to the corpus striatum. The figure of 50% is obtained by extrapolating to a 100% lesion of the C h A T and G A D markers. We have recently shown that approximately 12% of the total A C h E activity in the striatum is contained within the axons and terminals of the dopa-  223  minergic nigro-striatal projection (LEHMANN & FIBIGEE. 1978). Conceivably, cortical, thalamic and raphe afferents could make up the balance. However, two other possibilities must also be considered. First, some A C h E may be contained within non-neuronal elements of the striatum (but cf. BUTCHER et ai, 1975a). Second, denervation plasticity of the sort observed with A C h E in the superior cervical ganglion (SOMOGYI & CHUBB, 1976; GISIGER, VIGNY, GAUTRON  & RIEGER, 1978) must be considered as a contributing factor to biochemical changes which result from lesions. Our observation that kainic acid lesions, which produced extensive damage to the AChE-containing neurons in the striatum, did not result in a biochemically detectable change in A C h E activity in the substantia nigra suggests that these AChE-positive cells do not project to the substantia nigra. A C h E reactive neurons are few in number and appear aspiny at the light-microscopic level (BUTCHER et ai. 1975a; POIRIER et ai, 1977). Furthermore, in the monkey (POIRIER et ai, 1977) A C h E reactive neurons are larger ( > 25 /<m) and fewer in number than the medium spiny neuron (12-18 nm, KEMP & POWELL, 1971:13-20 / t m , GROFQVA,  1975). These observations suggest that neurons with high A C h E activity are not medium spiny neurons, and that medium spiny neurons have low, if any, A C h E activity. Although earlier work suggested that the large aspiny neurons were the source of striatal efferents to the globus pallidus and substantia nigra (Fox, RAFOLS & COWAN, 1975), more recent studies have demonstrated that the striatal efferents originate predominantly, if not exclusively, from the mediumsized, spiny neurons of KEMP & POWELL (1971) (GROFOVA, 1975; BUNNEY & AGHAJANIAN, 1976: Kocsis, PRESTON & KITAI. 1976: S. T. KITAI, personal com-  munication). Lack of A C h E staining in these numerous medium-sized, spiny efferent cells is consistent with the absence of a detectable change in A C h E activity in the substantia nigra after the kainic acid lesions. Inasmuch as the aspiny AChE-containing neurons appear not to project to the substantia nigra, this raises the speculation that some of the AChE-reactive cells may be the cholinergic neurons which are also thought to be intrinsic to the corpus striatum (MCGEER et ai, 1971b). Arguing against this speculation is the immunohistochemical observation by HATTORI et al. (1976) that some dendritic spines in the striatum contain C h A T , and that medium spiny neurons, as characterized by an unindented nucleus (KEMP & POWELL, 1971). contain C h A T (T. HATTORI,  personal communication). If this is the case, then on the basis of presently available evidence it would have to be concluded that these cholinergic, spiny neurons do not contain A C h E . Such a situation would be unprecedented for cholinergic neurons which typically contain very high levels of A C h E activity. Clearly, additional work is required to identify the nature of AChE-containing neurons in the corpus striatum.  51 224  J. LEHMANN, H. C. FIBIGER and L. L. BUTCHER  (U.C.L.A.) is thanked for performing the histochemical exAcknowledgements—Supported by the Medical Research Council (J.L. and H.C.F.) and by USPHS grant NS-10928 perirr.f-'s. The authors thank T. HATTORI for stimulating discussions and reviewing the manuscript. (L.L.B.). The excellent technical assistance of S. ATMADJA is gratefully acknowledged. Mr. KEN HIRABAYASHI  REFERENCES  BUNNEY B. S. & AGHAJANIAN G. K. (1976) The precise localization of nigral afferents in the rat as determined by a retrograde tracing technique. 117, 423-435. BUTCHER L. L. (1977) Nature and mechanism of cholinergic-monoaminergic interactions in the brain. 21, 1207-1226. BUTCHER L. L. 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Sci. 31, 181-198.  Acetylcholinesterase in corpus striatum and substantia nigra  225  RIBAK C. E. (1978) Immunocytochemical localization of glutamic acid decarboxylase (GAD) in the rat corpus striatum.  Anal. Rec. 190, 521. Brain The Biology of Cholinesteruses.  SHUTE C. C. D. & LEWIS P. R. (1967) The ascending cholinergic reticular system: cortical, olfactory and subcortical projections. 90, 497-520. SILVER A. (1974) Elsevier. New York. SOMOGYI P. & CHUBB 1. W. (1976) The recovery of acetylcholinesterase activity in the superior cervical ganglion of the rat following its inhibition by diisopropylphosphorofluoridate: a biochemical and cytochemical study. 1, 413-421. . •  Neuroscience  53  THE NUCLEUS BASALIS MAGNOCELLULARIS:  THE ORIGIN OF A CHOLINERGIC  PROJECTION TO THE NEOCORTEX OF THE RAT  by  John Lehmann, J . I . Nagy, S. Atmadja and H.C.  D i v i s i o n of N e u r o l o g i c a l  Sciences  Department of P s y c h i a t r y U n i v e r s i t y of B r i t i s h Columbia Vancouver, B.C., Canada  Please send correspondence t o : Dr. H.C.  Fibiger  D i v i s i o n of N e u r o l o g i c a l  Sciences  Department o f P s y c h i a t r y U n i v e r s i t y of B r i t i s h Columbia Vancouver, B.C., Canada  V6T 1W5  V6T 1W5  Fibiger  54 Abbreviations AChE, a c e t y l c h o l i n e s t e r a s e ; propylphosphorofluoridate; radish  peroxidase.  CAT, GAD,  choline acetyltransferase; glutamic a c i d decarboxylase;  DFP, HRP,  diisohorse  4  55  ABSTRACT The  cells  of o r i g i n of a n e o c o r t i c a l  been i d e n t i f i e d by peroxidase injected intense  a n t e r o g r a d e and  cholinergic afferent projection  r e t r o g r a d e methods i n the  i n t o neocortex labeled  rat.  Horseradish  large, acetylcholinesterase  neurons i n the v e n t r o m e d i a l e x t r e m i t y of  the  The  region  i n which c e l l  depletion  acetyltransferase  and  showed s i g n i f i c a n t  activities  electrolytic  k a i n i c a c i d l e s i o n s r e s t r i c t e d to the m e d i a l " p a r t  p a l l i d u s each r e s u l t e d  i n s i g n i f i c a n t depletions  transferase  H e m i t r a n s e c t i o n s c a u d a l to t h i s c e l l  in  and  AChE.  such d e p l e t i o n s .  intense  Taken t o g e t h e r  neurons l y i n g i n the  ventromedial extremity of  mapped i n t h i s s t u d y , c o n s t i t u t e projection treated  to the neocortex.'  a f t e r DFP  utility  of c h o l i n e r g i c  i n n o m i n a t a of p r i m a t e s , and (nBM)  i n the  t h a t n u c l e u s of  similarities  the  i n s i z e and  ergic perikarya  of  the  not  result  AChEas  cholinergic  i n DFP-prei n the  light  4-8  criterion  rat.  No  homologous t o  of  hrs  for  the  are  the n u c l e u s b a s a l i s  thus termed " n u c l e u s b a s a l i s  e v i d e n c e was  d i a g o n a l band p r o j e c t s  obtained  to s u p p o r t  to n e o c o r t e x .  AChE a c t i v i t y were o b s e r v e d among t h e  nBM,  acetyl-  perikarya.  neurons i n q u e s t i o n appear to be  nocellularis"  sufficient,  globus  globus p a l l i d u s ,  of AChE h i s t o c h e m i s t r y  not  the  the  o r i g i n of a major s u b c o r t i c a l  p r e t r e a t m e n t i s a n e c e s s a r y , but  substantia  thesis  that  S p e c i f i c a l l y , i t i s p r o p o s e d t h a t h i g h AChE a c t i v i t y  identification The  of  group d i d  suggest the  Discrete  choline  a n i m a l s i n i d e n t i f y i n g c h o l i n e r g i c neurons i s d i s c u s s e d  t h i s example.  the  The  the  AChE.  of n e o c o r t i c a l  these o b s e r v a t i o n s  This  cortical  d e c r e a s e s i n the and  of c h o l i n e  occurred a l s o  ~  (AChE)-  globus p a l l i d u s .  same group of neurons underwent r e t r o g r a d e d e g e n e r a t i o n f o l l o w i n g ablations..  have  the n u c l e u s of  the  the  of mag-  hypo-  However, s t r i k i n g putative  d i a g o n a l band, and  the  cholin-  medial  septal  nucleus. Kainic destruction.  acid lesions  of  These l e s i o n s  the n e o c o r t e x produced u n i f o r m and decreased n e o c o r t i c a l  glutamic a c i d  complete  perikaryal  decarboxylase  activity,  suggesting  ever,  same l e s i o n s d i d not  the  This observation cortex,  that  there  suggests that  a conclusion  that  are  GABAergic p e r i k a r y a  i n the n e o c o r t e x -  affect neocortical choline there  are  no  acetyltransferase.  cholinergic perikarya  i s c o n s i s t e n t w i t h the absence o f  r e a c t i v e neurons i n n e o c o r t e x .  How  i n the  neo-  i n t e n s e l y AChE-  57  INTRODUCTION Acetylcholinesterase  ( a c e t y l c h o l i n e hydrolase, EC 3.1.1.7; AChE) p e r s e  has not proven to be a r e l i a b l e marker f o r c h o l i n e r g i c neurons and t h e i r p r o j e c t i o n s i n the c e n t r a l nervous system (PHILLIS, 1976).  For example, i t i s  now known that high l e v e l s of AChE are contained i n c e r t a i n dopaminergic and noradrenergic neurons (SILVER, 1974; JACOBOWITZ & PALKOVTTS, 1974; BUTCHER, TALBOT & BILEZIKJIAN, 1975; LEHMANN & FIBIGER-, 1978). Despite t h i s lack of s p e c i f i c i t y f o r c h o l i n e r g i c neurons,  histochemical  studies of AChE have proven to be of value i n suggesting p o t e n t i a l c h o l i n e r g i c p r o j e c t i o n s because a l l neurons that have been unequivocally  characterized  as  c h o l i n e r g i c appear to contain very high l e v e l s of AChE (e.g., the septo-hippocampal p r o j e c t i o n : LEWIS, SHUTE, & SILVER, 1967; MESULAM, VAN HOESEN & ROSENE, 1977;  LYNCH, ROSE & GALL, 1978; the motoneuron, KREUTZBERG, TOTH & KAIYA, 1975;  v i s c e r a l e f f e r e n t neurons of the i n t e r m e d i o l a t e r a l MARCHAND, PARENT & POIRIER, 1977). a t i o n i t can therefore be postulated  s p i n a l cord,  BUTCHER,  On the basis of c u r r e n t l y a v a i l a b l e i n f o r m that a strong histochemical  reaction for  AChE i s a necessary (but not s u f f i c i e n t ) c r i t e r i o n f o r the i d e n t i f i c a t i o n o f c h o l i n e r g i c neurons. KODAMA (1929) f i r s t provided evidence f o r a p r o j e c t i o n from the magnoc e l l u l a r neurons of the basal f o r e b r a i n to the neocortex and t h i s  observation  has been confirmed and extended by others (DAS, 1971; KIEVET & KUYPERS, 1975; DIVAC, 1975; JONES, BURTON, SAPER & SWANSON, 1976). reported  MESULAM & VAN HOESEN (1976)  that neurons i n the basal forebrain of the rhesus monkey t h a t s t a i n  i n t e n s e l y for AChE are a l s o labeled with HRP a f t e r i n j e c t i o n s of the l a t t e r enzyme i n t o the neocortex.  