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GABA, substance P and the efferents of the striatum Vincent, Steven Robert 1980

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GABA, SUBSTANCE P AND THE EFFERENTS OF THE STRIATUM by STEVEN ROBERT VINCENT B.Sc.  ,  C a r l e t o n U n i v e r s i t y , 1976  A THESIS SUBMITTED  IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  THE FACULTY OF GRADUATE STUDIES (Interdisciplinary  We accept t h i s t h e s i s as  Studies)  conforming  to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA  J u l y 1980 (c^  Steven Robert  V i n c e n t , 1980  /  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives.  I t is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia 2075 Wesbropk Place Vancouver, Canada V6T 1W5  DE-6  BP  75-51  1 E  ii ABSTRACT The e f f e r e n t pathways from the s t r i a t u m t o t h e o t h e r n u c l e i o f t h e b a s a l g a n g l i a were examined b i o c h e m i c a l l y and h i s t o c h e m i c a l l y .  Acetylcho-  l i n e , GABA, e n k e p h a l i n and substance P have a l l been suggested t o o c c u r i n s t r i a t a l neurons,  and markers f o r these p o s s i b l e t r a n s m i t t e r s were t h e r e f o r e  measured i n v a r i o u s n u c l e i o f the b a s a l g a n g l i a f o l l o w i n g k n i f e c u t s o f the striatal  efferent fibres.  These s t u d i e s c o n f i r m e d t h e e x i s t e n c e o f GABA  p r o j e c t i o n s from t h e head of the s t r i a t u m t o t h e globus p a l l i d u s (GP), entopeduncular n u c l e u s  (EP) and s u b s t a n t i a n i g r a  (SN).  In a d d i t i o n the  presence o f s u b s t a n c e P i n t h e s t r i a t a l p r o j e c t i o n t o t h e EP was demonstr a t e d and t h e substance P p r o j e c t i o n t o t h e SN was c o n f i r m e d u s i n g a r a d i o immunoassay.  The f i r s t  evidence s u g g e s t i n g the p r e s e n c e o f b o t h substance P  and m e t h i o n i n e - e n k e p h a l i n i n t h e s t r i a t o p a l l i d a l f i b r e s was a l s o o b t a i n e d . A l s o t h e important o b s e r v a t i o n t h a t m e t h i o n i n e - e n k e p h a l i n i s n o t p r e s e n t i n the p r o j e c t i o n s from t h e head o f t h e s t r i a t u m t o t h e EP and SN was noted. In  o r d e r to v i s u a l i z e substance P f i b r e s i n t h e b r a i n a new method f o r  immunohistochemical  s t u d i e s o f the nervous  b i o t i n - a v i d i n system.  system was developed based on the  Using t h i s p o w e r f u l t e c h n i q u e s u b s t a n c e P f i b r e s and  t e r m i n a l s were observed i n t h e s t r i a t u m , GP, EP and SN, as w e l l as i n v a r i o u s o t h e r areas i n c l u d i n g t h e amygdaloid  complex, t h e habenula and the  interpeduncular nucleus. This represents the f i r s t f i b r e s i n t h e b a s a l g a n g l i a demonstrated The  r e p o r t o f substance P  u s i n g an immunoperoxidase procedure.  enzyme GABA-transaminase (GABA-T) was examined as a p o t e n t i a l  marker f o r t h e GABA neurons  of the b a s a l ganglia.  Using s e l e c t i v e  lesions  and a b i o c h e m i c a l assay procedure t h e enzyme was found t o be p r e s e n t i n t h e neurons  o f the s t r i a t u m and i n the s t r i a t o n i g r a l pathway.  a p p a r e n t l y n o t p r e s e n t i n the g l i a l  GABA-T was  elements o f t h e s t r i a t u m n o r was i t  p r e s e n t i n t h e n i g r o s t r i a t a l dopamine neurons.  H i s t o c h e m i c a l experiments  iii  demonstrated GABA-T t o be p r e s e n t  i n the t e r m i n a l s  o f t h e s t r i a t a l and  p a l l i d a l e f f e r e n t s which a r e thought to use GABA as a t r a n s m i t t e r . These experiments e s t a b l i s h the u s e f u l n e s s  of GABA-T h i s t o c h e m i s t r y as  a new method f o r t h e a n a l y s i s of the topography o f the GABA systems i n the b a s a l The  ganglia. response o f the GABA and substance P c e l l s i n t h e b a s a l  ganglia  to the s e l e c t i v e removal o f t h e dopamine c e l l s o f t h e SN was examined and  compared w i t h  disease.  t h e p a t h o l o g i c a l f i n d i n g s observed i n P a r k i n s o n ' s  In contrast with  the decrease r e p o r t e d  i n glutamate d e c a r b o x y l a s e  a c t i v i t y i n the b a s a l g a n g l i a i n P a r k i n s o n ' s d i s e a s e , an i n c r e a s e i n t h e a c t i v i t y of t h i s enzyme was observed i n the animal model.  Also, a  s i g n i f i c a n t d e c r e a s e i n n i g r a l and s t r i a t a l substance P l e v e l s following this lesion. and  pharmacological  occurred  The 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 t h e e t i o l o g y  therapy o f P a r k i n s o n i s m a r e d i s c u s s e d .  F i n a l l y , t h e n i g r o t e c t a l pathway was examined u l t r a s t r u c t u r a l l y and biochemically ganglia.  since i t represents  a major output pathway o f the b a s a l  A s e l e c t i v e decrease was found i n the glutamate d e c a r b o x y l a s e  a c t i v i t y o f the s u p e r i o r c o l l i c u l u s f o l l o w i n g l e s i o n s o f t h e SN. observation  provides  the f i r s t  may use GABA as a t r a n s m i t t e r . terminals myelinated  This  i n d i c a t i o n that the n i g r o t e c t a l p r o j e c t i o n Electron microscopic  e x a m i n a t i o n o f axon  of t h e n i g r o t e c t a l pathway i n d i c a t e d t h e axons were and t h a t the t e r m i n a l s  form symmetric synapses w i t h  d e n d r i t e s o f neurons i n the deep l a y e r s o f the s u p e r i o r  probably the major  colliculus.  iv  TABLE  OF  CONTENTS  ABSTRACT  i i  TABLE  iv  OF CONTENTS  LIST  OF T A B L E S  vi  LIST  OF F I G U R E S  x i i i  LIST  OF A B B R E V I A T I O N S  x  ACKNOWLEDGEMENTS  xi  INTRODUCTION  1  STATEMENT  OF THE PROBLEMS  EXPERIMENT  1:  TO B E E X A M I N E D  Neurotransmitters  9  contained  in  the  efferents  of  the  striatum EXPERIMENT  2:  The in  EXPERIMENT  3:  11  immunohistochemical the  The  basal  4:  localization  The in  EXPERIMENT  5:  Biochemical  animal  GENERAL  6:  GABA-transaminase  in  the 40  of  GABA-transaminase  ganglia  the  model  53 following  nigrostriatal  6-hydroxydopamine dopamine  neurons:  an 66  7 Parkinsonism'  of  The n i g r o t e c t a l  projection:  ultrastructural  study  a biochemical  and 76  DISCUSSION  Nigrostriatal  Substance  P  85  regulation  GABA r e g u l a t i o n  GABA,  P 27  localization  changes  of  substance  system  basal  lesions  EXPERIMENT  of  histochemical the  of  ganglia  striatonigral EXPERIMENT  demonstration  of  the  regulation  substance  P  and  85  nigrostriatal of the  the  dopamine  nigrostriatal  output  of  the  system  dopamine  striatum  89 system  . . .  92 94  V  Outputs of the s u b s t a n t i a n i g r a  97  A model of the b a s a l g a n g l i a  104  REFERENCES APPENDIX  :  107 133  vi  LIST OF TABLES T a b l e 1.  Glutamic a c i d d e c a r b o x y l a s e and c h o l i n e  acetyltransferase  a c t i v i t i e s i n v a r i o u s areas a f t e r l e s i o n s i s o l a t i n g t h e globus p a l l i d u s from t h e s t r i a t u m T a b l e 2.  (GP i s l a n d s )  Glutamic a c i d d e c a r b o x y l a s e and c h o l i n e a c t i v i t i e s i n various  areas a f t e r  16  acetyltransferase  hemitransections  a n t e r i o r t o t h e globus p a l l i d u s T a b l e 3.  Substance P l e v e l s i n v a r i o u s  18  areas a f t e r l e s i o n s  i s o l a t i n g t h e globus p a l l i d u s from t h e s t r i a t u m (GP islands) T a b l e 4.  20  The l e v e l s of substance P and met-enkephalin i n v a r i o u s areas a f t e r h e m i t r a n s e c t i o n s  a n t e r i o r to t h e globus  pallidus T a b l e 5.  21  The a c t i v i t i e s o f n e u r o t r a n s m i t t e r - r e l a t e d  enzymes i n  the s t r i a t u m and s u b s t a n t i a n i g r a one month a f t e r t h e i n j e c t i o n o f 6-0HDA i n t o t h e n i g r o s t r i a t a l bundle . . . T a b l e 6.  The a c t i v i t i e s of n e u r o t r a n s m i t t e r - r e l a t e d  44  enzymes i n  the s t r i a t u m and s u b s t a n t i a n i g r a two weeks a f t e r t h e i n j e c t i o n o f f i v e o r t e n nmoles o f k a i n i c a c i d i n t o the striatum T a b l e 7.  45  The a c t i v i t i e s o f n e u r o t r a n s m i t t e r - r e l a t e d  enzymes i n  the s t r i a t u m a f t e r t h e i n j e c t i o n o f 20 nmoles of k a i n i c a c i d i n t o the s t r i a t u m o f ten-day o l d r a t s Table 8.  Glutamic a c i d d e c a r b o x y l a s e a c t i v i t y  i n various  50 brain  areas a f t e r u n i l a t e r a l l e s i o n s o f t h e n i g r o s t r i a t a l pathway w i t h 6-OHDA  69  vii  T a b l e 9.  Enzyme a c t i v i t i e s and n e u r o p e p t i d e and  l e v e l s i n the s t r i a t u m  s u b s t a n t i a n i g r a t h r e e months a f t e r  the i n j e c t i o n  of s a l i n e or 6-OHDA i n t o the l e f t n i g r o - s t r i a t a l pathway T a b l e 10. D i s t r i b u t i o n  of s i l v e r g r a i n s i n the s u p e r i o r  71  colliculus  24 hr a f t e r the i n j e c t i o n of [ H ] l e u c i n e i n t o t h e 3  substantia nigra  80  T a b l e 11. B i o c h e m i c a l changes i n the s u p e r i o r c o l l i c u l u s a f t e r lesions  o f the s u b s t a n t i a n i g r a  83  V l l l  LIST OF FIGURES F i g u r e 1.  The k n i f e the  F i g u r e 2.  c u t s s e p a r a t i n g the globus p a l l i d u s  from  striatum  A comparison o f the b i o t i n - a v i d i n method w i t h the  peroxi-  d a s e - a n t i p e r o x i d a s e procedure f o r immunohistochemistry F i g u r e 3.  Control section  of s u s b t a n t i a  nigra  .  incubated with  pre-absorbed a n t i - s u b s t a n c e P s e r a F i g u r e 4.  Substance P immunohistochemistry projection  F i g u r e 5.  and i t s  areas  Substance P immunohistochemistry habenula and the amygdaloid  F i g u r e 6.  i n the s t r i a t u m  i n the l a t e r a l  complex  Substance P immunohistochemistry  i n the  substantia  nigra F i g u r e 7.  Correlation  between s t r i a t a l glutamate d e c a r b o x y l a s e  and GABA-transaminase kainic F i g u r e 8.  following  the i n j e c t i o n of  a c i d i n t o the s t r i a t u m  Correlation  between n i g r a l glutamate d e c a r b o x y l a s e  and GABA-transaminase  following  the i n j e c t i o n of  k a i n i c a c i d i n t o the s t r i a t u m F i g u r e 9.  Sagital section  through the r a t b r a i n  GABA-transaminase  stained  for  activity  F i g u r e 10. E f f e c t of k a i n i c a c i d on GABA-transaminase  staining  i n the s t r i a t u m F i g u r e 11. The e f f e c t of k a i n i c a c i d l e s i o n s of the s t r i a t u m GABA-transaminase  s t a i n i n g i n the p r o j e c t i o n  areas  on  ix  F i g u r e 12.  GABA-transaminase h i s t o c h e m i s t r y i n the nucleus  subthalamic  a f t e r k a i n i c a c i d l e s i o n s of the globus  pallidus F i g u r e 13.  59  GABA-transaminase h i s t o c h e m i s t r y i n the habenula f o l l o w i n g k a i n i c a c i d l e s i o n s of the entopeduncular nucleus  F i g u r e 14.  60  Increased  s t r i a t a l glutamate d e c a r b o x y l a s e  following  6-0HDA l e s i o n s of the n i g r o s t r i a t a l dopamine neurons . . F i g u r e 15.  Schematic r e p r e s e n t a t i o n of the s i t e and e x t e n t of the  F i g u r e 16.  68  [ % ] l e u c i n e i n j e c t i o n i n the s u b s t a n t i a n i g r a . . .  Labeled  79  t e r m i n a l s i n the s u p e r i o r c o l l i c u l u s f o l l o w i n g  the i n j e c t i o n of [ H ] l e u c i n e i n t o the s u b s t a n t i a a  nigra  82  F i g u r e 17.  Summary diagram of the outputs  F i g u r e 18.  H y p o t h e t i c a l mechanisms of a c t i o n f o r h a l o p e r i d o l and  amphetamine  o f the s t r i a t u m  86  101  X  LIST OF ABBREVIATIONS AOAA  amino-oxyacetic  acid  BA  biotin-avidin  CAT  choline acetyltransferase  DOPAC  dihydroxyphenylacetic acid  EOS  ethanolamine-o-sulphate  EP  entopeduncular  GABA  -y-aminobutyric a c i d  GABA-T  GABA-transaminase  GAD  glutamate  GP  globus p a l l i d u s  HVA  homovanillic acid  KA  kainic  leu-enkephalin  leucine-enkephalin  nucleus  decarboxylase  acid  met-enkephalin  methionine-enkephalin  6-OHDA  6-hydroxy dopamine  PAP  peroxidase anti-peroxidase  PBS  phosphate b u f f e r e d s a l i n e  SN  substantia nigra  SNC  s u b s t a n t i a n i g r a p a r s compacta  SNR  s u b s t a n t i a n i g r a pars  reticulata  xi  ACKNOWLEDGEMENTS I would l i k e to thank my s u p e r v i s o r Dr. E d i e McGeer f o r a l l o w i n g me a f r e e r e i n to e x p l o r e any o f the c r a z y i d e a s t h a t popped i n t o head d u r i n g the course o f t h i s work.  The f r i e n d l y i n t e r e s t ,  and encouragement of Dr. C h r i s F i b i g e r i s a l s o e s p e c i a l l y I would l i k e to acknowledge my debt to my  my  advice  appreciated.  two sen sei D r s . T. H a t t o r i  and H. Kimura, w i t h o u t whom none of the h i s t o l o g i c a l work would have been p o s s i b l e .  F i n a l l y , i t i s a p l e a s u r e to remember the many members  of the Kinsmen L a b o r a t o r y  and, i n p a r t i c u l a r , B i l l  J i m Nagy, f o r making t h i s work a p l e a s u r e .  S t a i n e s and  INTRODUCTION The b a s a l g a n g l i a a r e a group of s u b c o r t i c a l n u c l e i w h i c h i n c l u d e s  the  s t r i a t u m , g l o b u s p a l l i d u s (CP), e n t o p e d u n c u l a r n u c l e u s ( E P ) , s u b t h a l a m i c n u c l e u s and  s u b s t a n t i a n i g r a (SN).  Together t h e s e s t r u c t u r e s c o m p r i s e a  major p o r t i o n of what has been termed the e x t r a p y r a m i d a l are i n v o l v e d i n the c o n t r o l of p o s t u r e and  locomotion.  motor system Dysfunction  and  of  the  b a s a l g a n g l i a r e s u l t i n a v a r i e t y of c l i n i c a l c o n d i t i o n s i n v o l v i n g motor behaviour. and  Parkinson's disease  i s the b e s t u n d e r s t o o d of t h e s e d i s o r d e r s  i s c h a r a c t e r i z e d by a l o s s of the d o p a m i n e - c o n t a i n i n g neurons of the  and a c o r r e s p o n d i n g r e d u c t i o n i n s t r i a t a l dopamine l e v e l s  SN  (Hornykiewicz,  1973). H u n t i n g t o n ' s d i s e a s e i s a n o t h e r d i s o r d e r i n v o l v i n g the b a s a l I t i s c h a r a c t e r i z e d by n e u r o p a t h o l o g i c a l tum  (Lange et a l . , 1976).  ganglia.  changes i n the c o r t e x and  Although choreiform  the  stria-  movements a r e the h a l l m a r k  of  t h i s d i s e a s e , dementia i s a l s o a s t r i k i n g f e a t u r e o f H u n t i n g t o n ' s c h o r e a (Garron, The phrenia.  1973). b a s a l g a n g l i a have a l s o been i m p l i c a t e d i n the e t i o l o g y of Motor d i s t u r b a n c e s  including  c h o r e a , a k a t h i s i a , and  oral  d y s k i n e s i a s o c c u r f r e q u e n t l y i n p s y c h o t i c s , and  t h i s observation  (1955) t o propose o r i g i n a l l y t h a t s c h i z o p h r e n i a  i s a d i s o r d e r of the  ganglia.  schizo-  led Mettler basal  W i t h the i n t r o d u c t i o n of the n e u r o l e p t i c d r u g s f o r the symptomatic  treatment of t h i s d i s e a s e a t t e n t i o n has f o c u s e d p a r t i c u l a r l y on the r o l e of the n i g r a l dopamine neurons i n  schizophrenia.  These c l i n i c a l c o n d i t i o n s i l l u s t r a t e g a n g l i a may  provide  basal  be c h a r a c t e r i z e d not o n l y by impairments of motor b e h a v i o u r , but  a l s o by d i s r u p t i o n s of c o g n i t i v e f u n c t i o n . may  t h a t d i s o r d e r s of the  Thus, study of t h e b a s a l  ganglia  i n s i g h t i n t o the n e u r a l mechanisms i n v o l v e d i n b o t h of these  important processes.  I n p a r t i c u l a r , a s t u d y of the o u t p u t pathways of  the  2  basal the  ganglia  rest  of  The and As  the  it  is  the  including  motor  and memory  1970;  as  et  for  et  as  a  a l . , 1977;  (Kitai that  et  the  the  1976b)  the  inputs  and  and  1977; et  is of  Jones a l . ,  third  The  cortico-striatal  (Divac 1979;  et  the  a l . ,  anatomical  (Endo  parts  of  the  neocortex,  perception,  a l . ,  et  independent  parafasicular  and  centromedian  to  Kim  is  et  Kemp  the  a l . ,  a l . ,  and 1965;  striatum to  this  thought  1977)  et  1961;  terminals  a l . ,  and  nucleus  to  to  and  be  use  1977b;  McGeer  Electrophysiological  a l . ,  are  1977;  Webster,  pathway  association  Garcia-Rill  input  1976).  brain. basal  et  et  ganglia  the  1963;  1977;  Spencer,  with  of  afferent  Kocsis  basal  mammalian  1964;  the major  one  1976b;  interact  center  in  Smith,  the  the  a l l  implicated  Niimi This  in  in  from  fibers  nuclei  striatum  (Kemp  parafasicular  and  the  a l . ,  1975;  1973)  of  the  evidence  indicates  corticospinal  striatum  to  inputs  make up  one  quarter  have  lesion of  the  and L e a v i t t ,  of  1978)  (Jones the  or  et  striatum  in  a l l  of  a l . ,  and  terminals  the  1979; few  the  the  Leavitt,  choline in  receives  This  major  and  reported  (Saelens  the  afferent  changes  been  1974).  provide  Royce,  Although  levels  one-third  (Jones  the  1971b).  parafasicular  anterior  afferents  1978;  project  acetylcholine  nuclei  Kalil,  to  and P o w e l l ,  following  1977)  et  appear  these  (CAT)  striatum  Saelens,  1977;  1976a;  a l . ,  (Kuroda  Together  transferase the  et  included  integrative  DeVito  can  systems.  intralaminar  in  a l . ,  corticostriatal  Although  1974).  et  Reubi  corticobulbar  thalamic  1971b).  transmitter  a l . ,  areas  system  behaviour.  projections  as  this  cell-mass  the main  1978).  approximately  (Kitai  structure  1963;  1975;  accounts  glutamate  well  which  influence  Goldman and N a u t a ,  Kunzle,  and P o w e l l ,  at  subcortical  a l . ,  and van Hoesen,  excitatory  largest  receives  Yeterian  (Kemp  to  represents  areas  1944;  sites  system  the  striatum  (Carmen  Glees,  the  largest  probably The  Powell,  nervous  is  ganglia.  1979;  indicate  striatum  indeed, such  may  acetyl-  head  Simke if  lack  any of  of and  s t a i n i n g of p a r a f a s i c u l a r c e l l s f o r CAT  (H. Kimura, p e r s o n a l  r a i s e s doubt about t h i s b e i n g a c h o l i n e r g i c t r a c t .  communication)  Thus, a t p r e s e n t  the  b i o c h e m i c a l nature of t h i s i n p u t remains a matter of s p e c u l a t i o n , a l t h o u g h e l e c t r o p h y s i o l o g i c a l r e s u l t s suggest ( K o c s i s et a l . , 1977; The  M a l l i a n i and Purpura,  1967;  Purpura  and M a l l i a n i ,  t h i r d major s t r i a t a l a f f e r e n t a r i s e s i n the m i d b r a i n  the s t r i a t u m w i t h a p p r o x i m a t e l y (Kemp and P o w e l l , 1971b). both the SN and Beckstead 1974).  t h a t i t i s an e x c i t a t o r y pathway  the a d j a c e n t v e n t r a l tegmental  e t a l . , 1979;  The  F a l l o n and Moore, 1978;  n i g r o - s t r i a t a l system was  p r o j e c t i o n ; but t h e r e i s now  and s u p p l i e s  f i f t e e n p e r c e n t of i t s a f f e r e n t  T h i s pathway c o n t a i n s dopamine and area  arises  Richardson  e t a l . , 1977).  system has a l s o been suggested  o r i g i n a l l y thought  to be an  t h a t the dopamine  A second non-dopaminergic  F i b i g e r et a l . , 1972;  from the b r a i n s t e m have a l s o been i d e n t i f i e d .  K i t a i et a l . ,  nigrostriatal  L j u n d a h l et a l . , 1975;  Miller  et a l . , 1975).  t h i s p r o j e c t i o n i s most c o n c e n t r a t e d (Ternaux et a l . , 1977).  two  aminergic a f f e r e n t s  Serotonin i s contained  s t r i a t a l a f f e r e n t s from the d o r s a l raphe n u c l e u s  striatum  ter-  1978).  In a d d i t i o n to these major s t r i a t a l a f f e r e n t s ,  Jacobs et a l . , 1978;  inhibitory  on both p h y s i o l o g i c a l and b i o c h e m i c a l grounds  ( F e l t z and deChamplain, 1972; Guyenet and A g h a j a n i a n ,  from  Bjorklund,  m i n a l s d e p o l a r i z e s t r i a t a l neurons (Davies and Tongroach, 1978; 1975;  terminals  (Anden e t a l . , 1964;  L i n d v a l l and  c o n s i d e r a b l e evidence  1967).  ( A z m i t i a and  Segal,  1978;  B i o c h e m i c a l s t u d i e s suggest  i n the v e n t r o c a u d a l r e g i o n of  the  (Mason and  F i b i g e r , 1979;  noradrener-  Moore, 1978).  In c o n t r a s t t o the d i v e r s i t y of the a f f e r e n t s to the s t r i a t u m , the put of t h i s nucleus  that  A l s o , the u b i q u i t o u s axons of the l o c u s  c o e r u l e u s have been shown to p r o j e c t to the s t r i a t u m , p r o v i d i n g a g i c i n n e r v a t i o n to t h i s a r e a  i n the  i s quite r e s t r i c t e d .  d i r e c t l y o n l y t h r e e s t r u c t u r e s , the GP,  The EP and  out-  s t r i a t u m i s known to i n n e r v a t e SN.  Thus, the e n t i r e  output  of the striatum i s confined to other n u c l e i within the basal ganglia. parts of the striatum, including the head (Cowan and Powell,  All  1966;  Graybiel et a l . , 1979,  Nagy et a l . , 1978a, Tulloch et a l . , 1978)  and  (Tulloch et a l . , 1978)  project to the pallidum.  receives  Although the EP  fibers from the head of the striatum ( A d i n o l f i , 1969;  tail  Nagy et a l . , 1978a)  the extent to which i t receives afferents from the t a i l of the striatum i s uncertain.  The SN receives a r i c h innervation from diverse areas of the  striatum (Bunney and Aghajanian, 1976; 1976;  Grofova, 1975;  Nagy et a l . , 1978a; Richardson et a l . , 1977;  both to the pars compacta (SNC)  Kanazawa et a l . ,  Tulloch et a l . , 1978)  and to the pars r e t i c u l a t a (SNR).  Which of the neuronal types of the striatum gives r i s e to these efferent pathways has been a matter of some debate.  I t has been estimated  that over 95% of s t r i a t a l neurons are medium size spiny c e l l s (Kemp and Powell, 1971a).  The striatum also contains a few small neurons as well as  a population of giant aspiny c e l l s (Kemp and Powell, 1971a). c e l l s , which account for only about one percent  These giant  of the s t r i a t a l neurons  (Kemp and Powell, 1971a) were o r i g i n a l l y suggested on the basis of Golgi material to be the projection neurons of the striatum (Fox et a l . , 1971). However, with the introduction of retrograde transport as an anatomical t o o l , this conclusion has been questioned.  Thus, these giant c e l l s have  not been labeled following the i n j e c t i o n of horseradish peroxidase into the GP, EP or SN.  Recently, however, some large neurons have been labeled  following i n j e c t i o n s into the retrorubral area extension of the SN  (Beckstead et a l . , 1979;  (Grofova,  1979), a dorsal  Nauta et a l . , 1978).  Although horseradish peroxidase i n j e c t i o n s into the GP, EP or SN proper have f a i l e d to l a b e l the giant aspiny neurons of the striatum, many medium size c e l l s are labeled following these i n j e c t i o n s (Bunney and Aghajanian, 1976;  Graybiel et a l . , 1979;  Grofova, 1975;  Kanazawa et a l . , 1976;  Tulloch  et  a l . , 1978).  for  the  This  striatal  Evidence acid  (GABA)  1978; Nagy  is  1974;  al. ,  Gale  et  a l . ,  in  at  of  these  et  some  cells  these  cells  of  have  striatal  Nagy  SN  et  inhibitory  the  1973;  1977b;  Nagy  the  in  and  a l . ,  1977a;  glutamate  a l . ,  1978a)  least  that  contained  et  that  provide  the  major  pathways  outflow.  indicates  Hattori et  indicates  et  Hattori  a l . ,  et  1978a).  t h e medium  spiny  been  to  found  decarboxylase  (GAD)  in  to  1978a)  (Fonnum e t  et  EP  a l . ,  1973b;  This  suggests  stain  of  for  GP  _  Jessel that  the  the  et  immunohistochemical  m  i  nobutyric  a l . ,  GABA  Kim  contained  and  in  synthetic studies  1978a;  1978;  is  a l . ,  1978a;  a l . ,  al. ,  striatum,  GABA  a  (Fonnum e t  1977 ; Fonnum e t  a l . ,  cells  the  Y  afferents  a l . ,  (Brownstein  neurotransmitter  fact  some  enzyme  (Ribak  et  a l . ,  1979). Recently, is  also  1977c;  contained Kanazawa  Paxinos of  evidence  et  contain  Ljtfngdahl  the  a l . ,  1978a)  spiny  histochemical do  et  a l . ,  the medium  in  studies  and EP  have P  substance  GABA a n d  the  the  striatum  (Brownstein  populations The function the  of  as  in  et  opioid  a l . , et  the to  a l . ,  1977;  this  and medium (Cuello  Gale  et  are  et  In size and  1977;  probably  1978;  that  some  immuno-  striatal  cells  Kanazawa,  1978;  have  shown sites  Jessel  contained  that within  et  a l . ,  in  separate  neurons.  also  been  peptides  striatopallidal  found  to  contain  In particular methionine-  fibers  (Cuello  and  other  some  P  a l . ,  a l . ,  fact,  from different a l . ,  et  suggesting  experiments  arise  substance  (Hong  Palkovits  peptide.  material  transmitters  nigra  1978b),  biochemical  a l . ,  neuropeptide  the  1977;  small  P projections  neurotransmitters.  endogenous  present  has  Recent  these  striatal  striatum  some  immunoreactive  1978a).  that  et  contain  shown  that  projections  (Paxinos  should  a l . ,  indicating  obtained  1977a; Mroz  the  1978)  been  striatal  cells  substance et  has  peptides  evidence  that  suggests  leucine-enkephalin  and P a x i n o s , 1 9 7 8 b ;  Hong  may et  may that be a l . ,  6  1977a). A l t h o u g h the d i r e c t output of the s t r i a t u m i s c o n f i n e d t o t h e b a s a l g a n g l i a , pathways must e x i s t t o t r a n s m i t t h i s output beyond t h i s u l t i m a t e l y t o the motor neurons.  system,  Both the EP ( C a r p e n t e r , 1973; Kim e t a l . ,  1976; Kuo and C a r p e n t e r , 1973; L a r s e n and McBride, 1979; Nauta and 1966)  and SN (Beckstead e t a l . , 1979)  Mehler,  send p r o j e c t i o n s t o t h e p a r s compacta  of the n u c l e u s t e g m e n t i p e d u n c u l o p o n t i s , a m e s e n c e p h a l i c n u c l e u s l y i n g w i t h i n the b r a c h i u m c o n j u n c t i v u m .  r e t i c u l a r formation  The e f f e r e n t c o n n e c t i o n s  of t h i s n u c l e u s a r e unknown, but p r e l i m i n a r y r e p o r t s suggest t h a t i t s axons descend  to the s p i n a l c o r d (Ross e t a l . , 1979)  f o r e b r a i n , perhaps  and ascend towards  s y n a p s i n g i n the p a l l i d u m (Gonya-Magee and  the  Anderson,  1979). A p o p u l a t i o n of dopamine c e l l s i n the m e d i a l SN and the v e n t r a l tegment a l a r e a (Fuxe e t a l . , 1974; L i n d v a l l e t a l . , 1974; T h i e r r y e t a l . , 1973) and a p o p u l a t i o n of c h o l i n e r g i c c e l l s i n and around  t h e GP and EP  (Kelley  and Moore, 1978a; Lehmann e t a l . , 1980) p r o j e c t to t h e c e r e b r a l c o r t e x . However, t h e s e pathways have o n l y r e c e n t l y been d i s c o v e r e d and  their  c o n n e c t i o n s w i t h the s t r i a t u m and t h e i r r o l e i n m e d i a t i n g b a s a l g a n g l i a o u t p u t have n o t been examined. The pathways most commonly thought t o p r o v i d e t h e main o u t p u t f o r the b a s a l g a n g l i a a r i s e from the EP and from the SNR.  As d i s c u s s e d above t h e s e  two n u c l e i b o t h r e c e i v e d i r e c t i n p u t s from the s t r i a t u m .  I n a d d i t i o n the  GP p r o j e c t s d i r e c t l y t o the SN (Bunney and A g h a j a n i a n , 1976; G r o f o v a , H a t t o r i e t a l . , 1975; Kanazawa e t a l . , et a l . , 1978)  1975;  1976; Kim e t a l . , 1976; T u l l o c h  and t o t h e s u b t h a l a m i c n u c l e u s ( H a t t o r i e t a l . , 1975;  Kim  et a l . , 1976; Nauta and M e h l e r , 1966) which i n t u r n p r o j e c t s t o t h e EP ( C a r p e n t e r and S t r o m i n g e r , 1967; Nauta and C o l e , 1978; W h i t t i e r and M e t t l e r , 1949)  and the SN (Hammond et a l . , 1978; Kanazawa e t a l . , 1976; Nauta and  7 C o l e , 1978;  T u l l o c h et a l . , 1978;  W h i t t i e r and M e t t l e r , 1949).  A major p r o j e c t i o n from the EP to the l a t e r a l habenula F i b i g e r , 1978;  Herkenham and Nauta, 1977;  McBride,  Nauta, 1974)  1979;  have suggested 1977;  et a l . , 1976;  has been demonstrated  L a r s e n and  and b i o c h e m i c a l s t u d i e s  However, the l a t e r a l habenula  i s not thought  i n v o l v e d i n the c o n t r o l of motor b e h a v i o u r .  Rather,  p r o j e c t i o n from the EP to the thalamus has been suggested function  ( H a s s l e r , 1978).  C a r p e n t e r , 1973;  L a r s e n and McBride,  Kim  1979;  the  pallidal  and p a r a f a s i c u l a r n u c l e i ,  more i m p o r t a n t l y , the v e n t r o l a t e r a l - v e n t r o a n t e r i o r complex and ( C a r t e r and F i b i g e r , 1978;  to  to s e r v e such a  The p r i n c i p a l s i t e s of t e r m i n a t i o n o f  f i b e r s i n the thalamus a r e the centromedian  medial n u c l e u s  and  t h a t GABA i s a t r a n s m i t t e r i n t h i s pathway ( G o t t e s f e l d et a l . ,  Nagy et a l . , 1978b).  be c r i t i c a l l y  Kim  (Carter  et a l . , 1976;  the v e n t r o -  Kuo  Nauta and Mehler,  and  and  1966).  Since  the nucleus v e n t r a l i s l a t e r a l i s p r o j e c t s to the motor c o r t e x areas f o u r and s i x , the p a l l i d a l p r o j e c t i o n to t h i s nucleus p r o v i d e s a pathway f o r the b a s a l g a n g l i a to a f f e c t d i r e c t l y  the motor output of the c o r t e x .  A s u b s t a n t i a l p r o j e c t i o n to the thalamus from the SNR demonstrated.  T h i s i s a non-dopaminergic  p r o j e c t i o n and  the m e d i a l p a r t of the nucleus v e n t r a l i s l a t e r a l i s , of the n u c l e u s v e n t r a l i s a n t e r i o r and (Beckstead e t a l . ,  et a l . , 1976;  Carpenter and P e t e r , 1972;  F i b i g e r , et a l . , 1972;  i s distributed  the m a g n o c e l l u l a r p a r t  B e n t i v i g l i o et a l . , C o l e et a l . , 1964;  M e t t l e r , 1970;  R i n v i k , 1975).  1979;  et a l . , 1979;  C l a v i e r et a l . , 1976).  w i t h the n u c l e u s v e n t r a l i s l a t e r a l i s ,  Carpenter  F a u l l and  Mehler,  A projection  to the p a r a f a s i c u l a r n u c l e u s has a l s o been r e p o r t e d ( A h l e n i u s , Beckstead  to  the p a r a l a m i n a r p a r t of the n u c l e u s  dorsomedialis  1978;  1979;  has a l s o been  1978;  A g a i n , v i a the c o n n e c t i o n  t h i s output pathway p r o v i d e s the b a s a l  g a n g l i a w i t h a c c e s s to the motor c o r t e x . F i n a l l y , f i b e r s from the SNR,  a r i s i n g a t l e a s t i n p a r t as  collaterals  of the n i g r o t h a l a m i c  p r o j e c t i o n (Anderson and Y o s h i d a , 1977;  et a l . , 1979), i n n e r v a t e Beckstead et a l . , 1979;  the s u p e r i o r c o l l i c u l u s F a u l l and Mehler, 1978;  and N i e s s e n , 1976; Jayaraman  Bentivoglio  ( A f i f i and K a e l b e r , 1965; G r a y b i e l , 1978;  Hopkins  et a l . , 1977; R i n v i k e t a l . , 1976).  n i g r o t e c t a l f i b e r s t e r m i n a t e i n the deeper l a y e r s of the s u p e r i o r (Anderson and Y o s h i d a , 1977; Jayaraman  et a l . , 1977;  Beckstead et a l . , 1979;  Graybiel,  The colliculus  1978,  R i n v i k e t a l . , 1976), which a r e known to g i v e  to the t e c t o r e t i c u l a r and t e c t o s p i n a l pathways (Kuypers and Maisky,  rise  1975).  Indeed, e l e c t r o p h y s i o l o g i c a l experiments have i n d i c a t e d t h a t the p r o j e c t i o n from the SNR  to the s u p e r i o r  system which r e g u l a t e s and Faber, 1977).  the neck muscles i n v o l v e d  ganglia.  i n head o r i e n t a t i o n (York  Thus, t h i s system c o u l d w e l l be i n v o l v e d i n the  of the motor asymmetries basal  c o l l i c u l u s i n t e r a c t s w i t h the t e c t o s p i n a l  expression  observed a f t e r u n i l a t e r a l m a n i p u l a t i o n s of the  9  STATEMENT OF THE PROBLEMS TO BE EXAMINED In  the p r e s e n t study a b i o c h e m i c a l and h i s t o l o g i c a l a n a l y s i s o f t h e  output pathways o f t h e b a s a l g a n g l i a i s p r e s e n t e d . examination of  We b e g i n w i t h an  o f the e f f e r e n t s o f t h e caudate-putamen t o t h e o t h e r  the b a s a l g a n g l i a .  As mentioned i n t h e I n t r o d u c t i o n , GABA, a c e t y l c h o l i n e ,  e n k e p h a l i n and substance P have a l l been suggested striatal  cells.  nuclei  I n Experiment  t o be c o n t a i n e d i n  1 the existence o f these t r a n s m i t t e r s i n the  s t r i a t a l p r o j e c t i o n s to the GP, EP and SN was b i o c h e m i c a l l y examined following lesions of the s t r i a t a l  output pathways.  The b i o c h e m i c a l approach used i n Experiment  1 can p r o v i d e  evidence  t h a t a c h e m i c a l such as GABA o r substance P i s c o n t a i n e d i n a f i b r e  tract.  T h i s t e c h n i q u e w i l l n o t however g i v e i n f o r m a t i o n r e g a r d i n g t h e topography and s y n a p t i c r e l a t i o n s h i p s o f the f i b r e systems. methods a r e e s s e n t i a l .  T h e r e f o r e i n Experiment  For this  histological  2 the d i s t r i b u t i o n of the  substance P f i b r e s was examined u s i n g an immunohistochemical  approach.  To study t h e GABA systems o f the b a s a l g a n g l i a t h e l o c a l i z a t i o n o f t h e enzyme GABA-transaminase (GABA-T), which c a t a b o l i z e s GABA, was examined. The b i o c h e m i c a l s t u d i e s i n Experiment  3 suggest  t h a t GABA-T i s l o c a l i z e d  i n t h e GABA neurons o f t h e s t r i a t u m .  T h e r e f o r e , i n Experiment  h i s t o c h e m i c a l method f o r t h i s enzyme was adapted and p a l l i d a l The  efferents are intimately associated with the n i g r o s t r i a t a l  neurons.  I n o r d e r to examine t h e n a t u r e o f t h i s  the responses o f t h e s t r i a t a l  GABA and substance P c e l l s  l o s s of t h e n i g r a l dopaminergic Experiment  t o examine t h e s t r i a t a l  GABA e f f e r e n t s .  striatal  dopaminergic  A, a  5.  interaction  to the s e l e c t i v e  neurons were b i o c h e m i c a l l y examined i n  As t h i s l e s i o n o f the dopaminergic  neurons has been used as  a model o f P a r k i n s o n ' s d i s e a s e (Marsden, 1977), t h e r e s u l t s o f t h i s have been compared w i t h t h e b i o c h e m i c a l changes r e p o r t e d t o o c c u r i n  study  10  Parkinsonism. Finally  i n Experiment 6 the b i o c h e m i c a l  pathway, which p r o b a b l y  represents  nature of the n i g r o t e c t a l  a major output o f the b a s a l  was examined by measuring v a r i o u s n e u r o t r a n s m i t t e r colliculus following nigral lesions.  markers i n t h e s u p e r i o r  The morphology o f t h e n i g r o t e c t a l  p r o j e c t i o n was a l s o examined u s i n g e l e c t r o n m i c r o s c o p y . the f i r s t pathways.  ganglia,  The r e s u l t s p r o v i d e  i n d i c a t i o n t h a t GABA may be a t r a n s m i t t e r i n t h e n i g r a l The i m p l i c a t i o n s of t h i s o b s e r v a t i o n  are t h e r e f o r e  discussed.  output  f o r basal ganglia function  11  EXPERIMENT  1;  Three the  NEUROTRANSMITTERS  neurotransmitter  fibers  emanating  from  CONTAINED  candidates  the  I N THE EFFERENTS  have  striatum.  been  These  found  are  to  GABA,  OF THE be  STRIATUM  contained  substance  P  in  and  enkephalin. GABA h a s 1978a;  been  Hattori  (Fonnum e t  shown  et  a l . ,  a l . ,  Nagy  et  a l . ,  1977:  Fonnum e t  et  a l . ,  1978;  Kim  entopeduncular contain and  and P  1977c;  respectively.  A  1978)  and  kainic  comprehensive been  conducted.  contribution the  GP,  1974; et  1978a), and  a l . ,  a l . ,  et  each of  of  levels the  study  these  a l . ,  1978a)  fibers.  also et  1977;  a l . ,  et  striatum  shown  et  a l . ,  of  each  provides  substances  of  to  the  1977; techniques,  and to  has  Paxinos, decrease  a l . , 1977a).  these  further  1978b)  pathway  been observed et  to  1977a)  (Cuello  (Hong  striato-  1978a;  a l . ,  methods  Jessel  The  been  Gale  Kanazawa  have  a l . ,  (Brownstein  1973b;  leu-enkephalin-containing  examination  present  et  (Paxions  1978;  met-enkephalin lesions  striatonigral  have  a l . ,  (Fonnum e t  striatoentopeduncular  Hattori  projections  (Brownstein  systematic  and  a l . ,  1978a)  1978a;  et  striatopallidal  immunohistofluorescent  The  of  EP  a l . ,  in  immunofluorescent  acid  and  et  1 9 7 1 ; Nagy  Jessel  by  pallidal  following  et  striatopallidal  been demonstrated  to  by  radioimmunochemical a l . ,  Nagy  a l . ,  a l . ,  contained  striatonigral  substance  Hong e t  be  1973b;  1978a;  et  to  systems  information  striatal  A  has  on  not  the  projections  SN. METHODS  Male Wistar and  450  placed  g in in  separating in  radius  mounted  in  the  a Kopf the and a  GP  rats GP  weighing  island  group  stereotaxic from  three  the  mm i n  26 g a u g e  about  300  were  height  cannula.  the  hemitransection  anesthetized  instrument.  striatum  g in  (GP  For  the  islands)  was  fashioned  After  removal  with  of  pentobarbital  semicircular a Halasz  from  flap  and  cuts  knife  tungsten  a bone  group  four  wire and  mm  and deflection  12  of the dura the k n i f e was  lowered a t a p a r a s a g g i t a l o r i e n t a t i o n to  e m p i r i c a l l y determined set of c o o r d i n a t e s . k n i f e was The  3.5  mm  p o s t e r i o r to bregma and  t i p of the k n i f e was  p o s i t i o n e d the k n i f e was and  then r o t a t e d 130  8.1  mm  2.3  mm  a x i s o f r o t a t i o n of l a t e r a l to the  10-15  degrees l a t e r o c a u d a l l y . withdrawn and  the  midline.  v e n t r a l to the c r a n i a l s u r f a c e .  rotated slowly  entry o r i e n t a t i o n the k n i f e was 0.1%  The  an  Once  degrees towards the A f t e r r e t u r n i n g to  midline the  the wound t r e a t e d w i t h  Zephiran. H e m i t r a n s e c t i o n s j u s t a n t e r i o r to the GP were produced w i t h a  s t r a i g h t t u n g s t e n w i r e lowered at the m i d l i n e the b r a i n and  to the v e n t r a l s u r f a c e  drawn to the l a t e r a l extreme i n the p l a n e of the  of  anterior  commissure. Animals were s a c r i f i c e d c e r v i c a l f r a c t u r e and areas were d i s s e c t e d tome.  The  two  to t h r e e weeks p o s t o p e r a t i v e l y  the b r a i n s r a p i d l y removed and freehand from s e c t i o n s o b t a i n e d  head of the s t r i a t u m was  not v i s i b l y damaged by  the k n i f e .  dissected only The  GP was  which the k n i f e c u t encroached on t h i s a r e a . was  d i s s e c t e d from the same s e c t i o n s as the  placed  by  on  ice.  All  on a f r e e z i n g m i c r o -  from those  sections  not  assayed i n animals i n  The  t a i l o f the  striatum  GP.  For enzyme a c t i v i t y measurements t i s s u e s were homogenized i n 20 to volumes of 50 mM X-100.  T r i s - a c e t a t e b u f f e r , pH  P r o t e i n was  6.4,  containing  0.2%  v/v  determined on these homogenates a c c o r d i n g  to  50  Triton the  method of Lowry et a l . (1951). A m o d i f i c a t i o n of the method of A l b e r s the assay of GAD (20 y l ) was  activity  and  Brady  (Chalmers et a l . , 1970).  (1959) was  T i s s u e homogenate  added to 50 y l of i n c u b a t i o n m i x t u r e c o n t a i n i n g  concentrations)  2.0  mM  mmol, Amersham), 0.02%  [l-  lif  C]glutamate  used f o r  (final  ( s p e c i f i c a c t i v i t y 0.1  bovine serum albumin (Sigma) and  0.1  mM  to 0.2  mCi/  pyridoxal  13  phosphate small a  (Calbiochem)  glass  gelatin  0.1  ml  of  septums  were  incubated  of  0.2  ml  with  parts  gauge  vials  liquid  ul  0.05  stopped  zene  (POPOP)  and  was  counted  Substance immunoassay (see  then  a  liquid P  adding five  for  and  into  the  50  1 I +  yl  8.0  C]acetyl  of  a  by  the  tubes  mM c h o l i n e  coenzyme  A  a  solution  0.015% 1  pH  7.4.  vial.  buffer  toluene  The of pH  7.4  containing  p_-bis-[2-(5-phenyloxazolyl)]ben-  ^-acetylcholine  organic  of  chloride,  consisting  t e n mM s o d i u m p h o s p h a t e Ten ml  reaction  (Amersham,  buffer  of  boron.  The  mixture  seven ml  and m e t - e n k e p h a l i n 1.0  to  scintillation  the  rubber  the  (1975).  a  scintillation  on l y o p h i l i z e d  Fonnum  in  and  which  ACS  20 m i n  tetraphenyl  added  of  3 0 0 mM N a C l ,  parts  (PPO)  extracted  in  by  with  reaction in  half  to  stopped  5 0 mM s o d i u m p h o s p h a t e  37°C  sodium  2,5-diphenyl-oxazole  in  with  through  counted  2  homogenate  mM [ 1 -  at  and  0.4%  were  0.2  C0  the  a  counter.  the method  tissue  and  l t +  was  tube  in  paper  capped  reaction  hr  vial  filter  were  two  trapped  to  mCi/mmol)  incubated  two mg/ml  incubation  of  2 0 mM E D T A ,  then  the  The  After  concentrations)  acetonitrile  containing  and  of  incubation  scintillation  20  (final  was  needle.  pH 7.4  scintillation  The v i a l s  the  buffer,  a piece  30 m i n .  into  according  adding  activity  was  for  2M H^SO^  assayed  contained  was  37°C  19  the  a  by  The m i x t u r e  at  of a  from  mil e s e r i n e ,  two  a  added.  started  reaction  in  hydroxide  septum w i t h  specific  placed  hyamine  CAT was  0.1  was  containing  removed  which  tube  phosphate  (Parke-Davis)  (Amersham)  was  Each  2 0 mM p o t a s s i u m  capsule  and  injection rubber  tube.  in  phase  with  produced the  during  tetraphenyl  the boron  counter. levels  N acetic  acid  were  determined  extracts  of  by  radio-  weighed  tissue  Appendix). RESULTS The  (GP  extent  islands)  is  to  which  shown  in  the Fig.  knife 1.  cuts  isolated  Rostrally,  the  the  GP f r o m  lesions  the  started  striatum just  lateral  14  F i g u r e 1.  The k n i f e c u t s s e p a r a t i n g the globus p a l l i d u s from the striatum. The diagram on the l e f t r e p r e s e n t s a h o r i z o n t a l through the f o r e b r a i n showing circular  the s i t e s o f b o t h t h e  cut forming the GP i s l a n d  hemitransection  section  the  The photograph  on the  r i g h t i s a c o r o n a l s e c t i o n through the a n t e r i o r  globus  p a l l i d u s showing  (double arrow).  (arrow) and  the c i r c u l a r k n i f e c u t .  The  circular  c u t i s seen l a t e r a l to the globus p a l l i d u s which i s outl i n e d i n white dots.  Shrinkage of s t r i a t a l t i s s u e i s  evidenced by the d i l a t i o n o f the l a t e r a l v e n t r i c l e ( L V ) . AC n u c l e u s accumbens, GP globus p a l l i d u s , CP putamen.  Magnification:  25X.  caudate-  ita  15  to  the  septum and  anterior  to  From t h i s  point,  the  subscribe  an a r c  surrounding  to  avoid  isolate  damage one-half  Although  of  the  to  direct  occasionally and  to  lesions  of  the  lesions  to  the  some  the  lateral  Regions  the  anterior  laterally  and  then  capsule  of  the  and  lesions the  of  retrograde  although  were of  the  loss  of  the  gliosis  was  In  order  so  as  to  lesions Some  gliosis  from  some  and  to  striatum.  resulting  anterior  no  produced  structure.  probably  striatum  posteriorly  observed,  this  commissure.  laterally.  t a i l  rarely  evident  the  shrinkage  was  aspect  were and  the  GP f r o m  pallidum  terminals  neurons.  exhibited  the  of  GP b o t h d o r s a l l y  of  pallidum  striato-pallidal  projection  the  two-thirds  damage  decussation  proceeded  internal  approached  shrinkage  the  the  loss  pallidal  lateral  to  the  observed  in  these  areas. The brain  effect  areas  enzyme was  percent  these  examined  significantly  activity  of  by  not  of in  are  44  and  GP  not  following  a 49 p e r c e n t  anterior  to  the  is the of  shown  in  anterior  reduction  of 2.  pole  of  after  in the  observed  was  significantly  not  GAD w a s  Table  in  this  without  the  the  in  circular in  nigral  reduced  significantly  in  the  SN o r The  EP  cuts  just  GP  EP  but  to  explains  and  account  GP b u t in  in  the  unaltered the  tail  of  lesion.  was  reduced  the  the  posterior  this  As  GAD w a s  this  of  various  the  head  of  reduced  this The  by GP  35 islands  the  striatum  activity.  following could  the  activity  production in  in  a n d EP  The  rostral  were more  activity  SN b y  CAT  probably  GAD.  altered  the  GAD a c t i v i t y  affecting  This  GP a n d  in  the  lesions  GP.  In  lesion.  increase  These  1.  CAT  respectively.  hemitransections  Table  GAD o b s e r v e d  observed  lesion,  o n GAD a n d  decreased  affected  in  effect  in  22 p e r c e n t ,  resulted  The  cuts  shown  significantly  CAT w a s the  knife  and  the  lesion for  GP  o n CAT  encroached  greater  compared the  the  and  upon  reductions with  those  significant  case  of  GP i s l a n d s ,  in  the  EP  of  the  striatum  or  GAD  SN.  CAT  Neither lateral  CAT to  16  Table  1.  Glutamic  acid  decarboxylase  activity  in  pallidus  from  various the  Glutamic  areas  striatum  acid  nmol/mg Area  Head  of  % of  Globus % of  striatum  % of  indicate  **p  < .01,  isolating  the  globus  islands).  decarboxylase  protein/hr  Choline  acetyltransferase  nmol/mg  350±16**  498±30  protein/hr  Control  Lesion  168±9  173±10 103  279±26**  55.8+7.2  36.4±3.3* 65  56  nucleus  290±27  225±14*  16.9±1.3  20.2±1.5  84  78  nigra  Values  (GP  235±14  control  control  lesions  Lesion  control  % of  after  acetyltransferase  149  pallidus  Substantia  choline  Control  control  Entopeduncular  and  683±78  545±50 80  means  Student's  ± S.E.M. _t  test.  of eight  determinations.  *p  < .05,  17 the  GP.  This  simply  due  suggests  to  a posterior  lesions.  As  increased  GAD a c t i v i t y  activity  in  the  remained  Substance after  lesions  P  of  reduced  substance  percent  ed.  The  just  anterior  peptide those in  the  by  P  in  GP  by  The  GP b y  the  GP a r e  levels  the  in  head  case  but  lesions  head  of  and  islands  48  activities  after  the  the  was  not  hemitransection  hemitransections  of  significantly  striatum while  projection  are  P  of  in  in  the  these in  the  head same  GP  of  of  4.  areas  of  CAT  Table  3.  Expressed  striatum  were  after  as  significantly in  EP  and  unaffect-  hemitransections  reductions  striatum  islands.  striatum  58 p e r c e n t  the  The  the  lesions  GP,  areas  the  were  Similarly,  of  this  comparable substance  to  P  levels  unaltered.  significantly not  caused  in  these  the  Table  areas  were  did  the  in  shown  side,  percent  shown  striatum  20 p e r c e n t these  enzyme  damage  islands,  head  projection  the  the  addition,  tissue  the  levels  The h e m i t r a n s e c t i o n s the  pallidal  contralateral  substance  in  of  GP  the  levels  three  observed  the  the  SN.  of  to  the  in  of  54% i n  levels  the  levels  head  of  unlesioned  in  in  spread  case  producing  the  reduction  unchanged.  percent  47  the  alter  a 34  reduced  levels  percent  met-enkephalin  in  the  EP  increase  in  or  SN  levels  (Table  in  4).  In  met-enkephalin  striatum. DISCUSSION  Although the  striatum  it  are  has to  neurotransmitters transmitters  examined  the  projections  the  are  thought  acetylcholine,  GABA,  the  GP,  EP  involved to  be  and in  existence  striatum.  P, of  some  SN,  and  time  there  each  associated  substance  possible of  been known f o r  of with  that  is  these  s t i l l  striatal  of  these  outputs  controversy  systems.  enkephalin.  each  the major  At  neurons We h a v e ,  substances  over  present,  of which the  include therefore, in  the  various  18  Table  2.  Glutamic in  acid  various  decarboxylase  areas  after  and  choline  hemitransections  acetyltransferase anterior  to  the  activity globus  pallidus.  Glutamic  acid  nmol/mg  of  % of  Tail  of  % of  Globus % of  % of  Values the  326±19  199±18  224±15  89  139±18  167±12  417±44  83  128  control  215+24**  506±22  pallidus  41.4+2.9*  62.8±6.5  66  42  control  nucleus  338±25  196±21**  25.4±5.5  28.5±5.4  89  58  control  632±39  nigra  369±37* 58  control  indicate  remaining  Lesion  154  striatum  Substantia  protein/hr  Control  440±33*  control  Entopeduncular % of  acetyltransferase  nmol/mg  Lesion  285±23  striatum  Choline  protein/hr  Control  Area  Head  decarboxylase  means ±  tissues.  S.E.M. *p <  of  seven  .025,  **p  determinations <.005,  for  Student's  SN a n d _t  test.  five  for  19 (a)  The  Globus  The  decrease  separation may b e half  from  in  substance  the  striatum  a transmitter  the  while  Pallidus  substance  the  striatum  or  Substance  could  and Kanazawa, Huntington's  pallidum  In a  the  (Fonnum e t  In  present  the  less  after  that  the  GP  reduce  to  in  been  the  brain  have  been  injections the  KA  in  with  lesion  GP to  the  not  in  the  the  GP  to P  GP.  that  levels  previously  in  has  1973b;  been  Nagy  GAD w e r e  et  also  hemitransections. of  its  the  GABA  input  striatum. of  and  the  of  et  1979;  Hong  been presented (Cuello  for  the et  GP  other  hand,  1977a).  striatum  a l . ,  but  striatum  by  do  met-  1977b).  leu-enkephalin-  1978)  following  a l . ,  a  supported  of  and P a x i n o s ,  and P a x i n o s ,  On t h e  et  indicates  neurons  is  the  1978a).  slightly  from  levels  a l . ,  a l . ,  This  This  tail  demons-  only  the highest  the  the  (Cuello  note  contain  in  of  of  1980).  (Hong  only  have  pallidal  in  (Cuello  decrease  striatum  included  in  projection  study  that  striatum,  Fibiger,  has  appear  t a i l  neurons  substance  a l . ,  (Nagy  met-enkephalin. to  et  lesions  (Gramsch  evidence  that  observed into  the  in  fibers  lesions  the  interesting  P  1978).  portion  reported  striatopallidal  serum employed reactivity  in  been  is  after  electrolytic  GAD a c t i v i t y  Immunohistochemical containing  both  found  it  reductions  from  have  a l . ,  than  arise  its  substance  would  anterior  P  Hattori  the major  that  GP h a s  enkephalin  1978a;  the  following  that  It  substance  decrease  et  lesions  rostral  not  (Gale  the  island  observation  The  a l . ,  study  the  regard  GAD-containing  GP r e c e i v e s  originating  cells  50 p e r c e n t GP  Striatopallidal trated  this  from  either  intrinsic  evidence  pathway.  comes  could  GP o b s e r v e d  first  the  immunoreactive  in  the  in  50 p e r c e n t  chorea  the  striatopallidal  1978).  been observed  provides  in  the  represent  P-like  levels  in  P  remaining  P  1978).  had  The  anti-  l i t t l e  cross-  met-enkephalin  levels  kainic In  also  acid  that the  (KA)  study,  however,  GP a n d p a r t  of  20  Table  3.  Substance the  globus  P  levels  in  pallidus  various  from  the  areas  striatum  Substance pg/mg Area  Head  of  Globus  striatum pallidus  Entopeduncular Substantia  Values *p  are  < .025,  means **p  ± S.E.M. < .01,  isolating  islands).  P  % of  control  Control  Lesion  445±110  448±82  6  101  502±  206±45*  5  41  416±90**  6  42  1067±215**  6  53  90  2014±175  nigra  (GP  lesions  tissue  993+114  nucleus  after  of  the  Student's  number _t  of  test.  determinations  (N)  indicated.  21  Table  4.  The  levels  areas  of  after  substance  pg/mg Area  of  % of  Globus % of  striatum  % of  % of  Values  globus  pallidus  pg/mg  tissue  Lesion  N  Control  Lesion  322±16  301±24  15  1126±85  1531±110***  462±14  134  233±50***  16  2675±249*  3351±181  80  50  nucleus  614+99  9  300±56**  749±96  728±80  96  49  1503±237  nigra  710+204***  5  187±28  152±28  123  47  control  represent  the  < .05,  brain  Control  control  *p  the  various  Met-enkephalin  tissue  control  indicated.  to  in  94  pallidus  Substantia  anterior  P  control  Entopeduncular  and m e t - e n k e p h a l i n  hemitransections  Substance  Head  P  means **p  ± S.E.M.  of  the  < .025,  ***p  number  < .01,  of  determination  Student's  t  test.  22  the thalamus.  Thus, i n view of the demonstration o f d i s t i n c t p o p u l a t i o n s  of l e u c i n e - and m e t h i o n i n e - e n k e p h a l i n - c o n t a i n i n g neurons putamen and GP  (Larsson et a l . ,  1979),  i n the  the p r e s e n t r e s u l t s p r o v i d e the  f i r s t demonstration of the e x i s t e n c e of a met-enkephalin pallidal projection.  containing striato-  The s m a l l but s i g n i f i c a n t d e c r e a s e i n the l e v e l s of  t h i s p e p t i d e observed i n the GP a f t e r a l e s i o n which markedly GAD  caudate-  depleted  and substance P, suggests an a l t e r n a t e source f o r the m a j o r i t y of the  met-enkephalin  i n the GP than the head of the s t r i a t u m .  S i n c e KA  lesions  of the s t r i a t u m which i n c l u d e the GP l e a d to a 50% d e c r e a s e i n p a l l i d a l met-enkephalin  levels  (Hong et a l . , 1977a), i t i s p r o b a b l e t h a t t h e r e a r e  i n t r i n s i c p a l l i d a l met-enkephalin s u b s t a n t i a l met-enkephalin Although CAT  neurons  and/or  t h a t the p a l l i d u m r e c e i v e s  a f f e r e n t s from o t h e r s o u r c e s .  activity  i n the GP was  decreased by both l e s i o n s i n t h i s  study, these r e s u l t s a r e not s u f f i c i e n t  to demonstrate  cholinergic s t r i a t o p a l l i d a l projection.  the e x i s t e n c e of a  I t has p r e v i o u s l y been observed  t h a t c o r t i c a l l e s i o n s r e s u l t i n the r e t r o g r a d e d e g e n e r a t i o n of the i n t e n s e l y s t a i n i n g a c e t y l c h o l i n e s t e r a s e neurons are thought 1980).  l o c a t e d i n and around  the GP  which  to p r o v i d e the c h o l i n e r g i c i n p u t to the c o r t e x (Lehmann e t a l . ,  I t has a l s o been observed  (Wm.  S t a i n e s and H.C.  Fibiger, personal  communication) t h a t KA l e s i o n s c o n f i n e d w h o l l y t o the head of the s t r i a t u m do not decrease p a l l i d a l CAT. the p r e s e n t study may  The l o s s of p a l l i d a l CAT  a c t i v i t y observed i n  t h e r e f o r e be a t t r i b u t a b l e to the r e t r o g r a d e l o s s of  the c h o l i n e r g i c p a l l i d a l neurons which p r o j e c t t o a r e a s r o s t r a l to the l e s i o n s i t e s such as the c e r e b r a l c o r t e x (Johnston e t a l . , Moore, 1978a; Lehmann e t a l . , (b)  The Entopeduncular  1979;  Kelley  and  1980).  Nucleus  The EP has p r e v i o u s l y been shown to c o n t a i n substance P - l i k e immunor e a c t i v i t y by immunohistochemical  methods ( C u e l l o and Kanazawa, 1978;  Ljiing-  23  dahl  et  P  the  in  The  a l . , EP  levels  gation  1978a; have  of  and  from  not  similar  this  contain  P  it  higher  Paxinos  in to  substance  used  al.  of  the  substance  of  the  striatum.  In  of  (1978b)  present P  addition  in  to  a  a l . ,  1978a;  previous GAD t o  evidence  the  same  contrast, more  in  as  input the  the  anterior  leaving  Nagy  some  large to  GP.  lesions  a  EP  the  in  found  head  the  striatum  less  than  GAD  is  P  than  tissue  present  from  that  medial  indicates  with  to  trigeminal  progressively  complex  substance  to  confirm  that  afferents  et  GP  islands, wide  decreased  al.  lesion,  did  not  portion  of  laterally result  the  anterior  GABA  pole  Fonnum e t  lesions  In  started  GP  of  EP  (1978a).  which  the  head  the  the  bordering  the  (Fonnum  of  a major  majority  GAD-containing  support  Nagy  greater  the  demonstrated  by  posterior  to  from  tract,  results  the  P-like  similar  observations  more  1976)  lesions  in  swept  that  cells  the  successively  led  the  and  distribu-  and J e s s e l ,  that  recently  reported  striatal  investi-  The h e m i t r a n s e c t i o n s  produce  methods.  regional  the  data  The p r e s e n t  intact  This  and  following  been  a pathway.  to  the  striato-pallidal  also  previously  present  substance  EP.  The p r e s e n t  1978a).  the  of  SN,  of  biochemical  reported  probably  levels  Kanazawa  the  recently  have  agreement  who  the  hemitransections  arises  is  only  employed  decrease.  This  The  as  striatal  the  peduncular  such  extent  (1978a) of  a l . ,  for  than  that  disappeared  fibers  et  studies 1976;  GABA-containing  striatoentopeduncular et  EP  in  by  SN i n  a l . ,  study.  the  GP a n d  et  have  EP  However,  been q u a n t i f i e d  substance  the  1978b).  reported  appear  in  the  a l . ,  striatum,  those  levels  in  t  (Brownstein  immunofluorescence those  the  would  et  e  previously  measured  are  tion  Paxinos  al.  involving  reductions  in  of  the  ento-  activity.  levels  of  met-enkephalin  those  in  the  striatum;  in  the  however,  EP were they  found  were  not  to  be  only  affected  slightly by  either  24 type of l e s i o n .  T h i s suggests t h a t , i n c o n t r a s t to the  striatopallidal  p r o j e c t i o n , the a f f e r e n t s to the EP from the head o f the s t r i a t u m do  not  c o n t a i n met-enkephalin. F i n a l l y , s i n c e CAT  activity  i n the EP was  u n a f f e c t e d by  any  of  the  l e s i o n s i t would appear that a c e t y l c h o l i n e i s not a t r a n s m i t t e r i n the striato-entopeduncular projection. (c)  The  Substantia  The  SN was  Nigra  found to c o n t a i n very low  l e s i o n d a t a i n d i c a t e t h a t the SN, or m e t - e n k e p h a l i n - c o n t a i n i n g  l e v e l s of met-enkephalin.  l i k e the EP,  The  r e c e i v e s no c h o l i n e r g i c  i n p u t from the s t r i a t u m .  The  absence of a  c h o l i n e r g i c s t r i a t o n i g r a l p r o j e c t i o n i s i n agreement w i t h p r e v i o u s  reports  (Fonnum et a l . , 1978a; McGeer et a l . , 1971). Since the d i s c o v e r y of the e x i s t e n c e of a GABA-containing p r o j e c t i o n from the s t r i a t o p a l l i d a l complex to the SN, conducted to determine the exact pathway (Brownstein  e t a l . , 1977;  J e s s e l e t a l . , 1978).  numerous s t u d i e s have been  l o c a t i o n of the neurons g i v i n g r i s e to  Fonnum e t a l . , 1978a, Gale e t a l . , 1977;  These r e c e n t s t u d i e s and,  i n p a r t i c u l a r , that  Brownstein e t a l . (1977) have c o n s i d e r a b l y c l a r i f i e d  this issue.  p r o j e c t i o n neurons i n the s t r i a t u m j u s t a n t e r i o r to the GP. r e s u l t s are i n agreement w i t h t h i s p r o p o s a l . s t r i a t u m near the r o s t r a l t i p of the GP, However, the GP  island  Hemitransections  reduced n i g r a l GAD  r e s u l t s together with recent observations F i b i g e r , 1980)  a l t e r n i g r a l GAD  t h a t KA  nigral present  i n the  activity  l e s i o n s , which began at a more  r o s t r a l l e v e l , d i d not s i g n i f i c a n t l y decrease n i g r a l GAD  and  The  by  These  workers suggested t h a t t h e r e i s a c o n c e n t r a t i o n of GAD-containing  substantially.  this  activity.  ( D i C h i a r a e t a l . , 1980;  i n j e c t i o n s i n t o the GP  do not  These Nagy  significantly  are i n f u l l agreement w i t h Brownstein e t a l . (1977)  have suggested t h a t the o r i g i n of the m a j o r i t y of GAD  i n the SN  who  i s derived  25 from neurons  l o c a t e d o u t s i d e the p a l l i d u m b u t c l o s e l y  i s noteworthy  apposed  to i t .  It  t h a t w h i l e GP i s l a n d l e s i o n s d i d n o t d e c r e a s e GAD i n the SN  and o n l y produced  a s l i g h t decrease i n the EP GAD a c t i v i t y ,  i n the GP was o n l y s l i g h t l y  the decrease  l e s s than a f t e r the h e m i t r a n s e c t i o n .  i n d i c a t e s t h a t the GABA-containing at l e a s t p a r t i a l l y be d i s s o c i a t e d  neurons  This  i n n e r v a t i n g the SN and EP can  from those p r o j e c t i n g t o t h e GP.  Of a l l t h e b r a i n areas t h a t have been examined, the SN c o n t a i n s t h e h i g h e s t l e v e l s o f substance P (Brownstein e t a l . , J e s s e l , 1976).  I n Huntington's  1976; Kanazawa and  chorea substance P l e v e l s and  have both been found t o decrease i n the SN (Kanazawa e t a l . , s i m i l a r p a r a l l e l decrease has been observed f o l l o w i n g the l e v e l o f the l a t e r a l hypothalamus  (Mroz e t a l . ,  GAD a c t i v i t y  1977b).  A  hemitransections at  1977).  These  results  l e d to the s u g g e s t i o n t h a t both GABA and substance P a r e t r a n s m i t t e r s i n the s t r i a t o n i g r a l pathway (Hong e t a l . , 1977c; Kanazawa e t a l . , Mroz e t a l . ,  1977; Paxinos e t a l . , 1978b).  