On the basis of t h i s evidence these authors sup-  ported e a r l i e r suggestions (SHUTE 6. LEWIS, 1967; DIVAC, 1975) that there i s a c h o l i n e r g i c p r o j e c t i o n from the basal forebrain, mostly from the nucleus b a s a l i s of the substantia  innominata, which innervates  the p r e c e n t r a l neocortex.  Re-  58  cently, KELLY & MOORE (1978) have published r e s u l t s that appear to be c o n s i s t e n t with t h i s hypothesis.  They found  that e l e c t r o l y t i c l e s i o n s i n the v i c i n i t y of  the globus p a l l i d u s of the r a t produced s i g n i f i c a n t decreases.in c o r t i c a l c h o l i n e acetyltransferase a c t i v i t y .  EMSON & LINDVALL (1979) have a l s o r e c e n t l y com-  mented on various magnocellular  f o r e b r a i n n u c l e i as p o s s i b l e o r i g i n s of the  c h o l i n e r g i c innervation of the neocortex. was  The purpose of the present experiments  to provide further information concerning the o r i g i n and l o c a l i z a t i o n of  c h o l i n e r g i c neuronal elements i n the  neocortex.  METHODS Male Wis tar r a t s weighing approximately used i n a l l experiments. pentobarbital anesthesia.  A l l surgery was  conducted while animals were under  Horseradish peroxidase  i n j e c t e d i n t o various c o r t i c a l areas i n 0.1 30% i n 0.9%  saline.  300 gm at the time of surgery were  (HRP; .Sigma type VI)  y l volumes at a concentration of  The h i s t o l o g i c a l p r o t o c o l of MESULAM & VAN  MESULAM (1976a,b) ; MESULAM (.personal communication) was of HRP HRP  was  HOESEN (1976);  followed for v i s u a l i z a t i o n  with benzidene dihydrochloride: a l t e r n a t e sections were stained for both  and AChE, according to the. same procedure.  lowing HRP  Twenty-four to t h i r t y hours f o l -  i n j e c t i o n s , the animals were perfused under deep p e n t o b a r b i t a l anes-  thesia with 50 ml 0.9%  s a l i n e at room temperature; t h i s was  fusion of 400 ml cold  f i x a t i v e (1% paraformaldehyde and 1.25%  10% sucrose, 0.1 M sodium phosphate b u f f e r , pH 7.4).  The HRP  stored i n  M sodium phosphate  and kept overnight at 4°C.  r e a c t i o n was  c a r r i e d out e s s e n t i a l l y as p r e v i o u s l y described..  (MESULAM 1976a; MESULAM, personal communication). r i n s e d i n d i s t i l l e d water for 1 min. incubation medium containing 4 mM s u l f a t e , and  glutaraldehyde,  The b r a i n was  f i x a t i v e for 2-3 hrs, then t r a n s f e r r e d i n t o 5% sucrose- 0.1 buffer (pH 7.4)  followed by a per-  50 mM  sodium acetate.  Free f l o a t i n g s e c t i o n s were  They were then incubated  a c e t y l t h i o c h o l i n e , . 10 mM  for one hour i n  g l y c i n e , 2 mM  The pH of t h i s incubation medium was  copper main-  59  tained  a t 5.5.  30  each.  sec  0.1% for  Sections  were t h e n r i n s e d  Then they were t r a n s f e r r e d  n i t r o f e r r i c y a n i d e , 0.01 10 min.  The  t r a y was  t i s s u e s were r e i n s e r t e d 4.5  out  benzidine dihydrochloride pH  5.0,  10%  in  of t h i s l a s t medium b r i e f l y ,  added to an  incubation  into t h i s incubation  H R P - r e a c t i o n was  nitroferricyanide,  50%  s t o p p e d by  volume o f  m i x t u r e and  were r i n s e d  kept at  (Sigma),  ethanol,  while  100  5  ml  ml.  shaken g e n t l y  distilled slides, cresyl  water.  and  The for 4  to  T h i s was  0-4°C.  Following  sections followed  by  9%  sodium  5.0),  which  20 m i n u t e s i n t h i s medium,  water.  were immersed  Subsequently, sections  air-dried.  into  In  i n 10%  order  to v i s u a l i z e  potassium  ferri-  extensive r i n s i n g i n s i x changes were mounted from w a t e r o n t o  In some c a s e s the  sections  of  glass  were c o u n t e r s t a i n e d  with  violet.  Five cortical  t y p e s of b r a i n  l e s i o n s were u t i l i z e d ;  (1)  l e s i o n s were made i n s e v e n a n i m a l s by  damage any The  min.  the  sections  sodium a c e t a t e b u f f e r „(pH  i n s i x changes of d i s t i l l e d  AChE r e a c t i o n p r o d u c t ,  c y a n i d e f o r 15  t r a n s f e r r i n g the  e t h a n o l i n 40 mM  f r e s h l y p r e p a r e d and  sections the  lifted  water,  min. The  was  to 50%  M sodium a c e t a t e b u f f e r  H 2 O 2 was  f r e s h l y p r e p a r e d 0.3%  i n s i x changes o f d i s t i l l e d  subcortical structures  lesions included  such as  f r o n t a l , dorsal  and  the  Extensive u n i l a t e r a l  suction.  Care was  taken not  hippocampus, septum and  lateral  cortex  anterior  to  to  striatum.  the  level  of  (T) bregma.  Gelfoam  rats received lularis at  the  zero. with  was  The  i n c i s o r bar  nmoles of  + 8.1 was  mm,  ML  set at  + 2.6 -4.2  mm  (3)  i n a volume o f  inates  used  ceived  u n i l a t e r a l i n j e c t i o n s of k a i n i c a c i d  the  y l over a period  e l e c t r o l y t i c nBM  A second  a current  DV  + 3.6  mm  of  of 2 mA  (10  (4)  nine  magnocelf o r 20  sec  from s t e r e o t a x i c  (7.0)  seven m i n u t e s a t  lesions.  group o f  Eleven animals were  i n NaPOi, b u f f e r e d  saline  for  and  (2)  the n u c l e u s b a s a l i s  applying  mm.  kainic acid dissolved 0.2  the v o i d .  l e s i o n s of  l e s i o n s were made by  c o o r d i n a t e s AP  two  to f i l l  unilateral electrolytic  (nBM).  The  applied  injected  sterile the  same c o o r -  Another n i n e aminals  nmol i n 2 y l o f  "•' j .  the  re-  same v e h i c l e  9  60  over f i v e m i n u t e s ) i n t o t h e f r o n t a l c o r t e x ML + 2.0 mm, (5)  and DV -2.2 mm  a t the c o o r d i n a t e s  from s t e r e o t a x i c z e r o  mm,  ( i n c i s o r "bar s e t a t + 5.0 mm).  Hemitransections o f the b r a i n a t a l e v e l j u s t p o s t e r i o r to t h e entopeduncular  nucleus  (AP + 5.9 mm  from s t e r o t a x i c z e r o ,  seven a n i m a l s a c c o r d i n g All  i n c i s o r b a r a t - 4.2 mm)  a n i m a l s t h a t had r e c e i v e d u n i l a t e r a l  Animals t h a t had r e c e i v e d  were s a c r i f i c e d  Animals which r e c e i v e d  one week p o s t o p e r a t i v e l y .  on a f r e e z i n g m i c r o t o m e . sections sected  from u n f r o z e n b r a i n s  the s t r i a t u m .  surface The  kainic acid l e s i o n s of the cortex  Some b r a i n s were s e c t i o n e d  (see F i g . 1 ) .  by making a c o r o n a l  The f r o n t a l  activities  of choline  acetyltransferase  (Acetyl-CoA:  EC 2.3.1.6; CAT) and AChE were measured a c c o r d i n g  cortical  dis-  p o l e was.  choline-O-acetylto modifications  Glutamic a c i d decar- •  ( L - g l u t a m a t e 1 - c a r b o x y l a s e , EC 4.1.1.15; GAD) was a s s a y e d b y t h e method  LOWRY, ROSENSROUGH,  & McGEER (1970).  FARR & RANDALL,  histological verification  f r o n t a l cortex  o f the k a i n i c a c i d - i n d u c e d  and t h e n p e r f u s e d  were removed and p l a c e d  Where p o s s i b l e  to  l e s i o n s of the  l e s i o n s o f t h e nBM, t h e a n i m a l s were  w i t h 10% F o r m a l i n  i n 10% F o r m a l i n  a t 50 ym a t the l e s i o n s i t e s  P r o t e i n was m e a s u r e d a c c o r d i n g  (1951).  and nBM, and e l e c t r o l y t i c  deeply a n e s t h e t i z e d  violet.  these  cortex.  of CHALMERS, McGEER, WICKSON  For  coronally  c u t j u s t a n t e r i o r t o t h e head  (LEHMANN & FIBIGER, 1978) o f t h e method o f FONNUM (1969) . boxylase  two  Care was taken t o remove the o l f a c t o r y b u l b s f r o m t h e v e n t r a l  o f the f r o n t a l  transferase,  were s a c r i f i c e d  the m e d i a l and c e n t r a l g l o b u s p a l l i d u s , was  from each s i d e o f t h e b r a i n  obtained  electrolytic  The s e c t i o n s were k e p t c o l d on i c e and f r o m  the nBM, w h i c h i n c l u d e d  (1973).  c o r t i c a l l e s i o n s by s u c t i o n were  l e s i o n s , k a i n i c a c i d l e s i o n s o f nBM, and h e m i t r a n s e c t i o n s weeks p o s t o p e r a t i v e l y .  were made i n  t o t h e method o f McGEER, FIBIGER, McGEER & BROOKE  used a f t e r a s u r v i v a l time o f s i x months.  of  AP + 1 0 . 8  - 0.9% s a l i n e .  - 0.9% s a l i n e f o r two weeks,  The b r a i n s '  sectioned  on a f r e e z i n g microtome and s t a i n e d w i t h c r e s y l  these h i s t o l o g i c a l v e r i f i c a t i o n s  were c o n d u c t e d on t h e  61 same b r a i n s that were analyzed f o r the v a r i o u s enzymes.  For h i s t o c h e m i c a l  s t u d i e s of AChE, animals were i n j e c t e d i n t r a m u s c u l a r l y w i t h d i i s o p r o p y l p h o s p h o r o f l u o r i d a t e (DFP, 1.5 mg/kg, Sigma, d i s s o l v e d i n peanut o i l ) and s a c r i f i c e d a t v a r i o u s times l a t e r .  Histochemical s t a i n i n g f o r AChE was performed a c c o r d i n g  to KARNOVSKY & ROOTS (1964) on 25 um, f r e e - f l o a t i n g s e c t i o n s .  Perikaryal  dimensions were estimated both p h o t o g r a p h i c a l l y and w i t h the a i d o f a measuring eyepiece, both of which were c a l i b r a t e d w i t h a stage micrometer.  11  62  RESULTS TOPOGRAPHY OF INTENSELY AChE-REACTIVE NEURONS IN THE Nucleus  o f the d i a g o n a l band and m e d i a l  The and  2.  BASAL FOREBRAIN  septal nucleus  o r g a n i z a t i o n of i n t e n s e l y A C h E - r e a c t i v e neurons At  neurons  the most r o s t r a l l e v e l  i s given i n F i g s .  ( F i g . 1A-B), numerous i n t e n s e l y  AChE-reactive  i n the m e d i a l s e p t a l n u c l e u s and v e r t i c a l l i m b o f t h e n u c l e u s o f  d i a g o n a l band  (nD3)  can be  becomes more l a t e r a l l y  and  identified. ventrally  Moving c a u d a l l y  l o c a t e d , and  l i m b o f the nDB  (PRICE & POWELL, 1970).  this c e l l  becomes much l e s s  group  t e n s e l y A C h E - r e a c t i v e neurons n u c l e u s and  l a t e r a l hypothalamic  t h e s e i n t e n s e l y A C h E - r e a c t i v e neurons w i t h one  a n o t h e r , a l t h o u g h as n o t e d  t i o n d e n s i t i e s of t h e  nDB  horizontal  scattered, magnocellular i n -  the r e g i o n s termed  area  the  the  At more c a u d a l l e v e l s " ( F i g . 2 A - E ) ,  compact; now  occupy  ( F i g . 1C-D), t h e  is called  1  magnocellular  preoptic  (WYSS, SWANSON & COWAN, 1979). a r e more or l e s s  A l l of  c o n t i g u o u s a t some p o i n t  t h e r e a r e marked d i f f e r e n c e s  i n the  popula-  neurons.  Ventral pallidum A few seen  s c a t t e r e d , m a g n o c e l l u l a r , i n t e n s e l y AChE-reactive neurons  i n the r e g i o n termed  1B-E).  I t i s important  t o the v e n t r a l  to note  part  that  the term v e n t r a l p a l l i d u m r e f e r s innominata  of the g l o b u s p a l l i d u s  the m e d i a l b o r d e r of the g l o b u s p a l l i d u s neurons  w h i c h may  However, t h e s e neurons,  homologue of the p r i m a t e n u c l e u s b a s a l i s  The  to  the  does n o t  (HEIMER & WILSON, 1975) . of i n t e n s e l y  Along  AChE-reactive  a t the more d o r s a l and. c a u d a l which are b e l i e v e d  of the s u b s t a n t i a  c u s s i o n ) , have a c h a r a c t e r i s t i c morphology and Distribution  o f p r i m a t e s , and  i s a group  be c o n t i n u o u s w i t h the nDB  a s p e c t s o f the nDB.  be  " v e n t r a l p a l l i d u m " by HEIMER & WILSON (1975) ( F i g .  r a t ' s p u t a t i v e homologue o f s u b s t a n t i a refer  can  t o be  innominata  the (see  rat's dis-  topographical d i s t r i b u t i o n .  o f the n u c l e u s b a s a l i s m a g n o c e l l u l a r i s  r o s t r a l p o l e o f the n u c l e u s b a s a l i s m a g n o c e l l u l a r i s (nBM)  i s located  63  approximately j u s t caudal to the decussation of the a n t e r i o r commissure.  At  t h i s point i t i s situated along the v e n t r a l and medial boundaries of the globus p a l l i d u s ( F i g . IE) u n t i l the t a i l of the globus p a l l i d u s i s reached  ( F i g . 2D).  It should be noted, however, that a few scattered c h a r a c t e r i s t i c a l l y l a r g e , i n t e n s e l y AChE-reactive neurons  t y p i c a l of nBM are found deep w i t h i n the core  of the globus p a l l i d u s at a l l rostrocaudal  levels.  Furthermore, a t i t s most  caudal extent,the nBM extends v e n t r o l a t e r a l l y into" the medial h a l f of the t a i l of the globus p a l l i d u s ( F i g . 2E).  At t h i s point and f u r t h e r caudally  many of the intensely AChE-reactive neurons are located KREUTZBERG,1968), that i s , t h e i r perikarya  ( F i g . 2F),  interstitially  (DAS &  and dendrites are s i t u a t e d between  the f i b r e bundles of the i n t e r n a l capsule.  At the most caudal l e v e l s i n v e s t i -  gated i n t h i s study ( F i g . 2F) , these large intensely AChE-reactive neurons are located i n the v e n t r a l edge of the i n t e r n a l capsule, predominantly l a t e r a l to the entopeduncular nucleus, which would appear i n sections s l i g h t l y more caudal than F i g . 2F. Injections of HRP i n t o the f r o n t a l and antero-dorsal neocortex r e s u l t e d i n numerous HRP-labeled neurons  i n nBM ( F i g . 3A). With the r o s t r a l l y  located  i n j e c t i o n s used i n these experiments, the most a n t e r i o r l y labeled c e l l s were observed j u s t v e n t r a l to the GP i n nBM.  In those cases i n which there was d i f -  fusion of HRP from the i n j e c t i o n s i t e i n the neocortex to s u b c o r t i c a l a few labeled c e l l s were o c c a s i o n a l l y observed i n the nDB. HRP d i f f u s i o n remained within was observed.  regions,  However, when the  the confines of the neocortex, no l a b e l i n g o f nDB  The majority of neurons  labeled by KRP i n the present experiments  were found i n the group of large, multipolar neurons located medial regions of the p o s t e r i o r h a l f of the GP. the l o c a t i o n of the AChE-intense neurons  i n the v e n t r a l and  Tnese corresponded p r e c i s e l y to  seen i n F i g s . IF and 2A.  With the  r o s t r a l c o r t i c a l HRP i n j e c t i o n s employed i n t h i s study, the caudal extent of  64  labeled c e l l s i n nBM corresponded to the AChE-intense neurons  i n F i g . 2B.  subsequent preliminary experiments i t has been found that more p o s t e r i o r  In cortical  HRP i n j e c t i o n s (e.g., o c c i p i t a l cortex) l a b e l magnocellular neurons a t more p o s t e r i o r regions of the nBM, i . e . , at the l e v e l of the entopeduncular n u c l e u s . In order to determine i f the c o r t i c a l HRP i n j e c t i o n s d i d i n f a c t l a b e l these intensely AChE-reactive neurons of the nBM, the method of MESULAM & VAN HOESEN (1976) was u t i l i z e d to demonstrate HRP and AChE i n the same s e c t i o n .  Although  AChE s t a i n i n g was somewhat reduced by t h i s procedure, i t n e v e r t h e l e s s r e v e a l e d that some c e l l s i n the nBM with a high AChE content also contained HRP r e a c t i o n product. MORPHOLOGICAL OBSERVATIONS OF THE INTENSELY AChE-REACTIVE NEURONS IN THE BASAL FOREBRAIN Nucleus of the diagonal band, medial septal nucleus, and caudate-putamen . The AChE-intense neurons of the medial septal nucleus and nDB have s i m i l a r morphological features, with the size of the major axis o f the p e r i k a r y a ranging from 19-42 ym, and averaging 29 ym. putamen have s i m i l a r dimensions  AChE-intense aspiny neurons  of the caudate-  ( F i g . 3C, range, 23-47 ym; average, 34 ym) .  Since intensely AChE-reactive neurons i n the striatum are l e s s densely packed than the i n t e n s e l y AChE-reactive neurons of the medial s e p t a l nucleus and nDB, t h e i r dendrites are more e a s i l y v i s u a l i z e d , although f o r a l l three of these n u c l e i , the appreciable "background"  s t a i n i n g f o r AChE l i m i t s the d i s t a n c e from  the soma at which the dendrite may be seen c l e a r l y . Nucleus b a s a l i s m a g n o c e l l u l a r i s While the p e r i k a r y a of the nBM have s i m i l a r dimensions  to those  neurons  described above (major axis ranging from 25 to 45 ym, averaging 35 ym), t h e i r dendrites are e a s i l y seen against the background are u s u a l l y found.  of white matter i n which they  Thus the n e u r o p i l that these neurons occupy  distinguishes  them anatomically from the other intensely AChE-reactive neurons of the b a s a l  65  forebrain. their  The most s t r i k i n g m o r p h o l o g i c a l f e a t u r e o f t h e n e u r o n s o f nBM i s  " i s o d e n d r i t i c " nature  (RAMON-MOLINER & NAUTA,1966; DAS & KREUTZBERG,1968) :  t h e i r d e n d r i t e s t a p e r o f f v e r y g r a d u a l l y from difficult (Fig.  t o d e l i n e a t e e x a c t l y where t h e soma ends and t h e d e n d r i t e b e g i n s  3B) .  crepancy  This morphological feature i n a l l p r o b a b i l i t y  between t h e a b s o l u t e d i m e n s i o n s  PARENT, GRAVEL & OLIVIER ( 1 9 7 9 ) . with minimal in  the p e r i k a r y o n , i n d e e d making i t  clusters,  e a s i l y be a s c e r t a i n e d .  so t h a t  i n course.  Often these c e l l s  these l a r g e ,  to  t h o s e i n the m e d i a l  somatofugally, a r e arranged  the morphology o f i n d i v i d u a l n e u r o n s  These c l u s t e r s a r e q u i t e o f t e n so d e n s e l y p a c k e d  low m a g n i f i c a t i o n they appear t o form a g i a n t n e u r o n . of  f o r the^dis-  r e p o r t e d h e r e a n d t h o s e r e p o r t e d by  The d e n d r i t e s p r o j e c t d i r e c t l y  branching or deviation  t i g h t l y packed  accounts  interstitial  cannot that at  F u r t h e r , t h e morphology  n e u r o n s i s v a r i a b l e and i r r e g u l a r  compared  s e p t a l n u c l e u s and nDB.  LESION STUDIES Retrograde  degeneration of nucleus basalis magnocellularis  E x t e n s i v e l e s i o n s o f the n e o c o r t e x a n t e r i o r  t o bregma r e s u l t e d  l o s s o f l a r g e , i n t e n s e l y A C h E - r e a c t i v e neurons t h a t were l o c a t e d  i n a marked  i n t h e m e d i a l and  v e n t r a l a s p e c t s of t h e GP ( F i g . 2A-F) , i n agreement w i t h d a t a p r e s e n t e d by DAS (1971) f o r t h e r a b b i t . r e g i o n on t h e l e s i o n e d shrunken violet,  Of t h e few A C h E - i n t e n s e side of the b r a i n ,  and p y k n o t i c ( F i g . A ) . t h e r e was no a p p a r e n t  t h e v e n t r a l and m e d i a l GP.  remained  the m a j o r i t y a p p e a r e d  i n this  t o b e somewhat  I n s e c t i o n s t h a t were c o u n t e r s t a i n e d w i t h  decrease  neurons t h a t d i d n o t s t a i n i n t e n s e l y of  neurons t h a t  i n the p o p u l a t i o n o f t h e s m a l l e r  f o r AChE and t h a t were l o c a t e d  cresyl  diameter  i n the region  The i n t e n s e l y A C h E - r e a c t i v e n e u r o n s o f t h e m e d i a l  s e p t a l n u c l e u s and nDB d i d n o t appear t o undergo r e t r o g r a d e changes o r d e g e n e r ation after  the c o r t i c a l  acetyltransferase  lesions.  On the c o r t i c a l l y - l e s i o n e d  (CAT) and AChE a c t i v i t y were d e c r e a s e d  while glutamic acid  decarboxylase  side choline  i n t h e r e g i o n o f nBM,  (GAD) a c t i v i t y was n o r m a l  (Table I ) .  15  66  Anterograde degeneration f o l l o w i n g Electrolytic parallel  depletions  (Table I I ) . kainic the  and  acid  kainic acid  o f CAT and  lesions  o f nucleus  l e s i o n s o f the  AChE a c t i v i t i e s  F i g . 5 shows, d i a g r a m m a t i c a l l y , lesions.  d i s t r i b u t i o n of  the m e d i a l and  Comparison o f the the  ventral  large,  n i f i c a n t number o f t h e s e n e u r o n s . c u l a r n u c l e u s d i d not  nBM r e s u l t e d  i n the  the  in similar  ipsilateral  and  frontal  cortex  e x t e n t o f e l e c t r o l y t i c and  a r e a s encompassed by the  intensely  globus p a l l i d u s  basalis  lesions  with  AChE-reactive, i n t e r s t i t i a l neurons  indicated  that  the  lesions  damaged a s i g -  Hemitransections j u s t caudal t o the  r e s u l t i n s i g n i f i c a n t depletions  of either  in  entopedun-  CAT o r AChE  (Table I I ) . Cortical kainic acid Kainic  lesions  a c i d i n j e c t i o n s i n the  neuronal d e s t r u c t i o n logy.  CAT,  in a l l layers  assayed  f o r CAT,  a minor d e p l e t i o n  kainic acid lesioned  AChE and  i n AChE, and  tissues  between w e i g h t s o f l e s i o n e d , examination a l s o d i d not the  kainic  acid  GAD.  cortical  These t i s s u e s  a major d e p l e t i o n . i n  d i d not  shrink,  since  c o n t r a l a t e r a l , and  indicate  rat resulted  i n uniform  o f c o r t e x a s a s s e s s e d by c r e s y l v i o l e t  a r e a s composed r o u g h l y 50% o f the  Tne a f f e c t e d  s e c t e d and  f r o n t a l c o r t e x o f the  that  any  tissue  histo-  sample  dis-  showed n o d e p l e t i o n  in  GAD ( T a b l e I I I ) . T h e  t h e r e was no d i f f e r e n c e  control  s h r i n k a g e had  tissues.  