t h a t t h e substance P and GAD-containing The  substance P f i b e r s appear  Jessel et a l . ,  More r e c e n t work has shown  e f f e r e n t s appear  to be d i s s o c i a b l e .  to a r i s e from the a n t e r i o r s t r i a t u m and n o t  from the c a u d a l and l a t e r a l areas 1977;  1977b;  (Brownstein e t a l . ,  1978; P a l k o v i t s e t a l . ,  1978).  1977; Gale e t a l . , I n c o n t r a s t and as  d i s c u s s e d above, the GABA a f f e r e n t s to the SN a r i s e from s t r i a t a l c l o s e l y apposed  to t h e GP (Brownstein e t a l . ,  employed i n t h e p r e s e n t study produced  1977).  neurons  That the two l e s i o n s  a similar depletion i n nigral  substance P, but had d i f f e r e n t e f f e c t s on n i g r a l GAD a c t i v i t y i s i n agreement w i t h t h i s s e g r e g a t i o n o f these two n i g r a l a f f e r e n t (d)  systems,  The S t r i a t u m The i n c r e a s e s i n GAD a c t i v i t y and met-enkephalin  l e v e l s i n the s t r i a t u m  a n t e r i o r t o the h e m i t r a n s e c t i o n s and GP i s l a n d l e s i o n s c o u l d be due e i t h e r to the s h u n t i n g of enzyme and p e p t i d e to l o c a l c o l l a t e r a l branches o r  26 terminals  after severing  long axon e f f e r e n t c o l l a t e r a l s  1975), or to an a l t e r a t i o n i n the a c t i v i t y and met-enkephalin.  (Storm-Mathisen,  of the neurons  containing  That t h i s change does not merely r e f l e c t  GAD  tissue  shrinkage i s i n d i c a t e d by the unchanged l e v e l s of substance P and the normal CAT a c t i v i t y i n t h i s same t i s s u e .  At l e a s t a p o r t i o n of t h e GAD i n c r e a s e i n  the head of the s t r i a t u m can be a t t r i b u t e d t o the s e v e r i n g o f the dopaminerg i c input  t o t h i s s t r u c t u r e s i n c e we have found t h a t 6-hydroxydopamine  (6-OHDA) l e s i o n s o f the n i g r o s t r i a t a l pathway i n c r e a s e striatum  (Experiment 5 ) . H e m i t r a n s e c t i o n s caudal  GAD a c t i v i t y  i n the  to t h e GP do n o t appear  to r e s u l t i n an i n c r e a s e  i n the met-enkephalin l e v e l s i n the s t r i a t u m  ( P o l l a r d et a l . , 1978).  T h i s suggests t h a t t h i s i n c r e a s e  r u p t i o n o f the s t r i a t o p a l l i d a l met-enkephalin pathway. necessary t o determine t h e s i g n i f i c a n c e of these changes i n g o f the s t r i a t u m and i n i t s recovery  from i n j u r y .  i s due to d i s -  F u r t h e r work i s i n the f u n c t i o n -  27  EXPERIMENT 2:  THE  IMMUNOHISTOCHEMICAL DEMONSTRATION 0? SUBSTANCE P  IN THE The  undecapeptide  s t r i a t u m , where i t may  BASAL GANGLIA substance  f u n c t i o n as a n e u r o t r a n s m i t t e r .  the e x i s t e n c e of substance s t r i a t u m to the SN  P i s c o n t a i n e d i n the e f f e r e n t s of  (Brownstein  e t a l . , 1977;  confirmed  In a d d i t i o n , the presence  p r o j e c t i o n to the GP was  demonstrated.  h i s t o c h e m i c a l approach was of substance  Gale e t a l . , 1977;  1, the  Hong  Kanazawa et a l . , 1977a; Paxinos  (Paxinos e t a l . , 1978b) was  assay procedure.  I n Experiment  P - c o n t a i n i n g p r o j e c t i o n s from the head of  et a l . , 1977c; J e s s e l e t a l . , 1978; 1978a) and EP  the  et a l . ,  u s i n g a radioimmuno-  of substance  In the p r e s e n t  P i n the  striatal  study an immuno-  used to p r o v i d e i n f o r m a t i o n on the  distribution  P f i b e r s i n these and other b r a i n n u c l e i .  P r e v i o u s immunofluorescent s t u d i e s have demonstrated dense p l e x i of substance et a l . , 1979;  P t e r m i n a l s i n the SN H S k f e l t et a l . , 1977;  1978a; 1978b; Paxinos Ljungdahl GP  J e s s e l e t a l . , 1978;  et a l . , 1978a), EP  e t a l . , 1978a; Paxinos  (LjUngdahl  ( C u e l l o and Kanazawa, 1978;  et a l . , 1978a).  Cuello  Ljungdahl  ( C u e l l o and Kanazawa,  et a l . ,  1978;  et a l . , 1978b; J e s s e l e t a l . , 1978), and  These f l u o r e s c e n t s t u d i e s have been o f g r e a t  v a l u e i n the mapping of the substance  P systems of the b r a i n .  cent method does not however p r o v i d e the r e s o l u t i o n n e c e s s a r y p r e c i s e s y n a p t i c r e l a t i o n s h i p s of the substance In p a r t i c u l a r the method i s not compatible  The  fluores-  to d i s c e r n the  P t e r m i n a l s and  cells.  with e l e c t r o n microscopic studies.  For t h i s the immunoperoxidase method i s a v a i l a b l e . Immunoperoxidase s t a i n i n g f o r substance the l i g h t 1979;  1977)  and e l e c t r o n m i c r o s c o p i c l e v e l s i n the s p i n a l c o r d  H O k f e l t et a l . , 1977;  medulla  P has been performed a t  (Chan-Palay, 1978;  P i c k e l e t a l . , 1977; P i c k e l et a l . , 1979)  u s i n g the p e r o x i d a s e - a n t i p e r o x i d a s e  (PAP)  both  (Barber e t a l . ,  Vacca e t a l . , 1980), and amygdala ( P e l l e t i e r et a l . , method developed  by  28  Sternberger  et a l . (1970).  These s t u d i e s have p r o v i d e d  information  the l o c a l i z a t i o n of substance P i n nerve t e r m i n a l s i n s y n a p t i c s h i p s of v a r i o u s t y p e s . cedure was  In the present  study  on  relation-  an immunoperoxidase  pro-  used to demonstrate substance P i n some t e r m i n a l f i e l d s i n the  basal ganglia. METHODS Initially was  the t r a d i t i o n a l PAP  procedure o f S t e r n b e r g e r  e t a l . (1970)  attempted u s i n g the guinea p i g a n t i s e r a to substance P d e s c r i b e d i n  the Appendix.  T h i s method was  work a l e t t e r was guinea p i g PAP  abandoned when a f t e r a year  first  unsuccessful  r e c e i v e d from Cappel L a b o r a t o r i e s s t a t i n g t h a t  was  b e i n g d i s c o n t i n u e d due  (Heitzmann e t a l . , 1974;  (BA)  p r o c e d u r e has  Guesdon et a l . , 1979).  As  time t h a t t h i s method has been used i n immunohistochemical  d e s c r i b e d i n some d e t a i l and 2).  In both the BA  contrasted with  technique  and  p r o c e d u r e , the  i n c u b a t i o n of the t i s s u e i n guinea p i g a n t i - s u b s t a n c e same (Step 1 ) .  In the PAP  rabbit anti-guinea p i g sera.  in  order t o l e a v e one  will  be method  initial  P s e r a i s the  procedure the second step i n v o l v e s  in  studies  the more f a m i l i a r PAP  the PAP  been  t h i s i s the  of the nervous system, the t h e o r e t i c a l b a s i s of t h i s t e c h n i q u e  (Fig.  their  to " t e c h n i c a l problems".  As an a l t e r n a t i v e method, the b i o t i n - a v i d i n utilized  of  T h i s must be used at h i g h  incubation  concentration  b i n d i n g s i t e of the r a b b i t a n t i s e r a f r e e a f t e r  r e a c t i o n w i t h the primary a n t i b o d y .  The  use of h i g h c o n c e n t r a t i o n s  of  t h i s s e r a i n c r e a s e s the amount of n o n s p e c i f i c b i n d i n g of t h i s s e r a to the t i s s u e and  this  can i n c r e a s e the a r t i f a c t u a l s t a i n i n g .  I n the  BA  procedure, the b i o t i n y l a t e d r a b b i t a n t i - g u i n e a p i g s e r a i s used i n p l a c e of the r a b b i t a n t i s e r a used i n the PAP  procedure.  I t has  been found t h a t  e x t e n s i v e b i o t i n y l a t i o n of a n t i b o d i e s does not modify t h e i r a n t i g e n ing  capacity  (Guesdon et a l . , 1979).  I n the BA procedure the second  bindstep  29  F i g u r e 2:  A comparison  of the b i o t i n - a v i d i n  (BA) method (b) w i t h t h e  p e r o x i d a s e - a n t i p e r o x i d a s e (PAP) procedure histochemistry. (GPA)  (a) f o r immuno-  I n Step 1 guinea p i g - a n t i  b i n d s t o substance P i n the t i s s u e .  substance P s e r a I n Step 2 o f the  PAP procedure r a b b i t - a n t i guinea p i g IgG (RAGP) b i n d s t h e GPA and then the PAP i n Step 3.  I n the BA procedure the  B i o t i n r e s i d u e s a t t a c h e d t o the RAGP bound i n Step 2 a r e bound by a v i d i n peroxidase  (A) which has been c o u p l e d to h o r s e r a d i s h  (HRP).  30  can be used at h i g h d i l u t i o n s i n c e i t i s not n e c e s s a r y antibody b i n d i n g s i t e a v a i l a b l e f o r Step  3.  I t i s i n the t h i r d step where the BA the PAP  approach.  In the PAP  the second step b i n d s the PAP  system i s most d i f f e r e n t  procedure,  to the second a n t i b o d y  by a v i d i n to which h o r s e r a d i s h p e r o x i d a s e  has been c h e m i c a l l y  i n t e r a c t i o n between a v i d i n and b i o t i n i s n o n - c o v a l e n t ,  strong  (Kd = 10  15  M;  Green, 1963).  o r d e r s of magnitude h i g h e r than  from  the f r e e b i n d i n g s i t e l e f t i n  complex which a c t s as an a n t i g e n .  BA method the b i o t i n r e s i d u e s a t t a c h e d  The  to have a f r e e  In f a c t the a f f i n i t y  In  the  a r e bound coupled.  but  extremely  i s s i x to e i g h t  that of most a n t i b o d i e s f o r a n t i g e n .  This  means t h a t the a v i d i n - p e r o x i d a s e complex can be used at v e r y h i g h  dilution.  T h i s a g a i n g r e a t l y reduces the chances f o r n o n - s p e c i f i c b i n d i n g .  Also,  e x t e n s i v e washing can be performed a f t e r the i n c u b a t i o n w i t h o u t t i o n o f the a v i d i n - b i o t i n complex s i n c e the h a l f - t i m e of  dissocia-  dissociation  r e q u i r e s months. In the p r e s e n t was  study  the d i s t r i b u t i o n o f substance  examined i n the c a t b r a i n u s i n g the BA procedure.  were deeply  c o l d 4% paraformaldehyde and The  b r a i n was  0.3%  pH 7.4)  glutaraldehyde  removed, cut i n t o 5 mm  i n 4% paraformaldehyde at 4°C  f o r 6 hr.  o v e r n i g h t i n the b u f f e r c o n t a i n i n g 15% t h i c k s e c t i o n s on a c r y o s t a t a t -20°C. were washed i n PBS temperature and substance  saline  c o n t a i n i n g 0.01%  then i n c u b a t e d  The  The  through the  (PBS)  i n 0.1  followed  by  M phosphate b u f f e r  c o r o n a l s e c t i o n s and t i s s u e was  sucrose  and  then  kept  stored  cut i n t o 25 micron  S e c t i o n s f o r immunohistochemistry  T r i t o n X-100  o v e r n i g h t a t 4°C  f o r one h r at room i n guinea p i g - a n t i  P s e r a (see Appendix) d i l u t e d 1:2,000 i n PBS  T r i t o n X-100.  immunoreactivity  A d u l t male c a t s  a n e s t h e t i z e d w i t h p e n t o b a r b i t a l and p e r f u s e d  h e a r t w i t h phosphate b u f f e r e d (0.1 M,  pH 7.4.  P  s e c t i o n s were then washed i n PBS  and  containing incubated  0.05% for  two  31  F i g u r e 3.  C o n t r o l s e c t i o n of s u b s t a n t i a n i g r a i n c u b a t e d absorbed a n t i - s u b s t a n c e background e x i s t s , and occurs.  23X.  P sera. no  with  pre-  Only a l i g h t brown  s p e c i f i c substance P s t a i n i n g  32  hr a t room temperature w i t h b i o t i n y l a t e d r a b b i t a n t i - g u i n e a p i g IgG (E-Y Labs.) d i l u t e d 1:200 i n PBS c o n t a i n i n g 0.01% T r i t o n X-100. the s e c t i o n s were washed i n PBS and f i n a l l y  incubated  temperature i n h o r s e r a d i s h p e r o x i d a s e - a v i d i n 1:500 i n PBS. peroxidase  The s e c t i o n s were f i n a l l y  then r e a c t e d  (Sigma) and 0.006% H 0 2  z  f o r 20 min w i t h  in d i s t i l l e d with  f o r one h r a t room  (Vector Labs. Inc.) d i l u t e d  washed i n PBS f o r one h r and the 0.02%  (w/v) 3,3'-diaminobenzidine  i n 50 mM T r i s - C l b u f f e r , pH 7.6.  were then washed and mounted from PBS.  Again  The. s e c t i o n s  The mounted s e c t i o n s were soaked  water 30 min to remove s a l t s and d e t e r g e n t  and then t r e a t e d  0.01% (w/v) osmium t e t r o x i d e f o r t h r e e min to enhance c o n t r a s t . C o n t r o l s e c t i o n s were processed  i d e n t i c a l l y except t h a t the i n i t i a l  a n t i s e r a was preabsorbed w i t h 100 ug of s y n t h e t i c substance P (Beckman) per ml a t room temperature f o r f o u r h r p r i o r to use i n immunohistochemistry. Sections  incubated  staining  (Fig. 3).  with  t h i s pre-absorbed s e r a d i d not e x h i b i t any s p e c i f i c  RESULTS In s e c t i o n s from u n t r e a t e d P immunoreactivity not  a d u l t c a t s which were s t a i n e d f o r substance  u s i n g the BA procedure immunoreactive c e l l b o d i e s were  c o n s i s t e n t l y detected.  T h i s i s i n agreement w i t h p r e v i o u s  f l u o r e s c e n t s t u d i e s i n the r a t i n  which c e l l b o d i e s c o u l d o n l y be r e p r o -  d u c i b l y demonstrated f o l l o w i n g c o l c h i c i n e pretreatment 1978; L j i i n g d a h l e t a l . , 1978a). bodies normally  contain very  immuno-  ( C u e l l o and Kanazawa,  T h i s presumably i n d i c a t e s t h a t the c e l l  low l e v e l s of substance P.  A l t h o u g h p o s i t i v e c e l l b o d i e s were not apparent, widespread networks o f substance P - p o s i t i v e t e r m i n a l s and f i b e r s were observed through the b r a i n and  s p i n a l cord.  In the p r e s e n t  r e p o r t we s h a l l c o n f i n e our d i s c u s s i o n to  substance P - p o s i t i v e s t r u c t u r e s i n areas a s s o c i a t e d w i t h  the b a s a l  ganglia.  Both the putamen ( F i g . 4a) and the caudate ( F i g . 4b) c o n t a i n e d  a  33  d e l i c a t e network of f i n e f i b e r s which c o u l d be observed forming c l u s t e r s of axo-somatic c o n t a c t s  w i t h medium s i z e s t r i a t a l neurons.  A similar  network o f f i b e r s was a l s o observed i n the c l a u s t r u m , p a r t i c u l a r l y v e n t r o - m e d i a l p o r t i o n o f t h i s nucleus Within  i n the  (Fig. 4c).  the GP ( F i g . 4d) and the EP ( F i g . 4 f ) t h e n e u r o p i l was  densely s t a i n e d .  rather  I n both areas v a r i c o s e f i b e r s appeared t o o u t l i n e the  d e n d r i t i c p r o c e s s e s of the p a l l i d a l neurons, presumably f o r m i n g synapses along  their The  length.  ansa l e n t i c u l a r i s ,  are thought t o course  i n which the s t r i a t o n i g r a l substance P f i b e r s  ( P a l k o v i t s e t a l . , 1978), c o n t a i n e d  a dense network  of v a r i c o s e f i b e r s ( F i g . 4 e ) . A l t h o u g h not p r e v i o u s l y d i s c u s s e d  i n t h i s r e p o r t t h e amygdaloid  complex i s another t e l e n c e p h a l i c s t r u c t u r e o f t e n i n c l u d e d ganglia. The  C e r t a i n amygdaloid n u c l e i s t a i n v e r y  c o r t i c a l nucleus contains  s t r o n g l y f o r substance P.  an extremely dense network o f p o s i t i v e l y  s t a i n e d p u n c t a t e s t r u c t u r e s throughout i t s n e u r o p i l l e s s dense network i s present  i n the b a s a l  ( F i g . 5b).  A somewhat  i n t h e m e d i a l amygdala where l o n g  varicose  f i b e r s c o u l d o f t e n be seen ( F i g . 5 c ) . A dense punctate network o f substance P - p o s i t i v e observed i n the l a t e r a l habenula, p a r t i c u l a r l y t h i s nucleus  f i b e r s was a l s o  i n the m e d i a l p o r t i o n o f  ( F i g . 5a).  In the mesencephalon  a d i f f u s e network o f v a r i c o s e axons appears  to form a s h e l l over the d o r s a l and l a t e r a l b o r d e r s o f the nucleus ( F i g . 6 c ) .  The s t a i n i n g i s s t r o n g e s t  of t h i s s h e l l where i t a p p a r e n t l y i m m u n o r e a c t i v i t y i n t h e SN.  i n the v e n t r o - l a t e r a l p o r t i o n  i s i n continuum w i t h the substance P  Within  the SN the substance P - p o s i t i v e  s t r u c t u r e s form a massive, d i f f u s e and p u n c t i f o r m most i n t e n s e i n the SNR  interpeduncular  ( F i g . 6b).  pattern.  The s t a i n i n g i s  Bundles o f i n t e n s e l y s t a i n e d  neuropil  34  F i g u r e 4.  Substance  P i m m u n o r e a c t i v i t y i n c o r o n a l s e c t i o n s of t h e r e a t b a s a l  g a n g l i a demonstrated  In  w i t h the b i o t i n - a v i d i n method.  (a) a s p a r s e network o f p o s i t i v e l y s t a i n e d  apparent.  These can o f t e n be seen forming t e r m i n a l s about  the medium s i z e c e l l s  Figi  (arrows).  .(b) shows a s i m i l a r p a t t e r n o f substance P s t a i n i n g i n  the caudate n u c l e u s .  A g a i n the p u n c t a t e f i b e r s can o f t e n be  seen c o v e r i n g the u n s t a i n e d c e l l b o d i e s  Fig.  puncta i s  (arrows).  (c) shows t h e same s p a r s e p a t t e r n o f p u n c t a t e  e x i s t s i n the c l a u s t r u m . s p e c i f i c background  In  staining  The c e l l b o d i e s show a s l i g h t non-  staining.  (d) t h e n e u r o p i l o f the globus p a l l i d u s can be seen to  c o n t a i n a dense network o f substance P - p o s i t i v e f i b e r s . These appear t o cover t h e c e l l b o d i e s and major d e n d r i t e s (arrow)  Fig.  of t h e p a l l i d u m w i t h many v a r i c o s i t i e s .  (e) demonstrates  the substance P - l i k e  immunoreactivity  in  the f i b e r s o f t h e ansa l e n t i c u l a r i s .  to  have many v a r i c o s i t i e s a l o n g t h e i r l e n g t h .  The entopeduncular  nucleus  These f i b e r s  appear  (f)-. t h e p a t t e r n o f s t a i n i n g i s  v e r y s i m i l a r t o t h a t seen i n the globus p a l l i d u s ( d ) . A g a i n the s t a i n e d puncta cover the c e l l bodies  (arrow)  and appear t o f o l l o w the major d e n d r i t e s through t h e neuropil.  C a l i b r a t i o n bars r e p r e s e n t 20 microns  in. a l l  figures.  35  F i g u r e 5.  Substance P immunoreactive  f i b e r s demonstrated w i t h the  b i o t i n - a v i d i n immunoperoxidase p r o c e d u r e .  In (a) the dense network o f p o s i t i v e puncta found i n the m e d i a l p o r t i o n of the l a t e r a l habenula n u c l e u s i s shown i n a c o r o n a l  section.  A s i m i l a r dense network can a l s o be seen i n the n e u r o p i l of the c o r t i c o m e d i a l amygdala ( b ) .  In t h i s  coronal  s e c t i o n the v a r i c o s e f i b e r s appear to branch throughout the n e u r o p i l .  O c c a s i o n a l y , such as i n t h i s c o r o n a l s e c t i o n of the m e d i a l amygdala (c) substance P - c o n t a i n i n g v a r i c o s e axons can be f o l l o w e d f o r some d i s t a n c e .  The v a r i c o s i t i e s  to be e v e n l y spaced and about 2 microns  apart.  C a l i b r a t i o n bar r e p r e s e n t s 20 microns f o r a l l t h r e e photographs.  appear  35  a  36  can  be  the  SNC  seen  extending  from  the  substance  P  orientation. a n d do n o t  The  the  positive  unstained  appear  to  SNR i n t o  cell  receive  the  subadjacent  fibers  appear  to  bodies  of  SNC a r e  many  the  axo-somatic  run  crus  in  a  cerebri.  In  horizontal  quite  apparent  contacts.  DISCUSSION In  the  substance  present  study  we h a v e  P-immunoreactivity  technique. substance  The  findings  in  P-immunoreactivity  in  and Kanazawa,  studies  a network  of  the  striatum.  immunoreactivity of  the  cat.  P  and Kanazawa, In both represents  major  network  cell  bodies  1978; the  the  (see  in  which  Jessel  GP a n d  in  et  the of  Experiment processes  rat  the  brain  the  using et  immunofluorescent a l . ,  1978a). were  In  those  observed  in  substance  P-  nucleus  and  the  two  nuclei  between local  observed  1978;  Kanazawa  the  dense  plexus  striatopallidal  forming  of  caudate  a l . ,  perhaps  reports  of  from  fibers  immunoperoxidase  network  been  These  an  structures  have  the  with  of  fine  staining  arises  demonstration  previous  Ljungdahl  a similar  both  first  ganglia  fluorescent  the  EP  1).  the  extend  1978;  probably  terminals  dendritic  the  our m a t e r i a l observed  basal  and  weakly  differences  This  substance  pathways  was  No  apparent.  fine,  In  the  confirm  techniques(Cuello  rat  provided  these  collaterals  in  the  et  of  fibers  seemed  were  the (Cuello  1977). present  substance  often  of  striatum  a l . ,  putamen  likely  P-containing  to  outline  synapses  en passant  (Fox  Cuello  and Kanazawa  the et  a l . ,  1974) . In  previous  reported  a very  amygdaloid in the  the  compact  nuclei.  caudal  cortical  contain  immunofluorescent  very  Ljungdahl  portion  of  amygdala. high  network  levels  the  et  of  fibers  al.  medial  The m e d i a l of  studies  in  (1978a) nucleus,  nucleus  substance  P by  in  the  medial  report but the  and  very  only rat  dense  single has  the  cortical staining  fibers  been  radioimmunoassay,  (1978)  in  found  while  to  levels  37  F i g u r e 6.  Substance P - l i k e i m m u n o r e a c t i v i t y i n c o r o n a l s e c t i o n s of the v e n t r a l mesencephalon demonstrated  w i t h the b i o t i n -  a v i d i n immunoperoxidase t e c h n i q u e .  In (a) the dense f i b e r system o c c u r s throughout the n e u r o p i l o f t h e s u b s t a n t i a n i g r a pars compacta.  The  c e l l s o f the pars compacta a r e u n s t a i n e d (arrows) and do not appear synapses. dorsally  to r e c e i v e many axosomatic substance P  Some substance P - p o s i t i v e f i b e r s  extend  from t h e n i g r a i n t o t h e m e d i a l l e m n i s c u s ( m l ) .  In (b) t h e i n t e n s e s t a i n i n g which e x i s t s i n the n e u r o p i l of the s u b s t a n t i a n i g r a pars r e t i c u l a t a strated.  (snr) i s demon-  Bundles o f f i b e r s can be seen e x t e n d i n g  v e n t r a l l y from t h e n i g r a i n t o t h e c r u s c e r e b r i ( c c ) .  In (c) a d i s c r e t e network of substance P f i b e r s can be seen a t t h e v e n t r o - l a t e r a l border o f t h e i n t e r p e d u n c u l a r nucleus  ( i p ) a d j a c e n t to the v e n t r a l tegmental a r e a ( v t a )  C a l i b r a t i o n b a r r e p r e s e n t s 50 microns f o r a l l t h r e e figures.  38 i n the c o r t i c a l nucleus appear  q u i t e low  (Ben-Ari et a l . ,  1977).  the p r e s e n t study, the c o r t i c o m e d i a l n u c l e u s o f the cat was  In  found to s t a i n  most i n t e n s e l y f o r substance P, w i t h the f i b e r s o f t e n forming b a s k e t - l i k e s t r u c t u r e s about  the c e l l  bodies.  between the n u c l e u s amygdaloideus m e d i a l n u c l e u s of the c a t .  Perhaps  t h i s i n d i c a t e s a homology  m e d i a l i s of the r a t and the  In both s p e c i e s i t i s apparent  cortico-  t h a t most of  the substance P i s p r e s e n t i n the more p r i m i t i v e components of the amygdaloid  complex. The  i n t e n s e s t a i n i n g observed i n the l a t e r a l habenula  w i t h the p r e v i o u s immunofluorescent L j u n g d a h l et a l . ,  studies  1978a; C u e l l o e t a l . ,  i s consistent  ( C u e l l o and Kanazawa,  1978).  1978;  These s t u d i e s have shown  t h a t the substance P t e r m i n a l s l i e i n the m e d i a l p o r t i o n of the  lateral  habenula.  In a d d i t i o n ,  A s i m i l a r r e s u l t was  observed  i n the p r e s e n t study.  a p r e v i o u s l y unknown band of dense t e r m i n a l s o v e r l y i n g the d o r s o - l a t e r a l s u r f a c e of t h i s n u c l e u s was  observed.  t e r m i n a l s i n the l a t e r a l habenula  appears t o be q u i t e d i s t i n c t  of the GABA t e r m i n a l s which appear of the l a t e r a l habenula  1978)  from t h a t  to l i e i n the v e n t r o - l a t e r a l  (see Experiment  t e r m i n a l s have been suggested (Cuello et a l . ,  The l o c a t i o n of the substance P  4).  A l t h o u g h the substance P  to a r i s e from c e l l s i n the m e d i a l  a n a t o m i c a l s t u d i e s have f a i l e d  c o n n e c t i o n between these n u c l e i  portion  habenula  to demonstrate  (Herkenham and Nauta, 1979;  Iwahori,  a 1977).  Thus, the o r i g i n of the substance P i n the l a t e r a l habenula remains i n doubt. The m e d i a l habenula has a l s o been suggested to be the source of the substance P i n the i n t e r p e d u n c u l a r n u c l e u s et a l . , 1977;  Hong et a l . ,  the immunofluorescent  1976;  (Cuello et a l . ,  Mroz et a l . , 1976).  1978;  Emson  In agreement w i t h  s t u d i e s i n the r a t 'the substance P i m m u n o r e a c t i v i t y  found i n the c a t forms a s h e l l about  the d o r s a l and l a t e r a l b o r d e r s of the  39  i n t e r p e d u n c u l a r nucleus cells  (Dahlstrom  c u l a r nucleus  adjacent  t o the area o f the dopaminergic  and Fuxe, 1964).  A-10  The c e n t r a l core o f the i n t e r p e d u n -  i s r e l a t i v e l y b a r r e n of substance  P terminals.  As the m e d i a l  habenula i s known t o p r o v i d e the major i n n e r v a t i o n to t h i s p a r t o f the i n t e r p e d u n c u l a r nucleus  (Herkenham and Nauta, 1979), i t appears t h a t the  major t r a n s m i t t e r s i n t h i s system remain to be W i t h i n t h e SN the substance  determined.  P immunoreactivity  i s most i n t e n s e i n  the SNR, i n agreement w i t h radioimmunoassay d a t a o b t a i n e d SN  (Gauchy e t a l . , 1979).  i n the c a t  The s t a i n i n g i n the SNC was l e s s i n t e n s e but  many v a r i c o s e f i b e r s were s t i l l  apparent.  This may.indicate  that the  dopamine c e l l s o f the A9 group.and, i n p a r t i c u l a r , t h e i r d e n d r i t i c i n the SNR a r e d i r e c t l y i n n e r v a t e d by substance et a l . , 1978b).  T h i s i d e a i s supported  r e t i c a l l y a p p l i e d substance  P terminals  (Ljungdahl  by t h e o b s e r v a t i o n t h a t  P e x c i t e s the dopaminergic  (Davies and Dray, 1976; Walker e t a l . , 1976).  processes  iontopho-  c e l l s o f the SN  Behavioural  (James and  S t a r r , 1977; 1979; K e l l e y and I v e r s e n , 1978; 1979; K e l l e y e t a l . , 1979; Olpe and K o e l l a , 1977) and b i o c h e m i c a l  (Cheramy e t a l . , 1977; 1978;  Magnusson e t a l . , 1976; S t a r r , 1978b) o b s e r v a t i o n s a l s o support interaction. developed 1979)  H o p e f u l l y the e x t e n s i o n of the immunoperoxidase  i n the p r e s e n t  such an procedure  study t o the e l e c t r o n m i c r o s c o p i c l e v e l  (Pickel,  w i l l a l l o w the d i r e c t v i s u a l i z a t i o n o f the s y n a p t i c r e l a t i o n s h i p s  between the substance  P t e r m i n a l s and the dopamine c e l l s o f • t h e SN.  40  EXPERIMENT 3: THE LOCALIZATION OF GABA-TRANSAMINASE IN THE STRIATO-NIGRAL SYSTEM. In Experiment 1 the e x i s t e n c e  of GABA p r o j e c t i o n s from the s t r i a t u m  to the GP, EP and SN was c o n f i r m e d . ments p r o v i d e  A l t h o u g h these b i o c h e m i c a l  good evidence f o r the e x i s t e n c e  p r o v i d e no i n f o r m a t i o n innervations.  regarding  of GABA pathways, they  the t o p o g r a p h i c d i s t r i b u t i o n of the GABA  For t h i s a r e l i a b l e morphological technique i s required.  A f l u o r e s c e n t method f o r GABA has been d e v i s e d , w i t h t h i s t e c h n i q u e have not been p r o m i s i n g histochemical  but the r e s u l t s  (Wolman, 1971).  obtained  The immuno-  method f o r GAD has made p o s s i b l e the v i s u a l i z a t i o n of GAD-  c o n t a i n i n g nerve t e r m i n a l s has  experi-  and c e l l s  (Roberts,  1979); however, t h i s method,  not been used t o c h a r a c t e r i z e the topography o f GABA p r o j e c t i o n s and  the complexity of the t e c h n i q u e p r e c l u d e s simple h i s t o c h e m i c a l  i t s general  usage.  Although a  procedure f o r GABA-transaminase (GABA-T), the enzyme  which c a t a b o l i z e a GABA, has l o n g been known (Van G e l d e r , 1965) i t s use i n examining GABA systems has n o t been  explored.  High l e v e l s o f GABA-T have been found i n the b a s a l g a n g l i a and  (Salvador  A l b e r s , , 1959), and the s t r i a t u m and SN have been found to e x h i b i t  intense histochemical  s t a i n i n g f o r t h i s enzyme.(Robinson and W e l l s ,  However, t h e p r e c i s e l o c a l i z a t i o n o f GABA-T has not been c l e a r l y Histochemical  terminals  or g l i a l  elements i n the b a s a l g a n g l i a ,  which o f these elements a c t u a l l y c o n t a i n s (Robinson and W e l l s ,  High a f f i n i t y  directly  the enzyme has been  although  difficult  1973).  GABA uptake i s thought t o be p r i m a r i l y r e s p o n s i b l e f o r  the removal o f s y n a p t i c a l l y r e l e a s e d studies using  resolved.  s t u d i e s have suggested t h a t GABA-T a c t i v i t y c o u l d be p r e s e n t  i n nerve c e l l s ,  to d i s c e r n  1973).  GABA ( M a r t i n , 1976), b u t r e c e n t  s p e c i f i c GABA-T i n h i b i t o r s have i n d i c a t e d t h a t GABA-T may be  i n v o l v e d i n r e g u l a t i n g the t r a n s m i t t e r p o o l o f GABA.  Thus,  41  intracerebral  o r systemic i n j e c t i o n s  marked e l e v a t i o n and  of b r a i n GABA l e v e l s  Kamioka, 1978).  basal ganglia  of GABA-T i n h i b i t o r s r e s u l t i n a (Rando and B a n g e r t e r , 1977; M a t s u i  A l s o l o c a l i n j e c t i o n s o f GABA-T i n h i b i t o r s i n t o the  r e s u l t i n marked b e h a v i o u r a l e f f e c t s , i n v o l v i n g both GABA  systems and the n i g r o s t r i a t a l dopaminergic system (Pycock e t a l . , 1976; M a t s u i and Kamioka, 1978). ganglia  The l o c a l i z a t i o n of GABA-T i n the b a s a l  has t h e r e f o r e been b i o c h e m i c a l l y examined i n o r d e r t o e v a l u a t e the  s u i t a b i l i t y o f t h i s enzyme f o r t h e h i s t o c h e m i c a l d e m o n s t r a t i o n of GABA pathways. METHODS To  d e s t r o y the s t r i a t a l neurons, KA was i n j e c t e d  putamen as p r e v i o u s l y received NaPOi+j an  described  (McGeer and McGeer, 1976a).  pH 7, a t a r a t e  of 1 y l / 5 min.  s a c r i f i c e d by c e r v i c a l f r a c t u r e ,  An a d d i t i o n a l  SN was o b t a i n e d from s e c t i o n s  group of r a t s  injections  and the s t r i a t u m  was d i s s e c t e d  c u t on a f r e e z i n g  of KA i n t o t h e s t r i a t u m  d e p l e t e CAT as compared t o GAD. the  microtome. (1979) have found  of neonatal r a t s p r e f e r e n t i a l l y  In the p r e s e n t r e p o r t  e f f e c t of such l e s i o n s on GABA-T a c t i v i t y .  