Histological  o c c u r r e d one  week a f t e r  lesion. DISCUSSION  The  nucleus b a s a l i s In p r i m a t e s ,  straightforward, r o n s found w i t h i n appropriately  - substantia  the  delineation  and l i k e w i s e the  termed  the  substantia  innominata o f the  complex  substantia  innominata i s r e l a t i v e l y  c l u s t e r s of m a g n o c e l l u l a r , A C h E - i n t e n s e i n n o m i n a t a i s c l e a r ; hence t h i s c e l l  " n u c l e u s b a s a l i s o f the  substantia  group  innominata" i n  the  topography o f t h e s e n u c l e i  o b s e r v a t i o n s and  i s not  as c l e a r  PARENT, p e r s o n a l c o m m u n i c a t i o n ) .  i n c a t and  rat  HEIMER & WILSON  is  primates  (KIEVET & KUYPERS, 1975; JONES e t a l . , 1976; MESULAM & VAN HOESEN, 1976). ever,  neu-  How-  (present (1975)  have  67  attempted  to c l a r i f y the current understanding of the topography  of the r a t ' s  homologue to the substantia innominata c a l l e d the "ventral p a l l i d u m " i n the r a t , d e s c r i b i n g i t as an area which or o l f a c t o r y tubercle,  2)  1)  receives an input from nucleus accumbens and/  l i e s adjacent to s t r i a t a l s t r u c t u r e s , and  3)  has  n e u r o p i l i d e n t i c a l to the globus p a l l i d u s . According to d e s c r i p t i o n s of the nucleus b a s a l i s i n v a r i o u s s p e c i e s , these neurons are c h a r a c t e r i s t i c a l l y intensely AChE-reactive, l a r g e (25-45 ym),and p r o j e c t to the neocortex (DAS 1976; MESULAM & VAN  & KREUTZBERG,1968; DAS,1971; DIVAC,1975; JONES et a l . ,  HOESEN,1976 ; PARENT et al.,1979).  These c h a r a c t e r i s t i c s  have been used as operational c r i t e r i a f o r mapping nBM  i n F i g s . 1 & 2.  It i s  clear that i n the r a t , the n e u r o p i l surrounding these neurons does not  always  resemble that of the globus p a l l i d u s ; neither does the d i s t r i b u t i o n of these neurons always follow the region of the v e n t r a l pallidum o u t l i n e d by REIMER & WILSON (1975) and NAUTA, SMITH, "FAULL & DOMESICK (1978), e s p e c i a l l y i n the more caudal sections ( F i g s . 1E-2F).  This leads us to question the a p p l i c a b i l i t y o f  the term "nucleus b a s a l i s of the substantia innominata" f o r the r a t . we have adopted the more parsimonious nomenclature,  Hence,  "nucleus b a s a l i s magnocel-  l u l a r i s , " and i t i s suggested that i n the r a t t h i s c e l l group i s homologous to the nucleus b a s a l i s of the substantia innominata i n primates. the nBM  The homology o f  i n r a t and nucleus b a s a l i s of the substantia innominata i n primates i s  supported by t h e i r common c h a r a c t e r i s t i c c o r t i c a l p r o j e c t i o n s , morphology, and intense AChE a c t i v i t y . Organization of intensely AChE-reactive neurons  i n the basal f o r e b r a i n  The s i m i l a r i t i e s and apparent c o n t i n u i t y of large, i n t e n s e l y AChE-reactive neurons of the r a t f o r e b r a i n has already been noted (DIVAC ,1975). of d i f f e r e n c e s i n p r o j e c t i o n areas of these neurons the i n t e n s e l y AChE-reactive neurons  On the b a s i s  (EMSON & LINDVALL ,1979) ,  i n the basal f o r e b r a i n of DFP-pretreated r a t  can be d i v i d e d into at l e a s t three main groups:  (a)  the medial s e p t a l nucleus,  x/  68  (b)  the nucleus of the diagonal band of Broca, and  magnocellularis (nBM).  (c) the nucleus b a s a l i s  The intensely AChE-reactive neurons of the medial septum  are probably the o r i g i n of the v e i l known c h o l i n e r g i c septo-hippocampal j e c t i o n (see LYNCH et al.,1978).  The intensely AChE-reactive neurons  pro-  of the  medial septum are continuous with those of the r o s t r a l p o r t i o n of the nDB ( F i g . 1A,B).  Furthermore, l i k e the medial septal nucleus, some neurons  i n the nDB a l s o  project to the hippocampus (CONRAD & PFAFF, 1976; MEIBACH & SIEGEL, 1977).  This  also suggests that at l e a s t part of the nDB can be viewed as a caudal extension of the medial septal nucleus.  The other areas to which the nDB p r o j e c t s include  the habenula, the anteromedial nucleus of the thalamus,  the interpeduncular  nucleus and the mammillary nucleus (CONRAD & PFAFF,1976; MEIBACH & SIEGEL, 1977; KERKENKAM & NAUTA.1977). I t may be noted that the caudal border o f nDB i s i n d i s t i n c t from that of the magnocellular nucleus of the p r e o p t i c area.  Earlier  studies (JACOBOWITZ & PALKOVITS,1974) have suggested that the t i g h t l y  packed,  intensely AChE-reactive c e l l group of nDB continues i n t o a region not recognized as nDB but generally termed . BZN-ARI & SILVER,1979).  l a t e r a l p r e o p t i c area (EMSON, PAXINOS, LE GAL LA SALLE,  I t i s apparent from F i g s . 1 & 2 that the p o p u l a t i o n  density of the magnocellular, i n t e n s e l y AChE-reactive neurons decreases i n the l a t e r a l preoptic area ( F i g . 2A-C) and l a t e r a l hypothalamic area ( F i g . 2D). Desp i t e the present d i f f i c u l t i e s i n d e f i n i n g the boundary, biochemical evidence supports the concept that d i s t i n c t d i f f e r e n c e s between nDB and l a t e r a l p r e o p t i c area do e x i s t .  S p e c i f i c a l l y , nDB has much higher choline a c e t y l t r a n s f e r a s e  a c t i v i t y than the l a t e r a l preoptic area (HOOVER et al.,1978).  Further s t u d i e s ,  based on retrograde neuroanatomical techniques combined with h i s t o c h e m i c a l and morphological i d e n t i f i c a t i o n are required to c l a r i f y the d i s t i n c t i o n between the intensely AChE-reactive neurons  i n the nDB and those i n the magnocellular nucleus  of the p r e o p t i c area (WYSS et a l . , 1979).  ' , In a s i m i l a r v e i n , the pro-  j e c t i o n s of the intensely AChE-reactive neurons  i n the magnocellular nucleus of  69  the p r e o p t i c area and i n the l a t e r a l hypothalamic area are not p r e s e n t l y known _. and r e q u i r e i n v e s t i g a t i o n . _ The present r e s u l t s suggest that the nDB does not p r o j e c t to n e o c o r t e x . Thus, HRP i n j e c t i o n s confined  t o the neocortex d i d not r e s u l t i n l a b e l e d c e l l s  e i t h e r i n the medial septum or the nDB, i n agreement w i t h JONES e t a l . (1976). However, i t remains p o s s i b l e , of course, that the nDB may p r o j e c t t o n e o c o r t i c a l areas that were not i n v e s t i g a t e d i n the present experiments. DIVAC (1975) found some l a b e l e d c e l l s i n the medial septum and nDB a f t e r c o r t i c a l HRP i n j e c t i o n s but according to h i s F i g . 3, i t appears p o s s i b l e t h i s was due to d i f f u s i o n of the i n j e c t e d HRP to the hippocampus.  that  Consistent  w i t h our f a i l u r e to l a b e l c e l l s i n medial s e p t a l nucleus and nDB a f t e r c o r t i c a l HRP i n j e c t i o n s i s the f i n d i n g that the magnocellular, i n t e n s e l y A C h E - r e a c t i v e neurons that c h a r a c t e r i z e these n u c l e i d i d not appear to undergo r e t r o g r a d e degeneration or l o s s a f t e r the extensive  cortical ablations.  C h a r a c t e r i z a t i o n of the nBM  ' •  In c o n s t r a s t to the l a c k of l a b e l i n g of the medial septum and nDB, c o r t i c a l i n j e c t i o n s of HRP l a b e l e d many neurons i n nBM ( F i g . 3A). These r e s u l t s c o n f i r m the f i n d i n g s of DIVAC (1975).  The l o c a t i o n of these l a b e l e d neurons correspond  to the d i s t r i b u t i o n of the magnocellular, i n t e n s e l y AChE-reactive neurons w h i c h were found to undergo extensive retrograde atrophy or l o s s a f t e r c o r t i c a l a b l a tion.  Furthermore, i n one s e r i e s of animals i n which the t i s s u e s were processed  for both HRP and AChE h i s t o c h e m i s t r y ,  i t was found, t h a t a l l the c e l l s i n t h e  nBM that contained HRP r e a c t i o n product a l s o s t a i n e d i n t e n s e l y f o r AChE.  These  r e s u l t s are i n s u b s t a n t i a l agreement w i t h MESULAM & VAN HOESEN'S (1976) o b s e r v a t i o n s i n the monkey.  I t should be noted, however, that these l a t t e r a u t h o r s  found a few HRP-labeled, AChE-reactive neurons i n nDB a f t e r c o r t i c a l HRP i n jections.  Since nD3 l a b e l i n g was not observed i n the present experiments, i t i s  not known whether t h i s discrepancy i s due to species d i f f e r e n c e s o r t o o t h e r  70  factors sent be  s u c h as those d i s c u s s e d  observations  concluded  the o r i g i n  that  above.  I n any e v e n t , on t h e b a s i s  and i n agreement v i t h p r e v i o u s  reductions  Thus, e l e c t r o l y t i c  nucleus b a s a l i s - n e o c o r t i c a l p r o lesions of t h i s region  i n the n e o c o r t i c a l a c t i v i t y  of a r e l i a b l e  c h o l i n e r g i c neurons, c h o l i n e a c e t y l t r a n s f e r a s e .  k a i n i c a c i d , a neurotoxin  which d e s t r o y s  neuronal perikarya  choline acetyltransferase a c t i v i t y .  tion of neuronal perikarya  i n the r e g i o n  resulted i n  enzyme marker  Furthermore, l e s i o n s with  o f passage i n t a c t (MASON & FIBIGER.1979) y i e l d e d  neocortical  interstitial,  i n F i g s . 1 & 2.  l e s i o n experiments i n d i c a t e that'the  significant  fibres  1  o f t h i s p r o j e c t i o n i s t h e group o f m a g n o c e l l u l a r , l a r g e l y  jections i s cholinergic.  for  (DIVAC 1975) , i t c a n  the nBM p r o j e c t s w i d e l y upon the n e o c o r t e x o f t h e r a t and t h a t  i n t e n s e l y A C h E - r e a c t i v e neurons i d e n t i f i e d The  findings  o f the p r e -  This  but g e n e r a l l y  leaves  t h e same d e c r e a s e s i n  suggests that  of the e l e c t r o l y t i c  the destruc-  lesions,-and not  damage t o f i b r e s o f p a s s a g e , was r e s p o n s i b l e  f o r the d e c r e a s e i n c o r t i c a l  a c e t y l t r a n s f e r a s e caused by the e l e c t r o l y t i c  lesions. T h e observations  hemitransections  j u s t caudal  -acetyltransferase a c t i v i t y indicates  that neurons caudal  cholinergic  selective generation Whither  to t h i s  i n n e r v a t i o n of t h i s part  i s a s o u r c e of a c o r t i c a l  level  do n o t c o n t r i b u t e  of t h e n e o c o r t e x .  following c o r t i c a l  lesions  the b a l a n c e of c o r t i c a l  acetyltransferase activity,  present  l e s i o n s were s m a l l e r  isolation  The h y p o t h e s i s  that  to the nBM  i n nBM c a u s e d by r e t r o g r a d e  acetyltransferase? decreases i n n e o c o r t i c a l  i n no i n s t a n c e was t h i s  than t h o s e employed  smaller  de-  (Table I ) .  choline  choline  Cortical  significantly  c h o l i n e r g i c p r o j e c t i o n i s f u r t h e r s u p p o r t e d by t h e  decrease i n c h o l i n e a c e t y l t r a n s f e r a s e  i s not s u r p r i s i n g that  choline  s u p p o r t s t h i s c o n c l u s i o n and  W h i l e l e s i o n s o f the nBM r e s u l t e d i n s i g n i f i c a n t  it  that  t o the e n t o p e d u n c u l a r n u c l e u s d i d n o t a f f e c t  i n the f r o n t a l c o r t e x  choline  l o s s complete.  The  by KELLY & M00PJ£ (1978) ; t h u s  choline acetyltransferase depletions  a l s o p r o d u c e s much l a r g e r d e p l e t i o n s  in cortical  resulted.'  choline  71  acetyltransferase  (GREEN, HALPERN & VAN  reproduced  in this laboratory  f a i l u r e of  the p r e s e n t  transferase of and  2)  (LEHMANN, ATMADJA & FIBIGER, i n p r e p a r a t i o n ) .  could  the e x i s t e n c e  t h e r e f o r e be e x p l a i n e d of o t h e r  p r o j e c t to the n e o c o r t e x , or  the n e o c o r t e x i t s e l f . bility.  First,  Several  3)  lines  the  existence  These c o r t i c a l  the  significant  This l a t t e r cortex.  decrease i n c o r t i c a l  observation  This  the  and  cortex  glutamic  perikarya  c o n s i s t e n t with  stain  a completely  n e o c o r t e x of the r a t . chemical of t h i s The  observations discrepancy  small  tissues  are no  possi-  signifi-  region  damaged  histological  t h i s was  corroborated activity.  GABAer.gic p e r i k a r y a  in  the  neo-  of GABAergic  by  (HOKFELT & LJ7JNGDAKL, ~ perikarya  in  visual  intensely AChE-reactive-perikarya  in  Inasmuch as a l l i d e n t i f i e d  s t r o n g l y f o r AChE, t h i s a b s e n c e i n t h e n e o c o r t e x extrinsic  s o u r c e o f c h o l i n e r g i c i n n e r v a t i o n of i s at variance  with  initial  (McGEER, McGEER, SINGH & CHASE, 1974). been d i s c u s s e d  r e c e n t l y by  d e p l e t i o n o f AChE t h a t o c c u r r e d  i s c o n s i s t e n t w i t h b o t h our  observations  in  i d e n t i f i c a t i o n o f GABAergic p e r i k a r y a  This conclusion  has  last  acid.decarboxylase  the n e o c o r t e x of c o n t r o l or D F P - p r e t r e a t e d r a t s . cholinergic  this  i n the c o r t i c a l  f r o n t a l c o r t e x and  immunohistochemical i d e n t i f i c a t i o n  (RIBAK ,1978). - Second, t h e r e  lesion  of c h o l i n e r g i c p e r i k a r y a  a u t o r a d i o g r a p h y o f l a b e l e d GABA u p t a k e i n p a r i e t a l c o r t e x 1972)  incomplete  f r o n t a l cortex d i d not  suggests t h a t there are  i s consistent with  choline acetyl-  i n j e c t i o n s d i d , however, p r o d u c e  e v i d e n c e of m a s s i v e n e u r o n a l l o s s i n the by  1)  of e v i d e n c e argue a g a i n s t  k a i n i c a c i d i n j e c t i o n s i n t o the  the k a i n i c a c i d .  by  The  s u b c o r t i c a l neurons which a r e c h o l i n e r g i c  cantly affect choline acetyltransferase a c t i v i t y by  been  These r e s u l t s h a v e  l e s i o n s to produce a complete d e p l e t i o n of  i n the c o r t e x  the nBM;  NIEL,1970).  and  The  possible  EMSON & LINDVALL  i n the c o r t e x .  basis  (1979) . cortical  (KRNJEVIC & SILVER,  t h a t weakly A C h E - r e a c t i v e n e u r o n s e x i s t  the  immunohisto-  i n the k a i n i c a c i d l e s i o n e d  previous  is  1965) These  emit c o m m i s s u r a l f i b r e s which c o n t a i n AChE (KRNJEVIC & SILVER, 1965) .  may  72  Functional considerations The being  r e l a t i o n s h i p between nBM and the v e n t r a l p a l l i d u m , t h e l a t t e r  t h e r a t ' s homologue f o r the p r i m a t e  implications innominata  f o r a f u n c t i o n a l understanding  appears  (1975) .  stantia  i n primates,  facto-striatal  system.  S i n c e the n u c l e u s b a s a l i s  i s found w i t h i n t h e s u b -  i t may be f u n c t i o n a l l y  Whether t h i s a s s o c i a t i o n o c c u r s  integrated with the o l i n c a t and r a t i s l e s s  On t h e o t h e r hand, some e v i d e n c e has been p r e s e n t e d by DAS & KREUTZBERG  (1968) t h a t t h e s e A C h E - r i c h neurons may be a r o s t r a l formation.  Additional observations r e l a t i n g  r e c e n t comparative  study o f n u c l e u s b a s a l i s  et a l . ,  1979).  thought  t o s u b s e r v e , assignment  system  The s u b s t a n t i a  termed " o l f a c t o - s t r i a t a l , " a n d ' e l e g a n t l y d i s c u s s e d  by HEIMER & WILSON innominata  of these neurons.  has important  t o be a p a r t of t h e system w h i c h i s i n t e r m e d i a t e between  e x t r a p y r a m i d a l and l i m b i c ,  clear.  s u b s t a n t i a innominata,  apparently  ..functions t o t h i s the p o s s i b i l i t y  to t h i s q u e s t i o n a r e f o u n d  system  cortical  that  in a  i n r a t , c a t , and monkey (PARENT  Because o f the v e r y d i f f e r e n t  o r the r e t i c u l a r  extension of the r e t i c u l a r  f u n c t i o n s t h e s e systems a r e  of nBM t o e i t h e r  the descending  olfacto-striatal  w i l l be o f c o n s i d e r a b l e v a l u e i n a s s i g n i n g  cholinergic projection.  The p r e s e n t r e s u l t s  the d r a m a t i c d e p l e t i o n i n c o r t i c a l  choline acetyltransfer-  a s e and AChE i n c e r e b r a l c o r t e x o f v i c t i m s o f A l z h e i m e r ' s d i s e a s e may be due t o a l e s i o n o f t h e s e s u b c o r t i c a l  neurons.  raise  (DAVIES,1979),  73  ACKNOWLEDGEMENTS The a u t h o r s e x p r e s s deep a p p r e c i a t i o n  t o D r . Andre" P a r e n t  f o r making  f r e e l y a v a i l a b l e t o us h i s d a t a and e x p e r t i s e on t h e n u c l e u s b a s a l i s substantia  innominata  these s t u d i e s .  complex, w h i c h p r o v e d  Supported  invaluable  by t h e M e d i c a l R e s e a r c h  i n the course of  Council.  74  REFERENCES  BUTCHER L.L.,  MARCHAND R. , PARENT A. & POIRIER L . J . (1977) M o r p h o l o g i c a l the  c h a r a c t e r i s t i c s of a c e t y l c h o l i n e s t e r a s e - c o n t a i n i n g  neurons i n  CNS  s p i n a l cord.  of DFP-treated monkeys. 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(1977)' S u b s t a n t i a  and n u c l e i of t h e d i a g o n a l  band: o r g a n i z a t i o n  acetylcholinesterase histochemistry.  7th Annual  innominata, of efferents Meeting,  f o r N e u r o s c i e n c e A b s t r . . 630.  NAUTA W.J.H., SMITH G.P., FAULL R.L.M. & DOMESICK V.B. (1978) E f f e r e n t c o n n e c t i o n s and n i g r a l r a t . Neurosci.  3,  a f f e r e n t s o f the n u c l e u s accumbens s e p t i i n t h e  385-401.  PARENT A. & BUTCHER L . L .  (1976) O r g a n i z a t i o n  cholinesterase-containing of  and m o r p h o l o g i e s o f a c e t y l -  neurons i n t h e thalamus and h y p o t h a l a m u s  the r a t . _J- comp. Neur. 170, 205-226.  78  PARENT A.,  GRAVEL S. & OLIVIER A.  (1979) The extrapyramidal and l i m b i c  systems' r e l a t i o n s h i p at the globus p a l l i d u s l e v e l : a comparative h i s t o chemical study i n the r a t , cat, and monkey.  In Advances i n Neurology  (ed. P o i r i e r L . J . , Sourkes T.L. & Bedard P.J.) v o l . 24, pp. 1-11. Press, New P h i l l i s , J.W.  Raven  York. (1976) A c e t y l c h o l i n e and synaptic transmission i n the  nervous system.  In Chemical Transmission  System. (ed. Hockman C H .  and Bieger D.)  central  i n the Mammalian C e n t r a l Nervous pp. 159-213. U n i v e r s i t y Park P r e s s ,  Baltimore. PRICE J.L. & POWELL T.P.S. (1970). An experimental  study of the o r i g i n  and  the course of c e n t r i f u g a l f i b r e s to the o l f a c t o r y bulb i n the r a t . J . Anat. 107,215-237. RAMON-MOLINER E. & NAUTA W.J.H. (1966).The i s o d e n d r i t i c core of the b r a i n stem. J_. comp. Neur. 126, RIBAK C.E.  311-336.  (1978) Aspinous and  sparsely-spinous s t e l l a t e neurons i n the  v i s u a l cortex of r a t s contain glutamic a c i d decarboxylase.  J_. N e u r o c y t o l .  7_, 461-478. SILVER A (1974) The Biology of Cholinesterases. E l s e v i e r , New  York.  WALAAS I. & FONNUM F. (1979) The d i s t r i b u t i o n and o r i g i n of glutamate decarboxylase  and choline a c e t y l t r a n s f e r a s e i n v e n t r a l p a l l i d u m and  basal f o r e b r a i n regions. Brain Res. WYSS J.M.,  SWANSON L.W.  h COWAN W.M.  177,  other  325-336.  (1979) A study of  subcortical  a f f e r e n t s to the hippocampal formation i n the r a t . N e u r o s c i . 4_, 463-476.  79 Figure 1:  D i s t r i b u t i o n of AChE-reactibe perikarya i n r a t f o r e b r a i n  AChE s t a i n according to KARNOVSKY & ROOTS (1964) i n animals p r e t r e a t e d with 1.5 mg/kg DTP i.m. 5 hours preceding s a c r i f i c e .  A few s c a t t e r e d  intensely  AChE-reactive neurons can be seen i n the regions described by HEIKER & WILSON (1975) and NAUTA et a l . (1978) as v e n t r a l pallidum ( F i g . 1B-E).  The d i s t r i -  bution o f the intensely AChE-reactive neurons o p e r a t i o n a l l y d e f i n e d as nBM (see discussion) d i f f e r s from the topography  of the v e n t r a l p a l l i d u m , which i s  thought to represent the r a t ' s homologue to primate substantia innominata (HEIMER & WILSON,1975; WALAAS & FONNUM,1979).  Although many groups o f  intensely  AChE-reactive neurons are contiguous, i f not continuous, the h i s t o c h e m i c a l regimen employed here i s not s u f f i c i e n t by i t s e l f to c l a r i f y the t o p o g r a p h i c a l boundaries, for instance, of the nDB which merges with medial s e p t a l n u c l e u s along i t s v e r t i c a l limb ( F i g . IB) and with the magnocellular p r e o p t i c nucleus • at caudal l e v e l s ( F i g . 