i s o s m o l a r Ringer s o l u t i o n . the striatum  dissected  In an a d d i t i o n a l 0.9%  we have examined  Ten day o l d r a t s were (pH 7.4)  F o u r t e e n days l a t e r the animals were s a c r i f i c e d f o r enzyme a n a l y s e s .  group of a d u l t  animals, 2.9 yg o f 6-OHDA i n 1 y l of  s a l i n e , 0.1% a s c o r b a t e was i n j e c t e d  i n t o the l e f t n i g r o s t r i a t a l bundle  to d e s t r o y s e l e c t i v e l y the n i g r o s t r i a t a l dopaminergic neurons F i b i g e r , 1977).  received  on i c e .  i n j e c t e d w i t h 20 nmoles of KA i n 1 y l o f sodium phosphate b u f f e r e d  and  rats  Two weeks l a t e r , the r a t s were  Campochiara and C o y l e (1978) and Lehmann and F i b i g e r that  Nine  a u n i l a t e r a l i n j e c t i o n o f f i v e nmoles o f KA i n 0.5 y l 50 mM  i n j e c t i o n of t e n nmoles of KA i n 1 y l .  The  i n t o the caudate-  ( C l a v i e r and  The i n j e c t i o n s were made a t A + 4.4; L + 1.8; DV - 2.4 mm,  42  a c c o r d i n g t o the a t l a s of Kb'nig and K l i p p e l  (1963).  These animals r e c e i v e d  25 mg/kg d e s i p r a m i n e 30 min p r i o r to the 6-OHDA to prevent damage to the n o r a d r e n e r g i c neurons  (Roberts et a l . ,  1975).  s t r i a t u m and SN were d i s s e c t e d as d e s c r i b e d  One month a f t e r the l e s i o n  above.  For the b i o c h e m i c a l a n a l y s e s the t i s s u e s were homogenized i n 50 phosphate b u f f e r , pH 7.4,  c o n t a i n i n g 0.25%  T r i t o n X-100.  x y l a s e was measured as p r e v i o u s l y d e s c r i b e d  mM  T y r o s i n e hydro-  (McGeer e t a l . ,  1967).  Forty  y l of t i s s u e homogenate were added to 80 y l of a m i x t u r e c o n t a i n i n g c o n c e n t r a t i o n s ) 1.0 mM  2-amino-5-hydroxy-6,7-dimethyl  (DMPH , Sigma) , 0.1 mM  1- [ C (U) ] t y r o s i n e  4  mmol, New  lt+  England N u c l e a r ) , 0.3 mM  (Eastman) i n 0.2  M sodium  the  tetrahydropteridine  (specific activity  f e r r i c s u l p h a t e , 50 mM  a c e t a t e b u f f e r , pH 6.0.  (final  3 t o 5 mCi/  2-mercapto-ethanol  The r e a c t i o n was  incubated  f o r 12 min a t 37°C, then stopped by the a d d i t i o n of two ml of a s o l u t i o n c o n t a i n i n g 1.4% xyphenylalanine.  p e r c h l o r i c a c i d , 0.52%  a c e t i c a c i d and 0.5  yg/ml of d i h y d r o -  The r e a c t i o n tubes were c e n t r i f u g e d a t 1,000  min and the s u p e r n a t a n t s t r a n s f e r r e d r i n s e d w i t h two ml of 0.35  to 25 ml beakers.  M potassium phosphate  The p e l l e t s were  b u f f e r , pH 6.0,  as b e f o r e and the s u p e r n a t a n t s added to those i n the beakers. of 28 mM brought  Na EDTA were then added to each beaker and the samples 2  to pH 9 to 9.5  of alumina  g for five  centrifuged  Twelve ml were  and then poured onto columns packed w i t h about  (Calbiochem, a c i d , AG^,  100-200 mesh).  0.3  The columns were washed  w i t h 35 ml of d i s t i l l e d water, e l u t e d i n t o s c i n t i l l a t i o n v i a l s w i t h 2.0 of 0.5 M a c e t i c a c i d and counted i n 14 ml o f ACS The method of S t e r r i and Fonnum (1978) was activity.  Twenty y l of t i s s u e homogenate was  t i o n v i a l s i n an i c e bath. c o n c e n t r a t i o n s ) 5 mM mmol, Amersham), 2 mM  used  to assay GABA-T  F i f t y y l of r e a c t i o n m i x t u r e c o n t a i n i n g 11+  a-ketoglutaric acid  ml  (Amersham).  placed d i r e c t l y into  y-amino [U- C] b u t y r i c a c i d  g  (specific activity  (Sigma), 10 mM  scintilla(final 0.71  dithiothrietol  mCi/  43  (Sigma), 3 mM n i c o t i n e adenine d i n u c l e o t i d e (NAD, Calbiochem), 1 mM succinate  (Sigma), 0.3 mM p y r i d o x a l phosphate  (Calbiochem) i n 50 mM T r i s -  C l b u f f e r pH 8.2, c o n t a i n i n g 0.2% T r i t o n X-100 were then added and t h e v i a l s i n c u b a t e d a t 37°C f o r 20 min.  The r e a c t i o n was stopped by the  a d d i t i o n o f one ml of 0.1 M sodium phosphate succinate  formed  b u f f e r , pH 7.4.  i n t h e r e a c t i o n was e x t r a c t e d  The l a b e l e d  from the aqueous phase by  the a d d i t i o n o f one ml o f isoamyl a l c o h o l c o n t a i n i n g 0.2 M t r i - n - o c t y l ammonium phosphate, according  f r e s h l y p r e p a r e d from t r i - n - o c t y l a m i n e  to the method o f S t e r r i and Fonnum (1978).  (Sigma)  Ten ml o f t h e t o l u e n e -  based f l u o r used i n t h e CAT assay were then added and t h e r a d i o a c t i v i t y i n the o r g a n i c  phase counted.  B e f o r e use i n t h e assay, the commercial  sample  of r a d i o a c t i v e GABA was p u r i f i e d by e x t r a c t i n g once w i t h an e q u a l volume o f 20 mM tri-n-octylammonium CAT  phosphate  i n chloroform.  and GAD were assayed a c c o r d i n g  Experiment  t o t h e procedures o u t l i n e d i n  1, and p r o t e i n was determined by t h e method o f Lowry e t a l .  (1951). RESULTS One month a f t e r the i n j e c t i o n o f 6-OHDA, t h e a c t i v i t y o f t y r o s i n e h y d r o x y l a s e i n t h e s t r i a t u m was reduced by h a l f i n d i c a t i n g a s i g n i f i c a n t d e s t r u c t i o n o f the n i g r o s t r i a t a l dopamine neurons  (Table 5 ) .  The a c t i v i t i e s  of CAT and GAD i n t h e s t r i a t u m were n o t s i g n i f i c a n t l y a f f e c t e d n o r was GAD a c t i v i t y i n t h e SN a l t e r e d .  T h i s l e s i o n had no e f f e c t on t h e GABA-T  a c t i v i t y o f e i t h e r the s t r i a t u m o r t h e SN.  T h i s agrees w i t h the r e p o r t  o f Kim (1973) who found no change i n s t r i a t a l GABA-T a c t i v i t y  four  days  a f t e r i n t r a v e n t r i c u l a r 6-OHDA. Following  t h e i n j e c t i o n o f KA i n t o t h e s t r i a t u m ,  s t r i a t a l CAT and GAD were reduced i n a dose-dependent  dose-dependent  t h e a c t i v i t i e s of manner.  decrease was seen i n s t r i a t a l GABA-T a c t i v i t y  A similar  (Table 6 ) .  44  Table  5.  THE ACTIVITIES OF NEUROTRANSMITTER-RELATED  ENZYMES IN THE  STRIATUM AND SUBSTANTIA NIGRA ONE MONTH AFTER INJECTION OF 6-OHDA INTO THE NIGROSTRIATAL BUNDLE .  Control (nmol/mg p r o t e i n / h r  Lesion S.E.M.)  % of c o n t r o l  Striatum Tyrosine hydroxylase  82.0±3.0  39.0±7.8  140.4±6.7  142.5±2.7  101  48.2±1.4  54.3±3.3  113  109.3±5.0  110.5±5.0  101  Glutamate d e c a r b o x y l a s e  255.6±14.4  218.8±17.8  86  GABA-transaminase  101.0±1.6  99.2±6.7  98  Choline  acetyltransferase  Glutamate d e c a r b o x y l a s e GABA-transaminase Substantia  48*  nigra  n = 12 *p_ < .001, Student's t w o - t a i l e d t_ t e s t .  45  T a b l e 6.  The a c t i v i t i e s o f n e u r o t r a n s m i t t e r - r e l a t e d s t r i a t u m and s u b s t a n t i a n i g r a  enzymes i n t h e  two weeks a f t e r the i n j e c t i o n  of f i v e or t e n nmoles o f k a i n i c a c i d  5 nmol k a i n a t e (N=9) Control Lesion (nmol/mg p r o t e i n / h r  into the striatum.  10 nmol k a i n a t e (N=6) Control Lesion ± S.E.M.)  Striatum 105.9±3.2 18.9±1.7 (18% o f c o n t r o l ) * * *  CAT  GAD  GABA-T  Substantia  72.6±5.2 46.5±5.0 (64% of c o n t r o l ) * * *  91.2±4.5 22.6±2.6 (25% o f c o n t r o l ) * * *  107.7±3.2 66.8±4.7 (62% of c o n t r o l ) * * *  107.3±3.1 35.9±2.4 (34% o f c o n t r o l ) * * *  nigra  GAD  254.6+6.1 188.6±8.9 (74% o f c o n t r o l ) * *  260.0±8.2 86.6±7.6 (33% o f c o n t r o l ) * * *  GABA-T  109.7±4.7 110.0±4.8 (100% o f c o n t r o l )  117.6±2.8 104.6±3.4 (89% of c o n t r o l ) *  *p_ < .01;  **p_ < .005;  ***p_ < .001;  Student's t w o - t a i l e d jt t e s t  \  46  T h i s decrease i n s t r i a t a l GABA-T a c t i v i t y c o r r e l a t e d remarkably w e l l the d e c r e a s e i n s t r i a t a l GAD a c t i v i t y  with  ( F i g . 7 ) . The i n j e c t i o n o f f i v e  nmoles o f KA i n t o t h e s t r i a t u m which r e s u l t e d i n a s m a l l but s i g n i f i c a n t decrease i n n i g r a l GAD, d i d n o t s i g n i f i c a n t l y GABA-T i n t h e SN (Table 6 ) .  Following  reduce the a c t i v i t y o f  the i n j e c t i o n o f 10 nmoles o f KA,  however, n i g r a l GAD was reduced by 75% and n i g r a l GABA-T a c t i v i t y showed a s i g n i f i c a n t r e d u c t i o n observed i n GABA-T a c t i v i t y  (Table 6 ) .  also  As i n the s t r i a t u m , the decrease  i n t h e SN c o r r e l a t e d s i g n i f i c a n t l y w i t h the  decrease i n GAD ( F i g . 8 ) . Following  the i n j e c t i o n o f KA i n t o the s t r i a t u m o f t e n day o l d r a t s ,  the a c t i v i t y o f CAT i n the i n j e c t e d s t r i a t u m was reduced by h a l f (Table 7).  T h i s i n j e c t i o n had no e f f e c t on the a c t i v i t i e s o f GAD o r GABA-T  i n the s t r i a t u m . DISCUSSION The  dopamine neurons o f the SN a r e thought to r e c e i v e a major GABA  innervation.  Evidence suggests t h a t a massive GABA p r o j e c t i o n to the SN  a r i s e s i n the striatum Nagy et a l . , 1978a).  (Brownstein e t a l . , 1977; Fonnum e t a l . , 1978a; Immunocytochemical s t u d i e s have r e p o r t e d  of the boutons i n c o n t a c t w i t h n i g r a l d e n d r i t e s a l . , 1976). (Yoshida  s t a i n f o r GAD (Ribak e t  S t i m u l a t i o n o f the s t r i a t u m can i n h i b i t n i g r a l c e l l  and P r e c h t ,  1971), and t h i s e f f e c t  i s blocked  firing  by p i c r o t o x i n  (Precht and Y o s h i d a , 1971) and mimicked by i o n t o p h o r e t i c GABA 1971).  t h a t most  (Feltz,  A marked r e d u c t i o n i n n i g r a l H-GABA b i n d i n g has been observed 3  f o l l o w i n g 6-OHDA l e s i o n s of the n i g r o s t r i a t a l dopamine neurons ( G u i d o t t i et a l . , 1978).  H-GABA b i n d i n g has a l s o been found t o be reduced i n the  3  SN of P a r k i n s o n i a n rated  p a t i e n t s , i n which the dopamine neurons have degene-  ( L l o y d e t a l . , 1977b; Rinne e t a l . , 1978).  anatomical  A wealth o f biochemical,  and p h y s i o l o g i c a l evidence t h e r e f o r e i n d i c a t e s t h a t the dopamine  47  F i g u r e 7.  The c o r r e l a t i o n between glutamate d e c a r b o x y l a s e and GABAtransaminase a c t i v i t i e s  i n the s t r i a t u m f o l l o w i n g  a c i d i n j e c t i o n s of the s t r i a t u m , t r i a n g l e = 5 nmoles k a i n i c kainic acid. ion equation.  kainic  squares = c o n t r o l ;  acid; c i r c l e s  = 10 nmoles  The l i n e was drawn from the l i n e a r r e g r e s s -  *7  3  G A D (nmoles/mg protein/hr)  48 neurons r e c e i v e a major GABA i n n e r v a t i o n .  Thus, the l a c k of change i n  GABA-T a c t i v i t y f o l l o w i n g 6-OHDA l e s i o n s o f t h e dopamine neurons r a i s e s some i n t e r e s t i n g q u e s t i o n s  regarding  GABA  neurotransmission.  I t has p r e v i o u s l y been suggested t h a t GABA r e l e a s e d taken up and c a t a b o l i z e d i n the p o s t s y n a p t i c glial  a t the synapse i s  neuron and i n s u r r o u n d i n g  elements s i n c e these were the s t r u c t u r e s thought t o c o n t a i n most  of the GABA-T (Baxter,  1976).  However, the p r e s e n t  r e s u l t s i n d i c a t e that  the dopamine neurons do not c o n t a i n GABA-T, a l t h o u g h they a r e thought to be p o s t s y n a p t i c  to a major GABA system.  This observation  i n d i c a t e s that  a l l neurons which r e c e i v e GABA synapses do not n e c e s s a r i l y c o n t a i n GABA-T. T h i s s i t u a t i o n i s i n sharp c o n t r a s t w i t h the s i t u a t i o n f o r a c e t y l cholinesterase,  the enzyme which c a t a b o l i z e s a c e t y l c h o l i n e .  The dopa-  m i n e r g i c neurons o f t h e SN have been shown to c o n t a i n a p p r e c i a b l e of t h i s enzyme (Lehmann and F i b i g e r , 1978). little  However, a t p r e s e n t  levels there i s  evidence f o r the presence o f c h o l i n e r g i c synapses onto the dopamine  neurons.  Thus, the f u n c t i o n o f the a c e t y l c h o l i n e s t e r a s e i n these neurons  remains a mystery. Schwarz e t a l . , (1977) and N i c k l a s e t a l . (1979) have r e c e n t l y reported and  a d e c r e a s e i n GABA-T i n t h e s t r i a t u m f o l l o w i n g i n t r a s t r i a t a l KA  have suggested a n e u r o n a l l o c a l i z a t i o n f o r t h i s enzyme.  of the p r e s e n t GABA-T a c t i v i t y  The r e s u l t s  experiments w i t h KA suggest t h a t v i r t u a l l y a l l o f t h e i n the striatum i s neuronal.  Thus, the dose-dependent  decrease i n GABA-T a c t i v i t y c o r r e l a t e s w i t h t h a t o f the n e u r o n a l marker GAD ( F i g . 7 ) .  I n f a c t , the r e g r e s s i o n l i n e f o r t h i s c o r r e l a t i o n  approaches the o r i g i n , i n d i c a t i n g t h a t i f GAD a c t i v i t y was c o m p l e t e l y abolished,  the a c t i v i t y o f GABA-T i n the s t r i a t u m would a l s o approach  zero.  Recent s t u d i e s have shown t h a t t h e i n j e c t i o n o f KA i n t o the s t r i a t u m of n e o n a t a l r a t s p r e f e r e n t i a l l y depletes CAT compared w i t h GAD  (Campochiara  49  F i g u r e 8.  The c o r r e l a t i o n between glutamate d e c a r b o x y l a s e and GABAtransaminase i n the s u b s t a n t i a acid  nigra  i n j e c t i o n s of the s t r i a t u m ,  following  squares =  t r i a n g l e s = 5 nmoles k a i n i c a c i d ; c i r c l e s = kainic acid. regression  kainic  control; 10 nmoles  The l i n e was drawn from the l i n e a r  equation.  50  T a b l e 7.  The a c t i v i t i e s of n e u r o t r a n s m i t t e r - r e l a t e d striatum after into  the i n j e c t i o n of twenty nmoles of k a i n i c  acetyltransferase  Glutamate d e c a r b o x y l a s e GABA-Transaminase  n = 6;  acid  the s t r i a t u m of t e n day r a t s .  Control (nmol/mg p r o t e i n / h r  Choline  enzymes i n the  *p_ < .001;  Lesion S.E.M.)  % of C o n t r o l  286.3±5.4  124.1±24.9  102.1±6.8  95.0+ 6.8  93  88.812.6  97.9± 3.3  110  Student's two t a i l e d _t t e s t  43*  51  and  Coyle,  Fibiger  1978; Lehmann and F i b i g e r , 1979).  I n f a c t , Lehmann and  (1979) have found t h a t these i n j e c t i o n s s e l e c t i v e l y  the l a r g e a s p i n y  destroy  neurons of t h e s t r i a t u m , which a r e thought to be the  c h o l i n e r g i c neurons (Lehmann and F i b i g e r , 1979; Kimura e t a l . , 1980) and  do n o t a f f e c t  present  the d e n s i t y o f the medium and s m a l l c e l l s .  I n the  experiment t h i s p r e f e r e n t i a l decrease i n CAT a c t i v i t y was a g a i n  observed, w h i l e  GAD and GABA-T a c t i v i t i e s were u n a f f e c t e d .  t h a t the l a r g e a s p i n y  This  suggests  c h o l i n e r g i c neurons of t h e s t r i a t u m do not c o n t a i n  GABA-T. Both GABA and substance P neurons a r e known t o p r o j e c t t o the SN from the s t r i a t u m  (see Experiment 1 ) . T h e r e f o r e  d e s t r u c t i o n o f e i t h e r of  these systems c o u l d account f o r t h e d e c r e a s e i n n i g r a l GABA-T seen a f t e r s t r i a t a l KA i n j e c t i o n .  However-, i f GABA t r a n s m i s s i o n w i t h i n the s t r i a t u m  i s s i m i l a r i n mechanism t o t h a t d i s c u s s e d  above f o r the SN, then even i f  the s t r i a t o n i g r a l substance P neurons r e c e i v e a GABA.input i n the s t r i a t u m , t h i s would not n e c e s s i t a t e t h e i r c o n t a i n i n g GABA-T.  On the o t h e r hand, j u s t  as a c e t y l c h o l i n e s t e r a s e i s found i n v e r y h i g h c o n c e n t r a t i o n s  i n cholinergic  neurons (Lehmann and F i b i g e r , 1979), perhaps i t i s the GABA neurons thems e l v e s which c o n t a i n the GABA-T a c t i v i t y  i n t h i s system.  This  hypothesis  i s supported by the s i g n i f i c a n t c o r r e l a t i o n between GAD and GABA-T found i n the s t r i a t u m and SN a f t e r i n t r a s t r i a t a l KA. S t r i a t a l KA i n j e c t i o n s o n l y reduced n i g r a l GABA-T by t e n p e r c e n t . Kataoka e t a l . (1974) have r e p o r t e d  a 25% d e c r e a s e i n GABA-T a c t i v i t y i n  the baboon SN a f t e r h e m i t r a n s e c t i o n  between the s t r i a t u m and SN which  reduced n i g r a l GAD by 70%. GABA-T a c t i v i t y  Kim et a l . (1974) found a 30% r e d u c t i o n i n  i n the c a t SN f o l l o w i n g removal o f the caudate by s u c t i o n ,  a procedure which reduced n i g r a l GABA'levels by 50%.  The r e g r e s s i o n  line  f o r the c o r r e l a t i o n between n i g r a l GAD and GABA-T f o l l o w i n g s t r i a t a l KA  52  injections situation origin  ( F i g . 8) does not approach t h e o r i g i n .  This contrasts with the  i n the s t r i a t u m , where the r e g r e s s i o n l i n e does approach the  ( F i g . 7 ) . T h i s may i n d i c a t e t h a t GAD and GABA-T a r e i n d i f f e r e n t  c e l l u l a r compartments.  In the s t r i a t u m , KA i n j e c t i o n s  i n t r i n s i c GABA c e l l bodies striatum.  This results  and the t e r m i n a l s o f these  and GAD i s p r e f e r e n t i a l l y  the s t r i a t o n i g r a l t e r m i n a l s a r e  decreased  compared w i t h GABA-T.  suggests t h a t a h i g h GAD to GABA-T r a t i o may e x i s t but not i n GABA c e l l  c e l l s w i t h i n the  i n a p a r a l l e l d e c r e a s e i n s t r i a t a l GAD and GABA-T.  In the SN f o l l o w i n g the s t r i a t a l l e s i o n , destroyed  d e s t r o y both the  This  i n GABA t e r m i n a l s ,  bodies.  Most of the GABA-T i n the SN i s i n elements o t h e r than the s t r i a t o n i g r a l terminals.  As d i s c u s s e d above, the dopamine neurons do n o t appear  to c o n t a i n t h i s enzyme.  GABA-T c o u l d be c o n t a i n e d  i n other n i g r a l neurons, or i n g l i a l elements. the SN c o n t a i n s evidence  I t has been suggested t h a t  a p o p u l a t i o n of GABA neurons (Nagy e t a l . , 1978d).  i s presented  Also,  i n Experiment 6 f o r a GABA p r o j e c t i o n from the SNR  to the s u p e r i o r c o l l i c u l u s . i t would n o t be s u r p r i s i n g contained  i n other n i g r a l a f f e r e n t s ,  I n view o f the arguments presented  above,  i f most of the GABA-T a c t i v i t y i n the SN was  i n these GABA neurons.  53  EXPERIMENT 4:  THE HISTOCHEMICAL LOCALIZATION OF GABA-TRANSAMINASE IN THE BASAL GANGLIA.  As d i s c u s s e d p r e v i o u s l y , b i o c h e m i c a l  s t u d i e s have suggested the  e x i s t e n c e of many GABA pathways i n the b a s a l g a n g l i a . i t was demonstrated t h a t GABA-T i s c o n t a i n e d  In Experiment 3  i n the s t r i a t a l  p r o j e c t i o n to  the SN and i t was suggested t h a t t h i s enzyme may i n f a c t be c o n t a i n e d i n the s t r i a t o n i g r a l GABA neurons.  As a simple  GABA-T has l o n g been known (Van Gelder, for  h i s t o c h e m i c a l procedure f o r  1965) the use o f t h i s  technique  the h i s t o c h e m i c a l d e m o n s t r a t i o n o f some GABA pathways i n the b a s a l  g a n g l i a was t h e r e f o r e examined. METHODS Male W i s t a r  r a t s weighing about 300 g were o b t a i n e d  from Woodlyn  L a b o r a t o r i e s , Guelph, O n t a r i o and were used i n a l l the experiments. chemical  Histo-  s t a i n i n g f o r GABA-T was performed by a m o d i f i c a t i o n of the procedure  of Van Gelder  (1965).  Rats were p e r f u s e d  i n t r a c a r d i a l l y with  50 ml of i c e -  c o l d 0.1 M phosphate b u f f e r e d s a l i n e , pH 7.4, f o l l o w e d by 200 ml of 2.0% paraformaldehyde and 2.0% g l u t a r a l d e h y d e The  i n 0.1 M phosphate b u f f e r , pH 7.4.  f r e e - f l o a t i n g s e c t i o n s were p r e i n c u b a t e d  f o r 20 min a t 37°C i n a  r e a c t i o n mixture c o n t a i n i n g 5.0 mg/ml a - k e t o g l u t a r a t e and  0.05 mg/ml KCN i n 50 mM T r i s - C l , pH 8.6.  1.0 mg/ml malonate,  A f t e r the p r e i n c u b a t i o n the  s u b s t r a t e and dye were added t o g i v e f i n a l c o n c e n t r a t i o n s  of 5.0 mg/ml  GABA, 1.0 mg/ml n i t r o b l u e t e t r a z o l i u m and 0.01 mg/ml phenazine methosulphate, and t h e i n c u b a t i o n was continued  f o r 30 min i n the dark.  The  r e a c t i o n was stopped by d i l u t i n g i n phosphate b u f f e r , and the s e c t i o n s were then mounted, dehydrated and c o v e r - s l i p p e d . without  GABA, or w i t h  showed no r e a c t i o n In order  Control sections  incubated  the GABA-T i n h i b i t o r amino-oxyacetic a c i d (AOAA)  product.  to d e s t r o y  the s t r i a t a l  neurons, KA was i n j e c t e d i n t o the  54  s t r i a t u m as p r e v i o u s l y d e s c r i b e d  (McGeer and McGeer, 1976a).  Rats  r e c e i v e d a u n i l a t e r a l i n j e c t i o n of 5 nmoles o f KA i n 0.5 y l o f 50 mM NaPO^, pH 7 a t a r a t e o f 1 y l / 5 min. deeply a n e s t h e t i z e d  Two weeks l a t e r these animals were  with pentobarbital  GABA-T h i s t o c h e m i s t r y  and p e r f u s e d  and p r o c e s s e d f o r  a t the i n j e c t i o n s i t e and i n the p r o j e c t i o n areas  of the s t r i a t a l GABA e f f e r e n t s . In a second study the GABA-T s t a i n i n g o f the e f f e r e n t s of the p a l l i d u m was examined.  One group of animals was  given  a unilateral  stereotaxic  i n j e c t i o n o f KA (2.0 nmol. i n 0.25 y l ) i n t o the GP (AP + 8.0; ML + 2.8; DV -J- 3.6; w i t h r e s p e c t  t o s t e r e o t a x i c zero) w h i l e another group r e c e i v e d  the same i n j e c t i o n i n t o t h e EP (AP + 6.3; ML + 3.0; DV + 2.2). l a t e r these animals were a l s o p r o c e s s e d f o r GABA-T  One week  histochemistry.  RESULTS (a)  GABA-T s t a i n i n g i n c o n t r o l animals A l l o f the n u c l e i o f the b a s a l g a n g l i a s t a i n s t r o n g l y f o r GABA-T. i n c o n t r o l  animals.  T h i s i s r e a d i l y apparent i n s a g i t a l s e c t i o n s through t h i s  (Fig. 9).  I n f a c t , i n many areas the s t a i n i n g i s  which c e l l u l a r s t r u c t u r e s c o n t a i n f o r GABA-T.  the enzyme.  as t o obscure  The s t r i a t u m s t a i n s  The r e a c t i o n p r o d u c t i s absent i n t h e w h i t e matter  through the s t r i a t u m but i s d i f f u s e l y p r e s e n t ( F i g . 10).  so i n t e n s e  Stained  region  strongly  coursing  throughout the n e u r o p i l  neurons can not be c l e a r l y . d i s c e r n e d and f o r the most  p a r t a r e h i d d e n by the i n t e n s e n e u r o p i l s t a i n i n g . The  n e u r o p i l o f t h e GP a l s o s t a i n s s t r o n g l y f o r GABA-T a c t i v i t y and i n  a d d i t i o n some l a r g e , i n t e n s e l y s t a i n e d neurons can be seen i n t h i s ( F i g . 11a). The white matter p a s s i n g n a l capsule  a r e not s t a i n e d .  area  through the GP and the a d j a c e n t  An even more i n t e n s e  inter-  staining pattern i s  observed i n the n e u r o p i l o f the EP, a l t h o u g h s t a i n e d neurons a r e not apparent here ( F i g .  11c).  55  F i g u r e 9.  A s a g i t a l s e c t i o n through the r a t b r a i n s t a i n e d c a l l y f o r GABA-transaminase a c t i v i t y  histochemi-  (magnification =  9.2. x)  Note the r e g i o n a l d i f f e r e n c e s i n the i n t e n s i t y o f the staining.  In p a r t i c u l a r areas a s s o c i a t e d w i t h the  b a s a l g a n g l i a show an i n t e n s e r e a c t i o n , ep nucleus;  cp  caudate-putamen;  gp  entopeduncular  globus p a l l i d u s ;  lh  l a t e r a l habenula;  sc  superior  colliculus;  sn  substantia nigra;  st  subthalamic  nucleus.  £5 a  56  Figure  10.  F i f t y micron vibratome s e c t i o n through the r a t f o r e b r a i n stained histochemically The i n t e n s e out  f o r GABA-transaminase  r e a c t i o n p r o d u c t , which  activity.  i s p r e s e n t through-  the s t r i a t a l n e u r o p i l o f the c o n t r o l s i d e  (left),  i s almost c o m p l e t e l y absent on the c o n t r a l a t e r a l s i d e f o l l o w i n g the d e s t r u c t i o n o f the s t r i a t a l neurons by the i n j e c t i o n of f i v e nmoles of k a i n i c a c i d . s t a i n i n g can a l s o be seen i n the c o r t e x side.  This  (arrow).  Some l o s s of on the  i s most apparent a l o n g the n e e d l e Magnification  i s 10  x.  lesioned tract  SO a  57  Figure  11.  GABA-transaminase h i s t o c h e m i s t r y  f o l l o w i n g the i n j e c t i o n  of k a i n i c a c i d i n t o the head o f the s t r i a t u m .  (a)  The c o n t r o l u n i n j e c t e d  i n t h i s coronal Note the i n t e n s e  s i d e o f the b r a i n can be seen  s e c t i o n through the t a i l s t a i n i n g i n the t a i l  o f the caudate.  o f the caudate  and i n the globus p a l l i d u s m e d i a l t o i t . is  Magnification  16 x.  (b)  On t h e i n j e c t e d s i d e t h e s t a i n i n g i n t h e globus p a l l i d u s  i s markedly  reduced  (arrow) but the GABA-T a c t i v i t y  i n the  t a i l o f t h e caudate l a t e r a l t o the globus p a l l i d u s i s unaffected  by the k a i n a t e  striatum.  M a g n i f i c a t i o n i s 16 x.  (c)  In t h i s coronal  l e s i o n o f the head o f the  s e c t i o n s t a i n i n g i n t h e entopeduncular  n u c l e u s on t h e c o n t r o l s i d e can be compared w i t h t h a t on the l e s i o n e d s i d e .  The GABA-T a c t i v i t y on the l e s i o n e d  s i d e (arrow) shows a marked r e d u c t i o n s t r i a t a l kainic acid injection. ing  A s i m i l a r l o s s of s t a i n -  can be seen i n the l a t e r a l hypothalamic a r e a (*) as  well.  (d)  f o l l o w i n g the  Magnification  i s 15.1 x.  The GABA-transaminase a c t i v i t y i n the s u b s t a n t i a  n i g r a i s a l s o d r a s t i c a l l y reduced striatal  (arrow) f o l l o w i n g the  l e s i o n when compared w i t h the c o n t r a l a t e r a l  control nigra.  T h i s i s t r u e both i n the p a r s compacta  and the pars r e t i c u l a t a .  Note t h e s t a i n i n g i n the v e n t r a l  tegmental a r e a m e d i a l to t h e s u b s t a n t i a n i g r a i s u n a f f e c t ed by the s t r i a t a l  lesions.  Magnification  i s 17 x.  58  W i t h i n the SN, t h e GABA-T s t a i n i n g i s p r e s e n t as a dense band i n the r o s t r a l SNC ( F i g . l i d ) , w h i l e i n the SNR t h e p a t t e r n o f s t a i n i n g resembles  t h a t seen i n t h e GP ( F i g . l i d ) .  A few l a r g e , s t a i n e d  neurons  s i m i l a r t o . t h o s e i n t h e GP can a l s o be found i n t h e SNR. The subthalamic n u c l e u s shows the most i n t e n s e GABA-T s t a i n i n g o f any a r e a y e t examined.  The r e a c t i o n p r o d u c t i s so s t r o n g i n t h i s  nucleus  that m o r p h o l o g i c a l f e a t u r e s cannot be d i s c e r n e d ( F i g . 9 ) . A l t h o u g h the e n t i r e n u c l e u s shows h i g h GABA-T a c t i v i t y t h e m e d i a l h a l f i s the most intensely stained  ( F i g . 12). The c r u s c e r e b r i p a s s i n g v e n t r a l t o the  subthalamus i s u n s t a i n e d except f o r some f i n e bands o f r e a c t i o n product e x t e n d i n g from the subthalamic n u c l e u s i n t o the a d j a c e n t i n t r a p e d u n c u l a r area. F i n a l l y , t h e l a t e r a l habenula  s t a i n s q u i t e s t r o n g l y f o r GABA-T  w h i l e minimal a c t i v i t y i s p r e s e n t i n the m e d i a l habenula s t a i n i n g i n the l a t e r a l habenula  ( F i g . 13). The  i s most i n t e n s e i n the v e n t r o l a t e r a l  d i v i s i o n and i s n o t i c e a b l y weaker i n the more m e d i a l p o r t i o n o f t h i s nucleus.  The d o r s a l aspect of the l a t e r a l habenula  the m e d i a l habenula (b)  s t a i n s as weakly as  f o r GABA-T.  GABA-T s t a i n i n g a f t e r l e s i o n s of GABA pathways F o l l o w i n g the d e s t r u c t i o n o f t h e neurons  o f the s t r i a t u m by the  i n j e c t i o n of KA, the GABA-T s t a i n i n g i s almost a b o l i s h e d i n the s t r i a t a l neuropil  ( F i g . 1 0 ) . There i s f a i n t s t a i n i n g o f macrophages or monocytes  a l o n g t h e n e e d l e t r a c t , and of a s t r o c y t e s throughout  the a r e a o f damage.  I n f i l t r a t i o n by macrophages which s t a i n weakly f o r GABA-T was a l s o observed i n the corpus c a l l o s u m d o r s a l t o the l e s i o n e d s t r i a t a .  The l e s i o n  was c o n f i n e d t o the s t r i a t u m r o s t r a l t o the d e c u s s a t i o n o f t h e a n t e r i o r commissure but extended  v e n t r a l l y to i n c l u d e t h e n u c l e u s accumbens.  The i n j e c t i o n o f KA i n t o the s t r i a t u m a l s o reduced  the GABA-T s t a i n i n g  59  F i g u r e 12.  GABA-transaminase a c t i v i t y i n the s u b t h a l a m i c n u c l e u s of the r a t i s extremely s t r o n g on the c o n t r o l  side  (arrow), but i s d r a s t i c a l l y reduced on the s i d e i n which the globus p a l l i d u s was acid  (double arrow).  injected with k a i n i c  Note t h a t i n the c o n t r o l  sub-  thalamus the s t a i n i n g i s most i n t e n s e i n the m e d i a l p o r t i o n of the n u c l e u s .  A l s o , bands o f r e a c t i o n p r o -  duct can be seen extending v e n t r a l l y from the lamus i n t o the a d j a c e n t i n t r a p e d u n c u l a r a r e a . M a g n i f i c a t i o n i s 12.9  x.  subtha-  60  F i g u r e 13.  GABA-transaminase h i s t o c h e m i s t r y i n the habenula ing k a i n i c  a c i d l e s i o n s o f the entopeduncular  follow-  nucleus.  In the lower f i g u r e , which i s a c o r o n a l s e c t i o n  a t the  l e v e l o f the i n j e c t i o n s i t e , the l o s s o f s t a i n i n g i n the entopeduncular n u c l e u s f o l l o w i n g i o n s i s r e a d i l y apparent  the k a i n i c  (arrow).  acid  This lesion  reduced t h e GABA-T a c t i v i t y of t h e i p s i l a t e r a l habenula control.  dramatically lateral  (double arrow) compared w i t h the c o n t r a l a t e r a l Magnification  In the upper activity  inject-  i s 15.7 x.  f i g u r e the decrease i n habenular  following  i n greater d e t a i l .  