2A and further caudal).  Tne o r g a n i z a t i o n o f A C h E - r e a c t i v e  neurons i n the thalamus and hypothalamus of the r a t has been p r e v i o u s l y described  (PARENT & BUTCHER, 1976) .  Abbreviations:  AC, a n t e r i o r commissure;  GP, globus p a l l i d u s ; LPO,  diagonal band;  F, f o r n i x ;  IC, i n t e r n a l capsule;- LHA, l a t e r a l hypothalamic  l a t e r a l preoptic area;  septal nucleus;  CP, caudate-putamen;  MaPO, magnocellular p r e o p t i c nucleus;  area;  MS, medial  nBM, nucleus b a s a l i s magnocellularis; nDB, nucleus o f t h e OT, o l f a c t o r y tubercle;  dorsal thalamic nucleus; thalamic nucleus;  TAV, anteroventral thalamic nucleus;  TV, v e n t r a l thalamus;  according to HEIMER & WILSON (1975). C a l i b r a t i o n bar:  SM, s t r i a m e d u l l a r i s ;  1mm.  TAD, a n t e r o TR, r e t i c u l a r  VP, v e n t r a l pallidum, diagrammed  80  F i g u r e 2:  D i s t r i b u t i o n of AChE-reactive  perikarya i n r a t f o r e b r a i n (cont.)  At more c a u d a l l e v e l s , p e r i k a r y a o f nBM become more numerous.  I n each  s e c t i o n , neurons m o r p h o l o g i c a l l y t y p i c a l o f nBM a r e o c c a s i o n a l l y found the c o r e o f t h e GP ( F i g . 2A, B) w h i l e  t h e p o p u l a t i o n of n e u r o n s  the b u l k o f nBM i n c r e a s e s g r e a t l y between GP and the i n t e r n a l a network o f p e r i k a r y a and p r o c e s s e s (Fig.  2B-D) .  the t a i l  The i n t e r s t i t i a l  o f the GP ( r i g . 2E) .  the l e v e l  o f the e n t o p e d u n c u l a r  1.  capsule,  forming  as d e s c r i b e d by PARENT e t a l . (1979)  The p o p u l a t i o n o f nBM p e r i k a r y a d i m i n i s h e s a s nucleus  i s approached  ( F i g . 2F).  c o n d i t i o n s a s i n F i g . 1.  Note t h e  i n the r e g i o n o f t h e nBM compared  other r e g i o n s d e s c r i b e d which c o n t a i n i n t e n s e l y A C h E - r e a c t i v e  Fig.  comprising  c h a r a c t e r o f nBM i s seen most d r a m a t i c a l l y n e a r  v e r y low "background" AChE s t a i n i n g  perimental  within  A b b r e v i a t i o n s a r e found  neurons.  to a l l Ex-  i n legend to  MaPO, LPO, and LHA have been l o c a t e d a c c o r d i n g t o t h e d e s c r i p t i o n of.  WYSS, SWANSON & COWAN C a l i b r a t i o n bar:  (1979). 1 mm  81  F i g u r e 3:  HRP-labeled  (A) and A C h E - s t a i n e d  (B) neurons  of nucleus  basalis  magnocellularis  HRP identical  injected  into  f r o n t a l c o r t e x l a b e l e d neurons  t o t h o s e o f neurons  l o w i n g DFP p r e t r e a t m e n t . cresyl violet  i n nBM when s t a i n e d f o r AChE  The-HRP  DFP p r e t r e a t m e n t .  s e c t i o n s were s t a i n e d  the a r e a s l e s i o n e d  following cortical  s e c t i o n was n o t c o u n t e r s t a i n e d (B) . f o r b o t h AChE and HRP  (see F i g . 5) c o n t a i n e d HR? p r o d u c t .  injections  The topography  following always  No o t h e r These  neurons  micrographs  of HRP-labeled  f o l l o w e d t h e topography  l a b e l e d nBM i n F i g s . 1 and 2.  C a l i b r a t i o n b a r : 50 ym  fol-  s e c t i o n was c o u n t e r - s t a i n e d w i t h  H a t c h e t t ' s brown f o r AChE.  were t a k e n a t the l e v e l o f F i g . I F .  r e a c t i v e neurons  (B) 5 h o u r s  Neurons which c o n t a i n e d the b l u e HRP p r o d u c t  s t a i n e d w i t h the c h a r a c t e r i s t i c in  labeled  (A), while the AChE-stained  In some e x p e r i m e n t s ,  i n nBM w i t h m o r p h o l o g y  of i n t e n s e l y  neurons AChE-  82  F i g u r e 4:  Retrograde degeneration of nucleus b a s a l i s produced  A,C - l e s i o n e d  by c o r t i c a l  side;  are evident  (A).  2B.  KARNOVSKY  The m i c r o g r a p h  1.5 mg/kg DFP.  & ROOTS  s t a i n i n g neurons  do n o t a p p e a r  t o be  i s taken from a s e c t i o n a t t h e l e v e l o f 6 months a f t e r  the c o r t i c a l  l e s i o n s and  AChE s t a i n i n g was performed a c c o r d i n g t o  (1964).  C a l i b r a t i o n bar:  Under h i g h e r power and  (A,B), d e p l e t i o n and s h r i n k a g e o f nBM p e r i k a r y a  The a n i m a l was s a c r i f i c e d  12 h o u r s a f t e r  side.  The s m a l l e r , weakly  adversely affected. Fig.  lesions  B,D - c o n t r o l  In low power m i c r o g r a p h s  magnocellularis  200 ym  (C,D), t h e s e m i c r o g r a p h s r e v e a l  the somal s h r i n k a g e  a p p a r e n t d e n d r i t i c a t r o p h y s u s t a i n e d by s u r v i v i n g nBM n e u r o n s C a l i b r a t i o n bar:  50 ym  (C) .  83  F i g u r e 5:  The e x t e n t o f damage c a u s e d by e l e c t r o l y t i c (A) and k a i n i c (B) l e s i o n , as a s s e s s e d by c r e s y l v i o l e t h i s t o l o g y ,  is  acid  indicated  diagramatically.  Abbreviations: F, f o r n i x ; St,  GP,  AC, a n t e r i o r  globus p a l l i d u s ;  caudate-putamen.  commissure;  EP, e n t o p e d u n c u l a r  IC, i n t e r n a l capsule;  SM,  stria  nucleus; medullaris;  Table  I.  N e u r o t r a n s m l t t e r - r e l a t e d enzymes i n the r e g i o n of the nucleus b a s a l i s m a g n o c e l l u l a r i s s i x months a f t e r e x t e n s i v e u n i l a t e r a l c o r t i c a l l e s i o n s  %  Control  C o n t r o l a c t i v i t y ± S.E.M.  choline acetyltransferase  64.1 ± 6.0%  63.9 ± 2.9 nmol/mg  protein/h  acetylcholinesterase  80.6 ±  19.5 + 1.6 umol/mg  protein/h  glutamic  acid  107.0 ± 9.3%  decarboxylase  n=4 *P< .02; **P< .001, S t u d e n t ' s  3Ja  two-tailed  test.  296 J: 31  nmol/mg  protein/h  Table I I .  Choline  acetyltransferase after  and a c e t y l c h o l i n e s t e r a s e  l e s i o n s i n the r e g i o n  %  Kainic acid lesions choline  activities  of nucleus b a s a l i s  i n the f r o n t a l  cortex  magnocellularis  Control  Control a c t i v i t y  ± S.E.M.  (n=ll)  acetyltransferase  acetylcholinesterase  78.0%  ± 2.2%***  30.5  ± 0.70 nmol/mg  protein/h  79.3%  ± 4.0%***  5.51 ± 0.10 umol/mg  protein/h  76.2%  ± 4.9%*  27.8  ± 1.94 nmol/mg  protein/h  74.2%  ± 3.9%**  5.85 ± 0.36 umol/mg  protein/h  95.7%  ± 5.3%  26.9  ± 1.63 nmol/mg  protein/h  96.1%  ± 7.5%  5.20 ± 0.45 umol/mg  protein/h  E l e c t r o l y t i c l e s i o n s (n=9) choline  acetyltransferase  acetylcholinesterase  Hemitransections - caudal choline  to nBM (n=7)  acetyltransferase  acetylcholinesterase  *P  <.02;  **P.  <.0l;  ***P  <-001,  Student's two-tailed  test,  Table I I I . Neurotransmitter-related  enzymes i n the f r o n t a l c o r t e x a f t e r l o c a l k a i n i c a c i d i n j e c t i o n s  Lesioned s i d e , % Control  Contralateral, % Control  Control  (unoperated) a c t i v i t y , + S.E.M.  choline acetyltransferase  92.2%  ± 4.9%  96.4%  + 5.5%  29.1  ± 2.1 nmol/mg  protein/h  acetylcholines terase  83.4%  ± 5.3%*  86.6%  ± 4.6%  3.59 ± 0.22umol/mg  protein/h  glutamic  59.3%  ± 5.0%**  100.4% + 4.1%  190 ± 7 . 3 nmol/mg  protein/h  acid  decarboxylase  n=6 *P< .05; **P <.001, S t u d e n t ' s  two-tailed  test.  Fig.  1  88  Fig. 2  89  Fig.  3  Fig.  4  91  Fig:  5A  F i g . 5B  f  The for area  f o l l o w i n g two  the c o p i e s  93  f i g u r e s a r e appended to the m a n u s c r i p t  included  i n the  thesis only.  F i g . 6 depicts  of c o r t i c a l a b l a t i o n which r e s u l t e d i n r e t r o g r a d e  of the nBM. greatest  F i g . 7 depicts  two-dimensional  the HRP  size.  injection  the  degeneration  s i t e at i t s  94 /  Fig. 6  95  Fig. 7  96  L i f e Sciences, Vol. 25, pp. 1939-1947 Printed i n the U.S.A.  Pergamon Press  MINIREVIEW ACETYLCHOLINESTERASE AND THE CHOLINERGIC NEURON John Lehmann and H.C. Fibiger Division of Neurological Sciences University of B r i t i s h Columbia Vancouver, B r i t i s h Columbia, V6T 1W5 Canada  The study of acetylcholinesterase (AChE) dates as far back as the d i s covery of acetylcholine (1,2). It has widespread distribution and very high a c t i v i t y , generally two orders of magnitude higher than choline acetyltransferase (3). It is stable, and there are many simple assay and histochemical techniques for measuring its a c t i v i t y . For these reasons, AChE has been the subject of a vast amount of research. Yet today no teleologi c a l model has been found to explain the distribution of AChE in the central nervous system: The existence of AChE on a given neuron is not s u f f i c i e n t information to predict that neuron's relationship with acetylcholine. In general there is an excellent correlation between AChE and choline acetyltransferase a c t i v i t y in the rat forebrain on a regional basis (3). It has long been recognized, however, that AChE is radically disproportionate with acetylcholine and choline acetyltransferase in some brain regions (4). For instance, the cerebellum is high in AChE compared to i t s content of choline acetyltransferase (5), while the inverse holds true for the medi a l habenula (3) and median eminence (6). On a c e l l u l a r l e v e l , AChE is found in f a i r l y high a c t i v i t y on some neurons which are known not to be cholinergic and furthermore are not thought to be cholinoceptive. Two salient examples are the dopaminergic neurons of the substantia nigra (7-9) and the noradrenergic neurons of the locus coeruleus (10). It is clear from just these two examples, as Koelle pointed out in 1955 (11), that the presence of AChE in a given neuron is not sufficient evidence to indicate that such a neuron is cholinergic. Nonetheless, on occasion AChE has proven worthy of study in the pursuit which may be called "biochemical neuroanatomy", i . e . , the i d e n t i f i c a tion of neurons 1) morphologically, 2) by afferent and efferent connections, and 3) by the transmitter(s) used. The study of AChE is a useful adjunct to more specific enzyme markers and conventional neuroanatomical techniques as an arbitrary and characteristic marker of certain classes of neurons. For example, AChE histochemistry formed the link between the biochemical and anatomical characterization of the non-homogeneous organization of the striatum (12,13). It has also proven useful in the identification of similar classes of neurons within the CNS (14) and across species (15). Furthermore, when an irreversible inhibitor such as d i i s o propylphosphorofluoridate (DFP) is administered in vivo some time preceding s a c r i f i c e , the morphological features of neurons that contain AChE are revealed with detail exceeded only by the Golgi method. This powerful modi0024-3205/79/231939-09$02.00/0 C o p y r i g h t (c) 1979 Pergamon P r e s s L t d  97  1940  AChE and the C h o l i n e r g i c Neuron  V o l . 25, No. 23, 1979  fication of the AChE histochemical technique was introduced by Lynch and coworkers (16) and has been applied extensively with technical improvements by Butcher and collaborators, particularly in the striatum (7). Beyond these pragmatic applications, we have lately re-examined the v a l i d i t y of an hypothesis conceived as long ago as 1954 by Koelle (17). Having divided nuclei of the brain into four categories according to the intensity of AChE staining, Koelle generalized from the single example of the motoneuron ". . . i t may be postulated that neurons in the intensely and moderately stained categories are likewise cholinergic." In the light of our understanding 25 years l a t e r , i t is striking to note how many correct examples of cholinergic neurons are l i s t e d in the "intensely stained" category and how few correct examples are l i s t e d in the "moderately stained" category. Today, comparison of the density of AChE-staining in various perikarya, although s t i l l qualitative, is f a c i l i t a t e d by advances in histochemical technique. Thus, well-character!zed cholinergic projections o r i g i nating in the central nervous system have been shown to arise from somata which synthesize large amounts of AChE following OFP-pretreatment. Examples of such cholinergic neurons include the septo-hippocampal projection (18,19) and the motoneuron (20,21). Most importantly, there is no known example of a cholinergic neuron that does not have high levels of AChE. We may therefore form an empirical generalization based on cases in the central and peripheral nervous systems which adhere to the rule: high AChE a c t i v i t y is a necessary but not s u f f i cient characteristic for identifying cholinergic neurons. If indeed this rule holds true, the easily determined distribution of AChE w i l l greatly accelerate the elucidation of cholinergic neuroanatomy. The value of this rule depends upon i t s v a l i d i t y in each circumstance; the discovery of one cholinergic neuron without high levels of AChE will destroy the rule's u t i l i t y . This laboratory has made use of the rule in three cases in which i t seemed most l i k e l y to f a i l . Here we summarize the course of those investigations in three areas of the brain: the striatum, the cerebral cortex, and the globus pallidus. The Striatum There are six types of neurons described in the striatum of the cat by Kemp & Powell (22): the large, so-called "aspiny" neuron (22-30 um, mean of major and minor axes), comprising less than 1% of the total neuron population; the medium spiny (12-18- um), comprising 96% of the population; three other medium-sized neurons (16-18 pm, 16-18 um, and 12-14 um), together comprising 3% of the population; and the small neuron (5-9 um) f i l l i n g out the last 1%. Traditionally, the large aspiny neuron was considered to be the sole source of the descending projections from the striatum (23,24). This concept has been revised in the light of data gathered in the last decade; now at least 50% of the medium spiny neurons are known to have descending projections (25,26), while the large aspiny neuron is thought to be an interneuron (27). In the striatum from DFP-pretreated animals, the large aspiny neuron is unique in that i t stains intensely for AChE; the small neuron stains l i g h t l y , and the medium c e l l s are generally judged not to stain at a l l (7). However, immunohistochemical evidence,obtained with antibodies directed against purified choline acetyltransferase, previously suggested that medium spiny neurons were cholinergic (28,29). Similar results were recently obtained by Kaiya et a l . (30) employing a conventional histochemical reaction for l o c a l izing choline acetyltransferase ultrastructurally. These observations are  98  V o l . 25, No. 23, 1979  AChE and the C h o l i n e r g i c Neuron  1941  in disagreement with the hypothesis that high levels of AChE are necessarily contained in cholinergic neurons. This empirical rule led us to suspect that the large aspiny neurons, rather than the medium-sized neurons, were the elusive cholinergic interneurons of the striatum (27), the existence of which was o r i g i n a l l y proposed by McGeer et a l . (31). Data reported by Campochiara & Coyle (32) has indicated that kainic acid injected into the striatum of 10-21 day old rats preferentially depletes choline acetyltransferase, compared to the GABAergic marker glutamic acid decarboxylase. Kainic acid is a neurotoxin which in general destroys neuronal perikarya while leaving afferent axons and terminals intact (33,34). However, s t r i a t a l neurons appear to require a functional glutamateric innervation in order to be susceptible to kainic acid's neurotoxic action, as f i r s t demonstrated by McGeer et a l . (35,36). The simplest hypothesis to explain the preferential depletion of choline acetyltransferase at early postnatal times is that the glutamatergic corticostriatal projection (37,38) establishes a functional synaptic contact with cholinergic neurons s l i g h t l y e a r l i e r than i t does with the other neurons of the striatum. The data reported by Campochiaro & Coyle (32) thus presented an opportunity to test a prediction of the hypothesis that the cholinergic neuron of the striatum was the AChE-intense large aspiny neuron. TABLE I The selective depletion of striatal tylcholinesterase by intrastriatal % of Control CAT AChE GAD  74.7 ± 2.0** 78.4 ± 2.8* 96.5 ± 4.6  n = 5 *P < .002;  **p < .001,  choline acetyltransferase and aceinjections of kainic acid neonatally  Velocity, contralateral 153 ± 4.1 48.5 ± 2.8 102 ± 5.3  Student' s two tailed  striatum  ± S.E.M.  nmol/mg protein/h umol/mg protein/h nmol/mg protein/h  test.  10 nmol kainic acid in 0.5 ul sodium phosphate buffered (pH 7.4) isosmolar Ringer solution was injected unilaterally into the corpus striatum of rats 10 days post partum. 14 days after surgery, choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were s i g n i f i c a n t l y reduced, while glutamic acid decarboxylase (GAD) a c t i v i t y was unaffected. This laboratory reproduced the biochemical data of Campochiara & Coyle (32) in the ten-day old neonate (see Table I) and performed histochemical studies in p a r a l l e l . The kainic acid injections resulted in a striatum with large, irregularly shaped areas entirely void of the AChE-intense, large aspiny neuron, when visualized by AChE histochemistry following DFPpretreatment. In contrast, c r e s y l - v i o l e t stained neurons (being composed 96% of medium spiny neurons) and small, weakly AChE-reactive neuron populations were unaltered (Table II). These data argue strongly that neither the medium spiny nor the small neuron of the striatum is cholinergic. It is possible that one of the three minority medium-sized neurons identified by Kemp & Powell (22) may be cholinergic, but by far the favored candidate is the large, AChE-intense aspiny neuron. Furthermore, in recent developmental studies of the striatum pursued in this laboratory, the latero-medial progression of the postnatal development of large, AChE-intense neurons is  99  •1942  AChE and Cholinergic Neuron  Vol. 25, No. 23, 1979  paralleled exactly by the regional development of choline acetyltransferase a c t i v i t y (Lehmann & Fibiger, in preparation). F i n a l l y , i t should be noted that in more recent immunohistochemical experiments aimed at the neuronal localization of choline acetyltransferase, preliminary evidence has been obtained that implicates the large aspiny neuron of the striatum as a cholinergic neuron (39). These observations demonstrate that the criterion requiring high AChE activity as a necessary but not sufficient characterist i c of cholinergic neurons was capable of predicting the morphology of the cholinergic neuron in the striatum. TABLE  II  Quantitation of morphologically identifiable neurons in regions made void of putative cholinergic neuron by neonatal kainic acid lesions Type of neuron  Cresyl-violet stained Sml weakly AChE-reactive Large AChE-intense  % of Control  106.5 ± 8.1 n.s. 124.4 ± 10.6 n.s. Zero  Density, contralateral atal ± S.E.M. 1574 ± 5 7 . 4 17.2 ± 1.39 12.2 ± 0.60  stri-  neurons/mm neurons/mm neurons/mm  Identically lesioned rats from the same group on which biochemical assays were performed (Table I) were injected intramuscularly with 2.0 mg/kg DFP. 6 hr l a t e r , they were perfused and the brains processed for AChE histochemical staining. Alternate sections were c r e s y l - v i o l e t counterstained. At least 22 sample areas entirely void of large AChE-intense neurons were counted from 3 rats; the corresponding s t r i a t a l region contralaterally served as control. There were no significant changes in the density of either of the other morphologically identifiable neurons. Note that each of the AChE-reactive neurons represents roughly 1% of the total neuronal population as estimated by c r e s y l - v i o l e t staining.  The Cerebral Cortex There are three major lines of evidence that have argued for the existence of cholinergic perikarya in the neocortex: 1) Chronic isolation of cortical slabs results in a large (65% - 80%) but not complete depletion of choline acetyltransferase (40-42). Other workers have reported no decreases in cortical choline acetyltransferase activity following similar operations (38,43). 2) Local electrical stimulation of a c h r o n i c a l l y - i s o lated cortical slab results in a long-lasting inhibition of glutamate-induced f i r i n g which is blocked by atropine and mimicked by acetylcholine (44,45). 3) Antibodies directed against choline acetyltransferase stain large numbers of neurons in the cortex (46). Further support for the existence of cholinergic perikarya in the cortex has derived from the arguments that the decrease in choline acetyltransferase following cortical i s o lation may be due to retrograde degeneration of cholinergic perikarya (46) or a "secondary effect of denervation" (38), and the observation that the cortical inhibition evoked by surface stimulation of the cortex in the slab is identical to that found in intact cortex (44). Arguing against this hypothesis is the observation that there are no intensely AChE-reactive perikarya in the cerebral cortex (17,47,48). This observation becomes more striking in rat cerebral neocortex 5 hours follow-  2 2 2  100  V o l . 