GABA-T  the entopeduncular l e s i o n can be seen Note that  i n the c o n t r o l  habenula  the GABA-T a c t i v i t y i s most c o n c e n t r a t e d i n the v e n t r o l a t e r a l portion i s 36.6 x.  of the l a t e r a l habenula.  Magnification  61  in  the GP, EP and SN i p s i l a t e r a l t o the l e s i o n ( F i g . 11). The s t a i n i n g  in  the t a i l o f the caudate was not a f f e c t e d  This  suggests that  by the l e s i o n ( F i g . l i b ) .  t h e reduced p a l l i d a l s t a i n i n g was not due t o the  d i f f u s i o n o f KA to t h i s s t r u c t u r e .  L a r g e GABA-T p o s i t i v e neurons were  s t i l l v i s i b l e i n the GP and SN and a few s m a l l GABA-T p o s i t i v e c o u l d be seen i n the EP.  cells  In the c o n t r o l s i d e these c e l l s a r e s t i l l  c o n c e a l e d by the i n t e n s e s t a i n i n g of the n e u r o p i l .  W i t h i n the SN, both the  i n t e n s e s t a i n i n g o f the SNC and the more d i f f u s e SNR s t a i n i n g were markedly reduced by the s t r i a t a l l e s i o n ( F i g . l i d ) . F o l l o w i n g the i n j e c t i o n of KA i n t o the GP a marked r e d u c t i o n T a c t i v i t y was e v i d e n t . no longer v i s i b l e .  i n GABA-  I n p a r t i c u l a r , t h e GABA-T p o s i t i v e neurons were  The KA l e s i o n of the GP a l s o d r a s t i c a l l y reduced the  GABA-T s t a i n i n g i n t h e i p s i l a t e r a l subthalamic n u c l e u s . evident i n the medial portion  of the n u c l e u s  This  i s most  ( F i g . 1 2 ) . The s t a i n i n g i n  the  f i e l d s of F o r e l and the zona i n c e r t a around the s u b t h a l a m i c n u c l e u s was  not  affected  by the l e s i o n s .  Finally, in  the i n j e c t i o n o f KA i n t o the EP r e s u l t e d  the GABA-T s t a i n i n g of t h i s n u c l e u s , a l t h o u g h some d i f f u s e  product was s t i l l p r e s e n t i n t h e n e u r o p i l in  i n a marked  reduction  reaction  ( F i g . 1 3 ) . The GABA-T s t a i n i n g  the i p s i l a t e r a l l a t e r a l habenula was a l s o d r a m a t i c a l l y  d e c r e a s e d by t h i s  l e s i o n , w h i l e the s t a i n i n g o f the c o n t r a l a t e r a l habenula was n o t d i f f e r e n t from c o n t r o l  ( F i g . 13). DISCUSSION  In Experiment 3, b i o c h e m i c a l evidence was p r e s e n t e d s u g g e s t i n g GABA-T a c t i v i t y  i n the s t r i a t u m  histochemical studies destruction  i s c o n t a i n e d i n neurons.  support such a c o n c l u s i o n .  that  The p r e s e n t  Thus, f o l l o w i n g the  o f the s t r i a t a l neurons w i t h KA t h e GABA-T s t a i n i n g i n t h i s  nucleus was almost c o m p l e t e l y a b o l i s h e d .  The l e s i o n e d  striata did  62  c o n t a i n non-neuronal  elements which s t a i n e d weakly f o r GABA-T; however,  i n v a s i o n by these elements related  (monocytes,  fibrous a s t r o c y t e s ) i s probably  to the pathology of the l e s i o n and they would not n o r m a l l y be  p r e s e n t i n the s t r i a t u m . P r e v i o u s s t u d i e s have found that l e s i o n s of the s t r i a t u m r e s u l t r e d u c t i o n i n GAD a c t i v i t y The p r e s e n t experiments  ina  i n both the GP and the EP (see Experiment 1 ) .  clearly illustrate  t h a t f o l l o w i n g the i n j e c t i o n  of KA i n t o the head o f the s t r i a t u m a marked r e d u c t i o n i n the GABA-T activity  i n these a r e a s a l s o o c c u r s .  T h i s suggests t h a t GABA-T i s con-  t a i n e d w i t h i n the t e r m i n a l s of the s t r i a t o p a l l i d a l GABA neurons. neurons  i n the GP and EP s t a i n f o r GABA-T a f t e r t h e l e s i o n ,  Some  indicating  the presence of t h i s enzyme i n some p a l l i d a l c e l l b o d i e s as w e l l as i n afferent terminals.  Both the GP (Fonnum et a l . ,  1973b) and the EP (Nagy e t a l . , neurons,  1978b; H a t t o r i e t a l . ,  1978b) a r e thought  t o c o n t a i n GABA  s u g g e s t i n g t h a t i t i s these c e l l s which s t a i n f o r GABA-T.  I t i s i n t e r e s t i n g t o note t h a t the i n j e c t i o n o f KA i n t o t h e s t r i a t u m reduces the a c t i v i t y o f GABA-T i n the l a t e r a l hypothalamic m e d i a l t o the EP ( F i g . 11c). s i d e of the b r a i n  ( F i g . 11c).  T h i s r e g i o n s t a i n s i n t e n s e l y on the c o n t r o l The l a t e r a l hypothalamic  shown to possess the h i g h e s t GABA l e v e l s and GAD a c t i v i t y  (Kimura  area ventro-  a r e a has been  (Kimura and Kuriyama, 1975a)  and Kuriyama, 1975b) i n t h e hypothalamus.  A n a t o m i c a l s t u d i e s have shown t h a t the n u c l e u s accumbens p r o j e c t s m a s s i v e l y to t h i s r e g i o n (Nauta e t a l . ,  1978; P o w e l l and Leman, 1976).  Recently,  b i o c h e m i c a l s t u d i e s have i n d i c a t e d that t h e accumbens p r o j e c t i o n s to the s u b s t a n t i a innominata, GP (Walaas and Fonnum, 1979),  SN and v e n t r a l  teg-  mental a r e a (Waddington and C r o s s , 1978b; Walaas and Fonnum, 1980) may c o n t a i n GAD.  Together, these r e s u l t s suggest t h a t the d e c r e a s e i n t h e  GABA-T a c t i v i t y observed  i n the l a t e r a l hypothalamic  a r e a f o l l o w i n g KA  63  i n j e c t i o n s in  the  of  the  nucleus  The  s t r i a t u m may be d u e  accumbens  existence  of  a  demonstrated,  although  some  (see  debate  concentration apposed  to  of  the  anterior  SNC,  while  the  (see  Experiment et  although with  from  the  loss is  of  staining  damaged by for  neurons medial  thalamus  GP o r  1978; that could  be  et  GABA-T the  of  the  hypothesis  that  the  regulation  of  SNR  observation  been demonstrated SNC d o  in  the  finding As  in  the  cells  which  shown  in  action GABA-T  to  effects The  the  in  these  after to  GABA  of  neurons  the  effects.  cells  is  not  the  lesions.  Experiment  6).  inhibitors (Matsui  and  observations  striatal  terminals  are in  GABA  would  be  directly  The  lesions  the  behaviour  This  3).  and  histochemical of  the  striatal  GABA-T  P  projection  that  tectum  (see  the  correlates  GABA  striatal  the  to  staining  (Experiment  large  a  1975;  SNC  the  following  on motor  GABA N e r v e  transmission.  GABA-T  injections  terminals  for  and  project  present  of  dopaminergic  contain  that  the  of  1).  al. ,  GABA-T  in  is  substance  et  the  some  there  predominantly  biochemically  pallidum,  a matter  striatum  contains  that  compacta the  that  the  (Hattori  contain  animals  thought  1976).  GABA  since  in  marked  not  pars  control  have  present  site  which  terminals  the  neurons  been  Experiment  project  the  in  a l . ,  the  has  in  1977;  of  and a r e  studies  a l . ,  distribution  nigral  SN r e s u l t  Pycock the  the  neurons  neurons  to  GABA  repeatedly  suggests  intense  this  both  evidence  most  of  GABA-T  may b e  the  has  lesion.  Recent  et  of  been  pathway  is  observed  with  this  SN,  It  this  Recent the  throughout  neurons  of  projection,  the  has  origin  largely  Thus,  proposed  consistent  stain  1978).  GABA p a t h w a y  these  striatal  destruction  area.  GAD-containing  that  projects  striatum.  dopaminergic  1).  the  this  (Brownstein  suggest  1)  a l . ,  the  precise  GP  anterior  present  well  the  striatonigral  studies  to  striatonigral  Experiment  Anatomical  Tulloch  projecting  to  SNR These ventro-  into Kamioka, suggest  projections consistent involved  with in  64 L e s i o n s o f t h e EP have p r e v i o u s l y been found t o d e c r e a s e GABA l e v e l s and GAD a c t i v i t y i n t h e habenula i n d i c a t i n g t h a t t h i s i s a GABA pathway ( G o t t e s f e l d e t a l . , 1977; Nagy e t a l . , 1978b).  I n the present histochem-  c a l s t u d y , t h e a c t i v i t y o f t h e enzyme GABA-T i n t h e h a b e n u l a was a l s o found t o d e c r e a s e f o l l o w i n g EP l e s i o n s .  T h i s s u g g e s t s t h a t GABA-T i n t h e  habenula i s c o n t a i n e d i n t h e t e r m i n a l s o f t h e EP-habenula pathway.  The  GABA-T s t a i n i n g i n t h e l a t e r a l h a b e n u l a i s most i n t e n s e i n t h e v e n t r o l a t e r a l p o r t i o n o f t h i s n u c l e u s , and i s weakest i n t h e d o r s o - m e d i a l r e g i o n .  This  may i n d i c a t e t h a t t h e GABA t e r m i n a l s i n t h e l a t e r a l h a b e n u l a a r e most concentrated i n the v e n t r o l a t e r a l area.  The e v i d e n c e from a n a t o m i c a l  s t u d i e s of t h e EP-habenula pathway i s i n f u l l agreement w i t h t h i s h y p o t h e s i s . I n a u t o r a d i o g r a p h i c s t u d i e s of t h e o r t h o g r a d e t r a n s p o r t o f r a d i o l a b e l e d p r o t e i n from t h e EP t h e l a b e l has been found t o d i s t r i b u t e p r e d o m i n a n t l y i n t h e v e n t r o l a t e r a l p o r t i o n o f t h e l a t e r a l h a b e n u l a ( C a r t e r and F i b i g e r , 1978; L a r s e n and M c B r i d e , 1979; Nagy e t a l . , 1978b) and n o t i n t h e d o r s o m e d i a l p o r t i o n (Nauta, 1974).  A s i m i l a r topography has been o b t a i n e d from  r e t r o g r a d e s t u d i e s w i t h h o r s e r a d i s h p e r o x i d a s e (Herkenham and N a u t a , 1977; L a r s e n and M c B r i d e , 1979). The r e c e n t o b s e r v a t i o n of Fonnum e t a l . (1978b) t h a t GP l e s i o n s i n t h e c a t reduce s u b t h a l a m i c GAD a c t i v i t y i n d i c a t e s t h a t t h i s may a l s o be a GABA pathway.  L e s i o n s o f t h e GP i n t h e r a t reduce GABA-T a c t i v i t y i n t h e sub-  t h a l a m i c n u c l e u s , s u g g e s t i n g t h a t t h i s enzyme i s p r e s e n t i n t h e t e r m i n a l s of t h e p a l l i d a l a f f e r e n t s t o t h i s n u c l e u s .  Fonnum e t a l .  (1978b) have  found t h a t t h e c o n c e n t r a t i o n o f GAD i n c r e a s e s from t h e l a t e r a l t o t h e medial  p a r t o f t h e subthalamus.  I n the present experiments the i n t e n s i t y  of GABA-T s t a i n i n g was found t o f o l l o w a s i m i l a r p a t t e r n .  A g a i n t h i s agrees  w i t h t h e topography of t h e p a l l i d o - s u b t h a l a m i c pathway found i n a n a t o m i c a l studies.  A u t o r a d i o g r a p h i c s t u d i e s i n t h e r a t f o l l o w i n g GP i n j e c t i o n s o f  65  tritiated nucleus  amino a c i d s have found  the m e d i a l p o r t i o n o f the s u b t h a l a m i c  to be most h e a v i l y l a b e l e d  The p r e s e n t experiments  ( C a r t e r and F i b i g e r ,  1978).  i n d i c a t e t h a t GABA-T h i s t o c h e m i s t r y  may be  a u s e f u l a d j u n c t t o b i o c h e m i c a l s t u d i e s f o r the a n a l y s i s o f GABA pathways. The l o c a l i z a t i o n o f GABA-T s t a i n i n g i n the e f f e r e n t s of the s t r i a t u m corresponds  to the d i s t r i b u t i o n o f the s t r i a t a l GABA e f f e r e n t s .  d i s t r i b u t i o n of GABA-T s t a i n i n g i n the l a t e r a l habenula nucleus c o r r e l a t e s w e l l w i t h the known topography p r o j e c t i o n s to these a r e a s .  A l s o the  and subthalamic  of the p a l l i d a l GABA  Moreover, the combination  o f GABA-T h i s t o -  chemistry w i t h s e l e c t i v e l e s i o n s has p r o v i d e d a v e r y g r a p h i c summary o f what has been l e a r n e d from s e v e r a l years of b i o c h e m i c a l work. approach p r o v i d e s a d e t a i l e d p i c t u r e o f the topography  This  of these GABA  pathways p r e v i o u s l y o n l y h i n t e d a t from c o r r e l a t i o n s o f l e s i o n - b i o c h e m i c a l work w i t h a n a t o m i c a l s t u d i e s .  Thus, GABA-T h i s t o c h e m i s t r y may p r o v i d e a  simple t e c h n i q u e f o r t h e m o r p h o l o g i c a l a n a l y s i s o f known o r s u s p e c t e d GABA pathways.  66  EXPERIMENT 5:  BIOCHEMICAL CHANGES FOLLOWING 6-HYDROXYDOPAMINE LESIONS OF THE NIGROSTRIATAL DOPAMINE NEURONS: AN ANIMAL MODEL OF PARKINSONISM?  L e s i o n s of the n i g r o s t r i a t a l dopamine p r o j e c t i o n have been used animal models of P a r k i n s o n ' s d i s e a s e (see Marsden et a l . , i t was  d i s c o v e r e d t h a t t h i s system degenerates  ( E h r i n g e r and H o r n y k i e w i c z , 1960;  i n this  1975)  as  since  disorder  Hornykiewicz, 1973).  A detailed  study  of the e f f e c t s of such l e s i o n s on the o t h e r t r a n s m i t t e r systems of the b a s a l g a n g l i a has not been conducted.  T h i s i s of g r e a t importance f o r ,  a l t h o u g h the most s t r i k i n g a s p e c t o f P a r k i n s o n ' s d i s e a s e i s the marked decrease i n n i g r a l and s t r i a t a l dopamine, changes i n o t h e r systems have a l s o been r e p o r t e d .  A decrease i n GAD  activity  n u c l e i o f the b a s a l g a n g l i a has been c o n s i s t e n t l y observed disease 1974;  ( L l o y d and H o r n y k i e w i c z , 1973;  1979).  McGeer e t a l . ,  1971;  i n some  i n Parkinson's Rinne et a l . ,  T h i s d e c r e a s e has been r e p o r t e d to be r e v e r s e d by c h r o n i c  L-dopa therapy normal  transmitter  ( L l o y d and Hornykiewicz, 1973;  Rinne et a l . , 1979).  Also,  r a t s t r e a t e d c h r o n i c a l l y w i t h L-dopa have been r e p o r t e d to have an  i n c r e a s e d s t r i a t a l GAD  activity  ( L l o y d and H o r n y k i e w i c z , 1973).  These  r e s u l t s suggest an i n t i m a t e i n t e r a c t i o n between the n i g r o s t r i a t a l dopamine system and the GABA neurons  of the s t r i a t u m .  Although the l e v e l s of substance P and met-enkephalin  have not been  measured i n P a r k i n s o n ' s d i s e a s e , e x p e r i m e n t a l d a t a i n d i c a t e t h a t dopaminergic  n i g r o s t r i a t a l system p r o f o u n d l y i n f l u e n c e s the  c o n t a i n i n g these p e p t i d e s i n the s t r i a t u m .  the  neurons  Acute amphetamine treatment  has  been found t o d e c r e a s e the substance P l e v e l s i n the s t r i a t u m ( P e t t i b o n e et a l . ,  1978a; 1978b).  C h r o n i c n e u r o l e p t i c treatment d e c r e a s e s the  of substance P i n the SN (Hong and C o s t a , 1978)  (Hong and C o s t a , 1978)  and  levels  i n c r e a s e s the l e v e l s  and a c c e l e r a t e s the s y n t h e s i s o f  met-enkephalin  67  (Hong e t a l . , 1978b) i n the s t r i a t u m , The  GP and n u c l e u s accumbens.  p r e s e n t s e r i e s of experiments was designed t o examine f u r t h e r  the i n t e r a c t i o n s between the dopaminergic neurons and the t r a n s m i t t e r s contained  i n the e f f e r e n t s of t h e s t r i a t u m .  S e l e c t i v e l e s i o n s of t h e  n i g r o s t r i a t a l dopamine neurons were performed  w i t h 6-OHDA and t h e  e f f e c t s o f such l e s i o n s on t h e GABA, substance P and met-enkephalin systems of the b a s a l g a n g l i a  examined. METHODS  Male W i s t a r r a t s weighing about 300 g were p l a c e d anesthesia  and g i v e n  a u n i l a t e r a l s t e r e o t a x i c i n j e c t i o n o f f o u r yg 6-OHDA  i n t o the n i g r o s t r i a t a l pathway as d e s c r i b e d animals r e c e i v e d  under Nembutal  i n Experiment 3.  an i n j e c t i o n o f v e h i c l e o n l y .  A l l animals  Control received  desipramine (25 mg/kg) 30 min b e f o r e t h e 6-OHDA i n j e c t i o n s . Following  s u r g e r y the animals were s i n g l y housed i n a 12 h r l i g h t - d a r k  environment and g i v e n  food  and water ad l i b i t u m .  s a c r i f i c e d by c e r v i c a l f r a c t u r e a t v a r i o u s b r a i n s removed and d i s s e c t e d Tissues  The animals were  times a f t e r t h e o p e r a t i o n , t h e  for biochemical  analyses.  f o r t h e enzyme assays were homogenized i n 20 to 30 volumes o f  50 mM T r i s - a c e t a t e b u f f e r pH 6.4 c o n t a i n i n g assayed as d e s c r i b e d  0.2% T r i t o n X-100.  GAD was  i n Experiment 1, t y r o s i n e h y d r o x y l a s e as o u t l i n e d i n  Experiment 3, and p r o t e i n a c c o r d i n g  to Lowry e t a l . (1951).  The l e v e l s  of substance P and met-enkephalin were measured by radioimmunoassay f o l l o w i n g e x t r a c t i o n i n one N a c e t i c a c i d  (Appendix).  RESULTS In t h e f i r s t  experiment, animals were s a c r i f i c e d 25 days a f t e r the  i n j e c t i o n of 6-OHDA i n t o t h e l e f t n i g r o s t r i a t a l pathway. striatum,  i n c l u d i n g t h e accumbens and GP was d i s s e c t e d  SN was o b t a i n e d from s e c t i o n s  The whole  f r e e h a n d , w h i l e the  c u t on a f r e e z i n g microtome.  This  lesion  67a  Figure 1 4 .  Enzyme a c t i v i t i e s  i n the s t r i a t u m and s u b s t a n t i a n i g r a (SN)  2 5 days a f t e r u n i l a t e r a l l e s i o n s o f the n i g r o s t r i a t a l pathway, expressed  as p e r c e n t o f c o n t r o l .  Each column  r e p r e s e n t s t h e mean of the number of r a t s i n d i c a t e d i n brackets.  V e r t i c a l bars represent the standard e r r o r  of t h e mean. and  Absolute values f o r t y r o s i n e hydroxylase  glutamate d e c a r b o x y l a s e  (TH)  (GAD) i n c o n t r o l s t r i a t a were  4.2 - 0.2 and 98.9 - 3.7 nmoles/mg p r o t e i n / h r e s p e c t i v e l y . For c o n t r o l SN, the TH and GAD v a l u e s were 2.3 - 0.1 and 272 - 1 1 nmoles/mg p r o t e i n / h .  * JK.OOI, paired ^ - t e s t .  ENZYME ACTIVITY (% OF CONTROL)  68 r e s u l t e d i n an d e c r e a s e i n s t r i a t a l and a c t i v i t y of more than 90%,  n i g r a l tyrosine hydroxylase  r e s u l t i n g from the orthograde and  d e g e n e r a t i o n of the dopamine t e r m i n a l s  and  perikarya respectively.  T h i s l e s i o n was  associated with a highly s i g n i f i c a n t  a c t i v i t y of GAD  i n the s t r i a t u m on the i n j e c t e d s i d e .  a c t i v i t y i n the SN was  not  a f f e c t e d by  r e s u l t s have r e c e n t l y been p u b l i s h e d Since  and  the GP,  experiment i n c l u d e d  the experiment was  increase  (Vincent  i n the  In c o n t r a s t ,  the l e s i o n ( F i g . 14).  These  i n c r e a s e was  observed i n  the caudate-putamen, the accumbens  r e p l i c a t e d and  GAD  was  assayed i n  d i s c r e t e n u c l e i of the b a s a l g a n g l i a i n an attempt to l o c a l i z e the of t h i s change i n a c t i v i t y . were k i l l e d by the t a i l  One  s e c t i o n s obtained  the GP  the n u c l e u s accumbens, the head  and  the SN were d i s s e c t e d from  on a f r e e z i n g microtome and  assayed f o r GAD  activity. 92%  the head of the s t r i a t u m i n d i c a t i n g t h a t the l e s i o n of the dopamine  neurons was increased  e s s e n t i a l l y complete.  GAD  GAD  ficance  the GP  to i n c r e a s e (.05  As  seen i n T a b l e 8,  by about the same e x t e n t .  i n the SN but  t h i s d i d not  of  a tendency  reach s t a t i s t i c a l  i n s t r i a t a l GAD  t r a n s i e n t or a permanent change, the a c t i v i t y of GAD SN  signi-  was  a c t i v i t y was  a  measured i n the  t h r e e months a f t e r the u n i l a t e r a l i n j e c t i o n of 6-OHDA  i n t o the n i g r o s t r i a t a l bundle. P and  There was  the t a i l  < p < .1).  To determine i f the observed i n c r e a s e  s t r i a t u m and  these l e s i o n s  a c t i v i t y i n the n u c l e u s accumbens, the head and  the s t r i a t u m , and for  and  coronal  These l e s i o n s were found to reduce t y r o s i n e h y d r o x y l a s e more than in  site  month a f t e r the 6-OHDA^lesion animals  c e r v i c a l f r a c t u r e and  of the s t r i a t u m ,  GAD  et a l . , 1978b).  the s t r i a t a l sample i n which the GAD  the p r e l i m i n a r y  retrograde  The  l e v e l s of the n e u r o p e p t i d e s  substance  met-enkephalin were a l s o measured at t h i s s u r v i v a l time, and  a d d i t i o n a group r e c e i v i n g a u n i l a t e r a l i n j e c t i o n o f v e h i c l e o n l y  in was  69  T a b l e 8.  Glutamic a c i d d e c a r b o x y l a s e a c t i v i t y  i n v a r i o u s b r a i n areas  a f t e r u n i l a t e r a l l e s i o n s of the n i g r o s t r i a t a l pathway w i t h 6-OHDA.  Enzyme (nmol/mg p r o t e i n / h r ) Control  Lesioned  % of c o n t r o l  Accumbens  15815.3  19818.3  125**  Body o f caudate-putamen  10917.6  13517.9  124***  T a i l of caudate-putamen  99.4±6.7  12714.2  128**  Globus p a l l i d u s  22117.8  26615.7  120***  Substantia  29318.9  321113  110  Area  nigra  Enzyme a c t i v i t i e s a r e the mean ± S.E.M. o f the l e s i o n e d and c o n t r a l a t e r a l s i d e of 12 r a t s . the p a i r e d _t t e s t .  S t a t i s t i c a l e v a l u a t i o n was conducted  **p < .01, ***p < .001.  using  70  included i n the a n a l y s i s . As shown i n T a b l e 9, t h e decrease 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 t h i s study was a g a i n v i r t u a l l y complete.  T h i s i n d i c a t e s that the l e s i o n  was t o t a l and no r e g e n e r a t i o n of t h e dopamine neurons had o c c u r r e d .  The  v e h i c l e i n j e c t e d c o n t r o l group showed a s m a l l r e d u c t i o n i n mean t y r o s i n e hydroxylase a c t i v i t y  i n the s t r i a t u m , however, t h i s d i d not r e a c h  s t a t i s t i c a l s i g n i f i c a n c e at the f i v e percent l e v e l . i n t h i s regard that previous reports  have found  I t i s worth n o t i n g  ascorbate-  c o n t a i n i n g v e h i c l e t o be s l i g h t l y t o x i c . t o t h e n i g r a l dopamine c e l l s ( W o l f a r t h e t a l . , 1977). The  i n c r e a s e i n GAD a c t i v i t y observed  a t t h r e e months f o l l o w i n g  6-OHDA was i d e n t i c a l to t h a t seen a t 28 days. denervated  The GAD a c t i v i t y  i n the  s t r i a t u m was s i g n i f i c a n t l y h i g h e r than t h a t i n e i t h e r the  c o n t r a l a t e r a l u n i n j e c t e d s t r i a t a o r i n the s a l i n e i n j e c t e d s t r i a t a o f t h e control The  group. l e s i o n o f the n i g r o s t r i a t a l dopamine system w i t h 6-OHDA d r a m a t i -  c a l l y reduced and  t h e l e v e l s of substance P i n both t h e head o f t h e s t r i a t u m  i n the SN.  levels.  S a l i n e i n j e c t e d animals showed no change i n substance P  The l e v e l s of met-enkephalin  were u n a f f e c t e d i n e i t h e r t h e  s t r i a t u m o r t h e SN f o l l o w i n g t h e 6-OHDA l e s i o n s  (Table 9 ) .  DISCUSSION Although catecholamine  6-OHDA has been found  neurons ( H C k f e l t and U n g e r s t e d t , 1973; Maler e t a l . , 1973)  the changes observed study make  to be a s e l e c t i v e n e u r o t o x i n f o r  i n GAD a c t i v i t y and substance P l e v e l s i n the p r e s e n t  i t e v i d e n t t h a t secondary  and p r o b a b l y i n d i r e c t e f f e c t s on  o t h e r n e u r o n a l systems c a n a l s o occur f o l l o w i n g 6-OHDA i n j e c t i o n s .  Thus,  the s e l e c t i v e l e s i o n o f t h e n i g r o s t r i a t a l dopamine system  i n an  enhanced s t r i a t a l GAD a c t i v i t y .  resulted  As GAD a c t i v i t y was measured a t s u b s t r a t e  T a b l e 9.  Enzyme a c t i v i t i e s and n e u r o p e p t i d e l e v e l s i n the s t r i a t u m and s u b s t a n t i a n i g r a t h r e e months a f t e r the i n j e c t i o n of s a l i n e or 6-hydroxydopamine i n t o t h e l e f t n i g r o s t r i a t a l pathway.  Striatum R  L  Substantia R  nigra L  Saline i n j e c t i o n s : 1.62+ .33  0.98±.17  Glutamate d e c a r b o x y l a s e 60.9+ 4.3 (nmol/mg p r o t e i n / h r )  56.2±4.3  Tyrosine (nmol/mg  hydroxylase protein/hr)  Substance P (pg/mg t i s s u e )  369+ 26  353±25  1918H55  1854+189  6-OHDA i n j e c t i o n s : Tyrosine hydroxylase (nmol/mg p r o t e i n / h r )  1.181.12 0.01±.01* (.85% o f c o n t r o l )  66.7±4.0** Glutamate d e c a r b o x y l a s e 56.1 +3.7 (119% o f c o n t r o l ) (nmol/mg p r o t e i n / h r ) Substance P (pg/mg t i s s u e )  393121 232±12* (59% o f c o n t r o l )  Met-enkephalin (pg/mg t i s s u e )  533±84  696±128  1997+170 1013±67* (51% o f c o n t r o l ) 153±18  135±8  *p < .001 compared t o c o n t r a l a t e r a l s i d e . o r i p s i l a t e r a l s a l i n e i n j e c t i o n ( 2 - t a i l _t t e s t ) **p < .05 compared to c o n t r a l a t e r a l s i d e (1 t a i l e d s a l i n e i n j e c t i o n ( 2 - t a i l _t t e s t )  t t e s t ) or i p s i l a t e r a l  concentrations  w e l l above s a t u r a t i o n , i t appears t h a t t h e i n c r e a s e d  measured i n d i c a t e s an i n c r e a s e d  activity  enzyme v e l o c i t y , i n d i c a t i n g an a c t u a l  i n c r e a s e i n t h e amount o f enzyme r a t h e r than an a c t i v a t i o n o f e x i s t i n g enzyme.  T h i s has i n f a c t been found i n a r e c e n t  GAD i n c r e a s e  k i n e t i c a n a l y s i s of t h e  f o l l o w i n g 6-OHDA l e s i o n s ( F i b i g e r e t a l . , 1980).  It i s evident  from t h e r e g i o n a l examination t h a t these l e s i o n s  i n c r e a s e GAD a c t i v i t y  i n t h e caudate-putamen as w e l l as i n the n u c l e u s  accumbens and t h e GP.  A n o n - s i g n i f i c a n t tendency toward i n c r e a s e d  a c t i v i t y was a l s o observed i n t h e SN.  GAD  Inasmuch as i t i s known t h a t t h e  d i s t r i b u t i o n o f GAD w i t h i n t h e SN i s n o t homogeneous (Fonnum e t a l . , 1978a; Fonnum e t a l . , 1974) i t i s p o s s i b l e t h a t t h e r e o c c u r r e d cant  regional increases  signifi-  i n n i g r a l GAD which may have been obscured when  the whole SN was assayed.  Saavadra e t a l . (1978) have r e c e n t l y  evidence t h a t such r e g i o n a l i n c r e a s e s  provided  i n n i g r a l GAD do indeed occur a f t e r  6-OHDA l e s i o n s o f t h e dopamine neurons. These changes i n GAD a c t i v i t y contrast  i n t h e b a s a l g a n g l i a stand  i n marked  t o those observed i n P a r k i n s o n ' s d i s e a s e which t h e 6-OHDA l e s i o n  i s thought t o mimic.  I n P a r k i n s o n i s m , a c o n s i s t e n t decrease i n b a s a l  g a n g l i a GAD a c t i v i t y has been observed  (Lloyd and H o r n y k i e w i c z , 1973;  McGeer and McGeer, 1976b, McGeer, e t a l . , 1971; T h i s decrease has l e d t o t h e p r o p o s a l  Rinne e t a l . , 1974).  that i n Parkinson's disease the  d e c r e a s e s i n s t r i a t a l and n i g r a l GAD a c t i v i t y a r e compensatory changes and  represent  an attempt t o m a i n t a i n n i g r o s t r i a t a l dopamine  ( L l o y d and Davidson, 1979). t h i s hypothesis untenable. striatal  The p r e s e n t  observations  would seem t o make  Thus, complete d e s t r u c t i o n o f the  nigro-  dopamine system w i t h 6-OHDA r e s u l t s not i n a compensatory d e c r e a s e  i n GAD a c t i v i t y , but r a t h e r , i n a s i g n i f i c a n t The  transmission  present  increase.  f i n d i n g s thus have important i m p l i c a t i o n s f o r t h e e t i o l o g y  73  of P a r k i n s o n ' s d i s e a s e . demonstrated  They s t r o n g l y suggest t h a t i n a d d i t i o n to the  pathology o f the dopaminergic  systems i n t h i s d i s e a s e a  p a t h o l o g y of t h e s t r i a t a l GABA neurons may a l s o be p r e s e n t . observed 1973;  The d e c r e a s e  i n p a l l i d a l and n i g r a l GAD i n P a r k i n s o n i s m ( L l o y d and H o r n y k i e w i c z ,  McGeer and McGeer, 1976b; McGeer e t a l . ,  1971) i s e n t i r e l y  consis-  t e n t w i t h t h i s h y p o t h e s i s and suggests t h a t the s t r i a t o p a l l i d a l and s t r i a t o n i g r a l GABA t r a c t s a t r o p h y i n t h i s d i s e a s e . In t h i s r e g a r d , i t has been found t h a t the c e r e b r o s p i n a l f l u i d of GABA a r e markedly 1976)  reduced i n P a r k i n s o n i a n p a t i e n t s  levels  (Lakke and T e e l k e n ,  and i n f a c t a r e even lower than those found i n p a t i e n t s w i t h  Huntington's d i s e a s e where a d e g e n e r a t i o n o f s t r i a t a l GABA neurons i s c o n s i d e r e d t o be a main p a t h o l o g i c a l f i n d i n g  (Chase and Tammiga,  1979).  A l s o , Rinne e t a l . (1979) r e p o r t a s i g n i f i c a n t r e d u c t i o n i n GABA l e v e l s i n the c e r e b r a l and c e r e b e l l a r c o r t i c e s i n P a r k i n s o n i s m .  Rinne e t a l . (1979)  have a l s o observed s i g n i f i c a n t c o r r e l a t i o n s between GAD a c t i v i t y and the symptoms of P a r k i n s o n ' s d i s e a s e . These d e f i c i t s i n t h e GABA system may have some importance a p h a r m a c o l o g i c a l therapy f o r t h i s d i s e a s e . experiments neurons.  i n designing  We have seen t h a t i n the animal  s t r i a t a l GAD i n c r e a s e s i n response t o the l o s s o f the dopamine  In P a r k i n s o n ' s d i s e a s e t h i s a p p a r e n t l y i s not p o s s i b l e because of  the concomitant  atrophy of the GABA neurons.  Thus, j u s t as L-dopa has been  used t o r e p l a c e the l o s t dopamine i n n e r v a t i o n i n the s t r i a t u m , perhaps a GABA a g o n i s t c o u l d be of use i n r e p l a c i n g the GABA d e f i c i t , i n the SN where both GAD and GABA r e c e p t o r s a r e reduced Rinne e t a l . ,  1978; 1979).  particularly  (Lloyd et a l . ,  1977b;  In t h i s r e g a r d B a r t h o l i n i e t a l . (1979) have  found i n p r e l i m i n a r y s t u d i e s t h a t the GABA a g o n i s t and prodrug SL 76003, when combined w i t h L-dopa t h e r a p y , p r e v e n t s L-dopa-induced  involuntary  movements and i n c r e a s e s the dosage of L-dopa t h a t can be g i v e n , r e s u l t i n g  74  i n g r e a t l y a m e l i o r a t e d P a r k i n s o n i a n symptoms w i t h t h e absence o f i n v o l u n t a r y movements. Pharmacological have been found SN  manipulations  of the n i g r o s t r i a t a l dopamine system  t o a f f e c t the l e v e l s o f substance  P i n t h e s t r i a t u m and  (Hong and C o s t a , 1978; Hong e t a l . , 1978a; P e t t i b o n e e t a l . , 1978a;  1978b).  The p r e s e n t experiments have shown t h a t t h e d e s t r u c t i o n o f the  n i g r o s t r i a t a l dopamine system w i t h i n t r a c e r e b r a l 6-OHDA i s a s s o c i a t e d w i t h a marked r e d u c t i o n i n substance  P l e v e l s i n the s t r i a t u m and the SN.  T h i s c o u l d r e p r e s e n t a change i n substance it  P t u r n o v e r i n these a r e a s , o r  c o u l d be due t o t h e a c t u a l d e s t r u c t i o n o f s t r i a t o n i g r a l substance P  neurons by 6-OHDA. tation.  S e v e r a l o b s e r v a t i o n s argue a g a i n s t the l a t t e r  I n Experiment 1, complete h e m i t r a n s e c t i o n s  the GP reduced  substance  the l e v e l s o f substance d e s t r u c t i o n of substance result  interpre-  a t t h e a n t e r i o r p o l e of  P l e v e l s i n t h e EP, GP and SN, b u t d i d not a f f e c t P i n the head of t h e s t r i a t u m .  This i n d i c a t e s that  P axons p r o j e c t i n g t o t h e p a l l i d u m and SN does not  i n the death o f the s t r i a t a l , substance  they possess many i n t r i n s i c  collaterals  P neurons, presumably because  (Experiment  2).  A l s o , the 6-OHDA  i n j e c t i o n was made i n t o the n i g r o s t r i a t a l pathway a t a l e v e l a t which the descending fibers  s t r i a t o n i g r a l system i s s e p a r a t e d  ( T u l l o c h e t a l . , 1978).  from t h e a s c e n d i n g  I n a d d i t i o n , i t has been r e p o r t e d t h a t  i n t r a c i s t e r n a l i n j e c t i o n s of 250 ug of 6-OHDA does not a f f e c t substance  P levels  dopamine  s p i n a l cord  ( S i n g e r e t a l . , 1979) i n d i c a t i n g t h a t 6-OHDA i s not i n  g e n e r a l t o x i c t o substance  P neurons.  I n c i d e n t a l l y , these l e s i o n s , had no  e f f e c t on t h e met-enkephalin l e v e l s i n the s t r i a t u m . The 1978;  observations that pharmacological manipulations  Hong e t a l . , 1978a; P e t t i b o n e e t a l . , 1978a; 1978b) or l e s i o n s o f the  n i g r o s t r i a t a l dopamine neurons can reduce substance and  (Hong and C o s t a ,  SN suggest  P l e v e l s i n the striatum  t h a t g r e a t c a u t i o n should be e x e r c i s e d i n i n t e r p r e t i n g t h e  75  r e s u l t s of l e s i o n s t u d i e s where a decrease i n p e p t i d e l e v e l s i s observed. Although such d e c r e a s e s c o u l d i n d i c a t e the i n t e r r u p t i o n of a p e p t i d e pathway, i n the absence turnover due  o f o t h e r evidence, a secondary  change i n p e p t i d e  to d i s r u p t i o n o f some unknown system would seem an e q u a l l y  l i k e l y explanation. The decreased l e v e l s of substance P i n the SN p r o b a b l y r e p r e s e n t an i n c r e a s e i n the t u r n o v e r of substance P.  A s i m i l a r d e c r e a s e has been  observed a f t e r c h r o n i c h a l o p e r i d o l treatment Hong et a l . , 1978a).  (Hong and C o s t a ,  I n t r a n i g r a l substance P i n c r e a s e s the f i r i n g of the  n i g r o s t r i a t a l dopamine neurons  (Davies and Dray, 1976;  Walker et a l . , 1976)  thereby i n c r e a s i n g the r e l e a s e of dopamine i n the s t r i a t u m 1977) .  1978;  (Cheramy e t a l . ,  These o b s e r v a t i o n s suggest t h a t substance P t u r n o v e r i n c r e a s e s i n  the n i g r a i n response to a decrease i n dopamine t r a n s m i s s i o n i n the s t r i a t u m . The decrease i n s t r i a t a l  substance P l e v e l s seen i n the p r e s e n t study  be a m a n i f e s t a t i o n o f the prolonged a c t i v a t i o n o f t h e s e substance P and may  may  neurons  not have been apparent a t the s h o r t e r s u r v i v a l times used i n the  chronic haloperidol studies  (Hong and C o s t a , 1978;  Hong et a l . , 1978a).  T h i s h y p o t h e s i s suggests t h a t drugs which i n c r e a s e the e f f e c t i v e n e s s of substance P t r a n s m i s s i o n , p a r t i c u l a r l y i n the SN, may therapy f o r P a r k i n s o n ' s d i s e a s e .  be of v a l u e i n the  76 EXPERIMENT 6: THE  NIGROTECTAL PROJECTION: A BIOCHEMICAL AND  ULTRASTRUCTURAL  STUDY As  confirmed i n Experiment 1, the SN r e c e i v e s both a GABA and  substance P p r o j e c t i o n from the s t r i a t u m .  In r e t u r n , the SN  a  provides  the s t r i a t u m w i t h a dense dopaminergic i n n e r v a t i o n a r i s i n g from the The  non-dopaminergic SNR  c e l l s have a l s o been suggested to send a s p a r s e  p r o j e c t i o n to the s t r i a t u m area p r o j e c t s "massively G r a y b i e l , 1978; thalamus 1976;  F a u l l and  ( F i b i g e r et a l . , 1972)  to the s u p e r i o r  Hopkins and  (Carpenter  SNC.  and  colliculus  N i e s s e n , 1976;  Peters,  Mehler, 1978;  1972;  and  in addition  ( F a u l l and Mehler,  R i n v i k et a l . , 1976)  Carpenter et a l . , 1976;  R i n v i k , 1975).  this  and  1978;  the  C l a v i e r et a l . ,  L e s i o n s t u d i e s have shown  t h a t the s t r i a t o n i g r a l pathway i s e s s e n t i a l f o r the e x p r e s s i o n  of some types  of s t r i a t a l - m e d i a t e d b e h a v i o u r  Ungerstedt,  1971).  For  (Lee  et a l . , 1980;  Marshall  and  example, the r o t a t o r y behaviour induced by dopamine a g o n i s t s  in  r a t s w i t h u n i l a t e r a l l e s i o n s of the n i g r o s t r i a t a l dopamine c e l l s i s dependent upon the i n t e g r i t y of t h i s pathway ( M a r s h a l l and I t has  U n g e r s t e d t , 1971).  been suggested t h a t the p r o j e c t i o n from the SNR  to the  c o l l i c u l u s i n t e r a c t s w i t h the t e c t o s p i n a l system which r e g u l a t e s muscles i n v o l v e d  i n head o r i e n t a t i o n (York and  Faber, 1977).  the 'output of the b a s a l g a n g l i a i n v o l v e d  superior  the neck  Thus,  this  pathway may  be  behaviour.  In t h i s study, the e f f e c t s o f l e s i o n s of the SN on neurochemical  parameters i n the s u p e r i o r c o l l i c u l u s was the t r a n s m i t t e r s  i n t h i s system.  of t h i s p r o j e c t i o n was  The  in rotational  examined i n an attempt to determine  f i n e s t r u c t u r e of the t e r m i n a l  a l s o examined to understand f u r t h e r the  r e l a t i o n s h i p s of t h i s p r o j e c t i o n w i t h the c o l l i c u l a r  boutons  synaptic  cells.  METHODS Male W i s t a r r a t s weighing about 300 For  electron microscopic  g were used f o r a l l experiments.  analysis six rats received a stereotaxic i n j e c t i o n  of  10 y C i of [ H ] l e u c i n e  (New England N u c l e a r ,  3  s a l i n e i n t o the l e f t  SNR.  80 Ci/mmol) i n 0.5 V l  The c o o r d i n a t e s of the i n j e c t i o n were AP + 2.9;  ML + 2.1; DV + 3.0, w i t h r e s p e c t to s t e r e o t a x i c z e r o , w i t h the i n c i s o r bar 5 mm  above the h o r i z o n t a l plane.  t r a c t completely  avoids  With these c o o r d i n a t e s the needle  the s u p e r i o r c o l l i c u l u s .  and  the cannula was l e f t  The  r a t s were p e r f u s e d  The i n j e c t i o n was 20 min,  i n p l a c e an a d d i t i o n a l f i v e min a f t e r the i n j e c t i o n .  through the h e a r t 24 h r l a t e r w i t h 500 ml o f 4%  paraformaldehyde, 0.5% g l u t a r a l d e h y d e , and 0.6% d e x t r o s e A phosphate b u f f e r , pH 7.4.  One mm  i n 0.1 M sodium  cubes were cut from the deeper l a y e r s of  the i p s i l a t e r a l  s u p e r i o r c o l l i c u l u s and processed  f o r electron microscopic  autoradiography  a c c o r d i n g to the method of H a t t o r i et a l . (1973a).  The  b l o c k s were p o s t f i x e d i n the above s o l u t i o n o v e r n i g h t and then i n 1% b u f f e r e d osmium t e t r o x i d e (pH 7.4) f o r two hours. embeded i n e p o n - a r a l d i t e mixture  was a p p l i e d by the standard  copper g r i d s , and I l f o r d  loop t e c h n i q u e .  exposure a t 4°C the s e c t i o n s were developed Kodak Rapid citrate.  Fix.  then  and g o l d s e c t i o n s cut on an LKB microtome.  S e c t i o n s were p i c k e d up on formvar-coated emulsion  The t i s s u e was  L4  A f t e r one month  i n M i c r o d o l X and f i x e d i n  S e c t i o n s were c o u n t e r s t a i n e d w i t h u r a n y l a c e t a t e and l e a d  G r a i n d i s t r i b u t i o n and a r e a d e t e r m i n a t i o n s  were performed on  e l e c t r o n micrographs taken w i t h a P h i l i p s 201 e l e c t r o n microscope, and the r e l a t i v e g r a i n d e n s i t y over v a r i o u s c e l l to  S a l p e t e r and McHenry  s t r u c t u r e s was c a l c u l a t e d a c c o r d i n g  (1973).  T h i r t y m i c r o n s e c t i o n s through the i n j e c t i o n s i t e were c u t on a f r e e z i n g microtome and examined w i t h a l i g h t m i c r o s c o p i c technique.  S l i d e s were dipped  i n emulsion  autoradiographic  (Kodak NTB 3 ) , d r i e d and then  s t o r e d i n l i g h t - t i g h t boxes c o n t a i n i n g s i l i c a a t 4°C f o r two weeks. s l i d e s were developed  i n D-19, f i x e d and c o u n t e r s t a i n e d w i t h c r e s y l  In the b i o c h e m i c a l  s t u d i e s , the l e f t  The violet.  SN was i n j e c t e d w i t h f i v e nmoles  78  of  KA i n one  y l of b u f f e r e d s a l i n e .  k i l l e d by c e r v i c a l f r a c t u r e .  The  Three weeks l a t e r , the animals were  s u p e r i o r c o l l i c u l i were d i s s e c t e d  from c o r o n a l s l i c e s and homogenized i n 30 volumes of 0.32 A 75 y l a l i q u o t was buffer  removed and added to 75 y l of 50 mM  (pH 7) c o n t a i n i n g 0.2%  assay GAD  and CAT  T r i t o n X-100.  fresh  M sucrose.  Tris-acetate  A l i q u o t s of t h i s were used to  as d e s c r i b e d i n E x p e r i m e n t a l 1.  The P  fraction  2  was  o b t a i n e d from the o r i g i n a l homogenate by the method of Simon et a l . (1976) and was  resuspended  i n 50 volumes of 0.32  M sucrose.  A s p a r t a t e and  g l y c i n e uptake were measured i n 5 min i n c u b a t i o n s of 60 y l of t h i s s i o n i n Krebs-Ringer phosphate  b u f f e r w i t h 10 M  England N u c l e a r 30 Ci/mol) , or  [U-  moi).  6  1 L f  of  C ] g l y c i n e (New  saline.  c o n t r o l i n c u b a t i o n s sodium i o n was  the b u f f e r . for  P r o t e i n was  determined  (New  England N u c l e a r ; 68 C i /  The f i l t e r s were counted  ml water and 9 ml of a s o l u t i o n of 10% naphthalene In  [U-^C]aspartate  A f t e r i n c u b a t i o n , the samples were r i n s e d onto M i l l i p o r e  and washed w i t h 0.9%  suspen-  filters  i n a m i x t u r e of  and 0.4%  PPO  one  i n dioxane.  r e p l a c e d by c h o l i n e or potassium i n  on both the s u c r o s e homogenates used  uptake and on the samples used f o r the enzyme assays by the method of  Lowry et a l . (1951). RESULTS The r e s u l t s of t h i s study have r e c e n t l y been p u b l i s h e d ( V i n c e n t et a l . , 1978a).  In the a u t o r a d i o g r a p h i c experiments  s i t e was  c o n f i n e d t o SNR  ( F i g . 15).  to be d i s c u s s e d the i n j e c t i o n  The d i s t r i b u t i o n of s i l v e r g r a i n s i n  the s u p e r i o r c o l l i c u l u s one day a f t e r the i n j e c t i o n of SNR  i s summarized i n T a b l e 10.  compared to unmyelinated  axons.  [ H]leucine into 3  M y e l i n a t e d axons were p r e f e r e n t i a l l y  i n d i c a t i n g t h a t l a b e l t r a n s p o r t e d from the SN was t i a l manner i n these s t r u c t u r e s  labeled  Boutons forming symmetrical synapses  major d e n d r i t e s were l a b e l e d w i t h a h i g h r e l a t i v e g r a i n d e n s i t y of localized  the  with  2.4,  i n a preferen-  ( S a l p e t e r and McHenry, 1973).  The  labeled  79  F i g u r e 15.  Schematic r e p r e s e n t a t i o n of the s i t e and [H]leucine reticulata nucleus;  i n j e c t i o n s i n the (SNR).  CC  pars compacta.  of  the  s u b s t a n t i a n i g r a pars  crus c e r e b r i ; IP  LM m e d i a l l e m i n i s c u s ;  extent  SNC  interpeduncular  substantia nigra  '71a  80  Table  10.  D i s t r i b u t i o n of s i l v e r g r a i n s i n the s u p e r i o r c o l l i c u l u s a f t e r the i n j e c t i o n of [ H ] l e u c i n e 3  i n t o the substantia n i g r a .  Number o f grains  Percent grains  Percent area  51  15.9  6.5  2.4*  Asymmetric  5  1.4  4.4  0.32  Preterminal  8  2.5  2.6  0.96  Myelinated  98  30.5  21.5  1.4*  Unmyelinated  40  12.4  15.3  0.81  Soma  23  7.2  10.8  0.67  Dendrite  66  20.7  24.3  0.85  Glia  27  8.2  12.8  0.64  4  1.2  1.8  0.67  Structure  Relative grain density  Bouton Symmetric  Axon  Blood  vessel  Total  *RGD > 1.  322  100  24 hr  100  81  boutons were one to two microns packed  i n diameter  s l i g h t l y pleomorphic v e s i c l e s  and c o n t a i n e d moderately  (Fig.  16).  The r e s u l t s of the b i o c h e m i c a l s t u d i e s of the s u p e r i o r  colliculus  a f t e r u n i l a t e r a l KA l e s i o n s of the SN a r e summarized i n T a b l e 11.  No  s i g n i f i c a n t changes were observed i n a s p a r t a t e or g l y c i n e uptake o r i n the a c t i v i t y of CAT i n t h e s u p e r i o r c o l l i c u l u s a f t e r l e s i o n i n g the SN. GAD a c t i v i t y showed a s i g n i f i c a n t d e c r e a s e s i n the s u p e r i o r  However,  colliculus  i p s i l a t e r a l t o the l e s i o n when compared t o t h e c o n t r a l a t e r a l c o n t r o l  side.  DISCUSSION Glutamic a c i d d e c a r b o x y l a s e i s t h e r a t e l i m i t i n g enzyme i n the synt h e s i s of GABA and i s a u s e f u l marker f o r GABA nerve endings.  The  d i s t r i b u t i o n of GAD c o r r e l a t e s w e l l w i t h t h a t of GABA (Fahn, 1975) and GAD a c t i v i t y i s c o n c e n t r a t e d i n synaptosomes i n areas known t o r e c e i v e GABA afferents  (Fonnum and Walberg, 1973).  Thus, the s i g n i f i c a n t drop i n GAD  a c t i v i t y observed i n t h e s u p e r i o r c o l l i c u l u s  f o l l o w i n g the SN l e s i o n  suggests t h a t a GABA p r o j e c t i o n t o t h e tectum was l e s i o n e d .  I t has been  shown t h a t i n t r a n i g r a l i n j e c t i o n s of KA s i m i l a r t o those used i n the p r e s e n t study r e s u l t 1978d).  i n a s u b s t a n t i a l r e d u c t i o n i n n i g r a l GAD a c t i v i t y  (Nagy e t a l . ,  I n j e c t i o n s o f KA have been suggested t o damage o n l y neurons  c e l l b o d i e s a t the s i t e o f i n j e c t i o n , s p a r i n g f i b e r s of passage  with  (Coyle and  Schwarcz, 1976; McGeer and McGeer, 1976a).  Thus, the d e c r e a s e i n GAD  a c t i v i t y observed  t o be due t o the l o s s o f n i g r a l  i n the c o l l i c u l u s appears  GABA neurons which p r o j e c t to the tectum. The m o r p h o l o g i c a l d a t a o b t a i n e d i n t h e p r e s e n t study c o n f i r m e a r l i e r r e p o r t s o f a p r o j e c t i o n from the SNR t o t h e s u p e r i o r c o l l i c u l u s .  Further-  more, u l t r a s t r u c t u r a l examination r e v e a l e d t h a t the t e r m i n a l s l a b e l e d  after  SN i n j e c t i o n s form symmetrical synapses w i t h the major d e n d r i t e s of t e c t a l neurons  and c o n t a i n pleomorphic v e s i c l e s .  T h i s morphology i s i d e n t i c a l  82  F i g u r e 16.  Examples of l a b e l e d boutons i n the s u p e r i o r  colliculus  f o l l o w i n g i n j e c t i o n of  substantia  n i g r a pars r e t i c u l a t a . symmetrical s y n a p t i c dendrites  (D)  and  [ H ] l e u c i n e i n t o the 3  The  labeled terminals  contacts  form  (arrow heads) w i t h major  c o n t a i n s l i g h t l y pleomorphic v e s i c l e s  M a g n i f i c a t i o n A: X 46,309;  B: X 62,842.  83 T a b l e 11.  B i o c h e m i c a l changes i n the s u p e r i o r c o l l i c u l u s a f t e r l e s i o n s of the s u b s t a n t i a n i g r a . 5 min.  Uptake expressed as pmol/mg p r o t e i n /  Enzyme a c t i v i t y as nmol/mg p r o t e i n / h r .  n  Control  Lesion  % of c o n t r o l  G l y c i n e uptake  6  209127  223+29  107  Aspartate  6  352+40  302+30  86  18.211.3  18.5+1.2  Choline  uptake  a c e t y l t r a n s f e r a s e 12  Glutamate d e c a r b o x y l a s e  *p  < 0.001;  8  Student's _t t e s t  221+5.7  132+14^  102 60*  84 to t h a t o f P u r k i n j e  c e l l terminals  r a d i o a c t i v e GABA t r a n s p o r t  (McGeer  i n t h e deep c e r e b e l l a r n u c l e i l a b e l e d by et a l . , 1975) and w i t h presumed  GABA  boutons i n the SN l a b e l e d by uptake o f r a d i o a c t i v e GABA ( H a t t o r i e t a l . , 1973b).  They a r e a l s o s i m i l a r to those synapses o f the c e r e b e l l a r  cortex  or SN which s t a i n immunocytochemically f o r GAD (McLaughlin e t a l . , 1974; Ribak et a l . , 1976). Anderson and Y o s h i d a (1977) have r e c e n t l y  suggested t h a t some n i g r o -  t e c t a l neurons send c o l l a t e r a l s t o the v e n t r o m e d i a l thalamus, and have shown t h a t  SN s t i m u l a t i o n  r e s u l t s i n monosynaptic i n h i b i t i o n i n the thalamus.  A n a t o m i c a l s t u d i e s have a l s o found t h a t many n i g r a l neurons p r o j e c t to both the tectum and t h e v e n t r o m e d i a l thalamus and the morphology  o f the n i g r o t h a l a m i c  the n i g r o t e c t a l t e r m i n a l s  (Bentivoglio  boutons i s i d e n t i c a l t o t h a t o f  ( K u l t a s - I l i n s k y e t a l . , 1978).  the n i g r o t e c t a l p r o j e c t i o n may a l s o be i n h i b i t o r y . (1977) o r i g i n a l l y r e p o r t e d stimulation  studies  have demonstrated t h a t t e c t a l  c o l l i c u l u s and suggests t h a t  p r o j e c t i o n may u t i l i z e GABA as w e l l .  et a l . , 1979).  (Deniau e t a l . , 1978).  w i t h t h e p r e s e n t e v i d e n c e f o r a GABA-containing p r o j e c t i o n  from t h e SNR t o t h e s u p e r i o r  been p u b l i s h e d  suggests t h a t  t h a t a few t e c t a l u n i t s were a c t i v a t e d by  i n the SN, more r e c e n t  i s consistent  This  A l t h o u g h York and Faber  u n i t s a r e m o n o s y n a p t i c a l l y i n h i b i t e d by SN s t i m u l a t i o n This  e t a l . , 1979),  the n i g r o t h a l a m i c  In f a c t , evidence has v e r y  supporting t h i s hypothesis (DiChiara  recently  e t a l . , 1979; F e l t e r  85 GENERAL DISCUSSION The  s t r i a t u m has  cognitive functions.  been i m p l i c a t e d In the present  i n both motor behaviour and study the pathways by which  s t r i a t u m can a f f e c t these p r o c e s s e s have been examined u s i n g and  h i s t o l o g i c a l techniques.  a p a r a l l e l p a i r of s t r i a t a l EP  and  SN.  One  Evidence has  been p r o v i d e d  of these systems c o n t a i n s  Further  Fig.  substance  The  histochemical  little  These pathways a r e summarized i n  i s known c o n c e r n i n g  s t r i a t a l p r o j e c t i o n s to the GP on the s t r i a t o n i g r a l system and  and  GABA  evidence  on the  r o l e of these"pathways i n m e d i a t i n g the f u n c t i o n s of the b a s a l As  P.  results  the f i r s t  In the f o l l o w i n g d i s c u s s i o n some s p e c u l a t i o n s  w i l l be o f f e r e d .  GP,  a l s o been proposed  w i t h GABA-T support the h y p o t h e s i s of a p a l l i d o h a b e n u l a r  a n i g r o t e c t a l GABA p r o j e c t i o n . 17.  i n d i c a t i n g that  GABA, the other  pathway, w h i l e the r e s u l t s of Experiment 6 p r o v i d e d for  biochemical  experiments have suggested a r o l e for GABA i n  the output pathways of the b a s a l g a n g l i a . obtained  the  e f f e r e n t systems e x i s t s p r o j e c t i n g to the  A m e t - e n k e p h a l i n - c o n t a i n i n g s t r i a t o p a l l i d a l pathway has (Experiment 1).  i n more  EP,  the  possible ganglia  f u n c t i o n s of  the d i s c u s s i o n w i l l  the  concentrate  the n i g r a l output pathways.  NIGROSTRIATAL REGULATION The provides serves The  s t r i a t o n i g r a l pathway appears to serve  two  major f u n c t i o n s : ' i t  an output pathway from the s t r i a t u m and, . i n a d d i t i o n , i t ;  as a feedback system r e g u l a t i n g the n i g r o s t r i a t a l dopamine system.  r e g u l a t i o n of the n i g r o s t r i a t a l dopamine neurons has  i n t e n s e debate f o r almost twenty y e a r s . noted t h a t n e u r o l e p t i c drugs i n c r e a s e d s t r i a t u m and  suggested t h a t t h i s was  In 1963  C a r l s s o n and L i n q v i s t  the turnover  due  of dopamine i n the  to a compensatory a c t i v a t i o n of  the monoamine neurons subsequent to dopamine r e c e p t o r observations  been a s u b j e c t  blockade.  of Kehr et a l . (1972) t h a t the dopamine a g o n i s t  The  apomorphine  of  86  Figure 17:  Summary diagram of the inputs and outputs of the basal ganglia. G = GABA pathway P = Substance P pathway  C E R E B R A L CORTEX INTRALAMINAR [> STRIATUM [> E N T O P E D U N C U L A R THALAMUS NUCLEUS  SUBSTANTIA A VENTROLATERAL NIGRA 1/ THALAMUS G  ^  SUPERIOR COLLICULUS  &  o  MOTOR CORTEX  X^L  SPINAL CORD  87  and  a n t a g o n i s t h a l o p e r i d o l c o u l d i n f l u e n c e dopamine metabolism i n the  s t r i a t u m even a f t e r h e m i t r a n s e c t i o n s from t h e i r c e l l b o c i e s l e d C a r l s s o n of " a u t o r e c e p t o r s " Autoreceptors the SN  (Aghajanian  s e p a r a t i n g the dopamine t e r m i n a l s (1975) to p o s t u l a t e the  or dopamine r e c e p t o r s on the dopamine t e r m i n a l s . f o r dopamine have a l s o been p o s t u l a t e d to e x i s t i n and  Bunney, 1973;  Groves e t a l . , 1975).  t i c a l l y a p p l i e d dopamine or apomorphine i n h i b i t s  Iontophore-  the dopamine c e l l s i n  the n i g r a i n a h a l o p e r i d o l - r e v e r s i b l e manner (Aghajanian 1973).  existence  and  Bunney,  In f a c t the dopamine c e l l s have been found to be much more  s e n s i t i v e than s t r i a t a l neurons to the a c t i o n of dopamine a g o n i s t s ( S k i r b o l l et a l . , 1979). systemic  E l e c t r o p h y s i o l o g i c a l s t u d i e s have found  apomorphine i n h i b i t s dopamine c e l l  of the s t r i a t a l neurons w i t h KA  v a t i o n t h a t i n t r a n i g r a l and s t r i a t a l dopamine turnover The  f i r i n g even a f t e r d e s t r u c t i o n  ( B a r i n g et a l . , 1980), s u g g e s t i n g  SN i s the s i t e of a c t i o n f o r t h i s drug. systemic  T h i s i s supported  and  by  that  the  the  obser-  apomorphine have s i m i l a r e f f e c t s  on  (Maggi et a l . , 1977).  concept of n i g r a l dopamine r e c e p t o r s has  from a n a t o m i c a l  that  biochemical  also received  demonstrations of the mechanisms  support necessary  f o r dopamine t r a n s m i s s i o n i n the n i g r a .  Thus, n i g r a l d e n d r i t e s have been  shown to c o n t a i n dopamine ( B j o r k l u n d and  L i n d v a l l , 1975;  as w e l l as the s y n t h e t i c enzyme t y r o s i n e h y d r o x y l a s e  Felton,  ( P i c k e l et a l . , 1976).  A l s o s p e c i a l i z e d o r g a n e l l e s which accumulate the dopamine analog dopamine have been observed  ( H a t t o r i et a l . , 1979;  e t a l . , 1977)  and  both d e n d r o - d e n d r i t i c  Wilson  et a l . , 1977)  and  dendro-axonic ( H a t t o r i et a l . , 1979;  connections  have been r e p o r t e d .  (Hajdu et a l . , 1973;  In a d d i t i o n , a  dependent dopamine r e l e a s e ( H e f t i and L i c h t e n s t e i g e r , 1978; 1976;  Korf  et a l . , 1976;  5-hydroxy-  Mercer et a l . , 1979;  Wilson  S a n d r i , 1979)  1977)  N i e o u l l o n e t a l . , 1977;  Reubi  and  calcium-  Geffen et a l . ,  Paden e t a l . , 1976),  88  dopamine b i n d i n g s i t e s  (Nagy e t a l . , 1978c; Quick e t a l . , 1979) and a  dopamine-sensitive adenylate cyclase Phillipson  (Kebabian and Saavadra, 1976;  and Horn, 1976; Spano e t a l . , 1976; T r a f i c a n t e e t a l . , 1976)  have been shown t o e x i s t  i n the SN.  A l t h o u g h the e x i s t e n c e of dopamine a u t o r e c e p t o r s i n the s t r i a t u m and  SN appears to e x p l a i n the a c t i o n s o f s y s t e m i c apomorphine on dopamine  n e u r o n a l a c t i v i t y , some d i f f i c u l t i e s a r i s e when the a c t i o n s o f o t h e r dopaminergic drugs are c o n s i d e r e d .  Amphetamine i s thought to r e l e a s e  dopamine and to b l o c k the n e u r o n a l uptake of t h i s amine i n both t e r m i n a l regions  ( C a r l s s o n e t a l . , 1965; G l o w i n s k i e t a l . , 1966) and a t the  dopamine c e l l bodies et  a l . , 1976).  (Groves e t a l . , 1975; N i e o u l l o n e t a l . , 1977; Paden  T h i s i s accompanied by a d e p r e s s i o n of n e u r o n a l  firing  i n both the s t r i a t u m and SN (Bunney and A g h a j a n i a n , 1973; Rebec and Groves, 1975).  The i n h i b i t i o n of dopamine c e l l f i r i n g i s markedly  a t t e n u a t e d by l e s i o n s of the s t r i a t o n i g r a l pathway p r o v i d i n g  support  f o r the h y p o t h e s i s of C o r r o d i e t a l . (1967) t h a t amphetamine a c t s v i a a n e u r o n a l feedback loop to modulate dopamine c e l l a c t i v i t y .  However,  amphetamine a l s o appears to a c t i n the SN to i n h i b i t dopamine c e l l s s i n c e at h i g h doses i t s d e p r e s s a n t e f f e c t  on n i g r a l c e l l s s t i l l  occurs  i n the absence of the s t r i a t o n i g r a l system (Bunney and A g h a j a n i a n , 1978).  The e f f e c t  of amphetamine.on dopamine t u r n o v e r i n the s t r i a t u m i s  a l s o not a b o l i s h e d by the d e s t r u c t i o n of the s t r i a t o n i g r a l  pathway  ( A r g i o l a s e t a l . , 1978). Systemic dopamine a n t a g o n i s t s such as h a l o p e r i d o l i n c r e a s e the f i r i n g r a t e of the dopamine neurons of the SN.  I n c o n t r a s t to the i n h i b i t i o n of  dopamine c e l l f i r i n g by apomorphine and amphetamine which are r e s p e c t i v e l y u n a f f e c t e d o r p a r t i a l l y attenuated, the a c t i o n s of h a l o p e r i d o l on dopamine cell  f i r i n g a r e c o m p l e t e l y a b o l i s h e d by i n t e r r u p t i o n o f the s t r i a t o n i g r a l  89  pathway (Kondo and  Iwatsubo, 1980).  T h i s i n d i c a t e s t h a t the  striatonigral  feedback system mediates the a c t i o n s of n e u r o l e p t i c s on dopamine neuron activity. Strong  support  f o r t h i s hypothesis  comes from o b s e r v a t i o n s  k i n e t i c a c t i v a t i o n of s t r i a t a l t y r o s i n e h y d r o x y l a s e .  on  the  This e f f e c t i s  dependent upon an i n c r e a s e i n impulse flow i n the n i g r o s t r i a t a l neurons (Roth et a l . , 1975)  and  occurs  ( Z i v k o v i c e t a l . , 1974). i n dopamine c e l l  firing  f o l l o w i n g systemic  Neuroleptic-induced  n e u r o l e p t i c treatment  activation, like  i s a b o l i s h e d by l e s i o n s of the  pathway (Gale et a l . , 1978).  it  (Gale and  appears t h a t systemic  G u i d o t t i , 1976;  dopamine a n t a g o n i s t s  kept i n mind t h a t h a l o p e r i d o l can b l o c k  while  nigral  affect  Thus,  the dopamine neurons However, i t must  the a c t i o n s of apomorphine  and  NIGROSTRIATAL DOPAMINE SYSTEM  presence of a s t r i a t o n i g r a l pathway has  been known f o r q u i t e some  time, and much evidence i n d i c a t e s t h a t GABA i s a t r a n s m i t t e r i n t h i s (see Experiment 1 ) .  system  S t i m u l a t i o n of the s t r i a t u m r e s u l t s i n a p i c r o t o x i n -  r e v e r s i b l e i n h i b i t i o n of n i g r a l neurons ( F e l t z , 1971;  Precht  1971).  striatonigral  T h i s suggests t h a t GABA c o u l d f u n c t i o n i n the  feedback system c o n t r o l l i n g dopamine c e l l a c t i v i t y . comes from the o b s e r v a t i o n  that intravenous  and  Yoshida,  Support f o r t h i s  i n j e c t i o n s of p i c r o t o x i n  r e v e r s e the amphetamine-induced i n h i b i t i o n of n i g r a l c e l l s , and effect  i s dependent upon the s t r i a t o n i g r a l neurons (Bunney and  1978).  T h i s suggests t h a t amphetamine a c t s i n the s t r i a t u m to  the  be  independent of t h i s feedback system.  GABA REGULATION OF THE The  striatonigral  Gale e t a l . , 1978).  predominantly v i a the s t r i a t o n i g r a l feedback l o o p .  amphetamine which seem to be  increase  A l s o d i r e c t a p p l i c a t i o n of n e u r o l e p t i c s to  the s t r i a t u m r e s u l t s i n a c t i v a t i o n (Gale et a l . , 1978) a p p l i c a t i o n does not  the  this Aghajanian, increase  f i r i n g r a t e of s t r i a t o n i g r a l GABA neurons which i n t u r n i n h i b i t  the  90  dopamine c e l l s o f the SN. Intracerebroventricular peritoneal reported  (Biswas and C a r l s s o n , 1977b) i n j e c t i o n s of GABA have been  to i n c r e a s e s t r i a t a l dopamine l e v e l s s u g g e s t i n g  of dopamine c e l l and  (Biswas and C a r l s s o n , 1977a) and i n t r a -  firing.  T h i s has a l s o been observed w i t h  the GABA a g o n i s t  prodrug SL 76002 ( L l o y d e t a l . , 1979) or w i t h muscimol, another GABA  agonist  (Anden e t a l . , 1979; L l o y d et a l . , 1979).  GABA l e v e l s by systemic  AOAA r e v e r s e s  E l e v a t i o n o f endogenous  i n j e c t i o n o f GABA-T i n h i b i t o r s such as AOAA a l s o  i n c r e a s e s s t r i a t a l dopamine l e v e l s and  an i n h i b i t i o n  (Anden, 1974; Biswas and C a r l s s o n ,  t h e i n c r e a s e i n dopamine t u r n o v e r  (Anden, 1974; L a h t i and Losey, 1974), w h i l e enhance h a l o p e r i d o l - i n d u c e d (Gale et a l . , 1978).  induced  1977b)  by n e u r o l e p t i c s  i s o n i a z i d or p i c r o t o x i n  a c t i v a t i o n of 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  F i n a l l y , c h r o n i c e l e v a t i o n o f b r a i n GABA l e v e l s  r e s u l t s i n dopamine r e c e p t o r  s u p e r s e n s i t i v i t y i n the s t r i a t u m ,  suggesting  t h a t the n i g r o s t r i a t a l dopamine c e l l s have been c h r o n i c a l l y i n h i b i t e d (Ferkany e t a l . , 1980).  These r e s u l t s i n d i c a t e t h a t measures which  i n c r e a s e c e n t r a l GABA f u n c t i o n i n h i b i t  the f i r i n g o f n i g r a l dopamine  cells.  