25, No. 23, 1979  AChE and the C h o l i n e r g i c Neuron  1943  ing DFP-pretreatment (Lehmann, Atmadja & Fibiger, in preparation), a condition which causes known cholinergic neurons to stain intensely for AChE. The "necessary but not sufficient" rule therefore predicts that there are no cholinergic perikarya in the neocortex. Again, the opportunity to test the v a l i d i t y of the "necessary but not sufficient" criterion presented i t s e l f . Kainic acid was employed by this laboratory to effect a complete and uniform neuronal lesion in frontal cortex of rat, as assessed by cresylviolet histology. The volume of complete perikaryal depletion comprised approximately 50% of the assayed tissue. In this experiment i t was found that glutamic acid decarboxylase activity and high a f f i n i t y glutamate uptake were decreased in the lesioned tissue by approximately 50 percent. However, choline acetyltransferase activity did not change (Table III). In order to escape the conclusion that there are no cholinergic neurons in the cortex, i t would be necessary to invoke the condition that putative cholinergic interneurons of the cortex project heavily for distances exceeding one centimeter, rather than terminating l o c a l l y . This condition is in d i s agreement with arguments 1) and 2) cited above as support for the existence of cholinergic neurons in the cortex. Again, pending agreement, of future data, the necessary but not sufficient rule for AChE activity in cholinergic neurons predicted successfully the absence of cholinergic perikarya in the cerebral cortex suggested by the results of the kainic acid lesion experiment (Table III). It should be noted that an explanation for both residual choline acetyltransferase a c t i v i t y and stimulus-evoked release of acetylcholine following cortical isolation is s t i l l lacking. Similar largely unexplained failures to observe complete depletions of either choline acetyltransferase or acetylcholine release have been observed distal to peripheral cholinergic nerves following positively complete transection in several species (49-56). It is d i f f i c u l t of course to extrapolate across species and from peripheral to central nervous system in order to suggest that an analogous phenomenon does or does not occur in the neocortex of the rat. To perform such a comparison on a percentage basis (57) is a l l the more hazardous in view of the very low s p e c i f i c a c t i v i t y of choline acetyltransferase in the neocortex compared to the peripheral nerves studied. Globus Pallidus On the basis that AChE-rich axons projecting to the neocortex appeared to originate from the pallidum, Shute & Lewis (58) o r i g i n a l l y proposed the existence of a cholinergic pal 1ido-neocortical projection. This suggestion was subject to question, however, since i t was well-known at that time that AChE content was not s u f f i c i e n t to characterize a projection as cholinergic (11). Furthermore, there was no known projection from the globus pallidus to the cortex; and the globus pallidus was known to have extremely low levels of AChE and choline acetyltransferase (59). Yet in 1976, Kelly & Moore (60) found that pallidal lesions did indeed result in substantial decreases of choline acetyltransferase in large areas of neocortex. Reporting retrograde transport of HRP injected into the cortex, Divac (61) speculated that what Shute & Lewis (58) had identified as neurons in the globus pallidus were actually the rat's homologue of the primate nucleus basalis of the substantia innominata, which also projects to neocortex (6264). Mesulam & van Hoesen (63) demonstrated that in the primate, HRP was transported to AChE-rich neurons of the nucleus basalis of the substantia innominata, and joined Divac (61) in speculating that these were the source of a cholinergic projection to the neocortex. Ensuing experiments designed to prove that these AChE-rich neurons were the source of the cholinergic projection from the pallidal region in the rat became obvious.  101  1944  AChE and the Cholinergic Neuron  TABLE Neurotransmitter-related  CAT AChE GAD Glu-up n = 6 *P < .05;  Vol. 25, No. 23, 1979  III  enzymes in the frontal cortex after local kainic acid injections  Lesioned s i d e , % of Control  Control (unoperated) v e l o c i t y ,  92.2 83.4 59.3 56.6  29.1 3.59 190 1.23  **P  ± ± ± ±  4.9 5.3* 5.0** 4.5**  ± ± ± ±  2.1 0.22 7.3 0.04  nmol/mg umol/mg nmol/mg umol/mg  + S.E.M.  protein/h protein/h protein/h protein/h  < .001, Student's two-tailed test.  One week following injection of 10 nmol kainic acid in 2 ul sodium phosphate buffered (pH 7.4) saline into frontal cortex of rat, choline acetyltransferase (CAT) was not s i g n i f i c a n t l y decreased. Acetylcholinesterase (AChE) a c t i v i t y was s l i g h t l y decreased, while major decreases in glutamic acid decarboxylase (GAD) and higha f f i n i t y glutamate uptake (Glu-up) were observed. Introducing the minor refinement of suppressing non-perikaryal AChE staining by DFP-pretreatment to Mesulam & van Hoesen's protocol for simultaneously visualizing HRP and AChE, this laboratory replicated Mesulam & van Hoesen's (63) findings in the rat; HRP injected in the neocortex labelled only neurons of nucleus basalis magnocellularis (nBM) which stained heavily for AChE. Discrete lesions of nBM produced either e l e c t r o l y t i c a l l y or with kainic acid produced identical and parallel depletions in choline acetyltransferase and AChE in frontal cortex, while hemitransections s l i g h t ly caudal to nBM did not affect these enzymes (Lehmann, Nagy, Atmadja & Fibiger, submitted). Six months following cortical ablations, retrograde degeneration in nBM visualized by AChE-histochemistry following DFP pretreatment was paralleled by choline acetyltransferase and AChE depletions in nBM (Table IV). What had been speculation in 1967 became an obvious conclusion by the close of the seventies: the source of the cholinergic innervation of the neocortex arising from the p a l l i d a l region is the group of intensely AChE-reactive neurons, the nBM. Looking Forward The above have been but three examples where the predictive u t i l i t y of high AChE levels in identified neurons can be demonstrated. . Several other examples have been cited above where the necessary but not sufficient rule also applies, and there is no clear exception. It must be conceded, however, that relatively few cholinergic neurons in the CNS have been unequivocally characterized. In searching for an exception to the rule that a l l cholinergic neurons contain high levels of AChE, the habenular complex particularly presents a challenge. The tightly-packed cluster of neurons comprising the medial habenula stains very weakly for AChE following DFP pretreatment, while the lateral habenula contains only moderately-staining neurons (65; Lehmann & Fibiger, unpublished observations). Although at one time the medial habenula was thought to be the sole source of a massive cholinergic projection to the interpeduncular nucleus (66-71), is is now believed that  102  V o l . 25, No. 23, 1979  AChE and the C h o l i n e r g i c  Neuron  1945  the cholinergic input to the interpeduncular nucleus derives at lease 50% from the nucleus of the diagonal band (72), which does contain large, intensely AChE-reactive neurons (73) and probably projects along s t r i a medu l l a r i s (72i74), although others believe that the source is entirely from cholinergic perikarya residing in the lateral habenula (75,76). Kainic acid injections in the habenular region led to approximately 50% depletion of choline acetyltransferase in the interpeduncular nucleus (77), supporting the notion that the habenular complex was not the sole source of choline acetyltransferase in the interpeduncular nucleus. However, the necessary but not sufficient criterion of high AChE levels indicates that no choline acetyltransferase may originate from perikarya in the habenular complex. It may be possible to account for the data obtained with kainic acid by McGeer et a l . (77) on the basis of partial lesion of fibers of passage, which has been demonstrated in a similar dense fiber bundle, namely the dorsal noradrenergic bundle (78). Apart from the nucleus of the diagonal band, at present we are not able to suggest another candidate as an origin for the cholinergic innervation-of the AChE-rich interpeduncular nucleus. However, i t should be noted that the "simple" neuroanatomy of this complex remains to be c l a r i f i e d , and that the habenular complex represents the most striking anomaly in the central nervous system with regard to disproportionate AChE and choline acetyltransferase a c t i v i t i e s (3). TABLE IV Neurotransmitter-related  enzymes in the region of nBM six months after decortication Control velocity ± S.E.M.  % of Control  63.9 ± 2.9 19.5 ± 1.6 296 ± 31  64.1 ± 6.0** 80.6 + 3.4* 107.0 ± 9.3  CAT AChE GAD n = 4 *P < .02;  nmol/mg protein/h pmol/mg protein/h nmol/mg protein/h  **P < .001, Student's two-tai.led test.  The retrograde degeneration of AChE-intense neurons identified as nucleus basalis magnocellularis (nBM) was paralleled by decreases in choline acetyltransferase (CAT) and acetylcholinesterase (AChE), but not glutamic acid decarboxylase (GAD). Data from Lehmann, Nagy, Atmadja and Fibiger, submitted. Summary While the distribution of AChE in the central nervous system remains largely unexplained, neurons with very high levels of AChE are frequently identified as cholinergic, and cholinergic neurons always have high levels of AChE. This supports the u t i l i t y of the empirical rule, that i d e n t i f i c a tion of cholinergic neurons requires demonstration of high levels of AChE. The application of this rule in conjunction with neuroanatomical and biochemical data has already provided substantial new insights into the organization of central cholinergic systems. It is evident that AChE histochemistry continues to be a valuable neurobiological t o o l , particularly in characterizing neurons that may be cholinergic, and also in identifying those neurons that cannot be cholinergic.  103  1946  AChE and the Cholinergic Neuron  Vol. 25, No. 23, 1979  Acknowledgements The excellent technical assistance of Stella Admadja and Amelia Wong is gratefully acknowledged. Supported by the Medical Research Council. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.  —  27. 28. 29. 30. 31. 32. 33. 34.  H.H. DALE, J . Pharmacol. 6 147-189 (1914). 0. LOEWI and E. 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