Although GABA may have some a c t i o n on dopamine r e l e a s e d i r e c t l y i n the s t r i a t u m  (Doble e t a l . , 1980; G i o r g i e f f - C h e s s e l e t e t a l . , 1979;  S t a r r , 1978a; Stoof and Mulder, 1977; Stoof i n f l u e n c e c l e a r l y occurs  i n the SN.  e t a l . , 1979) i t s main  H-GABA r e c e p t o r s appear to occur  3  on the dopamine c e l l s o f the' SN, s i n c e t h e i r d e n s i t y decreases f o l l o w i n g 6-OHDA l e s i o n  ( G u i d o t t i e t a l . , 1978).  S i m i l a r decreases i n H-GABA  b i n d i n g have been observed i n the SN o f P a r k i n s o n i a n  3  patients (Lloyd et  al.', 1977b; Rinne e t a l . , 1978; 1979) i n which the n i g r o s t r i a t a l dopamine c e l l s a r e known t o degenerate.  Lesions  o f the s t r i a t o n i g r a l pathway  i n c r e a s e the d e n s i t y of H-GABA or H-muscimol b i n d i n g s i t e s 3  3  I a d o r o l a , 1980; G u i d o t t i et a l . , 1979; Waddington and Cross,  (Gale and 1978a) i n  91  a way s u g g e s t i v e  of denervation  s u p e r s e n s i t i v i t y . The o b s e r v a t i o n of  Bunney and A g h a j a n i a n (.1978) t h a t KA l e s i o n s of t h e s t r i a t o n i g r a l system g r e a t l y i n c r e a s e the responses of SNC c e l l s f u r t h e r supports  the i d e a that these  t o i o n t o p h o r e t i c GABA  r e c e p t o r s can d i s p l a y s u p e r s e n s i -  tivity. Anden and Stock (1973) and K e l l y and Moore (1978b) have found  that  the l o c a l a p p l i c a t i o n o f GABA onto n i g r a l dopamine neurons causes an i n c r e a s e i n s t r i a t a l dopamine l e v e l s s i m i l a r t o t h a t seen a f t e r GABA a g o n i s t s .  T h i s does not occur  (Andeh and Stock, 1973).  systemic  a f t e r i n t r a s t r i a t a l GABA a p p l i c a t i o n  These r e s u l t s suggest t h a t n i g r a l GABA r e c e p t o r s  a r e i n v o l v e d i n the r e g u l a t i o n of dopamine c e l l s .  Gale and G u i d o t t i  (1976) a l s o p o i n t t o t h e r o l e o f GABA i n the SN w i t h t h e i r t h a t i n t r a n i g r a l muscimol b l o c k s of 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 .  observation  the n e u r o l e p t i c - i n d u c e d a c t i v a t i o n B i c u c u l l i n e i n j e c t i o n s o f the SN  b l o c k t h i s e f f e c t i n d i c a t i n g t h a t s p e c i f i c GABA r e c e p t o r s a r e i n v o l v e d . W a l t e r s e t a l . (1979) have found t h a t muscimol or GABA-T i n h i b i t o r s also i n h i b i t hydroxylase  the h a l o p e r i d o l - i n d u c e d  a c t i v i t y i s measured i n v i v o .  n-dipropylacetate with  i n c r e a s e observed when t y r o s i n e  (Valproate)  Their observation  that  i s more e f f e c t i v e than AOAA i s c o n s i s t e n t  the o b s e r v a t i o n of I a d o r a o l a and Gale (1979) that V a l p r o a t e  selective-  l y i n c r e a s e s GABA l e v e l s i n the s t r i a t o n i g r a l t e r m i n a l s w h i l e AOAA appears to i n c r e a s e p r i m a r i l y other n i g r a l GABA p o o l s . the h y p o t h e s i s  put forward  These o b s e r v a t i o n s  i n . Experiment 3 t h a t the GABA-T present  s t r i a t o n i g r a l t e r m i n a l s may be d i r e c t l y i n v o l v e d i n c o n t r o l l i n g  support i n the  nigral  GABA t r a n s m i s s i o n . In summary, the r e s u l t s d e s c r i b e d above i n d i c a t e t h a t GABA r e l e a s e d from the s t r i a t o n i g r a l t e r m i n a l s a c t s t o i n h i b i t n i g r o s t r i a t a l dopamine neurons.  the f i r i n g of the  I n times o f i n c r e a s e d  dopaminergic  92  stimulation i n the striatum  ( i . e . a f t e r amphetamine o r L-dopa) the  s t r i a t o n i g r a l GABA neurons would show an i n c r e a s e i n t h e i r  activity,  thereby i n h i b i t i n g the n i g r a l dopamine c e l l s .  d u r i n g a time  Conversely,  of decreased dopaminergic a c t i v i t y i n the s t r i a t u m tics)  (i.e. after  neurolep-  t h e a c t i v i t y o f the s t r i a t o n i g r a l GABA neurons d e c r e a s e s ,  the dopamine c e l l s  of t h e SN t o i n c r e a s e t h e i r  allowing  f i r i n g rate.  SUBSTANCE P REGULATION OF THE NIGROSTRIATAL DOPAMINE SYSTEM Substance P i s a l s o p r e s e n t and  i n t h e s t r i a t o n i g r a l system (Experiment 1)  thus c o u l d be invoked as a p a r t i c i p a n t i n the feedback c o n t r o l o f  n i g r a l dopamine c e l l s . striatal  Systemic substance P has been r e p o r t e d  dopamine turnover  to increase  ( S t a r r et a l . , 1978) as have i n j e c t i o n s of  substance P i n t o the l a t e r a l v e n t r i c l e (Magnusson e t a l . , 1976).  Intra-  n i g r a l a p p l i c a t i o n of substance P has been found t o produce a s i m i l a r i n c r e a s e i n s t r i a t a l dopamine t u r n o v e r ,  as e v i d e n c e d by i n c r e a s e d  striatal  l e v e l s o f t h e dopamine metabolitesDOPAC and HVA (Waldemier e t a l . , 1978). James and S t a r r (1979) r e p o r t t h a t t h i s i n c r e a s e i n s t r i a t a l turnover  i s associated with  dopamine  substance P i n j e c t i o n s o f the SNC.  When the  i n j e c t i o n i s made i n t o the SNR a decrease i n s t r i a t a l HVA r e s u l t s . In v i v o s t u d i e s have a l s o shown t h a t i n t r a n i g r a l a p p l i c a t i o n of substance P i n c r e a s e s dopamine r e l e a s e i n the i p s i l a t e r a l (Cheramy et a l . , 1977).  caudate n u c l e u s  I n c o n t r a s t , n i g r a l a p p l i c a t i o n of a n t i b o d i e s  a g a i n s t substance P, which f u n c t i o n as substance P a n t a g o n i s t s , a decrease i n s t r i a t a l Behavioural  dopamine r e l e a s e  produces  (Cheramy et a l . , 1978).  experiments a l s o suggest a r o l e f o r t h e s t r i a t o n i g r a l  substance P system i n the r e g u l a t i o n of dopamine n e u r o n a l a c t i v i t y . and  Koella  Oipe  (1977) r e p o r t t h a t u n i l a t e r a l i n t r a n i g r a l substance P  i n j e c t i o n s induce c o n t r a l a t e r a l r o t a t i o n .  James and S t a r r  (1979) have  found that t h i s r o t a t i o n f o l l o w i n g SNC i n j e c t i o n s i s a s s o c i a t e d w i t h an  93  i n c r e a s e d t u r n o v e r o f dopamine i n t h e i p s i l a t e r a l s t r i a t u m , and were a b l e to b l o c k t h i s response w i t h h a l o p e r i d o l  (James and S t a r r , 1977).  These  o b s e r v a t i o n s agree w i t h the h y p o t h e s i s t h a t animals r o t a t e away from t h e s i d e w i t h i n c r e a s e d s t r i a t a l dopamine f u n c t i o n  (Ungerstedt e t a l . ,  1969).  I n j e c t i o n o f substance P e i t h e r i n t o t h e l a t e r a l v e n t r i c l e (Katz, 1979)  o r t h e SN ( K e l l e y e t a l . ,  1979) produces a grooming  response  which  can be b l o c k e d by l e s i o n s o f t h e dopamine t e r m i n a l s i n t h e s t r i a t u m w i t h 6-OHDA ( K e l l e y and I v e r s e n , 1978; 1979).  Bilateral nigral  application  r e s u l t s i n a s t r o n g s t e r e o t y p e d r e a r i n g and s n i f f i n g w i t h no c o n c u r r e n t enhancement o f l o c o m o t i o n ( K e l l e y and I v e r s e n , 1979). a l s o b l o c k e d by s t r i a t a l 6-OHDA l e s i o n s  T h i s response i s  ( K e l l y and I v e r s e n , 1979).  r e s u l t s a r e c o n s i s t e n t w i t h the h y p o t h e s i s t h a t s t e r e o t y p y r e s u l t s an a c t i v a t i o n of t h e n i g r o s t r i a t a l dopamine neurons  These from  ( K e l l y e t a l . , 1975)  and t h a t such an a c t i v a t i o n c a n be produced by substance P. I n t r a n i g r a l substance P has a l s o been found t o produce a d r a s t i c r e t r o g r a d e amnesia  f o r a p a s s i v e avoidance t a s k (Huston and S t a u b l i , 1978)  T h i s e f f e c t t o o can be e x p l a i n e d by a s t i m u l a t i o n o f t h e n i g r o s t r i a t a l dopamine neurons  ( F i b i g e r and P h i l l i p s ,  1976).  F i n a l l y , i n j e c t i o n s o f substance P i n t o t h e mesolimbic A-10 dopamine c e l l r e g i o n produce an i n c r e a s e d locomotor response which can be b l o c k e d by t h e i n f u s i o n o f n e u r o l e p t i c s i n t o t h e n u c l e u s accumbens o r by 6-OHDA l e s i o n s o f these neurons  (Kelley et a l . ,  1979).  A l s o t h e locomotor  response t o s y s t e m i c amphetamine i s p o t e n t i a t e d by these substance P injections  (Stinus et a l . ,  1978).  These r e s u l t s a r e c o n s i s t e n t w i t h t h e  h y p o t h e s i s t h a t enhanced locomotor a c t i v i t y r e s u l t s from a c t i v a t i o n o f the mesolimbic dopamine neurons  (Kelly et a l . ,  substance P c o u l d p l a y a r o l e i n t h i s  1975) and i n d i c a t e  that  response.  In summary i t appears t h a t t h e e f f e r e n t s of t h e s t r i a t u m can e x e r t  94  o p p o s i t e e f f e c t s on the n i g r a l dopamine neurons.  The GABA system p r o v i d e s  a n e g a t i v e feedback system i n h i b i t i n g dopaminergic  a c t i v i t y , w h i l e the  substance P system a c t s to i n c r e a s e the a c t i v i t y o f these neurons. through these two systems  Perhaps  the f i r i n g r a t e o f t h e dopamine neurons can  be c o n t r o l l e d w i t h g r e a t p r e c i s i o n . GABA, SUBSTANCE P AND THE OUTPUT OF THE STRIATUM In a d d i t i o n t o t h e i r r o l e i n the r e g u l a t i o n o f the n i g r a l dopamine cells,  the e f f e r e n t s of the s t r i a t u m must p r o v i d e output pathways f o r t h e  e x p r e s s i o n of t h e many f u n c t i o n s i n which dopamine and the s t r i a t u m have been i m p l i c a t e d .  D y s k i n e t i c d i s o r d e r s have been a t t r i b u t e d  t o a hyper-  dopaminergic a c t i v i t y or t o a c h o l i n e r g i c - d o p a m i n e r g i c imbalance i n the striatum.  GABA involvement i n d y s k i n e s i c syndromes has a l s o been suggested  due t o j t h e s i g n i f i c a n t r e d u c t i o n i n s t r i a t o n i g r a l GABA and GAD observed i n Huntington's d i s e a s e ( B i r d and I v e r s e n , 1974; Enna e t a l . , 1976; McGeer and McGeer, 1976; P e r r y e t a l . , i n cerebrospinal f l u i d sia patients  1973; Urquhart e t a l . ,  1975).  A reduction  l e v e l s o f GABA has a l s o been found i n t a r d i v e d y s k i n e -  (Neophytides e t a l . ,  1978).  I n animal s t u d i e s i t has been found  t h a t d e c r e a s i n g s t r i a t a l GABA f u n c t i o n by the i n t r a s t r i a t a l i n j e c t i o n o f GABA a n t a g o n i s t s r e s u l t s i n a G A B A - r e v e r s i b l e d y s k i n e s i a 1979).  (Robin e t a l . ,  These r e s u l t s suggest t h a t treatment w i t h drugs which  i n c r e a s e GABA  f u n c t i o n i n the s t r i a t u m c o u l d be o f b e n e f i t i n t r e a t i n g d y s k i n e s i a s i n man. Both muscimol (Chase and Tamminga, 1979) and V a l p r o a t e ( L i n n o i l a e l a l . , 1976) have been found t o be of some b e n e f i t i n t a r d i v e  dyskinesia.  A l s o , the d i r e c t GABA a g o n i s t SL 76002 has been found to be of b e n e f i t i n t r e a t i n g the d y s k i n e s i a s r e s u l t i n g from L-dopa therapy i n P a r k i n s o n ' s disease ( B a r t h o l i n i et a l . ,  1979).  I t has a l s o been found t o be o f some '  v a l u e i n t h e e a r l y stages o f Huntington's d i s e a s e .  However, V a l p r o a t e  has not been found b e n e f i c i a l i n Huntington's d i s e a s e (Lenman e t a l . , 1976;  95  Shoulson e t a l . , 1976).  The poor r e s u l t s o b t a i n e d  w i t h GABA drugs i n  Huntington's d i s e a s e , e s p e c i a l l y i n i t s l a t e r s t a g e s , l o s s o f s t r i a t a l GABA r e c e p t o r s i n t h i s d i s e a s e L l o y d e t a l . , 1977a).  c o u l d be due t o the  ( I v e r s e n e t a l . , 1979;  A l s o , as the u s u a l treatment f o r Huntington's  d i s e a s e i n v o l v e s n e u r o l e p t i c drugs, the d e c r e a s e i n GABA r e c e p t o r s by  these drugs (Trabbuchi  et a l . , 1978) c o u l d a l s o tend  agonists i n e f f e c t i v e i n this  a c t i o n o f dopamine i n the s t r i a t u m .  from an i n h i b i t i o n of the  Thus, the motor d e f i c i t s  by h a l o p e r i d o l or r e s e r p i n e a r e r e v e r s e d  produced  by L-dopa o r apomorphine.  It  that AOAA, muscimol and SL 76002 i n j e c t e d s y s t e m i c a l l y  potentiate neuroleptic-induced 1976;  GABA  disease.  A k i n e s i a and c a t a l e p s y a r e thought to r e s u l t  has been r e p o r t e d  t o render  produced  catalepsy  ( B i g g i o et a l . , 1977; K a a r i a i n e n ,  K e l l e r e t a l . , 1976; L l o y d and Davidson, 1979; Worms and L l o y d , 1978).  I t was suggested t h a t t h i s was due to a f a c i l i t a t i o n of the GABA i n h i b i t i o n of the dopamine neurons i n the SN ( K e l l e r et a l . , 1976; L l o y d and Davidson, 1979;  Matsui  and Deguchi, 1977).  However, t h e o b s e r v a t i o n  O l l e y , 1971) t h a t l e s i o n s o f t h e GP antagonize  ( C o s t a l l and  haloperidol catalepsy  i n d i c a t e s t h a t the s t r i a t o p a l l i d a l pathway may be i n v o l v e d i n t h i s  response.  Indeed, i t has been found t h a t d i r e c t e l e v a t i o n of p a l l i d a l GABA l e v e l s or the i n j e c t i o n of muscimol i n t o the GP p o t e n t i a t e s the c a t a l e p t i c a c t i o n o f haloperidol (EOS,  (Matsui and Kamioka, 1978).  Also  ethanolamine-o-sulphate  a GABA-T i n h i b i t o r ) i n j e c t i o n s o f the GP r e s u l t i n an a k i n e s i c s t a t e  which i s not b l o c k e d  by amphetamine (Pycock et a l . , 1976).  The EOS  i n j e c t i o n o f the GP a l s o b l o c k s t h e h y p e r a c t i v e response i n d u c e d - i n t h e r a t by d i r e c t s t i m u l a t i o n of t h e dopamine r e c e p t o r s (Pycock and Horton, 1976).  i n the accumbens  Perhaps t h i s i n d i c a t e s t h a t i n h i b i t i o n of  the r e c e n t l y d i s c o v e r e d GABA pathway from the accumbens t o t h e GP (Walaas and  Fonnum, 1979) mediates t h e i n c r e a s e d locomotor response t o amphetamine.  96  Neuroleptic  drugs have been found to i n c r e a s e the turnover  i n t h e GP and the nucleus accumbens (Marco e t a l . , 1976). turnover  t h a t t h i s change c o u l d be a b i o c h e m i c a l  f o r t h e mechanisms i n v o l v e d i n the symptomatic r e l i e f e l i c i t e d by these drugs (Costa e t a l . , 1978). develops t o the i n c r e a s e i n the turnover T h i s may be a b i o c h e m i c a l  to the c a t a l e p t o g e n i c 1977) .  marker  of s c h i z o p h r e n i a  In c o n t r a s t ,  tolerance  o f GABA i n the GP (Marco e t a l . ,  i n d i c a t o r o f the t o l e r a n c e t h a t  p r o p e r t i e s of n e u r o l e p t i c s  (Ezrin-Waters  A l s o , Moroni e t a l . (1979) have observed an i n c r e a s e d  turnover  i n the GP f o l l o w i n g e i t h e r systemic  Evidence a l s o e x i s t s i m p l y i n g  occurs  and Seeman, GABA  or i n t r a s t r i a t a l morphine and  suggest t h a t t h i s may a l s o be i n d i c a t i v e o f o p i a t e  catalepsy.  t h a t the e l e v a t i o n of n i g r a l GABA l e v e l s  does not p o t e n t i a t e n e u r o l e p t i c c a t a l e p s y . by  The i n c r e a s e d  i n t h e accumbens p e r s i s t s a f t e r c h r o n i c h a l o p e r i d o l (Marco  et a l . , 1976) s u g g e s t i n g  1976) .  o f GABA  E l e v a t i o n o f n i g r a l GABA l e v e l s  the l o c a l i n j e c t i o n o f EOS (Dray e t a l . , 1975) or g a b a c u l l i n e , another  GABA-T i n h i b i t o r i n d u c e s h y p e r a c t i v i t y which i s b l o c k e d picrotoxin  (Matsui and Kamioka, 1978).  by i n t r a n i g r a l  Furthermore, i n t r a n i g r a l  injection  of muscimol produces b e h a v i o u r a l  s t i m u l a t i o n and a n t a g o n i z e s h a l o p e r i d o l -  induced  e t a l . , 1977).  catalepsy  (Scheel-Kruger  E l e v a t i o n o f n i g r a l GABA  l e v e l s by l o c a l i n j e c t i o n s of g a b a c u l l i n e a l s o a n t a g o n i z e s h a l o p e r i d o l catalepsy  (Matsui and Kamioka, 1978).  e f f e c t s probably  As d i s c u s s e d below, these  depend upon t h e i n h i b i t i o n o f e f f e r e n t s from the SNR.  In summary, these r e s u l t s i n d i c a t e t h a t the systemic b r a i n GABA f u n c t i o n p o t e n t i a t e s n e u r o l e p t i c - i n d u c e d the h y p e r a c t i v e not  the SN.  behavioural  e l e v a t i o n of  c a t a l e p s y and a n t a g o n i z e s  response t o dopamine a g o n i s t s v i a an a c t i o n i n the GP and  T h i s suggest that some o f the b e h a v i o u r a l  f u n c t i o n s o f the  n i g r o s t r i a t a l dopamine system might be mediated through an a c t i o n on the s t r i a t o p a l l i d a l GABA pathway.  97 Rats w i t h u n i l a t e r a l 6-OHDA l e s i o n s of the n i g r o s t r i a t a l dopamine  system  r o t a t e away from t h e s i d e w i t h the l e s i o n when c h a l l e n g e d w i t h apomorphine (Ungerstedt e t a l . , 1969).  T h i s has been a t t r i b u t e d t o d e n e r v a t i o n super-  s e n s i t i v i t y of the dopamine r e c e p t o r s  f o l l o w i n g the l e s i o n .  This  has been found t o be reduced by e l e c t r o c o a g u l a t i o n of the caudate ipsilateral  response nucleus  t o the 6-OHDA l e s i o n ( M a r s h a l l and U n g e r s t e d t , 1977) i n d i c a t i n g  t h a t s t r i a t a l neurons  are involved.  As c r u s c e r e b r i l e s i o n s or h e m i t r a n s e c -  t i o n s j u s t r o s t r a l t o t h e SN a l s o reduce t h i s r o t a t i o n a l response, i t has been suggested  t h a t t h e s t r i a t o n i g r a l f i b e r s mediate  ( M a r s h a l l and U n g e r s t e d t , 1977).  this  behaviour  Garcia-Munoz e t a l . (1977) have found  t h a t l e s i o n s o f t h e s t r i a t o n i g r a l pathway r e s u l t s i n a marked response towards the l e s i o n e d  turning  s i d e w i t h b o t h apomorphine and amphetamine,  i n s p i t e of i n t a c t dopamine neurons.  S i m i l a r e f f e c t s have been found  a f t e r e x t e n s i v e u n i l a t e r a l a b l a t i o n s o f the s t r i a t u m  (Anden e t a l . , 1966)  or more r e c e n t l y f o l l o w i n g KA induced d e g e n e r a t i o n of s t r i a t a l c e l l b o d i e s (Schwarcz  e t a l . , 1979).  Also, e l e c t r i c a l stimulation of the striatum  has been found t o r e s u l t i n c o n t r a l a t e r a l head t u r n i n g , and t h i s i s abolished 1980).  by l e s i o n s of t h e GP o r SN on t h e s t i m u l a t e d  These r e s u l t s i n d i c a t e t h a t some  s i d e (Lee e t a l . ,  o f t h e motor asymmetries  observed a f t e r m a n i p u l a t i o n s o f s t r i a t a l dopaminergic mediated  behaviour  function are  through the s t r i a t o n i g r a l pathway. OUTPUTS OF THE SUBSTANTIA NIGRA  I t i s known t h a t the e f f e r e n t s which a r i s e i n t h e head o f t h e s t r i a t u m synapse p r e f e r e n t i a l l y i n t h e SNR on non-dopaminergic et  a l . , 1973b; T u l l o c h e t a l . , 1978).  predominantly t o the VM-VL thalamus et  neurons  (Hattori  The SNR c o n t a i n s neurons  projecting  (Beckstead e t a l . , 1979; Carpenter  a l . , 1976; Carpenter and P e t e r , 1972; C o l e e t a l . , 1964; F a u l l and  Mehler, 1978; F i b i g e r e t a l . , 1972; M e t t l e r , 1970; R i n v i k ,  1975) t h e  98  the s u p e r i o r c o l l i c u l u s  ( A f i f i and K a e l b e r ,  G r a y b i e l , 1978; Hopkins and N i e s s e n ,  1965; Beckstead e t a l . , 1979;  1976; Jayaraman e t a l . , 1977; R i n v i k  e t a l . , 1976) and t h e n u c l e u s tegmentus p e d u n c u l o p o n t i s , (Beckstead  e t a l . , 1979).  p a r compacta  E l e c t r o p h y s i o l o g i c a l and a n a t o m i c a l  evidence  suggests t h a t many SNR neurons p r o j e c t t o both the v e n t r o l a t e r a l thalamus and  the s u p e r i o r c o l l i c u l u s  et a l . , 1979).  (Anderson and Y o s h i d a , 1977; B e n t i v o g l i o  I t i s t h e r e f o r e o f i n t e r e s t t h a t Deniau e t a l . (1976)  have shown t h a t those neurons i n the SNR which a r e i n h i b i t e d a f t e r s t i m u l a t i o n of the s t r i a t u m a r e a l s o a n t i d r o m i c a l l y d r i v e n from t h e v e n t r o l a t e r a l thalamus. and  tectum may r e p r e s e n t The  SN  T h i s i n d i c a t e s t h a t t h e n i g r a l e f f e r e n t s t o the thalamus  behavioural  s t r o n g l y support  major output systems f o r the s t r i a t u m .  e f f e c t s seen f o l l o w i n g the i n j e c t i o n o f KA i n t o the t h i s suggestion.  Thus, u n i l a t e r a l ,  a d m i n i s t r a t i o n o f KA r e s u l t s i n c h r o n i c side  ( D i C h i a r a et a l . , 1977; O l i a n a s  t u r n i n g away from the l e s i o n e d  e t a l . , 1978a), w h i l e  n i g r a l i n j e c t i o n s produce c h r o n i c s t e r e o t y p e d 1978a).  intranigral  bilateral  b e h a v i o u r s ( O l i a n a s et a l . ,  These e f f e c t s mimic a s t r i a t a l dopamine r e c e p t o r  stimulation,  a l t h o u g h they a r e independent o f t h e n i g r o s t r i a t a l dopamine pathway ( O l i a n a s et a l . , 1978a).  Thus, the s t e r e o t y p y  h a l o p e r i d o l , which a l s o f a i l s ( O l i a n a s et a l . , 1978a).  i s not antagonized by  to produce c a t a l e p s y  i n these animals  These r e s u l t s suggest t h a t dopamine r e c e p t o r  s t i m u l a t i o n i n the s t r i a t u m r e s u l t s v i a the s t r i a t o n i g r a l pathway i n the i n h i b i t i o n of t h e SNR output c e l l s .  T h i s c o u l d be accomplished by e i t h e r  an i n c r e a s e i n the f i r i n g of t h e s t r i a t o n i g r a l GABA c e l l s o r by a decrease i n the a c t i v i t y o f t h e s t r i a t o n i g r a l ' substance P c e l l s . As d i s c u s s e d  above, the b e h a v i o u r a l  responses t o i n t r a n i g r a l  P appear to be dependent upon t h e a s c e n d i n g dopamine systems.  substance  In contrast,  i n t r a n i g r a l a d m i n i s t r a t i o n of GABA a g o n i s t s produces an acute syndrome-  99  s i m i l a r to t h a t seen a f t e r KA l e s i o n s of the SN.  That i s , u n i l a t e r a l  n i g r a l i n j e c t i o n s o f GABA, muscimol or GABA-T i n h i b i t o r s r e s u l t s i n c o n t r a l a t e r a l turning et  (Dray e t a l . , 1975; O l i a n a s  a l . , 1977; Scheel-KrOger et a l . , 1977), w h i l e  produces i p s i l a t e r a l  e t a l . , 1978b; Oberlander t h a t of GABA  antagonists  r o t a t i o n ( O l i a n a s e t a l . , 1978b; Scheel-Kruger e t a l . , t  1977).  B i l a t e r a l i n j e c t i o n s o f p i c r o t o x i n r e s u l t s i n c a t a l e p s y which i s  r e s i s t a n t t o apomorphine a d m i n i s t r a t i o n  ( ( l i n a n a s e t a l . , 1978b), w h i l e  b i l a t e r a l i n j e c t i o n s . o f muscimol produce i n t e n s e s t e r e o t y p y  t h a t i s un-  a f f e c t e d by h a l o p e r i d o l ( O l i a n a s e t a l . , 1978b; Scheel-Krttger It  can, t h e r e f o r e , be suggested that s t r i a t a l  r e s u l t s i n increased  activity  striatal  dopamine r e c e p t o r  stimulation  i n the s t r i a t o n i g r a l GABA neurons i n c r e a s i n g  n i g r a l GABA r e l e a s e and thereby i n h i b i t i n g cells.  e t a l . , 1977).  This leads to stereotypy.  the non-dopaminergic SNR output  Conversely,  catalepsy  i n response t o  dopamine r e c e p t o r blockade depends upon a r e d u c t i o n i n GABA  r e l e a s e w i t h i n t h e SN, w i t h t h e consequent a c t i v a t i o n of the n i g r a l nondopaminergic e f f e r e n t s . This hypothesis  suggests t h a t n e u r o l e p t i c s should  GABA r e l e a s e d u r i n g c a t a l e p s y .  decrease n i g r a l  However, we have a l r e a d y  seen t h a t neuro-  l e p t i c s i n c r e a s e GABA a c t i o n i n t h e GP (Marco e t a l . , 1976).  This  i m p l i e s t h a t the s t r i a t o p a l l i d a l and s t r i a t o n i g r a l GABA systems a r e functionally dissociable.  T h i s i s i n agreement w i t h  the r e s u l t s found  i n Experiment 1 t h a t these two systems a r e a n a t o m i c a l l y What n e u r o t r a n s m i t t e r s provide  are contained  distinct.  i n the n i g r a l neurons which  these output pathways from the ;basal g a n g l i a ?  Recently, a decrease  i n n i g r a l GAD has been observed f o l l o w i n g t h e d e s t r u c t i o n o f n i g r a l neurons w i t h KA (Nagy e t a l . , 1978d).  T h i s has l e d to t h e s u g g e s t i o n  t h a t the  n i g r a , i n a d d i t i o n t o r e c e i v i n g a GABA i n n e r v a t i o n from t h e s t r i a t u m , c o n t a i n s a p o p u l a t i o n o f GABA neurons (Nagy e t a l . , 1978d).  Could GABA be  100  contained  i n the n i g r a l output pathways?  T h i s p o s s i b i l i t y was examined  i n Experiment 6 where a s i g n i f i c a n t decrease i n GAD a c t i v i t y was observed i n t h e s u p e r i o r c o l l i c u l u s f o l l o w i n g KA l e s i o n s o f the SN. t h a t GABA i s a t r a n s m i t t e r Electrophysiological  This  suggests  i n the n i g r a l e f f e r e n t s t o the tectum. (Anderson and Y o s h i d a , 1977) and a n a t o m i c a l  ( B e n t i v o g l i o e t a l . , 1979) evidence i n d i c a t e s t h a t the n i g r o t e c t a l neurons send c o l l a t e r a l s t o the VM-VL thalamus. nigrothalamic  neurons may a l s o u t i l i z e GABA as a t r a n s m i t t e r .  Y o s h i d a and Omata (1978) have r e p o r t e d the v e n t r o m e d i a l t h a l a m i c blocked has  T h i s i m p l i e s t h a t the  by p i c r o t o x i n .  In f a c t ,  t h a t the monosynaptic i n h i b i t i o n o f  neurons i n response t o n i g r a l s t i m u l a t i o n i s  A l s o , a decrease i n GAD a c t i v i t y  i n the thalamus  r e c e n t l y been found f o l l o w i n g n i g r a l KA or e l e c t r o l y t i c  lesions  ( D i C h i a r a e t a l . , 1979a; F e l t e r et a l . , 1979). The  GABA n i g r o t e c t a l and n i g r o t h a l a m i c  p r o j e c t i o n s a r e thus  c a n d i d a t e s f o r the m e d i a t i o n of s t r i a t a l e f f e r e n t i n f o r m a t i o n .  likely In t h i s  scheme, dopamine a g o n i s t s , by i n c r e a s i n g the a c t i v i t y o f the s t r i a t o n i g r a l GABA system and thus i n h i b i t i n g the n i g r a l GABA e f f e r e n t s i n t h e SNR, would act to r e l e a s e c e r t a i n thalamic  and t e c t a l u n i t s from  C o n v e r s e l y , n e u r o l e p t i c s , by d e c r e a s i n g  the a c t i v i t y o f the s t r i a t o n i g r a l  GABA c e l l s would i n h i b i t c e r t a i n t e c t a l and t h a l a m i c pathways.  inhibition.  u n i t s v i a these  These two c o n d i t i o n s a r e summarized i n F i g . 18.  DiChiara  e t a l . (1979b) have examined the p o s s i b i l i t y t h a t t h e GABA  pathway to t h e v e n t r o m e d i a l thalamus may mediate b a s a l g a n g l i a  output.  These workers have found t h a t the i n j e c t i o n o f muscimol i n t o the v e n t r o m e d i a l thalamus produces a c a t a l e p s y which i s not b l o c k e d Also,  by apomorphine.  the i n t r a t h a l a m i c i n j e c t i o n of muscimol had no e f f e c t on the  stereotypy catalepsy  induced by apomorphine.  Thus, these animals d i s p l a y both  and.stereotypy simultaneously.  P i c r o t o x i n i n j e c t i o n s of the  101  F i g u r e 18.  H y p o t h e t i c a l mechanisms o f a c t i o n f o r h a l o p e r i d o l and amphetamine.  I n c o n d i t i o n A amphetamine causes an i n c r e a s e d of dopamine (DA) w i t h i n the s t r i a t u m .  release  This results  ina  decrease i n the f i r i n g o f the s t r i a t o n i g r a l substance P cells  (P) and an i n c r e a s e i n the a c t i v i t y o f the s t r i a t o -  n i g r a l GABA f i b e r s  (GABA^.  to d e c r e a s e the e f f e c t r e l e a s e by i n h i b i t i n g  Together these systems a c t  of amphetamine on s t r i a t a l dopamine the n i g r a l dopamine neurons.  In  a d d i t i o n , the i n c r e a s e d a c t i v i t y of t h e s t r i a t o n i g r a l GABA fibers  i n h i b i t s the GABA c e l l s of the SNR which p r o j e c t  to the thalamus and tectum (GABA ). 2  This i n h i b i t i o n i s  b e h a v i o u r a l l y expressed i n the form o f s t e r e o t y p y .  In c o n d i t i o n B h a l o p e r i d o l a c t s t o i n h i b i t dopamine activity  i n the s t r i a t u m .  This results  i n an i n c r e a s e  i n s t r i a t o n i g r a l substance P a c t i v i t y and a d e c r e a s e i n s t r i a t o n i g r a l GABA a c t i v i t y , e f f e c t s which a c t t o i n c r e a s e the a c t i v i t y of the n i g r o s t r i a t a l dopamine neurons.  The  decrease i n a c t i v i t y o f the s t r i a t o n i g r a l GABA neurons a l s o results  i n increased  neurons o f the SNR. catalepsy.  a c t i v i t y i n the GABA p r o j e c t i o n T h i s l e a d s t o t h e development of  io! a  B  A /  Striatum  9  Thalamus  Tectum  ^  9 /  Thalamus  Tectum  O stereotypy  catalepsy  102  ventromedial  thalamus produce hyperactivity but no stereotypy, and i n  addition reverse haloperidol catalepsy.  Thus, the catalepsy produced  by the blockade of s t r i a t a l dopamine receptors appears to r e s u l t from the a c t i v a t i o n of the nigrothalamic neurons. It i s noteworthy that manipulation  of GABA i n the  ventromedial  thalamus affects c a t a l e p t i c behaviour but not stereotypy.  As discussed  above, increasing GABA function i n the SN r e s u l t s i n profound  stereotyped  behaviours that are independent of the dopamine system (Olianas et a l . , 1978b; Scheel-Kruger et a l . , 1977).  Thus, we would expect that some  n i g r a l efferents would be involved i n this process. efferents are involved may  A clue to which  come from the work of Cools  found that the blockade of GABA action i n the  (1979) who  parafasicular-centromedian  complex produces stereotypy which i s not blocked by haloperidol. SNR neurons are known to project to t h i s region (Ahlenius, Beckstead et a l . , 1979;  Clavier et a l . , 1976)  As  1978;  this observation raises  the p o s s i b i l i t y that i n h i b i t i o n of a GABA projection from SNR parafasicular-centromedian  has  to the  complex could be the basis for the stereotypic  response to dopamine agonists. The e l e c t r o p h y s i o l o g i c a l evidence that the projection from the  SNR  to the superior c o l l i c u l u s interacts with the tectospinal system which regulates the neck muscles involved i n head Faber, 1977)  o r i e n t a t i o n (York and  suggests that this pathway may mediate the r o t a t i o n a l  behaviours associated with u n i l a t e r a l manipulations dopamine system.  of the n i g r o s t r i a t a l  Support for this concept comes from the observation  that lesions of the tectospinal pathway or the superior c o l l i c u l u s markedly attenuate apomorphine-induced rotation i n rats with u n i l a t e r a l 6-OHDA lesions (Wirtshafter et a l . , 1978).  However, other workers have questioned  this result (Crossman and Sanbrook, 1978;  Lee et a l . , 1980;  R e a v i l l et a l . ,  103  1979)  leaving  the  role  of  the  nigrotectal  system  in  these  responses  in  doubt. The deep optokinetic Thus,the the  of  grey  of  layer  the  saccades.  layer  the  impair  as  1976).  ganglia  act  initiation  1976)  the  role  "readiness"  and  the  feedback  (Anderson  et  superior  movements  the  of  a l . ,  basal  1979).  head  a  feedback  for  regulation  of  eye  this  of  discharge  with  the  recorded of  the  in  theory  the  (i.e.  the  basal lesions  (Chandler  the  the  (Denny-Brown  pathway  zone  Mohler  and  time-lead  responses  visuomotor that and  and  intermediate  shortest  neural  in  regard,  1969).  part  movement  1973).  provide In  (Bowen,  of  a l . ,  this  upper  view  involved  deviations  "readiness"  have been  spinal  involved  and body  a route  the  also  visually  The are  could  be  the  in system  basal  Yanagisawa,  generation  of  these  considered.  ganglia  system  p a t h w a y may p r o v i d e  in  of  may b e  colliculus the  Thus,  nigrotectal  potentials  Finally, internal  of  have  signalling  and W u r t z , the  in  colliculus  (Mohler  in  units  workers  interpreted  tracking  et  within  mechanisms. in  to  (Sprague  layer)  result  visual  that  These  to  appear  pathway  grey  occulomotor found  superior  tracking  nigrotectal  intermediate  found  colliculus  visual  the  to  to  superior  in  have been  have  of  and  the  and  (1976)  the  of  access  1975)  before this  part  striatum  Crosby, Wurtz  nystagmus  with  the  of  termination  upper  ganglia  layers  in  behaviour.  1973). basal  function guided  arising  controlling  the  to  somatically  projections  (Kuypers,  by which  motor  and  suggested  the  in  as  an  movements the  tegmentum  integrated  Thus,  the  nigrotectal  ganglia  can  act  in  such  104  A MODEL OF THE BASAL GANGLIA I t i s o f t e n u s e f u l to formulate  models t o e x p l a i n t h e b e h a v i o u r of  complex systems, even i f those models appear premature and o v e r s i m p l i f i e d . Roberts (1976) has employed the concept o f d i s i n h i b i t i o n i n d i s c u s s i n g the f u n c t i o n s o f t h e b a s a l g a n g l i a . discovered and  I n view of t h e many r e c e n t l y  GABA pathways i n t h i s system such a view may be a p p r o p r i a t e ,  i t may be u s e f u l t o expand upon t h i s model.  As shown i n F i g . 17, t h e  s t r i a t u m r e c e i v e s i n p u t from t h e e n t i r e c e r e b r a l c o r t e x and from the i n t r a laminar  thalamus, which r e c e i v e s a f f e r e n t s from t h e r e t i c u l a r  formation.  Thus, the s t r i a t u m i s i n a p o s i t i o n t o r e c e i v e i n f o r m a t i o n o f a g e n e r a l nature regarding  t h e emotional and p h y s i c a l s t a t e , t h e l e v e l o f c o n s c i o u s -  ness and the degree o f a l e r t n e s s of the animal. The  s t r i a t u m a l s o r e c e i v e s an i n p u t from t h e dopamine c e l l s o f t h e SNC.  In a h i g h l y s i m p l i f i e d "requests" The  scheme t h i s i n p u t might be thought o f as p r o v i d i n g  t o the s t r i a t u m f o r c e r t a i n movements or b e h a v i o u r s to o c c u r .  s t r i a t u m c o u l d then compare these r e q u e s t s  with  the general  state of  the animal and make a " d e c i s i o n " as t o whether t o a l l o w t h e response o r n o t . T h i s response would then be sent out v i a the s t r i a t a l e f f e r e n t s . s t r i a t o - n i g r a l pathways c o u l d p r o v i d e  a feedback v i a t h e GABA and substance  P neurons to the dopamine c e l l s i n f o r m i n g or has not been made.  them t h a t the response e i t h e r has  I f t h e response has been made t h e s t r i a t o n i g r a l  GABA f i b e r s would i n c r e a s e t h e i r f i r i n g r a t e , w h i l e substance  P c e l l s would be decreased.  the a c t i v i t y o f the  T h i s would p r e v e n t f u r t h e r  a c t i v a t i o n o f t h e a p p r o p r i a t e dopamine c e l l s u n t i l again arose.  The  t h e need f o r t h e response  C e r t a i n SNR c e l l s would a l s o be i n h i b i t e d as w e l l .  These  GABA p r o j e c t i o n neurons, which may be t o n i c a l l y a c t i v e , would then decrease t h e i r f i r i n g r a t e and thereby d i s i n h i b i t c e r t a i n t e c t a l and t h a l a m i c a l l o w i n g t h e requested  response t o o c c u r .  units  T h i s may be thought of as the  105  d i s i n h i b i t i o n o f a pre-programmed n e u r o n a l c i r c u i t f o r p a t t e r n e d p o s t u r a l control,  n o r m a l l y h e l d i n a t o n i c i n h i b i t i o n by these GABA neurons.  I f o t h e r s t r i a t a l a c t i v i t i e s a r e i n c o m p a t i b l e w i t h the response r e q u e s t e d and i t i s n o t complied w i t h a d i f f e r e n t  r e s u l t would  ensue.  In t h i s case the s t r i a t o n i g r a l substance P c e l l s would i n c r e a s e t h e i r and t h e GABA c e l l s d e c r e a s e t h e i r r a t e of f i r i n g , a c t i v i t y of the n i g r a l dopamine c e l l s .  t h e r e b y i n c r e a s i n g the  T h i s would a l s o r e s u l t  d i s i n h i b i t i o n o f t h e n i g r a l p r o j e c t i o n neurons.  rate  i n the  They would t h e r e f o r e  more and a c t t o i n h i b i t the a p p r o p r i a t e t e c t a l and t h a l a m i c c e l l s  fire  thus  b l o c k i n g the response. Can t h i s scheme be r e l a t e d of the  the b a s a l g a n g l i a ?  t o what i s known r e g a r d i n g t h e a c t i v i t y  In P a r k i n s o n ' s d i s e a s e , or i n an animal  following  i n j e c t i o n o f n e u r o l e p t i c s , the dopaminergic i n p u t t o t h e s t r i a t u m i s  reduced.  I n t h i s c o n d i t i o n t h e r e i s d i f f i c u l t y i n i n i t i a t i n g movements.  In the p r e s e n t model the dopaminergic  s i g n a l s n e c e s s a r y f o r such  initia-  t i o n would be v e r y weak and t h i s would be a s s o c i a t e d w i t h a d e c r e a s e i n the a c t i v i t y of t h e s t r i a t o n i g r a l GABA f i b e r s a c t i v i t y o f the substance P system.  and an i n c r e a s e i n the  As a feedback system t h i s  would a c t t o i n c r e a s e the a c t i v i t y of the dopamine neurons. a l s o i n c r e a s e t h e a c t i v i t y of the n i g r o t e c t a l neurons  response  I t would  and n i g r o t h a l a m i c GABA  and thereby prevent the a p p r o p r i a t e response p a t t e r n s from b e i n g  initiated. In c o n t r a s t , d u r i n g times o f dopaminergic h y p e r a c t i v i t y , i . e. i n Huntington's d i s e a s e or f o l l o w i n g amphetamine a d m i n i s t r a t i o n , an inappropriate release of action patterns results. suggest t h a t t h i s nigral  The model would  i s due t o a r e l a t i v e l y g r e a t e r a c t i v i t y o f t h e s t r i a t o -  GABA neurons  as compared t o the substance P c e l l s .  This  response  would tend t o d e c r e a s e the a c t i v i t y o f t h e n i g r a l dopamine c e l l s i n an  106  attempt to r e t u r n the s t r i a t a l would a l s o r e s u l t  dopaminergic a c t i v i t y  to normal.  i n the i n h i b i t i o n of the n i g r o t e c t a l and  thalamic  GABA neurons.  thalamic  and  T h i s would r e s u l t  i n the r e l e a s e of  t e c t a l u n i t s c o n t r o l l i n g posture,  It  nigrothe  r e s u l t i n g i n inappropriate  movements. Although models such as t h i s may of the complexity  appear o v e r l y s i m p l i s t i c  i n view  of the b a s a l g a n g l i a , i t i s u s e f u l to attempt to  gain  a p e r s p e c t i v e on the f u n c t i o n of the b a s a l g a n g l i a as a whole.  Such  an overview o f t e n y i e l d s new  result  p r e d i c t i o n s and  i n f u r t h e r t e s t a b l e hypotheses. b a s i c anatomy, p h y s i o l o g y  and  Hopefully,  chemistry  i n s i g h t s which may  as f u r t h e r knowledge of  of the b a s a l g a n g l i a becomes  a v a i l a b l e a b e t t e r u n d e r s t a n d i n g of the f u n c t i o n s of t h i s system be p o s s i b l e .  the  will  107  REFERENCES A d i n o l f i , A.M. (1969) Degenerative changes i n the entopeduncular n u c l e u s f o l l o w i n g l e s i o n s of the caudate n u c l e u s : an e l e c t r o n m i c r o s c o p i c study. Exp. N e u r o l . 25, 246-254. A f i f i , A.K. and K a c l b e r , W.W. 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(1965) The c o r t i c o - s t r i a t a l p r o j e c t i o n i n the c a t . J . Anat. (Lond.) 99, 329-337. W h i t t i e r , J.R. and M e t t l e r , F.A. (1949) S t u d i e s on t h e subthalamus o f the rhesus monkey. J . Comp. N e u r o l . 90, 281-317. W i l s o n , C.J., Groves, P.M. and F i f k o v a , E. (1977) Monoaminergic synapses, i n c l u d i n g d e n d r o - d e n d r i t i c synapses i n the r a t s u b s t a n t i a n i g r a . Exp. B r a i n Res. 30, 161-174. W o l f a r t h , S., C o e l l e , E.-F., Osborne, N.N. and Sontag, K.-H. (1977) Evidence f o r a n e u r o t o x i c e f f e c t of a s c o r b i c a c i d a f t e r an i n t r a n i g r a l i n j e c t i o n i n the c a t . N e u r o s c i . L e t t . 6, 183-186.  133  Wolman, M. (1971) A f l u o r e s c e n t h i s t o c h e m i c a l t y r i c a c i d . H i s t o c h e m i e 28, 118-130.  procedure f o r gamma-aminobu-  Worms, P. and L l o y d , K.G. (1978) I n f l u e n c e of GABA-agonists and a n t a g o n i s t s on n e u r o l e p t i c - i n d u c e d c a t a l e p s y i n r a t s . L i f e S i c . 23, 475-478. Y e t e r i a n , E.H. and van Hoesen, G.W. (1978) C o r t i c o - s t r i a t e p r o j e c t i o n s i n the rhesus monkey: the o r g a n i z a t i o n of c e r t a i n c o r t i c o - c a u d a t e connections. B r a i n Res. 139, 43-63. York, D.H. and Faber, J . E . (1977) An e l e c t r o p h y s i o l o g i c a l study o f n i g r o t e c t a l r e l a t i o n s h i p s : a possible role i n turning behavior. B r a i n Res. 130, 383-386. Y o s h i d a , M. and Omata, S. (1978) B l o c k i n g by p i c r o t o x i n o f n i g r a - e v o k e d i n h i b i t i o n o f neurons o f v e n t r o m e d i a l nucleus o f t h e thalamus. E x p e r i e n t i a 35, 794. Y o s h i d a , M. and P r e c h t , W. (1971) Monosynaptic i n h i b i t i o n of neurons of the s u b s t a n t i a n i g r a by c a u d a t o - n i g r a l f i b r e s . B r a i n Res. 32, 225-228. Z i v k o v i c , B., G u i d o t t i , A. and Costa, E. (1974) E f f e c t s of n e u r o l e p t i c s on 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 : changes i n a f f i n i t y f o r t h e p t e r i d i n e c o f a c t o r . Moi. Pharmacol. 10, 727-735.  134  APPENDIX THE  SUBSTANCE P RADIOIMMUNOASSAY  (a)  P r e p a r a t i o n of t h e A n t i g e n Substance P i s a r e l a t i v e l y  s m a l l molecule and must t h e r e f o r e be  couple to a l a r g e r c a r r i e r molecule t o g i v e an a n t i g e n l a r g e enough f o r immunization.  In t h e f i r s t  r e p o r t of a radioimmunoassay  f o r substance P,  P o w e l l e t a l . (1973) coupled s y n t h e t i c substance P to b o v i n e with a carbodiimide.  y-globulin  I n the p r e s e n t study s y n t h e t i c substance P  (Beckman) was coupled t o bovine serum albumin f o l l o w i n g t h e method developed by G o o d f r i e n d e t a l . (1964). 1.90  Albumin  (3.72 g., b o v i n e RIA grade,  mg s y n t h e t i c substance P (Beckman) and 100 mg l - c y c l o h e x y l - 3 ( 2 - m o r -  p h o l i n o e t h y l ) - c a r b o d i i m i d e metho-p-toluene-sulphonate d i s s o l v e d i n t h i s o r d e r i n 0.5 ml d i s t i l l e d water room temperature  f o r one h r .  a g a i n s t d i s t i l l e d water (b)  Sigma),  ( A l d r i c h ) were  and a g i t a t e d g e n t l y a t  The g e l a t i n o u s m i x t u r e was then d i a l y s e d  f o r 24 h r a t 4°C and l y o p h i l i z e d .  Immunization The immunization procedure o f V a i t u k a i t i s e t a l . (1971) was used t o  produce  s p e c i f i c a n t i s e r a to substance P.  Our i n i t i a l  a n t i s e r a t o substance P i n r a b b i t s met w i t h l i m i t e d p i g s were used i n t h e p r e s e n t s t u d i e s .  Freund's complete  adjuvant (Cappel L a b s ) .  to r a i s e  s u c c e s s and so g u i n e a  For t h e i n i t i a l  mg of a n t i g e n was d i s s o l v e d i n t h r e e ml water  attempts  immunization one  and e m u l s i f i e d w i t h t h r e e ml  One ml o f t h i s emulsion was  i n j e c t e d per guinea p i g i n t r a d e r m a l l y i n 20-30 wheals on t h e back. r e c e i v e d a b o o s t e r i n j e c t i o n o f 100 yg a n t i g e n i n 200 y l water Freund's (c)  incomplete adjuvant  Animals  and 200 y l  (Cappel Labs) every f o u r weeks.  S c r e e n i n g of A n t i s e r a Sera were examined f o r t h e i r a b i l i t y  a f t e r each b o o s t e r .  to b i n d substance P two weeks  The guinea p i g s were b l e d by c a r d i a c p u n c t u r e w h i l e  135  under halothane  anesthesia.  The blood was  t u r e f o r one h r , then o v e r n i g h t at 4°C c e n t r i f u g a t i o n at 3,000 g f o r 15 min f r o z e n a t -20°C. and  Each a n t i s e r a was  f o r the a b i l i t y  the t r a c e r .  allowed  to stand a t room tempera-  to f a c i l i t a t e  clotting.  to remove c e l l s , checked  the s e r a were kept  for i t s a b i l i t y  of a s m a l l amount of u n l a b e l e d substance  A range of a n t i s e r a d i l u t i o n s  cubated w i t h about 5,000 cpm  r e p o r t e d h e r e , about 30%  With the a n t i s e r a used  of the t r a c e r was  to bind t r a c e r , P to d i s p l a c e  (1:100 t o 1:312,500) was i n -  of t r a c e r both with and w i t h o u t  one ng u n l a b e l e d substance P.  Following  an  i n a l l the  additional experiments  bound a t a s e r a d i l u t i o n of  1:62,500 and  65% of t h i s c o u l d be d i s p l a c e d by one ng of u n l a b e l e d  P  The  (Fig. I ) .  e l e d o i s i n , two  substance  c r o s s - r e a c t i v i t y of t h i s a n t i s e r a w i t h physalamen and  nonmammalian p e p t i d e s s t r u c t u r a l l y s i m i l a r to substance  P  w i t h the p e p t i d e s s o m a t o s t a t i n , b a c i t r a c i n , and l e u - or met-enkephalin l e s s than one p e r c e n t .  Although  o n l y one  substance  P would c o n t r i b u t e to t h i s immunoreactivity.  stance P l e v e l s r e p o r t e d i n these experiments s t a i n i n g should p r o p e r l y be termed substance (d)  P r e p a r a t i o n of ^ I - s u b s t a n c e 1  The  5  chloramine T procedure  and  the  P-like  T h e r e f o r e the subimmunohistochemical  immunoreactivity.  of Greenwood et a l . (1963) can be used  phenylalanine r e s i d u e i n p o s i t i o n eight without  was  (Beckman) i n 100  added to one mCi  (#9820).  The  y l of 500 mM  r e a c t i o n was  to  Substance P  be s u b s t i t u t e d f o r the  seriously affecting  a c t i v i t y of the p e p t i d e .  of I o d i n e 125  than  P  does not c o n t a i n a t y r o s i n e group, however, one may  substance P  obtain-  peptides other  i o d i n a t e p e p t i d e s or p r o t e i n s p o s s e s s i n g a t y r o s i n e r e s i d u e .  b i o l o g i c a l or r a d i o i m m u n o l o g i c a l  was  immunoreactive f r a c t i o n was  ed from t i s s u e e x t r a c t s chromatographed on Sephadex G-25,  and  Ten  the  yg of  sodium phosphate b u f f e r pH  (Amersham IMS-30) i n a pyrex  (Tyr )8  7.4  tube  s t a r t e d by the a d d i t i o n of 52 yg chloramine  T  136  F i g u r e I.:  The e f f e c t of s e r a d i l u t i o n able  1 2 5  bound;  I-substance  P bound,  on the amount of d i s p l a c e closed c i r c l e s =  open c i r c l e s = b i n d i n g i n the presence  ng u n l a b e l e d  substance  P.  total of one  137  (Sigma) i n 20 y l H 0.  A f t e r 12 sec the r e a c t i o n was  2  of 185  yg sodium m e t a b i s u l p h i t e i n 50 y l  The  i o d i n a t e d substance P was  Yalow and Berson f o l l o w e d by 10 mg  (1966).  One  H 0. 2  then p u r i f i e d a c c o r d i n g to the method of  ml of water was  of m i c r o f i n e s i l i c a  g f o r f i v e min and  added to the r e a c t i o n  (QUSO G32,  A f t e r s t a n d i n g f o r 10 min a t room temperature at 1,000  stopped by the a d d i t i o n  P h i l a d e l p h i a Quartz  the s u s p e n s i o n was  the supernatant d i s c a r d e d .  buffer.  was  substance P  a c e t i c a c i d , or one ml of the  Both e x t r a c t i o n procedures y i e l d e d about  both showed equal apparent b i n d i n g i n the absence l a b e l ) of 5%.  Co.).  centrifuged  The p e l l e t  washed f i v e times w i t h one ml of d i s t i l l e d water and the l a b e l e d then e x t r a c t e d i n t o one ml of 20% acetone-1%  mixture  0.2  mCi of t r a c e r ,  of a n t i b o d y  and  (damaged  Upon s t o r a g e a t 4°C the "damage l a b e l " of the t r a c e r  stored  i n b u f f e r i n c r e a s e d more than t h a t i n a c i d - a c e t o n e but e i t h e r t r a c e r u s a b l e f o r a t l e a s t 10 weeks. was (e)  always  experiments  Sample P r e p a r a t i o n  f o r f i v e min,  philized.  The sample was  b u f f e r immediately 1 2 5  dure the r e c o v e r y was  resuspended  20 volumes of 1.0  N acetic  The s u p e r n a t a n t was  acid min  then l y o -  i n i n an a p p r o p r i a t e volume o f assay i f n e c e s s a r y , spun to remove  sediment  added to the o r i g i n a l homogenate i n t h i s p r o c e -  g r e a t e r than  90%.  Assay  The assay b u f f e r was to pH 8.6.  50 mM  sodium b a r b i t a l t i t r a t e d w i t h a c e t i c  I t c o n t a i n e d an a n t i b a c t e r i a l agent  protease i n h i b i t o r albumin  g f o r 10 min.  b e f o r e assay and,  I - s u b s t a n c e P was  The  i n about  p l a c e d on i c e and homogenized, b o i l e d a g a i n f o r f i v e  and c e n t r i f u g e d at 1,000  (f)  t r a c e r used i n the p r e s e n t  prepared w i t h i n one month of use.  T i s s u e samples were b o i l e d  When  The  was  acid  (0.001% m e r t h i o l a t e ) , a  (500 KlU/ml a p r o t i n i n , Sigma) and 0.2%  bovine serum  (RIA grade, Sigma) t o minimize the l o s s o f substance P onto  the  133 s u r f a c e of the i n c u b a t i o n tubes.  The assay was  b o r o s i l i c a t e tubes  A standard curve was  (10 x 75 mm).  s e t up on i c e i n d i s p o s a b l e s e t up i n t r i p l i c a t e  w i t h c o n c e n t r a t i o n s of u n l a b e l e d substance P r a n g i n g from 31.25 e i g h t ng.  pg t o  D e t e r m i n a t i o n of the c o n c e n t r a t i o n o f substance P i n p r e l i m i n a r y  e x t r a c t i o n s a l l o w e d the samples to be assayed to be taken up i n an  appro-  p r i a t e volume o f b u f f e r such t h a t the c o n c e n t r a t i o n o f s u b s t a n c e P i n each l a y a p p r o x i m a t e l y a t the midpoint of the s t a n d a r d c u r v e . d i l u t e d over a t h r e e - f o l d range gave a dose-response  Assay o f  tissues  curve p a r a l l e l to the  standard curve. The f i n a l volume of each i n c u b a t i o n was  0.5  ml.  Antisera  b u f f e r t o g i v e a f i n a l c o n c e n t r a t i o n of 1:60,000) was g i v e 300  y l si and sample or standard i n 100  f i v e hr p r e i n c u b a t i o n at 4°C the l a b e l e d (about 5,000 cpm/tube) i n 100  1 2 5  y l was  mixed w i t h b u f f e r to  then added.  I - s u b s t a n c e P was  u l and the tubes l e f t  (diluted i n  After a added  a t 4°C f o r 48 h r .  To s e p a r a t e the t r a c e r bound to a n t i s e r a from f r e e t r a c e r , the coated c h a r c o a l technique was charcoal  used  (Herbert e t a l . ,  A s u s p e n s i o n of 10 g  ( N o r i t A n e u t r a l d e c o l o r i s i n g carbon) and 0.1  (Pharmacia) was  g Dextran  y l of the c h a r c o a l - d e x t r a n s u s p e n s i o n  added to each i n c u b a t i o n tube, and, a f t e r s t a n d i n g a t 4°C  the tubes were spun f o r 10 min at 1,000 supernatant was in a liquid  T-40  p r e p a r e d i n 10 ml of the assay b u f f e r w i t h o u t the a p r o t i n i n .  A f t e r the i n c u b a t i o n of the assay, 200 was  1965).  g.  A 500  sampled and counted f o r f o u r min  for five  min  y l a l i q u o t o f each i n 10 ml o f ACS  (Amersham)  s c i n t i l l a t i o n counter a t a r e l a t i v e e f f i c i e n c y o f 80%  as  determined by the channels r a t i o method u s i n g c h l o r o f o r m quenched standards of  1 2 5  I - s u b s t a n c e P.  the determined  The c o n c e n t r a t i o n o f substance P i n each sample  from the standard curve ( F i g .  To p r a c t i c a l s e n s i t i v i t y of the assay was of t r a c e r  was  II). d e f i n e d as 10%  ( e q u i v a l e n t to 50 fmoles of substance P per sample).  displacement A  substance  139  Figure I I .  I n h i b i t i o n of substance P;  1 2 5  I - s u b s t a n c e P b i n d i n g by u n l a b e l e d  the substance P s t a n d a r d  curve.  ma  140  P c o n c e n t r a t i o n o f 186 fmol/assay gave a 50% d i s p l a c e m e n t o f bound  tracer.  In the absence o f a n t i s e r a about f i v e p e r c e n t o f the t r a c e r remained i n the supernatant and t h i s blank  (damaged l a b e l ) was s u b t r a c t e d from a l l  samples. I t i s i n t e r e s t i n g t o compare the p r e s e n t assay w i t h the o r i g i n a l stance P radioimmunoassay r e p o r t e d by P o w e l l e t a l . (1973).  sub-  The s e r a  d i l u t i o n i n that r e p o r t was 1:8,000 compared w i t h 1:60,000 i n the p r e s e n t assay.  The "damage l a b e l " o r blank i n both assays was f i v e p e r c e n t ,  however, the s e n s i t i v i t y o f the p r e s e n t assay i s an o r d e r o f magnitude g r e a t e r than t h a t r e p o r t e d by P o w e l l e t a l . (1973). THE METHIONINE-ENKEPHALIN RADIOIMMUNOASSAY Rabbit a n t i s e r a t o m e t h i o n i n e - e n k e p h a l i n was o b t a i n e d from immuno N u c l e a r C o r p o r a t i o n , S t i l l w a t e r , Minnesota. at a f i n a l d i l u t i o n o f 1:1,000 i n the assay  The a n t i s e r a c o u l d be used (Fig. III).  Methionine-  e n k e p h a l i n l a b e l e d w i t h t r i t i u m on the t y r o s i n e r e s i d u e t o a s p e c i f i c a c t i v i t y o f 18.77 Ci/mmol (New England N u c l e a r ) was used as t r a c e r a t about 5,000 cpm p e r assay.  Unlabeled methionine-enkephalin  used a t c o n c e n t r a t i o n s from 78 pg to 10 ng f o r a s t a n d a r d t h i s assay two pmoles o f s y n t h e t i c met-enkephalin  (Sigma) was  ( F i g . I V ) . In  caused displacement o f  50% o f t h e bound t r a c e r , w h i l e t h e p r a c t i c a l l i m i t o f s e n s i t i v i t y was 0.5 pmoles, which gave a 10% d i s p l a c e m e n t .  T h i s i s s i m i l a r to the  s e n s i t i v i t y r e p o r t e d by o t h e r s ( S u l l i v a n e t a l . , 1977; Yang e t a l . ,  1977; Wesche e t a l . ,  1977).  The t i s s u e p r e p a r a t i o n and assay c o n d i t i o n s were i d e n t i c a l t o those desscribed above f o r the substance P assay, except t h a t the i n c u b a t i o n was i n p o l y p r o p y l e n e tubes as recommended by Yang e t a l . (1977). t r a c e r was counted i n a l i q u i d 35% f o r t r i t i u m .  The bound  s c i n t i l l a t i o n counter a t an e f f i c i e n c y o f  The c r o s s - r e a c t i v i t y o f the assay w i t h l e u c i n e - e n k e p h a l i n  1 4 1  Figure I I I .  The  e f f e c t of s e r a d i l u t i o n  on the amount of  d i s p l a c e a b l e H-methionine e n k e p h a l i n bound, 3  c l o s e d c i r c l e s = t o t a l bound; i n the presence enkephalin.  open c i r c l e s = b i n d i n g  of one ng u n l a b e l e d  methionine  Serum  Dilution  142  F i g u r e IV.  I n h i b i t i o n of H-meLhionine e n k e p h a l i n b i n d i n g by 3  u n l a b e l e d methionine e n k e p h a l i n standard  e n k e p h a l i n ; the curve.  methionine-  143  (Sigma) was found  to be l e s s than two p e r c e n t .  REFERENCES Goodfriend, T.L., L e v i n e , L. and Fasman, G.D. A n t i b o d i e s to b r a d y k i n i n and a n g i o t e n s i n : a use o f c a r b o d i i m i d e s i n immunology. S c i e n c e , 144 (1964) 1344-1346. Greenwood, F.C., Hunter, W.M. and G l o v e r , J.S. The p r e p a r a t i o n o f I l a b e l e d human growth hormone o f h i g h s p e c i f i c r a d i o a c t i v i t y . Biochem. J . , 89 (1963) 114-123. 1  2  5  H e r b e r t , V., Lau, K.-S., G o t t l i e b , CW. and B l e i c h e r , S.J. Coated c h a r c o a l immunoassay of i n s u l i n . J . C l i n . E n d o c r i n o l . , 25 (1965) 1375-1384. Mroz, E.A. and Leeman, S.E. Substance P, I n : "Methods of Hormone Radioimmunoassay", B.M. J a f f e and H.R. Behram, eds., Academic P r e s s , New York, (1979), pp. 121-137. P o w e l l , D., Leeman, S.E., T r e g e a r , CW., N i a l l , H.D. and P o t t s , J.T. J r . Radioimmunoassay f o r substance P. Nature (Lond.), New B i o l . 241 (1973) 252-254. S u l l i v a n , S., A k i l , H., Watson, S.J. and Barchas, J.D. A n t i b o d i e s to e n k e p h a l i n s : c o u p l i n g o f a n t i g e n s and a s p e c i f i c m e t h i o n i n e - e n k e p h a l i n radioimmunoassay. Commun. Psychopharmacol. 1 (1977) 605-610. V a i t u k a i t i s , J . , Robbins, J.B., N i e s c h l a g , E. and Ross, C T . producing s p e c i f i c a n t i s e r a w i t h s m a l l doses o f immunogen. E n d o c r i n o l . 33 (1971) 988-991.  A method f o r J. Clin.  Wesch, D., H o l l t , V. and Herz, A. Radioimmunoassay of e n k e p h a l i n s . R e g i o n a l d i s t r i b u t i o n i n r a t b r a i n a f t e r morphine treatment and hypophysectomy. A r c h . Pharmacol. 301 (1977) 79-82. Yalow, R.S. and Berson, S.A. P u r i f i c a t i o n o f ^-I-parathyroid hormone with m i c r o f i n e granules of p r e c i p i t a t e d s i l i c a . Nature (Lond.) 212 (1966) 357-358. 1  3  Yang, H.-Y., Hong, J.S. and Costa, E. R e g i o n a l d i s t r i b u t i o n o f l e u and met e n k e p h a l i n i n r a t b r a i n . Neuropharmacology 16 (1977) 303-307.  PUBLICATIONS Gardner, D.R. and Vincent, S.R., I n s e c t i c i d a l and neuroexcitant actions of DDT analogs on the cockroach, Periplaneta americana, B u l l . Environ. Contam. T o x i c o l . 20 (1978) 294-302. Vincent, S.R., Nagy, J.I. and F i b i g e r , H.C, Increased s t r i a t a l glutamate decarboxylase a f t e r l e s i o n s of the n i g r o s t r i a t a l pathway, Brain Research, 143 (1978) 168-173. Nagy, J . I . , Vincent, S.R., Lehmann, J . , F i b i g e r , H.C. and McGeer, E.G., The use of k a i n i c acid i n the l o c a l i z a t i o n of enzymes i n the s u b s t a n t i a nigra, Brain Research, 149 (1978) 431-441. Vincent, S.R., H a t t o r i , T. and McGeer, E.G., The n i g r o t e c t a l p r o j e c t i o n : a biochemical and u l t r a s t r u c t u r a l c h a r a c t e r i z a t i o n , Brain Research, 151 (1978) 159-164. Nagy, J . I . , Vincent, S.R. and F i b i g e r , H.C, Altered neurotransmitter synthetic enzyme a c t i v i t y i n some extrapyramidal nucei a f t e r l e s i o n s of the n i g r o - s t r i a t a l dopamine p r o j e c t i o n , L i f e Sciences, 22 (1978) 1777-1782. Vincent, S.R. and McGeer, E.G., Kainic acid binding to membranes of s t r i a t a l neurons, L i f e Sciences, 24 (1979) 265-270. McGeer, E.G., McGeer, P.L., H a t t o r i , T. and Vincent, S.R., K a i n i c a c i d neurotoxicity and Huntington's Disease. In: Advances i n Neurology, V o l . 23: Huntington's Disease. T.N. Chase, N.S. Wexler and A. Barbeau, eds., Raven Press, New York, (1979) pp. 577-591. McGeer, E.G., McGeer, P.L. and Vincent, S.R., Morphine, naloxone and k a i n i c acid n e u r o t o x i c i t y , Res. Comm. Chem. Path. Pharmacol. 25 (1979) 411-414. Vincent, S.R. and McGeer, E.G., S t r u c t u r a l s p e c i f i c i t y of high a f f i n i t y H-glutamate uptake i n rat striatum, J . Pharm. Pharmacol., 31 (1979) 703704. Vincent, S.R. and McGeer, E.G., A comparison of sodium-dependent glutamate binding with h i g h - a f f i n i t y glutamate uptake i n rat striatum, B r a i n Research, 184 (1980) 99-108. Nagy, J . I . , Vincent, S.R., Staines, W.A., F i b i g e r , H.C, R e i s i n e , T.D. Yamamura, H.I., Neurotoxic a c t i o n of c a p s a i c i n on s p i n a l substance P neurons, Brain Research, 186 (1980) 435-444.  and  Vincent, S.R., Kimura, H. and McGeer, E.G., The pharmacohistochemical demonstration of GABA-transaminase, Neurosci. L e t t . , 16 (1980) 345-348. Fibiger, H.C, Nagy, J . I . , Staines, W.A. and Vincent, S.R., Organization and p l a s t i c i t y of GABAergic neurons i n some extrapyramidal n u c l e i of the rat, Brain Research B u l l . , i n press (1980). Staines, W.A., Nagy, J . I . , Vincent, S.R. and F i b i g e r , H.C, Neurotransmitters contained i n the e f f e r e n t s of the striatum, B r a i n Research, i n press (1980).  -2Vincent, S.R., Lehmann, J . and McGeer, E.G., The l o c a l i z a t i o n of GABAtransaminase i n the s t r i a t r n i g r a l system, L i f e Sciences, i n press (1980), Vincent, S.R., Staines, W.A., McGeer, E.G. and F i b i g e r , H.C., Neurotransmitters contained i n the efferents of the habenula, Brain Research, i n press (1980).  

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